RESUMO
The avian pathogen Mycoplasma gallisepticum, the etiological agent of chronic respiratory disease in chickens, exhibits enhanced pathogenesis in the presence of a copathogen such as low-pathogenic avian influenza virus (LPAIV). To further investigate the intricacies of this copathogenesis, chickens were monoinfected or coinfected with either virulent M. gallisepticum strain Rlow or LPAIV H3N8 (A/duck/Ukraine/1963), with assessment of tracheal histopathology, pathogen load, and transcriptomic host responses to infection by RNA sequencing. Chickens coinfected with M. gallisepticum Rlow followed by LPAIV H3N8 exhibited significantly more severe tracheal lesions and mucosal thickening than chickens infected with LPAIV H3N8 alone and greater viral loads than chickens infected first with H3N8 and subsequently with M. gallisepticum Rlow Recovery of live M. gallisepticum was significantly higher in chickens infected first with LPAIV H3N8 and then with M. gallisepticum Rlow, compared to chickens given a mock infection followed by M. gallisepticum Rlow The transcriptional responses to monoinfection and coinfection with M. gallisepticum and LPAIV highlighted the involvement of differential expression of genes such as Toll-like receptor 15, Toll-like receptor 21, and matrix metallopeptidase 1. Pathway and gene ontology analyses of these differentially expressed genes suggest that coinfection with virulent M. gallisepticum and LPAIV induces decreases in the expression of genes related to ciliary activity in vivo and alters multiple immune-related signaling cascades. These data aid in the understanding of the relationship between M. gallisepticum and LPAIV during copathogenesis in the natural host and may contribute to further understanding of copathogen infections of humans and other animals.
Assuntos
Coinfecção/patologia , Influenza Aviária/patologia , Infecções por Mycoplasma/patologia , Doenças das Aves Domésticas/patologia , Traqueia/patologia , Animais , Carga Bacteriana , Galinhas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Histocitoquímica , Interações Hospedeiro-Patógeno , Vírus da Influenza A/crescimento & desenvolvimento , Influenza Aviária/complicações , Infecções por Mycoplasma/complicações , Mycoplasma gallisepticum/crescimento & desenvolvimento , Carga ViralRESUMO
Mycoplasma gallisepticum, the primary etiologic agent of chronic respiratory disease, is a significant poultry pathogen, causing severe inflammation and leading to economic losses worldwide. Immunodominant proteins encoded by the variable lipoprotein and hemagglutinin (vlhA) gene family are thought to be important for M. gallisepticum-host interaction, pathogenesis, and immune evasion, but their exact role remains unknown. Previous work has demonstrated that vlhA phase variation is dynamic throughout the earliest stages of infection, with vlhA 3.03 being the predominant vlhA expressed during the initial infection, and that the pattern of dominant vlhA expression may be nonrandom and regulated by previously unrecognized mechanisms. To further investigate this gene family, we assessed the vlhA profile of two well-characterized vaccine strains, GT5 and Mg7, a vlhA 3.03 mutant strain, and an M. gallisepticum population expressing an alternative immunodominant vlhA Here, we report that two M. gallisepticum vaccine strains show different vlhA profiles over the first 2 days of infection compared to that of wild-type Rlow, while the population expressing an alternative immunodominant vlhA gene reverted to a profile indistinguishable from that of wild-type Rlow Additionally, we observed a slight shift in the vlhA gene expression profile but no reduction in virulence in a vlhA 3.03 mutant. Taken together, these data further support the hypothesis that M. gallisepticum vlhA genes change in a nonstochastic temporal progression of expression and that vlhA 3.03, while preferred, is not required for virulence. Collectively, these data may be important in elucidating mechanisms of colonization and overall pathogenesis of M. gallisepticum.
