Publications
316 results found
Dunning J, Blankley S, Hoang LT, et al., 2019, Author Correction: Progression of whole-blood transcriptional signatures from interferon-induced to neutrophil-associated patterns in severe influenza., Nature Immunology, Vol: 20, Pages: 373-373, ISSN: 1529-2908
In the version of this article initially published, a source of funding was not included in the Acknowledgements section. That section should include the following: P.J.M.O. was supported by EU FP7 PREPARE project 602525. The error has been corrected in the HTML and PDF version of the article.
James J, Smith N, Ross C, et al., 2019, The cellular localization of avian influenza virus PB1-F2 protein alters the magnitude of IFN2 promoter and NFκB-dependent promoter antagonism in chicken cells., J Gen Virol, Vol: 100, Pages: 414-430
The accessory protein, PB1-F2, of influenza A virus (IAV) functions in a chicken host to prolong infectious virus shedding and thus the transmission window. Here we show that this delay in virus clearance by PB1-F2 in chickens is accompanied by reduced transcript levels of type 1 interferon (IFN)-induced genes and NFκB-activated pro-inflammation cytokines. In vitro, two avian influenza isolate-derived PB1-F2 proteins, H9N2 UDL01 and H5N1 5092, exhibited the same antagonism of the IFN and pro-inflammation induction pathways seen in vivo, but to different extents. The two PB1-F2 proteins had different cellular localization in chicken cells, with H5N1 5092 being predominantly mitochondrial-associated and H9N2 UDL being cytoplasmic but not mitochondrial-localized. We hypothesized that PB1-F2 localization might influence the functionality of the protein during infection and that the protein sequence could alter cellular localization. We demonstrated that the sequence of the C-terminus of PB1-F2 determined cytoplasmic localization in chicken cells and this was linked with protein instability. Mitochondrial localization of PB1-F2 resulted in reduced antagonism of an NFκB-dependent promoter. In parallel, mitochondrial localization of PB1-F2 increased the potency of chicken IFN 2 induction antagonism. We suggest that mitochondrial localization of PB1-F2 restricts interaction with cytoplasmic-located IKKβ, reducing NFκB-responsive promoter antagonism, but enhances antagonism of the IFN2 promoter through interaction with the mitochondrial adaptor MAVS. Our study highlights the differential mechanisms by which IAV PB1-F2 protein can dampen the avian host innate signalling response.
Barclay WS, 2019, Receptor for bat influenza virus uncovers potential risk to humans, Nature, Vol: 567, Pages: 35-36, ISSN: 0028-0836
How bat influenza viruses infect cells has been unclear. The discovery that they bind to a cell receptor that is present in many different species raises concerns about their potential risk to humans.
Long JS, Mistry B, Haslam SM, et al., 2019, Host and viral determinants of influenza A virus species specificity (vol 17, pg 67, 2018), NATURE REVIEWS MICROBIOLOGY, Vol: 17, Pages: 124-124, ISSN: 1740-1526
Goldhill DH, Langat P, Xie H, et al., 2019, Determining the mutation bias of favipiravir in influenza using next-generation sequencing, Journal of Virology, Vol: 93, ISSN: 1098-5514
Favipiravir is a broad-spectrum antiviral drug that may be used to treat influenza. Previous research has identified that favipiravir likely acts as a mutagen but the precise mutation bias that favipiravir induces in influenza virus RNAs has not been described. Here, we use next-generation sequencing (NGS) with barcoding of individual RNA molecules to accurately and quantitatively detect favipiravir-induced mutations and to sample orders of magnitude more mutations than would be possible through Sanger sequencing. We demonstrate that favipiravir causes mutations and show that favipiravir primarily acts as a guanine analogue and secondarily as an adenine analogue resulting in the accumulation of transition mutations. We also use a standard NGS pipeline to show that the mutagenic effect of favipiravir can be measured by whole genome sequencing of virus.IMPORTANCE New antiviral drugs are needed as a first line of defence in the event of a novel influenza pandemic. Favipiravir is a broad-spectrum antiviral which is effective against influenza. The exact mechanism of how favipiravir works to inhibit influenza is still unclear. We used next-generation sequencing (NGS) to demonstrate that favipiravir causes mutations in influenza RNA. The greater depth of NGS sequence information over traditional sequencing methods allowed us to precisely determine the bias of particular mutations caused by favipiravir. NGS can also be used in a standard diagnostic pipeline to show that favipiravir is acting on the virus by revealing the mutation bias pattern typical to the drug. Our work will aid in testing whether viruses are resistant to favipiravir and may help demonstrate the effect of favipiravir on viruses in a clinical setting. This will be important if favipiravir is used during a future influenza pandemic.
