LIBRARY
Michigan State
University

 

 

 

This is to certify that the

thesis entitled

.TROTU‘ICROSCOI‘IC ECIZTIQUE TO
.3 PM’LTICLES III BATIEITS

.1

WITH END Lilffi RBXAL ISLQSE

1.1;

presented by

 

Charlotte A. Currie

has been accepted towards fulfillment
of the requirements for

1.1.3, degreein Clinical lelicrobiology

 

Major professor

Date February 25, 1981

0-7639

 

 

MSU

LIBRARIES
n

 

 

RETURNING MATERIALS:
Place in book drop to
remove this checkout from
your record. FINES will
be charged if book is
returned after the date
stamped below.

 

 

 

IMMUNOELECTROMICROSCOPIC TECHNIQUE TO DETECT

DANE PARTICLES IN PATIENTS WITH

END STAGE RENAL DISEASE

BY

Charlotte A. Currie

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

MASTER OF SCIENCE

Department of Microbiology and Public Health

1981

ABSTRACT

IMMUNOELECTRONMICROSCOPIC TECHNIQUE TO DETECT
DANE PARTICLES IN PATIENTS WITH END STAGE RENAL DISEASE

BY

Charlotte A. Currie

Urine and serum samples from patients with end stage
renal disease were examined by immunoelectronmicroscopy
(IBM) for the presence of Dane particles. These patients
had previously been included in a statewide survey to
determine prevalence, among chronic maintenance hemodialysis
and renal transplant patients, of hepatitis B virus (HBV)
markers (HBsAg: hepatitis B surface antigen, anti-HES:
antibody to HBsAg, anti-BBC: antibody to hepatitis B core
antigen, HBeAg: hepatitis B e antigen, anti-HBe: antibody
to HBeAg) detectable by radioimmunoassay.

Thirty one of thirty two urines from HBsAg seropositive
patients were negative for whole HBV virions by IBM. Sub-
sequent urinalysis data indicated that the positive specimen
contained occult blood, the probable source of virions
detected by the procedure.

Thirty three serum samples demonstrating anti-HBC in
the absence of HBsAg and anti-HES, were all negative for
Dane particles by IBM. Refinement of the pseudoreplica
technique for IBM provided a highly sensitive and practical

system to detect Dane particles.

ACKNOWLEDGEMENTS

I am indebted to my major professor, Dr. Maria J.
Patterson, for the tremendous amount of effort, attention,
and encouragement she gave me while serving as my research
and academic advisor. I would also like to thank Drs. Karen
Baker and Leland Velicer for their contributions in serving
on my graduate committee. Special gratitude must also be
extended to Mr. Michael Verrilli for providing his expertise
in the preliminary stages of this project.

Additional appreciation goes to Mr. H. Stuart Pankratz,
Tom Kelly, Drs. Ron Patterson, Betty Hamilton, and Paul
Engelkirk for technical assistance, and to everyone in Dr.
Velicer's and Dr. Moon's laboratories for allowing me the

use of their equipment.

PART 1.

TABLE OF CONTENTS

LITERATURE REVIEWOOOOOOOOOOOOOOOO0.0.0....

Historical Introduction of IEM...........

Current Methods. ... ......................

The Classical Technique.................

Agar Techniques.........................
The Agar-diffusion Filtration
Method...............................

The Serum-in-Agar Method.............

Methods for Agar Gel Precipitin Lines..

The Pseudoreplica Technique..........
The Derrick Technique...................
The Modified Derrick System..........
The Derrick Technique Combined with
Decoration...........................
Hepatitis B.... ............ . .......... ..
Viral Characteristics................

Humoral Immune Response to HBV

Infection. .............. .. ........ .....

Transmissj-on.........OOOOOOOOOOOOOOO...

Hepatitis in Renal Dialysis Units......

List of References........................

iii

Page

10
11
12
14

15

15
18

20

22
24
26
28

Page
PART II. HBe ANTIGEN AND MORPHOLOGIC FORMS OF

HEPATITIS B VIRUS IN URINES FROM PATIENTS

WITH ENDSTAGE RENAL DISEASE................. 39
Introduction................................ 39
Materials and Methods....................... 40
Results........... ......... ................. 47
Discussion.................................. 57
List of References.. ..... ................... 59

PART III. ABSENCE OF DANE PARTICLES IN PATIENTS'
SERA LACKING PROTECTIVE ANTIBODY TO HBV.... 62

Introduction............................... 62
Materials and Methods...................... 64
Results. ............ . ...................... 66
Discussion................................. 67
List of References ....................... .. 70

PART IV. APPENDICES
Appendix 1

Hepatitis B markers in urine from persistent
HBsAg seropositive ESRD patients.

Appendix 2
Serial urines.
Appendix 3
HBV antigen-antibody system markers detected

by radioimmunoassay and urinalysis data in
urines examined by immunoelectronmicroscopy.

iv

Appendix 4

HBe Antigen-antibody System Markers Detected
by Radioimmunoassay in 33 Sera (positive for
anti-HBC, negative for HBsAg and anti-HBs)
Examined by Immunoelectronmicroscopy.

LIST OF TABLES

Page
Table 1 Comparison of immunoelectronmicroscopic
techniques to examine HBsAg-positive
urines..................................... 50

vi

Figure 1A

Figure 18

Figure 1C

Figure 1D

Figure 2

LIST OF FIGURES

Typical negatively stained single anti-

body-coated hepatitis B virion and HBsAg
particles observed following incubation

of highly diluted serum with HBIG.

Negatively stained Dane particles in
aggregate.

Negatively stained Dane particles in
complex with spherical and tubular morpho-
logical forms of HBsAg.

Negatively stained Dane particles in
complex with spherical and tubular morpho-
logical forms of HBsAg.

Pseudoreplica Technique.

vii

Page

51

52

53

54
56

REVIEW OF THE LITERATURE

Historical Introduction of IBM

Techniques which detect, by electron microscopy, the
specific binding of antibody to antigen are generally re-
fered to as immune electron microscopy or immuno-electron-
microscopy (IEM) (5,63). Currently, many procedures have
been established and depending upon the nature of materials
under investigation, variations of these can be employed
(87). This review focuses on the development of methods
of IBM for detection of viral antigens, particularly
those found in clinical specimens.

Anderson and Stanley (8) in 1941, were the first to
use IBM to visualize the interaction between tobacco mosaic
virus (TMV) and its specific antibody. In an attempt to
observe immunological reactions directly, viral particles
were examined following treatment with specific rabbit
antiserum, normal serum and no serum added. Representative
micrographs demonstrated morphological differences between
the viruses incubated with specific rabbit antiserum, and
those treated with normal or no serum. Viruses which were
coated with specific antibody appeared wider and more dense
with a fuzzy, indistinct outer boundary. In contrast,
mixtures of TMV with antisera to tomato bushy stunt, potato
latent mosaic or tobacco ring spot viruses resulted in little

or no adsorption of molecules onto the virus particles.

2
Furthermore, prolonged reaction time resulted in immune
complex formation readily observed in the electron micro-
scope.

In a review of IBM, Almeida and Waterson (5) pointed
out two significant improvements to the original IEM scheme,
shadow-casting and negative staining, as well as some limi-
tations. In early transmission electron microscopy, image
contrasting was a result of density differences between the
supporting film and the virus particles, and definitive
studies were difficult. With the advent and refinement of
the shadow-casting technique (40, 52, 66, 116), three dimen-
sional studies became possible. In this technique, the
specimen was dried onto the grid, which was then placed
under vacuum and metal evaporated onto the surface. Major
shortcomings associated with shadow-casting included poor
resolution, due to limitations imposed by granular size of
the metal used, and structural damage to the specimen
resulting from the trauma of pressure changes.

The introduction of negative contrasting by Brenner and
Horne (21) in 1959, allowed much greater resolution and
visualization of fine structure. Viral particles were
embedded in an electron dense heavy metal, usually phospho-
tungstic acid (PTA), and contrast was thus provided by
negative staining in a fashion similar to bacterial negative
staining. In initial studies involving tobacco mosaic virus
and turnip yellow mosaic virus, equal volumes of virus
suspensions and 2.0% solutions of PTA, pH 7.4, were poured

into an atomizer and directly sprayed onto carbon coated

3
grids. This approach yielded high contrast and excellent
preservation of external ultrastructure. In addition,
any cavities accessible to the phosphotungstate were also
stained, thus revealing some internal structure.

As part of an interpretive study on structural detail
of polyoma viruses, Almeida and co-workers (6), demonstrated
that variations in particle diameter were more likely to
occur in shadowed preparations than in those prepared in
PTA. This phenomenon was a manifestation of flattening of
the virus. It appeared that in negative staining, the
particles were enveloped and supported by a protein-phos-
photungstate complex that helped to retain the original size
and shape of the viral structure or complex under investi-
gation. The implications, therefore, as to the usefulness
of such a technique in transmission electron microscopy were
substantial.

The early work with negative contrast staining provided
a means to reconfirm information about morphological ar-
rangement and orientation of TMV and turnip yellow mosaic
virus. Subsequently, Lafferty and Oertelis (70) and
Anderson and associates (9), in 1961, adopted this method of
negative staining for the sole purpose of visualizing and
studying the structure of immune complexes. Suspensions of
viruses and antisera were mixed with phosphotugstate and
drops were mounted on carbon-formvar-coated grids. Both
groups reported that at high magnification, aggregated

particles with thin fibers forming connective virion-virion

4

bridges were observed. Individual particles, not within
complexes, appeared to have fibers forming loops upon their
surfaces. Lafferty and Oertelis, in an investigation into
the mechanism of the stabilization reaction during the
combining of virus and antibody, observed two-site at-
tachment between rabbit antibody molecules and influenza
virus. Anderson and collegues, demonstrated the specificity
of agglutination of bacteriophages P22 and P221, and polio
virus, with their respective homologous antisera.

Immunoelectromicroscopy has undergone considerable
evolution since the establishment and incorporation of
negative staining. Of particular medical importance, was the
use of IBM in the elucidation of fine structure of viruses
of clinical significance. Some of the viruses so visualized
include: polyoma, human papilloma, and other papovaviruses
(2,96), influenza virus (71), poliovirus type 1 (57),
rubella virus (15), hepatitis A (43,44) and B (30) viruses,
herpes simplex (114), varicella and herpes zoster (41)
viruses, rhinoviruses (58), coronaviruses (59), Norwalk
agent (60), rotavirus (16,46), adenovirus type 3 (91), and
smallpox virus (111). For some of these viruses, laboratory
isolation has not been achieved and demonstration by IBM

remains an integral part of the definitive identification.

Current Methods

For routine clinical use, virus antibody reactions are
not generally visualized with embedded, thin sectioned
materials. Rather, virus remains suspended in its original
fluid or is resuspended for processing. As few as 106
virus particles are sufficient to yield suitable specimens,
making IEM one of the most sensitive methods of detecting
antigen-antibody complexes (5). Many methods reported in the
literature have the advantages of being rapid, easy and
adaptable to a variety of specimen types. Among these are

the classical or direct technique, agar techniques, the

Derrick technique and its modifications, and decoration.

The Classical Technique

 

The development of the classical or direct method has
been primarily attributed to Anderson and co-workers (9) and
Lafferty and Oertelis (70), as a result of some of the
preliminary work of Anderson and Stanley (8) and Williams
and Wyckoff (116).

Basically, antiserum or purified antibody is incubated
with the virus, the suspended immune complexes centrifuged,
and the pellet resuspended in distilled water and mixed with
PTA (neutral). The preparation is then applied to a grid
that has previously been coated with a support film (usually
Parlodion, Formvar, and/or vacuum evaporated carbon), excess

fluid is removed by filter paper, and the grid is air dried.

6
For a particular virus or specimen type, it may be necessary
to modify one or more steps in the technique. Almeida and
Waterson (5) found that 0.1 ml of antiserum, undiluted and

l and 10-2, provided suitable proportions

at dilutions of 10’
to react with 0.2 m1 of a suspension of wart virus con-
taining 108 particles/ml, when mixed in 0.7 ml of physio-
logical saline. Ratios are dependent upon the particular
virus concentration and the antiserum titer. These de-
terminations are usually made by conventional methods (e.g.
RIA, CF, HI, etc.), prior to preparation for immune electron
microscopy.

Incubation times vary from 30 minutes to overnight, but
one hour at 37 C followed by 4 C overnight (5), has received
routine acceptance. Feinstone and coworkers (43), using a
one hour incubation at room temperature in studies on stool
filtrates, found this quite satisfactory in the detection of
hepatitis A virus.

The centrifugation step is another source of con-
siderable variation in the classical technique. Different
centrifugal forces, dependent upon the size of the virus,
are used to pellet the immune complex. Almeida and Goffe
(3) used 480 X g for 30 minutes to sediment wart virus

6

(nucleic acid molecular weight, 5 x 10 daltons) immune

complexes. For smaller viruses, for example picornaviruses
(nucleic acid M.W., 2-3 x 106 daltons), centrifugation at

27,000 X g has been recommended. Best (15) pelleted rubella

7
(nucleic acid M.W., 4 x 106 daltons) virus-antibody
complexes twice at 12,000 X g, and used phosphate buffered
saline for resuspension.

