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Hepatitis a virus infection: Pathogenesis and serodiagnosis of acute disease
Journal of Virological Mefhods, @ Elsevier/North-Holland
2 (1980)
31
31-45
Biomedical Press
HEPATITIS A VIRUS INFECTION: PATHOGENESIS
AND SERODIAGNOSIS
OF
ACUTE DISEASE
DANIEL W. BRADLEY Hepatitis Laboratories
Division *, Bureau
Health Service, Department AZ 85014,
of Epidemiology.
of Health, Education,
Center for Disease Control,
U.S. Public
and Welfare, 4402 North Seventh Street, Phoenix,
USA.
The early development of immune electron microscopic (IEM) methods for the detection of HAV in acute-phase stool suspensions and antibody to HAV (~ti-HAV) in serum made it possible to serologically identify cases of hepatitis A using paired acute and convalescent phase sera. Introduction of less cumbersome and time-consuming serologic test methods, including complement Bxation (CF) and immune adherence hemagglutination (JAHA), made it feasible to rapidly assay larger numbers of specimens for HAV or anti-HAV. Subsequent development of sensitive immunofluorescence (IF) assays, solid-phase radioimmunoassays (RIA), and enzyme immunoassays @IA) for HAV and antiHAV heralded intensive laboratory studies of the biophysical and biochemical properties of the virus as well as efforts to define the pathogenesis and clinical course of disease. Results of the latter studies showed that the bulk of HAV was usually excreted in stool before the onset of clinical symptoms. Other serologic studies demonstrated that alI acutely Ill patients had circulating anti-HAV IgM, while all convalescent patients were positive for anti-HAV IgG. The development of sensitive serologic tests (RIA and EIA) that could differentiate between anti-HAV IgM and IgG made it possible to serodiagnose an acute case of hepatitis A using a single acute-phase serum specimen.
INTRODUCTION
Viral hepatitis is caused by at least three distinct agents and is a major health problem in the United States and abroad. Approximately 60,000 cases of viral hepatitis A are reported annually in the United States (MMWR Annual Report, 1977); however, this figure represents only a fraction of the actual occurrence. In fact, it has recently been estimated that at least 100,000 cases of icteric hepatitis A occur annually in the United States (Francis et al., in preparation) and that several times this number of people are subclinically infected. Hepatitis A was thought to be caused by an enteric agent well before hepatitis A virus (HAV) was recovered from stool. In early outbreaks, especially in military populations in World War I, transmission by the fecal-oral route was hypothesized (Blumer, 1923).
* World Health Org~ization
Collaborating Center for Reference and Research on Vlral Hepatitis.
32
Human experiments
undertaken
to document
feces from jaundiced
individuals,
when fed to humans,
later (Paul et al., 1945). Studies
conducted
196Os, at the Willowbrook
State School,
tinct types of viral hepatitis
(Krugman
fecal-oral
transmission
demonstrated
that
would cause hepatitis four weeks
by Krugman
firmly established
and colleagues
in the early
the existence
of two dis-
et al., 1962, 1967). The agent associated
with
the tr~smission of shop-incubation hepatitis was designated MS-l. This agent was later identified as a strain of HAV. This strain of HAV has been shown to be antigenically indistinguishable from that recovered from the stools of naturally infected individuals in the United States and in other countries throughout ISOLATION
AND CHARACTERIZATION
The visualization
of the hepatitis
OF HEPATITIS
the world. A VIRUS
A virus (HAV) particle by immune
electron
micro-
scopy (IEM) in acute-phase stools of both experimentally and naturally infected individuals (Feinstone et al., 1973; Gravelle et al., 1975) marked the beginning of intensive laboratory efforts to characterize this virus. Mo~hologic~ly, HAV is a 27-29 nm diameter spherical particle with an icosahedral symmetry. Both electron-lu~nt (‘empty’) and electron-dense (‘full’) virus particles can be visualized by IEM and EM in liver, bile and stool specimens obtained from acutely infected individuals. The empty virus particles are devoid of nucleic acid, while the full particles contain electron-dense nucleic acid that often gives the appearance of a core-like structure. Preliminary studies (Feinstone et al., 1974) showed that HAV recovered from the stool of an experimentally infected volunteer banded in a cesium chloride (C&l) density gradient at a buoyant density of approximately 1.40 g/cm3. HAV was postulated to be a parvovirus since it possessed the morphologic dimensions and buoyant density characteristic of this class of small, DNA-containing viruses. Additional investigations demonstrated that HAV was acid, ether, and heat stable, but sensitive to UV irradiation and formalin treatment (Provost et al., 1973, 1975b). One of these early studies (Provost et al., 1973) also showed that HAV infectivity banded in a CsCl density gradient primarily at a buoyant density of 1.34 g/cm3, a biophysical property characteristic of enteroviruses. This in the cytoplasm HAV could be an strated that HAV
same study also revealed the presence of 27 nm virus-like particles of infected hepatocytes, a finding consistent with the concept that RNA-contain~g virus. Another study (Bradley et al., 1975) demonparticles, recovered from the stools of human patients and experi-
mentally infected chimpanzees, possessed a primary buoyant density of approximately 1.33 g/cm3. Minor peaks of HAV were also detected in CsCl gradients at buoyant densities of 1.39- 1.41 and 1.50 g/cm”. These workers suggested that HAV could be an enterovims based on its small size and primary buoyant density of 1.32Ll.33 gJcm3 (Bradley et al., 1975, 1976). HAV with multiple buoyant stools (Bradley et al., 1977a); densities as low as 1.29 g/cm3
A subsequent report further documented the existence of density properties in CsCl in both human and chimpanzee major virus peaks were found in CsCl gradients at buoyant and as high as 1.48 g/cm3. This same study also established
33
the serologic identity
of virus particles banding
at 1.34 g/cm3 and 1.48 g/cm3. Another
study (Bradley et al., 1977b) showed that HAV was excreted in a cyclic fashion in stools of experimentally infected chimpanzees; the buoyant density property of HAV particles was found to be dependent upon the time at which they were excreted from the animal. The fmding of a temporal relationship between the time of excretion and the buoyant density property of the virus resolved the earlier discrepancy in the reported buoyant densities of HAV. More definitive biophysical and biochemical studies of light and heavy density HAV showed that both ‘species’ of HAV had a sedimentation coefficient of 160 S in neutral sucrose gradients (Bradley et al., 1978a; Siegl and Frosner, 1978a), a property characteristic of picornaviruses. Interestingly, heavy density HAV (1.45 g/cm3) was found to differ from light density HAV (1.34 g/cm3) in its unusual ability to bind Cs” ions. In sucrose gradients containing 1.5 M CsCl, heavy density virus was found to have a sedimentation coefficient of 230 S while light density HAV sedimented at 157 S (Bradley et al., 1978a). This observation suggested that the heavy density HAV was structurally different from the morphologically identical light density HAV; this difference was postulated to account for the increased binding of Cs’ ions to the dense particle, presumably to the nucleic acid itself. Treatment of light and heavy density HAV particles at alkaline pH was found to render them susceptible to degradation by ribonuclease (RNase) but not by deoxyribonuclease (DNase) (Bradley et al., 1978a). RNase digestion of treated particles resulted in a substantial loss of 157 S virus particles and antigen activity, as determined by IEM and radioimmunoassay, respectively. No losses in particles or antigen activity were seen after digestion with DNase. These findings experimentally confirmed the notion that HAV is a picorna (small, RNA-containing) virus. More detailed analysis of the genome of purified light density HAV revealed that it is comprised of a linear, single-stranded RNA molecule (Siegl and Frosner, 1978b). The RNA was shown to have a kinked appearance and was found to range in length from 0.5 to 3.5 pm; molecules of viral RNA were found distributed and 1.7 pm, respectively. HAV purified
in two major size classes with modal lengths of 1.2
from stool by polyethylene
in CsCl, and molecular
sieve chromatography
glycol precipitation,
isopycnic
was found to be infectious
banding
in a chimpanzee
and useful as an immunogen in rabbits for the production of ‘monospecific’ anti-HAV (Bradley et al., 1977~). Purification of HAV from stools has also been accomplished by a five-step procedure involving differential centrifugation, organic solvent extraction, molecular sieve chromatography, ion-exchange chromatography, and isopycnic banding in CsCl (Locarnini et al., 1978a). A modification of the latter procedure, in which ionexchange chromatography was omitted, was found to yield HAV of sufficient purity for the characterization of its constituent polypeptides. Analysis of purified HAV by discontinuous SDS-PAGE* revealed three major polypeptides with molecular weights of
*SDS, sodium
dodecyl
sulfate;
PAGE,
polyacrylamide
gel electrophoresis.
