Publications by authors named "Massimo Palmarini"

81 Publications

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research.

PLoS Biol 2021 02 25;19(2):e3001091. Epub 2021 Feb 25.

MRC-University of Glasgow Centre for Virus Research (CVR), Glasgow, United Kingdom.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.
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http://dx.doi.org/10.1371/journal.pbio.3001091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906417PMC
February 2021

Genomic epidemiology reveals multiple introductions of SARS-CoV-2 from mainland Europe into Scotland.

Nat Microbiol 2021 01 21;6(1):112-122. Epub 2020 Dec 21.

Victoria Hospital, Kirkcaldy, UK.

Coronavirus disease 2019 (COVID-19) was first diagnosed in Scotland on 1 March 2020. During the first month of the outbreak, 2,641 cases of COVID-19 led to 1,832 hospital admissions, 207 intensive care admissions and 126 deaths. We aimed to identify the source and number of introductions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into Scotland using a combined phylogenetic and epidemiological approach. Sequencing of 1,314 SARS-CoV-2 viral genomes from available patient samples enabled us to estimate that SARS-CoV-2 was introduced to Scotland on at least 283 occasions during February and March 2020. Epidemiological analysis confirmed that early introductions of SARS-CoV-2 originated from mainland Europe (the majority from Italy and Spain). We identified subsequent early outbreaks in the community, within healthcare facilities and at an international conference. Community transmission occurred after 2 March, 3 weeks before control measures were introduced. Earlier travel restrictions or quarantine measures, both locally and internationally, would have reduced the number of COVID-19 cases in Scotland. The risk of multiple reintroduction events in future waves of infection remains high in the absence of population immunity.
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http://dx.doi.org/10.1038/s41564-020-00838-zDOI Listing
January 2021

Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms.

Science 2020 12 15;370(6521). Epub 2020 Oct 15.

Quantitative Biosciences Institute (QBI) COVID-19 Research Group (QCRG), San Francisco, CA 94158, USA.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 ORF9b, an interaction we structurally characterized using cryo-electron microscopy. Combining genetically validated host factors with both COVID-19 patient genetic data and medical billing records identified molecular mechanisms and potential drug treatments that merit further molecular and clinical study.
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http://dx.doi.org/10.1126/science.abe9403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7808408PMC
December 2020

"Frozen evolution" of an RNA virus suggests accidental release as a potential cause of arbovirus re-emergence.

PLoS Biol 2020 04 28;18(4):e3000673. Epub 2020 Apr 28.

MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. Bluetongue virus serotype 8 (BTV-8), an arthropod-borne virus of ruminants, emerged in livestock in northern Europe in 2006, spreading to most European countries by 2009 and causing losses of billions of euros. Although the outbreak was successfully controlled through vaccination by early 2010, puzzlingly, a closely related BTV-8 strain re-emerged in France in 2015, triggering a second outbreak that is still ongoing. The origin of this virus and the mechanisms underlying its re-emergence are unknown. Here, we performed phylogenetic analyses of 164 whole BTV-8 genomes sampled throughout the two outbreaks. We demonstrate consistent clock-like virus evolution during both epizootics but found negligible evolutionary change between them. We estimate that the ancestor of the second outbreak dates from the height of the first outbreak in 2008. This implies that the virus had not been replicating for multiple years prior to its re-emergence in 2015. Given the absence of any known natural mechanism that could explain BTV-8 persistence over this long period without replication, we hypothesise that the second outbreak could have been initiated by accidental exposure of livestock to frozen material contaminated with virus from approximately 2008. Our work highlights new targets for pathogen surveillance programmes in livestock and illustrates the power of genomic epidemiology to identify pathways of infectious disease emergence.
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http://dx.doi.org/10.1371/journal.pbio.3000673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188197PMC
April 2020

DisCVR: Rapid viral diagnosis from high-throughput sequencing data.

Virus Evol 2019 Jul 26;5(2):vez033. Epub 2019 Aug 26.

MRC-University of Glasgow Centre for Virus Research, Sir Michael Stoker Building, 464 Bearsden Road, Glasgow G61 1QH, UK.

High-throughput sequencing (HTS) enables most pathogens in a clinical sample to be detected from a single analysis, thereby providing novel opportunities for diagnosis, surveillance, and epidemiology. However, this powerful technology is difficult to apply in diagnostic laboratories because of its computational and bioinformatic demands. We have developed DisCVR, which detects known human viruses in clinical samples by matching sample -mers (twenty-two nucleotide sequences) to -mers from taxonomically labeled viral genomes. DisCVR was validated using published HTS data for eighty-nine clinical samples from adults with upper respiratory tract infections. These samples had been tested for viruses metagenomically and also by real-time polymerase chain reaction assay, which is the standard diagnostic method. DisCVR detected human viruses with high sensitivity (79%) and specificity (100%), and was able to detect mixed infections. Moreover, it produced results comparable to those in a published metagenomic analysis of 177 blood samples from patients in Nigeria. DisCVR has been designed as a user-friendly tool for detecting human viruses from HTS data using computers with limited RAM and processing power, and includes a graphical user interface to help users interpret and validate the output. It is written in Java and is publicly available from http://bioinformatics.cvr.ac.uk/discvr.php.
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http://dx.doi.org/10.1093/ve/vez033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735924PMC
July 2019

TRIM69 Inhibits Vesicular Stomatitis Indiana Virus.

