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PERSISTENCE OF THE HEPATITIS B VIRAL GENOME
IN SELECTED HEMATOPHAGOUS INSECTS

BY

Jamie Ann Blow

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Department of Entomology

1 998

ABSTRACT

THE PERSISTENCE OF THE HEPATITIS B VIRAL GENOME
IN SELECTED HEMATOPHAGOUS INSECTS

By

Jamie Ann Blow

Hepatitis B virus (HBV) infection is a major cause of acute and chronic
hepatitis, cirrhosis, and primary hepatocellular cancer, with an estimated 300
million carriers world wide. In China, Southeast Asia, sub-Saharan Africa, parts
of the Middle East, and South America the primary modes of transmission are
perinatal infection of infants, vertical transmission, and horizontal transmission,
however, 30-40% of people with hepatitis B virus have no identifiable risk factors.

Numerous researchers have investigated the possible role of
hematophagous arthropods in the transmission of hepatitis B virus under varying
conditions. All of the studies to date have examined the persistence of the
HBsAg as the detection method for the virus. The surface antigen is found on all
three viral particles produced by the virus, only one of which is infectious. Thus,
the detection and persistence of the HBsAg is not a measure of infectiousness
but an indicator that the arthropod ingested a potentially infectious blood meal.

The purpose of this research was to evaluate the persistence and
dissemination of the hepatitis B viral genome, by ingestion and inoculation, in
Cimex Iectularius L. (Hemiptera: Cimicidae), Rhodnius prolixus Stal (Hemiptera:
Reduviidae), Aedes trisen’atus (Say) (Diptera: Culicidae), Culex quinquefasciatus

Say (Diptera: Culicidae), and Anopheles stephensi Liston (Diptera: Culicidae).

The hepatitis B viral genome was detect by polymerase chain reaction with
confirmation by Southern blot. The sensitivity of these detection methods was
determined for each insect by serial dilution of a known serum with added insect
tissue. The virus was detectable for 48 hours in An. stephensi and 72 hours in
A9. trisen'atus ingesting the virus. Virus was not detected at 7 days in inoculated
mosquitoes. The vims persisted in the bodies and feces of C. Iectulan'us
ingesting a infected blood meal for 35 days. Virus was also detectable in low
levels for 21 days in bodies but not feces of inoculated C. Iectularius. Hepatitis B
virus also persisted trans-stadially through one molt in all instar stages, but trans-
ovarial transmision was not shown. Virus was detectable in inoculated R.
prolixus bodies only at 7 days.

The results obtained in this laboratory study indicates that mosquitoes, C.
Iectularius, and R. prolixus do not serve as biological vectors of the hepatitis B
virus. However, the persistence of the virus for varying time period in the bodies
and feces of the various insects does raise the question of potential for
mechanical transmission. Future studies need to examine if crushed insects and
defecated material from insects ingesting a infected blood meal are infectious to

susceptible hosts.

Copyright by
JAMIE ANN BLOW
1 998

This dissertation is dedicated to my parents, Donald and Jean Blow, who have
supported and encouraged me in all my pursuits.

ACKNOWLEDGMENTS

There are many people who have influenced me throughout my life. I would like
to thank those who have encouraged me to always do my best. First there are
my parents who have never discouraged me in any of my pursuits. They have
been there for the triumphs as well as the falls, always ready to lend words of
encouragement and moral support. Without them I would not be what and
where I am today.

Second, are the teachers I have had throughout my education. I owe a great
debt to Mrs. Janet Lutz, who helped turn a slow 3’‘1 grader around. By telling me
that “I can’t” never did anything and that “I can” can do anything if I try hard
enough. There were numerous other teachers and coaches who have
encouraged me throughout my public school education, to all of them I say thank
you.

I feel a strong attachment to Alma College where I did my undergraduate degree.
A school steeped in academic excellence and tradition, Alma taught me to think
outside the box. I have found this to be a valuable asset in the ensuing years. I
will always remember Alma for the academic challenge, the chance to play
college basketball, and most of all the deep friendships that still endure.

The Army has been a major influence in my life. From my time as an enlisted
soldier in Germany to ROTC to today as an officer, the Army has taught me
responsibility, integrity, and leadership. Thank you to everyone who has
influenced me during my career, I only hope that I can be a positive influence for
others as I continue my career.

I want to thank my major Professor, Dr. Edward D. Walker, for taking a chance
on someone who only had 3 years to do a PhD. Ned has taught me many things
and I am eager for the opportunity to implement them. Dr. Suzanne Thiem,
deserves a special thanks, for not only agreeing to be a committee member but
for being so patient in answering my questions about molecular techniques and
virology. Dr. Rich Merritt, well what can one say, thanks for everything Rich. Dr.
Michael Kron, thank you for your advice and suggestions. Dr. Celeste
Mazzacano, for all her help and critical reading of this dissertation. Dr. Ann
Silvennan, MD, William Beaumont Research Institute for allowing me to work on
her research project. Last but not least the office staff, Linda, Alice, Ballory,
Heather, Jan, Barb and Jill, without them I would never have gotten everything
done.

The AMEDD Long Term Health Education and Training program provided
academic funding throughout this program. Research funding came primarily
through a Hutson Grant, Department of Entomology, with additional support from
William Beaumont Research Institute Grant # Rl-96-15.

vi

TABLE OF CONTENTS

List of Tables ......................................... viii
List of Figures ......................................... x

List of Abbreviations .................................... xii
INTRODUCTION ...................................... 1
Hepatitis B ...................................... 1
Eitology ................................... 2

Epidemiology ............................... 12

Hepatitis and Arthropods ........................... 19

Arboviral Transmission ............................ 20

Erythrocyte Digestion ........................ 22

Vector Competency .......................... 24

Disease Vectors .................................. 28

Culicidae .................................. 29

Cimicidae ................................. 32

Reduviidae ................................ 37

Purpose of Research .............................. 41

Bibliography ..................................... 43

CHAPTER 1: PERSISTENCE OF THE HEPATITIS B VIRAL GENOME IN
THREE SPECIES OF DIPTERA: CULICIDAE

Introduction ..................................... 61
Materials and Methods ............................. 64
Results ........................................... 78
Discussion ...................................... 88
Literature Cited .................................. 91

CHAPTER 2: PERSISTENCE OF THE HEPATITIS B VIRAL GENOME IN
CIMEX LECTULARIU§ L. AND RHODNIUS PROLIXQS STAL

Introduction ..................................... 95
Materials and Methods ............................. 98
Results ........................................... 1 14
Discussion ...................................... 125
Literature Cited .................................. 128
CHAPTER 3: RESEARCH SUMMARY AND CONCULSIONS
Summary and Conclusion .......................... 131
Literature Cited .................................. 135
APPENDICES
Appendix A: DNAzoI Extraction Protocol ............... 138
Appendix B: Southern Detection Protocol .............. 140
Appendix C: Data ................................. 148
Appendix D: Human and Animal Use ................. 156
Appendix D: Entomology Voucher Specimens .......... 158

vii

LIST OF TABLES

INTRODUCTION

Table 1: Currently accepted nomenclature and abbreviations for viruses,

antigens, and antibodies associated with hepatitis B. ............... 4

Table 2: The risk of developing chronic hepatitis based on the

age of infection .............................................

Table 3: Geographic patterns of the prevalence of hepatitis B markers . .

CHAPTER 2

Table 1: Quantity of DNAzoI and Polycarrier gel added to each

sample type for extraction of DNA ..............................

CHAPTER 3
Table 1: Sensitivity of PCR and Southern blot detection methods

for hepatitis B viral genome ...................................

APPENDIX A

Table 1: Quantity of DNAzol and Polycarrier gel added to each sample

type for extraction of DNA ....................................

Table 2: Time and temperature to incubate samples ................

APPENDIX B

Table 1: Additionally required solutions for Southern blotting ..........

Table 2: Additionaly required solutions for DIG nucleic acid detection.... .

APPENDIX C

Table 1: Persistence of ingested HBV in bodies of An. stephensi

and Ae. tn'seriatus ...........................................

Table 2: Persistence of ingested HBV in bodies of An. stephensi and

Ae. trisen’atus ..............................................

viii

14

16

107

133

138

138

140

143

149

150

Table 3: Persistence of ingested HBV in feces of An. stephensi and
Ae. tn'seriatus .............................................. 151

Table 4: Persistence of ingested HBV in feces of An. stephensi and

Ae. trisen'atus .............................................. 152
Table 5: Transmission potential of An. stephensi during refeeding ..... 153
Table 6: Persistence of ingested HBV in C. Iectularius bodies ......... 153
Table 7: Persistence of inoculated HBV in C. Iectulan'us bodies ....... 153
Table 8: Persistence of ingested HBV in C. Iectulan'us feces ......... 154
Table 9: Transstadial transmission of ingested HBV in C. Iectularius . . . 154

Table 10: Persistence of inoculated HBV in Ff. prolixus .............. 155

ix

LIST OF FIGURES

INTRODUCTION

Figure 1: Hepatitis B viral particles .............................. 6
Figure 2: Hepatitis B viral genome .............................. 7
Figure 3: Mosquito Digestive System ............................ 25
CHAPTER 1

Figure 1: Diagram of Hemotek 5W1 artificial feeder ................. 67
Figure 2: Diagram of primer location in hepatitis B viral genome ....... 74

Figure 3: Determination of the effect of mosquito tissue on the
sensitivity of HBV detection using PCR .......................... 80

Figure 4: Determination of the effect of filter paper on the
sensitivity of HBV detection using PCR .......................... 81

Figure 5: The persistence of ingested hepatitis B virus in the bodies
of An. stephensi and Ae. tn'seriatus ............................. 84

Figure 6: Persistence of ingested hepatitis B virus in the feces of

An. stephensi and Ae. triseriatus ............................... 85
Figure 7: Persistence of HBV in 24 and 48 hour An. stephensi ........ 86
Figure 8: Persistence of HBV in 48 and 72 hour An. stephensi ........ 87
CHAPTER 2

Figure 1: Diagram of Hemotek 5W1 artificial feeder ................. 101
Figure 2: Diagram of primer location in hepatitis B viral genome ....... 108

Figure 3: Determination of the effect of bed bug tissue on the
sensitivity of HBV detection using PCR .......................... 115

Figure 4: Determination of the effect of kissing bug tissue on the
sensitivity of HBV detection using PCR .......................... 116

Figure 5: Determination of the effect of filter paper on the
sensitivity of HBV detection using PCR .......................... 117

Figure 6: Persistence of ingested and inoculated hepatitis B virus
in C. Iectulan’us ................................................ 119

Figure 7: Persistence of ingested HBV in day 28 and 35
C. Iectulan’us bodies ......................................... 120

Figure 8: Persistence of ingested hepatitis B virus
in the feces of C. Iectulan’us .................................. 122

Figure 9: Persistence of HBV in day 14, 21, 28 and 35
C. Iectulan'us feces .......................................... 123

Figure 10: Trans-stadial transmission of HBV ..................... 124

xi

DNA
HBV
HBsAg
HBeAg
HBcAg
HBIG
Anti-HBs
Anti-HBc
Anti-HBe
HBV-DNA
ORF
pgRNA
PCR
PHC
RNA

LIST OF ABBREVIATIONS

deoxyribonucleic acid

Hepatitis B virus

Hepatitis B surface antigen
Hepatitis B e antigen

Hepatitis B core antigen

Hepatitis B immune globulin
Antibody to hepatitis B surface antigen
Antibody to hepatitis B core antigen
Antibody to hepatitis B e antigen
DNA of HBV

Open reading frame

pregenomic RNA

Polymerase chain reaction

Primary hepatocellular cancer

ribonucleic acid

xii

INTRODUCTION

HEPA11TIS B

Hepatitis is a disease of antiquity. Hippocrates recognized epidemic jaundice
in the fourth century BC. and incidences of jaundice possibly caused by viral
hepatitis are described in the Babylonian Talmud (Purcell 1985, Krugman 1989).
The contagious nature of jaundice was recognized as early as the eight century
AD. in Europe. The first recognized epidemic of what was probably hepatitis B,
began in Bremen, Germany, in October, 1883, and ended in April, 1884 (Purcell
1985, Krugman 1989). The Bremen epidemic is interesting not only because it
was the first report of presumed hepatitis B, but it also pointed to parenteral
exposure to blood or blood components as an important vehicle of transmission.

During the first half of the twentieth century, outbreaks of "long incubation
period" hepatitis were observed in many countries of the world among: patients
who attended clinics for venereal disease, diabetes, and tuberculosis; persons
who received blood transfusions; persons who were inoculated with mumps or
measles convalescent serum; and military personnel who received yellow fever
vaccine during World War II (Krugman 1989). The etiology of viral hepatitis was
established in the early 1940's through transmission studies in volunteers
(MacCallum 1947). More detailed studies followed which revealed that there
were two types of hepatitis virus, original identified as serum hepatitis (hepatitis
B virus) and infectious hepatitis (hepatitis A virus).

The modern era of viral hepatitis research began with the description by
Blumberg et al. (1965) of an isoprecipitant in the serum of Australian aborigines,

1

termed Australia antigen, that was later shown by Prince (1968) and others to be
related to the hepatitis B virus. The discovery of Australia antigen, now called
hepatitis B surface antigen (HBsAg) (See Table 1 for current Nomenclature), and
its association with post transfusion hepatitis resulted in the expansion of
hepatitis research.

Dane et al. (1970) discovered virus like particles, about 42 nm in diameter, in
multiple serum specimens from three Australia-antigen-positive hepatitis
patients. He suggested that these particles were complete virus particles, HBV,
and that the much more numerous 22 nm particles, HBsAg, were surplus viral
coat material.

Etiology. Hepatitis B is a unique partially double-stranded DNA virus which has
been classified as a hepadnavirus (Ganem and Varrnus 1987), a group which
includes woodchuck hepatitis virus (W HV) (Summers et al. 1978), Pekin duck
hepatitis B virus (DHBV) (Mason et al. 1980), and Beechy ground squirrel
hepatitis virus (GSHV) (Marion et al. 1980). Hepadnaviruses are typically
restricted to species closely related to their natural host. The barrier to cross
species infection is the lack of appropriate receptors to the virus (Galle et al.
1988, Shih et al. 1989). The only natural host for hepatitis B virus appears to be
humans, although several higher nonhuman primates, such as, the chimpanzee
(the primary experimental model) the gibbon, and the gorilla have been shown to
be susceptible to persistent infection in the laboratory (Hadler and Margolis
1989)

Clinical Manifestation. Although persistent or chronic virus infection is a
minority outcome of primary infection, it is important for several reasons.

2

Persistent infection with hepatitis B virus often results in morbidity and mortality
(Ganem 1996). The incubation period of hepatitis B is long (45-160 days;
average 120) and the onset of acute disease develops so gradually that infection
is well established before becoming apparent symptomatically (CDC 1990).
Clinical manifestations of infection vary greatly, with many patients being
relatively symptom-free (“asymptomatic carriers"), while others have varying
grades of chronic liver injury and inflammation (chronic hepatitis B). Acute
hepatitis is subclinical in about two thirds of the cases and most people with
chronic liver disease never experience symptoms of acute hepatitis with jaundice
(Hollinger 1996). These asymptomatic carriers may remain healthy for years, or
the disease may progress rapidly to cirrhosis and death. Asymptomatic carriers
are the primary epidemiological reservoir for the spread of hepatitis B vian to
susceptible hosts (Ganem 1996). In those with clinical illness the onset of is
usually marked by anorexia, vague abdominal pain, nausea and vomiting, and
occasionally a rash. The symptoms may progress to jaundice and a mild fever.
Chronic liver disease occurs in about 1-8% of adult hepatitis B patients (Redeker

1975, Hoofnagle et al. 1978, Beasley et al. 1983).

 

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Particle Types. Hepatitis B virus is unusual among animal viruses in that
infected cells produce multiple types of vims-related particles (Bayer et al. 1968,
Dane et al. 1970, Gerin et al. 1971, Kim and Tilles 1973, Robinson and Li 1976).
Ganem (1996) characterized hepatitis B virus as having three types of particles:
(a) 42 to 47-nm double shelled infectious particles (Dane particles or HBV), (b)
22-nm spheres, and (c) smaller quantities of filaments of 22-nm diameter and
variable length. The hepatitis B virus outer shell is a lipoprotein envelope
containing the viral surface glycoproteins, originally detected serologically as
HBsAg (Blumberg et al. 1965). The 22-nm spheres and filaments are composed
exclusively of HBsAg and host-derived lipid; their principal lipids include
phospholipids, cholesterol, cholesterol esters, and triglycerides (Peterson 1981,
Gavilanes et al. 1982, Aggerbeck and Peterson 1985, Peterson 1987). The 42-
nm diameter virion consists of an envelope and a nucleocapsid containing a
circular 3200 nucleotide DNA molecule, a DNA polymerase, a protein kinase
activity and a 'DNA-linked protein' (T iollais et al. 1985). The envelope carries the
HBsAg and the capsid, the hepatitis B core antigen (HBcAg) (Figure 1). The
presence of complete virions in the serum is indicative of active viral
multiplication in the liver (T iollais et al. 1985).

Both complete HBV (Dane) particles and incomplete particles (HBsAg 22 nm
spheres and filaments) are found in serum during the course of acute and
chronic infection. The HBV particle, infectious particle of hepatitis B virus, titer
ranges from less then 104/ml to greater than 109/ml (Blumberg et al. 1965). The
concentration of non-virion associated HBsAg (spheres and filaments) greatly
exceeds the concentration of complete virions by 10‘ or greater.

5

HBsAg

 

 

 

A.
Sphere
B.
L m... J
Dane Particle
Tubulc

Figure 1. Hepatitis B viral particles. A) HBV DNA particle, envelope carries
the HBsAg and the capsid, the hepatitis B core antigen (HBcAg) and viral DNA.
B) 22 nm spheres and tubules consisting of HBsAg and host derived lipid.

Viral Genome. Hepadnavirus genomes consist of small (3000-3300
nucleotides), circular DNA that is partly single stranded (Tiollais et al. 1981,
Summer 1984, Hadler and Margolis 1989). Although the viral DNA is circular,
both strands of the duplex are linear, and the circular conformation is maintained
by base pairing between 250 to 300 nucleotides located at the extreme 5' ends
of the two strands. The minus strand is uniform in length (about 3200
nucleotides) and has a protein covalently bound at its 5' end (Gerlich and

Robinson 1980).

2.1kb RNA

9'0'81 WG-82

 

0-7 kb RNA

Figure 2. Hepatitis B viral genome. The inner circle represents virion DNA,
with dashed lines signifying single-stranded regions. Boxes denote DNA codeing
regions, arrows, indicating the direction of translation. Wavy lines depict viral
RNA detected in cells (Ganem 1996).

The replication mechanism of hepatitis B virus, discovered by Summers and
Mason (1982) for duck hepatitis B virus and confirmed later for human hepatitis
B virus (Summer and Mason 1982, Blum et al. 1984, Fowler et al. 1984, Miller et
al. 1984a, Miller et al. 1984b), differs strikingly from that of other DNA viruses.
The central feature is the use of a RNA copy of the genome as an intermediate

in replication. The current model for HBV replication (T iollais et al. 1985)

consists of the following steps: (1) the virus penetrates a hepatocyte, the DNA
reaches the nucleus and is converted to an open, circular, double-stranded DNA
and then to supercoiled DNA; (2) the minus strand is transcribed by the cellular
RNA polymerase to a 3.5 kb pre-genomic (pg) RNA; (3) the pgRNA is
encapsulated with the DNA polymerase and the DNA-linked protein; (4) the
minus strand DNA is then synthesized by reverse transcription of the pgRNA,
with a covalently bound DNA-linked protein at 5' end of the minus strand acting
as a primer. Hepatitis B virus DNA in the hepatocyte can exist free or integrated
into the host cellular chromosome (Brechot et al. 1982, Shafritz 1982). Free
hepatitis B virus DNA, which represents intermediate forms of replication, is
detected during the acute and chronic stages of hepatitis B virus infection

(T iollais et al. 1985).

The HBV genome has four overlapping open reading frames (ORF) that code
for four genes. The polymerase (P) gene encodes a protein that has four
different activities: the terminal protein, reverse transcriptase, RNase-H, and
HBV DNA polymerase (Thomas and Carman 1994). The core (C) gene contains
two initiation codons that encode the structural proteins of the nucleocapsid
(Pasek et al. 1978). The first initiation codon product is the 25-kd precore/core
protein that produces the HBeAg. The second initiation codon product is the 21 -
kd core protein, that forms the viral nucelocapsid with the HBV DNA (Thomas
and German 1994). The X gene’s exact function is currently undefined. The
surface (S) gene contains three in frame ATG’s that produce the three envelope

proteins, designated as HBsAg. The two larger proteins have either pre-S1 and

pre-S2 regions or only a pre-S2. The pre-S1 appears to be involved in binding to
the hepatocytes (Thomas and Carman 1994).

Cell and Tissue Affinity. Hepatitis B virus enters the circulatory system by
direct inoculation, passage through mucous membranes, or through breaks in
the integument, and is then carried to the hepatocytes, where primary replication
occurs (Hadler and Margolis 1989). An important step in tissue affinity is the
binding of hepatitis B virus envelope proteins to specific receptors on the
hepatocytes, however, the mechanism for attachment and penetration has not
been elucidated (Neurath et al. 1986, Leender et al. 1992, Hertogs et al. 1993).
Intensive investigations have focused on the pre-S1 sequence of the large
HBsAg gene product for attachment and penetration of hepatitis B virus into the
hepatocyte plasma membranes (Neurath et al. 1986, Pontisso et al. 1989,
Pontisso et al. 1991, Leender et al. 1992, Hertogs et al. 1993). It has been
postulated that hematopoietic cells may initially support viral replication (Ganem
1996). Species-specific receptors have been recognized on hepatocytes,
monocytes, lymphocytes, fibroblasts, and in spleen tissue (Michalak et al. 1991,
Pontisso et al. 1991, Franco et al. 1992, Leender et al. 1992, Neurath et al.
1992). The absence of the HBsAg receptor precludes the virus from gaining
access to the cell and thus replicating.

Pathogenicity. Although HBsAg has been detected in a wide variety of body
fluids, only semen, serum, and saliva have been demonstrated to be infectious
(Alter and Mat 1994). To fully comprehend the infectiousness of the blood of an
HBeAg positive carrier, it is important to realize that each milliliter of blood may
contain greater than 108 infectious virions (Hollinger 1996). Hepatitis B virus

9

circulates at approximately 10‘° virions per milliliter in the blood of an acutely
infected or early-stage chronic carrier (Hadler and Margolis 1989). The quantity
of viral particles at infection influences the incubation time to the onset of
disease (Hollinger 1996). The greater the quantity of virus at infection the
shorter the time before the onset of disease. The number of virus particles
present even in small amounts of blood serum or other body fluids can be quite
high. One picogram of HBV-DNA contains approximately 2.9 x 105 genomic
copies of the virus per milliliter or 290 copies/ul (Hollinger 1996). Inoculation with
one microliter of infectious blood may cause an infection (Alter 1982).

The stability of HBV does not always coincide with that of HBsAg. Various
methods of inactivating the virus, such as ether, sodium hypochlorite, isopropyl
alcohol, heating, and autoclaving have all been examined. HBV retained its
ability to induce an immune response (immunogenicity) and to interact
specifically with free antibody and/or with antigen-binding receptors on
lymphocytes (antigenicity) after exposure to ether, acid (pH 2.4 for at least 6
hours), and heat (98 degrees C for 1 minute; 60 degrees C for 10 hours)
(Shikata et al. 1976, Bond et al. 1977, Hollinger 1996). However, hepatitis B
virus inactivation may be incomplete under these conditions if the concentration
of virus is excessively high. Hepatitis B virus has been shown to retain infectivity
when stored at 30 to 32 degrees C for at least 6 months, 60 degrees C for 4
hours, and frozen at -20 degrees C for 15 years (Bond et al. 1977, Hollinger
1 996) .