34
34,000, 25,500, and 23,000. These polypeptides are similar in molecular weight to three of the four major polypeptides characteristic of virus belonging to the genus Enterovirus within the family Picornaviridae (Coulepis et al., 1978). PATHOGENESIS
OF DISEASE
With the development of serologic tests for the detection of HAV particles, HAV antigen, and antibody to HAV (to be described in detail below) it became possible to study the pathogenesis of disease in humans and in experimenta~y infected animals, including marmosets and chimpanzees (Maynard et al., 1975a, b; Dienstag et al., 1975a; Schulman et al., 1976; Ebert et al., 1978). The earliest experiments employed IEM for the detection of HAV in serial (daily) stool specimens from experimentally infected individuals. One study showed that the bulk of HAV was excreted just prior to the peak of alanine ~notr~sferase (ALT) activity and appro~mately l--2 weeks before the onset of jaundice (Dienstag et al., 1975b). Other studies of the fecal excretion patterns of HAV in infected
chimpanzees and humans have shown similar results (Dienstag et al., 197Sa,b; Bradley et al., 1977b;Flehmig et al., 1977; Frosner et al., 1977; Rakela and Mosley, 1977), although some individuals have been found to excrete virus for as long as l-2 weeks after the onset of jaundice. A significant proportion of acutely ill patients do not have virus detectable in stool by IEM or by radio~munoassay (RIA), a finding that correlates well with the early infectivity of stools and epidemiology of hepatitis A. Preliminary studies of the pathogenesis of hepatitis A in experimentally infected chimpanzees (Murphy et al., 1978) and marmosets (Mathiesen et al., 1978a) suggest that HAV replicates solely in the liver and not in the gut. Presumably, HAV gains access to the gut via excretion into bile canahculi which empty through the terminal bile ducts into the common bile duct. The virus may then be diverted to either the gallbladder or the duodenum. This latter hypothesis is supported by the visualization of HAV particles in liver preparations and in gallbladder bile of acutely infected animals (Bradley et al., 1976; Schulman et al., 1976). HAV antigen has been detected by i~unofluorescence (IF) only in the cytoplasm of infected hepatocytes of humans, chimpanzees (Murphy et al., 1978), and marmosets (Bradley et al., 1978a; Mathiesen et al., 1978a) in keeping with the fact that RNA-containing enteroviruses
replicate in the cytoplasm
(Murphy et al., 1978) detected by IF in the inoculation, coincident that HAV antigen can
of infected cells. In infected chimpanzees
and marmosets (Mathiesen et al., 1978a) HAV antigen can be liver of intravenously inoculated animals as early as 7 days after with the appearance of HAV in stool. An important fading is be detected in stool and liver before elevations in ALT or SICD
activity are observed (Figs. 1 and 2). HAV antigen in liver is found in maximum concentration at the peak of ALT activity, while stool HAV is at a maximum approximately 2 weeks before the peak of enzyme activity. Yoshizawa et al. (1980) have reported Bnding anti-HAV IgA in the stools of patients with acute hepatitis A. Anti-HAV IgA was detected soon after the disappearance of HAV from stool, or about the time ALT activity
35
Chllrnl Hepoiltls /-\
1
hbc.
5
IO
15
20
25
_ALT
30
35
40
45
50
55
60
Day After lnoculotion
Fig, 1. The
temporal
relationship
IgM and IgG, and alanine infected
of stool
HAV
aminotransferase
J
o
1 I 0
‘* . . . . . 1 4
I 8
1 12
_.
I 16
Fig. 2. Markers acute-phase
of HAV
chimpanzee
its relationship
infection stool
I 24
T 28
I 32
ponent
IgA,
serum
anti-HAV
for a typical
patient
in liver and stool. in stool.)
(The
ALT
Panel
activity.
absence
iw+f+’ 40 44
52
56
60
intravenously
A shows
Panel
the early
B shows
63
70
inoculated development
the simultaneous
with
an HAV-positive
of serum appearance
anti-HAV of HAV
at a time when HAV is still detectable
of anti-HAV
the relationship
in concentration
48
of HAV in stool
to the development
Panel C illustrates
C’3. C’3 is depressed
I 36
lnaculotlon
in a chimpanzee
suspension.
to serum
FA in liver may be related excreted
anti-HAV
illness are shown
.. . . . *
I 20
Day After
antigen
to clinical
with HAV.
50
and
stool
excretion,
activity
between
[either
IgM or IgA] that
can
total serum IgM and complement
at a time when IgM concentration
by
mask HAV
is at a maximum.
com-
in serum was at a maximum. It is interesting to note that antibody to HAV often appears in serum before elevations in ALT activity occur and that anti-HAV antibody is found in the sera of all acutely infected
individuals.
creased in concentration
the time ALT activity
found to increase
during
The C’3 component
of complement
is elevated,
is de-
while total IgM is
during the same period of time. Since IgM-specific
anti-HAV
is the
predominant antibody type during the acute-phase of disease (see discussion below), it is reasonable to assume that circulating immune complexes comprised of anti-HAV IgM and HAV exist in acute-phase sera. The consumption of C’3, coupled with the increase in total IgM during the acute phase of disease, further suggests the involvement of circulating immune complexes (CICs) in the pathogenesis of disease. It is currently unclear whether or not replication of HAV in vivo exerts a direct cytopathic hepatocytes; liver damage may very well result from the combined cytolytic CICs and active viral replication.
effect on effects of
DI’AGNOSIS OF INFECTION
Although IEM is a relatively sensitive and specific procedure for the detection of HAV particles and anti-HAV, it is a time-consuming method and must be performed by an experienced electron microscopist. The development of simpler serologic tests for HAV and anti-HAV, including complement fixation (CF) (Provost et al., 1975a), immune adherence hemagglutination (IAHA) (Miller et al., 1975) solid-phase radioimmunoassay (RIA) (Hollinger et al., 1975, 1976; Purcell et al., 1976; Flehmig et al., 1978) and solid-phase enzyme-linked immunosorbent assay (ELISA) (Duermeyer et al., 1978; Mathiesen et al., 1978a) has made it feasible to routinely screen specimens for HAV or anti-HAV. RIA and ELISA procedures, in particular, are suitable for the analysis of large numbers of specimens and may be readily performed by a laboratory technician. The early application of IEM, IAHA, and RIA procedures to the diagnosis of hepatitis A virus infection clearly showed that not all acutely infected patients had detectable virus in stool, nor could useful levels of HAV antigen be detected in serum. The variable excretion patterns of HAV in stools from infected patients and the inability of even the most sensitive serologic tests to detect HAV antigen in all acute-phase stools made it necessary to resort to measurement of an individual’s serologic response. Seroconversion to HAV infection was first documented by an IEM technique in which serum anti-HAV was crudely quantitated by noting the amount of antibody that bound to reagent HAV particles (Feinstone et al., 1973; Gravelle et al., 1975). This early procedure, however, was not practically useful in the serodiagnosis of acute hepatitis A since paired acute and convalescent sera were required for the determination of an antibody response. Unfortunately, CF, IAHA, RIA and ELISA procedures also suffer from the marked disadvantage of requiring paired sera for the diagnosis of hepatitis A. As with most virus infections, infection with HAV induces a virus-specific IgM antibody response during the acute phase of disease (Bradley et al., 1977d, 1979; Locarnmi et al., 1977; Bradley and Maynard, 1978). This antibody response occurs in all in-
37
fected individuals, although the duration of anti-HAV IgM and the relative proportions of acute-phase IgM- and IgG-specific anti-HAV may vary from person to person. It is evident
from Fig. 1 that
any serologic
test that can discriminate
between
anti-HAV
IgM (acute-phase) and IgG (convalescent-phase) antibody is suitable for the diagnosis of acute viral hepatitis A (Bradley et al., 1979). A variety of RIA and ELISA procedures has been adapted for the purpose of differentially or specifically detecting anti-HAV IgG and/or IgM. Brief descriptions
of the basic principles of these tests follow.
HAV antigen or antibody to HAV can be detected by solid-phase RIA procedures (Hollinger et al., 1975; Purcell et al.> 1976; Bradley et al., 1977d) as shown in Fig. 3. IgG containing anti-HAV activity is used to coat the wells of a polyvinyl microtiter Uplate (Fig. 3A) when HAV antigen is to be determined in a specimen. After the wells of the U-plate have been coated with antibody, aliquots of the test specimens (normally stools) are loaded into the wells. HAV antigen present in stool will then combine with specific anti-HAV; after incubation, the il-plate is washed to remove unreacted materials. In the final step, aliquots of 1251-labeled IgG are added to the wells; HAV antigen present in any of the wells will react with the radiolabeled ‘probe.’ After the second incubation period, the wells are again washed to remove unreacted probe, cut out with scissors, and placed in a gamma spectrometer for counting. The positivity of a given test specimen is determined by computing the ratio of the test specimen’s c.p.m. (P) to that of the average c.p.m. of 5 negative stools (N). Statistical analysis of large numbers of P/N vaIues has shown that a ratio of 2.1 adequately discriminates between negative (P/N <
Coat Well With Anti-HAV IgG
Add Aliquot Of Test Stod
Add Radioiobeied Anti-HAV IgG
t
------+
. 0 Coat Well With Diluted Patient’s Serum
Q Add Aliquot Of Reagent HAV
Fig. 3. Radioimmunoassay
Count
0
Add Radiolobeled Anti-HAV IgG
for HAV antigen (A) and antibody
to HAV (B).
