RESUMO
The equine infectious anemia virus (EIAV) attenuated vaccine was developed by long-term passaging of a field-isolated virulent strain in cross-species hosts, followed by successive cultivation in cells in vitro To explore the molecular mechanism underlying the evolution of the EIAV attenuated vaccine, a systematic study focusing on long-terminal-repeat (LTR) variation in numerous virus strains ranging from virulent EIAV to attenuated EIAV was performed over time both in vitro and in vivo Two hypervariable regions were identified within the U3 region in the enhancer region (EHR) and the negative regulatory element (NRE) and within the R region in the transcription start site (TSS) and the Tat-activating region (TAR). Among these sites, variation in the U3 region resulted in the formation of additional transcription factor binding sites; this variation of the in vitro-adapted strains was consistent with the loss of pathogenicity. Notably, the same LTR variation pattern was observed both in vitro and in vivo Generally, the LTR variation in both the attenuated virus and the virulent strain fluctuated over time in vivo Interestingly, the attenuated-virus-specific LTR variation was also detected in horses infected with the virulent strain, supporting the hypothesis that the evolution of an attenuated virus might have involved branching from EIAV quasispecies. This hypothesis was verified by phylogenetic analysis. The present systematic study examining the molecular evolution of attenuated EIAV from EIAV quasispecies may provide an informative model reflecting the evolution of similar lentiviruses.IMPORTANCE The attenuated EIAV vaccine was the first lentiviral vaccine used to successfully control for equine infectious anemia in China. This vaccine provides an important reference for studying the relationship between EIAV gene variation and changes in biological characteristics. Importantly, the vaccine provides a model for the investigation of lentiviral quasispecies evolution. This study followed the "natural" development of the attenuated EIAV vaccine by use of a systematic analysis of LTR evolution in vitro and in vivo The results revealed that the increase in LTR variation with passaging was accompanied by a decrease in virulence, which indicated that LTR variability might parallel the attenuation of virulence. Interestingly, the attenuated-virus-specific LTR variation was also detected in virulent-strain-infected horses, a finding consistent with those of previous investigations of gp90 and S2 evolution. Therefore, we present a hypothesis that the evolution of the attenuated virus may involve branching from EIAV quasispecies present in vivo.
Assuntos
Anemia Infecciosa Equina/genética , Evolução Molecular , Vírus da Anemia Infecciosa Equina/genética , Sequências Repetidas Terminais , Animais , Anemia Infecciosa Equina/metabolismo , Cavalos , Vírus da Anemia Infecciosa Equina/metabolismoRESUMO
Adenosine deaminases that act on RNA (ADARs) have been reported to be functional on various viruses. ADAR1 may exhibit antiviral or proviral activity depending on the type of virus. Human immunodeficiency virus (HIV)-1 is the most well-studied lentivirus with respect to its interaction with ADAR1, and variable results have been reported. In this study, we demonstrated that equine ADAR1 (eADAR1) was a positive regulator of equine infectious anemia virus (EIAV), another lentivirus of the Retroviridae family. First, eADAR1 significantly promoted EIAV replication, and the enhancement of viral protein expression was associated with the long terminal repeat (LTR) and Rev response element (RRE) regions. Second, the RNA binding domain 1 of eADAR1 was essential only for enhancing LTR-mediated gene expression. Third, in contrast with APOBEC proteins, which have been shown to reduce lentiviral infectivity, eADAR1 increased the EIAV infectivity. This study indicated that eADAR1 was proviral rather than antiviral for EIAV.
