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Metagenomic next-generation sequencing has transformed the discovery and diagnosis of infectious disease, with the power to characterise the complete 'infectome' (bacteria, viruses, fungi, parasites) of an individual host organism. However, the identification of novel pathogens has been complicated by widespread microbial contamination in commonly used laboratory reagents. Using total RNA sequencing ("metatranscriptomics") we documented the presence of contaminant viral sequences in multiple 'blank' negative control sequencing libraries that comprise a sterile water and reagent mix. Accordingly, we identified 14 viral sequences in 7 negative control sequencing libraries. As in previous studies, several circular replication-associated protein encoding (CRESS) DNA virus-like sequences were recovered in the blank control libraries, as well as contaminating sequences from the Totiviridae, Tombusviridae and Lentiviridae families of RNA virus. These data suggest that viral contamination of common laboratory reagents is likely commonplace and can comprise a wide variety of viruses.
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Vírus de DNA/genética , Contaminação de Equipamentos/estatística & dados numéricos , Indicadores e Reagentes/análise , Laboratórios/estatística & dados numéricos , Vírus/isolamento & purificação , Vírus de DNA/isolamento & purificação , Metagenoma , Vírus/classificação , Vírus/genéticaRESUMO
Meta-transcriptomic next-generation sequencing has transformed virus discovery, dramatically expanding our knowledge of the known virosphere. Nevertheless, the use of meta-transcriptomics for virus discovery faces important challenges. As this technology becomes more widely adopted, the proportion of viral sequences in public databases with incorrect (e.g. mis-assignment of host) or limited information (e.g. lacking taxonomic classification) is likely to grow, limiting their utility in bioinformatic pipelines for virus discovery. In addition, we currently lack the bioinformatic tools that can accurately identify viruses showing little or no sequence similarity to database viruses or those that represent likely reagent contaminants. Herein, we outline some of the challenges to effective meta-transcriptomic virus discovery as well as their potential solutions.
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Transcriptoma/genética , Virologia/métodos , Vírus/classificação , Vírus/genética , Biologia Computacional , Regulação Viral da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Vírus/isolamento & purificaçãoRESUMO
A segmented genome enables influenza virus to undergo reassortment when two viruses infect the same cell. Although reassortment is involved in the creation of pandemic influenza strains and is routinely used to produce influenza vaccines, our understanding of the factors that drive the emergence of dominant gene constellations during this process is incomplete. Recently, we defined a spectrum of interactions between the gene segments of the A/Udorn/307/72 (H3N2) (Udorn) strain that occur within virus particles, a major interaction being between the NA and PB1 gene segments. In addition, we showed that the Udorn PB1 is preferentially incorporated into reassortant viruses that express the Udorn NA. Here we use an influenza vaccine seed production model where eggs are coinfected with Udorn and the high yielding A/Puerto Rico/8/34 (H1N1) (PR8) virus and track viral genotypes through the reassortment process under antibody selective pressure to determine the impact of Udorn NA-PB1 co-selection. We discovered that 86% of the reassortants contained the PB1 from the Udorn parent after the initial co-infection and this bias towards Udorn PB1 was maintained after two further passages. Included in these were certain gene constellations containing Udorn HA, NA, and PB1 that confered low replicative fitness yet rapidly became dominant at the expense of more fit progeny, even when co-infection ratios of the two viruses favoured PR8. Fitness was not compromised, however, in the corresponding reassortants that also contained Udorn NP. Of particular note is the observation that relatively unfit reassortants could still fulfil the role of vaccine seed candidates as they provided high haemagglutinin (HA) antigen yields through co-production of non-infectious particles and/or by more HA molecules per virion. Our data illustrate the dynamics and complexity of reassortment and highlight how major gene segment interactions formed during packaging, in addition to antibody pressure, initially restrict the reassortant viruses that are formed.
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Italy's second wave of SARS-CoV-2 has hit hard, with more than three million cases and over 100,000 deaths, representing an almost ten-fold increase in the numbers reported by August 2020. Herein, we present an analysis of 6515 SARS-CoV-2 sequences sampled in Italy between 29 January 2020 and 1 March 2021 and show how different lineages emerged multiple times independently despite lockdown restrictions. Virus lineage B.1.177 became the dominant variant in November 2020, when cases peaked at 40,000 a day, but since January 2021 this is being replaced by the B.1.1.7 'variant of concern'. In addition, we report a sudden increase in another documented variant of concern-lineage P.1-from December 2020 onwards, most likely caused by a single introduction into Italy. We again highlight how international importations drive the emergence of new lineages and that genome sequencing should remain a top priority for ongoing surveillance in Italy.
