RESUMO
We previously identified a subset of interferon-stimulated genes (ISGs) upregulated by West Nile virus (WNV) infection in wild-type mouse embryo fibroblasts (MEFs) after viral proteins had inhibited type I interferon (IFN)-mediated JAK-STAT signaling and also in WNV-infected RIG-I-/-, MDA5-/-, STAT1-/-, STAT2-/-, IFNAR-/-, IRF3-/-, IRF7-/-, and IRF3/7-/- MEFs. In this study, ISG upregulation by WNV infection in IFNAR-/- MEFs was confirmed by transcriptome sequencing (RNA-seq). ISG upregulation by WNV infection was inhibited in RIG-I/MDA5-/- MEFs. ISGs were upregulated in IRF1-/- and IRF5-/- MEFs but only minimally upregulated in IRF3/5/7-/- MEFs, suggesting redundant IRF involvement. We previously showed that a single proximal interferon-stimulated response element (ISRE) in the Oas1a and Oas1b promoters bound the ISGF3 complex after type I IFN treatment. In this study, we used wild-type and mutant promoter luciferase reporter constructs to identify critical regions in the Oas1b and Ifit1 promoters for gene activation in infected IFNAR-/- MEFs. Two ISREs were required in both promoters. Mutation of these ISREs in an Ifit1 promoter DNA probe reduced in vitro complex formation with infected nuclear extracts. An NF-κB inhibitor decreased Ifit1 promoter activity in cells and in vitro complex formation. IRF3 and p50 promoter binding was detected by chromatin immunoprecipitation (ChIP) for upregulated ISGs with two proximal ISREs. The data indicate that ISREs function cooperatively to upregulate the expression of some ISGs when type I IFN signaling is absent, with the binding complex consisting of IRF3, IRF5, and/or IRF7 and an NF-κB component(s) as well as other, as-yet-unknown factors. IMPORTANCE Type I IFN signaling in mammalian cells induces formation of the ISGF3 transcription factor complex, which binds to interferon stimulated response elements (ISREs) in the promoters of interferon-stimulated genes (ISGs) in the cell nucleus. Flavivirus proteins counteract type I IFN signaling by preventing either the formation or nuclear localization of ISGF3. A subset of ISRE-regulated ISGs was still induced in West Nile virus (WNV)-infected mouse embryo fibroblasts (MEFs), indicating that cells have an alternative mechanism for activating these ISGs. In this study, cellular components involved in this ISG upregulation mechanism were identified using gene knockout MEFs and ChIP, and critical promoter regions for gene activation were mapped using reporter assays. The data indicate a cooperative function between two ISREs and required binding of IRF3, IRF5, and/or IRF7 and an NF-κB component(s). Moreover, type I IFN signaling-independent ISG activation requires different additional promoter activation regions than type I IFN-dependent activation.
Assuntos
Fibroblastos , Regulação da Expressão Gênica/imunologia , Interferon Tipo I/imunologia , Febre do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/imunologia , Animais , Fibroblastos/imunologia , Fibroblastos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Elementos de Resposta/imunologiaRESUMO
A human adenovirus (HAdV) species D, was isolated from a hospitalised child with severe lower respiratory infection. It was initially detected in the nasopharyngeal aspirate of the child followed by conventional PCR amplification of the hexon, penton base, and fibre genes. Sanger DNA sequencing and phylogenetic analyses showed characteristics of a recombinant genome not described before. Next Generation Sequencing analysis was performed to reconstruct its complete DNA genome after viral isolation in adenocarcinoma human cell line (A549). A complete genomic sequence of 35.2 kb in length, with a G+C content of 57â% was obtained, related to HAdV-D29 (96â% identity). Imputed serology analysis demonstrated its novel type with a nucleotide sequence identity of 95.3â% (hexon loop 1) and 96â% (hexon loop 2) to HAdV-D9. The penton base gene showed a novel sequence, distantly related to HAdV-D44. The E3 and E4 regions evolved significantly from their ancestors. The fibre gene was almost identical to the knob region of HAdV-D15 but showed an unrelated shaft sequence. In conclusion the genomics of this novel HAdV, designated the HAdV-D83 [P83H9F15] prototype and bearing a new penton base gene, supports the importance of viral evolution to understand modified tissue tropism, enhanced transmission, or altered virulence.
