Identification and characterization of incomplete hepatitis A virus particles.
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The range of hepatitis A virus (HAV) particles generated during persistent infection of different cell lines was studied. Buoyant density and sedimentation analyses of cell extracts revealed a uniform profile of particles in all cell lines analysed except for BS-C-1 cells. The virion itself usually represented less than 50% of the total mass of virus antigen. A major portion of the antigen was associated with non-infectious, empty particles, which banded at 1.305 g/ml and 1.20 g/ml CsCl, and sedimented in sucrose gradients at 76S and 59S. Empty HAV particles were similar to those of poliovirus with respect to their physical stability and had the characteristic capsid protein content (VP0, VP1 and VP3). An additional RNA-containing particle, probably the provirion, represented only a minor species characterized by a buoyant density of 1.32 g/ml in CsCl and sedimenting at 130S. (+info)
A recombinant vaccinia virus expressing hepatitis A virus structural polypeptides: characterization and demonstration of protective immunogenicity.
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A recombinant vaccinia virus containing most of the P1 region of hepatitis A virus (HAV) was constructed. Cell lysates of cultures infected with the virus contained HAV proteins detectable by radioimmunoassay. Western blot analysis revealed the presence of a single protein of Mr 60K to 62K, bearing epitopes from structural polypeptides VP4, -3 and -2, and the N terminus of VP1. The size of the protein suggests that at least some of the vaccinia virus thymidine kinase is also expressed. Inoculation of tamarin monkeys with the recombinant virus resulted in the development of a specific anti-HAV immune response which was protective against challenge with a virulent strain of HAV. Recombinant viruses expressing the above region of HAV or the proteins expressed by such viruses may be useful in the development of a vaccine suitable for use in man. (+info)
Protease digestion of hepatitis A virus: disparate effects on capsid proteins, antigenicity, and infectivity.
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High concentrations of either trypsin or chymotrypsin caused nearly complete cleavage of capsid protein VP2 of hepatitis A virus but did not significantly reduce the infectivity, thermostability, or antigenicity of the virus. Chymotrypsin also had a lesser effect on VP1. These findings indicate the presence of a protease-accessible VP2 surface site which neither contributes significantly to the dominant antigenic site nor plays a role in the attachment of the virus to putative cell receptors. (+info)
The 5' nontranslated region of hepatitis A virus RNA: secondary structure and elements required for translation in vitro.
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Although the lengthy 5' nontranslated regions (5'NTRs) of other picornaviral RNAs form highly ordered structures with important functions in viral translation, little is known about the 5'NTR of hepatitis A virus (HAV). We determined the nearly complete 5'NTR nucleotide sequences of two genetically divergent HAV strains (PA21 and CF53) and included these data in a comparative phylogenetic analysis of the HAV 5'NTR. We identified covariant nucleotide substitutions predictive of conserved secondary structures and used this information to develop a model of the 5'NTR secondary structure, which was further refined by thermodynamic predictions and nuclease digestion experiments. According to this model, the 5'NTR comprises six major structural domains. Domains I and II (bases 1 to 95) contain a 5'-terminal hairpin and two stem-loops followed by a single-stranded and highly variable pyrimidine-rich tract (bases 96 to 154). The remainder of the 5'NTR (domains III to VI, bases 155 to 734) contains several complex stem-loops, one of which may form a pseudoknot, and terminates in a highly conserved region containing an oligopyrimidine tract preceding the putative start codon by 13 bases. To determine which structural elements might function as an internal ribosome entry site, RNA transcripts representing the HAV 5'NTR with progressive 5' deletions were translated in rabbit reticulocyte lysates. The translation product was truncated, unprocessed P1 polyprotein. Removal of the 5'-terminal 354 bases of the 5'NTR had little effect on translation. However, deletion to base 447 slightly decreased translation, while deletion to base 533 almost completely abolished it. These data indicate that sequences 3' of base 355 play an important role in the translation mechanism utilized by genomic-length HAV RNA. Significantly, this region shares several conserved structural features with the internal ribosome entry site element of murine encephalomyocarditis virus. (+info)
Concentration and detection of hepatitis A virus and rotavirus from shellfish by hybridization tests.