Assuntos
Adenosina Desaminase/metabolismo , Anemia Infecciosa Equina/enzimologia , Vírus da Anemia Infecciosa Equina/fisiologia , Vírus da Anemia Infecciosa Equina/patogenicidade , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , Replicação Viral , Adenosina Desaminase/genética , Animais , Linhagem Celular , Anemia Infecciosa Equina/genética , Anemia Infecciosa Equina/virologia , Cavalos , Interações Hospedeiro-Patógeno , Vírus da Anemia Infecciosa Equina/genética , Estrutura Terciária de Proteína , RNA de Cadeia Dupla/genética , RNA Viral/química , RNA Viral/genética , Sequências Repetidas Terminais , VirulênciaRESUMO
Equine lentivirus receptor 1 (ELR1) has been identified as a functional cellular receptor for equine infectious anemia virus (EIAV). Herein, recombinant ELR1 and EIAV surface glycoprotein gp90 were respectively expressed in Drosophila melanogaster S2 cells, and purified to homogeneity by Ni-NTA affinity chromatography and gel filtration chromatography. Gel filtration chromatography and analytical ultracentrifugation (AUC) analyses indicated that both ELR1 and gp90 existed as individual monomers in solution and formed a complex with a stoichiometry of 1:1 when mixed. The structure of ELR1 was first determined with the molecular replacement method, which belongs to the space group P42 21 2 with one molecule in an asymmetric unit. It contains eight antiparallel ß-sheets, of which four are in cysteine rich domain 1 (CRD1) and two are in CRD2 and CRD3, respectively. Alignment of ELR1 with HVEM and CD134 indicated that Tyr61, Leu70, and Gly72 in CRD1 of ELR1 are important residues for binding to gp90. Isothermal titration calorimetry (ITC) experiments further confirmed that Leu70 and Gly72 are the critical residues.
Assuntos
Lentivirus Equinos/química , Glicoproteínas de Membrana/química , Estrutura Secundária de Proteína , Receptores Virais/química , Proteínas Recombinantes/química , Animais , Drosophila melanogaster , Anemia Infecciosa Equina/genética , Anemia Infecciosa Equina/virologia , Cavalos/virologia , Vírus da Anemia Infecciosa Equina/patogenicidade , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Complexos Multiproteicos/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
The Chinese attenuated equine infectious anemia virus (EIAV) vaccine has successfully protected millions of equine animals from EIA disease in China. Given that the induction of immune protection results from the interactions between viruses and hosts, a better understanding of the characteristics of vaccine strain infection and host responses would be useful for elucidating the mechanism of the induction of immune protection by the Chinese attenuated EIAV strain. In this study, we demonstrate in equine monocyte-derived macrophages (eMDM) that EIAVFDDV13, a Chinese attenuated EIAV strain, induced a strong resistance to subsequent infection by a pathogenic strain, EIAVUK3. Further experiments indicate that the expression of the soluble EIAV receptor sELR1, Toll-like receptor 3 (TLR3) and interferon ß (IFNß) was up-regulated in eMDM infected with EIAVFDDV13 compared with eMDM infected with EIAVUK3. Stimulating eMDM with poly I:C resulted in similar resistance to EIAV infection as induced by EIAVFDDV13 and was correlated with enhanced TLR3, sELR1 and IFNß expression. The knock down of TLR3 mRNA significantly impaired poly I:C-stimulated resistance to EIAV, greatly reducing the expression of sELR1 and IFNß and lowered the level of infection resistance induced by EIAVFDDV13. These results indicate that the induction of restraining infection by EIAVFDDV13 in macrophages is partially mediated through the up-regulated expression of the soluble viral receptor and IFNß, and that the TLR3 pathway activation plays an important role in the development of an EIAV-resistant intracellular environment.
