RESUMEN
Molnupiravir, an antiviral medication widely used against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), acts by inducing mutations in the virus genome during replication. Most random mutations are likely to be deleterious to the virus and many will be lethal; thus, molnupiravir-induced elevated mutation rates reduce viral load1,2. However, if some patients treated with molnupiravir do not fully clear the SARS-CoV-2 infections, there could be the potential for onward transmission of molnupiravir-mutated viruses. Here we show that SARS-CoV-2 sequencing databases contain extensive evidence of molnupiravir mutagenesis. Using a systematic approach, we find that a specific class of long phylogenetic branches, distinguished by a high proportion of G-to-A and C-to-T mutations, are found almost exclusively in sequences from 2022, after the introduction of molnupiravir treatment, and in countries and age groups with widespread use of the drug. We identify a mutational spectrum, with preferred nucleotide contexts, from viruses in patients known to have been treated with molnupiravir and show that its signature matches that seen in these long branches, in some cases with onward transmission of molnupiravir-derived lineages. Finally, we analyse treatment records to confirm a direct association between these high G-to-A branches and the use of molnupiravir.
Asunto(s)
Antivirales , COVID-19 , Citidina , Hidroxilaminas , Mutagénesis , Mutación , SARS-CoV-2 , Humanos , Antivirales/farmacología , Antivirales/uso terapéutico , COVID-19/epidemiología , COVID-19/transmisión , COVID-19/virología , Citidina/análogos & derivados , Citidina/farmacología , Citidina/uso terapéutico , Genoma Viral/efectos de los fármacos , Genoma Viral/genética , Hidroxilaminas/farmacología , Hidroxilaminas/uso terapéutico , Mutación/efectos de los fármacos , Filogenia , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , Carga Viral , Replicación Viral/efectos de los fármacos , Replicación Viral/genética , Evolución Molecular , Mutagénesis/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
The SARS-CoV-2 Delta (Pango lineage B.1.617.2) variant of concern spread globally, causing resurgences of COVID-19 worldwide1,2. The emergence of the Delta variant in the UK occurred on the background of a heterogeneous landscape of immunity and relaxation of non-pharmaceutical interventions. Here we analyse 52,992 SARS-CoV-2 genomes from England together with 93,649 genomes from the rest of the world to reconstruct the emergence of Delta and quantify its introduction to and regional dissemination across England in the context of changing travel and social restrictions. Using analysis of human movement, contact tracing and virus genomic data, we find that the geographic focus of the expansion of Delta shifted from India to a more global pattern in early May 2021. In England, Delta lineages were introduced more than 1,000 times and spread nationally as non-pharmaceutical interventions were relaxed. We find that hotel quarantine for travellers reduced onward transmission from importations; however, the transmission chains that later dominated the Delta wave in England were seeded before travel restrictions were introduced. Increasing inter-regional travel within England drove the nationwide dissemination of Delta, with some cities receiving more than 2,000 observable lineage introductions from elsewhere. Subsequently, increased levels of local population mixing-and not the number of importations-were associated with the faster relative spread of Delta. The invasion dynamics of Delta depended on spatial heterogeneity in contact patterns, and our findings will inform optimal spatial interventions to reduce the transmission of current and future variants of concern, such as Omicron (Pango lineage B.1.1.529).