Assuntos
Proteínas de Bactérias/biossíntese , Regulação Bacteriana da Expressão Gênica , Hemaglutininas/biossíntese , Lipoproteínas/biossíntese , Infecções por Mycoplasma/veterinária , Mycoplasma gallisepticum/genética , Doenças das Aves Domésticas/microbiologia , Animais , Variação Antigênica , Proteínas de Bactérias/genética , Galinhas , Perfilação da Expressão Gênica , Hemaglutininas/genética , Lipoproteínas/genética , Família Multigênica , Infecções por Mycoplasma/microbiologia , Mycoplasma gallisepticum/metabolismo , Doenças das Aves Domésticas/patologiaRESUMO
Mycoplasma gallisepticum, the primary etiologic agent of chronic respiratory disease (CRD) in poultry, leads to prolonged recruitment and activation of inflammatory cells in the respiratory mucosa. This is consistent with the current model of immune dysregulation that ostensibly allows the organism to evade clearance mechanisms and establish chronic infection. To date, studies using quantitative reverse transcription-PCR (qRT-PCR) and microarrays have shown a significant transient upregulation of cytokines and chemokines from tracheal epithelial cells (TECs) in vitro and tracheal tissue ex vivo in response to virulent strain Rlow that contributes to the infiltration of inflammatory cells into the tracheal mucosa. To expand upon these experiments, RNA was isolated from tracheas of 20 chickens infected with M. gallisepticum Rlow and 20 mock-infected animals at days 1, 3, 5, and 7 postinoculation, and samples were analyzed for differential gene expression using Illumina RNA sequencing. A rapid host response was observed 24 h postinfection, with over 2,500 significantly differentially expressed genes on day 3, the peak of infection. Many of these genes have immune-related functions involved in signaling pathways, including Toll-like receptor (TLR), mitogen-activated protein kinase, Jak-STAT, and the nucleotide oligomerization domain-like receptor pathways. Of interest was the increased expression of numerous cell surface receptors, including TLR4 and TLR15, which may contribute to the production of cytokines. Metabolic pathways were also activated on days 1 and 3 postinfection, ostensibly due to epithelial cell distress that occurs upon infection. Early perturbations in tissue-wide gene expression, as observed here, may underpin a profound immune dysregulation, setting the stage for disease manifestations characteristic of M. gallisepticum infection.
Assuntos
Galinhas/microbiologia , Redes e Vias Metabólicas/genética , Infecções por Mycoplasma/veterinária , Mycoplasma gallisepticum/imunologia , Mycoplasma gallisepticum/patogenicidade , Doenças das Aves Domésticas/imunologia , Traqueia/microbiologia , Animais , Quimiocinas/genética , Quimiocinas/imunologia , Galinhas/imunologia , Citocinas/genética , Citocinas/imunologia , Perfilação da Expressão Gênica/métodos , Infecções por Mycoplasma/imunologia , Infecções por Mycoplasma/microbiologia , Mycoplasma gallisepticum/genética , Doenças das Aves Domésticas/microbiologia , Reação em Cadeia da Polimerase em Tempo Real , Mucosa Respiratória/imunologia , Mucosa Respiratória/microbiologia , Análise de Sequência de RNA , Transdução de Sinais , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia , Traqueia/imunologiaRESUMO
Mycoplasma gallisepticum, known primarily as a respiratory pathogen of domestic poultry, has emerged since 1994 as a significant pathogen of the house finch (Haemorhousmexicanus) causing severe conjunctivitis and mortality. House finch-associated M. gallisepticum (HFMG) spread rapidly and increased in virulence for the finch host in the eastern United States. In the current study, we assessed virulence in domestic poultry with two temporally distant, and yet geographically consistent, HFMG isolates which differ in virulence for house finches-Virginia 1994 (VA1994), the index isolate of the epidemic, and Virginia 2013 (VA2013), a recent isolate of increased house finch virulence. Here we report a significant difference between VA1994 and VA2013 in their levels of virulence for chickens; notably, this difference correlated inversely to the difference in their levels of virulence for house finches. VA1994, while moderately virulent in house finches, displayed significant virulence in the chicken respiratory tract. VA2013, while highly virulent in the house finch, was significantly attenuated in chickens relative to VA1994, displaying less-severe pathological lesions in, and reduced bacterial recovery from, the respiratory tract. Overall, these data indicate that a recent isolate of HFMG is greatly attenuated in the chicken host relative to the index isolate, notably demonstrating a virulence phenotype in chickens inversely related to that in the finch host.
