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1.
Cell ; 181(4): 865-876.e12, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32353252

RESUMO

The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.


Assuntos
Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Sistemas CRISPR-Cas , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , RNA Viral/antagonistas & inibidores , Células A549 , Antibioticoprofilaxia/métodos , Sequência de Bases , Betacoronavirus/genética , Betacoronavirus/crescimento & desenvolvimento , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Simulação por Computador , Sequência Conservada , Coronavirus/efeitos dos fármacos , Coronavirus/genética , Coronavirus/crescimento & desenvolvimento , Infecções por Coronavirus/tratamento farmacológico , Células Epiteliais/virologia , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Pulmão/patologia , Pulmão/virologia , Proteínas do Nucleocapsídeo/genética , Pandemias , Filogenia , Pneumonia Viral/tratamento farmacológico , RNA Replicase/genética , Proteínas não Estruturais Virais/genética
2.
PLoS One ; 15(2): e0228029, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32045419

RESUMO

Influenza A (H1N1) pdm09 virus emerged in North America in 2009 and has been established as a seasonal strain in humans. After an antigenic stasis of about six years, new antigenically distinct variants of the virus emerged globally in 2016 necessitating a change in the vaccine formulation for the first time in 2017. Herein, we analyzed thirty-eight HA sequences of influenza A (H1N1) pdm09 strains isolated in Kenya during 2015-2018 seasons, to evaluate their antigenic and molecular properties based on the HA1 sub-unit. Our analyses revealed that the A (H1N1) pdm09 strains that circulated in Kenya during this period belonged to genetic clade 6B, subclade 6B.1 and 6B.2. The Kenyan 2015 and 2016 isolates differed from the vaccine strain A/California/07/2009 at nine and fourteen antigenic sites in the HA1 respectively. Further, those isolated in 2017 and 2018 correspondingly varied from A/Michigan/45/2015 vaccine strain at three and fifteen antigenic sites. The predicted vaccine efficacy of A/California/07/2009 against Kenyan 2015/2016 was estimated to be 32.4% while A/Michigan/45/2015 showed estimated vaccine efficacies of 39.6% - 41.8% and 32.4% - 42.1% against Kenyan 2017 and 2018 strains, respectively. Hemagglutination-inhibition (HAI) assay using ferret post-infection reference antiserum showed that the titers for the Kenyan 2015/2016 isolates were 2-8-fold lower compared to the vaccine strain. Overall, our results suggest the A (H1N1) pdm09 viruses that circulated in Kenya during 2015/2016 influenza seasons were antigenic variants of the recommended vaccine strains, denoting sub-optimal vaccine efficacy. Additionally, data generated point to a swiftly evolving influenza A (H1N1) pdm09 virus in recent post pandemic era, underscoring the need for sustained surveillance coupled with molecular and antigenic analyses, to inform appropriate and timely influenza vaccine update.


Assuntos
Antígenos Virais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/imunologia , Filogenia , Subunidades Proteicas/imunologia , Sequência de Aminoácidos , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vacinas contra Influenza/imunologia , Quênia , Homologia de Sequência de Aminoácidos , Organização Mundial da Saúde
3.
Int J Infect Dis ; 90: 84-96, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31669593

RESUMO

BACKGROUND: This study compared the genomes of influenza viruses that caused mild infections among outpatients and severe infections among hospitalized patients in Singapore, and characterized their molecular evolution and receptor-binding specificity. METHODS: The complete genomes of influenza A/H1N1, A/H3N2 and B viruses that caused mild infections among outpatients and severe infections among inpatients in Singapore during 2012-2015 were sequenced and characterized. Using various bioinformatics approaches, we elucidated their evolutionary, mutational and structural patterns against the background of global and vaccine strains. RESULTS: The phylogenetic trees of the 8 gene segments revealed that the outpatient and inpatient strains overlapped with representative global and vaccine strains. We observed a cluster of inpatients with A/H3N2 strains that were closely related to vaccine strain A/Texas/50/2012(H3N2). Several protein sites could accurately discriminate between outpatient versus inpatient strains, with site 221 in neuraminidase (NA) achieving the highest accuracy for A/H3N2. Interestingly, amino acid residues of inpatient but not outpatient isolates at those sites generally matched the corresponding residues in vaccine strains, except at site 145 of hemagglutinin (HA). This would be especially relevant for future surveillance of A/H3N2 strains in relation to their antigenicity and virulence. Furthermore, we observed a trend in which the HA proteins of influenza A/H3N2 and A/H1N1 exhibited enhanced ability to bind both avian and human host cell receptors. In contrast, the binding ability to each receptor was relatively stable for the HA of influenza B. CONCLUSIONS: Overall, our findings extend our understanding of the molecular and structural evolution of influenza virus strains in Singapore within the global context of these dynamic viruses.


