RESUMEN
A virus isolated from dead Chaerephon plicata bats collected near Kampot, Cambodia, was identified as a member of the family Bunyaviridae by electron microscopy. The only bunyavirus previously isolated from Chaerephon species bats in South-East Asia is Kaeng Khoi (KK) virus (genus Orthobunyavirus), detected in Thailand over 30 years earlier and implicated as a public health problem. Using RT-PCR, nucleotide sequences from the M RNA segment of several virus isolates from the Cambodian C. plicata bats were found to be almost identical and to differ from those of the prototype KK virus by only 2.6-3.2 %, despite the temporal and geographic separation of the viruses. These results identify the Cambodian bat viruses as KK virus, extend the known virus geographic range and document the first KK virus isolation in 30 years. These genetic data, together with earlier serologic data, show that KK viruses represent a distinct group within the genus Orthobunyavirus.
Asunto(s)
Infecciones por Bunyaviridae/veterinaria , Bunyaviridae/clasificación , Bunyaviridae/aislamiento & purificación , Quirópteros/virología , Animales , Encéfalo/virología , Bunyaviridae/genética , Bunyaviridae/patogenicidad , Infecciones por Bunyaviridae/virología , Cambodia , Chlorocebus aethiops , Ratones , Filogenia , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células VeroRESUMEN
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.
Asunto(s)
Virus Sin Nombre/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Animales , Línea Celular , Cricetinae , Retículo Endoplásmico/metabolismo , Endosomas/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Aparato de Golgi/metabolismo , Humanos , Lisosomas/metabolismo , Microscopía Inmunoelectrónica , Transporte de Proteínas , Ratas , Fracciones Subcelulares/metabolismo , Transfección , Proteínas del Envoltorio Viral/análisis , Proteínas del Envoltorio Viral/genéticaRESUMEN
The ultrastructure of Hendra and Nipah viruses is described in cultured cells, pigs, horses and humans. Differences in ultrastructure between the viruses are evident within infected cell cultures and lungs from infected amplifier hosts. These differences are important in viral identification and differentiation and understanding the pathogenesis of disease.
Asunto(s)
Infecciones por Paramyxoviridae/virología , Paramyxovirinae/ultraestructura , Animales , Encéfalo/virología , Células Cultivadas , Enfermedades de los Caballos/virología , Caballos , Humanos , Pulmón/virología , Microscopía Electrónica , Infecciones por Paramyxoviridae/veterinaria , Porcinos , Enfermedades de los Porcinos/virologíaRESUMEN
In late 1997 and early 1998, a large outbreak of hemorrhagic fever occurred in East Africa. Clinical samples were collected in Kenya and southern Somalia, and 27 of 115 (23%) hemorrhagic fever patients tested showed evidence of acute infection with Rift Valley fever (RVF) virus as determined by IgM detection, virus isolation, detection of virus RNA by reverse transcription-polymerase chain reaction (RT-PCR), or immunohistochemistry. However, two patients (one from Kenya and the other from Somalia) whose illness met the hemorrhagic fever case definition yielded virus isolates that were not RVF. Electron microscopy suggested these two virus isolates were members of the family Bunyaviridae. RT-PCR primers were designed to detect bunyavirus RNA in these samples. Regions of the S and L segments of the two isolates were successfully amplified, and their nucleotide sequences exhibited nearly complete identity with Bunyamwera virus, a mosquito-borne virus not previously associated with severe human disease. Unexpectedly, the virus M segment appeared to be reassorted, as the sequences detected exhibited 32-33% nucleotide and 28% amino acid differences relative to the corresponding M segment sequence of Bunyamwera virus. The association of this reassortant bunyavirus, proposed name Garissa virus, with severe disease is supported by the detection of the virus RNA in acute-phase sera taken from 12 additional hemorrhagic fever cases in the region.