Assuntos
Galinhas/microbiologia , Tentilhões/microbiologia , Infecções por Mycoplasma/epidemiologia , Mycoplasma gallisepticum/isolamento & purificação , Mycoplasma gallisepticum/patogenicidade , Animais , Feminino , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/veterinária , Fenótipo , Filogenia , Virginia , VirulênciaRESUMO
Mycoplasma gallisepticum is the primary etiologic agent of chronic respiratory disease in poultry, a disease largely affecting the respiratory tract and causing significant economic losses worldwide. Immunodominant proteins encoded by members of the variable lipoprotein and hemagglutinin (vlhA) gene family are thought to be important for mechanisms of M. gallisepticum-host interaction, pathogenesis, and immune evasion, but their exact role and the overall nature of their phase variation are unknown. To better understand these mechanisms, we assessed global transcriptomic vlhA gene expression directly from M. gallisepticum populations present on tracheal mucosae during a 7-day experimental infection in the natural chicken host. Here we report differences in both dominant and minor vlhA gene expression levels throughout the first week of infection and starting as early as day 1 postinfection, consistent with a functional role not dependent on adaptive immunity for driving phase variation. Notably, data indicated that, at given time points, specific vlhA genes were similarly dominant in multiple independent hosts, suggesting a nonstochastic temporal progression of dominant vlhA gene expression in the colonizing bacterial population. The dominant expression of a given vlhA gene was not dependent on the presence of 12-copy GAA trinucleotide repeats in the promoter region and did not revert to the predominate vlhA gene when no longer faced with host pressures. Overall, these data indicate that vlhA phase variation is dynamic throughout the earliest stages of infection and that the pattern of dominant vlhA expression may be nonrandom and regulated by previously unrecognized mechanisms.
Assuntos
Proteínas de Bactérias/biossíntese , Hemaglutininas/biossíntese , Lectinas/biossíntese , Infecções por Mycoplasma/veterinária , Mycoplasma gallisepticum/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Sequência de Bases , Galinhas , Feminino , Hemaglutininas/genética , Lectinas/genética , Lipoproteínas/biossíntese , Lipoproteínas/genética , Infecções por Mycoplasma/imunologia , Infecções por Mycoplasma/microbiologia , Mycoplasma gallisepticum/imunologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/microbiologia , Regiões Promotoras Genéticas , Doenças Respiratórias/imunologia , Doenças Respiratórias/microbiologia , Análise de Sequência de DNARESUMO
African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available and progress is hindered by lack of knowledge concerning the extent of ASFV strain diversity and the viral antigens conferring type-specific protective immunity in pigs. Available data from vaccination/challenge experiments in pigs indicate that ASF protective immunity may be haemadsorption inhibition (HAI) serotype-specific. Recently, we have shown that two ASFV proteins, CD2v (EP402R) and C-type lectin (EP153R), are necessary and sufficient for mediating HAI serological specificity (Malogolovkin et al., 2015).. Here, using ASFV inter-serotypic chimeric viruses and vaccination/challenge experiments in pigs, we demonstrate that serotype-specific CD2v and/or C-type lectin proteins are important for protection against homologous ASFV infection. Thus, these viral proteins represent significant protective antigens for ASFV that should be targeted in future vaccine design and development. Additionally, these data support the concept of HAI serotype-specific protective immunity.
