RESUMO
Equine infectious anemia (EIA) is listed by the World Organization for Animal Health (OIE) as one of the equine diseases that must be notified. No effective treatment or vaccine is available. EIA control is based on segregation and euthanasia of positive equids. The disease is caused by the equine infectious anemia virus (EIAV), a member of the genus Lentivirus of the Retroviridae family. Despite the importance of this disease in equids, EIA has been poorly studied in donkeys (Equus asinus). We evaluate the sanitary conditions related to EIAV in donkeys from a shelter of abandoned animals captured on the roads of the Ceará. A total of 124 donkeys were randomly selected, and three horses lived at the same shelter. The animals were clinically evaluated, and a group of the 20 animals was submitted to hematological tests. Three diagnostic tests for EIA were used, agar gel immunodiffusion (AGID), enzyme-linked immunosorbent assay (ELISA) using EIAV recombinant protein gp90 (rgp90) and recombinant protein p26 (rp26) ELISA, and polymerase chain reaction (PCR) for detection of the EIAV tat-gag gene. From the donkeys, only 1 animal was positive using AGID 0.81% (1/124), compared to 21.8% (27/124) in the rgp90 and 10.5% (13/124) in the rp26 ELISA. Proviral DNA was detected by PCR tat-gag in 8.8% (11/124), and phylogenetic analysis confirms that the EIAV sequences of donkeys from the Brazilian Northeast grouped with Pantanal Brazilian sequences. Thus, in light of the results, we conclude that donkeys are carriers of EIAV and could be sources of infection.
Assuntos
Anemia Infecciosa Equina , Vírus da Anemia Infecciosa Equina , Animais , Equidae , Anemia Infecciosa Equina/diagnóstico , Eutanásia Animal , Cavalos , Vírus da Anemia Infecciosa Equina/genética , FilogeniaRESUMO
The aetiological agent of equine infectious anaemia (EIA) is the retrovirus equine infectious anemia virus (EIAV) that infects all members of the Equidae family. The EIA is widely disseminated in the Brazilian territory with a high seroprevalence in the Brazilian Pantanal and is mainly diagnosed using agar gel immunodiffusion (AGID). There are few complete EIAV genome sequences available in GenBank, which had an impact on molecular detection studies. In this study, we conducted molecular detection and sequencing of EIAV proviral DNA from Brazilian horses. We analysed the genomic region from exon 1 of tat to gag (tat-gag). Comparative serological tests, comprising AGID and two enzyme-linked immunosorbent assays (ELISAs), were also conducted. Of the 133 samples, 58 were positive in the tat-gag PCR, and 49 nucleotide sequences of 272 bp were obtained. Using this developed tat-gag PCR EIAV proviral DNA was detected in 7% of the AGID-negative samples and 26% of the AGID-negative samples were positive in at least one of the ELISA tests used. Using phylogenetic analysis, the Brazilian Pantanal EIAV sequences grouped in a different clade of EIAV sequences from other countries. Thus, the EIAV sequences can contribute to the knowledge of the tat-gag genomic region in the circulating viruses in the Brazilian Pantanal, in addition to providing new information about the genetic diversity. In addition, the serological results demonstrate the greater sensitivity of the ELISAs used in this study compared to AGID for EIA diagnosis.