J Virol 2019 10 30;93(20). Epub 2019 Sep 30.

MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom

Vesicular stomatitis Indiana virus (VSIV), formerly known as vesicular stomatitis virus (VSV) Indiana (VSV), is a model virus that is exceptionally sensitive to the inhibitory action of interferons (IFNs). Interferons induce an antiviral state by stimulating the expression of hundreds of interferon-stimulated genes (ISGs). These ISGs can constrain viral replication, limit tissue tropism, reduce pathogenicity, and inhibit viral transmission. Since VSIV is used as a backbone for multiple oncolytic and vaccine strategies, understanding how ISGs restrict VSIV not only helps in understanding VSIV-induced pathogenesis but also helps us evaluate and understand the safety and efficacy of VSIV-based therapies. Thus, there is a need to identify and characterize the ISGs that possess anti-VSIV activity. Using arrayed ISG expression screening, we identified TRIM69 as an ISG that potently inhibits VSIV. This inhibition was highly specific as multiple viruses, including influenza A virus, HIV-1, Rift Valley fever virus, and dengue virus, were unaffected by TRIM69. Indeed, just one amino acid substitution in VSIV can govern sensitivity/resistance to TRIM69. Furthermore, TRIM69 is highly divergent in human populations and exhibits signatures of positive selection that are consistent with this gene playing a key role in antiviral immunity. We propose that TRIM69 is an IFN-induced inhibitor of VSIV and speculate that TRIM69 could be important in limiting VSIV pathogenesis and might influence the specificity and/or efficacy of vesiculovirus-based therapies. Vesicular stomatitis Indiana virus (VSIV) is a veterinary pathogen that is also used as a backbone for many oncolytic and vaccine strategies. In natural and therapeutic settings, viral infections like VSIV are sensed by the host, and as a result the host cells make proteins that can protect them from viruses. In the case of VSIV, these antiviral proteins constrain viral replication and protect most healthy tissues from virus infection. In order to understand how VSIV causes disease and how healthy tissues are protected from VSIV-based therapies, it is crucial that we identify the proteins that inhibit VSIV. Here, we show that TRIM69 is an antiviral defense that can potently and specifically block VSIV infection.
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http://dx.doi.org/10.1128/JVI.00951-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6798119PMC
October 2019

Immunophenotyping of Sheep Paraffin-Embedded Peripheral Lymph Nodes.

Front Immunol 2018 11;9:2892. Epub 2018 Dec 11.

MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.

Sheep are not only a major livestock species globally, they are also an important large animal model for biomedical research and have contributed to our understanding of the ontogeny and architecture of the mammalian immune system. In this study, we applied immunohistochemistry and multicolor immunofluorescence in fixed and paraffin-embedded lymph nodes to phenotype the key populations of antigen presenting cells, lymphocytes, and stromal cells that orchestrate the host adaptive immune response. We used an extensive panel of antibodies directed against markers associated with dendritic cells (MHC class II, CD83, and CD208), macrophages (CD11b, CD163, and CD169), stromal cells (CNA.42, S100, and CD83), and lymphocytes (CD3, Pax5, CD4, CD8). Using different methods of tissue fixation and antigen retrieval, we provide a detailed immunophenotyping of sheep lymph nodes including the identification of potential subpopulations of antigen presenting cells and stromal cells. By characterizing cells expressing combinations of these markers in the context of their morphology and location within the lymph node architecture, we provide valuable new tools to investigate the structure, activation, and regulation of the sheep immune system in health and disease.
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http://dx.doi.org/10.3389/fimmu.2018.02892DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297804PMC
October 2019

Genome Sequences of Five African Swine Fever Virus Genotype IX Isolates from Domestic Pigs in Uganda.

Microbiol Resour Announc 2018 Oct 4;7(13). Epub 2018 Oct 4.

MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.

Complete genome sequences of five African swine fever virus isolates were determined directly from clinical material obtained from domestic pigs in Uganda. Four sequences were essentially identical to each other, and all were closely related to the only known genome sequence of p72 genotype IX.
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http://dx.doi.org/10.1128/MRA.01018-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6256554PMC
October 2018

Testicular Degeneration and Infertility following Arbovirus Infection.

J Virol 2018 10 12;92(19). Epub 2018 Sep 12.

