RESUMO
We evaluated the performance of 12 lateral flow devices by assessing their analytical sensitivity for SARS-CoV-2 variant BA.2.86. Kits from ACON, Orient Gene, Xiamen Biotime, Getein, and SureScreen detected variant BA.2.86 to sufficient sensitivity levels, comparable to those observed with previous Omicron variants. The stocks of lateral flow devices currently held by the UK government do not currently need changing for deployment for this variant.
Assuntos
COVID-19 , Humanos , SARS-CoV-2 , GovernoRESUMO
Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic "swine" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.
Assuntos
Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Influenza Humana/virologia , Rimantadina/farmacologia , Proteínas da Matriz Viral/antagonistas & inibidores , Zanamivir/farmacologia , Antivirais/farmacologia , Farmacorresistência Viral , Sinergismo Farmacológico , Quimioterapia Combinada , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Influenza Humana/tratamento farmacológico , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismoRESUMO
The transmissibility and pandemic potential of influenza viruses depends on their ability to efficiently replicate and be released from an infected host, retain viability as they pass through the environment, and then initiate infection in the next host. There is a significant gap in knowledge about viral properties that enable survival of influenza viruses between hosts, due to a lack of experimental methods to reliably isolate viable virus from the air. Using a novel technique, we isolate and characterise infectious virus from droplets emitted by 2009 pandemic H1N1-infected ferrets. We demonstrate that infectious virus is predominantly released early after infection. A virus containing a mutation destabilising the haemagglutinin (HA) surface protein displayed reduced survival in air. Infectious virus recovered from droplets exhaled by ferrets inoculated with this virus contained mutations that conferred restabilisation of HA, indicating the importance of influenza HA stability for between-host survival. Using this unique approach can improve knowledge about the determinants and mechanisms of influenza transmissibility and ultimately could be applied to studies of airborne virus exhaled from infected people.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A Subtipo H1N1/metabolismo , Ar/análise , Microbiologia do Ar , Animais , Linhagem Celular , Transmissão de Doença Infecciosa , Furões/virologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Hemaglutininas/imunologia , Hemaglutininas/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/virologia , Viabilidade Microbiana/imunologia , Infecções por Orthomyxoviridae/virologiaRESUMO
Influenza virus infection is a significant cause of morbidity and mortality worldwide. The surface antigens of influenza virus change over time blunting both naturally acquired and vaccine induced adaptive immune protection. Viral antigenic drift is a major contributing factor to both the spread and disease burden of influenza. The aim of this study was to develop better infection models using clinically relevant, influenza strains to test vaccine induced protection. CB6F1 mice were infected with a range of influenza viruses and disease, inflammation, cell influx, and viral load were characterized after infection. Infection with circulating H1N1 and representative influenza B viruses induced a dose-dependent disease response; however, a recent seasonal H3N2 virus did not cause any disease in mice, even at high titers. Viral infection led to recoverable virus, detectable both by plaque assay and RNA quantification after infection, and increased upper airway inflammation on day 7 after infection comprised largely of CD8 T cells. Having established seasonal infection models, mice were immunized with seasonal inactivated vaccine and responses were compared to matched and mismatched challenge strains. While the H1N1 subtype strain recommended for vaccine use has remained constant in the seven seasons between 2010 and 2016, the circulating strain of H1N1 influenza (2009 pandemic subtype) has drifted both genetically and antigenically since 2009. To investigate the effect of this observed drift on vaccine induced protection, mice were immunized with antigens from A/California/7/2009 (H1N1) and challenged with H1N1 subtype viruses recovered from 2009, 2010, or 2015. Vaccination with A/California/7/2009 antigens protected against infection with either the 2009 or 2010 strains, but was less effective against the 2015 strain. This observed reduction in protection suggests that mouse models of influenza virus vaccination and infection can be used as an additional tool to predict vaccine efficacy against drift strains.