Asunto(s)
COVID-19 , SARS-CoV-2 , COVID-19/epidemiología , COVID-19/prevención & control , COVID-19/transmisión , COVID-19/virología , Ciudades/epidemiología , Trazado de Contacto , Inglaterra/epidemiología , Genoma Viral/genética , Humanos , Cuarentena/legislación & jurisprudencia , SARS-CoV-2/genética , SARS-CoV-2/crecimiento & desarrollo , SARS-CoV-2/aislamiento & purificación , Viaje/legislación & jurisprudenciaRESUMEN
The ongoing SARS-CoV-2 epidemic was marked by the repeated emergence and replacement of "variants" with genetic and phenotypic distance from the ancestral strains, the most recent examples being viruses of the Omicron lineage. Here, we describe a hamster direct contact exposure challenge model to assess protection against reinfection conferred by either vaccination or prior infection. We found that two doses of self-amplifying RNA vaccine based on the ancestral Spike ameliorated weight loss following Delta infection and decreased viral loads but had minimal effect on Omicron BA.1 infection. Prior vaccination followed by Delta or BA.1 breakthrough infections led to a high degree of cross-reactivity to all tested variants, suggesting that repeated exposure to antigenically distinct Spikes, via infection and/or vaccination drives a cross-reactive immune response. Prior infection with ancestral or Alpha variant was partially protective against BA.1 infection, whereas all animals previously infected with Delta and exposed to BA.1 became reinfected, although they shed less virus than BA.1-infected naive hamsters. Hamsters reinfected with BA.1 after prior Delta infection emitted infectious virus into the air, indicating that they could be responsible for onwards airborne transmission. We further tested whether prior infection with BA.1 protected from reinfection with Delta or later Omicron sublineages BA.2, BA.4, or BA.5. BA.1 was protective against BA.2 but not against Delta, BA.4, or BA.5 reinfection. These findings suggest that cohorts whose only immune experience of COVID-19 is Omicron BA.1 infection may be vulnerable to future circulation of reemerged Delta-like derivatives, as well as emerging Omicron sublineages.
Asunto(s)
COVID-19 , Hepatitis D , Animales , Cricetinae , Infección Irruptiva , Reinfección , Reacciones Cruzadas , Anticuerpos Neutralizantes , Anticuerpos AntiviralesRESUMEN
Detection of viral DNA by cyclic GMP-AMP synthase (cGAS) is a first line of defence leading to the production of type I interferon (IFN). As HIV-1 replication is not a strong inducer of IFN, we hypothesised that an intact capsid physically cloaks viral DNA from cGAS. To test this, we generated defective viral particles by treatment with HIV-1 protease inhibitors or by genetic manipulation of gag. These viruses had defective Gag cleavage, reduced infectivity and diminished capacity to saturate TRIM5α. Importantly, unlike wild-type HIV-1, infection with cleavage defective HIV-1 triggered an IFN response in THP-1 cells that was dependent on viral DNA and cGAS. An IFN response was also observed in primary human macrophages infected with cleavage defective viruses. Infection in the presence of the capsid destabilising small molecule PF-74 also induced a cGAS-dependent IFN response. These data demonstrate a protective role for capsid and suggest that antiviral activity of capsid- and protease-targeting antivirals may benefit from enhanced innate and adaptive immunity in vivo.
Asunto(s)
ADN Viral/inmunología , Infecciones por VIH/inmunología , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/inmunología , Macrófagos/metabolismo , Nucleotidiltransferasas/metabolismo , Replicación Viral/genética , Inmunidad Adaptativa , Factores de Restricción Antivirales , Sistemas CRISPR-Cas , Cápside/metabolismo , Línea Celular , ADN Viral/genética , Edición Génica , Productos del Gen gag/genética , Infecciones por VIH/enzimología , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , VIH-1/genética , VIH-1/metabolismo , VIH-1/patogenicidad , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata , Indoles/farmacología , Interferones/metabolismo , Interferones/farmacología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Transducción de Señal/inmunología , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
ANP32 proteins, which act as influenza polymerase cofactors, vary between birds and mammals. In mammals, ANP32A and ANP32B have been reported to serve essential but redundant roles to support influenza polymerase activity. The well-known mammalian adaptation PB2-E627K enables influenza polymerase to use mammalian ANP32 proteins. However, some mammalian-adapted influenza viruses do not harbor this substitution. Here, we show that alternative PB2 adaptations, Q591R and D701N, also allow influenza polymerase to use mammalian ANP32 proteins, whereas other PB2 mutations, G158E, T271A, and D740N, increase polymerase activity in the presence of avian ANP32 proteins as well. Furthermore, PB2-E627K strongly favors 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 the preferred cofactor. Using an experimental evolution approach, we show that the passage of viruses containing avian polymerases in human cells drove acquisition of PB2-E627K, but not in the absence of ANP32B. Finally, we show that the strong pro-viral support of ANP32B for PB2-E627K maps to the low-complexity acidic region (LCAR) tail of ANP32B. IMPORTANCE Influenza viruses naturally reside in wild aquatic birds. However, the high mutation rate of influenza viruses allows them to rapidly and frequently adapt to new hosts, including mammals. Viruses that succeed in these zoonotic jumps pose a pandemic threat whereby the virus adapts sufficiently to efficiently transmit human-to-human. The influenza virus polymerase is central to viral replication and restriction of polymerase activity is a major barrier to species jumps. ANP32 proteins are essential for influenza polymerase activity. In this study, we describe how avian influenza viruses can adapt in several different ways to use mammalian ANP32 proteins. We further show that differences between mammalian ANP32 proteins can select different adaptive changes and are responsible for some of the typical mutations that arise in mammalian-adapted influenza polymerases. These different adaptive mutations may determine the relative zoonotic potential of influenza viruses and thus help assess their pandemic risk.
