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This is to certify that the

thesis entitled

ANTIBODIES TO HUMAN HERPES VIRUS-6 (HHV-6)
IN PREGNANT WOMEN IN MICHIGAN

presented by

Mohanrned-Saleh Ali Islaih

has been accepted towards fulfillment
of the requirements for

Master's of Science degree in Clinical Laboratory
Sc1ence

 

 

 

 

Major p . fessor

Norma Phelps :arratt, Ph. D.

Date May 5, 1993

 

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__ _. ._ .._

ANTIBODIES TO BENIN EERPES VIRUS-6 (KEV-6)
IN PREGNANT IONEN IN KICEIGAN

BY

uehenned-aeleh Ali Ieleih

A THESIS

Submitted to
nichigen State University
in pertiel tultillnent of the requirements
for the degree of

NESTER OF SCIENCE

Clinicel Leheretery Sciencee

Hedicel Technology Progren

1993

ABSTRACT

ANTIBODIES TO HUMAN HERPES VIRUS-6 (HHV-S)
IN PREGNANT 'OHEN IN MICHIGAN

BY

HOMED-SAL‘ ALI ISLAIH

Antibodies to human herpesvirus-6 and cytomegalovirus
(CMV) were measured in sera of four groups of pregnant women,
from 34 counties in Michigan, to investigate the possible
association of HHV-6 with neural tube defects (NTD) and
premature deliveries.

Eighty sera from pregnant women with normal gestation and
normal alpha-fetoprotein (AFP) , 10 sera from women with neural
tube defects babies, 18 sera from women with high AFP and
normal gestation, and 35 sera from women with premature
gestation*were tested.by neutralization test to measure HHV-e
antibodies, and by Latex Agglutination test to measure
cytomegalovirus antibodies.

12.5% of the sera from women with normal gestation and
normal AFP were seropositive, while 100% of all the sera from
the other three groups of women showed seropositivity to HHV-6
antibodies. This suggests that.HHV-6, in some way is connected
to NTD, high AFP, and prematurity. In this study no antibody

cross-reactivity between.HHV-6 and cytomegalovirus was found.

Copyright by
Mohemmed-Seleh Ali Islaih

1993

ACKNOWLEDGMENTS

My sincere thanks go to my research advisor, Dr. Norma
Barratt. Her ideas, encouragement, support, and advice have
been essential in this study and are greatly appreciated.

I would like to thank the members of my committee, Dr.
Marcia Murry, Dr. David Thorne, and Dr. Louis Guskey for their
counseling, and encouragement in the preparation of this
thesis.

This project was made possible with the help of St.
Lawrence Hospital, Lansing, MI, and the Prenatal Screening
Clinic at Michigan State University, E. Lansing, MI.

Most importantly, I would like to thank my parents and my
wife for their love, understanding, and moral support which

made possible the completion of this work.

iv

TABLE OE CONTENTS

LIST OF TABLES........................................vi
LIST OF FIGURES......................................vii
INTRODUCTION...........................................1
MATERIALS AND METHODS.................................14
THE STUDY pOPULATION.............................14
SPECIMENS........................................14
ISOLATION OF CORD BLOOD MONONUCLEAR CELLS........15
CELLS AND MEDIUM.................................16
VIRUS POOLS......................................16
VIRUS POOLS TITRATION............................17
NEUTRALIZATION TEST..............................18
CMV ANTIBODY TESTING.. ...................... .....19
RESULTS...............................................20
DISCUSSION AND CONCLUSION.............................36

LIST OFREFERENCES...OOOOOOOOOOIOOOOOOOOOOOOO000......42

LIST OF TABLES

Page

Sera from Women with Normal AFP and Normal
GestationOOOOOOOOOOO...O0.000000000000000000000000. 27

Sera from Women with Neural Tube Defects Babies.... 31
Sera from Women with High AFP and Normal Gestation. 32
Sera from Women with Premature Gestation........... 33

The Distributions of HHV-6 in Michigan............. 35

LIST OF FIGURES

Figure Page
1. Clumps of stimulated cord blood mononuclear cells.. 23
2. Characteristic large cell formation of,

cord blood mononuclear cells infected with HHV-G....24
3. Neutralization test for KEV-6. The antibody-
negative serum permitted the cytopathic effect..... 25

4. Neutralization test for HHV-G. The virus
was neutralized with an antibody to the virus...... 26

Introduction

Herpes virus particles are large DNA-containing viruses
that share common biological characteristics and architectural
features of their virion. Within a population of virions,
which have diameters from 180nm - 200nm , many particles do
not possess envelopes, and others are empty capsids. The
virion components are arranged in a DNA-containing toroidal
core about 75nm in diameter which is composed of 162 elongated
hexagonal prisms (capsomers) . Each capsomer has a small,
central hole, an icosahedral capsid 95nm to 105nm in diameter,
a surrounding granular zone (tegument) composed of globular
proteins and an encompassing envelope possessing periodic
short projections (14).

In addition to structural similarities, the herpes
viruses also share biological characteristics of latency,
persistence, and reactivation. The molecular bases for these
biological features is not yet understood. But their
importance in disease processes is being examined. During
latency the viral genome is in inactive state. The latent
genomes are replicated, but in a limited, highly regulated
fashion, probably without production of viruses. Persistence

means a chronic low level of virus replication that is

_ 2

asymptomatic. This silent replication is distinctive and
puzzling, especially from the vantage point of the cell, which
is ordinarily destroyed by replication of productive herpes
virus infection. Latency and persistence may shade into one
another. That is, latency may be a constant production of a
few virus particles in an indolent process marked by complete
or partial expression of all the viral genes. Reactivated
infection, in contrast to persistent infection, is often
symptomatic, and the symptoms that accompany reactivated
infection may be more severe and quite different from the
symptoms of primary infection. A variety of stimuli can
trigger this recurrent viral activity, e.g., exposure to
ultraviolet light, fever, menstruation, nerve injury,
emotional disturbances and stress. The precise ways in which
these factors induce virus replication are unknown, however,
stress is known to result in immunosuppression (5,48).

