RESUMO
Mortality in ducks and geese caused by highly pathogenic avian influenza A(H5N1) infection had not been previously identified in Bangladesh. In June-July 2011, we investigated mortality in ducks, geese and chickens with suspected H5N1 infection in a north-eastern district of the country to identify the aetiologic agent and extent of the outbreak and identify possible associated human infections. We surveyed households and farms with affected poultry flocks in six villages in Netrokona district and collected cloacal and oropharyngeal swabs from sick birds and tissue samples from dead poultry. We conducted a survey in three of these villages to identify suspected human influenza-like illness cases and collected nasopharyngeal and throat swabs. We tested all swabs by real-time RT-PCR, sequenced cultured viruses, and examined tissue samples by histopathology and immunohistochemistry to detect and characterize influenza virus infection. In the six villages, among the 240 surveyed households and 11 small-scale farms, 61% (1789/2930) of chickens, 47% (4816/10 184) of ducks and 73% (358/493) of geese died within 14 days preceding the investigation. Of 70 sick poultry swabbed, 80% (56/70) had detectable RNA for influenza A/H5, including 89% (49/55) of ducks, 40% (2/5) of geese and 50% (5/10) of chickens. We isolated virus from six of 25 samples; sequence analysis of the hemagglutinin and neuraminidase gene of these six isolates indicated clade 2.3.2.1a of H5N1 virus. Histopathological changes and immunohistochemistry staining of avian influenza viral antigens were recognized in the brain, pancreas and intestines of ducks and chickens. We identified ten human cases showing signs compatible with influenza-like illness; four were positive for influenza A/H3; however, none were positive for influenza A/H5. The recently introduced H5N1 clade 2.3.2.1a virus caused unusually high mortality in ducks and geese. Heightened surveillance in poultry is warranted to guide appropriate diagnostic testing and detect novel influenza strains.
Assuntos
Galinhas , Surtos de Doenças/veterinária , Patos , Gansos , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/epidemiologia , Influenza Humana/epidemiologia , Doenças das Aves Domésticas/epidemiologia , Adolescente , Adulto , Idoso , Animais , Bangladesh/epidemiologia , Criança , Feminino , Humanos , Influenza Aviária/mortalidade , Influenza Aviária/virologia , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Filogenia , Doenças das Aves Domésticas/mortalidade , Doenças das Aves Domésticas/virologia , Análise de Sequência de DNA/veterinária , Adulto JovemRESUMO
New members of the influenza A virus genus have been detected recently in bats from South America. By molecular investigations, using a generic real-time RT-PCR (RT-qPCR) that detects all previously known influenza A virus subtypes (H1-H16) and a newly developed RT-qPCR specific for the South American bat influenza-like virus of subtype H17, a total of 1571 samples obtained from 1369 individual bats of 26 species from Central Europe were examined. No evidence for the occurrence of such influenza viruses was found. Further attempts towards a more comprehensive evaluation of the role of bats in the ecology and epidemiology of influenza viruses should be based on more intense monitoring efforts. However, given the protected status of bats, not only in Europe, such activities need to be embedded into existing pathogen-monitoring programs.
Assuntos
Quirópteros/virologia , Vírus da Influenza A/isolamento & purificação , Influenza Humana/epidemiologia , Infecções por Orthomyxoviridae/veterinária , Animais , Monitoramento Epidemiológico , Europa (Continente)/epidemiologia , Humanos , Vírus da Influenza A/genética , Influenza Humana/virologia , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Óvulo/virologia , Saúde Pública , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Sensibilidade e Especificidade , ZoonosesRESUMO
Pigs and humans have shared influenza A viruses (IAV) since at least 1918, and many interspecies transmission events have been documented since that time. However, despite this interplay, relatively little is known regarding IAV circulating in swine around the world compared with the avian and human knowledge base. This gap in knowledge impedes our understanding of how viruses adapted to swine or man impacts the ecology and evolution of IAV as a whole and the true impact of swine IAV on human health. The pandemic H1N1 that emerged in 2009 underscored the need for greater surveillance and sharing of data on IAV in swine. In this paper, we review the current state of IAV in swine around the world, highlight the collaboration between international organizations and a network of laboratories engaged in human and animal IAV surveillance and research, and emphasize the need to increase information in high-priority regions. The need for global integration and rapid sharing of data and resources to fight IAV in swine and other animal species is apparent, but this effort requires grassroots support from governments, practicing veterinarians and the swine industry and, ultimately, requires significant increases in funding and infrastructure.