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Virus de la Influenza A , Gripe Aviar , Gripe Humana , Proteínas Nucleares , Animales , Perros , Humanos , Ratones , Proteínas de Ciclo Celular/metabolismo , Caballos , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Gripe Aviar/genética , Gripe Humana/genética , Mamíferos , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleotidiltransferasas/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Porcinos , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Vaccines are proving to be highly effective in controlling hospitalisation and deaths associated with SARS-CoV-2 infection but the emergence of viral variants with novel antigenic profiles threatens to diminish their efficacy. Assessment of the ability of sera from vaccine recipients to neutralise SARS-CoV-2 variants will inform the success of strategies for minimising COVID19 cases and the design of effective antigenic formulations. Here, we examine the sensitivity of variants of concern (VOCs) representative of the B.1.617.1 and B.1.617.2 (first associated with infections in India) and B.1.351 (first associated with infection in South Africa) lineages of SARS-CoV-2 to neutralisation by sera from individuals vaccinated with the BNT162b2 (Pfizer/BioNTech) and ChAdOx1 (Oxford/AstraZeneca) vaccines. Across all vaccinated individuals, the spike glycoproteins from B.1.617.1 and B.1.617.2 conferred reductions in neutralisation of 4.31 and 5.11-fold respectively. The reduction seen with the B.1.617.2 lineage approached that conferred by the glycoprotein from B.1.351 (South African) variant (6.29-fold reduction) that is known to be associated with reduced vaccine efficacy. Neutralising antibody titres elicited by vaccination with two doses of BNT162b2 were significantly higher than those elicited by vaccination with two doses of ChAdOx1. Fold decreases in the magnitude of neutralisation titre following two doses of BNT162b2, conferred reductions in titre of 7.77, 11.30 and 9.56-fold respectively to B.1.617.1, B.1.617.2 and B.1.351 pseudoviruses, the reduction in neutralisation of the delta variant B.1.617.2 surpassing that of B.1.351. Fold changes in those vaccinated with two doses of ChAdOx1 were 0.69, 4.01 and 1.48 respectively. The accumulation of mutations in these VOCs, and others, demonstrate the quantifiable risk of antigenic drift and subsequent reduction in vaccine efficacy. Accordingly, booster vaccines based on updated variants are likely to be required over time to prevent productive infection. This study also suggests that two dose regimes of vaccine are required for maximal BNT162b2 and ChAdOx1-induced immunity.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Vacuna BNT162 , COVID-19 , Inmunización Secundaria , SARS-CoV-2/inmunología , Eficacia de las Vacunas , Deriva y Cambio Antigénico/inmunología , Vacuna BNT162/administración & dosificación , Vacuna BNT162/inmunología , COVID-19/inmunología , COVID-19/mortalidad , COVID-19/prevención & control , Células HEK293 , HumanosRESUMEN
To detect new and changing SARS-CoV-2 variants, we investigated candidate Delta-Omicron recombinant genomes from Centers for Disease Control and Prevention national genomic surveillance. Laboratory and bioinformatic investigations identified and validated 9 genetically related SARS-CoV-2 viruses with a hybrid Delta-Omicron spike protein.