Members of the herpesviridae family have been isolated
from more than 80 different species. Seven different human
herpesviruses have been described to date. They have been
placed into three subfamilies based on biological and physical
properties. The alphaherpesviruses include herpes simplex
virus type-1 (HSV-l), herpes simplex virus type-2 (HSV-2) and
varicella zoster virus (VZV). The betaherpesviruses includes
cytomegalovirus (CMV) . The gammaherpesviruses includes
epstein—barr virus (EBV) (13,14) I

The recognition of a new herpes virus was announced to

3

the world by Salahuddin et al. (1986) from the National Cancer
Institute, NIH (36). These investigators were involved in
studies of the mechanism regulating human hematopoiesis,
especially defects leading to malignancy. In these studies,
mononuclear cells from patients with various
lymphoproliferative disorders were established in culture.
Occasionally short lived, large, refractile cells, containing
intranuclear and intracytoplasmic inclusion bodies were
Observed.

Electron microscope (EM) examination of infected cells
demonstrated herpes-type virus particles in these cultures.
Unenveloped icosahedral nucleocapsids developed in the nucleus
and enveloped virions were seen in cytoplasmic vesicles,
cisternae and at the cell surface markers of infected cells.
The virus was called human B-lymphotropic virus (HBLV)
(36,40). Similar isolates were obtained during attempts to
isolate the human immunodeficiency virus type-1 (HIV-1) from
AIDS patients in Uganda, Gambia and in the U.S.A at the Center
for Disease Control (CDC). DNA hybridization studies showed
that the isolates were closely related to the original NIH
isolate, HBLV (12,28,41,46). Since that time viruses with
similar properties have been isolated from children with
roseola (42) , AIDS patients, patients with a variety of
lymphoid abnormalities, and healthy adults (4,27,38).

Antigenic and molecular studies showed the new HBLV

is clearly distinct from the other known human herpes viruses,

4
because of its unique cell tropism and the absence of antigen
cross-reactivity with other herpes viruses, although minimal
DNA homology with CMV was recently found by Southern blotting
analysis (40). Because the genes of HHV-6 have a number of
distinctive features i.e. they are often smaller than their
CMV homologs, they occur closer together, and they overlap
more often, this virus was proposed as human herpes virus-6
(HHV-6) (25). The HHV-6 genome is contained on a 170 kb
linear, double-stranded DNA molecule. Endonuclease restriction
mapping and hybridization experiments were used to develop a
model for the gross structure of HHV-6 (44). The organization
and direction of transcription of HHV-6 genes show more
homology to cytomegalovirus (CMV) genome than to other herpes
viruses. It is likely that HHV-6 will be classified in the
beta-herpes virus subfamily along with cytomegalovirus
(25,34).

Based.on‘these'observations, as well as the similarity of
predicted amino acid sequences to ‘those of CMV, it 'was
concluded that CMV and HHV-6 are as closely related as HSV-l
and VZV (12) or EBV and herpesvirus saimiri (HVS) (25,14).
In accordance with the provisional classification of the
international committee on the taxonomy of viruses this new
virus was classified in the family Herpesviridae, and called
human herpes virus-6 (HHV-6), a nomenclature that is
independent of its tropism i.e. ability to grow in particular

cell (40).

5

Human herpes viruses (even within subfamilies) differ
significantly in their pathogenic potential and mode of
spread. Herpes simplex virus type-1 is primarily associated
with oral and ocular lesions and is transmitted in oral and
respiratory secretions. Type 2 is isolated primarily from
genital and anal lesions and passed through sexual contact.
Mothers with genital herpes (a painful, persistent, recurrent
infection) are likely to present an increased risk of
neonatal infections with type 2 virus. Neonatal herpes is now
known to cover a wide spectrum of severity, from the
relatively mild to the most severe. The grave feature is
dissemination to crucial organs such as brain or adrenals
(3.6) I

Involvement of the of the central nervous system with
herpes simplex viruses, though rare, accounts for most cases
of sporadic encephalitis in the western world. Herpes simplex
encephalitis (HSE) may be caused by either type 1 or type 2
and both produce similar effects of acute necrotization with
associated inflammation and a consequent rise in intracranial
pressure. In the neonate most cases are caused by type 2 HSV
through congenital infection. After the neonatal period,
infection is usually caused by type 1. Neonatal HSE is usually
part of viraemic dissemination of the virus. Cerebral
involvement is widespread and mortality is close to 100%
(18,21).

Varicella (chickenpox), the primary disease produced by

j 6

the varicella virus in hosts without immunity is usually a
mild, self limited illness of young children. Herpes zoster
(shingles) is the recurrent form of the disease, occurring
predominantly in adults. The pathogenesis of varicella is not
fully understood. The behavior of VZV in pregnancy does
however, shed. some light. on. this. First, the fact. that
varicella produce intrauterine infection provides further
evidence that primary infection must have a viraemic phase
even though this is difficult to detect. 0f greater
significance is the observation that serious congenital
varicella will occur only if the onset of the maternal rash is
within four days of delivery (15)

Epstein—Barr virus is unusual in that it is associated
with several disease states. In some cases it is the direct
etiological agent while in other situations the virus acts as
a cofactor in a complex series of events that lead to
disease. Infectious mononucleosis is caused by primary EBV
infection, while Burkitt's lymphoma and nasopharyngeal
carcinoma occur in seropositive individuals as a result of a
series of alteration in a cell type infected by EBV.
Infectious mononucleosis during pregnancy is uncommon and does
not result in damage to the fetus (6).

