Characterization of a simian hepatitis A virus (HAV): antigenic and genetic comparison with human HAV.

PA21, a strain of hepatitis A virus (HAV) recovered from a naturally infected captive owl monkey, is indistinguishable from human HAV in polyclonal radioimmunoassays and cross-neutralization studies. However, cDNA-RNA hybridization has suggested a significant difference at the genomic level between PA21 and a reference human virus, HM175. Further characterization of this unique HAV was undertaken in an effort to determine the extent of genetic divergence from human HAV and its relation to the conserved antigenic structure of the virus. The close similarity between PA21 and HM175 antigens was confirmed with an extended panel of 18 neutralizing murine monoclonal antibodies: a reproducible difference in binding to the two viruses was detected with only one antibody (B5-B3). The nucleotide sequence of the P1 region of the PA21 genome had only 83.2% identity with HM175 virus, a difference approximately twice as great as that found between any two human strains. Most nucleotide changes were in third base positions, and the amino acid sequences of the capsid proteins were largely conserved. Amino acid replacements were clustered in the carboxy terminus of VP1 and the amino-terminal regions of VP2 and VP1. These data indicate that PA21 virus represents a unique genotype of HAV and suggest the existence of an ecologically isolated niche for HAV among feral owl monkeys.     

A technology of preparing hepatitis A virus (HAV) from human stools

Two-step differential centrifugation through 40% and 20% sucrose of 20% water extracts of HAV-positive stools yielded specific defined antigens for hepatitis A serological diagnosis by 3rd-generation methods. The antigens would be suitable also for other purposes. Products of comparable quality were obtained from HAV-positive stools yielding different amounts of virus. Optimal were incubation-period stools, where the loss in HAV yield was minimal; these preparations displayed the highest antigenic capacity and could be diluted up to 150 fold for serological reactions. Results obtained in extensive preliminary experiments, outlined in the Discussion, corroborated the advantages and efficiency of the proposed technology.     

Mutations responsible for adaptation of hepatitis A virus to efficient growth in cell culture.

Chimeric genomes of hepatitis A virus strain HM-175 were constructed from cDNA clones of the wild-type virus and its cell culture-adapted variant. RNA transcribed in vitro from each construct was assayed for infectivity by transfection of cultured cells. RNA transcribed from the wild-type cDNA clone was minimally infectious and produced virus that grew inefficiently in vitro, whereas that transcribed from certain chimeric genomes consistently produced virus that grew efficiently in cultured cells. Mutations in the P2 region were found to be necessary for efficient virus growth in vitro, while mutations in the 5' noncoding region imparted a conditional enhancement of growth in vitro.     

Mutations in hepatitis C virus RNAs conferring cell culture adaptation

As an initial approach to studying the molecular replication mechanisms of hepatitis C virus (HCV), a major causative agent of acute and chronic liver disease, we have recently developed selectable self-replicating RNAs. These replicons lacked the region encoding the structural proteins and instead carried the gene encoding the neomycin phosphotransferase. Although the replication levels of these RNAs within selected cells were high, the number of G418-resistant colonies was reproducibly low. In a search for the reason, we performed a detailed analysis of replicating HCV RNAs and identified several adaptive mutations enhancing the efficiency of colony formation by several orders of magnitude. Adaptive mutations were found in nearly every nonstructural protein but not in the 5' or 3' nontranslated regions. The most drastic effect was found with a single-amino-acid substitution in NS5B, increasing the number of colonies approximately 500-fold. This mutation was conserved with RNAs isolated from one cell line, in contrast to other amino acid substitutions enhancing the efficiency of colony formation to a much lesser extent. Interestingly, some combinations of these nonconserved mutations with the highly adaptive one reduced the efficiency of colony formation drastically, suggesting that some adaptive mutations are not compatible.     

Intermolecular cleavage of hepatitis A virus (HAV) precursor protein P1-P2 by recombinant HAV proteinase 3C.

Active proteinase 3C of hepatitis A virus (HAV) was expressed in bacteria either as a mature enzyme or as a protein fused to the entire polymerase 3D or to a part of it, and their identities were shown by immunoblot analysis. Intermolecular cleavage activity was demonstrated by incubating in vitro-translated and radiolabeled HAV precursor protein P1-P2 with extracts of bacteria transformed with plasmids containing recombinant HAV 3C. Identification of cleavage products P1, VP1, and VPO-VP3 by immunoprecipitation clearly demonstrates that HAV 3C can cleave between P1 and P2 as well as within P1 and thus shows an activity profile similar to that of cardiovirus 3C.     