Asunto(s)
COVID-19 , SARS-CoV-2 , COVID-19/epidemiología , Biología Computacional , Humanos , SARS-CoV-2/genética , Estados Unidos/epidemiologíaRESUMEN
Following the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in PR China in late 2019 a number of variants have emerged, with two of these - alpha and delta - subsequently growing to global prevalence. One characteristic of these variants are changes within the spike protein, in particular the receptor-binding domain (RBD). From a public health perspective, these changes have important implications for increased transmissibility and immune escape; however, their presence could also modify the intrinsic host range of the virus. Using viral pseudotyping, we examined whether the variants of concern (VOCs) alpha, beta, gamma and delta have differing host angiotensin-converting enzyme 2 (ACE2) receptor usage patterns, focusing on a range of relevant mammalian ACE2 proteins. All four VOCs were able to overcome a previous restriction for mouse ACE2, with demonstrable differences also seen for individual VOCs with rat, ferret or civet ACE2 receptors, changes that we subsequently attributed to N501Y and E484K substitutions within the spike RBD.
Asunto(s)
COVID-19 , SARS-CoV-2 , Enzima Convertidora de Angiotensina 2/genética , Animales , Hurones , Especificidad del Huésped , Humanos , Ratones , Peptidil-Dipeptidasa A/química , Ratas , SARS-CoV-2/genéticaRESUMEN
Receptor recognition and binding is the first step of viral infection and a key determinant of host specificity. The inability of avian influenza viruses to effectively bind human-like sialylated receptors is a major impediment to their efficient transmission in humans and pandemic capacity. Influenza H9N2 viruses are endemic in poultry across Asia and parts of Africa where they occasionally infect humans and are therefore considered viruses with zoonotic potential. We previously described H9N2 viruses, including several isolated from human zoonotic cases, showing a preference for human-like receptors. Here we take a mutagenesis approach, making viruses with single or multiple substitutions in H9 haemagglutinin and test binding to avian and human receptor analogues using biolayer interferometry. We determine the genetic basis of preferences for alternative avian receptors and for human-like receptors, describing amino acid motifs at positions 190, 226 and 227 that play a major role in determining receptor specificity, and several other residues such as 159, 188, 193, 196, 198 and 225 that play a smaller role. Furthermore, we show changes at residues 135, 137, 147, 157, 158, 184, 188, and 192 can also modulate virus receptor avidity and that substitutions that increased or decreased the net positive charge around the haemagglutinin receptor-binding site show increases and decreases in avidity, respectively. The motifs we identify as increasing preference for the human-receptor will help guide future H9N2 surveillance efforts and facilitate our understanding of the emergence of influenza viruses with increased zoonotic potential.IMPORTANCE As of 2020, over 60 infections of humans by H9N2 influenza viruses have been recorded in countries where the virus is endemic. Avian-like cellular receptors are the primary target for these viruses. However, given that human infections have been detected on an almost monthly basis since 2015, there may be a capacity for H9N2 viruses to evolve and gain the ability to target human-like cellular receptors. Here we identify molecular signatures that can cause viruses to bind human-like receptors, and we identify the molecular basis for the distinctive preference for sulphated receptors displayed by the majority of recent H9N2 viruses. This work will help guide future surveillance by providing markers that signify the emergence of viruses with enhanced zoonotic potential as well as improving understanding of the basis of influenza virus receptor-binding.