Assuntos
Adenovírus Humanos/classificação , Adenovírus Humanos/isolamento & purificação , Células A549 , Infecções por Adenoviridae/virologia , Adenovírus Humanos/genética , Composição de Bases , DNA Viral/química , DNA Viral/genética , Genoma Viral , Humanos , Lactente , Masculino , Nasofaringe/virologia , Filogenia , Reação em Cadeia da Polimerase , Infecções Respiratórias/virologia , Análise de Sequência de DNA , Homologia de Sequência , Infecções Tumorais por Vírus/virologia , Proteínas Estruturais Virais/genética , Cultura de VírusRESUMO
Human rhinovirus (HRV), a single-stranded, positive-sense RNA virus, is associated with mild upper respiratory tract infections in children. The aim of this study was to carry out a molecular characterization and phylogeny-based classification of the circulating genotypes of HRV in hospitalized children with clinical manifestations of acute lower respiratory infection in Paraguay. Nasopharyngeal aspirates were collected from 101 children under 5 years of age, hospitalized with symptoms of acute lower respiratory infection, between May 2010 and December 2011, at the largest public pediatric hospital in the Central Department of Paraguay. Detection was performed by a real-time polymerase chain reaction, followed by conventional amplification of the VP4/VP2 genomic region, sequencing, and phylogenetic analysis. Rhinovirus was detected in 33.7% of the samples. Amplification of 18 samples showed the presence of all three species (HRV-A, -B, and -C). Different genotypes were found for each species: 11 for HRV-A (-9, -12, -22, -30, -36, -43, -59, -61, -68, -88, and -89), one for HRV-B (-4), and four for HRV-C (-C2, -C3, -C6, and -C9). In South America, information about HRV diversity is scarce. This is the first report on HRV genotype diversity in South America.
Assuntos
Variação Genética , Filogenia , Infecções Respiratórias/virologia , Rhinovirus/classificação , Rhinovirus/genética , Criança Hospitalizada , Pré-Escolar , Feminino , Genótipo , Humanos , Lactente , Masculino , Epidemiologia Molecular , Dados de Sequência Molecular , Nasofaringe/virologia , Paraguai/epidemiologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Infecções Respiratórias/epidemiologia , Rhinovirus/isolamento & purificação , Análise de Sequência de DNARESUMO
BACKGROUND: The National Program for Chagas disease was implemented in Bolivia in 2006, and it greatly decreased the number of infections through vector control. Subsequently, a treatment regimen of benznidazole (BNZ) was started in seropositive school-age children living in certified vector control areas. METHODS AND FINDINGS: We conducted a 12-month follow-up study and seven blood samples were taken during and after the treatment. Serology, conventional diagnostic PCR (cPCR) and quantitative Real-time PCR (qPCR) were performed. Plasma Th1/Th2/Th17 cytokines levels were also determined. Approximately 73 of 103 seropositive children complied with BNZ, with three interruptions due to side effects. To evaluate each individual's treatment efficacy, the cPCR and qPCR values during the final 6 months of the follow-up period were observed. Among 57 children who completed follow-up, 6 individuals (11%) showed both cPCR(+) and qPCR(+) (non reactive), 24 (42%) cPCR(-) but qPCR(+) (ambiguous) and 27 (47%) cPCR(-) and qPCR(-) (reactive). Within 14 Th1/Th2/Th17 cytokines, IL-17A showed significantly higher levels in seropositive children before the treatment compared to age-matched seronegative children and significantly decreased to the normal level one-year after. Moreover, throughout the follow-up study, IL-17A levels were positively co-related to parasite counts detected by qPCR. At the 12 months' time point, IL-17A levels of non-reactive subjects were significantly higher than either those of reactive or ambiguous subjects suggesting that IL-17A might be useful to determine the reactivity to BNZ treatment. CONCLUSIONS: Plasma levels of IL-17A might be a bio-marker for detecting persistent infection of T. cruzi and its chronic inflammation.
