Hepatitis a virus infection: Pathogenesis and serodiagnosis of acute disease

The early development of immune electron microscopic (IEM) methods for the detection of HAV in acute-phase stool suspensions and antibody to HAV (anti-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 fixation (CF) and immune adherence hemagglutination (IAHA), 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 (EIA) for HAV and anti-HAV 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 all 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-phase serum specimen.     

Non-A, non-B hepatitis in chimpanzees: interference with acute hepatitis A virus and chronic hepatitis B virus infections

Two chimpanzees with persistent non-A, non-B (NANB) hepatitis were superinfected with marmoset-passaged MS-1 HAV. Two control chimpanzees were also infected with marmoset-passaged HAV. Neither animal with persistent NANB hepatitis developed elevated alanine aminotransferase (ALT) activity, whereas both control chimpanzees exhibited ALT elevations within 3 weeks after inoculation. In addition, both NANB-infected chimpanzees demonstrated a delayed anti-HAV antibody response in which one animal failed to produce detectable IgM anti-HAV. With the exception of one stool, all serial liver biopsy specimens and daily stool suspensions from the superinfected chimpanzees were negative for HAV antigen. One chimpanzee with a chronic HBV infection was superinfected with non-A, non-B hepatitis and was shown to develop elevated ALT activity and hepatocyte ultrastructural alterations accompanied by a marked reduction in the titer of serum HBsAg. Our combined findings indicate that acute and persistent non-A, non-B hepatitis infections are capable of interferring with two distinctly different hepatotropic viruses. These results also suggest that in vitro detection of non-A, non-B hepatitis infection or virus(es) may be achieved by antibody-independent methodologies that employ the basic principle of viral interference.     

Hepatitis a virus: Growth characteristics of in vivo and in vitro propagated wild and attenuated virus strains

Serial passage of the MS-1 strain hepatitis A virus (HAV) in marmosets was shown to increase the yield of virus and to shorten the incubation period from approximately 55 days in the first passage to 3–7 days in the ninth and higher passages. Intravenous inoculation of susceptible chimpanzees with MS-1 HAV was found to result in a typical course of disease in two animals who had received eighth marmoset-passage virus, including the occurrence of elevated ALT activity, presence of HAV antigen in liver and stool, and seroconversion to anti-HAV. Two chimpanzees inoculated with 20th passage MS-1 HAV (M001 liver homogenate) exhibited normal or nearly normal ALT activity and had no demonstrable or significant HAV in weekly liver biopsy specimens or in serial stool suspensions obtained during 64 days of observation. However, both animals seroconverted to anti-HAV within 2 weeks after inoculation, as did the animals who had received eighth passage MS-1 HAV. These findings suggest that subpassage of the MS-1 strain of HAV in marmosets resulted in the generation of an attenuated virus strain that was still capable of inducing a vigorous antibody response in intravenously infected chimpanzees. Serial propagation of wild and attenuated strains of HAV (HAS-15 and MS-1/M001, respectively) in FRhK-4 cells was associated with a significant decrease in the growth period for both viruses. Our studies have also shown that HAS-15 HAV can be recovered in maximum yield in later passages as early as 2 to 3 days after inoculation.     

Serodiagnosis of viral hepatitis A: detection of acute-phase immunoglobulin M anti-hepatitis A virus by radioimmunoassay.

A modified micro solid-phase radioimmunoassay (RIA) for antibody to hepatitis A virus (anti-HAV) was developed. This double antibody procedure was performed by coating the surface of a polyvinyl microtiter plate "well" with 200 microliter of a 1:1,000 dilution of a patient's test serum. Purified HAV and 125I-labeled immunoglobulin G (IgG) anti-HAV were then sequentially added to form an antibody sandwich. The specificity and sensitivity of the RIA procedure for anti-HAV were verified by examination of coded human and chimpanzee serum specimens. Radioimmunoassay of early-acute-phase serum specimens from human cases of hepatitis A revealed the presence of anti-HAV activity. Differential examination by RIA of IgG and IgM fractions of acute-phase sera from experimentally infected chimpanzees demonstrated that IgM contained the bulk of the anti-HAV activity. A modification of the RIA procedure for anti-HAV (RIA-IgM blocking), incorporating an incubation step with anti-IgM (Mu chain specific), was further shown to differentiate acute- from convalescent-phase hepatitis A sera. This adapted RIA-IgM blocking procedure required less than 1 microliter of a single acute-phase serum specimen for the diagnosis of viral hepatitis A.     