RESUMEN
MOTIVATION: Analysis of the T-cell receptor repertoire is rapidly entering the general toolbox used by researchers interested in cellular immunity. The annotation of T-cell receptors (TCRs) from raw sequence data poses specific challenges, which arise from the fact that TCRs are not germline encoded, and because of the stochastic nature of the generating process. RESULTS: In this study, we report the release of Decombinator V4, a tool for the accurate and fast annotation of large sets of TCR sequences. Decombinator was one of the early Python software packages released to analyse the rapidly increasing flow of T-cell receptor repertoire sequence data. The Decombinator package now provides Python 3 compatibility, incorporates improved sequencing error and PCR bias correction algorithms, and provides output which conforms to the international standards proposed by the Adaptive Immune Receptor Repertoire Community. AVAILABILITY AND IMPLEMENTATION: The entire Decombinator suite is freely available at: https://github.com/innate2adaptive/Decombinator. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Receptores de Antígenos de Linfocitos T , Programas Informáticos , Algoritmos , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
SARS-CoV-2 is thought to have originated in the human population from a zoonotic spillover event. Infection in humans results in a variety of outcomes ranging from asymptomatic cases to the disease COVID-19, which can have significant morbidity and mortality, with over two million confirmed deaths worldwide as of January 2021. Over a year into the pandemic, sequencing analysis has shown that variants of SARS-CoV-2 are being selected as the virus continues to circulate widely within the human population. The predominant drivers of genetic variation within SARS-CoV-2 are single nucleotide polymorphisms (SNPs) caused by polymerase error, potential host factor driven RNA modification, and insertion/deletions (indels) resulting from the discontinuous nature of viral RNA synthesis. While many mutations represent neutral 'genetic drift' or have quickly died out, a subset may be affecting viral traits such as transmissibility, pathogenicity, host range, and antigenicity of the virus. In this review, we summarise the current extent of genetic change in SARS-CoV-2, particularly recently emerging variants of concern, and consider the phenotypic consequences of this viral evolution that may impact the future trajectory of the pandemic.
Asunto(s)
Adaptación Fisiológica/genética , COVID-19/virología , SARS-CoV-2/genética , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , COVID-19/epidemiología , COVID-19/inmunología , COVID-19/transmisión , Humanos , Evasión Inmune/genética , Mutación , ARN Viral/biosíntesis , ARN Viral/genética , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/patogenicidad , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Internalización del Virus , Replicación Viral , Zoonosis/transmisión , Zoonosis/virologíaRESUMEN
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 wild-type ANP32B and binds FluPol with lower affinity. Interestingly, ANP32B-D130A exerts a dominant negative effect over wild-type ANP32B and interferes with the functionally redundant paralogue ANP32A. FluPol activity and virus replication are attenuated in CRISPR-edited cells expressing wild-type 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.
Asunto(s)
Subtipo H3N2 del Virus de la Influenza A/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleótido Simple , ARN Polimerasa Dependiente del ARN/metabolismo , Línea Celular , Humanos , Subtipo H3N2 del Virus de la Influenza A/enzimología , Modelos Moleculares , Proteínas Nucleares/química , Conformación Proteica , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , ARN Polimerasa Dependiente del ARN/química , Replicación ViralRESUMEN
Influenza A viruses encode several accessory proteins that have host- and strain-specific effects on virulence and replication. The accessory protein PA-X is expressed due to a ribosomal frameshift during translation of the PA gene. Depending on the particular combination of virus strain and host species, PA-X has been described as either acting to reduce or increase virulence and/or virus replication. In this study, we set out to investigate the role PA-X plays in H9N2 avian influenza viruses, focusing on the natural avian host, chickens. We found that the G1 lineage A/chicken/Pakistan/UDL-01/2008 (H9N2) PA-X induced robust host shutoff in both mammalian and avian cells and increased virus replication in mammalian, but not avian cells. We further showed that PA-X affected embryonic lethality in ovo and led to more rapid viral shedding and widespread organ dissemination in vivo in chickens. Overall, we conclude PA-X may act as a virulence factor for H9N2 viruses in chickens, allowing faster replication and wider organ tropism.