Assuntos
Doença de Chagas/tratamento farmacológico , Interleucina-17/sangue , Nitroimidazóis/uso terapêutico , Resultado do Tratamento , Adolescente , Biomarcadores/sangue , Bolívia , Doença de Chagas/sangue , Criança , Pré-Escolar , Citocinas , Feminino , Seguimentos , Humanos , Masculino , Nitroimidazóis/sangue , Reação em Cadeia da Polimerase/métodos , Tripanossomicidas/sangue , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/isolamento & purificaçãoRESUMO
The introduction of different multiplex RT-PCR strategies for the characterization of field rotavirus strains has led to improvements of surveillance systems worldwide. Nevertheless, the failure or incorrect characterization of rotavirus strains by these PCR strategies, mainly due to accumulation of point mutations in the VP4 and VP7 genes, has been reported. In this work, sequence analyses of the VP4 and VP7 genes from Paraguayan G1P[8] and G4P[8] strains revealed that the high degree of similarity with the primers pNCDV and ET10 could lead to the incorrect characterization of these strains as P[1] and G10 types. Moreover, the nucleotide diversity of the VP4 gene at the 1T-1 primer binding site could be one, although not the only, reason of the failure of the P[8] typing. Therefore, the typing methods utilized by surveillance programs should be constantly evaluated and sequencing of atypical strains should become a current practice in order to confirm their real nature.
Assuntos
Antígenos Virais/genética , Proteínas do Capsídeo/genética , Variação Genética , Rotavirus/classificação , Rotavirus/genética , Sequência de Bases , Genótipo , Humanos , Paraguai , Filogenia , RNA Viral/genética , Infecções por Rotavirus/virologiaRESUMO
BACKGROUND: Group A rotaviruses are the main cause of severe gastroenteritis in children worldwide. OBJECTIVES: To survey human rotavirus strains circulating in Paraguay. STUDY DESIGN: One hundred ninety-six rotavirus-positive fecal samples collected from children up to 5 years old, from 2002 to 2005, were characterized. RESULTS: The most common G genotype detected was G9 (36.2%), followed by G1 (34.2%), G2 (11.7%) and G4 (8.7%). Changes in the G genotype frequency were observed from year to year. The G4 genotype was predominant in 2002; G1 in 2003; and G9 from 2004 to 2005. Sequence and phylogenetic analysis of the VP7 gene from Paraguayan G1 strains suggested that the high frequency of G1 in 2003 could be due to the introduction of an atypical sub-lineage. In addition, there were amino acid changes in the variable/antigenic regions of the VP7 gene from G4 and G9 strains detected in different years. CONCLUSIONS: This study further indicates that antigenic pressure can drive the evolution of rotaviruses, and also suggests that a vaccine that protects against the most prevalent strains and its variants, will be necessary to elicit a protective immune response against the range of rotavirus types currently circulating in Paraguay.
Assuntos
Epidemiologia Molecular , Infecções por Rotavirus/epidemiologia , Rotavirus/genética , Sequência de Aminoácidos , Antígenos Virais/genética , Proteínas do Capsídeo/genética , Pré-Escolar , Diarreia/epidemiologia , Fezes/virologia , Variação Genética , Humanos , Lactente , Dados de Sequência Molecular , Paraguai/epidemiologia , Filogenia , Vigilância de Evento Sentinela , Alinhamento de SequênciaRESUMO
In recent years it was reported that the accumulation of point mutations in VP4 and VP7 genes of rotavirus strains was the main cause of the failure of the G or P-typing. Failures in the correct genotyping of G1, G2, G8, G9 and G10 rotavirus strains were reported in the most commonly used reverse transcription (RT)-PCR strategies. Collecting VP7 gene sequences of G1 rotavirus strains from databases we found that 74 (61.2 %) out of 121 G1 strains from lineage I showed the four specific mismatches at the 5' end of the 9T1-1 primer, previously associated with the failure of G1-typing. Thus, a great percentage of the G1 strains from lineage I worldwide reported could not have been typed if the Das's RT-PCR strategy were used. This analysis shows that the failure on the detection of the G1 strains could be due to the diversification of rotavirus strains in phylogenetic lineages. Therefore, the use of different RT-PCR strategies with different primer binding locations on the VP7 gene or new typing methodologies -like microarrays procedures- could be a better option to avoid the failure of the G-typing of rotavirus strains detected during surveillance programs.