Some investigators have omitted pelleting entirely.
Norrby and associates (91) used dialysis on membrane filters
in an analysis of the specificity of, and interaction
between adenovirus type 3 and IgG antibodies against dif-
ferent viral antigenic moieties. In preliminary experiments
on serological crossreactivity among picornaviruses,
Chaudhary and fellow workers (29) noted a tendency of
centrifugation to cause clumping in control preparations.

To avoid this, a prolonged incubation in the cold (4 C) for
24-48 hours, following the initial 37 C incubation, was
substituted for the centrifugation step, and good complex
formation was obtained.

Specimens are mounted onto the grid in the classical
technique either directly in droplets or via a modification
of the method of Doane (39). A commonly employed method
follows: one drop of the immune reaction mixture is added
to deionized water on parafilm; a coated grid is quickly
touched to the surface, one drop of PTA, pH 7.0 is placed on
top; excess is removed by blotting with filter paper; the
grid is air dried. Alternative stains, other than 1-3 % PTA
at a pH of 6-7, include 24% sodium silicotungstic acid, pH 7
(91,109) or 0.5—l% uranyl acetate in methanol or water, pH
4.6 (10,74).

Milne and Luisoni (87) have described the advantages

and disadvantages of the classical technique in a recent

8
review of IBM. The favorable aspects included: 1) good
resolution of both virus and antibody, 2) virion trapping
in recognizable clumps allowing specific identification, in
circumstances where the concentration of virus is below
levels of detectability by conventional transmission
electron microscopy, and 3) increased sensitivity due to
the concentration and partial purification provided in the
pelleting step. Potentially unfavorable features included:
1) the additional time required for the incubation and
centrifugation steps, 2) non-specific clumping associated
with centrifugation, 3) unsuitability of the initial virus
suspension for immune complex formation, and 4) degradation
of the quality of the image due to co-pelleting of

impurities in the virus-antibody preparation.

Agar Techniques

 

The development of agar techniques followed from at-
tempts to eliminate two of the problems associated with the
classical technique: contamination and lengthy preparation
time. Agar filtration was first used by Kellenberger and
Arber (64) for particle counting by conventional electron
microscopy. The basic technique included the following
general steps. Solutions of Parlodion were poured over the
surface of dried agar plates. Agar, prepared in the same
medium as that used to suspend the viral particles under
investigation, was dried in an oven to remove added water.

This increased absorption capacity, while maintaining the

9
same medium concentration of the particle suspension. The
Parlodion film served as a filter through which the liquids
and salts diffused into the agar gel. After drops of the
preparation were spread over the surface and filtration was
completed, the film was fixed and floated off at the surface
of La(N03)3. Specimens were mounted onto grids by approach-
ing the floating membrane from below and securing it. By
addition of a step including the mixing of known quantities
of latex spheres with particles of unknown concentration and
calculating the resulting ratios from electron micrographs,
this method was suitable for particle counting.

The Agar-diffusion Filtration Method- Steere (107)

 

showed that an agar film acts as a molecular sieve absorbing
fluid together with interferring salts, macromolecules, and
tissue debris of :15 nm in diameter, while retaining the
larger particles on its surface. Kelen and associates (62,
63) developed this basic observation, as well as the ideas
of Kellenberger and Arber, for use in IBM. Agar surfaces
were employed to trap immune complexes and absorb impuri-
ties. In 1971, the agar-diffusion filtration method (ADF)
(62) was introduced for demonstrating the presence of the
hepatitis B surface antigen and its corresponding antibody
in serum specimens. Serial dilutions of specimens were
prepared in a range to include the predetermined complement
fixing surface antigen or antibody titer, depending upon the
assay, and equal volumes of the appropriate concentrations

of serum and "detector reagents" (either antigen or antibody)

10
were incubated in microtiter plates with U-shaped wells.
Thin drops were deposited on agar covered slides, and an
inverted coated grid was left floating on top. As the fluid
phase of the drop containing normal serum components dif-
fused into the agar layer, the drop gradually flattened
until the grid settled onto the surface of the agar, thus
trapping the antigen-antibody complexes between the two
surfaces. Grids were removed with forceps and negatively
stained as described above. In addition to simplicity and
speed of preparation (less than one hour), this method was
reported to have both high specificity and sensitivity equal
to or greater than that of complement-fixation (62).

The main advantage of the ADF technique over the
classical method (63) was that it incorporated partial
purification and concentration in one step. Additionally,
with the elimination of the blotting step of direct methods,
all of the virus particles contained in the original drop of
fluid became attached to the grid.

The Serum-Agar Method - The ADF method was modified by

 

Anderson and Doane (7) for rapid type-specific identifi-
cation of crude cell culture isolates of enteroviruses (Echo
6, 7, 9 and 11, Coxsackie A9, Bl, B4, and BS, and Polio l,
2, and 3). In this technique, agar was used as a reservoir
of antibody, and the formation of immune complexes was
achieved by rapid diffusion of homologous antibodies into
the viral specimen. The technique included the following

steps. Dilutions of antisera were added to a solution of

ll
cooled molten agar (1%). This was transferred into wells of
microtiter plates, allowed to solidify at room temperature,
and coated grids were placed on the surface. One to two
small drops of viral specimens were added, and the grids
were air dried for 30-60 minutes, and negatively stained.

In comparison to the classical technique, the serum-in—
agar technique had several practical advantages (38). It
was rapid and simple to perform, required only small amounts
of virus and antiserum, and the antibody-coated wells could
be stored at 4 C for several months prior to their use.
Also, since specific virus identification was not dependent
on optimal dilutions, this technique appeared especially
suitable for detection of viruses directly from clinical
specimens.

Methods for Agar Gel Precipitin Lines - Several methods

 

have been described in the literature to study the nature of
the components forming precipitin lines in the Ouchterlony
gel diffusion technique. Watson and co-workers (113), in an
attempt to examine polyoma virus-antibody precipitates by
electron microscopy, used an extraction method developed by
Weintraub, Rigetli, and Townsley (115). The entire length
of the precipitin line was cut from the agar plate and
suspended in a drop of distilled water in a test tube. The
gel was mixed into the fluid by stirring, followed by
immersion in an electrosonic bath for 30 seconds. Using a
platinum loop, a droplet of the suspension was placed on the

coated grid and the excess blotted. The grid was washed and

12
negatively stained. Examination of the extracted material
revealed aggregates of polyoma and antibody, that had a
similar appearance to those complexes formed in the direct
method. Beale and Mason (13) modified this technique by
freezing the fragments of agar containing the precipitin
lines corresponding to the D and C antigens of polio virus
type 1. The agar fragments were cut into small pieces,
thawed and negatively stained. By this method, it was
confirmed that the D and C lines were associated with com-
plete and empty virus particles, respectively. Other in-
vestigators fixed and embedded precipitin lines in studies
on mouse mammary tumor virus (17), bovine papilloma virus
(75), and the configuration of IgM antibodies attached to
polio virus type 1 (109). However, certain disadvantages
were recognized in this method also. Svehag and Bloth (109)
reported that impurities in the gel, together with the
thickness of the antibody layer, tended to obscure specimen
detail.

The Pseudoreplica Technique - In a study on the ultra-

 

structure of disrupted herpesvirus capsids, Palmer, Martin
and Gary (93) reintroduced the drop pseudoreplica technique,
which had been described earlier. Briefly, a drop of viral
material was allowed to air dry on a small block of 2.0%
agarose and then overlaid with 0.5%, liquid Formvar. After
excess fluid was drained, the resulting membrane, with

entrapped virus particles, was floated off at the surface of

13
a 2.0% solution of PTA, pH 5. Copper grids were placed on
the film which was retrieved using a metal peg, and excess
stain was removed with filter paper. Uranyl acetate, 0.5%,
was substituted for PTA by Nahmias (10) and Lee and as-
sociates (74), in studies directly examining clinical
specimens of body fluids for herpes viruses.

The major advantage of this method was its rapidity,
since processing required less than 30 minutes. In ad-
dition, viruses were concentrated approximately tenfold.
This technique was used for rapid detection of viruses
easily demonstrated by routine clinical methods, and also
facilitated detection and identification of virus groups not
easily demonstrable in body fluids by other methods.

Nahmias (10) visualized adenoviruses, rotaviruses, papova-
viruses, cytomegaloviruses, herpes zoster and herpes simplex
viruses. He also reported that specimen volumes of only 25
microliters were satisfactory when examining urine, stool
and fecal suspensions, oral secretions, tears, vesicular
fluids, serum, cerebrospinal, ventricular and other body
fluids.

Lee, Nahmias and Stagno (74) detected cytomegalovirus
(CMV) particles in urines and oral specimens, obtained from
symptomatic and asymptomatic infants who had congenitally or
postnatally acquired CMV infections. The virus were readily
detectable in specimens that had been stored or shipped at
4 C for several days, again supporting the value of pseudo-

replication as a rapid diagnostic tool.

14

The Derrick Technique

 

Derrick (35, 36, 37) described a method in which grids
coated with specific antiserum were used for electron micro-
scopy of plant viruses. Potato virus Y (PVY), tobacco
mosaic virus (TMV), cucumber mosaic and tobacco etch viruses
were specifically attached to, and concentrated on, specimen
grids coated with a film of specific antiserum. One of the
advantages of this technique (87) was that the grids could
be washed at two points, after antibody was adsorbed to the
grid and after virus was adsorbed to the antibody. These
washes helped to remove salts and other impurities, without
disturbing antibody or viral antigen.

Coated grids were floated for 30 minutes at 24 C on
antiserum diluted in 0.05 M Tris buffer, pH 7.2. Im-
mediately after a wash in the same buffer, the grids were
floated on 0.1 m1 drops of crude virus extracts for one hour
at 24 C. Grids were washed in the same buffer used to
prepare the viral extracts (Tris buffer containing 0.9%
NaCl), followed by a wash in water, and were air dried and
shadowed. Positive staining with 1.0% uranyl acetate could
be substituted for shadow-casting. In a quantitive assay,
Derrick (37) reported that approximately 50 times more TMV
and 20 times more PVY were trapped on grids coated with
their specific antisera, than on control grids which were
coated with unrelated antibody. Other refinements in the

same study showed that the number of entrapped virus

15
particles could lengthening the incubation time of the virus
step, or by raising the incubation temperature to 37 C.

The Modified Derrick System - Milne and Luisoni (87)

simplified the Derrick procedure and shortened incubation
times so that processing was completed in less than 25
minutes, rather than over 90 minutes. Phosphate buffer, 0.1
M, pH 7.0 was used instead of Tris buffer, and uranyl
acetate was replaced by PTA. Diluted serum was incubated on
a grid (held in forceps) in a humid box for 5 minutes at 24
C. The grid was washed with 20 consecutive drops of buffer
followed by 30 drops of distilled water, and stained.
Thirty to fifty times more plant viruses, from crude and
purified preparations, were trapped on grids coated with
homologous antiserum, than on those coated with unrelated
antiserum.

The Derrick Technique Combined with Decoration - A1-

 

though the terminology was introduced by Milne and Luisoni,
the technique of decoration was first described by Yanagida
and Ahmad-Zadeh (118) in a study to locate bacteriophage
structural proteins. Virus was adsorbed onto coated grids,
which were washed and floated (specimen side inverted) on
diluted antiserum. Antigenic sites were thus coated or
"haloed" by antibody molecules (decoration). Clumping was
prevented since the virus particles were fixed in place and
not free to move. This method has also been used to visu-
alize specific antigenic sites on viral capsids (110, 117,

118).

16

To confirm a specific immune reaction between plant
viruses and their respective homologous antisera, Milne and
Luisoni decorated each particle first trapped by the Derrick
system (87). The procedure required about 35 minutes and
trapped 50 times more virus particles than the control
(coated with normal serum). A grid was incubated with di-
luted antiserum for 5 minutes, washed with 20 drops of
phosphate buffer, incubated with the virus for 15 minutes
and washed again with 20 drops of phosphate buffer. Another
drop of diluted antiserum was added and the grid was incu-
bated 15 minutes longer. This was followed by the routine
wash of buffer and distilled water, as previously described,
and the grid was stained for examination.

This method had the shared advantages of both systems.
Decoration was useful in high resolution work, in which
antibody attachment sites must be clearly visible. It was
also an attractive method in those situations in which
samples contained components which interfered with immune
reactions. Modified Derrick methods could be used on crude
materials and were useful in trapping viruses present in low
concentrations (87).

The use of many electron microscopic techniques as
research and diagnostic tools, is becoming increasingly more
practical. The transmission electron microscope has con-
tributed to many areas of clinical medicine such as hema-
tology, nephrology, gastroenterology, and the study of

metabolic disease, as an aid in diagnostic procedures, a

l7

guide to treatment, and the characterization of pathogenic
mechanisms. More specifically, viral diseases can be diag-
nosed in minutes, under ideal conditions, from the time the
sample is taken until identification under the electron
microscope (24). In those circumstances in which in vitro
cultivation of some viruses has not been possible, IBM has
proved to be invaluable. One such elucive virus is he-

patitis B virus.

