Establishment Of Reverse Genetics Of H1N1 Subtype Swine Influenza Virus

Influenza is a common respiratory and immunosuppressive disease of swine in swine-producing regions throughout the world, with clinical signs, including coughing, nasal discharge, sneezing, depression, lethargy, laboured breathing, and fever. Once occurs, it may cause great economic lost to swine industry, especially, co-infecting with other respiratory disease, such as PRRS. Besides its severe veterinary importance, swine influenza also poses serious public health threats.In 2004, a virus strain was isolated from tissue samples and cotton swabs collected in pig farm with respiratory tract problems in Guangdong. After propagation in SPF embryonated chicken's eggs and purification, the virus was determinted to be a H1N1 subtype SIV by HI and NI, designated A/Swine/ Guangdong/LM/2004(H1N1), (short for LM). EID50 of LM was 10-6.8/mL, TCID50 was 10-7.2/mL.The entire segments of LM were amplified by RT- PCR, and then all the genes were respectively cloned into pMD18-T vector and sequenced. The sequencing results showed that neither insertion nor deletion was observed in nucleotides of LM. The amino acids sequence of cleavage site of HA is IPSIQSR↓G, suggesting that LM does not have the molecular characteristics of high pathogen. HA has highly conservative N-glycosylation site at the 10, 11, 23, 87, 276 and 287 sites of HA1. However, there are two others at the 154 and 213 sites of HA2. NA has highly conservative N-glycosylation site at the 58, 63, 68, 88 and 146, however, there are three others at the 44, 235 and 386 sites, which may be one molecular characteristics of H1N1 subtype SIV recently. The results of blast show that: HA gene has high homology with the strain of'human-like'SIV(99%), while others have high homology with 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.Meanwhile, the entire segments of LM were respectively cloned into the bidirection transcriptional vector pHW2000. The recombinant plasmids of polymerase genes and nucleotide protein gene were detected by EGFP reporter plasmid. A/Goose/Dalian/3/01(H9N2) was used as backbone to identify the activities of all recombinant plasmids of LM. Then, the eight recombinant positive plasmids were transfected into MDCK/293T cells and then the rH1N1 was rescued. The HA titer of rH1N1 in embryonated chicken's eggs was 1:64, EID50 and TCID50 of rH1N1 was 10-7.1/mL and 10-7.6/mL, respectively. So far, it is the first time to succeed in establishing the reverse genetics syetem of H1N1 subtype swine influenza virus by plasmid-based system worldwide. It settles the foundation for the further research in pathogenicity mechanism and the production of cell grown influenza vaccine of H1N1 in future. After 15 passages in SPF embryonated chicken's eggs, HA and NA of 5th, 10th and 15th of rH1N1 were sequenced. The result showed that all the sequences were completely consistent with the parental strain, demonstrating the stability of rH1N1. Furtherly, another recombinant virus was obtained which contained hemagglutinin (HA) and neuraminidase (NA) genes derived from LM, and the other six internal genes from A/Goose/Dalian/3/01(H9N2). The rescued virus titer was measured by hemagglutination assay and the maximum virus titre of 1:1024 hemagglutination unit was obtained after infection of MDCK cell for 60h, which owned the characteristics of high-yield. A formalin-inactivated oil-emulsion vaccine was prepared from this virus. When pigs were inoculated with 1mL of the vaccine, the hemagglutinin-inhibition (HI) antibody became detectable after two weeks post-vaccination with re-LM and the average titers of HI were 1:32, 1:128 and 1:512 after three times vaccinations, respectively, demonstrating its favourable immunogenicity.