38
2.1) and HAV-positive
(P/N > 2.1) stools. The RIA for anti-HAV is performed
in basic-
ally the same manner (Fig. 3B), except that the patient’s serum is used to coat the U-plate well. Test sera are normally diluted 1 : 1000 in phosphate-buffered saline (PBS) and then aliquoted
into U-p!ate wells. A standard HAV antigen (either a stool suspension
homogenate)
or a liver
is used in place of the stool specimen described above. The rest of the test
procedure is the same as that of the RIA for HAV antigen. Sera that contain anti-HAV activity (either IgM- or IgG-specific) will react with the reagent HAV antigen; radiolabeled probe will subsequently combine with the HAV antigen retained in wells coated with antiHAV positive sera. The positivity of a serum is determined by computing aP/N ratio, where N is the average c.p.m. of 5 negative sera. Sera are considered to be positive for anti-HAV ifP/N>2.1. Enzyme-linked immunosorbent assay (ELlSA) for HA V and anti-HA V In place of the radiolabeled
IgG probe used in RIA procedures,
ELISA techniques
employ IgG conjugated to an enzyme, such as alkaline phosphatase or horseradish peroxidase (HRP). The enzyme-conjugated IgG serves as indicator in the assay by producing a colored reaction product from a suitable substrate (Locarnini et al., 1978b; Mathiesen et al., 1978b). One of the most commonly used indicator enzymes is HRP; substrates for this enzyme include 5-aminosalicylic acid (S’-AS), ortho-phenylenediamine (OPD), and [2,2’-azino-di-(3-ethylbenzthiazoline-6-sulfonate)] (ABTS). 5’-AS and OPD both yield yellow reaction products, while ABTS gives a blue-green product. As can be seen in Fig. 4, the ELISA procedure, shown here in the configuration of an antibody sandwich, is basically the same as that of the solid-phase RIA procedure (described above). Although the sensitivity of the ELISA procedures may be somewhat less than a comparably configured RIA, ELISA procedures in general have several distinct advantages over their RIA counterparts. These advantages include long shelf-life of kit components (conjugate, substrate, etc.) as well as the economy and simplicity of the test itself, since a relatively inexpensive spectrophotometer can be used to measure the colored reaction product.
f+
-
j-+#
a
0 Coot Well With Ant)-HAV IgG (Y Pat~enti Serum
fi-+
0%
c3 Add IgG-Enzyme Conjugate
Add Aliquot Of Test Stool or Reagent HAV
Enzyme + Substrate
@ Add Substrote 6-AS.OPD,or
Fig. 4.
@@p-E”zyme,HRp~ -
-
ABTS)
-
Colored Reaction Product 0 Measure Colored Product (Visually or Spectrophotometricolly)
Enzyme-linked immunosorbent assay (ELISA) for HAV antigen and anti-HAV.
39
Alternative
configurations
of the basic RIA and ELISA procedures
discussed above
have been described, including a commercially produced RIA kit (HAVAB, Abbott Laboratories) for the detection of anti-HAV (Bradley et al., 1979). As of April 1980, this kit is the only one that is commercially available. However, other manufacturers are soon expected
to market
RIA or ELISA kits for the detection
of anti-HAV.
The
following discussion of the determination of IgM- and IgC-specific anti-HAV by RIA and ELISA procedures includes a description of a modified HAVAB procedure that can be used to differentially detect anti-HAV IgM and IgG. p-Chain (IgM) blocking RIA A modification of the solid-phase RIA procedure for the detection of anti-HAV was shown to be useful in the differentiation of acute- and convalescent-phase hepatitis A sera (Bradley et al., 1977d). The modified RIA employed an additional step in which goat anti-human IgM @-chain-specific) was used to block binding of reagent HAV to test serum IgM coated on the U-plate well. Acute-phase sera, containing primarily antiHAV IgM, were shown to be blocked by anti-IgM antibody, yielding reduced c.p.m. compared to the unblocked control. Convalescent sera, containing primarily, if not entirely, anti-HAV IgC, were not blocked by the addition of anti-IgM antibody to the U-plate well. Although this p-chain-blocking RIA procedure was found to be useful in the serodiagnosis of acute viral hepatitis A, it required the analysis of two or more dilutions of the test serum and use of a highly specific blocking antibody. It should be noted that many commercial preparations of goat, rabbit, or horse anti-human IgM were also found to contain anti-human IgG activity. Modified competitive binding RIA for IgM anti-HA V Abbott
Laboratories
recently
introduced
a radioimmunoassay
kit (HAVAB*)
for
the determination of anti-HAV in serum. The standard HAVAB procedure, however, does not differentially detect anti-HAV IgM and IgG and cannot be used to serodiagnose acute viral hepatitis A using a single serum specimen. The principle of the standard HAVAB procedure is shown in Fig. 5. The HAVAB test is a solid-phase competitive binding RIA based on the use of a polystyrene bead coated with HAV derived from infected primate (marmoset) liver. The solid-phase HAV can combine with anti-HAV IgM or IgG in a test serum as well as with the 1251-labeled IgG anti-HAV used as the test ‘probe.’ If a patient’s serum is bead to a mixture of this serum and of the ‘*‘I-IgG probe. If a patient’s of a HAV-coated bead to a mixture
*All
commercial
endorsement
names
are
used
by the U.S. Department
for
negative for anti-HAV, addition of a HAV-coated ‘*‘I-IgG anti-HAV will result in maximum binding serum is positive for anti-HAV, however, addition of test serum and ‘251-IgG anti-HAV will result in
identification
of Health,
only
Education,
and
their
and Welfare.
mention
does
not
constitute
40
Mixture: +
I.wiient Serum, Anti-HAV Neg. _
High CPM Mixture: Polystyrene Beads Coated Wii HAV
+
I. Patient Serum, An&t/IV Pas. _
LoY; CPM
Fig. 5. Commercially available test (HAVAB) for the detection of anti-HAV in serum.
a decreased binding of the 12’I-IgG probe. The patient’s anti-HAV, whether it is of the IgM or IgG class of antibody, will compete with 12’I-IgG for HAV binding sites. Presumably, higher titred sera will result in greater competition with 1251-IgC for HAV binding sites. A modified
form
of the standard
HAVAB test was developed
at our Division for dif-
ferentiation of IgM- and IgC-specific anti-HAV in a single acute-phase serum specimen (Bradley et al., 1979). The modified HAVAB procedure uses protein A from StaphyZococcus aureus cells to preferentially absorb anti-HAV IgG from a patient’s serum. It is a well-known fact that protein A has a much higher affinity for IgG than for IgM and that it will react with approximately 95% of the IgG species (subclasses 1, 2, and 4) present in serum. Under appropriate test conditions the predominant anti-HAV antibody type in a patient’s serum can be readily determined by the modified HAVAB procedure (referred to as HAVAM). Invalid test results (i.e. false positive anti-HAV IgM) may result if improper dilutions or aliquots of serum are used. Protein A used for the absorption of serum must also be titred to determine its potency, since different lots and S. aureus strains vary in their capacity to absorb IgG. One of the advantages of the HAVAM procedure is its ability to determine the predominant anti-HAV antibody type, whether it is IgM or IgG, in contrast to p-chain-specific anti-HAV procedures that yield positive test results only if anti-HAV IgM is present (to be discussed in detail below). Another potential advantage of the HAVAM test is its relatively moderate sensitivity for anti-HAV IgM. Sera collected from infected individuals more than 4 weeks after the onset of symptoms are often negative for anti-HAV IgM and almost all sera collected 2-3 months after onset of illness are negative for anti-HAV IgM. This relatively narrow anti-HAV IgM ‘window’ facilitates diagnosis of a patient’s illness, since there is a clear temporal relationship of illness to positivity for anti-HAV IgM. The HAVAM procedure has been successfully used to define major epidemics of viral hepatitis A in Burma, France, and the United States, and has proved to be an accurate and reliable laboratory test when properly performed.