Diagnosis. Serological tests allow for diagnosis of hepatitis B infection and
differentiation between acute and chronic disease by serological markers, whose

10

appearance and disappearance signals a change in the clinical stage of the
disease. A serum sample positive for HBsAg indicates that an active hepatitis B
virus infection is present (Hollinger 1996) but does not determine the stage or
infectivity of the host. In acute hepatitis B, HBsAg is detectable in the serum and
usually persists through convalescence. The presence of HBeAg correlates with
viral replication in the liver and is detectable in the early course of acute hepatitis
B. As acute hepatitis B resolves, HBsAg and HBeAg become undetectable and
neutralizing antibodies, anti-HBs, appear. The presence of anti-HBs is
associated with the resolution of infection and correlates with immunity.
However, the persistence of HBsAg and HBeAg in serum suggests a transition
from acute to chronic hepatitis infection and indicates continuing viral replication.
Patients who are HBeAg positive are more likely to transmit hepatitis B sexually,
percutaneously, or perinatally.
Serological Assays. A number of serological assays are available
commercially to detect the hepatitis B virus and aid the clinician, epidemiologist
or researcher in differentiating acute from chronic infections, evaluating relative
infectivity and prognosis, and assessing the immune status of the patient.
Radioimmunoassays (RIA) and enzyme-linked immunoassays (EIA) are the
most sensitive and specific of the various commercially licensed assays
available. These assays, performed both as a “sandwich” and competitive
binding assays, can detect HBsAg in the 0.25 to 0.5 ng/ml range (Hollinger
1996).

The infectiousness of blood or body fluids of a HBsAg positive patient is
determined by testing for HBeAg, anti-HBe, or HBV DNA. HBeAg positive

11

samples may contain up to 10° infectious virions per milliliter of blood (Krugman
et al. 1974, Krugman et al. 1979, van Ditzhuijsen et al. 1985, Hollinger1996),
whereas, patients who are anti—HBe positive circulate fewer than 100 infectious
virions per milliliter of blood.

The polymerase chain reaction (PCR) technique is a method for amplifying
nucleic acid by repeated cycles of high temperature denaturization,
oligonucleotide primer hybridization, and DNA polymerase extension. A PCR
based assay is used for the detection of hepatitis B virus DNA in serum and
tissue and can detect as few as 250 HBV DNA molecules/ml (Hollinger 1996).
Due to its greater sensitivity and detection of specific nucleotide sequences of
the viral genome, PCR has been important in elucidating the intricate
immunopathogenic mechanisms of hepatitis B virus.

Epidemiology

Hepatitis B infection is a major cause of acute and chronic hepatitis, cirrhosis,
and primary hepatocellular carcinoma (PHC) world wide (CDC 1990). It is
estimated that there are as many as 300 million carriers world wide and an
estimated 2 million people are killed each year by this virus (Hamilton 1994).
There are three main outcomes when an individual is infected with the hepatitis
B virus: (1) symptomatic acute hepatitis with development of anti-HBs antibody in
the blood leading to long term immunity; (2) a chronic carrier state of HBV; (3)
asymptomatic infection leading to either immunity or a chronic carrier state (Kane
1995). It is estimated that 6-1 0% of acute HBV infections become chronic

(Mehaeus 1995).

12

Persons at high risk for exposure to the hepatitis B virus are: intravenous
drug users; sexually active homosexual and bisexual men; sexually active
heterosexual men and women; inmates of long term correction facilities; health
care and public safety workers who are exposed to blood and bodily fluids;
transfusion recipients; hemodialysis patients; household contacts and sexual
partners of HBV carriers; and international travelers. An important feature of the
epidemiology of hepatitis B in the United States is that 30% to 40% of patients
cannot be definitively associated with an identifiable risk factor, which places
them out of reach of immunization strategies that target high risk groups (Alter
and Mat 1994).

Incidence. The frequency of hepatitis B infection and patterns of transmission
vary markedly in different parts of the world. It is estimated that 300 million
people across the globe carry the hepatitis B virus (Kuhns 1995) with three
million cases of acute viral hepatitis diagnosed each year in Africa alone (Kuhns
1995). The number of reported cases of hepatitis B is only a fraction of the
actual cases, since symptomatic cases make up only 33-50% of all hepatitis B
virus infections (Mehaeus 1995). To obtain an estimate of the actual number of
cases the reported number of cases must be multiplied by a factor between 2
and 10 depending on the country.

Beasley et al. (1982) conducted a study of 1,510 Chinese preschool children
(1-4 years of age) and found that 118 (7.8%) were HBsAg positive and 122
(8.1%) were anti-HBs positive at enrollment. A year later 33 of the susceptible
children had become HBsAg positive and 65 anti-HBs positive. In a subsequent
follow up of 25 of the 33 HBsAg positive children, 17 (68%) were still positive.

13

The study found there was no difference in incidence of infection in relation to
age but among children infected at older ages fewer children became carriers
among (Table 2). Beasley et al. (1982) also noted a strong correlation between
HBV markers in the child and the HBsAg positive mothers. Infants born to HBV
carrier mothers had a 95% incidence of becoming infected with HBV (Beasley et

al. 1981).

Table 2: The risk of developing chronic hepatitis based on the age of infection
Baesle et al. 1982, Hadler and Mr-

  
 
  

 

 

 

 

e of Chroilctin

     

         

e of Infection j _ Percenta

 

 

 

 

 

 

 

Infant 90-95 %
1-5 25-50%
Older children 5-10%
Adults 1 -8%

 

 

Prevalence. The highest prevalence of hepatitis B virus infection is found
among people born in areas of high endemicity and their descendants,
intravenous drug users, and homosexual men (Alter and Mat 1994). The
endemicity of hepatitis B infection is considered high in those areas of the world
where the prevalence of HBsAg is 8-15% and where 70-90% of the population
have serological evidence of previous infection (Table 3) (Hadler and Margolis
1989)

Public Health Implications. Primary hepatocellular carcinoma (PHC) is one of
the most common cancers in Asia and sub-Saharan Africa (Beasley 1988).

Epidemiological evidence has established a strong association between chronic

14

HBV infection and PHC (Rogers et al. 1997). Hepatitis B virus is the etiological
agent for 70% - 90% of the cases of PHC world wide (Beasley 1988, Benenson
1995). An estimated 15-25% of people with chronic HBV infection die
prematurely of either cirrhosis or PHC (Alter and Mat 1994).

Modes of Transmission. The efficiency of the various mechanisms of hepatitis
B transmission is related to the amount or titer of circulating infectious virus in
the blood of infected persons in either the acute phase of infection, or in chronic
HBsAg carriers positive for hepatitis B e antigen (HBeAg) (Favero et al. 1979).
Mechanisms of transmission, in order of efficiency, are: direct percutaneous
inoculation by needle of contaminated serum or plasma; percutaneous transfer
of infective serum or plasma in the absence of overt needle puncture;
contamination of mucosal surfaces by infective serum or plasma; contamination
of mucosal surfaces by infective secretions other than serum or plasma; and,
transfer of infective material via vectors or inanimate environmental surfaces
(Favero et al. 1979). Perinatal and transplacental transmission may represent
the most important mechanism for the maintenance of a carrier population in
certain regions of the world (Wright et al. 1970, Merrill et al. 1972, Ohbayashi et
al. 1972, Schweitzer et al. 1973, Mazzur et al. 1974, Schweitzer 1975). Women
with acute hepatitis late in pregnancy or during the neonatal period readily infect
their infants (Mirick et al. 1965, Merrill et al. 1972, Schweitzer 1975). The infants
are usually HBsAg negative at birth but develop antigenemia within 1 to 3
months (Hollinger1996). This time period corresponds to the incubation period
for HBV if the infection occurred during delivery, although the method of
transmission during delivery is unknown. However, HBsAg has been detected

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in cord blood, amniotic fluid, breast milk and vaginal secretions as well as
maternal blood (Lennemann and Goldberg 1974, Papaevangelou et al. 1974,
Boxall 1975, Schweitzer 1975, Stevens et al. 1975, Derso et al. 1978, Lee et al.
1978) to that transmission could occur when the placental barrier breaks down,
via amniotic fluid, minor abrasions, or mucous membranes.

Person to person transmission of HBV can occur in settings involving
interpersonal contact over a long period of time via inapparent contact of
infective secretions with skin lesions or mucosal surfaces (CDC 1990). Foster et
al. (1984) found that exudates from skin lesions to be HBsAg positive and a
history of skin disease is known to be a risk factor for infection (Bemier et al.
1982). In China, Southeast Asia, sub-Saharan Africa, parts of the Middle East,
and South America, the primary modes of transmission are perinatal infection of
infants from HBeAg positive mothers, vertical transmission, and horizontal
transmission between children. Bond et al. (1981) found that inanimate objects
contaminated with hepatitis B virus could contribute to disease transmission for
periods of time up to one week and possibly longer. Tattooing, circumcision, ear
piercing and bites of hematophagous arthropods have also been proposed as
mechanisms of transmission although supporting evidence is weak (Fox et al.
1988). The HBsAg has been found to be very stable on surfaces and could be
readily detected in an environment contaminated with blood or fluid containing
high levels of surface antigen (Favero et al. 1973). The presence of HBsAg on a
surface indicates contamination with infectious blood at some previous time;

however, it does not prove the presence of viable HBV (Bond et al. 1977).

17

Methods of Control. The most effective control measure is to immunize
susceptible populations. There are currently two inactivated vaccines licensed
in the United States and Canada, both of which have been shown to be safe and
effective in protecting against all serotypes of the hepatitis B virus. The vaccines
are currently part of the childhood immunization program (CDC 1990) and are
commonly administered in 3 injections over the course of 6 months (Benenson
1995). The vaccine is highly immunogenic in neonates, children, and
immunocompetent adults with an immune response rate of 95-97.5% (Rogers et
al. 1997). In persons not responding to the initial vaccine series, approximately
40% will acquire anti-HBs after revaccination with another three shot series.
Only wide scale immunization of Infants and children can reduce the
incidence of disease in endemic areas of the world. Infants who are infected
vertically or perinatally have the greatest risk of becoming a carrier. The
administration of hepatitis B immune globulin (HBIG) after birth is approximately
70-80% effective in reducing infant carrier rates, however, 20-25% of infants
treated with HBIG became carriers (Beasley et al. 1981, Beasley et al. 1983).
The administration of the hepatitis B vaccine to infants treated with HBIG
increased the efficacy to 94% in the prevention of perinatal transmission of
hepatitis B and in providing long term immunity (Beasley et al. 1983). The World
Health Organization (WHO) (1997) has recommended the addition of hepatitis B
vaccine in the national immunization programs for routine infant and/or
adolescent immunization. In other areas with low prevalence, the initial strategy
was the immunization of high risk groups with the first available vaccine.
However, immunization of high-risk adults was not feasible and many of these

18

countries have begun to implement routine infant and adolescent immunization.
Other methods of stopping the spread of HBV include: screening of all donated
blood for the HBsAg; use of disposable needles and syringe or sterilization of
reusable equipment; routine immunization of all persons falling into a high risk
group; screening of all pregnant women; and surveillance for post-transfusion
hepatitis.
HEPATITIS AND ARTHROPODS

Neefe (1949) was the first to consider the possibility that heamtophagous
arthropods could transmit the hepatitis virus. Prince (1970) found an increased
prevalence of SH (hepatitis B surface antigen) antigenemia in the tropics but was
unable to account for this by known mechanisms of transmission, suggesting
transmission in nature by nonparenteral routes. The subsequent detection of the
hepatitis B surface antigen (HBsAg) by solid-phase radioimmunoassay in wild-
caught mosquitoes supported this hypothesis (Prince et al. 1972). The detection
of the hepatitis B surface antigen in wild caught mosquitoes (Prince et al. 1972,
Blumberg et al. 1973, Brotman et al. 1973, Dick et al. 1974, Wills et al. 1976),
Cimex spp. (Newkirk et al. 1975, Wills et al. 1977, Jupp et al. 1978, Jupp and
McElligott 1979, Ogston et al. 1979, Jupp et al. 1980, Ogston and London 1980,
Taylor and Morrison 1980, Jupp et al. 1983, El-Masry and Kotkat 1990), tampan
tick, Omithodoros moubata Murray, (Joubert et al. 1985, Jupp et al. 1987) and
Hhodnius prolixus Stal (Villarejos et al. 1975) implicated these arthropods as
potential vectors. Numerous studies have since been conducted utilizing both

wild collected and laboratory-fed arthropods. However, all of these studies used

19

detection assays for the HBsAg, which is not indicative of viral replication or
antigenicity.

Arboviral Transmission. Hundreds of species of hematophagous arthropods
transmit viral, bacterial, protozoan, and filarial microogansims pathogenic to
humans and animals. The relationship between arthropods and microorganisms
has been, and still is, the subject of intensive research to define factors
responsible for making the arthropod an efficient vector of the microorganism.
The lntemational Catalogue of Arboviruses (Karabatsos 1985) lists over 500
viruses that are definite, probable, or possible arboviruses. The World Health
Organization (1985) based its definition of an arbovirus on the ecological
interrelationships of virus, vertebrate host, and arthropod:

"arboviruses are viruses which are maintained in nature principally, or to an
important extent, through biological transmission between susceptible vertebrate
hosts by hematophagous arthropods or through transovarian and possibly
veneral transmission in arthropods; they multiply and produce viremia in the
vertebrates, multiply in the tissues of arthropods, and are passed on to new
vertebrates by the bites of arthropods after a period of extrinsic incubation.“
This definition does not include viruses that are transmitted mechanically since
they do not multiply in the arthropod during transmission. Arboviruses vary
greatly in size and structure and belong to various families and genera, although
most have RNA genomes and enveloped virions (Woodring et al 1996). They
are united by their ability to replicate in hematophagous arthropods, to infect a
vertebrate host when the arthropod vector feeds on that host, and to replicate in
the vertebrate producing viremia (WHO 1985).

Arthropod-bome diseases have the following characteristics: the infectious

agent is found in the blood of an infected vertebrate host; the agent can be

20

transmitted from the host to an arthropod in a blood meal; the arthropod can
transmit the agent to another host; and, an effective vector must in some way
support the pathogen it is transmitting (Harwood 1981). The relationship
between arthropod and pathogen depends to a great extent upon the nature of
the pathogen. It is not necessary for the pathogen to multiply within the vector,
but it must be stabilized for a sufficient time to allow the vector to transport it to a
susceptible host (Harwood 1981 ).

Two methods of transmission are recognized, mechanical and biological.
Mechanical transmission occurs when the vector is merely a carrier, usually via
contaminated mouthparts, from infectious source to a susceptible host [e.g.,
equine infectious anemia by stable flies (Hawkins et al., 1976)]. Biological
transmission occurs when the pathogen multiplies in the vector and is
subsequently transmitted following the invasion of the salivary glands (e.g.,
yellow fever virus by Aedes mosquitoes) (Mitchell 1983) or excreted in the feces
during feeding (e.g., Trypanosoma cruzi by Triatoma). Biological and mechanical
transmission are not mutually exclusive nor mutually inclusive. Vectors that
transmit viruses biologically may be capable of mechanical transmission for a
period immediately after the blood meal.

Hanlvood (1981) found that frequent feeding or probing could greatly increase
the effectiveness of a vector. This is particularly true where mechanical
contamination of the mouthparts is the means of transfer. The frequent
interruption of feeding due to annoyance and restless movement of the host
greatly aids the spread of the causal agents of anthrax, tularemia, equine
infectious anemia, and anaplasmosis by tabanid flies (Harwood 1981).

21

Some viruses are transmitted vertically from parent organisms to progeny
(Japanese encephalitis by Cx. tritaeniomynchus, St. Louis encephalitis by Cx.
tarsalis, Yellow fever by Aedes aegyptii )(Fine 1985). Vertical transmission
includes transovarial, transovum, and transvenereal passage (Nuttal et al.
1991). To be transmitted vertically the virus has to persist throughout the
embryonic, larval and pupal stages, withstanding the enzymatic autolysis of
larval tissue during metamorphosis (Fouche et al. 1989). Vertical transmission of
virus allows the arthropod to be the reservoir host for the virus, thereby
maintaining a potential source of infection without a vertebrate host.

Arboviruses spend a significant portion of their life cycle in the arthropod.
Their movement into vertebrate hosts is usually short-tenn for amplification and
subsequent transmission to more vectors (Nuttal et al. 1991). Upon transmission
to a susceptible vertebrate host, the virus usually undergoes rapid amplification
causing a high viremia. This viremia is causes morbidity and mortality in the
host, reduces host defenses making the host more vulnerable to vectors, and
allows the high viral titers necessary to infect competent vectors feeding on the
host. Many arboviruses are zoonotic (e.g., encephaitides) maintained in animal-
mosquito cycles with humans being an incidental dead-end host (Leake 1998).
Erythrocyte Digestion. Blood borne pathogens gain access to hematophagous
arthropods during the ingestion of the blood meal. Blood is pumped via the
mouthparts from the host to the digestive tract. There are two basic types of
digestive tracts found in hematophagous insects, a simple tube in which the
blood is stored in the midgut or a complex gut having between one and three
diverticulae which may be used in addition to the midgut for storage of the blood

22

meal (Lehane 1996). The blood meal is separated from the midgut epithelium by
the peritrophic matrix. The function of the peritrophic matrix is not fully
understood. It is composed of mucopolysaccharides, with some protein and lipid
components (Lehane 1996) and is semi-permeable to allow the digestion and
subsequent absorption of the blood meal. Two types of peritrophic matrices are
recognized based on their method of production (Waterhouse 1953). Type I
peritrophic matrices form from the secretions of cells along the complete length
of the midgut and gradually develop into the peritrophic matrix and are found in
adult mosquitoes, blackflies, sandflies, and tabanids. Type II peritrophic
matrices are produced by the proventriculus, and pass backwards down the
length of the midgut forming an unbroken cylinder which contains and separates
the food from the midgut epithelium (Lehane 1996). Type II matrices are found
in adult muscids, tsetse flies and hippoboscids. Hemipteran insects do not
produce a peritrophic matrix but they do produce a extracellular coating in
response to ingestion of the blood meal (Lane and Harrison 1979, Billingsley and
Downe 1985, Billingsley and Downe 1986)

There are two general patterns of erythrocyte digestion (Vaughan and Azad
1993). The first pattern (Type I), observed for fleas and lice, is characterized by
the rapid hemolysis and liquefaction of the blood meal within 6 hours after
feeding. No peritrophic matrix was formed and the blood meal remained liquid
throughout the 48 hour observation period. In the second pattern (Type 2),
observed for bedbugs, sandflies, and mosquitoes, there was a time lag of 6-18
hours before substantial breakdown of erythrocytes began. A well-defined

peritrophic matrix surrounded the blood meal of sand flies and mosquitoes at 18

23

to 24 hours after feeding, and blood meals were clotted. Peritrophic matrix
formation and blood meal clotting was much less pronounced in bed bugs.
Vector Competency. Not all hematophagous arthropods are competent vectors
for all pathogens. Vector competency, a term that has recently gained usage, is
the intrinsic ability of a vector to biologically transmit a disease agent (Woodring
et al. 1996). Intrinsic aspects of vector competency include susceptibility to
infection, ability of the pathogen to reproduce or develop, and transmission
efficiency. Four basic criteria have been used to incriminate vectors in the
transmission of arboviruses: (1) the isolation of the virus from naturally infected
vectors; (2) laboratory demonstration of vector infection by feeding on a viremic
host; (3) laboratory demonstration of infected vectors transmitting the virus
during blood feeding; and, (4) evidence of blood feeding contact between the
suspected vector and host under natural conditions.

Vector competence is complex involving several barrier systems with in the
arthropod. Chamberlain and Sudia (1961) recognized that one of the main
factors in determining infection of the mosquito and its importance as a disease
vector, was the susceptibility of the mid-gut area to infection. Ingested virus
must first infect and replicate in the arthropod's midgut epithelial cells, and
progeny vims must then disseminate via the hemocoel before the virus can be
subsequently transmitted horizontally (via salivary glands) or vertically (via the
egg or sperm) (Nuttal et al. 1991) (Figure 3). Although only a few days are
required after virus ingestion for high viral concentrations to be attained in the
mosquito, two to four times that incubation period are generally needed before
efficient transmission by bite can occur (Chamberlain and Sudia 1961 ).

24

Hemocoel

  
  

 

   

Proventriculus
Salivary glands

Figure 3. Mosquito Digestive System. Ingested blood passes through the
proboscus into the mid-gut were digestion takes place. Virus must infect,
reproduce and escape from the mid-gut cells to move into the hemocoel.

Successful infection of the midgut depends partly on the amount of virus in
the host's blood and the volume of blood ingested (Nuttal et al. 1991). The
concept of “minimum dose“ or “infection thresholds” permeates the literature on
virus-vector systems (Rehacek 1965, Burgdorfer and Vanna 1967, Murphy et al.
1975, Nuttal 1984). The concentration of virus necessary to overcome the
threshold of infection varies in different arthropod species, and for individuals of
the same arthropod or virus (Hardy et al. 1983, DeFoliart et al. 1987). Studies
with yellow fever showed that ingestion of a definite concentration of virus was
required to infect Aedes aegypti and Haemagogus sp. (Anderson and Osomo-

Mesa 1946, Bates and Roca—Garcia 1946a, Bates and Roco-Garcia 1946b,

25

Waddell and Taylor 1947, Anderson 1985). Mosquitoes fed virus in lower
concentration failed to become infected but an increase in the viral concentration
resulted in a higher proportion of infected specimens. Others (Chamberlain et al.
1954, Barnett 1956, Chamberlain et al. 1956, Hurlburt 1956, Chamberlain and
Sudia 1957, Hale et al. 1957, Gresser et al. 1958, Chamberlain et al. 1959)
working with a number of viruses demonstrated that the threshold virus
concentration varied considerably between vector mosquito species for different
viruses as measured by infection of susceptible hosts.

Once an infected blood meal has been ingested, studies indicate that the
midgut is the anatomical site of viral interference (Nuttal et al. 1991 ). Following
infection, viral titers in the midgut cells initially decrease during which time the
virions penetrate the host cell becoming an intracellular (replication) stage know
as the eclipse phase. Viral titers then increase rapidly to reach maximum levels
within a few days as new virions are produced. The next barrier to infection is
the basal lamina which is found in both the midgut and surrounding the salivary
glands. This barrier is often referred to as the ”midgut escape barrier' or
"salivary gland infection barrier' (Hardy et al. 1983).

Salivary glands appear to become infected from viremic hemolymph
(Chamberlain and Sudia 1961, Murphy et al. 1975, Beaty and Thompson 1978,
McLintock 1978). Chamberlain and Sudia (1961) proposed that the absence of
salivary gland infection was due to failure of virus to reach the salivary glands or,
the inability to penetrate the glandular tissue to establish infection. Secondary
amplification of virus in extramesenteronal cells and tissues is a prerequisite for

infection of the salivary glands of some arboviruses (Nuttal et al. 1991) especially

26

when low concentrations of virus are ingested. In some virus-vector
associations, a “leaky gut" has been described whereby the virus passes into the
hemocoel more rapidly than replication could occur. If the virus is ingested in
sufficient quantity when reaching the hemocoel it may infect the salivary glands
without undergoing prior multiplication (Hardy et al. 1983). The passive
movement of virus from the gut lumen to the hemocoel may relate to a
potentially explosive cycling and vertebrate epizootics, and reflect a response of
the virus to changes in selection pressures (Nuttal et al. 1991). Whatever the
mechanism of the barrier to salivary gland infection, its presence or absence,
together with the gut infection threshold, is of primary concern in determining the
epidemiological importance of a vector species (Chamberlain and Sudia 1961).
The time interval between acquisition of virus in a blood meal and the ability
to transmit the virus is known as the "extrinsic incubation period“. During this
period the vector is incapable of biological transmission because sufficient time
has not elapsed for the virus to establish an infection of the salivary glands.
Hence, the extrinsic incubation period is an important factor in the epidemiology
and epizootiology of arthropod-home viruses, as it dictates how long the vector
must survive before it is a competent transmitter. Extrinsic conditions such as
ambient temperature can affect the extrinsic incubation period (T urell et al. 1985,
Watts et al. 1987, Kay et al. 1989). Intrinsic vector competence includes both
physiological factors that govern infection of the vector and ability to transmit the
virus, and innate behavioral traits such as host preference and probing activity

(Nuttal et al. 1991).

27

The interplay of both intrinsic and extrinsic factors in vector disease
transmission is termed the vectorial capacity, which is the overall ability of the
vector at a given place and time to transmit a pathogen (Woodring et al. 1996).
It encompasses the interactions between the vector, pathogen and vertebrate
host, as well as ecological and behavioral factors such as vector population size,
longevity, length and number of gonotrophic cycles, feeding behavior, and diel
activity. Vector competency is the component of vectorial capacity that is
restricted to the vector-pathogen relationship.

