Molecular Epidemic Investigation Of Canine Parvovirus And Establishment Of A Newly Isothermal Assay For Detection Of The Virus

Since the discovery of canine parvovirus,serotypes such as CPV-2a,CPV-2b,CPV-2c,CPV-2c(a),CPV-2c(b),New CPV-2a and New CPV-2b have appeared successively.These genotypes have brought great difficulties to the breeding of economic animals and the raising of family pets in China.Therefore,the isolation identification and genetic evolution analysis of canine parvovirus in different regions are of great significance for the analysis and prediction of the epidemic trend of canine parvovirus and the development of targeted vaccines.At the same time,the emergence of new variants has resulted in significant changes in antigenicity and gene sequence,and the accuracy of the virus detection method put forward higher requirements.In order to understand the genetic variation of CPV in recent years,132 suspected canine samples collected from Henan,Hubei,Anhui and Jiangsu provinces from 2016 to2017 were tested.The results showed that the detection rate of CPV was 42.42%.The genome of CPV VP2 in 56 clinically positive samples was sequenced and analysis,named as: CN/HN1601-CN/HN1723,CN/HB1601-CN/HB1715,CN/AH1601-CN/AH1710 and CN/JS1601-CN/JS1708.The results showed the 67.85%(38/56)of the CPV-2c subtype,21.42%(12/56)of the new CPV-2a subtype and 8.9%(5/56)of the new CPV-2b subtype.The strain CN/HB1714 was CPV-2a strain with obvious genetic difference.New CPV-2a and CPV-2c have become the dominant strains in current region.New CPV-2a mainly discovered in Henan and Hubei provinces.The nucleotide homology between isolates strains was 97.3%-99.9%,and 97.4%-99.8% with 87 reference strains.The sequence analysis of VP2 protein showed that there were five major amino acid mutations(N426D/E,T440 A,Q370R,Y324 I and S297A).The mutation rates of N426D/E,T440 A,Y324I and S297 A were 78.57%(46/56),26.78%(15/56),96.42%(54/56)and 69.64%(39/56),respectively.Among all 56 strains,55 isolates had S297 A mutation.The mutation of type2a/2b showed that most of the strains in this study were new CPV-2a/2b subtype.The mutation sites were in the potential antigenic epitope region of VP2 protein gene,had veryimportant biological significance.The phylogenetic tree analysis showed that the strains showed a stable genetic trend.All 55 strains were in the same branch.They were closely related to Wuhan,Henan,Guangxi,Guiyang and Heilongjiang and other provinces,but far related to the isolates from the US,Germany and Japan.This study enriched the epidemiological data of canine parvovirus and provided references for the epidemiology,pathogenesis and new vaccines of canine parvovirus in China.In addition,in this study,specificity primers for CPV VP2 gene were designed and synthesized for nucleic acid amplification,and an improved polymerase cross-linking spiral reaction(PCLSR)method for early diagnosis of canine parvovirus was established,and the reaction conditions were optimized.The results showed that the isothermal amplification reaction had the best amplification efficiency when incubated at 62? for 50 min,and had no cross reaction with other canine infectious diseases.Reaction results can be directly judged by naked eyes with the positive amplification tube was luminous yellow,while the negative tube remained bright purple.The amplified products were identified by restriction endonuclease Eco R I,and the target bands were obtained in accordance with the expected size with high specificity.Compared with the established PCR technology,its sensitivity is 100 times higher than that of conventional PCR.The positive rate of 132 clinical samples collected is 42.42%,which is identical with convertional PCR method,slightly higher than that of colloidal gold strip method(39.39%).Compared with the previously reported PSR method,special primer improvements and establish newly dye identification methods were carried out in this study.The established CPV-PCLSR detection method has the advantages of fast and intuitive results.It can complete the whole reaction process in a constant temperature water bath.It is suitable for rapid diagnosis of inspection and quarantine departments,primary veterinary surveillance and primary laboratories.