RESUMO
Manufactured influenza vaccines have to contain a defined amount of hemagglutinin (HA) antigen. Therefore, vaccine viruses with a high HA antigen yield (HAY) are preferable for manufacturing vaccines, particularly vaccines in response to a pandemic, when vaccines need to be rapidly produced. However, the viral properties associated with a high HAY have not yet been fully clarified. To identify the HAY-associated traits, we first propagated 26 H5 candidate vaccine viruses (CVVs) in eggs, which were previously developed based on genetic reassortment methods using master viruses, to determine their total protein yield (TPY), ratio of HA to total viral protein (%-HA content) and HAY. The results revealed that the HAY was correlated with the TPY but not with the %-HA content. We further found that altering the sequences of the 3' noncoding region of HA vRNA or replacing the master virus improved the HAYs and TPYs of the low-HAY CVVs to approximately double the values of the original CVVs but did not change the %-HA content, which a previous study suggested was associated with the HAY. Analyses based on real-time PCR assays and scanning electron microscopy revealed that the virus samples with an improved HAY contained more copies of the virus genome and viral particles than the original samples. The results suggest that an improvement in virus growth (i.e., an increase in the amount of viral particles) leads to an increase in the TPY and thus in the HAY, regardless of the %-HA content. The approximately twofold increase in the HAY shown in this study may not appear to represent a large improvement, but the impact will be significant given the millions of chicken eggs used to produce vaccines. These findings will be informative for developing high-HAY vaccine viruses.
Assuntos
Vacinas contra Influenza , Orthomyxoviridae , Animais , Hemaglutininas/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Galinhas , Anticorpos AntiviraisRESUMO
The practical use of cell-based seasonal influenza vaccines is currently being considered in Japan. From the perspective of adventitious virus contamination, we assessed the suitability of NIID-MDCK cells (NIID-MDCK-Cs) as a safe substrate for the isolation of influenza viruses from clinical specimens. We first established a sensitive multiplex real-time PCR system to screen for 27 respiratory viruses and used it on 34 virus samples that were isolated by passaging influenza-positive clinical specimens in NIID-MDCK-Cs. Incidentally, the limit of detection (LOD) of the system was 100 or fewer genome copies per reaction. In addition to influenza viruses, human enterovirus 68 (HEV-D68) genomes were detected in two samples after two or three passages in NIID-MDCK-Cs. To further investigate the susceptibility of NIID-MDCK-Cs to adventitious viruses, eight common respiratory viruses were subjected to passages in NIID-MDCK-Cs. The genome copy numbers of seven viruses other than parainfluenza 3 decreased below the LOD by passage 4. By passaging in NIID-MDCK-Cs, the genome numbers of the input HEV-D68, 1 × 108 copies, declined to 102 at passage 3 and to under the LOD at passage 4, whereas those of the other six viruses were under the LOD by passage 3. These results implied that during the process of isolating influenza viruses with NIID-MDCK-Cs, contaminating viruses other than parainfluenza 3 can be efficiently removed by passages in NIID-MDCK-Cs. NIID-MDCK-Cs could be a safe substrate for isolating influenza viruses that can be used to develop cell-based influenza vaccine candidate viruses.
