EIAV-infected Horses Research Articles

Differences of immune Env-specific responses between newly EIAV infected and inapparent carrier horses

Differences of immune Env-specific responses between newly EIAV infected and inapparent carrier horses

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 by nucleotide sequencing. The complete provirus of Miyazaki2011-A strain is 8208 bp in length with an overall genomic organization typical of EIAV. However, this field isolate possesses just 77.2 and 78.7 % nucleotide sequence identity with the EIAV(Wyoming) and EIAV(Liaoning) strains, respectively, while similarity plot analysis suggested all three strains arose independently. Furthermore, phylogenetic studies using sequences obtained from all EIAV-infected Misaki horses against known viral strains strongly suggests these Japanese isolates comprise a separate monophyletic group.

Stability differences of envelope-specific T cells responses between newly EIAV infected and inaparent carrier horses

Stability differences of envelope-specific T cells responses between newly EIAV infected and inaparent carrier horses

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. Here, we have developed a method to monitor EIAV-envelope specific cellular immunity in vivo. An EIA carrier horse with no clinical signs infected 7 years ago and 4 related experimental ponies infected 6 months previously were used in this study. Forty-four 20-mer peptides, representing the entire surface unit protein (gp90) of EIAV, were combined into 14 peptide pools and intradermally injected into the neck of EIAV-infected horses. An identical volume of saline alone was injected into a fifteenth site as a negative control. After 48h, those sites with palpable infiltrations were measured prior to the collection of 2mm and 4mm punch biopsies. Total RNA was extracted from each 2mm biopsy for determination of CD3 and interferon-γ (IFN-γ) mRNA expression by real-time PCR. The 4mm skin biopsies were formalin-fixed and paraffin-embedded for immunohistochemistry (IHC) staining for CD3, CD20, CD25 and MAC387 (macrophage marker). Peripheral blood mononuclear cells (PBMC) were obtained prior to the injection and tested for in vitro reactivity against the same peptides. Histological examination showed that some of the envelope peptides elicited a lymphocytic cellular infiltration at the injection site, as evidenced by positive staining for CD3. Gp90 peptide-specific increases in CD3 and IFN-γ gene expression were also detected in the injection sites. Furthermore, differences were found between in vivo and in vitro responses to gp90 specific peptides. These results demonstrate a novel method for detecting in vivo cell-mediated immune responses to EIAV-specific peptides that is readily applicable to other host/pathogen systems.

Effect of two synthetic peptides mimicking conserved regions of equine infectious anemia virus proteins gp90 and gp45 upon cytokine mRNA expression

Gp90 and gp45 synthetic peptides, which mimic conserved sequences of native viral proteins, are recognized by antibodies to equine infectious anemia virus (EIAV) in asymptomatic carrier horses and generate humoral and cellular responses in immunized mice. Cytokine mRNA levels were evaluated in equine peripheral blood mononuclear cells (PBMCs) after in vitro stimulation with gp90 and gp45 with the aim of determining the cytokine profile associated with the proliferative response. Stimulation index (SI) values indicate that 100 and 60% of EIAV-infected horses recognized gp90 and gp45, respectively. A strong positive correlation was found between IL-12p40 and SI, IFN-gamma and SI, and IL-12p40 and IFN-gamma (p < 0.001). These results suggest the presence of specific memory cells that would contribute to maintain reinfection resistance and that conserved viral regions represented by gp90 and gp45 synthetic peptides may be good candidates for inclusion in vaccine strategies against EIAV.

Early detection of dominant Env-specific and subdominant Gag-specific CD8+ lymphocytes in equine infectious anemia virus-infected horses using major histocompatibility complex class I/peptide tetrameric complexes

Cytotoxic T lymphocytes (CTL) are critical for control of lentiviruses, including equine infectious anemia virus (EIAV). Measurement of equine CTL responses has relied on chromium-release assays, which do not allow accurate quantitation. Recently, the equine MHC class I molecule 7-6, associated with the ELA-A1 haplotype, was shown to present both the Gag-GW12 and Env-RW12 EIAV CTL epitopes. In this study, 7-6/Gag-GW12 and 7-6/Env-RW12 MHC class I/peptide tetrameric complexes were constructed and used to analyze Gag-GW12- and Env-RW12-specific CTL responses in two EIAV-infected horses (A2164 and A2171). Gag-GW12 and Env-RW12 tetramer-positive CD8+ cells were identified in nonstimulated peripheral blood mononuclear cells as early as 14 days post-EIAV inoculation, and frequencies of tetramer-positive cells ranged from 0.4% to 6.7% of nonstimulated peripheral blood CD8+ cells during the 127-day study period. Although both horses terminated the initial viremic peak, only horse A2171 effectively controlled viral load. Neutralizing antibody was present during the initial control of viral load in both horses, but the ability to maintain control correlated with Gag-GW12-specific CD8+ cells in A2171. Despite Env-RW12 dominance, Env-RW12 escape viral variants were identified in both horses and there was no correlation between Env-RW12-specific CD8+ cells and control of viral load. Although Gag-GW12 CTL escape did not occur, a Gag-GW12 epitope variant arose in A2164 that was recognized less efficiently than the original epitope. These data indicate that tetramers are useful for identification and quantitation of CTL responses in horses, and suggest that the observed control of EIAV replication and clinical disease was associated with sustained CTL recognition of Gag-specific epitopes.