Assuntos
Anemia Infecciosa Equina/imunologia , Regulação da Expressão Gênica , Doenças dos Cavalos/imunologia , Vírus da Anemia Infecciosa Equina/fisiologia , Vacinas Virais/imunologia , Animais , Resistência à Doença , Ensaio de Imunoadsorção Enzimática/veterinária , Anemia Infecciosa Equina/genética , Anemia Infecciosa Equina/virologia , Doenças dos Cavalos/genética , Doenças dos Cavalos/virologia , Cavalos , Vírus da Anemia Infecciosa Equina/genética , Interferon beta/genética , Interferon beta/metabolismo , Macrófagos/imunologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Receptores Virais/genética , Receptores Virais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismoRESUMO
Human tetherin is a host restriction factor that inhibits replication of enveloped viruses by blocking viral release. Tetherin has an unusual topology that includes an N-terminal cytoplasmic tail, a single transmembrane domain, an extracellular domain, and a C-terminal glycosylphosphatidylinositol anchor. Tetherin is not well conserved across species, so it inhibits viral replication in a species-specific manner. Thus, studies of tetherin activities from different species provide an important tool for understanding its antiviral mechanism. Here, we report cloning of equine tetherin and characterization of its antiviral activity. Equine tetherin shares 53%, 40%, 36%, and 34% amino acid sequence identity with feline, human, simian, and murine tetherins, respectively. Like the feline tetherin, equine tetherin has a shorter N-terminal domain than human tetherin. Equine tetherin is localized on the cell surface and strongly blocks human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIAV) release from virus-producing cells. The antiviral activity of equine tetherin is neutralized by EIAV envelope protein, but not by the HIV-1 accessory protein Vpu, which is a human tetherin antagonist, and EIAV envelope protein does not counteract human tetherin. These results shed new light on our understanding of the species-specific tetherin antiviral mechanism.
Assuntos
Antígenos CD/metabolismo , Anemia Infecciosa Equina/metabolismo , Vírus da Anemia Infecciosa Equina/fisiologia , Proteínas do Envelope Viral/metabolismo , Liberação de Vírus , Sequência de Aminoácidos , Animais , Antígenos CD/química , Antígenos CD/genética , Sequência de Bases , Gatos , Linhagem Celular , Clonagem Molecular , Anemia Infecciosa Equina/genética , Anemia Infecciosa Equina/virologia , Cavalos , Humanos , Vírus da Anemia Infecciosa Equina/genética , Camundongos , Dados de Sequência Molecular , Retroviridae/genética , Retroviridae/fisiologia , Alinhamento de Sequência , Proteínas do Envelope Viral/genéticaRESUMO
EIA (Equine Infectious Anemia) is a blood-borne disease primarily transmitted by haematophagous insects or needle punctures. Other routes of transmission have been poorly explored. We evaluated the potential of EIAV (Equine Infectious Anemia Virus) to induce pulmonary lesions in naturally infected equids. Lungs from 77 EIAV seropositive horses have been collected in Romania and France. Three types of lesions have been scored on paraffin-embedded lungs: lymphocyte infiltration, bronchiolar inflammation, and thickness of the alveolar septa. Expression of the p26 EIAV capsid (CA) protein has been evaluated by immunostaining. Compared to EIAV-negative horses, 52% of the EIAV-positive horses displayed a mild inflammation around the bronchioles, 22% had a moderate inflammation with inflammatory cells inside the wall and epithelial bronchiolar hyperplasia and 6.5% had a moderate to severe inflammation, with destruction of the bronchiolar epithelium and accumulation of smooth muscle cells within the pulmonary parenchyma. Changes in the thickness of the alveolar septa were also present. Expression of EIAV capsid has been evidenced in macrophages, endothelial as well as in alveolar and bronchiolar epithelial cells, as determined by their morphology and localization. To summarize, we found lesions of interstitial lung disease similar to that observed during other lentiviral infections such as FIV in cats, SRLV in sheep and goats or HIV in children. The presence of EIAV capsid in lung epithelial cells suggests that EIAV might be responsible for the broncho-interstitial damages observed.