Assuntos
Modelos Animais de Doenças , Vacinas contra Influenza/administração & dosagem , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas Atenuadas/administração & dosagem , Animais , Antígenos Virais/imunologia , Feminino , Pulmão/virologia , Camundongos , Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/virologia , RNA Viral/análise , Estações do AnoRESUMO
In each influenza season, a distinct group of young, otherwise healthy individuals with no risk factors succumbs to life-threatening infection. To better understand the cause for this, we analyzed a broad range of immune responses in blood from a unique cohort of patients, comprising previously healthy individuals hospitalized with and without respiratory failure during one influenza season, and infected with one specific influenza A strain. This analysis was compared with similarly hospitalized influenza patients with known risk factors (total of n = 60 patients recruited). We found a sustained increase in a specific subset of proinflammatory monocytes, with high TNF-α expression and an M1-like phenotype (independent of viral titers), in these previously healthy patients with severe disease. The relationship between M1-like monocytes and immunopathology was strengthened using murine models of influenza, in which severe infection generated using different models (including the high-pathogenicity H5N1 strain) was also accompanied by high levels of circulating M1-like monocytes. Additionally, a raised M1/M2 macrophage ratio in the lungs was observed. These studies identify a specific subtype of monocytes as a modifiable immunological determinant of disease severity in this subgroup of severely ill, previously healthy patients, offering potential novel therapeutic avenues.
Assuntos
Influenza Humana/imunologia , Macrófagos/imunologia , Monócitos/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Idoso , Animais , Feminino , Humanos , Virus da Influenza A Subtipo H5N1 , Influenza Humana/patologia , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fenótipo , Carga Viral , Adulto JovemRESUMO
Pancreatic ductal adenocarcinoma (PDA) is one of the leading causes of cancer-related deaths worldwide and the development of new treatment strategies for PDA patients is of crucial importance. Virotherapy uses natural or engineered oncolytic viruses (OVs) to selectively kill tumour cells. Due to their genetic heterogeneity, PDA cells are highly variable in their permissiveness to various OVs. The avian influenza A virus (IAV) H7N3 A/turkey/Italy/2962/03 is a potent inducer of apoptosis in PDA cells previously shown to be resistant to other OVs (Kasloff et al., 2014), suggesting that it might be effective against specific subclasses of pancreatic cancer. To improve the selectivity of the avian influenza isolate for PDA cells, here confirmed deficient for IFN response, we engineered a truncation in the NS1 gene that is the major virus-encoded IFN antagonist. The recombinant virus (NS1-77) replicated efficiently in PDA cells, but was attenuated in non-malignant pancreatic ductal cells, in which it induced a potent IFN response that acted upon bystander uninfected cancer cells, triggering their death. The engineered virus displayed an enhanced ability to debulk a PDA-derived tumour in xenograft mouse model. Our results highlight the possibility of selecting an IAV strain from the diverse natural avian reservoir on the basis of its inherent oncolytic potency in specific PDA subclasses and, through engineering, improve its safety, selectivity and debulking activity for cancer treatment.
Assuntos
Adenocarcinoma/terapia , Carcinoma Ductal Pancreático/terapia , Vírus da Influenza A Subtipo H7N3/isolamento & purificação , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/isolamento & purificação , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Xenoenxertos , Humanos , Fatores Imunológicos/genética , Fatores Imunológicos/metabolismo , Vírus da Influenza A Subtipo H7N3/genética , Vírus da Influenza A Subtipo H7N3/fisiologia , Camundongos , Camundongos SCID , Vírus Oncolíticos/genética , Deleção de Sequência , Resultado do Tratamento , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação ViralRESUMO
Influenza viruses cause annual seasonal epidemics and occasional pandemics. It is important to elucidate the stringency of bottlenecks during transmission to shed light on mechanisms that underlie the evolution and propagation of antigenic drift, host range switching or drug resistance. The virus spreads between people by different routes, including through the air in droplets and aerosols, and by direct contact. By housing ferrets under different conditions, it is possible to mimic various routes of transmission. Here, we inoculated donor animals with a mixture of two viruses whose genomes differed by one or two reverse engineered synonymous mutations, and measured the transmission of the mixture to exposed sentinel animals. Transmission through the air imposed a tight bottleneck since most recipient animals became infected by only one virus. In contrast, a direct contact transmission chain propagated a mixture of viruses suggesting the dose transferred by this route was higher. From animals with a mixed infection of viruses that were resistant and sensitive to the antiviral drug oseltamivir, resistance was propagated through contact transmission but not by air. These data imply that transmission events with a looser bottleneck can propagate minority variants and may be an important route for influenza evolution.