Assuntos
Doenças Endêmicas , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A/isolamento & purificação , Influenza Humana/epidemiologia , Infecções por Orthomyxoviridae/veterinária , Doenças dos Suínos/epidemiologia , Animais , Pesquisa Biomédica , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A/fisiologia , Influenza Humana/transmissão , Cooperação Internacional , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/transmissão , Saúde Pública , Vigilância em Saúde Pública , Suínos , Doenças dos Suínos/transmissão , Doenças dos Suínos/virologia , ZoonosesRESUMO
Continued circulation and geographical expansion of highly pathogenic avian influenza H5N1 virus have led to the emergence of numerous clades in Vietnam. Although viral RNA sequencing and phylogenetic analysis are the gold standard for H5N1 HA clade designation, limited sequencing capacity in many laboratories precludes rapid H5N1 clade identification and detection of novel viruses. Therefore, a Taqman real-time RT-PCR assay for rapid differentiation of the four major H5N1 clades detected in Vietnam was developed. Using HA sequence alignments of clades 1.1, 2.3.2.1, 2.3.4, and 7 viruses, primers and FAM-labeled probes were designed to target conserved regions characteristic of each clade. The assay was optimized and evaluated using circulating clades of H5N1 collected in Vietnam from 2007 to 2012 and shown to be both sensitive and specific for the differentiation of the four H5N1 clades. The assay provides a useful tool for screening of large specimen collections for HA gene sequencing and phylogenetic analysis and for the rapid identification of molecular clade signatures to support outbreak investigations and surveillance activities. Finally, this assay may be useful to monitor for the emergence of novel or variant clades of H5N1 in Vietnam in the future or in other countries where these particular clades may circulate.
Assuntos
Virus da Influenza A Subtipo H5N1/classificação , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Virologia/métodos , Animais , Aves , Primers do DNA/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Virus da Influenza A Subtipo H5N1/genética , Influenza Humana/virologia , Epidemiologia Molecular/métodos , Sensibilidade e Especificidade , VietnãRESUMO
An H3N8 influenza virus closely related to equine influenza virus was identified in racing greyhound dogs with respiratory disease in 2004 and subsequently identified in shelter and pet dogs. Pathologic findings in dogs spontaneously infected with canine influenza virus were compared with lesions induced in beagle and mongrel dogs following experimental inoculation with influenza A/canine/Florida/43/2004. BALB/c mice were inoculated with canine influenza virus to assess their suitability as an experimental model for viral pathogenesis studies. All dogs inoculated with virus developed necrotizing and hyperplastic tracheitis and bronchitis with involvement of submucosal glands as well as mild bronchiolitis and pneumonia. Viral antigen was identified in bronchial and tracheal epithelial cells of all dogs and in alveolar macrophages of several dogs. Many dogs that were spontaneously infected with virus also developed bacterial pneumonia, and greyhound dogs with fatal spontaneous infection developed severe pulmonary hemorrhage with hemothorax. Virus-inoculated BALB/c mice developed tracheitis, bronchitis, bronchiolitis, and mild pneumonia in association with viral antigen in airway epithelial cells and in type 2 alveolar epithelial cells. Virus was not detected in extrarespiratory sites in any animals. The results indicate that canine influenza virus infection consistently induces acute tracheitis and bronchitis in dogs. Mice may be a useful model for some pathogenesis studies on canine influenza virus infection.
Assuntos
Doenças do Cão/patologia , Doenças do Cão/virologia , Vírus da Influenza A Subtipo H3N8 , Infecções por Orthomyxoviridae/veterinária , Animais , Brônquios/ultraestrutura , Brônquios/virologia , Bronquiolite/complicações , Bronquiolite/veterinária , Bronquiolite/virologia , Modelos Animais de Doenças , Cães , Feminino , Vírus da Influenza A Subtipo H3N8/isolamento & purificação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/patologia , Pneumonia Viral/complicações , Pneumonia Viral/veterinária , Traqueíte/complicações , Traqueíte/veterinária , Traqueíte/virologiaRESUMO
Previously, we identified bovine CD18 as the receptor for leukotoxin secreted by Mannheimia (Pasteurella) haemolytica. In this study, we constructed bovine-murine CD18 chimeras to locate the leukotoxin binding domain on CD18. Leukotoxin specifically lysed transfectants expressing bovine CD18 fragment encompassing amino acids 1 to 291, indicating that leukotoxin binding domain lies within amino acids 1 to 291 of bovine CD18.