Excretion of hepatitis A virus in the stools of hospitalized hepatitis patients

A study was carried out to determine whether hepatitis A virus (HAV) can be detected in the stools of patients hospitalized for HAV infection. Acute phase samples of whole blood and stool, as well as completed questionnaires, were obtained from 31 patients hospitalized at any of 13 hospitals in the Phoenix metropolitan area. Blood specimens were tested for hepatitis B surface antigen (HBsAg), IgG antibody to HAV (IgG anti-HAV), and IgM antibody to HAV (IgM anti-HAV). Stools were tested for HAV by radioimmunoassay. Five patients (16.1%) had acute hepatitis B, five (16.1%) had acute non-A/non-B hepatitis, and 21 (67.7%) had acute hepatitis A. Of these 21 patients with acute hepatitis A, 11 (52.4%) were found to have HAV in their stools. These results confirm the potential for infectivity of stools of patients hospitalized for hepatitis A and emphasizes the need for caution when dealing with such stools.     

FULMINANT TYPE A VIRAL HEPATITIS IN A CHIMPANZEE

A case of a chimpanzee with fulminant hepatitis caused by spontaneous hepatitis A virus (HAV) infection was reported. The liver at autopsy revealed massive liver cell necrosis with mononuclear and polymorphonuclear cell infiltration. Aggregation of HAV-like particles (22–25 nm in diameter) were found within the vesicles of hepatocytes under the electron microscope. Immunofluorescent examination of the liver showed positive staining for HAV antigen, C₁q, C3, C4, immunoglobulin M (IgM), and immunoglobulin G (IgG) in the hepatocytes and/or Kupffer cells in a granular fashion. The anti-HAV antibody (IgM type) and circulating immune complexes were detected in the postmortem serum. The present study suggests the possibility that the deposition of immune complexes of HAV and anti-HAV antibody in the liver cell plays an important role in the pathogenesis of massive liver cell necrosis in fulminant type A viral hepatitis.     

Diagnosis of hepatitis A infection: Comparative specificity of IgM capture assays using antigens derived from tissue cultures and marmoset faeces

Hepatitis A virus (HAV) antigens from two tissue culture sources were compared with that from the faeces of infected marmosets to determine whether the former were satisfactory substitutes. Sera from 313 healthy blood donors and 417 patients with various clinical conditions were tested for IgM class antibody to HAV (anti-HAV IgM) using an IgM antibody capture assay (MACRIA) with each of the 3 antigens.Forty-eight specimens, all from cases of acute hepatitis, were positive in MACRIA with all 3 antigens. Only 2 of the 313 blood donors' sera reacted at all. These reactions were weak and did not arise with all antigens. Weakly reactive specimens were also found in 3 out of the 13 clinical categories. Overall 12 weak reactions arose with the faecal antigen and 8 and 7 with the two tissue culture antigens. Rheumatoid factor (RhF) was detected in 8 of the weakly reactive specimens and these had significantly higher titres of anti-HAV than sera known to contain RhF that were unreactive in MACRIA.It is concluded that tissue culture derived HAV antigen should replace that from primates on the grounds of quality, economy and convenience: also that non-specific activity in HAV MACRIA is usually due to a combination of RhF and high anti-HAV titres, but is infrequently strong enough to cause reactions interpreted as positive.     

Aetiology of acute sporadic hepatitis in adults in Kenya

Markers for acute hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis non-A, non-B (HNANB) infections were examined in the sera of 94 patients presenting with acute hepatitis in Kenya. Hepatitis B virus was responsible for 70% of cases, HNANB for 18%, and HAV for only 12%. The use of an IgM anti-HBc assay increased the rate of diagnosis of acute HBV infection, thereby reducing the proportion of cases designated as NANB.     

Further evaluation of a live hepatitis A vaccine in marmosets.