Human herpes virus-7 (HHV-7) has been recently isolated
from. activated CD4+ T lymphocytes. Its isolation raises
questions about the prevalence of this virus in humans and its

immunological relationship to other herpes virus. HHV-7 is a

7
ubiquitous virus which is immunologically distinct from the
highly prevalent T-lymphotropic HHV-6. In some instances HHV-7
infection occurred in the presence of high titer of HHV-6
antibodies. This suggests the lack of apparent protection of
children who are seropositive for HHV-6 against subsequent
infection of HHV-7. HHV-7 is a prevalent human herpes virus
which like other human herpes viruses, infects during
childhood. The age of infection appears to be some what later
than the very early age documented for HHV-6 (13,49).

CMV is the leading cause of congenital viral infection
occurs with an average incidence of 1% of all live births. 5%
of congenitally infected babies are born with symptoms. They
are said to have "cytomegalic inclusion disease" (CID) and
their prognosis are poor. The remaining 95% appear to be
normal at birth but a proportion develop sequelae on follow-
up. The classic presentation of these symptoms is one of
intrauterine growth, retardation, jaundice,
hepatosplenomegaly, thrombocytopenia and encephalitis with or
without microcephaly. In 1% of all those congenitally
infected, the damage caused by the virus is so severe that
they die during infancy. If a neonate survives it is almost
certain to have serious abnormalities for the rest of its
'life. Long term follow-up of the asymptomatic children has
revealed that approximately 15% are likely to have hearing
defects or impaired intellectual performances.

Intrauterine infection is assumed to follow maternal

8

viraemia and subsequent placental infection, congenital CMV
infection is thus assumed to be acquired transplacentally
although this has not been proven formally. Intrauterine
transmission of CMV occurs in only one third of pregnant women
with primary infection but it is not known how the virus
prevented from infecting the fetus in the other two thirds
cases.

Congenital infection also results from recurrent maternal
CMV infection. Primary infection occurs in 19% to 20% of
seronegative pregnant women, with subsequent infection of
about 50% of the fetuses. In contrast, only 10%-20% of
pregnant women with latent infection transmit CMV to their
fetuses. A mother with a primary or a latent infection may
transmit CMV to the fetus, either by transplacental transfer
during pregnancy or by excreting virus into the genital tract
at the time of birth. Earlier reports showed that virus
excretion from the cervix increased as pregnancy progressed
and this was interpreted as being a response to some
”immunosuppressive" effect of pregnancy induced stress
(14,16,39).

HHV-6 is the causative agent of exanthem subitum (roseola
infantum) , a childhood disease characterized by spiking fever
and skin rash (50,51) . Infection is commonly acquired early in
life but the proportion of seropositive persons appears to
decrease after 40 years of age. HHV-6 is lymphotropic, appears

to undergo latency and may be reactivated in immunocompromised

9

persons. The reported prevalence rates have varied between 20%
and 100% in normal population, one of the studies shows that
almost all infants were infected by HHV-6 by one year of age
and that approximately 80% of adults of all age groups have
antibody against this virus.

other investigators found the presence of the genome of
HHV—6 and expressed proteins in human parotid and
submandibular salivary glands. These findings are consistent
with the transmission of HHV-6 by saliva and suggest that the
tissue of the salivary glands is a site of replication of the
virus and a potential site for persistence of HHV-6 (17).

After its discovery, illnesses linked to HHV-6 have been
reported and include roseolla infantum, hepatitis,
lymphadenitis, mononucleosis, atypical polyclonal
lymphoproliferative disorder, hemophagocytic syndrome(33) ,
myalgic encephalomyelitis, also called chronic fatigue
syndrome (47) and meningitis (18). HHV-6 infects glioblastoma
cells and may be a potential cause of nervous system disease.
Although the number of diseases linked to HHV-6 are
increasing, the connection of HHV-6 with nervous system
illness has been demonstrated only in a 13 month old girl
suffering from encephalitis and experiencing brain atrophy
(7,18,43).

Alpha-fetoprotein (AF?) is a plasma protein made by the
fetal liver. In the nonpregnant state, women's plasma contains

virtually no detectable AFP. During pregnancy, the maternal

10

serum AFP level is related to the level of AFP in the amniotic
fluid. Measurement of the AFP level in maternal serum is used
as a screening test for fetal condition associated with
abnormal levels of amniotic fluid AFP. Any fetal condition
that results in increased. passage of fetal plasma into
amniotic fluid will increase the maternal serum AFP level.
Such conditions include open neural tube defects, such as
anencephaly or meningomyelocele, abdominal wall defects and
congenital nephrotic syndrome.

Elevated levels of maternal serum AFP are also associated
of pregnancy complications in the absence of fetal
malformation. Such complications include fetomaternal
transfusion, increased risk of premature delivery, increased
risk of fetal death and the presence of twins. AFP
determinations are also measured when amniotic fluid is
sampled, such as with amniocentesis in women over the age of
35 years. A close correlation also exists between the level of
amniotic fluid AFP and the fetal conditions cited above.
Amniotic fluid levels of APP, however, also vary with
gestation age (22,32).

