RESUMEN
Influenza A/H2N2 viruses caused a pandemic in 1957 and continued to circulate in humans until 1968. The antigenic evolution of A/H2N2 viruses over time and the amino acid substitutions responsible for this antigenic evolution are not known. Here, the antigenic diversity of a representative set of human A/H2N2 viruses isolated between 1957 and 1968 was characterized. The antigenic change of influenza A/H2N2 viruses during the 12 years that this virus circulated was modest. Two amino acid substitutions, T128D and N139K, located in the head domain of the H2 hemagglutinin (HA) molecule, were identified as important determinants of antigenic change during A/H2N2 virus evolution. The rate of A/H2N2 virus antigenic evolution during the 12-year period after introduction in humans was half that of A/H3N2 viruses, despite similar rates of genetic change.IMPORTANCE While influenza A viruses of subtype H2N2 were at the origin of the Asian influenza pandemic, little is known about the antigenic changes that occurred during the twelve years of circulation in humans, the role of preexisting immunity, and the evolutionary rates of the virus. In this study, the antigenic map derived from hemagglutination inhibition (HI) titers of cell-cultured virus isolates and ferret postinfection sera displayed a directional evolution of viruses away from earlier isolates. Furthermore, individual mutations in close proximity to the receptor-binding site of the HA molecule determined the antigenic reactivity, confirming that individual amino acid substitutions in A/H2N2 viruses can confer major antigenic changes. This study adds to our understanding of virus evolution with respect to antigenic variability, rates of virus evolution, and potential escape mutants of A/H2N2.
Asunto(s)
Sustitución de Aminoácidos , Antígenos Virales , Evolución Molecular , Glicoproteínas Hemaglutininas del Virus de la Influenza , Subtipo H2N2 del Virus de la Influenza A , Gripe Humana , Pandemias , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , Perros , Hurones , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Subtipo H2N2 del Virus de la Influenza A/genética , Subtipo H2N2 del Virus de la Influenza A/inmunología , Gripe Humana/epidemiología , Gripe Humana/genética , Gripe Humana/inmunología , Células de Riñón Canino Madin Darby , Mutación Missense , Factores de TiempoRESUMEN
RNA structures are increasingly recognized to be of importance during influenza A virus replication. Here, we investigated a predicted conserved hairpin in the M gene segment (nt 967-994) within the region of the vRNA 5' packaging signal. The existence of this RNA structure and its possible role in virus replication was investigated using a compensatory mutagenesis approach. Mutations were introduced in the hairpin stem, based on natural variation. Virus replication properties were studied for the mutant viruses with disrupted and restored RNA structures. Viruses with structure-disrupting mutations had lower virus titers and a significantly reduced median plaque size when compared with the wild-type (WT) virus, while viruses with structure restoring-mutations replicated comparable to WT. Moreover, virus replication was also reduced when mutations were introduced in the hairpin loop, suggesting its involvement in RNA interactions. Northern blot and FACS experiments were performed to study differences in RNA levels as well as production of M1 and M2 proteins, expressed via alternative splicing. Stem-disruptive mutants caused lower vRNA and M2 mRNA levels and reduced M2 protein production at early time-points. When the RNA structure was restored, vRNA, M2 mRNA and M2 protein levels were increased, demonstrating a compensatory effect. Thus, this study provides evidence for functional importance of the predicted M RNA structure and suggests its role in splicing regulation.