Assuntos
Transmissão de Doença Infecciosa , Farmacorresistência Viral , Infecções por Orthomyxoviridae/transmissão , Sistema Respiratório/virologia , Animais , Antivirais/farmacologia , Cães , Feminino , Furões , Genoma Viral/genética , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Influenza Humana/transmissão , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Mutação , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Oseltamivir/farmacologiaRESUMO
The influenza B virus encodes a unique protein, NB, a membrane protein whose function in the replication cycle is not, as yet, understood. We engineered a recombinant influenza B virus lacking NB expression, with no concomitant difference in expression or activity of viral neuraminidase (NA) protein, an important caveat since NA is encoded on the same segment and initiated from a start codon just 4ânt downstream of NB. Replication of the virus lacking NB was not different to wild-type virus with full-length NB in clonal immortalized or complex primary cell cultures. In the mouse model, virus lacking NB induced slightly lower IFN-α levels in infected lungs, but this did not affect virus titres or weight loss. In ferrets infected with a mixture of viruses that did or did not express NB, there was no fitness advantage for the virus that retained NB. Moreover, virus lacking NB protein was transmitted following respiratory droplet exposure of sentinel animals. These data suggest no role for NB in supporting replication or transmission in vivo in this animal model. The role of NB and the nature of selection to retain it in all natural influenza B viruses remain unclear.
Assuntos
Furões , Vírus da Influenza B/fisiologia , Influenza Humana/transmissão , Influenza Humana/virologia , Proteínas Virais/metabolismo , Replicação Viral , Animais , Modelos Animais de Doenças , Feminino , Humanos , Vírus da Influenza B/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Virais/genéticaRESUMO
Influenza nucleoprotein (NP) is a major component of the ribonucleoprotein (vRNP) in influenza virus, which functions for the transcription and replication of viral genome. Compared to the nucleoprotein of influenza A (ANP), the N-terminal region of influenza B nucleoprotein (BNP) is much extended. By virus reconstitution, we found that the first 38 residues are essential for viral growth. We further illustrated the function of BNP by mini-genome reconstitution, fluorescence microscopy, electron microscopy, light scattering and gel shift. Results show that the N terminus is involved in the formation of both higher homo-oligomers of BNP and BNP-RNA complex.
Assuntos
Vírus da Influenza B/química , RNA Viral/química , Proteínas do Core Viral/química , Vírus da Influenza A/química , Vírus da Influenza A/metabolismo , Vírus da Influenza B/metabolismo , Proteínas do Nucleocapsídeo , Estrutura Terciária de Proteína , RNA Viral/metabolismo , Proteínas do Core Viral/metabolismoRESUMO
Ferrets have become the model animal of choice for influenza pathology and transmission experiments as they are permissive and susceptible to human influenza A viruses. However, inoculation of ferrets with mumps virus (MuV) did not lead to successful infections. We evaluated the use of highly differentiated ferret tracheal epithelium cell cultures, FTE, for predicting the potential of ferrets to support respiratory viral infections. FTE cultures supported productive replication of human influenza A and B viruses but not of MuV, whereas analogous cells generated from human airways supported replication of all three viruses. We propose that in vitro strategies using these cultures might serve as a method of triaging viruses and potentially reducing the use of ferrets in viral studies.
Assuntos
Células Epiteliais/virologia , Furões , Vírus da Influenza B/fisiologia , Influenza Humana/virologia , Vírus da Caxumba/fisiologia , Traqueia/citologia , Replicação Viral , Animais , Técnicas de Cultura de Células , Modelos Animais de Doenças , Furões/virologia , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/fisiologia , Vírus da Influenza B/genética , Vírus da Influenza B/crescimento & desenvolvimento , Caxumba/virologia , Vírus da Caxumba/genética , Vírus da Caxumba/crescimento & desenvolvimento , Traqueia/virologia , Cultura de VírusRESUMO
UNLABELLED: The influenza pandemic that emerged in 2009 provided an unprecedented opportunity to study adaptation of a virus recently acquired from an animal source during human transmission. In the United Kingdom, the novel virus spread in three temporally distinct waves between 2009 and 2011. Phylogenetic analysis of complete viral genomes showed that mutations accumulated over time. Second- and third-wave viruses replicated more rapidly in human airway epithelial (HAE) cells than did the first-wave virus. In infected mice, weight loss varied between viral isolates from the same wave but showed no distinct pattern with wave and did not correlate with viral load in the mouse lungs or severity of disease in the human donor. However, second- and third-wave viruses induced less alpha interferon in the infected mouse lungs. NS1 protein, an interferon antagonist, had accumulated several mutations in second- and third-wave viruses. Recombinant viruses with the third-wave NS gene induced less interferon in human cells, but this alone did not account for increased virus fitness in HAE cells. Mutations in HA and NA genes in third-wave viruses caused increased binding to α-2,6-sialic acid and enhanced infectivity in human mucus. A recombinant virus with these two segments replicated more efficiently in HAE cells. A mutation in PA (N321K) enhanced polymerase activity of third-wave viruses and also provided a replicative advantage in HAE cells. Therefore, multiple mutations allowed incremental changes in viral fitness, which together may have contributed to the apparent increase in severity of A(H1N1)pdm09 influenza virus during successive waves. IMPORTANCE: Although most people infected with the 2009 pandemic influenza virus had mild or unapparent symptoms, some suffered severe and devastating disease. The reasons for this variability were unknown, but the numbers of severe cases increased during successive waves of human infection in the United Kingdom. To determine the causes of this variation, we studied genetic changes in virus isolates from individual hospitalized patients. There were no consistent differences between these viruses and those circulating in the community, but we found multiple evolutionary changes that in combination over time increased the virus's ability to infect human cells. These adaptations may explain the remarkable ability of A(H1N1)pdm09 virus to continue to circulate despite widespread immunity and the apparent increase in severity of influenza over successive waves of infection.