Developmental abnormalities of the central nervous system
(CNS), malformations and destructive lesions, are associated
with incidents that occur within the first half of pregnancy
and may be the result of infectious agents including TORCH
pathogens Toxoplasma, Rubella, CMV and HSV (30).

The process of neurulation in which the primordium of the

11

central nervOus system, the neural tube, is formed is one of
the earliest organogenetics events occurring in embryonic
development. Abnormalities of neurulation lead to a variety of
congenital malformations, referred to collectively as neural
tube defects (NTD) (9) . Spina bifida and anencephaly are
relatively common neural tube defects (NTD) and constitute a
large portion of the serious congenital malformations in
humans. The causes of neural tube defect is currently unknown.
To examine a potential link of HHV-6 infection to neural tube
defects, serum specimens from a group of women who have NTD
babies were tested for HHV-6 antibodies to see if there is an
association between HHV-6 infection and NTD. HHV-6 antibody in
serum from women with elevated AFP were also measured, since
AFP is the marker used to test for NTD in pregnant women.

Spina bifida is one of the most common central nervous
system malformations. The posterior arches and spines of some
vertebrae are absent and there is a midline defect of the
spine that results in a protrusion of the meninges or spinal
cord or other neural elements. Similarly meningomyelocele is
a defect in the spinal column in which spinal cord and
meninges protrude into the skin of the back. Anencephaly is a
malformation in ‘which the entire forebrain is missing.
Anencephaly may result from maternal infections (toxoplasma)
or reactivation during pregnancy. The cause of spina bifida
and meningomyelocele is not known (31).

Prematurity is defined as the birth of a viable infant

12
before the thirty-seventh week of gestation. Premature birth
may result from almost any microbe capable of establishing
fetal infection during the last trimester of pregnancy.
Cytomegalovirus and herpes simplex infections have been
associated with stillbirth, abortion and prematurity, but
there are no studies concerning the association of HHV-6
infection with prematurity (23).

Virus neutralization is defined as the decrease in virus
infectivity following the interaction of virus with specific
antibody. The mechanism by which antibody neutralizes virus
infectivity and ultimately effects its destruction depends
upon the virus, the class of antibody, the ratio of antibody
to virus, and.hOW'a particular host cell.handles the antigen -
antibody complex. Inhibition of virus adsorption to cells and
enhancing virus degradation are important mechanisms by which
neutralizing antibody effects neutralization.

Early IgM and IgG possess low neutralizing activity for
herpesvirus in vitro in the absence of complement (C). When C
is added, both IgM: and IgG antibodies exhibit enhanced
neutralizing activity. Late IgM antibody can neutralize
herpesvirus in the absence of C, but its activity is enhanced
by the addition of C. The quantitative effectiveness of late
IgG antibody in neutralizing virus does not appear to be
influenced by C; however, the rate at which virus is
neutralized by 196 is enhanced in the presence of C (2).

Based on observations concerning association of CMV’with

. 13
disease status and because of the homology of CMV to HHV-6, a
seroepidemiological study among pregnant women in Michigan was
conducted to correlate the occurrence of CMV and, HHV-6
antibodies in the serum of pregnant women in Michigan. The
study also involved examining a possible association of HHV-6
infection with neural tube defects (NTD), abnormal alpha-
fetoprotein and premature delivery. The objective was to
conduct an epidemiological survey among pregnant women in
Michigan to determine the prevalence of HHV-6 infection, and
to investigate HHV-6 infection during pregnancy which is
associated with neural tube defects and premature deliveries.
HHV-6 infection was determined by measuring the antibody titer
in maternal serum using a neutralization test which can detect

both 196 and IgM.

KATERIALS AND HETHODS

THE STUDY POPULATION

The study population consisted of four groups of pregnant
women between ages of 20 - 40 years. The first group were
pregnant women who had normal babies and normal level of serum
AFP; the second group were pregnant women who had NTD babies;
the third group were pregnant women with high serum alpha-
fetoprotein and normal gestation and apparently normal babies;
and the fourth group were pregnant women with premature
deliveries.

Sera were tested for antibodies to HHV-6 using the
neutralization test described below and for cytomegalovirus
antibodies by using the CMVscan card test (Becton Dickinson,
Cockeysville, MD), a commercial kit based upon the principle
of passive latex agglutination.

SPECIMENS

Maternal sera samples were collected between 16-18 weeks
of gestation at the Prenatal Screening Clinic at Michigan
State University, E.Lansing, MI. and were stored at - 80°C

until tested.

14

15

ISOLATION OP CORD BLOOD HONONUCLEAR CELLS

Heparinized cord blood was provided by Labor and Delivery
at St. Lawrence Hospital, Lansing, MI (usually 45 — 50 ml were
received) . The mononuclear cells were separated by Histopaque-
1077 (Sigma Chemical Co. St. Louis, MO) gradient
centrifugation, according to the method recommended by the
manufacturers. Six (6.0) ml of Histopaque-1077 were added to
a.15 ml conical centrifuge tube (Corning Glass'Works, Corning,
N.Y), an equal volume.of cord.blood was carefully layered onto
the Histopaque-1077, and the tube centrifuged at 1300 rpm for
30 minutes, at room temperature using an International
Portable Refrigerated Centrifuge Model PR-2 (International
Equipments Co., Needham HTS., MA). Centrifugation produced 4
layers: an upper layer of plasma, a white cell layer, clear
Histopaque and a bottom layer of red blood cells. After
centrifugation, the plasma layer above the white interface
containing mononuclear cells (MNCs) was removed by careful
aspiration with a sterile Pasteur pipet and discarded. The
interface containing the MNCs were aspirated with a sterile
Pasteur pipet and pooled in a 50 ml centrifuge tube. The
volume was brought to 40-45 ml with phosphate buffer saline
(PBS), ph 7.4, mixed and centrifuged at 1000 rpm for 10
minutes. The supernatant fluid was removed and the cells
(MNCs) were resuspended in cord blood growth medium, viable
MNCs concentration was determined using a hemacytometer and

trypan blue.

