RESUMO
Equine herpesvirus 1 (EHV-1) causes respiratory disease, abortion, neonatal death and neurological disease in equines and is endemic in most countries. The viral factors that influence EHV-1 disease severity are poorly understood, and this has hampered vaccine development. However, the N752D substitution in the viral DNA polymerase catalytic subunit has been shown statistically to be associated with neurological disease. This has given rise to the term "neuropathic strain," even though strains lacking the polymorphism have been recovered from cases of neurological disease. To broaden understanding of EHV-1 diversity in the field, 78 EHV-1 strains isolated over a period of 35 years were sequenced. The great majority of isolates originated from the United Kingdom and included in the collection were low passage isolates from respiratory, abortigenic and neurological outbreaks. Phylogenetic analysis of regions spanning 80% of the genome showed that up to 13 viral clades have been circulating in the United Kingdom and that most of these are continuing to circulate. Abortion isolates grouped into nine clades, and neurological isolates grouped into five. Most neurological isolates had the N752D substitution, whereas most abortion isolates did not, although three of the neurological isolates from linked outbreaks had a different polymorphism. Finally, bioinformatic analysis suggested that recombination has occurred between EHV-1 clades, between EHV-1 and equine herpesvirus 4, and between EHV-1 and equine herpesvirus 8.
Assuntos
Aborto Animal/virologia , Encefalopatias/veterinária , Variação Genética , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/genética , Doenças dos Cavalos/virologia , Transtornos Respiratórios/veterinária , Animais , Sequência de Bases , Encefalopatias/virologia , DNA Viral/genética , DNA Polimerase Dirigida por DNA/genética , Surtos de Doenças/veterinária , Equidae , Feminino , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/virologia , Herpesvirus Equídeo 1/isolamento & purificação , Doenças dos Cavalos/epidemiologia , Cavalos , Filogenia , Gravidez , Transtornos Respiratórios/virologia , Reino UnidoRESUMO
Equine influenza virus is a major respiratory pathogen in horses, and outbreaks of disease often lead to substantial disruption to and economic losses for equestrian industries. The hemagglutinin (HA) protein is of key importance in the control of equine influenza because HA is the primary target of the protective immune response and the main component of currently licensed influenza vaccines. However, the influenza virus HA protein changes over time, a process called antigenic drift, and vaccine strains must be updated to remain effective. Antigenic drift is assessed primarily by the hemagglutination inhibition (HI) assay. We have generated HI assay data for equine influenza A (H3N8) viruses isolated between 1968 and 2007 and have used antigenic cartography to quantify antigenic differences among the isolates. The antigenic evolution of equine influenza viruses during this period was clustered: from 1968 to 1988, all isolates formed a single antigenic cluster, which then split into two cocirculating clusters in 1989, and then a third cocirculating cluster appeared in 2003. Viruses from all three clusters were isolated in 2007. In one of the three clusters, we show evidence of antigenic drift away from the vaccine strain over time. We determined that a single amino acid substitution was likely responsible for the antigenic differences among clusters.
Assuntos
Evolução Molecular , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H3N8/genética , Vírus da Influenza A Subtipo H3N8/imunologia , Infecções por Orthomyxoviridae/virologia , Substituição de Aminoácidos , Animais , Antígenos Virais/classificação , Antígenos Virais/imunologia , Western Blotting , Células Cultivadas , Cães , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Hemaglutininas/imunologia , Hemaglutininas/metabolismo , Cavalos , Vírus da Influenza A Subtipo H3N8/isolamento & purificação , Rim/citologia , Rim/metabolismo , Rim/virologia , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/imunologia , Filogenia , RNA Mensageiro/genética , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
An Autographa californica nuclear polyhedrosis virus gene encoding an occlusion-derived virus (ODV) envelope protein of 56 kDa was identified and sequenced. Transcription initiates from a conserved baculovirus late motif (ATAAG) with transcripts detected from 16 through 72 hr p.i. The protein is detected in infected cell extracts from 36 hr p.i. Western blot assay of ODV, BV, viral envelope, and nucleocapsid preparations coupled with immunoelectron microscopy reveal that this protein localizes to the ODV envelope. This protein is named ODV-E56 to identify its viral origin, envelope location, and apparent molecular weight. ODV-E56 is enriched in viral induced intranuclear microvesicles as determined by immunogold labeling. A mutant was constructed with the C-terminal portion of the protein replaced with beta-galactosidase. The fusion protein, E56-beta-gal, locates to the viral nucleocapsids and not to the ODV envelope or intranuclear microvesicles. This suggests that the signals necessary for transport and/or retention into these structures lies within the C-terminal portion of ODV-E56. Additionally, both ODV-E56 and E56-beta-gal are enriched in electron dense regions that cluster around the inner nuclear membrane and within the nucleoplasm.