Neutralization of hepatitis A virus (HAV) by an immunoadhesin containing the cysteine-rich region of HAV cellular receptor-1

Hepatitis A virus (HAV) infects African green monkey kidney (AGMK) cells via the HAV cellular receptor-1 (havcr-1), a mucin-like type 1 integral-membrane glycoprotein of unknown natural function. The ectodomain of havcr-1 contains an N-terminal immunoglobulin-like cysteine-rich region (D1), which binds protective monoclonal antibody (MAb) 190/4, followed by an O-glycosylated mucin-like threonine-serine-proline-rich region that extends D1 well above the cell surface. To study the interaction of HAV with havcr-1, we constructed immunoadhesins fusing the hinge and Fc portion of human IgG1 to D1 (D1-Fc) or the ectodomain of the poliovirus receptor (PVR-Fc) and expressed them in CHO cells. These immunoadhesins were secreted to the cell culture medium and purified through protein A-agarose columns. In a solid-phase assay, HAV bound to D1-Fc in a concentration-dependent manner whereas background levels of HAV bound to PVR-Fc. Binding of HAV to D1-Fc was blocked by treatment with MAb 190/4 but not with control MAb M2, which binds to a tag epitope introduced between the D1 and Fc portions of the immunoadhesin. D1-Fc neutralized approximately 1 log unit of the HAV infectivity in AGMK cells, whereas PVR-Fc had no effect in the HAV titers. A similarly poor reduction in HAV titers was observed after treating the same stock of HAV with murine neutralizing MAbs K2-4F2, K3-4C8, and VHA 813. Neutralization of poliovirus by PVR-Fc but not by D1-Fc indicated that the virus-receptor interactions were specific. These results show that D1 is sufficient for binding and neutralization of HAV and provide further evidence that havcr-1 is a functional cellular receptor for HAV.     

Alteration of hepatitis A virus (HAV) particles by a soluble form of HAV cellular receptor 1 containing the immunoglobin-and mucin-like regions

Hepatitis A virus (HAV) infects African green monkey kidney cells via HAV cellular receptor 1 (havcr-1). The ectodomain of havcr-1 contains an N-terminal cysteine-rich immunoglobin-like region (D1), followed by a mucin-like region that extends D1 well above the cell surface. D1 is required for binding of HAV, and a soluble construct containing D1 fused to the hinge and Fc portions of human immunoglobin G1 (IgG1), D1-Fc, bound and neutralized HAV inefficiently. However, D1-Fc did not alter the virions. To determine whether additional regions of havcr-1 are required to trigger uncoating of HAV, we constructed D1muc-Fc containing D1 and two-thirds of the mucin-like region fused to the Fc and hinge portions of human IgG1. D1muc-Fc neutralized 10 times more HAV than did D1-Fc. Sedimentation analysis in sucrose gradients showed that treatment of HAV with 20 to 200 nM D1muc-Fc disrupted the majority of the virions, whereas treatment with 2 nM D1muc-Fc had no effect on the sedimentation of the particles. Treatment of HAV with 100 nM D1muc-Fc resulted in low-level accumulation of 100- to 125S particles. Negative-stain electron microscopy analysis revealed that the 100- to 125S particles had the characteristics of disrupted virions, such as internal staining and diffuse edges. Quantitative PCR analysis showed that the 100- to 125S particles contained viral RNA. These results indicate that D1 and the mucin-like region of havcr-1 are required to induce conformational changes leading to HAV uncoating.     

Synthesis and testing of azaglutamine derivatives as inhibitors of hepatitis A virus (HAV) 3C proteinase.

Hepatitis A virus (HAV) 3C proteinase is a picornaviral cysteine proteinase that is essential for cleavage of the initially synthesized viral polyprotein precursor to mature fragments and is therefore required for viral replication in vivo. Since the enzyme generally recognizes peptide substrates with L-glutamine at the P1 site, four types of analogues having an azaglutamine residue were chemically synthesized: hydrazo-o-nitrophenylsulfenamides A (e.g. 16); frame-shifted hydrazo-o-nitrophenylsulfenamides B (e.g. 25-28); the azaglutamine sulfonamides C (e.g. 7, 8, 11, 12); and haloacetyl azaglutamine analogues 2 and 3. Testing of these compounds for inhibition of the HAV 3C proteinase employed a C24S mutant in which the non-essential surface cysteine was replaced with serine and which displays identical catalytic parameters to the wild-type enzyme. Sulfenamide 16 (type A) showed no significant inhibition. Sulfenamide 27 (type B) had an IC50 of ca 100 microM and gave time-dependent inactivation of the enzyme due to disulfide bond formation with the active site cysteine thiol, as demonstrated by electrospray mass spectrometry. Sulfonamide 8 (type C) was a weak competitive inhibitor with an IC50 of approximately 75 microM. The haloacetyl azaglutamine analogues 2 and 3 were time-dependent irreversible inactivators of HAV 3C proteinase with rate constants k(obs)/[I] of 680 M(-1) s(-1) and 870 M(-1) s(-1), respectively, and were shown to alkylate the active site thiol.     

