Establishment Of Reverse Genetics System For An Equine Influenza Virus

Equine Influenza virus(EIV) is the pathogen of equine influenza, which is a kind of important equine infectious diseases. Its genome consists of 8 segmented negative-sense RNAs that encode at least 10 proteins. A/equine/Jilin/1/1989(H3N8)(JL89) isolated from the Heilongjiang and Jilin province, China in 1989 caused the outbreak of equine influenza and is significantly different from other EIVs. In terms of phylogenetices, the genetic relationsip of JL89 is between the EIVs and Avian Influenza viruses(AIVs) and shows a closer relationship with AIVs. These signify that this virus may originate from avian, break the species barriers and transmit to equine. In addition, this virus caused high morbidity and mortality in herds. Thus, to study the molecular evolution and pathogenesis of this virus is meaningful to understand and control the equine influenza disease. As a powerful tool of virus research, reverse genetics plays an important role in discovering the mechanism of cross-species transmission, pathogenesis and virulence determinants of Influenza virus. Therefore, to establish a reverse genetic system for JL89 will provide a platform to explore the above scientific issues.First of all, a plasmid named pEZ containing EF1-α and polI promoter was constructed. The recombinant plasmid pEZ-vEGFP and pEZ were transfected into HEK 293 T cells, respectively. The cells with pEZ-vEGFP transfected issued green fluorescence while cells with pEZ transfected could not. On the other hand, the coding region and non-coding region can be amplified from the cells with pEZ-vEGFP transfected while nothing can be amplified from cells with pEZ transfected. These illustrate that pEZ can transcripe +RNA and –RNA from the same templet. Secondly, the coding region and non-coding region of 8 segments were cloned and sequenced for acquiring the squences of JL89’s genome. After that, 8 cDNAs were cloned into pEZ to construct 8 recombinant plasmids, and pEZ-HA was testified by western blot. There is a good expression of HA in HEK 293 Tcells. Further study showed that EIV was rescued by co-transfection of 8 recombinant plasmids. The titer of rescued virus was 5.34±0.22 log10TCID50/mL. HA titer was 8 log2/50μL after amplification in SPF embryonated eggs. And the growth kinetics of the rescued virus in MDCK cells was similar to the parent virus. The identity of sequences was 100%. These proved that the reverse genetics system of JL89 was estabilished successfully. Furthermore, to distinguish rescued virus and parent virus, HA obtained HindIII and NdeI restriction sites by site mutations. The rescued virus was confirmed to contain these sites after RFLP analysis. Finally, the ability of rescuing virus of this system was evaluated. It had a higer titer than the pBD system containing CMV promoter(3.76±0.69 log10TCID50/mL).In this study, a bidirectional transcription plasmid pEZ was constructed. The reverse genetics was established and this system have good performance in rescuing virus. The system will be a powerful tool for discovering the mechanism of cross-species transmission, pathogenesis and virulence determinants of JL89.