| Equine influenza (El) is one of the most important contagious respiratory diseases which endangered horse raising and racing competition. The disease is characterized by sneeze, fever, depression, anorexia, dyspnoea, and a harsh cough caused by equine influenza virus (EIV). EIV, a member of influenza A viruses, the genus influenzavirus A, B, of the family Orthomyxoviridae, has relatively more close relation with avian influenza viruses, swine influenza viruses, and human influenza viruses which belong to the same species as EIV. EIV complete genome is about 13. 6kb, consisting eight gene segments encoding about ten proteins. Those proteins were PB2, FBI, PA, HA, NA, NP, M, and NS respectively. Once there were three epidemics of equine influenza in China generally, from one of which, a virus named A/Equine/Qinghai/1/94 was isolated and identified. To illustrate the genetic background and origin of EIV, and to develop an ideal vaccine to prevent or control the disease, the following experiments were carried out.After 7 or 8 passages in 10-day embyronated eggs, the HA titer of A/Equine /Qinghai/1/94 tended to keep constant, as high as 1:512. Under the electro-microscope, the virus displayed the morphology of Orthomyxoviridae, appearing almost spherical with envelope, sometimes with pleomorphic forms and lOOnra in diameter. When the virus was heated to over 73℃ for more than 10 minutes or over 71 ℃ for more than 30 minutes, the haemagglutinining activity was completely inactivated. The virus could induce HI antibodies production in mouse, rat, guinea pig, and rabbit with experimental infection via nose drop and lung inoculation. Only in mouse, visibly clinical signs like depression, trembling, and dyspnoea, accompanied by congestion and enlargement of disease lungs. From the lungs a virus was isolated and showed to be EIV like with envelope and haemagglutinining activity. There were not any visible signs or pathological changes in rat, guinea pig, and rabbit, no viruses isolated from the lungs. The titer level of HI antibody in rabbit was much higher than that of mouse, rat and guinea pig.HA was the most important structural protein and protective antigen on the EIV. The mutation frequency of HA gene, faster than any other genes, almost represented the whole virus. To clarify the relation of EIV in China, the HA genes of EIV (A/Equine/Qinghai/1/94, A/Equine/Jilin/1/89 and A/Equine/Heilongjiang /1/89) were cloned and sequenced. Phylogenetic analysis of the HA genes showed the HA of A/Equine/Qinghai/1/94 had 97. 3%~84.7% similarity with the reference strains like A/Equine/Kentucky/2/86, A/Equine/Hong Kong/1/92, and A/Equine/ Miami/1/63, great similarity with Europe H3N8 EIVs, but great difference with A/Equine/Jilin/1/89 and A/Equine/Heilongjiang/1/89 derived from avian influenzavirus.The total RNA was extracted from allantoic fluid of 10-day embyronated eggs infected with the virus. The cDNA of all genes of A/Equine/Qinghai/1/94 were amplified by reverse transcription and polymerase chain reaction (RT-PCR), and then the fragments were ligated with the plasmid pGEM-T-easy. After transforming the recombinant plasmids to DH5a , positive clones were selected. About the same size as expected were identified by cutting the extracted plasmids. Phylogenetic analysis of most genes had close relation with Europe reference EIV strains.A pair of primers with Hindlll restriction digest site and Kozak translation initiation sequence with an ATG initiation codon in forward primer and BamHI restriction digest site in reserve primer were designed to amplify HA gene of the A/Equine/Qinghai/1/94 from pGEM-HA. The amplified HA and the pcDNA 5/TO vector were digested with Hindlll and BamHI and then a eukaryotic expression plasmid was constructed. After transforming and identifying, the plasmid was sequenced to display the ligated open reading frames was correct.In brief, related characterization A/Equine/Qinghai/1/94 was determined, HI test to diagnose EIV was established, a eukaryotic plasmid expressing A/Equine /Qin...
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