19 results found
Goldhill DH, Yan A, Frise R, et al., 2021, Favipiravir-resistant influenza A virus shows potential for transmission, PLoS Pathogens, Vol: 17, Pages: 1-17, ISSN: 1553-7366
Favipiravir is a nucleoside analogue which has been licensed to treat influenza in the event of a new pandemic. We previously described a favipiravir resistant influenza A virus generated by in vitro passage in presence of drug with two mutations: K229R in PB1, which conferred resistance at a cost to polymerase activity, and P653L in PA, which compensated for the cost of polymerase activity. However, the clinical relevance of these mutations is unclear as the mutations have not been found in natural isolates and it is unknown whether viruses harbouring these mutations would replicate or transmit in vivo. Here, we infected ferrets with a mix of wild type p(H1N1) 2009 and corresponding favipiravir-resistant virus and tested for replication and transmission in the absence of drug. Favipiravir-resistant virus successfully infected ferrets and was transmitted by both contact transmission and respiratory droplet routes. However, sequencing revealed the mutation that conferred resistance, K229R, decreased in frequency over time within ferrets. Modelling revealed that due to a fitness advantage for the PA P653L mutant, reassortment with the wild-type virus to gain wild-type PB1 segment in vivo resulted in the loss of the PB1 resistance mutation K229R. We demonstrated that this fitness advantage of PA P653L in the background of our starting virus A/England/195/2009 was due to a maladapted PA in first wave isolates from the 2009 pandemic. We show there is no fitness advantage of P653L in more recent pH1N1 influenza A viruses. Therefore, whilst favipiravir-resistant virus can transmit in vivo, the likelihood that the resistance mutation is retained in the absence of drug pressure may vary depending on the genetic background of the starting viral strain.
Lee LYY, Zhou J, Koszalka P, et al., 2021, Evaluating the fitness of PA/I38T-substituted influenza A viruses with reduced baloxavir susceptibility in a competitive mixtures ferret model, PLOS PATHOGENS, Vol: 17, ISSN: 1553-7366
Moshe M, Daunt A, Flower B, et al., 2021, SARS-CoV-2 lateral flow assays for possible use in national covid-19 seroprevalence surveys (REACT2): diagnostic accuracy study, BMJ: British Medical Journal, Vol: 372, Pages: 1-8, ISSN: 0959-535X
Objective: To evaluate the performance of new lateral flow immunoassays (LFIAs) suitable for use in a national COVID-19 seroprevalence programme (REACT2).Design: Laboratory sensitivity and specificity analyses were performed for seven LFIAs on a minimum of 200 sera from individuals with confirmed SARS-CoV-2 infection, and 500 pre-pandemic sera respectively. Three LFIAs were found to have a laboratory sensitivity superior to the finger-prick sensitivity of the LFIA currently used in REACT2 seroprevalence studies (84%). These LFIAs were then further evaluated through finger-prick testing on participants with confirmed previous SARS-CoV-2 infection. Two LFIAs (Surescreen, Panbio) were evaluated in clinics in June-July, 2020, and a third LFIA (AbC-19) in September, 2020. A Spike protein enzyme-linked immunoassay (S-ELISA) and hybrid double antigen binding assay (DABA) were used as laboratory reference standards.Setting: Laboratory analyses were performed at Imperial College, London and University facilities in London, UK. Research clinics for finger-prick sampling were run in two affiliated NHS trusts.Participants: Sensitivity analysis on sera were performed on 320 stored samples from previous participants in the REACT2 programme with confirmed previous SARS-CoV-2 infection. Specificity analysis was performed using 1000 pre-pandemic sera. 100 new participants with confirmed previous SARS-CoV-2 infection attended study clinics for finger-prick testing.Main outcome measures: The accuracy of LFIAs in detecting IgG antibodies to SARS-CoV-2 in comparison to two in-house ELISAs.Results: The sensitivity of seven new LFIAs using sera varied between 69% and 100% (vs S-ELISA/hybrid DABA). Specificity using sera varied between 99.6% and 100%. Sensitivity on finger-prick testing for Panbio, Surescreen and AbC-19 was 77% (CI 61.4 to 88.2), 86% (CI 72.7 to 94.8) and 69% (CI 53.8 to 81.3) respectively vs S-ELISA/hybrid DABA. Sensitivity for sera from matched clinical samples performe