Hepatitis B

Despite intensive efforts to characterize the agents
causing different forms of hepatitis, the understanding that
hepatitis B was a separate entity did not become clear until
the 1930's. At that time the disease was referred to as
catarrhal or homologous serum jaundice, and was recognized
as a complication of injections and serotherapy (45, 101).
Early epidemiological studies suggested its transmission was
associated with the inoculation of blood products from one
individual to another, but little importance was attached to
observations of transfer by physical contact from servicemen
(88) and experimentally infected volunteers (20) to their
spouses. Following the transmission experiments at the
Willowbrook State School for the mentally retarded (69), the
concept of transmission by modes other than serum took on
new significance. Krugman, Giles and Hammond, in 1967,
reported evidence that two clinically, epidemiologically and
immunologically distinct types of infection (subsequently
designated hepatitis A and B) were occurring in the insti-
tution. Plasma (designated MS-l) from a patient suffering
from a first attack of hepatitis, caused disease after an
incubation period of approximately 30 days in experimental
groups. Other experimental groups received plasma (MS-2)
from the same patient during a second attack of hepatitis,

and developed hepatitis following a much longer incubation

18

19
time of about 90 days. The investigators suggested that
MS—l was infectious or short-incubation hepatitis
(hepatitis A) and MS-2 was serum or long-incubation
hepatitis (hepatitis B). Subsequent studies at Willowbrook
defined other properties of these two agents, one of the
most significant being that hepatitis B could be transmitted
by oral ingestion of serum (69). In 1968, Prince (99)
recognized a relationship between the Australia antigen, a
lipoprotein discovered by Blumberg, Alter and Visnich (18)
in 1965, and the viremic phase of hepatitis B. The antigen,
later renamed Hepatitis B surface antigen (HBsAg), received
its first name following its detection in the sera of
Australian aborigines, during a study of varients in serum
proteins. Prior to Prince's studies, the association of
Australia antigen with hepatitis B had been suggested by
Blumberg and co—workers, but this was not fully established
(19). Giles (51) in 1969, tested sera from patients in the
Willowbrook studies for HBsAg and showed the complete cor-
relation between the presence of the antigen and MS-2
infection.

HBsAg has been used as a marker of hepatitis B in-
fection in many clinical and epidemiological studies. These
studies have provided a more complete understanding of the
nature of the virus, in spite of the fact that all efforts
to cultivate it in vitro have been unsuccessful. In 1970,
Dane, Cameron and Briggs (30) identified the hepatitis B

virion (subsequently called the Dane particle) by immune

20
electron microscopy of HBsAg positive serum. The following
year Almeida, Rubenstein and Scott (4) described the
nucleocapsid core or hepatitis B core antigen (HBcAg), also
by using IBM. Experiments to develop animal models by
Maynard and coworkers (84), showed that chimpanzees were
susceptible to infection, although clinical effects are

minimal.

Viral Characteristics — Three morphological forms are as-

 

sociated with the hepatitis B virus (HBV): 1) the Dane
particle, 2) the spherical form of HBsAg, and 3) filamentous
or tubular forms of HBsAg. The complete virion, or Dane
particle, exists as a complex double layered structure
measuring 42 nm in diameter (30). It has a 27 nm core
containing circular, double stranded DNA with a molecular

weight of 1.6 x 106

daltons, and DNA polymerase. Single
stranded regions may be present on the DNA molecule (72).
Studies suggest the core has icosahedral symmetry, subunit
capsomeres of variable size (appx. 4 nm) and 8-10 nm long
spikelike structures projecting from the surface (23). The
existence of subpopulations of Dane particles has been
demonstrated (61) in cesium chloride gradients. Full
core particles containing DNA have buoyant densities of 1.36
g/ml, and empty cores, densities of 1.30 g/ml.

A 7 nm envelope surrounds the core, which is

antigenically distinct from antigens on the 27 nm core

(HBcAg). This material, the hepatitis B surface antigen

21
(HBsAg), is produced in excess by infected hepatocytes and
is released into the blood as spherical particles and as
tubular or tadpole-like filaments. Both forms have diameters
between 17 and 25 nm (averaging 22 nm), but the tubular
forms exhibit a variable length from 100-700 nm. It has been
suggested that the tubular forms are stacked discs,
which dissemble into the spherical particles under appropri-
ate conditions (54).

The constitution of HBsAg has been studied extensively
(67). Estimates of its molecular weight range between 2.4 to
4.6 x 106 daltons (27, 65). It has a density of 1.21 g/ml
in CsCl (80), and has the chemical characteristics of lipo-
protein. Approximately 20-30% is lipid (cholesterol, phos-
phatidylcholine, sphingomyelin, and lysophosphatidylcholine
are predominant) (108), and 5% is carbohydrate (28). A
protein content of 40-60% has been suggested (56), with 14%
tryptophan (102) and large amounts of proline and leucine.

HBsAg is antigenically complex and its immunoreactivity
is not fully established (67). It appears to carry a
common or group-specific determinant designated "a" (76) on
its surface, and at least two other subdeterminants "d" or
"y" (73) and "wl", "w2", "w3", "w4", or "r" (12). Eight
subdeterminant categories or subtypes are recognized, aywl,
ayw2, ayw3, ayw4, adr, ayr, adw2, adw4, as well as two mixed
subtypes adyw and adyr (67). There appears to be no cor—

relation between subtype and severity of illness (50) and

22
the distribution is probably related to geographic factors
(86), and thus is of epidemiological importance.

Antibodies against HBcAg and HBsAg, are designated
anti-HBc and anti-HBs, respectively. A third antigen,
immunologically and chemically distinct from HBcAg and
HBsAg, was identified by Magnius and Espmark in 1972 (80,
81). Hepatitis B e antigen (HBeAg) appears to be of viral
origin, and closely associated with HBV DNA polymerase and
circulating Dane particles in serum (53). It exists in
serum as a complex of three molecular species of soluble
proteins varying in size. One component has been charac-
terized and has a buoyant density of 1.29 g/ml in CsCl (79),
and an S-value of 11.6 (89). In urine, HBeAg is a heat
labile soluble protein with a buoyant density of 1.34 g/ml,
S-value of 4 and molecular weight of approximately 40,000

daltons (89).

Humoral Immune Response to HBV Infection - The appearance

 

and disappearance of HBc-, HBs-, and HBe-antigens and the
appearance of their respective antibodies, generally follow
a characteristic pattern, during the course of and conva-
lescence from most HBV infections (77). The first sero-
marker to appear is HBsAg, followed by HBeAg, and both reach
maximum titer at approximately two to three months post-
infection. Anti-HBe is the first antibody produced by the
host. It appears about three months after infection,

usually during the early acute phase of illness. It may

23
persist for several years, and is considered to be one of
the best markers of current or past infection. Disap-
pearance of the IgM class of anti-HBc probably indicates HBV
clearance, while persistence may signal continuing viral
replication and chronic disease (33). Seroconversion to
anti-HBe follows the appearance of HBeAg and precedes the
disappearance of HBsAg. Eventually, anti-HBs, the protective
antibody, is elicited and the host convalesces. Anti-HBs is
also long lasting, persisting similarly to anti-HBc, and
confers immunity to future infections.

There is an "immunologic window period" between the
disappearance of detectable HBsAg and appearance of anti-HES
in which anti-HBC and anti-HBe, or anti-HBc only, may be
detectable. Studies (55) have shown that the patient's blood
may be infectious during this period.

The antigen-antibody system markers have been related
to the status or severity of infection. HBsAg seroposi-
tivity indicates current infection and presence of HBeAg
suggests severe liver damage, high infectivity and a rela-
tively poor prognosis. The presence of Dane particles or
HBcAg are also an indication of continuing viral replication
and infectivity. Testing for these markers however, is not
routine, due to limitation in the sensitivity of detection
methods (33). Failure to seroconvert to anti-HBe during the
carrier state implies on-going active infection and chronic
liver disease is a probable outcome (90). The presence of

anti-HBe may indicate a reduced level of infectious virions

24
because of decreased viral replication. In the acute stage,
the presence of anti—HBe indicates the beginning of conva-
lescence, and in the chronic HBsAg carrier, its appearance
is often associated with improvement in the patient's

underlying liver disease (33).

Transmission - Human chronic carriers are the main reser-

 

voir of hepatitis B virus. Plasma, serum and their products
serve as the principle source of infection; however, it is
clear that transmission is accomplished by routes other than
direct inoculation. As reviewed by Maynard (83), several
mechanisms are currently recognized.

Direct percutaneous parenteral inoculation of con-
taminated serum, plasma or transfusion of infective blood or
blood products remains the major mode of transfer. Included
also are other procedures involving use of needles, such as
tattooing, acupuncture, ear-piercing, indiscriminate drug
use, improper decontamination of syringes and needles during
mass immunization, and accidental needle punctures among
health care employees. Second in importance as a route of
transmission, is non-needle percutaneous transfer of in-
fective serum or plasma through minute breaks in the skin.
Larger skin lesions from impetigo, burns, or scratches serve
as entry sites as well.

Mucosal surfaces, especially oral and conjunctival
mucosa, appear to provide the third portal of entry for HBV

from infective serum or plasma. Serum/blood-oral and

25
serum/blood-eye mechanisms are implicated in certain oc-
cupational groups. At high risk are hospital personnel,
laboratory technicians and dentists.

The fourth mechanism, the introduction of infectious
body secretions onto mucosal surfaces, or through breaks in
skin, is a more recently recognized route of infection.
Secretions have been studied from patients with acute
disease and chronic carriers. HBsAg has been detected in
feces (112), urine (11, 112), saliva (112), nasopharyngeal
secretions (92), menstrual blood (85), vaginal secretions
(32), bile (97), breast milk (78), cerebrospinal fluid (34),
sweat (22), and synovial fluid (98). Recently, HBsAg
positive saliva and semen have been shown to cause infection
by both of the parenteral transmission routes in experi-
mental studies in gibbons (104). This substantiates
epidemiological data implicating sexual activity with
transmission and high rates of hepatitis B among prostitutes
(94), homosexuals (48), and spouses of partners with the
disease (103).

Indirect transfer of infective serum or plasma via
inanimate objects or arthropods, has been suggested as the
fifth specific mode of transmission. HBsAg has been de-
tected in mosquitoes in Africa (100), however attempts
experimentally to transfer the disease from mosquitoes to

chimpanzees have been unsuccessful (14).

26
Airborne transmission may be a possible but compara-
tively insignificant mechanism of transfer. Similarly oral

transmission via saliva is probably infrequent (104).

Hepatitis in renal dialysis units - Outbreaks of hepatitis B

 

in hemodialysis units were recognized in the 1960's as a
major hazard affecting both patients and staff (82).

Because dialysis patients may receive a renal transplant,
and move back and forth between the dialysis and transplant
populations the disease has also been associated with trans-
plantation units (47).

Studies (49, 82) have shown that the virus is usually
introduced into dialysis units through blood transfusions.
Efforts to prevent this entry have included restricted blood
transfusion (26) and the substitution of reconstituted
frozen red cells for whole blood, when transfusion of
patients or priming of dialysis machines is necessary (25).
The virus may also enter the hemodialysis unit via new
patients or staff members who are infectious, or by the
infection of patients or staff in other parts of the
hospital followed by transmission into the unit, although
these are less common mechanisms (68).

Evidence suggests that there are multiple routes of
infection from infectious carriers and acutely ill viremic
patients to fellow patients or staff, once the virus has

been introduced into the unit (68). Strongly implicated are

‘J‘:O.l_.; (1.:

27
malfunctioning dialysis machines (e.g. rupture or leaks in
dialysis membranes) (105), accidental needle penetration
among the staff, and transmission of blood through breaks in
the skin (106). Other investigators have speculated that
contaminated surfaces in the unit or airborne transmission
may play some role (1, 31, 42). Household contacts, es-
pecially spouses, are also at risk of developing a hepatitis
B infection (95).

As previously mentioned, HBsAg has been detected with
some frequency, in urine of HBsAg seropositive patients.
Recent development of a very sensitive solid phase radio-
immunoassay (RIA) for HBeAg has made possible the detection
of HBeAg in urine of HBsAg seropositive endstage renal
disease (ESRD) patients (89). One purpose of this study,
was to assess the infectivity of ESRD patient urines by

detection of Dane particles by immunoelectronmicroscopy.

LIST OF REFERENCES

1.

2.

3.

4.

5.

6.

7.

8.

9.

LI ST OF REFERENCES

Almeida, J.D., G.D. Chisholm, A.B. Kulatilake, A.B.
MacGregor, D.H. MacKay, B.P.N. O'Donoghue, R.
Shackman, and A.P. Waterson. 1971. Possible air-
borne spread of serum-hepatitis virus within a
heamodialysis unit. Lancet 2: 849-850.

Almeida, J., B. Cinader, and A. Howatson. 1963. The
structure of antigen-antibody complexes. J. Exptl.
Med. 118: 327-340.

Almeida, J.D., and A.P. Goffe. 1965. Antibody to wart
virus in human sera demonstrated by electron micros-
copy and precipitin tests. Lancet 1: 1205-1207.

