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development and characterization of an equine infectious anemia virus env-pseudotyped reporter virus.we developed a replication-defective reporter virus pseudotyped with the envelope glycoprotein of equine infectious anemia virus (eiav). the in vitro host range and neutralization phenotype of eiav env-pseudotyped virus were similar to those of replication-competent virus. an eiav env pseudovirus will improve antigenic characterization of viral variants and evaluation of lentivirus vaccines.200818448619
correction of the disease phenotype in the mouse model of stargardt disease by lentiviral gene therapy.autosomal recessive stargardt disease (stgd1) is a macular dystrophy caused by mutations in the abca4 (abcr) gene. the disease phenotype that is most recognized in stgd1 patients, and also in the abca4-/- mouse (a disease model), is lipofuscin accumulation in retinal pigment epithelium. here, we tested whether delivery of the normal (wt) human abca4 gene to the subretinal space of the abca4 -/- mice via lentiviral vectors would correct the disease phenotype; that is, reduce accumulation of the l ...200818463687
the hiv-1 p6/eiav p9 docking site in alix is autoinhibited as revealed by a conformation-sensitive anti-alix monoclonal antibody.alix [alg-2 (apoptosis-linked gene 2)-interacting protein x], a component of the endosomal sorting machinery, contains a three-dimensional docking site for hiv-1 p6(gag) or eiav (equine infectious anaemia virus) p9(gag), and binding of the viral protein to this docking site allows the virus to hijack the host endosomal sorting machinery for budding from the plasma membrane. in the present study, we identified a monoclonal antibody that specifically recognizes the docking site for p6(gag)/p9(gag) ...200818476810
refined study of the interaction between hiv-1 p6 late domain and alix.the interaction between the hiv-1 p6 late budding domain and alix, a class e vacuolar protein sorting factor, was explored by using the yeast two-hybrid approach. we refined the alix binding site of p6 as being the leucine triplet repeat sequence (lxx)4 (lypltslrslfg). intriguingly, the deletion of the c-terminal proline-rich region of alix prevented detectable binding to p6. in contrast, a four-amino acid deletion in the central hinge region of p6 increased its association with alix as shown by ...200818477395
[construction of an infectious clone of equine infectious anemia virus by n-glycosylation reverse-mutations].to elucidate the role of n-glycosylation in fetal donkey dermal cell (fdd)-attenuated equine infectious anemia virus (eiav), we constructed an n-glycosylation reverse-mutation molecular clone, plgn191n236n246. this viral molecular clone was derived from the infectious clone plgfd3-8 by site-directed mutagenesis. this clone was used to transfect fetal donkey dermal (fdd) cells. infectious characteristics of transfectants were monitored by rt-pcr, indirect immune fluorescence and reverse transcrip ...200818479052
analysis of the eiav rev-responsive element (rre) reveals a conserved rna motif required for high affinity rev binding in both hiv-1 and eiav.a cis-acting rna regulatory element, the rev-responsive element (rre), has essential roles in replication of lentiviruses, including human immunodeficiency virus (hiv-1) and equine infection anemia virus (eiav). the rre binds the viral trans-acting regulatory protein, rev, to mediate nucleocytoplasmic transport of incompletely spliced mrnas encoding viral structural genes and genomic rna. because of its potential as a clinical target, rre-rev interactions have been well studied in hiv-1; however ...200818523581
binding of equine infectious anemia virus to the equine lentivirus receptor-1 is mediated by complex discontinuous sequences in the viral envelope gp90 protein.the identification and characterization of a functional cellular receptor for equine infectious anemia virus (eiav), designated equine lentivirus receptor-1 (elr1), a member of the tumour necrosis factor receptor protein family, has been reported previously [zhang, b. et al. (2005). proc natl acad sci u s a, 102 , 9918-9923]. the finding of a single receptor for eiav is distinct from feline, simian and human immunodeficiency viruses, which typically utilize two co-receptors for infection, but is ...200818632973
an equine infectious anemia virus variant superinfects cells through novel receptor interactions.wild-type strains of equine infectious anemia virus (eiav) prevent superinfection of previously infected cells. a variant strain of virus that spontaneously arose during passage, eiav(vma-1c), can circumvent this mechanism in some cells, such as equine dermis (ed) cells, but not in others, such as equine endothelial cells. eiav(vma-1c) superinfection of ed cells results in a buildup of unintegrated viral dna and rapid killing of the cell monolayer. here, we examined the mechanism of resistance t ...200818667522
amino acid preferences of retroviral proteases for amino-terminal positions in a type 1 cleavage site.the specificities of the proteases of 11 retroviruses were studied using a series of oligopeptides with amino acid substitutions in the p1, p3, and p4 positions of a naturally occurring type 1 cleavage site (val-ser-gln-asn-tyr downward arrowpro-ile-val-gln) in human immunodeficiency virus type 1 (hiv-1). previously, the substrate specificity of the p2 site was studied for the same representative set of retroviral proteases, which included at least one member from each of the seven genera of the ...200818701588
[a flow cytometric assay for the expression of interferon gamma in t lymphocytes and its application in the study of eiav-induced immune response].the attenuated vaccine of equine infectious anemia virus (eiav) is the first lentiviral vaccine that provides solid protection against the infection of eiav virulent strains. study of the immune response induced by eiav vaccine is an important approach to understand the immunity to other lentiviruses. ifn-gamma expressed by specifically stimulated lymphocytes is an important indicator for the evaluation of t cell-mediated immunity. a flow cytometry based assay was established in this study to ac ...200818720846
mapping of equine lentivirus receptor 1 residues critical for equine infectious anemia virus envelope binding.the equine lentivirus receptor 1 (elr1), a member of the tumor necrosis factor receptor (tnfr) protein family, has been identified as a functional receptor for equine infectious anemia virus (eiav). toward defining the functional interactions between the eiav su protein (gp90) and its elr1 receptor, we mapped the gp90 binding domain of elr1 by a combination of binding and functional assays using the eiav su gp90 protein and various chimeric receptor proteins derived from exchanges between the fu ...200818032504
analysis of factor viii mediated suppression of lentiviral vector titres.effective gene therapy for haemophilia a necessitates a vector system that is not subject to a pre-existing immune response, has adequate coding capacity, gives long-term expression and preferably can target non-dividing cells. vector systems based on lentiviruses such as equine infectious anaemia virus (eiav) fulfil these criteria for the delivery of factor viii (fviii). we have found that b domain-deleted (bdd) fviii protein inhibits functional viral particle production when co-expressed with ...200818046428
