Molecular Epidemiological Investigation And Establishment Of Multienzyme-isothermal-rapid-amplification Detection Method For Chicken Chaphamaparvovirus

Chicken chaphamparvovirus(CkChpV)is a new type of parvovirus belonging to the genus Chaphamparvovirus in the family Parvoviridae.The virus is often detected in vertebrates exhibiting diarrhea symptoms.In order to investigate the prevalence of CkChpV in domestic chicken flocks,478 chicken samples(including 357 diarrheal chickens and 121 healthy chickens)were collected from 25 domestic chicken farms and conducted pathogen screening in this study.The collected samples were screened for CkChpV,avian nephritis virus(ANV),rotavirus(RTV),chicken parvovirus(Ck PV),Newcastle disease virus(NDV),infectious bronchitis virus(IBV),chicken adenogastric necrosis virus(CPNV)and chicken circovirus(CCV),and the obtained positive rates for them were 32%,9%,6%,2%,2%,1%,0%,and 0%,respectively.Statistical analysis showed a correlation between CkChpV infection and diarrhea symptoms(P <0.05).Finaly,a total of 9 complete genomes of CkChpV were amplified from the 9 positive samples detected,with a total genome length of 4432 nt.Compared these 9 CkChpV strains with the reference strain RS/BR/15/2S in GenBank,the amino acid sequence similarities of NS1 and VP1 were 97.2-98.7%,98.1-99.1%,and 98.2-99.2%,respectively.The phylogenetic analysis shows that the CkChpV strains obtained in this study has a close genetic relationship with the reference strain RS/BR/15/2S,but clustered into a different branch from the classical type Ck PV with a distant genetic relationship.These results indicated that CkChpV is commonly infected in China and undergoes independent and rapid mutations,broadening our new understanding of the host spectrum and genetic diversity of chaphamaparvovirus strains.In order to diagnose CkChpV more conveniently and quickly,this study established a multienzyme isothermal rapid amplification(MIRA)method for detecting CkChpV and optimized the conditions.The developed MIRA detection method need only 15 minutes,and its optimal reaction temperature is 38 ℃.Combined with a lateral flow dipstick(LFD),the detection of CkChpV via MIRA assay can be completed within 20 minutes.Compared with nested PCR method,the detection limit of MIRA using standard plasmids as templates can be as low as 21.3copies,and its sensitivity is 100 times higher than that of nested PCR.Moreover,the primer set designed in this experiment can only detect CkChpV specifically,and there is no cross reaction with ANV,RTV,Ck PV,NDV,or IBV with similar clinical symptoms.The above experimental results indicates that MIRA diagnostic method for CkChpV which established in this study is convenient,sensitive,and specific,and does not require a high detection environment or sophisticated instruments.Compared to PCR detection technology,it is more suitable for clinical detection of the virus.In this study,the infection of chicken CkChpV in flocks in some regions of China was investigated on the collecting clinical samples.At the same time,a new MIRA detection method for diagnosing CkChpV has been established,providing a new theoretical basis and technical support for better exploring the prevalence and evolution of CkChpV in domestic chickens.