Almeida, J., D. Rubenstein, and B.J. Stott. 1971. New
antigen-antibody system in Australia-antigen-posi-
tive hepatitis. Lancet 2: 1225-1227.

Almeida, J.D., and A.P. Waterson. 1969. The morphology
of virus-antibody interaction. Adv. Virus Res.
15: 307-338.

Almeida, J.D., A.P. Waterson, and E.W.L. Fletcher. 1966.
Interpretation of structural detail in viruses of

the polyoma group. Progr. Exp. Tumor Res.
8: 95-124.

Anderson, N., and F.W. Doane. 1973. Specific identifi-
cation of enteroviruses by immuno-electron micros-
copy using a serum-in-agar diffusion method. Can.
J. Microbiol. 19: 585-589.

Anderson, T.F., and W.M. Stanley. 1941. A study by
means of the electron microscope of the reaction

between tobacco mosaic virus and its antiserum.
J. Biol. Chem. 139: 339-344.

Anderson, T.F., N. Yamamoto, and K. Hummeler. 1961.
Specific agglutination of phages and polio virus
by antibody molecules as seen in the electron
microscope. Abstract. J. Appl. Phys. 32: 1639.

28

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

29

Annexton, M. 1978. New microscope techniques take
the stage. J. Am. Med. Assoc. 240: 2233.

Apostolov, K., D.J. Bauer, J.W.T. Selway, R.A. Fox,
F.J. Dudley, and S. Sherlock. 1971. Australia
antigen in urine. Lancet 1: 1274-1275.

Bancroft, W.H., F.K. Mundon, and P.K. Russell. 1972.
Detection of additional antigenic determinants
of hepatitis B antigen. J. Immunol. 109: 842-848.

Beale, A.J., and P.J. Mason . 1968. Electron micros-
copy of poliovirus antigen antibody precipitates
extracted from gel diffusion plates. J. Gen.
Virol. 2: 203-204.

Berquist, K.R., J.E. Maynard, D.B. Francy, M.J. Sheller,
and C.A. Schable. 1976. Experimental studies
on the transmission of hepatitis B by mosquitoes.
Am. J. Trop. Med. Hyg. 25: 237-732.

Best, J.M., J.E. Banatvala, J.D. Almeida, and A.P.
Waterson. 1967. Experimental studies on the
transmission of hepatitis B by mosquitoes.
Am. J. Trop. Med. Hyg. 25: 730-732.

Bishop, R.F., G.P. Davidson, I.H. Holmes, and B.J.
Ruck. 1973. Virus particles in epithelial
cells of duodenal mucosa from children with
acute non-bacterial gastroenteritis. Lancet
2: 1281-1283.

Blair, P.B., D.W. Weiss, and D.R. Pitelka. 1966.
Immunology of the mouse mammary tumor virus.
Correlation of the immunodiffusion precipitate
line with type-B virus particles. J. Nat. Cancer
Inst. 37: 261-277.

Blumberg, B.S., H.J. Alter, and S. Visnich. 1965.
A "new" antigen in leukemia sera. J. Am. Med.
Assoc. 191: 541-545.

Blumberg, B.S., B.J.S. Gerstley, D.A. Hungerford,
W.T. London, and A.I. Sutnick. 1967. A serum
antigen (Australia antigen) in Down's Syndrome,
leukemia, and hepatitis. Ann. Intern. Med.

66: 924-931.

Bradley, W.H. 1946. Homologous serum jaundice. Proc.
R. Soc. Med. 39: 649-654.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

30

Brenner, S., and R.W. Horne. 1959. A negative staining
method for high resolution electron microscopy
of viruses. Biochim. Biophys. Acta 34: 103-110.

Brodersen, M., and M. Wirth. 1976. Detection of HBsAg
and HBsAb in sweat. Acta Hepato-Gastroenterol.
23: 194-201.

Budkowska, A. 1977. Immunochemical and morphological
studies of hepatitis B core antigen from the nuclei
of hepatocytes. J. Infect. Dis. 135: 463-467.

Burns, W.A., H.J. Zimmerman, J. Hammond, A. Howatson,
A. Katz, and J. White. 1975. The clinician's
view of diagnostic electron microscopy. Human
Path. 6: 467-478.

Carr, J.B., A.M. deQuesada, and D.L. Shires. 1973.
Decreased incidence of transfusion hepatitis
after exclusive transfusion with reconstituted
frozen erythrocytes. Studies in a dialysis
unit. Ann. Int. Med. 78: 693-695.

Center for Disease Control. 1971. Hepatitis Sur-
veillance Report. 33: 6-17.

Chairez, R., F. B. Hollinger, J. P. Brunschurg, and
G. B. Dreesman. 1975. Comparative biophysical
studies of hepatits B antigen, subtypes adw and
ayw. J. Virol. 15: 182-190.

Chairez, R., S. Steiner, J.L. Melnick, and G.R.
Dreesman. 1973. Glycoproteins associated
with hepatitis B antigen. Intervirol. 1: 224-228.

Chaudhary, R.K., D.A. Kennedy, and J.C.N. Westwood.
1971. Serological cross-reactivity within the
picornaviruses as studied by electron microscopy.
Can J. Microbiol. 17: 477-480.

Dane, D.S., C.H. Cameron, M. Briggs. 1970. Virus-
1ike particles in serum of patients with Australia-
antigen-associated hepatitis. Lancet. 1: 695-698.

Dankert, J., J. Uitentuis, B. Houwen, A.M. Tegzess,
and G.R. van der Hem. 1976. Hepatitis B surface
antigen in environmental samples from hemodialysis
units. J. Infect. Dis. 134: 123-127.

Darani, M., and M. Gerber. 1974. Hepatitis B antigen
in vaginal secretions. Lancet 2: 1008.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

31

Deinhardt, F. 1980. Predictive value of markers of
hepatitis virus infection. J. Infect. Dis.
141: 299-305.

Denes, A.B., J.W. Ebert, R.E. Moncada, K.R. Berquist,
E.H. Cook, Jr., and J.E. Maynard. 1975. Hepatitis-
B surface antigen in cerebrospinal fluid. Lancet
2: 1038.

Derrick, K.S. 1972. Immuno-specific grids for electron
microscopy of plant viruses. Abstract. Phyto-
pathology 62: 753-754.

Derrick, K.S. 1973. Detection and identification of
plant viruses by serologically specific electron
microscopy. Abstract. Phytopathology 63: 441.

Derrick, K.S. 1973. Quantitative assay for plant
viruses using serologically specific electron
microscopy. Virology 56: 652-653.

Doane, F.W. 1974. Identification of viruses by immuno-
electron microscopy, p. 237-255. In E. Kurstak
and R. Morisset (ed.) Viral Immunodiagnosis.
Academic Press, Inc. New York.

Doane, M.A., N. Anderson, A. Zbitnew, and A.J. Rhodes.
1969. Application of electron microscopy to the
diagnosis of virus infections. Canad. Med. Ass.
J. 100: 1043-1049.

Easty, G.C., and E.H. Mercer. 1958. Electron micros-
copic studies of the antigen-antibody complex.
Immunol. 1: 353-364.

Evans, A.S., and J.L. Melnick. 1949. Electron
microscopic studies of the vesicle and spinal
fluids from a case of herpes zoster. Proc. Soc.
Exptl. Biol. Med. 283-286.

Favero, M.S., J.E. Maynard, N.J. Petersen, K.M. Boyer,
W.W. Bond, and K.R. Berquist. 1973. Hepatitis-
B antigen on environmental surfaces. Lancet
2: 1455.

Feinstone, S.M., A.Z. Kapikian, and R.H. Purcell.
1973. Hepatitis A: Detection by immune electron
microscopy of a virus-like antigen associated with
acute illness. Science 182: 1026-1028.

Feinstone, S.M., A.Z. Kapikian, and R.H. Purcell.
1974. Buoyant density of the hepatitis A virus-
like particle in cesium chloride. J. Virol.

13: 1412-1414.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

32

Findlay, G.M., J.L. Dunlop, and H.C. Brown. 1937.
Observations on epidermic catarrhal jaundice.
Trans. R. Soc. Trop. Med. Hyg. 25: 7-28.

Flewett, T.H., A.S. Bryden, and H. Davies. 1973.
Virus particles in gastroenteritis. Lancet
2: 1497.

Franksson, C. 1969. Hepatitis and liver damage among
patients and staff in a transplantation unit.
Transplant. Proc. 1: 209-212.

Fulford, K.W.M., D.S. Dane, R.D. Catteral, R. Woof,
and J.V. Denning. 1973. Australia antigen and
antibody among patients attending a clinic for
sexually-transmitted diseases. Lancet 1: 1470-
1473.

Garibaldi, R.A., J.N. Forrest, J.A. Bryan, B.F. Hanson,
and W.E. Dismukes. 1973. Hemodialysis-associated
hepatitis. J. Am. Med. Assoc. 225: 384-389.

Gerety, R.J., J.H. Hoofnagle, D.F. Nortman, and L.F.
Barker. 1975. Hepatitis B surface antigen
(HBsAg) subtypes and indices of clinical disease.
Gastroenterology 68: 1253-1260.

Giles, J.P., R.W. McCollum, L.W. Berndtson, and S.
Krugman. 1969. Relation of Australia/SH antigen
to the Willowbrook MS-2 strain. N. Engl. J. Med.
281: 119-122.

Hall, C.B., A. Nisonoff, and H.S. Slayter. 1959.
Electron microscopic observations of rabbit anti-
bodies. J. Biophysic. and Biochem. Cytol.

6: 407-411.

Hess, G., W. Arnold, and K.H. Myer. 1980. Comparison
of indicators for Dane particles. Klin.
Wochenschr. 58:371-376.

Hirschman, 8.2., J. Schwartz, S. Vernace, F. Schaffner,
and C. Ganz. 1973. An electron microscopic study
of the structural polymorphism of hepatitis B
antigen. J. Infect. Dis. 128: 605-617.

Hoofnagle, J.H., R.J. Gerety, L.Y.N.I., and L.F. Barker.
1974. Antibody to hepatitis B core antigen.
A sensitive indicator of hepatitis B virus repli-
cation. N. Engl. J. Med. 290: 1336-1340.

Howard, C.R., and C.J. Burrell. 1976. Structure and
nature of hepatitis B antigen. Prog. Med. Virol.
22: 36-103.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

33

Hummeler, K., T.F. Anderson, and R.A. Brown. 1962.
Identification of poliovirus particles of different
antigenicity by specific agglutination as seen
in the electron microscope. Virology 16: 84-90.

Kapikian, A.Z., J.D. Almeida, and E.J. Stott. 1972.
Immune electron microscopy of rhinoviruses.
J. Virol. 10: 142-146.

Kapikian, A.Z., H.D. James, Jr., S.J. Kelly, and A.L.
Vaughn. 1973. Detection of coronavirus strain

692 by immune electron microscopy. Infect. Immun.
7: 111-116.

Kapikian, A.Z., R.G. Wyatt, R. Dolin, T.S. Thornhill,
A.R. Kalica, and R.M. Chanock. 1972. Visualiza-
tion by immune electron microscopy of a 27-nm
particle associated with acute infectious gastro-
enteritis. J. Virol. 10: 1075-1081.

Kaplan, P.M., E.C. Ford, R.H. Purcell, and J.L. Gerin.
1976. Demonstration of subpopulations of Dane
particles. J. Virol. 17: 885-893.

Kelen, A.B., A.B. Hathaway, and D.A. McLeod. 1971.
Rapid detection of Australia/SH antigen and anti-
body by a simple technique of immunoelectron-
microscopy. Can. J. Microbiol. 17:993-1000.

Kelen, A.B., and D.A. McLeod. 1974. Differentiation
of myxoviruses by electronmicroscopy and immuno-
electronmicroscopy, p. 257-275. In E. Kurstak
and R. Morisset (ed.), Viral Immunodiagnosis.
Academic Press, Inc. New York.

Kellenberger, E., and W. Arber. 1957. Electron micros-
copical studies of phage multiplication. 1. A
method for quantitative analysis of particle sus-
pensions. Virology 3: 245-255.

Kim, C.Y., and J.G. Tilles. 1973. Purification and
biophysical characterization of hepatitis B
antigen. J. Clin. Invest. 52: 1176-1186.

Kleczkowski, A. 1961. Serological behaviour of tobacco
mosaic virus and of its protein fragments.
Immunology 4: 130-141.

Koff, R.S. 1978. Viral Hepatitis, p. 12-34.
John Wiley & Sons, Inc., New York.

Koff, R.S. 1978. Viral Hepatitis, p. 119-123.
John Wiley & Sons, Inc., New York.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

34

Krugman, S., J.P. Giles, and J. Hammond. 1967.
Infectious hepatitis. Evidence for two distinctive
clinical, epidemiological, and immunolocial types
of infection. J. Am. Med. Assoc. 200:365-373.

Lafferty, K.J., and S.J. Oertelis. 1961. Attachment
of antibody to influenza virus. Nature 192:
764-765.

