Isolation And Identification Of H3 Subtype Canine Influenza Virus And Establishment Of Fluorescence Quantitative RT-PCR Method

Canine influenza was a contagious respiratory infection caused by Canine Influenza Virus(CIV).Dogs were mainly characterized by fever,cough,runny nose and other respiratory symptoms.At present,there were mainly H3N2 and H3N8 subtypes CIV in dogs.Dogs were closely related to human beings as partners,and influenza virus was likely to be transmitted to people through dogs.Therefore,it had important public health significance to strengthen the monitoring of canine influenza and establish a rapid detection method for canine influenza.In this study,3758 canine nasal swabs collected from Heilongjiang Province,Jiangsu Province,Zhejiang Province,Hebei Province,Yunnan Province and Ningxia Hui Autonomous Region from 2018 to 2020 were inoculated with chicken embryos,and four H3N2 subtype CIV strains were successfully isolated.The genetic evolution,receptor binding characteristics and antigenicity of the isolated viruses were analyzed.The results of virus genetic evolution analysis showed that the isolated strains were in the same branch with H3N2 subtype CIVs in Korea and China,and formed an independent branch with H3N2 subtype CIVs in China after 2016.The nucleotide homology of each gene fragment of the four strains was more than 98%,and two amino acid deletions were found in the head of NA gene of each strain.The results of antigenicity analysis showed that there was good cross reaction between the positive sera of each virus in this study,and there was cross hemagglutination with the positive sera of three H3N2 subtype avian influenza viruses,but there was no reaction with the positive sera of H3N2 subtype swine influenza virus and H3N2 subtype human influenza vaccine strains recommended by WHO.The results of receptor binding analysis showed that four H3N2 subtype canine influenza viruses could bind ?-2,6 sialic acid receptor and ?-2,3 sialic acid receptor at the same time,but mainly was ?-2,3 sialic acid receptor.In view of the fact that the symptoms of some dogs infected with CIVs were not obvious or the amount of detoxification was less,it was difficult for clinical diagnosis of CIVs.In this study,specific primers and probes were designed and synthesized according to the nucleotide sequences of M gene and HA gene of CIV subtypes at home and abroad.The fluorescence quantitative PCR detection methods of general and H3 subtypes of CIVs were established by optimizing the reaction system and conditions.The two methods were highly specific,sensitive,reproducible and easy to operate,not only can we analyze the virus qualitatively,but also analyze the virus quantitatively by establishing the standard curve.The sensitivity results showed that the detection limits of the two methods were 150 copies/?Land 19 copies/?L,respectively.The repeatability test results showed that the coefficients of variation of the two methods were less than 2%.Compared with the results of virus isolation,the coincidence rates of the two methods were more than 95%.In conclusion,the genetic evolution of four H3N2 subtype CIVs were systematically analyzed,and a fluorescence quantitative PCR detection method was established for rapid detection of general and H3 subtype CIVs.It will be of great significance to effectively control the epidemic of CIV in dogs and prevent the pandemic of human influenza.