Molecular Epidemiologic Surveillance Of Equine Influenza Virus In China And Primary Study Of Fowlpox Virus-vectored Vaccine Against EIV

Equine influenza (EI) is a common viral respiratory disease of Equus caused by orthomyxovirus of the genus influenza A. As we known, influenza A viruses could be divided into different subtypes according to the difference of the surface glycoproteins (H1-H16, N1-N9), while the viruses infecting Equus are of the H7N7 or H3N8 subtype. Though the H7N7 subtype equine influenza viruses have not been detected from the horse herds for a long time, vaccine inoculation does not prevent the transmission of H3N8 subtype equine influenza viruses around the world. China is a big country of horse breeding, and has experienced many equine influenza epizootics, which caused a serious economic loss to the horse breeding industry. Meanwhile, the horse racing industry in China is booming. Hence, the topic of this study is research of the genetic evolution of equine influenza virus, selection of equine influenza field strains isolated in China as vaccine strains, and preparation of candidate vaccine to prevent the equine influenza epizootics in China effectively.In 2007, a new outbreak of equine influenza appeared in Hetian of Xinjiang, China. Cases of severe respiratory disease occurred in donkeys. Approximately 13600 donkeys were infected and some died after onset of clinical signs. Sick animals were mainly local species of Xinjiang donkey with clinical signs of fever, couch, nasal discharge, and anorexia. We collect nasal samples from infected donkeys. The result of virus isolation showed that the isolation could propagate in 9-10-day old embryonated chicken eggs. The hemagglutination assay titer of the allantoic fluid was 128 and the EID₅₀ was 10^7.5/ml. Hemagglutination activity of the virus could be inhibited by the equine influenza positive sera. A field strain, designated as A/donkey/Xinjiang/5/2007, was isolated from a sick donkey in Xinjiang. Cluster analysis of the genomic fragments of equine influenza virus showed that A/donkey/Xinjiang/5/2007 belonged to equine influenza virus. Hence, the present study described the isolation and genetic characterization of an equine influenza virus isolated from sick donkeys for the first time in China. This study provided a detailed analysis of the genetic variation of equine influenza virus in China that is likely to be helpful to guide efficient diagnostic, preventive and control strategies against equine influenza in China.China experienced an outbreak of equine influenza during 2007–2008. Meanwhile, its neighbor countries, such as Mongolia, India and Japan, have also been affected by various influenza virus strains in each country. Phylogenetic analysis showed that the newly emerging Chinese strains belong to Florida sublineage clade 2, as well as the Indian strain Jammu-Katra/6/08 and the Mongolian strain Mongolia/1/08. All of these strains were derived from European strains of this clade, such as the Newmarket/1/07 and Cheshire/1/07 strains, but these were not related to Japanese strains isolated around the same time (Florida sublineage clade 1) or to Chinese strains isolated in the 1990s (European lineage). Some unique amino acid changes were found in the antigenic sites in Asian strains of Florida sublineage clade 2. Moreover, the loss of a glycosylation site was found in the Liaoning/9/08 strain. From these studies, we have determined that equine influenza viruses in China have evolved with some new characteristics during recent years, and this emphasizes the importance of continued equine influenza virus surveillance in China.During recent years, China is always threatened by the equine influenza. Unfortunately, most horses were not inoculated by the equine influenza vaccine, and there was no available equine influenza vaccine in China, which mainly rely on import. Hence, we decided to develop a fowlpox virus-vectored equine influenza vaccine expressing the hemagglutinin gene of both lineages of H3N8 equine influenza viruses. First, we successfully constructed two recombinant fowlpox viruses (rFPV-XJ and rPFV-QH), which can express the protective antigen genes of American lineage virus strain A/equine/Xinjiang/3/2007 and European lineage virus strain A/equine/Qinghai/1/1994. The recombinant viruses could be reactive with equine influenza positive sera by western blot or indirect immunofluorescent assay, which indicated that the recombinant virus could express the foreign gene correctly.In order to identify the efficacy of the recombinant vaccine in experiment animals, we made a research on the recombinant fowlpox virus vaccine. First, the grouped mice were immunized with different recombinant vaccine or control, and challenged with A/equine/Xinjiang/3/2007 or A/equine/Qinghai/1/1994. The results showed that two recombinant equine influenza vaccines could induce virus specific antibodies in mouse. The recombinant vaccine could protect mouse from the challenge of equine influenza virus, while all the control groups were infected by the virus. Pony experiment showed that the recombinant vaccine could induce protective antibodies in pony. Virus isolation was occasionally positive in the immune group, while the control group could shed the virus continuously. In conclusion, the animal experiment study showed that the recombinant vaccine could protect animals from challenge of the virus. The recombinant vaccine has potential application in the prevention of equine influenza epizootics in China.