Lafferty, K.J., and S.J. Oertelis. 1963. The inter-
action between virus and antibody. III. Examination
of virus-antibody complexes with the electron
microscope. Virology 21: 91-99.

Landers, T.A., H.B. Greenberg, and W.S. Robinson. 1977.
Structure of hepatitis B Dane particle DNA and
nature of the endogenous DNA polymerase reaction.
J. Virol. 23: 368-376.

LeBouvier, G.L. 1971. The heterogeniety of Australia
antigen. J. Infect. Dis. 123: 671-675.

Lee, F.K., A.J. Nahmias, and S. Stagno. 1978. Rapid
diagnosis of cytomegalovirus infection in infants
by electron microscopy. N. Engl. J. Med. 299:
1266-1270.

Lee, K.P., and C. Olson. 1969. A gel diffusion precipi-
tin test for bovine papilloma virus. Am. J.
Vet. Res. 30: 725-729.

Levene, C., and 8.5. Blumberg. 1969. Additional speci-
ficities of Australia antigen and the possible
identification of hepatitis carriers. Nature

221: 195-196.

Ling, C. 1980. Radioimmunoassays for hepatitis B
markers, p. 372-379. £2.N-R° Rose and H. Friedman
(ed.), Manual of Clinical Immunology, 2nd edition.
American Society for MicrobiologY: Washington,
D.C.

Linnemann, C.C., and S. Goldberg. 1974. HBsAg in
breast milk. Lancet 2: 155.

Magnius, L.O. 1975. Characterization of a new antigen-
antibody system associated with hepatitis B.
Clin. Exp. Immunol. 20: 209-216.

Magnius, L.O., and S.A. Espmark. 1972. A new antigen
complex co-occurring with Australia antigen.
Acta Path. Microbiol. Scand. Section B.
80: 335-337.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

35

Magnius, L.O., and J.A. Espmark. 1972. New specifi-
cities in Australia antigen positive sera dis-
tinct from the LeBouvier determinents. J. Immunol.
109: 1017-1021.

Marmion, B.P., and R.W. Tonkin. 1972. Control of
hepatitis in dialysis units. Br. Med. Bull.
28: 169-179.

Maynard, J.E. 1976. Modes of hepatitis B virus
transmission, p. 125-137. In T. Oda (ed.),
Hepatitis Viruses, Proceedings of the Inter-
national Symposium on Hepatitis Viruses. Uni-
versity Park Press, Baltimore.

Maynard, J.E., K.R. Berquist, and D.H. Krushak. 1972.
Studies of hepatitis candidate agents in tissue
cultures and in animal models, p. 393-402. In
G.N. Vyas, H.A. Perkins, and R. Schmid (ed.),
Hepatitis and Blood Transfusion. Grune & Stratton,
New York.

Mazzur, S. 1973. Menstrual blood as a vehicle of
Australia-antigen transmission. Lancet 1:749.

Mazzur, S., S. Burgert, and 8.8. Blumberg. 1974.
Geographical distribution of Australia antigen
determinants d, y and w. Nature 247: 38-40.

Milne, R.G., and E. Luisoni. 1977. Rapid immune
electron microscopy of virus preparations, p.
265-281. In K. Maramorsch, and H. Koprowski
(ed.), Methods in Virology, Vol. VI. Academic
Press, Inc., New York.

Mirick, 6.8., and R.E. Shank. 1959. An epidemic of
serum hepatitis studied under controlled con-
ditions. Trans. Am. Clin. Ass. 71: 176-190.

Mushahwar, I.K., L.R. Overby, C.M. Ling, T.J. Kelly, and
M.J. Patterson. 1980. Characterization of urinary
hepatitis B e-antigen. Abstract. Fed. Proc.

39: 2207.

Norkrans, G., G. Frosner, and S. Iwarson. 1979.
Determination of HBeAg by radioimmunoassay: prog-
nostic implications in hepatitis B. Scand. J.
Gastroenterol. 14: 289-293.

Norrby, E., H. Marusyk, and M. Hammarskjold. 1969.
The relationship between the soluble antigens and
the virion of adenovius type 3. V. Identification of
antigen specificities available at the surface of
virions. Virology 38: 477-482.

92.

93.

94.

95.

96.

97.

98.

99.

100.

101.

102.

103.

36

Ogra, P.L. 1973. Immunologic aspects of hepatitis-
associated antigen and antibody in human body
fluids. J. Immunol. 110: 1197-1205.

Palmer, E.L., M.L. Martin, and G.W. Gary, Jr. 1975.
The ultrastructure of disrupted herpesvirus nucleo-
capsids. Virology 65: 260-265.

Papaevangelou, G., D. Trichopoulos, T. Kremastinou,
and G. Papoutsakis. 1974. Prevalence of hepatitis
B antigen and antibody in prostitutes. Br. Med.
J. 2: 256-258.

Pattison, C.P., J.E. Maynard, K.R. Berquist, and H.M.
Webster. 1973. Serological and epidemiological
studies of hepatitis B in haemodialysis units.
Lancet 2: 172-174.

Penney, J.E., and O. Narayan. 1973. Studies of the
antigenic relationships of the new human papova-
viruses by electron microscopy agglutination.
Infect. Immun. 8: 299-300.

Piazza, M., L. Cacciatore, V. Molinari, V. Guadagnino,
P. T. Cataldo, G. Borgia, E. Gravina, G. Manzillo,
and G. Maio. 1975. Presence of HBsAg in the bile
of subjects with acute HBsAg viral hepatitis.
Path. Microbiol. 43: 307-316.

Primack, W.A., M. Schoeneman, A. Spitzer, and I. Greifer.
1977. Hepatitis-B surface antigen in synovial
fluid during chronic renal failure: a potential
danger to medical personnel. Transpl. Proc.

9: 1673-1674.

Prince, A.M. 1968. An antigen detected in the blood
during the incubation period of serum hepatitis.
Proc. Nat. Acad. Sci. 60: 814-821.

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

Propert, S.A. 1938. Hepatitis after prophylactic
serum. Br. Med. J. 2: 677-678.

Rao, K.R., and G.N. Vyas. 1974. Hepatitis B surface
antigen (HBsAg): tryptophan content and biological
activity. J. Gen. Virol. 54: 571-573.

Redeker, A.G., J.W. Mosley, D.J. Gocke, A.P. McKee,
and W. Pollack. 1975. Hepatitis B immune globu-
lin as a prophylactic measure for Spouses exposed
to acute type B hepatitis. N. Engl. J. Med.

293: 1055-1059.

104.

105.

106.

107.

108.

109.

110.

111.

112.

113.

114.

37

Scott, R.M., R. Snitbhan, W.H. Bancroft, H.J. Alter,
and M. Tingpalapong. 1980. Experimental trans-
mission of hepatitis B virus by semen and saliva.
J. Infect. Dis. 142: 67-71.

Snydman, D.R., J.A. Bryan, W.T. London, E. Werner, D.
Bregman, B.S. Blumberg, and M.G. Gregg. '1976.
Transmission of hepatitis B associated with hemo-
dialysis: role of malfunction (blood leaks) in
dialysis machines. J. Infect. Dis. 134: 562-570.

Snydman, D.R., J.A. Bryan, B.J. Macon, and M.B. Gregg.
1976. Hemodialysis-associated hepatitis: report
of an epidemic with further evidence on mechanisms
of transmission. Am. J. Epidemiol. 104: 563-570.

Steere, R.L., and G.K. Ackers. 1962. Purification and
separation of tobacco mosaic virus and southern
bean mosaic virus by agar-gel filtration. Nature
194: 114-116.

Steiner, S., M.T. Huebner, and G.R. Dreesman. 1974.
Major polar lipids of hepatitis B antigen prepara-
tions: evidence for the presence of a glycosphingo-
lipid. J. Virol. 14: 572-577.

Svehag, S., and B. Bloth. 1967. Examination of virus-
M antibody complexes by electron microscopy. I.
Configuration of M antibodies and mode of attach-
ment to antigen. Virology 31: 676-687.

Tosi, M., and D.L. Anderson. 1973. Antigenic properties
of bacteriophage 029 structural proteins. J. Virol.
12: 1548-1559.

van Rooyen, C.B., and G.D. Scott. 1948. Smallpox
diagnosis with special reference to electron micros-
copy. Can. J. Pub. Hlth. 39: 467-477.

Villarejos, V.M., A.K. Visona, A. Gutierrez, and A.
Rodriguez. 1974. Role of saliva, urine and feces
in the transmission of type B hepatitis. N. Engl.
J. Med. 291: 1375-1378.

Watson, D.H., G.L. LeBouvier, J.A. Tomlinson, and D.G.A.
Walkey. 1966. Electron microscopy of antigen
precipitates extracted from gel diffusion plates.
Immunology 10: 305-308.

Watson, D.H., and P. Wildy. 1963. Some serological
properties of herpes virus particles studied with
the electron microscope. Virology 21: 100-111.

115.

116.

117.

118.

38

Weintraub, M., H.W.J. Ragetli, and P.M. Townsley.
1962. The use of agar gel electrophoresis for
the electron microscopic analysis of virus
particles. Virology 17:196-198.

Williams, R.C., and R.W.G. Wyckoff. 1946. Applications
of metal shadow-casting to microscopy. J. Appl.
Phys. 17:23-33.

Yanagida, M. 1972. Identification of some antigenic
precursors of bacteriophage T4. J. Mol. Biol.
65: 501-517.

Yanagida, M., and C. Ahmad-Zadeh. 1970. Determination
of gene product positions in bacteriophage T4 by

specific antibody association. J. Mol. Biol. 51:
411-421.

ABSTRACT
HEPATITIS B e ANTIGEN AND MORPHOLOGIC FORMS OF HEPATITIS
B VIRUS IN URINES FROM PATIENTS WITH END STAGE RENAL
DISEASE
BY
C.A. Currie, I.K. Mushahwar, T.J. Kelly, G.H. Mayor,

and M.J. Patterson

Urines from hepatitis B surface antigen (HBsAg)
persistently seropositive patients with end stage renal
disease were studied for markers of hepatitis B e antigen
(HBeAg) and antibody to HBeAg (anti-HBe) by radioimmuno-
assay (RIA) and for Dane particles by immunoelectron-
microscopy (IBM). Fifty-one percent of the HBsAg positive
urines were positive for HBeAg and 14 percent for anti-HBe.
The over-all prevalence of HBeAg and anti-HBe was 59 per-
cent. Some urines were positive for HBeAg by RIA while
not having detectable HBsAg. When serial urines from these
patients were inspected, HBe antigenuria was an intermit-
tent and persistent marker. Despite the presence of HBsAg
and HBeAg in the urines, careful examination by transmis-
sion electron microscopy in samples prepared by the

pseudoreplica technique for IBM were negative for complete

hepatitis B virions.

HBe ANTIGEN AND MORPHOLOGIC FORMS OF
HEPATITIS B VIRUS IN URINES FROM PATIENTS

WITH END STAGE RENAL DISEASE.

BY

C.A. Currie, I.K. Mushahwar, T.J. Kelly, G.H. Mayor, and

M.J. Patterson

From the Departments of Micr0biology and Public Health, and
Medicine, Michigan State University, East Lansing, Michigan,

and Abbott, Laboratories, North Chicago, IL.

This research was supported by a grant from the Michigan

Department of Public Health.

Reprint requests should be sent to M.J. Patterson, Department
of Microbiology and Public Health, Michigan State University,

East Lansing, Michigan 48824

INTRODUCTION

The finding of hepatitis B e antigen (HBeAg) in the
presence of hepatitis B surface antigen (HBsAg) in serum,
generally correlates with hepatitis B virus (HBV)
infectivity (1,2,3). Patients on chronic maintenance
hemodialysis or recipients of renal transplants have been
shown to have HBsAg and HBeAg in serum (4,5). Additionally,
almost all such patients persistently HBsAg seropositive
have detectable HBsAg in their urines when two or more
randomly timed urine samples are assayed (6). The link
between surface antigenuria and infectivity in unclear but
transmission of HBV by other fluids has been documented (7),
and HBV remains a significant risk for patients with end
stage renal disease. Thus, it is important to evaluate
urine as a vehiCle of HBV transmission.

Accordingly, in this study, urines from renal dialysis
and transplant patients who are persistantly HBsAg sero-
positive were examined for HBeAg and Dane particles.

A sensitive solid phase radioimmunoassay (RIA) was used for
HBeAg detection and immunolectronmicroscopy (IBM) was

employed for virion detection.

39

MATERIALS AND METHODS

Patients and Specimens - Fifty-one urine samples were

 

obtained from end stage renal disease (ESRD) patients in
Michigan. These patients were selected from a population
previously surveyed to determine prevalence of various
hepatitis B serum markers (HBsAg, anti-HBs, anti—HBe, HBeAg,
and anti-HBe) detectable by radioimmunoassay (RIA) (5,8,9).

Twenty-four urine samples examined in this study were
obtained from patients on chronic maintenance hemodialysis,
while 27 samples were from renal transplant recipients. All
samples were obtained from patients who were HBsAg sero-
positive at the time of sampling. Multiple serial urine
samples were obtained from ten patients, while 23 patients
contributed single specimens. All urines were coded and
stored at -20 C until processing.