Asunto(s)
Virus Bunyamwera/genética , Infecciones por Bunyaviridae/virología , Fiebres Hemorrágicas Virales/virología , Virus Reordenados/genética , Animales , Anticuerpos Antivirales/sangre , Secuencia de Bases , Virus Bunyamwera/clasificación , Virus Bunyamwera/aislamiento & purificación , Virus Bunyamwera/ultraestructura , Infecciones por Bunyaviridae/epidemiología , Infecciones por Bunyaviridae/inmunología , Chlorocebus aethiops , Brotes de Enfermedades , Fiebres Hemorrágicas Virales/epidemiología , Fiebres Hemorrágicas Virales/inmunología , Humanos , Kenia/epidemiología , Datos de Secuencia Molecular , Filogenia , ARN Viral/análisis , Virus Reordenados/clasificación , Virus Reordenados/aislamiento & purificación , Virus Reordenados/ultraestructura , Recombinación Genética , Somalia/epidemiología , Células VeroRESUMEN
Current treatment of HIV-1-infected individuals involves the administration of several drugs, all of which target either the reverse transcriptase or the protease activity of the virus. Unfortunately, the benefits of such treatments are compromised by the emergence of viruses exhibiting resistance to the drugs. This situation warrants new approaches for interfering with virus replication. Considering the activation of protease in the virus particles, a novel strategy to inhibit HIV-1 replication was tested targeting the dimerization domain of the protease. To test this idea, we have selected four residues from the C terminus of HIV-1 protease that map to the dimer interface region of the enzyme. We have exploited Vpr to display the peptides in the virus particles. The chimeric Vpr exhibited expression and virion incorporation similar to wildtype Vpr. The virus derived from the HIV-1 proviral DNA containing chimeric Vpr sequences registered a reduced level of replication in CEM and CEM X 174 cells in comparison with viruses containing wildtype Vpr. Similar results were observed in a single-round replication assay. These results suggest that the intravirion display of peptides targeting viral proteins is a powerful approach for developing antiviral agents and for dissecting the dynamic interactions between structural proteins during virus assembly and disassembly.
Asunto(s)
Fármacos Anti-VIH/farmacología , Productos del Gen vpr/farmacología , Proteasa del VIH/metabolismo , VIH-1/fisiología , Proteínas Recombinantes de Fusión/farmacología , Replicación Viral/efectos de los fármacos , Secuencia de Bases , Línea Celular , Productos del Gen vpr/química , Humanos , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Ensayo de Radioinmunoprecipitación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes/farmacología , Productos del Gen vpr del Virus de la Inmunodeficiencia HumanaRESUMEN
A paramyxovirus virus termed Nipah virus has been identified as the etiologic agent of an outbreak of severe encephalitis in people with close contact exposure to pigs in Malaysia and Singapore. The outbreak was first noted in late September 1998 and by mid-June 1999, more than 265 encephalitis cases, including 105 deaths, had been reported in Malaysia, and 11 cases of encephalitis or respiratory illness with one death had been reported in Singapore. Electron microscopic, serologic, and genetic studies indicate that this virus belongs to the family Paramyxoviridae and is most closely related to the recently discovered Hendra virus. We suggest that these two viruses are representative of a new genus within the family Paramyxoviridae. Like Hendra virus, Nipah virus is unusual among the paramyxoviruses in its ability to infect and cause potentially fatal disease in a number of host species, including humans.
Asunto(s)
Encefalitis Viral/virología , Infecciones por Paramyxoviridae/epidemiología , Infecciones por Paramyxoviridae/virología , Paramyxovirinae , Animales , Anticuerpos Antivirales/sangre , Brotes de Enfermedades , Encefalitis Viral/epidemiología , Endotelio Vascular/patología , Endotelio Vascular/virología , Genes Virales , Células Gigantes/patología , Células Gigantes/virología , Humanos , Malasia/epidemiología , Microscopía Electrónica , Datos de Secuencia Molecular , Nucleocápside/ultraestructura , Infecciones por Paramyxoviridae/transmisión , Infecciones por Paramyxoviridae/veterinaria , Paramyxovirinae/clasificación , Paramyxovirinae/genética , Paramyxovirinae/aislamiento & purificación , Paramyxovirinae/ultraestructura , Filogenia , Sistema Respiratorio/virología , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/veterinaria , Infecciones del Sistema Respiratorio/virología , Análisis de Secuencia de ADN , Singapur/epidemiología , Porcinos , Enfermedades de los Porcinos/epidemiología , Enfermedades de los Porcinos/virología , Vasculitis/virología , Proteínas Virales/genéticaRESUMEN
In the spring of 1996, multiple cases of an acute febrile illness resulting in several deaths in remote locations in Peru were reported to the Centers for Disease Control and Prevention (CDC). The clinical syndromes for these cases included dysphagia and encephalitis. Because bat bites were a common occurrence in the affected areas, the initial clinical diagnosis was rabies. However, rabies was discounted primarily because of reported patient recovery. Samples of brain tissue from two of the fatal cases were received at CDC for laboratory confirmation of the rabies diagnosis. An extensive array of tests on the formalin-fixed tissues confirmed the presence of both rabies viral antigen and nucleic acid. The virus was shown to be most closely related to a vampire bat rabies isolate. These results indicate the importance of maintaining rabies in the differential diagnosis of acute febrile encephalitis, particularly in areas where exposure to vampire bats may occur.