equine infectious anemia virus entry occurs through clathrin-mediated endocytosis.entry of wild-type lentivirus equine infectious anemia virus (eiav) into cells requires a low-ph step. this low-ph constraint implicates endocytosis in eiav entry. to identify the endocytic pathway involved in eiav entry, we examined the entry requirements for eiav into two different cells: equine dermal (ed) cells and primary equine endothelial cells. we investigated the entry mechanism of several strains of eiav and found that both macrophage-tropic and tissue culture-adapted strains utilize c ...200818057237
structural and functional studies of alix interactions with ypx(n)l late domains of hiv-1 and eiav.retrovirus budding requires short peptide motifs (late domains) located within the viral gag protein that function by recruiting cellular factors. the ypx(n)l late domains of hiv and other lentiviruses recruit the protein alix (also known as aip1), which also functions in vesicle formation at the multivesicular body and in the abscission stage of cytokinesis. here, we report the crystal structures of alix in complex with the ypx(n)l late domains from hiv-1 and eiav. the two distinct late domains ...200818066081
failure of low-dose recombinant human il-2 to support the survival of virus-specific ctl clones infused into severe combined immunodeficient foals: lack of correlation between in vitro activity and in vivo efficacy.although ctl are important for control of lentiviruses, including equine infectious anemia virus (eiav), it is not known if ctl can limit lentiviral replication in the absence of cd4 help and neutralizing antibody. adoptive transfer of eiav-specific ctl clones into severe combined immunodeficient (scid) foals could resolve this issue, but it is not known whether exogenous il-2 administration is sufficient to support the engraftment and proliferation of ctl clones infused into immunodeficient hor ...200817727961
hiv-1 exploits importin 7 to maximize nuclear import of its dna genome.nuclear import of the hiv-1 reverse transcription complex (rtc) is critical for infection of non dividing cells, and importin 7 (imp7) has been implicated in this process. to further characterize the function of imp7 in hiv-1 replication we generated cell lines stably depleted for imp7 and used them in conjunction with infection, cellular fractionation and pull-down assays.200919193229
[receptors for animal retroviruses].diseases caused by animal retroviruses have been recognized since 19th century in veterinary field. most livestock and companion animals have own retroviruses. to disclose the receptors for these retroviruses will be useful for understanding retroviral pathogenesis, developments of anti-retroviral drugs and vectors for human and animal gene therapies. of retroviruses in veterinary field, receptors for the following viruses have been identified; equine infectious anemia virus, feline immunodefici ...200920218331
equine infectious anemia viral vector-mediated codelivery of endostatin and angiostatin driven by retinal pigmented epithelium-specific vmd2 promoter inhibits choroidal neovascularization.equine infectious anemia virus (eiav) is a nonprimate lentivirus that does not cause human disease. subretinal injection into mice of a recombinant eiav lentiviral vector in which lacz is driven by a cmv promoter (eiav cmv lacz) resulted in rapid and strong expression of lacz in retinal pigmented epithelial (rpe) cells and some other cells including ganglion cells, resulting in the presence of 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside within the optic nerve. substitution of the rpe-spe ...200920377369
development of inducible eiav-based lentiviral vector packaging and producer cell lines.large-scale production of gene therapeutics comprising equine infectious anaemia virus (eiav) -based lentiviral vectors (lvs) would benefit from the development of producer cell lines enabling the generation of larger quantities of vector than achievable by transient systems. such cell lines would contain three vector components (gag/pol, vsv-g envelope and genome expression constructs). as the vesicular stomatitis virus (vsv-g) envelope protein is cytotoxic, its expression must be regulated. it ...200919262613
[equine infectious anemia (eia)].equine infectious anemia (eia) is a reportable, eradicable epizootic disease caused by the equine lentivirus of the retrovirus family which affects equids only and occurs worldwide. the virus is transmitted by blood, mainly by sanguivorous insects. the main symptoms of the disease are pyrexia, apathy, loss of body condition and weight, anemia, edema and petechia. however, infected horses can also be inapparent carriers without any overt signs. the disease is diagnosed by serological tests like t ...200919333901
[serological and clinical proof of freedom from equine infectious anemia (eia) in imported and domestic horses in switzerland].since 1991, no cases of equine infectious anemia (eia) have been reported in switzerland. risk factors for introduction of the virus into switzerland are still present or have even increased as frequent inapparent infections, large numbers of imported horses, (since 2003) absence of compulsory testing prior to importation, eia cases in surrounding europe, possible illegal importation of horses, frequent short-term stays, poor knowledge of the disease among horse owners and even veterinarians. th ...200919333902
in vivo evolution of the gp90 gene and consistently low plasma viral load during transient immune suppression demonstrate the safety of an attenuated equine infectious anemia virus (eiav) vaccine.to study the in vivo evolution of the attenuated chinese equine infectious anemia virus (eiav) vaccine, viral gp90 gene variation and virus replication in immunosuppressed hosts were investigated. the results showed that after vaccination, the gp90 gene followed an evolutionary trend of declining diversity. the trend coincided with the maturation of immunity to eiav, and eventually, the gp90 gene became highly homologous. the sequences of these predominant quasispecies were consistently detected ...200919363668
viral load and clinical disease enhancement associated with a lentivirus cytotoxic t lymphocyte vaccine regimen.effective dna-based vaccines against lentiviruses will likely induce ctl against conserved viral proteins. equine infectious anemia virus (eiav) infects horses worldwide, and serves as a useful model for lentiviral immune control. although attenuated live eiav vaccines have induced protective immune responses, dna-based vaccines have not. in particular, dna-based vaccines have had limited success in inducing ctl responses against intracellular pathogens in the horse. we hypothesized that priming ...200919368787
embryonic substantia nigra grafts in the mesencephalon send neurites to the host striatum in non-human primate after overexpression of gdnf.in spite of partial success in treating parkinson's disease by using ectopically placed grafts of dopamine-producing cells, restoration of the original neuroanatomical circuits, if possible, might work better. previous evidence of normal anatomic projections from ventral mesencephalic (vm) grafts placed in the substantia nigra (sn) has been limited to neonatal rodents and double grafting or bridging procedures. this study attempted to determine whether injection of a potent growth-promoting fact ...200919399891
