Glòria Sánchez

Molecular Characterization of Hepatitis A Virus Isolates from a Transcontinental Shellfish-Borne Outbreak

ABSTRACT One hundred eighty-four serologically confirmed cases of hepatitis A were reported in eastern Spain in 1999. A matched case-control study implicated imported coquina clams complying with European Union shellfish standards as the source of infection; this implication was confirmed by the detection by reverse transcription-PCR of hepatitis A virus (HAV) RNA in shellfish samples. In spite of the recognized low variability of HAV, genetic characterization of the complete capsid region of virus isolates from patient serum samples revealed the existence of both synonymous and nonsynonymous variants. Two antigenic variants were detected, one in a discontinuous epitope defined by monoclonal antibody K3-4C8 and a second in a linear VP1 epitope of the virus. In spite of these antigenic variants, all isolates were assigned to genotype IB, providing further evidence that the outbreak originated from a common source, although multiple strains were likely to be involved.

Genome Variability and Capsid Structural Constraints of Hepatitis A Virus

ABSTRACT The number of synonymous mutations per synonymous site (Ks ), the number of nonsynonymous mutations per nonsynonymous site (Ka ), and the codon usage statistic (Nc ) were calculated for several hepatitis A virus (HAV) isolates. While Ks was similar to those of poliovirus (PV) and foot-and-mouth disease virus (FMDV), Ka was 1 order of magnitude lower. The Nc parameter provides information on codon usage bias and decreases when bias increases. The Nc value in HAV was about 38, while in PV and FMDV, it was about 53. The emergence of 22 rare codons in front of 8 in PV and 7 in FMDV was detected. Most of the conserved rare codons of the P1 region were strategically located at the carboxy borders of β barrels and α helices, their potential function being the assurance of proper folding of the capsid proteins through a decrease in the translation speed. This strategic location was not observed for amino acids encoded by the conserved rare codons of the 3D region. The percentage of bases with low pairing number values was higher in the latter region, suggesting a role of the conserved rare codons in the maintenance of RNA structure. Many of the rare codons in HAV are among the most frequent in humans, unlike in PV or in FMDV. This fact may be explained by the lack of cellular shutoff in HAV. One hypothesis is that HAV has evolved in order to avoid competition with its host for cellular tRNAs.

Prevalence of enterovirus and hepatitis A virus in bivalve molluscs from Galicia (NW Spain): inadequacy of the EU standards of microbiological quality.

A study of the presence of hepatitis A virus (HAV) and enterovirus (EV) in shellfish from the northwestern coast of Spain, one of the most important mussel producers in the world, was carried out employing dot-blot hybridization and RT-PCR techniques. In addition, bacterial contamination of the samples was evaluated by Escherichia coli (EC) counts, according to the European Union (EU) standards of shellfish microbiological quality. Shellfish samples included raft-cultured and wild mussels, as well as wild clams and cockles. Bacterial counts showed that the majority of samples (40.8%) could be classified as moderately polluted following the EU standards, and therefore should undergo depuration processes. However, differences in bacterial contamination were observed between cultured mussel and wild shellfish. Thus, percentage of clean samples (<230 EC/100 g shellfish) was clearly higher in cultured mussels (49.1%) than in wild mussels (22.8%) or clams and cockles (10.7%). HAV was detected in 27.4% and EV in 43.9% of the samples that were analyzed. Simultaneous detection of both viral types occurred in 14.1% of the samples. Statistical tests of dependence (chi-square test) showed no relationship either between viral and bacterial contamination, or between the presence of HAV and EV. Comparative analysis of hybridization and RT-PCR for viral detection yielded different results depending on the virus type that was studied, RT-PCR being effective for HAV but not for EV detection. The obtained results reinforce once again the inadequacy of bacteriological standards to assess viral contamination and suggest that although virological analysis of shellfish is possible by molecular techniques, interlaboratory standardization and validation studies are needed before the routine use in monitoring shellfish microbiological safety.

Evidence for quasispecies distributions in the human hepatitis A virus genome.

