Genetic variability of hepatitis A virus strain HAF-203 isolated in Brazil and expression of the VP1 gene in Escherichia coli

Abstract

The hepatitis A virus (HAV) HAF-203 strain was isolated from an acute case of HAV infection. The primary isolation of HAF-203 in Brazil and its adaptation to the FRhK-4 cell lineage allowed the production of large amounts of viral particles enabling molecular characterization of the first HAV isolate in Brazil. The aim of our study was to determine the nucleotide sequence of the HAF-203 strain genome, compare it to other HAV genomes and highlight its genetic variability. The complete nucleotide sequence of the HAF-203 strain (7472 nucleotides) was compared to those obtained earlier by others for other HAV isolates. These analyses revealed 19 HAF-specific nucleotide sequence differences with 10 amino acid substitutions. Most of the non-conservative changes were located at VP1, 2C, and 3D genes, but the 3B region was the most variable. The availability of HAF-203 complementary DNA was useful for the production of the recombinant VP1 protein, which is a major determinant of viral infectivity. This recombinant protein was shown by enzyme-linked immunoassay and blotting, to be immunogenic and resemble the native protein, therefore suggesting its value as a reagent for incorporation into diagnostic tests. Key words: hepatitis A virus (HAV) -HAF-203 strain -complete genome (Brazil) -VP1 gene recombinant proteinEscherichia coli expression -HAV diagnosisThe hepatitis A virus (HAV) is the causative agent of an old and common human disease, with high incidence rates, still present in many countries throughout the world. In Brazil, this disease has been responsible for 50% of the acute hepatitis cases referred to the National Reference Center for Viral Hepatitis/Oswaldo Cruz Institute/Ministry of Health. The etiological agent was first identified by Feinstone et al. (1973), and the virus is currently classified as the first and unique member of the genus Hepatovirus, Picornaviridae family (Minor 1991).HAV is a 27-nm non-enveloped icosahedral particle, which comprises a single stranded plus-sense RNA (Coulepis et al. 1981). The viral genome is composed of approximately 7500 nucleotides, edged by a covalently linked VPg protein at the 5' terminal and a polyadenylated 3' end. The viral RNA encodes a single polyprotein of 2224 amino acids, which is later cleaved into 11 viral polypeptides (Cohen et al. 19847a).The Brazilian HAF-203 strain investigated here was obtained from a faecal specimen of an acute hepatitis A patient. The viral isolation and serial passage history was previous reported by Gaspar et al. (1992). The HAF-203 HAV strain was inoculated into fetal Rhesus kidney cells (Baptista ML et al. unpublished). In the present study, we have sequenced the complete genome of the HAF-203 HAV strain and compared it with other HAV isolates. Several groups have adopted recombinant viral proteins for diagnostic purposes (Ostermayer et al. 1987, Johnston et al. 1988, LaBrecque et al. 1998, Ternovai et al. 2001, Di Napoli et al. 2004. A fragment of the HAF-203 strain encoding the complete VP1 gene was cloned and expressed in E...

First full-length genomic sequence of a hepatitis A virus isolated in Argentina shows recombination between subgenotypes IA and IB

“…The identity of HAV-Arg/06 nucleotide sequence with the two other full-length sequences available from South America was 95.3% with Uruguayan subgenotype IA HAV5 strain (García-Aguirre and Cristina, 2008) and 91.7% with the Brazilian subgenotype IB HAF-203 strain (Baptista et al, 2006;Gaspar et al, 1992). At the amino acid level, the identity of the HAV-Arg/06 sequence was 99.0% and 98.2% with the same strains, respectively (Table 3).…”

Detection of Antibodies against Hepatitis A Virus (HAV) by a Surface Plasmon Resonance (SPR) Biosensor: A New Diagnosis Tool Based on the Major HAV Capsid Protein VP1 (SPR-HAVP1)

“…The immunodominant neutralization site of HAV mainly involves residues of VP1 and VP3 and a potentially independent site involving residue 221 of VP1 [ 24 ]. Due to the recognized role of VP1 in the humoral immune response during infection, this protein has been the main target of interest for application in the diagnosis of hepatitis A [ 21 , 25 ]. The identification of B cell epitopes in target antigens is one of the crucial steps in immunodiagnostic tests based on epitope [ 26 ].…”

Optimization of Methods for Detecting Hepatitis A Virus in Food

“…Virus used in seeded experiments presented a titer of 10 4 TCID 50 /ml (6.9 9 10 3 PFU/ml) determined by EIA and 6 9 10 6 copies/ml as determined by quantitative PCR. HAF-203 strain was used because it is the only strain isolated in Brazil and it was propagated and characterized previously (Gaspar et al, 1993;Villar et al 2004b) and presented the same genotype and high similarity to HM175 strain (Baptista et al 2006).…”