Assuntos
Antígenos CD18/genética , Antígenos CD18/metabolismo , Exotoxinas/metabolismo , Mannheimia haemolytica/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Antígenos CD18/imunologia , Bovinos , Linhagem Celular Tumoral , DNA Complementar/genética , DNA Complementar/metabolismo , Exotoxinas/genética , Mannheimia haemolytica/genética , Mannheimia haemolytica/imunologia , Camundongos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/genéticaRESUMO
Numerous studies have shown the potential of Salmonella typhimurium as a vector for delivery of heterologous proteins for vaccination against other pathogens. Earlier studies showed that the inefficient elicitation of MHC class I-restricted responses could limit the use of S. typhimurium as a heterologous antigen delivery vector for vaccination. We recently developed an approach to overcome this limitation by using a bacterial-encoded specialized protein secretion system, termed type III, to deliver proteins into the class I antigen presenting pathways. Thus, peptides of interest fused to proteins bearing the type III secretion signal, which can elicit protective CTL responses. Because protective immunity is usually assessed a few weeks after vaccination, there is a paucity of information regarding duration of protective immunity induced by this system. We show here that mice immunized orally with S. typhimurium vectors expressing a MHC class I-restricted epitope of the lymphocytic choriomeningitis virus (LCMV) nucleoprotein developed specific antiviral CTL responses. CD8+ T cells were found to be necessary for this CTL activity against targets presenting the LCMV epitope. The survival of mice challenged with lethal doses of LCMV 60 or 135 days after vaccination was as complete as the survival of mice challenged 2 weeks after immunization with the same vectors. By demonstrating their ability to induce prolonged protective immunity after oral delivery, S. typhimurium vectors have met an essential requirement in support of their development as vectors for heterologous vaccination.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T , Antígenos de Histocompatibilidade Classe I/imunologia , Memória Imunológica , Vírus da Coriomeningite Linfocítica/imunologia , Salmonella typhimurium/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/imunologia , Animais , Células Apresentadoras de Antígenos/fisiologia , Células Apresentadoras de Antígenos/virologia , Linhagem Celular , Feminino , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Influenza A virus expresses three viral polymerase (P) subunits-PB1, PB2, and PA-all of which are essential for RNA and viral replication. The functions of P proteins in transcription and replication have been partially elucidated, yet some of these functions seem to be dependent on the formation of a heterotrimer for optimal viral RNA transcription and replication. Although it is conceivable that heterotrimer subunit interactions may allow a more efficient catalysis, direct evidence of their essentiality for viral replication is lacking. Biochemical studies addressing the molecular anatomy of the P complexes have revealed direct interactions between PB1 and PB2 as well as between PB1 and PA. Previous studies have shown that the N-terminal 48 amino acids of PB1, termed domain alpha, contain the residues required for binding PA. We report here the refined mapping of the amino acid sequences within this small region of PB1 that are indispensable for binding PA by deletion mutagenesis of PB1 in a two-hybrid assay. Subsequently, we used site-directed mutagenesis to identify the critical amino acid residues of PB1 for interaction with PA in vivo. The first 12 amino acids of PB1 were found to constitute the core of the interaction interface, thus narrowing the previous boundaries of domain alpha. The role of the minimal PB1 domain alpha in influenza virus gene expression and genome replication was subsequently analyzed by evaluating the activity of a set of PB1 mutants in a model reporter minigenome system. A strong correlation was observed between a functional PA binding site on PB1 and P activity. Influenza viruses bearing mutant PB1 genes were recovered using a plasmid-based influenza virus reverse genetics system. Interestingly, mutations that rendered PB1 unable to bind PA were either nonviable or severely growth impaired. These data are consistent with an essential role for the N terminus of PB1 in binding PA, P activity, and virus growth.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Vírus da Influenza A/enzimologia , Vírus da Influenza A/fisiologia , RNA Polimerase Dependente de RNA , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , RNA Polimerases Dirigidas por DNA/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Testes de Precipitina , Transcrição Gênica , Técnicas do Sistema de Duplo-Híbrido , Proteínas Virais/química , Proteínas Virais/genética , Replicação ViralRESUMO
Infectious transcripts from the full-length infectious clone of the NADL strain of bovine viral diarrhea virus (BVDV) were used to vaccinate cattle and sheep against BVDV. In vitro synthesized RNA delivered by microparticle bombardment with a Helios Gene Gun initiated replication of BVDV and consequently induced humoral immunity against type I BVDV (serum neutralization titers, SNT > 2(12)) and type II BVDV (SNT > 2(7)). The quality and long-term stability of the RNA-carrier complexes was assessed by microparticle bombardment of tissue culture monolayers. The RNA cartridges were found to be stable for at least 8 months upon storage. This is the first report on successful RNA vaccination of large ruminants.