Assuntos
Células Epiteliais/patologia , Anemia Infecciosa Equina/patologia , Doenças dos Cavalos/patologia , Doenças Pulmonares Intersticiais/veterinária , Pulmão/patologia , Proteínas do Core Viral/genética , Animais , Western Blotting/veterinária , Células Epiteliais/virologia , Anemia Infecciosa Equina/genética , Anemia Infecciosa Equina/virologia , Feminino , França , Doenças dos Cavalos/genética , Doenças dos Cavalos/virologia , Cavalos , Vírus da Anemia Infecciosa Equina/fisiologia , Pulmão/virologia , Doenças Pulmonares Intersticiais/genética , Doenças Pulmonares Intersticiais/patologia , Doenças Pulmonares Intersticiais/virologia , Masculino , Microscopia de Fluorescência/veterinária , Romênia , Proteínas do Core Viral/metabolismoRESUMO
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 to produce evasion genotypes that lead to late re-emergence of disease. There is a high rate of genetic variation in the EIAV surface envelope glycoprotein (SU) and in the region of the transmembrane protein (TM) overlapped by the major exon of Rev. This review examines genetic and phenotypic variation in Rev during EIAV disease and a possible role for Rev in immune evasion and virus persistence.
Assuntos
Anemia Infecciosa Equina/genética , Genes env/genética , Genes rev/genética , Vírus da Anemia Infecciosa Equina , Animais , Anemia Infecciosa Equina/imunologia , Produtos do Gene rev/imunologia , HIV-1/genética , HIV-1/imunologia , Cavalos , Humanos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , Vírus da Anemia Infecciosa Equina/genética , Vírus da Anemia Infecciosa Equina/imunologia , Modelos Biológicos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Transcrição Gênica/imunologia , Replicação ViralRESUMO
OBJECTIVE: To study some factors that may influence DNA sequencing by analyzing 2040 sequencing samples retrospectively. METHODS: The effects of DNA templates, primers, cycle sequencing reactions as well as purification methods were comparatively analyzed. RESULTS: 70 percent of sequencing failure is caused by DNA template. Characteristics of data from poor template preparation include top-heavy with a short read length, acceptable signal with some noise peaks (Ns), weak signal, overall noise and flat lines or no signal. The ineffective primers can cause 15.4 percent sequencing failure. Possible explanations include primer mismatches and low Tm value. CONCLUSIONS: The quality of template DNA can have a major impact on the quality of the DNA sequencing. Purification of the extension products is absolutely necessary in DNA sequencing, and isopropanol precipitation is highly recommended for this procedure.
Assuntos
DNA Viral/genética , Anemia Infecciosa Equina/genética , HIV-1/genética , Animais , Sequência de Bases , Cavalos , Humanos , Dados de Sequência Molecular , Receptores de Quimiocinas/genética , Estudos Retrospectivos , Análise de Sequência de DNA/métodosRESUMO
Six strains of equine infectious anemia virus (EIAV) were recovered from febrile and non-febrile stages of a horse experimentally infected with the P337-V70 strain given once to a horse. The env gp90 genes of the isolates, the P337-V70 and P337-V26, avirulent virus derived from the P337-V70 strain, were sequenced. A comparison of the gp90 gene sequences revealed that amino acid variations among the viruses tested showed as high as 8.2 to 11.5%. In addition, the comparison also indicated that the isolates that recovered from the non-febrile stage were contained in nucleotide insertions in the principal neutralizing domain (PND) region. The insertions were arranged regularly with smaller segments. The nucleotide sequence of the P337-V26 gp90 gene was found to contain a six-nucleotides insertion and seven nucleotide substitutions outside the PND region, when compared with that of the P337-V70 strain.