Vectors characteristically exhibit much lower levels of infection than the
primary host in endemic regions. There are several possible explanations for
this observation. One likely possibility is that the rate of transmission from
primary host to vector is low in comparison to the transmission between two host
species (Anderson 1985). High population densities in endemic regions often
result in frequent contacts between vectors and susceptible hosts (Harwood
1981) which permits some vectors that are otherwise poor hosts of a pathogen
to be of epidemiological significance. A striking feature of the epidemiology of
indirectly transmitted disease agents is that the prevalence of infection within
intermediate host (or vector) population is characteristically low, even in areas
where the primary host population is heavily infected (endemic regions)
(Anderson 1985).

DISEASE VECTORS
The ability of various Diptera to transmit disease pathogens has been

extensively researched and studied. Several species of Hemiptera are also

28

known to transmit disease or have been implicated in the transmission of
disease due to their intimate relationship with man.
Culicidae. There are approximately three and a half thousand species of
mosquitoes, or Culicidae, in the order Diptera (Clements 1992). The family
Culicidae is subdivide into 3 subfamilies: Culicinae, Toxorhynchitinae, and
Anophelinae. The subfamilies Culicinae and Anophelinae contain primarily
hematophagous species which are of medical importance as disease vectors.
The subfamily Toxorhynchitinae consists of non-blood feeding mosquitoes.
Biology. All mosquitoes undergo complete metamorphosis. Females
mosquitoes lay between 50 and 500 eggs at one time. The eggs are deposited
on water or other damp substrates that are likely to be flooded. Eggs laid in the
water hatch within 1-3 days while eggs laid on damp substrates diapause until
appropriate conditions signal hatching. Mosquito larvae are legless, with a well
formed head capsule and siphon for breathing. Larvae feed by either filtering
microorganisms and detritus from the water column or scraping material off
surfaces. A pupae is formed after four larval instars. During the pupal stage the
larval organs degenerate and are replaced with adult organs from
undifferentiated cells in the imaginal discs. When metamorphisis is complete the
pupae swallows air to increase intemal pressure causing the pupal cuticle to split
along the cleavage lines. Adults slowly emerge and unfold the legs and body to
stand on the water surface. The soft adult cuticle sclerotizes within 10-15
mintues and the adult is able to fly away. The time from pupation to eclosion is

between 2-4 days.

29

Mosquitoes do not associate with a vertebrate host except for feeding and
some use the host as a mating site. Mosquitoes actively search with variety of
sensory stimuli for a suitable host from which to obtain a bloodmeal. After
locating a host the mosquito begins to search for a suitable site to probe for
blood. Chemoreceptors are used to locate the appropriate site to insert the
proboscus. Mosquitoes have piercing sucking mouthparts which they use for
feeding either from subderrnal hematomas caused by the action of the
mouthparts or directly from blood vessels. During probing the mosquito releases
saliva through the proboscis. The saliva in some species contains
anticoagulants, anti-platelet, and aggregation factors that keep the blood from
clotting, counteracting the host's immune response to feeding. Mosquitoes
withdraw and move to a new site to probe if unable to locate an appropriate
feeding site.

Hearing. The reproductive ability of female mosquitoes is dependent on their
nutrition in both the larval and adult stages. Large, well fed larvae have the
greatest fecundity as adults. The actual fertility of the female is dependent on
her ability to locate a blood source, obtain an adequate blood meal, and find a
mate to produce eggs. There is great variation in fecundity and fertility between
genera and species. The number of eggs produced by female mosquitoes has
been shown to be influenced by maternal body size (Hawley 1985, Briegel
1990a, Briegel 1990b), the number of previous gonotrophic cycles (Roubaud
1934, Detinova 1 949, Detinova 1 955, Hien 1 976, Hawley 1 985), seasonal
variation (Shannon and Hadjinicalao 1941, Detinova 1955), and blood meal type
(Shelton 1972, Jalil 1974, Downe and Archer 1975, Nayar and Sauerrnan 1975).

30

Laboratories rearing mosquitoes for research all use some type of larval diet.
Many use highly specific food that is mixed for optimum concentration of protein,
carbohydrates and other essential amino acids and nutrients while others utilize
ground fish food or other dry food. Larva need to be kept in rearing pans that
are age specific and not crowded to limit competition for food resources.
Different mosquitoes require different larval densities, nutritional requirements,
and water quality. Larval rearing temperature is important in producing optimum
size adults. The relationship between larval growth and temperature has been
investigated extensively and can be summarized as: larval growth and
development occur within a temperature range, with a lower developmental
threshold and an upper lethal temperature; temperature ranges vary with
species; larval growth and development is positively correlated with an increase
in temperature for most of the temperature range (Clements 1992).

Feeding Mechanisms. Most female mosquitoes require blood, for egg
production, and plant juices, for energy. Plant sugars are a major food source
providing an important energy source during the adults life span. Ingested plant
juices are stored in the crop and digested in the midgut. Mosquitoes commonly
ingest more than their own weight in blood, the mean blood meal weight can be
from two to four times the weight of the female (Nayar and Sauerrnan 1975).
The mosquito abdomen is able to stretch to accommodate the ingestion of the
large quantity of blood. Anopheline mosquitoes are unable to grossly distend
their midgut and abdomens with ingested blood. Certain species use diuresis to
increase the protein intake of the limited blood meal. Certain species discharge
fluid from the anus during feeding. Since the erythrocytes contain the protein

31

requried for egg production, the discharge of excess fluid and hence the
concentration of the erythrocyctes increases the amount of protein a female
ingests in a single blood meal.

Disease Transmission. Anopheline mosquitoes are vectors of malaria
parasites of humans, human filarial nematodes and very few arboviruses (Gillies
and DeMeillon 1968, Gillies and Coetzee 1987). The relationship between
Anopheline mosquitoes and the human malaria parasites is exclusive. The
malaria parasite undergoes sexual reproduction within the mosquito, thus without
this intimate relationship the malaria cycle would be broken. Human pathogens
vectored by members of the Culicinae subfamily include: filarial nematodes and
arboviruses.

Cimicidae. The family Cimicidae includes bed bugs, swallow bugs, poultry
bugs, bat bugs, and others. The wings are reduced to inconspicuous pads. The
bodies are broad and flat, enabling the bugs to creep into narrow crevices.
These insects are night prowling and hematophagous, feeding on birds and bats,
but some occasionally fed on humans. Usinger (1966) described 74 species
representing 22 genera and 6 subfamilies. Others have been described since
then (Hanlvood and James 1979). Many are local in distribution and are of little
medical importance, but two have followed man, a unusual host for the family,
over a large part of the world. These are Cimex Iectularius, the common bed
bug, a cosmopolitan species of both hemispheres but particularly occurring in the
temperate regions, and Cimex hemiptems, the tropical bed bug, found in both

hemispheres but essentially a species of the tropics (Harwood and James 1979).

32

Biology. The bed bug is nocturnal in its feeding habits, hiding in cracks and
crevices during the day. In its nighttime activity it leaves its hiding place, often
traveling considerable distances to feed on hosts. Ordinarily, the bugs stay close
to wooden bedsteads. Seams in mattresses and box springs commonly afford
harborage. Bed bugs are gregarious; great assemblages may be found in some
convenient crevice or beneath some nearby loose wallpaper. Infestations of bed
bugs are traceable to introductions of eggs, young, or adults. One impregnated
female might furnish the nucleus for a well developed colony within a few
months. Bugs in all stages are easily carried in clothing, traveling bags,
suitcases, or laundry, and they may be introduced with second hand beds,
bedding and furniture. They may pass from the clothing of one person to
another on crowded public vehicles (Harwood and James 1979).

The adult 0. lectularius is reddish brown in color, whereas the young are
yellowish white. The female deposits eggs in batches of 10 to 50, totaling 200 to
500, spread out in a yellowish patch (Harwood and James 1979). The eggs are
relatively large and yellowish white incolor. The young hatch in 4 to 21 (usually
about 10) days. The time required for development from egg to maturity is 37 to
128 days, under favorable laboratory conditions; these, however, are seldom, if
ever, realized in nature. All instars, particularly the later ones, can endure
prolonged starvation (80 to 140 days, depending on the instar and sex; the
higher figure being for adult males); this of course lengthens the life cycle
(Harwood and James 1979). Bed bugs molt five times, and the minute wing
pads characteristic of the adult insect make their appearance with the last molt.
Ordinarily one meal is taken between each molt and one before egg deposition;

33

an average of 8 days is required between each molting (Harwood and James
1979)

Hearing. For maintenance purposes colonies may be limited to one blood
meal per week. However, for maximum egg production, rapid development of
the nymphal stage and availability of large numbers of bugs for experiments,
blood meals should be offered two or three times per week (Harwood and James
1979). After a full blood-meal has been ingested the time required until molting
depends on the temperature of the environment, the amount of blood ingested
above the minimum required for molting, the particular instar, individual
variations of the insects themselves, and perhaps also on the amount of activity
subsequent to feeding: for the more active insect will utilize a greater proportion
of blood to supply energy for movement and less will remain for growth (Johnson
1941 ). The lower threshold for hatching, nymphal development, and adult
activity in C. Iectularius is between 13 degrees C and 15 degrees C (Hase 1930,
Mellanby 1935, Kemper 1936, Johnson 1941), although the insect can tolerate
extremes as low as -15 degrees C for brief periods (Usinger 1966). The thermal
death point is between 44 degrees C and 45 degrees C (Usinger 1966), but
Omori (1941) found that development ceased, especially with C. Iectularius, at
36 to 37 degrees C. Johnson (1941) found the total period of development to
range from 36.6 days at 33 degrees C to 127.9 days at 18 degrees C. Burden
(1966) found in searching the literature that temperatures of 23.9 to 29.4
degrees C and relative humidities of 26% to 70% have been used for

colonization.

34

In C. Iectularius the frequency of food intake is closely correlated with egg-
laying, molting, longevity, and temperature, which affects all other processes
including rate of digestion (Usinger 1966). Larger bugs take larger blood meals
and produce more eggs. A mated female will feed to repletion in about 5 to 10
minutes and then retreat to a harborage to remain quiescent while digesting the
blood meal and developing eggs. At 23 degrees C, the time after feeding until
the first eggs were laid was 5 to 6 days, and oviposition lasted for 6 days,
producing 6 to 10 eggs (Johnson 1941, Usinger 1966). Kemper (1936) found
that young adults, at room temperatures 18 to 20 degrees C, were stimulated to
search for another blood meal at approximately weekly intervals, while at 27
degrees C the time was reduced to about 3 days.

Various wann-blooded animals and feeding methods (Girault 1910, Janisch
1933, Rendtorff 1938, Busvine 1958, Gratz 1958, Adkins and Arant 1959, Gilbert
1964) have been used in the laboratory colonization of bed bugs. The relative
values of the blood of man, fowl, rabbit, and mouse have been discussed by
Johnson (1937, 1940). Johnson (1941) found that under laboratory conditions
larger meals were taken from mice (probably on account of its high body
temperature) than from man or fowl, and it was this, presumably, that induced a
relatively rapid rate of development. Adult bugs bred on mice, perhaps because
of the larger meals taken, are heavier than those bred on man or fowl and
associated with this is a slight increase in fecundity (Johnson 1941).

Feeding Mechanism. The bite of the bed bug is produced by piercing organs
of the hemipteran type. It is probable that punctures by these stylets,
unattended by contamination or specific poisons, would produce little pain.

35

Dickerson and Lavoipierre (1959) described the feeding mechanics of the bed
bug. The labium, a sheath around the fascile of piercing stylets, does not enter
the skin, although it plays an important part in the act of feeding, as its lip-like
tips grasp the fascicle in a pincer-like grip and helping to steady it as it is thrust
into the tissues. Having entered the skin, the fascicle, which is extremely
flexible, readily probes in various directions, moving forwards and backwards and
at times bending well over 90 degrees. While probing, the fascicle often
pierces, cuts across, and sometimes enters, minute capillaries and larger
vessels, without ceasing its restless movement. This active probing results in the
formation of small and large hemorrhages in the tissues, but bugs have seldom
been observed to feed from them. The fascicle continues its active movements
in the tissues until it encounters and enters a vessel of suitable calibre, from
which the blood meal is taken up. The bed bug is able to engorge itself
completely with blood in 3 to 10 minutes (Harwood and James 1979).

Persons bitten by bed bugs are affected in various ways. In some the bite
produces marked swellings and considerable irritation; in others not the slightest
inconvenience is caused (Harwood and James 1979). The welts and local
inflammation are caused by allergic reactions to the saliva that is introduced
early in the act of feeding.

Disease Transmission. Bed bugs meet all the criteria for human disease
transmission, i.e., they are obligate bloodsucking parasites that have close and
frequent contact with humans (Gubler 1991). The fact that bed bugs must feed
at least four and usually five times, upon either the same or different hosts, to
reach maturity, has placed these insects under suspicion as potential vectors of

36

disease causing organisms (Hanrvood and James 1979). Bed bugs have been
suspected in the transmission of 41 human diseases, the agents, or causes of
which coincide with 10 disease categories (Burton 1963, Usinger 1966). Many of
the causative agents of disease have developed or remained alive within the bed
bug after experimental infection for varying numbers of days (Burton 1963). This
does not necessarily mean that these organisms are transmitted by bed bugs
under natural conditions. Experimentally they have become infected with a
variety of pathogens, but to date, they have not been incriminated in
transmission of any human disease (Strickland 1991).

Reduviidae. The family Reduviidae is composed of two biological groups, the
hematophagous species of the subfamily Triatominae which are ectoparasites
on vertebrates, and the nineteen or more additional subfamilies which are
predaceous on arthropods (Ryckman and Ryckman 1966, Gubler1991). The
subfamily Triatominae consists of 100 species in 15 genera (McClelland 1992).
Most species of bugs are sylvatic and live in close association with a variety of
wild animals, e.g., arrnadillos, opossums, mice, rats, bats, and squirrels. Others
live in close association with humans and domestic animals. The bugs usually
lay their eggs in or near the habitation of the host. Flhodnius prolixus is the most
commonly studied species of Triatominae.

Biology. The Triatominae have five nymphal instars and need to feed only
once to repletion before each molt. The life cycle of Fl. prolixus takes about six
months at 280 (Gardiner and Maddrell 1972). In instars, one blood meal up to
nine times their body weight is taken per molt cycle; adults take meals of up to
three times their body weight each time they feed (Friend et al. 1965b) A blood

37

meal of this size will cause the insect to molt if it is a instar, or to produce eggs if
it is a fertilized female adult; it will also sustain the insect for periods of at least
90 days (Friend 1965a).

Rearing. Laboratory culture methods have been described by Larrousse
(1927), Galliard (1935), Dias and Philip (1938), Usinger (1944), Grundemann
(1947), Abalos and Wygodzinsky (1951), Ames et al. (1954), and Ryckman
(1952, 1954, 1965, 1966). Artificial feeding experiments by Nt’rr'iez and Segura
(1987) observed no significant difference between heparinized guinea pig or
sheep blood and excluded citrate as an anticoagulant. Greater adult size and
consequently higher fecundity resulted when feeding occurred on two successive
days (Patterson 1979). The mechanical damage to erythrocytes and the
potential alteration of the ionic balance probably essential for this type of
hematophagous insect must be taken into account when using defibrinated
blood (McGuire et al. 1973).

The voluminous meal needs to be initially digested before being absorbed,
as in other animals. Blood is the only nutrient the triatomines need for molting,
metabolism, and egg production (Buxton 1930). Since more than 50% of the
blood proteins are inside the erythrocytes, the mechanism of hemolysis is an
important step for digestion (Garcia 1987). Normally, within 4 days after the
meal, most erythrocytes are lysed and the hemoglobin is crystallized in the crop.
This process is observed in all instars and adults (Baur et al. 1979, Smit 1983).
Garcia (1987) found free amino acids are found in small quantities in the blood
meal. Amino acids are constituents of proteins, which must be hydrolyzed
before the amino acids are available to insect cells. Protein digestion occurs in

38

the anterior intestine of the triatomines, which has a quite different mixture of
proteolytic enzymes with pH optima in the acidic range. The proteolytic enzymes
(proteases) attack peptide bonds in proteins and peptides. They are usually
divided in proteinases (endopeptidases) and peptidases (exopeptidases). By the
sequential action of these proteases, ingested proteins are hydrolyzed and their
complete breakdown is ensured to yield a mixture of free amino acids which are
then rapidly absorbed and incorporated into insect proteins.

Feeding Mechanism. Triatomines are bloodsucking bugs of immoderate
feeding habits. Given the opportunity, these insects quickly, skillfully, and
painlessly obtain a blood meal from a host (Garcia 1987). Factors known or
suspected to affect feeding behavior in R. prolixus include: (a) external cues
(e.g., temperature gradients, carbon dioxide, odors, movement, vibration,
surface texture, standing liquid) (Wigglesworth and Gillett 1934a, Wigglesworth
and Gillett 1934b, Lavoipierre et al. 1959, Friend 1965a, Friend and Smith 1971,
Friend and Smith 1972, McGuire et al. 1973); (b) diet composition (e.g., the
nature and concentration of phagostimulatory nucleotides, osmotic pressure, pH,
ion species and concentrations), which determines whether or not engorging
occurs (Friend 1965a, Friend and Smith 1971, Friend and Smith 1972, Smith
and Friend 1972, Smith and Friend 1976a, Smith and Friend 1976b, Friend and
Smith 1977a); (c) length of time since previous meal (Friend and Smith 1975);
and (d) degree of abdominal stretch (Maddrell 1963, Anwyl 1972). Consequently
rigidly controlled and uniform conditions of rearing and handling need to be

employed in the rearing and handling of H. prolixus.

39

The orientation and feeding process in H. prolixus has been described
(Wigglesworth and Gillett 1934b) as a "chain of reflexes,“ in which the initiation
of the response is conditioned by the physiological state of the insect. The
insect is aroused from a state of akinesis by the antennae responding to air
currents or heat (Friend and Smith 1977a). It is then attracted to the host,
natural or artificial, by heat and perhaps odor stimuli perceived by receptors on
the antennae. During probing, the tip of the labium is applied to the skin surface.
The mandibles penetrate the surface, and the maxillae, which form the food and
salivary canals, are then inserted in a series of thrusts and withdrawals during
which saliva is continuously ejected from between their tips (Friend and Smith
1977a). After one or more samplings of the available fluid, ingestion of the diet
commences in the presence of suitable phagostimulants. Feeding stops when a
critical abdomen volume is reached; this is controlled by abdominal stretch
receptors (Maddrell 1963, Anwyl 1972) rather than by back pressure on the
pharyngeal pumps.

Disease Transmission. Triatomine are known to transmit Trypanosoma cruzi,
causative agent of Chagas' Disease, by the defecation of infective material on a
susceptible host. All known vectors of T. cmzr' comprise about 50 species (out of
100) in nine (of 15) genera in the subfamily Triatominae of the family
Reduviidae(McClelland 1992). Transmission is usually associated with the bite
of the bug, which often takes 10 to 20 minutes to engorge and frequently
defecates during or shortly after the feeding process (Gubler 1991). Infection of
the new host is usually accomplished by scratching the infective trypanosomes
into the bite wound or into other skin abrasions or, most commonly, by

40

transferring the agents on fingers to the highly receptive conjunctiva of the eye or
to the mucosa of the mouth or nose (Gubler1991). Bugs which are slow to
defecate will leave the host before excreting urine rich in infective metacyclic
trypanosomes (McClelland 1992).
PURPOSE OF RESEARCH

The overall goal of this research project was to determine the persistence of
HBV genome in several hematophagous arthropods. Previous studies, utilized
primarily ElA/RIA methodologies testing for the presence of HBsAg. The
detection of HBsAg showed ingestion of the viral particles during feeding on
either a viremic hosts or artificial feeders. However, the significance of the
continued detection of the HBsAg over varying time periods is debatable. Is it an
artifact of the original blood meal? or, New viral particles? Due to the unique
constraints of the virus, a vector competency study was impractical as it would
require an animal model with a hepadnavirus. With the use of molecular
detection methods regions of the HBV genome can be detected. By testing for
pre-C and C regions of the genome, only regions of the genome coding for the
infectious particle regions would be detected. However, the detection of the viral
genome does not signify infectivity or transmissibility. The persistence of the
virus and its dissemination in the insect may signify that the vector has been
infected. The insect must then be able to mechanically or biologically transmit
the virus to another susceptible host to be a competent vector.

The purpose of this research is to evaluate the persistence and dissemination
of, ingested and inoculated, hepatitis B virus, by PCR and Southern blot, in
Cimex Iectularius L. (Hemiptera: Cimicidae), Rhodnius prolixus Stal (Hemiptera:

41

Reduviidae), Aedes trisen'atus (Say) (Diptera: Culicidae), Culex quinquefasciatus
Say (Diptera: Culicidae) and Anopheles stephensi Liston (Diptera: Culicidae).
The specific objectives to be tested are: (1) persistence of hepatitis B virus in the
saliva or on mouthparts of R. prolixus, Ae. tn'seratus, Cx. quinquefasciatus and
An. stephensi, (2) persistence of hepatitis B virus in the legs of R. prolixus, Ae.
trisen'atus, Cx. quinquefasciatus An. stephensi. and C. Iectularius“, (3)
persistence of hepatitis B virus in the feces of C. Iectularius, Fl. prolixus, Ae.
trisen'atus, Cx. quinquefasciatus and An. stephensi, (4) persistence of hepatitis B
virus in the bodies of C. Iectularius, Fl. prolixus, Ae. trisen'atus, Cx.
quinquefasciatus and An. stephensi, and (5) persistence of hepatitis B virus

trans-stadially and trans-ovarially in C. Iectularius.

42

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Woodring, J., S. Higgs and B. Beaty. 1996. Natural cycles of vector-bome
pathogens. pp. 51 -72. In B. Beaty and C. Marquardt [eds.], The Biology of
Disease Vectors, University Press of Colorado, Niwot.

Wright, R., J. Perkins, B. Bower and D. Jerropme. 1970. Cirrhosis associated

with the Australia antigen in an infant who acquired hepatitis from her mother.
Br Med J.4: 719-721 .

6O

Chapter 1

Persistence of the Hepatitis B viral genome in

Three Species of Diptera: Culicidae

INTRODUCTION

Hepatitis B infection is a major cause of acute and chronic hepatitis, cirrhosis,
and primary hepatocellular carcinoma (PHC) world wide (CDC 1990) killing an
estimated 2 million people each year (Hamilton 1994). Primary hepatocellular
cancer is one of the most common cancers in Asia and sub-Saharan Africa
(Beasley 1988) where 845% of the population carry markers of HBV infection
(Maynard 1981). Persons known to be at risk of contracting hepatitis B include:
sexually active men and women; prison inmates; health care and public safety
workers; hemodialysis patients; international travelers; and household contacts
of HBV carriers. Primary modes of transmission are parenteral, perinatal, and
sexual contact, although, 30 - 40% of people infected with HBV have no
identifiable risk factor (Alter and Mat 1994).

Neefe (1949) was the first to consider the possibility that hematophagous
arthropods could transmit the hepatitis virus. Prince (1970) found an increased
prevalence of SH (hepatitis B surface antigen) antigenemia in the topics but was
unable to account for this by known mechanisms of transmission, and suggested
transmission in nature by nonparenteral routes. Of the potential routes
discussed, the possibility of transmission by hematophagous arthropods was
thought the most probable. The subsequent detection of the hepatitis B surface

61

antigen (HBsAg) by solid-phase radioimmunoassay in wild caught mosquitoes
supported this hypothesis (Prince et al. 1972).