Assuntos
Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/imunologia , RNA Viral/genética , RNA Viral/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/biossíntese , Biolística , Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Bovinos , Células Cultivadas , Microesferas , Estabilidade de RNA , RNA Viral/administração & dosagem , Ovinos , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagemRESUMO
Three Brazilian isolates of bovine viral diarrhea virus (BVDV), antigenically distinct from the standard North American isolates, were selected to immunize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV monoclonal antibodies (mAbs). Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11), were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidine-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA) assay with all 39 South and North American BVDV field isolates and reference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions up to 1:25,600 (ascitic fluid) and 1:100 (hybridoma culture supernatant) in IFA and immunoperoxidase (IPX) staining of BVDV-infected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to recognize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggesting they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cells transiently expressing each of the BVDV proteins: mAb 3. 1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural protein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our knowledge, these are the first mAbs produced against South American BVDV isolates and will certainly be useful for research and diagnostic purposes.
Assuntos
Anticorpos Monoclonais/biossíntese , Vírus da Diarreia Viral Bovina/imunologia , Hibridomas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Variação Antigênica/imunologia , Bovinos , Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/isolamento & purificação , Técnica Indireta de Fluorescência para Anticorpo , Genótipo , Cavalos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas RecombinantesRESUMO
Three Brazilian isolates of bovine viral diarrhea virus (BVDV), antigenically distinct from the standard North American isolates, were selected to immunize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV monoclonal antibodies (mAbs). Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11), were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidine-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA) assay with all 39 South and North American BVDV field isolates and reference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions up to 1:25,600 (ascitic fluid) and 1:100 (hybridoma culture supernatant) in IFA and immunoperoxidase (IPX) staining of BVDV-infected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to recognize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggesting they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cells transiently expressing each of the BVDV proteins: mAb 3.1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural protein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our knowledge, these are the first mAbs produced against South American BVDV isolates and will certainly be useful for research and diagnostic purposes
Assuntos
Animais , Camundongos , Bovinos , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Vírus da Diarreia Viral Bovina/imunologia , Hibridomas , Variação Antigênica , Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/isolamento & purificação , Técnica Indireta de Fluorescência para Anticorpo , Genótipo , Cavalos , Camundongos Endogâmicos BALB C , Proteínas RecombinantesRESUMO
Non-cytopathic (NCP) and cytopathic (CP) parent-daughter pairs are often isolated from cattle with bovine viral diarrhea virus (BVDV) induced mucosal disease. Alignment of these pair genomes revealed that genetic changes in CP BVDV involve the NS2-3 coding region and correlate with expression of NS3. However, additional mutations are present elsewhere in the genomes of these natural pairs, precluding unambiguous mapping of this function to the NS2-3 region. To evaluate this phenomenon in identical genetic backgrounds, we have constructed an NCP isogenic pair of the NADL by deletion of the cIns from NS2 region. The levels of viral protein synthesis in infected cells revealed no marked difference between the CP and the isogenic NCP BVDV mutant. In contrast, RNA accumulation in cells infected with CP virus was up to 25 times higher than that in cells infected with NCP BVDV. No significant difference in growth kinetics and viral yields were observed between the CP BVDV and the isogenic NCP pair. Analyses of additional NCP/CP parent-daughter field BVDV isolates revealed a similar pattern of macromolecular synthesis, suggesting the generality of this phenomenon. These results implicate increased levels of RNA accumulation in CP BVDV infected cells, along with the production of NS3 as potential contributors to viral cytopathogenicity.
