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Hepatitis A virus (HAV) encodes a single polyprotein which is posttranslationally processed into the functional structural and nonstructural proteins. Only one protease, viral protease 3C, has been implicated in the nine protein scissions. Processing of the capsid protein precursor region generates a unique intermediate, PX (VP1-2A), which accumulates in infected cells and is assumed to serve as precursor to VP1 found in virions, although the details of this reaction have not been determined. Coexpression in transfected cells of a variety of P1 precursor proteins with viral protease 3C demonstrated efficient production of PX, as well as VP0 and VP3; however, no mature VP1 protein was detected. To identify the C-terminal amino acid residue of HAV VP1, we performed peptide sequence analysis by protease-catalyzed [18O]H2O incorporation followed by liquid chromatography ion-trap microspray tandem mass spectrometry of HAV VP1 isolated from purified virions. Two different cell culture-adapted isolates of HAV, strains HM175pE and HM175p35, were used for these analyses. VP1 preparations from both virus isolates contained heterogeneous C termini. The predominant C-terminal amino acid in both virus preparations was VP1-Ser274, which is located N terminal to a methionine residue in VP1-2A. In addition, the analysis of HM175pE recovered smaller amounts of amino acids VP1-Glu273 and VP1-Thr272. In the case of HM175p35, which contains valine at amino acid position VP1-273, VP1-Thr272 was found in addition to VP1-Ser274. The data suggest that HAV 3C is not the protease responsible for generation of the VP1 C terminus. We propose the involvement of host cell protease(s) in the production of HAV VP1.

Utilization of internal ribosome entry segment (IRES) structures in the 5' noncoding region (5'NCR) of picornavirus RNAs for initiation of translation requires a number of host cell factors whose distribution may vary in different cells and whose requirement may vary for different picornaviruses. We have examined the requirement of the cellular protein poly(rC) binding protein 2 (PCBP2) for hepatitis A virus (HAV) RNA translation. PCBP2 has recently been identified as a factor required for translation and replication of poliovirus (PV) RNA. PCBP2 was shown to be present in FRhK-4 cells, which are permissive for growth of HAV, as it is in HeLa cells, which support translation of HAV RNA but which have not been reported to host replication of the virus. Competition RNA mobility shift assays showed that the 5'NCR of HAV RNA competed for binding of PCBP2 with a probe representing stem-loop IV of the PV 5'NCR. The binding site on HAV RNA was mapped to nucleotides 1 to 157, which includes a pyrimidine-rich sequence. HeLa cell extracts that had been depleted of PCBP2 by passage over a PV stem-loop IV RNA affinity column supported only low levels of HAV RNA translation. Translation activity was restored upon addition of recombinant PCBP2 to the depleted extract. Removal of the 5'-terminal 138 nucleotides of the HAV RNA, or removal of the entire IRES, eliminated the dependence of HAV RNA translation on PCBP2.

Hepatitis A virus (HAV), unlike other picornaviruses, has a slow-growth phenotype in permissive cell lines and in general does not induce host cell cytopathology. Although there are no published reports of productive infection of HeLa cells by HAV, HAV RNA appears to be readily translated in HeLa cells when transcribed by T7 RNA polymerase provided by a recombinant vaccinia virus. The 5' noncoding region of HAV was fused to poliovirus (PV) coding sequences to determine the effect on translation efficiency in HeLa cell extracts in vitro. Conditions were optimized for utilization of the HAV internal ribosome entry segment (IRES). Transcripts from chimeric constructs fused precisely at the initiation codon were translated very poorly. However, chimeric RNAs which included 114 or more nucleotides from the HAV capsid coding sequences downstream of the initiation codon were translated much more efficiently than those lacking these sequences, making HAV-directed translation efficiency similar to that directed by the PV IRES. Sixty-six nucleotides were insufficient to confer increased translation efficiency. The most 5'-terminal HAV 138 nucleotides, previously determined to be upstream of the IRES, had an inhibitory effect on translation efficiency. Constructs lacking these terminal sequences, or those in which the PV 5'-terminal sequences replaced those from HAV, translated three- to fourfold better than those with the intact HAV 5'-terminal end.