Numerous researchers have since investigated the possible role of
mosquitoes in the transmission of hepatitis B virus under varying conditions.
However, all of these studies used detection assays for the hepatitis B surface
antigen (HBsAg) which is present on the surface of all three particles produced
by the hepatitis B virus. Only one of the three particles contains virions and is
thus infectious (Blumberg et al. 1965). The concentration of non-virion
associated HBsAg greatly exceeds the concentration of complete virions by 104
per milliliter or greater. The results and interpretations from these experiments
are contradictory. Some authors (Hawkes et al. 1972, Leevy et al. 1972, Prince
et al. 1972, Brotman et al. 1973, Muniz and Micks 1973, lshii et al. 1974, Wills et
al. 1976, Chen et al. 1987, Fouche et al. 1989, Fouche et al. 1990, Jupp et al.
1991) considered mosquitoes to be potential mechanical vectors of hepatitis B
virus, while others (Smith et al. 1972, Metselaar et al. 1973, Wills et al. 1976)
claimed that they are biological vectors of the virus. No evidence implicating
mosquitoes as potential vectors of hepatitis B virus could be found by other
authors (Skinhoj 1972, Byrom et al. 1973, Newkirk et al. 1975, Berquist et al.
1976). Still other authors (Blumberg et al. 1973, Tin et al. 1973, Dick et al. 1974,
Papaevangelou and Kourea-Kremastinou 1974, Hyams 1989) found the results
to be inconclusive, requiring more in detailed study.

Several investigators (Leevy et al. 1972, Brotman et al. 1973, Newkirk et al.
1975, Chen et al. 1987) found a direct correlation between antigen
disappearance and blood meal digestion. Other investigators have detected

62

HBsAg after 6 days (Muniz and Micks 1973), 7 days (Tin et al. 1973), 10 days
(Smith et al. 1972), and 5 weeks (Metselaar et al. 1973).

Due to the host specificity of the virus few transmission studies have been
conducted. The results of the studies that have been conducted are
contradictory. No evidence of mechanical or biological transmission was found
in studies using human volunteers (Neefe 1949) and chimpanzees (Berquist et
al. 1976), however, evidence of transmission was found in studies using
chickens (Smith et al. 1972) and monkeys (Yuhuang et al. 1995). Smith (1972)
detected HBsAg by immunofluorescent staining in chicken subcutis immediately
after the chickens were bitten by infected mosquitoes. Yuhuang et al. (1995)
reported that 9 of 29 monkeys bitten by infected mosquitoes were positive for
HBV serum markers. The validity of this study, however, is questionable. The
mosquitoes were fed an infectious blood meal via an artificial feeder and after
blood meal digestion were allowed to feed on the abdomen of a Tupaia
Belangeri monkey. The study gives the detection of hepatitis markers for the
monkeys but not for the mosquitoes. Nor does the study address the question of
potential mechanical transmission of the virus due to infectious blood on the
mosquito cages. To date a conclusive study has not been published that
explicitly shows mosquitoes are capable of transmitting the hepatitis B virus.

Still other researchers (Whittle et al. 1983, Foster et al. 1984, Tabor et al.
1985, Mayans et al. 1990) have examined the problem from an epidemiological
perspective. Hepatitis B infections in Africa commonly occur after one year of
age (Tabor et al. 1985), whereas, in Asia many children are infected at birth by
their mother (Stevens et al. 1975). Tattooing, circumcision, ear piercing,

63

traditional scarring, exudative skin lesions, and hematophagous arthropods have
all been proposed as mechanisms of transmission. Mayans (1990) found a
significant association between hepatitis e (HBe) antigenaemia and the presence
of bed bugs in children’s beds. However, a later intervention study (Mayans et
al. 1994) found little epidemiological evidence to support bedbugs as the major
mode of transmission of hepatitis B virus in Gambian children.

The purpose of this study was to determine the persistence of the hepatitis B
virus in Anopheles stephensi, Aedes triseriatus, and Culex quinquefasciatus that
had ingested or been inoculated intrahemocoelly with the virus. Polymerase
chain reaction and Southern blotting was used to detect a segment of the
hepatitis B viral genome.

MATERIALS AND METHODS

Insect Rearing. Anopheles stephensi and Aedes triseriatus colonies were
reared at 24 degrees C, with 30% humidity and a photoperiod of 16:8 (L:D) h.
Stock colony adults were held in 12 X 12 X 12 screened cages with raisin water
for a sugar source, and blood fed weekly on anesthetized rats. Larvae were
reared in 500 ml plastic pans with lids and given ground fish food.

Anopheles stephensi: Three days post blood feeding, a wet oviposition paper
was placed in the cage. Eggs were rinsed off the paper into a pan of distilled
water to hatch. First and second instars were fed a diet of suspended yeast.
Older larvae were fed Tetra Min Baby fish Food 'E.‘ Larval density was
monitored in the pans and larvae were sorted into new pans when they were
gauged to be too dense. Pupae were collected and placed in an emergence
cage at 6 days prior to the start of an experiment. Females, one to six days old,

64

were transferred to experimental feeding containers on the day prior to an
experiment and wet gauze was placed on the mesh lids to provide humidity.

Aedes triseriatus: A oviposition cup with egg paper as a liner was placed in
the stock colony cage and left for 3-5 days after blood feeding. The cup was
removed from the cage and allowed to sit with water for another 1-3 days to
allow embryonation to occur. The egg paper was removed from the cup and
allowed to dry. Dried egg papers were stored at room temperature with low
humidity. Egg papers were placed on wet paper towels 24 hours prior to
hatching. The egg paper was then placed in deoxygenated water, as a hatching
stimulus, and suspended liver powder was added. Newly hatched larvae were
transferred to 500 ml pans the next day and fed the suspended liver powder until
pupation. Larval density was monitored in the pans and larvae were sorted into
new pans when they were gauged to be too dense. Pupae were collected and
placed in an emergence cage at 6 days prior to the start of an experiment.
Females, one to six days old, were transferred to experimental feeding
containers on the day prior to an experiment. Wet gauze was placed on the
mesh lids of these containers to provide humidity.

Culex quinquefasciatus: The Culex quinquefasciatus were obtained from the
stock colony at the US Army Medical Research Institute for Infectious Disease
(USAMRIID), Ft. Detrick, Maryland. Adult females were collected and placed in
an experimental container immediately prior to intrathoracic inoculation.

Blood Feeding on Animals. Stock colonies were fed weekly on rats. The All
University Committee on Animal Use and Care approved the use of rats to feed
the insect colonies, AUF# 05/96-053-00. Rats where selected based on

65

willingness of both Diptera and Hemiptera to feed on them and the ability to
maintain a sufficient number of rats to feed the all the insect colonies necessary
to conduct this research.

Cages of An. stephensi and Ae. triseriatus were transported from the Natural
Science Building to the Giltner animal room, a University Laboratory Animal
Resource (ULAR) facility. Cages were placed directly in front of the room vent.
Rats were restrained in surgical stocking and wrapped loosely in a towel and
placed under the scavenger hood. Methoxyflurane was administered with a
nose cone until the rat became fully relaxed at which time the towel and surgical
stocking were removed. The abdominal area was shaved and sterile eye
ointment administered to the eyes in preparation for feeding. The rat was then
laid on its stomach on the mosquito cage with its nose in the nose cone for
approximately 20 minutes. Rats’ respiration rate was monitored throughout the
feeding period. Rats were removed from the cage and allowed to expire the
anesthetic before being placed in a recovery cage. Recovering rats’ respiration
and state of consciousness were monitored until each rat was awake, then it was
returned to its own cage.

Rats were used once every two weeks for feeding. Any rat not appearing
active and healthy was removed from the feeding rotation until examined by
ULAR personnel. Any rat that developed a suspected tumor or possible irritation
due to feeding was removed from the feeding rotation and the ULAR veterinarian
notified immediately.

Artificial Blood Feeding. Stock mosquito colonies were fed by the artificial
feeder and all infective feedings were done using the artificial feeder. A

66

Hemotek 5W1 artificial feeding system (Discovery Workshop, England) was
used for all feedings. The system consists of 5 independently controlled, heated
feeding chambers. Each feeding chamber holds 5 milliliters of blood. Three
different types of feeding membranes (parafilm, latex, and cellulose) were used
for colony maintenance. A cellulose membrane was used exclusively during
infected feedings. The membrane was stretched across the feeding chamber
and secured with an O-ring. The chambers were filled with blood, capped and
screwed into the heating element, then placed on the cage for feeding.

unit can supply power to five independently controlled heating elements.

A. B
H - ating element
Power Unit
‘ Heating
O O O 0 element

 

Feeding Chamber

Figure 1. Diagram of Hemotek 5W1 artificial feeding system. A) The power
unit can supply power to five independently controlled heating elements. B) Each
feeding chamber was covered with a membrane and filled holds 5 milliliters of
blood.

67

Bovine or equine blood was used for all feedings. Mature bovine blood
preserved with heparin was obtained in 100 ml aliquots from Pel-Freez
Biologicals, (PO. Box 68, Rogers AR 72757). Mature equine blood preserved
with heparin was obtained in 100 ml aliquots from Veterinary Dynamics Inc.,
(1535 Templeton Road, Templeton CA 93465). Blood was collected and
shipped over night on wet ice at 4 degrees C and then stored at 4 degrees C in
the laboratory. Blood was kept no longer than 30 days from date of drawing from
the bovine or equine.

Hepatitis B Virus Infected Blood Collection and Transport. Blood was
collected by venipuncture from a HBV positive patient by the Department of
Gastroenterology-Hepatology, William Beaumont Hospital, Royal Oak, Michigan,
after obtaining informed consent. The collection has been approved by both
William Beaumont Hospital and Michigan State University, University Committee
on Research Involving Human Subjects (UCRIHS) IRB # 96-039. Non-infected
blood was collected by the Medical Entomology Laboratory, Department of
Entomology, Michigan State University after receiving informed consent. The
blood was transported on ice from William Beaumont Hospital to Michigan State
University. The blood was centrifuged and serum removed and frozen at -70
degrees C until use.

Quantitative hepatitis B viral levels were determined by the Abbott hepatitis B
viral DNA Assay (Abbott Laboratories, Abbott Park, IL) a liquid phase molecular
hybridization assay for quantifying hepatitis B virus DNA in human serum.

Results are reported in picograms of DNA per milliliter (pg/ml) of serum.

68

Experimental Insect Feeding, Handling and Containment. All cohort feeding
was conducted using a single Hemotek feeding chamber in a Labconco glove
box rated to Biosafety Level III (inspected by Michigan State University, ORCBS,
Environmental Services 17 April 1996). The chamber was loaded with infectious
blood in the same manner as in routine feeding. The chamber used for the
infected feeding was soaked in 10% bleach solution and then autoclaved to
sterilize it after use. Engorged mosquitoes were transferred by bulb aspirator
from the feeding container to a clean container with filter paper in the bottom. All
feeding containers were autoclaved, placed in biohazard bags, and autoclaved
again prior to disposal. Infected cohorts were kept in the sealed Labconco glove
box with humidity pads for the duration of the experiment.

All cohorts, control and infected, were handled in the same manner. Control
samples were always collected before the infected samples. A mechanical
aspirator was used to collect the sample from the infected cohort containers.
The aspirator chamber was sealed with tape and placed in a -4 degrees C
freezer to chill the mosquitoes. Immobilized mosquitoes were transferred to a
labeled petri dish in the freezer. The petri dish was then placed on ice and taken
to the bench top work space. Individual mosquitoes were removed from the petri
dish with heat sterilized forceps and placed on a clean microscope glass slide.
Wings and legs were removed with a sterile scalpel and the mosquito was then
placed on a sticky piece of tape to restrain movement. Legs were collected and
placed in a labeled 1.5 ml eppendorf tube. The proboscis was then inserted into
a microcapillary tube containing tissue culture broth, either E-199 or TM100, to
stimulate salivation. After 5 minutes the microcapillary tube was removed from

69

the proboscis and the tissue culture broth was expelled into a labeled 1.5 ml
eppendorf tube. Finally, the body was placed in a labeled 1.5 ml eppendorf tube.
All samples were then frozen at -70 degrees C until DNA extraction. Filter paper
used to collect feces was removed at the designated time points, placed in
labeled petri dishes, and frozen at -70 degrees C. Clean filter paper was then
placed in the containers.

Experiments

Sensitivity of PCR and Southern blot assays for the hepatitis B virus. To
determine the sensitivity of the PCR and Southem blot assays a ten-fold serial
dilutions of the HBV serum (1485 pg/ml) were made. One picogram (pg) of HBV
DNA equates to 2.8 x 105 genomic equivalents thus, approximately 1 picogram
of HBV DNA per milliliter of serum equals 2.9 x 105 copies/ ml or 290 copies/ul
(Hollinger1996). Therefore, 1 ul of the 1485 pg/ml serum used in the dilution
contained approximately 400 copies of the HBV DNA. This serum was then
serially diluted beyond the theoretical and point of one viral particle. A second
set of serial dilutions was made and a whole mosquito was added to each tube
to determine the level of detection in mosquitoes. A third set of serial dilutions
was made and dispensed onto a piece of fitter paper to determine the level of
detection from filter paper. The dilution tubes were then handled in accordance
with extraction and detection methods.

Persistence of ingested Hepatitis B virus. Anopheles stephensi and Aedes
triseriatus cohorts were fed either an infective or control blood meal by artificial
feeder. The HBV blood meal consisted of 1 ml HBV serum (1495 pg/ml HBV-

DNA), 2 ml horse serum and 2 ml horse red blood cells. The control blood meal

70

consisted of 1 ml HBV negative human sera, 2 ml horse serum and 2 ml horse
red blood cells. Both mixtures were loaded into separate Hemotek W51 feeding
chambers and respective mosquito cohorts allowed to feed.

Experiment 1: Persistence of ingested HBV in mosquitoes. An. stephensi
and Ae. triseriatus that ingested a HBV infected blood meal were collected at 3,
7 and 14 days post infected feeding. Collections were made within 2 hours of
the time of the experimental feeding.

Experiment 2: Persistence of ingested HBV in mosquito feces. Collection of
fecal paper from An. stephensi and Ae. triseriatus cohorts from experiment 1.
Filter paper was identified by its collection time. The fecal filter paper contained
feces only from the previous collection time to the labeled collection time.

Experiment 3: Persistence of ingested HBV in mosquitoes. An. stephensi
and Ae. triseriatus that engorged on a HBV infected blood meal were collected
immediately after feeding (time point 0), 6, 12, 24, 48, and 72 hours post feeding.

Experiment 4: Persistence of ingested HBV in mosquitoes feces. Collection
of fecal paper from An. stephensi and Ae. triseriatus cohorts from experiment 3.
Filter paper was identified by its collection time. The fecal filter paper contained
feces only from the previous collection time to the labeled collection time.

Experiment 5: Transmission potential of refeeding An. stephensi. An.
stephensi that engorged on a HBV infected blood meal were held for 3 days and
then offered the opportunity to fed on a negative blood meal. Engorging
mosquitoes, remaining blood, feeding membrane, and fecal paper were all

collected.

71

Persistence of inoculated hepatitis B virus. Anopheles stephensi, Aedes
triseriatus, and Culex quinquefasciatus were given intrathoracic inoculations of
0.3 ul of either an HBV or control suspension. The HBV suspension consisted of
1 part HBV serum (>2000 pg/ml HBV-DNA) and 5 parts E-199 tissue culture
broth mixture. Thus, each 0.3 ul HBV inoculation theoretically contained 30 viral
particles. The control suspension consisted of 1 part negative serum and 5 parts
E-199 tissue culture broth mixture. Inoculations were performed by Dr. Michael
Turrell, USAMRIDD, Ft. Detrick, Maryland.

Experiment 6: Inoculated Anopheles stephensi, Aedes triseriatus, and Culex
quinquefasciatus were collected at time 3, 7, 14 and 21 days as described
above.

Experiment 7: Collection of fecal paper from Anopheles stephensi, Aedes
triseriatus, and Culex quinquefasciatus cohorts from experiment 6. Filter paper
was identified by its collection time. The fecal filter paper contained feces only
from the previous collection time to the labeled collection time.

Methods

DNA Extraction and Detection Procedure. DNA was extracted from the insect
using DNAzolTM and Polyacryl Carrier (Molecular Research Center). DNAzolTM
is a ready to use reagent for the isolation of genomic DNA. The DNAzolT'“
procedure is based on the use of a novel guanidine-detergent lysing solution
which hydrolizes RNA and allows the selective precipitation of DNA from a cell
Iysate. Polyacryl Carrier is a molecular biology grade solution of acryl polymer

designed for use in the isolation of small amounts of DNA with DNAzoIeT".

72

Polyacryl carrier does not affect the activity of restriction enzyme, reverse
transcriptases, TAQ polymerases, DNA polymerase, Iigase and other enzymes
use for DNA analysis.

Two hundred fifty microliters (ul) of DNAzoleTM and 2.5 ul of Polyacryl
CarrierTM were added to the tubes containing mosquito legs and bodies. The
samples were homogenized with a pestle. Five hundred microliters of
DNAzoleTM and 5 ul of Polyacryl CarrierTM were added to the tubes containing
tissue culture broth and saliva. Individual fecal spots were cut out of the filter
paper. Filter paper that contained numerous spots was cut into pieces no larger
than 2 mm X 2 mm. Four to five fecal spots or one piece of paper were placed in
a Iabled eppendorf tube. Seven hundred and fifty microliters of DNAzoleT'“ and
15 ul of Polyacryl CarrierTM were added to tubes containing fecal filter paper,
tubes were centrifuged for 10 seconds at 10,000 rpms, and placed in a 4
degrees C refrigerator for overnight extraction of the feces from the filter paper.
Genomic DNA was precipitated from the lysate with an equal amount of ice cold
100% ethanol. The precipitate was then washed twice with 95% ethanol and
allowed to dry. Precipitated DNA was reconstituted with 10 ul HPLC water,
concentrated by centrifugation and frozen at -20 degrees C until use. In this
condition the isolated DNA was ready for analysis without additional purification.
(See Appendix A for detailed procedure).

Oligonucleotide primers for PCR. The majority of the primers used for
polymerase chain reaction (PCR) provide amplification of the C (HBc) and S
(HBs) genome regions. Analysis of the hepatitis B virus genome revealed
regions that appear to be highly conserved (nucleotides 241 to 270, 1891 to

73

1920, 2431 to 2460). (Robertson 1995). The oligonucleotide primers used in
these experiments amplify the conserved pre-core and core region,
nucleotides1891 to 1920 (Lauder et al. 1993). Primer 1763 5'-GCTTTGGG-
GCATGGACATTGACCCGTATAA-3' begins at map position 1763 and primer
2032B 5'-CTGACTACTAATI'CCCTGGATGCTGGGTCT—3' (from the
complementary reverse [R] DNA strand) begins at map position 2032 of the
hepatitis B virus genome (Fujiyama et al. 1983) producing a 270 bp band

fragment (Figure 2).

 

PreS1/82 ORF S

 

 

 

 

 

 

ORF P 1763

J?
ORF x 2032a

 

 

 

 

 

 

ti
FPre-c l orig I

Figure 2. Diagram of primer location in hepatitis B viral genome. Four
overlapping open reading frames are present in the DNA. The ORF C (core) region
encodes the structural protein of the nucleocapsid and the ORF S/pre-S encodes the viral
surface glycoproteins. Primers amplify a conserved core region, producing a 270 bp size

fragment.

74

Polymerase Chain Reaction. Polymerase chain reactions were run in
accordance with the procedures outlined by Kaneko et al. (1989). Each run
contained at least one positive control, negative control and a blank. A 2.5uL
aliquot of DNA extracted from each sample was amplified in a 50uL reaction
volume containing 2.5 U of Taq DNA polymerase (Perkin Elmer), 200 pmoVL
each of the four deoxyribonucleic triphosphates (Boehringer Mannheim, 100mM
pH7, dNTP's Li-salt), 1 umol/L of the primer pair (primers 1763 and 2032B), 50
mmoVL Tris-HCI ( pH 8.3) (Perkin EImer10X PCR Buffer II), 1.5 mmoVL MgClz,
and 0.01% (wt/vol) gelatin (25mM MgClz' Perkin Elmer). The reaction was
performed for 30 cycles in a programmable DNA Thermal Cycler (Perkin Elmer
GeneAmp® PCR System 2400). Samples were heated at 94 degrees C for 1.5
minutes , cooled to 42 degrees C for 1.5 minutes and extended for 3 minutes at
72 degrees C (DNA polymerase extension).

Contamination of the PCR reaction mixture with minute amounts of
recombinant hepatitis B virus DNA can result in spurious positive results. To
eliminate potential sources of DNA contamination, all reagents were prepared
with pipettors designated for reagent use only and dispensing was done in a
laminar flow hood. No infectious samples were processed in the hood, and the
ultra-violet light was turned on prior to preparing PCR reagents in the hood. The
PCR reaction mixture was dispensed into PCR tubes in the hood and the tubes
were closed. Tubes were then taken to the bench top and sample DNA was
added there. Counter tops were disinfected with 10% bleach solution before and
after any procedures. Autoclavable Oxford” Benchmate digital pipettors were
used with aerosol resistant tips to dispense reagents and specimen.

75

All PCR products were separated on 2% agarose gels with 0.5 pg/ml
ethidium bromide in a electrophoresis chamber containing 1X TBE (0.9 mM Tris,
0.9 mM boric Acid, .02 mM EDTA) with 0.5 [1ng ethidium bromide. Amplified
DNA was mixed by gently aspirating with the pipettor. Eight microliters of the
PCR reaction mixture was mixed with 2 uL of gel loading buffer III 6X (0.25%
bromophenol blue, 0.25% xylene cyanol FF, and 30% glycerol in H20) and
dispensed into the appropriate gel well. Two microliters of a 123bp lambda DNA
ladder (Gibco BRL) was mixed with 2 uL of gel loading buffer III and 6uL of
HPLC water. The electrophoresis apparatus was adjusted to a voltage of 100
between 60-80 amps of current, allowed to run for at least 1.5 hour, and then the
DNA was visualized under ultraviolet light.

Southern Analysis. DNA products were transferred from the 2% agarose gels
to nylon membranes by a BioRad Trans-Blot SD semi dry electrophoretic
transfer cell (Cat. Nos. 170-3940, 170-3948, 170-3949). Two pieces of extra
thick BioRad blotting paper and one piece of nylon membrane (Boehringer
Mannheim Cat Nos. 1209-272, 1209-299) were cut to the size of the gel. The
gel, membrane, and blotting paper were soaked in 0.5X TBE with no ethidium
bromide for 10 minutes then formed into a sandwich built on the anode plate.
Each layer was rolled to remove air bubbles prior to adding the next layer in the
sandwich. The sandwich, from bottom to top, was composed of blotting paper,
nylon membrane, gel, and blotting paper. The sandwich was then saturated
with 0.5x TBE and excess TBE was wiped up. The gel frame was placed
around the sandwich, the top plate latched down, and the top cover secured
completing the circuit. The Trans-blotter was then plugged into the power pack.

76

The amperage required for blotting was calculated by taking the product of:
(length of the gel) X (width of gel) X 0.3. The power pack was set to run on
constant amperage and the calculated amperage was set at a 40 minute run
time. Voltage was monitored throughout the transfer. A voltage that increased
over 25 indicated that the buffer capacity had expired and the run should be
stopped. When this occurred, the amperage was either reduced or the process
was halted depending upon the amount of transfer time remaining.

When the transfer was completed, the system was disassembled in the
reverse order. A pencil was used to mark the wells on the nylon membrane
before removing the gel. The membrane was then removed, rinsed in 2X SSC
(3mM NaCI and .3 mM sodium citrate), and placed on a piece of extra thick filter
paper soaked in 0.4 N NaOH for 5 minutes to break the DNA bonds. The
membrane was rinsed again in fresh 2 X SSC and then placed on piece of filter
paper that was folded into a pocket and labeled. The DNA was then fixed to the
membrane by drying at either 80EIC for 2 hours or 1200C for 1 hour. After drying
all membranes were stored in a ziplock bag in a 40 refrigerator.

Digoxigenin oligonucleotide labeling and detection. A oligonucleotide
internal to the PCR oligonucleotides was used to probe PCR products. Primer
1913 5' -TGTTCACCTCACCATACAGC- 3' began at map position 1913 of the
hepatitis B viral genome and was 3'-end labeled with digoxigenin-ddUTP
(Boehringer Mannheim Cat. No. 1362-372). The steroid hapten digoxigenin
(DIG) was linked via a spacer arm to the primer. The oligonucleotide was
enzymatically labeled at its 3' end with terminal transferase using a single DIG-
labeled dideoxyuridine-triphosphate (DIG-ddUTP).