Giotis E, Carnell G, Young E, et al., 2019, The MHC class-II HLA-DR receptor mediates bat influenza A-like H17N10 virus entry into mammalian cells
Bats are notorious reservoirs of diverse, potentially zoonotic viruses, exemplified by the evolutionarily distinct, influenza A-like viruses H17N10 and H18N11 (BatIVs). The surface glycoproteins [haemagglutinin (H) and neuraminidase (N)] of BatIVs neither bind nor cleave sialic acid receptors, which suggests that these viruses employ cell attachment and entry mechanisms that differ from those of classical influenza A viruses (IAVs). Identifying the cellular factors that mediate entry and determine susceptibility to infection will help assess the host range of BatIVs. Here, we investigated a range of cell lines from different species for their susceptibility to infection by pseudotyped viruses bearing bat H17 and/or N10 envelope proteins. We show that a number of human haematopoietic cancer cell lines and the canine kidney MDCK II (but not MDCK I) cells are susceptible to H17-pseudotypes (H17-PV). We observed with microarrays and qRT-PCR that the dog leukocyte antigen DLA-DRA mRNA is overexpressed in late passaged parental MDCK and commercial MDCK II cells, compared to early passaged parental MDCK and MDCK I cells, respectively. The human orthologue HLA-DRA encodes the alpha subunit of the MHC class II HLA-DR antigen-binding heterodimer. Small interfering RNA- or neutralizing antibody-targeting HLA-DRA, drastically reduced the susceptibility of Raji B cells to H17-PV. Conversely, overexpression of HLA-DRA and its paralogue HLA-DRB1 on the surface of the unsusceptible HEK293T/17 cells conferred susceptibility to H17-PV. The identification of HLA-DR as an H17N10 entry mediator will contribute to a better understanding of the tropism of the virus and will elucidate its zoonotic transmission.
Long J, Idoko-Akoh A, Mistry B, et al., 2019, Avian ANP32B does not support influenza A virus polymerase and influenza A virus relies exclusively on ANP32A in chicken cells.: Supplementary Information
Influenza A viruses (IAV) are subject to species barriers that prevent frequent zoonotic transmission and pandemics. One of these barriers is the poor activity of avian IAV polymerases in human cells. Differences between avian and mammalian ANP32 proteins underlie this host range barrier. Human ANP32A and ANP32B homologues both support function of human-adapted influenza polymerase but do not support efficient activity of avian IAV polymerase which requires avian ANP32A. We show here that avian ANP32B is evolutionarily distinct from mammalian ANP32B, and that chicken ANP32B does not support IAV polymerase activity even of human-adapted viruses. Consequently, IAV does not replicate in chicken cells that lack ANP32A. Amino acid differences in LRR5 domain accounted for the inactivity of chicken ANP32B. Transfer of these residues to chicken ANP32A abolished support of IAV polymerase. Understanding ANP32 function will help develop antiviral strategies and aid the design of influenza virus resistant genome edited chickens.
