Isolation And Identification Of Canine Parvovirus 2c And Preparation Of Equine Immunoglobulin Against CPV-2c

Canine parvovirus（CPV）is a member of the Parvovirus family and the genus Parvovirus.It mainly infects carnivorous animals such as Canine,Mustelidae and Procyonidae.The main cause of death of carnivores such as the family.At present,CPV has become a worldwide epidemic,seriously threatening the health of domestic and wild carnivores,and also causing serious economic losses to fur animal breeding.Vaccination is the main means to prevent the disease,but due to the frequent mutation of CPV,the vaccine cannot completely prevent CPV infection.Due to the amino acid variation of the VP2 gene,canine parvovirus has gradually evolved from the original CPV-2 to CPV-2a,CPV-2b,CPV-2c and other subtypes,and has formed different epidemic trends in different regions.Therefore,understanding the local epidemiological characteristics of CPV is of great significance to the prevention and treatment of the disease.In addition,there is no specific treatment for the disease,and symptomatic treatment is commonly used in clinical practice.Therefore,safe and rapid specific therapeutic preparations are the first choice for CPV treatment.Compared with other antibodies,the preparation of horse anti-antibodies has the advantages of low cost,large blood collection,and high antibody yield.Therefore,it is of great significance to carry out research on horse anti-immunoglobulin against CPV.First,this study conducted CPV detection on stool samples of dogs from different pet hospitals in Changchun City,and performed genotype analysis and virus isolation.Fifteen of the 20 stool samples were positive for CPV,and the VP2 gene amino acid site and genetic evolution analysis showed that the 15 samples were all CPV-2c subtype.One strain of virus was successfully isolated using F81 cells,which was identified as CPV by indirect immunofluorescence,transmission electron microscopy and PCR,and named CC/JS-01.The strain was passaged stably for 8 generations,the hemagglutination titer was 1:2⁹,and the virus titer was 10^5.24TCID₅₀/m L.VP2 gene amplification and genetic evolution analysis showed that this strain belongs to the same branch as the reference strain of CPV-2c subtype,and is closely related to the Chinese epidemic strain.Then,the immunogen was prepared by mixing and emulsifying virus-like particles of CPV-2c subtype with Freund’s adjuvant,and the horse was immunized.The neutralization test was performed to observe the changes in the titer of neutralizing antibodies in the horse serum.CPV hyperimmune serum was precipitated with saturated ammonium sulfate to extract immunoglobulin IgG,and its purity and CPV-2c subtype neutralizing antibody titer were tested.The results showed that the IgG extracted by ammonium sulfate precipitation had a complete protein band at about 55KDa by SDS-PAGE analysis,and its purity was 91.63%as measured by thin-layer scanning.The prepared equine anti-CPV-2c refined immunoglobulin was neutralized.The potency is1:6451.6.In addition,the sterility test of the prepared IgG is qualified,which proved that the prepared immunoglobulin IgG is safe.In summary,this study proved that the CPV-2c subtype is the dominant strain in Changchun in recent years,and one strain of CPV-2c was successfully isolated,and a horse anti-immunoglobulin with high neutralization potency against CPV-2c was prepared.It provided a theoretical basis for the comprehensive prevention and control of canine parvovirus in Changchun area,and also provided a reference technical method for the development of CPV therapeutic antibodies.