Brown JC, Goldhill DH, Zhou J, et al., 2021, Increased transmission of SARS-CoV-2 lineage B.1.1.7 (VOC 2020212/01) is not accounted for by a replicative advantage in primary airway cells or antibody escape
<jats:title>Abstract</jats:title><jats:p>Lineage B.1.1.7 (Variant of Concern 202012/01) is a new SARS-CoV-2 variant which was first sequenced in the UK in September 2020 before becoming the majority strain in the UK and spreading worldwide. The rapid spread of the B.1.1.7 variant results from increased transmissibility but the virological characteristics which underpin this advantage over other circulating strains remain unknown. Here, we demonstrate that there is no difference in viral replication between B.1.1.7 and other contemporaneous SARS-CoV-2 strains in primary human airway epithelial (HAE) cells. However, B.1.1.7 replication is disadvantaged in Vero cells potentially due to increased furin-mediated cleavage of its spike protein as a result of a P681H mutation directly adjacent to the S1/S2 cleavage site. In addition, we show that B.1.1.7 does not escape neutralisation by convalescent or post-vaccination sera. Thus, increased transmission of B.1.1.7 is not caused by increased replication, as measured on HAE cells, or escape from serological immunity.</jats:p>
Peacock TP, Goldhill DH, Zhou J, et al., 2020, The furin cleavage site of SARS-CoV-2 spike protein is a key determinant for transmission due to enhanced replication in airway cells
<jats:title>Summary</jats:title><jats:p>SARS-CoV-2 enters cells via its spike glycoprotein which must be cleaved sequentially at the S1/S2, then the S2’ cleavage sites (CS) to mediate membrane fusion. SARS-CoV-2 has a unique polybasic insertion at the S1/S2 CS, which we demonstrate can be cleaved by furin. Using lentiviral pseudotypes and a cell-culture adapted SARS-CoV-2 virus with a S1/S2 deletion, we show that the polybasic insertion is selected for in lung cells and primary human airway epithelial cultures but selected against in Vero E6, a cell line used for passaging SARS-CoV-2. We find this selective advantage depends on expression of the cell surface protease, TMPRSS2, that allows virus entry independent of endosomes thus avoiding antiviral IFITM proteins. SARS-CoV-2 virus lacking the S1/S2 furin CS was shed to lower titres from infected ferrets and was not transmitted to cohoused sentinel animals. Thus, the polybasic CS is a key determinant for efficient SARS-CoV-2 transmission.</jats:p>
Peacock T, Sheppard C, Staller E, et al., 2020, Mammalian ANP32A and ANP32B proteins drive alternative avian influenza virus polymerase adaptations, biorxiv
Abstract ANP32 proteins, which act as influenza polymerase co-factors, vary between birds and mammals. The well-known mammalian adaptation, PB2-E627K, enables influenza polymerase to use mammalian ANP32 proteins. However, some mammalian-adapted influenza viruses do not harbour this adaptation. Here, we show that alternative PB2 adaptations, Q591R and D701N also allow influenza polymerase to use mammalian ANP32 proteins. PB2-E627K strongly favours use of mammalian ANP32B proteins, whereas D701N shows no such bias. Accordingly, PB2-E627K adaptation emerges in species with strong pro-viral ANP32B proteins, such as humans and mice, while D701N is more commonly seen in isolates from swine, dogs and horses where ANP32A proteins are more strongly pro-viral. In an experimental evolution approach, passage of avian viruses in human cells drives acquisition of PB2-E627K, but not when ANP32B is ablated. The strong pro-viral support of ANP32B for PB2-E627K maps to the LCAR region of ANP32B.