CTL from EIAV carrier horses with diverse MHC class I alleles recognize epitope clusters in Gag matrix and capsid proteins

Cytotoxic T lymphocytes (CTL) are important for controlling equine infectious anemia virus (EIAV). Because Gag matrix (MA) and capsid (CA) are the most frequently recognized proteins, the hypothesis that CTL from EIAV-infected horses with diverse MHC class I alleles recognize epitope clusters (EC) in these proteins was tested. Four EC were identified by CTL from 15 horses and 8 of these horses had diverse MHC class I alleles. Two of the eight had CTL to EC1, six to EC2, five to EC3, and four to EC4. Because EC2–4 were recognized by CTL from >50% of horses with diverse alleles, the hypothesis was accepted. EC1 and EC3 were the most conserved EC and these more conserved broadly recognized EC may be most useful for CTL induction, helping overcome MHC class I polymorphism and antigenic variation.

Epitope specificity is critical for high and moderate avidity cytotoxic T lymphocytes associated with control of viral load and clinical disease in horses with equine infectious anemia virus

Equine infectious anemia virus (EIAV) is a lentivirus that causes persistent infections in horses. We hypothesized that high-avidity CTL specific for nonvariable epitopes might be associated with low viral load and minimal disease in EIAV-infected horses. To test this hypothesis, memory CTL (CTLm) responses were analyzed in two infected horses with high plasma viral loads and recurrent disease (progressors), and in two infected horses with low-to-undetectable viral loads and mild disease (nonprogressors). High-avidity CTLm in one progressor recognized an envelope gp90 epitope, and the data documented for the first time in EIAV that viral variation led to CTL escape. Each of the nonprogressors had high-to-moderate avidity CTLm directed against epitopes within Rev, including the nuclear export and nuclear localization domains. These results suggested that the epitope specificity of high- and moderate-avidity CTLm was an important determinant for disease outcome in the EIAV-infected horses examined.

Identification of broadly recognized, T helper 1 lymphocyte epitopes in an equine lentivirus.

Equine infectious anaemia virus (EIAV) is a horse lentivirus causing lifelong, persistent infection. During acute infection, CD8(+) cytotoxic T lymphocytes (CTL) are probably involved in terminating plasma viraemia. However, only a few EIAV CTL epitopes, restricted to fewer horse major histocompatibility complex (MHC) class I alleles, are known. As interferon-gamma (IFN-gamma)-secreting CD4(+), T helper 1 (Th1) lymphocytes promote CTL activity and help maintain memory CTL, identifying broadly recognized EIAV Th1 epitopes would contribute significantly to vaccine strategies seeking to promote strong CTL responses among horses with varying class I haplotypes. To this end, peripheral blood mononuclear cells (PBMC) from 10 MHC disparate, EIAV-infected horses were tested in T-lymphocyte proliferation assays for recognition of peptides from the Gag p26 capsid region and a portion of Pol. Both regions are highly conserved among EIAV isolates, and this Pol region is 51-63% homologous to other lentiviral Pol proteins. Seven of 10 horses recognized peptide Gag 221-245, and peptides Gag 242-261 and Pol 323-344 were recognized by five and four horses, respectively. Furthermore, the Gag peptides were recognized by two additional horses after resolving their initial plasma viraemia, indicating that these two peptides can be immunodominant early in infection. Gag peptide-responsive PBMC produced only IFN-gamma, indicating a Th1 response, while Pol 323-344-responsive PBMC produced IFN-gamma both with and without interleukin-4. PBMC from uninfected horses failed to either proliferate or secrete cytokines in response to peptide stimulation. Finally, CD4(+) T lymphocytes were required for proliferation responses, as shown by assays using CD4- versus CD8-depleted PBMC.

Gag protein epitopes recognized by CD4(+) T-helper lymphocytes from equine infectious anemia virus-infected carrier horses.