restriction of equine infectious anemia virus by equine apobec3 cytidine deaminases.the mammalian apobec3 (a3) proteins comprise a multigene family of cytidine deaminases that act as potent inhibitors of retroviruses and retrotransposons. the a3 locus on the chromosome 28 of the horse genome contains multiple a3 genes: two copies of a3z1, five copies of a3z2, and a single copy of a3z3, indicating a complex evolution of multiple gene duplications. we have cloned and analyzed for expression the different equine a3 genes and examined as well the subcellular distribution of the cor ...200919458006
analysis of neuronal proliferation, migration and differentiation in the postnatal brain using equine infectious anemia virus-based lentiviral vectors.ongoing neurogenesis in discrete sectors of the adult central nervous system depends on the mitotic activity of an elusive population of adult stem cells. the existence of adult neural stem cells provides an alternative approach to transplantation of embryonic stem cells in cell-based therapies. owing to the limited intrinsic fate of adult stem cells and inhibitory nature of the adult brain for neurogenesis, accommodation for circuit replacement in the brain will require genetic and epigenetic m ...200919474809
risk of equine infectious anemia virus disease transmission through in vitro embryo production using somatic cell nuclear transfer.prevention and regulation of equine infectious anemia virus (eiav) disease transmission solely depend on identification, isolation, and elimination of infected animals because of lack of an effective vaccine. embryo production via the somatic cell nuclear transfer (scnt) technology uses oocytes collected mainly from untested animals, which creates a potential risk of eiav transmission through infected embryos. the current review examines the risk of eiav disease transmission through scnt embryo ...200919482352
[construction and in vitro evaluation of an infectious clone of the equine infectious anemia virus vaccine strain eiav(fddv) with four reverse-mutated vaccine-specific sites in the s2 gene].to elucidate the function of the s2 gene in equine infectious anemia virus (eiav) and its role in the attenuation of the chinese attenuated eiav vaccine strains, the s2 in the eiav vaccine strain eiav (fddv) was reverse-mutated and the in vitro replication character of the resultant virus was evaluated. based on the sequence variation of the s2 gene between the eiav virulent strains and vaccine strains, all the four vaccine-specific sites in the s2 of an eiav(fddv) infectious clone, pfddv3-8, we ...200919769166
defects in cellular sorting and retroviral assembly induced by gga overexpression.we previously demonstrated that overexpression of golgi-localized, gamma-ear containing, arf-binding (gga) proteins inhibits retrovirus assembly and release by disrupting the function of endogenous adp ribosylation factors (arfs). gga overexpression led to the formation of large, swollen vacuolar compartments, which in the case of gga1 sequestered hiv-1 gag.200919788741
an eiav field isolate reveals much higher levels of subtype variability than currently reported for the equine lentivirus family.equine infectious anemia virus (eiav), a lentivirus that infects horses, has been utilized as an animal model for the study of hiv. furthermore, the disease associated with the equine lentivirus poses a significant challenge to veterinary medicine around the world. as with all lentiviruses, eiav has been shown to have a high propensity for genomic sequence and antigenic variation, especially in its envelope (env) proteins. recent studies have demonstrated env variation to be a major determinant ...200919843328
solution structure of the equine infectious anemia virus p9 protein: a rationalization of its different alix binding requirements compared to the analogous hiv-p6 protein.the equine infection anemia virus (eiav) p9 gag protein contains the late (l-) domain required for efficient virus release of nascent virions from the cell membrane of infected cell.200920015412
characterization of an equine macrophage cell line: application to studies of eiav infection.eiav is a monocyte/macrophage tropic virus. to date, even though eiav has been under investigation for numerous years, very few details have been elucidated about eiav/macrophage interactions. this is largely due to the absence of an equine macrophage cell line that would support viral replication. herein we describe the spontaneous immortalization and generation of a clonal equine macrophage-like (eml) cell line with the functional and immunophenotype characteristics of differentiated equine mo ...200919038510
replication of equine infectious anemia virus in engineered mouse nih 3t3 cells.we employed the equine lentivirus equine infectious anemia virus (eiav) to investigate the cellular restrictions for lentivirus replication in murine nih 3t3 cells. the results of these studies demonstrate that nih 3t3 cells expressing the eiav receptor elr1 and equine cyclin t1 supported productive replication of eiav and produced infectious virions at levels similar to those found in a reference permissive equine cell line. the studies presented here demonstrate, for the first time, differenti ...200919073738
genetic variation in the long terminal repeat associated with the transition of chinese equine infectious anemia virus from virulence to avirulence.a highly virulent strain of equine infectious anemia virus (eiav) lost its fatal virulence but retained the desired antigens during serial passage over 130 generations in leukocytes in vitro. we compared the long terminal repeat (ltr) sequences of the different generations and found that three stable genetic variations occurred in the transcriptional start site, the initial base of tar, and the pre-mrna cleavage site at the r-u5 boundary, respectively. these three mutations happened at the infle ...200919130201
structural model of the rev regulatory protein from equine infectious anemia virus.rev is an essential regulatory protein in the equine infectious anemia virus (eiav) and other lentiviruses, including hiv-1. it binds incompletely spliced viral mrnas and shuttles them from the nucleus to the cytoplasm, a critical prerequisite for the production of viral structural proteins and genomic rna. despite its important role in production of infectious virus, the development of antiviral therapies directed against rev has been hampered by the lack of an experimentally-determined structu ...200919137065
comparative analysis of hiv-1-based lentiviral vectors bearing lyssavirus glycoproteins for neuronal gene transfer.the delivery of therapeutic genes to the central nervous system (cns) using viral vectors represents an appealing strategy for the treatment of nerve injury and disorders of the cns. important factors determining cns targeting include tropism of the viral vectors and retrograde transport of the vector particles. retrograde transport of equine anemia virus (eiav)-based lentiviral vectors pseudotyped with the glycoprotein derived from the rabies virus rabera strain from peripheral muscle to spinal ...200919144125
an invariant surface patch on the trim5alpha pryspry domain is required for retroviral restriction but dispensable for capsid binding.trim5alpha is a retrovirus restriction factor in the host cell cytoplasm that blocks infection before provirus establishment. restriction activity requires capsid (ca)-specific recognition by the pryspry domain of trim5alpha. to better understand the restriction mechanism, nine charge-cluster-to-triple-alanine mutants in the trim5alpha pryspry domain were assessed for ca-specific restriction activity. five mutants distributed along the trim5alpha pryspry primary sequence disrupted restriction ac ...200919153241