Retrospective detection of a subclinical hepatitis A virus (HAV) epidemic affecting juvenile cohorts of the Hungarian population

Sero-epidemiological surveys of serum samples taken in 1982, 1987, 1994 and 1999 have been performed with hepatitis A virus-specific (HAV-specific) serological tests. Results obtained during these surveys show that the proportion of seropositive blood donors decreased from 69% to 18% within 17 years. The authors have recognised a (mainly subclinical) epidemic, affecting about 115000 teenagers in 1992-1994 in Hungary, is a threatening phenomenon. It was calculated that only about 3600 clinical diseases were associated with the epidemic, recognised retrospectively from the findings of the four sero-epidemiological surveys. Epidemiological data indicated that the excess clinical diseases caused by HAV concentrated in the southern counties of Hungary, which have been affected by the social and military activities between 1992 and 1994. Due to the decrease of subjects seropositive for HAV, sera from preselected or actively immunised donors will be required in the future and vaccination against HAV with killed virus is likely to be recommended for risk groups. Furthermore, health authorities might promote active immunisation of young children against HAV infection; for that, promotion of manufacturing combination vaccines of HAV/HBV/DPT or, for certain countries, HAV/DPT would be desirable.     

Adaptation of hepatitis A virus strain (JaM-55) originally pathogenic for monkeys to a cell culture

The results of adaptation of hepatitis A viral strain JaM-55 to the culture of embryo kidney cells FRhk-4 from macaque Rhesus are presented. The viral strain was isolated from a M. fascicularis suffering from spontaneous hepatitis. Before inoculating the cell culture the virus was passaged twice in the M. arctoides capable of reproducing hepatitis. FRhk-4 cell line inoculation by the monkey liver extract, containing the strain HAV-YaM-55, resulted in isolation of single viral particles of hepatitis A in the preparations obtained at the first 3 passages by the 28-31 day of cultivation. Beginning from the fourth passage the abrupt increase in the number of viral particles and hepatitis A antigen was registered. There were no traces of cytopathogenic effect at any level of viral passages in the inoculated cell culture. The adapted virus contains hepatitis A viral RNA identified by spot hybridization with the cloned cDNA of hepatitis A virus.     

Physico-chemical properties of mouse hepatitis virus (MHV-2) grown on DBT cell culture.

Some properties of a strain of mouse hepatitis virus, MHV-2, grown on DBT cells were determined using a plaque assay on the cells. Viral growth was not inhibited by the presence of actinomycin D or 5-iodo-2-deoxyuridine. MHV-2 was completely inactivated by ether, chloroform, sodium deoxycholate or beta-propiolactone, but showed a moderate resistance to trypsin. Heating at 56 C for 30 min did not completely abolish the virus infectivity. The virus was stable after heating at 50 C for 15 min in 1M-MgCl2 or 1M-MgSO4 as well as at 37 C for 60 min at pH 3.0 to 9.0. Infectivity was decreased to 1/100 and 1/400 after storing at 4 C for 30 days and 37 C for 24 hr, respectively. The virus passed through a 200-nm but not a 50-nm Sartorius membrane filter. The buoyant density of MHV-2 was 1.183 g/cm3 in sucrose gradient, and the fraction contained coronavirus-like particles measuring 70 to 130 nm in diameter. Survival rate was 10% after exposure to ultraviolet at 150 ergs/mm2. Freezing and thawing or sonication at 20 kc for 3 min did not affect the virus titer. No hemagglutinin was demonstrable with red blood cells of the chicken, Japanese quail, mouse, rat, hamster, guinea pig, sheep, bovine or human.     

Restricted replication of human hepatitis A virus in cell culture: intracellular biochemical studies.

When hepatitis A virus was inoculated into Vero cells, virus-specified protein and RNA synthesis was detected. Production of viral protein was detected by electrophoretic analysis in polyacrylamide gels by using a double-label coelectrophoresis and subtraction method which eliminated the contribution of host protein components from the profiles of virus-infected cytoplasm. Eleven virus-specified proteins were detected in the net electrophoretic profiles of hepatitis A virus-infected cells. The molecular weights of these proteins were very similar to those detected in cells infected with poliovirus type 1. Virus-specified protein synthesis could be detected at 3 to 6 h and continued for at least 48 h postinfection, but no significant effect on host-cell macromolecular synthesis was observed. Limited viral RNA replication occurred between 2 and 6 h postinfection. The genomic RNA of hepatitis A virus was extracted and shown to be capable of infecting cells and inducing the same set of proteins as intact virus, indicating that the RNA genome is positive stranded. Progeny virus was never detected in the supernatant fluids of infected cell cultures, and the cells showed no observable cytopathology, even though hepatitis A virus-specific proteins and antigens were being produced. The nature of the defect in the replicative cycle of hepatitis A virus in this system remains unknown.     

Magnetic immunoseparation PCR assay (MIPA) for detection of hepatitis a virus (HAV) in American oyster (Crassostrea virginica)

In order to detect the low numbers of hepatitis A viral (HAV) particles which may potentially be present in food and cause a serious illness, an original procedure which combines immunomagnetic separation and PCR is described. The use of streptavidin magnetic beads coated with biotinylated human anti-HAV IgG allows virus capture and the removal of the RT-PCR inhibitory compounds which usually are present in shellfish extracts. Following immunomagnetic capture, the separated HAV were lysed, the beads discarded, and the supernatant containing the viral RNA subjected to the RT-PCR protocol. Levels of HAV ranging from 10 to 10⁵ pfu were successfully detected in artificially contaminated samples of shucked American oyster ( Crassostrea virginica ).