Goldhill D, Yan A, Frise R, et al., 2020, Favipiravir-resistant influenza A virus shows potential for transmission, Publisher: Cold Spring Harbor Laboratory
Abstract Favipiravir is a nucleoside analogue which has been licensed to treat influenza in the event of a new pandemic. We previously described a favipiravir resistant influenza A virus generated by in vitro passage in presence of drug with two mutations: K229R in PB1, which conferred resistance at a cost to polymerase activity, and P653L in PA, which compensated for the cost of polymerase activity. However, the clinical relevance of these mutations is unclear as the mutations have not been found in natural isolates and it is unknown whether viruses harbouring these mutations would replicate or transmit in vivo. Here, we infected ferrets with a mix of wild type p(H1N1) 2009 and corresponding favipiravir-resistant virus and tested for replication and transmission in the absence of drug. Favipiravir-resistant virus successfully infected ferrets and was transmitted by both contact transmission and respiratory droplet routes. However, sequencing revealed the mutation that conferred resistance, K229R, decreased in frequency over time within ferrets. Modelling revealed that due to a fitness advantage for the PA P653L mutant, reassortment with the wild-type virus to gain wild-type PB1 segment in vivo resulted in the loss of the PB1 resistance mutation K229R. We demonstrated that this fitness advantage of PA P653L in the background of our starting virus A/England/195/2009 was due to a maladapted PA in first wave isolates from the 2009 pandemic. We show there is no fitness advantage of P653L in more recent pH1N1 influenza A viruses. Therefore, whilst favipiravir-resistant virus can transmit in vivo, the likelihood that the resistance mutation is retained in the absence of drug pressure may vary depending on the genetic background of the starting viral strain. Author Summary In the event of a new influenza pandemic, drugs will be our first line of defence against the virus. However, drug resistance has proven to be particularly problematic to drugs against influenza. Favipir
Flower B, Brown JC, Simmons B, et al., 2020, Clinical and laboratory evaluation of SARS-CoV-2 lateral flow assays for use in a national COVID-19 sero-prevalence survey, Thorax, Vol: 75, Pages: 1082-1088, ISSN: 0040-6376
BackgroundAccurate antibody tests are essential to monitor the SARS-CoV-2 pandemic. Lateral flow immunoassays (LFIAs) can deliver testing at scale. However, reported performance varies, and sensitivity analyses have generally been conducted on serum from hospitalised patients. For use in community testing, evaluation of finger-prick self-tests, in non-hospitalised individuals, is required.MethodsSensitivity analysis was conducted on 276 non-hospitalised participants. All had tested positive for SARS-CoV-2 by RT-PCR and were ≥21d from symptom-onset. In phase I we evaluated five LFIAs in clinic (with finger-prick) and laboratory (with blood and sera) in comparison to a) PCR-confirmed infection and b) presence of SARS-CoV-2 antibodies on two “in-house” ELISAs. Specificity analysis was performed on 500 pre-pandemic sera. In phase II, six additional LFIAs were assessed with serum.Findings95% (95%CI [92.2, 97.3]) of the infected cohort had detectable antibodies on at least one ELISA. LFIA sensitivity was variable, but significantly inferior to ELISA in 8/11 assessed. Of LFIAs assessed in both clinic and laboratory, finger-prick self-test sensitivity varied from 21%-92% vs PCR-confirmed cases and 22%-96% vs composite ELISA positives. Concordance between finger-prick and serum testing was at best moderate (kappa 0.56) and, at worst, slight (kappa 0.13). All LFIAs had high specificity (97.2% - 99.8%).InterpretationLFIA sensitivity and sample concordance is variable, highlighting the importance of evaluations in setting of intended use. This rigorous approach to LFIA evaluation identified a test with high specificity (98.6% (95%CI [97.1, 99.4])), moderate sensitivity (84.4% with fingerprick (95%CI [70.5, 93.5])), and moderate concordance, suitable for seroprevalence surveys.