Assuntos
Vacinas contra Influenza , Influenza Humana , Orthomyxoviridae , Infecções por Paramyxoviridae , Vírus , Animais , Cães , Humanos , Vacinas contra Influenza/genética , Influenza Humana/prevenção & controle , Células Madin Darby de Rim Canino , Desenvolvimento de Vacinas , Cultura de Vírus/métodosRESUMO
The influenza A(H1N1)pdm09 virus emerged in April 2009 with an unusual incidence of severe disease and mortality, and currently circulates as a seasonal influenza virus. Previous studies using consensus viral genome sequencing data have overlooked the viral genomic and phenotypic diversity. Next-generation sequencing (NGS) may instead be used to characterize viral populations in an unbiased manner and to measure within-host genetic diversity. In this study, we used NGS analysis to investigate the within-host genetic diversity of influenza A(H1N1)pdm09 virus in the upper and lower respiratory samples from nine patients who were admitted to the intensive care unit (ICU). A total of 47 amino acid substitution positions were found to differ between the upper and lower respiratory tract samples from all patients. However, the D222G/N substitution in hemagglutinin (HA) protein was the only amino acid substitution common to multiple patients. Furthermore, the substitution was detected only in the six samples from the lower respiratory tract. Therefore, it is important to investigate influenza A(H1N1)pdm09 virus populations using multiple paired samples from the upper and lower respiratory tract to avoid overlooking potentially important substitutions, especially in patients with severe disease.IMPORTANCE The D222G/N substitution in the hemagglutinin (HA) protein of influenza A(H1N1)pdm09 virus has been reported to be associated with disease severity and mortality in numerous previous studies. In the present study, 75% of lower respiratory samples contained heterogeneous influenza populations that carried different amino acids at position 222 of the HA protein, whereas all upper respiratory samples only contained the wild-type 222D. These results suggest the influenza A(H1N1)pdm09 virus has diversified inside the host owing to differences in tissue specificity. In this study, the within-host genetic diversity of influenza A(H1N1)pdm09 virus was investigated for the first time using next-generation sequencing analysis of the viral whole-genome in samples extracted from the upper and lower respiratory tracts of patients with severe disease.
Assuntos
Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/virologia , Mutação de Sentido Incorreto , Filogenia , Sistema Respiratório/virologia , Adulto , Idoso , Substituição de Aminoácidos , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/classificação , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Sistema Respiratório/anatomia & histologia , Índice de Gravidade de Doença , Adulto JovemRESUMO
In Japan, the practical application of completely cell-based seasonal influenza vaccines is under consideration. Considering the good correlation between the immunogenicity of egg-based influenza vaccines and the hemagglutinin (HA) content determined by the single radial immunodiffusion (SRD) assay, we determined the potency of the first cell-based quadrivalent vaccine experimentally generated in Japan using the SRD assay in this study. A primary liquid standard (PLS) and reference antigen were generated from the purified vaccine virus, and a sheep antiserum was produced against the HA of the vaccine virus. Since the purity of the PLS affects the reliability of vaccine potency testing, the purification steps are significant. We successfully prepared a purified PLS nearly free of cell debris. The HA content in the PLS was first estimated from the total amount of viral protein and the percentage of HA content determined by SDS-PAGE analysis. The HA content in the reference antigen was calibrated to that in the PLS via the SRD assay. The vaccine potency, that is, the HA content in each vaccine, was finally measured using the corresponding reference antigen. Ultimately, the measured vaccine potency of the monovalent vaccine was similar to that of the quadrivalent vaccine.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Estações do Ano , Tecnologia Farmacêutica/métodos , Potência de Vacina , Animais , Anticorpos Antivirais/imunologia , Cães , Humanos , Soros Imunes/imunologia , Vacinas contra Influenza/normas , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Padrões de Referência , Ovinos , Tecnologia Farmacêutica/normasRESUMO
Influenza A(H1N1)pdm09 viruses carrying a dual neuraminidase (NA) substitution were isolated from immunocompromised patients after administration of one or more NA inhibitors. These mutant viruses possessed an H275Y/I223R, H275Y/I223K, or H275Y/G147R substitution in their NA and showed enhanced cross-resistance to oseltamivir and peramivir and reduced susceptibility to zanamivir compared to single H275Y mutant viruses. Baloxavir could be a treatment option against the multidrug-resistant viruses because these dual H275Y mutant viruses showed susceptibility to this drug. The G147R substitution appears to stabilize the NA structure, with the fitness of the H275Y/G147R mutant virus being similar or somewhat better than that of the wild-type virus. Since the multidrug-resistant viruses may be able to transmit between humans, surveillance of these viruses must continue to improve clinical management and to protect public health.
