Biological Characteristics And Attenuation Of Chicken Anemia Virus Epidemic Isolates

Chicken anemia virus(CAV),as a member of the the circovirus genus of the anelloviridae,mainly harms chicks at the age of 2-3 weeks.CAV is an important immunosuppressive disease virus,the chicks showed aplastic anemia and systemic lymphatic tissue atrophy after infection.CAV can be transmitted vertically or horizontally.In addition,CAV has been detected in a variety of avian attenuated vaccine,which brings great difficulty to the prevention and control of CAV and seriously affects the healthy development of the poultry industry.Therefore,it is of great significance for the basic research,prevention and control of CAV to carry out the epidemiological investigation of CAV in some areas of China in recent years,the study on the biological characteristics of the epidemic strains and the weakening caused by in vitro passage.In this study,1677 liver samples from chickens with suspected infection in 13 provinces and cities were detected from January 2016 to September 2019.It was found that the CAV positive samples detection rate was 14.43%and the positive chicken flocks detection rate was 22.31%,indicating that CAV infection was prevalent in the investigated areas.The mixed infection of CAV with Marek's disease virus(MDV),Avian leukemia virus(ALV)and other immunosuppressant viruses were commonly found in the detected chickens,accounting for 52.94%of the total CAV infection.Among them,co-infection of CAV and MDV was the most common type of mixed infection.37 CAV-positive liver samples were used for virus isolation.After PCR,indirect immunofluorescence and electron microscope identification,32 CAV c ell-adapted strains were successfully isolated.Analys is of the VP1 gene sequence of 32 virus strains found that the VP1 gene homology among the strains was high;the genetic evolution analysis of the amino acid sequence deduced from the VP1 gene found that the isolated strains were located in the A1 subgroup(24 strains)and A3 subgroup(8 strains).In addition,12 amino acid mutation sites were found in the isolated strains.In order to understand the pathogenicity characteristics of these new isolates,CAV-He N19/3001(A1subgroup)and CAV-JL16/8901(A3 subgroup)were selected as the representative strains and Cux-1 as the reference strain to perform pathogenicity assessment in SPF chickens.14 days after challenge,the SPF chickens in each challenge group showed different degree of thymus atrophy and anemia symptoms.Among them,chickens in the challenge group with isolated strains CAV-He N19/3001 and CAV-JL16/8901 showed 20%and 30%mortality,respectively.No chickens died in the challenge group with Cux-1,and the CAV-He N19/3001 and CAV-JL16/8901 challenge groups showed thymic atrophy and anemia symptoms lasting longer than the reference strain Cux-1 challenge group,indicating that the pathogenicity of isolated strains was stronger than that of Cux-1.Align analys is of amino acid sequence derived from VP1 gene indicted that the epidemic strain showed different mutation characteristics from the reference strain,which may be the reason for the increased pathogenicity.However,further studies are needed to explore the specific amino acid sites that affect pathogenicity.In order to explore safe and effective prevention and control measures,in this study,an isolated strain CAV-JL17/1204 was selected to carry out the attenuation study on MDCC-MS B 1 cells.A highly passaged strain(CAV-JL17/1204-F120)was obtained after being cultured in vitro for 120 generations.In order to understand the pathogenicity of the highly passaged strain,SPF chickens were infected with10 times the dose of the parental strain CAV-JL17/1204-F8(1×10^3.5TCID50/100?L).The results showed that the parental strain caused 50%morbidity of SPF chickens at 14 days after challenge.However,no obvious clinical symptoms were observed in chickens in the highly passaged strain challenge group,indicating that the strain is an attenuated strain,and the pathogenicity of the highly passaged strain to 1-day-old chicks had been greatly reduced.We further studied the characteristics of the amino acid sequence deduced from the VP1 gene of CAV-JL17/1204 of different generations,and found that compared with the parental strain,the high ly passaged strains had two amino acid mutations,which may be the reason for the decline in pathogenicity of the highly passaged strain,but further experimental verification is needed.This study investigated the prevalence of CAV in some areas of China in recent years,isolated and identified 32 CAV cell-adapted viruses,and obtained the VP1 gene sequence of the epidemic strains,and initially explored the reasons for the difference in pathogenicity between the epidemic strain and Cux-1.Through in vitro passage,an attenuated strain was obtained,and its attenuated characteristics were studied,which enriched the CAV biological information database in China and laid the foundation for developing CAV vaccines.