Asunto(s)
Virus de la Influenza A/genética , ARN Mensajero/química , ARN Viral/química , Proteínas de la Matriz Viral/química , Replicación Viral , Empalme Alternativo , Animales , Emparejamiento Base , Secuencia Conservada , Perros , Células HEK293 , Humanos , Virus de la Influenza A/crecimiento & desarrollo , Virus de la Influenza A/metabolismo , Secuencias Invertidas Repetidas , Células de Riñón Canino Madin Darby , Mutagénesis , Conformación de Ácido Nucleico , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Relación Estructura-Actividad , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Ensamble de VirusRESUMEN
Many subtypes of low-pathogenicity avian influenza (LPAI) virus circulate in wild bird reservoirs, but their prevalence may vary among species. We aimed to compare by real-time reverse-transcriptase polymerase chain reaction, virus isolation, histology, and immunohistochemistry the distribution and pathogenicity of 2 such subtypes of markedly different origins in Mallard ducks (Anas platyrhynchos): H2N3 isolated from a Mallard duck and H13N6 isolated from a Ring-billed Gull (Larus delawarensis). Following intratracheal and intraesophageal inoculation, neither virus caused detectable clinical signs, although H2N3 virus infection was associated with a significantly decreased body weight gain during the period of virus shedding. Both viruses replicated in the lungs and air sacs until approximately day 3 after inoculation and were associated with a locally extensive interstitial, exudative, and proliferative pneumonia. Subtype H2N3, but not subtype H13N6, went on to infect the epithelia of the intestinal mucosa and cloacal bursa, where it replicated without causing lesions until approximately day 5 after inoculation. Larger quantities of subtype H2N3 virus were detected in cloacal swabs than in pharyngeal swabs. The possible clinical significance of LPAI virus-associated pulmonary lesions and intestinal tract infection in ducks deserves further evaluation.
Asunto(s)
Charadriiformes/virología , Patos/virología , Virus de la Influenza A/fisiología , Gripe Aviar/virología , Replicación Viral , Sacos Aéreos/virología , Animales , Cloaca/patología , Cloaca/virología , Modelos Animales de Enfermedad , Femenino , Interacciones Huésped-Patógeno , Inmunohistoquímica/veterinaria , Virus de la Influenza A/clasificación , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza A/patogenicidad , Gripe Aviar/patología , Mucosa Intestinal/patología , Mucosa Intestinal/virología , Pulmón/patología , Pulmón/virología , Masculino , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Esparcimiento de VirusRESUMEN
Between June and September 2013, sera from 11 dromedary camels, 150 goats, 126 sheep and 91 cows were collected in Jordan, where the first human Middle-East respiratory syndrome (MERS) cluster appeared in 2012. All sera were tested for MERS-coronavirus (MERS-CoV) specific antibodies by protein microarray with confirmation by virus neutralisation. Neutralising antibodies were found in all camel sera while sera from goats and cattle tested negative. Although six sheep sera reacted with MERS-CoV antigen, neutralising antibodies were not detected.
Asunto(s)
Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Camelus/sangre , Coronavirus/inmunología , Animales , Bovinos , Coronavirus/aislamiento & purificación , Infecciones por Coronavirus/sangre , Femenino , Cabras/sangre , Humanos , Jordania , Ganado , Análisis por Micromatrices , Medio Oriente , Pruebas de Neutralización , Reacción en Cadena en Tiempo Real de la Polimerasa , Infecciones del Sistema Respiratorio/etiología , Ovinos/sangre , SíndromeRESUMEN
We present two real-time reverse-transcription polymerase chain reaction assays for a novel human coronavirus (CoV), targeting regions upstream of the E gene (upE) or within open reading frame (ORF)1b, respectively. Sensitivity for upE is 3.4 copies per reaction (95% confidence interval (CI): 2.56.9 copies) or 291 copies/mL of sample. No cross-reactivity was observed with coronaviruses OC43, NL63, 229E, SARS-CoV, nor with 92 clinical specimens containing common human respiratory viruses. We recommend using upE for screening and ORF1b for confirmation.
