RESUMO
Crimean-Congo hemorrhagic fever virus (CCHFV; family Nairoviridae) is a tick-borne pathogen that frequently causes lethal disease in humans. CCHFV has a wide geographic distribution, and cases have been reported in Africa, Asia, the Middle East, and Europe. Availability of a safe and efficacious vaccine is critical for restricting outbreaks and preventing disease in endemic countries. We previously developed a virus-like replicon particle (VRP) vaccine that provides complete protection against homologous and heterologous lethal CCHFV challenge in mice after a single dose. However, the immune responses induced by this vaccine are not well characterized, and correlates of protection remain unknown. Here we comprehensively characterized the kinetics of cell-mediated and humoral immune responses in VRP-vaccinated mice, and demonstrate that they predominantly target the nucleoprotein (NP). NP antibodies are not associated with protection through neutralizing activity, but VRP vaccination results in NP antibodies possessing Fc-mediated antibody effector functions, such as complement activation (ADCD) and antibody-mediated cellular phagocytosis (ADCP). This suggests that Fc-mediated effector functions may contribute to this vaccine's efficacy.
Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo , Vacinas , Humanos , Animais , Camundongos , Vacinação , Anticorpos , Nucleoproteínas , Linfócitos TRESUMO
We report here the complete genome sequences for all three segments of the New York hantavirus (New York 1). This is the first reported L segment sequence for hantaviruses maintained in Peromyscus spp. endemic to the eastern United States and Canada.
RESUMO
Sin Nombre virus (SNV) is a major representative of the New World hantaviruses and the most common cause of hantavirus pulmonary syndrome (HPS) with high mortality in North America. Unlike other members of the family Bunyaviridae which mature in the Golgi complex, New World hantaviruses have been previously reported to mature at the cell surface. For family Bunyaviridae viruses, retention of the viral glycoproteins at the Golgi complex is thought to be responsible for their Golgi maturation. In our studies, the majority of SNV glycoproteins, G1 and G2, was localized in the Golgi complex when expressed from a full-length GPC clone or in SNV-infected cells, in agreement with data for other members of the family Bunyaviridae, including the Old World hantaviruses. However, the SNV glycoproteins could also be detected at the cell surface at advanced posttransfection or postinfection time points. G1 expressed in the absence of G2 did not accumulate in the Golgi, but remained predominantly associated with the endoplasmic reticulum (ER). Overexpressed amounts of apparently misfolded G1 were aggregated in a subcellular compartment likely to represent the aggresome. Unexpectedly, an additional major pool of G1 was detected intracellularly in SNV-infected and GPC-expressing transfected cells, by using a SNV G1-specific Fab antibody. This pool of G1 is predominantly localized in late endosomes-lysosomes.
Assuntos
Vírus Sin Nombre/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Linhagem Celular , Cricetinae , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Complexo de Golgi/metabolismo , Humanos , Lisossomos/metabolismo , Microscopia Imunoeletrônica , Transporte Proteico , Ratos , Frações Subcelulares/metabolismo , Transfecção , Proteínas do Envelope Viral/análise , Proteínas do Envelope Viral/genéticaRESUMO