Assuntos
Apoptose , Vírus da Diarreia Viral Bovina/genética , Genoma Viral , RNA Viral/análise , Animais , Northern Blotting , Western Blotting , Bovinos , Técnicas de Cultura de Células , Linhagem Celular , Efeito Citopatogênico Viral , Elementos de DNA Transponíveis , Vírus da Diarreia Viral Bovina/química , Vírus da Diarreia Viral Bovina/patogenicidade , Dados de Sequência Molecular , Mutação Puntual , Proteínas não Estruturais Virais/análise , Proteínas não Estruturais Virais/genéticaRESUMO
We generated a recombinant vesicular stomatitis virus (VSV-E2) encoding the bovine viral diarrhea virus (BVDV) E2 glycoprotein with the VSV-G protein signal peptide. Infection of BHK21 cells with VSV-E2 induced the synthesis of a recombinant E2 (rE2) that comigrated with authentic BVDV-E2 in PAGE-SDS gels. Non-reducing immunoblots showed that rE2 is a disulfide bond-linked homodimer with at least 10-fold higher avidity for conformation-dependent anti-BVDV-E2 antibodies than its reduced monomeric counterpart. Immunofluorescence microscopy also showed that rE2 was transported to the plasma membrane of infected cells and analysis of purified particles demonstrated that dimeric rE2 was incorporated into VSV-E2 virions in approximately 1:10 ratio with respect to the G glycoprotein. BALB/c mice inoculated intranasally with VSV-E2 doses of up to 10(7) plaque forming units (pfu) showed no symptoms of viral-induced disease and developed a specific BVDV neutralizing response that lasted for at least 180 days post inoculation.
Assuntos
Anticorpos Antivirais/biossíntese , Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/imunologia , Glicoproteínas de Membrana , Vírus da Estomatite Vesicular Indiana/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Sequência de Bases , Bovinos , Linhagem Celular , Quimera/genética , Quimera/imunologia , Cricetinae , DNA Recombinante/genética , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas Sintéticas/genética , Vacinas Virais/genéticaRESUMO
Unique to pestiviruses, the N-terminal protein encoded by the bovine viral diarrhea virus (BVDV) genome is a cysteine protease (Npro) responsible for a self-cleavage that releases the N terminus of the core protein (C). This unique protease is dispensable for viral replication, and its coding region can be replaced by a ubiquitin gene directly fused in frame to the core. To develop an antiviral assay that allows the assessment of anti-hepatitis C virus (HCV) NS3 protease inhibitors, a chimeric BVDV in which the coding region of Npro was replaced by that of an NS4A cofactor-tethered HCV NS3 protease domain was generated. This cofactor-tethered HCV protease domain was linked in frame to the core protein of BVDV through an HCV NS5A-NS5B junction site and mimicked the proteolytic function of Npro in the release of BVDV core for capsid assembly. A similar chimeric construct was built with an inactive HCV NS3 protease to serve as a control. Genomic RNA transcripts derived from both chimeric clones, P(H/B) (wild-type HCV NS3 protease) and P(H/B(S139A)) (mutant HCV NS3 protease) were then transfected into bovine cells (MDBK). Only the RNA transcripts from the P(H/B) clone yielded viable viruses, whereas the mutant clone, P(H/B(S139A)), failed to produce any signs of infection, suggesting that the unprocessed fusion protein rendered the BVDV core protein defective in capsid assembly. Like the wild-type BVDV (NADL), the chimeric virus was cytopathic and formed plaques on the cell monolayer. Sequence and biochemical analyses confirmed the identity of the chimeric virus and further revealed variant viruses due to growth adaptation. Growth analysis revealed comparable replication kinetics between the wild-type and the chimeric BVDVs. Finally, to assess the genetic stability of the chimeric virus, an Npro-null BVDV (BVDV-Npro in which the entire Npro coding region was deleted) was produced. Although cytopathic, BVDV-Npro was highly defective in viral replication and growth, a finding consistent with the observed stability of the chimeric virus after serial passages.
