Molecular Characterization of the Major Open Reading Frames (ORFs) and Enhancer Elements From Four Geographically Distinct North American Equine Infectious Anemia Virus (EIAV) Isolates.

Although the equine lentivirus (equine infectious anemia virus [EIAV]) poses a major threat to equid populations throughout most regions of the world, detailed knowledge concerning its molecular epidemiology is still in its infancy. Such information is important because the few studies conducted to date suggest there is extensive genetic variation between viral isolates that if confirmed has significant implications for future vaccine design and development of newer diagnostic procedures. Here, we avoid potential assembly artifacts inherent in composite sequencing techniques by using long-range PCR in conjunction with next-generation sequencing for the rapid molecular characterization of all major open reading frames (ORFs) and known transcription factor binding motifs within the long terminal repeats (LTRs) of four North American EIAV isolates from Pennsylvania (EIAV), Tennessee (EIAV), North Carolina (EIAV), and Florida (EIAV).

Detection of West Nile Virus and other common equine viruses in three locations from the Leeward Islands, West Indies.

Equines in the West Indies are used for recreational purposes, tourism industry, racing and agriculture or can be found in feral populations. Little is known in the Caribbean basin about the prevalence of some major equine infectious diseases, some with zoonotic potential, listed as reportable by the OIE. Our objective was to study the prevalence of antibodies for West Nile Virus (WNV), Equine Herpes Virus-1 and 4 (EHV-1 and EHV-4), Equine Influenza (EI), Equine Viral Arteritis (EVA) and Equine Infectious Anemia Virus (EIAV) using a retrospective serological convenience study.

Equine infectious anemia prevalence in feral donkeys from Northeast Brazil.

Equine infectious anemia virus (EIAV) is an important cause of morbidity and mortality throughout the world. Although the virus infects all members of the Equidae the vast majority of studies have been conducted in horses (Equus caballus) with comparatively little information available for other equid species. Brazil has one of the most abundant donkey (E.

Evaluation of six serological ELISA kits available in Italy as screening tests for equine infectious anaemia surveillance.

Background: ELISAs are known to have a higher diagnostic sensitivity than the agar gel immunodiffusion (AGID) when employed for serological diagnosis of equine infectious anaemia (EIA). For this purpose, an "in-house" and five commercial ELISAs available in Italy were assessed by the National Reference Centre for EIA for their analytic specificity (Sp); precocity, defined as capability of detecting first antibodies produced during a new infection; precision based on repeatability and reproducibility, estimated from the coefficient of variation (CV); accuracy, estimated from multiple K and relative Sp and sensitivity (Se). Two serum panels, positive for non-equine retroviruses and the most frequent equine viruses, were employed to measure analytic Sp.

Protective efficacy of centralized and polyvalent envelope immunogens in an attenuated equine lentivirus vaccine.

Lentiviral Envelope (Env) antigenic variation and related immune evasion present major hurdles to effective vaccine development. Centralized Env immunogens that minimize the genetic distance between vaccine proteins and circulating viral isolates are an area of increasing study in HIV vaccinology. To date, the efficacy of centralized immunogens has not been evaluated in the context of an animal model that could provide both immunogenicity and protective efficacy data.

Equine infectious anemia in 2014: live with it or eradicate it?

In the absence of an effective vaccine, the success of the test and removal approach for the control of equine infectious anemia (EIA) cannot be overstated, at least in those areas where testing has been traditionally routine. This article addresses 4 main aspects: what has been learned about EIA virus, host control of its replication, and inapparent carriers; international status regarding the control of EIA; diagnostic and laboratory investigation; and reducing the spread of blood-borne infections by veterinarians. An attempt is made to put these issues into practical contemporary perspectives for the equine practitioner.

Epitope shifting of gp90-specific cellular immune responses in EIAV-infected ponies.

Unlike other lentiviruses, EIAV replication can be controlled in most infected horses leading to an inapparent carrier state free of overt clinical signs which lasts for many years. While the resolution of the initial infection is correlated with the appearance of virus specific cellular immune responses, the precise immune mechanisms responsible for control of the infection are not yet identified. Since the virus undergoes rapid mutation following infection, the immune response must also adapt to meet this challenge.

Envelope determinants of equine lentiviral vaccine protection.

Lentiviral envelope (Env) antigenic variation and associated immune evasion present major obstacles to vaccine development. The concept that Env is a critical determinant for vaccine efficacy is well accepted, however defined correlates of protection associated with Env variation have yet to be determined. We reported an attenuated equine infectious anemia virus (EIAV) vaccine study that directly examined the effect of lentiviral Env sequence variation on vaccine efficacy.