Asunto(s)
Infecciones por Coronavirus/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/aislamiento & purificación , Coronavirus Humano 229E/genética , Coronavirus Humano 229E/aislamiento & purificación , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/genética , Coronavirus Humano NL63/genética , Coronavirus Humano NL63/aislamiento & purificación , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/aislamiento & purificación , Humanos , Sistemas de Lectura Abierta , Arabia Saudita , Sensibilidad y Especificidad , Viaje , Proteínas del Envoltorio Viral , Proteínas ViroporinasRESUMEN
Influenza B virus is a human pathogen whose origin and possible reservoir in nature are not known. An influenza B virus was isolated from a naturally infected harbor seal (Phoca vitulina) and was found to be infectious to seal kidney cells in vitro. Sequence analyses and serology indicated that influenza virus B/Seal/Netherlands/1/99 is closely related to strains that circulated in humans 4 to 5 years earlier. Retrospective analyses of sera collected from 971 seals showed a prevalence of antibodies to influenza B virus in 2% of the animals after 1995 and in none before 1995. This animal reservoir, harboring influenza B viruses that have circulated in the past, may pose a direct threat to humans.
Asunto(s)
Virus de la Influenza B/aislamiento & purificación , Infecciones por Orthomyxoviridae/veterinaria , Phocidae/virología , Animales , Anticuerpos Antivirales/sangre , Línea Celular , Células Cultivadas , Reservorios de Enfermedades , Perros , Ensayo de Inmunoadsorción Enzimática , Genes Virales , Pruebas de Inhibición de Hemaglutinación , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Virus de la Influenza B/clasificación , Virus de la Influenza B/genética , Virus de la Influenza B/inmunología , Pruebas de Neutralización , Infecciones por Orthomyxoviridae/epidemiología , Infecciones por Orthomyxoviridae/virología , Faringe/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas no Estructurales Virales/genética , Esparcimiento de VirusRESUMEN
Using reverse transcription/polymerase chain reaction (RT-PCR), we have screened more than 8500 wild birds in Northern Europe in 1999 and 2000 for the presence of influenza A virus. Although our primary focus was on ducks, geese, and shorebirds, we have also tested thousands of samples from other bird species. Approximately 1% of our samples were positive for influenza A virus by RT-PCR, and from half of these we were able to isolate influenza A virus in embryonated chicken eggs. A wide variety of isolates was obtained representing hemagglutinin (HA) subtypes 1 through 7, 10, 11, 13, an unidentifiable HA, and neuraminidase (NA) subtypes 1 through 8.
Asunto(s)
Aves/virología , Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/epidemiología , Animales , Animales Salvajes , Embrión de Pollo/virología , Patos , Europa (Continente)/epidemiología , Gansos , Virus de la Influenza A/clasificación , Virus de la Influenza A/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cynomolgus macaques (Macaca fascicularis) infected with influenza virus A/HongKong/156/97 (H5N1) developed acute respiratory distress syndrome (ARDS) with fever. Reverse transcriptase/polymerase chain reaction (RT/PCR) and virus isolation showed that the respiratory tract is the major target of the virus. The main lesion observed upon necropsy, performed 4 or 7 days postinfection, was a necrotizing bronchointerstitial pneumonia, similar to that found in primary influenza pneumonia in human beings. By immunohistochemistry, influenza virus antigen proved to be limited to pulmonary tissue and tonsils. The data indicate that ARDS and multiple organ dysfunction syndrome (MODS), observed in both humans and monkeys infected with this virus, are caused by diffuse alveolar damage from virus replication in the lungs alone.