inhibition of lentivirus replication by aqueous extracts of prunella vulgaris.various members of the mint family have been used historically in chinese and native american medicine. many of these same family members, including prunella vulgaris, have been reported to have anti-viral activities. to further characterize the anti-lentiviral activities of p. vulgaris, water and ethanol extractions were tested for their ability to inhibit equine infectious anemia virus (eiav) replication.200919154592
virulence determinants of equine infectious anemia virus.equine infectious anemia virus (eiav) is a macrophage-tropic lentivirus that rapidly induces disease in experimentally infected horses. because eiav infection and replication is centered on the monocyte/macrophage and has a pronounced acute disease stage, it is a useful model system for understanding the contribution of monocyte/macrophages to other lentivirus-induced diseases. genetic mapping studies utilizing chimeric proviruses in which parental viruses are acutely virulent or avirulent have ...201020210781
eiav envelope diversity: shaping viral persistence and encumbering vaccine efficacy.equine infectious anemia virus (eiav) and its associated disease have presented a considerable challenge to veterinary medicine worldwide ever since its identification in the 19th century. furthermore eiav, along with its fellow animal lentiviruses, has been utilized as an animal model of hiv-1/aids research since the latters identification in the late 20th century. like all lentiviruses, eiav has been shown to have a high propensity for genomic sequence and antigenic variation, principally in i ...201020210783
molecular and biological characterization of equine infectious anemia virus rev.equine infectious anemia virus (eiav) is one of the most divergent members of the lentivirus subfamily of retroviruses and is considered a useful comparative model for molecular studies of lentivirus replication. the rev protein of eiav is functionally homologous with other lentiviral revs and facilitates export of incompletely spliced viral mrnas through a crm1-dependent pathway. the trans- and cis-acting elements that mediate eiav rev function are similar to, but distinct from, the well-charac ...201020210784
the requirement for cellular transportin 3 (tnpo3 or trn-sr2) during infection maps to human immunodeficiency virus type 1 capsid and not integrase.recent genome-wide screens have highlighted an important role for transportin 3 in human immunodeficiency virus type 1 (hiv-1) infection and preintegration complex (pic) nuclear import. moreover, hiv-1 integrase interacted with recombinant transportin 3 protein under conditions whereby moloney murine leukemia virus (mlv) integrase failed to do so, suggesting that integrase-transportin 3 interactions might underscore active retroviral pic nuclear import. here we correlate infectivity defects in t ...201019846519
eiav s2 enhances pro-inflammatory cytokine and chemokine response in infected macrophages.equine infectious anemia virus (eiav) infection is distinctive in that it causes a rapid onset of clinical disease relative to other retroviruses. in order to understand the interaction dynamics between eiav and the host immune response, we explored the effects of eiav and its s2 protein in the regulation of the cytokine and chemokine response in macrophages. eiav infection markedly altered the expression pattern of a variety of pro-inflammatory cytokines and chemokines monitored in the study. c ...201019945727
differential inhibition of homotrimeric dutpases by the 3'-azido derivative of dideoxy-utp.the inhibitory effects of 3'-azido-2',3'-dideoxyuridine-5'-triphosphate in complex with the mg2+ ion (azido-ddutp.mg) on the dutpases of the human, e. coli, and equine infectious anemia virus have been compared. azido-ddutp is analogous to drugs used in the treatment of hiv. here it is shown to inhibit the bacterial dutpase in a competitive manner (ki = 9.3 microm), but to exhibit only marginal or no binding to the human and viral dutpases, respectively. this is the first demonstration of an inh ...201019761402
an lypsl late domain in the gag protein contributes to the efficient release and replication of rous sarcoma virus.the efficient release of newly assembled retrovirus particles from the plasma membrane requires the recruitment of a network of cellular proteins (escrt machinery) normally involved in the biogenesis of multivesicular bodies and in cytokinesis. retroviruses and other enveloped viruses recruit the escrt machinery through three classes of short amino acid consensus sequences termed late domains: pt/sap, ppxy, and lypx(n)l. the major late domain of rous sarcoma virus (rsv) has been mapped to a pppy ...201020392845
selection of a rare neutralization-resistant variant following passive transfer of convalescent immune plasma in equine infectious anemia virus-challenged scid horses.vaccines preventing hiv-1 infection will likely elicit antibodies that neutralize diverse strains. however, the capacity for lentiviruses to escape broadly neutralizing antibodies (nabs) is not completely understood, nor is it known whether nabs alone can control heterologous infection. here, we determined that convalescent immune plasma from a horse persistently infected with equine infectious anemia virus (eiav) neutralized homologous virus and several envelope variants containing heterologous ...201020392850
[infectious anemia in belgium]. 201020415032
identification of cellular proteins interacting with equine infectious anemia virus s2 protein.the macrophage-tropic lentivirus, equine infectious anemia virus (eiav), encodes the small auxiliary protein s2 from a short open reading frame that overlaps the amino terminus of env eiav s2 is dispensable for virus replication in cultured cells but is required for disease production. s2 is approximately 7 kda and has no overall amino acid sequence homology to other cellular or viral proteins. therefore it is likely that s2 plays a role as an adaptor protein. to further investigate s2 function ...201020417672
[study of the correlation between the plasma viral load and protective immunity induced by the equine infectious anemia attenuated vaccine and its parental virulent strain].the threshold hypothesis of attenuated lentiviral vaccine considers that the type of host response to infections of lentiviruses depends on the viral load. to evaluate the correlation between viral loads of the attenuated vaccine strain of equine infectious anemia virus (eiav) and their effects to induce protective immunity, longitudinal plasma viral loads in groups of horses inoculated with either an attenuated eiav vaccine strain (eiav(dlv125)) or sub-lethal dose of an eiav virulent strain (ei ...201020480642
lentiviral vif degrades the apobec3z3/apobec3h protein of its mammalian host and is capable of cross-species activity.all lentiviruses except equine infectious anemia virus (eiav) use the small accessory protein vif to counteract the restriction activity of the relevant apobec3 (a3) proteins of their host species. prior studies have suggested that the vif-a3 interaction is species specific. here, using the apobec3h (z3)-type proteins from five distinct mammals, we report that this is generally not the case: some lentiviral vif proteins are capable of triggering the degradation of both the a3z3-type protein of t ...201020519393