Assuntos
Cisteína Endopeptidases/genética , Vírus da Diarreia Viral Bovina/genética , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Western Blotting , Linhagem Celular , Efeito Citopatogênico Viral , Vírus da Diarreia Viral Bovina/metabolismo , Vírus da Diarreia Viral Bovina/patogenicidade , Vírus da Diarreia Viral Bovina/fisiologia , Dados de Sequência Molecular , RNA Viral/análise , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas não Estruturais Virais/metabolismo , Ensaio de Placa Viral , Montagem de VírusAssuntos
Vacinas Bacterianas , Animais , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Humanos , Infecções por Mycobacterium/prevenção & controle , Infecções por Salmonella/prevenção & controle , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
We describe a new reverse-genetics system that allows one to efficiently generate influenza A viruses entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with eight plasmids, each encoding a viral RNA of the A/WSN/33 (H1N1) or A/PR/8/34 (H1N1) virus, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator-together with plasmids encoding viral nucleoprotein and the PB2, PB1, and PA viral polymerases. This strategy yielded >1 x 10(3) plaque-forming units (pfu) of virus per ml of supernatant at 48 hr posttransfection. The addition of plasmids expressing all of the remaining viral structural proteins led to a substantial increase in virus production, 3 x 10(4)-5 x 10(7) pfu/ml. We also used reverse genetics to generate a reassortant virus containing the PB1 gene of the A/PR/8/34 virus, with all other genes representing A/WSN/33. Additional viruses produced by this method had mutations in the PA gene or possessed a foreign epitope in the head of the neuraminidase protein. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and in the production of vaccines and gene therapy vectors.
Assuntos
DNA Complementar , DNA Viral/genética , Vírus da Influenza A/genética , RNA Viral/genética , Animais , Linhagem Celular , Embrião de Galinha , Cães , Proteína HN/genética , Humanos , Vírus da Influenza A/fisiologia , Rim , Camundongos , Plasmídeos , RNA Polimerase I/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Proteínas Estruturais Virais/genética , Replicação ViralRESUMO
To ascertain the virulence of bovine viral diarrhea virus (BVDV) genotype II, isolate NY-93 was inoculated intranasally into 3 calves, 2 of which were treated with a synthetic glucocorticoid prior to and after virus inoculation. Anorexia, fever (up to 42 C), dyspnea, and hemorrhagic diarrhea developed 6 days after intranasal inoculation with BVDV NY-93. The condition of all calves deteriorated further until the end of the study on day 14 postinoculation. The most significant postmortem macroscopic changes in all calves were limited to the gastrointestinal tract and consisted of moderate to severe congestion of the mucosa with multifocal hemorrhages. Microscopic lesions found in the gastrointestinal tract were similar to those observed in mucosal disease, including degeneration and necrosis of crypt epithelium and necrosis of lymphoid tissue throughout the ileum, colon, and rectum. The basal stratum of the epithelium of tongue, esophagus, and rumen had scattered individual necrotic cells. Spleen and lymph nodes had lymphocytolysis and severe lymphoid depletion. Severe acute fibrinous bronchopneumonia was present in dexamethasone-treated calves. Abundant viral antigen was detected by immunohistochemistry in the squamous epithelium of tongue, esophagus, and forestomachs. BVDV antigen was prominent in cells of the media of small arteries and endothelial cells. The presence of infectious virus in tissues correlated with an absence of circulating neutralizing antibodies. These findings highlight the potential of BVDV genotype II to cause severe disease in normal and stressed cattle.