41
p-Chain specific RIA and ELISA procedures for the detection of IgM anti-HA V Anti-HAV IgM can be specifically detected by RIA and ELISA procedures that incorporate the use of a p-chain-specific antibody that will bind human IgM. Several recent reports, in fact, have described p-chain-specific anti-HAV tests; the basic principles of these tests are briefly reviewed below. Solid-phase RIA for anti-HAV IgM (Flehmig
et al., 1979). The principles
of this test
are shown in Fig. 6. Rabbit antiserum to human IgM @-chain-specific) is used to coat the wells of a polyvinyl microtiter plate. Diluted patient serum is then added to one of the U-plate wells. IgM antibody present in the patient’s serum will bind to the p-chainspecific antibody coated on the well, forming a double-antibody sandwich. A specified quantity of reagent HAV is added to the well; anti-HAV IgM bound to the solid-phase anti-p antibody will then bind HAV. Finally, radiolabeled IgC, containing anti-HAV activity, is added to the well where it will bind to HAV. Test sera containing anti-HAV IgM will yield high c.p.m., whereas sera negative for anti-HAV IgM will yield low c.p.m. Positivity of a serum for acute-phase (IgM) anti-HAV able negative and positive control sera. The solid-phase
can be evaluated by using suitRIA for anti-HAV IgM has been
shown to be specific and highly sensitive. In fact, of 60 sera collected from different patients at various times after the onset of icterus, 25 (42%) were found to be positive for anti-HAV IgM as long as 6 months after onset, while 13 (22%) were judged to be positive for anti-HAV IgM 52 weeks after onset (Flehmig et al., 1979). These findings demonstrate that the sensitivity of the RlA test for IgM anti-HAV is greater than that of the modified HAVAB procedure. A potential disadvantage of this test procedure, however, would appear to be related to its extreme sensitivity, since a patient with acute nonA hepatitis could conceivably be positive for anti-HAV IgM a year or more after infection with HAV. In this regard, it would be necessary to couple the test results with the patient’s history of viral hepatitis to resolve the question
Ck+3 Well With Rabbit Anti-Human IgM
Add Diluted Patient Serum
of acute hepatitis A virus infection.
Add Aliquit Of Reagent HAV
(mu-chain specific)
b --@!-““I
-
Count
@
Add ‘251-lgG Anti-HAV
Fig. 6. Radioimmunoassay
8 Evaluate Test Results
for anti-HAV
IgM (~-chain-specific)
in acute-phase
hepatitis
A serum.
42
Alternatively,
test conditions
could be altered to reduce the inherent
sensitivity
of the
RIA procedure and eliminate the possibility of a ‘false positive’ result and subsequent m~diagnosis. It should also be noted that rheumatoid factor, a potentially interfering antibody,
was not found to interfere in the above RIA for anti-HAV IgM.
ELI,!24 for anti-N. V IgM (Moller and Mathiesen, 1979). The principles of this test are shown in Fig. 7, and are basically the same as those described for the RIA procedure, except that HRP-conjugated IgG containing anti-HAV activity is used as the test probe. Test sera positive for anti-HAV Igh4 will yield a ye~ow-colored reaction product in the ELISA procedure, while sera negative for anti-HAV IgM will not yield a colored reaction product. Rheumatoid factor was not found to give a positive reaction in the ELISA test for anti-HAV IgM. It is presently unclear whether or not the sensitivity of the ELISA procedure for anti-HAV IgM will be problematic in the interpretation of some test results. As previously mentioned, however, intentional reduction in the sensitivity of this test procedure could be used to circumvent such difficulties. ELKSA for anti-HAV
IgM (Locarnini et al., 1979). A variation of the above ELISA procedure is shown in Fig. 8. The wells of a microtiter plate are coated with a standard IgG ~ont~~g anti-IIAV activity. Reagent HAV is then added to each well where it will bind to anti-HAV. Aliquots of diluted patient sera are added to the wells; anti-HAV Igh4 or IgG present in the sera will bind to the solid-phase HAV. Anti-HAV IgM bound to HAV is detected by an enzyme conjugate consisting of HRP linked to a goat anti-
human &M-specific h-chain) IgG. Sera positive for anti-HAV IgM wi.Il yield a yellowcolored reaction product that can be quantitated visually or spectrophotomet~c~y.
CoatWell With Add Diluted Robblt Anti-Human Patient Serum IgM (mu-chain specific)
Add Aliquot Of Reagent HAV
+ Substrate
Add Anti-YAV IgGEnzyme Conjugate
Fig. 7. Enzyme-iinked
-
Add Substrate
~munosorben~ phase hepatitis A serum.
Colored Reaction Product
Evaluate Test Results
assay (ELBA) for anti-HAV IgM (p-chain-specific)
in acute-
43
Coot Well With Antl-HAV IgG
Add Aliquot Of Reagent HAV
l
>-T:;RP
+ Substrate
Add Aliquot Of Diluted Patient Serum
-
Colwed ReactIon Product
ti @ Add HRP-Conjugated Goot IgG Anti-Humon IgM (mu-chain specific) Fig. 8. Alternative
Q Evoluate Test Results
@ Add Substrate
ELISA method for the detection of anti-HAV IgM (M-chain-specific) acute-phase
hepatitis A serum. CONCLUSIONS Although much has been learned about the basic biophysical and biochemical properties of HAV, relatively little is known about the molecular structure and composition of the viral capsid polypeptides and RNA genome. Furthermore, no virus-specific RNAdependent RNA polymerase (replicase) has been identified. It appears that detailed studies of the molecular fine structure of HAV and its replication must await successful cultivation of the virus in vitro. Indeed, several encouraging reports (Provost and Hilleman, 1979; Purcell, personal communication; Maynard, personal communication), describing the propagation of HAV in tissue culture, offer some hope for future detailed studies of the virus as well as the eventual production of a vaccine. Preliminary studies of the pathogenesis of hepatitis A infection that HAV replicates
support
solely in the liver and not in other organs, including
the notion the gut. Al-
though HAV can occasionally be found by FA in kidney, spleen and lymph nodes of experimentally infected animals, it is thought that localization of antigen in these tissues is related to the process of clearance of virus from the liver. Rapid advances in our understanding
of the serologic responses of infected individuals
has led to the development of a variety of tests for IgG- and/or IgM-specific anti-HAV. There is little doubt that several commercially produced kits will eventually appear on the market and will make serodiagnosis of acute viral hepatitis A a routine procedure.
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8,277. Locarnini,
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S.M. Feinstone,
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D. Lorenz,
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and R.H.
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and D.H.
Krushak,
1978b, 1975a,
1978a,
J. Infect.
J. Clin. MicroJ. Infect.
Dis.
45
Maynard, J.E., D. Lorenz, D.W. Bradley, S.M. Feinstone, D.H. Krushak, L.F. Barker and R.H. Purcell, 1975b, Am. J. Med. Sci. 270,81. Miller, W.J., P.J. Provost, W.J. McAleer, O.L. Ittensohn, V.M. Villarejos and MR. HiIIeman, 1975, Proc. Sot. Exp. Biol. Med. 149,254. Moller, A.M. and L.R. Mathiesen, 1979, J. Clin. Microbial. 10,628. Morbidity and Mortality Weekly Report, Annual Summary, 1977, Center for Disease Control: Reported Morbidity and Mortality in the United States. Murphy, B.L., J.E. Maynard, D.W. Bradley, J.W. Ebert, L.R. Mathiesen and R.H. Purcell, 1978, Infect. Immun. 21,663. Paul, J.R., W.P. Havens, A& Sabin and C.B. PhiIips, 1945, J. Am. Med. Assoc. 128,91 I. Provost, P.J., O.L. Ittensohn, V.M. VilIarejos, J.A. Arguedas and M.R. Hilleman, 1973, Proc. Sot. Exp. Biol. Med. 142, 1257. Provost, P. J., O.L. Ittensohn, V.M. Villarejos and M.R. HiIleman, 1975a, Proc. Sot. Exp. Biol. Med. 148,962. Provost, P. J., B.S. Wolanski, W. J. Miller, O.L. Ittensohn, W. J. McAleer and M.R. HilIeman, 1975b, Proc. Sot. Exp. Biol. Med. 148,532. Provost, P. J. and M.R. Hilleman, 1979, Proc. Sot. Exp. Biol. Med. 160, 213. Purcell, RH., D.C. Wong, Y. Moritsugu, J.L. Dienstag, JA. Routenberg and J.D. Boggs, 1976, J. Immunol. 116,349. Rakela, J. and J.W. Mosley, 1977, J. Infect. Dis. 135,933. Schulman, A.N., J.L. Dienstag, DR. Jackson, JH. Hoofnagle, R.J. Gerety, R.N. Purcell and L.F. Barker, 1976, J. Infect. Dis. 134,80. Siegl, G . and G .G. Frosner, 1978a, J. Viral. 26,40. Siegl, G. and G.G. Frosner, 1978b, J. Virol. 26,48. Yoshizawa, H., Y. Itho, S. Iwakiri, F;. Tsuda, S. Nakano, Y. Miyakawa and M. Mayumi, 1980, Gastroenterology 78, 114.