Assuntos
DNA Viral/genética , Anemia Infecciosa Equina/genética , Genes Virais/genética , Variação Genética , Vírus da Anemia Infecciosa Equina/genética , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Viral/análise , DNA Viral/química , Feminino , Cavalos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , Proteínas do Envelope Viral/análise , Proteínas do Envelope Viral/químicaRESUMO
Transcription of pathogenic equine infectious anemia virus (EIAV) in an acutely infected horse was examined by using the polymerase chain reaction and nucleotide sequencing. Four spliced transcripts were identified in liver tissue, in contrast to the multiplicity of alternatively spliced messages reported for in vitro-propagated human immunodeficiency virus, simian immunodeficiency virus, and, to a lesser extent, EIAV. Nucleotide sequence analysis demonstrated that three of these mRNAs encode known viral proteins: the envelope precursor, the product of the S2 open reading frame, and the regulatory proteins Tat and Rev. The fourth transcript encodes a novel Tat-TM fusion protein, Ttm. Ttm is a 27-kDa protein translated from the putative tat CTG initiation codon and containing the carboxy-terminal portion of TM immediately downstream from the membrane-spanning domain. p27ttm is expressed in EIAV-infected canine cells and was recognized by peptide antisera against both Tat and TM. Cells transfected with ttm cDNA also expressed p27ttm, which appeared to be localized to the endoplasmic reticulum or Golgi apparatus by indirect immunofluorescence. The carboxy terminus of lentiviral TM proteins has previously been shown to influence viral infectivity, growth kinetics, and cytopathology, suggesting that Ttm plays an important role in the EIAV life cycle.
Assuntos
Anemia Infecciosa Equina/genética , Proteínas de Membrana , RNA Mensageiro/genética , RNA Viral/genética , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Compartimento Celular , Retículo Endoplasmático , Éxons/genética , Produtos do Gene rev , Produtos do Gene tat , Complexo de Golgi , Cavalos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Splicing de RNA , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
Equine infectious anemia virus (EIAV) is a lentivirus that infects and persists in the monocyte/macrophage populations of blood and tissues. We employed polymerase chain reaction to investigate the distribution and the level of genome variability of EIAV DNA in different tissues of a horse infected with a highly virulent variant of the Wyoming strain of the virus. Long terminal repeat, gag, and pol primer pairs were used to direct the amplification of EIAV DNA from the peripheral blood mononuclear cells and from cells, presumably the macrophage subtypes, of the kidney, spleen, liver, lymph node, and cerebellum and periventricular regions of the brain. Polymerase chain reaction analysis of four domains within the envelope showed that viral subtypes with particular envelope domains segregated to different tissues as defined by the presence or absence of a given type of domain. Collectively, these results show that virus variants in the env gene home to different tissues, presumably because of selection for tissue-specific envelope determinants.
Assuntos
Anemia Infecciosa Equina/genética , Genes env/genética , Vírus da Anemia Infecciosa Equina/genética , Animais , Sequência de Bases , Variação Genética , Cavalos , Vírus da Anemia Infecciosa Equina/classificação , Vírus da Anemia Infecciosa Equina/patogenicidade , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Reação em Cadeia da PolimeraseRESUMO
The unique periodic nature of equine infectious anaemia (EIA) is believed to result from the ability of the infecting virus. EIAV, to undergo relatively rapid antigenic variations which circumvent host immune responses resulting in distinct virus populations in sequential clinical episodes in the persistently infected horse. This model was examined by oligonucleotide mapping comparisons of the RNA genomes of selected isolates of EIAV. Variations in oligonucleotide maps could be reproducibly demonstrated (i) after adaptation of the laboratory strain of EIAV to replication in a pony, (ii) after serial passage of virus between two ponies, and (iii) after a prolonged persistent infection in a single pony. In the latter case, the two isolates examined were recovered from different clinical episodes and were shown to be antigenic variants. In contrast, no variations in RNA structure could be detected in oligonucleotide maps of virus isolated after prolonged passage in tissue culture. Thus, these results support our concept that EIAV is a highly mutable virus, which may given rise to antigenic variants in the presence of immune pressures. The degree of variation observed between oligonucleotide maps is similar to that observed previously between variants of visna virus. These similarities between EIAV an visna suggest that genomic point mutations producing antigenic variants may be a more important mechanism of retrovirus persistence than was previously recognized.