RESUMO
BACKGROUND: Neurogenic pulmonary edema is a rare but serious complication of febrile status epilepticus in children. Comprehensive screening for viral pathogens is seldomly performed in the work-up of febrile children. CASE PRESENTATION: A 22-month-old girl presented with her first episode of febrile status epilepticus, after which she developed acute pulmonary edema and respiratory failure. After the termination of seizure activity, the patient was intubated and managed on mechanical ventilation in the emergency room. The resolution of respiratory failure, as well as the neurological recovery, was achieved 9 h after admission, and the patient was discharged 6 days after admission without any complications. Molecular biological diagnostic methods identified the presence of human coronavirus HKU1, influenza C virus, and human parainfluenza virus 2 from the patient's nasopharyngeal specimens. CONCLUSIONS: Neurogenic pulmonary edema following febrile status epilepticus was suspected to be the etiology of our patient's acute pulmonary edema and respiratory failure. Timely seizure termination and rapid airway and respiratory intervention resulted in favorable outcomes of the patient. Molecular biological diagnostic methods identified three respiratory viruses; however, their relevance and association with clinical symptoms remain speculative.
Assuntos
Edema Pulmonar/etiologia , Infecções Respiratórias/virologia , Doenças do Sistema Nervoso Central/etiologia , Doenças do Sistema Nervoso Central/terapia , Coronavirus/isolamento & purificação , Infecções por Coronavirus , Feminino , Febre/complicações , Humanos , Lactente , Influenza Humana , Gammainfluenzavirus/isolamento & purificação , Técnicas de Diagnóstico Molecular , Nasofaringe/virologia , Vírus da Parainfluenza 2 Humana/isolamento & purificação , Edema Pulmonar/diagnóstico por imagem , Edema Pulmonar/terapia , Infecções Respiratórias/complicações , Estado EpilépticoRESUMO
A community outbreak of human influenza A(H1N1)pdm09 virus strains was observed in Myanmar in 2017. We investigated the circulation patterns, antigenicity, and drug resistance of 2017 influenza A(H1N1)pdm09 viruses from Myanmar and characterized the full genome of influenza virus strains in Myanmar from in-patients and out-patients to assess the pathogenicity of the viruses. Nasopharyngeal swabs were collected from out-patients and in-patients with acute respiratory tract infections in Yangon and Pyinmana City in Myanmar during January-December 2017. A total of 215 out-patients and 18 in-patients infected with A(H1N1)pdm09 were detected by virus isolation and real-time RT-PCR. Among the positive patients, 90.6% were less than 14 years old. Hemagglutination inhibition (HI) antibody titers against A(H1N1)pdm09 viruses in Myanmar were similar to the recommended Japanese influenza vaccine strain for 2017-2018 seasons (A/Singapore/GP1908/2015) and WHO recommended 2017 southern hemisphere vaccine component (A/Michigan/45/2015). Phylogenetic analysis of the hemagglutinin sequence showed that the Myanmar strains belonged to the genetic subclade 6B.1, possessing mutations of S162N and S164T at potential antigenic sites. However, the amino acid mutation at position 222, which may enhance the severity of disease and mortality, was not found. One case with no prior history of oseltamivir treatment possessed H275Y mutated virus in neuraminidase (NA), which confers resistance to oseltamivir and peramivir with elevated IC50 values. The full genome sequence of Myanmar strains showed no difference between samples from in-patients and out-patients, suggesting no additional viral mutations associated with patient severity. Several amino acid changes were observed in PB2, PB1, and M2 of Myanmar strains when compared to the vaccine strain and other Asian strains. However, no mutations associated with pathogenicity were found in the Myanmar strains, suggesting that viral factors cannot explain the underlying reasons of the massive outbreak in Myanmar. This study reported the first detection of an oseltamivir-resistant influenza virus in Myanmar, highlighting the importance of continuous antiviral monitoring and genetic characterization of the influenza virus in Myanmar.
