| Immunocapture-RT-PCR (IC-PCR) may be used in detecting viruses with degenerate primers, without prior isolation of total nucleic acids. It is especially useful for detecting viruses that exist in low or variable titers en planta and in plant species which contain various forms of PCR amplification inhibitors (polysaccharides, tannins, polyphenolics etc.).A multiplex, immunocapture PCR (M-IC-PCR) was developed for the detection of HBV,HCV and HEV. Eight pairs primers were selected. Furthermore, immunocapture conditions were optimized in order to prevent from interfering with other genome during the PCR step. This improved detection method successfully allowed the accurate,specific and sensitive detection of HBV,HCV and HEV only. It has been used many samples detection. And the PCR products were sequenced and analyzed by ClusterX v1.81 ,MEGA and PHYLIP WIN2.0 software.The full-length genomic sequence of a Changchun swine hepatitis E virus (HEV) isolate (Ch-S-1) recovered from bile of a pig indigenous infected was determined. Four overlapping fragments of genome were amplified with reverse transcription nested polymerase chain reaction (RT-nPCR) and the5'and 3'ends were amplified with RACE method. The PCR products were cloned into pMD18-T vector and sequenced. The result showed that Ch-S-1(GenBank ID EF077630) genome consisted of 7261 nucleotides,excluding the poly (A) tail of at least 22 residues and contained three open reading frames (ORFs), ORF-1 encoding 1706 amino acids, ORF-2 encoding 674 amino acids and ORF-3 encoding 114 amino acids. Comparative full-length genome sequence and phylogenetic analyses suggested that the Changchun swine HEV represents a distinct variant among the genotype 4 isolates with a divergence of 17.5–6.1%. Analyses based on ORF-1, 2 and 3 yielded similar results. The ORF-1 consisted of 5121 nucleotides capable of coding for a protein of 1706 amino acids (a.a.). It was 1 a.a. shorter than that of other genotype4, while identical in length to JYI-ChiSai01C and JKO-ChiSai98C human isolates genotype4. Ch-S-1 showed 93.7%-97.6% a.a. identity with other genotype 4 isolates. The ORF-2 showed 85.3% nucleotide and 97.2% amino acid identity with JYI-ChiSai01C genotype 4 isolates. ORF-3 of Ch-S-1 had a coding capacity of 114 amino acids. As seen in all genotype 4 sequences,the single nucleotide insertion at position 5159nt shifted the initiation codon of ORF-3 of Changchun swine HEV further 28 nt downstream resulting in 9 aminoacids shorter ORF-3 when compared with that of genotypes 1–3. This inserted nucleotide was T in all the other genotype 4 isolates. The PNI was 91.6–97.6 with with other isolates of genotype 4. At a.a. level Ch-S-1 ORF-3 was 95.8- 99.2% similar to genotype 4 human and swine isolates.As compared to type 4 HEV isolates, 36 unique amino acid substitutions were recorded. Ch-S-1 showed absence of'C(T)CC'at 2406 to 2408 position while all other type 4 isolates have insertion of'C(T)CC'at the same position. Whether these changes contribute towards observed absence of type 4 HEV infections in Changchun swine needs to be determined.For future studies of HEV replication and pathogenesis a full-length cDNA clone of swine HEV was constructed .The availability of this cDNA clone for a swine strain of HEV now affords an opportunity to study the mechanisms of HEV cross-species infection and tissue tropism by constructing chimeric viruses among human, swine and avian HEVs.
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