Propagation and assay of hepatitis A virus in vitro

Ten strains of hepatitis A virus (HAV) originating from far distant geographical locations were adapted to growth in PLC/PRF/5 (human hepatoma derived) and/or MRC-5 (human embryonic lung) cells. In the course of primary adaptation some of these strains exhibited a predilection for distinct cultural conditions such as type of host cell and temperature of incubation. With progressive passage, variant viruses with quite different requirements could be selected; yet, it proved impossible to isolate a virus which replicated equally well in both types of cells and at both 32 and 37°C without at least one preceding passage under the new conditions. Analysis of the virus/cell relationship of well adapted HAV strains revealed that the replication cycle of HAV extends over about 24 h. Moreover, replication evidently passes from a state of active production of infectious virus to a phase during which hepatitis A antigen (HAAg) is synthesized and terminates in the state of persistent infection with markedly reduced synthetic activity. In all three phases replication of HAV is non-cytolytic and the vast majority of both infectious virus and of HAAg remains cell associated. The observations concerning the growth characteristics of HAV were used to develop two rapid in vitro assay systems for HAV infectivity (fluorescent focus assay and in situ RIA). Finally, the conditions for large scale production of infectious HAV and of HAAg in a cell factory system were analysed.     

Enhancement of hepatitis A propagation in tissue culture with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole

The adenosine analog 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) was found to increase the production of hepatitis A (HAV) antigen in two monkey kidney cell lines (Frhk-4 and Vero cells). DRB, a known inhibitor of the synthesis of messenger RNA, caused moderate changes in cell morphology. However, Frhk-4 cells could be maintained for several weeks at 80 microM of DRB, the concentration that caused maximal enhancement on HAV. DRB should be present from about the time of virus inoculation and its strongest effect was seen at low multiplicities of infection. Using radioimmunofocus assay it could be shown that DRB increased the amount of infectious virus. DRB treatment was applied in primary isolation of HAV from feces. In nine of ten strains HAV antigen expression was strongly increased and in six of the ten strains infectivity of harvested material increased by one 10log or more. DRB thus seems to be a useful enhancer of HAV growth in tissue culture.     

Internalisation of hepatitis C virus core protein by human conjunctival fibroblasts

Recent studies indicate that hepatitis C virus (HCV) proteins can mediate innate immune response and inflammation in conjunctival fibroblasts which contributes to the pathology of dry eye condition associated with chronic HCV infection. The present study investigates the phagocytic potential of human conjunctival fibroblasts (HCFj) for HCV core protein. HCFj cells were incubated with HCV core antigen for different periods of time, and fluorescent micrographs were taken to observe protein internalisation. HCFj cells were capable of internalising HCV core antigen within 1 h; this gives an insight into another molecular mechanism which may contribute towards HCV-associated conjunctival inflammation.     

Propagation of human hepatitis A virus in conventional cell lines

Fecal extracts of hepatitis A (HA) patients were selected for the presence of hepatitis A virus (HAV) by radioimmunoassay (RIA) and immune electron microscopy (IEM). When FL and Vero cells were inoculated with fecal extracts containing HAV, development of hepatitis A antigen (HAAg) was evident in the cytoplasm of the two cell lines by the indirect immunofluorescence (IF) test. The antigen was detectable in the cells 12 hr postinoculation (pi), and reached a plateau within two days pi. FL cell cultures inoculated with a specimen containing HAV were harvested and passaged four times. During the passages, efficient production of HAAg was confirmed in the infected cultures by three different serological tests: The indirect IF test, RIA using fixed cells, and RIA by the sandwich method. At the second and fourth passages, HAV particles were recovered in abundance from infected FL cell cultures by IEM. Throughout these experiments, no cytopathic effect (CPE) was discernible in the cultures.     