Staller E, Baillon L, Frise R, et al., 2020, A rare variant in ANP32B impairs influenza virus replication in human cells, biorxiv
Viruses require host factors to support their replication, and genetic variation in such factors can affect susceptibility to infectious disease. Influenza virus replication in human cells relies on ANP32 proteins, which are involved in assembly of replication-competent dimeric influenza virus polymerase (FluPol) complexes. Here, we investigate naturally occurring single nucleotide variants (SNV) in the human Anp32A and Anp32B genes. We note that variant rs182096718 in Anp32B is found at a higher frequency than other variants in either gene. This SNV results in a D130A substitution in ANP32B, which is less able to support FluPol activity than wildtype ANP32B and binds FluPol with lower affinity. Interestingly, ANP32B-D130A exerts a dominant negative effect over wildtype ANP32B and interferes with the functionally redundant paralogue ANP32A. FluPol activity and virus replication are attenuated in CRISPR-edited cells expressing wildtype ANP32A and mutant ANP32B-D130A. We propose a model in which the D130A mutation impairs FluPol dimer formation, thus resulting in compromised replication. We suggest that both homozygous and heterozygous carriers of rs182096718 may have some genetic protection against influenza viruses.
Lee LYY, Zhou J, Frise R, et al., 2020, Baloxavir treatment of ferrets infected with influenza A(H1N1)pdm09 virus reduces onward transmission, PLOS PATHOGENS, Vol: 16, ISSN: 1553-7366
Singanayagam A, Zhou J, Elderfield RA, et al., 2020, Characterising viable virus from air exhaled by H1N1 influenza-infected ferrets reveals the importance of haemagglutinin stability for airborne infectivity, PLoS Pathogens, Vol: 16, Pages: 1-21, ISSN: 1553-7366
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.
Artarini A, Meyer M, Shin YJ, et al., 2019, Regulation of influenza a virus mRNA splicing by CLK1, Antiviral Research, Vol: 168, Pages: 187-196, ISSN: 0166-3542
Influenza A virus carries eight negative single-stranded RNAs and uses spliced mRNAs to increase the number of proteins produced from them. Several genome-wide screens for essential host factors for influenza A virus replication revealed a necessity for splicing and splicing-related factors, including Cdc-like kinase 1 (CLK1). This CLK family kinase plays a role in alternative splicing regulation through phosphorylation of serine-arginine rich (SR) proteins. To examine the influence that modulation of splicing regulation has on influenza infection, we analyzed the effect of CLK1 knockdown and inhibition. CLK1 knockdown in A549 cells reduced influenza A/WSN/33 virus replication and increased the level of splicing of segment 7, encoding the viral M1 and M2 proteins. CLK1-/- mice infected with influenza A/England/195/2009 (H1N1pdm09) virus supported lower levels of virus replication than wild-type mice. Screening of newly developed CLK inhibitors revealed several compounds that have an effect on the level of splicing of influenza A gene segment M in different models and decrease influenza A/WSN/33 virus replication in A549 cells. The promising inhibitor KH-CB19, an indole-based enaminonitrile with unique binding mode for CLK1, and its even more selective analogue NIH39 showed high specificity towards CLK1 and had a similar effect on influenza mRNA splicing regulation. Taken together, our findings indicate that targeting host factors that regulate splicing of influenza mRNAs may represent a novel therapeutic approach.
