Genome Sequence And Env Immunity Characterization Of Equine Infectious Anemia Virus EIAV_YN

Equine Infectious Anemia virus?EIAV?is the pathogen of Equine Infectious Anemia?EIA?,an infectious disease of equine.It is similar to human immunodeficiency virus?HIV-1/2?,simian immunodeficiency virus?SIV?,bovine immunodeficiency virus?BIV?and feline immunodeficiency virus?FIV?and they all belong to the members of retrovirus lentivirus.EIA is strictly controlled all over the world since it seriously damaged the health of equine animals.Comprehensive and detail epidemiological information is essential for the effective prevention and control of infectious disease.However,until now,only four whole genome of EIAV field isolates have been sequenced worldwide,they are EIAV_WYO?America?,EIAV_LIA?China?,EIAV_IRE?Ireland?and EIAV_MIY?Japan?.Limit genomic information restricted the understanding of global EIAV evolution and epidemic characters,and more genome sequences information of EIAV field isolates were required to enrich the EIAV genome data.In our study,we completed the whole genome sequencing of EIAV proviral DNA in the spleen lymph nodes from the EIAV infection serum positive mule at Yunnan,China.This new EIAV field isolate was named EIAV_YN.By completing genetic analysis for EIAV_YN genome sequence,we found that it showed a lower genomic homology,with an average of about 80%,compared with the EIAV reference strain that had published the whole genome sequence.It has the highest similarity with EIAV_MIY isolated from Japanese wild horses,but only 82.5%.Genetic analysis based on genome sequence analysis showed that the strain had formed a relatively independent branch,and had a distant genetic distance from the reference strain.Further Simplot analysis revealed no gene fragments of other reference strains in the genome of the strain.We also compared the differences between EIAV_YN and reference strains in the LTR,Gag,Pol and Env genes and predicted the corresponding amino acid sequences in the main structural proteins,respectively.More specific,for structure proteins,EIAV_YN Env showed the highest variation level?approximately 35%?compared with the reference strains,while the highest viriation happened for non-structural proteins was on S2.Gene mutation can affect the corresponding biological function of the virus.Previous studies have proved that EIAV Env mutation,on the one hand,caused significant immune escape characteristics,mainly to escape the neutralization of the host neutralizing antibodies;on the other hand,it may also affect the counteract effect against host innate immune restriction factor Tetherin,which shows an ability to suppress EIAV replication.In order to determine the effect of EIAV_YN Env mutation on escaping neutralizing antibody and antagonizing host restriction factor,we carried out the following two experiments.First,Env protein expression plasmids?pVR1012-3-8-Env,pVR1012-LN-Env,pVR1012-UK-Env,p VR1012-YN-Env?,Pony8.1 and UKGP were co-transfected with 293T cells to rescue pseudovirus,and the pseudovirus was incubated with vaccine immunized horse?5#,8#?serum,and then infected with 293T cell line stably expressing EIAV receptor ELR1.By detecting the Luc activity in cells,the serum dilution titer of vaccine immunized horse serum to neutralize 50%pseudovirus infection was calculated.The results showed that the vaccine immunized horse serum dilution value for 50%neutralization of each strain was:pVR1012-3-8-Env>pVR1012-LN-Env>pVR1012-UK-Env>pVR1012-YN-Env.And there was also a significant correlation between neutralization escaping and Env's variation.In addition,although the vaccine serum still had a neutralizing effect on the EIAV_YN strain,EIAV_YN partially escaped the neutralization effect of the vaccine-immunized horse serum to homologous strains.The ability of immune sera to neutralize different strains was correlated with the variation of different strains.Second,for testing antagonizing effect against the fuction of Tetherin by EIAV_YN Env protein,different viral envelope plasmids,VR-GP2and pcDNA3.1-Eq-Tetherin were co-transfected into 293T cells to compare the interaction differenc between Env and Tetherin molecules of EIAV_YN and the reference EIAV strains.The results showed that the effect of Env to antagonize Tetherin was declined as follows:pVR1012-LN-Env>pVR1012-UK-Env>pVR1012-3-8-Env>pVR1012-YN-Env.In conclusion,we completed the whole genome sequencing and analysis of a novel EIAV field isolate EIAV_YN,which showed a high level of genetic variation relative to the reference EIAV strains those whole gene sequences had been published already.This discovery enriched the genetic diversity of EIAV field isolate strains worldwide.In addition,the difference between EIAV_YN and reference strains on escaping host immune restriction we found in this study will be useful for uncovering the interaction between EIAV infection and host responds even for the understanding of EIAV immune mechanism.