Assuntos
Anemia Infecciosa Equina , Doenças dos Cavalos , Vírus da Anemia Infecciosa Equina , Animais , Anemia Infecciosa Equina/epidemiologia , Variação Genética , Genômica , Cavalos , Vírus da Anemia Infecciosa Equina/genética , Filogenia , Estudos SoroepidemiológicosRESUMO
Equine infectious anemia virus (EIAV) is a persistent lentivirus that causes equine infectious anemia (EIA). In Brazil, EIAV is endemic in the Pantanal region, and euthanasia is not mandatory in this area. All of the complete genomic sequences from field viruses are from North America, Asia, and Europe, and only proviral genomic sequences are available. Sequences from Brazilian EIAV are currently available only for gag and LTR regions. Thus, the present study aimed for the first time to sequence the entire EIAV genomic RNA in naturally infected horses from an endemic area in Brazil. RNA in plasma from naturally infected horses was used for next-generation sequencing (NGS), and gaps were filled using Sanger sequencing methodology. Complete viral genomes of EIAV from two horses were obtained and annotated (Access Number: MN560970 and MN560971). Putative genes were analyzed and compared with previously described genes, showing conservation in gag and pol genes and high variations in LTR and env sequences. Amino acid changes were identified in the p26 protein, one of the most common targets used for diagnosis, and p26 molecular modelling showed surface amino acid alterations in some epitopes. Brazilian genome sequences presented 88.6% nucleotide identity with one another and 75.8 to 77.3% with main field strains, such as EIAV Liaoning, Wyoming, Ireland, and Italy isolates. Furthermore, phylogenetic analysis suggested that this Brazilian strain comprises a separate monophyletic group. These results may help to better characterize EIAV and to overcome the challenges of diagnosing and controlling EIA in endemic regions.
Assuntos
Anemia Infecciosa Equina/virologia , Variação Genética , Genoma Viral , Vírus da Anemia Infecciosa Equina/genética , Animais , Brasil/epidemiologia , Doenças Endêmicas/veterinária , Anemia Infecciosa Equina/epidemiologia , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Cavalos/virologia , Vírus da Anemia Infecciosa Equina/classificação , Filogenia , RNA Viral/sangueRESUMO
The capsid domain (CA) of the lentiviral Gag polyproteins has two distinct roles during virion morphogenesis. As a domain of Gag, it mediates the Gag-Gag interactions that drive immature particle assembly, whereas as a mature protein, it self-assembles into the conical core of the mature virion. Lentiviral CA proteins are composed of an N-terminal region with seven α-helices and a C-terminal domain (CA-CTD) formed by four α-helices. Structural studies performed in HIV-1 indicate that the CA-CTD helix 9 establishes homodimeric interactions that contribute to the formation of the hexameric Gag lattice in immature virions. Interestingly, the mature CA core also shows inter-hexameric associations involving helix 9 residues W184 and M185. The CA proteins of feline immunodeficiency virus (FIV) and equine infectious anemia virus (EIAV) exhibit, at equivalent positions in helix 9, the motifs Y176/L177 and L169/F170, respectively. In this paper, we investigated the relevance of the Y176/L177 motif for FIV assembly by introducing a series of amino acid substitutions into this sequence and studying their effect on in vivo and in vitro Gag assembly, CA oligomerization, mature virion production, and viral infectivity. Our results demonstrate that the Y176/L177 motif in FIV CA helix 9 is essential for Gag assembly and CA oligomerization. Notably, mutations converting the FIV CA Y176/L177 motif into the HIV-1 WM and EIAV FL sequences allow substantial particle production and viral replication in feline cells.