serosurveillance for equine infectious anaemia in the ardahan province of turkey.equine infectious anaemia is a retroviral infection of horses. all infected horses, including those that are asymptomatic, become carriers and are infectious for life. in this study, blood samples of all equines in the province of ardahan were collected. the material consisted of 8,947 equines, including 8,769 horses and 178 donkeys, from ardahan province in northeastern turkey. blood was collected from all horses and donkeys and the sera were analysed for the presence of antibodies to equine in ...201020521104
genomic analysis of an effective lentiviral vaccine-attenuated equine infectious anemia virus vaccine eiav fddv13.chinese equine infectious anemia virus (eiav) attenuated vaccine is the first lentiviral vaccine with a successful application. in order to understand the correlation of viral genomic mutations with viral attenuation and with induced immunoprotective properties, we analyzed the proviral genome sequences of the eiav-attenuated vaccine strain eiav(fddv13) (eiav fetal donkey dermal cell-adapted vaccine) and its highly virulent parental strain eiav(ln40). the sequences of these strains were compared ...201020526660
murine double minute 2 as a modulator of retroviral restrictions mediated by trim5alpha.in human cells, endogenous trim5alpha strongly inhibits n-tropic strains of murine leukemia virus (n-mlv) but does not target the closely related b-mlv. we have used a shrna-based loss-of-function screen to isolate factors other than trim5alpha involved in the restriction of n-mlv. in one of the isolated clones, the shrna expressed was found to target the murine double minute-2 mrna. knocking down mdm2 increased n-mlv and eiav infection of human cells by 2- to 5-fold while having little effect o ...201020619429
multiplex immunoassays of equine virus based on fluorescent encoded magnetic composite nanoparticles.a new detection format for multiplexed analysis based on fluorescent encoded magnetic composite nanoparticles is presented. two kinds of virus were analyzed by this new method: equine influenza virus (eiv) and equine infectious anemia virus (eiav). firstly, eiv antigen and eiav antigen were conjugated to two kinds of fluorescent encoded magnetic composite nanoparticles, while the green-emitting cdte quantum dots (qds) were attached to the antibody of eiv and eiav. then both green-emitting cdte q ...201020652548
broader hiv-1 neutralizing antibody responses induced by envelope glycoprotein mutants based on the eiav attenuated vaccine.in order to induce a potent and cross-reactive neutralizing antibody (nab), an effective envelope immunogen is crucial for many viral vaccines, including the vaccine for the human immunodeficiency virus (hiv). the chinese equine infectious anemia virus (eiav) attenuated vaccine has controlled the epidemic of this virus after its vaccination in over 70 million equine animals during the last 3 decades in china. data from our past studies demonstrate that the env protein of this vaccine plays a piv ...201020807451
progress towards a sars vaccine. 201020883159
amino acid mutations in the env gp90 protein that modify n-linked glycosylation of the chinese eiav vaccine strain enhance resistance to neutralizing antibodies.the chinese eiav vaccine is an attenuated live-virus vaccine obtained by serial passage of a virulent horse isolate (eiav(l)) in donkeys (eiav(d)), and subsequently in donkey cells in vitro. in this study, we compare the env gene of the original horse virulent virus (eiav(l)) with attenuated strains serially passaged in donkey mdm (eiav(dlv)), and donkey dermal cells (eiav(fddv)). genetic comparisons among parental and attenuated strains found that vaccine strains contained amino acid substituti ...201020883167
decoding the intrinsic mechanism that prohibits alix interaction with escrt and viral proteins.the adaptor protein alix [alg-2 (apoptosis-linked-gene-2 product)-interacting protein x] links retroviruses to escrt (endosomal sorting complex required for transport) machinery during retroviral budding. this function of alix requires its interaction with the escrt-iii component chmp4 (charged multivesicular body protein 4) at the n-terminal bro1 domain and retroviral gag proteins at the middle v domain. since cytoplasmic or recombinant alix is unable to interact with chmp4 or retroviral gag pr ...201020929444
divergence, not diversity of an attenuated equine lentivirus vaccine strain correlates with protection from disease.we recently reported an attenuated eiav vaccine study that directly examined the effect of lentiviral envelope sequence variation on vaccine efficacy. the study [1] demonstrated for the first time the failure of an ancestral vaccine to protect and revealed a significant, inverse, linear relationship between envelope divergence and protection from disease. in the current study we examine in detail the evolution of the attenuated vaccine strain utilized in this previous study. we demonstrate here ...201020955830
molecular detection, epidemiology, and genetic characterization of novel european field isolates of equine infectious anemia virus.the application of molecular diagnostic techniques along with nucleotide sequence determination to permit contemporary phylogenetic analysis of european field isolates of equine infectious anemia virus (eiav) has not been widely reported. as a result, of extensive testing instigated following the 2006 outbreak of equine infectious anemia in italy, 24 farms with a history of exposure to this disease were included in this study. new pcr-based methods were developed, which, especially in the case o ...201021084503
a proviral derivative from a reference attenuated eiav vaccine strain failed to elicit protective immunity.to investigate essential factors that determine the efficacy of vaccines against lentiviruses, an effective attenuated equine infectious anemia virus (eiav) vaccine strain and a proviral derivative of the vaccine were compared with respect to differences in inducing protective immunity. although these two strains replicated equally well in vitro and in vivo, the proviral strain induced significantly less protection from disease and infection caused by viral challenge and significantly lower spec ...201021094511
genomic comparison between attenuated chinese equine infectious anemia virus vaccine strains and their parental virulent strains.a lentiviral vaccine, live attenuated equine infectious anemia virus (eiav) vaccine, was developed in the 1970s, and this has made tremendous contributions to the control of equine infectious anemia (eia) in china. four key virus strains were generated during the attenuation of the eiav vaccine: the original liao-ning strain (eiav(ln40)), a donkey-adapted virulent strain (eiav(dv117)), a donkey-leukocyte-attenuated vaccine strain (eiav(dlv121)), and a fetal donkey dermal cell (fdd)-adapted vacci ...201021136127
immunochromatographic lateral flow test for detection of antibodies to equine infectious anemia virus.the purpose of this study was to develop and evaluate a simple immunochromatographic lateral flow (iclf) test for specific detection of equine infectious anemia virus (eiav) antibodies in equine sera. viral recombinant p26 capsid protein (rp26) was used as the capture protein in the test line and as the detector reagent conjugated to colloidal gold. the performance of rp26-iclf was evaluated, and the results obtained were compared with a commercially available agar gel immunodiffusion (agid) tes ...201020362005
