Evolutionary Patterns of the Proviral gp90 V3 to V5 Regions of Equine Infectious Anemia Virus Associated with Immune Selection in Progressors and Nonprogressors

The aim of this study was to determine the genomic evolutionary pattern of virulent equine infectious anemia virus （EIAV） during persistent infection. The evolutionary dynamics of proviral genomes were examined by challenging an EIAV seronegative equine （pony 1） and three EIAV vaccinated equines （ponies 4, 7, and 8） with the Chinese virulent strain EIAV- L. Ponies 1 and 7 succumbed to disease and were called progressors, while ponies 4 and 8 lacked clinical symptoms and were considered nonprogressors. Sequences spanning the V3, V4, and V5 hyper-variable regions of the EIAV-L envelope gp90 gene were sequenced from each pony as evolutionary markers of the provirus. The proviral genome of the EIAV-L inoculum evolved during persistent infection and displayed different patterns between EIA progressors and nonprogressors. Inoculum-like variants were isolated from nonprogressors during persistent infection, but only from progressors during acute infection. Variant mutations from nonprogressors were dispersed throughout the sequenced region, while those from progressors were predominantly localized to V3. Humoral immunity and virus variant population selection analyses indicated that immune selection was positive in chronically infected progressors and weak in nonprogressors. In-frame stop codons were frequently localized to a defect ＂hot spot＂. The high number of defective variants in nonprogressors may promote disease survival.

Complete Sequence of Proviral DNA of Equine Infectious Anemia Virus Strain L

Equine infectious anemia virus strain L (EIAV-L) is the parental virulent virus of equine infectious anemia donkey leukocyte attenuated vaccine (DLA EIAV). In this study, peripheral blood leukocytes(PBL) were collected from a horse infected with EIAV-L.The PBL DNAs were extracted.The EIAV-L proviral DNA was amplified in four parts covering the entire proviral genomic sequence by polymerase chain reaction (PCR). Each of the four parts was cloned into the plasmid pBluescript SK, and the recombinant plasmids were designated as p2.8, p2.4. p3.1, and p1.2 respectively. After identification with restriction digestion, the inserts within the four plasmids were sequenced. The complete nucleotide sequence of EIAV-L provirus was determined by analyzing each of the four parts and connecting them as a whole. The genome of EIAV-L is 8235 bp in length, and G + C content is 38%. The comparison analysis by the computer software DNASIS showed that the sequence of EIAV-L shares 98.4% and 96.9% identities with that of D-A EIAV and DLA EIAV respectively. The high homology between these strains showed that they were genetically related. The homology between EIAV-L and D-A EIAV is higher than that between EIAV-L and DLA EIAV, and this is consistent with the derivation progress of DLA EIAV. At both ends of EIAV-L provirus, there is an identical long terminal repeat (LTR) sequence of 316bp in length. The LTR consists of U3, R, and U5 regions. The genome of EIAV-L provirus has three long open reading frames(ORF) corresponding to gag, pol and env genes respectively. The gag gene is 1200bp and located at position 613-1912nt. The pol gene is 3402bp and located at position 1708-5109nt. There is a termination codon within the env dividing it into two parts, envl of 699bp (position 5305-6003nt)and env2 of 1827bp (position 6073-7899nt). The provirus has three additional small ORFs: S1, S2 and S3 with sizes of 153bp(position 5113-5265nt), 204bp(position 5279-5482nt)and 402bp(position 7245-7646nt) respectively.

Host cell restriction factors of equine infectious anemia virus

Equine infectious anemia virus(EIAV)is a member of the lentivirus genus in the Retroviridae family and is considered an animal model for HIV/AIDS research.An attenuated EIAV vaccine,which was successfully developed in the 1970s by classical serial passage techniques,is the first and only lentivirus vaccine that has been widely used to date.Restriction factors are cellular proteins that provide an early line of defense against viral replication and spread by interfering with various critical steps in the viral replication cycle.However,viruses have evolved specific mechanisms to overcome these host barriers through adaptation.The battle between the viruses and restriction factors is actually a natural part of the viral replication process,which has been well studied in human immunodeficiency virus type 1(HIV-1).EIAV has the simplest genome composition of all lentiviruses,making it an intriguing subject for understanding how the virus employs its limited viral proteins to overcome restriction factors.In this review,we summarize the current literature on the interactions between equine restriction factors and EIAV.The features of equine restriction factors and the mechanisms by which the EIAV counteract the restriction suggest that lentiviruses employ diverse strategies to counteract innate immune restrictions.In addition,we present our insights on whether restriction factors induce alterations in the phenotype of the attenuated EIAV vaccine.