Istituto Zooprofilattico Sperimentale della Sardegna, Sassari, Italy

Arboviruses can cause a variety of clinical signs, including febrile illness, arthritis, encephalitis, and hemorrhagic fever. The recent Zika epidemic highlighted the possibility that arboviruses may also negatively affect the male reproductive tract. In this study, we focused on bluetongue virus (BTV), the causative agent of bluetongue and one of the major arboviruses of ruminants. We show that rams that recovered from bluetongue displayed signs of testicular degeneration and azoospermia up to 100 days after the initial infection. Importantly, testicular degeneration was induced in rams experimentally infected with either a high (BTV-1)- or a low (BTV-1)-virulence strain of BTV. Rams infected with the low-virulence BTV strain displayed testicular lesions in the absence of other major clinical signs. Testicular lesions in BTV-infected rams were due to viral replication in the endothelial cells of the peritubular areas of the testes, resulting in stimulation of a type I interferon response, reduction of testosterone biosynthesis by Leydig cells and destruction of Sertoli cells and the blood-testis barrier in more severe cases. Hence, BTV induces testicular degeneration and disruption of spermatogenesis by replicating solely in the endothelial cells of the peritubular areas unlike other gonadotropic viruses. This study shows that a naturally occurring arboviral disease can cause testicular degeneration and affect male fertility at least temporarily. During the recent Zika epidemic, it has become apparent that arboviruses could potentially cause reproductive health problems in male patients. Little is known regarding the effects that arboviruses have on the male reproductive tract. Here, we studied bluetongue virus (BTV), an arbovirus of ruminants, and its effects on the testes of rams. We show that BTV was able to induce testicular degeneration in naturally and experimentally infected rams. Testicular degeneration was caused by BTV replication in the endothelial cells of the peritubular area surrounding the seminiferous tubules (the functional unit of the testes) and was associated with a localized type I interferon response, destruction of the cells supporting the developing germinal cells (Sertoli cells), and reduction of testosterone synthesis. As a result of BTV infection, rams became azoospermic. This study highlights that problems in the male reproductive tract caused by arboviruses could be more common than previously thought.
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http://dx.doi.org/10.1128/JVI.01131-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6146814PMC
October 2018

A new era of virus bioinformatics.

Virus Res 2018 06 8;251:86-90. Epub 2018 May 8.

European Virus Bioinformatics Center, Jena, Germany; RNA Bioinformatics and High Throughput Analysis Jena, Friedrich Schiller University Jena, Jena, Germany. Electronic address:

Despite the recognized excellence of virology and bioinformatics, these two communities have interacted surprisingly sporadically, aside from some pioneering work on HIV-1 and influenza. Bringing together the expertise of bioinformaticians and virologists is crucial, since very specific but fundamental computational approaches are required for virus research, particularly in an era of big data. Collaboration between virologists and bioinformaticians is necessary to improve existing analytical tools, cloud-based systems, computational resources, data sharing approaches, new diagnostic tools, and bioinformatic training. Here, we highlight current progress and discuss potential avenues for future developments in this promising era of virus bioinformatics. We end by presenting an overview of current technologies, and by outlining some of the major challenges and advantages that bioinformatics will bring to the field of virology.
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http://dx.doi.org/10.1016/j.virusres.2018.05.009DOI Listing
June 2018

Fundamental properties of the mammalian innate immune system revealed by multispecies comparison of type I interferon responses.

PLoS Biol 2017 12 18;15(12):e2004086. Epub 2017 Dec 18.

MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.

The host innate immune response mediated by type I interferon (IFN) and the resulting up-regulation of hundreds of interferon-stimulated genes (ISGs) provide an immediate barrier to virus infection. Studies of the type I 'interferome' have mainly been carried out at a single species level, often lacking the power necessary to understand key evolutionary features of this pathway. Here, using a single experimental platform, we determined the properties of the interferomes of multiple vertebrate species and developed a webserver to mine the dataset. This approach revealed a conserved 'core' of 62 ISGs, including genes not previously associated with IFN, underscoring the ancestral functions associated with this antiviral host response. We show that gene expansion contributes to the evolution of the IFN system and that interferomes are shaped by lineage-specific pressures. Consequently, each mammal possesses a unique repertoire of ISGs, including genes common to all mammals and others unique to their specific species or phylogenetic lineages. An analysis of genes commonly down-regulated by IFN suggests that epigenetic regulation of transcription is a fundamental aspect of the IFN response. Our study provides a resource for the scientific community highlighting key paradigms of the type I IFN response.
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http://dx.doi.org/10.1371/journal.pbio.2004086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5747502PMC
December 2017

Bluetongue virus spread in Europe is a consequence of climatic, landscape and vertebrate host factors as revealed by phylogeographic inference.