Asunto(s)
Subtipo H9N2 del Virus de la Influenza A/metabolismo , Gripe Aviar/virología , Gripe Humana/virología , Proteínas Represoras/metabolismo , Proteínas no Estructurales Virales/metabolismo , Factores de Virulencia/metabolismo , Animales , Línea Celular , Pollos , Citocinas/genética , Citocinas/inmunología , Humanos , Subtipo H9N2 del Virus de la Influenza A/genética , Subtipo H9N2 del Virus de la Influenza A/patogenicidad , Gripe Aviar/genética , Gripe Aviar/inmunología , Gripe Humana/genética , Gripe Humana/inmunología , Pulmón/inmunología , Pulmón/virología , Ratones , Proteínas Represoras/genética , Proteínas no Estructurales Virales/genética , Factores de Virulencia/genética , Replicación Viral , Esparcimiento de VirusRESUMEN
H9N2 avian influenza viruses (AIVs) circulate in poultry throughout much of Asia, the Middle East, and Africa. These viruses cause huge economic damage to poultry production systems and pose a zoonotic threat both in their own right and in the generation of novel zoonotic viruses, for example, H7N9. In recent years, it has been observed that H9N2 viruses have further adapted to gallinaceous poultry, becoming more highly transmissible and causing higher morbidity and mortality. Here, we investigate the molecular basis for this increased virulence, comparing a virus from the 1990s and a contemporary field strain. The modern virus replicated to higher titers in various systems, and this difference mapped to a single amino acid polymorphism at position 26 of the endonuclease domain shared by the PA and PA-X proteins. This change was responsible for increased replication and higher morbidity and mortality rates along with extended tissue tropism seen in chickens. Although the PA K26E change correlated with increased host cell shutoff activity of the PA-X protein in vitro, it could not be overridden by frameshift site mutations that block PA-X expression and therefore increased PA-X activity could not explain the differences in replication phenotype. Instead, this indicates that these differences are due to subtle effects on PA function. This work gives insight into the ongoing evolution and poultry adaptation of H9N2 and other avian influenza viruses and helps us understand the striking morbidity and mortality rates in the field, as well as the rapidly expanding geographical range seen in these viruses.IMPORTANCE Avian influenza viruses, such as H9N2, cause huge economic damage to poultry production worldwide and are additionally considered potential pandemic threats. Understanding how these viruses evolve in their natural hosts is key to effective control strategies. In the Middle East and South Asia, an older H9N2 virus strain has been replaced by a new reassortant strain with greater fitness. Here, we take representative viruses and investigate the genetic basis for this "fitness." A single mutation in the virus was responsible for greater fitness, enabling high growth of the contemporary H9N2 virus in cells, as well as in chickens. The genetic mutation that modulates this change is within the viral PA protein, a part of the virus polymerase gene that contributes to viral replication as well as to virus accessory functions-however, we find that the fitness effect is specifically due to changes in the protein polymerase activity.
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Subtipo H9N2 del Virus de la Influenza A , Gripe Aviar , Enfermedades de las Aves de Corral , ARN Polimerasa Dependiente del ARN , Proteínas Virales , Tropismo Viral , Animales , Pollos , Perros , Células HEK293 , Humanos , Subtipo H9N2 del Virus de la Influenza A/patogenicidad , Subtipo H9N2 del Virus de la Influenza A/fisiología , Gripe Aviar/genética , Gripe Aviar/metabolismo , Gripe Aviar/patología , Células de Riñón Canino Madin Darby , Enfermedades de las Aves de Corral/genética , Enfermedades de las Aves de Corral/metabolismo , Enfermedades de las Aves de Corral/patología , Enfermedades de las Aves de Corral/virología , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Avian influenza viruses occasionally infect and adapt to mammals, including humans. Swine are often described as "mixing vessels," being susceptible to both avian- and human-origin viruses, which allows the emergence of novel reassortants, such as the precursor to the 2009 H1N1 pandemic. ANP32 proteins are host factors that act as influenza virus polymerase cofactors. In this study, we describe how swine ANP32A, uniquely among the mammalian ANP32 proteins tested, supports the activity of avian-origin influenza virus polymerases and avian influenza virus replication. We further show that after the swine-origin influenza virus emerged in humans and caused the 2009 pandemic, it evolved polymerase gene mutations that enabled it to more efficiently use human ANP32 proteins. We map the enhanced proviral activity of swine ANP32A to a pair of amino acids, 106 and 156, in the leucine-rich repeat and central domains and show these mutations enhance binding to influenza virus trimeric polymerase. These findings help elucidate the molecular basis for the mixing vessel trait of swine and further our understanding of the evolution and ecology of viruses in this host.IMPORTANCE Avian influenza viruses can jump from wild birds and poultry into mammalian species such as humans or swine, but they only continue to transmit if they accumulate mammalian adapting mutations. Pigs appear uniquely susceptible to both avian and human strains of influenza and are often described as virus "mixing vessels." In this study, we describe how a host factor responsible for regulating virus replication, ANP32A, is different between swine and humans. Swine ANP32A allows a greater range of influenza viruses, specifically those from birds, to replicate. It does this by binding the virus polymerase more tightly than the human version of the protein. This work helps to explain the unique properties of swine as mixing vessels.