Assuntos
Epidemias , Vírus da Influenza A Subtipo H1N1 , Influenza Humana/epidemiologia , Adolescente , Adulto , Substituição de Aminoácidos , Antígenos Virais , Antivirais/farmacologia , Criança , Pré-Escolar , Farmacorresistência Viral/genética , Feminino , Genoma Viral , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Lactente , Vírus da Influenza A Subtipo H1N1/classificação , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Mianmar/epidemiologia , Oseltamivir/farmacologia , Filogenia , Adulto JovemRESUMO
BACKGROUND: The novel cap-dependent endonuclease inhibitor baloxavir marboxil was approved in February 2018 for the treatment of influenza virus infection in Japan. In vitro studies have revealed that an I38T substitution in the polymerase acidic subunit (PA) is associated with reduced susceptibility of influenza viruses to baloxavir. OBJECTIVES: Development of a rapid and simple method for monitoring influenza A(H1N1)pdm09, A(H3N2), and B viruses possessing the I38T substitution in PA. METHODS: Three assays were developed based on RNase H2-dependent PCR (rhPCR) and named A/H1pdm PA_I38T rhPCR, A/H3 PA_I38T rhPCR, and B PA_I38T rhPCR. The assays were evaluated using cDNAs synthesized from in vitro-transcribed PA gene RNA controls, RNAs purified from viruses isolated in the 2017/2018 and 2018/2019 influenza seasons, and RNAs purified from clinical specimens collected in the 2018/2019 influenza season. RESULTS: The assays developed in this study accurately discriminated PA I38 and PA T38 with high sensitivity. CONCLUSIONS: Our assays should be considered a powerful tool for monitoring the emergence of baloxavir-resistant influenza viruses.
Assuntos
Substituição de Aminoácidos/efeitos dos fármacos , Antivirais/farmacologia , Dibenzotiepinas/farmacologia , Farmacorresistência Viral/genética , Técnicas de Diagnóstico Molecular/métodos , Morfolinas/farmacologia , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/genética , Piridonas/farmacologia , Triazinas/farmacologia , Substituição de Aminoácidos/genética , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/enzimologia , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/enzimologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza B/efeitos dos fármacos , Vírus da Influenza B/enzimologia , Vírus da Influenza B/genética , Influenza Humana/diagnóstico , Influenza Humana/virologia , Japão , Limite de Detecção , Técnicas de Diagnóstico Molecular/normas , Orthomyxoviridae/enzimologia , Ribonuclease H/genética , Sensibilidade e EspecificidadeRESUMO
Intranasally administered influenza vaccines could be more effective than injected vaccines, because intranasal vaccination can induce virus-specific immunoglobulin A (IgA) antibodies in the upper respiratory tract, which is the initial site of infection. In this study, immune responses elicited by an intranasal inactivated vaccine of influenza A(H5N1) virus were evaluated in healthy individuals naive for influenza A(H5N1) virus. Three doses of intranasal inactivated whole-virion H5 influenza vaccine induced strong neutralizing nasal IgA and serum IgG antibodies. In addition, a mucoadhesive excipient, carboxy vinyl polymer, had a notable impact on the induction of nasal IgA antibody responses but not on serum IgG antibody responses. The nasal hemagglutinin (HA)-specific IgA antibody responses clearly correlated with mucosal neutralizing antibody responses, indicating that measurement of nasal HA-specific IgA titers could be used as a surrogate for the mucosal antibody response. Furthermore, increased numbers of plasma cells and vaccine antigen-specific Th cells in the peripheral blood were observed after vaccination, suggesting that peripheral blood biomarkers may also be used to evaluate the intranasal vaccine-induced immune response. However, peripheral blood immune cell responses correlated with neutralizing antibody titers in serum samples but not in nasal wash samples. Thus, analysis of the peripheral blood immune response could be a surrogate for the systemic immune response to intranasal vaccination but not for the mucosal immune response. The current study suggests the clinical potential of intranasal inactivated vaccines against influenza A(H5N1) viruses and highlights the need to develop novel means to evaluate intranasal vaccine-induced mucosal immune responses.