Proc Biol Sci 2017 Oct;284(1864)

College of Medical, Veterinary and Life Sciences, Institute of Biodiversity, Animal Health and Comparative Medicine, Boyd Orr Centre for Population and Ecosystem Health, University of Glasgow, Glasgow, UK

Spatio-temporal patterns of the spread of infectious diseases are commonly driven by environmental and ecological factors. This is particularly true for vector-borne diseases because vector populations can be strongly affected by host distribution as well as by climatic and landscape variables. Here, we aim to identify environmental drivers for bluetongue virus (BTV), the causative agent of a major vector-borne disease of ruminants that has emerged multiple times in Europe in recent decades. In order to determine the importance of climatic, landscape and host-related factors affecting BTV diffusion across Europe, we fitted different phylogeographic models to a dataset of 113 time-stamped and geo-referenced BTV genomes, representing multiple strains and serotypes. Diffusion models using continuous space revealed that terrestrial habitat below 300 m altitude, wind direction and higher livestock densities were associated with faster BTV movement. Results of discrete phylogeographic analysis involving generalized linear models broadly supported these findings, but varied considerably with the level of spatial partitioning. Contrary to common perception, we found no evidence for average temperature having a positive effect on BTV diffusion, though both methodological and biological reasons could be responsible for this result. Our study provides important insights into the drivers of BTV transmission at the landscape scale that could inform predictive models of viral spread and have implications for designing control strategies.
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http://dx.doi.org/10.1098/rspb.2017.0919DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5647287PMC
October 2017

Sensitivity to BST-2 restriction correlates with Orthobunyavirus host range.

Virology 2017 09 17;509:121-130. Epub 2017 Jun 17.

MRC-University of Glasgow Centre for Virus Research, 464 Bearsden Road, Glasgow G61 1QH, Scotland, United Kingdom.

Orthobunyaviruses include several recently emerging viruses of significant medical and veterinary importance. There is currently very limited understanding on what determines the host species range of these pathogens. In this study we discovered that BST-2/tetherin restricts orthobunyavirus replication in a host-specific manner. We show that viruses with human tropism (Oropouche virus and La Crosse virus) are restricted by sheep BST-2 but not by the human orthologue, while viruses with ruminant tropism (Schmallenberg virus and others) are restricted by human BST-2 but not by the sheep orthologue. We also show that BST-2 blocks orthobunyaviruses replication by reducing the amount of envelope glycoprotein into viral particles egressing from infected cells. This is the first study identifying a restriction factor that correlates with species susceptibility to orthobunyavirus infection. This work provides insight to help us dissect the adaptive changes that bunyaviruses require to cross the species barrier and emerge into new species.
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http://dx.doi.org/10.1016/j.virol.2017.06.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5526858PMC
September 2017

Heparan Sulfate Proteoglycan Is an Important Attachment Factor for Cell Entry of Akabane and Schmallenberg Viruses.

J Virol 2017 08 12;91(15). Epub 2017 Jul 12.

Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan

(AKAV) and Schmallenberg virus (SBV) are members of the genus , which are transmitted by arthropod vectors with a broad cellular tropism as well as Both AKAV and SBV cause arthrogryposis-hydranencephaly syndrome in ruminants. The main cellular receptor and attachment factor for entry of these orthobunyaviruses are unknown. Here, we found that AKAV and SBV infections were inhibited by the addition of heparin or enzymatic removal of cell surface heparan sulfates. To confirm this finding, we prepared heparan sulfate proteoglycan (HSPG)-knockout (KO) cells by using a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system and measured the quantities of binding of these viruses to cell surfaces. We observed a substantial reduction in AKAV and SBV binding to cells, limiting the infections by these viruses. These data demonstrate that HSPGs are important cellular attachment factors for AKAV and SBV, at least , to promote virus replication in susceptible cells. AKAV and SBV are the etiological agents of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic losses in the livestock industry. Here, we identified heparan sulfate proteoglycan as a major cellular attachment factor for the entry of AKAV and SBV. Moreover, we found that heparin is a strong inhibitor of AKAV and SBV infections. Revealing the molecular mechanisms of virus-host interactions is critical in order to understand virus biology and develop novel live attenuated vaccines.
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http://dx.doi.org/10.1128/JVI.00503-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5512253PMC
August 2017

Nonstructural Protein NSs of Schmallenberg Virus Is Targeted to the Nucleolus and Induces Nucleolar Disorganization.

J Virol 2017 Jan 16;91(1). Epub 2016 Dec 16.

ANSES, UMR1161 Virologie, Laboratory for Animal Health, Maisons-Alfort, France

Schmallenberg virus (SBV) was discovered in Germany in late 2011 and then spread rapidly to many European countries. SBV is an orthobunyavirus that causes abortion and congenital abnormalities in ruminants. A virus-encoded nonstructural protein, termed NSs, is a major virulence factor of SBV, and it is known to promote the degradation of Rpb1, a subunit of the RNA polymerase II (Pol II) complex, and therefore hampers global cellular transcription. In this study, we found that NSs is mainly localized in the nucleus of infected cells and specifically appears to target the nucleolus through a nucleolar localization signal (NoLS) localized between residues 33 and 51 of the protein. NSs colocalizes with nucleolar markers such as B23 (nucleophosmin) and fibrillarin. We observed that in SBV-infected cells, B23 undergoes a nucleolus-to-nucleoplasm redistribution, evocative of virus-induced nucleolar disruption. In contrast, the nucleolar pattern of B23 was unchanged upon infection with an SBV recombinant mutant with NSs lacking the NoLS motif (SBVΔNoLS). Interestingly, unlike wild-type SBV, the inhibitory activity of SBVΔNoLS toward RNA Pol II transcription is impaired. Overall, our results suggest that a putative link exists between NSs-induced nucleolar disruption and its inhibitory function on cellular transcription, which consequently precludes the cellular antiviral response and/or induces cell death.