Assuntos
Antígenos Virais/análise , Doença das Mucosas por Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/patogenicidade , Sistema Digestório/virologia , Animais , Doença das Mucosas por Vírus da Diarreia Viral Bovina/patologia , Bovinos , Vírus da Diarreia Viral Bovina/genética , Sistema Digestório/patologia , Hemorragia Gastrointestinal/etiologia , Hemorragia Gastrointestinal/veterinária , Índice de Gravidade de Doença , Distribuição TecidualRESUMO
Pestiviruses and hepaciviruses are atypical members of the Flaviviridae due to their unique biological properties, including the utilization of internal ribosome entry for translation initiation. In contrast to internal initiation in picornaviruses, which depends on numerous canonical initiation factors, the mode of internal ribosome entry in pestiviruses and hepaciviruses resembles prokaryotic translation initiation. To identify functionally important elements within the bovine viral diarrhea virus (BVDV) internal ribosome entry segment (IRES), we carried out a mutational analysis of the 5' untranslated region (5' UTR) of BVDV cloned in the intercistronic region of a bicistronic reporter plasmid. IRES function was assessed in a bicistronic transcript by inserting the 5' 901 nucleotides of BVDV genome, which correspond to the 385 nucleotides of the 5' UTR and 515 nucleotides of the open reading frame (ORF) encoding for Npro and 4 amino acids from the capsid protein. The resulting Npro-luciferase fusion encoded by the 3' cistron was cleaved efficiently to release the luciferase reporter. In vivo translation analyses showed that stem-loops Ia and Ib in the 5' UTR were completely dispensable for efficient translation, whereas stem-loop IIIe and the hairpin end of IIIb were only partially required. In contrast, deletions or insertions in any of other four stem-loop structures, including domains II, IIIa, IIIc, and IIId, caused nearly 10-fold reductions of in vivo IRES activity. The tolerance of structural modifications within the distal portion of domain IIIb and IIIe correlated with a low level of sequence conservation in these regions among pestiviruses. The 5' boundary of the IRES resides at the 5' end of stem-loop II near nucleotide 75. The 3' of the IRES extends into the 5' end of the polyprotein ORF because removal of the Npro coding region reduced translation by 21%.
Assuntos
Doença das Mucosas por Vírus da Diarreia Viral Bovina/genética , Vírus da Diarreia Viral Bovina/genética , Ribossomos/genética , Animais , Células COS , Bovinos , Linhagem Celular , Chlorocebus aethiops , Códon de Iniciação , Vetores Genéticos , Masculino , Mutagênese Insercional , Conformação de Ácido Nucleico , Poliovirus , Biossíntese de Proteínas , Ribossomos/virologia , Deleção de Sequência , Transcrição GênicaRESUMO
The M1 protein of influenza virus inhibits the in vitro transcriptase activity of ribonucleoprotein cores from virions. This inhibitory activity is thought to be relevant in vivo because accumulation of M1 at the late stages of viral replication may be the cue to halt viral mRNA production. A model influenza reporter genome was used to explore the effect of M1 on the activity of the influenza virus transcriptase complex within cultured cells. Expression of M1 in cells bearing the model influenza virus reporter genome was accompanied by a reduction of CAT gene expression to 12% of control levels. Quantification of RNA by ribonuclease protection assay revealed that the influenza reporter genome mRNA levels in M1-expressing cells were reduced by approximately 74% compared with those of cells expressing a control protein. These findings are consistent with the proposed model in which M1 is responsible for limiting viral transcription during late stages of infection. By expressing truncated forms of M1, the inhibitory activity was found to reside within the amino-terminal half of the M1 protein. Two independent inhibitory domains were identified in this region: one between amino acid residues 1-90 and the other spanning residues 91-127.
Assuntos
Regulação Viral da Expressão Gênica , Genoma Viral , Vírus da Influenza A/genética , Proteínas da Matriz Viral/genética , Animais , Sequência de Bases , Linhagem Celular , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/genética , Dados de Sequência Molecular , Proteínas da Matriz Viral/metabolismoRESUMO
The matrix (M) and nonstructural (NS) genes of influenza A viruses each encode two overlapping proteins. In the M gene, evolution of one protein affects that of the other. To determine whether or not this evolutionary influence operating between the two M proteins also occurs in the NS gene, we sequenced the NS genes of 36 influenza A viruses isolated from a broad spectrum of animal species (wild and domestic birds, horses, pigs, humans, and sea mammals) and analyzed them phylogenetically, together with other previously published sequences. These analyses enabled us to conclude the following host species-related points that are not found in the other influenza A virus genes and their gene products. (1) The evolution of the two overlapping proteins encoded by the NS gene are lineage-dependent, unlike the M gene where evolutionary constraints on the Ml protein affect the evolution of the M2 protein (Ito et al.. J. Virol. 65 (1991) 5491 5498). (2) The gull-specific lineage contained nonH13 gull viruses and the non-gull avian lineage contained H13 gull viruses, indicating that the gull-specific lineage does not link to the H13 HA subtype in the NS gene unlike findings with other genes. (3) The branching topology of the recent equine lineage (H7N7 viruses isolated after 1973 and H3N8) indicates recent introduction of the NS, M, and PB2 genes into horses from avian sources by genetic reassortment.