2 (1980)
31
31-45
Biomedical Press
HEPATITIS A VIRUS INFECTION: PATHOGENESIS
AND SERODIAGNOSIS
OF
ACUTE DISEASE
DANIEL W. BRADLEY Hepatitis Laboratories
Division *, Bureau
Health Service, Department AZ 85014,
of Epidemiology.
of Health, Education,
Center for Disease Control,
U.S. Public
and Welfare, 4402 North Seventh Street, Phoenix,
USA.
The early development of immune electron microscopic (IEM) methods for the detection of HAV in acute-phase stool suspensions and antibody to HAV (~ti-HAV) in serum made it possible to serologically identify cases of hepatitis A using paired acute and convalescent phase sera. Introduction of less cumbersome and time-consuming serologic test methods, including complement Bxation (CF) and immune adherence hemagglutination (JAHA), made it feasible to rapidly assay larger numbers of specimens for HAV or anti-HAV. Subsequent development of sensitive immunofluorescence (IF) assays, solid-phase radioimmunoassays (RIA), and enzyme immunoassays @IA) for HAV and antiHAV heralded intensive laboratory studies of the biophysical and biochemical properties of the virus as well as efforts to define the pathogenesis and clinical course of disease. Results of the latter studies showed that the bulk of HAV was usually excreted in stool before the onset of clinical symptoms. Other serologic studies demonstrated that alI acutely Ill patients had circulating anti-HAV IgM, while all convalescent patients were positive for anti-HAV IgG. The development of sensitive serologic tests (RIA and EIA) that could differentiate between anti-HAV IgM and IgG made it possible to serodiagnose an acute case of hepatitis A using a single acute-phase serum specimen.
INTRODUCTION
Viral hepatitis is caused by at least three distinct agents and is a major health problem in the United States and abroad. Approximately 60,000 cases of viral hepatitis A are reported annually in the United States (MMWR Annual Report, 1977); however, this figure represents only a fraction of the actual occurrence. In fact, it has recently been estimated that at least 100,000 cases of icteric hepatitis A occur annually in the United States (Francis et al., in preparation) and that several times this number of people are subclinically infected. Hepatitis A was thought to be caused by an enteric agent well before hepatitis A virus (HAV) was recovered from stool. In early outbreaks, especially in military populations in World War I, transmission by the fecal-oral route was hypothesized (Blumer, 1923).
* World Health Org~ization
Collaborating Center for Reference and Research on Vlral Hepatitis.
32
Human experiments
undertaken
to document
feces from jaundiced
individuals,
when fed to humans,
later (Paul et al., 1945). Studies
conducted
196Os, at the Willowbrook
State School,
tinct types of viral hepatitis
(Krugman
fecal-oral
transmission
demonstrated
that
would cause hepatitis four weeks
by Krugman
firmly established
and colleagues
in the early
the existence
of two dis-
et al., 1962, 1967). The agent associated
with
the tr~smission of shop-incubation hepatitis was designated MS-l. This agent was later identified as a strain of HAV. This strain of HAV has been shown to be antigenically indistinguishable from that recovered from the stools of naturally infected individuals in the United States and in other countries throughout ISOLATION
AND CHARACTERIZATION
The visualization
of the hepatitis
OF HEPATITIS
the world. A VIRUS
A virus (HAV) particle by immune
electron
micro-
scopy (IEM) in acute-phase stools of both experimentally and naturally infected individuals (Feinstone et al., 1973; Gravelle et al., 1975) marked the beginning of intensive laboratory efforts to characterize this virus. Mo~hologic~ly, HAV is a 27-29 nm diameter spherical particle with an icosahedral symmetry. Both electron-lu~nt (‘empty’) and electron-dense (‘full’) virus particles can be visualized by IEM and EM in liver, bile and stool specimens obtained from acutely infected individuals. The empty virus particles are devoid of nucleic acid, while the full particles contain electron-dense nucleic acid that often gives the appearance of a core-like structure. Preliminary studies (Feinstone et al., 1974) showed that HAV recovered from the stool of an experimentally infected volunteer banded in a cesium chloride (C&l) density gradient at a buoyant density of approximately 1.40 g/cm3. HAV was postulated to be a parvovirus since it possessed the morphologic dimensions and buoyant density characteristic of this class of small, DNA-containing viruses. Additional investigations demonstrated that HAV was acid, ether, and heat stable, but sensitive to UV irradiation and formalin treatment (Provost et al., 1973, 1975b). One of these early studies (Provost et al., 1973) also showed that HAV infectivity banded in a CsCl density gradient primarily at a buoyant density of 1.34 g/cm3, a biophysical property characteristic of enteroviruses. This in the cytoplasm HAV could be an strated that HAV
same study also revealed the presence of 27 nm virus-like particles of infected hepatocytes, a finding consistent with the concept that RNA-contain~g virus. Another study (Bradley et al., 1975) demonparticles, recovered from the stools of human patients and experi-
mentally infected chimpanzees, possessed a primary buoyant density of approximately 1.33 g/cm3. Minor peaks of HAV were also detected in CsCl gradients at buoyant densities of 1.39- 1.41 and 1.50 g/cm”. These workers suggested that HAV could be an enterovims based on its small size and primary buoyant density of 1.32Ll.33 gJcm3 (Bradley et al., 1975, 1976). HAV with multiple buoyant stools (Bradley et al., 1977a); densities as low as 1.29 g/cm3
A subsequent report further documented the existence of density properties in CsCl in both human and chimpanzee major virus peaks were found in CsCl gradients at buoyant and as high as 1.48 g/cm3. This same study also established
33
the serologic identity
of virus particles banding
at 1.34 g/cm3 and 1.48 g/cm3. Another
study (Bradley et al., 1977b) showed that HAV was excreted in a cyclic fashion in stools of experimentally infected chimpanzees; the buoyant density property of HAV particles was found to be dependent upon the time at which they were excreted from the animal. The fmding of a temporal relationship between the time of excretion and the buoyant density property of the virus resolved the earlier discrepancy in the reported buoyant densities of HAV. More definitive biophysical and biochemical studies of light and heavy density HAV showed that both ‘species’ of HAV had a sedimentation coefficient of 160 S in neutral sucrose gradients (Bradley et al., 1978a; Siegl and Frosner, 1978a), a property characteristic of picornaviruses. Interestingly, heavy density HAV (1.45 g/cm3) was found to differ from light density HAV (1.34 g/cm3) in its unusual ability to bind Cs” ions. In sucrose gradients containing 1.5 M CsCl, heavy density virus was found to have a sedimentation coefficient of 230 S while light density HAV sedimented at 157 S (Bradley et al., 1978a). This observation suggested that the heavy density HAV was structurally different from the morphologically identical light density HAV; this difference was postulated to account for the increased binding of Cs’ ions to the dense particle, presumably to the nucleic acid itself. Treatment of light and heavy density HAV particles at alkaline pH was found to render them susceptible to degradation by ribonuclease (RNase) but not by deoxyribonuclease (DNase) (Bradley et al., 1978a). RNase digestion of treated particles resulted in a substantial loss of 157 S virus particles and antigen activity, as determined by IEM and radioimmunoassay, respectively. No losses in particles or antigen activity were seen after digestion with DNase. These findings experimentally confirmed the notion that HAV is a picorna (small, RNA-containing) virus. More detailed analysis of the genome of purified light density HAV revealed that it is comprised of a linear, single-stranded RNA molecule (Siegl and Frosner, 1978b). The RNA was shown to have a kinked appearance and was found to range in length from 0.5 to 3.5 pm; molecules of viral RNA were found distributed and 1.7 pm, respectively. HAV purified
in two major size classes with modal lengths of 1.2
from stool by polyethylene
in CsCl, and molecular
sieve chromatography
glycol precipitation,
isopycnic
was found to be infectious
banding
in a chimpanzee
and useful as an immunogen in rabbits for the production of ‘monospecific’ anti-HAV (Bradley et al., 1977~). Purification of HAV from stools has also been accomplished by a five-step procedure involving differential centrifugation, organic solvent extraction, molecular sieve chromatography, ion-exchange chromatography, and isopycnic banding in CsCl (Locarnini et al., 1978a). A modification of the latter procedure, in which ionexchange chromatography was omitted, was found to yield HAV of sufficient purity for the characterization of its constituent polypeptides. Analysis of purified HAV by discontinuous SDS-PAGE* revealed three major polypeptides with molecular weights of
*SDS, sodium
dodecyl
sulfate;
PAGE,
polyacrylamide
gel electrophoresis.