Live, attenuated F' hepatitis A vaccine virus was studied in vivo in Saguinus labiatus marmosets for possible reversion to virulence, for possible establishment of persistent infection and for its capacity as a parenterally administered vaccine to induce immunity to oral infection. Serial transmission of the virus in S. labiatus, using infectious stool extracts for the second and third passages, produced no evidence of reversion of the F' vaccine virus to virulence. Monitoring for live HAV in stools over a 135-day period post-inoculation of marmosets with the F' vaccine revealed no evidence of persistent infection. Vaccinated animals were also shown to be resistant to infection on challenge by the oral route as well as by the previously demonstrated parenteral route.     

甲型肝炎病毒IgM AlphaLISA检测方法的初步建立

目的 建立甲型肝炎病毒抗体IgM的AlphaLISA检测.方法 浓缩甲型肝炎病毒抗原并生物素化,通过dot-Blot法摸索出生物素化的甲型肝炎病毒的最适量为6 × 10-8ng.优化供体珠、受体微珠、血清等实验反应条件,建立了甲型肝炎病毒的AlphaLISA的IgM检测方法.并对23份感染甲型肝炎病毒的IgM阳性患者和70份健康人的血清进行检测.结果 AlphaLISA甲型肝炎病毒的IgM方法检测:灵敏度(真阳性率)为78％;特异度(真阴性率)为98.5％;假阳性率为1％;假阴性率为22％;阳性预测值为95％;阴性预测值为93％;准确度为94％.结论 建立了均相、快速检测AlphaLISA甲型肝炎病毒IgM检测方法并得到初步应用.     

Application of a solid-phase radioimmunoassay and immune electron microscopy for hepatitis A in diagnosis and research

With crude virus suspensions from stool and antibodies from hepatitis-A patients, a solid-phase radioimmunoassay (RIA) for detection of hepatitis virus A (HVA) and antibodies against hepatitis virus A (anti-HVA) has been developed. Examples for the application of this test are demonstrated. Virus particles from the stools of the two patients were further characterized. Serologically, they were identical or very similar to the MS-1 strain. Isopycnic CsCl-gradient centrifugation of both strains revealed two peaks, but the particles of different densities did not differ in size or serologically. A modification of the RIA was also useful for determination of IgM antibodies in patients' sera fractionated by sucrose-density centrifugation.The application of the RIA method for serologic epidemiology is demonstrated by a comparison of anti-HVA prevalence in German and non-German women residing in Germany.     

用核酸序列分析鉴别甲肝病毒疫苗株与野毒株

目的鉴别在接种甲肝减毒活疫苗（Ｈ２株）后２３天发生的一例甲型肝炎,其病原体是疫苗株还是野毒株。方法用细胞培养、ＩＥＭ等确认病原体,接种普通狨猴检定病毒毒力,测定核苷酸序列分析病毒基因型。结果甲型肝炎的确诊依据有：抗ＩｇＭ（＋）,双份血清ＨＡＶ总抗体效价１６倍增长以及粪便排出ＨＡＶ（郑株）。该粪便悬液攻击普通狨猴后,肝脏呈现持续性组织病理改变,证明郑株是强毒ＨＡＶ。郑株的３个片段共１３７７个核苷酸序列分析表明,与１９８８年上海甲肝流行株合－５２的同源性高达（９８．３０～９９．４０）％,判定为ＩＡ基因亚型,与Ｈ２株疫苗病毒的基因亚型不同。结论该例甲型肝炎是接种甲肝减毒活疫苗中交叉感染野毒株引发的偶合病例,与疫苗接种无关     

Propagation of hepatitis A virus in human embryo fibroblasts

human diploid fibroblasts and human primary liver cell carcinoma cells (PLC/PRF/5) were infected with hepatitis A virus (HAV) adapted to growth in cell culture or derived directly from human stool. Viral antigen was expressed in PLC/PRF/5 cells 28 days after infection with cell culture-adapted HAV, and 50 days after infection with virus from human stool. In human fibroblasts the periods until first expression of viral antigen were 90 and 210 days, respectively. During further passages of HAV in fibroblasts the time of first appearance of antigen decreased to about 28 days. Biophysical properties of HAV extracted from infected fibroblasts were comparable to those of HAV derived directly from human stool. Immunofluorescence studies showed that the antigen was located exclusively within the cytoplasm of the infected fibroblasts. Kinetics of antigen production indicated that an equilibrium between virus multiplication and cell metabolism was reached in persistently infected fibroblasts.     

