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.     

Utilization of Chimeras between Human (HM-175) and Simian (AGM-27) Strains of Hepatitis A Virus To Study the Molecular Basis of Virulence

Chimeras between human (HM-175) and simian (AGM-27) strains of hepatitis A virus (HAV) were constructed to evaluate the effect of the 2C gene of AGM-27 on HAV replication in cell culture and virulence in tamarins (Saguinus mystax) and chimpanzees (Pan troglodytes). Kinetic studies and radioimmunofocus assays demonstrated that replacement of the 2C gene of HAV/7, a cell culture-adapted strain of HM-175, with that of AGM-27 drastically reduced the ability of the virus to replicate in cultured cells. Intragenic chimeras containing AGM-27 sequences in either the 5′ or 3′ half of the 2C gene replicated in cell culture at an intermediate level. Whereas HAV/7 is attenuated for tamarins, a chimera containing the simian virus 2C gene in the HAV/7 background was virulent in tamarins, demonstrating that the simian virus 2C gene alone can confer the phenotype of virulence to an otherwise attenuated virus. Clusters of AGM-27-specific residues near both ends of the 2C protein were required for virulence since a chimera containing AGM-27 sequences in the carboxy-terminal half of 2C was partially attenuated for tamarins while one containing AGM-27 sequences only in the amino-terminal half of 2C was even more attenuated. Chimeras containing either the entire or only the 3′ half of the simian virus 2C gene in the HAV/7 background were attenuated for chimpanzees.     

Progress toward the development of a genetically engineered attenuated hepatitis A virus vaccine.

Mutations which positively affect growth of hepatitis A virus in cell culture may negatively affect growth in vivo. Therefore, development of an attenuated vaccine for hepatitis A may require a careful balancing of mutations to produce a virus that will grow efficiently in cells suitable for vaccine production and still maintain a satisfactory level of attenuation in vivo. Since such a balance could be achieved most directly by genetic engineering, we are analyzing mutations that accumulated during serial passage of the HM-175 strain of hepatitis A virus in MRC-5 cell cultures in order to determine the relative importance of the mutations for growth in MRC-5 cells and for attenuation in susceptible primates. Chimeric viral genomes of the HM-175 strain were constructed from cDNA clones derived from a virulent virus and from two attenuated viruses adapted to growth in African green monkey kidney (AGMK) and MRC-5 cells, respectively. Viruses encoded by these chimeric genomes were recovered by in vitro or in vivo transfection and assessed for their ability to grow in cultured MRC-5 cells or to cause hepatitis in primates (tamarins). The only MRC-5-specific mutations that substantially increased the efficiency of growth in MRC-5 cells were a group of four mutations in the 5' noncoding (NC) region. These 5' NC mutations and a separate group of 5' NC mutations that accumulated during earlier passages of the HM-175 virus in primary AGMK cells appeared, independently and additively, to result in decreased biochemical evidence of hepatitis in tamarins. However, neither group of 5' NC mutations had a demonstrable effect on the extent of virus excretion or liver pathology in these animals.     

Mutations within the 5'' nontranslated region of hepatitis A virus RNA which enhance replication in BS-C-1 cells.

Passage of human hepatitis A virus (HAV) in cell culture results in attenuation of the virus as well as progressive increases in the efficiency of virus replication in cell culture. Because the presence of identical mutations within the 5' nontranslated regions (5'NTRs) of several independently isolated cell culture-adapted HAV variants suggests that the 5'NTR may play a role in determining this change in virus host range, we constructed chimeric infectious cDNA clones in which portions of the 5'NTR of cell culture-adapted HM175/p35 virus were replaced with cDNA from either wild-type virus (HM175/wt) or a second independently isolated, but closely related cell culture-adapted virus (HM175/p16). Substitution of the complete 5'NTR of HM175/p35 with the 5'NTR of HM175/wt resulted in virus with very small replication foci in continuous African green monkey kidney (BS-C-1) cells, indicating that 5'NTR mutations in HM175/p35 virus are required for optimal growth in these cells. A chimera with the 5'NTR sequence of HM175/p16 retained the large foci of HM175/p35 virus, while the growth properties of other viruses having chimeric 5'NTR sequences indicated that mutations at bases 152 and/or 203 to 207 enhance replication in BS-C-1 cells. These findings were confirmed in one-step growth experiments, which also indicated that radioimmunofocus size is a valid measure of virus replication competence in cell culture. An additional mutation at base 687 of HM175/p16 had only a minor role in enhancing growth. In contrast to their effect in BS-C-1 cells, these 5'NTR mutations did not enhance replication in continuous fetal rhesus monkey kidney (FRhK-4) cells. Thus, mutations at bases 152 and/or 203 to 207 enhance the replication of HAV in a highly host cell-specific fashion.     