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COVID-19 , Surtos de Doenças , SARS-CoV-2/isolamento & purificação , COVID-19/epidemiologia , COVID-19/virologia , Humanos , Itália/epidemiologia , Quarentena , Estudos SoroepidemiológicosRESUMO
Unraveling and exploiting mechanisms of disease resistance in cereal crops is currently limited by their large repeat-rich genomes and the lack of genetic recombination or cultivar (cv)-specific sequence information. We cloned the first leaf rust resistance gene Rph1 (Rph1 a) from cultivated barley (Hordeum vulgare) using "MutChromSeq," a recently developed molecular genomics tool for the rapid cloning of genes in plants. Marker-trait association in the CI 9214/Stirling doubled haploid population mapped Rph1 to the short arm of chromosome 2H in a physical region of 1.3 megabases relative to the barley cv Morex reference assembly. A sodium azide mutant population in cv Sudan was generated and 10 mutants were confirmed by progeny-testing. Flow-sorted 2H chromosomes from Sudan (wild type) and six of the mutants were sequenced and compared to identify candidate genes for the Rph1 locus. MutChromSeq identified a single gene candidate encoding a coiled-coil nucleotide binding site Leucine-rich repeat (NLR) receptor protein that was altered in three different mutants. Further Sanger sequencing confirmed all three mutations and identified an additional two independent mutations within the same candidate gene. Phylogenetic analysis determined that Rph1 clustered separately from all previously cloned NLRs from the Triticeae and displayed highest sequence similarity (89%) with a homolog of the Arabidopsis (Arabidopsis thaliana) disease resistance protein 1 protein in Triticum urartu In this study we determined the molecular basis for Rph1-mediated resistance in cultivated barley enabling varietal improvement through diagnostic marker design, gene editing, and gene stacking technologies.
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Hordeum/fisiologia , Interações Hospedeiro-Patógeno , Proteínas NLR/fisiologia , Mapeamento Cromossômico , Genes de Plantas , Proteínas de Plantas/fisiologia , Análise de Sequência de DNARESUMO
The occurrence of a spontaneous nephropathy with intranuclear inclusions in laboratory mice has puzzled pathologists for over 4 decades, because its etiology remains elusive. The condition is more severe in immunodeficient animals, suggesting an infectious cause. Using metagenomics, we identify the causative agent as an atypical virus, termed "mouse kidney parvovirus" (MKPV), belonging to a divergent genus of Parvoviridae. MKPV was identified in animal facilities in Australia and North America, is transmitted via a fecal-oral or urinary-oral route, and is controlled by the adaptive immune system. Detailed analysis of the clinical course and histopathological features demonstrated a stepwise progression of pathology ranging from sporadic tubular inclusions to tubular degeneration and interstitial fibrosis and culminating in renal failure. In summary, we identify a widely distributed pathogen in laboratory mice and establish MKPV-induced nephropathy as a new tool for elucidating mechanisms of tubulointerstitial fibrosis that shares molecular features with chronic kidney disease in humans.
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Nefrite Intersticial/virologia , Parvovirus/isolamento & purificação , Parvovirus/patogenicidade , Animais , Austrália , Progressão da Doença , Feminino , Fibrose/patologia , Fibrose/virologia , Humanos , Rim/metabolismo , Rim/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nefrite Intersticial/fisiopatologia , América do Norte , Infecções por Parvoviridae/metabolismoRESUMO
Human enteroviruses (EV) pose a major risk to public health. This is especially so in the Asia-Pacific region where increasing numbers of hand, foot and mouth disease (HFMD) cases and large outbreaks of severe neurological disease associated with EV-A71 have occurred. Despite their importance, key aspects of the emergence, epidemiology and evolution of EVs remain unclear, and most studies of EV evolution have focused on a limited number of genes. Here, we describe the genomic-scale evolution of EV-A viruses sampled from pediatric patients with mild disease attending a single hospital in western Sydney, Australia, over an 18-month period. This analysis revealed the presence of eight viral serotypes-Coxsackievirus (CV) A2, A4, A5, A6, A8, A10, A16 and EV-A71-with up to four different serotypes circulating in any 1 month. Despite an absence of large-scale outbreaks, high levels of geographical and temporal mixing of serotypes were identified. Phylogenetic analysis revealed that multiple strains of the same serotype were present in the community, and that this diversity was shaped by multiple introductions into the Sydney population, with only a single lineage of CV-A6 exhibiting in situ transmission over the entire study period. Genomic-scale analyses also revealed the presence of novel and historical EV recombinants. Notably, our analysis revealed no association between viral phylogeny, including serotype, and patient age, sex, nor disease severity (for uncomplicated disease). This study emphasizes the contribution of EV-A viruses other than EV-A71 to mild EV disease including HFMD in Australia and highlights the need for greater surveillance of these viruses to improve strategies for outbreak preparedness and vaccine design.