Asunto(s)
Encefalopatías/diagnóstico , Encéfalo/virología , Quirópteros/virología , Virus de la Rabia/aislamiento & purificación , Rabia/diagnóstico , Animales , Anticuerpos Monoclonales , Anticuerpos Antivirales/sangre , Antígenos Virales/análisis , Secuencia de Bases , Encéfalo/ultraestructura , Encefalopatías/virología , Cartilla de ADN/química , Brotes de Enfermedades , Vectores de Enfermedades , Femenino , Técnica del Anticuerpo Fluorescente Directa , Histocitoquímica , Humanos , Hibridación in Situ , Ratones , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Perú , Reacción en Cadena de la Polimerasa , Rabia/mortalidad , Rabia/virología , Virus de la Rabia/genética , Virus de la Rabia/inmunología , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
Human herpesvirus-8 (HHV-8) is a gammaherpesvirus that is present primarily in a state of low level persistence in primary effusion lymphoma cell lines. Using BCBL-1 cells that harbour HHV-8 but lack Epstein-Barr virus, we demonstrate that sodium butyrate is much more effective than the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) at inducing high levels of class II and III virus transcription and viral DNA replication, but also initiates apoptosis. Apoptosis occurs prior to assembly of virions when high concentrations of butyrate (1-3 mM) are used, whereas reduction of butyrate concentration to 0.3 mM decreases the rate of apoptosis and results in production and secretion of enveloped virions that are visualized at high number by electron microscopy in approximately 20% of BCBL-1 cells. Butyrate induces much higher levels of multiple class II and class III transcripts than does TPA, including v-MIPI, v-IL-6, v-Bcl-2, vGPCR and ORF26. A decrease in concentration of butyrate from 3 to 0.3 mM delays the peak induction of these genes, but peak levels remain higher than peak levels in response to TPA. These studies indicate that the massive apoptosis induced by 3 mM butyrate could be diminished and delayed by reduction of butyrate concentration to 0.3 mM, thereby allowing expression of high levels of lytic-associated genes and production of high yields of HHV-8 virions.
Asunto(s)
Butiratos/farmacología , Replicación del ADN , ADN Viral , Regulación Viral de la Expresión Génica/efectos de los fármacos , Herpesvirus Humano 8/genética , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , Herpesvirus Humano 8/efectos de los fármacos , Herpesvirus Humano 8/fisiología , Humanos , ARN Mensajero , ARN Viral , Células Tumorales CultivadasRESUMEN
Laboratory diagnosis of Ebola hemorrhagic fever (EHF) is currently performed by virus isolation and serology and can be done only in a few high-containment laboratories worldwide. In 1995, during the EHF outbreak in the Democratic Republic of Congo, the possibility of using immunohistochemistry (IHC) testing of formalin-fixed postmortem skin specimens was investigated as an alternative diagnostic method for EHF. Fourteen of 19 cases of suspected EHF met the surveillance definition for EHF and were positive by IHC. IHC, serologic, and virus isolation results were concordant for all EHF and non-EHF cases. IHC and electron microscopic examination showed that endothelial cells, mononuclear phagocytes, and hepatocytes are main targets of infection, and IHC showed an association of cellular damage with viral infection. The finding of abundant viral antigens and particles in the skin of EHF patients suggests an epidemiologic role for contact transmission. IHC testing of formalin-fixed skin specimens is a safe, sensitive, and specific method for laboratory diagnosis of EHF and should be useful for EHF surveillance and prevention.