Genomic heterogeneity among human and nonhuman strains of hepatitis A virus.

Cloned cDNA probes derived from the P1 and P2 regions of the genome of HM175 virus, a reference strain of human hepatitis A virus (HAV), failed to hybridize under standard stringency criteria with RNA from PA21 and PA33 viruses, two epizootiologically related HAV strains recovered from naturally infected New World owl monkeys. Hybridization of these probes to PA21 RNA was only evident under reduced stringency conditions. However, cDNA representing the 5' nontranslated region of the HM175 genome hybridized equally to HM175 and PA21 RNA under standard stringency conditions, while a probe derived from the 3' 1,400 bases of the genome yielded a reduced hybridization signal with PA21 RNA. In contrast, no differences could be discerned between HM175 virus and three other HAV strains of human origin (GR8, LV374, and MS1) in any region of the genome, unless increased stringency conditions were used. These results suggest that PA21 and PA33 are unique among HAV isolates and may represent a virus native to the owl monkey. Despite extremely poor homology within the P1 region, which encodes capsid polypeptides, monoclonal antibody analysis confirmed that the immunodominant neutralization epitopes of HAV were highly conserved between HM175 and PA21 viruses. Furthermore, experimental challenge of the owl monkey with successive PA33 and HM175 inocula confirmed a high but incomplete degree of cross-protection. Only one of six monkeys previously infected with PA33 developed recurrent hepatitis 28 days after intravenous HM175 challenge, while none of six monkeys previously infected with HM175 had demonstrable hepatitis following PA33 challenge. These data provide molecular evidence for the existence of HAV strains unique to nonhuman primate species and indicate that strict conservation of antigenic function may accompany substantial genetic divergence in HAV.     

Complete nucleotide sequence of wild-type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses.

The complete nucleotide sequence of wild-type hepatitis A virus (HAV) HM-175 was determined. The sequence was compared with that of a cell culture-adapted HAV strain (R. Najarian, D. Caput, W. Gee, S.J. Potter, A. Renard, J. Merryweather, G.V. Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82:2627-2631, 1985). Both strains have a genome length of 7,478 nucleotides followed by a poly(A) tail, and both encode a polyprotein of 2,227 amino acids. Sequence comparison showed 624 nucleotide differences (91.7% identity) but only 34 amino acid differences (98.5% identity). All of the dipeptide cleavage sites mapped in this study were conserved between the two strains. The sequences of these two HAV strains were compared with the partial sequences of three other HAV strains. Most amino acid differences were located in the capsid region, especially in VP1. Whereas changes in amino acids were localized to certain portions of the genome, nucleotide differences occurred randomly throughout the genome. The most extensive nucleotide homology between the strains was in the 5' noncoding region (96% identity for cell culture-adapted strains versus wild type; greater than 99% identity among cell culture-adapted strains). HAV proteins are less homologous with those of any other picornavirus than the latter proteins are when compared with each other. When the sequences of wild-type and cell culture-adapted HAV strains are compared, the nucleotide differences in the 5' noncoding region and the amino acid differences in the capsid region suggest areas that may contain markers for cell culture adaptation and for attenuation.     

甲型肝炎重组痘苗病毒(VMS11HAV25)的构建及其某些生物学性质

将5′端经过剪切的甲型肝炎病毒全部开放读码框架cDNA连接于痘苗病毒晚期启动子P11下游,重组于痘苗病毒天坛株的HiodⅢM片段Spb Ⅰ位点获得了重组病毒VMS11HAV25。对其生物学性质的研究表明,该重组病毒诱生痘苗抗体的能力、对鸡红细胞的血凝性质、空斑大小及对温度的稳定性等均与原天坛株相同。重要的区别是,重组病毒在家兔皮内和小鼠脑内的毒力都比原天坛株低约1个对数。病毒在人胚肺二倍体细胞连续传15代的表达水平与传代早期者相同。连续传20代后提取病毒DNA做Southern blot杂交表明,甲型肝炎病毒基因仍稳定地存在于原插入位置。     

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 hepatitis A virus deletion mutant which lacks the first pyrimidine-rich tract of the 5' nontranslated RNA remains virulent in primates after direct intrahepatic nucleic acid transfection.