Antigen-specific T-helper (Th) lymphocytes are critical for the development of antiviral humoral responses and the expansion of cytotoxic T lymphocytes (CTL). Identification of relevant Th lymphocyte epitopes remains an important step in the development of an efficacious subunit peptide vaccine against equine infectious anemia virus (EIAV), a naturally occurring lentivirus of horses. This study describes Th lymphocyte reactivity in EIAV carrier horses to two proteins, p26 and p15, encoded by the relatively conserved EIAV gag gene. Using partially overlapping peptides, multideterminant and possibly promiscuous epitopes were identified within p26. One peptide was identified which reacted with peripheral blood mononuclear cells (PBMC) from all five EIAV-infected horses, and three other peptides were identified which reacted with PBMC from four of five EIAV-infected horses. Four additional peptides containing both CTL and Th lymphocyte epitopes were also identified. Multiple epitopes were recognized in a region corresponding to the major homology region of the human immunodeficiency virus, a region with significant sequence similarity to other lentiviruses including simian immunodeficiency virus, puma lentivirus, feline immunodeficiency virus, Jembrana disease virus, visna virus, and caprine arthritis encephalitis virus. PBMC reactivity to p15 peptides from EIAV carrier horses also occurred. Multiple p15 peptides were shown to be reactive, but not all infected horses had Th lymphocytes recognizing p15 epitopes. The identification of peptides reactive with PBMC from outbred horses, some of which encoded both CTL and Th lymphocyte epitopes, should contribute to the design of synthetic peptide or recombinant vector vaccines for EIAV.

Detection and induction of equine infectious anemia virus-specific cytotoxic T-lymphocyte responses by use of recombinant retroviral vectors.

Cytotoxic T lymphocytes (CTL) appear to be critical in resolving or reducing the severity of lentivirus infections. Retroviral vectors expressing the Gag/Pr or SU protein of the lentivirus equine infectious anemia virus (EIAV) were constructed and used to evaluate EIAV-specific CTL responses in horses. Three promoters, cytomegalovirus, simian virus SV40, and Moloney murine sarcoma virus (MoMSV) long terminal repeat (LTR), were used, and there was considerable variation in their ability to direct expression of Gag/Pr and SU. Vectors expressing EIAV proteins under the direction of MoMSV LTR and using the gibbon ape leukemia virus (GALV) Env for internalization were efficient at transducing equine kidney (EK) target cells and were effective targets for EIAV-specific CTL lysis. CTL from EIAV-infected horses caused lysis of retroviral vector-transduced EK cells expressing either Gag/Pr or SU in an ELA-A-restricted manner. In contrast, lysis of recombinant vaccinia virus-infected EK cells expressing Gag/Pr and SU/TM was often non-LA-A restricted. Five horses were immunized by direct intramuscular injection with a mixture of retroviral vectors expressing Gag/Pr or SU, and one responded with EIAV-specific CTL. This result indicates that retroviral vector stimulation of CTL in horses needs to be optimized, perhaps by inclusion of appropriate cytokine genes in the constructs. However, the studies demonstrated that retroviral vector-transduced target cells were very effective for in vitro dissection of EIAV-specific CTL responses.

Gag protein epitopes recognized by ELA-A-restricted cytotoxic T lymphocytes from horses with long-term equine infectious anemia virus infection.

Most equine infectious anemia virus (EIAV)-infected horses have acute clinical disease, but they eventually control the disease and become lifelong carriers. Cytotoxic T lymphocytes (CTL) are considered an important immune component in the control of infections with lentiviruses including EIAV, but definitive evidence for CTL in the control of disease in carrier horses is lacking. By using retroviral vector-transduced target cells expressing different Gag proteins and overlapping synthetic peptides of 16 to 25 amino acids, peptides containing at least 12 Gag CTL epitopes recognized by virus-stimulated PBMC from six long-term EIAV-infected horses were identified. All identified peptides were located within Gag matrix (p15) and capsid (p26) proteins, as no killing of target cells expressing p11 and p9 occurred. Each of the six horses had CTL recognizing at least one Gag epitope, while CTL from one horse recognized at least eight different Gag epitopes. None of the identified peptides were recognized by CTL from all six horses. Two nonamer peptide epitopes were defined from Gag p26; one (18a) was likely restricted by class I equine leukocyte alloantigen A5.1 (ELA-A5.1) molecules, and the other (28b-1) was likely restricted by ELA-A9 molecules. Sensitization of equine kidney target cells for CTLm killing required 10 nM peptide 18a and 1 nM 28b-1. The results demonstrated that diverse CTL responses against Gag epitopes were generated in long-term EIAV-infected horses and indicated that ELA-A class I molecules were responsible for the diversity of CTL epitopes recognized. This information indicates that multiple epitopes or whole proteins will be needed to induce CTL in horses with different ELA-A alleles in order to evaluate their role in controlling EIAV.