Sin Nombre virus (SNV) and Hantaan virus (HTN) infect endothelial cells and are associated with different patterns of increased vascular permeability during human disease. It is thought that such patterns of increased vascular permeability are a consequence of endothelial activation and subsequent dysfunction mediated by differential immune responses to hantavirus infection. In this study, the ability of hantavirus to directly induce activation of human lung microvascular endothelial cells (HMVEC-Ls) was examined. No virus-specific modulation in the constitutive or cytokine-induced expression of cellular adhesion molecules (CD40, CD54, CD61, CD62E, CD62P, CD106, and major histocompatibility complex classes I and II) or in cytokines and chemokines (eotaxin, tumor necrosis factor alpha, interleukin 1beta [IL-1beta], IL-6, IL-8, MCP-1, MIP-1alpha, and MIP-1beta) was detected at either the protein or message level in hantavirus-infected HMVEC-Ls. Furthermore, no virus-specific enhancement of paracellular or transcellular permeability or changes in the organization and distribution of endothelial intercellular junctional proteins was observed. However, infection with either HTN or SNV resulted in detectable levels of the chemokines RANTES and IP-10 (the 10-kDa interferon-inducible protein) in HMVEC-Ls within 72 h and was associated with nuclear translocation of interferon regulatory factor 3 (IRF-3) and IRF-7. Gamma interferon (IFN-gamma)-induced expression of RANTES and IP-10 could also be detected in uninfected HMVEC-Ls and was associated with nuclear translocation of IRF-1 and IRF-3. Treatment of hantavirus-infected HMVEC-Ls with IFN-gamma for 24 h resulted in a synergistic enhancement in the expression of both RANTES and IP-10 and was associated with nuclear translocation of IRF-1, IRF-3, IRF-7, and NF-kappaB p65. These results reveal a possible mechanism by which hantavirus infection and a TH1 immune response can cooperate to synergistically enhance chemokine expression by HMVEC-Ls and trigger immune-mediated increases in vascular permeability.
Assuntos
Permeabilidade Capilar , Quimiocina CCL5/biossíntese , Quimiocinas CXC/biossíntese , Endotélio Vascular/metabolismo , Orthohantavírus/fisiologia , Animais , Células Cultivadas , Quimiocina CCL2/biossíntese , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CXCL10 , Proteínas de Ligação a DNA/genética , Endotélio Vascular/citologia , Endotélio Vascular/ultraestrutura , Humanos , Junções Intercelulares/metabolismo , Junções Intercelulares/ultraestrutura , Fator Regulador 7 de Interferon , Interferon gama/farmacologia , Pulmão/irrigação sanguínea , Proteínas Inflamatórias de Macrófagos/biossíntese , Camundongos , Coelhos , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
A retrospective serological and genetic study of hantaviruses responsible for hemorrhagic fever with renal syndrome (HFRS) in Greece during the last 17 years is presented. Fifty-one serum samples taken from 30 HFRS cases previously diagnosed by immunofluorescence assay were tested by ELISA for IgG (Hantaan, Dobrava, and Puumala) and IgM antibodies (Hantaan and Puumala). Results were compatible with the majority of infections being related to hantaviruses carried by rodents of the subfamily Murinae. RNA was extracted from 26 selected samples and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using primers specifically designed for the detection of hanta-viruses associated with murine (MS-N-specific, MM-G1-specific primers) or arvicoline rodents (PPT-N-specific primers). In addition, primers previously designed for the detection of the G2 coding region of the Murine-associated hanta-viruses were also used. Sequencing of the PCR products was then performed, followed by phylogenetic analysis of nucleotide sequence differences. Eleven out of the 26 serum samples tested were found to be positive by PCR with the MS-N primers, whereas four were positive with the MM-G1 primers, and only two with the G2 primers. None of the samples was found positive with the PPT primers. The sequence analysis showed that the virus that was responsible for these 11 HFRS cases was the Dobrava virus, which is endemic throughout the Balkans.