Assuntos
Adaptação Biológica , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/virologia , Mutação , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Modelos Animais de Doenças , Feminino , Genoma Viral , Humanos , Lactente , Recém-Nascido , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Influenza Humana/epidemiologia , Interferons/metabolismo , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Masculino , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Filogenia , RNA Viral , Análise de Sequência de DNA , Reino Unido/epidemiologia , Ligação Viral , Replicação Viral , Adulto JovemRESUMO
Following the emergence and global spread of a novel H1N1 influenza virus in 2009, two A(H1N1)pdm/09 influenza vaccines produced from the A/California/07/09 H1N1 strain were selected and used for the national immunisation programme in the United Kingdom: an adjuvanted split virion vaccine and a non-adjuvanted whole virion vaccine. In this study, we assessed the immune responses generated in inbred large white pigs (Babraham line) following vaccination with these vaccines and after challenge with A(H1N1)pdm/09 virus three months post-vaccination. Both vaccines elicited strong antibody responses, which included high levels of influenza-specific IgG1 and haemagglutination inhibition titres to H1 virus. Immunisation with the adjuvanted split vaccine induced significantly higher interferon gamma production, increased frequency of interferon gamma-producing cells and proliferation of CD4(-)CD8(+) (cytotoxic) and CD4(+)CD8(+) (helper) T cells, after in vitro re-stimulation. Despite significant differences in the magnitude and breadth of immune responses in the two vaccinated and mock treated groups, similar quantities of viral RNA were detected from the nasal cavity in all pigs after live virus challenge. The present study provides support for the use of the pig as a valid experimental model for influenza infections in humans, including the assessment of protective efficacy of therapeutic interventions.
Assuntos
Adjuvantes Imunológicos/farmacologia , Vírus da Influenza A Subtipo H1N1 , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Subpopulações de Linfócitos T/imunologia , Animais , Linhagem Celular , Proliferação de Células , Cães , Ensaio de Imunoadsorção Enzimática , ELISPOT , Citometria de Fluxo , Fluoresceínas , Imunoglobulina G/sangue , Leucócitos Mononucleares , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estatísticas não Paramétricas , Succinimidas , Sus scrofaAssuntos
Vírus da Influenza A/patogenicidade , Influenza Humana/epidemiologia , Influenza Humana/virologia , Infecções por Orthomyxoviridae/epidemiologia , Infecções por Orthomyxoviridae/virologia , Pandemias , Animais , Humanos , Influenza Humana/transmissão , Infecções por Orthomyxoviridae/transmissãoRESUMO
Recovery of an infectious virus of defined genetic structure entirely from cDNA and the deduction of information about the virus resulting from phenotypic characterization of the mutant is the process of reverse genetics. This approach has been possible for a number of negative-strand RNA viruses since the recovery of rabies virus in 1994. However, the recovery of recombinant orthomyxoviruses posed a greater challenge due to the segmented nature of the genome. It was not until 1999 that such a system was reported for influenza A viruses, but since that time our knowledge of influenza A virus biology has grown dramatically. Annual influenza epidemics are caused not only by influenza A viruses but also by influenza B viruses. In 2002, two groups reported the successful recovery of influenza B virus entirely from cDNA. This has allowed greater depth of study into the biology of these viruses. This review will highlight the advances made in various areas of influenza B virus biology as a result of the development of reverse genetics techniques for these viruses, including (i) the importance of the non-coding regions of the influenza B virus genome; (ii) the generation of novel vaccine strains; (iii) studies into the mechanisms of drug resistance; (iv) the function(s) of viral proteins, both those analogous to influenza A virus proteins and those unique to influenza B viruses. The information generated by the application of influenza B virus reverse genetics systems will continue to contribute to our improved surveillance and control of human influenza.