Asunto(s)
Ebolavirus/aislamiento & purificación , Fiebre Hemorrágica Ebola/diagnóstico , Inmunohistoquímica/métodos , Piel/virología , Adolescente , Adulto , Anciano , Antígenos Virales/metabolismo , República Democrática del Congo/epidemiología , Brotes de Enfermedades , Ebolavirus/inmunología , Ebolavirus/ultraestructura , Femenino , Fiebre Hemorrágica Ebola/epidemiología , Fiebre Hemorrágica Ebola/transmisión , Humanos , Inmunohistoquímica/estadística & datos numéricos , Cuerpos de Inclusión Viral/ultraestructura , Lactante , Hígado/patología , Hígado/virología , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Estudios Retrospectivos , Sensibilidad y Especificidad , Piel/patologíaRESUMEN
Two new isolates of Ehrlichia chaffeensis (designated Jax and St. Vincent) were obtained from patients with fatal ehrlichial infections. Patients developed characteristic manifestations of severe disease due to E. chaffeensis, including marked thrombocytopenia, pulmonary insufficiency, and encephalopathy. Primary isolation was achieved in DH82 cells; the Jax and St. Vincent isolates were detected within 19 and 8 days postinoculation, respectively. The isolates were characterized by molecular evaluation of the 16S rRNA gene, the groESL heat shock operon, a 120-kDa immunodominant protein gene, and an incompletely characterized repetitive-motif sequence (variable-length PCR target [VLPT]). The sequences were compared with those of the corresponding molecular regions in the type isolate (Arkansas). St. Vincent contained one fewer repeat unit in both the 120-kDa protein gene and the VLPT compared with corresponding sequences of the Jax and Arkansas isolates. 16S rRNA gene sequences from the two new isolates had 100% identity to the corresponding sequences of the 91HE17 and Sapulpa isolates of E. chaffeensis, and to the corrected 16S rRNA gene sequence of the Arkansas isolate. The Jax isolate grew more slowly than the St. Vincent isolate in DH82 cells, and both of the new isolates grew more slowly than the extensively passaged Arkansas isolate. Although specific associations between ehrlichial pathogenicity and genotype were not identified from these comparisons, recovery of this organism from a spectrum of clinical presentations remains an integral step in understanding mechanisms of disease caused by E. chaffeensis.
Asunto(s)
Ehrlichia chaffeensis/aislamiento & purificación , Ehrlichiosis/microbiología , Análisis de Varianza , Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Chaperonina 10/genética , Chaperonina 60/genética , Ehrlichia chaffeensis/genética , Ehrlichia chaffeensis/ultraestructura , Ehrlichiosis/sangre , Ehrlichiosis/etiología , Ehrlichiosis/mortalidad , Femenino , Humanos , Epítopos Inmunodominantes/genética , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNAsunto(s)
Virus Bunyamwera/aislamiento & purificación , Infecciones por Bunyaviridae/virología , Encefalitis Viral/virología , Adulto , Virus Bunyamwera/clasificación , Resultado Fatal , Humanos , Masculino , Microscopía Electrónica , Insuficiencia Multiorgánica/virología , North Carolina , Viremia/virologíaRESUMEN
The growth characteristics of human herpesvirus 7 strain SB (HHV-7 (SB)) were studied in human umbilical cord blood lymphocyte (CBL) cultures. The virus has approximately a 4-day growth cycle, as measured by immunofluorescence analysis, quantitation of the relative viral DNA concentration, and examination of infected cells by electron microscopy on consecutive days post-infection. By systematically varying the culture media components, improved culturing conditions were established. Activated lymphocytes were required for virus growth. HHV-7(SB) grew best in phytohemagglutinin-stimulated CBL cultured in media containing 0.01 mg/ml hydrocortisone. Addition of recombinant human interleukin 2 (IL-2) at concentrations exceeding 1-10 U/ml inhibited virus growth in most CBL cultures. Addition of exogenous IL-2 to the culture media had no effect on viral DNA production. However, the percentage of virus antigen-positive cells was highest when 0.1-1 U/ml was added to the media. Differences in the ability of individual CBL cultures to replicate HHV-7(SB) was not explained by differing CD4+ cell concentrations. However, individual cultures varied in the level of endogenous IL-2 production, which may contribute to the virus growth variability in CBL. HHV-7(SB) grew in the CD4-positive T-cell line SupT1, but not in a variety of other lymphocyte, fibroblast, or epithelial cell lines. Nine compounds were tested for antiviral activity against HHV-7 in vitro. Phosphonoformic acid inhibited virus growth with a 50% effective concentration of 4.8 microM. Ganciclovir (200 microM) and phosphonoacetic acid (100 microM) inhibited more than 90% of virus production. None of the compounds were cytotoxic at concentrations which inhibited the virus. A generalized increase in host cell protein synthesis was also observed in virus-infected cells similar to that seen in CBL infected with human herpesvirus 6.