Risk analysis of quarantine station performance: a case study of the importation of equine infectious anemia virus-infected horses into California.

We examined the risk of importing and mistakenly releasing equine infectious anemia virus (EIAV)-infected horses into California. A computer simulation model was constructed to evaluate current and alternative quarantine station procedures; 150,000 iterations were performed to simulate 15 different scenarios of 10,000 horses imported into the state over a 14-year period. Simulation results showed that under current conditions of low EIAV prevalence in exporting countries, increasing the quarantine period would not decrease the number of EIAV-infected horses mistakenly released from quarantine. In a worst case scenario of high EIAV prevalence in exporting countries, the model predicted 10 EIAV-infected horses would be imported, of these 1 or none would escape detection and would be released mistakenly if quarantine duration were 3 or 14 days, respectively. This model may be applied to other quarantine station situations for evaluating the importation risk for EIAV and other diseases.

A versatile synthetic peptide-based ELISA for identifying antibody epitopes

A simple, versatile and very inexpensive procedure for cross-linking synthetic peptides to the polystyrene surfaces of micro-well assay plates for use in ELISA was developed. The method is based on the use of poly- L-lysine (PLL) as the anchor protein for synthetic peptides which were then easily and covalently linked to the PLL using glutaraldehyde. The synthetic peptides used for the study were based on the amino acid sequence of the equine infectious anemia virus (EIAV) envelope sequence and evaluated as antigens in an ELISA designed to detect antibodies in serum of EIAV-infected horses and ponies. The ELISA using cross-linked peptides proved to be significantly more sensitive when compared to assays where passively coated peptides were used. In one instance, a peptide was identified that was not recognized by any of our antisera and appeared not to bind to the assay plates. However, once this peptide was cross-linked to the assay plate it proved to be very useful for detecting EIAV-specific antibodies. This cross-linking approach functioned equally well with peptides of various charges and sizes and did not appear to alter epitopes contained in the peptides.

Major histocompatibility complex-restricted CD8+ cytotoxic T lymphocytes from horses with equine infectious anemia virus recognize Env and Gag/PR proteins.

Cytotoxic T lymphocytes (CTL) can control some viral infections and may be important in the control of lentiviruses, including human immunodeficiency virus type 1. Since there is limited evidence for an in vivo role of CTL in control of lentiviruses, dissection of immune mechanisms in animal lentiviral infections may provide needed information. Horses infected with equine infectious anemia virus (EIAV) a lentivirus, have acute plasma viremia which is terminated in immunocompetent horses. Viremic episodes may recur, but most horses ultimately control infection and become asymptomatic carriers. To begin dissection of the immune mechanisms involved in EIAV control, peripheral blood mononuclear cells (PBMC) from infected horses were evaluated for CTL to EIAV-infected cells. By using noninfected and EIAV-infected autologous equine kidney (EK) cells in 51Cr-release assays, EIAV-specific cytotoxic activity was detected in unstimulated PBMC from three infected horses. The EIAV-specific cytotoxic activity was major histocompatibility complex (MHC) restricted, as determined by assaying EIAV-infected heterologous EK targets, and was mediated by CD8+ T lymphocytes, as determined by depleting these cells by a panning procedure with an anti-CD8 monoclonal antibody. MHC-restricted CD8+ CTL in unstimulated PBMC from infected horses caused significant specific lysis of autologous EK cells infected with recombinant vaccinia viruses expressing EIAV genes, either env or gag plus 5' pol. The EIAV-specific MHC-restricted CD8+ CTL were detected in two EIAV-infected horses within a few days after plasma viremia occurred and were present after viremia was terminated. The detection of these immune effector cells in EIAV-infected horses permits further studies to determine their in vivo role.

High prevalence of serum antibodies to equine infectious anemia virus reverse transcriptase.

The immunogenicity of the equine infectious anemia virus (EIAV) reverse transcriptase (RT) was examined by immunoblot assay with recombinant EIAV RT. All of the 19 sera from EIAV-infected horses tested contained antibodies that recognized EIAV RT and directly inhibited the polymerase activity of the enzyme. An examination of sera obtained sequentially from two experimentally infected animals revealed that anti-RT antibodies arise early in infection and increase in level. The appearance of the antibodies correlated with progression toward the asymptomatic period of infection.