Assuntos
Vírus da Febre Suína Africana/imunologia , Febre Suína Africana/imunologia , Febre Suína Africana/prevenção & controle , Anticorpos Antivirais/imunologia , Doenças dos Suínos/prevenção & controle , Proteínas Virais/imunologia , Vacinas Virais/imunologia , Febre Suína Africana/virologia , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Lectinas Tipo C/imunologia , Sorogrupo , Suínos/virologia , Doenças dos Suínos/virologia , VacinaçãoRESUMO
African swine fever (ASF) is an emerging disease threat for the swine industry worldwide. No ASF vaccine is available and progress is hindered by lack of knowledge concerning the extent of ASF virus (ASFV) strain diversity and the viral antigens responsible for protection in the pig. Available data from vaccination/challenge experiments in pigs indicate ASF protective immunity is haemadsorption inhibition (HAI) serotype-specific. A better understanding of ASFV HAI serological groups and their diversity in nature, as well as improved methods to serotype ASFV isolates, is needed. Here, we demonstrated that the genetic locus encoding ASFV CD2v and C-type lectin proteins mediates HAI serological specificity and that CD2v/C-type lectin genotyping provides a simple method to group ASFVs by serotype, thus facilitating study of ASFV strain diversity in nature, and providing information necessary for eventual vaccine design, development and efficacious use.
Assuntos
Vírus da Febre Suína Africana/genética , Loci Gênicos , Subtipos Sorológicos de HLA-DR/genética , Lectinas Tipo C/genética , Animais , Antígenos Virais/genética , DNA Viral/genética , Genótipo , Lectinas Tipo C/imunologia , Sensibilidade e Especificidade , Suínos , Proteínas Virais/genética , Vacinas Virais/genética , Vacinas Virais/imunologiaRESUMO
The genus Macavirus of the subfamily Gammaherpesvirinae includes viruses that infect lymphoid cells of domestic and wild ruminants and swine, causing asymptomatic latent infections in reservoir hosts. Here, we describe the genome of bovine herpesvirus 6 (BoHV-6), a macavirus ubiquitous in healthy cattle populations. The BoHV-6 genome exhibited architecture conserved in macaviruses, including a repetitive H-DNA region and unique 141 kbp L-DNA region predicted to encode 77 genes. BoHV-6 encoded, in variable genomic regions, a novel complement of genes relative to other characterized macaviruses, probably contributing to distinctive aspects of BoHV-6 infection biology and host range. Most notably, BoHV-6 encoded the first herpesviral protein (Bov2.b2) similar to cellular ornithine decarboxylase, an enzyme that catalyses the first and rate-limiting step in the biosynthesis of polyamines. Bov2.b2 conceivably mediates a novel mechanism by which BoHV-6 promotes cell-cycle-dependent viral replication.
Assuntos
DNA Viral/química , DNA Viral/genética , Gammaherpesvirinae/genética , Genoma Viral , Animais , Bovinos , Gammaherpesvirinae/isolamento & purificação , Ordem dos Genes , Dados de Sequência Molecular , Ornitina Descarboxilase/genética , Análise de Sequência de DNA , Sintenia , Proteínas Virais/genéticaRESUMO
Mycoplasma pneumoniae is a significant human respiratory pathogen that causes high morbidity worldwide. No vaccine to prevent M. pneumoniae infection currently exists, since the mechanisms of pathogenesis are poorly understood. To this end, we constructed a P30 cytadhesin mutant (P-130) with a drastically reduced capacity for binding to erythrocytes and an inability to glide on glass substrates. This mutant was determined to be avirulent and cannot survive in the lungs of BALB/c mice. We also ascertained that the previously identified P30 gliding motility mutant II-3R is avirulent and also cannot be recovered from the lungs of mice after infection. Mutant P130 was then assessed for its efficacy as a live attenuated vaccine candidate in mice after challenge with wild-type M. pneumoniae. After vaccination with the P-130 P30 mutant, mice showed evidence of exacerbated disease upon subsequent challenge with the wild-type strain PI1428, which appears to be driven by a Th17 response and corresponding eosinophilia. Our results are in accordance with other reports of vaccine-induced disease exacerbation in rodents and emphasize the need to better understand the basic mechanisms of M. pneumoniae pathogenesis.