formation of tat-tar containing ribonucleoprotein complexes for biochemical and structural analyses.viruses manipulate multiple processes of the host cell machinery in order to replicate successfully in the infected cell. among these, stimulation of transcription of the viral genes is crucial for lentiviruses such as hiv for increased protein expression levels and generation of escape mutants. the transactivation response (tar) element at the 5'-end of hiv, siv, biv, eiav or jdv retroviruses forms a unique rna based promoter element that together with the transcription activator protein tat st ...201120385237
recombinant envelope protein (rgp90) elisa for equine infectious anemia virus provides comparable results to the agar gel immunodiffusion.equine infectious anemia (eia) is an important viral infection affecting horses worldwide. the course of infection is accompanied generally by three characteristic stages: acute, chronic and inapparent. there is no effective eia vaccine or treatment, and the control of the disease is based currently on identification of eiav inapparent carriers by laboratory tests. recombinant envelope protein (rgp90) was expressed in escherichia coli and evaluated via enzyme-linked immunosorbent assay (elisa). ...201122227617
phosphoinositides direct equine infectious anemia virus gag trafficking and release.phosphatidylinositol 4,5-biphosphate [pi(4,5)p(2) ], the predominant phosphoinositide (pi) on the plasma membrane, binds the matrix (ma) protein of human immunodeficiency virus type 1 (hiv-1) and equine infectious anemia virus (eiav) with similar affinities in vitro. interaction with pi(4,5)p(2) is critical for hiv-1 assembly on the plasma membrane. eiav has been shown to localize in internal compartments; hence, the significance of its interaction with pi(4,5)p(2) is unclear. we therefore inves ...201121176037
unique evolution characteristics of the envelope protein of eiav(ln40), a virulent strain of equine infectious anemia virus.the chinese equine infectious anemia virus (eiav) virulent strain eiav(ln40) is derived from a naturally occurring virus by continuously passing in horses for 16 generations. its genome sequence is 23% different from that of the american strains or the japanese strains, and the variation of envelope gp90 surface unit (su) is as high as 41%. in this study, evolutions of the eiav(ln40) gp90 gene in four infected horses were analyzed. results showed that new quasispecies arose in the early stage of ...201121369830
dynamics of escrt protein recruitment during retroviral assembly.the escrt (endosomal sorting complex required for transport) complexes and associated proteins mediate membrane scission reactions, such as multivesicular body formation, the terminal stages of cytokinesis and retroviral particle release. these proteins are believed to be sequentially recruited to the site of membrane scission, and then complexes are disassembled by the atpase vps4a. however, these events have never been observed in living cells, and their dynamics are unknown. by quantifying th ...201121394083
a pilot study on an attenuated chinese eiav vaccine inducing broadly neutralizing antibodies.the attenuated chinese equine infectious anemia virus (eiav) vaccine has successfully protected millions of equine animals from eia disease in china. in this pilot study, to determine whether this attenuated vaccine can induce broadly neutralizing antibodies, we immunized four horses with the attenuated chinese vaccine strain eiavfddv and then observed the evolution of neutralizing antibodies against different eiav strains. during the vaccination phase, all vaccinees rapidly developed high level ...201121499906
cloning, expression and preliminary crystallographic analysis of the equine infectious anaemia virus (eiav) gp45 ectodomain.like human immunodeficiency virus (hiv), equine infectious anaemia virus (eiav) belongs to the lentivirus genus. the first successful lentiviral vaccine was developed for eiav. thus, eiav may serve as a valuable model for hiv vaccine research. eiav glycoprotein 45 (gp45) plays a similar role to gp41 in hiv by mediating virus-host membrane fusion. the gp45 ectodomain was constructed according to the structure of hiv gp41, with removal of the disulfide-bond loop region. the protein was expressed i ...201121505247
c-terminal truncation of the transmembrane protein of an attenuated lentiviral vaccine alters its in vitro but not in vivo replication and weakens its potential pathogenicity.preliminary studies revealed that the gene of the gp45 transmembrane protein (tm) of the attenuated equine infectious anemia virus (eiav) vaccine strain eiav(fddv13) had a high frequency of a premature stop codon at position 261w, which generated a 154-residue truncation at the c-terminus. eiav(fddv-tm36), a recombinant virus with the tm truncated at the intracytoplasmic (ct) domain due to the presence of a stop codon, was constructed based on eiav(fddv)3-8, which is a proviral derivative of the ...201121539871
protective effects of broadly neutralizing immunoglobulin against homologous and heterologous equine infectious anemia virus infection in horses with severe combined immunodeficiency.using the equine infectious anemia virus (eiav) lentivirus model system, we previously demonstrated protective effects of broadly neutralizing immune plasma in young horses (foals) with severe combined immunodeficiency (scid). however, in vivo selection of a neutralization-resistant envelope variant occurred. here, we determined the protective effects of purified immunoglobulin with more potent broadly neutralizing activity. overall, protection correlated with the breadth and potency of neutrali ...201121543497
equine infectious anemia virus infection and immunity: lessons for aids vaccine development. 201121643555
the distribution properties of lentiviral vectors administered into the striatum by convection-enhanced delivery.prior to the successful use of lentiviral vectors in clinical trials it is essential that strategies for direct vector delivery into the brain are evaluated in vivo, particularly as these vectors are significantly larger than the brain extracellular space. to date no such studies have been undertaken. in this study, convection enhanced delivery (ced) was employed in an attempt to achieve widespread lentiviral delivery in the striatum. infusions of eiav and hiv vector constructs expressing the re ...201121793715
the pathogenic and vaccine strains of equine infectious anemia virus differentially induce cytokine and chemokine expression and apoptosis in macrophages.the attenuated equine infectious anemia virus (eiav) vaccine was the first attenuated lentivirus vaccine to be used in a large-scale application and has been used to successfully control the spread of equine infectious anemia (eia) in china. to better understand the potential role of cytokines in the pathogenesis of eiav infection and resulting immune response, we used branched dna technology to compare the mrna expression levels of 12 cytokines and chemokines, including il-1a, il-1ß, il-4, il-1 ...201121782860
decreased infectivity of a neutralization-resistant equine infectious anemia virus variant can be overcome by efficient cell-to-cell spread.two variants of equine infectious anemia virus (eiav) that differed in sensitivity to broadly neutralizing antibody were tested in direct competition assays. no differences were observed in the growth curves and relative fitness scores of eiav(pnd-1) and eiav(pnd-5); however, the neutralization-resistant eiav(pnd-5) variant was less infectious in single round replication assays. infectious center assays indicated similar rates of cell-to-cell spread, which was approximately 1,000 fold more effic ...201121752904