Asunto(s)
Subtipo H5N1 del Virus de la Influenza A , Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/fisiopatología , Animales , Modelos Animales de Enfermedad , Macaca fascicularis , Infecciones por Orthomyxoviridae/virología , Síndrome de Dificultad Respiratoria/fisiopatología , Síndrome de Dificultad Respiratoria/virologíaRESUMEN
Newcastle disease virus (NDV) is an avian paramyxovirus with oncolytic potential. Detailed preclinical information regarding the safety of oncolytic NDV is scarce. In this study, we evaluated the toxicity, biodistribution and shedding of intravenously injected oncolytic NDVs in non-human primates (Macaca fascicularis). Two animals were injected with escalating doses of a non-recombinant vaccine strain, a recombinant lentogenic strain or a recombinant mesogenic strain. To study transmission, naive animals were co-housed with the injected animals. Injection with NDV did not lead to severe illness in the animals or abnormalities in hematologic or biochemistry measurements. Injected animals shed low amounts of virus, but this did not lead to seroconversion of the contact animals. Postmortem evaluation demonstrated no pathological changes or evidence of virus replication. This study demonstrates that NDV generated in embryonated chicken eggs is safe for intravenous administration to non-human primates. In addition, our study confirmed results from a previous report that naïve primate and human sera are able to neutralize egg-generated NDV. We discuss the implications of these results for our study and the use of NDV for virotherapy.
Asunto(s)
Antineoplásicos/farmacocinética , Antineoplásicos/toxicidad , Virus de la Enfermedad de Newcastle/genética , Viroterapia Oncolítica/métodos , Alantoides/virología , Animales , Antineoplásicos/administración & dosificación , Línea Celular , Embrión de Pollo , ADN Complementario/genética , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inyecciones Intravenosas , Macaca fascicularis , Masculino , Mutagénesis Sitio-Dirigida , Pruebas de Neutralización , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vacunas Virales/administración & dosificación , Esparcimiento de VirusRESUMEN
Infections with low pathogenic avian influenza (LPAI) A(H7N9) viruses have caused more than 100 hospitalized human cases of severe influenza in China since February 2013 with a case fatality rate exceeding 25%. Most of these human infections presented with severe viral pneumonia, while limited information is available currently on the occurrence of mild and subclinical cases. In the present study, a ferret model for this virus infection in humans is presented to evaluate the pathogenesis of the infection in a mammalian host, as ferrets have been shown to mimic the pathogenesis of human infection with influenza viruses most closely. Ferrets were inoculated intratracheally with increasing doses (>10 e5 TCID50) of H7N9 influenza virus A/Anhui/1/2013 and were monitored for clinical and virological parameters up to four days post infection. Virus replication was detected in the upper and lower respiratory tracts while animals developed fatal viral pneumonia. This study illustrates the high pathogenicity of LPAI-H7N9 virus for mammals. Furthermore, the intratracheal inoculation route in ferrets proofs to offer a solid model for LPAI-H7N9 virus induced pneumonia in humans. This model will facilitate the development and assessment of clinical intervention strategies for LPAI-H7N9 virus infection in humans, such as preventive vaccination and the use of antivirals.
Asunto(s)
Modelos Animales de Enfermedad , Subtipo H7N9 del Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/mortalidad , Infecciones por Orthomyxoviridae/virología , Estructuras Animales/virología , Animales , Aves , China , Femenino , Hurones , Humanos , Gripe Aviar/virología , Gripe Humana/virología , Infecciones por Orthomyxoviridae/patología , Sistema Respiratorio/virología , Análisis de SupervivenciaRESUMEN
Reverse genetics can be used to produce recombinant influenza A viruses containing virtually every desired combination of hemagglutinin (HA) and neuraminidase (NA) genes using the virus backbone of choice. Here, a repository of plasmids and recombinant viruses representing all contemporary Eurasian HA and NA subtypes, H1-H16 and N1-N9, was established. HA and NA genes were selected based on sequence analyses of influenza virus genes available from public databases. Prototype Eurasian HA and NA genes were cloned in bidirectional reverse genetics plasmids. Recombinant viruses based on the virus backbone of A/PR/8/34, and containing a variety of HA and NA genes were produced in 293T cells. Virus stocks were produced in MDCK cells and embryonated chicken eggs. These plasmids and viruses may be useful for numerous purposes, including influenza virus research projects, vaccination studies, and to serve as reference reagents in diagnostic settings.