Importance: Schmallenberg virus (SBV) is an emerging arbovirus of ruminants that spread in Europe between 2011 and 2013. SBV induces fetal abnormalities during gestation, with the central nervous system being one of the most affected organs. The virus-encoded NSs protein acts as a virulence factor by impairing host cell transcription. Here, we show that NSs contains a nucleolar localization signal (NoLS) and induces disorganization of the nucleolus. The NoLS motif in the SBV NSs is absolutely necessary for virus-induced inhibition of cellular transcription. To our knowledge, this is the first report of nucleolar functions for NSs within the Bunyaviridae family.
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http://dx.doi.org/10.1128/JVI.01263-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5165206PMC
January 2017

Follicular dendritic cell disruption as a novel mechanism of virus-induced immunosuppression.

Proc Natl Acad Sci U S A 2016 10 26;113(41):E6238-E6247. Epub 2016 Sep 26.

MRC-University of Glasgow Centre for Virus Research, Glasgow G61 1QH, Scotland;

Arboviruses cause acute diseases that increasingly affect global health. We used bluetongue virus (BTV) and its natural sheep host to reveal a previously uncharacterized mechanism used by an arbovirus to manipulate host immunity. Our study shows that BTV, similarly to other antigens delivered through the skin, is transported rapidly via the lymph to the peripheral lymph nodes. Here, BTV infects and disrupts follicular dendritic cells, hindering B-cell division in germinal centers, which results in a delayed production of high affinity and virus neutralizing antibodies. Moreover, the humoral immune response to a second antigen is also hampered in BTV-infected animals. Thus, an arbovirus can evade the host antiviral response by inducing an acute immunosuppression. Although transient, this immunosuppression occurs at the critical early stages of infection when a delayed host humoral immune response likely affects virus systemic dissemination and the clinical outcome of disease.
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http://dx.doi.org/10.1073/pnas.1610012113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5068271PMC
October 2016

Bluetongue virus serotype 27: detection and characterization of two novel variants in Corsica, France.

J Gen Virol 2016 09 19;97(9):2073-2083. Epub 2016 Jul 19.

Université Paris Est, ANSES, ENVA, INRA, UMR 1161 VIROLOGIE, Laboratoire de Santé Animale d'Alfort, Maisons-Alfort, France.

During the compulsory vaccination programme against bluetongue virus serotype 1 (BTV-1) in Corsica (France) in 2014, a BTV strain belonging to a previously uncharacterized serotype (BTV-27) was isolated from asymptomatic goats. The present study describes the detection and molecular characterization of two additional distinct BTV-27 variants found in goats in Corsica in 2014 and 2015. The full coding genome of these two novel BTV-27 variants show high homology (90-93 % nucleotide/93-95 % amino acid) with the originally described BTV-27 isolate from Corsican goats in 2014. These three variants constitute the novel serotype BTV-27 ('BTV-27/FRA2014/v01 to v03'). Phylogenetic analyses with the 26 other established BTV serotypes revealed the closest relationship to BTV-25 (SWI2008/01) (80 % nucleotide/86 % amino acid) and to BTV-26 (KUW2010/02) (73-74 % nucleotide/80-81 % amino acid). However, highest sequence homologies between individual segments of BTV-27/FRA2014/v01-v03 with BTV-25 and BTV-26 vary. All three variants share the same segment 2 nucleotype with BTV-25. Neutralization assays of anti-BTV27/FRA2014/v01-v03 sera with a reassortant virus containing the outer capsid proteins of BTV-25 (BTV1VP2/VP5 BTV25) further confirmed that BTV-27 represents a distinct BTV serotype. Relationships between the variants and with BTV-25 and BTV-26, hypotheses about their origin, reassortment events and evolution are discussed.
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http://dx.doi.org/10.1099/jgv.0.000557DOI Listing
September 2016

Late Ebola virus relapse causing meningoencephalitis: a case report.

Lancet 2016 Jul 18;388(10043):498-503. Epub 2016 May 18.

MRC-University of Glasgow Centre for Virus Research, Glasgow, UK.

Background: There are thousands of survivors of the 2014 Ebola outbreak in west Africa. Ebola virus can persist in survivors for months in immune-privileged sites; however, viral relapse causing life-threatening and potentially transmissible disease has not been described. We report a case of late relapse in a patient who had been treated for severe Ebola virus disease with high viral load (peak cycle threshold value 13.2).

Methods: A 39-year-old female nurse from Scotland, who had assisted the humanitarian effort in Sierra Leone, had received intensive supportive treatment and experimental antiviral therapies, and had been discharged with undetectable Ebola virus RNA in peripheral blood. The patient was readmitted to hospital 9 months after discharge with symptoms of acute meningitis, and was found to have Ebola virus in cerebrospinal fluid (CSF). She was treated with supportive therapy and experimental antiviral drug GS-5734 (Gilead Sciences, San Francisco, Foster City, CA, USA). We monitored Ebola virus RNA in CSF and plasma, and sequenced the viral genome using an unbiased metagenomic approach.

