Construction Of A Bivalent Vaccine For Canine Distemper And Canine Parvovirus Based On Canine Distemper Virus

Canine distemper(CD)is an acute and severe infectious disease caused by canine distemper virus(CDV)infection and is characterized by dysfunction of the respiratory system,digestive system and nervous system.Canine parvovirus disease is an acute and violent gastrointestinal infectious disease characterized by vomiting and diarrhea caused by canine parvovirus(CPV)infection.The prevention and control of CDV and CPV is currently mainly based on vaccination,but the failure of immunization with commercialized CPV vaccines often occurs.For CPV,the VP2 protein is the main neutralizing antigen.In order to produce new vaccine for CPV,we summarized the genomes of all CPV wild strains published in the NCBI database in China from 2010 to2019,and analyzed them through genetic evolution.It was found that 2a,2b and 2c were the main subtypes of CPV epidemic in my country.At the same time,analysis of the amino acid level of VP2 found that the vaccine strain and the wild strains have extremely large mutations at key amino acid positions such as 87,93,101,300,323,and 426,and the mutation rates could reach 98.8%,100%,98.8%,98.8%,100%,100% and 100%respectively.Therefore,developing a new type of canine parvovirus vaccine for the current epidemic subtypes will be a powerful tool to provide the prevention and control of the disease.In order to prevent and control canine distemper and canine parvovirus disease at the same time,this study planned to construct a bivalent vaccine for canine distemper and canine parvovirus based on canine distemper vector.We inserted the VP2 gene of the representative strain of the main subtypes of CPV currently circulating between P and M of the CDV genome,the reverse genetic manipulation technique was used to successfully rescue the recombinant CDV expressing different subtypes of CPV VP2 protein,named rCDV-2a,rCDV-2b,and rCDV-2c;Indirect immunofluorescence assay and western blotting results showed that the three recombinant CDVs can express VP2 protein,and the insertion of VP2 gene had no significant effect on the biological characteristics of CDV-11 replication and amplification in vitro.We further determined the immunogenicity of rCDV-2a,rCDV-2b and rCDV-2c in a mouse model.The neutralizing antibody assay results showed that rCDV-2a,rCDV-2b and rCDV-2c can simultaneously produce anti-CDV and anti-CPV neutralizing antibody after immunization.In addition,the genetic stability of rCDV-2a,rCDV-2b and rCDV-2c was determined by continuous passage.In short,this study found that canine parvovirus 2a,2b and 2c were the main popular subtypes in China through bioinformatics analysis,and successfully constructed three canine distempers,rCDV-2a,rCDV-2b and rCDV-2c,using CDV as a carrier,and the three vaccines had good genetic stability and immunogenicity,laying the foundation for the research of a new canine distemper-canine small recombinant dual vaccine.