Asunto(s)
Vectores Genéticos , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Virus de la Influenza A/genética , Neuraminidasa/genética , Recombinación Genética , Animales , Línea Celular , Pollos , Perros , Genes Virales , Humanos , Virus de la Influenza A/enzimología , Óvulo/virología , Filogenia , Plásmidos , Cultivo de VirusRESUMEN
BACKGROUND: Rapid and specific molecular tests for identification of the recently identified pandemic influenza A/H1N1 2009 virus as well as rapid molecular tests to identify antiviral resistant strains are urgently needed. OBJECTIVES: We have evaluated the performance of two novel reverse transcriptase polymerase chain reactions (RT-PCRs) targeting specifically hemagglutinin and neuraminidase of pandemic influenza A/H1N1 virus in combination with a conserved matrix PCR. In addition, we investigated the performance of a novel discrimination RT-PCR for detection of the H275Y resistance mutation in the neuraminidase gene. STUDY DESIGN: Clinical performance of both subtype specific RT-PCR assays was evaluated through analysis of 684 throat swaps collected from individuals meeting the WHO case definition for the novel pandemic influenza virus. Analytical performance was analyzed through testing of 10-fold serial dilutions of RNA derived from the first Dutch sequenced and cultured confirmed case of novel pandemic influenza infection. Specificity and discriminative capacities of the H275Y discrimination assay were performed by testing wild type and recombinant H275Y pandemic influenza. RESULTS: 121 throat swaps collected from April 2009 to July 2009 were positive by at least two out of three RT-PCRs, and negative for the seasonal H3/H1 subtype specific RT-PCR assays. 117 of these were tested positive for all three (Ct-values from 15.1 to 36.8). No oseltamivir resistance was detected. CONCLUSIONS: We present a sensitive and specific approach for detection of pandemic influenza A/H1N1 2009 and a rapid RT-PCR assay detecting a primary oseltamivir resistance mutation which can be incorporated easily into clinical virology algorithms.
Asunto(s)
Brotes de Enfermedades , Subtipo H1N1 del Virus de la Influenza A/genética , Gripe Humana/epidemiología , Gripe Humana/virología , Neuraminidasa/genética , Oseltamivir/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Algoritmos , Antivirales/farmacología , Antivirales/uso terapéutico , Farmacorresistencia Viral , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/tratamiento farmacológico , Modelos Lineales , Oseltamivir/uso terapéutico , Mutación Puntual , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Proteínas Virales/genéticaRESUMEN
Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children. Immunopathology may play a role in RSV-induced disease and a severe RSV infection may also be associated with an increased risk of developing asthma. Vaccination with formalin-inactivated RSV (FI-RSV) prior to infection resulted both in human and in the mouse model in extensive lung pathology. In the mouse model, it has been shown that this aggravation of disease was associated with a shift in the balance between Th1 and Th2 cytokines towards a Th2-type response. The aim of the present study was to characterise the immunological and inflammatory responses in BALB/c mice upon RSV infection with or without prior vaccination with aluminium-adjuvanted FI-RSV or control antigens (FI-Mock). As previously reported by others, we also observed that a primary RSV infection in BALB/c mice resulted in a predominant Th1-type cytokine response, which was associated with slight bronchiolitis and alveolitis. FI-RSV vaccination prior to RSV challenge prevented virus replication and was associated with an aggravation of pulmonary histopathology and a shift towards a Th2-type response. Vaccination with FI-Mock did not prevent RSV replication in the lung but resulted in an even more pronounced Th2 response after infection while these mice were not sensitised to specific viral antigens. Thus, viral replication in a Th2 responding animal (induced by aluminium-adjuvanted mock vaccine) appears to boost the Th2 response upon RSV infection.