Assuntos
Adesinas Bacterianas/genética , Vacinas Bacterianas/efeitos adversos , Vacinas Bacterianas/imunologia , Progressão da Doença , Técnicas de Inativação de Genes , Mycoplasma pneumoniae/imunologia , Pneumonia por Mycoplasma/prevenção & controle , Animais , Aderência Bacteriana , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Eosinofilia , Eritrócitos/microbiologia , Feminino , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana , Mycoplasma pneumoniae/genética , Pneumonia por Mycoplasma/imunologia , Pneumonia por Mycoplasma/microbiologia , Células Th17/imunologia , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , VirulênciaRESUMO
Mycoplasma gallisepticum, a significant respiratory and reproductive pathogen of domestic poultry, has since 1994 been recognized as an emergent pathogen of the American house finch (Carpodacus mexicanus). Epizootic spread and pathognomonic characteristics of house finch-associated Mycoplasma gallisepticum (HFMG) have been studied as a model of an emergent to endemic pathogen in a novel host. Here we present comparative analysis of eight HFMG genomes, including one from an index isolate and seven isolates separated spatially and temporally (1994-2008) across the epizootic, and notably having differences in virulence. HFMG represented a monophyletic clade relative to sequenced poultry isolates, with genomic changes indicating a novel M. gallisepticum lineage and including unique deletions of coding sequence. Though most of the HFMG genome was highly conserved among isolates, genetic distances correlated with temporal-spatial distance from the index. The most dramatic genomic differences among HFMG involved phase-variable and immunodominant VlhA lipoprotein genes, including those variable in presence and genomic location. Other genomic differences included tandem copy number variation of a 5 kbp repeat, changes in and adjacent to the clustered regularly interspaced short palindromic repeats, and small-scale changes affecting coding potential and association of genes with virulence. Divergence of monophyletic isolates from similar time/space in the epizootic indicated local diversification of distinct HFMG sublineages. Overall, these data identify candidate virulence genes and reveal the importance of phase-variable lipoproteins during the evolution of M. gallisepticum during its emergence and dissemination in a novel host in nature, likely mediating an important role at the interface between pathogen virulence and host immunity.
Assuntos
Proteínas de Bactérias/genética , Doenças das Aves/microbiologia , Evolução Molecular , Variação Genética , Lipoproteínas/genética , Infecções por Mycoplasma/veterinária , Mycoplasma gallisepticum/genética , Passeriformes/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Sequência de Bases , Genoma Bacteriano , Genômica , Lipoproteínas/metabolismo , Dados de Sequência Molecular , Infecções por Mycoplasma/microbiologia , Mycoplasma gallisepticum/classificação , Mycoplasma gallisepticum/isolamento & purificação , Mycoplasma gallisepticum/patogenicidade , Filogenia , Virulência , Zoonoses/microbiologiaRESUMO
Mycoplasma gallisepticum is a significant respiratory and reproductive pathogen of domestic poultry. While the complete genomic sequence of the virulent, low-passage M. gallisepticum strain R (R(low)) has been reported, genomic determinants responsible for differences in virulence and host range remain to be completely identified. Here, we utilize genome sequencing and microarray-based comparative genomic data to identify these genomic determinants of virulence and to elucidate genomic variability among strains of M. gallisepticum. Analysis of the high-passage, attenuated derivative of R(low), R(high), indicated that relatively few total genomic changes (64 loci) occurred, yet they are potentially responsible for the observed attenuation of this strain. In addition to previously characterized mutations in cytadherence-related proteins, changes included those in coding sequences of genes involved in sugar metabolism. Analyses of the genome of the M. gallisepticum vaccine strain F revealed numerous differences relative to strain R, including a highly divergent complement of vlhA surface lipoprotein genes, and at least 16 genes absent or significantly fragmented relative to strain R. Notably, an R(low) isogenic mutant in one of these genes (MGA_1107) caused significantly fewer severe tracheal lesions in the natural host compared to virulent M. gallisepticum R(low). Comparative genomic hybridizations indicated few genetic loci commonly affected in F and vaccine strains ts-11 and 6/85, which would correlate with proteins affecting strain R virulence. Together, these data provide novel insights into inter- and intrastrain M. gallisepticum genomic variability and the genetic basis of M. gallisepticum virulence.