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Subtipo H1N1 del Virus de la Influenza A/genética , Proteínas Nucleares/genética , Infecciones por Orthomyxoviridae/genética , Proteínas de Unión al ARN/genética , ARN Polimerasa Dependiente del ARN/genética , Proteínas Virales/genética , Animales , Sitios de Unión , Línea Celular , Pollos , Células Epiteliales/metabolismo , Células Epiteliales/virología , Regulación de la Expresión Génica , Especificidad del Huésped , Humanos , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Modelos Moleculares , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Transducción de Señal , Porcinos , Proteínas Virales/química , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Internal gravity waves, the subsurface analogue of the familiar surface gravity waves that break on beaches, are ubiquitous in the ocean. Because of their strong vertical and horizontal currents, and the turbulent mixing caused by their breaking, they affect a panoply of ocean processes, such as the supply of nutrients for photosynthesis, sediment and pollutant transport and acoustic transmission; they also pose hazards for man-made structures in the ocean. Generated primarily by the wind and the tides, internal waves can travel thousands of kilometres from their sources before breaking, making it challenging to observe them and to include them in numerical climate models, which are sensitive to their effects. For over a decade, studies have targeted the South China Sea, where the oceans' most powerful known internal waves are generated in the Luzon Strait and steepen dramatically as they propagate west. Confusion has persisted regarding their mechanism of generation, variability and energy budget, however, owing to the lack of in situ data from the Luzon Strait, where extreme flow conditions make measurements difficult. Here we use new observations and numerical models to (1) show that the waves begin as sinusoidal disturbances rather than arising from sharp hydraulic phenomena, (2) reveal the existence of >200-metre-high breaking internal waves in the region of generation that give rise to turbulence levels >10,000 times that in the open ocean, (3) determine that the Kuroshio western boundary current noticeably refracts the internal wave field emanating from the Luzon Strait, and (4) demonstrate a factor-of-two agreement between modelled and observed energy fluxes, which allows us to produce an observationally supported energy budget of the region. Together, these findings give a cradle-to-grave picture of internal waves on a basin scale, which will support further improvements of their representation in numerical climate predictions.