Long JS, Mistry B, Haslam SM, et al., 2018, Host and viral determinants of influenza A virus species specificity, NATURE REVIEWS MICROBIOLOGY, Vol: 17, Pages: 67-81, ISSN: 1740-1526
Goldhill DH, te Velthuis AJW, Fletcher RA, et al., 2018, The mechanism of resistance to favipiravir in influenza, Proceedings of the National Academy of Sciences of the United States of America, Vol: 115, Pages: 11613-11618, ISSN: 0027-8424
Favipiravir is a broad-spectrum antiviral that has shown promise in treatment of influenza virus infections. While emergence of resistance has been observed for many antiinfluenza drugs, to date, clinical trials and laboratory studies of favipiravir have not yielded resistant viruses. Here we show evolution of resistance to favipiravir in the pandemic H1N1 influenza A virus in a laboratory setting. We found that two mutations were required for robust resistance to favipiravir. We demonstrate that a K229R mutation in motif F of the PB1 subunit of the influenza virus RNA-dependent RNA polymerase (RdRP) confers resistance to favipiravir in vitro and in cell culture. This mutation has a cost to viral fitness, but fitness can be restored by a P653L mutation in the PA subunit of the polymerase. K229R also conferred favipiravir resistance to RNA polymerases of other influenza A virus strains, and its location within a highly conserved structural feature of the RdRP suggests that other RNA viruses might also acquire resistance through mutations in motif F. The mutations identified here could be used to screen influenza virus-infected patients treated with favipiravir for the emergence of resistance.
Te Velthuis AJW, Long JS, Barclay WS, 2018, Assays to Measure the Activity of Influenza Virus Polymerase., Methods Mol Biol, Vol: 1836, Pages: 343-374
Influenza viruses use an RNA-dependent RNA polymerase (RdRp) to transcribe and replicate their segmented negative-stranded RNA genomes. The influenza A virus RdRp consists of a heterotrimeric complex of the proteins PB1, PB2, and PA. The RdRp is associated with the incoming influenza A viral RNA (vRNA) genome bound by the viral nucleoprotein (NP), in complexes called viral ribonucleoproteins, vRNPs. During the viral replication cycle, the RdRp snatches capped primers from nascent host mRNAs to carry out primary viral transcription. Viral mRNA translation produces new copies of the RdRp subunits and NP, which are required to stabilize and encapsidate complementary copies of the genome (cRNAs), forming cRNPs. These cRNPs then use the cRNAs to make new vRNAs, which are encapsidated into new vRNPs. Secondary transcription by new vRNPs results in further viral mRNAs and an increase of the viral protein load in the cell. The activities of the RdRp (mRNA, cRNA, and vRNA synthesis) in the influenza virus replication cycle can be measured on several levels, ranging from assessment of the accumulation of RNA products in virus-infected cells, through in situ reconstitution of the RdRp from cloned cDNAs, to in vitro biochemical assays that allow the dissection of individual functions of the RdRp enzyme. Here we describe these assays and point out the advantages and drawbacks of each.
Barclay W, Openshaw P, 2018, The 1918 Influenza Pandemic: one hundred years of progress, but where now?, Lancet Respiratory Medicine, Vol: 6, ISSN: 2213-2600
Goldhill DH, Langat P, Xie H, et al., 2018, Determining the mutation bias of favipiravir in influenza using next-generation sequencing, Publisher: Cold Spring Harbor Laboratory
Favipiravir is a broad spectrum antiviral drug that may be used to treat influenza. Previous research has identified that favipiravir likely acts as a mutagen but the precise mutation bias that favipiravir induces in influenza virus RNAs has not been described. Here, we use next-generation sequencing (NGS) with barcoding of individual RNA molecules to accurately and quantitatively detect favipiravir-induced mutations and to sample orders of magnitude more mutations than would be possible through Sanger sequencing. We demonstrate that favipiravir causes mutations and show that favipiravir primarily acts as a guanine analogue and secondarily as an adenine analogue resulting in the accumulation of transition mutations. We also use a standard NGS pipeline to show that the mutagenic effect of favipiravir can be measured by whole genome sequencing of virus.