Assuntos
Proteínas de Membrana , Nucleopoliedrovírus/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Viral , Genes Virais , Glicosilação , Corpos de Inclusão Viral/genética , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Nucleopoliedrovírus/química , Nucleopoliedrovírus/ultraestrutura , Biossíntese de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/ultraestrutura , Spodoptera , Proteínas do Envelope Viral/análise , beta-Galactosidase/genéticaRESUMO
Restriction enzyme digests of DNA from 22 unselected isolates of EHV-1 were analysed by hybridization with cloned DNA fragments covering the genome. In addition to a small amount of inter-strain variation, heterogeneity within strains was observed, caused by loss of specific restriction endonuclease sites in the DNA of a proportion of the virus particles of any one stock. Fifteen strains demonstrated the same intra-strain variation involving loss of the BamHI L-M site which was shown to lie within coding sequence for the large subunit of ribonucleotide reductase. This particular mutation may therefore be selected for by passage in RK13 cells.
Assuntos
Variação Genética , Genoma Viral , Herpesvirus Equídeo 1/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Linhagem Celular , Clonagem Molecular , Sondas de DNA , DNA Viral , Cavalos , Dados de Sequência Molecular , Mapeamento por RestriçãoRESUMO
A portion of equine herpesvirus type 1 (EHV-1) gene 28, which is homologous to herpes simplex virus type 1 gene UL32, was expressed using a prokaryotic system to yield a fusion protein which reacted on Western blots with P19, a monoclonal antibody (MAb) that reacts with EHV-1 glycoprotein 300 (gp300), confirming that this gene encodes gp300. Hydrophobicity analysis showed that gp300 is a glycoprotein with multiple hydrophobic domains that might interact with, or span, the membrane several times. As such, it may represent the first member of a new family of herpesvirus glycoproteins to be identified as a virus structural component. Gp300 was also shown to be modified by palmitic acid residues, and a second MAb (1G12) directed against gp300 inhibited fusion between EHV-1-infected cells.
Assuntos
Genes Virais/genética , Herpesvirus Equídeo 1/genética , Proteínas do Envelope Viral/genética , Acilação , Sequência de Aminoácidos , Anticorpos Monoclonais/farmacologia , Anticorpos Antivirais/farmacologia , Sequência de Bases , Fusão Celular/efeitos dos fármacos , Escherichia coli/genética , Dados de Sequência Molecular , Plasmídeos/genética , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Virais de Fusão/genética , Proteínas Virais/genéticaRESUMO
The DNA sequence of the equine herpesvirus type 1 (EHV-1) gD gene homologue has been determined for the strain Ab1 and compared with previously published sequences. A portion of the gene has been located to a region of the genome which also encodes homologues of the herpes simplex virus type 1 genes for gE and gI and is known to encode an epitope of the virion protein gp17/18. Analysis of the EHV-1 strain Kentucky A (KyA) by DNA hybridization showed the presence of a gD gene homologue and established the absence of genes for gI and gE. Western blot analysis, however, showed that KyA virus particles contain gp17/18, thus indicating that this protein is encoded by the gD gene homologue. The KyA gp17/18 was found to be smaller than that detected in other strains and this is accounted for by a frameshift mutation in the KyA sequence relative to Ab1. The mutation in the KyA strain results in an altered C-terminal sequence and could explain the apparent structural differences suggested by the reactivities with monoclonal antibodies (MAbs). We have also expressed part of the Ab1 gD gene as a fusion protein with glutathione S-transferase in Escherichia coli and shown that this reacts with the MAb 5H6 originally used to map gp17/18. These experiments establish that gp17/18 is encoded by the gD gene homologue.