Increased circulation of hepatitis A virus genotype IIIA over the last decade in St Petersburg, Russia

The current study, covering the period 2004–2009, is a part of long‐term monitoring for hepatitis A virus (HAV) strains circulating in St Petersburg, Russia. The HAV RNA was isolated directly from the sera of hepatitis A patients and RT‐PCR was carried out using primer pairs for VP1/2A and VP1 genomic regions. PCR products were sequenced and 324 nucleotides from VP1/2A and 332 from the VP1 region were used for phylogenetic analysis. The results show that the IA subtype was the most common circulating subtype during the follow‐up period, as found in the previous study: almost 90% of the isolated HAV strains belonged to the IA subtype. The large hepatitis A food‐borne outbreak in St Petersburg in 2005 was caused by HAV IA. However, the proportion of HAV isolates belonging to subtype IIIA significantly increased in the period 2001–2009 (7.9%) compared to the period 1997–2000 (none found). The subtype IIIA was first found in St Petersburg in 2001 among a group of intravenous drug users. The increase in its circulation during the decade suggests that this previously unusual genotype has been permanently introduced into the general population of St Petersburg. These results indicate the usefulness of molecular epidemiological methods for studying changes in the circulation of HAV strains. J. Med. Virol. 84:1528–1534, 2012. © 2012 Wiley Periodicals, Inc.     

An experimental model of hepatitis A inMacaca mulatta infected with human hepatitis a virus

Institute of Experimental Pathology and Therapy, Academy of Medical Sciences of the USSR, Sukhumi. D. I. Ivanovskii Institute of Virology, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 109, No. 6, pp. 536–539, June, 1990.     

Time course of hepatitis A viremia and viral load in the blood of human hepatitis A patients

The hepatitis A virus (HAV) is the most common etiological cause of acute hepatitis infections in humans in industrialized countries. Investigations into the viral load during HAV viremia, however, are rare. Therefore, correlation studies between viral load, biochemical, and specific serological markers have been undertaken. The group of sera comprised a series of multiple consecutive blood samples drawn from 11 patients at different times after onset of the disease. During the period up to 70 days after the onset of icterus, the individual range was at 1 x 10(3) to 3 x 10(4) HAV genome equivalents/ml. From day 75 until 120 after onset of the disease, the levels traced were at 10(3). In one case, it was possible to trace 1.25 x 10(4) genome equivalents/ml up to 180 days after onset of icterus and in two cases even up to 408 and 490 days viral load levels of 5 x 10(3) and 4 x 10(4) were detected, respectively. The same sera were used to measure IgM class antibodies to hepatitis A virus and the total anti-HAV. The results demonstrate that a direct correlation to peak levels of viral load exists with peak serum transaminase levels, but neither with peak anti-HAV IgM levels nor with total anti-HAV. Decreasing amounts of anti-HAV IgM tend to occur with decreasing amounts of HAV genome equivalents; and, vice versa, increasing amounts of total anti-HAV are accompanied by decreasing amounts of HAV genome equivalents. The longest duration of viremia was found in patients infected with HAV genotype IA.     

Lack of complement-dependent cytolytic antibodies in hepatitis A virus infection

Sera collected from patients with acute hepatitis A virus (HAV) infection and convalescent sera were examined for cytolytic activity against HAV-infected human-embryo lung fibroblasts (HAV carrier fibroblasts). Using the 51chromium release assay, no complement dependent antibody mediated cytolytic activity against HAV carrier cells could be detected. In control experiments with identical cell strains, anti-herpes simplex virus (HSV) positive sera and complement caused specific lysis of HSV type 1 infected target cells. The data presented here do not support the hypothesis that in the possible immunopathogenesis of HAV infection, complement-dependent cytolytic antibodies play an essential role.     

Incidence of hepatitis A virus (HAV) infection in rural Liberia

To provide background for future hepatitis A vaccine trials, sera were collected from 0- to 4-year-old Liberian infants and their mothers on two occasions an average of 14.75 months apart and tested for antibody to hepatitis A virus (anti-HAV). The prevalence of anti-HAV rose from 2.5% in infants 0-6 months of age to 70% in children 3-4 years of age and did not differ between male and female infants. The annual incidence of new infections was slightly lower in the first year of life (35%) than in the subsequent 3 years, when it averaged 45%. The presence of HBV infection did not affect the incidence of HAV seroconversion. No clinical hepatitis was recognized in the subjects who seroconverted. Dual hepatitis A and B virus infection were observed; these were all clinically inapparent. The extraordinary incidence of HAV infection documented in the present study offers an opportunity for vaccine efficacy trials requiring minimal numbers of subjects.     