Assuntos
Proteínas do Capsídeo/metabolismo , Produtos do Gene gag/metabolismo , Vírus da Imunodeficiência Felina/fisiologia , Montagem de Vírus , Motivos de Aminoácidos , Animais , Células COS , Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Chlorocebus aethiops , Produtos do Gene gag/genética , HIV-1/genética , Vírus da Imunodeficiência Felina/química , Vírus da Imunodeficiência Felina/metabolismo , Vírus da Anemia Infecciosa Equina/genética , Mutação , Conformação Proteica em alfa-Hélice , Vírion/genética , Vírion/metabolismoRESUMO
A codon-optimized equine infectious anemia virus p26 gene was fused to a maltose-binding protein (MBP) and expressed in Escherichia coli for use as an antigen in agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA) for diagnosis of equine infectious anemia. An analysis of analytical sensitivity and specificity showed that the antigen MBP-p26rec reacted positively with a reference World Organization for Animal Health serum and demonstrated no cross-reaction against sera from vaccinated animals in either test. The diagnostic characteristics were evaluated and presented excellent values. The AGIDrec showed 100% sensitivity and specificity, and the ELISArec showed 100% sensitivity and 99.64% specificity. In addition, MBP-p26rec was stabile after three years of storage at 4 °C, maintaining its immunoreactivity.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Anemia Infecciosa Equina/virologia , Imunodifusão/métodos , Vírus da Anemia Infecciosa Equina/isolamento & purificação , Proteínas Ligantes de Maltose/análise , Proteínas do Core Viral/análise , Animais , Ensaio de Imunoadsorção Enzimática/instrumentação , Anemia Infecciosa Equina/diagnóstico , Anemia Infecciosa Equina/imunologia , Cavalos , Imunodifusão/instrumentação , Vírus da Anemia Infecciosa Equina/genética , Vírus da Anemia Infecciosa Equina/imunologia , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/imunologia , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologiaRESUMO
Equine infectious anemia (EIA) has a worldwide distribution, and is widespread in Brazil. The Brazilian Pantanal presents with high prevalence comprising equine performance and indirectly the livestock industry, since the horses are used for cattle management. Although EIA is routinely diagnosed by the agar gel immunodiffusion test (AGID), this serological assay has some limitations, so PCR-based detection methods have the potential to overcome these limitations and act as complementary tests to those currently used. Considering the limited number of equine infectious anemia virus (EIAV) sequences which are available in public databases and the great genome variability, studies of EIAV detection and characterization molecular remain important. In this study we detected EIAV proviral DNA from 23 peripheral blood mononuclear cell (PBMCs) samples of naturally infected horses from Brazilian Pantanal using a semi-nested-PCR (sn-PCR). The serological profile of the animals was also evaluated by AGID and ELISA for gp90 and p26. Furthermore, the EIAV PCR amplified DNA was sequenced and phylogenetically analyzed. Here we describe the first EIAV sequences of the 5' LTR of the tat gene in naturally infected horses from Brazil, which presented with 91% similarity to EIAV reference sequences. The Brazilian EIAV sequences also presented variable nucleotide similarities among themselves, ranging from 93,5% to 100%. Phylogenetic analysis showed that Brazilian EIAV sequences grouped in a separate clade relative to other reference sequences. Thus this molecular detection and characterization may provide information about EIAV circulation in Brazilian territories and improve phylogenetic inferences.
Assuntos
Anemia Infecciosa Equina/virologia , Vírus da Anemia Infecciosa Equina/isolamento & purificação , Animais , Brasil , DNA Viral/genética , Anemia Infecciosa Equina/imunologia , Cavalos , Vírus da Anemia Infecciosa Equina/classificação , Vírus da Anemia Infecciosa Equina/genética , Leucócitos Mononucleares/virologia , Filogenia , Reação em Cadeia da PolimeraseRESUMO
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. asinus) populations of any nation although the economic importance of these animals is declining as transportation becomes increasingly mechanized. As a result, considerable numbers of donkeys especially in the Northeast of the country have been released and allowed pursue an almost feral existence. Consequently, this large and growing population constitutes a significant risk as a reservoir for the maintenance and transmission of important equine infectious diseases such as glanders and equine arteritis virus in addition to EIAV. This study examines the prevalence of EIA in a semi-wild donkey population from Mossoró city, in Northeast Brazil, using AGID followed by cELISA, rgp90 ELISA and immunoblot (IB). Serum samples were collected from 367 donkeys without obvious EIA clinical signs. Subsequent testing revealed seropositive rates of 1.6% (6/367) in officially approved AGID tests, 3.3% (12/367) in cELISA and 14.4% (53/367) in the rgp90 ELISA. However, 88.7% (47/53) of the rgp90 ELISA positive samples were almost certainly false reactions because they failed to react with two or more antigens in IB. Consequently, the rpg90 ELISA has a similar sensitivity to AGID with donkey serum samples. Such high false positive rates have not been observed previously with serum samples from horses. Another highly significant finding is that 56.9% (33/58) of the donkey serum samples tested in IB had reactivity to EIAV p26 only. Although this could result from recent infection with the virus, it has been found that in some equids p26 only reactivity persists for extensive periods of time suggesting exposure to antigens possessing cross-reactive determinants or EIAV strains with envelope glycoproteins that are different from any that have been previously characterized and so undetectable by current IB techniques.