Asunto(s)
Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/prevención & control , Virus Sincitiales Respiratorios/inmunología , Virus Sincitiales Respiratorios/patogenicidad , Vacunas Virales/administración & dosificación , Vacunas Virales/toxicidad , Animales , Anticuerpos Antivirales/biosíntesis , Preescolar , Citocinas/biosíntesis , Citocinas/genética , Femenino , Formaldehído , Humanos , Inmunización , Lactante , Inflamación/etiología , Inflamación/patología , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , ARN Mensajero/genética , ARN Mensajero/metabolismo , Infecciones por Virus Sincitial Respiratorio/patología , Virus Sincitiales Respiratorios/fisiología , Células TH1/inmunología , Células Th2/inmunología , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/toxicidad , Replicación ViralRESUMEN
Mycoplasma contamination of biological materials remains a major problem. Most contaminations are caused by the use of Mycoplasma-contaminated cell lines. We adapted a Mycoplasma group-specific PCR to detect Mycoplasma contamination in cell lines and demonstrate its use in monitoring decontamination procedures with Mycoplasma-contaminated suspensions of Chlamydia spp. Three different methods were investigated: the use of Mycoplasma-specific antiserum in cell culture, physical separation by the combined use of enzymatic treatment and differential centrifugation, and the use of detergents. With these methods only incubation with Triton X-100 resulted in decontamination of Mycoplasma-contaminated suspensions of several laboratory strains of Chlamydia pneumoniae, C. pecorum, and C. trachomatis. Only one C. pneumoniae strain, UZG-1, was sensitive to Triton X-100 treatment. Since 39 of 40 throat swabs from patients with symptoms of an upper respiratory tract infection had positive reactions in the Mycoplasma group-specific PCR, this procedure could also have clinical significance in attempts to propagate C. pneumoniae strains from clinical specimens.
Asunto(s)
Chlamydia/genética , Mycoplasma/genética , Mycoplasma/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Chlamydia/crecimiento & desarrollo , Chlamydia/aislamiento & purificación , Chlamydia trachomatis/genética , Chlamydia trachomatis/crecimiento & desarrollo , Chlamydia trachomatis/aislamiento & purificación , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/crecimiento & desarrollo , Chlamydophila pneumoniae/aislamiento & purificación , Chlamydophila psittaci/genética , Chlamydophila psittaci/crecimiento & desarrollo , Chlamydophila psittaci/aislamiento & purificación , Cartilla de ADN/genética , Sondas de ADN/genética , ADN Bacteriano/genética , Estudios de Evaluación como Asunto , Células HeLa , Humanos , Datos de Secuencia MolecularRESUMEN
The recently raised awareness of the threat of a new influenza pandemic has stimulated interest in the detection of influenza A viruses in human as well as animal secretions. Virus isolation alone is unsatisfactory for this purpose because of its inherent limited sensitivity and the lack of host cells that are universally permissive to all influenza A viruses. Previously described PCR methods are more sensitive but are targeted predominantly at virus strains currently circulating in humans, since the sequences of the primer sets display considerable numbers of mismatches to the sequences of animal influenza A viruses. Therefore, a new set of primers, based on highly conserved regions of the matrix gene, was designed for single-tube reverse transcription-PCR for the detection of influenza A viruses from multiple species. This PCR proved to be fully reactive with a panel of 25 genetically diverse virus isolates that were obtained from birds, humans, pigs, horses, and seals and that included all known subtypes of influenza A virus. It was not reactive with the 11 other RNA viruses tested. Comparative tests with throat swab samples from humans and fecal and cloacal swab samples from birds confirmed that the new PCR is faster and up to 100-fold more sensitive than classical virus isolation procedures.
