| Hepatitis A virus (HAV) infection is the leading cause of acute viral hepatitis throughout the world. The distribution patterns of hepatitis A in different geographical areas of the world are closely related to socioeconomic development. Although through the improvement of sanitary conditions and the use of HAV vaccines, hepatitis A outbreak in China has declined in recent years, HAV is still one of the main causes of acute viral hepatitis in China.Hepatitis A is transmitted via the fecal-oral route either by person to person contact or by contaminated food or water. Molecular epidemiological methods provide a better understanding of the virus genotypes circulating in a particular territory and the mechanisms of virus transmission.There is only one serotype identified so far for HAV. Studies have shown that the HAV neutralizing antigenic sites are highly conserved and mainly located within structural protein VP1 and VP3. Many HAV strains have been identified by sequencing the VP1-2A junction region in the world, but the VP3-VP1 region, especially the neutralizing antigenic sites at the nucleotide and amino acid level were rarely sequenced. In this study, anti-HAV IgM positive serum samples were collected from provinces of Ningxia, Henan, Hebei and Xinjiang during 2005-2008, HAV structural protein VP3-VP1-2A region were characterized and analyzed by nucleic acid (RNA) extraction, RT and Nested PCR,42 sera samples were sequenced, Phylogenetic analysis showed that 42 strains belonged to genotype I,40 belonged to sub-genotype IA,2 belonged to sub-genotype IB. In the VP1-2A junction region, the nucleotide and amino acid homology for the 42 HAV strains were 89.1%-100% and 97.3% - 100% respectively; in the full-length structural protein VP3-VP1 region, the nucleotide and amino acid homology were 87.6%-100% and 98.8%-100%.Strains with identical nucleotide sequences in the VP1-2A junction region were 98.4% to 100% identities in the complete VP3-VP1 region, or 0 to 2 nucleotide differences in this region. There were no amino acid changes at neutralizng antigenetic sites of VP3-VP1 region for the 42 HAV strains. All the 42 HAV strains belonged to genotype I, with 40 strains clustering to sub-genotype IA and 2 to sub-genotype IB. Different HAV strains analysed in this paper differ on the nucleotide sequences in the VP3-VP1 region, but the amino acid sequences were highly conserved with no changes at neutralizng antigenetic sites. Both the nucleotide and amino acid sequences of the strains with the same VP1-2A junction region had identical or closely related sequences when compared in the complete VP3-VP1 region.Real-time RT-PCR technology offers many advantages over conventional RT-PCR for rapid and specific detection of viral particles, including high sensitivity, the possibility of simultaneously amplifying and detecting the targeted nucleic acids in a single step, et al. Considering their higher analytical sensitivity, the proposed real-time RT-PCR assay could therefore represent valuable tools for the detection of HAV in clinical, environment and food samples.In this study, a Taq man realtime RT-PCR assay for the quantitative detection of HAV was developed, using primer and probe system from HAV 5'NCR region, which is the most conserved region of the genome.Using a single-stranded RNA synthetic transcript (in vitro transcription) of a cloned HAV cDNA corresponding to the amplimer, the best performance in terms of sensitivity and reproducibility was achieved by a one-step TaqMan RT-PCR, and the detection limit of this TaqMan real time RT-PCR assay was 10 copies/reaction or 0.03TCID50/reaction respectively, while the sensitivity of conventional nested RT-PCR was 0.1TCID50. The test proved to be highly specific after a broad panel of virus was tested. The maximum CV of intra-sample Ct values was 2.0% and inter-sample Ct value was 2.6%, revealing its good reproducibility.87 HAV IgM positive serum samples and 2 attenuated live Hepatitis A vaccines were tested in this study, the titers of HAV found in serum samples were5.18 x 102-4.93 x 107 copies/ml. In some serum samples (7-50 days after onset and treatment), the HAV titers were 5.18×102-2.5 x 104 copies/ml, while for the live attenuated Hepatitis A vaccines,1TCID50 contains about 3.3 x 102 copies of HAV RNA.In summary, a Taqman Realtime RT-PCR assay for detection of HAV has been established. This method is sensitive, specific, reproducible, and is suitable for detection and quantification of most prevalent HAV subtypes. |
PDF Full Text Request