Assuntos
Herpesvirus Equídeo 1/genética , Simplexvirus/genética , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Mapeamento Cromossômico , Dados de Sequência Molecular , Família Multigênica/genética , Proteínas Recombinantes de Fusão/biossíntese , Homologia de Sequência do Ácido NucleicoRESUMO
Monoclonal antibodies (MAbs) specific for equine herpesvirus type 1 (EHV-1) glycoprotein 60 (gp60) and gp 17/18 (F3132 and 5H6 respectively) were found to react with the same protein, which was identified as a homologue of herpes simplex virus type 1 gD. MAb F3132 strongly neutralized virus infectivity and inhibited the penetration of the virus into the cell. The effects on penetration were shared with three other MAbs against this protein (P68, F3116 and F3129), but no effect on virus penetration was found with any other anti-EHV-1 MAb tested. The level of glycosylation of gp60 was analysed using glycanase enzymes and glycosylation inhibitors, and consisted of mainly N-linked carbohydrate. The M(r) of non-N-glycosylated gp60 was 50K.
Assuntos
Herpesvirus Equídeo 1/genética , Simplexvirus/genética , Proteínas do Envelope Viral/genética , Replicação Viral/genética , Animais , Anticorpos Monoclonais , Carboidratos/análise , Glicosilação , Infecções por Herpesviridae/metabolismo , Herpesvirus Equídeo 1/patogenicidade , Dados de Sequência Molecular , Testes de Neutralização , Homologia de Sequência do Ácido Nucleico , Proteínas do Envelope Viral/imunologia , VirulênciaRESUMO
The DNA fragments representing the entire short unique region and part of the repeat sequences of the equine herpesvirus type-1 genome were cloned into plasmid vectors. The approximate positions of the junctions between the short unique region and the inverted repeats were then located by restriction endonuclease mapping. Two open reading frames coding for potential glycoproteins have been identified within the short unique region, using DNA sequence analysis. The predicted amino acid sequences of these open reading frames had extensive homology to the herpes simplex virus glycoproteins gE and gI and the related glycoproteins of pseudorabies virus and varicella-zoster virus.
Assuntos
DNA Viral/química , Glicoproteínas/genética , Herpesvirus Equídeo 1/genética , Fases de Leitura Aberta , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Vetores Genéticos , Glicoproteínas/química , Dados de Sequência Molecular , Plasmídeos , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Simplexvirus/genética , Moldes Genéticos , Proteínas do Envelope Viral/químicaRESUMO
To localize gene that may encode immunogens potentially important for recombinant vaccine design, we have analysed a region of the equine herpesvirus type-1 (EHV-1) genome where a glycoprotein-encoding gene had previously been mapped. The 4707-bp BamHI-EcoRI fragment from the short unique region of the EHV-1 genome was sequenced. This sequence contains three entire open reading frames (ORFs), and portions of two more. ORF1 codes for 161 amino acids (aa), and represents the C terminus of a possible membrane-bound protein. ORF2 (424 aa) and ORF3 (550 aa) are potential glycoprotein-encoding genes; the predicted aa sequences contain possible signal sequences, N-linked glycosylation sites and transmembrane domains; they also show homology to the glycoproteins gI and gE of herpes simplex virus type-1 (HSV-1), and the related proteins of pseudorabies virus and varicella-zoster virus. The predicted aa sequence of ORF4 shares no homology with other known herpesvirus proteins, but the nucleotide sequence shows a high level of homology with the corresponding region of the EHV-4 genome. ORF5 may be related to US9 of HSV-1.