Previous sequence analysis of consecutive passages of the hepatitis A virus (HAV) strain GBM/WT in human embryonic kidney cells (HEK cells), human embryonic lung fibroblasts (HFS cells) and in FRhK-4 cells (foetal rhesus monkey kidney cells) pointed to a host cell dependent cell culture adaptation of GBM/WT in HFS cells involving mutations in the 5' noncoding region (5'NCR). Multiple nucleotide changes occurred in the 5'NCR of the GBM genome after the cell line used for virus passage was changed from HEK cells to HFS cells. In contrast, no mutations in the 5'NCR occurred during the first 20 passages of GBM/WT in FRhK-4 cells. In order to analyse the influence of the 5'NCR on host cell specific adaptation of HAV strain GBM in different cell cultures, GBM/HM175 chimeras were constructed which contained 5'NCRs from different GBM variants by replacing the 5'NCR of the infectious clone pHAV/7. Parallel transfection assays in FRhK-4 and HFS cells, performed with transcripts from the chimeric GBM/HM175 constructs, showed that the 5'NCR of the GBM variant GBM/HFS is essential for virus growth in HFS cells. The GBM/HM175 chimeric RNA, which contained the 5'NCR of GBM/HFS, exclusively, was able to produce infectious virus after transfection of HFS cells. The growth of the different GBM/HM175 chimeras in FRhK-4 cells, in contrast, did not seem to be strongly influenced by a specific sequence of the 5'NCR.

The relationship of hepatitis A virus (HAV) isolates associated with an outbreak in Genoa, Italy, in 1993 was examined using direct sequencing of amplicons derived by antigen capture PCR (AC/PCR) from faecal samples of the infected persons. Forty samples recovered from 38 primary and two secondary cases were examined. The latter were household contacts of the primary cases. In addition, faecal material of 2 unrelated persons infected simultaneously with hepatitis A in Genoa were tested. The PCR products derived from the P1/P2 junction of the HAV genome were analysed. A 100% nucleotide identity was detected between the viral isolates originating from the primary as well as the secondary cases. The viral isolates recovered from the faecal samples of the two unrelated cases differed from the virus causing the outbreak as well as from each other. These results indicate that a single HAV strain caused the outbreak. The virus might have been transmitted by ingestion of contaminated food or water since all hepatitis A infected employees of the factory had eaten in the same canteen. Definitions of HAV genotypes are based on numerous genetic comparisons of different strains. The sequence comparison of the investigated isolates with published HAV sequences of the P1/P2 genome region revealed that the virus associated with the outbreak belongs to HAV subgenotype IA, whereas the strains recovered from the viral isolates of the unrelated cases belong to subgenotype IB.

Hepatitis A infection among patients receiving solvent/detergent inactivated factor VIII preparations in various locations in Europe have been documented recently. In investigations in Italy, Germany and Ireland, polymerase chain reaction (PCR) amplification was used to detect hepatitis A virus in frozen plasma pools, purified factor VIII, patient sera and samples from animal transmission studies; nucleic acid sequencing was used to clarify and identify the virus responsible based upon genotype analysis. Unique virus strains were found among the cases in Italy and Germany, and identical virus sequences were also found in some factor VIII lots. However, with the exception of the Italian investigation, lack of appropriate samples have precluded the identification of virus in these outbreaks. In addition, animal infectivity studies have not been successful in demonstrating infectivity under laboratory conditions. We discuss the limitations of PCR amplification with respect to detecting virus within these situations, and the necessity for the corresponding epidemiologic investigations.

In order to study the adaptation of hepatitis A virus (HAV) in cell culture, we examined the mutational events of the genome in early passages of HAV strain GBM propagated either in FRhK-4 cells (fetal rhesus monkey kidney-derived) or in human embryonic kidney (HEK) and human embryonic fibroblast cells (HFS) in relation to their growth characteristics. Sequence analysis of the nucleotide region encoding 2B, 2C, and the beginning of 3A as well as the nucleotide region encompassing the 5' noncoding region (5'NCR) of the genome was performed on consecutive virus passages after amplification of the viral RNA from the cell culture supernatant by antigen-capture PCR. By the 2nd passage of the GBM variants cultured in FRhK-4 or in HEK cells we found a mutation at nucleotide position 3889 (2B coding region) which results in an amino acid substitution from alanine to valine. Further mutations present in the 2B/2C region of the cell culture-adapted GBM variants differ from each other and occur after the 10th or even the 40th virus passage. Another early change, an in frame deletion of nine nucleotides in the 3A region, appeared in the 5th virus passage only in GBM cultured on FRhK-4 cells. This genome region showed different mutations in the virus passages on HEK and HFS cells. The 5'NCR of the cell culture-adapted GBM variants, in contrast, did not show any mutations before the 8th virus passage. The faster and more efficient growth of the HAV strain GBM during successive propagation on cell cultures seems to correlate with the appearance of mutations in the investigated genome regions.

