Molecular Epidemiology Of Marek's Disease Viruses During 2017-2019 And The Pathogenicity Analysis Of GX18NNM4 Strain

Marek's disease?MD?is an infectious malignant T-cell lymphomas proliferative disease caused by Marek's diseases virus?MDV?.Over the past half-century,with the successful development of MD vaccine and its worldwide application,MD has been effectively controlled.However,in recent years,the emergence of very virulent?vv?or/and very virulent plus?vv+?strains in the field has been suggested as the main cause of the vaccination failure,which caused new threat and greater losses to the poultry industry.Therefore,it is necessary to keep the epidemiological surveillance of MDV infection in the commercial chicken flocks,which is of great significance for understanding the current evolutionary trend of the virus and the protective efficacy of the MD commercial vaccines.Guangxi is a major province in the production of high-quality chickens in China and has rich resource of indigenous chicken breeds that are usually slow-growing chickens and have a considerable long feeding period,and results in a higher risk of MD outbreak.In this study,a molecular epidemiological investigation for the MDV isolates coming2017-2019 in Guangxi and the neighbour areas was carried out.Through the virus isolation on the cell cultures and the consequent immunoflurence assay?IFA?and the oncogene meq sequence identification,totally 23 MDV strains were isolated from the clinical MD suspected chickens.The results of gene sequencing and comparison showed that the 23 strains had high homology each other and were closely related to the early Chinese Guangxi-origin isolate GX0101.According to the deducted amino acid sequences and the phylogenetic analysis,it was indicated that these isolates belonged to vv or vv+MDV strains.The analysis on the host-origin of the isolates showed that the chickens had all been vaccinated with the serotype1 commercial vaccines and most of the cases were found to be co-infected with Avian leukosis virus?ALV?or reticuloendotheliosis virus?REV??73.9%,17/23?.Based on the meq sequences of the 23 isolates from this study and other 149 reference strains isolated in the last 55 years?1964-2019?from the Gen Bank database,three data sets were constructed respectively,the Bayesian method is used for analysis.The phylogenetic tree constructed showed that 172 strains were classified into two clusters:Cluster 1 had 71strains and included mild strains,vaccine strains and virulent strains from other countries,while Cluster 2 had 101 strains and the Chinese virulent strains were dominated and the 23 isolates of the study constituted a relatively independent evolutionary sub-cluster.The phylogeographic analysis showed that the early Chinese MDV strains were originated from abroad?US and Japan?,and then spread from the north to the south in domestic;The population dynamics revealed that the genetic variation of the Chinese strains was in a continuous increase since 2005 and with a sharp increase in 2007-2012 and 2015-2019,respectively.The pathogenicity of a MDV strain GX18NNM4,isolated from a clinical outbreak in broiler breeder flock that had already been vaccinated MD vaccine was investigated.Through the challenge and the commercial vaccines protection tests and by the evaluations on the clinical tumor incidence and the histopathological observation,fluorescence quantitative PCR detection of virus load in the peripheral blood lymphocytes?PBLs?,the m RNA expression levels of cytokines PD-1 and PD-L1 in the tissues and the protection index?PI?,to classify the pathogeniesis of GX18NNM4and the protection provided by the current commercial vaccines.The clinical symptoms like wings drooping,feather disorder,depression and the reduction in weight gain etc.were found in the unvaccinated challenged birds.The earliest death was found on 16 days post challenge?dpc?and significant tumor-like lesions were observed in heart,liver,spleen and ceca etc.in the necropsy.While the earliest death was found on 22 and 18 dpc in the CVI988 and 814 vaccinated challenged birds,respectively,and tumor-like lesions were found also in heart,pancreas etc.in some of the birds.The viral load in PBLs of the unvaccinated challenged birds reached a peak value of 10^3.7copies/10⁶ cells at 28 dpc and was higher than those of the vaccines CVI988 and 814 vaccinated challenged birds at every time points detected.PD-1 and PD-L1 m RNA expression levels were detected in the spleen and caecal tonsil?CT?of MDV-infected chickens,we observed a significantly higher expression of the PD-1 or PD-L1 m RNA in the spleen than in the CT?P<0.05?,but the general trend is basically the same,and the expression of PD-1 and PD-L1 were significantly increased in the unvaccinated challenged birds?P<0.05?.The tumor incidence rate in the unvaccinated challenged birds was 70.7%?41/58?,while those in the CVI988 and 814 vaccinated challenged birds were 42.5%?17/40?and 27.5%?11/40?,with the PI values as 39.9%and 61.1%,respectively.The results of the study have revealed the origin of MDV and the transmission route in China,as well as the diversity evolution and phylogenetics of meq gene in Chinese isolates.It also has proved that MD is still an epidemic disease recently in the Yellow-chickens in southern China and has caused seriously loss to the poultry industry.vv or vv+MDV strain has been emerged and the current commercial vaccines cannot provide complete protection.