34
34,000, 25,500, and 23,000. These polypeptides are similar in molecular weight to three of the four major polypeptides characteristic of virus belonging to the genus Enterovirus within the family Picornaviridae (Coulepis et al., 1978). PATHOGENESIS
OF DISEASE
With the development of serologic tests for the detection of HAV particles, HAV antigen, and antibody to HAV (to be described in detail below) it became possible to study the pathogenesis of disease in humans and in experimenta~y infected animals, including marmosets and chimpanzees (Maynard et al., 1975a, b; Dienstag et al., 1975a; Schulman et al., 1976; Ebert et al., 1978). The earliest experiments employed IEM for the detection of HAV in serial (daily) stool specimens from experimentally infected individuals. One study showed that the bulk of HAV was excreted just prior to the peak of alanine ~notr~sferase (ALT) activity and appro~mately l--2 weeks before the onset of jaundice (Dienstag et al., 1975b). Other studies of the fecal excretion patterns of HAV in infected
chimpanzees and humans have shown similar results (Dienstag et al., 197Sa,b; Bradley et al., 1977b;Flehmig et al., 1977; Frosner et al., 1977; Rakela and Mosley, 1977), although some individuals have been found to excrete virus for as long as l-2 weeks after the onset of jaundice. A significant proportion of acutely ill patients do not have virus detectable in stool by IEM or by radio~munoassay (RIA), a finding that correlates well with the early infectivity of stools and epidemiology of hepatitis A. Preliminary studies of the pathogenesis of hepatitis A in experimentally infected chimpanzees (Murphy et al., 1978) and marmosets (Mathiesen et al., 1978a) suggest that HAV replicates solely in the liver and not in the gut. Presumably, HAV gains access to the gut via excretion into bile canahculi which empty through the terminal bile ducts into the common bile duct. The virus may then be diverted to either the gallbladder or the duodenum. This latter hypothesis is supported by the visualization of HAV particles in liver preparations and in gallbladder bile of acutely infected animals (Bradley et al., 1976; Schulman et al., 1976). HAV antigen has been detected by i~unofluorescence (IF) only in the cytoplasm of infected hepatocytes of humans, chimpanzees (Murphy et al., 1978), and marmosets (Bradley et al., 1978a; Mathiesen et al., 1978a) in keeping with the fact that RNA-containing enteroviruses
replicate in the cytoplasm
(Murphy et al., 1978) detected by IF in the inoculation, coincident that HAV antigen can
of infected cells. In infected chimpanzees
and marmosets (Mathiesen et al., 1978a) HAV antigen can be liver of intravenously inoculated animals as early as 7 days after with the appearance of HAV in stool. An important fading is be detected in stool and liver before elevations in ALT or SICD
activity are observed (Figs. 1 and 2). HAV antigen in liver is found in maximum concentration at the peak of ALT activity, while stool HAV is at a maximum approximately 2 weeks before the peak of enzyme activity. Yoshizawa et al. (1980) have reported Bnding anti-HAV IgA in the stools of patients with acute hepatitis A. Anti-HAV IgA was detected soon after the disappearance of HAV from stool, or about the time ALT activity
35
Chllrnl Hepoiltls /-\
1
hbc.
5
IO
15
20
25
_ALT
30
35
40
45
50
55
60
Day After lnoculotion
Fig, 1. The
temporal
relationship
IgM and IgG, and alanine infected
of stool
HAV
aminotransferase
J
o
1 I 0
‘* . . . . . 1 4
I 8
1 12
_.
I 16
Fig. 2. Markers acute-phase
of HAV
chimpanzee
its relationship
infection stool
I 24
T 28
I 32
ponent
IgA,
serum
anti-HAV
for a typical
patient
in liver and stool. in stool.)
(The
ALT
Panel
activity.
absence
iw+f+’ 40 44
52
56
60
intravenously
A shows
Panel
the early
B shows
63
70
inoculated development
the simultaneous
with
an HAV-positive
of serum appearance
anti-HAV of HAV
at a time when HAV is still detectable
of anti-HAV
the relationship
in concentration
48
of HAV in stool
to the development
Panel C illustrates
C’3. C’3 is depressed
I 36
lnaculotlon
in a chimpanzee
suspension.
to serum
FA in liver may be related excreted
anti-HAV
illness are shown
.. . . . *
I 20
Day After
antigen
to clinical
with HAV.
50
and
stool
excretion,
activity
between
[either
IgM or IgA] that
can
total serum IgM and complement
at a time when IgM concentration
by
mask HAV
is at a maximum.
com-
in serum was at a maximum. It is interesting to note that antibody to HAV often appears in serum before elevations in ALT activity occur and that anti-HAV antibody is found in the sera of all acutely infected
individuals.
creased in concentration
the time ALT activity
found to increase
during
The C’3 component
of complement
is elevated,
is de-
while total IgM is
during the same period of time. Since IgM-specific
anti-HAV
is the
predominant antibody type during the acute-phase of disease (see discussion below), it is reasonable to assume that circulating immune complexes comprised of anti-HAV IgM and HAV exist in acute-phase sera. The consumption of C’3, coupled with the increase in total IgM during the acute phase of disease, further suggests the involvement of circulating immune complexes (CICs) in the pathogenesis of disease. It is currently unclear whether or not replication of HAV in vivo exerts a direct cytopathic hepatocytes; liver damage may very well result from the combined cytolytic CICs and active viral replication.
effect on effects of
DI’AGNOSIS OF INFECTION
Although IEM is a relatively sensitive and specific procedure for the detection of HAV particles and anti-HAV, it is a time-consuming method and must be performed by an experienced electron microscopist. The development of simpler serologic tests for HAV and anti-HAV, including complement fixation (CF) (Provost et al., 1975a), immune adherence hemagglutination (IAHA) (Miller et al., 1975) solid-phase radioimmunoassay (RIA) (Hollinger et al., 1975, 1976; Purcell et al., 1976; Flehmig et al., 1978) and solid-phase enzyme-linked immunosorbent assay (ELISA) (Duermeyer et al., 1978; Mathiesen et al., 1978a) has made it feasible to routinely screen specimens for HAV or anti-HAV. RIA and ELISA procedures, in particular, are suitable for the analysis of large numbers of specimens and may be readily performed by a laboratory technician. The early application of IEM, IAHA, and RIA procedures to the diagnosis of hepatitis A virus infection clearly showed that not all acutely infected patients had detectable virus in stool, nor could useful levels of HAV antigen be detected in serum. The variable excretion patterns of HAV in stools from infected patients and the inability of even the most sensitive serologic tests to detect HAV antigen in all acute-phase stools made it necessary to resort to measurement of an individual’s serologic response. Seroconversion to HAV infection was first documented by an IEM technique in which serum anti-HAV was crudely quantitated by noting the amount of antibody that bound to reagent HAV particles (Feinstone et al., 1973; Gravelle et al., 1975). This early procedure, however, was not practically useful in the serodiagnosis of acute hepatitis A since paired acute and convalescent sera were required for the determination of an antibody response. Unfortunately, CF, IAHA, RIA and ELISA procedures also suffer from the marked disadvantage of requiring paired sera for the diagnosis of hepatitis A. As with most virus infections, infection with HAV induces a virus-specific IgM antibody response during the acute phase of disease (Bradley et al., 1977d, 1979; Locarnmi et al., 1977; Bradley and Maynard, 1978). This antibody response occurs in all in-
37
fected individuals, although the duration of anti-HAV IgM and the relative proportions of acute-phase IgM- and IgG-specific anti-HAV may vary from person to person. It is evident
from Fig. 1 that
any serologic
test that can discriminate
between
anti-HAV
IgM (acute-phase) and IgG (convalescent-phase) antibody is suitable for the diagnosis of acute viral hepatitis A (Bradley et al., 1979). A variety of RIA and ELISA procedures has been adapted for the purpose of differentially or specifically detecting anti-HAV IgG and/or IgM. Brief descriptions
of the basic principles of these tests follow.
HAV antigen or antibody to HAV can be detected by solid-phase RIA procedures (Hollinger et al., 1975; Purcell et al.> 1976; Bradley et al., 1977d) as shown in Fig. 3. IgG containing anti-HAV activity is used to coat the wells of a polyvinyl microtiter Uplate (Fig. 3A) when HAV antigen is to be determined in a specimen. After the wells of the U-plate have been coated with antibody, aliquots of the test specimens (normally stools) are loaded into the wells. HAV antigen present in stool will then combine with specific anti-HAV; after incubation, the il-plate is washed to remove unreacted materials. In the final step, aliquots of 1251-labeled IgG are added to the wells; HAV antigen present in any of the wells will react with the radiolabeled ‘probe.’ After the second incubation period, the wells are again washed to remove unreacted probe, cut out with scissors, and placed in a gamma spectrometer for counting. The positivity of a given test specimen is determined by computing the ratio of the test specimen’s c.p.m. (P) to that of the average c.p.m. of 5 negative stools (N). Statistical analysis of large numbers of P/N vaIues has shown that a ratio of 2.1 adequately discriminates between negative (P/N <
Coat Well With Anti-HAV IgG
Add Aliquot Of Test Stod
Add Radioiobeied Anti-HAV IgG
t
------+
. 0 Coat Well With Diluted Patient’s Serum
Q Add Aliquot Of Reagent HAV
Fig. 3. Radioimmunoassay
Count
0
Add Radiolobeled Anti-HAV IgG
for HAV antigen (A) and antibody
to HAV (B).