Assuntos
Evolução Molecular , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Influenzavirus B/genética , Adolescente , Adulto , Idoso , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Hospitalização , Humanos , Influenza Humana/virologia , Pessoa de Meia-Idade , Mutação , Neuraminidase/genética , Pacientes Ambulatoriais , Filogenia , Receptores Virais/química , Singapura , Proteínas Virais/genética , Adulto Jovem
4.
Emerg Microbes Infect ; 8(1): 1465-1478, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31608791

RESUMO

The ANP32A is responsible for mammalian-restricted influenza virus polymerase activity. However, the mechanism of ANP32A modulation of polymerase activity remains poorly understood. Here, we report that chicken ANP32A (chANP32A) -X1 and -X2 stimulated mammalian-restricted PB2 627E polymerase activity in a dose-dependent manner. Distinct effects of ANP32A constructs suggested that the 180VK181 residues within chANP32A-X1 are necessary but not sufficient to stimulate PB2 627E polymerase activity. The PB2 N567D, T598V, A613V or F636L mutations promoted PB2 627E polymerase activity and chANP32A-X1 showed additive effects, providing further support that species-specific regulation of ANP32A might be only relevant with the PB2 E627K mutation. Rescue of cycloheximide-mediated inhibition showed that ANP32A is species-specific for modulation of vRNA but not mRNA and cRNA, demonstrating chANP32A-X1 compensated for defective cRNPs produced by PB2 627E virus in mammalian cells. The promoter mutations of cRNA enhanced the restriction of PB2 627E polymerase in mammalian cells, which could be restored by chANP32A-X1, indicating that ANP32A is likely to regulate the interaction of viral polymerase with RNA promoter. Coimmunoprecipitation showed that ANP32A did not affect the primary cRNPs assembly. We propose a model that chANP32A-X1 regulates PB2 627E polymerase for suitable interaction with cRNA promoter for vRNA replication.


Assuntos
Vírus da Influenza A Subtipo H1N1/enzimologia , Subtipo H7N9 do Vírus da Influenza A/enzimologia , Vírus da Influenza A Subtipo H9N2/enzimologia , Influenza Aviária/metabolismo , Influenza Humana/metabolismo , Doenças das Aves Domésticas/metabolismo , RNA Replicase/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Galinhas , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/fisiologia , Vírus da Influenza A Subtipo H9N2/genética , Vírus da Influenza A Subtipo H9N2/fisiologia , Influenza Aviária/genética , Influenza Aviária/virologia , Influenza Humana/genética , Influenza Humana/virologia , Mutação , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , Ligação Proteica , RNA Replicase/genética , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Alinhamento de Sequência , Especificidade da Espécie , Proteínas Virais/genética , Replicação Viral
5.
Vet Res ; 50(1): 77, 2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31590684

RESUMO

This report describes the detection of a triple reassortant swine influenza A virus of H1avN2 subtype. It evolved from an avian-like swine H1avN1 that first acquired the N2 segment from a seasonal H3N2, then the M segment from a 2009 pandemic H1N1, in two reassortments estimated to have occurred 10 years apart. This study illustrates how recurrent influenza infections increase the co-infection risk and facilitate evolutionary jumps by successive gene exchanges. It recalls the importance of appropriate biosecurity measures inside holdings to limit virus persistence and interspecies transmissions, which both contribute to the emergence of new potentially zoonotic viruses.


Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H1N2/fisiologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Vírus Reordenados/fisiologia , Doenças dos Suínos/virologia , Animais , França , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N2/genética , Vírus da Influenza A Subtipo H3N2/genética , Sus scrofa , Suínos
6.
MMWR Morb Mortal Wkly Rep ; 68(40): 880-884, 2019 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-31600182

RESUMO

During May 19-September 28, 2019,* low levels of influenza activity were reported in the United States, with cocirculation of influenza A and influenza B viruses. In the Southern Hemisphere seasonal influenza viruses circulated widely, with influenza A(H3) predominating in many regions; however, influenza A(H1N1)pdm09 and influenza B viruses were predominant in some countries. In late September, the World Health Organization (WHO) recommended components for the 2020 Southern Hemisphere influenza vaccine and included an update to the A(H3N2) and B/Victoria-lineage components. Annual influenza vaccination is the best means for preventing influenza illness and its complications, and vaccination before influenza activity increases is optimal. Health care providers should recommend vaccination for all persons aged ≥6 months who do not have contraindications to vaccination (1).


Assuntos
Saúde Global/estatística & dados numéricos , Vacinas contra Influenza/química , Influenza Humana/epidemiologia , Vigilância da População , Farmacorresistência Viral , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Vírus da Influenza B/efeitos dos fármacos , Vírus da Influenza B/genética , Vírus da Influenza B/isolamento & purificação , Influenza Humana/virologia , Estações do Ano , Estados Unidos/epidemiologia
7.
Emerg Microbes Infect ; 8(1): 1535-1545, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31661383

RESUMO

Influenza A virus infections occur in different species, causing mild to severe respiratory symptoms that lead to a heavy disease burden. Eurasian avian-like swine influenza A(H1N1) viruses (EAS-H1N1) are predominant in pigs and occasionally infect humans. An influenza A(H1N1) virus was isolated from a boy who was suffering from fever and headache and designated as A/Tianjin-baodi/1606/2018(H1N1). Full-genome sequencing and phylogenetic analysis revealed that A/Tianjin-baodi/1606/2018(H1N1) is a novel reassortant EAS-H1N1 containing gene segments from EAS-H1N1 (HA and NA), classical swine H1N1(NS) and A(H1N1)pdm09(PB2, PB2, PA, NP and M) viruses. The isolation and analysis of A/Tianjin-baodi/1606/2018(H1) provide further evidence that EAS-H1N1 poses a threat to human health and greater attention should be paid to surveillance of influenza virus infection in pigs and humans.


Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/virologia , Infecções por Orthomyxoviridae/veterinária , Vírus Reordenados/isolamento & purificação , Doenças dos Suínos/virologia , Animais , Criança , China , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/genética , Influenza Aviária/virologia , Masculino , Infecções por Orthomyxoviridae/virologia , Filogenia , Aves Domésticas , Doenças das Aves Domésticas/virologia , Vírus Reordenados/classificação , Vírus Reordenados/genética , Suínos
8.
Emerg Microbes Infect ; 8(1): 1456-1464, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31603050

RESUMO

Pigs play an important role in interspecies transmission of the influenza virus, particularly as "mixing vessels" for reassortment. Two influenza A/H1N1 virus strains, A/swine/Siberia/1sw/2016 and A/swine/Siberia/4sw/2017, were isolated during a surveillance of pigs from private farms in Russia from 2016 to 2017. There was a 10% identity difference between the HA and NA nucleotide sequences of isolated strains and the most phylogenetically related sequences (human influenza viruses of 1980s). Simultaneously, genome segments encoding internal proteins were found to be phylogenetically related to the A/H1N1pdm09 influenza virus. In addition, two amino acids (129-130) were deleted in the HA of A/swine/Siberia/4sw/2017 compared to that of A/swine/Siberia/1sw/2016 HA.


Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Infecções por Orthomyxoviridae/veterinária , Vírus Reordenados/genética , Doenças dos Suínos/epidemiologia , Suínos/microbiologia , Animais , Genoma Viral , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Infecções por Orthomyxoviridae/epidemiologia , Filogenia , Vírus Reordenados/isolamento & purificação , Federação Russa/epidemiologia , Doenças dos Suínos/virologia
9.
BMC Res Notes ; 12(1): 628, 2019 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-31551085

RESUMO

OBJECTIVE: We conducted four cross-sectional studies over 1 year among humans and pigs in three slaughterhouses in Central and Western Kenya (> 350 km apart) to determine infection and exposure to influenza A viruses. Nasopharyngeal (NP) and oropharyngeal (OP) swabs were collected from participants who reported acute respiratory illness (ARI) defined as fever, cough or running nose. Nasal swabs and blood samples were collected from pigs. Human NP/OP and pig nasal swabs were tested for influenza A virus by real-time reverse transcriptase polymerase chain reaction (PCR) and pig serum was tested for anti-influenza A antibodies by ELISA. RESULTS: A total of 288 participants were sampled, 91.3% of them being male. Fifteen (5.2%) participants had ARI but the nine swabs collected from them were negative for influenza A virus by PCR. Of the 1128 pigs sampled, five (0.4%) nasal swabs tested positive for influenza A/H1N1/pdm09 by PCR whereas 214 of 1082 (19.8%) serum samples tested for Influenza A virus antibodies. There was higher seroprevalence in colder months and among pigs reared as free-range. These findings indicate circulation of influenza A/H1N1/pdm09 among pigs perhaps associated with good adaptation of the virus to the pig population after initial transmission from humans to pigs.


Assuntos
Matadouros , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/diagnóstico , Infecções por Orthomyxoviridae/diagnóstico , Doenças dos Suínos/diagnóstico , Adulto , Animais , Anticorpos Antivirais/sangue , Estudos Transversais , Feminino , Geografia , Humanos , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/transmissão , Influenza Humana/virologia , Quênia/epidemiologia , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Orofaringe/virologia , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Pandemias , Suínos , Doenças dos Suínos/epidemiologia , Doenças dos Suínos/virologia , Adulto Jovem
10.
Emerg Microbes Infect ; 8(1): 1428-1437, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31560252

RESUMO

Switching of receptor binding preference has been widely considered as one of the necessary mutations for avian influenza viruses, enabling efficient transmissions between human hosts. By stably overexpressing B4GalNT2 gene in MDCK cells, surface α2,3-siallylactose receptors were modified without affecting α2,6-receptor expression. The cell line MDCK-B4GalNT2 was used as a tool to screen for α2,3-receptor requirements in a panel of influenza viruses with previously characterized glycan array data. Infection of viruses with α2,3-receptor binding capability was inhibited in MDCK-B4GalNT2 cells, with the exception of A/WSN/33 (WSN). Infection with the 2009 pandemic H1N1 strains, A/California/04/2009 (Cal04) and A/Hong Kong/415742/2009 (HK09), despite showing α2,6-receptor binding, was also found to be inhibited. Further investigation showed that viral inhibition was due to a reduction in viral entry rate and viral attachment. Recombinant WSN virus with the neuraminidase (NA) gene swapped to A/Puerto Rico/8/1934 (PR8) and Cal04 resulted in a significant viral inhibition in MDCK-B4GalNT2 cells. With oseltamivir, the NA active site was found to be important for the replication results of WSN, but not Cal04.


Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , N-Acetilgalactosaminiltransferases/genética , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/metabolismo , Ligação Viral , Internalização do Vírus , Animais , Antivirais/farmacologia , Linhagem Celular , Cães , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/genética , Células Madin Darby de Rim Canino , N-Acetilgalactosaminiltransferases/metabolismo , Neuraminidase/genética , Oseltamivir/farmacologia , Replicação Viral/efeitos dos fármacos
11.
Int J Infect Dis ; 89: 87-95, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31493523

