Genetic Evolution Analysis Of HA Genes Of H3N8Subtype EIV In China From2010-2011and Preliminary Studies Of Multiform Vaccines

In recent years, H3N8subtype equine influenza (EI) outbreaks are very frequent, even theimmunized horses sometimes can't defend against the attack of the circulating strains. At present therecombinant canarypox-based vaccines of Proteq-Flu1(Merial) recommended by OIE are only used inGuangzhou Specific Equine Disease-free Zone in China, while the vaccine strain ofA/equine/Kentucky/1/1994(H3N8)(Ken94) does not match with the epidemic strains isolated in Chinafrom2007-2008in antigenicity. Therefore, the purpose of this present study is researching the geneticevolution of EI viruses and screening the vaccine strains, at the same time developing the inactivatedvaccine utilizing the reverse genetics technology and also attempting to prepare the EI recombinantadenovirus vaccine and Virus-like particles (VLPs) vaccine to prevent and control EI outbreaks inChina.Twenty two equine influenza viruses (EIV) were isolated in China from2010-2011. The HA genesof11among22EIV strains were cloned and sequenced, the results showed that the2010-2011and2007-2008isolates were all belong to Asian sub-clade of Florida clade2, but present the graduallydiverging trend. The current vaccine strain of Ken94belongs to Kentucky sub-lineage, comparing with2010-2011China isolates there are eight amino acids difference located in four antigen sites, whichwarned us China EIV isolates took new genetic and antigenic variation.Based on the molecular epidemiology, the HA and NA genes ofA/Equine/Xingjiang/3/2007(H3N8)(XJ3) EIV w as reassorted with the internal genes of PR8utilizingthe reverse genetics technology and quickly rescued the EI vaccine strains rH3N8-PR with thecharacteristic of low toxicity, stability, and high yield in chicken embryonated eggs. Immunologicaltests showed that the vaccine strains rH3N8-PR remained the immunogenicity of parental XJ3, andelicited the hemagglutination inhibiting (HI) and virus neutralizing (VN) antibody responsepost-vaccination on mice and guinea pigs, and provided complete protection against the challenge ofXJ3. The experiments also indicated that the rH3N8-PR vaccine with dose of30μg HA protein caninduce all horses developing HI and VN antibody in a short period of time, although body temperatureof individual horse has a rising phenomenon after challenge, but the horses didn't shed viruses and couldprovide100%protection. While the Proteq-Flu1vaccine (Merial) only provided75%protection againstthe XJ3virus challenge.In order to construct the recombinant adenovirus vaccine rAd-XJ3-HA, the XJ3HA gene wasinserted into an adenovirus vector. The experimental results on mice model showed that therAd-XJ3-HA vaccine has good immunological activity. The rAd-XJ3-HA vaccine could elicit HI andVN antibodies in muscle vaccination, provide100%protection against the attack of parental strain XJ3,and effectively clear viruses in mice. The preliminary research results on horses showed that108TCID50rAd-XJ3-HA could induce50%horse to produceHI and VN antibody, though another50%horse didn'tdevelop detectable HI antibody, but could produce VN antibodies in low level. H3N8subtype EI VLPs vaccine contain HA and M1protein was constructed successfully usingbaculovirus/insect cell expression system. The protein expressed by insect cells infected with therecombinant baculovirus rBV-XJ3-HA-M1shows hemagglutinin activity and the VLPs can beobserved by electronic microscopy. The experimental results on mice model showed that the HA-M1VLPs vaccine could effectively induced HI and NV antibodies and could provide100%protectionagainst the parental XJ3virus attack.In present study, the new genetic and antigenic variation was found in China EIV isolates, whichsuggests that it's necessary to strengthen EI epidemiological surveillance and develop the safe andeffective vaccine using various platforms and systems to control EI outbreaks in China. The reversegenetics technology has the advantages in rescuing the EI vaccine strain. The inactivated vaccineprepared by this technology can provide complete protection to the horses, and is the first choice ofcurrent vaccine research and application. The preliminary research data indicated that the rAd-XJ3-HAvaccine could provide50%protection on horses, so the further research work focuses on optimizing thevaccine to improve the immunity effect. The EI VLPs vaccine shows good immune effect on micemodel. The further experiments should evaluate the immunity effect on horses. This pilot opens up anew train of thought for the development of EI vaccine, and fills the blank of EI vaccine research inChina. This study also provides theoretical foundation and technical reserves for development of EIvaccine with independent intellectual property rights and fitting to our country and for prevention andcontrol EI in China, which has important theoretical and practical significance.