Development Of H3N8 Subtype Equine Influenza Virus Inactivated Vaccine

Equine influenza vaccines were first developed in the 1960s, and are used widely for control of equine influenza. Therefore, equine influenza outbreak has been effectively controlled in the word. Until now, the commercial equine influenza vaccines developed by our country are unavailable, and majority horses in the mainland of China have never been vaccinated. Thus, Chinese horse industry continues to be threatened by equine influenza. From 2007 to 2008, Chinese mainland areas experienced a horse flu pandemic, which brought great disaster once again to the equine industry, gave a heavy blow to Chinese horse racing industry which will rise, and threatened the 2008 Beijing Olympic Equestrian Competition held at the project. With frequent contacts with the international competition and the development of the Chinese horse racing industry, the equine influenza prevention and control work in China has a long way to go, and vaccine development in China is imperative.This dissertation studied the biological characteristics of two American lineage vaccine prototype, A/equine/xinjiang/3/2007(H3N8) (XJ isolation) and A/equine/Hubei/6/2008(H3N8) (HB isolation), which were isolated by our laboratory, and analyzed genetic characterization of HA gene of HB isolation, which provided references to the control of equine influenza in China. After 7 continuous passages in SPF chicken embryos, the HAU of XJ isolation stabilized at 27 to 28, while the HB isolation can be stabilized at 28 starting from the 1st passage. Two isolations can be neutralized by H3N8 subtype special antiserum; can agglutinate the red blood cell of pigs, horse, cattle, sheep, goat, donkey, rabbit, guinea pig, rat, mice and chicken; both were thermostabilization influenza virus; the infectivity still existed after treated by acid, alkali, chloroform and ether. After infected XJ isolation and HB isolation, foals can appear complete influenza clinical symptoms. The virus can be isolated from nasal swabs about during 1 week after infection, and specific antibody can be detected by HI assay about 1 week later after infection.HA gene of HB isolation was cloned using RT-PCR and sequenced by Invitrogen Company. Comparing homology of H3N8 EIV HA gene By NCBI Blast, we found that the HA gene of HB isolation and A/equine/Newmarket/5/2003 (H3N8) showed a homology as high as 98.7%. HA protein amino acids sequence phylogenetic analysis showed that HB isolation belonged to Florida sub-lineage in American lineage equine H3N8.The virus was inactivated by adding formalin to a final concentration of 0.05% and adding BPL to a final concentration of 0.025% shaking at 4°C for 48 to 72 hours. The results showed that equine H3N8 can be completely inactivated by both of two virus inactivators, and the virus HAU detected by HA assay unchanged during inactivation. The result of optimizing virus inactivation time of BPL implied that the inactivation time was relevant to HAU of EIV. Comprehensive considering all factors, we suggested that EIV inactivation time needs 48 to 72 hours. Using molecular weight cut off technology to purify equine H3N8, the results indicated that total protein content of viral suspension purified by 1000kD ultrafiltration membrane was lower than by 350kD ultrafiltration membrane. The aim of purification can be achieved by repeated purified three times using 1000kD ultrafiltration membrane, and virus recovery was up to 100% roughly estimated by HAU. When ponies were vaccinated purified vaccine and unpurified vaccine, the results suggested that the potency of vaccines purified by Millipore tangential flow filtration is much better than non-purified vaccines.OIE suggests that both American lineage prototype and European lineage prototype should be included in the equine influenza vaccine. Equine H3N8 inactivated vaccine was prepared by combing 10% alhydrogel adjuvant with viruses mixture which was equally mixed American lineage vaccine prototype A/equine/xinjiang /3/2007(H3N8) and European lineage vaccine prototype A/equine/Qinghai/1/94(H3N8) (QH isolation) when both of virus isolations were purified by 1000kD ultrafiltration membrane. Inactivation vaccines had a graded HAU of 210, 29, 28, 27 and 26, The animal experiments showed that the inactivated vaccine was safety to guinea pigs and ponies. Serological responses of guinea pigs indicated that similar HI antibody level which was stimulated by graded level of XJ isolation and QH isolation can be produced. Serological responses in horses implied that the HI antibody titers were above in 6log2 at the second week after the second vaccination of all inactivated vaccines, which can meet the requirements of OIE that horses can be completely protected when the HI antibody titers were not less than 6log2. All inactivated vaccines can stimulate higher geometric antibody titers than Virus-based vector ProteqFlu vaccine which has been shown previously to protect horses against challenge infection. Antibody dynamics showed that antibody which was stimulated by all kinds of vaccines can be produced at the first week after the first vaccination, and reached peak at the first or second week after the second vaccination. Then, the antibody was slowly down, and persisted in 25 weeks after the second vaccination. Antibody dynamics after the third vaccination was the similar to the second vaccination.