16
CELLS AND HEDIUN

Mononuclear cells isolated from the cord blood were
cultured (2 x 10‘ cells/ml) in RPMI 1640 culture medium (Sigma
Chemical CO. St. Louis, MO) supplemented with 10% (V/V) heat-
inactivated fetal bovine serum (Sigma Chemical Co. St. Louis,
MO) and 0.1% (V/V) of jphytohemagglutinin (PHA)-P (DIFCO
Laboratories, Detroit, MI) and 1% of a commercial mixture of
penicillin, streptomycin and amphotercine-B (Sigma Chemical
Co. St. Louis, MO) (PHA-RPMI) in 75 cm2 plastic tissue culture
flasks (Corning Glass Works, Corning, N.Y).

Cell cultures were maintained in 75 on2 tissue flasks
with a loose cap at 37°C in a 5% C02, 95% air mixture and 57%
humidity for 3 days to stimulate cell division by PHA-P. Cells
were observed daily using an inverted microscope (Photo Zoom,
Inverted microscope, Cambridge Instruments) (1,52).

Cells for neutralization tests and virus titration were
grown in 96 well microtiter plates. 2 x 10‘ cells were
incubated in 100 pl PHA-P RPMI per well and maintained for 3
days at 37°C, 5% C02, 95% air mixture and 57% humidity (53) .
VIRUS POOLS .

Mononuclear cells which had been stimulated with PHA-P
for 3 days, were used for the preparation of the HHV-6 pools.
Two-thirds of the medium was removed from the culture, without
disturbing the cells. Cells were infected.with an inoculum of
HHV-6 (HHV—6 was kindly provided by Dr. P. Pellett, CDC,

Atlanta, GA) at 1% (V/V) of total volume of the medium of the

17

uninfected culture. The inoculum was allowed to adsorb 1-2
hours at 37°C. Fresh RPMI medium was added to restore the
original volume. The inoculated cells were again incubated
under the same conditions and observed daily microscopically
for cytopathic effect (CPE) over a post-inoculation period of
7-12 day. Signs of cytopathic effect (CPE) included large
cells with a pleomorphic and balloon-like appearance (45).

When the CPE was seen in more than 70% of the infected
cells, the cells were sedimented by centrifugation.at 1000 rpm
for 10 minutes. Medium was removed and the cells containing
the virus were resuspended in 10% (V/V) of the original
culture volume using a 1:1 mixture of 3X skim milk and the
original medium. The virus pools were stored at - 80°C in 1 ml
aliquots.
VIRUS POOLS TITRATION

To facilitate virus release from infected cells, viral
infected cells were frozen and thawed twice before titration.
Ten fold dilutions of the virus preparation from 10'1 to 10*
were prepared in fresh cord blood medium. Fifty pl of medium
were removed from each well and discarded. One hundred ul Of
each dilution of virus were added to 6 separate wells. An
additional six separate wells containing uninfected cells
received 100 pl of fresh virus-free cord blood medium, these
wells were used as controls. Virus infected cells were allowed
to adsorb for 1 hour at 37°C. After 1 hour, 100 pl of fresh

cord blood medium were added to each well and the plates were

18
incubated for 7 days and monitored daily for CPE.

If 25% or less of the cells in each well demonstrated CPE
the score was considered negative. If more than 25% of cells
were damaged in each well, the score was considered positive.

The Karber method was used to calculate the titer of the
virus based on these observations by using the following
formula:

-log(TCID),,o = [-logH] - [ S/100 - 0.5) x logD]

Where TCID= Tissue culture infective dose

H The highest virus concentration used.
S = Sum of the mortality percentages.
D = Interval between dilutions (26).
NEUTRALIZATION TEST
Basic procedures for the neutralization test (NT) were
followed. Serial two fold serum dilutions were prepared (1/2,
1/4 ...etc) in disposable plastic microtiter plate (Costar,
Cambridge, MA) containing 96 wells. Serum dilution were mixed
with an equal volume (100 pl) of the titrated virus
preparation containing 10” tissue culture infective doses
(TCID),0 (the amount of virus required to cause a demonstrable
infection in 50% of the inoculated tissue culture cells) per
0.1 ml of the virus suspension. After 1 hour of incubation at
37°C, the mixture of the serum and the virus were transferred
to a second microtiter plate contain 100 pl of stimulated cord

blood mononuclear cells in each well. Plates were reincubated

for 7 days at 37°C.

19

The antibody titer was determined as the reciprocal of
the highest dilution of serum that prohibited large cell
formation in more than 25% of the cells in each well (35,53).
CNV ANTIBODY TESTING

All sera were tested for antibody to CMV by means of the
CMV scan card test (Becton Dickinson, Cockeyville, MD). The
test is based upon the principle of passive latex
agglutination. Latex particles which have previously been
sensitized with CMV viral antigens will agglutinate in the
presence of antibody to CMV.