Urine specimens were evaluated for HBsAg by RIA (AUSRIA
II, Abbott Laboratories, North Chicago, Illinois) and for
the presence of occult blood or proteinuria by colorometric
tests (Multi Stix, Ames Company, Elkart, Indiana).

Forty-four (86 percent) of the 51 urines were evaluated
for HBeAg and anti-HBe by RIA (HBe, Abbott Laboratories,
North Chicago, Illinois) and 32 (63 percent) of the 51 were

selected for IBM.

40

41

RIn - HBeAg and anti-HBe were monitored using sensitive
solid phase radioimmunoassays. To detect HBeAg, unconcen-
trated urine (0.2 ml) was incubated overnight at room
temperature with an anti-HBe pre-coated polystyrene bead.

1251-1abe1ed

Bound antigen was quantitated using 0.2 m1
antiHBe, incubated for 4 hours at 45 C with the washed
bead. Urine samples with i 2.1 times the net negative
control counts per minute (cpm) of 1251 were presumptive
positives for HBeAg. These specimens were then confirmed
as positive for HBeAg by specific antibody inhibition.
Anti-HBe was detected using a two-step inhibition
assay. Urine (0.1 ml) was incubated for 2 hours at room
temperature with known HBeAg (0.1 ml), quantitated to yield
5-10 x 103 cpm at the end of the assay in the presence of
urine negative for anti-HBe. Following this initial in-
cubation step, an anti-HBe precoated polystyrene head was
added and the procedure continued as for HBeAg above. Urine
samples were positive for anti-HBe when cpm were <50 percent
of the net negative control cpm.

Clarification Procedures - Prior to treatment for immuno-

 

electronmicroscopy (IBM), all urines were clarified by
ultracentrifugation (Beckman L3-50) at 20,000 rpm and 4 C
for 30 minutes, using a fixed angle rotor (Beckman 50 Ti).
Alternative methods of clarification included centrifugation
at 3000 rpm (Beckman L5-50) for two hours at 4 C or
filtration through a 0.2 micron filter unit (Nalge Company,

Rochester, New York) with applied vacuum.

42

Controls for IBM - Positive controls were obtained by adding

 

serum, positive for HBsAg by RIA and known to contain Dane
particles, to aliquots of urine. The positive serum was
diluted 10’2, 10'3, and 10'4 in urines from both a HBsAg
seronegative individual with normal renal function and

an ESRD patient with proteinuria. Normal urine, without
serum added, was employed as the negative control.

Aqnn - The agar blocks used in the pseudoreplica technique
were prepared with Bacto-Agar (Difco Laboratories, Detroit,
Michigan). Agar was dissolved in distilled water to a
final concentration of 2 percent (w/v). This was
autoclaved, delivered to standard size Petri plates, and
refrigerated until needed. Blocks (approximately 12 x

12 mm) were cut with a scalpel and allowed to warm to room
temperature before use.

Buffers - Sterile 0.01 M phosphate buffered saline (PBS),
(0.01 M phosphate, 0.13 N NaCl) pH 6.9 was the diluent in
all procedures in which serial dilutions are indicated. In
the modified Derrick system, 0.1 M phosphate buffer, pH 7.0
was included as a routine wash.

Plastic Support Film - Parlodion (Ted Pella, Inc., Tustin,

 

California) was used to manufacture specimen support films
in all procedures. Film solutions (0.05 percent) were made
by dissolving broken nitrocellulose strips (Parlodion) in
amyl acetate. After being tightly covered with parafilm and

wrapped in aluminum foil, the solutions were placed in a

43

dessicator for 48 to 72 hours to allow complete dissolution
of the plastic. The solution was then filtered through
Whatman #1 filter paper and used immediately, either to
coat grids directly or to be used in the pseudoreplica
technique.

To coat grids, the Parlodion solution was cast upon
clean microsc0pe slides and floated off onto distilled
water. Four hundred mesh copper grids (Ted Pella, Inc.,
Tustin, California) were placed upon the membrane. The
entire film was retrieved by parafilm, placed in Petri
plates and stored in a dessicator.

Negative Stain - A two percent solution of phosphotungstic

 

acid was adjusted to pH 6.0-6.7 by titrating with 5 N KOH.
Following filtration through a 0.2 micron Nalgene filter
unit (Nalge Company, Rochester, New York), 0.05 percent
powdered bacitracin (Sigma Chemical Company, Saint Louis,
Missouri) was added to facilitate spreading.

Pseudoreplica Technique - Grids were prepared for IBM

 

according to a modification of the pseudoreplica technique
described by Palmer and associates (10).

Antiserum - Dilutions of hyperimmune hepatitis B immune

 

globulin (HBIG)(John R. Kateley, Jr., Ph.D., Department of
Laboratories, E. W. Sparrow Hospital) were prepared in

0.01 M PBS for urine reaction mixtures prior to concen—
tration for electron microscopy. Measurements of antibody
titers showed that undiluted HBIG contained approximately 5-

6 x 103 RIA units of anti-HES and small amounts of anti-HBc.

44

Immune CompIex Reaction Mixure - Two drops of diluted

 

HBIG were mixed with an equal volume of clarified urine in
12 x 75 mm test tubes and covered with parafilm. Tubes were
incubated for one hour, 37 C. Alternative incubation
methods evaluated, included refrigeration at 4 C overnight
following incubation for one hour at 37 C, or incubation for
one hour at room temperature (24 C) only.

Grid Preparation - One drop of each reaction mixture was

 

deposited on an agar block, positioned at a corner of a
microscope slide, and spread with the edge of a Pasteur
pipette. Excess fluid was drained from the agar onto filter
paper by tilting the slide, and the remaining thin fluid
layer was allowed to air dry. One drop of liquid Parlodion
was carefully delivered and spread by slowly rotating the
slide. This was drained as above and the film was air dried
resting at an angle of ninety degrees. The entire membrane
was floated off onto the surface of the negative stain
solution (PTA). Two four hundred mesh copper grids were
placed on areas with an even appearance using forceps, and
the film was removed with parafilm. To avoid pooling of
PTA, excess moisture was blotted with filter paper. Grids
were removed when completely dry and examined by transmis-

sion electron microscopy (TEM).

45

Modified Derrick System - The technique to form immune

 

complexes directly on grids, described by Derrick (11) and
modified by Milne and Luisoni (12), was further modified
here to detect HBsAg and Dane particles in urine.

One drop of HBIG was delivered to a grid held by
forceps and previously coated with Parlodion. For each
specimen, dilutions of HBIG formed the initial layer. After
a five minute incubation period in a 37C humid box, the
grid was washed with 20 consecutive drops of 0.1 M phos-
phate buffer, pH 7.0, and drained by touching the grid edge
with filter paper. One drop of urine was placed on the
grid, incubated under the same conditions for fifteen
minutes, washed and drained as above. One drop of HBIG

(10'2

dilution) was layered on top, creating a sandwich
effect, and the grid was incubated 5 minutes as above.

The grid was again washed with 20 drops of buffer followed
by 30 drops of filtered distilled water. The grid was then
drained and negatively stained with one drop of PTA. Excess
fluid was removed with filter paper, and the grid was air

dried.

Transmission Electron Microscopy - Grids were examined in a

 

Philips 300 Transmission Electron Microscope operated at 80
kV. To determine the presence of Dane particles, 5 to 6

squares per grid were scanned at magnifications of approxi-
mately 25,000x and 67,000x for a total of 20 to 30 minutes.
Micrographs were taken using Kodak Electron Image Film 4463

or Kodak Electron Microscope Film 4489 (Eastman Kodak Co.).

46

Negatives were developed in Kodak D-19 Developer and fixed
in Kodak Rapid Acid Fixer. After a 20 to 30 minute wash in
running water, negatives were briefly immersed in Kodak

Photo-Flo and dried in a hot air drier.

 

RESULTS

RIA - The results of HBeAg and anti-HBe testing on the 44
urines using solid phase radioimmunoassays are as follows.
Nineteen of the 37 HBsAg positive urines had concomitant

HBeAg. Five had anti-HBe, and 13 had neither marker. In

no instance were HBeAg and anti-HBe detected simultaneously.

Unlike the patterns observed in serum, where HBsAg always
coexists with HBe or anti-HBe, two of seven urines negative
for HBsAg showed HBeAg. The remaining five were negative
for both e antigen and antibody.

When the total ESRD urines were separated into two
subgroups, urines obtained from patients on dialysis and
those who received transplants, the results shown in Ap-
pendix 1 were obtained. Of 18 urines from dialysis
patients, 12 had HBsAg. Nine of these 12 had HBeAg and 2
anti-HBe. One of the 6 HBsAg-negative urines had HBeAg.
Among urines obtained from transplant patients, 25 of 26
were surface antigen positive, with 10 of these having
detectable HBeAg and 3 others anti-HBe. The one HBsAg-
negative urine was HBeAg-positive.

The over-all prevalence of HBe markers in urines from
ESRD patients (59 percent) was not significantly different

when patient subgroups were examined; 67 percent of the

47

48

dialySis urines and 54 percent of transplant urines
demonstrated one or the other marker. No significant
differences in urine frequency ratios (HBeAg to anti-HBe)
were seen among dialysis versus transplant patient urines.

Ten ESRD patients were followed by collection of serial
urine samples 5 to 16 months after initial urine sampling.
The results are shown in Appendix 2. Tests for HBsAg in
these urine samples were positive in all but one instance
(designated by the open circle). Urines designated with the
letter D were taken during a time when the patient was being
maintained on chronic hemodialysis. Where not marked, the
patient has received a kidney transplant. Some patients
have moved from the dialysis to the transplant population
during the course of the study. HBeAg and anti-HBe results
are shown within the symbols. Appearance and disappearance
of detectable HBeAg in urine is seen (top patient) as well
as the disappearance of detectable levels of pre-existing
anti-HBe (third patient, coincident with movement from the
dialysis to the transplant population).

Develqpment of IBM Techniqne - Results of the examination by

 

IBM of known HBsAg-positive urines using modifications of
the pseudoreplica and Derrick techniques are shown in

Table 1. HBsAg was readily detected in patient urines using
the pseudoreplica technique following incubations for one
hour at 24C or 37C. In control preparations, Dane
particles, as well as HBsAg, occurred singly and in com-

plexes with or without the excess HBsAg spherical and

49

filamentous morphological forms. Fewer complexes were
formed during room temperature incubations, and single
particles of HBsAg appeared to be surrounded by hazy mat-
erial. Incubation at 37 C followed by overnight incubation
at 4 C, was an unsatisfactory procedure, since prepara-
tions appeared cloudy and any complexes that may have been
formed were totally obscured.

When incubated for one hour at 37 C, Dane particles and
excess HBsAg were abundant and easily detected in 10'2
and 10"3 dilutions of positive control serum. Fewer morpho-
logical forms were visualized at the higher dilution (10'4),
although they were still present in moderate to abundant
amounts. At all three dilutions, most virions were highly
complexed with antibody, but the most dilute serum had more
antibody coated single virions and HBsAg particles (Fig.1A).
Representative micrographs demonstrating all three morph-
ological forms (ie. Dane particles, spherical and tubular
forms) in control urines are shown in Figures 1B, 1C, and
1D.

Fewer immune complexes were formed in patient urine
using the Derrick system. For all specimens, two dilutions
of HBIG (10'1 and 10'2, representing 500-600 and 50-60 RIA
units) were chosen to form the initial layer, and more
virions and/or HBsAg were trapped on grids coated with the
more concentrated antiserum. While control preparations
were comparable, patient specimens did not appear as clean

as those processed by pseudoreplication.

.oowsomno maouoHQEoo ouoz ucomoum macauw> no moummmnmmm
was coflnz CH mowum wpsoHo\osommo >HHMfiucommo GA pwuaomou m pocuoe coaumwmmonm «

 

.oumswm pHHm
mom mmaofluumm mammm mamCHm
calm .mcoHHH> o: mcflcwmucoo
moummoummm mflmmm HMCOflmmooo

«pouoombo

.oumsvm

pflum nod moaofluumm mammm mmmoxm
mamcfim Hmcoflmmooo .mcofiufi>
usonuflz moummoummm 04mmm oniom

50

.oumsvm Ufium

mom moHOHuumm mammm mmooxm
mamcfim cm on oa cam .mcofiuw>
usozuflz moummoummm m<mmm calm

.oumovm

capo mom mcoHHH> omumoo
uponwucm mamcflm Hmcoammooo
“moaofluumm mdmmm mamcflm oaum

.mconw> mauo mcwcwmucoo mouscHE om Mom ohm
mmumooummm omuom onmme is
muson NH How 0v
.uooc H mom Ohm
«nousomno «UHAmmmooDmmm Am
.oumsvm
owum mom mc0fiwa> pounce
soonauqm mamcflm oaum
moHOADMMQ mmmmm mmmoxo
mamcwm Hmcoflmmooo .nomo
mc0fiwa> mane mcacfimucoo woo: a how Ohm
moummmummm mammm coauom mqummmoaommm Am
.oumsvm ofium mom mcoanw>
poumoo xponflucm mamswm
chowmmooo .moaowuumm mmmmm
mmooxo mamcam omloa can
oummoummm woo mcofluw> Nio noon H wow Dem

mcacwmucoo mouwooummm calm

duHAmmmoaommm AH

 

mmZHmD
BZmHBdm

mszs m>HeHmoa mammm so

mmZHmD

domBZOU z

ZOHB¢ZHS¢xm mom mMDOHZEUMB ZmH hO ZOmHmdeOU

maomfimz
OHBdMfimmmm

“H mqmfifi

Figure 1A:

51

 

Typical negatively stained single antibody-
coated hepatitis B virion and HBsAg particles
observed following incubation of highly
diluted serum with HBIG. Bar represents
approximately 112 nm.