Efficient neutralizing activity of cocktailed recombinant human antibodies against hepatitis A virus infection in vitro and in vivo

Hepatitis A virus (HAV) is the major pathogen responsible for acute infectious hepatitis A, a disease that is prevalent worldwide. Although HAV immunization effectively prevents infection, primary immunizations must be administered at least 2 weeks prior to HAV exposure. In contrast, passive immunization with pooled human immunoglobulin (Ig) can provide immediate and rapid protection from HAV infection. Because the use of human sera-derived Igs carries the risk of contamination, we sought to develop recombinant HAV-neutralizing human antibodies. We prepared a combinatorial phage display library of recombinant human anti-HAV antibodies from RNA extracted from the blood lymphocytes of a convalescent hepatitis A patient. Two recombinant human IgG antibodies, HAIgG16 and HAIgG78, were screened from the antibody library by their ability to bind with high affinity to purified, inactivated HAV virions. These antibodies recognized different epitopes of the HAV virion capsid, and competed with both patient sera and well-characterized neutralizing mouse monoclonal antibodies. A cocktailed mixture of HAIgG16 and HAIgG78 at a 3:1 ratio was prepared to compare its combined biological activity with that conferred by each antibody individually. The cocktailed antibodies displayed a stronger neutralizing activity in vitro than that observed with either HAIgG16 and HAIgG78 alone. To determine the in vivo neutralizing abilities of these antibodies, rhesus monkeys were inoculated with cocktailed antibodies and challenged with HAV. Whereas control animals developed hepatitis A and seroconverted to the HAV antibody, animals receiving cocktailed antibodies were protected either from viral infection or from developing clinical hepatitis. These results demonstrate that recombinant human antibody preparations could be used to prevent or treat early-stage HAV infection.     

Genotype I hepatitis A virus introduction in Italy: Bayesian phylogenetic analysis to date different epidemics

Despite a significant decrease in acute hepatitis A in the last 2 decades in Italy, outbreaks were observed occurring mostly in southern Italy. In this study, Bayesian phylogenetic analysis was used to analyze the origin of these epidemics. With this aim, 5 different data sets of hepatitis A virus sequences were built to perform genotyping by the neighbor‐joining method to estimate the evolutionary rates by using a Bayesian Markov chain Monte Carlo approach and to investigate the demographic history by independent Markov chain Monte Carlo runs enforcing both a strict and relaxed clock. The estimated mean value of the evolutionary rate, representing Ia and Ib strains, was 1.21 × 10^?3 and 2.0 × 10^?3 substitutions/site/year, respectively. The Bayesian maximum clade credibility tree of hepatitis A virus (HAV) Ia and Ib strains showed that Italian sequences mostly formed separate clusters. The root of the time for the most recent common ancestor (tMRCA) for HAV Ia and Ib strains dated back to 1981 and to 1988, respectively, showing in both cases different epidemic entrances. Phylodynamic analysis showed that genotype Ia increased in 1997, when the Apulia epidemic started, then suffered a bottleneck, probably consequent to vaccination and to the herd immunity, followed by a new increase in virus population in the years 2013‐2014 consequent to the epidemic caused by the ingestion of mixed frozen berries. A similar trend without an evident bottleneck was observed also in the case of genotype Ib. In conclusion, the Bayesian phylogenetic analysis represents a good tool to measure the effectiveness of the public health plans used for HAV control.     

Emerging hepatitis E virus compared with hepatitis A virus: A new sanitary challenge

Hepatitis A (HAV) and E (HEV) viruses are able to cause liver disease in humans. Among the five classical hepatotropic viruses, they are mainly transmitted via the fecal‐oral route. Historically, many similarities have thus been described between them according to their incidence and their pathogenicity, especially in countries with poor sanitary conditions. However, recent advances have provided new insights, and the gap is widening between them. Indeed, while HAV infection incidence tends to decrease in developed countries along with public health improvement, HEV is currently considered as an underdiagnosed emerging pathogen. HEV autochthonous infections are increasingly observed and are mainly associated with zoonotic transmissions. Extra hepatic signs resulting in neurological or renal impairments have also been reported for HEV, as well as a chronic carrier state in immunocompromised patients, arguing in favor of differential pathogenesis between those two viruses. Recent molecular tools have allowed studies of viral genome variability and investigation of links between viral plasticity and clinical evolution. The identification of key functional mutations in viral genomes may improve the knowledge of their clinical impact and is analyzed in depth in the present review.     