38
2.1) and HAV-positive
(P/N > 2.1) stools. The RIA for anti-HAV is performed
in basic-
ally the same manner (Fig. 3B), except that the patient’s serum is used to coat the U-plate well. Test sera are normally diluted 1 : 1000 in phosphate-buffered saline (PBS) and then aliquoted
into U-p!ate wells. A standard HAV antigen (either a stool suspension
homogenate)
or a liver
is used in place of the stool specimen described above. The rest of the test
procedure is the same as that of the RIA for HAV antigen. Sera that contain anti-HAV activity (either IgM- or IgG-specific) will react with the reagent HAV antigen; radiolabeled probe will subsequently combine with the HAV antigen retained in wells coated with antiHAV positive sera. The positivity of a serum is determined by computing aP/N ratio, where N is the average c.p.m. of 5 negative sera. Sera are considered to be positive for anti-HAV ifP/N>2.1. Enzyme-linked immunosorbent assay (ELlSA) for HA V and anti-HA V In place of the radiolabeled
IgG probe used in RIA procedures,
ELISA techniques
employ IgG conjugated to an enzyme, such as alkaline phosphatase or horseradish peroxidase (HRP). The enzyme-conjugated IgG serves as indicator in the assay by producing a colored reaction product from a suitable substrate (Locarnini et al., 1978b; Mathiesen et al., 1978b). One of the most commonly used indicator enzymes is HRP; substrates for this enzyme include 5-aminosalicylic acid (S’-AS), ortho-phenylenediamine (OPD), and [2,2’-azino-di-(3-ethylbenzthiazoline-6-sulfonate)] (ABTS). 5’-AS and OPD both yield yellow reaction products, while ABTS gives a blue-green product. As can be seen in Fig. 4, the ELISA procedure, shown here in the configuration of an antibody sandwich, is basically the same as that of the solid-phase RIA procedure (described above). Although the sensitivity of the ELISA procedures may be somewhat less than a comparably configured RIA, ELISA procedures in general have several distinct advantages over their RIA counterparts. These advantages include long shelf-life of kit components (conjugate, substrate, etc.) as well as the economy and simplicity of the test itself, since a relatively inexpensive spectrophotometer can be used to measure the colored reaction product.
f+
-
j-+#
a
0 Coot Well With Ant)-HAV IgG (Y Pat~enti Serum
fi-+
0%
c3 Add IgG-Enzyme Conjugate
Add Aliquot Of Test Stool or Reagent HAV
Enzyme + Substrate
@ Add Substrote 6-AS.OPD,or
Fig. 4.
@@p-E”zyme,HRp~ -
-
ABTS)
-
Colored Reaction Product 0 Measure Colored Product (Visually or Spectrophotometricolly)
Enzyme-linked immunosorbent assay (ELISA) for HAV antigen and anti-HAV.
39
Alternative
configurations
of the basic RIA and ELISA procedures
discussed above
have been described, including a commercially produced RIA kit (HAVAB, Abbott Laboratories) for the detection of anti-HAV (Bradley et al., 1979). As of April 1980, this kit is the only one that is commercially available. However, other manufacturers are soon expected
to market
RIA or ELISA kits for the detection
of anti-HAV.
The
following discussion of the determination of IgM- and IgC-specific anti-HAV by RIA and ELISA procedures includes a description of a modified HAVAB procedure that can be used to differentially detect anti-HAV IgM and IgG. p-Chain (IgM) blocking RIA A modification of the solid-phase RIA procedure for the detection of anti-HAV was shown to be useful in the differentiation of acute- and convalescent-phase hepatitis A sera (Bradley et al., 1977d). The modified RIA employed an additional step in which goat anti-human IgM @-chain-specific) was used to block binding of reagent HAV to test serum IgM coated on the U-plate well. Acute-phase sera, containing primarily antiHAV IgM, were shown to be blocked by anti-IgM antibody, yielding reduced c.p.m. compared to the unblocked control. Convalescent sera, containing primarily, if not entirely, anti-HAV IgC, were not blocked by the addition of anti-IgM antibody to the U-plate well. Although this p-chain-blocking RIA procedure was found to be useful in the serodiagnosis of acute viral hepatitis A, it required the analysis of two or more dilutions of the test serum and use of a highly specific blocking antibody. It should be noted that many commercial preparations of goat, rabbit, or horse anti-human IgM were also found to contain anti-human IgG activity. Modified competitive binding RIA for IgM anti-HA V Abbott
Laboratories
recently
introduced
a radioimmunoassay
kit (HAVAB*)
for
the determination of anti-HAV in serum. The standard HAVAB procedure, however, does not differentially detect anti-HAV IgM and IgG and cannot be used to serodiagnose acute viral hepatitis A using a single serum specimen. The principle of the standard HAVAB procedure is shown in Fig. 5. The HAVAB test is a solid-phase competitive binding RIA based on the use of a polystyrene bead coated with HAV derived from infected primate (marmoset) liver. The solid-phase HAV can combine with anti-HAV IgM or IgG in a test serum as well as with the 1251-labeled IgG anti-HAV used as the test ‘probe.’ If a patient’s serum is bead to a mixture of this serum and of the ‘*‘I-IgG probe. If a patient’s of a HAV-coated bead to a mixture
*All
commercial
endorsement
names
are
used
by the U.S. Department
for
negative for anti-HAV, addition of a HAV-coated ‘*‘I-IgG anti-HAV will result in maximum binding serum is positive for anti-HAV, however, addition of test serum and ‘251-IgG anti-HAV will result in
identification
of Health,
only
Education,
and
their
and Welfare.
mention
does
not
constitute
40
Mixture: +
I.wiient Serum, Anti-HAV Neg. _
High CPM Mixture: Polystyrene Beads Coated Wii HAV
+
I. Patient Serum, An&t/IV Pas. _
LoY; CPM
Fig. 5. Commercially available test (HAVAB) for the detection of anti-HAV in serum.
a decreased binding of the 12’I-IgG probe. The patient’s anti-HAV, whether it is of the IgM or IgG class of antibody, will compete with 12’I-IgG for HAV binding sites. Presumably, higher titred sera will result in greater competition with 1251-IgC for HAV binding sites. A modified
form
of the standard
HAVAB test was developed
at our Division for dif-
ferentiation of IgM- and IgC-specific anti-HAV in a single acute-phase serum specimen (Bradley et al., 1979). The modified HAVAB procedure uses protein A from StaphyZococcus aureus cells to preferentially absorb anti-HAV IgG from a patient’s serum. It is a well-known fact that protein A has a much higher affinity for IgG than for IgM and that it will react with approximately 95% of the IgG species (subclasses 1, 2, and 4) present in serum. Under appropriate test conditions the predominant anti-HAV antibody type in a patient’s serum can be readily determined by the modified HAVAB procedure (referred to as HAVAM). Invalid test results (i.e. false positive anti-HAV IgM) may result if improper dilutions or aliquots of serum are used. Protein A used for the absorption of serum must also be titred to determine its potency, since different lots and S. aureus strains vary in their capacity to absorb IgG. One of the advantages of the HAVAM procedure is its ability to determine the predominant anti-HAV antibody type, whether it is IgM or IgG, in contrast to p-chain-specific anti-HAV procedures that yield positive test results only if anti-HAV IgM is present (to be discussed in detail below). Another potential advantage of the HAVAM test is its relatively moderate sensitivity for anti-HAV IgM. Sera collected from infected individuals more than 4 weeks after the onset of symptoms are often negative for anti-HAV IgM and almost all sera collected 2-3 months after onset of illness are negative for anti-HAV IgM. This relatively narrow anti-HAV IgM ‘window’ facilitates diagnosis of a patient’s illness, since there is a clear temporal relationship of illness to positivity for anti-HAV IgM. The HAVAM procedure has been successfully used to define major epidemics of viral hepatitis A in Burma, France, and the United States, and has proved to be an accurate and reliable laboratory test when properly performed.