Assuntos
Antígenos Virais/genética , Pareamento Incorreto de Bases , Proteínas do Capsídeo/genética , Primers do DNA , Rotavirus/classificação , Rotavirus/genética , Genótipo , Humanos , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Mixed viral and bacterial infections are widely described in community-acquired pneumonia; however, the clinical implications of co-infection on the associated immunopathology remain poorly studied. In this study, microRNA, mRNA and cytokine/chemokine secretion profiling were investigated for human monocyte-derived macrophages infected in-vitro with Influenza virus A/H1N1 and/or Streptococcus pneumoniae. We observed that the in-vitro co-infection synergistically increased interferon-γ-induced protein-10 (CXCL10, IP-10) expression compared to the singly-infected cells conditions. We demonstrated that endogenous miRNA-200a-3p, whose expression was synergistically induced following co-infection, indirectly regulates CXCL10 expression by targeting suppressor of cytokine signaling-6 (SOCS-6), a well-known regulator of the JAK-STAT signaling pathway. Additionally, in a subsequent clinical pilot study, immunomodulators levels were evaluated in samples from 74 children (≤5 years-old) hospitalized with viral and/or bacterial community-acquired pneumonia. Clinically, among the 74 cases of pneumonia, patients with identified mixed-detection had significantly higher (3.6-fold) serum IP-10 levels than those with a single detection (P = 0.03), and were significantly associated with severe pneumonia (P < 0.01). This study demonstrates that viral and bacterial co-infection modulates the JAK-STAT signaling pathway and leads to exacerbated IP-10 expression, which could play a major role in the pathogenesis of pneumonia.
Assuntos
Infecções Bacterianas/imunologia , Quimiocina CXCL10/metabolismo , Coinfecção/microbiologia , Coinfecção/virologia , Imunidade Inata , Pneumonia/imunologia , Pneumonia/microbiologia , Viroses/imunologia , Adulto , Idoso , Infecções Bacterianas/microbiologia , Infecções Bacterianas/virologia , Sequência de Bases , Coinfecção/imunologia , Feminino , Perfilação da Expressão Gênica , Humanos , Imunidade Inata/genética , Lactente , Vírus da Influenza A Subtipo H1N1 , Macrófagos/imunologia , Macrófagos/microbiologia , Macrófagos/virologia , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Pneumonia/virologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Streptococcus pneumoniae , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Pesquisa Translacional Biomédica , Adulto JovemRESUMO
Influenza virus is associated with upper respiratory tract infections. The fourth influenza pandemic was declared in 2009. The aim of this study was to determine the genetic variability of the 2009 H1N1 pandemic virus circulating in Paraguay. Nasal swabs were collected from 181 patients with flu symptoms managed at the Hospital of the Medical School in Asunción, Paraguay, between August and October 2009. Virus detection was carried out by real-time reverse transcription-polymerase chain reaction, followed by sequencing of the hemagglutinin and neuraminidase genes, and phylogenetic analysis. H1N1pdm09 was detected in 14.9% (27/181) of the suspected cases. Analysis of 13 samples showed that these viruses the clustered in a single genetic group. Neither the mutation related to exacerbation of disease (D239G in hemagglutinin) nor that related to antiviral resistance (H275Y in neuraminidase), both detected in neighboring countries, were found. This genetic analysis of H1N1pdm09 will help to understand the spread of the disease.
RESUMO
Influenza A virus (H1N1), which arose in 2009, constituted the fourth pandemic after the cases of 1918, 1957, and 1968. This new variant was formed by a triple reassortment, with genomic segments from swine, avian, and human influenza origins. The objective of this study was to analyze sequences of hemagglutinin (n=2038) and neuraminidase (n=1273) genes, in order to assess the extent of diversity among circulating 2009-2010 strains, estimate if these genes evolved through positive, negative, or neutral selection models of evolution during the pandemic phase, and analyze the worldwide percentage of detection of important amino acid mutations that could enhance the viral performance, such as transmissibility or resistance to drugs. A continuous surveillance by public health authorities will be critical to monitor the appearance of new influenza variants, especially in animal reservoirs such as swine and birds, in order to prevent the potential animal-human transmission of viruses with pandemic potential.