Assuntos
Anemia Infecciosa Equina/diagnóstico , Anemia Infecciosa Equina/epidemiologia , Testes Imunológicos/veterinária , Animais , Animais Selvagens , Anticorpos Antivirais/sangue , Antígenos Virais/sangue , Brasil/epidemiologia , Ensaio de Imunoadsorção Enzimática/veterinária , Equidae/sangue , Anemia Infecciosa Equina/sangue , Análise Fatorial , Cavalos , Testes Imunológicos/métodos , Vírus da Anemia Infecciosa Equina/genética , Vírus da Anemia Infecciosa Equina/isolamento & purificação , Prevalência , Sensibilidade e EspecificidadeRESUMO
Equine infectious anemia is an important infectious disease that affects equids worldwide. Control of the disease is currently based on detection of anti-p26 EIAV by Agar Gel Immunodiffusion (AGID). In this work, 62 animals were examined by AGID and nested-PCR using primers for the gag gene. Fifty-three samples (85.5%) were positive by nested-PCR, whereas only 33 samples (53%) were positive for AGID. Fifteen amplicons obtained by nested-PCR were sequenced and the aligned results subjected to phylogenetic analysis. The analysis suggests that the Brazilian EIAV form a cluster with WSU5, EIAVUK and Wyoming strains from United States.
Assuntos
Anemia Infecciosa Equina/virologia , Vírus da Anemia Infecciosa Equina/isolamento & purificação , Animais , Brasil , Cavalos , Vírus da Anemia Infecciosa Equina/classificação , Vírus da Anemia Infecciosa Equina/genética , Filogenia , Reação em Cadeia da Polimerase , Proteínas do Core Viral/genéticaRESUMO
Molecular and serological techniques for Equine Infectious Anemia Virus (EIAV) diagnosis were compared using samples from 59 clinically normal horses stabled on five farms in the Santa Fe Province of Argentina. Of these 26 (44.1%) were positive in official AGID tests and/or gp45/gp90-based ELISA. Surprisingly 18 of the 33 seronegative horses were positive in a PCR against viral sequences encoding gp45 (PCR-positive/AGID-negative) with all but one remaining EIAV-antibody negative throughout a two year observation period. The gp45 PCR results are supported by fact that 7/18 of these horses were positive in the Office International des Epizooties (OIE) recommended EIAV gag gene specific PCR plus 2 of this 7 also reacted in a PCR directed predominantly against the 5' untranslated region of the viral genome. Furthermore sufficient quantities of serum were available from 8 of these horses to verify their seronegative status in sensitive Western Blot tests and demonstrate by ELISA the absence of EIAV-specific antibodies was not attributable to abnormalities in total IgG concentration. Studies involving 7 of the PCR-positive/AGID-negative horses to measure lymphocyte proliferation in the presence of PHA showed no significant differences between this group and control animals. In addition, lymphocytes from 2 of these 7 horses responded to peptides derived from gp90 and gp45. Together these results demonstrate that apparently clinically normal horses with no gross signs of immunodeficiency in terms of total IgG concentration or T helper-cell function can remain seronegative for at least 24 months while harboring EIAV specific nucleic acid sequences.