52

   

Figure 1B: Negatively stained Dane particles in aggregate.
Bar represents approximately 100 nm.

53

 

Figure 1C: Negatively stained particles in complex with
spherical and tubular morphological forms of
HBsAg. Bar represents approximately 100 nm.

54

 

Figure 1D:

Negatively stained Dane Particles in complex
with spherical and tubular morphological

forms of HBsAg. Bar represents approximately
100 nm.

55

The visual quality of specimens clarified by altern-
ative methods of centrifugation or filtration was poor, and
these techniques were not employed further.

To determine if HBV virions were pelleted during the

ultracentrifugation step, a 10'-2

dilution of serum positive
for Dane particles was made in an unclarified aliquot of
urine from an ESRD patient with proteinuria. This was
subsequently divided into two equal volumes. One fraction
was clarified as earlier described by ultracentrifugation,
the other was not further processed until preparation for
IBM. Examination by IBM showed that pelleting of Dane
particles did not occur at the centrifugal force employed
for clarification. Dane particles were detected in both
preparations and virions were significantly more visible in
the clarified specimen.

Since the pseudoreplica technique modified for immuno-
electronmicroscopy by incubation with 10"1 and 10'”2 di-
lutions of HBIG for one hour at 37 C, consistently allowed
visualization of whole HBV virions in urines with serum
positive for Dane particles added, all subsequent urines
were examined by that method. A flow diagram of this
protocol is shown in Figure 2.

IBM Survey of Patient Urines - Of 32 urines examined,

 

31 were negative for Dane particles. Urinalysis data
(Appendix 3) revealed that the urine specimen positive for
Dane particles by IBM contained occult blood, the probable

source of virions.

56

2 DROPS SAMPLE + 2 DROPS HBIG

INCUBATE l H, 37 C
l
1 DROP SPREAD ON AGAR BLOCK, DRAINED AND DRIED
l
1 DROP PARLODION SPREAD ON AGAR BLOCK, DRAINED AND DRIED
MEMBRANE FLOATED ONTO 2% KPTA
GRIDS PLACED ON MEMBRANE
RETRIEVED WITH PARAFILM, DRAINED AND DRIED

TRANSMISSION ELECTRON MICROSCOPY

Figure 2: Pseudoreplica Technique

 

DISCUSS ION

HBsAg positive body fluids and excretions include urine
(13,14), saliva (14), feces (l4), nasopharyngeal secretions
(15), menstrual blood (16), vaginal secretions (l7), bile
(18), breast milk (19), cerebrospinal fluid (20), sweat
(21), semen (22), and synovial fluid (23), all of which have
been suggested to be potentially infective. Only saliva and
semen have unequivocably been shown to be infective in
transmission experiments (7). Blood components, including
frozen blood, are uniformly recognized as the most important
vehicle of transmission (24,25,26,27).

In an assessment of the potential for transmissibility
of HBV infections by urine, 51 urines from HBsAg seropositive
hemodialysis or transplant patients were examined. A

sensitive solid phase radioimmunoassay (RIA) enabled detec-

 

tion of HBe-markers in these urines. Urinary HBeAg was :7
confirmed by specific antibody inhibition. When the urines I
most likely to contain Dane particles, those positive for g

'1
both HBsAg and HBeAg, were inspected by immunoelectron- é
microscopy, whole virions were not demonstrated. The single é;

urine in which Dane particles were observed also showed
hematuria, further confirming the link between blood com-

ponents and infectivity.

57

58

The presence of HBeAg in urine, here-to-fore not
described, has been documented. Development of an easy,
rapid and highly sensitive technique for detection of Dane
particles by immunoelectronmicroscopy has been described.
Incorporation of the pseudoreplica technique yielded good
particle concentration while removing salts and impurities
from the specimen. Use of hyperimmune hepatitis B immune
globulin allowed increased specificity for HBsAg, free and
virion-associated. Refinement of the system resulted in
consistent visualization of Dane particles in positive
control specimens.

While serum HBeAg is associated with HBV DNA polymerase
and circulating Dane particles(28), HBeAg positive urine
samples were negative for Dane particles using IEM. HBe-
antigenuria is common among patients with end stage renal
disease, both on hemodialysis and renal transplant patients.
The chronic maintenance hemodialysis and transplant pop-
ulations do not appear different when evaluated for HBe
markers, and HBe antigenuria is both intermittent and
persistent. Despite the detection of HBsAg and HBeAg in the
urine samples, the absence of observable Dane particles by
IBM suggests that these urines are not infectious. More
definitive studies, using a primate model for experimental

transmission, remain to be done.

F -

LIST OF REFERENCES

I'A; -

 

lbs.

LIST OF REFERENCES

Alter, H.J., Seeff, L.B., Kaplan, P.M., McAuliffe,
V.J., Wright, E.C., Gerin, J.L., Purcell, R.H.,
Holland, P.V., Zimmerman, H.J. Type B hepatitis:
the infectivity of blood positive for e antigen
and DNA polymerase after accidental needlestick
exposure. N. Engl. J. Med. 295: 909-913, 1976.

Okada, K., Kamiyama, I., Inomata, M., Mitsunobu,

I., Miyakawa, Y., Mayumi, M. e antigen and anti-

e in the serum of asymptomatic carrier mothers as
indicators of positive and negative transmission

of hepatitis B virus to their infants. N. Engl.

J. Med. 294: 746-749, 1976.

Takahashi, K., Imai, M., Tsuda, F., Takahashi,

T., Miyakawa, Y., Mayumi, M. Association of Dane
particles with e antigen in the serum of asymptomatic
carriers of hepatitis B surface antigen. J. Immunol.
117: 102-105, 1976.

Aldershvile, J., Skinhoj, P., Frosner, G.G., Black,
F., Deinhardt, F., Hardt, F., Neilson, J.O. The
expression pattern of hepaitits B e antigen and
antibody in different ethnic and clinical groups

of hepatitis B surface antigen carriers. J. Infect.
Dis. 142: 18-22, 1980.

Kelly, T.J., Sanchez, T.V., Patterson, M.J., Mayor,
G.H. Hepatitis B markers among dialysis patients
without hepatitis B surface antigen or antibody.
Proceedings of the Clinical Dialysis and Transplant
Forum. (In Press).

Patterson, M.J., Mayor, G.H., Hourani, M.R., Kelly,
T.J. Rosenbaum, R.W. Serial urine sampling for
hepatitis B surface antigen (HBsAg) in hemodialysis
patients. Presented to the American Society of
Microbiology, Annual Meeting, Los Angeles, California.
May 4-11, 1979. (Abstract).

Scott, R.M., Snitbhan, R., Bancroft, W.H., Alter,
H.J., Tingpalapong, M. Experimental transmission
of hepatitis B virus by semen and saliva. J. Inf.
Dis. 142:67-71, 1980.

59

 

10.

ll.

12.

13.

14.

15.

16.

17.

18.

19.

20.

60

Kelly, T.J., Hourani, M.R., Patterson, M.J., Mayor,
G.H. Serological markers of hepatitis in hemodialysis
patients and staff. Proc. Dialysis Transplant Forum.
179-182, 1978.

Mayor, G.H., Hourani, M.R., Greenbaum, D.S., Patterson,
M.J. Prevalence of hepatitis B in 27 Michigan
hemodialysis centers. Am. J. Pub. Hlth. 69: 581-584,
1979.

Palmer, E.L., Martin, M.L., Gary, G.W., Jr. The

ultrastructure of disrupted herpesvirus nucleocapsids.
Virology 65: 260-265, 1975.

Derrick, K.S. Immuno-specific grids for electron
microscopy of plant viruses. Abstract. Phytopathology
62: 753-754, 1972.

Milne, R.G., Luisoni, E. Rapid immune electron
microscopy of virus preparations, p. 265-281, In

K. Maramorsch, and H. Koprowski (ed.), Methods—In
Virology, Vol, VI. Academic Press, New York, 1977.

Apostolov, K., Bauer, D.J., Selway, J.W.T., Fox,
R.A., Dudley, F.J., Sherlock, 8. Australia antigen
in urine. Lancet 1: 1274-1275, 1971.

Villarejos, V.M., Visona, A.K., Gutierrez, A.,
Rodriguez, A. Role of saliva, urine, and feces in
the transmission of type B hepatitis. N. Engl. J.
Med. 291: 1375-1378, 1974.

Ogra, P.L. Immunologic aspects of hepatitis-associated
antigen and antibody in human body fluids. J. Immunol. r
110: 1197-1205, 1973.

Mazzur, S. Menstrual blood as a vehicle of Australia-
antigen transmission. Lancet 1: 749, 1973. K

Darani, M., Gerber, M. Hepatitis B antigen in vaginal
secretions. Lancet 2: 1008, 1974.

 

Piazza, M., Cacciatore, L., Molnari, V., Guadagnino, i
V., Cataldo, P.T., Borgia, G., Gravina, E., Manzillo,

G., Maio, G. Presence of HBsAg in the bile of subjects

with acute HBsAg viral hepatitis. Path. Microbiol.

43: 307-316, 1975.

Linnemann, C.C., Goldberg, S. HBsAg in breast milk.
Lancet 2: 155, 1974.

Denes, A.B., Ebert, J.W., Moncada, R.E., Berquist,
K.R., Cook,E.H., Jr., Maynard, J.E. Hepatitis-B
surface antigen in cerebrospinal fluid. Lancet

2: 1038, 1975.

21.

22.

23.

24.

25.

26.

27.

28.

61

Brodensen, M., Wirth, M. Detection of HBsAg and
HBsAb in sweat. Acta Hepato-Gastroenterol. 23:
194-201, 1976.

Heathcote, J., Cameron, C.H., Dane, D.S. Hepatitis-B
antigen in saliva and semen. Lancet 1: 71-73,
1974.

Primack, W.A., Schoeneman, M., Spitzer, A., Greifer,
I. Hepatitis-B surface antigen in synovial fluid
during chronic renal failure: a potential danger
to medical personnel. Transpl. Proc. 9: 1673-1674,
1977.

Alter, H.J., Tabor, E., Meryman, H.T., Hoofnagle,
J.H., Kahn, R.A., Holland, P.V., Gerety, R.J.,
Baker, L.F. Transmission of hepatitis B virus
infection by transfusion of frozen-deglycerolized
red blood cells. N. Engl. J. Med. 298: 637-642,
1978.

Craske, J., Dilling, N., Stern, D. An outbreak of
hepatitis associated with intravenous injection
of factor VIII concentrate. Lancet 2: 221-223, 1975.

Favero, M.S., Maynard, J.E., Leger, R.T., Graham,
D.R., Dixon, R.E. Guidelines for the care of patients
hospitalized with viral hepatitis. Ann. Intern.

Med. 91: 872-876, 1979.

Shikata, T., Karasawa, T., Abe, K., Uzawa, T.,

Suzuki, H., Oda, T., Imai, M., Mayami, M., Moritsugu,
Y. Hepatitis B e antigen and infectivity of hepatitis
B virus. J. Infect. Dis. 136: 571-576, 1977.

Deinhardt, F. Predictive value of markers of hepatitis
virus infection. J. Infect. Dis. 141: 299-305, 1980.

 

ABSTRACT
ABSENCE OF DANE PARTICLES IN PATIENTS' SERA
LACKING PROTECTIVE ANTIBODY TO HBV
BY

C.A. Currie, T.J. Kelly, G.H. Mayor, and M.J. Patterson

Serum samples from patients with end stage renal
disease (ESRD) were examined by immunoelectronmicroscopy
(IBM) for the presence of Dane particles. These patients
had previously been included in a statewide survey to
determine prevalence, among chronic maintenance hemodialysis
and renal transplant patients, of various hepatitis B virus
(HBV) markers (HBsAg: hepatitis B surface antigen, anti-HBe:
antibody to hepatitis B surface antigen, anti-HBe: antibody
to hepatitis B core antigen, HBeAg: hepatitis B e antigen,
anti-HBe: antibody to hepatitis B e antigen) detectable by
radioimmunoassay (RIA). Thirty three serum samples positive
for anti-HBC but lacking HBsAg and anti-HES, were all

negative for Dane particles when examined by IBM.