Asunto(s)
Virus de la Influenza A/aislamiento & purificación , Gripe Humana/virología , Reacción en Cadena de la Polimerasa/métodos , Proteínas de la Matriz Viral/genética , Animales , Enfermedades de las Aves/virología , Aves/virología , Secuencia Conservada/genética , Cartilla de ADN/genética , Humanos , Virus de la Influenza A/genética , Gripe Humana/diagnóstico , Faringe/virología , Sensibilidad y EspecificidadRESUMEN
Cynomolgus macaques (Macaca fascicularis) infected with influenza virus A/Hong Kong/156/97 (H5N1) developed acute respiratory distress syndrome and fever associated with a necrotizing interstitial pneumonia. Reverse transcription PCR, virus isolation, and immunohistochemistry showed that the respiratory tract is the major target of the virus.
Asunto(s)
Subtipo H5N1 del Virus de la Influenza A , Virus de la Influenza A , Infecciones por Orthomyxoviridae/virología , Animales , Modelos Animales de Enfermedad , Fiebre/patología , Inmunohistoquímica , Virus de la Influenza A/aislamiento & purificación , Enfermedades Pulmonares Intersticiales/patología , Enfermedades Pulmonares Intersticiales/virología , Macaca fascicularis , Necrosis , Síndrome de Dificultad Respiratoria/patología , Vísceras/virologíaRESUMEN
Viruses exploit different strategies to escape immune surveillance, including the introduction of mutations in cytotoxic T-lymphocyte (CTL) epitopes. The sequence of these epitopes is critical for their binding to major histocompatibility complex (MHC) class I molecules and recognition by specific CTLs, both of which interactions may be lost by mutation. Sequence analysis of the nucleoprotein gene of influenza A viruses (H3N2) isolated in The Netherlands from 1989 to 1999 revealed two independent amino acid mutations at the anchor residue of the HLA-B27-specific CTL epitope SRYWAIRTR (383 to 391). A R384K mutation was found in influenza A viruses isolated during the influenza season 1989-1990 but not in subsequent seasons. In the influenza season 1993-1994, a novel mutation in the same CTL epitope at the same position was introduced. This R384G mutation proved to be conserved in all influenza A viruses isolated from 1993 onwards. Both mutations R384K and R384G abrogated MHC class I presentation and allowed escape from recognition by specific CTLs.
Asunto(s)
Variación Antigénica , Subtipo H3N2 del Virus de la Influenza A , Virus de la Influenza A/inmunología , Nucleoproteínas/genética , Nucleoproteínas/inmunología , Proteínas de Unión al ARN , Linfocitos T Citotóxicos/inmunología , Proteínas del Núcleo Viral/genética , Proteínas del Núcleo Viral/inmunología , Secuencia de Aminoácidos , Antígenos Virales/inmunología , Epítopos de Linfocito T , Antígeno HLA-B27/inmunología , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/aislamiento & purificación , Gripe Humana/virología , Datos de Secuencia Molecular , Mutación , Proteínas de la Nucleocápside , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Filogenia , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/metabolismoRESUMEN
A monoclonal antibody LMBH5 was derived from mice which had been immunized with A/Victoria/3/75 (H3N2)-type recombinant, secreted hemagglutinin (HA), and were subsequently challenged with a potentially lethal dose of X31 [A/Aichi/2/68 (H3N2) x A/PR/8/34 (H1N1)] virus. LMBH5 reacted strongly with the native and low-pH-induced conformations of the HA of A/Aichi (X31 strain) and A/Victoria (X47 strain), but very weakly with the native structure of the HA of A/Philippines/2/82 (X79 strain) and not at all with the HA of A/Guizhou/54/89 H3 (NIB25 strain). However, the acid-induced conformations of the latter two viruses were recognized by LMBH5. The antibody prevented infection of MDCK cells with X31 and X47, whereas X79 virus was partially neutralized by LMBH5. X31 monoclonal escape variants had single amino acid substitutions (Ser 227-->Pro) near the interface. The data obtained suggest that the neutralizing LMBH5 reacts with a fairly conserved epitope of influenza A (H3N2) virus, which as a result of antigenic drift becomes inaccessible in the native state of the HA.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antivirales , Epítopos/análisis , Hemaglutininas Virales/inmunología , Subtipo H3N2 del Virus de la Influenza A , Virus de la Influenza A/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/metabolismo , Epítopos/química , Genes Virales/genética , Pruebas de Hemaglutinación , Hemaglutininas Virales/genética , Hemaglutininas Virales/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ratones , Mutación , Pruebas de Neutralización , Conformación Proteica , Especificidad de la Especie , Tripsina , Proteínas Estructurales Virales/genéticaRESUMEN