77

The digoxigenin labeled probe was used with the digoxigenin nucleic acid
detection kit (Boehringer Mannheim Cat. No. 1175 041) to detect hepatitis B
nucleic acids on the Southern blots, by enzyme linked immunoassay using an
anti-digoxigenin alkaline phosphatase conjugated antibody. The nylon
membranes were labeled and placed in pre-hybridized solution (concentrations)
in a shaker over at 39 degrees C for 6 hours. One picomole of DIG-labeled
probe was added for every 10 ml’s of pre-hybridization solution. The
membranes were returned to the 39 degrees C shaker oven for overnight
hybridization. After blocking of the membrane, binding of antibody-conjugate to
hybrized DIG labeled nucleic acid occurs. The color reaction is initiated alkaline
pH by the addition of BCIP and NBT. A blue precipitate starts to form within a
few minutes in the positive control bands. Color development was stopped after
1 hour and the membranes were photographed with 35mm 160 Tungsten film on
a copy table (See Appendix B for complete protocol).

RESULTS
Sensitivity of PCR and Southern blot detection of HBV.

The addition of mosquito bodies had no effect on the sensitivity of the PCR or
Southern detection methods. The PCR had visible bands present at the 1:1000
dilution, 4.0 viral particles, in both the serum and serum plus mosquito lanes
(Figure 3). The Southern blot had visible bands at the 1:10000 dilution, 0.4 viral
particles, in both the serum and serum plus mosquito lanes. This level of
sensitivity was considered adequate as theoretically we were able to detect a
single viral particle. The addition of fecal filter paper decreased the sensitivity of
the PCR and Southern detection methods hundred fold. The PCR and Southern

78

blot had visible bands at the 1:10 dilution, 400 viral particles (Figure 4). Nested
PCR was not done due to a high rate of false positives and the inability to
establish a consistent lower limit of viral detection.

Determination of Virus Persistence and Dissemination. Viral persistence
was determined by the detection of virus in the mosquito bodies. A mosquito
that had virus detected in its body, but not its legs, was considered to have a
nondisseminated viral persistence. The detection of virus in the legs of the

mosquitoes indicated that the virus had disseminated (T urrell et al. 1984).

79

Size

EN12 3 4 5 GBL
7' "'(bp)

EN123456BL

 

Figure 3. Determination of the effect of mosquito tissue on the sensitivlty
of HBV detection using PCR. Serial dilution of 1485 pg/ml HBV DNA serum
plus mosquito. A) PCR detection. B) Southern blot detection. Lanes: E- empty,
N- negative control, 1- straight serum (400 virions), 2- 1:10 (400 virions), 3- 1:100
(40 virions), 4-1:1000 (4 virions), 5-1210000 (0.4 virions), 6-12100000 (0.04
virions), B - reagent blank, L-123 bp lambda ladder.

80

123456789101112L

A)

3)

 

Figure 4. Determination of the effect of filter paper on the sensitivity of
HBV detection using PCR. Serial dilution of 1485 pg/ml HBV DNA serum.
Lanes: E- empty. A) Agarose gel of PCR products, 270 bp band indicates
positive samples. B) Southern blot detection. Lanes:1- empty, 2-1 :10 (400
virions), 3-12100 (40 virions), 4-1:1000 (4.0 virions), 5-1:10000 (0.4 virions), 6-
1:100000 (0.04 virions), 7- 1:10 (400 virions), 8- 1:100 (40 virions), 9-1:1000 (4.0
virions), 10-1:10000 (0.4 virions), 11-1:100000 (0.04 virions), 12- positive control,
L-123 bp lambda ladder.

81

HBV Infected Feeding Results. In the first experiment all results for both An.
stephensi and Ae. triseriatus were negative at days 3, 7 and 14 (Table 1).
Therefore, a second experiment was conducted to examine the HBV persistence
at shorter intervals after feeding. Mosquito legs, saliva, bodies and fecal paper
were collected immediately after feeding, 6,12, 24, 48 and 72 hours post feeding.
Virus was detected by PCR in 100% of An. stephensi bodies at 0 and 6 hours.
At 12 hours 60% of the bodies had detectable virus by PCR but 100% of bodies
were positive by Southern blot. At 24 hours 70% of the bodies were positive by
PCR and Southern blot. At 48 hours only 40% of the bodies were positive by
PCR and Southern blot, however, one mosquitoes legs were also positive by
PCR and Southern blot (Figure 5 and Figure 7). All mosquitoes were negative
by 72 hours and all subsequent collection times (Figure 5 and Figure 8).

Virus was detected by PCR in 100% Ae. triseriatus bodies immediately after
feeding. The 2 mosquitoes collected at 6 hours were both negative. At 12, 24,
48 and 72 hours, 100% of the mosquitoes had virus detectable by PCR and
Southem blot. One 12 hour mosquito saliva and legs and one 48 hour mosquito
legs were positive by Southern blot (Appendix C, Table 2).

Persistence of HBV in Feces. In the first experiment, hepatitis B virus was
detected on 27% of the An. stephensi pieces of 3 day fecal filter paper by PCR
and 45% by Southern blot. Hepatitis B virus was detected on 3% of the A9.
triseriatus pieces of 3 day fecal filter paper by PCR and Southern blot, and 23%
of the pieces of 7 day fecal filter paper by PCR and and 30% by Southern blot

(Figure 6).

82

In the second An. stephensi feeding experiment, 33% of the pieces of fecal
filter paper collected immediately after feeding were positive by PCR and 44% by
Southern blot. No virus was detected in the feces at 6 hours by either PCR or
Southern blot. At 12 hours, 25% of the pieces of fecal paper were positive by
Southern blot only. At 24 hours, 16% of the pieces of fecal filter paper were
positive by PCR and Southern blot. At 48 hours, 100% of the pieces of fecal
filter paper were positive by PCR and Southern blot. At 72 hours, 71% of the
pieces of fecal filter paper were positive by PCR and 86% by Southern blot
(Figure 6).

In the second Ae. triseriatus feeding experiment, no virus was detected on
the fecal filter paper immediately after feeding, 12 hours and 48 hours. There
was no feces on the 6 hour filter paper. At 24 hours, 16% of the pieces of fecal
filter paper were positive by PCR and Southern blot and at 72 hours 11% of the
pieces of fecal filter paper were positive by PCR and Southern blot (FigUre 6).
Transmission potential of An. stephensi during refeeding. All mosquitoes
that refed were negative for the virus by both PCR and Southern blot. The
membrane and all blood used in the refeeding negative by PCR and Southern
blot for virus and virus was not detected on the filter paper collected after

immediately after refeeding. (Appendix C, Table 5).

83

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L13 5 7911131517192123L

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Figure 7. Persistence of HBV in 24 and 48 hour An. stephensi. A) Agarose
gel of PCR products, 270 bp bands indicate positive samples. B) Southern blot
detection. Lanes L 123 lambda ladder, Lane 1 and 23 positive controls, Lanes 2
and 22 negative controls, lane 21 reagent blank. All control and infected sample
run in the order legs, saliva, and body. Lanes 3-5 48 hour control, lanes 6-8 one
24 hour infected sample, lanes 9-20 four 48 hour infected samples. Lanes 8, 11,
14, 17, and 20 contain bodies.

86

A)

3)

 

Figure 8. Persistence of HBV In 48 and 72 hour An. stephensi. A) Agarose
gel of PCR products, 270 bp bands indicate positive samples. B) Southern blot
detection. Lanes L - 123 bp lambda ladder, Lanes 1 and 23 positive controls,
Lane 2 and 22 negative controls, lane 21 reagent blank. All control and infected
sample run in the order legs, saliva, and body. Lanes 3-5 72 hour control, lanes
6-8 one 48 hour infected sample, lanes 9-20 four 72 hour infected samples.
Lanes 8, 11, 14, 17, and 20 contain bodies.

87

HBV intrathoracic inoculation results. No virus was detected, by PCR or
Southern blot, in any samples of saliva, legs, bodies or feces of Ae. triseriatus,
An. stephensi, and Cx. quinquefasciatus collected at 3, 7,and 14 days post HBV
intrathoracic inoculation.
All raw data from this results section are shown in Appendix C, Tables 1-5.
DISCUSSION

The results obtained in this laboratory study indicate that the mosquitoes
studied are unlikely to serve as biological vectors of the hepatitis B virus. Viral
nucleic. acid was detected in An. stephensi for 48 hours post infected feeding
and in As. triseriatus for 72 hours post infected feeding, indicating that the
disappearance of the virus correlates with the digestion of the blood meal. This
observation agrees with the findings of Brotman (1973), Chen (1987), Leevy
(1972), and Newkirk (1975), who found a direct correlation between HBsAg
disappearance and blood meal digestion. The lack of reappearance of virus in
the later infected feeding collection time points and the lack of detectable vims in
the inoculated mosquitoes indicates that the virus does not infect mosquitoes.

The detection of virus on the legs of one 48 hour An. stephensi legs, saliva
and legs of one 12 hour Ae. triseriatus, and the legs of one 48 hour Ae.
triseriatus poses several questions. The detection of virus in the legs of 3
mosquitoes may indicate virus dissemination, however, the legs were positive
prior to the digestion of the blood meal so it is unlikely that there was sufficient
time for the virus to disseminate into the legs. One possible explanation is the

contamination of the legs and saliva during the collection process. The handling

88

and removal of the legs and wings from the body may have resulted in the
rupture of the gut or defecation of virus, contaminating the legs and saliva during
collection. Due to the sensitivity of the PCR method these results need to be
examined carefully for potential contamination. The small sample size does not
allow a determination of the significance of these positive samples.

The presence of detectable virus in the feces of the mosquitoes raise the
question as to whether the virus may be mechanically transmitted. Mosquitoes
only associate with a host during feeding, therefore, the significance of virus in
the feces is debatable. Our results indicate that anopheline mosquitoes defecate
virus during feeding on an infected host, however, the host is the source of the
virus and thus cannot be reinfected. There is potential that a anopheline
mosquito ingesting a partial infected blood meal and subsequently feeding on a
susceptible host may transmit the virus during feeding by excretion of virus
during pre-diruess. A second potential means of mechanical transmission is
when a partially engorged infected mosquito is killed during a subsequent
feeding attempt resulting in deposition of virus on a susceptible host. A third
possibility is that mosquitoes are resting in human dwellings after feeding and
excreting virus into the environment, providing a potential route of contact
infection.

This study agrees with earlier studies (Hawkes et al. 1972, Leevy et al. 1972,
Prince et al. 1972, Brotman et al. 1973, Muniz and Micks 1973, lshii et al. 1974,
Wills et al. 1976, Chen et al. 1987, Fouche et al. 1989, Fouche et al. 1990, Jupp
et al. 1991) in finding no evidence of biological transmission by mosquitoes but

they may potential be mechanical vectors. The use of PCR and Southern blot

89

techniques, allowed us to detect a segment of the viral genome and not just the
surface antigen which is found on all three particle types. The specificity and
sensitivity of these methods, theoretically allowed us to detect as few a 0.4
hepatitis B virions in the mosquito and 40 hepatitis B virions in the feces. The
detection of a segment of the pre-core and core region of the viral nucleic acid
does not correlate to infectivity. Hence, no definitive conclusions can be drawn
from the detection of viral genome in the feces of the mosquitoes. A carefully
controlled transmission study is needed to answer these lingering questions.
However, due to the host specificity of the hepatitis B virus and the cost
associated with working with primates, the use of a related animal hepadnavirus
and its animal host would be recommended to address the question of

mechanical transmission of the virus by mosquitoes.

9O

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States. Viral Hepatitis.23:(3); 437-455.

Beasley, R. 1988. Hepatitis B Virus: The major etiology of hepatocellular
carcinoma. Cancer.61:(10); 1942-1956.

Berquist, K., J. Maynard, D. Francy, M. Sheller and C. Schable. 1976.
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Blumberg, B., H. Alter and S. Visnich. 1965. A'new" antigen in leukemia sera.
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Blumberg, B., W. Wills, I. Millman and W. London. 1973. Australia antigen in
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Brotman, B., A. Prince and H. Godfrey. 1973. Role of arthropods in
transmission of Hepatitis B virus in the tropics. Lancet.i: 1305-1308.

Byrom, N., G. Davidson, C. Draper and A. Zuckerman. 1973. Role of
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Chen, C., L. Shou, L. Hwang, P. Tang and W. Tu. 1987. Persistence of
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Dick, 8., C. Tamburro and C. Leevy. 1974. Hepatitis B antigen in urban-caught
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Foster, 0., A. Ajdukiewicz, R. Ryder, H. Whittle and A. Zuckerman. 1984.
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Fouche, A., R. Crewe and I. Windsor. 1989. Vertical transmission of hepatitis B

virus in Culex quinquefasciatus. Medical and Veterinary Entomology.3: 385-
389.

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Fouche, A., R. Crewe, I. Windsor and S. Karim. 1990. Persistence of hepatitis
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Fujiyama, A., A. Miyanohara, C. Nozaki, T. Yoneyama, N. Ohtomo and K.
Matsubara. 1983. Cloning and structural analyses of hepatitis B virus DNA,
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Hamilton, J. 1994. Huge bounty on a global killer. Business Week. April 4, 1994.
92-94.

Hawkes, R., T. Vale, I. Marshall and R. Maclennan. 1972. Contrasting sero-
epidemiology of Australia antigen and arbovirus antibodies in New Guinea.
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Hollinger, F. 1996. Hepatitis B Virus. pp. 2739-2807. In B. Fields, E. Knipe, P.
Howley, R. Chanock, T. Monath, J. Melnick, B. Roizman and S. Straus [eds.],
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Hyams, K. 1989. Mosquito transmission of hepatitis B. Tropical and
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lshii, A., A. Yago, H. Nariuchl, A. Shirasake, Y. Wada and T. Matuhasi. 1974.
Some aspects of the transmission of hepatitis B antigen; Model experiments
by mosquitoes with Murine hepatitis virus. Japanese Joumal of Experimental
Medicine.44:(6); 495-501.

Jupp, P., R. Purcell, M. Shapiro and J. Gerin. 1991. Attempts to transmit
hepatitis B virus to chimpanzees by arthropods. South African Medical
Joumal.79: 320-322.

Kaneko, S., R. Miller, S. Feinstone, M. Unoura, K. Kobayashi, N. Hattori and
R. Purcell. 1989. Detection of serum hepatitis b virus DNA in patients with
chronic hepatitis using the polymerase chain reaction assay. Proceedings of
the National Academy of Science, USA.86: 312-316.

Lauder, l., H.-J. Lin, J. Lau, T.-S. Siu and C.-L Lai. 1993. The variability of the
hepatitis B virus genome: Statistical analysis and biological implications.
Molecular Biology and Evolution.10:(2); 457-470.

Leevy, C., M. Howard, W. Crane, E. Evans and P. A. 1972. Viral hepatitis and
the mosquito. Gastroenterology.62:(4); 872.

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Mayans, M., A. Hall, I. HM, J. Chotard, S. Lindsay, E. Coromina, M. Mendy,
P. Alonso and H. Whittle. 1990. Risk factors for transmission of hepatitis B
virus to Gambian children. Lancet.336: 1107-1 109.

Mayans, M., A. Hall, H. Inskip, S. Lindsay, J. Chotard, M. Mendy and W. HC.
1994. Do bedbugs transmit hepatitis B? Lancet.343: 761-763.

Maynard, J. 1981. Hepatitis B vaccine: Strategies for utilization. pp. 13-19. In
P. Maupas and P. Guesry [eds.], Hepatitis B Vaccine,, vol. Inserrn
Symposium No. 18. Elsevier/ North Holland Biomedical Press, Amsterdam.

Metselaar, D., B. Blumberg, l. Millman, A. Parker and A. Bagshawe. 1973.
Hepatitis B antigen in colony mosquitoes. Lancet.2: 758-759.

Muniz, F. and D. Micks. 1973. The persistence of hepatitis B antigen in Aedes
aegypti. Mosquito News.33:(4); 509-511.

Neefe, J. 1949. Viral Hepatitis: Problems and Progress. Annals of Intemal
Medicine.31:(5); 857-870.

Newkirk, M., A. Downe and J. Simon. 1975. Fate of ingested hepatitis B
antigen in blood-sucking insects. Gastroenterology.69: 982-987.

Papaevangelou, G. and T. Kourea-Kremastinou. 1974. Role of mosquitoes in
transmission of hepatitis B virus infection. Joumal of Infectious
Disease.1 30:(1 ); 78-80.

Prince, A. 1970. Prevalence of serum-hepatitis-related antigen (SH) in different
geographic regions. American Journal of Tropical Medicine and
Hygiene.19:(3); 872-879.

Prince, A., D. Metselaar, G. Kafuko, L. Mukwaya, C. Ling and L Overby.
1972. Hepatitis B antigen in wild-caught mosquitoes in Africa. Lancet.2: 247-
250.

Robertson, B. 1995. PCR detection and analysis of hepatitis viruses. pp. 57-80.
In J. Clewley [ed.] The polymerase chain reaction (PCR) for human viral
diagnosis, CRC Press, Boca Raton.

Skinhoj, P. 1972. Hepatitis B antigen in mosquitoes. Lancet.2: 715.

Smith, J., E. Ogunba and T. Francis. 1972. Transmission of Australia Au (1)
antigen by Culex mosquitoes. Nature.237: 231 -232.

Stevens, C., R. Beasley, J. Tsui and W.-C. Lee. 1975. Vertical transmission of
hepatitis B antigen in Taiwan. N Engl J Med.292: 771 -774.

93

Tabor, E., A. Bayley, L. Cairns, L. Pelleu and R. Gerety. 1985. Horizontal
transmission of hepatitis B virus among children and adults in five rural
villages in Zambia. Joumal of Medical Virology.15: 113-120.

Tin, K., S. Sebastian and A. Kyaw. 1973. Hepatitis B antigen in mosquitoes.
Lancet.2:259.

Turrell, M., T. Gargan and C. Bailey. 1984. Replication and dissemination of
Rift Valley fever virus in Culex pipiens. American Journal of Tropical
Medicine.19: 335-341 .

Whittle, H., K. McLaughlan, A. Bradley, A. Ajdukiewicz, C. Howard, A.
Zuckerman and I. McGregor. 1983. Hepatitis B virus infection in two
Gambian villages. Lancet. 1203-1206.

Wills, W., G. Saimot, C. Brochard, B. Blumberg, W. London, R. Dechene and
I. Millman. 1976. Hepatitis B surface antigen (Australia antigen) in
mosquitoes collected in Senegal, West Africa. The American Journal of
Tropical Medicine and Hygiene.25:(1); 186-190.

Yuhuang, Z., L. Duo, F. Deyun, T. Hong, L. Yuzhang and Y. Xueke. 1995. An

animal study on transmission of hepatitis B virus through mosquitoes.
Chinese Medical Joumal.108:(12); 895-897.

94

CHAPTER 2

PERSISTENCE OF THE HEPATITIS B VIRAL GENOME IN
CIMEX LECTULAFIIUS L. AND RHODNIUS PROLIXUS Stal

INTRODUCTION

Hepatitis B infection is a major cause of acute and chronic hepatitis, cirrhosis,
and primary hepatocellular carcinoma (PHC) world wide (CDC 1990) killing
estimated 2 million people each year (Hamilton 1994). Primary hepatocellular
cancer is one of the most common cancers in Asia and sub-Saharan Africa
(Beasley 1988) where 8- 15% of the population carry markers of HBV infection
(Maynard 1981). Persons known to be at risk of contracting hepatitis B include:
sexually active men and women; prison inmates; health care and public safety
workers; hemodialysis patients; lntemational travelers; and household contacts
of HBV carriers. Primary modes of transmission are parenteral, perinatal, and
sexual contact, however, 30 - 40% of people infected with HBV have no
identifiable risk factor (Alter and Mat 1994).

Neefe (1949) was the first to consider the possibility that hematophagous
arthropods could transmit the hepatitis virus. Prince (1970) found an increased
prevalence of SH (hepatitis B surface antigen) antigen in the Tropics. Unable to
account for the increase by known mechanisms of transmission, Prince (1970)
suggested transmission in nature by nonparenteral routes. Of the potential
routes discussed, the possibility of transmission by hematOphagous arthropods
was thought the most probable. The subsequent detection of the hepatitis B

95

surface antigen (HBsAg) by solid-phase radioimmunoassay (RIA) in wild caught
mosquitoes supported this hypothesis (Prince et al. 1972) and stimulated
research on other anthropophilic hematophagous arthropods.

The detection of HBsAg in unengorged nymphs and adult Cimex hemipterus
and C. Iectularius from huts in Africa (Jupp et al. 1978, Ogston et al. 1979, Wills
et al. 1977) suggested that bed bugs could be potential mechanical or biological
vectors of hepatitis B virus. Bed bugs must feed at least four and usually five
times, upon either the same or different hosts, to reach maturity. Trans-stadial
transmission of the hepatitis B virus was shown for one molt but not two molts
and detected 52 days in bugs only undergoing a single molt (Jupp and McElligott
1979). Neither Jupp (1979) nor Taylor (1980) found evidence of transovarial
transmission of the virus. Laboratory experiments showed that HBsAg could be
detected in the insect bodies for 6-7.5 weeks post infected feeding (Jupp and
McElligott 1979) and in the feces for 5-6 weeks (Jupp et al. 1983, Taylor and
Morrison 1980). Jupp et al. (1980) and Jupp et al. (1983) demonstrated that the
virus was not replicating by serial passage of the virus through three successive
cohorts of bed bugs during which hepatitis B surface antigen was detected in the
first and second passages but not the third. Jupp et al. (1983) concluded that
there was no indication of virus multiplication in C. Iectularius but that mechanical
transmission from insects to man could occur by contamination of a person when
crushing infective bugs; contamination from infected feces; or, infection by bite
due to regurgitation or interrupted feeding.

Villajeros et al. (1975) examined the vector potential of Rhodnius prolixus for
hepatitis B virus in South and Central America by feeding colony cohorts infected

96

blood and collecting them at weekly intervals. Antigen was detected in 100% of
the stomachs and feces for the first two weeks and persisted up to four weeks in
50% of the insects. At no stage during the 6 week experiment did they detect
HBsAg in the hemolymph or salivary glands.

All of these studies used detection assays for the hepatitis B surface antigen
(HBsAg) which is present on the surface of all three particles produced by the
hepatitis B virus. Of the three particles, only the Dane particle contains virions
and is infectious (Blumberg et al. 1965). The concentration of non-virion
associated HBsAg greatly exceeds the concentration of complete virions by 104
or greater.

The epidemiological significance of the association between suspected
arthropod vectors, hepatitis B virus, and humans provides a different perspective
of the potential importance of arthropod vectors. Hepatitis B infections in Africa
commonly occur after one year of age (Tabor et al. 1985), whereas, in Asia
many children are infected at birth by their mother (Stevens et al. 1975).
Tattooing, circumcision, ear piercing, traditional scarring, exudative skin lesions,
and hematophagous arthropods have all been proposed as mechanisms of
transmission. Mayans (1990) found a significant association between hepatitis e
(HBe) antigenaemia and the presence of bed bugs in children’s beds. However,
a later intervention study (Mayans et al. 1994) found no significant association to
support bed bugs as a major mode of transmission of hepatitis B in Gambian
children.

Due to the host specificity of the virus few transmission studies have been

conducted. No evidence of mechanical or biological transmission was found in

97

studies using mosquitoes and human volunteers (Neefe 1949) or chimpanzees
(Berquist et al. 1976) but evidence of transmission was found in studies using
mosquitoes and chickens (Smith et al. 1972) or monkeys (Yuhuang et al. 1995).
Jupp et al. (1991) found no evidence of transmission by C. Iectularius with
chimpanzees however, he did not test for mechanical transmission by interrupted
feeding or fecal contamination.

The purpose of this study was to determine the persistence of the hepatitis B
virus in Cimex Iectularius and Rhodnius prolixus that had ingested or been
inoculated with the virus. Polymerase chain reaction and Southern blotting was
used to detect a segment of the hepatitis B viral genome.

MATERIALS AND METHODS
Insect Rearing

Cimex Iectularius were obtained from a colony maintained by LTC Harold
Harlan, MSC, USA (Ret.) that was originally collected from a basic training billet
at Fort Dix, NJ, on 20 March 1973 (Bartley 1974). One liter Nalgene jars with .
mesh screened lids and pieces of file folder for harborage were used for rearing.
The cohort jars were maintained at 240 with 30% humidity and a 16:8 (L:D) h
photoperiod When container density became to great to safely fed on a rat, the
jar was opened and part of the paper substrate removed and placed in a new jar.
Fresh paper substrate was added to both jars and the jars closed. Colony jars
were not age graded due to the difficulty in handling and containing the larvae.