Assuntos
Imunidade nas Mucosas , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Administração Intranasal , Adulto , Anticorpos Neutralizantes/análise , Anticorpos Antivirais/análise , Feminino , Humanos , Imunoglobulina A Secretora/análise , Imunoglobulina G/sangue , Virus da Influenza A Subtipo H5N1 , Vacinas contra Influenza/administração & dosagem , Masculino , Pessoa de Meia-Idade , Mucosa Nasal/imunologia , Vacinação , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Adulto JovemRESUMO
BACKGROUND: Cell-based influenza vaccines can solve the problem of the frequent occurrence of egg adaptation-associated antigenic changes observed in egg-based vaccines. Seed viruses for cell-based vaccines can be prepared from clinical specimens by cell culture; however, clinical samples risk harboring respiratory viruses other than influenza virus. Therefore, it is necessary to investigate the patterns of co-infection in clinical samples and explore whether cell culture technology can selectively propagate influenza viruses from samples containing other respiratory viruses. METHODS: A total of 341 clinical specimens were collected from patients with influenza or influenza-like illness and analyzed by ResPlex II assay to detect 18 respiratory viruses. The patterns of co-infection were statistically analyzed with Fisher's exact test. The samples with double or triple infections were passaged in suspension MDCK cells (MDCK-S), adherent MDCK cells (MDCK-A), and LLC-MK2D cells. Cell-passaged samples were analyzed by ResPlex II assay again to investigate whether each cell line could amplify influenza viruses and eliminate other respiratory viruses. RESULTS: Double infections were detected in 8.5% and triple infections in 0.9% of the collected clinical specimens. We identified four pairs of viruses with significant correlation. For all samples with double and triple infection, MDCK-S and MDCK-A could selectively propagate influenza viruses, while eliminating all contaminating viruses. In contrast, LLC-MK2D showed lower isolation efficiency for influenza virus and higher isolation efficiency for coxsackievirus/echovirus than MDCK-S and MDCK-A. CONCLUSIONS: Both MDCK-S and MDCK-A are considered suitable for the preparation of influenza vaccine seed viruses without adventitious agents or egg-adaptation mutations.
Assuntos
Células Madin Darby de Rim Canino/virologia , Orthomyxoviridae/isolamento & purificação , Cultura de Vírus/métodos , Animais , Linhagem Celular , Cães , Humanos , Orthomyxoviridae/crescimento & desenvolvimento , Vacinas ViraisRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Suspension Madin-Darby canine kidney (MDCK) cells (MDCK-N), adherent MDCK cells (MDCK-C), and adherent rhesus monkey kidney LLC-MK2 cells (LLC-MK2D) were systematically evaluated for the preparation of influenza vaccine seed viruses for humans on the basis of primary virus isolation efficiency, growth ability, genetic stability of the hemagglutinin (HA) and neuraminidase (NA) genes, and antigenic properties in hemagglutination inhibition (HI) test of each virus isolate upon further passages. All the subtypes/lineages of influenza viruses (A(H1N1), A(H1N1)pdm09, A(H3N2), B-Victoria, and B-Yamagata) were successfully isolated from clinical specimens by using MDCK-N and MDCK-C, whereas LLC-MK2D did not support virus replication well. Serial passages of A(H1N1) viruses in MDCK-N and MDCK-C induced genetic mutations of HA that resulted in moderate antigenic changes in the HI test. All A(H1N1)pdm09 isolates from MDCK-C acquired amino acid substitutions at the site from K153 to N156 of the HA protein, which resulted in striking antigenic alteration. In contrast, only 30% of MDCK-N isolates showed amino acid changes at this site. The frequency of MDCK-N isolates with less than two-fold reduction in the HI titer was as high as 70%. A(H3N2) and B-Yamagata isolates showed high antigenic stability and no specific amino acid substitution during passages in MDCK-N and MDCK-C. B-Victoria isolates from MDCK-N and MDCK-C acquired genetic changes at HA glycosylation sites that greatly affected their antigenicity. When these cell isolates were applied to passages in hen eggs, A(H1N1), B-Victoria, and B-Yamagata viruses grew well in eggs, while none of the cell isolates of A(H3N2) viruses did. Thus, we demonstrate that MDCK-N might be useful for the preparation of influenza vaccine seed viruses.