The antigen capture/PCR (AC/PCR) has been applied in the analysis of various factor VIII preparations, which were suspected to be contaminated with hepatitis A virus (HAV). AC/PCR involves capturing the antigen, i.e. the intact virus particles, by binding to the HAV monoclonal antibody mAb 7e7, reverse transcription of the viral RNA and amplification of the cDNA with HAV-specific primer pairs. The PCR analysis of one factor VIII concentrate yielded an HAV-specific DNA product, which could be confirmed by Southern blot analysis. The HAV strain recovered by AC/PCR from this factor VIII concentrate could be classified into genotype III after solid-phase sequencing of the product and comparison with the consensus sequences for the known HAV genotypes. Analysis of the sera from 3 haemophiliacs treated with this batch has not resulted in a reliable product. However, the results obtained indicate that HAV can be detected in purified factor VIII preparations by AC/PCR.

Outbreaks and sporadic cases of hepatitis A have been observed in 4 European countries in hemophilia patients receiving factor VIII preparations. PCR amplification of potential hepatitis A virus (HAV) nucleic acid present in plasma pools, purified factor VIII and acute-phase sera from infected individuals has been performed and the nucleic acid sequence determined for those samples that resulted in a positive PCR product. HAV sequences were detected in the serum of 2 German patients, but not in the factor VIII lots administered to these individuals. Screening of plasma pools and the corresponding 5 lots of factor VIII associated with the outbreak in Ireland did not reveal any HAV sequences. In contrast, a study of samples from Italy detected HAV sequences in 5 of 12 lots and in 2 hemophilia patients who developed hepatitis A. These data suggest that implicated factor VIII preparations might have been involved in the outbreaks of HAV infection among Italian hemophiliacs. However, no molecular evidence was obtained for a similar association in Germany or Ireland. The preliminary data from these two investigations must be verified by animal inoculation studies and supported by epidemiologic analysis.

In order to study cell tropism and attenuation of hepatitis A virus (HAV), the genome of HAV wild-type GBM and two cell culture-adapted variants, GBM/FRhK and GBM/HFS, were cloned and sequenced after amplification by reverse transcriptase-PCR. During virus cultivation, the HAV variant GBM/FRhK had a strict host range for FRhK-4 cells, in contrast to GBM/HFS, which can be grown in HFS and FRhK-4 cells. The HAV variant GBM/HFS was shown to be attenuated when inoculated into chimpanzees (B. Flehmig, R. F. Mauler, G. Noll, E. Weinmann, and J. P. Gregerson, p. 87-90, in A. Zuckerman, ed., Viral Hepatitis and Liver Disease, 1988). On the basis of this biological background, the comparison of the nucleotide sequences of these three HAV GBM variants should elucidate differences which may be of importance for cell tropism and attenuation. The comparison of the genome between the GBM wild type and HAV wild types HM175 (J. I. Cohen, J. R. Ticehurst, R. H. Purcell, A. Buckler-White, and B. M. Baroudy, J. Virol. 61:50-59, 1987) and HAV-LA (R. Najarian, O. Caput, W. Gee, S. J. Potter, A. Renard, J. Merryweather, G. Van Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82:2627-2631, 1985) showed a 92 to 96.3% identity, whereas the identity was 99.3 to 99.6% between the GBM variants. Nucleotide differences between the wild-type and the cell culture-adapted variants, which were identical in both cell culture-adapted GBM variants, were localized in the 5' noncoding region; in 2B, 3B, and 3D; and in the 3' noncoding region. Our result concerning the 2B/2C region confirms a mutation at position 3889 (C-->T, alanine to valine), which had been shown to be of importance for cell culture adaptation (S. U. Emerson, C. McRill, B. Rosenblum, S. M. Feinstone, and R. H. Purcell, J. Virol. 65:4882-4886, 1991; S. U. Emerson, Y. K. Huang, C. McRill, M. Lewis, and R. H. Purcell, J. Virol. 66:650-654, 1992), whereas other mutations differ from published HAV sequence data and may be cell specific. Further comparison of the two cell culture-adapted GBM variants showed cell-specific mutations resulting in deletions of six amino acids in the VP1 region and three amino acids in the 3A region of the GBM variant GBM/FRhK.

Antigen capture polymerase chain reaction (PCR) was tested as a sensitive and rapid method for detecting hepatitis A virus (HAV) in raw sewage sludge. The antigen capture PCR was performed both with and without solid-phase virus-catching monoclonal antibodies. Similar results proved that both methods were equally sensitive. Sewage sludge samples from different regions in Germany were examined for evidence of HAV contamination by antigen capture PCR. This method of detection was compared with that used in a previous study of these sewage sludge samples, in which the HAV was detected through indirect immunofluorescence after cell culture inoculation. The results obtained by antigen capture PCR matched those obtained in the earlier cell culture investigations, when HAV was detected in raw as well as digested sewage sludge samples. The advantage of the PCR method, however, lies in the fact that it needs only two days while the cell culture propagation of HAV takes about 8 to 10 weeks.