Assuntos
DNA Bacteriano/genética , Genoma Bacteriano , Infecções por Mycoplasma/veterinária , Mycoplasma gallisepticum/genética , Mycoplasma gallisepticum/patogenicidade , Doenças das Aves Domésticas/microbiologia , Fatores de Virulência/genética , Animais , Galinhas , Hibridização Genômica Comparativa , DNA Bacteriano/química , Feminino , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
African swine fever virus (ASFV) is a large, intracytoplasmically-replicating DNA arbovirus and the sole member of the family Asfarviridae. It is the etiologic agent of a highly lethal hemorrhagic disease of domestic swine and therefore extensively studied to elucidate the structures, genes, and mechanisms affecting viral replication in the host, virus-host interactions, and viral virulence. Increasingly apparent is the complexity with which ASFV replicates and interacts with the host cell during infection. ASFV encodes novel genes involved in host immune response modulation, viral virulence for domestic swine, and in the ability of ASFV to replicate and spread in its tick vector. The unique nature of ASFV has contributed to a broader understanding of DNA virus/host interactions.
Assuntos
Vírus da Febre Suína Africana/fisiologia , Febre Suína Africana/virologia , Animais , SuínosRESUMO
Mycoplasma gallisepticum, a significant poultry pathogen, has evolved rapidly in its new passerine host since its first reported isolation from house finches in the US in 1994. In poultry, M. gallisepticum infects the upper respiratory tract, causing tracheal mucosal thickening and inflammation, in addition to inflammation of the reproductive tract. However, in house finches M. gallisepticum primarily causes inflammation of the conjunctiva. Given that different tissues are primarily affected by the same pathogen in different hosts, we have compared the early changes in gene expression of the phase-variable lipoproteins (vlhA) gene family of M. gallisepticum collected directly from target tissues in both hosts. Previous data have demonstrated that vlhA genes may be related to virulence, exhibiting changes in expression in a non-stochastic, temporal progression and we hypothesize that this may be influenced by differences in the target host tissue. If this is true, we would expect M. gallisepticum to display a different vlhA gene expression pattern in the chicken trachea compared to its expression pattern in house finch conjunctiva. Here we report significant differences in vlhA gene expression patterns between M. gallisepticum collected from chicken tracheas compared to those collected from house finch conjunctiva. While many of the predominant vlhA genes expressed in the input population showed an increase in expression in the chicken trachea at day one postinfection, those same vlhA genes decreased in expression in the house finch. These data suggest that discrete suites of vlhA genes may be involved in M. gallisepticum pathogenesis and tropism for unique tissues in two disparate avian hosts.
Assuntos
Proteínas de Bactérias/genética , Expressão Gênica , Interações entre Hospedeiro e Microrganismos/genética , Infecções por Mycoplasma/veterinária , Mycoplasma gallisepticum/genética , Doenças das Aves Domésticas/microbiologia , Animais , Galinhas/microbiologia , Túnica Conjuntiva/microbiologia , Feminino , Tentilhões/microbiologia , Doenças das Aves Domésticas/patologia , Análise de Sequência de RNA , Organismos Livres de Patógenos Específicos , Traqueia/microbiologia , VirulênciaRESUMO
Current work is beginning to reveal the complex mechanisms by which African swine fever virus interacts with its swine and tick hosts. This work includes the identification of novel viral genes that mediate virulence and host range, and influence important cellular regulatory pathways.
Assuntos
Vírus da Febre Suína Africana/patogenicidade , Febre Suína Africana/virologia , Proteínas Virais/genética , Vírus da Febre Suína Africana/genética , Animais , Suínos , Virulência/genéticaRESUMO
Despite a basic understanding of many aspects of FMD biology, much information regarding FMDV virulence, host range, and virus transmission remains poorly understood. Here we present how the use of high throughput sequencing for complete genome sequences of foot-and mouth disease virus (FMDV) led to a series of new insights into viral genome sequence conservation and variability, genetic diversity in nature and phylogenetic classification of isolates, including the first complete sequences of the South African Territories type 1 and 3 (SAT1 and SAT3) genomes. Comparative genomic analysis of full-length sequences of FMDV isolates did allow: (i) the identification of highly conserved regulatory or coding regions which are critical for aspects of virus biology as well as novel viral genomic motifs with likely biological relevance; (ii) characterization of the first complete sequences of the SAT1 and SAT3 genomes; (iii) identification of a novel SAT virus lineage genetically distinct from other SAT and Euro-Asiatic lineages; (iv) precise identification of strains circulating around the world for epidemiological and forensic attribution; (v) assessment of mutation and recombination processes as mechanisms equally involved in evolution; (vi) mutation rates, tolerance and constraints of genes and proteins during evolution of FMD viruses during in vivo replication and (vi) support for the hypothesis of a new evolutionary model.