Assuntos
Vírus da Influenza A/classificação , Vírus da Influenza A/fisiologia , Cultura de Vírus/métodos , Replicação Viral , Animais , Linhagem Celular , Cães , Hemaglutininas Virais/genética , Vacinas contra Influenza , Rim/citologia , Rim/virologia , Macaca mulatta , Células Madin Darby de Rim Canino , Mutação , Neuraminidase/genética , RNA Viral/genéticaRESUMO
In 2019, influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic gene, which confers reduced susceptibility to baloxavir, were detected in Japan in an infant without baloxavir exposure and a baloxavir-treated sibling. These viruses' whole-genome sequences were identical, indicating human-to-human transmission. Influenza virus isolates should be monitored for baloxavir susceptibility.
Assuntos
Antivirais/farmacologia , Suscetibilidade a Doenças , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Influenza Humana/transmissão , Influenza Humana/virologia , Oxazinas/farmacologia , Piridinas/farmacologia , Tiepinas/farmacologia , Triazinas/farmacologia , Adolescente , Adulto , Antivirais/uso terapêutico , Criança , Dibenzotiepinas , Humanos , Lactente , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/tratamento farmacológico , Influenza Humana/epidemiologia , Japão/epidemiologia , Pessoa de Meia-Idade , Morfolinas , Mutação , Oxazinas/uso terapêutico , Piridinas/uso terapêutico , Piridonas , Tiepinas/uso terapêutico , Triazinas/uso terapêutico , Adulto JovemRESUMO
This study examined the protective efficacy of and immune response to a nasal influenza vaccine combined with a novel mucosal oligodeoxynucleotide (ODN) adjuvant, CpG ODN G9.1 (G9.1), in a model of infection limited to the upper respiratory tract (URT) and a model of infection in the lower respiratory tract (LRT). Mice were nasally primed with an A/California/7/2009 (Cal7) split vaccine (X179A) plus G9.1 and were then nasally given a booster with X179A alone. When mice were challenged with either a large (infection of the LRT) or small (infection limited to the URT) volume of live Cal7 influenza virus, mice nasally given G9.1 combined with X179A had a markedly higher rate of protection against infection limited to the URT. Moreover, this group of mice promptly recovered from an infection of the LRT. When mice were subcutaneously (s.c.) given X179A as a current form of vaccination, they had no protection from an infection limited to the URT but they did recover from an infection of the LRT. The patterns of protection were closely correlated with influenza virus-specific mucosal secretory IgA (SIgA) or serum IgG antibody (Ab) responses. Thus, SIgA Abs responses play an important role in protection from an infection limited to the URT while influenza virus-specific serum IgG Ab responses help to protect from an infection of the LRT. A finding of note is that lungs from mice nasally given G9.1 had low levels of type I IFN-associated protein- and transcription factor-specific mRNA expression. These results suggest that nasal G9.1 can be used as an effective and safe mucosal adjuvant for influenza vaccines since this nasal vaccine system elicits both mucosal SIgA and serum IgG Ab responses that provide complete protection without inducing potent inflammatory responses.