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A modified polyethylene glycol precipitation method for concentration of virus followed by a new method to recover nucleic acid was used to detect hepatitis A virus (HAV) and rotavirus (SA11) in shellfish (oysters and hard-shell clams) by hybridization tests. Infectious virus, seeded into relatively large quantities of shellfish, was recovered consistently, with greater than 90% efficiency as measured by either in situ hybridization (HAV) or plaque assay (rotavirus SA11). Viral nucleic acid for dot blot hybridization assays was extracted and purified from virus-containing polyethylene glycol concentrates. Separation of shellfish polysaccharides from nucleic acid was necessary before viral RNA could be detected by dot blot hybridization. Removal of shellfish polysaccharides was accomplished by using the cationic detergent cetyltrimethylammonium bromide (CTAB). Use of CTAB reduced background interference with hybridization signals, which resulted in increased hybridization test sensitivity. After polysaccharide removal, dot blot hybridization assays could detect approximately 10(6) physical particles (corresponding to approximately 10(3) infectious particles) of HAV and 10(4) PFU of SA11 rotavirus present in 20-g samples of oyster and clam meats. These studies show continuing promise for the development of uniform methods to directly detect human viral pathogens in different types of shellfish. However, practical applications of such methods to detect noncultivatable human viral pathogens of public health interest will require additional improvements in test sensitivity. (+info)
A serologically confirmed, case-control study, of a large outbreak of hepatitis A in China, associated with consumption of clams.
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A matched and serologically confirmed case-control study was carried out to investigate the source of an outbreak of acute hepatitis involving 290,000 cases in the suburbs of Shanghai, in January 1988. A total of 132 patients with acute hepatitis from six different hospitals were chosen as cases and the same number of control patients without hepatitis were matched for gender, age, admission date and area of residence. Serum specimens from both case and control patients were detected for specific anti-hepatitis A (HA) IgM antibody and a questionnaire was used to investigate probable risk factors related to the outbreak. The positive rate of anti-HA IgM was 98.48% in the case group and only 0.76% in the control, indicating that the infection was caused by HA virus. The results revealed that the source and mode of transmission were due to the consumption of contaminated and inadequately cooked clams (Anadara subcrenata lischke). There was a highly positive dose-response relationship between the odds ratio of contracting HA and the quantity or frequency of clam consumption. The odds ratios of acquiring HA from clams were up to 62.4-63.4 by both group stratification and multiple unconditional logistic regression analyses. (+info)
A rapid method for determination of endoproteinase substrate specificity: specificity of the 3C proteinase from hepatitis A virus.
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The preferred amino acid residues at the P'1 and P'2 positions of peptide substrates of the 3C proteinase from hepatitis A virus (HAV-3C) have been determined by a rapid screening method. The enzyme was presented with two separate mixtures of N-terminal acetylated peptides, which were identical in sequence except for the amino acids at the P'1 or P'2 positions, where a set of 15 or 16 amino acids was introduced. Enzyme-catalyzed hydrolysis of the peptide mixtures generated free amino termini, which allowed direct sequence analysis by Edman degradation. The relative yield of each amino acid product in the appropriate sequencing cycle gave the amount of each substrate mixture component hydrolyzed. This allowed the simultaneous evaluation of the relative kcat/Km values for each component in the mixture. The peptide substrates preferred by the HAV-3C proteinase in the P'1 mixture were glycine, alanine, and serine. The enzyme has little specificity at P'2; only arginine and proline peptides were excluded as substrates. This method provides a rapid determination of the preferred residues for a peptide substrate and should be applicable to other endoproteinases. (+info)
Outbreak of hepatitis A in two federal states of Germany: bakery products as vehicle of infection.
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In April 2004, increased numbers of hepatitis A were noted in six neighbouring districts in Germany. Exploratory interviews showed that patients had consumed bakery products from company X where two employees had been diagnosed with hepatitis A in February. A case-control study of consumption of products of company X was carried out through telephone interviews. Altogether, 64 cases were identified. Fifty-two cases and 112 controls aged >or=16 years were included in the case-control study. In total, 46/52 cases and 37/112 controls had consumed company X products [odds ratio (OR) 15.5, 95% confidence interval (CI) 6.1-39.7]. Of these, 36/46 cases and 16/37 controls had consumed pastries (OR 4.7, 95% CI 1.8-12.3), 25/46 cases and 12/37 controls had consumed filled doughnuts (OR 2.5, 95% CI 1.0-6.1). Sequence analysis of the VP1-2A junction region indicated 100% strain homology between cases and an infected employee of company X. We recommended reinforcement of hygiene precautions, and consideration of a prolongation of compulsory work absence after post-exposure vaccination. (+info)