Is a diagnostic system based exclusively on agar gel immunodiffusion adequate for controlling the spread of equine infectious anaemia?

To improve the efficiency of the National equine infectious anaemia (EIA) surveillance program in Italy, a three-tiered diagnostic system has been adopted. This procedure involves initial screening by ELISA (Tier 1) with test-positive samples confirmed by the agar gel immunodiffusion test (AGIDT) (Tier 2) and, in the case of ELISA positive/AGIDT negative results, final determination by immunoblot (IB) (Tier 3). During this evaluation, 74,880 samples, principally collected from two Regions of Central Italy (Latium and Abruzzo) were examined, with 44 identified as negative in AGIDT but positive in both ELISA and IB.

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.

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.

An EIAV field isolate reveals much higher levels of subtype variability than currently reported for the equine lentivirus family.

Background: 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.

Full-length and internally deleted forms of interleukin-7 are present in horse (Equus caballus) lymph node tissue.

Horse IL-7 (HIL-7) cDNA was isolated from adult lymph node tissue by reverse transcription polymerase chain reaction (RT-PCR) using oligonucleotide primers based on horse genomic sequences (The Broad Institute). In addition, to the full-length (FL) 531bp reading frame encoding 176 amino acids, shorter open-reading frames of 477, 396 and 264bp were also amplified. Nucleotide sequence analysis of these RT-PCR products demonstrated they were homologous except the shorter species were missing internal sequences consistent with multiple RNA splicing events.

Envelope determinants of equine infectious anemia virus vaccine protection and the effects of sequence variation on immune recognition.

A highly effective attenuated equine infectious anemia virus (EIAV) vaccine (EIAV(D9)) capable of protecting 100% of horses from disease induced by a homologous Env challenge strain (EIAV(PV)) was recently tested in ponies to determine the level of protection against divergent Env challenge strains (J. K. Craigo, B.

Envelope variation as a primary determinant of lentiviral vaccine efficacy.

Lentiviral envelope antigenic variation and associated immune evasion are believed to present major obstacles to effective vaccine development. Although this perception is widely assumed by the scientific community, there is, to date, no rigorous experimental data assessing the effect of increasing levels of lentiviral Env variation on vaccine efficacy. It is our working hypothesis that Env is, in fact, a primary determinant of vaccine effectiveness.

Envelope-specific T-helper and cytotoxic T-lymphocyte responses associated with protective immunity to equine infectious anemia virus.

Equine infectious anemia virus (EIAV) infection of horses provides a valuable model for examining the natural immunological control of lentivirus infection and disease and the mechanisms of protective and enhancing vaccine immunity. We have previously hypothesized that the EIAV envelope (Env) proteins gp90 and gp45 are major determinants of vaccine efficacy, and that the development of protective immunity by attenuated viral vaccines may be associated with the progressive redirection of immune responses from immunodominant, variable Env segments to immunorecessive, conserved Env sequences. Whilst the antibody-neutralization determinants of Env have been defined, there are to date no comprehensive analyses of the lymphoproliferative (T-helper, Th) and cytotoxic T-cell (CTL) epitopes of the EIAV Env proteins.

Immune suppression of challenged vaccinates as a rigorous assessment of sterile protection by lentiviral vaccines.

We previously reported that an experimental live-attenuated equine infectious anemia virus (EIAV) vaccine, containing a mutated S2 accessory gene, provided protection from disease and detectable infection after virulent virus (EIAV(PV)) challenge [Li F, Craigo JK, Howe L, Steckbeck JD, Cook S, Issel C, et al. A live-attenuated equine infectious anemia virus proviral vaccine with a modified S2 gene provides protection from detectable infection by intravenous virulent virus challenge of experimentally inoculated horses. J Virol 2003;77(13):7244-53; Craigo JK, Li F, Steckbeck JD, Durkin S, Howe L, Cook SJ, et al.

Apparent elimination of EIAV ancestral species in a long-term inapparent carrier.

Equine infectious anemia virus (EIAV) envelope variation produces newly dominant quasispecies with each sequential disease cycle; new populations arise, and previous plasma quasispecies, including the original inoculum, become undetectable. The question remains whether these ancestral variants exist in tissue reservoirs or if the immune system eliminates quasispecies from persistent infections. To examine this, an EIAV long-term inapparent carrier was immune suppressed with dexamethasone.

