Molecular Detection And Pathogenic Biological Characteristics Of Canine Parvovirus In Chengdu

Canine parvovirus?CPV?infection of dogs is mainly manifested as hemorrhagic enteritis,and the mortality rate of puppies is as high as 70%,which is a global epidemic.CPV is extremely susceptible to mutation.Since its emergence in the late1970s,there have been a variety of epidemic strains,including CPV-2a,CPV-2b,CPV-2c,New CPV-2a,and New CPV-2b.The epidemiological investigation and pathogenic characteristics of epidemic strains are helpful for the prevention and control of the disease.The purpose of this study is to investigate the prevalence and pathogenic biological characteristics of CPV in Chengdu.The main findings are as follows:1.Distribution of CPV and its mixed infectionIn order to understand the CPV infection in Chengdu,using CPV-specific PCR method,176 stool samples of diarrhea of puppies collected from 6 pets in Chengdu from 2016 to 2018 were collected.),Canine distemper virus?CDV?,canine rotavirus?CRV?,canine parvovirus?Minute virus of canines,MVC?,Canine Adenovirus type 2,CAV-2)PCR detection of CanineCircovirus?CanineCV?,Canine astrovirus?CaAstv?and Canine Kobuviruses?CaKoV?.The results showed that the CPV positive rate was72.73%,of which only 11.72%CPV positive samples were single infections,88.28%CPV positive samples were mixed infections of 2 to 6 viruses,and the positive rates of other viruses in the mixed infection samples were CCoV 50.78%,CDV 28.13%,CAV-2 3.13%,CRV 10.94%,MVC 8.59%,CanineCV 17.97%,CaAstv 40.63%and CaKoV 46.88%,the mixed infection rate of 2 to 6 heavy viruses is 20.31%,34.38%,19.53%,respectively,11.72%and 2.34%.The results of this study show that CPV is the main cause of canine diarrhea in Chengdu,and the mixed infection with multiple viruses is common,which seriously affects the diagnosis and treatment of canine diarrhea.2.Antigen type of CPVIn order to understand the antigenic type of CPV epidemic strains in Chengdu,randomly select 60 CPV positive samples?20 per year?,PCR amplify their VP2 gene fragments,product sequencing and sequence analysis,intercept 501bp in each target fragment for analysis Based on the amino acid substitutions at the key positions 297and 426.The results showed that New CPV-2a type was 71.67%,New CPV-2b type was 11.67%,CPV-2c type was 16.67%,and the positive rates of New CPV-2a,New CPV-2b,and CPV-2c types in 2016 They are 75%,25%and 0%respectively,in 2017they were 85%,5%and 10%,and in 2018 they were 55%,5%and 40%.The results of this study show that there are three types of antigenic CPV prevalent in Chengdu from 2016 to 2018.New CPV-2a type is the dominant antigen type,but it is declining,and CPV-2c type has rapidly increased from 2017 to 2018.40%is one of the dominant epidemic types,indicating that the antigenic type of CPV epidemic strains in Chengdu is complex and diverse.The emergence of CPV-2c type and its relationship with the protection of existing vaccines deserves further study.3.Three types of antigenic CPV we re successfully isolated and identifiedTo further understand the biological characteristics of different epidemic strains,6 selected CPV positive samples?New CPV-2a,New CPV-2b and 2 CPV-2c positive samples each?were inoculated into F81 cells and observed by subculture For at least 3generations,continue to subculture the cells with lesions.After stable lesions appear,perform PCR and indirect immunofluorescence identification.Isolate the strains and determine the TCID₅₀ after plaque purification.The results showed that when the 6infected samples were blindly passed to the third generation,one of the three antigen-positive samples was exposed to cytopathy?CPE?.After continuous transmission to the fifth generation,New CPV-2a Type,New CPV-2b type and CPV-2c CPE appeared steadily at 96h,96h and 72h after inoculation.The cells showed shrinkage,disintegration and shedding and typica l reticular lesions.The antigenic type of the poisoned samples is the same,and indirect immunofluorescence shows cell-specific bright green fluorescence,which confirms the successful isolation of three antigenic CPV,named SC-16/2017/China?New CPV-2a?,SC-13/2017/China?New CPV-2b?and SC-14/2017/China?CPV-2c?,the TCID₅₀ is 10^3.5/0.1mL,10^3.5/0.1mL and 10^4.7/0.1mL respectively.In this study,one strain of New CPV-2a,New CPV-2b and CPV-2c strains were successfully obtained,which la id a material foundation for the basic research and applied research of the virus.4.Genomic characteristics of three CPV isolatesIn order to further understand the genomic characteristics and genetic evolution relationship of different CPV epidemic strains,using the entire CPV gene sequence in GenBank as the template?genome length is about 5200 bp?,using Premier 5.0software to design 6 pairs of primers to amplify 3 The entire gene sequence of the antigenic isolates was cloned and sequenced using DNAStar for splicing,and the genomes obtained by Megalign and MEGA 7.0 were used for