Hepatitis A virus in the liver and intestine of marmosets after oral inoculation.

A total of 12 seronegative marmosets (Saguinus mystax) were inoculated orally with hepatitis A virus (HAV) and sacrificed at 3- to 4-day intervals. Tissues from the livers, intestines, mesenteric lymph nodes, and spleens were obtained for immunofluorescence studies, and bile and intestinal contents were obtained for enzyme-linked immunosorbent assay studies. Two marmosets sacrificed on days 34 and 41 after inoculation developed antibody to HAV and demonstrated HAV in their livers but not in any part of their intestinal tissues. None of the remaining marmosets sacrificed from days -3 to 31 survived long enough to develop antibody to HAV, but an additional two marmosets, which were sacrificed on days 21 and 31, demonstrated HAV in their livers and also in bile but not in the intestinal tissues or their contents. The mesenteric lymph nodes and spleens were negative for HAV by immunofluorescence in all the marmosets. No evidence of HAV replication was demonstrated in any part of the intestine at any time during the incubation period or during acute illness in the marmosets inoculated orally with HAV. The shedding of HAV in stools in the late incubation period can be explained by excretion of HAV from the livers with the bile.     

Variation among hepatitis A virus strains. I. Genomic variation detected by T1 oligonucleotide mapping

The genomes of eight hepatitis A virus (HAV) strains originating from far distant geographic regions such as Europe, North Africa, Middle and North America, Australia and The People's Republic of China were compared by RNase T1 oligonucleotide mapping. For this purpose, the viruses were propagated in cell cultures and viral RNA was isolated from highly purified mature virions. It could be shown that variation in nucleotide sequence is common among HAV isolates, but is in the order of magnitude reported for other picornaviruses. For viruses isolated in cell culture directly from stool samples of diseased individuals, changes usually amounted to 1-4% of RNA genome sites. Genomic differences between two virus strains derived from one fecal sample but replicating at either 32 or 37 degrees C were in the same order of magnitude. Thereby, the number of consecutive in vitro passages proved to have only limited influence on the development of genetic variation. For two HAV strains, however, adaptation to and passage in marmosets evidently had imposed highly selective conditions which had favored the appearance of viral genomes differing in up to 75% of their large oligonucleotides (about 10% in sequence) from the oligonucleotide map of a reference HAV strain.     

Hepatitis a diagnosis in man: radioimmunoassay for hepatitis a antigen detection in faeces

A new radioimmunoassay (RIA) procedure using a double-sandwich technique was developed for the detection of hepatitis A antigen (HAV) in crude faecal extracts from patients involved in three outbreaks of type A hepatitis. Stools were obtained from 24 residents suffering from acute hepatitis A and from six children who remained asymptomatic throughout the epidemic. In addition, the HAV detection was performed in sera from 13 patients with hepatitis. HAV was detected in stools as early as five days before and as late as five days after the onset of jaundice. In this procedure, positive activity was only found in stools from patients with type A hepatitis, but not in negative controls. HAV was not detected in acute-phase sera. The double-sandwich RIA test used appears to be a reliable test for the large-scale screening of HAV in stool samples from patients suffering from type A hepatitis.     

Thermal treatment and infectivity of hepatitis A virus in human feces.

The susceptibility of white-lipped marmoset monkeys (Saguinus sp) to human hepatitis A virus (HAV) provides a system for evaluation of thermal inactivation of HAV in feces and contaminated shellfish. Intramuscular or oral administration of HAV derived from feces of four patients with acute hepatitis A induced hepatitis in 28--100% of the inoculated marmosets. A 10% (w/v) fecal pool (GBG-BM) prepared from two patients (GBG and GBM) induced hepatitis in marmosets (2/4 with 1 ml; 2/2 with 3 ml) when given orally as a 1 : 3 dilution. A HAV-baby food raw oyster mixture fed to fasted marmosets induced hepatitis in 1/4 and seroconversion in 2/4 animals. Two groups of oysters were injected with HAV (concentrated 3 : 1 by centrifugation of the GBG-BM pool); one group was treated at 140 degrees F for 19 minutes and the other served as an untreated control. In animals fed the untreated inoculum, 4/6 developed hepatitis and 6/6 seroconverted, whereas of those fed the heat-treated inoculum 1/7 developed hepatitis and 2/7 seroconverted. These data suggest that pasteurization methods could be developed that would eliminate shellfish-associated hepatitis A and retain the palatability of the shellfish.     