RESUMO

OBJECTIVES: To evaluate the performance of rapid influenza diagnostic tests (RIDT) and influenza vaccines' effectiveness (VE) during an outbreak setting. METHODS: We compared the performance of a RIDT with RT-PCR for influenza virus detection in influenza-like illness (ILI) patients enrolled during the 2016/17 season in Mexico City. Using the test-negative design, we estimated influenza VE in all participants and stratified by age, virus subtype, and vaccine type (trivalent vs quadrivalent inactivated vaccines). The protective value of some clinical variables was evaluated by regression analyses. RESULTS: We enrolled 592 patients. RT-PCR detected 93 cases of influenza A(H1N1)pdm09, 55 of AH3N2, 141 of B, and 13 A/B virus infections. RIDT showed 90.7% sensitivity and 95.7% specificity for influenza A virus detection, and 91.5% sensitivity and 95.3% specificity for influenza B virus detection. Overall VE was 33.2% (95% CI: 3.0-54.0; p = 0.02) against any laboratory-confirmed influenza infection. VE estimates against influenza B were higher for the quadrivalent vaccine. Immunization and occupational exposure were protective factors against influenza. CONCLUSIONS: The RIDT was useful to detect influenza cases during an outbreak setting. Effectiveness of 2016/17 influenza vaccines administered in Mexico was low but significant. Our data should be considered for future local epidemiological policies.


Assuntos
Vacinas contra Influenza/administração & dosagem , Influenza Humana/diagnóstico , Adolescente , Adulto , Criança , Testes Diagnósticos de Rotina/métodos , Surtos de Doenças , Feminino , Humanos , Imunização , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza B/genética , Vírus da Influenza B/imunologia , Vírus da Influenza B/isolamento & purificação , Vacinas contra Influenza/imunologia , Influenza Humana/epidemiologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Masculino , México/epidemiologia , Pessoa de Meia-Idade , Estações do Ano , Vacinação , Adulto Jovem
12.
Acta Biochim Pol ; 66(3): 329-336, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31531422

RESUMO

The potential emergence of deadly pandemic influenza viruses is unpredictable and most have emerged with no forewarning. The distinct epidemiological and pathological patterns of the Spanish (H1N1), pandemic-2009 (H1N1), and avian influenza (H5N1), known as bird flu, viruses may allow us to develop a 'template' for possible emergence of devastating pandemic strains. Here, we provide a detailed molecular dissection of the structural and nonstructural proteins of this triad of viruses. GenBank data for three representative strains were analyzed to determine the polymorphic amino acids, genetic distances, and isoelectric points, hydrophobicity plot, and protein modeling of various proteins. We propose that the most devastating pandemic strains may have full-length PB1-F2 protein with unique residues, highly cleavable HA, and a basic NS1. Any newly emerging strain should be compared with these three strains, so that resources can be directed appropriately.


Assuntos
Simulação por Computador , Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/genética , Influenza Aviária/virologia , Influenza Humana/virologia , Proteínas Virais/química , Animais , Aves , Transmissão de Doença Infecciosa , Genoma Viral , Humanos , Influenza Pandêmica, 1918-1919 , Vacinas contra Influenza , Influenza Aviária/epidemiologia , Influenza Aviária/prevenção & controle , Influenza Humana/epidemiologia , Influenza Humana/prevenção & controle , Pandemias , Conformação Proteica em alfa-Hélice , Proteínas Virais/genética
13.
Indian J Med Res ; 149(6): 783-789, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31496532

RESUMO

Background & objectives: Influenza virological surveillance is an essential tool for the early detection of novel genetic variants of epidemiologic and clinical significance. This study was aimed to genetically characterize A(H1N1)pdm09 virus circulating in 2017 and to compare it with the global data. Methods: The regional/State Viral Research and Diagnostic Laboratories (VRDLs) provided influenza diagnosis for referred clinical samples and shared influenza A(H1N1)pdm09 positives with the Indian Council of Medical Research-National Institute of Virology (ICMR-NIV), Pune, India, for hemagglutinin (HA) gene phylogenetic analysis. Sites at Manipal, Jaipur and Dibrugarh performed the sequencing and shared the sequence data for analysis. The antiviral susceptibility of influenza viruses was assessed for known molecular marker H275Y at the ICMR-NIV, Pune. Results: All the eight VRDLs had well-established influenza diagnostic facilities and showed increased activity of influenza A(H1N1)pdm09 during 2017. Phylogenetic analysis showed that the viruses from the different regions of the country were similar to A/Michigan/45/2015 strain which was the 2017-2018 recommended vaccine strain and were clustered with the globally circulating clade 6B.1 with signature mutations S84N, S162N and I216T. The clade 6B.1 showed further subgrouping with additional mutations S74R, S164T and I295V; however, there was no significant association between the presence of these mutations and severity of disease due to influenza. All the study viruses were sensitive to oseltamivir. Interpretation & conclusions: During the study period, all the study sites reported globally circulating A/Michigan/45/2015 vaccine strain of influenza A(H1N1)pdm09 viruses and remained sensitive to oseltamivir. Further genetic and antigenic characterization of influenza viruses is recommended to address public health concerns.


Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/genética , Oseltamivir/uso terapêutico , Filogenia , Antivirais/uso terapêutico , Farmacorresistência Viral/genética , Humanos , Índia/epidemiologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Vacinas contra Influenza/uso terapêutico , Influenza Humana/tratamento farmacológico , Influenza Humana/patologia , Influenza Humana/virologia , Mutação de Sentido Incorreto/genética , RNA Viral/genética , Análise de Sequência de DNA
14.
PLoS Pathog ; 15(9): e1008036, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31525249

RESUMO

Cytomegalovirus (CMV) is a ubiquitous ß-herpesvirus that establishes life-long latent infection in a high percentage of the population worldwide. CMV induces the strongest and most durable CD8+ T cell response known in human clinical medicine. Due to its unique properties, the virus represents a promising candidate vaccine vector for the induction of persistent cellular immunity. To take advantage of this, we constructed a recombinant murine CMV (MCMV) expressing an MHC-I restricted epitope from influenza A virus (IAV) H1N1 within the immediate early 2 (ie2) gene. Only mice that were immunized intranasally (i.n.) were capable of controlling IAV infection, despite the greater potency of the intraperitoneally (i.p.) vaccination in inducing a systemic IAV-specific CD8+ T cell response. The protective capacity of the i.n. immunization was associated with its ability to induce IAV-specific tissue-resident memory CD8+ T (CD8TRM) cells in the lungs. Our data demonstrate that the protective effect exerted by the i.n. immunization was critically mediated by antigen-specific CD8+ T cells. CD8TRM cells promoted the induction of IFNγ and chemokines that facilitate the recruitment of antigen-specific CD8+ T cells to the lungs. Overall, our results showed that locally applied MCMV vectors could induce mucosal immunity at sites of entry, providing superior immune protection against respiratory infections.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunidade nas Mucosas , Vacinas contra Influenza/imunologia , Muromegalovirus/imunologia , Administração Intranasal , Sequência de Aminoácidos , Animais , Linhagem Celular , Quimiocinas/biossíntese , Epitopos de Linfócito T/administração & dosagem , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Produtos do Gene env/administração & dosagem , Produtos do Gene env/genética , Produtos do Gene env/imunologia , Vetores Genéticos , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/genética , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Pulmão/imunologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Muromegalovirus/genética , Células NIH 3T3 , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/virologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
15.
Dokl Biochem Biophys ; 486(1): 201-205, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31367821

RESUMO

Infection of mice with influenza A viruses led to the formation of clones of lymphocytes that specifically recognizes viral domains in the central zone of the NSP protein (amino acid positions 83-119). Computer analysis of the primary structure of the NSP protein showed the presence of T-cell epitopes in the central part of the NSP molecule. The findings indicate that the viral NSP gene is expressed in the infected animals and verify the concept of the bipolar strategy (ambisense strategy) of the influenza A virus genome.


Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/fisiologia , Leucócitos/imunologia , RNA Viral/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Leucócitos/virologia , Camundongos , Camundongos Endogâmicos BALB C , Domínios Proteicos , Proteínas Virais/química , Proteínas Virais/metabolismo
16.
BMC Infect Dis ; 19(1): 676, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31370782