Assuntos
Febre Hemorrágica com Síndrome Renal/virologia , Orthohantavírus/classificação , Adulto , Idoso , Anticorpos Antivirais/sangue , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Feminino , Genoma Viral , Grécia , Orthohantavírus/genética , Orthohantavírus/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Reação em Cadeia da Polimerase , RNA Viral/genética , Estudos Retrospectivos , Análise de Sequência de RNARESUMO
In 1993 a number of cases of unexplained adult respiratory syndrome occurred in the southwestern United States. The illness was characterized by a prodrome of fever, myalgia, and other symptoms followed by the rapid onset of a capillary leak syndrome with hemoconcentration, thrombocytopenia, and pulmonary edema. Viral RNA sequences in the lungs identified a new member of the hantavirus genus, Sin Nombre virus (SNV), unique to North America. Pulmonary endothelial cells were heavily infected but were not necrotic. We speculated that this capillary leak syndrome was initiated by immune responses to the SNV-infected pulmonary endothelial cells. We isolated a CD8+ cytotoxic T lymphocyte (CTL) clone directly from the blood of a patient with the acute hantavirus pulmonary syndrome (HPS) which recognizes a SNV specific epitope on the virus nucleocapsid protein (aa 234-242) that is restricted by HLA C7 and produces IFN gamma but not IL-4. We identified a second CD8+ CTL epitope located within another site aa 131-139 on the nucleocapsid protein, which is HLA B35 restricted, and a CD4+ CTL epitope located on a third site on nucleocapsid protein aa 372-380 using lymphocytes obtained during HPS from another patient that were stimulated in vitro. Hantavirus specific CD8+ and CD4+ CTL may contribute to the immunopathology and capillary leak syndrome observed in the HPS.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Síndrome Pulmonar por Hantavirus/imunologia , Nucleocapsídeo/imunologia , Orthohantavírus/imunologia , Linfócitos T Citotóxicos/imunologia , Doença Aguda , Adulto , Antígenos Virais/genética , Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Células Cultivadas , Epitopos de Linfócito T/genética , Feminino , Variação Genética , Antígenos HLA-C , Síndrome Pulmonar por Hantavirus/sangue , Humanos , Pessoa de Meia-Idade , Nucleocapsídeo/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Blood samples were collected from an Albanian and a Greek patient with hemorrhagic fever with renal syndrome and tested by reverse transcriptase-polymerase chain reaction. The genetic detection assay amplified hantavirus-specific DNA fragments from RNA extracted from the blood of the patients; nucleotide sequence analysis revealed that the causative agent of the disease was Dobrava virus. These findings suggest that Dobrava virus (which was originally isolated from the lungs of an Apodenws flavicollis mouse in Slovenia) is endemic throughout the Balkan States and causes overt human disease.
Assuntos
Febre Hemorrágica com Síndrome Renal/virologia , Orthohantavírus/genética , Adulto , Albânia/etnologia , Variação Genética , Grécia/epidemiologia , Orthohantavírus/classificação , Orthohantavírus/isolamento & purificação , Febre Hemorrágica com Síndrome Renal/sangue , Febre Hemorrágica com Síndrome Renal/epidemiologia , Humanos , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNARESUMO
This study reports completion of the genetic characterization of the entire genome of Sin Nombre (SN) virus (NMH10) detected in autopsy tissues from a patient who died of hantavirus pulmonary syndrome (HPS). The large (L) genome segment was found to be 6,562 nucleotides in length and encoded a putative L polymerase that was 2,153 amino acids in length. No evidence of segment reassortment with other well-characterized hantaviruses was obtained. The sequence of the entire S, M, and L genome segments of SN virus (strain NMR11) isolated from a mouse (trapped in the residence of the patient infected with SN virus [NMH10]) by passage two times in Peromyscus maniculatus and then by five passages in E6 Vero cells was determined and compared with that of the virus detected in autopsy tissues. Only 16 nucleotide differences were detected between the virus genomes, and none of these resulted in virus protein amino acid substitutions. Determination of the exact 5'- and 3'-terminal sequences of all genome segments of SN virus and representatives of other serologic groups in the Hantavirus genus, family Bunyaviridae, showed the existence of conserved nucleotide domains that may be involved in important regulatory mechanisms, such as RNA encapsidation, polymerase binding, and control of transcription and replication.