Equine infectious anemia virus gene expression: characterization of the RNA splicing pattern and the protein products encoded by open reading frames S1 and S2.

The utilization of predicted splice donor and acceptor sites in generating equine infectious anemia virus (EIAV) transcripts in fetal donkey dermal cells (FDD) was examined. A single splice donor site identified immediately upstream of the gag coding region joins the viral leader sequence to all downstream exons of spliced EIAV transcripts. The predominant 3.5-kb transcript synthesized in EIAV-infected FDD cells appears to be generated by a single splicing event which links the leader sequence to the first of two functional splice acceptor sites near the 5' end of the S1 open reading frame (ORF). The translation products encoded by the 3.5-kb transcript were examined by producing in vitro transcripts from a cDNA corresponding to this RNA followed by in vitro translation in wheat germ extracts. These transcripts directed the synthesis of three proteins: the virus trans-activator protein (EIAV Tat) encoded by ORF S1, a protein of unknown function encoded by ORF S2, and the virus envelope glycoprotein. When transfected into FDD cells, this cDNA also directed expression of EIAV Tat. Amino-terminal sequence analysis of the in vitro-synthesized S1 protein supports the suggestion that translation of EIAV Tat is initiated at a CUG codon within the virus leader region. Both in vitro-synthesized S2 protein and synthetic peptides corresponding to S2 are shown to react positively with sera obtained from EIAV-infected horses, providing the first direct evidence of expression of this protein in infected animals.

Feline immunodeficiency virus infection

Feline immunodeficiency virus (FIV) (formerly feline T-lymphotropic lentivirus or FTLV) was first isolated from a group of cats in Petaluma, California in 1986. The virus is a typical lentivirus in gross and structural morphology. It replicates preferentially but not exclusively in feline T-lymphoblastoid cells, where it causes a characteristic cytopathic effect. The major structural proteins are 10, 17 (small gag), 28 (major core), 31 (endonuclease?), 41 (transmembrane?), 52 (core precursor polyprotein), 54 62 (reverse transcriptase?), and 110 130 (major envelope) kilodaltons in size. The various proteins are antigenically distinguishable from those of other lentiviruses, although serum from EIAV-infected horses will cross-react with some FIV antigens. Kittens experimentally infected with FIV manifest a transient (several days to 2 weeks) fever and neutropenia beginning 4 to 8 weeks after inoculation. This is associated with a generalized lymphadenopathy that persists for up to 9 months. Most cats recover from this initial phase of the disease and become lifelong carriers of the virus. Complete recovery does not occur to any extent in nature or in the laboratory setting. One experimentally infected cat died from a myeloproliferative disorder several months after infection. The terminal AIDS-like phase of the illness has been seen mainly in naturally infected cats. It appears a year or more following the initial infection in an unknown proportion of infected animals. FIV has been identified in cats from all parts of the world. It is most prevalent in high density populations of free roaming cats (feral and pet), and is very uncommon in closed purebred catteries. Male cats are twice as likely to become infected as females. Older male cats adopted as feral or stray animals are at the highest risk of infection, therefore. The infection rate among freely roaming cats rises throughout life, and reaches levels ranging from less than 1% to 12% or more depending on the area. Clinically affected cats tend to be 5 years or older at the time of hospitalization. Experimental and seroepidemiologic studies suggest that FIV is transmitted mainly by bites. Intimate, non-traumatic contact (mutual grooming, shared use of food, water and litter pans) is inefficient in transmitting the infection. In utero and venereal transmission could not be demonstrated in laboratory settings. There is no statistical linkage between FIV and feline leukemia virus (FeLV) infections in nature. The FeLV infection rate in FIV-infected animals is the same as it is for non-FIV-infected cats. Cats with both infections tend to die more quickly and at a younger age than animals infected with FIV alone. There is a statistical linkage between FIV infection and infection with a third retrovirus of cats, feline syncytium-forming virus (FeSFV). This is probably due to the common mode of transmission of these two retroviruses. The clinical manifestations of terminal FIV infection in nature are varied. One group of cats succumbs over a period of months or years to progressive secondary or opportunistic infections of the oral cavity, upper respiratory tract, gastrointestinal tract, skin, or urinary tract. Others suffer from vague signs of illness, unexplainable anemias, FeLV negative myeloproliferative or lymphoproliferative neoplasms, or neurologic disorders. Neurologic signs are usually of a cortical nature, with dementia and behavioral abnormalities predominating. Similarities between FIV infection of cats and HIV infection of man are striking, making FIV infection of cats a good model for human AIDS. In spite of the similarities between FIV and HIV, there is no evidence that FIV is a health hazard to man or other species of animals.