RESUMO

BACKGROUND: In addition to seasonal influenza viruses recently circulating in humans, avian influenza viruses (AIVs) of H5N1, H5N6 and H7N9 subtypes have also emerged and demonstrated human infection abilities with high mortality rates. Although influenza viral infections are usually diagnosed using viral isolation and serological/molecular analyses, the cost, accessibility, and availability of these methods may limit their utility in various settings. The objective of this study was to develop and optimized a multiplex detection system for most influenza viruses currently infecting humans. METHODS: We developed and optimized a multiplex detection system for most influenza viruses currently infecting humans including two type B (both Victoria lineages and Yamagata lineages), H1N1, H3N2, H5N1, H5N6, and H7N9 using Reverse Transcriptional Loop-mediated Isothermal Amplification (RT-LAMP) technology coupled with a one-pot colorimetric visualization system to facilitate direct determination of results without additional steps. We also evaluated this multiplex RT-LAMP for clinical use using a total of 135 clinical and spiked samples (91 influenza viruses and 44 other human infectious viruses). RESULTS: We achieved rapid detection of seasonal influenza viruses (H1N1, H3N2, and Type B) and avian influenza viruses (H5N1, H5N6, H5N8 and H7N9) within an hour. The assay could detect influenza viruses with high sensitivity (i.e., from 100 to 0.1 viral genome copies), comparable to conventional RT-PCR-based approaches which would typically take several hours and require expensive equipment. This assay was capable of specifically detecting each influenza virus (Type B, H1N1, H3N2, H5N1, H5N6, H5N8 and H7N9) without cross-reactivity with other subtypes of AIVs or other human infectious viruses. Furthermore, 91 clinical and spiked samples confirmed by qRT-PCR were also detected by this multiplex RT-LAMP with 98.9% agreement. It was more sensitive than one-step RT-PCR approach (92.3%). CONCLUSIONS: Results of this study suggest that our multiplex RT-LAMP assay may provide a rapid, sensitive, cost-effective, and reliable diagnostic method for identifying recent influenza viruses infecting humans, especially in locations without access to large platforms or sophisticated equipment.


Assuntos
Colorimetria/métodos , Vírus da Influenza A/genética , Influenza Humana/virologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Reações Cruzadas , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Virus da Influenza A Subtipo H5N1/genética , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/isolamento & purificação , Vírus da Influenza A/patogenicidade , Transcrição Reversa
17.
Indian J Med Microbiol ; 37(1): 42-49, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31424009

RESUMO

Introduction: Influenza A(H1N1)pdm09 virus, since its identification in April 2009, has continued to cause significant outbreaks of respiratory tract infections including pandemics in humans. In the course of its evolution, the virus has acquired many mutations with an ability to cause increased disease severity. A regular molecular surveillance of the virus is essential to mark the evolutionary changes that may cause a shift to the viral behavior. Materials and Methods: Samples of Throat/Nasal swabs were collected from a total of 3715 influenza-like illness cases and screened by Real-time Reverse Transcription-Polymerase Chain Reaction for influenza viruses. Nucleotide sequence analysis was done to identify changes in antigenicity of the virus strains. Results: The present study describes the molecular characteristics of influenza A(H1N1)pdm09 viruses detected in Assam of Northeast India during 2009-2015. Influenza A viruses were detected in 11.4% (425/3715), of which influenza A(H1N1)pdm09 viruses were detected in 41.4% (176/425). The nucleotide sequencing of influenza A(H1N1)pdm09 viruses revealed a total of 17 and 22 amino acid substitutions in haemagglutinin (HA) and neuraminidase (NA) genes of the virus, respectively, compared to contemporary vaccine strain A/California/07/2009. The important mutations detected in HA genes of A/Assam(H1N1)pdm09 strains included E391K, K180Q and S202T. Mutation 'N248D' which has an ability to develop oseltamivir resistance was also detected in NA gene of A/Assam(H1N1)pdm09 strains. Conclusions: Regular molecular surveillance of influenza A(H1N1)pdm09 is important to monitor the viral behavior in terms of increase virulence, drug resistance pattern and emergence of novel strains.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/epidemiologia , Neuraminidase/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antivirais/farmacologia , Sequência de Bases , Criança , Pré-Escolar , Feminino , Variação Genética/genética , Humanos , Índia/epidemiologia , Lactente , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Oseltamivir/farmacologia , Filogenia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , Adulto Jovem
18.
Zhonghua Yu Fang Yi Xue Za Zhi ; 53(8): 840-842, 2019 Aug 06.
Artigo em Chinês | MEDLINE | ID: mdl-31378046