Rhodnius prolixus were obtained from the colony maintained by Dr. John
Edman, University of Massachusetts. Age cohorts are maintained in 1 liter

Nalgene jars with mesh screened lids and folded Whitman’s number 1 filter

98

paper to provide harborage and egg laying substrate. The jars are surrounded
by a dark material cover to minimize light. Cohort jars were maintained at 28
degrees with 84% humidity and a 12:12 (L:D) h photoperiod. Adults were
removed from immature cohort jars and placed into adult cohort jars to promote
mating and egg laying. A few adults were left in the jars with the eggs and larva
to provide digestive symbionts. Larvae were sorted into age graded jars when
jar density and age range became to great . Exuviae and dead insects were
removed and fresh filter paper placed in the jars.

Blood Feeding on Animals. Stock colonies of bugs were fed weekly on rats.
The All University Committee on Animal Use and Care approved the use of rats
to feed the insect colonies, AUF# 05/96-053-00. Rats where selected based on
willingness of both Diptera and Hemiptera to feed on them and the ability to
maintain a sufficient number of rats to feed the all the insect colonies necessary
to conduct this research.

Jars of C. Iectularius and R. prolixus were transported from the Natural
Science Building to the Giltner animal room, a University Laboratory Animal
Resource (ULAR) facility. Rats were restrained in surgical stocking and wrapped
loosely in a towel and placed under the scavenger hood. Methoxyflurane was
administered with a nose cone until the rat became fully relaxed at which time
the towel and surgical stocking were removed. The abdominal area was shaved
and sterile eye ointment administered to the eyes in preparation for feeding. The
rat was then laid on its back on a rolled towel with its head in the scavenger hood
and its nose in the nose cone. The colony jars were placed on the abdomen
supported by the rolled towel for approximately 20 minutes. Rats’ respiration

99

rate was monitored throughout the feeding period. Rats were removed from the
cage and allowed to expire the anesthetic before being placed in a recovery
cage. Recovering rats’ respiration and state of consciousness were monitored
until each rat was awake, then it was returned to its own cage.

Rats were used once every two weeks for feeding. Any rat not appearing
active and healthy was removed from the feeding rotation until examined by
ULAR personnel. Any rat that developed a suspected tumor or possible irritation
due to feeding was removed from the feeding rotation and the ULAR veterinarian
notified immediately.

Artificial Blood Feeding. Stock R. prolixus colonies were also fed by artificial
feeder, however, C. Iectularius would not fed on the artificial feeder. A Hemotek
5W1 artificial feeding system (Discovery Workshops, England) was used for all
feedings. The system consists of 5 independently controlled, heated feeding
chambers. Each feeding chamber holds 5 milliliters of blood. Three different
types of feeding membranes (parafilm, latex, and cellulose) were used for colony
maintenance. A cellulose membrane was used exclusively during infected
feedings. The membrane was stretched across the feeding chamber and
secured with an O-ring. The chambers were filled with blood, capped and

screwed into the heating element, then placed on the cage for feeding (Figure 1).

100

H - ating element

Power Unit
‘ Heating
0 O O O 9 element

 

Feeding Chamber

Figure 1. Diagram of Hemotek 5W1 artificial feeding system. A) The power
unit can supply power to five independently controlled heating elements. B)
Each feeding chamber was covered with a membrane and filled holds 5 milliliters
of blood.

Bovine or equine blood was used for all feedings. Mature bovine blood
preserved with heparin was obtained in 100 ml aliquots from Pel-Freez
Biologicals,(P.O. Box 68, Rogers AR 72757). Mature equine blood preserved
with heparin was obtained in 100 ml aliquots from Veterinary Dynamics Inc.,
(1535 Templeton Road, Templeton CA 93465). Blood was collected and
shipped over night on wet ice at 4 degrees C and then stored at 4 degrees C in
the laboratory. Blood was kept no longer than 30 days from date of drawing from

the bovine or equine.

101

Experimental Insect Transport, Feeding, Handling and Containment. A HBV
positive volunteer, HBV DNA titer 1485 pg/ml, was arranged by the Department
of Gastroenterology-Hepatology, William Beaumont Hospital, Royal Oak,
Michigan and the negative volunteer was the researcher transporting
experimental insects. The human feeding was approved by both William
Beaumont Hospital and Michigan State University, University Committee on
Research Involving Human Subjects (UCRIHS) IRB # 96-039. Cimex Iectularius
adults and larva were collected from the stock colonies and placed in two clean
jars with fresh filter paper one week prior to the infected feeding. The filter paper
was changed the moming of the infected feeding and the Cimex transported to
William Beaumont Hospital for experimental feeding. The HBV volunteer was
shown the insects and the feeding procedure explained prior to obtaining
informed consent. The control and infected feeding were then done on the
forearm of both volunteers. The feeding time was approximately one hour to
allow for a high engorgement rate.

Quantitative hepatitis B viral levels were determined by the Abbott hepatitis B
viral DNA Assay (Abbott Laboratories, Abbott Park, IL) a liquid phase molecular
hybridization assay for quantifying hepatitis B virus DNA in human serum.
Results are reported in picograms of DNA per milliliter (pg/ml) of serum.
Hepatitis B virus infected insect processing and collection. Experimental
cohort jars were placed in a white enamel lab pan with petroleum gel applied
around the lip of the pan. That pan was then placed in a larger pan containing
ice. Three empty clean1 liter Nalgene jars and the experimental feeding jar were
placed in the first pan. The feeding jar was opened and engorged C. Iectularius

102

were collected with fine tipped forceps and placed into either the adult or
immature cohort jars. Non-engorged insects were placed in the third jar. After
all the bed bugs were sorted into the labeled jars, fresh filter paper was placed in
engorged adult and immature jars so that the paper did not reach the screened
lid. The non-engorged insects were killed by freezing and then autoclaved prior
to disposal. Infected cohorts were kept in the sealed Labconco glove box with
humidity pads for the duration of the experiment. All cohorts, control and
infected, were handled in the same manner. Control samples were always
collected before the infected samples.

Immature C. Iectularius were held for 14 days to allow for molting. The cohort
jar was then placed in the freezer to chill the insects. Individuals were then
removed from the jar, the instar noted, and placed in an appropriately labeled 1.5
ml eppendorf tube. All samples were frozen at -70 degrees C at the end of the
collection.

Five adult C. Iectularius were collected with fine tip forceps at each collection
time and placed in labeled individual 1.5 ml eppendorf tubes. The remaining
insects were gently removed from the filter paper and placed back in the jar. The
filter paper was carefully examined eggs and a portion containing eggs was
placed in a clean 100 ml Nalgene jar to hatch. The remaining filter paper was
placed in a labeled petri dish and frozen at -70 degrees C until analysis. A fresh
piece of filter paper was then placed in the cohort jar.

The infected insects were then placed on ice to chill them prior to processing.
Individual bed bugs were removed from the eppendorf tube with heat sterilized
forceps and placed on a clean microscope glass slide. The legs were removed

103

with a sterile scalpel and collected and placed in a labeled 1.5 ml eppendorf
tube. The body was the returned to the labeled 1.5 ml eppendorf tube. All
samples were then frozen at -70 degrees C until DNA analysis.
Experiments
Sensitivity of PCR and Southern blot assays for the hepatitis B virus. To
determine the sensitivity of the PCR and Southern blot assays, PCR and
Southern blot was done on a series of different template concentrations. One
picogram (pg) of HBV DNA equates to 2.8 x 105 genomic equivalents thus,
approximately 1 picogram of HBV DNA per milliliter of serum equals 2.9 x 105
copies/ ml or 290 copies/ul (Hollinger1996). Therefore, 1 ul of the 1485 pg/ml
serum used in the dilution contained approximately 400 copies of the HBV DNA.
This serum was then serially diluted beyond the theoretical end point of one viral
particle. Whole bed bugs or kissing bugs were added to separate replicate
serial dilutions to investigate the effect of insect tissue on the sensitivity of the
testing A fourth set of serial dilutions was made and dispensed onto a piece of
filter paper to determine the level of detection from filter paper. The dilution
tubes were then handled in accordance with extraction and detection methods.
Persistence of ingested Hepatitis B virus. C. Iectularius were fed on the
forearm of either a HBV positive (1495 pg/ml HBV-DNA) or negative volunteer.
The experimental cohorts were then handled in the manner described above.
Experiment 1: Persistence of HBV genome in adult C. Iectularius. One
engorged adult C. Iectularius was collected during the sorting process to serve
as a control. Cohorts of 5 adults were then collected on days, 3, 7, 14, 21, 28,

and 35 post HBV infected feeding.
104

Experiment 2. Persistence of HBV genome in C. Iectularius feces. The HBV
infected feeding paper was collected and then the filter paper was collected on
days 3, 7, 14, 21, 28, and 35 post HBV infected feeding. The fecal filter paper
contained feces only from the previous collection time to the labeled collection
time.

Experiment 3. Trans-stadial Transmission of ingested hepatitis B virus.
Immature C. Iectularius were placed in a separate jar and held 14 days to allow
for molting. At the time of collection, their instar stage was noted they were
placed into individually labeled 1.5 ml eppendorf tubes and frozen at -70 degrees
C until analysis.

Experiment 4. Trans-ovarial Transmission of ingested hepatitis B virus. A
portion of the fecal filter that contained eggs was placed into a separate 100 ml
Nalgene jar to hatch. All larva were collected and placed in one 1.5 ml labeled
eppendorf tube and frozen at -70 degrees C until analysis.

Persistence of inoculated hepatitis B virus. C. Iectularius were given a
intrathoracic inoculation with 0.5 ul of either an HBV or control suspension and
R. prolixus with 1.0 ul of either an HBV or control suspension in a ratio of 1 part
HBV serum (>2000 pglml HBV-DNA) or control serum and 5 parts E-199 tissue
culture broth mixture. Thus, each 0.5 ul HBV inoculation theoretically contained
45 viral particles and each 1.0 ul HBV inoculation theoretically contained 90 viral
particles. The control suspension consisted of 1 part negative serum and 5 parts
E-199 tissue culture broth mixture. Inoculations were performed by Dr. Michael
Turell, USAMRIDD, Ft. Detrick, Maryland. The experimental cohorts were then

handled in the manner described above.

105

Experiment 5. Persistence of inoculated HBV in C. Iectularius. Cohorts of 5
adults were then collected on days 7, 14, and 21 post HBV inoculation.

Experiment 6. Detection of inoculated HBV in C. Iectularius feces. The filter
paper was collected on days 7, 14, and 21 post HBV inoculation. Filter paper
was identified by its collection time. The fecal filter paper contained feces only
from the previous collection time to the labeled collection time.

Experiment 7. Persistence of inoculated HBV in R. prolixus. Cohorts of 5
adults were then collected on days 7, 14, 21, 28, and 35 post HBV inoculation.

Experiment 8. Detection of inoculated HBV in R. prolixus feces. The filter
paper was collected on days 7, 14, 21, 28, and 35 post HBV inoculation. The
fecal filter paper contained feces only from the previous collection time to the
labeled collection time.
Methods
DNA Extraction and Detection Procedure. DNA was extracted from the insect
using DNAzolTM and Polyacryl Carrier (Molecular Research Center). DNAzolTM
is a ready to use reagent for the isolation of genomic DNA. The DNAzolW‘
procedure is based on the use of a novel guanidine-detergent lysing solution
which hydrolizes RNA and allows the selective precipitation of DNA from a cell
lysate. Polyacryl Carrier is a molecular biology grade solution of acryl polymer
designed for use in the isolation of small amounts of DNA with DNAzoleT".
Polyacryl carrier does not affect the activity of restriction enzymes, reverse
transcriptases, TAQ polymerases, DNA polymerase, Iigase and other enzymes

use for DNA analysis.

106

Table 1. Quantity of DNAzol and Polycarrier gel added to each sample type for
extraction of DNA.

 

 

 

 

 

 

 

 

 

 

Sample DNAzol (ul) Polycarrier Gel (ul)
Cimex body 250 5
Cimex legs 250 5
Rhodnius body 750 15
Rhodnius legs 500 10
Rhodnius saliva 500 10
Fecal Filter Paper 750 15

 

DNAzoleTM and Polyacryl CarrierTM were added to the tubes containing
saliva, bodies, and legs and the samples were homogenized with a pestle (Table
1 ). Individual fecal spots were cut out of the filter paper. Filter paper that
contained numerous spots was cut into pieces no larger than 2 mm X 2 mm.
Four to five fecal spots or one piece of paper was placed in a labeled eppendorf
tube. DNAzoleTM and Polyacryl CarrierT" were added to tubes containing fecal
filter paper, tubes were centrifuged for 20 seconds at 10,000 rpm and placed in a
400 refrigerator for overnight extraction of the feces from the filter paper.
Genomic DNA was precipitated from the lysate with an equal amount of ice cold
100% ethanol. The precipitate was then washed twice with 95% ethanol and
allowed to dry. Precipitated DNA was reconstituted with 10 ul HPLC water,
concentrated by centrifugation and frozen at -20 degrees C until use. In this
condition the isolated DNA was ready for analysis without additional purification.

(See Appendix A for detailed procedure).

107

 

Oligonucleotide primers for PCR. The majority of primers used for PCR
provide amplification of the C (HBc) and S (HBs) genome regions. Analysis of
the hepatitis B virus genome revealed regions that appear to be highly
conserved (nucleotides 241 to 270, 1891 to 1920,2431 to 2460) (Roberston
1995). The primers used in these experiments amplify the conserved pre-core
and core region, mucleotides 1891 to 1920 (Lauder et al. 1993). Primer 1763
5'-GCTTI'GGGGCATGGACATTGACCCGTATAA-3' begins at map position 1763
and primer 2032R 5'-CTGACTACTAATTCCCTGGATGCTGGGTCT-3' (from the
complementary reverse [R] DNA strand) begins at map position 2032 of the

hepatitis B virus genome (Figure 2) (Fujiyama et al. 1983).

 

Pre-S1/SZ ORF S

 

 

 

 

 

 

ORF P 1763

 

 

 

 

 

 

lPre-C l ORFC |

Figure 2. Diagram of Primer location in Hepatitis B viral genome. Four
overlapping open reading frames are present in the DNA. The ORF C (core)
region encodes the structural protein of the nucleocapsid and the ORF S/pre-S
encodes the viral surface glycoproteins. Primers amplify a conserved core
region, producing a 270 bp size fragment.

108

Polymerase Chain Reaction. Polymerase chain reactions were run in
accordance with the procedures outlined by Kaneko (1989). Each run contained
at least one positive control, negative control and a blank. A 2.5uL aliquot of
DNA extracted from each sample was amplified in a 50pL reaction volume
containing 2.5 U of Taq DNA polymerase (Perkin Elmer), 200 pmol/L each of the
four deoxyribonucleic triphosphates (Boehringer Mannheim, 100mM pH7,
dNTP's Li-salt), 25 pmol of each of the primer pair (primers 1763 and 2032R), 50
mmol/L Tris-HCI (pH 8.3)[10X PCR Buffer ll (Perkin Elmer)] , 1.5 mmoVL MgClz
and 0.01% (wt/vol) gelatin (25mM MgClz Perkin Elmer). The reaction was
performed for 30 cycles in a programmable DNA Thermal Cycler (Perkin Elmer
GeneAmp® PCR System 2400). Samples were heated at 94 degrees C for 1.5
minutes (denaturization), cooled to 42 degrees C for 1.5 minutes (oligonucleotide
primer hybridization) and incubated for 3 minutes at 72 degrees C (DNA
polymerase extension). Contamination of the PCR reaction mixture with minute
amounts of hepatitis B virus DNA can result in spurious positive results (i.e.,
false positives). To eliminate potential sources of DNA contamination, all
reagents were prepared with pipettors designated for reagent use only and
dispensing was done in a laminar flow hood. No infectious samples were
processed in the hood, and the ultra-violet light was turned on prior to preparing
PCR reagents in the hood. The PCR reaction mixture was dispensed into PCR
tubes in the hood and the tubes were closed. Tubes were then taken to the
bench top and sample DNA was added there. Counter top’s were disinfected

with 10% bleach solution before and after any procedures. Autoclavable Oxford0

109

Benchmate digital pipettors were used with aerosol resistant tips to dispense
reagents and specimen.

All PCR products were separated on 2% agarose gels with 0.5 pglml
ethidium bromide in an electrophoresis chamber containing 1X TBE (0.9 mM
Tris Base, 0.9 mM Boric Acid, .02 mM EDTA) with 0.5 pglml ethidium bromide.
Amplified DNA was mixed by gently aspirating with the pipettor. Eight microliters
of the PCR reaction mixture was mixed with 2 (L of gel loading buffer III 6X
(0.25% bromophenol blue, 0.25% xylene cyanol FF, and 30% glycerol in H20)
and dispensed into the appropriate gel well. Two microliters of a 123 bp lambda
DNA ladder (Gibco BRL) was mixed with 2 uL of gel loading buffer III and 6 uL of
HPLC water. The electrophoresis apparatus was adjusted to a voltage of 100
between 60-80 amps of current, allowed to run for at least 1.5 hour, and then the
gel was examined for separation of DNA fragments. More time was occasionally
required to obtain complete DNA fragment separation.

After electrophoresis the gel was examined with ultraviolet light fluorescence.
The presence of a 270 bp band in the positive control lanes indicated that the
PCR amplification had performed as expected. The presence of 270 bp bands
in other lanes on the gel indicated positive samples. Gels were viewed and
photographed on an ultraviolet light box.

Southern Analysis. DNA products were transferred from the 2% agarose gels
to nylon membranes by a BioRad Trans-Blot SD semi dry electrophoretic
transfer cell (Cat. Nos. 170-3940, 170-3948, 170-3949). Two pieces of extra
thick BioRad blotting paper and one piece of nylon membrane (Boehringer
Mannheim Cat Nos. 1209-272, 1209-299) were cut to the size of the gel. The

110

gel, membrane, and blotting paper were soaked in 0.5X TBE with no ethidium
bromide for 10 minutes, then formed into a sandwich built on the anode plate.
Each layer was rolled to remove air bubbles prior to adding the next layer in the
sandwich. The sandwich, from bottom to top, was composed of blotting paper,
nylon membrane, gel, and blotting paper. The sandwich was then saturated
with 0.5x TBE and excess TBE was removed. The gel frame was placed around
the sandwich, the top plate latched down, and the top cover secured completing
the circuit. The Trans-blotter was then plugged into the power pack. The
amperage required for blotting was calculated by taking the product of: (length
of the gel) X (width of gel) X 0.3. The power pack was set to run on constant
amperage and the calculated amperage was set at a 40 minute run time.
Voltage was monitored throughout the transfer. A voltage that increased over 25
indicated that the buffer capacity had expired and the run should be stopped.
When this occurred, the amperage was either reduced or the process was
halted depending upon the amount of transfer time remaining.

When the transfer was completed, the system was disassembled in the
reverse order. A pencil was used to mark the wells on the nylon membrane
before removing the gel. The membrane was then removed, rinsed in 2X SSC
(3mM NaCl and .3 mM sodium citrate), and placed on a piece of extra thick filter
paper soaked in 0.4 N NaOH for 5 minutes to break the DNA bonds. The
membrane was rinsed again in fresh 2 X SSC and then placed on piece of filter
paper that was folded into a pocket and labeled. The DNA was then fixed to the

membrane by drying at either 80 degrees C for 2 hours or 120 degrees C for 1

111

hour. After drying, all membranes were stored in a ziplock bag in a 4 degrees C
refrigerator.

Digoxigenin oligonucleotide labeling and detection. A oligonucleotide
internal to the PCR oligonucleotides was selected to confirm that the product
amplified by PCR was actually part of the hepatitis B genome and not a random
DNA amplification. Primer 1913 5'-TG‘ITCACCTCACCATACAGC- 3' began at
map position 1913 of the hepatitis B viral genome and was 3'-end labeled with
digoxigenin-ddUTP (Boehringer Mannheim Cat. No. 1362-372). The steroid
hapten digoxigenin (DIG) was linked via a spacer arm to the primer. The
oligonucleotide was enzymatically labeled at its 3' end with terminal transferase
using a single DIG-labeled dideoxyuridine-triphosphate (DIG-ddUTP). The
labeled oligonucleotide was then used in the DIG nucleic acid detection.

The digoxigenin labeled probe was used with the digoxigenin nucleic acid
detection kit (Boehringer Mannheim Cat. No. 1175 041) to detect hepatitis B
nucleic acids on the Southern blots, by enzyme linked immunoassay using an
anti-digoxigenin alkaline phosphatase conjugated antibody. The nylon
membranes were labeled, placed in pre-hybridized solution in a 39 degree C
shaker oven, and then hybridized with the DIG-labeled 1913 oligonucleotide at
39 degree C in the shaker oven overnight. After blocking the membrane, binding
of antibody-conjugate to hybrized DIG labeled nucleic acid occurs. The color
reaction is initiated alkaline pH by the addition of BCIP and NBT. A blue
precipitate starts to form within a few minutes in the positive control bands.

Color development was stopped after 1 hour and the membranes were

112

photographed with 35mm 160 Tungsten film on a copy table (See Appendix B for
complete protocol).

Determination of Virus Persistence and Dissemination. Viral persistence
was determined by the detection of virus in the bugs bodies. A bed bug that had
virus detected in its body, but not its legs, was considered to have a
nondisseminated viral persistence. The detection of virus in the legs indicated
that the virus had disseminated (T urrell et al. 1984).

Adult bed bugs ingest 5 -10 pl (Newkirk et al. 1975) of blood during feeding.
The amount of virus ingested during the HBV infected blood meal was estimated
by multiplying 400 (number of viral copies/ul of serum) by amount of blood
ingested (5 or 10) and then multiplying the product times 55% (the estimated

percentage of serum in blood).

113

RESULTS
Sensitivity of PCR and Southern blot detection of HBV.

The addition of bed bug bodies decreased PCR sensitivity 10 fold and
Southern blot sensitivity 100 fold. The PCR had visible bands present at the
1:1000 dilution, 4 viral particles, in the serum and 1:100 dilution, 40 viral
particles, in the serum plus bed bug lanes (Figure 3). The Southern blot had
visible bands at the 1:10000 dilution, 0.40 viral particles, in the serum lanes and
1:100 dilution, 40 viral particles, in the serum plus bed bug lanes. The addition
of kissing bodies decreased PCR and Southern blot sensitivity 100 fold. The
PCR had visible bands present at the 1:10 dilution, 400 viral particles, in serum
plus kissing bug lanes (Figure 4). The Southern blot had visible bands at the
1:100 dilution, 40 viral particles, in serum plus kissing bug lanes. The addition of
fecal filter paper decreased the sensitivity of the PCR and Southern detection
methods 100 fold. The PCR and the Southern blot had visible bands at the 1:10
dilution, 400 viral particles (Figure 5). Nested PCR was not done due to a high
rate of false positives and the inability to establish a consistent lower limit of viral

detection.

114

L12345678910111213141516L

A)

B)

 

Figure 3. Determination of the effect of bed bug tissue on the sensitivity
of HBV detection. Serial dilution of 1485 pglml HBV DNA semm A) Agarose
gel of PCR products, 270 bp bands indicate positive samples. B) Southern blot
detection. Lanes: 1-positive control, 2 Negative control, 3 -straight serum (400
virions), 4- 1:10 (400 virions), 5— 1:100 (40 virions), 6-1:1000 (4.0 virions), 7-
1:10000 (0.4 virions), 8-1:100000 (0.04 virions), 9 -serum plus bug (400 virions),
10- 1:10 (400 virions), 11- 1:100 (40 virions), 12-121000 (4.0 virions), 13-1:10000
(0.4 virions), 14-1:100000 (0.04 virions), 15-negative control, 16-positive control,
L-123 bp lambda ladder.

115

1234 567891011L

A)

3)

 

Figure 4. Determination of the effect of kissing bug tissue on the
sensitivity of HBV detection. Serial dilution of 1485 pglml HBV DNA serum.

A) Agarose gel of PCR products, 270 bp band indicates positive samples. B)
Southern blot detection. Lanes: 1-1:10 (400 virions), 2-1:100 (40 virions), 3-
1:1000 (4.0 virions), 4-1210000 (0.4 virions), 5-1:100000 (0.04 virions), 6- 1:10
(400 virions), 7- 1:100 (40 virions), 8-121000 (4.0 virions), 9-1:10000 (0.4 virions),
10-1:100000 (0.04 virions), 11- positive control, L-123 bp lambda ladder.