Nucleotide sequence analysis of multiple molecular clones of the hepatitis A virus (HAV), generated by reverse transcription-PCR of two capsid-coding regions, revealed a degree of heterogeneity compatible with a quasispecies structure in three clinical samples. Passage of plaque-purified reference strain HAV pHM175 43c in FRhK-4 cells documented the generation of a mutant distribution of HAV genomes. The mutant spectra showed mutation frequencies in the range of 1 x 10(-3) to 1 x 10(-4) substitutions per nucleotide, with a dominance of transition over transversion mutations. While in the VP3-coding region, nonsynonymous mutations were predominant; in the VP1-coding region they were uncommon. Around 50% of the amino acid replacements involved residues located at or near antigenic sites. Most of the detected mutations occurred at or in the vicinity of rare codons, suggesting a dynamics of mutation-selection, predominantly at and around rare codons. The results indicate that despite antigenic conservation, HAV replicates as a complex distribution of mutants, a feature of viral quasispecies.

Hepatitis A virus detection in food : current and future prospects

Hepatitis A virus (HAV) is responsible for around half of the total number of hepatitis infections diagnosed worldwide. HAV infection is mainly propagated via the faecal‐oral route and as a consequence of globalisation, transnational outbreaks of foodborne infections are reported with increasing frequency. Molecular procedures are now available and should be employed for the direct surveillance of HAV in food and environmental samples.

Hepatitis A virus in urban sewage from two Mediterranean countries

Molecular methods for the detection and typing of hepatitis A virus (HAV) strains in sewage were applied to determine its distribution in Cairo and Barcelona. The study revealed the occurrence of different patterns of hepatitis A endemicity in each city. The circulating strains characterized, whether in Cairo or Barcelona, were genotype IB. The effects of a child vaccination programme and the increase in the immigrant population on the overall hepatitis A occurrence in Barcelona were evaluated. While vaccination contributed to a significant decrease in the number of clinical cases, the huge recent immigration flow has probably been responsible for the re-emergence of the disease in the last year of study, in the form of small outbreaks among the non-vaccinated population.

Capsid Region Involved in Hepatitis A Virus Binding to Glycophorin A of the Erythrocyte Membrane

ABSTRACT Hepatitis A virus (HAV) has previously been reported to agglutinate human red blood cells at acidic pHs. Treatment of erythrocytes with different enzymes and chemical reagents indicated that HAV attachment is mediated through an interaction with sialylglycoproteins. HAV hemagglutination could be blocked by incubating the virus with glycophorin A, indicating that this sialylglycoprotein is the erythrocyte receptor. The number of receptors used was estimated to be around 500 per cell. At the same time, HAV-induced hemagglutination could also be blocked by either monoclonal antibody H7C27 or an anti-VP3(102-121) ascitic fluid, indicating that lysine 221 of VP1 and the surrounding VP3 residues lining the capsid pit are involved in HAV binding to erythrocytes.

Enhancement of the immunogenicity of a synthetic peptide bearing a VP3 epitope of hepatitis A virus

The immune responses elicited in mice by different forms of the VP3(110–121) B‐epitope of the hepatitis A virus (HAV) were studied. Different forms of incorporation in liposomes were tested, encapsulation, rather than surface exposure, being the best antigenic preparation. Three larger peptides of the VP3 epitope, two of them containing a hepatitis B virus T‐epitope, and a third containing a putative T‐epitope of HAV (VP3(102–121)) were assayed. While this latter T‐epitope induced an enhancement of the response against the VP3 B‐epitope, the artificially coupled T‐epitopes failed to induce a significant increase. The administration of two multiple antigenic peptide (MAP) constructs, the first containing the VP3(110–121) and VP1(11–25) HAV sequences and the second only the VP1(11–25) sequence, also suggested the presence of a T‐epitope, since the response against the VP1 peptide was increased in the first construct.

Hepatitis A virus polyprotein processing by Escherichia coli proteases

Hepatitis A virus (HAV) encodes a single polyprotein, which is post-translationally processed. This processing represents an essential step in capsid formation. The virus possesses only one protease, 3C, responsible for all cleavages, except for that at the VP1/2A junction region, which is processed by cellular proteases. In this study, data demonstrates that HAV polyprotein processing by Escherichia coli protease(s) leads to the formation of particulate structures. P3 polyprotein processing in E. coli is not dependent on an active 3C protease: the same processing pattern is observed with wild-type 3C or with several 3C mutants. However, this processing pattern is temperature-dependent, since it differs at 37 or 42 degrees C. The bacterial protease(s) cleave scissile bonds other than those of HAV; this contributes to the low efficiency of particle formation.

Presence of Norovirus Sequences in Bottled Waters Is Questionable

Beuret and colleagues reported the detection and identification of noroviruses (NV) in 53 out of 159 samples of mineral water using reverse transcription-PCR (RT-PCR) ([1][1]). This sensitive technique is prone to false-positive results, but these can be identified as such by performing sequence