Assuntos
Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Oligodesoxirribonucleotídeos/imunologia , Adjuvantes Imunológicos , Administração Intranasal , Animais , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Feminino , Humanos , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/uso terapêutico , Cinética , Camundongos , Camundongos Endogâmicos BALB C , Orthomyxoviridae/imunologia , Orthomyxoviridae/patogenicidade , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/uso terapêuticoRESUMO
In January 2019, two influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic subunit (PA), which confers reduced susceptibility to baloxavir, were detected from epidemiologically unrelated hospitalised children in Japan. The viruses exhibited reduced susceptibility to baloxavir but were susceptible to neuraminidase inhibitors. Only one of the two children had been treated with baloxavir. An epidemiological analysis suggests possible transmission of the PA I38T mutant A(H3N2) virus among humans.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral/genética , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/tratamento farmacológico , Oxazinas/farmacologia , Piridinas/farmacologia , Tiepinas/farmacologia , Triazinas/farmacologia , Adolescente , Adulto , Antivirais/uso terapêutico , Criança , Dibenzotiepinas , Inibidores Enzimáticos/farmacologia , Humanos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Influenza Humana/diagnóstico , Pacientes Internados , Japão , Pessoa de Meia-Idade , Morfolinas , Oxazinas/uso terapêutico , Reação em Cadeia da Polimerase , Piridinas/uso terapêutico , Piridonas , Tiepinas/uso terapêutico , Resultado do Tratamento , Triazinas/uso terapêutico , Adulto JovemRESUMO
Rhinoviruses (RVs) are classified into three species: RV-A, B, and C. Unlike RV-A and -B, RV-C cannot be propagated using standard cell culture systems. In order to isolate RV-Cs from clinical specimens and gain a better understanding of their biological properties and pathogenesis, we established airâ»liquid-interface (ALI) culture methods using HBEC3-KT and HSAEC1-KT immortalized human airway epithelial cells. HBEC3- and HSAEC1-ALI cultures morphologically resembled pseudostratified epithelia with cilia and goblet cells. Two fully sequenced clinical RV-C isolates, RV-C9 and -C53, were propagated in HBEC3-ALI cultures, and increases in viral RNA ranging from 1.71 log10 to 7.06 log10 copies were observed. However, this propagation did not occur in HSAEC1-ALI cultures. Using the HBEC3-ALI culture system, 11 clinical strains of RV-C were isolated from 23 clinical specimens, and of them, nine were passaged and re-propagated. The 11 clinical isolates were classified as RV-C2, -C6, -C9, -C12, -C18, -C23, -C40, and -C53 types according to their VP1 sequences. Our stable HBEC3-ALI culture system is the first cultivable cell model that supports the growth of multiple RV-C virus types from clinical specimens. Thus, the HBEC3-ALI culture system provides a cheap and easy-to-use alternative to existing cell models for isolating and investigating RV-Cs.
Assuntos
Diferenciação Celular , Enterovirus/crescimento & desenvolvimento , Células Epiteliais/virologia , Cultura de Vírus , Contagem de Células , Linhagem Celular Transformada , Enterovirus/genética , Enterovirus/fisiologia , Humanos , Sistema Respiratório/citologiaRESUMO
Influenza virus and respiratory syncytial virus cause acute upper and lower respiratory tract infections, especially in children and the elderly. Early treatment for these infections is thought to be important, so simple and sensitive detection methods are needed for use at clinical sites. Therefore, in this study, real-time reverse transcription loop-mediated isothermal amplification assays with quenching primer for influenza virus and respiratory syncytial virus were developed. Evaluation of a total of 113 clinical specimens compared to real-time RT-PCR assays showed that the novel assays could distinguish between the types and subtypes of influenza virus and respiratory syncytial virus and had 100% diagnostic specificity. The diagnostic sensitivity of each assay exceeded 85.0% and the assays showed sufficient clinical accuracy. Furthermore, positive results could be obtained in around 15 min using the novel assays in cases with high concentrations of virus. The developed assays should be useful for identifying influenza virus and respiratory syncytial virus cases not only in experimental laboratories but also in hospital and quarantine laboratories.
