Pathogenicity Of Equine Influenza Virus H3N8 Subtype And Development Of Inactivated Vaccine Against Equine Influenza

Equine influenza is one kind of acute contagious respiratory disease in Equus animals caused by the equine influenza virus, and also it is in notifiable form of OIE. There are currently two subtypes of equine influenza viruses in circulation, the outbreaks of equine influenza caused by Equine -1 influenza virus (H7N7) or Equine-2 influenza virus (H3N8) is very common in the world. The equine influenza occurred in the world was caused only H3N8 subtype EIV in the last 30 years. Frequent outbreaks of H3N8 subtype equine influenza results in interruption of horse industry, and in some cases, particularly in the racing industry, with significant financial impact.In 2007, Outbreaks of equine influenza have been reported in Australia, Mongolia, Japan and other countries, an outbreak of respiratory disease with the similar influenza symptoms occurred in racehorses in Xinjiang and Northern China. According to the results of epidemiological investigation, we got one H3N8subtype equine influenza virus. Based on the data of isolation and identification, in order to further exploring pathogenesis, interspecies spread and the inactivated vaccine against Equine influenza, following experiments have been conducted in this study:(1) Isolation and identification of H3N8 EIV occurred in Northern China, determination the pathogenicity, exploring and analysis the molecular mechanism of attenuation with embryo propagations of virus seed lot of H3N8 EIV strains.One isolates denominated A/Equine/Huabei/1/07(H3N8) of lower pathogenic Equine influenza virus was isolated from nasal discharge of sick racing horse, it belongs to the American lineage and it is original horse strain.Twelve to 20-month-day-old Mongolia Horses were experimentally inoculated with the F2, F3, F4, F5 and F10 propagation of EIV H3N8 by spraying routes at the dose of 5×106.7EID50 per horse. All inoculated horses with F2 propagation virus exhibited the typical clinical signs such as continuity pyrexia and watery nasal discharge of EI. In contrast, all the horses inoculated with other propagation virus showed mild and subclinical signs of EI, the data from the experiment clearly suggest that the F10 propagation virus seed lot may not serve as a challenge virus for develop the model of El.By means of RT-PCR with specific primers from the noncoding sequence of each gene segment and sequencing, full coding sequences of eight viral genes of the F2, F3, F4, F5 and F10 propagation of EIV H3N8 were amplified and sequenced. Phylogenetic analysis based on HA gene and deduced amino acid showed that (1) the four propagation of virus (F2, F3, F4, F5) possessed an HA cleavage site sequence identical to each other with HA homology of 100% and 99.90%,99.80% to F10 respectively.The variation site is located in the 539, the nucleotide of F2, F3, F4, F5 propagation is Adenine and Guanine from F10 propagation, corresponding the amino acid of Asparagine (A) is changed to acidity Aspartic Acid (D), the variation of HA gene primarily interpret the mechanism of pathogenicity of 5 propagations of EIV.(2) Infectivity analysis of canine to isolates of EIV H3N8, providing the experimental data for further exploring interspecies spread regarding the infectivity of canine to EIV H3N8.Seven greyhounds and 12 beagles were experimentally inoculated with F2 propagation isolates of EIV H3N8 by intranasal (IN) routes at the dose of 106.7 EID50 per dog or 2×106.7 EID50 per dog. Of the 19 inoculated canines, none of them exhibited any clinical signs of illness and all remained healthy during the observation period (on days 14-21 post inoculation) and showed no gross lesions. While the trachea exhibited very mild lesions such as falling off of cilia. Both viruses were recovered and HI antibody was detected from most of the experimentally infected canines. Results from the experiment clearly suggest that the H3N8 subtype EIV prevalence now may not threaten to the dogs.(3) Inactivated vaccine against Equine influenza was primarily developed with isolated strain of H3N8 EIV.The inactivated whole virus vaccine against equine influenza containing Aluinium Hydroxide adjuvant was manufactured, the data of safety test indicated that the guinea pigs vaccinated with killed vaccine exhibited very little adverse reaction. Both horses and guinea pigs which received different vaccines containing 64 or 128 HA unit from F6, F8 and F10 propagation antigen had high levels of HI antibody on the 28 days post vaccination, furthermore, the serum HI titers from horse and guinea pigs showed direct correlation, guinea pigs is one of the ideal experimental animals available for equine influenza vaccine efficacy.