Cell culture-adapted variants of hepatitis A virus (HAV) in which the first pyrimidine-rich tract (pY1; nucleotides 99 to 138) of the 5' nontranslated region has been deleted (delta 96-137 or delta 96-139) replicate as well as parental virus in cultured cells (D.R. Shaffer, E.A. Brown, and S.M. Lemon, J. Virol. 68:5568-5578, 1994). To determine whether viruses with such large deletion mutations are able to replicate and to produce acute hepatitis in primates, we reconstructed the delta 96-137 deletion in the genetic background of a virulent virus which differs from the wild type by only one mutation in the 2B-coding region (HM175/8Y). Full-length synthetic delta 96-137/8Y RNA was injected into the livers of two HAV-seronegative marmosets (Saguinus mystax). Both animals developed serum liver enzyme elevations and inflammatory changes in serial liver biopsies within 3 to 4 weeks of inoculation which were comparable in magnitude to those observed previously following intrahepatic inoculation of marmosets with HM175/8Y RNA. Sequencing of RNA from virus shed in feces demonstrated the presence of the delta 96-137 deletion. These results indicate that the pY1 sequence of HAV is not required for efficient viral replication in hepatocytes in situ or for production of acute hepatic injury following intrahepatic RNA transfection in primates.     

Detection of hepatitis a virus by using a combined cell culture-molecular beacon assay

Rapid and efficient methods for the detection and quantification of infectious viruses are required for public health risk assessment. Current methods to detect infectious viruses are based on mammalian cell culture and rely on the production of visible cytopathic effects (CPE). For hepatitis A virus (HAV), viral replication in cell culture has been reported to be nonlytic and relatively slow. It may take more than 1 week to reach the maximum production and subsequent visualization of CPE. A molecular beacon (MB), H1, specifically targeting a 20-bp 5' noncoding region of HAV, was designed and synthesized. MB H1 was introduced into fixed and permeabilized fetal rhesus monkey kidney (FRhK-4) cells infected with HAV strain HM-175. Upon hybridizing with the viral mRNA, fluorescent cells were visualized easily under a fluorescence microscope. Discernible fluorescence was detected only in infected cells by using the specific MB H1. A nonspecific MB, which was not complementary to the viral RNA sequence, produced no visible fluorescence signal. This MB-based fluorescence assay enabled the direct counting of fluorescent cells and could achieve a detection limit of 1 PFU at 6 h postinfection, demonstrating a significant improvement in viral quantification over current infectivity assays.     

Replication of hepatitis A viruses with chimeric 5' nontranslated regions.

The role of the 5' nontranslated region in the replication of hepatitis A virus (HAV) was studied by analyzing the translation and replication of chimeric RNAs containing the encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES) and various lengths (237, 151, or 98 nucleotides [nt]) of the 5'-terminal HAV sequence. Translation of all chimeric RNAs, truncated to encode only capsid protein sequences, occurred with equal efficiency in rabbit reticulocyte lysates and was much enhanced over that exhibited by the HAV IRES. Transfection of FRhK-4 cells with the parental HAV RNA and with chimeric RNA generated a viable virus which was stable over continuous passage; however, more than 151 nt from the 5' terminus of HAV were required to support virus replication. Single-step growth curves of the recovered viruses from the parental RNA transfection and from transfection of RNA containing the EMCV IRES downstream of the first 237 nt of HAV demonstrated replication with similar kinetics and similar yields. When FRhK-4 cells infected with recombinant vaccinia virus producing SP6 RNA polymerase to amplify HAV RNA were transfected with plasmids coding for these viral RNAs or with subclones containing only HAV capsid coding sequences downstream of the parental or chimeric 5' nontranslated region, viral capsid antigens were synthesized from the HAV IRES with an efficiency equal to or greater than that achieved with the EMCV IRES. These data suggest that the inherent translation efficiency of the HAV IRES may not be the major limiting determinant of the slow-growth phenotype of HAV.     

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.     

Studies in chimpanzees of live, attenuated hepatitis A vaccine candidates

Human hepatitis A virus was attenuated in virulence for chimpanzees by passage in FRhK6 and human diploid lung fibroblast cell cultures. A number of variants were developed by passage in cell cultures which showed different levels of virulence/attenuation for chimpanzees. These results were compared to those obtained with marmosets and reported previously. In general, most variants behaved similarly in the two animal types. Two chimpanzees which gave vaccine-like responses following inoculation with HAV cell culture variants were challenged with virulent HAV. Both animals were immune to HAV infection. These findings provide further evidence for the feasibility of developing live, attenuated vaccines against human hepatitis A.