116

123456789101112L

A)

 

123456789101112L

wt”.

    

3)

Figure 5. Determination of the effect of filter paper on the sensitivity of
HBV detection using PCR. Serial dilution of 1485 pglml HBV DNA serum.
Lanes: E- empty, A) Agarose gel of PCR products, 270 bp band indicates
positive samples. B) Southern blot detection. Lanes:1- empty, 2-1:10 (400
virions), 3-1:100 (40 virions), 4-1:1000 (4.0 virions), 5-1:10000 (0.4 virions), 6-
1:100000 (0.04 virions), 7- 1:10 (400 virions), 8- 1 :100 (40 virions), 9-1:1000 (4.0
virions), 10-1:10000 (0.4 virions), 11-1:100000 (0.04 virions), 12- positive control,
L-123 bp lambda ladder.

117

Cimex Iectularius HBV Infected Feeding Results. Legs and bodies were
collected at 1, 7, 14, 21, 28 and 35 days post infected feeding (Table 2). Virus
was detected by PCR and Southern blot in the single bed bug collected on day
1. Virus was not detected by PCR on day 7 but 60% were positive by Southern
blot. At 14, 21, 28 and 35 days, virus was detected in 100% of the bodies by
both PCR and Southern blot (Figure 6 and Figure 7). Legs from one bug were
positive on day 21 and legs from another bug were positive on day 28 by
Southern blot.

Persistence of inoculated HBV in C. Iectularius bodies. Hepatitis B virus was
not detectable by PCR at any collection time but was detectable in 80% of the
day 7 and 14 bodies and 40% of the day 21 bodies by Southern blot. Two day 7

legs were positive by Southern blot (Figure 6).

118

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B) L1 3 5 7 91113151719212325L

 

Figure 7. Persistence of Ingested HBV In day 28 and 35 C. Iectularius
bodies. A) Agarsose gel of PCR products, 270 bp band indicates positive
samples. B) Southern blot products. Lanes L are 123 bp lambda ladders.
Lanes1 and 26 are positive controls, lanes 2 and 25 are negative controls and
lane 23 is a reagent blank. Each sample was run body then legs. Lanes 3 and 4
and 16 and 17 are control bugs. Lanes 5-15 were collected on day 28 and lanes
5,7,9, 11 and 13 are bodies. Lanes 18-24 were collected on day 35 and lanes
18, 20, and 22 are bodies.

120

Persistence of HBV in C. Iectularius feces.

Ingested. Hepatitis B virus was not detected on the filter paper that was in
the jars during infected feeding. Virus was detected in 5% of the filter paper
samples by Southern blot on Day 1. On day 7, 39% of the fecal samples were
positive by PCR and 52% by Southern blot. Sixty-seven percent of the day 14
fecal samples were positive by PCR and 83% by Southern blot. Day 21, 28 and
35 fecal samples were all 100% positive by both PCR and Southern blot (Figure
8 and Figure 9).

Inoculated. Hepatitis B virus was not detected by either PCR or Southern
blot in the feces from bed bugs inoculated with hepatitis B virus.

Transstadial and Transovarial transmission of ingested hepatitis B virus in
C. Iectularius. Hepatitis B virus was detected by both PCR and Southern blot in
all larval stages undergoing one molt (Figure 10). Hepatitis B virus was not
detected, by either PCR or Southern blot, in any of the larvae that hatched from
eggs laid on day 7 or 14 after the infected blood meal.

Persistence of inoculated HBV in R. prolixus.

Bodies. Hepatitis B virus was detected in 60% of bodies, 40% of saliva, and
20% of legs by Southern blot in day 7 samples. Virus was not detected, by
either PCR or Southem blot, in any sample at any of the later collection times .

Feces. Hepatitis B virus was not detected by PCR or Southern blot in any
fecal samples at any of the sampling times.

All raw data from this results section are shown in Appendix C, Tables 6-10.

121

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L13 5 7 9111315171921L

     

Figure 9. Persistence of HBV in day 14, 21, 28 and 35 C. Iectularius feces.
A) Agarose gel of PCR products, 270 bp band indicates positive samples. B)
Southern blot detection. Lanes L- 123 bp lambda ladders, lanes 1 and 22
positive controls, lanes 2 and 21 negative controls, and lane 21 reagent blank.
Lanes 3, 7,8, and 13 control feces. Lane 4- day 35, Lanes 5 and 6 day 28,
Lanes 9-12 day 21, and lanes 14-19 day 14 feces.

123

L1357911131517192123L

 

Figure 10. Trans-stadial transmission of HBV. Agarsoe gel of PCR products.
Lanes 1 and 24 positive controls, lanes 2 and 23 negative controls, lane 22
reagent blank, lane 3 sample control, lane 4-21 infected instars from youngest to
oldest.

DISCUSSION.

All the studies to date examined the persistence of the hepatitis B surface
antigen in Cimex spp. and R. prolixus. The surface antigen is found on all three
viral particles produced by the virus, only one of which is infectious. The use of
PCR and Southern blot detection methods here allowed for the detection of the
viral genome. Analysis of the hepatitis B virus genome revealed regions that
appear to be highly conserved (nucleotides 241 to 270, 1891 to 1920, 2431 to
2460). (Robertson 1995). The oligonucleotide primers used in these
experiments amplified the conserved pre-core and core region, from
nucleotides1891 to 1920 (Lauder et al. 1993). The core gene contains two
initiation codons that encode the structural proteins of the nucleocapsid (Pasek
et al. 1978). The first initiation codon product is the 25-kd precore/core protein
that produces the HBeAg. The second initiation codon product is the 21 -kd core
protein, that forms the viral nucelocapsid with the HBV DNA (Thomas and
Cannan 1994). Thus, the detection of this gene region indicates that the insects
in my experiments did ingest an infectious blood meal and not just noninfectious
particles with surface antigen.

The results obtained in this laboratory study indicate that C. Iectularius and Ff.
prolixus are unlikely to serve as biological vectors of the hepatitis B virus. Viral
nucleic acid was detected for 35 days post feeding and 21 days post inoculation
in C. Iectularius and 7 days in inoculated R. prolixus, however, no corresponding
infected feeding study was done due to colony numbers. The detection of

inoculated virus only by Southern blot, indicates that the less than 40 viral copies

125

were present. Since there was no subsequent detection by PCR at later time
points it is unlikely that the virus had infected and replicated in the insects.

The detection of virus from C. Iectularius legs on day 21 and 28 post infected
feeding poses several questions. The positive legs from inoculated insects
would be expected since hemolymph circulates throughout the insects body and
into the legs and the virus was inoculated into the insects body. The detection of
virus in day 7 Fl. prolixus saliva suggest the virus was able to penetrate the
salivary glands. The detection of virus in the legs of bed bugs ingesting the
virus, may indicate viral dissemination or possibly contamination during
collection, sample processing, or defecation. The removal of the legs from the
body may have resulted in the rupture of the gut or defecation of virus, thus
contaminating the legs dun‘ng collection. Due to the sensitivity of the PCR
method these results need to be considered carefully for potential contamination
and false positive results. The small sample size does not allow a determination
of the significance of these positive samples.

The presence of detectable virus in the bodies and feces of the C. Iectularius
raises the question as to whether the virus may be mechanically transmitted.
Bed bugs live in close proximity to a host, often found in bedding, furniture, and
cracks and crevices in walls and thus defecate shedding virus in the hosts
environment, providing a potential source for inapparent infection. A second
potential means of mechanical transmission is when a partially engorged or
unengorged infected bed bug is killed during a subsequent feeding attempt,

resulting in deposition of virus on a susceptible host.

126

The persistence of the virus after an infected blood meal in bed bug bodies
and feces agrees with studies that found the persistence of the HBsAg in bed
bugs ingesting infected blood meals (Jupp and McElligott 1979, Jupp et al. 1983,
Ogston and London 1980, Ogston et al. 1979, Taylor and Morrison 1980).
Further, the virus was shown to persist trans-stadially, in all stages of C.
Iectularius, confirming an earlier studies by Jupp and McElligott (1979).
However, trans-ovarian transmission was not detected, also confirming earlier
studies (Jupp and McElligott 1979, Taylor and Morrison 1980). The use of PCR
and Southern blot techniques, allowed me to detect a segment of the viral
genome and not just the surface antigen which is found on all three particle
types. The specificity and sensitivity of these methods, theoretically allowed
detection of as few a 4.0 hepatitis B virions in the bed bug, 4O virions in the
kissing bug, and 40 virions in the feces. It is important to note here that the
detection of a segment of the pre-core and core region of the viral nucleic acid
does not correlate to infectivity. Hence, no definitive conclusions can be drawn
about infectivity potential from the detection of viral genome. A carefully
controlled transmission study is needed to answer these lingering questions.
However, due to the host specificity of the hepatitis B virus and the cost
associated with working with primates, the use of a related animal hepadnavirus
and its animal host would be recommended to address the question of

mechanical transmission of the virus by bed bugs and kissing bugs.

127

LITERATURE CITED

Alter, J. and E. Mat. 1994. The epidemiology of viral hepatitis in the United
States. Viral Hepatitis.23:(3); 437-455.

Beasley, R. 1988. Hepatitis B Virus: The major etiology of hepatocellular
carcinoma. Cancer.61:(10); 1942-1956.

Berquist, K., J. Maynard, D. Francy, M. Sheller and C. Schable. 1976.
Experimental studies on the transmission of hepatitis B by mosquitoes.
American Journal of Tropical Medicine and Hygiene.25: 730-732.

Blumberg, B., H. Alter and S. Visnich. 1965. A'new“ antigen in leukemia sera.
JAMA.191:(7); 541-546.

CDC. 1990. Protection against viral hepatitis. Morbidity and Mortality Weekly
Report.39:(RR-2); 25.

Fujiyama, A., A. Miyanohara, C. Nozaki, T. Yoneyama, N. Ohtomo and K.
Matsubara. 1983. Cloning and structural analyses of hepatitis B virus DNA,
subtype adr. Nucleic Acids Research.11: 4601-4610.

Hamilton, J. 1994. Huge bounty on a global killer. Business Week. April 4, 1994.
92-94.

Hollinger, F. 1996. Hepatitis B Virus. pp. 2739-2807. In B. Fields, E. Knipe, P.
Howley, R. Chanock, T. Monath, J. Melnick, B. Roizman and S. Straus [eds.],
Fields Virology,, vol. 2. Lippincott-Raven Publishers, Philadelphia.

Jupp, P., O. Prozesky, S. McElligott and L. Van Wyk. 1978. Infection of the
Common Bedbug (Cimex Iectularius) with hepatitis B virus in South Africa.
South African Medical Joumal.53: 598-600.

Jupp, P. and S. McElligott. 1979. Transmission experiments with hepatitis B
surface antigen and the common bedbug (Cimex Iectularius L.). South
African Medical Joumal.56: 54-57.

Jupp, P., O. Prozesky and S. McElligott. 1980. Correspondence: Absence of
biological multiplication of hepatitis B virus in the common bedbug. South
African Medical Joumal.57: 36.

Jupp, P., S. McElligott and G. Lecatsas. 1983. The mechanical transmission of
hepatitis B virus by the common bedbug (Cimex Iectularius L.) in South
Africa. South African Medical Joumal.63: 77-81.

128

Jupp, P., R. Purcell, M. Shapiro and J. Gerin. 1991. Attempts to transmit
hepatitis B virus to chimpanzees by arthropods. South African Medical
Joumal.79: 320-322.

Kaneko, S., S. Feinstone and R. Miller. 1989. Rapid and sensitive method for
the deteciton of serum hepatitis B virus DNA using the polymerase chain
reaction technique. Journal of Clinical Microbiology.27:(9); 1930-1933.

Lauder, I., H.-J. Lin, J. Lau, T.-S. Siu and C.-L. Lai. 1993. The variability of the
hepatitis B virus genome: Statistical analysis and biological implications.
Molecular Biology and E volution.1 0:(2); 457-470.

Mayans, M., A. Hall, H. Inskip, J. Chotard, 8. Lindsay, E. Coromina, M.
Mendy, P. Alonso and H. Whittle. 1990. Risk factors for transmission of
hepatitis B virus 'to Gambian children. Lancet.336: 1107-1109.

Mayans, M., A. Hall, H. Inskip, 8. Lindsay, J. Chotard, M. Mendy and H.
Whittle. 1994. Do bedbugs transmit hepatitis B? Lancet.343: 761-763.

Maynard, J. 1981. Hepatitis B vaccine: Strategies for utilization. pp. 13-19. In
P. Maupas and P. Guesry [eds.], Hepatitis B Vaccine,, vol. lnserrn
Symposium No. 18. Elsevier/ North Holland Biomedical Press, Amsterdam.

Neefe, J. 1949. Viral Hepatitis: Problems and Progress. Annals of Intemal
Medicine.31:(5); 857-870.

Newkirk, M., A. Downe and J. Simon. 1975. Fate of ingested hepatitis B
antigen in blood-sucking insects. Gastroenterology.69: 982-987.

Ogston, C. and W. London. 1980. Excretion of hepatitis B surface antigen by
the bedbug Cimex hemipterus Fabr. Transactions of the Royal Society of
Tropical Medicine and Hygiene.74:(6); 823-825.

Ogston, C., F. Wittenstein, W. London and l. Millman. 1979. Persistence of
hepatitis B surface antigen in the bedbug Cimex hemipterus (Fabr). Joumal
of Infectious Diseases.140:(3); 41 1-414.

Pasek, M., T. Goto, W. Gilbert, 8. Zink, H. Schaller, P. Mackay, G. Ledbetter
and K. Murrary. 1978. Hepatitis B vian genes and their expression in E. coli.
Nature.282: 575-579.

Prince, A. 1970. Prevalence of serum-hepatitis-related antigen (SH) in different

geographic regions. American Journal of Tropical Medicine and
Hygiene.19:(3); 872-879.

129

Prince, A., D. Metselaar, G. Kafuko, L. Mukwaya, C. Ling and L. Overby.
1972. Hepatitis B antigen in wild-caught mosquitoes in Africa. Lancet.2: 247-
250.

Robertson, B. 1995. PCR detection and analysis of hepatitis vimses. pp. 57-80.
In J. Clewley [ed.] The polymerase chain reaction (PCR) for human viral
diagnosis, CRC Press, Boca Raton.

Smith, J., E. Ogunba and T. Francis. 1972. Transmission of Australia Au (1)
antigen by Culex mosquitoes. Nature.237: 231-232.

Stevens, C., R. Beasley, J. Tsui and W.-C. Lee. 1975. Vertical transmission of
hepatitis B antigen in Taiwan. N Engl J Med.292: 771-774.

Tabor, E., A. Bayley, L. Cairns, L. Pelleu and R. Gerety. 1985. Horizontal
transmission of hepatitis B virus among children and adults in five nJral
villages in Zambia. Journal of Medical Virology.15: 113-120.

Taylor, P. and J. Morrison. 1980. Cimex Iectularius as a vector of hepatitis B.
The Central African Joumal of Medicine.26: 198-200.

Thomas, H. and W. Carman. 1994. Envelope and precore/core variants of
Hepatitis B virus. Gastroenterology Clinics of North America.23:(3); 499-514.

Turrell, M., T. Gargan and C. Bailey. 1984. Replication and dissemination of
Rift Valley fever virus in Culex pipiens. American Journal of Tropical
Medicine.19: 335-341 .

Villareios, V., A. Zuniga and A. Gutierrez. 1975. Possible role of
haematophagous insects in the transmission of type B hepatitis. Transactions
of the Royal Society of Tropical Medicine and Hygiene.69: 368-369.

Wills, W., W. London, B. Werner, M. Pourtaghva, B. Blumberg, B. Larouze, I.
Millman, W. Ogston and S. Diallo. 1977. Hepatitis B virus in bedbugs
(Cimex hemipterus) from Senegal. Lancet.2: 217-219.

Yuhuang, Z., L. Duo, F. Deyun, T. Hong, L. Yuzhang and Y. Xueke. 1995. An

animal study on transmission of hepatitis B virus through mosquitoes.
Chinese Medical Joumal.1 08:(12); 895-897.

130

CHAPTER 3

RESEARCH SUMMARY AND CONCLUSIONS

Hepatitis B virus (HBV) infection is a major cause of acute and chronic
hepatitis, cirrhosis, and primary hepatocellular cancer world wide (CDC 1990). In
the United States 30-40% of patients with hepatitis B infection cannot be
definitively associated with an identifiable risk factor. Neefe (1949) was the first
to consider the possibility that hematophagous arthropods could transmit the
hepatitis virus. The subsequent detection of the hepatitis B surface antigen
(HBsAg) by radioimmunoassay in wild caught mosquitoes supported this
hypothesis (Prince et al. 1972). The detection of the HBsAg in wild caught
mosquitoes (Berquist et al. 1976, Blumberg et al. 1973, Brotman et al. 1973,
Dick et al. 1974, Prince et al. 1972, Wills et al. 1976), Cimex spp. (El-Masry and
Kotkat 1990, Jupp and McElligott 1979, Jupp et al. 1983, Jupp et al. 1980, Jupp
et al. 1978, Newkirk et al. 1975, Ogston and London 1980, Ogston et al. 1979,
Taylor and Morrison 1980, Wills et al. 1977), tampan tick, Omithodoros moubata
Murray, (Joubert et al. 1985, Jupp et al. 1987) and Rhodnius prolixus Stal
(Villarejos et al. 1975) implicated these arthropods as potential vectors. .

The purpose of this research project was to study the persistence of HBV
genome in several species of hematophagous arthropods. Previous studies
utilized primarily ElA/RIA methodologies to test for the presence of HBsAg. The
detection of HBsAg by those methods showed ingestion of the viral particles
during feeding on either a viremic host or artificial feeder. However, the medical
and epidemiological significance of the persistence of the HBsAg over varying

131

time periods post infected feeding is debatable. Is it a mere artifact of the
original blood meal or, does it represent viral replication and generation of new
viral particles? The use of molecular methods allowed for the detection of
specific regions of the HBV genome. Specifically, by testing for pre-C and C
regions of the genome, using PCR, only regions of the genome coding for the
infectious particle regions would be detected. The persistence of the virus and
its dissemination in the insect may indicate that the vector has been infected and
may be able to mechanically or biologically transmit the virus.

I studied the persistence and dissemination of ingested and inoculated
hepatitis B virus in Cimex Iectularius L. (Hemiptera: Cimicidae), Rhodnius
prolixus Stal (Hemiptera: Reduviidae), Aedes triseriatus (Say) (Diptera:
Culicidae), Culex quinquefasciatus Say (Diptera: Culicidae) and Anopheles
stephensi Liston (Diptera: Culicidae). The hepatitis B viral genome was detected
by polymerase chain reaction with confirmation by Southern blot. The sensitivity
of these detection methods was determined by serial dilution of a known serum
with the addition of insect tissue or filter paper and was shown to vary with
sample source (Table 1).

Viral nucleic acid was detected in An. stephensi for 48 hours and Ae.
triseriatus for 72 hours post infected feeding, indicating that the disappearance of
the virus correlates with the digestion of the blood meal (Brotman et al. 1973,
Chen et al. 1987, Leevy et al. 1972, Newkirk et al. 1975). The lack of
reappearance of virus in the later time points and the lack of detectable virus in
the intrathoracically inoculated mosquitoes indicated that the virus did not infect
mosquitoes. The presence of detectable vims in the feces of those mosquitoes

132

Table 1. Sensitivity of PCR and Southern blot detection methods for
hepatitis B viral genome. Sensitivity determined by serial dilution of known
serum with corresponding sample. Results are given in viral particles detected.

 

 

 

 

 

 

 

 

 

Sample PCR Southern blot
Mosquito 4 0.4
Cimex 40 < 4
Rhodnius 400 40

Filter paper 400 < 40

 

that ingested an infected blood meal raises the possibility that the virus may be
mechanically transmitted by mosquitoes by excretion of infectious material.

Viral nucleic acid was detected in C. Iectularius bodies and feces at 35 days
post infected feeding and at 21 days post inoculation. The persistence of the
virus after an infected blood meal in bodies and feces agrees with earlier studies
that found persistence of HBsAg in bed bugs that had ingested an infected blood
meal (Jupp and McElligott 1979, Jupp et al. 1983, Taylor and Morrison 1980).
Further, the virus was shown to persist trans-stadially, in all stages of C.
Iectularius, confirming an earlier study by Jupp and McElligott (1979). However,
trans-ovarian transmission was not detected, again confirming earlier studies
(Jupp et al. 1978, Taylor and Morrison 1980).

The presence of detectable virus in the bodies and feces of bed bugs raises
the question as to whether the virus may be mechanically transmitted. Bed bugs
live in close proximity to a host and are often found in bedding, furniture, and
cracks and crevices in walls. Thus, they may defecate infectious virus into the

human living environment, providing a potential source for inapparent infection.

133

A second potential means of mechanical transmission could be when a partially
engorged or unengorged infected bed bug is killed during a subsequent feeding
attempt, resulting in deposition of virus on a susceptible host.

It is important to note that the detection of a segment of the pre-core and core
region of the viral nucleic acid does not correlate to infectivity. Hence, no
definitive conclusions can be drawn about potential infectivity from the detection
of the viral genome. Further studies need to be conducted to examine the
infectivity of feces and bodies of the various insects. However, due to the host
specificity of the hepatitis B virus and the cost associated with working with
primates, the use of a related animal hepadnavirus and its animal host would be

recommended to address the question of mechanical transmission.

134

LITERATURE CITED

Berquist, K., J. Maynard, D. Francy, M. Sheller and C. Schable. 1976.
Experimental studies on the transmission of hepatitis B by mosquitoes.
American Joumal of Tropical Medicine and Hygiene.25: 730-732.

Blumberg, B., W. Wills, I. Millman and W. London. 1973. Australia antigen in
mosquitoes. Feeding experiments and field studies. Research
Communications in Chemical Pathology and Pharrnacolcgy.6:(2); 719-732.

Brotman, B., A. Prince and H. Godfrey. 1973. Role of arthropods in
transmission of Hepatitis B virus in the tropics. Lancet.i: 1305-1308.

CDC. 1990. Protection against viral hepatitis. Morbidity and Mortality Weekly
Report.39:(RR-2); 25.

Chen, C., L. Shou, L. Hwang, P. Tang and W. Tu. 1987. Persistence of
hepatitis B viral antigens in Culex quinquefasciatus. Southeast Asian Joumal
of Tropical Medicine and Public Health.18:(1); 44-51. ’

Dick, 5., C. Tamburro and C. Leevy. 1974. Hepatitis B antigen in urban-caught
mosquitoes. JAMA.229:(1 2); 1 627-1629.

El-Masry, S. and A. Kotkat. 1990. Hepatitis B surface antigen in Cimex
Iectularius. Journal of the Egyptian Public Health Association.65: 230-236.

Joubert, J., P. Jupp, D. Prozesky and J. Lourens. 1985. Infection of wild
populations of the Tampan, Omithordoros moubata Murray, with hepatitis B
virus in the Kavango, Northern Namibia. South African Joumal of Science.81:
167-168.

Jupp, P., J. Jourbert, A. Comel, C. Swanevelder and O. Prozesky. 1987. An
experimental assessment of the tampan tick, Omithodoros moubata, as
vector of hepatitis B virus. Medical and Veterinary Entomology.1: 361-368.

Jupp, P. and S. McElligott. 1979. Transmission experiments with hepatitis B
surface antigen and the common bedbug (Cimex Iectularius L.). South
African Medical Joumal.56: 54-57.

Jupp, P., S. McElligott and G. Lecatsas. 1983. The mechanical transmission of

hepatitis B virus by the common bedbug (Cimex Iectularius L.) in South
Africa. South African Medical Joumal.63: 77-81.

135

Jupp, P., O. Prozesky and S. McElligott. 1980. Correspondence: Absence of
biological multiplication of hepatitis B virus in the common bedbug. South
African Medical Jouranl.57: 36.

Jupp, P., 0. Prozesky, S. McElligott and L. Van Wyk. 1978. Infection of the
Common Bedbug (Cimex Iectularius) with hepatitis B virus in South Africa.
South Africain Medical Joumal.53: 598-600.

Leevy, C., M. Howard, W. Crans, E. Evans and P. A. 1972. Viral hepatitis and
the mosquito. Gastroenterology.62:(4); 872.

