| Canine adenovirus type 2(CAV-2)is a mammalian adenovirus that causes laryngeal tracheitis and diarrhea in dogs.Animal adenovirus infection occurs all over the world.CAV-2 has a high infection rate in clinical practice,but its onset is mostly transient,and it is easy to be mixed with other viruses,which is often ignored by clinicians.In addition,adenovirus vector is becoming a new hotspot in recombinant vaccine technology and future gene transplantation therapy because of its high efficiency,safety,simplicity and reliability.In this study,a strain of canine adenovirus type 2(CAV-HN45)was isolated from the secretion of an ill dog in Henan Province.The isolate was identified by PCR,electron microscopy,gene sequencing,and phylogenetic tree establishment and analysis of related genes.The strain was compared with other circulating adenovirus strains in the world.It is suggested that vaccination may have caused superinfection in dogs.The growth characteristics of CAV-HN45 strain were studied and the sensitivity of different cell lines to CAV-HN45 was compared.It was found that CAV-HN45 could infect PK15,3D4/21,ST of swine origin and MDCK cells of canine origin,as well as Hela cells of human origin.These studies provide data for monitoring the epidemiology of CAV-2 infection,and help to understand the genetic evolution of CAV-2 in China and the development of new vaccines,and provide a reference for future research on adenovirus vectors with highly selective expression.Most of the kits for CAV-2 detection and the serum and vaccine for CAV-2 treatment in China were imported,which were not specific to the virus epidemiological situation in China.Adenovirus can be widely transmitted in various animal groups in the wild and domestic,so it is particularly important to develop rapid and effective detection methods and monitor its prevalence.However,the accuracy of the kits used in clinical diagnosis is not high,and it is easy to be misdiagnosed or undiagnosed early.Only the clinical samples can be sent to the laboratory to confirm the diagnosis,but this method has low detection efficiency and can not meet the rapid detection of a large number of clinical samples.The aim of this study is to obtain monoclonal antibodies that can be used for laboratory detection,clinical diagnosis and treatment of CAV-2,and related detection and treatment reagents.The CAV-HN45 strain was concentrated and inactivated and used as antigen to immunize BALB/c mice.The spleen cells of immunized mice were fused with SP2/0 cells by cell fusion technology.Two monoclonal hybridomas secreting antibodies against CAV fiber protein were screened by immunoperoxidase monolayer assay and indirect immunofluorescence assay.Two monoclonal antibodies against CAV-2 fiber protein with high sensitivity and specificity were prepared,1-A12-C6 and 8-A12-B10,and the antibody subclasses were IgG2a and IgG2b,respectively.The indirect immunofluorescence titer of 1 mg/mL purified ascites antibody was 1-A12-C6:1:8192.8-A12-B10:1:2048.The neutralizing titers of 1-A12-C6 and 8-A12-B10 antibodies to CAV-2 were 1:256 and 1:64,respectively.These results provide materials for the establishment of rapid and efficient immunological detection technology of CAV-2 and basic virological research,and also provide alternative antibody drugs and reference materials for the prevention,control and treatment of CAV-2 in China. |