Findings: On admission, reverse transcriptase PCR identified Ebola virus RNA at a higher level in CSF (cycle threshold value 23.7) than plasma (31.3); infectious virus was only recovered from CSF. The patient developed progressive meningoencephalitis with cranial neuropathies and radiculopathy. Clinical recovery was associated with addition of high-dose corticosteroids during GS-5734 treatment. CSF Ebola virus RNA slowly declined and was undetectable following 14 days of treatment with GS-5734. Sequencing of plasma and CSF viral genome revealed only two non-coding changes compared with the original infecting virus.

Interpretation: Our report shows that previously unanticipated, late, severe relapses of Ebola virus can occur, in this case in the CNS. This finding fundamentally redefines what is known about the natural history of Ebola virus infection. Vigilance should be maintained in the thousands of Ebola survivors for cases of relapsed infection. The potential for these cases to initiate new transmission chains is a serious public health concern.

Funding: Royal Free London NHS Foundation Trust.
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http://dx.doi.org/10.1016/S0140-6736(16)30386-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967715PMC
July 2016

Bluetongue Virus NS4 Protein Is an Interferon Antagonist and a Determinant of Virus Virulence.

J Virol 2016 06 12;90(11):5427-39. Epub 2016 May 12.

MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland, United Kingdom

Unlabelled: Bluetongue virus (BTV) is the causative agent of bluetongue, a major infectious disease of ruminants with serious consequences to both animal health and the economy. The clinical outcome of BTV infection is highly variable and dependent on a variety of factors related to both the virus and the host. In this study, we show that the BTV nonstructural protein NS4 favors viral replication in sheep, the animal species most affected by bluetongue. In addition, NS4 confers a replication advantage on the virus in interferon (IFN)-competent primary sheep endothelial cells and immortalized cell lines. We determined that in cells infected with an NS4 deletion mutant (BTV8ΔNS4), there is increased synthesis of type I IFN compared to cells infected with wild-type BTV-8. In addition, using RNA sequencing (RNA-seq), we show that NS4 modulates the host IFN response and downregulates mRNA levels of type I IFN and interferon-stimulated genes. Moreover, using reporter assays and protein synthesis assays, we show that NS4 downregulates the activities of a variety of promoters, such as the cytomegalovirus immediate-early promoter, the IFN-β promoter, and a promoter containing interferon-stimulated response elements (ISRE). We also show that the NS4 inhibitory activity on gene expression is related to its nucleolar localization. Furthermore, NS4 does not affect mRNA splicing or cellular translation. The data obtained in this study strongly suggest that BTV NS4 is an IFN antagonist and a key determinant of viral virulence.

Importance: Bluetongue is one of the main infectious diseases of ruminants and is caused by bluetongue virus (BTV), an arthropod-borne virus transmitted from infected to susceptible animals by Culicoides biting midges. Bluetongue has a variable clinical outcome that can be related to both virus and host factors. It is therefore critical to understand the interplay between BTV and the host immune responses. In this study, we show that a nonstructural protein of BTV (NS4) is critical to counteract the innate immune response of the host. Infection of cells with a BTV mutant lacking NS4 results in increased synthesis of IFN-β and upregulation of interferon-stimulated genes. In addition, we show that NS4 is a virulence factor for BTV by favoring viral replication in sheep, the animal species most susceptible to bluetongue.
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http://dx.doi.org/10.1128/JVI.00422-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4934764PMC
June 2016

Mutations in the Schmallenberg Virus Gc Glycoprotein Facilitate Cellular Protein Synthesis Shutoff and Restore Pathogenicity of NSs Deletion Mutants in Mice.

J Virol 2016 06 12;90(11):5440-5450. Epub 2016 May 12.

MRC-University of Glasgow Centre for Virus Research, Glasgow, Scotland

Unlabelled: Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild-type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach high titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild-type SBV, as it has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate for both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an interferon (IFN) antagonist. Therefore, SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, for the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins, while the ability to control the production of IFN maps to the NSs protein.

Importance: The identification of viral determinants of pathogenesis is key to the development of prophylactic and intervention measures. In this study, we found that the bunyavirus Gc glycoprotein is a virulence factor. Importantly, we show that mutations in the Gc glycoprotein can restore the pathogenicity of attenuated mutants resulting from deletions or mutations in the nonstructural protein NSs. Our findings highlight the fact that careful consideration should be taken when designing live attenuated vaccines based on deletions of nonstructural proteins since single mutations in the viral glycoproteins appear to revert attenuated mutants to virulent phenotypes.
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http://dx.doi.org/10.1128/JVI.00424-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4934738PMC
June 2016

Characterization of a second open reading frame in genome segment 10 of bluetongue virus.