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Outbreaks of respiratory virus infection at mass gatherings pose significant health risks to attendees, host communities, and ultimately the global population if they help facilitate viral emergence. However, little is known about the genetic diversity, evolution, and patterns of viral transmission during mass gatherings, particularly how much diversity is generated by in situ transmission compared to that imported from other locations. Here, we describe the genome-scale evolution of influenza A viruses sampled from the Hajj pilgrimages at Makkah during 2013 to 2015. Phylogenetic analysis revealed that the diversity of influenza viruses at the Hajj pilgrimages was shaped by multiple introduction events, comprising multiple cocirculating lineages in each year, including those that have circulated in the Middle East and those whose origins likely lie on different continents. At the scale of individual hosts, the majority of minor variants resulted from de novo mutation, with only limited evidence of minor variant transmission or minor variants circulating at subconsensus level despite the likely identification of multiple transmission clusters. Together, these data highlight the complexity of influenza virus infection at the Hajj pilgrimages, reflecting a mix of global genetic diversity drawn from multiple sources combined with local transmission, and reemphasize the need for vigilant surveillance at mass gatherings.IMPORTANCE Large population sizes and densities at mass gatherings such as the Hajj (Makkah, Saudi Arabia) can contribute to outbreaks of respiratory virus infection by providing local hot spots for transmission followed by spread to other localities. Using a genome-scale analysis, we show that the genetic diversity of influenza A viruses at the Hajj gatherings during 2013 to 2015 was largely shaped by the introduction of multiple viruses from diverse geographic regions, including the Middle East, with only little evidence of interhost virus transmission at the Hajj and seemingly limited spread of subconsensus mutational variants. The diversity of viruses at the Hajj pilgrimages highlights the potential for lineage cocirculation during mass gatherings, in turn fuelling segment reassortment and the emergence of novel variants, such that the continued surveillance of respiratory pathogens at mass gatherings should be a public health priority.
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Variação Genética , Vírus da Influenza A/genética , Influenza Humana/epidemiologia , Influenza Humana/virologia , Islamismo , Infecções Respiratórias/virologia , Viagem , Surtos de Doenças , Evolução Molecular , Humanos , Influenza Humana/transmissão , Comportamento de Massa , Oriente Médio/epidemiologia , Mutação , Filogenia , Saúde Pública , Infecções Respiratórias/epidemiologia , Arábia Saudita/epidemiologiaRESUMO
Current split influenza virus vaccines that induce strain-specific neutralising antibodies provide some degree of protection against influenza infection but there is a clear need to improve their effectiveness. The constant antigenic drift of influenza viruses means that vaccines are often not an exact match to the circulating strain and so levels of relevant antibodies may not be sufficiently high to afford protection. In the situation where the emergent influenza virus is completely novel, as is the case with pandemic strains, existing vaccines may provide no benefit. In this study we tested the concept of a combination vaccine consisting of sub-optimal doses of split influenza virus vaccine mixed with a cross-protective T-cell inducing lipopeptide containing the TLR2 ligand Pam2Cys. Mice immunised with combination vaccines showed superior levels of lung viral clearance after challenge compared to either split virus or lipopeptide alone, mediated through activation of enhanced humoral and/or additional cellular responses. The mechanism of action of these vaccines was dependent on the route of administration, with intranasal administration being superior to subcutaneous and intramuscular routes, potentially through the induction of memory CD8+ T cells in the lungs. This immunisation strategy not only provides a mechanism for minimising the dose of split virus antigen but also, through the induction of cross-protective CD8+ T cells, proves a breadth of immunity to provide potential benefit upon encounter with serologically diverse influenza isolates.