Belser JA, Barclay W, Barr I, et al., 2018, Ferrets as Models for Influenza Virus Transmission Studies and Pandemic Risk Assessments, EMERGING INFECTIOUS DISEASES, Vol: 24, Pages: 965-971, ISSN: 1080-6040
Dunning J, Blankley S, Hoang LT, et al., 2018, Progression of whole-blood transcriptional signatures from interferon-induced to neutrophil-associated patterns in severe influenza, Nature Immunology, Vol: 19, Pages: 625-635, ISSN: 1529-2916
Transcriptional profiles and host-response biomarkers are used increasingly to investigate the severity, subtype and pathogenesis of disease. We now describe whole-blood mRNA signatures and concentrations of local and systemic immunological mediators in 131 adults hospitalized with influenza, from whom extensive clinical and investigational data were obtained by MOSAIC investigators. Signatures reflective of interferon-related antiviral pathways were common up to day 4 of symptoms in patients who did not require mechanical ventilator support; in those who needed mechanical ventilation, an inflammatory, activated-neutrophil and cell-stress or death (‘bacterial’) pattern was seen, even early in disease. Identifiable bacterial co-infection was not necessary for this ‘bacterial’ signature but was able to enhance its development while attenuating the early ‘viral’ signature. Our findings emphasize the importance of timing and severity in the interpretation of host responses to acute viral infection and identify specific patterns of immune-system activation that might enable the development of novel diagnostic and therapeutic tools for severe influenza.
Groves HT, McDonald JU, Langat P, et al., 2018, Mouse Models of Influenza Infection with Circulating Strains to Test Seasonal Vaccine Efficacy, Frontiers in Immunology, Vol: 9, ISSN: 1664-3224
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 predic
Li H, Bradley KC, Long JS, et al., 2018, Internal genes of a highly pathogenic H5N1 influenza virus determine high viral replication in myeloid cells and severe outcome of infection in mice., PLoS Pathogens, Vol: 14, ISSN: 1553-7366
The highly pathogenic avian influenza (HPAI) H5N1 influenza virus has been a public health concern for more than a decade because of its frequent zoonoses and the high case fatality rate associated with human infections. Severe disease following H5N1 influenza infection is often associated with dysregulated host innate immune response also known as cytokine storm but the virological and cellular basis of these responses has not been clearly described. We rescued a series of 6:2 reassortant viruses that combined a PR8 HA/NA pairing with the internal gene segments from human adapted H1N1, H3N2, or avian H5N1 viruses and found that mice infected with the virus with H5N1 internal genes suffered severe weight loss associated with increased lung cytokines but not high viral load. This phenotype did not map to the NS gene segment, and NS1 protein of H5N1 virus functioned as a type I IFN antagonist as efficient as NS1 of H1N1 or H3N2 viruses. Instead we discovered that the internal genes of H5N1 virus supported a much higher level of replication of viral RNAs in myeloid cells in vitro but not in epithelial cells and that this was associated with high induction of type I IFN in myeloid cells. We also found that in vivo during H5N1 recombinant virus infection cells of haematopoetic origin were infected and produced type I IFN and proinflammatory cytokines. Taken together our data infer that human and avian influenza viruses are differently controlled by host factors in alternative cell types; internal gene segments of avian H5N1 virus uniquely drove high viral replication in myeloid cells, which triggered an excessive cytokine production, resulting in severe immunopathology.
Singanayagam A, Zambon M, Lalvani A, et al., 2017, Can defective interfering RNAs affect the live attenuated influenza vaccine? Reply, Lancet Infectious Diseases, Vol: 17, Pages: 1235-1236, ISSN: 1473-3099
Singanayagam A, Zambon M, Lalvani A, et al., 2017, Urgent challenges in implementing live attenuated influenza vaccine., Lancet Infectious Diseases, Vol: 18, Pages: e25-e32, ISSN: 1473-3099
Conflicting reports have emerged about the effectiveness of the live attenuated influenza vaccine. The live attenuated influenza vaccine appears to protect particularly poorly against currently circulating H1N1 viruses that are derived from the 2009 pandemic H1N1 viruses. During the 2015-16 influenza season, when pandemic H1N1 was the predominant virus, studies from the USA reported a complete lack of effectiveness of the live vaccine in children. This finding led to a crucial decision in the USA to recommend that the live vaccine not be used in 2016-17 and to switch to the inactivated influenza vaccine. Other countries, including the UK, Canada, and Finland, however, have continued to recommend the use of the live vaccine. This policy divergence and uncertainty has far reaching implications for the entire global community, given the importance of the production capabilities of the live attenuated influenza vaccine for pandemic preparedness. In this Personal View, we discuss possible explanations for the observed reduced effectiveness of the live attenuated influenza vaccine and highlight the underpinning scientific questions. Further research to understand the reasons for these observations is essential to enable informed public health policy and commercial decisions about vaccine production and development in coming years.