Seroepidemiology of hepatitis B virus, hepatitis D virus, and human immunodeficiency virus infections among parenteral drug abusers in southern Taiwan

A total of 390 parenteral drug abusers (PDAs) at the Kaohsiung Municipal Narcotics Abstention Institute were examined for markers of hepatitis B virus (HBV), hepatitis D virus (HDV), and human immunodeficiency virus (HIV). All sera were tested for hepatitis B surface antigen (HBsAg), surface antibody (anti-HBs), and core antibody (anti-HBc) by radioimmunoassay (RIA) and for antibody to HIV (anti-HIV) by enzyme-linked immunosorbent assay (ELISA). Hepatitis B e antigen (HBeAg) and antibody to HDV (anti-HDV) were also tested for HBsAg-positive serum samples. Although the HBsAg-positive rate (22.1%) among PDAs was similar to that of the general population in southern Taiwan, the HBV infection rate (99.2%) and the anti-HDV-positive rate (78.5%) among HBsAg-positive subjects were significantly higher than those of the general population in southern Taiwan (P less than 0.0001). None of the PDAs studied were positive for anti-HIV. The levels of serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) among PDAs were significantly higher than those of the general population in southern Taiwan (P less than 0.0001). The more frequent the institutionalisation, the higher the infection rates with HBV and HDV and elevated levels of SGOT and SGPT. Horizontal transmission through parenteral drug abuse may be considered a possible reason for the significantly higher rates of HBV and HDV among parenteral drug abusers.     

Replication of enhancer-deficient amphotropic murine leukemia virus in human fibrosarcoma but not in primary human fibroblasts

Amphotropic murine leukemia virus (MLV) replicates in cells from various mammalian species including humans and is a potential contaminant in MLV vector preparations for human gene transfer studies. In general, MLV replication depends on the expression of viral genes under the control of 75 bp enhancer elements in the long terminal repeat. However, in specific human fibrosarcoma and lymphoma lines replication of amphotropic MLV is possible without these enhancers. Fibrosarcomas are malignant tumors of fibroblast origin. To test the replication potential of intact and enhancerless amphotropic MLV in untransformed cells, infection studies with these viruses were carried out in three types of primary human fibroblasts. Replication of amphotropic MLV is observed in two of three tested fibroblast strains. None of these primary human fibroblasts is permissive for enhancer-deficient MLV, suggesting that replication of this virus may be limited to transformed cells.     

Acute type A hepatitis during chronic hepatitis B virus infection: association of depressed hepatitis B virus replication with appearance of endogenous alpha interferon

Two patients with chronic type B hepatitis and intercurrent episodes of acute type A hepatitis are presented. Serological markers of hepatitis B virus replication decreased or became undetectable in both patients during the acute illness, while interferon activity was transiently detected in serum. The presence of serum leukocyte (alpha) interferon was confirmed by neutralization with specific antisera and tests of pH2 stability. These observations suggest a role for natural leukocyte (alpha) interferon in the modulation and control of hepatitis B virus infection and provide further evidence to support trials of exogenous leukocyte (alpha) interferon in the chronic infection.     

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.     

Prevalence of hepatitis A virus,hepatitis B virus,hepatitis C virus,hepatitis D virus and hepatitis E virus as causes of acute viral hepatitis in North India: A hospital based study

Context: Acute viral hepatitis (AVH) is a major public health problem and is an important cause of morbidity and mortality. Aim: The aim of the present study is to determine the prevalence of hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis E virus (HEV) as causes of AVH in a tertiary care hospital of North India. Settings and Design: Blood samples and clinical information was collected from cases of AVH referred to the Grade I viral diagnostic laboratory over a 1-year period. Subjects and Methods: Samples were tested for hepatitis B surface antigen, anti-HCV total antibodies, anti-HAV immunoglobulin M (IgM) and anti-HEV IgM by the enzyme-linked immunosorbent assay. PCR for nucleic acid detection of HBV and HCV was also carried out. Those positive for HBV infection were tested for anti-HDV antibodies. Statistical Analysis Used: Fisher’s exact test was used and a P < 0.05 was considered to be statistically significant. Results: Of the 267 viral hepatitis cases, 62 (23.22%) patients presented as acute hepatic failure. HAV (26.96%) was identified as the most common cause of acute hepatitis followed by HEV (17.97%), HBV (16.10%) and HCV (11.98%). Co-infections with more than one virus were present in 34 cases; HAV-HEV co-infection being the most common. HEV was the most important cause of acute hepatic failure followed by co-infection with HAV and HEV. An indication towards epidemiological shift of HAV infection from children to adults with a rise in HAV prevalence was seen. Conclusions: To the best of our knowledge, this is the first report indicating epidemiological shift of HAV in Uttar Pradesh.