J Gen Virol 2015 Nov 19;96(11):3280-3293. Epub 2015 Aug 19.

MRC-University of Glasgow Centre for Virus Research, Glasgow, UK.

Viruses have often evolved overlapping reading frames in order to maximize their coding capacity. Until recently, the segmented dsRNA genome of viruses of the Orbivirus genus was thought to be monocistronic, but the identification of the bluetongue virus (BTV) NS4 protein changed this assumption. A small ORF in segment 10, overlapping the NS3 ORF in the +1 position, is maintained in more than 300 strains of the 27 different BTV serotypes and in more than 200 strains of the phylogenetically related African horse sickness virus (AHSV). In BTV, this ORF (named S10-ORF2 in this study) encodes a putative protein 50-59 residues in length and appears to be under strong positive selection. HA- or GFP-tagged versions of S10-ORF2 expressed from transfected plasmids localized within the nucleoli of transfected cells, unless a putative nucleolar localization signal was mutated. S10-ORF2 inhibited gene expression, but not RNA translation, in transient transfection reporter assays. In both mammalian and insect cells, BTV S10-ORF2 deletion mutants (BTV8ΔS10-ORF2) displayed similar replication kinetics to wt virus. In vivo, S10-ORF2 deletion mutants were pathogenic in mouse models of disease. Although further evidence is required for S10-ORF2 expression during infection, the data presented provide an initial characterization of this ORF.
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http://dx.doi.org/10.1099/jgv.0.000267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806581PMC
November 2015

Widespread Reassortment Shapes the Evolution and Epidemiology of Bluetongue Virus following European Invasion.

PLoS Pathog 2015 Aug 7;11(8):e1005056. Epub 2015 Aug 7.

Vector-Borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Woking, United Kingdom.

Genetic exchange by a process of genome-segment 'reassortment' represents an important mechanism for evolutionary change in all viruses with segmented genomes, yet in many cases a detailed understanding of its frequency and biological consequences is lacking. We provide a comprehensive assessment of reassortment in bluetongue virus (BTV), a globally important insect-borne pathogen of livestock, during recent outbreaks in Europe. Full-genome sequences were generated and analysed for over 150 isolates belonging to the different BTV serotypes that have emerged in the region over the last 5 decades. Based on this novel dataset we confirm that reassortment is a frequent process that plays an important and on-going role in evolution of the virus. We found evidence for reassortment in all ten segments without a significant bias towards any particular segment. However, we observed biases in the relative frequency at which particular segments were associated with each other during reassortment. This points to selective constraints possibly caused by functional relationships between individual proteins or genome segments and genome-wide epistatic interactions. Sites under positive selection were more likely to undergo amino acid changes in newly reassorted viruses, providing additional evidence for adaptive dynamics as a consequence of reassortment. We show that the live attenuated vaccines recently used in Europe have repeatedly reassorted with field strains, contributing to their genotypic, and potentially phenotypic, variability. The high degree of plasticity seen in the BTV genome in terms of segment origin suggests that current classification schemes that are based primarily on serotype, which is determined by only a single genome segment, are inadequate. Our work highlights the need for a better understanding of the mechanisms and epidemiological consequences of reassortment in BTV, as well as other segmented RNA viruses.
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http://dx.doi.org/10.1371/journal.ppat.1005056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4529188PMC
August 2015

Turnover Rate of NS3 Proteins Modulates Bluetongue Virus Replication Kinetics in a Host-Specific Manner.

J Virol 2015 Oct 5;89(20):10467-81. Epub 2015 Aug 5.

UMR754, Université Claude Bernard Lyon 1, Institut National de la Recherche Agronomique, Ecole Pratique des Hautes Etudes, SFR BioSciences Gerland, Lyon, France

Unlabelled: Bluetongue virus (BTV) is an arbovirus transmitted to livestock by midges of the Culicoides family and is the etiological agent of a hemorrhagic disease in sheep and other ruminants. In mammalian cells, BTV particles are released primarily by virus-induced cell lysis, while in insect cells they bud from the plasma membrane and establish a persistent infection. BTV possesses a ten-segmented double-stranded RNA genome, and NS3 proteins are encoded by segment 10 (Seg-10). The viral nonstructural protein 3 (NS3) plays a key role in mediating BTV egress as well as in impeding the in vitro synthesis of type I interferon in mammalian cells. In this study, we asked whether genetically distant NS3 proteins can alter BTV-host interactions. Using a reverse genetics approach, we showed that, depending on the NS3 considered, BTV replication kinetics varied in mammals but not in insects. In particular, one of the NS3 proteins analyzed harbored a proline at position 24 that leads to its rapid intracellular decay in ovine but not in Culicoides cells and to the attenuation of BTV virulence in a mouse model of disease. Overall, our data reveal that the genetic variability of Seg-10/NS3 differentially modulates BTV replication kinetics in a host-specific manner and highlight the role of the host-specific variation in NS3 protein turnover rate.