Assuntos
Anemia Infecciosa Equina/sangue , Anemia Infecciosa Equina/epidemiologia , Vírus da Anemia Infecciosa Equina/imunologia , Animais , Anticorpos Antivirais/sangue , Argentina/epidemiologia , Sequência de Bases , DNA Viral/sangue , DNA Viral/genética , Monitoramento Epidemiológico/veterinária , Genes env/genética , Cavalos , Imunidade Celular/imunologia , Vírus da Anemia Infecciosa Equina/classificação , Vírus da Anemia Infecciosa Equina/genética , Mesterolona/sangue , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Proteínas do Envelope Viral/genéticaRESUMO
Equine Infectious Anemia (EIA) is a persistent lentivirus infection of horses which causes a chronic clinical condition with worldwide importance in veterinary medicine. The p26 protein is usually prepared for use as an antigen in serological tests for EIA diagnosis since it is a well-conserved gene sequence and very immunogenic. In view of the ability of yeast to make post-translational modifications of proteins, this study was carried out to allow Pichia pastoris to be used for the expression of a synthetic codon-optimized EIAV p26 gene. The gene was cloned into pPICZαA vector after appropriate enzymatic digestion. P. pastoris clones transformed with the pPICZαAp26 construction were induced to produce the recombinant p26 protein (rp26) under the regulation of alcohol oxidase 1 promoter by adding methanol to the culture medium. The p26 gene expression was detected by RT-PCR and the production of rp26 was confirmed by dot blotting, Western blotting, ELISA and AGID. The P. pastoris expression system was capable of producing a functional EIAV p26 protein that can be used directly in the functionality tests without requiring laborious purification or recovery steps. This is the first reported study of EIAV p26 protein production in yeast cells.
Assuntos
Antígenos Virais/metabolismo , Vírus da Anemia Infecciosa Equina/genética , Antígenos Virais/genética , Western Blotting , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Perfilação da Expressão Gênica , Vetores Genéticos , Pichia/genética , Plasmídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We analyzed the performance of a single-band Western blot (WB) test using recombinant p26 (rp26) capsid protein of equine infectious anemia virus. According to the results obtained, the rp26 WB test is a reliable confirmatory diagnostic tool to be used as a complementary test after an enzyme-linked immunosorbent assay or agar gel immunodiffusion test yielding doubtful results.
Assuntos
Anticorpos Antivirais/análise , Western Blotting/veterinária , Vírus da Anemia Infecciosa Equina/isolamento & purificação , Proteínas do Core Viral/química , Animais , Western Blotting/métodos , Western Blotting/normas , Ensaio de Imunoadsorção Enzimática/veterinária , Escherichia coli/genética , Cavalos , Imunodifusão/métodos , Imunodifusão/normas , Imunodifusão/veterinária , Vírus da Anemia Infecciosa Equina/genética , Vírus da Anemia Infecciosa Equina/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Reprodutibilidade dos Testes , Testes Sorológicos/veterinária , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologiaRESUMO
We developed and validated an agar gel immunodiffusion test (AGID) test for the diagnosis of equine infectious anemia (EIA) using as antigen the p26 protein of equine infectious anemia virus (EIAV) produced in the Escherichia coli expression system. The developed rp26-AGID test showed an excellent diagnostic relative sensitivity (100%) and specificity (100%) compared to a commercial AGID assay when 1855 field serum samples were analyzed. In addition, the rp26-AGID demonstrated to be a precise assay with excellent repeatability and reproducibility. In the analytical sensitivity trial, positive sera showed nearly the same endpoint dilutions for both compared tests. No positive-reactions were observed with 35 serum samples with antibodies related to other endemic agents and also with severely hemolysed samples, demonstrating that the rp26-AGID has an excellent analytical specificity. Complete concordance with blind previous results from five proficiency test panels confirmed the capability of the assay of accurate detection of EIAV antibodies. This is the first time that a recombinant AGID assay able to identify EIAV infections has been standardized and validated in Argentina according to international guidelines. Taking into account the results obtained, the p26-AGID could be adopted as an official test method for the diagnosis and control of EIA in this country.