The test was performed according to the manufacturers
instructions. Briefly, 50 ul of serum were placed onto the
appropriate circle and were distributed over the entire circle
on the test card. Latex reagent was mixed thoroughly before
one free-falling drop was added onto each circle containing
the serum. The card was rotated manually 3 to 4 times to
distribute the latex antigen throughout each circle. The card
was rotated for 8 minutes after which the card was read
macroscopically.

Reactive control and nonreactive control were tested with
each set of serum samples. The results were reported as
positive (+) if there was visible latex antigen agglutination
and negative (-) if there was no agglutination of the latex

antigen.

RESULTS

The cord blood mononuclear cells from each cord blood
sample used to grow the virus were sensitive to HHV-6. Titers
of 104 were achieved in the virus pools and CPE was typically
as described by other authors (35,36) and as shown in (Figures
1-4) .

Eighty sera from pregnant women with normal gestation and
normal AFP were tested by neutralization test for HHV-6
antibodies and by latex agglutination for CMV antibodies.

Ten sera from pregnant women with neural tube defects
were tested for HHV-6 antibodies using neutralization test,
and Latex agglutination to test for CMV antibodies.

Eighteen serum samples from women with high AFP and
normal gestation were tested for HHV-6 antibodies by
neutralization test and for CMV antibodies by latex
agglutination test.

Thirty five serum specimens from pregnant women with
premature gestation were tested by neutralization test to
detect HHV-6 antibodies, and a latex agglutination test was
used to detect CMV antibodies.

12.5% (10/80) of the sera from women with normal

gestation, and. normal AFP ‘tested. positive by ‘the

20

. 21
neutralization test for antibodies to HHV—6. Within the HHV-6
positive of this group 40% (4/10) were also positive for CMV
antibodies. Titers of HHV-6 antibodies ranged from 1:2 to
1:128 with a mean titer of 1:64 as shown in (Table 1).

100% (10/10) of the sera from women with neural tube
defects tested positive in neutralization test for antibodies
to HHV-6. None (0/10) of these tested positive to CMV
antibodies. Titers of HHV-6 antibodies ranged from 1:8 to
1:128 with a mean titer of 1:50 as shown in (Table 2).

All (18/18) of the sera from women with high AFP and
normal gestation tested positive by virus neutralization to
HHV-6 antibodies. Of the 100% positive 61% (11/18) were also
positive for CMV antibodies. The titer of HHV-6 ranged from
1:16 to 1:512 with a mean titer of 1:115 as shown in (Table
3).

100% (35/35) of the sera from women with premature
gestation tested positive by virus neutralization to HHV-6
antibodies. Of the 100% positive, 60% (21/35) were also
positive for CMV antibodies. The titer of HHV-6 antibodies
ranged from 1:4 to 1:512 with a mean of 1:127 as shown in
(Table 4).

The HHV-6 infections were clustered in the following
counties 75% (12/16) in Kent, 53.8% (7/13) in Genesee, 42.1%
(8/19) in Ingham. The other positive were distributed among
other counties as shown in (Table 5).

The distribution of the data was analyzed by the

22
Quantile-Quantile-Plot (Q-Q-Plot) using S-plus software from
Statsci, which showed that the data were not normally
distributed, therefore a chi-square test ()8) , which is a
distribution-free (nonparametric) test, was used to determine

the statistical significance.

23

 

Figure 1: Clumps of stimulated cord blood mononuclear cells.

 

Figure 2: Characteristic large cell formation of cord blood
mononuclear cells infected with KEV-6.

25

 

Figure 3: Neutralization test for KEV-6: The antibody-negative
serum permitted the cytopathic effect.

26

 

Figure 4: Neutralization test for REV-6: The virus was
neutralized with an antibody to the virus.

27

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 1: Sera from women with normal AFP and normal
Gestation.
a
NO. Titer of REV-6 CMV Antibody County
Antibody (Agglutination)
(Neutralization)
1 - - Isabella
2 - + Genesee
3 - + Not Reported u
4 - - Not Reported
5 - + St. Joseph
H 6 - + Not reported
7 - + Kent
8 - - Kalamazoo
9 1:64 + Not reported
10 1:128 - Saginaw
11 - - Otsego
12 - - St. Joseph
13 1:2 - Ingham
l4 - + Ingham
15 - + Lenawee
16 - + Eaton
17 - + Ingham
18 - + Washtenaw
19 - - Genesee
‘ 20 _ + =ggsana

 

 

28

Table 1: (cont'd).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

NO. Titer of HSV-6 CMV Antibody County
Antibody (Agglutination)
(Neutralization)
21 1:128 - Ingham
22 - + Kent
23 - - Calhoun l
I24 - + Ingham
I25 - - Washtenaw
'26 1:128 - Kalamazoo I
I27 - - Livingston I
I28 - - Saginaw I
29 - - Ingham I
30 - - Not Reported I
31 - - Genesee I
32 - - Not Reported
33 - - Kent
34 - - Muskegon I
35 - - Washtenaw
36 - - Van Buren
37 1:128 - Ingham
38 - + Tuscola
39 - - . Genesee
4 0 - + IIgalamazoo J

 

 

Table 1:

(cont'd).