BACKGROUND: Th2 lymphocyte responses are associated with inflammation and disease during allergic responses. Exposure to particular environmental factors during the expression of allergy could result in more pronounced Th2-like immune responses and more severe disease. One factor might be a respiratory virus infection. OBJECTIVE: The aim of our study was to investigate the influence of respiratory syncytial virus (RSV) infection on the expression of ovalbumin (OVA)-induced allergy in BALB/c mice. METHODS: We determined OVA-specific IgE in serum, cytokine profiles and histopathological lesions in lungs of OVA-allergic mice after RSV infection. RESULTS: OVA sensitization and challenge induced OVA-specific IgE in serum, Th2 cytokine mRNA expression, and mononuclear and eosinophilic inflammation in the lungs. RSV inoculation during the challenge period enhanced OVA-induced IL-4 and IL-5 mRNA expression in lung tissue. RSV further enhanced the OVA-induced hypertrophy of mucous cells and eosinophilic infiltration in lung tissue. Surprisingly, RSV infection decreased Th2 cytokine secretion and eosinophilic influx in bronchoalveolar lavage of OVA-allergic mice. Because inactivated RSV did not influence these responses, replication of RSV appeared essential for the modification of OVA-induced Th2 cytokine expression. RSV did not change OVA-specific IgE levels in serum. Furthermore, the RSV-induced IL-12 mRNA expression in lung tissue of OVA-allergic mice was diminished, but IFN-gamma mRNA expression was not affected. CONCLUSION: RSV infection enhanced particular OVA-induced Th2 cytokine mRNA responses and pulmonary lesions in allergic mice and thus aggravated allergic respiratory disease.
Asunto(s)
Citocinas/biosíntesis , Citocinas/inmunología , Neumonía/inmunología , Neumonía/fisiopatología , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/fisiopatología , Virus Sincitial Respiratorio Humano , Animales , Especificidad de Anticuerpos/inmunología , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Modelos Animales de Enfermedad , Femenino , Hipersensibilidad/inmunología , Hipersensibilidad/fisiopatología , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Pulmón/irrigación sanguínea , Pulmón/citología , Pulmón/metabolismo , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/efectos adversos , Ovalbúmina/inmunología , ARN Mensajero/biosíntesis , ARN Mensajero/inmunología , Índice de Severidad de la Enfermedad , Factores de Tiempo , Rayos UltravioletaRESUMEN
This paper describes the development and evaluation of a new nested reverse transcription (RT)-PCR for the detection of rhinovirus in clinical samples. The nucleotide sequences of the 5' noncoding regions of 39 rhinoviruses were determined in order to map the most conserved subregions. We designed a set of rhinovirus-specific primers and probes directed to these subregions and developed a new nested RT-PCR. The new assay includes an optimal RNA extraction method and amplicon identification with probe hybridization to discriminate between rhinoviruses and the closely related enteroviruses. It proved to be highly sensitive and specific. When tested on a dilution series of cultured viruses, the new PCR protocol scored positive at 10- to 100-fold-higher dilutions than a previously used nested RT-PCR. When tested on a collection of clinical samples obtained from 1,070 acute respiratory disease patients who had consulted their general practitioners, the new assay demonstrated a rhinovirus in 24% of the specimens, including all culture-positive samples, whereas the previously used PCR assay or virus culture detected a rhinovirus in only 3.5 to 6% of the samples. This new assay should help determine the disease burden associated with rhinovirus infections.