Establishment Of Detection Method Of Equine Influenza Virus

Equine influenza (EI) is a severe acute upper respiratory infection of the horse, just as with human influenza, and typical symptoms include pyrexia, dyspnoea, anorexia and coughing. Spread of equine influenza virus is very rapid and explosive. Equine influenza virus (EIV) is a type A influenza virus, a member of family Orthomyxoviridae, and there are two distinct subtypes, H7N7 and H3N8. It is generally accepted that the former has been no longer existence in the world the last outbreak caused by this virus was in 1979 in Italy, however, outbreaks of EIVcaused by H3N8 viruses occur annually. Currently, there are no effective therapies against EIV infection. Therefore, it is important to establish detection methods to detect and monitor EIV.1. Establishment of indirect immunofluorescent assay for EIVIn this study, EIV was purified and concentrated from infected allantoic fluid of embryonic egg by centrifugation and ultracentrifugation. Hyper-immunized rabbit serum was prepared by immunizing rabbit for three times with inactive purified EIV mixed with Freund's adjuvant or incomplete Freund's adjuvant. Then, the indirect immunofluorescent assay (IFA) was established for EIV. The optimal conditions for IFA were determined as follows: EIV was fixed by 80% acetone 30 min at 4℃, anti-sera was diluted for 1:80 and the FITC-labeled antibody was diluted for 1:40. The optimal reactive time of the detecting antibody and conjugate were done at 37℃for 60 min. The IFA established here was sensitive and can be used for detecting EIV.2. RT-PCR to detect the equine influenza virusTwo pairs of RT-PCR primers for HA and NA genes were designed and synthesized based on the nucleotide sequences of the equine H3N8 influenza virus in GenBank. After optimization of the detection methods, they were used to amplify EIV virus allantoic fluid. The result showed that all primers could get the specific bands. Then the two fragments from the RT-PCR products were extracted by Agarose Gel DNA Extraction Kit, respectively, sequenced and confirmed to be EIV gene. Sensitivity to detection of highly purified influenza virus by RT-PCR reached approximately 10 TCID₅₀. The high specificity of the methods was demonstrated by detecting 6 viruses, such as H3N1 virus, Pi virus, H3N2 virus, and so on. Therefore, two RT-PCR methods developed here for EIV detection had the characteristic of high efficiency, speediness, specificity and sensitivity. They can be used to detect and monitor EIV.3. Real time fluorescence RT-PCR to detect the equine influenza virusOne pairs of RT-PCR primers and probes for NA gene were designed and synthesized by DNAStar and Primer Expression 3.0 based on the nucleotide sequences of EIV (H3N8) in GenBank. After optimization of the detection methods, NA real-time fluorescence RT-PCR techniques were developed to amplify EIV. The high specificity of the methods was demonstrated by detecting 6 viruses, such as AIV (H5, H7, H9), H3N1, H3N2, Pi and SO on. The minimal detection limits of NA real-time RT-PCR were approximately 0.1 TCID₅₀ and 20 copies of double-stranded DNAs. Therefore, NA real-time fluorescence RT-PCR on the basis of the TaqMan technique developed here for EIV detection had the characteristic of high efficiency, speediness, specificity and sensitivity, which could be used to detect and monitor EIV.