Neefe, J. 1949. Viral Hepatitis: Problems and Progress. Annals of Intemal
Medicine.31:(5); 857-870.

Newkirk, M., A. Downe and J. Simon. 1975. Fate of ingested hepatitis B
antigen in blood-sucking insects. Gastroenterology.69: 982-987.

Ogston, C. and W. London. 1980. Excretion of hepatitis B surface antigen by
the bedbug Cimex hemipterus Fabr. Transactions of the Royal Society of
Tropical Medicine and Hygiene.74:(6); 823-825.

Ogston, C., F. Wittenstein, W. London and I. Millman. 1979. Persistence of
hepatitis B surface antigen in the bedbug Cimex hemipterus (Fabr). Journal
of Infectious Diseases.140:(3); 411-414.

Prince, A., D. Metselaar, G. Kafuko, L. Mukwaya, C. Ling and L Overby.
1972. Hepatitis B antigen in wild-caught mosquitoes in Africa. Lancet.2: 247-
250.

Taylor, P. and J. Morrison. 1980. Cimex Iectularius as a vector of hepatitis B.
The Central African Journal of Medicine.26: 198-200.

Villarejos, V., A. Zuniga and A. Gutierrez. 1975. Possible role of
haematophagous insects in the transmission of type B hepatitis. Transactions
of the Royal Society of Tropical Medicine and Hygiene.69: 368-369.

Wills, W., W. London, B. Werner, M. Pourtaghva, B. Blumberg, B. Larouze, l.
Millman, W. Ogston and S. Diallo. 1977. Hepatitis B virus in bedbugs
(Cimex hemipterus) from Senegal. Lancet.2: 217-219.

Wills, W., G. Saimot, C. Brochard, B. Blumberg, W. London, R. Dechene and
I. Millman. 1976. Hepatitis B surface antigen (Australia antigen) in
mosquitoes collected in Senegal, West Africa. The American Journal of
Tropical Medicine and Hygiene.25:(1); 186-190.

136

APENDICES

137

DNAzol Extraction Protocol

APPENDIX A

1. Add DNAzol and Poly Carrier gel to samples.

Table 1: Quantfiy of DNAzol and Poly Carrier gel to add each sample tube.

 

 

 

 

 

 

 

 

 

 

Sample type DNAzol (ul) Carrier Gel (ul)
serum, blood 250 5
saliva 500 1 0
mosquito bodies and legs 250 5
fecal filter paper 750 15
bed bugs bodies and legs 250 5
kissing bugs bodies and legs 750 15

 

1. Homogenize all samples except bed bug and kissing bug bodies in tube with
sterile pestle. Bed bug and kissing bug bodies are soaked in DNAzol and
carrier gel first then homogenized. Soaking softens the exoskeleton allowing
the bug to be homogenized. Samples were then soaked a second time.

2. Incubate samples:

Table 2: Time and temperature to incubate samples.

 

 

 

 

 

 

 

 

 

 

Sample type Time Temperature
serum, blood 10 minutes room
saliva 10 minutes room
mosquito bodies and legs 10 minutes room
fecal filter paper overnight 4 degrees C
bed bugs bodies and legs 1 hour 4 degrees C
kissing bugs bodies and legs 1 hour 4 degrees C

 

138

 

 

.‘1 9’

9°

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

Centrifuge samples for 15 minutes at 4 degrees C and 10,000 x 9.

While centrifuging, label another set of tubes with corresponding sample
labels. Add the same amount of ice cold 100% ethanol as DNAzol to each
tube.

Replace tubes in freezer until centrifugation is complete.

Transfer supernatant from specimen tubes to corresponding ETOH tubes.
Invert tubes 3-4 times and incubate for 5 minutes at room temperature.

Centrifuge 5 minutes at 4 degrees C and 1,000 x 9.

At end of centrifugation, examine tubes for presence of pellet or precipitate in
tube. If no visible pellet/precipitate centrifuge the tubes a second time.

Pour off supernatant, being careful not to lose the pellet/precipitate.
Add 750 ul of ice cold 95% ETOH, vortex the samples.

Centrifuge 5 minutes at 4 degrees C and 1,000 x 9.

Pour off supernatant, being careful not to lose the pellet/precipitate.
Add 500 ul of ice cold 95% ETOH, invert samples 3-4 times. .
Centrifuge 5 minutes at 4 degrees C and 1,000 x 9.

Pour off supematant, set tubes upside down on kim wipes until dry.
When dry, add 10 ul of deionized water to re-suspended the DNA.

Spin at room temperature for 20 seconds at 10,000 rpm to concentrated the
DNA in the bottom of the tube.

Incubate 3-5 minutes to allow DNA to dissolve.

DNA can be stored at -20 degrees C for 1 month, or kept on ice for
immediate use.

139

 

APPENDIX B

SOUTHERN DETECTION PROTOCOL

Table 1. Additionally required solutions for Southern blottinl

 

Additionally Required Reagents

Description

 

HCL

250 mM HCL

 

H20

Sterile, Distilled water

 

Denaturation solution 1

0.5 M NaOH, 1.5 M NaCI

 

Neutralization solution 1

0.5 M Tris-Hcl, pH7.5; 3 M NaCI

 

20 X SSC Buffer

3 M NaCl, 75 mM Sodium Citrate, pH 7.0

 

5 X SSC Buffer

750 mM NaCl, 75 mM Sodium Citrate, pH
7.0

 

Prehybridization Solution

5 X SSC, 1% Blocking Reagent, 0.1% N-
lauralysarconsine, 0.02% SDS (Store at -
2000

 

Hybridization solution

DIG-labeled probe, diluted in
Prehybridization solution

 

2 X Wash solution

2 X SSC, containing 0.1% SDS

 

 

0.5 X Wash solution

 

0.5 X SSC, containing 0.1 "/0 SDS

 

DENATURATION, NEUTRALIZATION AND BLOTI'ING BY TRANS BLO'ITER

1. Cut a piece of nylon membrane and two pieces of extra thick blotting paper to
the dimensions of the gel with a scalpel blade or scissors. The transfer
membrane and blotting paper must have the same dimensions as the gel for

proper transfer to occur.

2. Calculate the power conditions for transfer. For my samples the transfer
conditions are 3.00mA/cm2 of gel agarose for 40 minutes. Preset the BioRad
power supply to the calculated mA's and time. Run the power pack on

constant Amps.

3. Saturate two pieces of precut Extra thick blot paper and precut nylon
membrane in 0.5 X TBE buffer. Equilibrate the transfer membrane for at

least 10 minutes.

4. Remove one piece of blotting paper from 0.5 X TBE and allow the excess
buffer to drain off. Lay the blotting paper flat on the platinum anode. Using a

140

 

clean pipet roll out any air bubbles that may be trapped under the blotting
paper with a top to bottom, left to right rolling motions.

. Place the equilibrated transfer membrane on top of the blotting paper and roll
out air bubbles.

. Carefully place the agarose gel on top of the membrane, well side up. Make
sure all the edges are aligned and air bubbles are rolled out.

. Take the other piece of wetted blot paper and allow excess buffer to drain off,
place it on top of the agarose gel. Roll out the blot paper to remove air
bubbles and add approximately 15 ml's of 0.5 X TBE to top of sandwich to
saturate. Remove any excess buffer that is present on the anode surface.

. Place the safety cover onto the unit and plug the Trans-Blot SD cell into the
power supply. Plug red into red and black into black.

9. Turn on the power supply and verify your calculated settings are pre-set.

Press the run button and verify that the gel is running at your preset
conditions. Monitor the voltage during the transfer for any significant
fluctuations. The voltage will slowly increase during the transfer to maintain
constant mA. If the voltage is lower, increase the length of the transfer time.
If the voltage increases significantly (>25V) the buffer capacity has expired
and the run should be stopped.

10. Following transfer, tum off the power supply and disconnect the leads.

Remove the safety cover and the cathode electrode. Remove and discard
the top blot paper. Using a fine tip pencil mark the wells on the blot paper
through the gel. Remove the gel and restain with EtBr to check for complete
DNA transfer.

11. Rinse the nylon membrane in 2x SSC.

12. To fix the DNA, saturate a piece of blot paper with 0.4 N NaOH. Place the

transfer membrane on top of the saturated pad (DNA side up) for 5 minutes.
Rinse the membrane briefly in 2 X SSC, and bake for 30 minutes at 80
degrees C in a vacuum oven or 1 hour at 120 degrees C with no vacuum.

141

PREHYBRIDIZATION AND HYBRIDIZATION:

1.

Place the blot in a hybridization tube containing 20 ml prehybridization
solution per 100 cm2 of membrane surface area. Seal the tube, place in the
rotator, and prehybridize at 39 degrees C (T m = 47.9 degrees C) for 6 hours.
Longer hybridization times are acceptable. Several membranes can be
processed in the same tubes as long as there is sufficient prehybridization
solution ot cover all the membranes and the membranes can move freely in
the bag.

Add the DIG labeled 1913 probe (concentration 10 pmoVul) to
prehybridization solution to obtain a 10 pmol/ml concentration in the
hybridization solution. (Example: 30 ml's prehybridization solution add 3 ul of
probe 1913)

Allow the probe to hybridize for 1-6 hours at 39 degrees C. Longer is
acceptable.

At the end of the hybridization, pour the hybridization solution from the tube
into a 50 ml polypropylene tube. This hybridization solution contains
unannnealed DIG- labeled 1913 probe. The entire solution can be reused in
future hybridization experiments. Label and store oligo probe solutions at
20 degrees C. DIG-labeled probes stored in this manner are stable for at
least 1 year For reuse, thaw and denature by heating to 95 degrees C for 10
minutes.

. Wash the membrane twice, 5 minutes per wash, in 2 X Wash Solution at

room temperature. These washes remove unbound probe, which will lead to
high backgrounds if not removed.

Wash the membrane twice in 0.5 X Wash Solution at 39 degrees C for 5
minutes.

142

COLORMET RIC DETECTION WITH NBT AND BCIP:

PRODUCTS REQUIRED: DIG Nucleic Acid Detection Kit (Cat. No. 1175041 ).

Table 2. Additionally required re_agents for DIG Nucleic Acid Detection.

 

Additionally required
reagents

Description

 

 

 

 

 

 

 

 

Buffer 1 100 mM maleic acid, 150 mM NaCl; pH 7.5
(2013C adjusted with NaOH, autoclaved.

Buffer 2 1% Blocking reagent diluted in buffer 1 (final
concentration = 1% blocking reagent)

Washing Buffer Buffer 1 plus 0.3% (v/v) Tween-20

Buffer 3 100 mM Tris-HCI, 100 mM NaCl, 50 mM
MgCl2; pH 9.5 (2000)

Buffer 4 (TE Buffer) 10 mM Tris-HCl, 1 mM EDTA; pH 8.0 (+20 C)

Color-substrate solution 200 ul NBT/BCIP stock solution (vial 4) are

(freshly prepared) added to 10 ml of Buffer 3

 

Procedure: (Perform all incubations at room temperature.)

1.

After hybridization and post-hybridization washes, equilibrate the membrane

in Washing Buffer for 1 minute.

antibody solution

Using a freshly washed dish or bag, block the membrane by gently agitating it
in Buffer 2 for 30 minutes. Near the end of the blocking period, prepare the

Dilute the Anti-Digoxigenin-AP (vial 3 in refrigerator) 1:5,000 (after

centrifugation) in Buffer 2 for a working concentration of 150 mU/ml. Mix
gently by inversion. (Example add 6 ul Anti-Digoxigenin-AP to 30 ml of

blocking solution.)

Pour off the Buffer 2 and incubate the membrane for 30 minutes in the

Antibody Solution prepared in step 3. Agitate the tray gently to ensure that
the membrane is always covered.

Discard the antibody solution. Wash twice, 15 minutes per wash, in 100 ml's

Buffer 1. This wash removes unbound antibody.

Added 200 ul of NBT/BCIP solution to 10 ml's of Buffer 3. This will be used in

step 8 and needs to be protected from light.

143

 

 

7. Equilibrate the membrane in 20 ml Buffer 3 for 2 minutes.

8. Pour off Buffer 3, and add approximately 10 ml's color substrate solution
directly to the membranes surface by pipet. Incubate the membrane in a box
in the dark. Do not shake the container while the color is developing. The
color precipitate starts to form within a few min, and the reaction is usually
complete after 12 hours.

9. Once the desired spots or bands are detected wash the membrane with
Buffer 4 to prevent over-development.

10. The membrane can be stored in a ziplock bag containing buffer 4 to hold the
color.

11. Take a picture of the membrane using Ektachrome 160 Tungsten film on the
tungsten light table.

144

WORKING STOCK SOLUTIONS:

0.5 M EDTA (pH 8.0)
93.05 g disodium EDTA
500 ml MQ water
adjust pH to 8.0 with NaOH
pellets
autoclave to sterilize

1 M Tris (pH 7.4)
60.55 g Tris Base
500 ml's MQ Water
adjust pH to 7.4 with 6 N HCl
autoclaved to sterilize

1 M Tris-HCI, pH 7.0 (GMW 157.56)
15.756 9 Tris-HCI
100 ml's MQ water

2.5 M NaOH (40.00 GMW)
50 grams NaOH
500 mls MQ water
autoclave to sterilize

6 M NaCI
175.32 9 NaCI
500 ml MQ water
autoclave to sterilize

1% N- Lauroylsarkosine
1 g N-lauroylsarkosine
100 ml's MQ water

145

20 X SSC
175.3 9 NaCl
88.2 g Sodium citrate
1000 ml's MQ water
adjusted pH to 7.0
autoclave to sterilize

10% TWEEN-20
10 ml Tween-20
90 ml's MO water

0.5 M MgCl2 (FW 203.39)
10.1 g MgCl2
100 ml's MQ water

10X TBE
108 g Tris Base
55 g Boric Acid
40 ml’s 0.5M EDTA (pH8.0)
960 ml’s MO water

 

 

HYBRIDIZATION WORKING SOLUTIONS:

Pre-hybridization and [5X SSC; blocking reagent 1% (w/v); N-lauroylsarkosine
0.1% (w.v); SDS 0.02% (w/v)] freeze at -20C

25 mls 20 X SSC

1 gram Blocking reagent

0.2 mls 10% SDS

10 mls 1 M N-lauroylsarkosine

64.8 mls MQ water

Hybridization
prehybridization solution
10 pmonl of labeled primer

2 X Wash Solution (2 X SSC, containing 0.1 % SDS)
100 mls 20 X SSC
10 mls 10% SDS
890 mls MQ water

0.5 X Wash Solution (0.5 X SSC containing 0.1 % SDS)
25 mls 20 X SSC
10 mls 10% SDS
965 mls MQ water

146

FW‘

COLORMETIC DETECTION SOLUTIONS:

Buffer 1 (0.1 M Malic Acid; 0.15 M NaCl; pH 7.5)
13.4 g Malic Acid (FW 134.6)
25 mls 6 M NaCI
975 mls MQ H2O
Autoclave

Buffer 2 (1% blocking stock in Buffer 1) Make Fresh
0.5 grams Blocking stock
50 mls Butter 1

Buffer 3 (100 mM Tris-HCI; 100 mM NaCl; 50 mM MgCl2; pH 9.5)
50 mls 1 M Tris-HCI

8.4 mls of 6 M NaCl
50 mls 0.5 M MgCl2
391.6 mls MQ water

Butter 4 (10mM Tris-HCl; 1 mM EDTA; pH 8.0)
5 mls 1 M Tris-HCl
1 ml 0.5 M EDTA
494 ml's MQ water

Washing Butter:

3 mls 10% Tween-20
97 mls Buffer 1

147

 

 

APPENDIX C

DATA

148

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Table 1: Persistence of ingested HBV in bodies of An. stephensi and Ae.
triseriatus. Engorged mosquitoes ingested between 2-3 ul of infected blood
during feeding. Each ul of the HBV blood suspension contained approximately
116-174 copies of HBV DNA.

 

 

 

 

 

 

 

 

 

 

 

PCR Southern blot

An. stephensi

3 days 0/3 0/3

7 days 0/3 0/3

14 days 0/3 0/3
Ae. triseriatus

3 days 0/3 0/3

7 days 0/3 0/3
14 days 0/3 0/3

 

 

 

 

149

Table 2: Persistence of ingested HBV In bodies of An. stephensi and Ae.
triseriatus. Engorged mosquitoes ingested between 2-3 ul of infected blood
during feeding. Each ul of the HBV blood suspension contained approximately
116-174 copies of HBV DNA.

 

 

 

 

 

 

.l‘.—

 

 

 

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l

 

 

 

 

 

 

 

Bodies PCR Southern blot
An. Stephensi
0 hours 4/4 4/4
6 hours 5/5 5/5
12 hours 3/5 5/5
24 hours 7/10 7/10
48 hours 4/10* 4/10*
72 hours 0/10 0/10
Ae. triseriatus
0 hours 2/2 2/2
6 hours 0/2 0/2
12 hours 2/2 2/ **
24 hours 2/2 2/2
48 hours 2/2 2/ ***
72 hours 2/2 2/2

 

 

 

 

* one mosquitoes legs were positive by both PCR and Southern blot
“one mosquitoes body, saliva and legs all positive by Southern blot
***one mosquitoes legs were positive by Southern blot

150

 

Table 3. Persistence of ingested HBV in feces of An. stephensi and Ae.
triseriatus. Detection of the HBV genome in the feces of An. stephensi and Ae.
triseriatus. Number of positive samples over total number of samples collected.

 

 

 

 

 

 

 

 

 

 

 

 

 

PCR Southern blot
An. stephensi
3 days 6/22 10/22
7 days on 0 0/10
14 days 0/10 0/10
As. triseriatus
3 days 1/30 1/30
7 days 3/13 4/13
14 days 0/11 0/11

 

 

151

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um"- . ..-

Table 4. Persistence of ingested HBV in feces of An. stephensi and Ae.

triseriatus. Detection of the HBV genome in the feces of An. stephensi and Ae.
triseriatus. Number of positive samples over total number of samples collected.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PCR Southern blot
An. stephensi
0 hours 6/1 8 8/1 8
6 hours 0/9 0/9
12 hours 0/4 1/4
24 hours 1/6 1/6
48 hours 6/6 6/6
72 hours 5/7 6/7
Ae. triseriatus
0 hours 0/18 0/18
6 hours no feces no feces
12 hours 0/3 0/3
24 hours 1/6 1/6
48 hours 0/12 0/12
72 hours 1/9 1/9

 

 

152

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.‘~ .,'..‘

 

Table 5: Transmission potential of An. stephensi during refeeding. An.
stephensi fed an HBV infected blood meal and then held 72 hours and allowed
to oviposit. A second non-infectious blood meal was provided for feeding.
Engorged mosquitoes, feeding membrane, blood and fecal filter paper were

collected immediately after feeding.

 

 

 

 

 

 

 

 

Sample PCR Southern blot
mosquito bodies 0/4 0/4
blood Neg Neg
membrane Neg Neg
fecal filter paper 0/2 0/2

 

 

Table 6. Persistence of ingested HBV in C. Iectularius bodies. Detection of
the HBV virus genome in C. Iectularius bodies. Engorged Cimex ingested 3-5 ul
of infected blood (1485 pglml) during feeding. Each ul of HBV blood contained

approximately 720-1000 copies of HBV DNA.

 

 

 

 

 

 

 

 

 

 

Sample PCR Southern Blot
Day 1 1/1 1/1

Day 7 0/5 3/5

Day 14 5/5 5/5

Day 21 5/5 5/5*

Day 28 5/5 5/5*

Day 35 3/3 3/3

 

 

*one bed bugs legs were positive by Southern blot on each of these days

Table 7. Persistence of inoculated HBV in C. Iectularius bodies. Detection
of the HBV virus genome in C. Iectularius bodies. Bed bugs were inoculated with
0.5 ul of HBV suspension that theoretically contained 45 viral particles.

 

 

 

 

 

 

Bodies PCR Southern blot
Day 7 0/5 4/5”
Day 14 0/5 4/5
Day 21 0/5 2/5

 

 

 

*2 bed bug’s legs were positive by Southern blot at this time point

153

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Table 8. Persistence of ingested HBV in C. Iectularius feces. Detection of
the HBV genome in the feces. Number of positive samples over total number of

samples collected.

 

 

 

 

 

 

 

 

 

 

 

Collection Day PCR Southern blot
Immediately after 0/2 0/2
feeding

Day 1 0/18 1/18

Day 7 9/23 12/23

Day 14 4/6 5/6

Day 21 4/14 4/4

Day 28 2/2 2/2

Day 35 1/1 1/1

 

Table 9. Trans-stadial transmission of Ingested HBV. Larva were allowed to
molt following an HBV infected blood meal. They were collected 14 days post
infected feeding for testing. All exuviae were collected and divided into 2

 

 

 

 

 

 

 

samples.

Stage Tested PCR Southern blot
2"“I instar 1/1 1/1

3'‘1 instar 4/4 4/4

4th instar 8/8 8/8

5th instar 1/1 1/1

adult 8/8 8/8
exuviae POS POS

 

 

 

 

154

 

 

Table 10. Persistence of inoculated HBV in R. prolixus. Detection of the
HBV virus genome in R. prolixus samples. R. prolixus were inoculated with 1.0
ul of HBV suspension that theoretically contained 90 viral particles

 

 

 

 

 

 

 

 

 

 

 

Bodies PCR Southern blot

Day 7 0/5 3/ *”

Day 14 0/5 0/5

Day 21 0/5 0/5

Day 28 0/5 0/5

Day 35 0/3 0/3 F
* 2 kissing bug’s saliva was positive by Southern blot

’ 1 kissing bug’s legs were positive by Southern blot r' '

 

155

APPENDIX D

Human and Animal Use

I ,

 

156

 

UNIVERSITY COMMITTEE ON RESEARCH INVOLVING .33, ,1;
~ HUMAN 0R ANIMAL SUBJECTS . ,. ,

 

 

The Graduate School
Michigan State University
118 Linton Hall

University and federal policies and procedures require that all research involving human or
animal subjects receive prior approval from the appropriate review board. (See Faculty
Handbook, p. 116-117 and the Academic Programs book, p. 60.)

~ - . HUMAN SUBJECTS
Does the thesis or dissertation you are submitting include research involving human
subjects or materials of human origin? (Research involving human subjects includes
surveys and telephone interviews used for research; materials of human origin include

human blood and /or tissue.)
Yes 13] No D

If yes, indicate UCRIHS log number for the approved protocol and attach the UCRIHS
approval letter for that protocol to this form.

W‘T‘T‘fi 31.3!

 

 

 

UCRIHS Log Number: 133;; 95-039

 

~ - . ANIMAL SUBJECT S
Does the theSis or dissertat10n you are pr0posing to smeit include research involving
vertebrate animals 1n any way?
Yes E No D

If yes, and an animal use form was submitted to the All-University Committee on Animal
Use and Care (AUCAUC), please list the approval number below and attach a c0py of the
AUCAUC approval letter to this form.

 

 

 

 

AUF Number: 05/96-053-00

 

If yes, but your project did not need an animal use form, provide a copy of the letter from
the AUCAUC which cites the relevant exclusionary policy.

 

 

What
Maj or Professor’s Name (print)

(2.2?)

Student’s Signature 2 Major Professor's Signature

 

 

157
4197

APPENDIX E

Entomology Vouch Specimens

158

 

Record of Deposition of Voucher Specimens*

The specimens listed on the following sheet(s) have been deposited in
the named museum(s) as samples of those species or other taxa which were
used in this research. Voucher recognition labels bearing the Voucher
No. have been attached or included in fluid-preserved specimens.

Voucher No.: 1998—3

Title of thesis or dissertation (or other research projects):

Persistence of the Hepatitis 8 viral genome in selected
hematOphagous insects.

Museum(s) where deposited and abbreviations for table on following sheets:
Entomology Museum, Michigan State university (MSU)

Other Museums:

Investigator's Name (3) (typed)
Jamie A. Blow

 

 

Date July 21, 1998

*Reference: Yoshimoto, C. M. 1978. Voucher Specimens for Entomology in
North America. Bull. Entomol. Soc. Amer. 24:141-42.

Deposit as follows:

Original: Include as Appendix 1 in ribbon copy of thesis or
dissertation.
Copies: Included as Appendix 1 in copies of thesis or dissertation.

Museum(s) files.
Research project files.

This form is available from and the Voucher No. is assigned by the Curator,
Michigan State University Entomology Museum.

159

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