evidence of a role for soluble n-ethylmaleimide-sensitive factor attachment protein receptor (snare) machinery in hiv-1 assembly and release.retrovirus assembly is a complex process that requires the orchestrated participation of viral components and host-cell factors. the concerted movement of different viral proteins to specific sites in the plasma membrane allows for virus particle assembly and ultimately budding and maturation of infectious virions. the soluble n-ethylmaleimide-sensitive factor attachment protein receptor (snare) proteins constitute the minimal machinery that catalyzes the fusion of intracellular vesicles with th ...201121680744
productive replication and evolution of hiv-1 in ferret cells.a rodent or other small animal model for hiv-1 has not been forthcoming, with the principal obstacles being species-specific restriction mechanisms and deficits in hiv-1 dependency factors. some carnivorans may harbor comparatively fewer impediments. for example, in contrast to mice, the domestic cat genome encodes essential non-receptor hiv-1 dependency factors. all feliformia and at least one caniformia species also lack a major lentiviral restriction mechanism (trim5α/trimcyp proteins). here ...201122171279
geographic structuring of global eiav isolates: a single origin for new world strains?equine infectious anaemia virus (eiav) is classified within the retroviridae and, like other lentivirus, has the propensity for considerable antigenic variation. an extensive phylogenetic analysis in bayesian fashion, with significant amounts of new eiav gag sequence information, revealed a strong geographic compartmentalization clearly related to the phylogeographic history of modern horses, pointing out that new world eiav strains form a distinct group with a potentially common origin. this ev ...201122119901
Rev variation during persistent lentivirus infection.The ability of lentiviruses to continually evolve and escape immune control is the central impediment in developing an effective vaccine for HIV-1 and other lentiviruses. Equine infectious anemia virus (EIAV) is considered a useful model for immune control of lentivirus infection. Virus-specific cytotoxic T lymphocytes (CTL) and broadly neutralizing antibody effectively control EIAV replication during inapparent stages of disease, but after years of low-level replication, the virus is still able ...201121994723
the risk of introduction of equine infectious anemia virus into usa via cloned horse embryos imported from canada.deriving horse oocytes in the usa is hampered by the lack of abattoirs processing horse carcasses which could provide abundant quantities of ovaries from slaughtered mares. therefore, several cloning industries in the usa are attempting to import cloned horse embryos from canada. like any agricultural commodity, cloned embryos pose a risk of introduction of exotic animal diseases into the importing country. under such circumstances, risk assessment could provide an objective, transparent, and in ...201121958631
oxidant-antioxidant imbalance in horses infected with equine infectious anaemia virus.this study assesses the impact of equine infectious anaemia virus (eiav) infection on the oxidant/antioxidant equilibrium of horses. blood samples from 96 romanian horses aged 1-25years, were divided into different groups according to their eiav-infection status, age, and time post-seroconversion. the effect of infection on oxidative stress was estimated by measuring enzymatic antioxidants (superoxide dismutase [sod], glutathione peroxidase [gpx] and catalase), non-enzymatic antioxidants (uric a ...201121962828
the cargo-binding domain of transportin 3 is required for lentivirus nuclear import.lentiviruses, unlike the gammaretroviruses, are able to infect nondividing cells by transiting through nuclear pores to access the host genomic dna. several nuclear import and nuclear pore components have been implicated as playing a role in nuclear import, including transportin 3 (tnpo3), a member of the importin-β family of nuclear import proteins. we demonstrated that tnpo3 was required by several lentiviruses, with simian immunodeficiency virus mac239 (sivmac239) and equine infectious anemia ...201121976643
the cellular protein lyric interacts with hiv-1 gag.human immunodeficiency virus type 1 (hiv-1) gag is the main structural protein driving assembly and release of virions from infected cells. gag alone is capable of self-assembly in vitro, but host factors have been shown to play a role in efficient viral replication and particle morphogenesis within the living cell. in a series of affinity purification experiments, we identified the cellular protein lyric to be an hiv-1 gag-interacting protein. lyric was previously described to be an hiv-inducib ...201121957284
an attenuated eiav vaccine strain induces significantly different immune responses from its pathogenic parental strain although with similar in vivo replication pattern.the eiav (equine infectious anemia virus) multi-species attenuated vaccine eiav(dlv121) successfully prevented the spread of equine infectious anemia (eia) in china in the 1970s and provided an excellent model for the study of protective immunity to lentiviruses. in this study, we compared immune responses induced by eiav(dlv121) to immunity elicited by the virulent eiav(ln40) strain and correlated immune responses to protection from infection. horses were randomly grouped and inoculated with ei ...201121893100
a stable producer cell line for the manufacture of a lentiviral vector for gene therapy of parkinson's disease.prosavin is an equine infectious anemia virus vector-based gene therapy for parkinson's disease for which inducible hek293t-based producer cell lines (pcls) have been developed. these cell lines demonstrate stringent tetracycline-regulated expression of the packaging components and yield titers comparable to the established transient production system. a prerequisite for the use of pcl-derived lentiviral vectors (lvs) in clinical applications is the thorough characterization of both the lv and r ...201121070114
expression, refolding and preliminary x-ray crystallographic analysis of equine mhc class i molecule complexed with an eiav-env ctl epitope.in order to clarify the structure and the peptide-presentation characteristics of the equine major histocompatibility complex (mhc) class i molecule, a complex of equine mhc class i molecule (ela-a1 haplotype, 7-6 allele) with mouse β(2)-microglobulin and the cytotoxic t lymphocyte (ctl) epitope env-rw12 (rvedvtntaeyw) derived from equine infectious anaemia virus (eiav) envelope protein (residues 195-206) was refolded and crystallized. the crystal, which belonged to space group p2(1), diffracted ...201222232164
assessment of integration-defective hiv-1 and eiav vectors in vitro and in vivo.the interest in integrase-defective lentiviral vectors (idlvs) stems from their potential advantage of large cloning capacity and broad cell tropism while avoiding the possibility of insertional mutagenesis. here, we directly compared the transducing potential of idlvs based on the equine infectious anemia virus (eiav) to the more commonly described hiv-1 idlvs. idlvs were constructed by introducing equivalent single/triple mutations into the integrase catalytic triad. we show that both the sing ...201223232328