Peacock TP, Benton DJ, James J, et al., 2017, Immune Escape Variants of H9N2 Influenza Viruses Containing Deletions at the Hemagglutinin Receptor Binding Site Retain Fitness In Vivo and Display Enhanced Zoonotic Characteristics, Journal of Virology, Vol: 91, ISSN: 1098-5514
H9N2 avian influenza viruses are enzootic in poultry across Asia and North Africa, where they pose a threat to human health as both zoonotic agents and potential pandemic candidates. Poultry vaccination against H9N2 viruses has been employed in many regions; however, vaccine effectiveness is frequently compromised due to antigenic drift arising from amino acid substitutions in the major influenza virus antigen hemagglutinin (HA). Using selection with HA-specific monoclonal antibodies, we previously identified H9N2 antibody escape mutants that contained deletions of amino acids in the 220 loop of the HA receptor binding sites (RBSs). Here we analyzed the impact of these deletions on virus zoonotic infection characteristics and fitness. We demonstrated that mutant viruses with RBS deletions are able to escape polyclonal antiserum binding and are able to infect and be transmitted between chickens. We showed that the deletion mutants have increased binding to human-like receptors and greater replication in primary human airway cells; however, the mutant HAs also displayed reduced pH and thermal stability. In summary, we infer that variant influenza viruses with deletions in the 220 loop could arise in the field due to immune selection pressure; however, due to reduced HA stability, we conclude that these viruses are unlikely to be transmitted from human to human by the airborne route, a prerequisite for pandemic emergence. Our findings underscore the complex interplay between antigenic drift and viral fitness for avian influenza viruses as well as the challenges of predicting which viral variants may pose the greatest threats for zoonotic and pandemic emergence.
Barclay WS, 2017, Influenza: a world of discoveries, outbreaks and controversy, JOURNAL OF GENERAL VIROLOGY, Vol: 98, Pages: 892-894, ISSN: 0022-1317
Cole SL, Dunning J, Kok WL, et al., 2017, M1-like monocytes are a major immunological determinant of severity in previously healthy adults with life-threatening influenza., JCI Insight, Vol: 2, ISSN: 2379-3708
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.
Peacock TP, Benton DJ, Sadeyen J-R, et al., 2017, Variability in H9N2 haemagglutinin receptor-binding preference and the pH of fusion, EMERGING MICROBES & INFECTIONS, Vol: 6, ISSN: 2222-1751
H9N2 avian influenza viruses are primarily a disease of poultry; however, they occasionally infect humans and are considered a potential pandemic threat. Little work has been performed to assess the intrinsic biochemical properties related to zoonotic potential of H9N2 viruses. The objective of this study, therefore, was to investigate H9N2 haemagglutinins (HAs) using two well-known correlates for human adaption: receptor-binding avidity and pH of fusion. Receptor binding was characterized using bio-layer interferometry to measure virus binding to human and avian-like receptor analogues and the pH of fusion was assayed by syncytium formation in virus-infected cells at different pHs. We characterized contemporary H9N2 viruses of the zoonotic G1 lineage, as well as representative viruses of the zoonotic BJ94 lineage. We found that most contemporary H9N2 viruses show a preference for sulphated avian-like receptor analogues. However, the ‘Eastern’ G1 H9N2 viruses displayed a consistent preference in binding to a human-like receptor analogue. We demonstrate that the presence of leucine at position 226 of the HA receptor-binding site correlated poorly with the ability to bind a human-like sialic acid receptor. H9N2 HAs also display variability in their pH of fusion, ranging between pH 5.4 and 5.85 which is similar to that of the first wave of human H1N1pdm09 viruses but lower than the pH of fusion seen in zoonotic H5N1 and H7N9 viruses. Our results suggest possible molecular mechanisms that may underlie the relatively high prevalence of human zoonotic infection by particular H9N2 virus lineages.