RESUMO
El género Enterovirus es un grupo viral que afecta a un amplio rango de hospederos, entre ellos los humanos (especies A, B, C, y D), causan enfermedades respiratorias, gastrointestinales, neurológicas, y otras, y son altamente contagiosos. Los síntomas pueden ser leves o graves. El objetivo del trabajo fue analizar la variación nucleotídica, filogenética y de presión evolutiva de secuencias nucleotídicas del gen VP4 de las cuatro especies que afectan a los humanos. Se emplearon 92 secuencias nucleotídicas disponibles en la base de datos GenBank; éstas se editaron con el software BioEdit y se alinearon con Clustal W; las relaciones filogenéticas se determinaron con MEGA6, y las presiones evolutivas con los algoritmos SNAP y SLAC. Se encontró que la identidad nucleotídica mínima intra-especie fue de 43,2% (especie B) a 72,6% (especie D). Los genotipos más variables por especie fueron EV-71 (A), Echovirus 2 (B), EV-118 (C), y EV-94 (D). El análisis de presión evolutiva mostró que el gen VP4 en las cuatro especies evoluciona bajo presión selectiva negativa. Esto indicaría que la alta tasa mutacional y eventos de recombinación no tienen un rol significativo en la evolución de este gen, debido probablemente a la localización interna de la proteína VP4.
The Enterovirus genus is a viral group that affects a wide host range, including humans (species A, B, C and D), cause respiratory, gastrointestinal, and neurologic disease, amongothers, and are highly contagious. The symptoms range from mild to severe. The objectiveof this study was to perform a nucleotidic variation, phylogenetic and selective pressureanalyses of the VP4 gene from the four enterovirus species that affect humans. Ninety-twonucleotide sequences (available in the GenBank database) were employed; they were edited with Bio Edit software and aligned with Clustal W; the phylogenetic relationships weredetermined with MEGA6, and the evolutive pressures with SNAP and SLAC algorithms. Itwas found an intra-species nucleotide identity of at least 43,2% (species B) to 72,6% (species D). The more variable genotypes by species were EV-71 (A), Echovirus 2 (B), EV-118 (C), and EV-94 (D). The selective pressure analysis showed that VP4 gene of the fourspecies evolves by negative pressure. This would indicate that the high mutation rate andrecombination events do not have a significant role in the evolution of this gene, probablydue to the internal localization of the VP4 protein.
Assuntos
Humanos , Enterovirus Humano A , Infecções por EnterovirusAssuntos
Genótipo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/classificação , Vírus Sincicial Respiratório Humano/genética , Pré-Escolar , Análise por Conglomerados , Humanos , Lactente , Recém-Nascido , Dados de Sequência Molecular , Paraguai/epidemiologia , Filogenia , Infecções por Vírus Respiratório Sincicial/epidemiologia , Vírus Sincicial Respiratório Humano/isolamento & purificação , Análise de Sequência de DNA , Homologia de SequênciaAssuntos
Infecções por Adenovirus Humanos/epidemiologia , Adenovírus Humanos/genética , Criança Hospitalizada/estatística & dados numéricos , Variação Genética , Infecções Respiratórias/epidemiologia , Doença Aguda , Infecções por Adenovirus Humanos/fisiopatologia , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/classificação , Adenovírus Humanos/isolamento & purificação , Proteínas do Capsídeo/genética , Pré-Escolar , Humanos , Lactente , Dados de Sequência Molecular , Paraguai/epidemiologia , Filogenia , Reação em Cadeia da Polimerase , Infecções Respiratórias/fisiopatologia , Infecções Respiratórias/virologia , Análise de Sequência de DNARESUMO
BACKGROUND: Rotavirus is considered the main viral cause of acute gastroenteritis in children in both developed and developing countries. The aim of the present study was to continue the surveillance of rotavirus in the Paraguayan population in anticipation of a rotavirus vaccination in children. MATERIAL/METHODS: Fecal samples from infants (< or =5 years of age) and adults with diarrhea (912 and 801 samples, respectively) were collected in Paraguay during 2004-2005. Rotavirus incidence was screened by PAGE and genotyping was performed by reverse transcription (RT)-PCR. RESULTS: Rotavirus incidence was 23.8% and 19.4% for children and adults, respectively. The rotavirus incidence was higher in the coolest and driest months of the year. Five different group A rotavirus electropherotypes were detected. Rotaviruses with a long electropherotype were the most frequently detected in children in 2004 and 2005. However, in 2005 (after six years of absence in Paraguay) rotaviruses with a short electropherotype were detected at high frequency in both children and adults. Of these, 14 samples were genotyped (11 from children and 3 from adults) and all of them showed the G2P[4] type. CONCLUSIONS: This study reinforces the importance of continuous survey of rotavirus infection, extended to all age groups, in order to increase our knowledge about the complexity of rotavirus epidemiology.