 

 

ABSENCE OF DANE PARTICLES IN PATIENTS' SERA
LACKING PROTECTIVE ANTIBODY TO HBV

BY

C.A. Currie, T.J. Kelly, G.H. Mayor, and M.J. Patterson

 

INTRODUCTION

The development of solid phase radioimmunoassays (RIA)
for the antigen-antibody marker systems of hepatitis B virus
(HBV) infection has allowed precise interpretation of the
events in this virus-host interaction (1,2,3,4). Since
patients on chronic maintenance hemodialysis remain at high
risk of infection with HBV, this population has provided a
unique pool to study the serum prevalence of the marker
systems. It has recently been reported that, utilizing RIA
for five separate markers, 29.9% of hemodialysis patients
had hepatitis B surface antigen (HBsAg) and/or its associated
antibody (anti-HBs) in their sera, inclucing 23.8% of the
population which also had antibody to hepatitis B core
antigen (anti-HBC) (2). However, an additional 7.9% of
these hemodialysis patients showed anti-HBc in the absence
of HBsAg or anti-HES. When hepatitis B e antigen (HBeAg)
and antibody (anti-HBe) were assessed in these patients with
only anti-HBc detectable in their serological profile, 8.6%
had HBeAg and 25.7% anti-HBe, while 65.7% had no detectable
HBe marker (2). It was important to define further the
state of clinical infection and to assess potential
transmission from the blood of such patients, characterized
by RIAs as negative for HBsAg and anti-HBs.

62

 

63

and positive for anti-HBc with or without a marker of the
HBe system. This study was undertaken to examine sera of
these patients for the presence of circulating morphologic
forms of HBV by a highly sensitive pseudoreplica immuno-

electronmicroscopic technique.

 

r!

MATERIALS AND METHODS

Patient and Materials - Thirty three serum samples were

 

obtained from end stage renal disease (ESRD) patients in
Michigan. These patients were part of the population
previously surveyed to determine prevalence of various
hepatitis B markers (HBsAg, anti-HBs, anti-HBc, HBeAg, anti-
HBe) detectable by radioimmunoassay (RIA) (2,5,6).

All patients in this study were being maintained on
hemodialysis. The sera examined were selected from a
subpopulation demonstrating hepatitis B core antibody
(anti-HBC) in the absence of HBsAg and anti-HBs. The sera
were aliquoted and frozen at -20 C until use for immuno-
electronmicroscopy (IBM). Immediately prior to treatment
for IBM, sera were diluted 10'1 and 10"2 in 0.01 M sterile

phosphate buffered saline (PBS), 0.01 M phosphate, 0.13 N

 

r-

NaCl, pH 6.9. 2
Three sera positive for HBsAg by radioimmunoassay, were

used as positive controls for IBM. These sera were used in

final dilutions of 10'1 and 10'2 respectively, in patient

serum which was diluted in PBS as described. 4-

64

65

Immunoelectronmicroscopy

Antiserum - Varying dilutions of goat anti-human IgG

 

(Meloy, Springfield, VA) were used for immune complex
formation. Prior to pseudoreplication, goat antiserum was
added to patient sera at concentrations determined by solid
phase RIA (Corab, Abbott Laboratories, North Chicago,
Illinois) to remove detectable anti-HBc.

Immune Complex Reaction Mixture - Two drops of diluted

 

goat antiserum were mixed with an equal volume of prediluted
patient serum in test tubes. Tubes were sealed with par-
afilm and incubated for one hour, 37 C.

Grid Preparations - Grids were prepared for the pseudo-

 

replica technique and negatively stained with 2% phospho-
tungstic acid (KPTA), pH 6.0-6.7 as described in detail
previously (7).

Transmission Electron Microscopy - A Philips 300 TEM (80
kV) was used to examine grids. Grids were scanned (five to
six squares per grid) at magnifications of approximately F
26,000x and 67,000x for 20-30 minutes, for the presence of

immune complexes containing morphologic subunits of HBV. l

 

RESULTS

Determinations by solid phase radioimmunoassay of
concentrations of anti-human IgG necessary to remove de-
tectable anti-HBc from sera, were used as a guideline to
incorporate goat anti-human IgG into the pseudoreplica

technique. Dilutions of 10'1 and 10"2

were selected, and
immune complex formation was readily achieved in all speci-
mens prepared with HBsAg positive serum added. Dane par—
ticles occurred in clumps and singly, and appeared to have
fuzzy outer boundaries. Few to moderate virions were
detected in positive control sera containing high concen-
trations of added HBsAg positive serum, and rare to few
virions were detected in the more dilute control prepar-
ations. Comparatively more virions were trapped using

higher concentrations of goat anti-human IgG.

Since this modification of the pseudoreplica technique

. - . . l‘V'f'L!
. .

consistently allowed visualization of Dane particles in sera
prepared with morphological markers, all patient sera were

processed and examined by this method. The results of HBeAg

 

and anti-HBe assays for those patients are shown in Appendix
4. All 33 sera which contained anti-HBc in the absence of
HBsAg and anti-HBs and with or without e system markers,

were negative for Dane particles.

66

DISCUSSION

With the development of highly sensitive immunologic
assays for antigen-antibody markers of HBV infection,
serologic profiles have emerged as correlates of the stage
of clinical disease (8). When only HBsAg is detectable, the
infection generally is early, often presymptomatic. Se-
quential serum sample studies show that HBsAg is present for
3-5 weeks prior to the appearance of the first detectable
antibody, anti-HBc (9). HBsAg in the presence of anti-HBc,
indicates either acute infection or the presence of the
chronic carrier state. Appearance of anti-HBs signals
disappearance of HBsAg and recovery from HBV infection (8).
Usually anti-HBC and anti-HES remain detectable long after
infection. HBeAg appears transiently, shortly after HBsAg
is detectable. HBeAg in serum is regarded as indicative of

infectivity, since the concentration of HBeAg correlates g

.‘nw

closely with the number of circulating Dane particles
(8,10). Persistent HBeAg is associated with continuing in-

fectivity and chronic liver disease (8). However, more

 

commonly anti-HBe is found shortly after the appearance of
anti-HBc, during the acute phase of the clinical illness.
As with the other antibody moieties associated with HBV
infection, presence of anti-HBe may be prolonged in some

patients.

67

68

Hemodialysis patients present a unique population with
respect to HBV infection, since they remain at high risk for
HBV, infection frequently results in a persistent HBsAg
carrier state, and some of these patients are likely to
become renal transplant recipients, subject to immunosup-
pressive therapy. The present study examines that par-
ticular group of hemodialysis patients whose serologic
profile shows them to be negative for both surface antigen
and antibody, but positive for anti-HBc, that is, a group of
patients who have had HBV infection but show no protective
antibody.

Presence of anti-HBc in the absence of HBsAg or anti—
HBs may indicate the "immunologic window", that period
during acute infection following disappearance of HBsAg and
prior to detection of anti-HBs (1,11). Alternatively, this
profile might represent a period long after an infection
that had been apprOpriately terminated by anti-HES, but at a
point when anti-HES has fallen to undetectable levels. In 7"
addition, this category can also signal the presence of
persistent HBV infection where the low level of HBsAg is

undetectable.

 

 

The infectivity of patients with markers of prior
infection with HBV (anti-HBc with or without an e system
marker) without evidence of protective antibody (anti-HES),
remain suspect with regard to infectivity. In particular,

patients seropositive for anti-HBc and HBeAg may be

 

 

69

potentially infectious. However, careful examination of
sera from hemodialysis patients with previous HBV infection
and without anti-HBs, by a sensitive pseudoreplica immuno-
electronmicroscopic technique, shows lack of any circulating

morphologic evidence of HBV.

 

 

 

LI ST OF REFERENCES

 

LIST OF REFERENCES

Hoofnagle, J.H., Gerety, R.J., Ni, L.Y., Barker,

L.F. Antibody to hepatitis B core antigen. A
sensitive indicator of hepatitis B virus replication.
N. Engl. J. Med. 290: 1336-1340, 1974.

Kelly, T.J., Sanchez, T.V., Patterson, M.J., Mayor,
G.H. Hepatitis B markers among dialysis patients
without hepatitis B surface antigen or antibody. Proc.
Dialysis Transplant Forum. (In Press)

Mushahwar, I.K., Overby, L.R., Frosner, G., Deinhardt,
F., Ling, C.M. Prevalence of hepatitis B e antigen
and its antibody as detected by radioimmunoassays. J.
Med. Virol. 2:77-87, 1978.

Purcell, R.H., Gerin, J.L., Almeida, J.E., Holland,
P.V. Radioimmunoassay for the detection of the core
of the Dane particle and antibody to it.
Intervirology 2: 231-243, 1974.

Kelly, T.J., Hourani, M.R., Patterson, M.J., Mayor,
G.H. Serological markers of hepatitis in hemodialysis
patients and staff. Proc. Dialysis Transplant.
Forum. 179-182, 1978.

Mayor, G.H., Hourani, M.R., Greenbaum, D.S., Patterson,
M.J. Prevalence of hepatitis B in 27 Michigan
hemodialysis centers. Am. J. Pub. Hlth. 69: 581-

584, 1979.

Currie, C.A., Mushahwar, I.K., Kelly, T.J., Mayor,
G.H., Patterson, M.J. HBe antigen and morphologic
forms of hepatitis B virus in urines from patients
with end stage renal disease (Submitted for publication)

Deinhardt, F. Predictive value of markers of hepatitis
Virus infection. J. Infect. Dis. 141:299-305, 1980.

Hoofnagle, J.H, Seeff, L.B., Bales, Z.B., Gerety,
R.J., Tabor, E.: Serologic responses in HE, p.219-
242. In Viral Hepatitis, G.N. Vyas (ed.), The Franklin
Institute Press, Philadelphia, PA., 1978.

70

 

W“!

 

10.

ll.

71

Hess, G., Arnold, W., Myer, K.H. Comparison of
indicators for Dane particles. Klin. Wochenschr.

58:

371-376,

Fife, K.H.,

of hepatitis.

1980.

Corey,

L.

Recent advances in the diagnosis

Lab. Med. 11: 649-654, 1980.

 

“11

APPENDICES

 

 

APPENDIX 1

Hepatitis B Markers in Urine From
Persistent HBsAg SerOpositive ESRD Patients

POSITIVE FOR POSITIVE FOR

HBsAg HBeAg ANTI-HBe
ALL ESRD 44 PCS-37 19(51%) 5(14%)

NEG- 7 2(28%) 0
DIALYSIS 18 PCS-12 9(75%) 2(17%)

NEG- 6 1(17%) 0
TRANSPLANT 26 PCS-25 10(40%) 3(12%)

NEG- l l 0

 

 

 

E
E

APPENDIX 2

SERIAL URINES

2ND 4TH 5TH

o .15 m
L; a: + LLLLL
Lam—[m
m—E—LZ—l

L / L—L-L—I

E] '-"- HBSAG POSITIVE

O =2 HBSAG NEGATIVE
(HBEAG
ANTI—HBE

D =DIALYSIS (VS.
TRANSPLANT)

 

 

 

 

 

 

 

 

 

 

 

 

 

T\'
i

 

 

""1;
SEE
q §
Iifil

 

 

 

 

APPENDIX 3
HBV Antigen-antibody System Markers Detected by
Radioimmunoassay and Urinalysis Data in Urines examined by
Immunoelectronmicroscopy.

Occult Protein

HBsAg HBeAg Anti-HBe Blood mg/dl
5001 - - - - 100
5002 - - - - trace
5003 - - - - 30
5005 - Pos - - 100
5006 - NA* NA - 100
5007 - NA NA NA NA
5011 - - - - 100
5012 P05 P03 - - 30
5013 - NA NA - 100
5014 Pos - Pos - 100
5015 - NA NA - 100
5016 P03 P05 - - 300
5017 P05 P03 - - trace
5018 - NA NA - 100
5020 - NA NA - trace
5030 - Pos - - trace
5031 P03 P03 - - trace
5032 Pos - Pos 2+ trace
5033 P03 P05 - - 30
5038 Pos - - - -
5041 P03 P05 - - 30
5043 P03 P05 - - trace
5101 Pos - Pos - 30
5102 P05 P05 - - 30
5112 P03 P05 - - trace
5113 Pos - - - trace
5114 Pos - - - trace
5115 Pos - - - 300
5116 Pos - - - 100
5118 - Pos - - —
5120 Pos - Pos - -
5121 - NA NA NA NA

NA= Not assayed

Pos=

Positive

 

APPENDIX 4

HBe Antigen-antibody System Markers Detected by
Radioimmunoassay in 33 Sera (positive for anti-
HBc, negative for HBsAg and anti-HES) Examined
by Immunoelectronmicroscopy.

Patient # HBeAg Anti-HBe
0307 - —
0312 - -
0905 Pos -
0918 - -
0920 - -
0932 - -
1102 - -
1104 - -
1111 - Pos
1140 - -
1163 - Pos
1207 Pos -
1401 - -
1404 . - -
1505 Pos -
1702 — -
1921 - -
1940 - -
2004 - —
2115 - -
2134 - Pos
2401 - Pos
2409 - -
2427 - Pos
2430 - Pos
2434 Pos -
2603 - -
2610 - -
2618 - —
2623 - -
2704 - Pos
2718 - -
3212 - Pos