Assuntos
Primers do DNA/genética , Orthomyxoviridae/isolamento & purificação , Vírus Sincicial Respiratório Humano/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Humanos , Influenza Humana/diagnóstico , Influenza Humana/virologia , Infecções por Vírus Respiratório Sincicial/diagnóstico , Infecções por Vírus Respiratório Sincicial/virologia , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/virologia , Sensibilidade e Especificidade , TemperaturaRESUMO
Human rhinoviruses (RVs) belong to the genus Enterovirus of the family Picornaviridae, and are classified into RV-A, -B, and -C species. Two assays were developed to detect RVs by a real-time fluorescent reverse transcription loop-mediated isothermal amplification method: one was designed based on the 5'-untranslated regions (UTRs) of RV-A and -B, and the other was designed based on the 5'-UTR of RV-C. The competence of both assays for the diagnosis of RV infection was tested using isolated viruses and compared with real-time reverse transcription polymerase chain reaction assays on clinical specimens. Neither assay demonstrated cross-reactivity with other tested enteroviruses, and they detected 19 out of 21 tested RV-As and seven out of eight tested RV-Cs. The specificity of the assays was 100% for the detection of RVs and their sensitivity for RV-A and RV-C was 86.3% and 77.3%, respectively, on clinical specimens by the combined use of both assays. Considering that both developed assays were highly specific and detected the majority of recently circulating RVs, they are helpful for the diagnosis of RV infection. Consequently, the results generated by these assays will enhance the surveillance of respiratory illness and the study of the roles of RVs associated with clinical features and disease severity.
Assuntos
Fluorescência , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções por Picornaviridae/diagnóstico , Rhinovirus/genética , Temperatura , Regiões 5' não Traduzidas/genética , Primers do DNA , Humanos , Infecções por Picornaviridae/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e EspecificidadeRESUMO
The novel cap-dependent endonuclease inhibitor baloxavir marboxil was approved for the treatment of influenza virus infection in Japan in February 2018. Two influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic subunit (PA) were detected in baloxavir-treated children in December 2018. This mutation is known to confer reduced susceptibility to baloxavir, and the two mutant viruses exhibited 76- and 120-fold reduced susceptibility to baloxavir.
Assuntos
Antivirais/uso terapêutico , Endonucleases/antagonistas & inibidores , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/tratamento farmacológico , Oxazinas/uso terapêutico , Piridinas/uso terapêutico , Tiepinas/uso terapêutico , Triazinas/uso terapêutico , Substituição de Aminoácidos/genética , Antivirais/administração & dosagem , Dibenzotiepinas , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Endonucleases/genética , Humanos , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Influenza Humana/diagnóstico , Japão , Testes de Sensibilidade Microbiana , Morfolinas , Piridonas , Resultado do TratamentoRESUMO
A new reassortant H7N3 avian influenza virus (AIV) was isolated from a duck meat product that was illegally taken on board a passenger flight from China to Japan in March 2018. Sequencing analysis revealed that the H7N3 isolate, A/duck/Japan/AQ-HE30-1/2018 (Dk/HE30-1) (H7N3), was a reassortant highly pathogenic avian influenza virus (HPAIV) that contained the haemagglutinin (HA) gene of Chinese H7N9 HPAIV. Dk/HE30-1 (H7N3) possessed a novel polybasic sequence motif PEVPKRRRTAR/GLF at the HA cleavage site that has never previously been reported in H7 HPAIVs. The HA antigenicity of Dk/HE30-1 (H7N3) slightly differed from that of H7N9 HPAIVs previously reported. These findings will help further our knowledge of the circulation and genetic evolution of emerging AIVs in endemic areas.