Assuntos
Genoma Viral , Síndrome Pulmonar por Hantavirus/virologia , Orthohantavírus/genética , Filogenia , Sequência de Aminoácidos , Animais , Autopsia , Sequência de Bases , Bunyaviridae/classificação , Bunyaviridae/genética , Chlorocebus aethiops , Evolução Fatal , Orthohantavírus/classificação , Orthohantavírus/isolamento & purificação , Humanos , Camundongos , Dados de Sequência Molecular , Peromyscus , Homologia de Sequência do Ácido Nucleico , Células VeroRESUMO
A fatal case of hantaviral illness occurred in Louisiana, outside of the range of P. maniculatus, the rodent reservoir for Sin Nombre virus. Hantavirus RNA and antigens were detected in patient autopsy tissues, and nucleotide sequence analysis of amplified polymerase chain reaction (PCR) products identified a newly recognized unique hantavirus, provisionally named Bayou virus. Prominent features of the clinical illness are compatible with hantavirus pulmonary syndrome (HPS), but several features such as renal insufficiency and intraalveolar hemorrhage are more compatible with hemorrhagic fever with renal syndrome (HFRS), a disease associated with Eurasian hantaviruses.
Assuntos
Infecções por Hantavirus/virologia , Orthohantavírus/isolamento & purificação , Antígenos Virais/imunologia , Sequência de Bases , Evolução Fatal , Orthohantavírus/classificação , Orthohantavírus/genética , Orthohantavírus/imunologia , Infecções por Hantavirus/imunologia , Infecções por Hantavirus/fisiopatologia , Humanos , Imuno-Histoquímica , Louisiana , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , RNA Viral/análiseRESUMO
Genetic analysis of virus detected in autopsy tissues of a fatal hantavirus pulmonary syndrome-like case in Louisiana revealed the presence of a previously unrecognized hantavirus. Nucleotide sequence analysis of PCR fragments of the complete S and M segments of the virus amplified from RNA extracted from the tissues showed the virus to be novel, differing from the closest related hantavirus, Sin Nombre virus, by approximately 30%. Both genome segments were unique, and there was no evidence of genetic reassortment with previously characterized hantaviruses. The primary rodent reservoir of Sin Nombre virus, the deer mouse Peromyscus maniculatus, is absent from Louisiana. Thus, the virus detected in Louisiana, referred to here as Bayou virus, must possess a different rodent reservoir.
Assuntos
Síndrome Pulmonar por Hantavirus/microbiologia , Orthohantavírus/isolamento & purificação , Sequência de Bases , Primers do DNA/química , Orthohantavírus/genética , Humanos , Louisiana , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
To determine if Muerto Canyon Virus (MCV) RNA is present in the peripheral blood of patients with hantavirus pulmonary syndrome, a reverse transcriptase-polymerase chain reaction (RT-PCR) assay for MCV RNA was used on blood samples from 20 seropositive case-patients. RNA was prepared from peripheral blood mononuclear cells (PBMC) or blood clot (or both) from 19 and from plasma from 11 case-patients. All 12 blood clot, all 13 PBMC, and 8 of 11 plasma preparations produced an MCV amplification product after RT-PCR with primers from the G2 gene. All of 5 PBMC RNA preparations tested were positive using unnested primers in S segment. Nucleotide sequences were determined for 16 G2 amplimers and 4 S segment amplimers, verifying that unique MCV cDNA sequences were amplified. Viral RNA became undetectable in 5 of 7 convalescent samples tested but was present up to day 23 of illness in 2 case-patients.
Assuntos
Síndrome Pulmonar por Hantavirus/diagnóstico , Orthohantavírus/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , RNA Viral/sangue , Sequência de Bases , Primers do DNA , Síndrome Pulmonar por Hantavirus/sangue , Síndrome Pulmonar por Hantavirus/virologia , Humanos , Leucócitos Mononucleares/virologia , Dados de Sequência Molecular , Neutrófilos/virologiaRESUMO
Investigation of a recent outbreak of acute respiratory illness in the southwestern United States resulted in the recognition of a new disease, hantavirus pulmonary syndrome (HPS) with high mortality. Different animals and cell lines were used in attempts to isolate the causative agent. A previously unknown hantavirus was passaged in laboratory-bred deer mice, recovered from lung tissues of a deer mouse, Peromyscus maniculatus, and propagated in the E6 clone of Vero cells. Virus antigen was readily detected in the infected cells by an indirect immunofluorescence assay, using convalescent-phase sera from HPS patients. By electron microscopy, the virus was shown to have the typical morphologic features of members of the genus Hantavirus, family Bunyaviridae. Virus sequences corresponded to those previously detected by a nested reverse transcriptase-polymerase chain reaction assay of hantavirus-infected specimens from rodents and humans. This newly recognized virus, the etiologic agent of HPS, has been tentatively named Muerto Canyon virus.