Assuntos
Vírus da Febre Aftosa/genética , Genoma Viral , Genômica/métodos , Animais , Febre Aftosa/virologia , Vírus da Febre Aftosa/fisiologia , Mutação/genética , Filogenia , Vírus Reordenados/genética , Recombinação Genética/genética , Ruminantes , Suínos , Replicação ViralRESUMO
The immediate early protein (IE180) of pseudorabies virus (PrV) was generated by cycloheximide (CHX) reversal procedures in PrV-infected swine skin cells. Using this IE180 preparation as antigen, specific proliferation was detected in mononuclear cells (PBMNCs) from swine vaccinated with a modified live virus (MLV) PrV vaccine. Two sets of data support this conclusion. (a) PBMNCs of c/cSLA inbred swine vaccinated with an MLV vaccine a year before the test exhibited significant responses against structural virion proteins and IE180. (b) Vaccination of PrV-negative swine with the same MLV vaccine induced a conversion from an unresponsive state against IE180 to one of specific antigen-driven responsiveness. The responses of vaccinated swine against IE180 were significantly higher than their preimmune responses (p < or = 0.003) or to the response of control swine (p < or = 0.045). Moreover, IE180 antigens obtained from lysates of CHX-reversed, PrV-infected cells by heparin/agarose affinity separation also stimulated specific proliferation of PBMNCs from MLV-vaccinated swine, as their proliferative responses were significantly higher than those of unvaccinated swine (p < or = 0.05). These data suggest that PrV IE180 contributes at least in part to the overall cellular response to PrV.
Assuntos
Antígenos Virais/imunologia , Herpesvirus Suídeo 1/imunologia , Proteínas Imediatamente Precoces/imunologia , Leucócitos Mononucleares/imunologia , Vacinas Atenuadas/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Cromatografia de Afinidade , Cromatografia em Agarose , Cicloeximida/farmacologia , Feminino , Heparina/química , Imunização , Leucócitos Mononucleares/citologia , Ativação Linfocitária , Estudos Prospectivos , Estudos Retrospectivos , Suínos , Porco MiniaturaRESUMO
Pseudorabies virus (PrV) encodes for a single and essential immediate early phosphoprotein designated IE180. In this study, IE180 was examined in lysates from various cell lines infected at high multiplicities under cycloheximide inhibition of protein synthesis and subsequent reversal. Three distinct protein patterns of IE180 which were cell-specific and dependant on the extraction procedure were revealed. Detergent lysates of PrV infected MDBK cells yielded almost exclusively wild type IE molecule (180 kDa). In contrast, SSG/94 cells, VERO or CV-1 cells did not yield 180 kDa molecules but predominantly a shorter variant of approximately 60 kDa in molecular mass. Additional bands of about 50/55 kDa were also detected in lysates of SSG/94 and VERO cells by immunoprecipitation. Lysates of CV-1 and MDBK cells also yielded a 120 kDa molecule. The smaller molecular mass bands occurred in the presence of PMSF and aprotinin however, cleavage was blocked completely by addition of N alpha-p-Tosyl-L-lysine chloromethyl ketone (TLCK) into the lysis buffer. Moreover, an ability of the shorter IE180 variants to bind heparin was also revealed in the study. These data provide useful insights on protease profiles encountered among different PrV susceptible cells and indicates the use of appropriate protease inhibitors such as TLCK to protect IE180 under these experimental conditions.