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Visón , Pandemias , Animales , Pandemias/prevención & control , Granjas , Agricultura , Anticuerpos AntiviralesRESUMEN
ANP32 proteins have been implicated in supporting influenza virus replication, but most of the work to date has focused on the ability of avian Anp32 proteins to overcome restriction of avian influenza polymerases in human cells. Using a CRISPR approach, we show that the human acidic nuclear phosphoproteins (ANPs) ANP32A and ANP32B are functionally redundant but essential host factors for mammalian-adapted influenza A virus (IAV) and influenza B virus (IBV) replication in human cells. When both proteins are absent from human cells, influenza polymerases are unable to replicate the viral genome, and infectious virus cannot propagate. Provision of exogenous ANP32A or ANP32B recovers polymerase activity and virus growth. We demonstrate that this redundancy is absent in the murine Anp32 orthologues; murine Anp32A is incapable of recovering IAV polymerase activity, while murine Anp32B can do so. Intriguingly, IBV polymerase is able to use murine Anp32A. We show, using a domain swap and point mutations, that the leucine-rich repeat (LRR) 5 region comprises an important functional domain for mammalian ANP32 proteins. Our approach has identified a pair of essential host factors for influenza virus replication and can be harnessed to inform future interventions.IMPORTANCE Influenza virus is the etiological agent behind some of the most devastating infectious disease pandemics to date, and influenza outbreaks still pose a major threat to public health. Influenza virus polymerase, the molecule that copies the viral RNA genome, hijacks cellular proteins to support its replication. Current anti-influenza drugs are aimed against viral proteins, including the polymerase, but RNA viruses like influenza tend to become resistant to such drugs very rapidly. An alternative strategy is to design therapeutics that target the host proteins that are necessary for virus propagation. Here, we show that the human proteins ANP32A and ANP32B are essential for influenza A and B virus replication, such that in their absence cells become impervious to the virus. We map the proviral activity of ANP32 proteins to one region in particular, which could inform future intervention.
Asunto(s)
Betainfluenzavirus/fisiología , Proteínas de Ciclo Celular/genética , Virus de la Influenza A/fisiología , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Proteínas de Unión al ARN/genética , ARN Polimerasa Dependiente del ARN/metabolismo , Animales , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Humanos , Ratones , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Mutación Puntual , Dominios Proteicos , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
This work was undertaken to understand what would happen if a high-activity radioactive fragment became embedded in an individual following the use of a crude radiological dispersal device ('dirty bomb'). Two areas were addressed: how would a high-activity fragment be viewed on modern digital x-ray imaging systems; and, what would be the impact on medical management for the patient? A set of experimental trials were undertaken using an iridium-192 source and a DRagon mobile x-ray set equipped with a Canon CXDI-50G portable flat panel digital detector plate. In addition, the potential doses to a surgical team were calculated and potential doses to a patient were assessed using a Monte Carlo code, in which a radioactive point source of nil volume was located within a limb of an anthropomorphic voxel phantom. Three distinct effects on the digital imaging systems were observed, referred to in this paper as a localised 'bloom' effect, a 'discontinuity' effect towards the middle of the image and 'fogging' across the entire image. The first two of these effects were unexpected, and possible reasons for their appearance are discussed. The Monte Carlo modelling showed that the patient exposure can potentially lead to very high localised absorbed doses, which may result in symptoms associated with acute radiation syndrome. While the dose clearly depends upon the activity of the fragment and the length of time that the fragment is present inside the patient, it is clear that radiation necrosis of bone, muscle and other tissues may threaten the medium term viability of the limb. The dose rates associated with high-activity fragments may also restrict the time a surgeon has to operate, leading to challenging ethical and surgical decisions. Low-activity fragments allow for conventional surgical management to be considered with appropriate control measures.
Asunto(s)
Armas Nucleares , Traumatismos por Radiación , Radiactividad , Humanos , Fantasmas de Imagen , Intensificación de Imagen RadiográficaRESUMEN
We characterized 55 influenza A(H9N2) viruses isolated in Pakistan during 2014-2016 and found that the hemagglutinin gene is of the G1 lineage and that internal genes have differentiated into a variety of novel genotypes. Some isolates had up to 4-fold reduction in hemagglutination inhibition titers compared with older viruses. Viruses with hemagglutinin A180T/V substitutions conveyed this antigenic diversity and also caused up to 3,500-fold greater binding to avian-like and >20-fold greater binding to human-like sialic acid receptor analogs. This enhanced binding avidity led to reduced virus replication in primary and continuous cell culture. We confirmed that altered receptor-binding avidity of H9N2 viruses, including enhanced binding to human-like receptors, results in antigenic variation in avian influenza viruses. Consequently, current vaccine formulations might not induce adequate protective immunity in poultry, and emergence of isolates with marked avidity for human-like receptors increases the zoonotic risk.