Assuntos
Infecções por Bunyaviridae/microbiologia , Orthohantavírus/isolamento & purificação , Infecções Respiratórias/microbiologia , Animais , Ensaio de Imunoadsorção Enzimática , Gerbillinae , Cobaias , Orthohantavírus/genética , Orthohantavírus/ultraestrutura , Humanos , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica , Peromyscus , Reação em Cadeia da Polimerase , Inoculações Seriadas , Células VeroRESUMO
An outbreak of hantavirus pulmonary syndrome (HPS) in the southwestern United States was etiologically linked to a newly recognized hantavirus. Knowledge that hantaviruses are maintained in rodent reservoirs stimulated a field and laboratory investigation of 1696 small mammals of 31 species. The most commonly captured rodent, the deer mouse (Peromyscus maniculatus), had the highest antibody prevalence (30%) to four hantavirus antigens. Antibody also was detected in 10 other species of rodent and in 1 species of rabbit. Reverse transcriptase-polymerase chain reaction (RT-PCR) products of hantavirus from rodent tissues were indistinguishable from those from human HPS patients. More than 96% of the seropositive P. maniculatus were positive by RT-PCR, suggesting chronic infection. Antibody prevalences were similar among P. maniculatus trapped from Arizona (33%), New Mexico (29%), and Colorado (29%). The numeric dominance of P. maniculatus, the high prevalence of antibody, and the RT-PCR findings implicate this species as the primary rodent reservoir for a new hantavirus in the southwestern United States.
Assuntos
Reservatórios de Doenças , Orthohantavírus/isolamento & purificação , Peromyscus/microbiologia , Animais , Anticorpos Antivirais/sangue , Sequência de Bases , DNA Viral , Feminino , Orthohantavírus/genética , Masculino , Mamíferos/microbiologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Sudoeste dos Estados UnidosRESUMO
A previously unrecognized hantavirus (family Bunyaviridae) has recently been detected and shown to be associated with a severe respiratory illness with high mortality, termed hantavirus pulmonary syndrome (HPS). This disease has now been identified throughout the western United States. We present nucleotide sequence characterization of the three RNA segments composing the HPS virus genome and address the question of the apparent emergence of this highly lethal virus. No evidence of genetic reassortment with previously recognized hantaviruses was found, each RNA segment being unique and approximately 30% different at the nucleotide level to the segments of the closest relative, Prospect Hill virus. These findings, together with the observed extensive genetic diversity of HPS viruses and examples of geographic clustering of distinct virus genotypes, suggest that HPS and associated virus have likely existed undetected for many years. The virus genome M segment was determined to be 3696 nucleotides in length and encode G1 and G2 proteins, 652 and 488 amino acids in length. The S segment was found to be 2059 nucleotides in length and to encode a nucleocapsid protein, 428 amino acids in length. S segment analysis also revealed an unusually long noncoding region with numerous repeats and evidence for a potential NSS protein encoded in an overlapping frame.