Genetic immunization with codon-optimized equine infectious anemia virus (EIAV) surface unit (SU) envelope protein gene sequences stimulates immune responses in ponies.

In the context of DNA vaccines the native equine infectious anemia virus (EIAV)-envelope gene has proven to be an extremely weak immunogen in horses probably because the RNA transcripts are poorly expressed owing to an unusual codon-usage bias, the possession of multiple RNA splice sites and potential adenosine-rich RNA instability elements. To overcome these problems a synthetic version of sequences encoding the EIAV surface unit (SU) envelope glycoprotein was produced (SYNSU) in which the codon-usage bias was modified to conform to that of highly expressed horse and human genes. In transfected COS-1 cell cultures, the steady state expression levels of SYNSU were at least 30-fold greater than equivalent native SU sequences.

Specificity of serum neutralizing antibodies induced by transient immune suppression of inapparent carrier ponies infected with a neutralization-resistant equine infectious anemia virus envelope strain.

It has been previously reported that transient corticosteroid immune suppression of ponies experimentally infected with a highly neutralization resistant envelope variant of equine infectious anemia virus (EIAV), designated EIAV(DeltaPND), resulted in the appearance of type-specific serum antibodies to the infecting EIAV(DeltaPND) virus. The current study was designed to determine if this induction of serum neutralizing antibodies was associated with changes in the specificity of envelope determinants targeted by serum antibodies or caused by changes in the nature of the antibodies targeted to previously defined surface envelope gp90 V3 and V4 neutralization determinants. To address this question, the envelope determinants of neutralization by post-immune suppression serum were mapped.

Serological method using recombinant S2 protein to differentiate equine infectious anemia virus (EIAV)-infected and EIAV-vaccinated horses.

We recently reported a highly protective attenuated live virus vaccine for equine infectious anemia virus (EIAV) based on a proviral construct (EIAVUKDeltaS2) with a genetically engineered mutation in the viral S2 gene that eliminates expression of this accessory protein. While the EIAVUKDeltaS2 vaccine provides protection from detectable infection by experimental challenge with highly virulent virus, the potential for commercial application of this vaccine is complicated by the fact that horses inoculated with the EIAVUKDeltaS2 vaccine strain become seropositive in various reference diagnostic assays based on detection of antibodies to virion core or envelope proteins. To address this issue, we describe here the development and optimization of a new serologic EIAV diagnostic enzyme-linked immunosorbent assay (ELISA) to detect serum antibodies to the EIAV S2 protein that are produced in infected horses but not in horses inoculated with the EIAVUKDeltaS2 vaccine virus.

Enhancement of equine infectious anemia virus virulence by identification and removal of suboptimal nucleotides.

Pathogenicity was reportedly restored to an avirulent molecular clone of equine infectious anemia virus (EIAV) by substitution of 3' sequences from the pathogenic variant strain (EIAV(PV)). However, the incidence of disease in horses/ponies was found to be significantly lower (P = 0.016) with the chimeric clone (EIAV(UK)) than with EIAV(PV).

Characterization of EIAV LTR variability and compartmentalization in various reservoir tissues of long-term inapparent carrier ponies.

Dynamic genomic variation resulting in changes in envelope antigenicity has been established as a fundamental mechanism of persistence by equine infectious anemia virus (EIAV), as observed with other lentiviruses, including HIV-1. In addition to the reported changes in envelope sequences, however, certain studies indicate the viral LTR as a second variable EIAV gene, with the enhancer region being designated as hypervariable. These observations have lead to the suggestion that LTR variation may alter viral replication properties to optimize to the microenvironment of particular tissue reservoirs.

Equine infectious anemia virus envelope evolution in vivo during persistent infection progressively increases resistance to in vitro serum antibody neutralization as a dominant phenotype.

Equine infectious anemia virus (EIAV) infection of horses is characterized by well-defined waves of viremia associated with the sequential evolution of distinct viral populations displaying extensive envelope gp90 variation; however, a correlation of in vivo envelope evolution with in vitro serum neutralization phenotype remains undefined. Therefore, the goal of the present study was to utilize a previously defined panel of natural variant EIAV envelope isolates from sequential febrile episodes to characterize the effects of envelope variation during persistent infection on viral neutralization phenotypes and to define the determinants of EIAV envelope neutralization specificity. To assess the neutralization phenotypes of the sequential EIAV envelope variants, we determined the sensitivity of five variant envelopes to neutralization by a longitudinal panel of immune serum from the source infected pony.