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Imunidade Adaptativa , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Cães , Vírus da Influenza A/imunologia , Vacinas contra Influenza/uso terapêutico , Lipopeptídeos/imunologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/prevenção & controle , Carga ViralRESUMO
UNLABELLED: Egg-grown influenza vaccine yields are maximized by infection with a seed virus produced by "classical reassortment" of a seasonal isolate with a highly egg-adapted strain. Seed viruses are selected based on a high-growth phenotype and the presence of the seasonal hemagglutinin (HA) and neuraminidase (NA) surface antigens. Retrospective analysis of H3N2 vaccine seed viruses indicated that, unlike other internal proteins that were predominantly derived from the high-growth parent A/Puerto Rico/8/34 (PR8), the polymerase subunit PB1 could be derived from either parent depending on the seasonal strain. We have recently shown that A/Udorn/307/72 (Udorn) models a seasonal isolate that yields reassortants bearing the seasonal PB1 gene. This is despite the fact that the reverse genetics-derived virus that includes Udorn PB1 with Udorn HA and NA on a PR8 background has inferior growth compared to the corresponding virus with PR8 PB1. Here we use competitive plasmid transfections to investigate the mechanisms driving selection of a less fit virus and show that the Udorn PB1 gene segment cosegregates with the Udorn NA gene segment. Analysis of chimeric PB1 genes revealed that the coselection of NA and PB1 segments was not directed through the previously identified packaging sequences but through interactions involving the internal coding region of the PB1 gene. This study identifies associations between viral genes that can direct selection in classical reassortment for vaccine production and which may also be of relevance to the gene constellations observed in past antigenic shift events where creation of a pandemic virus has involved reassortment. IMPORTANCE: Influenza vaccine must be produced and administered in a timely manner in order to provide protection during the winter season, and poor-growing vaccine seed viruses can compromise this process. To maximize vaccine yields, manufacturers create hybrid influenza viruses with gene segments encoding the surface antigens from a seasonal virus isolate, important for immunity, and others from a virus with high growth properties. This involves coinfection of cells with both parent viruses and selection of dominant progeny bearing the seasonal antigens. We show that this method of creating hybrid viruses does not necessarily select for the best yielding virus because preferential pairing of gene segments when progeny viruses are produced determines the genetic makeup of the hybrids. This not only has implications for how hybrid viruses are selected for vaccine production but also sheds light on what drives and limits hybrid gene combinations that arise in nature, leading to pandemics.
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Neuraminidase/genética , Fases de Leitura Aberta/genética , Orthomyxoviridae/genética , Vírus Reordenados/genética , Proteínas Virais/genética , Animais , Linhagem Celular , Cães , Genes Virais/genética , Células HEK293 , Humanos , Vacinas contra Influenza/genética , Células Madin Darby de Rim Canino , Estudos Retrospectivos , Transfecção/métodosRESUMO
The yields of egg-grown influenza vaccines are maximized by the production of a seed strain using a reassortment of the seasonal influenza virus isolate with a highly egg-adapted strain. The seed virus is selected based on high yields of viral hemagglutinin (HA) and expression of the surface antigens from the seasonal isolate. The remaining proteins are usually derived from the high-growth parent. However, a retrospective analysis of vaccine seeds revealed that the seasonal PB1 gene was selected in more than 50% of reassortment events. Using the model seasonal H3N2 virus A/Udorn/307/72 (Udorn) virus and the high-growth A/Puerto Rico/8/34 (PR8) virus, we assessed the influence of the source of the PB1 gene on virus growth and vaccine yield. Classical reassortment of these two strains led to the selection of viruses that predominantly had the Udorn PB1 gene. The presence of Udorn PB1 in the seed virus, however, did not result in higher yields of virus or HA compared to the yields in the corresponding seed virus with PR8 PB1. The 8-fold-fewer virions produced with the seed virus containing the Udorn PB1 were somewhat compensated for by a 4-fold increase in HA per virion. A higher HA/nucleoprotein (NP) ratio was found in past vaccine preparations when the seasonal PB1 was present, also indicative of a higher HA density in these vaccine viruses. As the HA viral RNA (vRNA) and mRNA levels in infected cells were similar, we propose that PB1 selectively alters the translation of viral mRNA. This study helps to explain the variability of vaccine seeds with respect to HA yield.