Full-genome Sequences Anlaysis Of Five Reticuloendotheliosis Virus Strains And The Virus Isolation Of The Antibody Positive Chickens

Reticuloendotheliosis(RE) is a pathological syndrome caused by reticuloendotheliosis virus(REV),which mainly affects chickens,turkeys,ducks and other birds.REV belongs to retrovirus,which is one of the pathogens that cause tumorous diseases in poultry.Its transmission modes mainly include horizontal transmission,vertical transmission and vaccine transmission.On the one hand,the immune suppression caused by REV can result in a decline in the diseases resistance of the host,which can easily lead to the secondary infections of other diseases.On the other hand,it can result in a reduction in the level of the vaccination response and that may cause vaccine breaks,in severe cases and bring greater economic losses to the poultry industry.REV rarely causes the clinical disease alone under natural conditions,mainly in the form of co-infections.In the study,samples collected from clinical suspected tumor disease cases during 2018-2020,were used for the virus isolation via the cell cultures,and the subsequently detected by PCR and immunofluorescent assay(IFA).And totally5 REV strains were successfully isolated and named as GX18NNR1,GX19YLR1,GX19NNR1,GD20R1 and GX20YLR1,respectively.The full-genome sequences analysis was performed on these isolates.The results showed that the full-genome of these 5 isolates were 8284 bp-8416 bp in length and the isolate GX20YLR1 had the highest similarity with the REV American duck-derived classic strain SNV(nt: 99%,aa: 96.5%),and had the least similarity with the vaccine isolate FA(nt: 90.5%,aa: 88.4%).The remaining four strains had the highest similarity with the reference strain 104865(nt>99.8%,aa>97.4%),and had the least similarity with the ATCC-VR775 strain(nt<91.6%,aa<87.1%;The comparison of gene sequences showed that each gene segment of REV was relatively conservative and the pol gene is the most conservative.Based on the sequence analysis of the full-genome and the gene segments of REV,REV strain could be classified into three major evolutionary branches,namely REV subtypes I,II and III respectively.The four isolates GX18NNR1,GX19YLR1,GX19NNR1 and GD20R1 belong to REV subtype III,which was the major epidemic genotypes worldwide,and the isolate GX20YLR1 belongs to REV subtype II.Based on the Bayesian method to analyze REV strains in terms of time,population dynamics,geography and host,it was found that the genetic diversity of REV in China showed a trend of rising first and then leveling off,and its genetic diversity has not been increased significantly in the past ten years.The REV subtype III was introduced into China from the United States in the early stage,and Heilongjiang was the first province to be detected in China's mainland,and it became popular over China from the 1990 s.It was confirmed that chicken was the natural host of REV,and the infection of other birds was most likely to be originated from chicken.In this study,the growth curve of REV was tested by the culturing on the conventional chicken embryo fibroblasts(CEF)and the common laboratory cell line DF1,respevtively.The results showed that the viral load of REV in the cell cultures was positively correlated with its infection time.The viral propagation of the DF1 culture group was relatively slow,and the viral load began to reach a plateau at 4 dpi and the highest level at 5 days post-infection(dpi).The CEF culture group had a faster speed of the virus propagation,and reached a plateau at 3 dpi and the highest level at 6 dpi.The viral load of the CEF culture group was significantly higher than that of the DF1 culture group at all of the same time points.In the study,584 serum samples were collected from three local Yellow-chicken breeding companies in Guangxi and were detected by using the REV antibody detection EILISA kit.361 of the samples were positive and showed a mean positive rate of 61.82%,of which company DGGX11 had the highest positive rate of 84.15%,while company DGGX22 had the lowest of8.89%;Peripheral blood lymphocytes and plasma were collected from 181 antibody positive and 206 antibody negative chickens,and inoculated on to the CEF cell cultures for the isolation of REV,and the results of the PCR detection showed that no REV was isolated.In summary,5 REV strains were successfully isolated from the clinical cases,and the subtypes prevailing in China were identified by the full-genome sequencing and analysis.And the origin,the transmission route and the time of REV in China have also been revealed.The genome of REV is relatively conservative,and there has not obviously been change in the genetic diversity during the past decade.The optimal cells and the optimal culture time for REV were determined.It was proved that there was no correlation between the positive of the serum antibody and the existence of REV.