Asunto(s)
Herpesvirus Humano 7/crecimiento & desarrollo , Adulto , Antivirales/farmacología , Técnicas de Cultivo de Célula/métodos , Línea Celular , Células Cultivadas , Sangre Fetal , Herpesvirus Humano 7/efectos de los fármacos , Herpesvirus Humano 7/ultraestructura , Humanos , Linfocitos , Pruebas de Sensibilidad MicrobianaRESUMEN
The cytopathic effects (CPE) resulting from the infection of CD4+ T cells by human immunodeficiency virus (HIV) have generally been characterized as single-cell killing associated with apoptosis and/or the generation of syncytia resulting from the direct cell-to-cell transmission of the virus. Little is known, however, about the cellular factors influencing host cell susceptibility to HIV-mediated CPE. Because expression of the antiapoptosis gene, bcl-2, enhances cell viability after exposure to cytotoxic agents or stimuli, the effect of bcl-2 expression on HIV infection of stably transfected T-cell clones was investigated. Unexpectedly, bcl-2 expression by these cells accelerated the kinetics of an acute spreading HIV infection, as evidenced by a rapid loss of culture viability associated with the appearance of CPE and reverse transcriptase activity in the culture supernatant. This unexpected effect of bcl-2 expression results from the arrest of syncytial apoptosis, directly facilitating the cell-to-cell transmission of HIV. In addition, bcl-2 expression is associated with enhanced HIV replication as determined by HIV type 1-specific Western blot (immunoblot) analysis. These results suggest that the inhibition of apoptosis is essential for this mode of viral transmission.
Asunto(s)
VIH-1/patogenicidad , Proteínas Proto-Oncogénicas/fisiología , Línea Celular , Efecto Citopatogénico Viral , Expresión Génica , Células Gigantes/virología , VIH-1/fisiología , Humanos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2 , Células Tumorales CultivadasRESUMEN
A recent outbreak of a severe pulmonary disease in the southwestern United States was etiologically linked to a previously unrecognized hantavirus. The virus has been isolated from its major reservoir, the deer mouse, Peromyscus maniculatus, and recently named Sin Nombre virus. Clinically, the disease has become known as the hantavirus pulmonary syndrome (HPS). Since May 1993, 44 fatal cases of HPS have been identified through clinicopathological review and immunohistochemical (IHC) testing of tissues from 273 patients who died of an unexplained noncardiogenic pulmonary edema. In 158 cases for which suitable specimens were available, serological testing and/or reverse transcription-polymerase chain reaction (RT-PCR) amplification of extracted RNA was also performed. IHC, serological, and PCR results were concordant for virtually all HPS and non-HPS patients when more than one assay was performed. The prodromal illness of HPS is similar to that of many other viral diseases. Consistent hematological features include thrombocytopenia, hemoconcentration, neutrophilic leukocytosis with a left shift, and reactive lymphocytes. Pulmonary histopathological features were similar in most of the fatal HPS cases (40/44) and consisted of an interstitial pneumonitis with a variable mononuclear cell infiltrate, edema, and focal hyaline membranes. In four cases, however, pulmonary features were significantly different and included diffuse alveolar damage and variable degrees of severe air space disorganization. IHC analysis showed widespread presence of hantaviral antigens in endothelial cells of the microvasculature, particularly in the lung. Hantaviral antigens were also observed within follicular dendritic cells, macrophages, and lymphocytes. Hantaviral inclusions were observed in endothelial cells of lungs by thinsection electron microscopy, and their identity was verified by immunogold labeling. Virus-like particles were seen in pulmonary endothelial cells and macrophages. HPS is a newly recognized, often fatal disease, with a spectrum of microscopic morphological changes, which may be an important cause of severe and fatal illness presenting as adult respiratory distress syndrome.