29

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

NO. Titer of REV-6 CMV Antibody County
Antibody (Agglutination)
(Neutralization)

41 - - Jackson

42 - + Muskegon I

43 - + Muskegon I

44 - - Saginaw I

45 - + Washtenaw

46 - + Ingham

47 - - Branch

48 - + Ingham I
I49 - + Ingham I
I50 ‘- - Ottawa

51 - + Muskegon
I 52 - - Muskegon

53 - + Clare I

54 - - Calhoun

55 - + Not Reported

56 - - Ingham

57 - - Genesee

58 - + Saginaw I
I 59 - + Alger
I560 1:16 - Branch

 

 

 

 

Table 1:

(cont,d)

30

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

NO. Titer of HSV-6 CMV Antibody County
Antibody (Agglutination)
(Neutralization)
61 - - Ingham
I62 - - Saginaw
63 - - Ingham
I64 - + St. Joseph
I 65 - - Epeer
66 - + Muskegon
I67 1:32 + Branch
68 - + Oakland
69 - + Kent I
70 - + Berrien I
71 - + Arenac
72 - + Muskegon
73 - — Muskegon
74 - - Genesee
75 - - Jackson
76 - - Washtenaw
77 - - Saginaw
78 - - Ingham
79 - - Berrien
l£!L__ 1:8 + Branch I

 

 

31

Table 2: Sera from women with neural tube defects babies.

   

    

 

 

 

 

 

 

 

 

 

 

Titer of HSV-6 CMV Antibody County
Antibody (Agglutination)
(Neutralization)
I1. 1:32 - Kent
I2 1:32 - Oakland
I3 1:8 - Clinton
I4 1:16 - Kent
5 1:64 - Genesee
6 1:64 - St. Joseph]
I7 1:32 - Eaton
I8 1:64 - Shiawassee
9 1:64 - Mackinac
I10 1:128 - Ingham

 

 

 

 

 

 

32

Table 3: Sera from women with high AFP and normal gestation.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

a—-=-—= .—-—-—-—-—-—-—-—-——-—.

No. Titer of REV-6 CMV Antibody County

Antibody (Agglutination)
(Neutralization)

1 1:256 - Gladwin

2 1:512 + Genesee

3 1:32 + Saginaw

4 1:32 + Eaton

5 1:32 + Washtenaw

6 1:16 + Ingham

7 1:256 + Washtenaw

8 1:32 + Muskegon

9 1:64 - Eaton

10 1:256 + Genesee

11 1:16 + Muskegon

12 1:32 - Genesee

13 1:32 - Ingham

14 1:32 + Hilledale I

15 1:64. - Kent

16 1:128 - Kalamazoo

17 1:16 + Saginaw
I18 1:256 - Schoolcraft

 

33

Table 4: Sera from women with premature gestation.

 

—

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

No. Titer of REV-6 CMV antibody County
Antibody (Agglutination)
(Neutralization)

1 1:4 Berrien

2 1:32 Berrien

3 1:512 - St. Joseph

4 1:64 - Grand Traverse

5 1:16 + Branch

6 1:256 + Branch
I7 1:512 - Ingham I
I8 1:256 + Ingham
I9 1:256 + Saginaw

10 1:128 + Washtenaw

11 1:128 - Saginaw

12 1:32 + Kent

13 1:32 + Kent

14 1:128 - Calhoun

15 1:256 + Kalamazoo

16 1:256 + Wayne

17 1:128 - Kent I

18 1:64 - Oakland u

19 1:32 Genesee

20 1:32

 

 

 

Wayne

 

 

34

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 4: (cont'd).
No. Titer-OT-HHV-G CMV Antibody County I
Antibody (Agglutination)
(Neutralization)
I21 1:64 - Genesee
I22 1:8 - Clare
I23 1:16 + Kalamazoo
24 1:128 - Kent
25 1:64 - Kent
26 1:128 + Muskegon ‘
27 1:128 + Kent
'28 1:256 + Genesee
29 1:32 + Kent
30 1:16 + Saginaw
31 1:32 - Kent
32 1:128 + Oakland
[33 1:64 + Kent I
I34 1:128 - Muskegon I
I35 1:128 - _gakland

 

 

 

 

 

Table 5: The Distributions of REV-6 in Michigan.

L

 

I County

No.of serum

No. of positive

(%)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

samples to HHV-6
Genesee 13 7 53.8
St. Joseph 3 2 66.6
I Kent 16 12 75 . 0
Kalamazoo 6 4 66.6
I Saginaw 11 6 54 . 5
Ingham 19 8 42.1
I Eaton 3 3 100
Washtenaw 3 100
Oceana 1 1 100
Calhoun 1 1 100
Muskegon 12 4 33.3
Branch 5 5 100
Oakland 5 5 100
Clinton 1 1 100
Shiawassee 1 ’ 1 100
Mackinac 1 1 100
Gladwin 1 1 100
Hilledale 1 1 100
Schoolcraft 1 1‘ 100
Berrien 2 2 100
Grand Traverse 1 1 100
Wayne 2 2 100
_ 1“ 1 100

(Clare

DISCUSSION AND CONCLUSION

The frequency of HHV-6 antibodies in the sera of the
pregnant women with normal gestation and normal alpha-
fetoprotein was (12.5%). This is lower value than those
reported by others for the general population (37,40,46) , but
consistent with values (10%) measured in the serum of young
men between ages 18-45 years (unpublished data, Dr. N.
Barrett) . The lower prevalence of HHV-6 observed in our
laboratory is in line with currently accepted views of the
production and degradation of antibodies in humans. Following
primary infection there is an initial lag phase when no
antibody can be detected. This is followed by phases in which
the antibody titer rises logarithmically to a plateau and then
declines again as the antibodies are naturally degraded or
bind to the antigen and are cleared from the circulation. In
contrast, the synthesis of IgG antibodies rapidly accelerates
to a much higher titer on secondary challenge followed by a
relatively slow loss in serum antibody level.

