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.
Subject(s)
Animals , Dogs , Cell Line , Hemagglutination Tests , Influenza A Virus, H3N2 Subtype , Classification , Genetics , Influenza Vaccines , Plasmids , Virus CultivationABSTRACT
Highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtypes caused enormous economical loss to poultry farms in China and Southeastern Asian countries. The vaccination program is a reliable strategy in controlling the prevalence of these disastrous diseases. The six internal genes of the high-yield influenza virus A/Goose/Dalian/3/01 (H9N2), the hemagglutinin (HA) gene of A/Goose/HLJ/QFY/04 (H5N1) strain, and the neuraminidase gene from A/Duck/Germany/1215/73 (H2N3) reference strain were amplified by RT-PCR technique. The HA gene was modified by the deletion of four basic amino acids of the connecting peptide between HA1 and HA2. Eight gene expressing plasmids were constructed, and the recombinant virus rH5N3 was generated by cells transfection. The infection of chicken embryos and the challenge tests involving chickens demonstrated that the recombinant H5N3 (rH5N3) influenza virus is avirulent. The allantoic fluids of rH5N3-infected eggs contain high-titer influenza viruses with hemagglutination unit of 1:2048, which are eight times those of the parental H5N1 virus. The rH5N3 oil-emulsified vaccine could induce hemagglutination inhibition (HI) antibodies in chickens in 2 weeks post-vaccination, and maximum geometric mean HI-titer were observed 4 approximately 5 weeks post-vaccination and were kept under observation for 18 weeks. The rH5N3-vaccinated chickens were fully protected against morbidity and mortality of the lethal challenge of the H5N1 HPAI viruses, A/Goose/Guangdong/1/96 and A/Goose/HLJ/QFY/04, which had 8 years expansion and differences among multiple amino acids in HA protein. The N3 neuraminidase protein marker makes it possible to distinguish between H5N1 infected- and H5N3 vaccinated animals.
Subject(s)
Animals , Chick Embryo , Chickens , Hemagglutination Inhibition Tests , Hemagglutinin Glycoproteins, Influenza Virus , Genetics , Allergy and Immunology , Influenza A Virus, H5N1 Subtype , Allergy and Immunology , Influenza Vaccines , Allergy and Immunology , Influenza in Birds , Plasmids , Vaccines, Synthetic , Allergy and ImmunologyABSTRACT
The 1.7 kb full-length hemagglutinin (HA) gene fragment of H5N1 subtype avian influenza virus was amplified by RT-PCR and then cloned into the pFastBacHT donor plasmids. The recombinant plasmid pFastBac-H5 was identified by restriction enzyme digestion and sequencing. Following the transposition pFastBac-H5 into the bacmid in DH10Bac E.coli competent cells, the colonies were identified by blue and white selection. The recombinant bacmid (rBacmid-H5) was verified by PCR analysis. Transfection of rBacmid-H5 DNA into sf9 cells using Cellfectin reagent results in the production of recombinant viral stock. Cells were harvested 72h post infection and analyzed by SDS-PAGE, Western-blot, hemagglutination and hemagglutination inhibition test;the expressed HA protein (rH5)shows hemaggluting activity and can be inhibited by H5N1 virus immunized chicken sera. On the other hand, immunization of chickens with rH5 protein results in high titers of H5N1 virus specific hemagglutation inhibition antibodies, which proved its biological activity.