Assuntos
Infecções por Bunyaviridae/microbiologia , Genoma Viral , Pneumopatias/microbiologia , Orthohantavírus/classificação , Orthohantavírus/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Infecções por Bunyaviridae/epidemiologia , Variação Genética , Humanos , Pneumopatias/epidemiologia , Dados de Sequência Molecular , North Dakota/epidemiologia , Noroeste dos Estados Unidos/epidemiologia , Filogenia , RNA Viral/genética , Roedores/microbiologia , Homologia de Sequência de Aminoácidos , Sudoeste dos Estados Unidos/epidemiologia , Síndrome , Proteínas Virais/genéticaRESUMO
Several field isolates, strains, mutants, and revertants of vesicular stomatitis virus (VSV), Indiana (IN) serotype, were studied that differed greatly in their capacity to induce interferon (IFN) in aged chick embryo cells. The predicted M-protein amino acid sequence of a wild-type field isolate that induced > or = 10,000 units/ml IFN in chicken embryo cells was identical to that of a wild-type field isolate that induced < 2 units/ml and of a noninducing wild-type laboratory strain. The 47-base plus-strand leader RNA sequences were the same for five IFN-inducing, and eight noninducing independent isolates of wild-type VSV IN. Our data show that the M-protein and plus-strand leader RNA do not of themselves regulate the induction of IFN in this system. Because the capacity of VSV IN to induce IFN resides in virion-associated elements (Marcus and Sekellick, 1987, J. Interferon Res. 7, 269-284), the differences in IFN yield observed with various isolates must result from changes in other virion components that remain to be determined.
Assuntos
Indutores de Interferon , Proteínas Musculares , Proteínas do Mieloma , RNA Mensageiro/fisiologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Proteínas Virais/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Galinha , Conectina , Dados de Sequência Molecular , Mutação , Fenótipo , Temperatura , Vírus da Estomatite Vesicular Indiana/genéticaRESUMO
A newly recognized hantavirus was recently found to be associated with an outbreak of acute respiratory illness in the southwestern United States. The disease, which has become known as hantavirus pulmonary syndrome, has an unusually high mortality (64%). Virus isolation attempts have been unsuccessful thus far, resulting in a lack of homologous antigen for use in diagnostic assays. For this reason, a molecular approach was initiated to produce recombinant homologous antigen. The virus nucleocapsid (N) protein was selected, since N has been shown to be a sensitive antigenic target in other hantavirus systems. The N protein open reading frame of the virus S genome segment was synthesized from frozen autopsy tissue by polymerase chain reaction amplification, followed by cloning and expression in Hela cells (vaccinia-T7 RNA polymerase system) and Escherichia coli. N protein-expressing Hela cells served as excellent antigens for an improved indirect immunofluorescence assay. Use of the E. coli-expressed N protein in an enzyme-linked immunosorbent assay improved the sensitivity and specificity when compared with heterologous antigens used previously. Preliminary analysis also indicates that the higher sensitivity could result in earlier detection of infected persons. These data demonstrate that even in the absence of a virus isolate, the necessary homologous antigen can be produced and can serve to improve the detection and diagnostic capabilities needed to combat this newly recognized fatal respiratory illness in the United States.
Assuntos
Infecções por Bunyaviridae/microbiologia , Pneumopatias/microbiologia , Orthohantavírus/genética , RNA Viral/genética , RNA Viral/isolamento & purificação , Sequência de Aminoácidos , Antígenos Virais/biossíntese , Antígenos Virais/genética , Autopsia , Sequência de Bases , Infecções por Bunyaviridae/diagnóstico , Capsídeo/biossíntese , Capsídeo/genética , Capsídeo/imunologia , Primers do DNA/genética , Escherichia coli/genética , Expressão Gênica , Genes Virais , Orthohantavírus/imunologia , Orthohantavírus/isolamento & purificação , Células HeLa , Humanos , Pneumopatias/diagnóstico , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Síndrome , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologiaRESUMO
A mysterious respiratory illness with high mortality was recently reported in the southwestern United States. Serologic studies implicated the hantaviruses, rodent-borne RNA viruses usually associated elsewhere in the world with hemorrhagic fever with renal syndrome. A genetic detection assay amplified hantavirus-specific DNA fragments from RNA extracted from the tissues of patients and deer mice (Peromyscus maniculatus) caught at or near patient residences. Nucleotide sequence analysis revealed the associated virus to be a new hantavirus and provided a direct genetic link between infection in patients and rodents.