homology and genetic evolution analysis.The results show that this study successfully obtained three antigenic isolates containing partial UTR and two complete coding frame gene sequences,the sequence length is 4611 bp?New CPV-2a type?,4595 bp?New CPV-2b type?and 4619 bp?CPV-2c type?,GenBank accession number is MT165694?New CPV-2a/SC-16/2017/China?,MT165692?New CPV-2b/SC-13/2017/China?,MT165693?CPV-2c/SC-14/2017/China?.The nucleotide homology between the three antigenic isolates ranged from 98.8%to 99.8%,and the nucleotide homology to the CPV genome in GenBank was 98.5%to 99.8%.The deduced amino acid sequence showed that the three antigenic isolates only had a common site mutation at the 267th amino acid site of the VP2 protein.New CPV-2a isolates have mutations at the 572th and 624th positions of the NS1 protein,and amino acid positions at the 324th and440th positions of the VP2 protein;New CPV-2b isolates have the 19th,544th,545th and 583th positions of the NS1 protein Position,a mutation at the 116th amino acid position in the unique region of VP1;CPV-2c isolates were found at positions 60,544,545,and 630 of the NS1 protein,positions 5,324,and 370 of the VP2 protein,and in the unique region of VP1 The amino acid at position 131 has a mutation.These site changes on the NS1 protein may affect the gene expression in the early stages of viral infection.The site changes on the unique region of VP1 may affect the virus's infectivity,and whether the site changes on the VP2 protein will affect its antigenicity And the production of neutralizing antibodies affecting the host is worthy of further study.Genetic evolution analysis shows that the New CPV-2a type isolates in this study and two domestic New CPV-2a type strains?BJL-1,21-Sichuan?are grouped together,and these strains are all in 2015.The subsequent strains have a certain genetic distance from the Chinese strains before 2015 and the foreign strains,indicating that this type of strain in China has seen a unique evolutionary trend in recent years;the New CPV-2b isolate in this study The three New CPV-2b strains?HB-1,JL-6,LZ2?of China are clustered together,and there is a certain genetic distance from the New CPV-2b strains abroad,indicating that this type of strain There are different evolutionary trends in China and abroad;the CPV-2c isolates in this study and two CPV-2c strains?08-Sichuan,16-Sichuan?from Sichuan are grouped together separately,while they are different from other domestic ones.This type of strain in the region has a certain genetic distance,indicating that the CPV-2c strain has a unique evolutionary trend in Sichuan.This study revealed the genome structure characteristics of epidemic strains by analyzing the genomes of different antigenic isolates,and provided an important reference for further research on the genetic evolution of CPV.5.CPV antigenicity of three antigenic isolatesIn this study,by comparing the antigenicity of the three antigenic strains of CPV,it provides a reference for the development of vaccine strains with multiple antigenic CPV protection.Adjust the TCID₅₀ of the isolated strains SC-16/2017,SC-13/2017and SC-14/2017 to 10^3.5/0.1mL respectively,and prepare 201 adjuvant-inactivated vaccine after 1.5‰formaldehyde inactivation,3 antigen types All vaccines were immunized with subcutaneous injections in the legs and neck to immunize 5 groups of laying hens in each group,1 mL/mouse,and then two and three immunizations were performed every 14 days,2 mL/mouse.Eggs of different immunization periods were collected,and the hemagglutination inhibition test?HI?and antibody cross-neutralization test were used to determine the yolk antibody titer.The results showed that the antibodies were detected about 7 days after the first immunization in the three experimental groups,and the yolk antibody HI titer reached the highest peak10 days after the third immunization(2¹⁴ in SC-16/2017 group,2¹⁴ in SC-13/2017group,(SC-14/2017 group is 2¹³).After the three exemptions,the HI titer remained at around 2¹⁰ after 84 days.The neutralizing titers of three antigenic yolk antibodies against New CPV-2a,New CPV-2b and CPV-2c viruses showed that the neutralizing titers of New CPV-2a yolk antibodies were 1:890 and 1:110 and 1:890;New CPV-2b yolk antibody neutralization titers are 1:890,1:220 and 1:1780,CPV-2c yolk antibody neutralization titers are 1:890 and 1:450 respectively And>1:2560.The results of this study show that the three antigenic isolates have good immunogenicity and can induce the body to produce stable,high-cost antibodies.The three antigenic yolk antibodies have cross-protection titers against the same type strains,and also have cross-protection titers to the other two different antigen-type strains.Among them,the CPV-2c isolates induce high cross-protection antibodies For both types,it is recommended that the CPV-2c strain can be used as a candidate vaccine for the development of vaccines that protect multiple CPV antigens.