Isolation And Identification Of Chicken-derived Avian Reovirus And Its Pathogenicity To Broilers

Avian Reovirus(ARV)infection is an infectious disease characterized by swollen chicken gizzards and ankle joints,which caused by Avian reovirus.The disease has a high rate of spread and a wide range of incidence.Chickens of different ages and breeds can infect,especially for commercial broilers.The early onset of the disease mainly characterized by a higher fever,depression,appetite waste and malabsorption and so on.As the disease progressed,the chicken gradually characterized inflammation of the joint,claudication;Autopsy characterized by the typical viral arthritis symptoms such as fibrous exudation,tenosynovitis,and peroneal tendon rupture,which has caused severe economic losses for the poultry industry in China.It is necessary to strengthen the research on the disease to promote the healthy development of poultry industry in China.In this study,an ARV variant was isolated from a chicken muscle tendon.The sequence analysis and pathogenicity of the isolated strains were performed.The established fluorescence quantitative RT-PCR method was used to monitor the distribution and detoxification patterns of virus in different tissues and viremia of infected chickens,which provided a theoretical basis for the prevention and control of ARV.A strain virus(383)isolated from a suspected case of ARV infection through chicken embryo and the Chicken Embryonic Liver Cells inoculation was identified as avian reovirus(ARV)by PCR identification and animal experimental infection.The time of death of SPF chicken embryos infected with the virus was concentrated 72 to 96 hours after inoculation,and the yolk sac of the dead embryonic body was ruptured;the Chicken Embryonic Liver Cells infected with this virus exhibit lesions characterized by cell fusion,which is consistent with the reported ARV characteristics.The results of RT-PCR showed that the amplified product was the same size as the expected product of the primer.Genomic sequence analysis indicated the 383 strain had the highest homology with the Reo/PA/Broiler/15511/13 isolate and shared 95.2%nucleotide identities and 92.2%amino acid identities in?C gene with it.But it had the low homology with the standard strain 1733 and S1133 shared 51.3%,51.2%nucleotide identities and 29.6%amino acid identities.Pathogencity study showed that the clinical symptoms and necropsy changes of 1-day-old healthy chickens infected with 383strain are consistent with natural incidences.The above tests showed that the isolate was a novel ARV and was named 383 strains.To establish a method detected Avian Reovirus by TaqMan fluorescence quantitative RT-PCR.Special primers and probe based on Avian Reovirus M1 gene were designed.Then these amplified fragments were cloned into pMD18-T.Using the plasmids M1-pMD18-T as standard products,a real-time quantitative reverse transcription-polymerase chain reaction(RT-PCR)was performed to construct the standard curves of M1 gene and detected the sensitivity,specificity and repeatability.The standard curve of this method has a good linear relationship.The linear regression equation is y=-3.361x+45.635,and the correlation coefficient is R~2=0.9958.Only ARV can be detected by the method and there is no cross reaction.The minimum detectable template concentration is approximately 10 Copies/?L,which is 1000 times that of ordinary PCR.The variation coefficient was less than 0.5%by within and between the groups of repeatability tests.The fluorescence quantitative PCR method established in this study can be used for clinical detection of ARV with high sensitivity,specificity and repeatability.160 healthy Ross chicks were randomly separated into four groups which were infected with 0.1 mL five-fold-diluted virus solution by the ways of footpad inoculation,eye drop inoculation and leg muscle inoculation at 1 days old.And at the same time,40 chicks were infected with saline as control group.The clinical symptoms were detected every day.Three cotton swabs were collected every two days for the detoxification of broilers in four groups.Three anticoagulants were taken every two days for detection of viremia of broilers in four groups.Three chicks were sacrificed after weighed on 4,8,12,16,20,24,28 day post-inoculation(dpi)and organs were collected for the study of pathohistological changes.The immune organs including spleen,thymus and bursa were weighed to study the immune organ index.Three non-anticoagulant blood samples were collected at 4,8,12,14,20,24,28 day post-inoculation(dpi)for the determination of serum biochemical markers and ARV antibody levels.The results showed that the broilers in the three groups had different levels of arthritis and enteritis,with the most serious symptoms in the footpad injection group,and the lightest in the eyedrops group.The weight of broiler chickens in the experimental group increased slowly,which the group of footpad-innocation is the slowest.The thymus index of the experimental group was slightly higher than that of the control group,and the spleen and Bursa's index did not change significantly.The results of indirect ELISA showed that there was no significant change in antibody levels in the three experimental groups,but slightly higher than that in the control group.Alanine aminotransferase levels in serum indicated that383 strain may cause liver damage.The results of cotton swabs showed that all three groups had detoxification at the initial stage of infection,and the subsequent detoxification was completed.Viremia results show that artificial infection with 383 strains does not cause viremia in broilers.Viruses were detected in bursa of Fabricius,pancreas,and duodenum during the initial stage of infection.In summary,the pathogenicity test of the 383 strain indicated that the strain can cause viral arthritis,malabsorption,and immunosuppression in broilers.