Development In Reverse Genetics System For Equine Influenza Virus

Equine influenza is an acute contagious respiratory disease in equus animals caused by the equine influenza virus, and Equine influenza is listed in the notifiable diseases list of OIE. The outbreak of equine influenza has a bad influence of our country's economic, so the basic research for equine influenza virus has been the focus of research in virology.The first H3N8 subtype equine influenza isolate of A/equine/Miami/1/1963 was chosen to investigate in our study. After extracting RNA, reverse transcription and PCR, the eight segments of the influenza virus was amplified and inserted into the p HW2000 bidirectional expression plasmid. Finally, the recombination plasmids containing the eight segments was obtained and the equine influenza virus reverse genetics system was constructed. The plasmids were then transfected into the 293 T cell. The cell supernatant was collected and added into the MDCK cell, and at last the rescued influenza virus of r H3N8 was acquired. The sequencing results showed that the nucleotide sequence of r H3N8 was as same as the donor virus. The results of the HA assay, TCID50 and the virus growth curve was same between r H3N8 and the donor virus.Based on the constructed eight-plasmid DNA transfection system, the H3N8 subtype equine influenza isolate of A/equine/Heilongjiang/SS1/2013 isolated in our lab was chosen to investigate. After extracting RNA, reverse transcription and PCR, the PB1, PB2, PA and NP segments of the influenza virus was amplified and inserted into the p HW2000 bidirectional expression plasmid. And then, the p HW2000 bidirectional expression plasmids containing the PB1, PB2, PA and NP segments of A/equine/Miami/1/1963(H3N8)were replaced with that of A/equine/Heilongjiang/SS1/2013(H3N8). And the recombination equine influenza virus was rescued and named t H3N8. The sequencing results indicated that the nucleotide sequence of the rescued equine influenza virus was as same as the segments contained in the transfected plasmids. The HA assay results revealed that thechanging rate of t H3N8 was higher than that of r H3N8. The results of TCID50 and the virus growth curve suggested that t H3N8 had a higher replication rate compared with that of r H3N8. These results revealed that the polymerase proteins of A/equine/Heilongjiang/SS1/2013(H3N8) has a higher adaptability in mammalian cells than that of A/equine/Miami/1/1963(H3N8) virus.In this study, we establish the eight-plasmid DNA transfection system and successful rescue the virus. It provided a foundation for further searching of equine influenza virus,and establish a platform for the genetic engineering vaccine of equine influenza.