Lipsitch M, Barclay W, Raman R, et al., 2016, Viral factors in influenza pandemic risk assessment, eLife, Vol: 5, ISSN: 2050-084X
The threat of an influenza A virus pandemic stems from continual virus spillovers from reservoir species, a tiny fraction of which spark sustained transmission in humans. To date, no pandemic emergence of a new influenza strain has been preceded by detection of a closely related precursor in an animal or human. Nonetheless, influenza surveillance efforts are expanding, prompting a need for tools to assess the pandemic risk posed by a detected virus. The goal would be to use genetic sequence and/or biological assays of viral traits to identify those non-human influenza viruses with the greatest risk of evolving into pandemic threats, and/or to understand drivers of such evolution, to prioritize pandemic prevention or response measures. We describe such efforts, identify progress and ongoing challenges, and discuss three specific traits of influenza viruses (hemagglutinin receptor binding specificity, hemagglutinin pH of activation, and polymerase complex efficiency) that contribute to pandemic risk.
Dowall SD, Bewley K, Watson RJ, et al., 2016, Antiviral Screening of Multiple Compounds against Ebola Virus, Viruses, Vol: 8, ISSN: 1999-4915
In light of the recent outbreak of Ebola virus (EBOV) disease in West Africa, there have been renewed efforts to search for effective antiviral countermeasures. A range of compounds currently available with broad antimicrobial activity have been tested for activity against EBOV. Using live EBOV, eighteen candidate compounds were screened for antiviral activity in vitro. The compounds were selected on a rational basis because their mechanisms of action suggested that they had the potential to disrupt EBOV entry, replication or exit from cells or because they had displayed some antiviral activity against EBOV in previous tests. Nine compounds caused no reduction in viral replication despite cells remaining healthy, so they were excluded from further analysis (zidovudine; didanosine; stavudine; abacavir sulphate; entecavir; JB1a; Aimspro; celgosivir; and castanospermine). A second screen of the remaining compounds and the feasibility of appropriateness for in vivo testing removed six further compounds (ouabain; omeprazole; esomeprazole; Gleevec; D-LANA-14; and Tasigna). The three most promising compounds (17-DMAG; BGB324; and NCK-8) were further screened for in vivo activity in the guinea pig model of EBOV disease. Two of the compounds, BGB324 and NCK-8, showed some effect against lethal infection in vivo at the concentrations tested, which warrants further investigation. Further, these data add to the body of knowledge on the antiviral activities of multiple compounds against EBOV and indicate that the scientific community should invest more effort into the development of novel and specific antiviral compounds to treat Ebola virus disease.
James J, Howard W, Iqbal M, et al., 2016, Influenza A virus PB1-F2 protein prolongs viral shedding in chickens lengthening the transmission window, Journal of General Virology, Vol: 97, Pages: 2516-2527, ISSN: 1465-2099
Avian influenza is a significant economic burden on the poultry industry in geographical regions where it is enzootic. It also poses a public health concern when avian influenza subtypes infect humans, often with high mortality. Understanding viral genetic factors which positively contribute to influenza A virus (IAV) fitness – infectivity, spread and pathogenesis – is of great importance both for human and livestock health. PB1-F2 is a small accessory protein encoded by IAV and in mammalian hosts has been implicated in a wide range of functions that contribute to increased pathogenesis. In the avian host, the protein has been understudied despite high-level full-length conservation in avian IAV isolates, which is in contrast to the truncations of the PB1-F2 length frequently found in mammalian host isolates. Here we report that the presence of a full-length PB1-F2 protein, from a low pathogenicity H9N2 avian influenza virus, prolongs infectious virus shedding from directly inoculated chickens, thereby enhancing transmission of the virus by lengthening the transmission window to contact birds. As well as extending transmission, the presence of a full-length PB1-F2 suppresses pathogenicity evidenced by an increased minimum lethal dose in embryonated chicken eggs and increasing survival in directly infected birds when compared to a virus lacking an ORF for PB1-F2. We propose that there is a positive pressure to maintain a full-length functional PB1-F2 protein upon infection of avian hosts as it contributes to the effective transmission of IAV in the field.