Asunto(s)
Infecciones por Hantavirus/patología , Neumonía Viral/patología , Adolescente , Adulto , Anciano , Reacciones Antígeno-Anticuerpo , Antígenos Virales/análisis , Femenino , Orthohantavirus/genética , Orthohantavirus/inmunología , Orthohantavirus/aislamiento & purificación , Infecciones por Hantavirus/complicaciones , Infecciones por Hantavirus/virología , Humanos , Inmunohistoquímica , Inmunofenotipificación , Masculino , Microscopía Electrónica , Microscopía Inmunoelectrónica , Persona de Mediana Edad , Neumonía Viral/complicaciones , Neumonía Viral/virología , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , SíndromeRESUMEN
A previously unrecognized disease, hantavirus pulmonary syndrome, was described following an outbreak of severe, often lethal, pulmonary illness in the southwestern United States in May-June, 1993. We have now studied the morphologic features of the causative agent, Sin Nomber virus (SNV), by thin section electron microscopy and immunoelectron microscopy of infected Vero E6 cells. SNV virions were roughly spherical and had a mean diameter of 112 nm. They had a rather dense envelope and closely apposed fine surface projections, 7 nm in length. Filamentous nucleocapsids were present within virions. Viral inclusion bodies were present in the cytoplasm of infected cells; these appeared granular or filamentous, depending on the plane of section. All of these characteristics were similar to published descriptions of other hantaviruses; however, unlike all other hantaviruses and virtually all other member viruses of the family Bunyaviridae which bud upon smooth intracytoplasmic membranes, SNV budding occurred almost entirely upon the plasma membrane of infected cells. Virus budding was associated with the formation of long 28 nm diameter tubular projections. Occasional elongated 47 nm diameter virus-like particles were seen to bud upon intracytoplasmic membranes. As shown by immunoelectron microscopy, viral antigens were localized over virions, inclusions, and tubular projections associated with virion morphogenesis.
Asunto(s)
Bunyaviridae/ultraestructura , Síndrome Pulmonar por Hantavirus/virología , Orthohantavirus/ultraestructura , Animales , Anticuerpos Monoclonales , Cápside/análisis , Chlorocebus aethiops , Brotes de Enfermedades , Orthohantavirus/aislamiento & purificación , Síndrome Pulmonar por Hantavirus/epidemiología , Humanos , Microscopía Electrónica , Microscopía Inmunoelectrónica , Peromyscus , Sudoeste de Estados Unidos/epidemiología , Células Vero , Proteínas del Núcleo Viral/análisis , Virión/ultraestructuraRESUMEN
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.
Asunto(s)
Infecciones por Bunyaviridae/microbiología , Orthohantavirus/aislamiento & purificación , Infecciones del Sistema Respiratorio/microbiología , Animales , Ensayo de Inmunoadsorción Enzimática , Gerbillinae , Cobayas , Orthohantavirus/genética , Orthohantavirus/ultraestructura , Humanos , Ratones , Ratones Endogámicos ICR , Microscopía Electrónica , Peromyscus , Reacción en Cadena de la Polimerasa , Pase Seriado , Células VeroRESUMEN
African filoviruses have caused outbreaks of fulminating hemorrhagic fever among humans. In 1989, related filoviruses were isolated from cynomolgus monkeys imported into the United States from the Philippines. The pathogenic potential of these new filoviruses was compared in 16 Asian monkeys (Macaca fascicularis-cynomolgus) and 16 African monkeys (Cercopithecus aethiops-African green) using African filoviruses from Zaire (Ebola virus) and Sudan or Asian filoviruses (Reston and Pennsylvania). African filovirus infections resulted in earlier death (P = .005), had a shorter duration of disease and median incubation period (3-4 vs. 7 days), and had earlier peak viremia (5-7 vs. 7-9 days). African green monkeys showed significantly higher survival than cynomolgus monkeys (P less than .01), and some were asymptomatic as have been humans accidentally infected with Asian filovirus. Rechallenge experiments showed that protection in survivors of filovirus infections against fatal challenge with Ebola (Zaire) virus is unpredictable. The minimal clinical disease observed in humans infected with the Reston strain is consistent with host- and virus-dependent pathogenicity.