41
p-Chain specific RIA and ELISA procedures for the detection of IgM anti-HA V Anti-HAV IgM can be specifically detected by RIA and ELISA procedures that incorporate the use of a p-chain-specific antibody that will bind human IgM. Several recent reports, in fact, have described p-chain-specific anti-HAV tests; the basic principles of these tests are briefly reviewed below. Solid-phase RIA for anti-HAV IgM (Flehmig
et al., 1979). The principles
of this test
are shown in Fig. 6. Rabbit antiserum to human IgM @-chain-specific) is used to coat the wells of a polyvinyl microtiter plate. Diluted patient serum is then added to one of the U-plate wells. IgM antibody present in the patient’s serum will bind to the p-chainspecific antibody coated on the well, forming a double-antibody sandwich. A specified quantity of reagent HAV is added to the well; anti-HAV IgM bound to the solid-phase anti-p antibody will then bind HAV. Finally, radiolabeled IgC, containing anti-HAV activity, is added to the well where it will bind to HAV. Test sera containing anti-HAV IgM will yield high c.p.m., whereas sera negative for anti-HAV IgM will yield low c.p.m. Positivity of a serum for acute-phase (IgM) anti-HAV able negative and positive control sera. The solid-phase
can be evaluated by using suitRIA for anti-HAV IgM has been
shown to be specific and highly sensitive. In fact, of 60 sera collected from different patients at various times after the onset of icterus, 25 (42%) were found to be positive for anti-HAV IgM as long as 6 months after onset, while 13 (22%) were judged to be positive for anti-HAV IgM 52 weeks after onset (Flehmig et al., 1979). These findings demonstrate that the sensitivity of the RlA test for IgM anti-HAV is greater than that of the modified HAVAB procedure. A potential disadvantage of this test procedure, however, would appear to be related to its extreme sensitivity, since a patient with acute nonA hepatitis could conceivably be positive for anti-HAV IgM a year or more after infection with HAV. In this regard, it would be necessary to couple the test results with the patient’s history of viral hepatitis to resolve the question
Ck+3 Well With Rabbit Anti-Human IgM
Add Diluted Patient Serum
of acute hepatitis A virus infection.
Add Aliquit Of Reagent HAV
(mu-chain specific)
b --@!-““I
-
Count
@
Add ‘251-lgG Anti-HAV
Fig. 6. Radioimmunoassay
8 Evaluate Test Results
for anti-HAV
IgM (~-chain-specific)
in acute-phase
hepatitis
A serum.
42
Alternatively,
test conditions
could be altered to reduce the inherent
sensitivity
of the
RIA procedure and eliminate the possibility of a ‘false positive’ result and subsequent m~diagnosis. It should also be noted that rheumatoid factor, a potentially interfering antibody,
was not found to interfere in the above RIA for anti-HAV IgM.
ELI,!24 for anti-N. V IgM (Moller and Mathiesen, 1979). The principles of this test are shown in Fig. 7, and are basically the same as those described for the RIA procedure, except that HRP-conjugated IgG containing anti-HAV activity is used as the test probe. Test sera positive for anti-HAV Igh4 will yield a ye~ow-colored reaction product in the ELISA procedure, while sera negative for anti-HAV IgM will not yield a colored reaction product. Rheumatoid factor was not found to give a positive reaction in the ELISA test for anti-HAV IgM. It is presently unclear whether or not the sensitivity of the ELISA procedure for anti-HAV IgM will be problematic in the interpretation of some test results. As previously mentioned, however, intentional reduction in the sensitivity of this test procedure could be used to circumvent such difficulties. ELKSA for anti-HAV
IgM (Locarnini et al., 1979). A variation of the above ELISA procedure is shown in Fig. 8. The wells of a microtiter plate are coated with a standard IgG ~ont~~g anti-IIAV activity. Reagent HAV is then added to each well where it will bind to anti-HAV. Aliquots of diluted patient sera are added to the wells; anti-HAV Igh4 or IgG present in the sera will bind to the solid-phase HAV. Anti-HAV IgM bound to HAV is detected by an enzyme conjugate consisting of HRP linked to a goat anti-
human &M-specific h-chain) IgG. Sera positive for anti-HAV IgM wi.Il yield a yellowcolored reaction product that can be quantitated visually or spectrophotomet~c~y.
CoatWell With Add Diluted Robblt Anti-Human Patient Serum IgM (mu-chain specific)
Add Aliquot Of Reagent HAV
+ Substrate
Add Anti-YAV IgGEnzyme Conjugate
Fig. 7. Enzyme-iinked
-
Add Substrate
~munosorben~ phase hepatitis A serum.
Colored Reaction Product
Evaluate Test Results
assay (ELBA) for anti-HAV IgM (p-chain-specific)
in acute-
43
Coot Well With Antl-HAV IgG
Add Aliquot Of Reagent HAV
l
>-T:;RP
+ Substrate
Add Aliquot Of Diluted Patient Serum
-
Colwed ReactIon Product
ti @ Add HRP-Conjugated Goot IgG Anti-Humon IgM (mu-chain specific) Fig. 8. Alternative
Q Evoluate Test Results
@ Add Substrate
ELISA method for the detection of anti-HAV IgM (M-chain-specific) acute-phase
hepatitis A serum. CONCLUSIONS Although much has been learned about the basic biophysical and biochemical properties of HAV, relatively little is known about the molecular structure and composition of the viral capsid polypeptides and RNA genome. Furthermore, no virus-specific RNAdependent RNA polymerase (replicase) has been identified. It appears that detailed studies of the molecular fine structure of HAV and its replication must await successful cultivation of the virus in vitro. Indeed, several encouraging reports (Provost and Hilleman, 1979; Purcell, personal communication; Maynard, personal communication), describing the propagation of HAV in tissue culture, offer some hope for future detailed studies of the virus as well as the eventual production of a vaccine. Preliminary studies of the pathogenesis of hepatitis A infection that HAV replicates
support
solely in the liver and not in other organs, including
the notion the gut. Al-
though HAV can occasionally be found by FA in kidney, spleen and lymph nodes of experimentally infected animals, it is thought that localization of antigen in these tissues is related to the process of clearance of virus from the liver. Rapid advances in our understanding
of the serologic responses of infected individuals
has led to the development of a variety of tests for IgG- and/or IgM-specific anti-HAV. There is little doubt that several commercially produced kits will eventually appear on the market and will make serodiagnosis of acute viral hepatitis A a routine procedure.
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Maynard, J.E., D. Lorenz, D.W. Bradley, S.M. Feinstone, D.H. Krushak, L.F. Barker and R.H. Purcell, 1975b, Am. J. Med. Sci. 270,81. Miller, W.J., P.J. Provost, W.J. McAleer, O.L. Ittensohn, V.M. Villarejos and MR. HiIIeman, 1975, Proc. Sot. Exp. Biol. Med. 149,254. Moller, A.M. and L.R. Mathiesen, 1979, J. Clin. Microbial. 10,628. Morbidity and Mortality Weekly Report, Annual Summary, 1977, Center for Disease Control: Reported Morbidity and Mortality in the United States. Murphy, B.L., J.E. Maynard, D.W. Bradley, J.W. Ebert, L.R. Mathiesen and R.H. Purcell, 1978, Infect. Immun. 21,663. Paul, J.R., W.P. Havens, A& Sabin and C.B. PhiIips, 1945, J. Am. Med. Assoc. 128,91 I. Provost, P.J., O.L. Ittensohn, V.M. VilIarejos, J.A. Arguedas and M.R. Hilleman, 1973, Proc. Sot. Exp. Biol. Med. 142, 1257. Provost, P. J., O.L. Ittensohn, V.M. Villarejos and M.R. HiIleman, 1975a, Proc. Sot. Exp. Biol. Med. 148,962. Provost, P. J., B.S. Wolanski, W. J. Miller, O.L. Ittensohn, W. J. McAleer and M.R. HilIeman, 1975b, Proc. Sot. Exp. Biol. Med. 148,532. Provost, P. J. and M.R. Hilleman, 1979, Proc. Sot. Exp. Biol. Med. 160, 213. Purcell, RH., D.C. Wong, Y. Moritsugu, J.L. Dienstag, JA. Routenberg and J.D. Boggs, 1976, J. Immunol. 116,349. Rakela, J. and J.W. Mosley, 1977, J. Infect. Dis. 135,933. Schulman, A.N., J.L. Dienstag, DR. Jackson, JH. Hoofnagle, R.J. Gerety, R.N. Purcell and L.F. Barker, 1976, J. Infect. Dis. 134,80. Siegl, G . and G .G. Frosner, 1978a, J. Viral. 26,40. Siegl, G. and G.G. Frosner, 1978b, J. Virol. 26,48. Yoshizawa, H., Y. Itho, S. Iwakiri, F;. Tsuda, S. Nakano, Y. Miyakawa and M. Mayumi, 1980, Gastroenterology 78, 114.