Assuntos
Antígenos Virais/metabolismo , Cicloeximida/farmacologia , Herpesvirus Suídeo 1/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/efeitos dos fármacos , Bovinos , Linhagem Celular , Chlorocebus aethiops , Endopeptidases/metabolismo , Heparina/metabolismo , Herpesvirus Suídeo 1/efeitos dos fármacos , Proteínas Imediatamente Precoces/efeitos dos fármacos , Camundongos , Testes de Precipitina , Suínos , Células VeroAssuntos
Genes Precoces , Herpesvirus Suídeo 1/imunologia , Pseudorraiva/imunologia , Vacinas de DNA , Vacinas Virais , Animais , Herpesvirus Suídeo 1/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Pseudorraiva/prevenção & controle , Vacinas contra Pseudorraiva , SuínosRESUMO
Sheeppox virus (SPPV), a member of the Capripoxvirus genus of the Poxviridae, is the etiologic agent of a significant disease of sheep in the developing world. Genomic analysis of pathogenic and vaccine capripoxviruses identified genes with potential roles in virulence and host range, including three genes with similarity to kelch-like genes of other poxviruses and eukaryotes. Here, a mutant SPPV with a deletion in the SPPV-019 kelch-like gene, DeltaKLP, was derived from the pathogenic strain SPPV-SA. DeltaKLP exhibited in vitro growth characteristics similar to those of SPPV-SA and revertant virus (RvKLP). DeltaKLP-infected cells exhibited a reduction in Ca(2+)-independent cell adhesion, suggesting that SPPV-019 may modulate cellular adhesion. When inoculated in sheep by the intranasal or intradermal routes, DeltaKLP was markedly attenuated, since all DeltaKLP-infected lambs survived infection. In contrast, SPPV-SA and RvKLP induced mortality approaching 100%. Lambs inoculated with DeltaKLP exhibited marked reduction or delay in fever response, gross lesions, viremia, and virus shedding compared to parental and revertant viruses. Together, these findings indicate that SPPV-019 is a significant SPPV virulence determinant in sheep.
Assuntos
Capripoxvirus/genética , Capripoxvirus/patogenicidade , Proteínas Virais/fisiologia , Animais , Genes Virais/fisiologia , Mutação , Ovinos , Virulência/genéticaRESUMO
Here, we present the genome sequence, with analysis, of a poxvirus infecting Nile crocodiles (Crocodylus niloticus) (crocodilepox virus; CRV). The genome is 190,054 bp (62% G+C) and predicted to contain 173 genes encoding proteins of 53 to 1,941 amino acids. The central genomic region contains genes conserved and generally colinear with those of other chordopoxviruses (ChPVs). CRV is distinct, as the terminal 33-kbp (left) and 13-kbp (right) genomic regions are largely CRV specific, containing 48 unique genes which lack similarity to other poxvirus genes. Notably, CRV also contains 14 unique genes which disrupt ChPV gene colinearity within the central genomic region, including 7 genes encoding GyrB-like ATPase domains similar to those in cellular type IIA DNA topoisomerases, suggestive of novel ATP-dependent functions. The presence of 10 CRV proteins with similarity to components of cellular multisubunit E3 ubiquitin-protein ligase complexes, including 9 proteins containing F-box motifs and F-box-associated regions and a homologue of cellular anaphase-promoting complex subunit 11 (Apc11), suggests that modification of host ubiquitination pathways may be significant for CRV-host cell interaction. CRV encodes a novel complement of proteins potentially involved in DNA replication, including a NAD(+)-dependent DNA ligase and a protein with similarity to both vaccinia virus F16L and prokaryotic serine site-specific resolvase-invertases. CRV lacks genes encoding proteins for nucleotide metabolism. CRV shares notable genomic similarities with molluscum contagiosum virus, including genes found only in these two viruses. Phylogenetic analysis indicates that CRV is quite distinct from other ChPVs, representing a new genus within the subfamily Chordopoxvirinae, and it lacks recognizable homologues of most ChPV genes involved in virulence and host range, including those involving interferon response, intracellular signaling, and host immune response modulation. These data reveal the unique nature of CRV and suggest mechanisms of virus-reptile host interaction.