Asunto(s)
Filoviridae/patogenicidad , Virosis/fisiopatología , África , Animales , Asia , Chlorocebus aethiops , Hígado/patología , Macaca fascicularis , Ensayo de Radioinmunoprecipitación , Especificidad de la Especie , Proteínas Virales/análisis , Virosis/sangre , Virosis/mortalidadRESUMEN
Volepox virus (VPX) from skin lesions on a vole and a piñon mouse caught in California and raccoon poxvirus (RCN) from raccoons trapped in Maryland were examined to begin elucidating their relationship to other orthopoxviruses, most of which are not known to be indigenous to the Americas. VPX and RCN produced pinpoint, nonhemorrhagic pocks on chick embryo chorioallantoic membranes. In cell cultures both viruses produced 1-mm diameter, irregular plaques, A-type inclusions (ATIs), and despite production of hemagglutinin, both viruses caused syncytia formation. Considerable cross-hybridization was seen between VPX and RCN DNA and the DNAs of other orthopoxviruses; however, HindIII cleavage site maps showed marked central and terminal region differences between VPX (222.8 kbp) and RCN (224.8 kbp) DNA and mapped DNAs of other orthopoxviruses. Cognate DNAs of the ATI 160-kDa protein and 38-kDa serine protease inhibitor homologue of cowpox virus (CPV) and the 14-kDa fusion protein of vaccinia virus (VAC) were present within the right end of VPX and RCN DNA, matching their location in CPV and VAC. VPX and RCN, respectively, expressed a 150- and a 155-kDa ATI major protein and a 20- and an 18-kDa fusion protein. Low stringency annealing suggested that cognate DNAs for the VAC growth factor and the alpha-amanitin target protein were present within the left end of VPX and RCN DNA, matching their location in VAC. Terminal tandem repeat sequences of VAC and RCN did not cross-hybridize with each other or with VPX DNA end fragments. Together, the data suggested that VPX and RCN are phylogenetically rather distant from orthopoxviruses not indigenous to the Americas, although genetic information is arranged as in other examined orthopoxviruses.
Asunto(s)
Variación Genética , Poxviridae/genética , Animales , Southern Blotting , Western Blotting , Embrión de Pollo , ADN Viral , Desoxirribonucleasa HindIII/metabolismo , Electroforesis en Gel de Campo Pulsado , Mapaches , Mapeo Restrictivo , Células VeroRESUMEN
In an effort to understand the contribution of the primer-binding site (PBS) region to human immunodeficiency virus (HIV) replication, we have constructed a mutant HIV proviral DNA with an alteration in the 5' end of the PBS. The PBS mutant proviral DNA was characterized by transfection of the viral DNA into CD4+ and non-CD4+ target cells. The results indicate that mutation in the PBS reduced the level of viral particles released into the medium of transfected cells in comparison to wild-type proviral DNA. The viral particles were noninfectious upon transmission to established CD4+ cell lines and phytohemagglutinin-stimulated peripheral blood lymphocytes. Electron microscopic analysis of the transfected cells revealed no abnormalities in the structure of the virion directed by the mutant proviral DNA. Also, the protein and RNA contents of the mutant virions were similar to the wild type. The quantitation of intracellular viral structural protein in the transfected cells, however, indicated that the PBS mutation may have an effect on the assembly of viral particles in addition to completely abolishing reverse transcription of viral RNA into DNA. These results provide evidence that the PBS region of the viral genome has multiple functions in HIV-1 replication.