ABSTRACT
To study the genetic variation and evolutionary characteristics of H1N1 swine influenza virus, all the eight genes of LM were amplified by RT- PCR, cloned into pMD18-T vector and sequenced respectively. The results showed that neither insertion nor deletion was observed in nucleotides of LM. The amino acids sequence of cleavage site of HA is IPSIQSR decrease G, suggesting that LM did not have the molecular characteristics of high pathogen. HA had highly conservative N-glycosylation site at position 11, 23, 87 and 276 sites of HA1, and two more at position 154 and 213 sites of HA2. NA had highly conservative N-glycosylation site at position 58, 63, 68, 88, 146, and two more at position 44 and 235 sites, which might be one molecular characteristics of H1N1 subtype of SIV. The results of Bast showed HA gene had high homology to the strain of 'human-like' SIV (99%), while others had high homology to the 'classical' SIV. So it is inferred that HA of LM might originate from human-like linage swine influenza virus, while others might originate from 'classical' swine influenza virus.
Subject(s)
Animals , Cloning, Molecular , Hemagglutinin Glycoproteins, Influenza Virus , Chemistry , Genetics , Influenza A Virus, H1N1 Subtype , Classification , Genetics , Neuraminidase , Chemistry , Genetics , Phylogeny , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
High-yield H3N2 subtype swine influenza virus for large-scale vaccine production in cell culture was generated by reverse genetics. The rescued H3N2 (rH3N2) candidate virus contained hemagglutinin (HA) and neuraminidase (NA) genes derived from a field isolate A/Swine/Henan/S4/01 (H3N2), PB2 gene from A/PR/8/34, and the other five internal genes from A/Goose/Dalian/3/01 (H9N2). The rH3N2 virus titer in MDCK cell culture were measured by hemagglutination assay and the maximum virus titre of 1:512 hemagglutination unit was obtained after infection of MDCK cell for 60 h. The results of the present study indicated that rH3N2 virus was suitable for growth in MDCK cell culture and is feasible to be used for the production of cell grown influenza vaccine.