Comparison of serological tests for antibody to hepatitis A antigen, using coded specimens from individuals infected with the MS-1 strain of hepatitis A virus.

To compare serological tests for antibody to hepatitis A antigen (anti-HA), we tested 15 paired serum specimens, submitted under code, from individuals infected with the MS-1 strain of hepatitis A virus. Immune electron microscopy (IEM), immune adherence hemagglutination (IAHA), and solid-phase radioimmunoassay (RIA) tests for anti-HA were performed with hepatitis A antigen (HA Ag) derived from human stool; results were also compared with previously reported titers determined by IAHA with HA Ag derived from marmoset liver. Antibody titers (IAHA and RIA) and ratings (IEM) determined with stool-derived HA Ag compared favorably, and a seroresponse to HA Ag was detected by all three methods for every serum pair tested. Differences in titers were noted between IAHA tests with liver-derived and with stool-derived HA Ag, but the discrepancies could be accounted for by differences in test technique. The agreement found in this study among the three techniques was quite good and confirms the specificity and sensitivity of tests for anti-HA that are done with stool-derived HA-Ag.     

Acute hepatitis A is the chief etiology of acute hepatitis in Egyptian children: a single-center study

Acute hepatic illness is an important health issue in children. Our work aimed to determine the prevalence of viral hepatitis in symptomatic children. It is a prospective cohort study of 268 children presented with acute hepatitis. Complete blood count, liver panel, and anti-hepatitis A virus (HAV) IgM were done initially. Cases negative for HAV were tested for anti-hepatitis E (HEV) IgM, anti-Epstein-Barr virus viral capsid antigen (EBV VCA) IgM, anti-cytomegalovirus virus IgM, hepatitis B surface antigen, anti-hepatitis B core IgM antibody, and anti-HCV antibody. Anti-HCV was repeated after 12 weeks to exclude seroconversion. In cases with negative viral serology, ceruloplasmin, total immunoglobulin G, antinuclear antibody, and abdominal ultrasound were done. Follow-up visits were bimonthly till recovery, then after 6 months. The mean age?±?SD was 7.1?±?3.7 years (1.5–18), and 56% were males. Acute HAV infection was diagnosed in 260 (97%) of cases and acute EBV infection in one case (0.4%). HAV/HEV coinfection was excluded in 70 HAV-positive cases. Six (2.2%) children remain undiagnosed and one child lost follow-up. About 80% of HAV-cases had normal laboratory results within 45 days. Unusual presentation of HAV infection was noticed in six children: four (1.5%) were relapsing, one had a cholestatic course, and one case had severe hemolytic anemia. Acute HAV infection was the chief etiology of acute hepatitis in our Egyptian children. The majority of the presentations were mild and children recover within a few weeks. An unusual pattern of HAV in children can be observed in endemic areas.     

Fulminant hepatitis in asymptomatic hepatitis B surface antigen carriers in Greece

Eleven male fulminant hepatitis (FH) patients (mean age: 47.7 +/- 16 years) positive for hepatitis B surface antigen (HBsAg) but negative for IgM antibody to hepatitis B core antigen (IgM anti-HBc) were admitted consecutively to the Athens Hospital for Infectious Diseases between May 1981 and November 1983. Because of the absence of IgM anti-HBc, determined by an enzyme immunoassay, these patients were considered to be HBsAg carriers with a superimposed acute hepatitis. Three of the 11 patients received immunosuppressive chemotherapy during the six months before the onset of the acute hepatitis. None of the patients was homosexual or a drug addict. Infection with hepatitis A virus (HAV), hepatitis B virus (HBV), or hepatitis delta virus (HDV) was detected with serologic markers and/or molecular hybridization techniques. Fulminant hepatitis was attributed to spontaneous reactivation of chronic hepatitis B in four patients, chemotherapy-induced reactivation of chronic hepatitis B in three patients, HDV superinfection in one patient and possible superinfection by non-A, non-B agent(s), HDV, or HDV-like agents in three patients. Reactivation of chronic hepatitis B was an important cause of apparent acute hepatitis in heterosexual male HBsAg carriers from an area with a high prevalence of HBV infection.