a pilot study comparing the development of eiav env-specific antibodies induced by dna/recombinant vaccinia-vectored vaccines and an attenuated chinese eiav vaccine.data from successful attenuated lentiviral vaccine studies indicate that fully mature env-specific antibodies characterized by high titer, high avidity, and the predominant recognition of conformational epitopes are associated with protective efficacy. although vaccination with a dna prime/recombinant vaccinia-vectored vaccine boost strategy has been found to be effective in some trials with non-human primate/simian/human immunodeficiency virus (shiv) models, it remains unclear whether this vacc ...201223171359
development of an equine-tropic replication-competent lentivirus assay for equine infectious anemia virus-based lentiviral vectors.the release of lentiviral vectors for clinical use requires the testing of vector material, production cells, and, if applicable, ex vivo-transduced cells for the presence of replication-competent lentivirus (rcl). vectors derived from the nonprimate lentivirus equine infectious anemia virus (eiav) have been directly administered to patients in several clinical trials, with no toxicity observed to date. because eiav does not replicate in human cells, and because putative rcls derived from vector ...201223121195
the comparison of genetic variation in the envelope protein between various immunodeficiency viruses and equine infectious anemia virus.the envelope protein (env) of lentiviruses such as hiv, siv, fiv and eiav is larger than that of other retroviruses. the chinese eiav attenuated vaccine is based on env and has helped to successfully control this virus, demonstrating that envelope is crucial for vaccine. we compared env variation of the four kinds of lentiviruses. phylogenetic analysis showed that the evolutionary relationship of env between hiv and siv was the closest and they appeared to descend from a common ancestor, and the ...201222899432
recent progress in gene therapy for parkinson's disease.parkinson's disease (pd) is an age-related and the second most common neurodegenerative disorder beyond alzheimer's disease. a neuropathological hallmark of pd is a prominent loss of dopaminergic neurons in the substantia nigra projecting into the caudate and putamen. oral administration of l-dopa and/or dopamine agonists ameliorates cardinal motor symptoms of pd. however, an intermittent and long-term treatment with l-dopa frequently induces adverse side effects such as motor fluctuations and d ...201222834832
development of a high throughput, semi-automated, infectious center cell-based elisa for equine infectious anemia virus.a faster semi-automated 96-well microtiter plate assay to determine viral infectivity titers, or viral focal units (vfu), of equine infectious anemia virus (eiav) stocks is described. optimization of the existing method modernizes a classic virological technique for viral titer determination by quantitating eiav in experimentally infected cells via a cell-based elisa. to allow for automation, multiple parameters of the current assay procedures were modified resulting in an assay that required on ...201222820072
the determination of in vivo envelope-specific cell-mediated immune responses in equine infectious anemia virus-infected ponies.distinct from human lentivirus infection, equine infectious anemia virus (eiav)-infected horses will eventually enter an inapparent carrier state in which virus replication is apparently controlled by adaptive immune responses. although recrudescence of disease can occur after immune suppression, the actual immune correlate associated with protection has yet to be determined. therefore, eiav provides a model for investigating immune-mediated protective mechanisms against lentivirus infection. he ...201222795699
mechanisms of equine infectious anemia virus escape from neutralizing antibody responses define epitope specificity.determining mechanisms of viral escape to particular epitopes recognized by virus-neutralizing antibody can facilitate characterization of host-neutralizing antibody responses as type- versus group-specific, and provides necessary information for vaccine development. our study reveals that a single n-glycan located in the 5' region of the wyoming wild-type equine infectious anemia virus (eiav) principal neutralizing domain (pnd) accounts for the differences in neutralization phenotype observed b ...201222746986
safety and biodistribution of an equine infectious anemia virus-based gene therapy, retinostat(®), for age-related macular degeneration.retinostat(®) is an equine infectious anemia virus-based lentiviral gene therapy vector that expresses the angiostatic proteins endostatin and angiostatin that is delivered via a subretinal injection for the treatment of the wet form of age-related macular degeneration. we initiated 6-month safety and biodistribution studies in two species; rhesus macaques and dutch belted rabbits. after subretinal administration of retinostat the level of human endostatin and angiostatin proteins in the vitreou ...201222716662
selection of peptides for serological detection of equine infectious anemia.equine infectious anemia caused by equine infectious anemia virus is an important disease due to its high severity and incidence in animals. we used a phage display library to isolate peptides that can be considered potential markers for equine infectious anemia diagnosis. we selected peptides using igg purified from a pool comprised of 20 sera from animals naturally infected with equine infectious anemia virus. the diagnostic potential of these peptides was investigated by elisa, western blot a ...201222653674
budding of retroviruses utilizing divergent l domains requires nucleocapsid.we recently reported that human immunodeficiency virus type 1 (hiv-1) carrying ptap and lypx(n)l l domains ceased budding when the nucleocapsid (nc) domain was mutated, suggesting a role for nc in hiv-1 release. here we investigated whether nc involvement in virus release is a property specific to hiv-1 or a general requirement of retroviruses. specifically, we examined a possible role for nc in the budding of retroviruses relying on divergent l domains and structurally homologous nc domains tha ...201222345468
detection, molecular characterization and phylogenetic analysis of full-length equine infectious anemia (eiav) gag genes isolated from shackleford banks wild horses.the genetically distinct wild horse herds inhabiting shackleford banks, north carolina are probably the direct descendents of spanish stock abandoned after failed attempts to settle mid-atlantic coastal regions of north america in the sixteenth century. in a 1996 island survey, 41% of the gathered horses were discovered seropositive for equine infectious anemia virus (eiav) with additional cases identified in 1997 and 1998. as a result of their unique genetic heritage, eiav seropositive individu ...201222310073
identification of a novel equine infectious anemia virus field strain isolated from feral horses in southern japan.although equine infectious anemia (eia) was described more than 150 years ago, complete genomic sequences have only been obtained from two field strains of eia virus (eiav), eiav(wyoming) and eiav(liaoning). in 2011, eia was detected within the distinctive feral misaki horse population that inhabits the toi-cape area of southern japan. complete proviral sequences comprising a novel field strain were amplified directly from peripheral blood of one of these eiav-infected horses and characterized b ...201323100364
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