Pizzuto MS, Silic-Benussi M, Ciminale V, et al., 2016, An engineered avian-origin influenza A virus for pancreatic ductal adenocarcinoma virotherapy, JOURNAL OF GENERAL VIROLOGY, Vol: 97, Pages: 2166-2179, ISSN: 0022-1317
Frise R, Bradley K, van Doremalen N, et al., 2016, Contact transmission of influenza virus between ferrets imposes a looser bottleneck than respiratory droplet transmission allowing propagation of antiviral resistance, Scientific Reports, Vol: 6, ISSN: 2045-2322
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.
Kobayashi Y, Dadonaite B, van Doremalen N, et al., 2016, Computational and molecular analysis of conserved influenza A virus RNA secondary structures involved in infectious virion production, RNA Biology, ISSN: 1547-6286
As well as encoding viral proteins, genomes of RNA viruses harbor secondary and tertiary RNA structures that have been associated with functions essential for successful replication and propagation. Here, we identified stem-loop structures that are extremely conserved among 1,884 M segment sequences of influenza A virus (IAV) strains from various subtypes and host species using computational and evolutionary methods. These structures were predicted within the 3' and 5' ends of the coding regions of M1 and M2, respectively, where packaging signals have been previously proposed to exist. These signals are thought to be required for the incorporation of a single copy of eight different negative-strand RNA segments (vRNAs) into an IAV particle. To directly test the functionality of conserved stem-loop structures, we undertook reverse genetic experiments to introduce synonymous mutations designed to disrupt secondary structures predicted at three locations and found them to attenuate infectivity of recombinant virus. In one mutant, predicted to disrupt stem loop structure at nucleotide positions 219-240, attenuation was more evident at increased temperature and was accompanied by an increase in the production of defective virus particles. Our results suggest that the conserved secondary structures predicted in the M segment are involved in the production of infectious viral particles during IAV replication.
Elderfield RA, Koutsakos M, Frise R, et al., 2016, NB protein does not affect Influenza B virus replication in vitro and is not required for replication in or transmission between ferrets., Journal of General Virology, Vol: 97, Pages: 593-601, ISSN: 1465-2099
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 protein, an important caveat since NA is encoded on the same segment and initiated from a start codon just 4 nucleotides 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 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.
Peacock T, Reddy K, James J, et al., 2016, Antigenic mapping of an H9N2 avian influenza virus reveals two discrete antigenic sites and a novel mechanism of immune escape, Scientific Reports, Vol: 6, ISSN: 2045-2322
H9N2 avian influenza virus is a major cause of poultry production loss across Asia leading to the wide useof vaccines. Efficacy of vaccines is often compromised due to the rapid emergence of antigenic variants.To improve the effectiveness of vaccines in the field, a better understanding of the antigenic epitopesof the major antigen, hemagglutinin, is required. To address this, a panel of nine monoclonal antibodieswere generated against a contemporary Pakistani H9N2 isolate, which represents a major Asian H9N2viral lineage. Antibodies were characterized in detail and used to select a total of 26 unique ‘escape’mutants with substitutions across nine different amino acid residues in hemagglutinin including seventhat have not been described as antigenic determinants for H9N2 viruses before. Competition assaysand structural mapping revealed two novel, discrete antigenic sites “H9-A” and “H9-B”. Additionally,a second subset of escape mutants contained amino acid deletions within the hemagglutinin receptorbinding site. This constitutes a novel method of escape for group 1 hemagglutinins and could representan alternative means for H9N2 viruses to overcome vaccine induced immunity. These results will guidesurveillance efforts for arising antigenic variants as well as evidence based vaccine seed selection andvaccine design.
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