Muniz JRC, Szeto NW-S, Frise R, et al., 2019, Role of protein structure in variant annotation: structural insight of mutations causing 6-pyruvoyl-tetrahydropterin synthase deficiency, Pathology, Vol: 51, Pages: 274-280, ISSN: 0031-3025
Lesch M, Luckner M, Meyer M, et al., 2019, RNAi-based small molecule repositioning reveals clinically approved urea-based kinase inhibitors as broadly active antivirals, PLoS Pathogens, Vol: 15, Pages: 1-34, ISSN: 1553-7366
Influenza viruses (IVs) tend to rapidly develop resistance to virus-directed vaccines and common antivirals targeting pathogen determinants, but novel host-directed approaches might preclude resistance development. To identify the most promising cellular targets for a host-directed approach against influenza, we performed a comparative small interfering RNA (siRNA) loss-of-function screen of IV replication in A549 cells. Analysis of four different IV strains including a highly pathogenic avian H5N1 strain, an influenza B virus (IBV) and two human influenza A viruses (IAVs) revealed 133 genes required by all four IV strains. According to gene enrichment analyses, these strain-independent host genes were particularly enriched for nucleocytoplasmic trafficking. In addition, 360 strain-specific genes were identified with distinct patterns of usage for IAVs versus IBV and human versus avian IVs. The strain-independent host genes served to define 43 experimental and otherwise clinically approved drugs, targeting reportedly fourteen of the encoded host factors. Amongst the approved drugs, the urea-based kinase inhibitors (UBKIs) regorafenib and sorafenib exhibited a superior therapeutic window of high IV antiviral activity and low cytotoxicity. Both UBKIs appeared to block a cell signaling pathway involved in IV replication after internalization, yet prior to vRNP uncoating. Interestingly, both compounds were active also against unrelated viruses including cowpox virus (CPXV), hantavirus (HTV), herpes simplex virus 1 (HSV1) and vesicular stomatitis virus (VSV) and showed antiviral efficacy in human primary respiratory cells. An in vitro resistance development analysis for regorafenib failed to detect IV resistance development against this drug. Taken together, the otherwise clinically approved UBKIs regorafenib and sorafenib possess high and broad-spectrum antiviral activity along with substantial robustness against resistance development and thus constitute attractive hos
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.
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.
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.
Long J, Efstathios SG, Moncorge O, et al., 2016, Species difference in ANP32A underlies influenza A virus polymerase host restriction, Nature, Vol: 529, Pages: 101-104, ISSN: 1476-4687
Influenza pandemics occur unpredictably when zoonotic influenza viruses with novel antigenicity acquire the ability to transmit amongst humans1. Host range breaches are limited by incompatibilities between avian virus components and the human host. Barriers include receptor preference, virion stability and poor activity of the avian virus RNA-dependent RNA polymerase in human cells2. Mutants of the heterotrimeric viral polymerase components, particularly PB2 protein, are selected during mammalian adaptation, but their mode of action is unknown3, 4, 5, 6. We show that a species-specific difference in host protein ANP32A accounts for the suboptimal function of avian virus polymerase in mammalian cells. Avian ANP32A possesses an additional 33 amino acids between the leucine-rich repeats and carboxy-terminal low-complexity acidic region domains. In mammalian cells, avian ANP32A rescued the suboptimal function of avian virus polymerase to levels similar to mammalian-adapted polymerase. Deletion of the avian-specific sequence from chicken ANP32A abrogated this activity, whereas its insertion into human ANP32A, or closely related ANP32B, supported avian virus polymerase function. Substitutions, such as PB2(E627K), were rapidly selected upon infection of humans with avian H5N1 or H7N9 influenza viruses, adapting the viral polymerase for the shorter mammalian ANP32A. Thus ANP32A represents an essential host partner co-opted to support influenza virus replication and is a candidate host target for novel antivirals.
Ito K, Ashcroft J, Brookes D, et al., 2013, Inhibitory Effects Of Rv1088, A Narrow Spectrum Kinase Inhibitor, On Cytokine Production In Response To Pandemic Flu Infections Of Primary Respiratory Cell Cultures, AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE, Vol: 187, ISSN: 1073-449X
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