RESUMO

In this study, the swabs were collected among patients with an influenza-like illness (ILI) admitted to 2 sentinel surveillance hospitals of Yantai from April 2014 to August 2017. All specimen were cultured and identified by hemagglutination inhibition assay. Complete sequences of Hemagglutinin (HA) of influenza A were amplified, sequenced and analyzed using molecular and phylogenetic methods. The potential vaccine efficacy were calculated using Pepitope model. The results showed that the antigenicity of A (H3N2) had changed greatly. 8 strains of influenza A (H1N1) pdm09 belonged to subclade 6B.1 and 14 strains clustered in 6B.2. 12 strains of influenza A (H3N2) fell into subgroup 3C.3a and 33 strains clustered in 3C.2a. Several residues at antigen sites and potential glycosylation sites had changed in influenza A strains. Vaccine efficacy of influenza A (H1N1) pdm09 in 2015/2016 and 2016/2017 seasons were 77.29% and 79.11% of that of a perfect match with vaccine strain, meanwhile vaccine efficacy of influenza A (H3N2) in 2014/2015, 2015/2016 and 2016/2017 were-5.18%, 16.97% and 42.05% separately. In conclusion, the influenza A virus circulated in Yantai from 2014 to 2017 presented continual genetic variation. The recommended vaccine strains still afforded protection against influenza A (H1N1) pdm09 strains and provided suboptimal protection against influenza A (H3N2) strains.


Assuntos
Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Vacinas contra Influenza/uso terapêutico , Influenza Humana/prevenção & controle , Potência de Vacina , China , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Humanos , Filogenia , RNA Viral
19.
Adv Exp Med Biol ; 1222: 63-68, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31352577

RESUMO

The SENTINEL influenza surveillance system is an important tool for monitoring influenza in Poland. Data from this system are necessary to determine the dynamics of seasonal infections and to announce the epidemic by the country level. For the 2017/18 epidemic season, the dominance of influenza type B was recorded and the highest percentage of infections was recorded in the age group 45-64 years. Among the subtypes of influenza type A, A/H1N1/pdm09 was the predominated subtype. Most cases were reported in the age group of 26-44 and 0-4 years. The influenza virus frequently undergoes modifications. Therefore, it is necessary to constantly monitor the emerging strains around the world.


Assuntos
Epidemias , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza B/isolamento & purificação , Influenza Humana/epidemiologia , Vigilância de Evento Sentinela , Adolescente , Adulto , Distribuição por Idade , Idoso , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza B/genética , Pessoa de Meia-Idade , Polônia/epidemiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estações do Ano , Adulto Jovem
20.
Emerg Microbes Infect ; 8(1): 989-999, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31267843

RESUMO

It has recently been proposed that the Eurasian avian-like H1N1 (EA H1N1) swine influenza virus (SIV) is one of the most likely zoonotic viruses to cause the next influenza pandemic. Two main genotypes EA H1N1 viruses have been recognized to be infected humans in China. Our study finds that one of the genotypes JS1-like viruses are avirulent in mice. However, the other are HuN-like viruses and are virulent in mice. The molecular mechanism underlying this difference shows that the NP gene determines the virulence of the EA H1N1 viruses in mice. In addition, a single substitution, Q357K, in the NP protein of the EA H1N1 viruses alters the virulence phenotype. This substitution is a typical human signature marker, which is prevalent in human viruses but rarely detected in avian influenza viruses. The NP-Q357K substitution is readily to be occurred when avian influenza viruses circulate in pigs, and may facilitate their infection of humans and allow viruses also carrying NP-357K to circulate in humans. Our study demonstrates that the substitution Q357K in the NP protein plays a key role in the virulence phenotype of EA H1N1 SIVs, and provides important information for evaluating the pandemic risk of field influenza strains.


Assuntos
Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/virologia , Mutação de Sentido Incorreto , Infecções por Orthomyxoviridae/veterinária , Proteínas de Ligação a RNA/genética , Doenças dos Suínos/virologia , Proteínas do Core Viral/genética , Animais , China , Feminino , Genótipo , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/virologia , Filogenia , Proteínas de Ligação a RNA/metabolismo , Suínos , Proteínas do Core Viral/metabolismo , Virulência , Replicação Viral
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