Assuntos
Infecções por Bunyaviridae/microbiologia , Surtos de Doenças , Reservatórios de Doenças , Genoma Viral , Pneumopatias/microbiologia , Orthohantavírus/genética , Peromyscus/microbiologia , Animais , Sequência de Bases , Infecções por Bunyaviridae/epidemiologia , Infecções por Bunyaviridae/veterinária , Primers do DNA , Orthohantavírus/classificação , Orthohantavírus/isolamento & purificação , Humanos , Pneumopatias/epidemiologia , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Doenças dos Roedores/epidemiologia , Doenças dos Roedores/microbiologia , Homologia de Sequência do Ácido Nucleico , Sudoeste dos Estados Unidos/epidemiologiaRESUMO
Vesicular stomatitis virus of the New Jersey serotype (VSV-NJ) causes vesicular disease in cattle, pigs, and horses throughout the Americas. Vesicular disease is clinically indistinguishable from foot-and-mouth disease (FMD). Therefore, outbreaks of vesicular disease in FMD-free areas must be rapidly diagnosed by laboratory methods and affected farms must be quarantined until laboratory results confirm the absence of FMD. Diagnosis is currently performed in high-containment (biosafety level 3) laboratories by using complement fixation and virus isolation in tissue culture. We describe here an alternative method for the detection of VSV-NJ RNA in clinical samples. This method includes a rapid acid guanidine-phenol RNA extraction procedure coupled with a one-tube polymerase chain reaction (PCR) using reverse transcriptase. By using this test, we were able to detect the largest number of positive samples (53 of 58), followed by complement (48 of 58) and isolation in tissue culture (43 of 58). The primers chosen for this assay amplify a 642-nucleotide region of the phosphoprotein gene of VSV-NJ but not of VSV-IN. Sequencing of the PCR product enables genetic typing of virus isolates and epidemiological studies. Since no infectious materials are necessary to perform this test and any infectious virus in clinical samples is destroyed by acid guanidine-phenol treatment, diagnosis can be safely performed in regular diagnostic laboratories.
Assuntos
Doenças dos Bovinos/diagnóstico , Reação em Cadeia da Polimerase , Vesiculovirus/isolamento & purificação , Viroses/veterinária , Animais , Sequência de Bases , Bovinos , Dados de Sequência Molecular , RNA Viral/análise , Sensibilidade e Especificidade , Sorotipagem , Estomatite/diagnóstico , Estomatite/veterinária , Vesiculovirus/genética , Viroses/diagnósticoRESUMO
Vesicular stomatitis virus (VSV) has served for several decades as the prototype rhabdovirus and a model RNA virus. Extensive studies upheld the original view of VSV genetics with simply five genes (N, P, M, G, and L), each encoding a single unique protein. We now report the first unambiguous demonstration of the existence of an additional unique protein encoded in an overlapping frame within the virus P gene. Experiments using antipeptide sera specific for the predicted second open reading frame have demonstrated the synthesis of two N-terminally nested forms of the protein in virus-infected cells. The major form is 55 amino acids in length, whereas the minor form has 10 additional N-terminal amino acids. Ribosome initiation of synthesis of these proteins appears to occur at AUG codons, 68 and 41 bases, respectively, downstream of the P protein AUG initiation codon. The proteins are found in the cytoplasm of the infected cell but are undetectable in purified virions, consistent with their being nonstructural proteins. Both the major and minor forms of the protein are highly basic and arginine rich, reminiscent of the C and C' proteins encoded in overlapping frame close to the 5' terminus of the P mRNA of several paramyxoviruses. The potential to encode small, highly basic proteins within the P mRNA 5' terminus is highly conserved among the vesiculoviruses.