In contrast, in sera from women with either neural tube
defects babies, or high alpha-fetoprotein, or premature
gestation, the incidence of antibodies to HHV-6 was very high

(100%) and differed significantly from women with normal

36

. 37

babies (P<0.005). This would lead one to believe that this
virus (HHV-6) was in some way connected to neural tube
defects, high alpha-fetoprotein, and to prematurity.

Infections are relatively common during pregnancy. A wide
variety of organisms may infect pregnant women in the first
few weeks of gestation culminating in death of the embryo.
Since these infections may occur before the women realizes
she is pregnant, statistical analysis of causative agents is
conservative. The earliest recognizable effects of fetal
infection are seen after the sixth to eighth week of pregnancy
and include abortion and stillbirth. Intrauterine death may
result from overwhelming fetal infection, or the
microorganisms may interfere with embryonic development. A
number of factors probably determine the ultimate out-come of
intrauterine infection. These include virulence or tissue
tropism of the infectious agent, the stage of pregnancy,
associated placental damage and the severity of the maternal
illness. Primary infection is likely to have a more severe
effect on the fetus than recurrent infection.

Cytomegalovirus, rubella virus and varicella-zoster virus
cause developmental anomalies in the human fetus. The
pathogenic mechanisms responsible for the fetal abnormalities
produced by infectious agents remain obscure. Careful
histologic studies of aborted fetuses and congenitally
infected infants have suggested, however, that the ultimate

mode of action of the viruses rests in their ability to cause

38

cell death, alteration in cell growth or chromosomal damage.
The gestation age is important in determining the consequences
of prenatal infection. For instance when congenital rubella
and toxoplasmosis are acquired during the last trimester of
pregnancy, the incidence of clinical disease in the infected
infants is lower than when infection occurs during the first
or second trimester (23).

Based on these observations, two explanations can be
formulated to explain the high percentage of HHV-6 antibody in
sera from women having with of neural tube defects, high
levels of alpha-fetoprotein and premature gestation. The first
is that the mothers of infants with neural tube defects were
infected with human herpesvirus-6 during the first 3-4 weeks
of pregnancy. The second explanation is that stress during
pregnancy triggered reactivation of latent HHV-6.

The first hypothesis is supported by the facts that
formation of the neural tube occurs during the third and
fourth weeks of gestation (9,31) and that the mean titer
(1:50) of the sera from women with neural tube defects is less
than the mean titer (1:115) of the sera from women with high
alpha-fetoprotein but normal babies and the mean titer (1: 127)
of the sera from women with premature gestation. The lower
mean titer against HHV-6 supports the hypothesis that HHV-6
infection took place in women with neural tube defects early
in the third and fourth weeks of pregnancy. Since the sera

were taken after 16-18 weeks of pregnancy, the level of the

39

antibodies presumably declines by the time of serum sampling.

To explain the correlation of HHV-6 antibody and the
premature deliveries and high level of AFP, but with babies
of apparently normal gestation, one can speculate that HHV-6
infection took place later during the pregnancy, mitigating
severe damage to the central nervous system. Although HHV-6
was associated with prematurity and high level of AFP it may
not be the causitive agent. Prematurity associated with other,
members of the herpesvirus family has been reported (23). The
difference between the mean titer of the antibody to HHV-6 in
sera from women with premature deliveries (1:127) and the mean
titer of the antibody to HHV-6 in sera from women with high
AFP (1:115) was similar and higher than the mean titer in
women with NTD. This supports the notion that HHV-6 infection
took place later during the pregnancy resulting in less damage
to the fetus than infection by HHV-6 in the first trimester.
Perhaps enough stress was placed upon the mother and infant,
that high AFP was produced, in one group, and in the other
group prematurity resulted. Elevated levels of maternal serum
AFP are indicative of pregnancy complications in the absence
of a fetal malformation, such as increased risk of premature
delivery, increase risk of fetal death, and the presence of
twins (22).

Human herpesvirus-6 is clearly antigenically distinct
from other herpesviruses, including closely related

cytomegalovirus (25) . Initially, there was concern about

40
serological cross-reactivity between CMV and HHV-6. However,
subsequent adsorption studies with either CMV or HHV-6 removed
only homologous 196 indicating that cross-reactivity is
unlikely (29).

To insure that serum CMV antibody were not cross-reacting
in our assay, all sera were tested for CMV antibodies using a
latex agglutination test and some of the results were
confirmed by using an ELISA. Surprisingly, none of the HHV-6
positive sera from womenwwith.babies with.neural tube defects,
tested positive to CMV antibodies. Although 40% to 68% of the
sera from the other three groups tested positive to CMV. The
prevalence of CMV antibody in our test population is in
agreement ‘with. data previously’ reported for CMV in ‘the
population in large (30).

Thus we conclude that HHV-6 not CMV is in some way
associated to neural tube defects, and this group in
particular showed that there is no cross-reactivity between
HHV-6 and cytomegalovirus antibodies.

Due to the small number of specimens received from each
county, it is not possible to determine whether or not HHV-6
is more prevalent in a specific area. It seems that infections
are clustered in Kent 75% (12/16), Genesee 53.8% (7/13) and
Ingham 42.1% (8/19), where each of these counties have large
urban areas. It would be interesting to conduct further
epidemiologic studies to determine exactly how the virus is

distributed in Michigan.

_ 41
There was no clustering of NTD according to counties. But
nine of the thirty five (9/35) premature deliveries, which
tested positive, were collected from women from Kent. The

other positive were distributed among other counties.

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