Importance: BTV is the causative agent of a severe disease transmitted between ruminants by biting midges of Culicoides species. NS3, encoded by Seg-10 of the BTV genome, fulfills key roles in BTV infection. As Seg-10 sequences from various BTV strains display genetic variability, we assessed the impact of different Seg-10 and NS3 proteins on BTV infection and host interactions. In this study, we revealed that various Seg-10/NS3 proteins alter BTV replication kinetics in mammals but not in insects. Notably, we found that NS3 protein turnover may vary in ovine but not in Culicoides cells due to a single amino acid residue that, most likely, leads to rapid and host-dependent protein degradation. Overall, this study highlights that genetically distant BTV Seg-10/NS3 influence BTV biological properties in a host-specific manner and increases our understanding of how NS3 proteins contribute to the outcome of BTV infection.
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http://dx.doi.org/10.1128/JVI.01541-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580187PMC
October 2015

Transcriptome analysis reveals the host response to Schmallenberg virus in bovine cells and antagonistic effects of the NSs protein.

BMC Genomics 2015 Apr 19;16:324. Epub 2015 Apr 19.

MRC-University of Glasgow Centre for Virus Research, Glasgow G61 1QH, Scotland, UK.

Background: Schmallenberg virus (SBV) is a member of the Orthobunyavirus genus (Bunyaviridae family) causing malformations and abortions in ruminants. Although, as for other members of this family/genus, the non-structural protein NSs has been shown to be an interferon antagonist, very little is known regarding the overall inhibitory effects and targets of orthobunyavirus NSs proteins on host gene expression during infection. Therefore, using RNA-seq this study describes changes to the transcriptome of primary bovine cells following infection with Schmallenberg virus (SBV) or with a mutant lacking the non-structural protein NSs (SBVdelNSs) providing a detailed comparison of the effect of NSs expression on the host cell.

Results: The sequence reads from all samples (uninfected cells, SBV and SBVdelNSs) assembled well to the bovine host reference genome (on average 87.43% of the reads). During infection with SBVdelNSs, 649 genes were differentially expressed compared to uninfected cells (78.7% upregulated) and many of these were known antiviral and IFN-stimulated genes. On the other hand, only nine genes were differentially expressed in SBV infected cells compared to uninfected control cells, demonstrating the strong inhibitory effect of NSs on cellular gene expression. However, the majority of the genes that were expressed during SBV infection are involved in restriction of viral replication and spread indicating that SBV does not completely manage to shutdown the host antiviral response.

Conclusions: In this study we show the effects of SBV NSs on the transcriptome of infected cells as well as the cellular response to wild type SBV. Although NSs is very efficient in shutting down genes of the host innate response, a number of possible antiviral factors were identified. Thus the data from this study can serve as a base for more detailed mechanistic studies of SBV and other orthobunyaviruses.
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http://dx.doi.org/10.1186/s12864-015-1538-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4404599PMC
April 2015

Multiple genome segments determine virulence of bluetongue virus serotype 8.

J Virol 2015 May 11;89(10):5238-49. Epub 2015 Mar 11.

MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom

Unlabelled: Bluetongue virus (BTV) causes bluetongue, a major hemorrhagic disease of ruminants. In order to investigate the molecular determinants of BTV virulence, we used a BTV8 strain minimally passaged in tissue culture (termed BTV8L in this study) and a derivative strain passaged extensively in tissue culture (BTV8H) in in vitro and in vivo studies. BTV8L was pathogenic in both IFNAR(-/-) mice and in sheep, while BTV8H was attenuated in both species. To identify genetic changes which led to BTV8H attenuation, we generated 34 reassortants between BTV8L and BTV8H. We found that partial attenuation of BTV8L in IFNAR(-/-) mice was achieved by simply replacing genomic segment 2 (Seg2, encoding VP2) or Seg10 (encoding NS3) with the BTV8H homologous segments. Fully attenuated viruses required at least two genome segments from BTV8H, including Seg2 with either Seg1 (encoding VP1), Seg6 (encoding VP6 and NS4), or Seg10 (encoding NS3). Conversely, full reversion of virulence of BTV8H required at least five genomic segments of BTV8L. We also demonstrated that BTV8H acquired an increased affinity for glycosaminoglycan receptors during passaging in cell culture due to mutations in its VP2 protein. Replication of BTV8H was relatively poor in interferon (IFN)-competent primary ovine endothelial cells compared to replication of BTV8L, and this phenotype was determined by several viral genomic segments, including Seg4 and Seg9. This study demonstrated that multiple viral proteins contribute to BTV8 virulence. VP2 and NS3 are primary determinants of BTV pathogenesis, but VP1, VP5, VP4, VP6, and VP7 also contribute to virulence.

Importance: Bluetongue is one of the major infectious diseases of ruminants, and it is listed as a notifiable disease by the World Organization for Animal Health (OIE). The clinical outcome of BTV infection varies considerably and depends on environmental and host- and virus-specific factors. Over the years, BTV serotypes/strains with various degrees of virulence (including nonpathogenic strains) have been described in different geographical locations. However, no data are available to correlate the BTV genotype to virulence. This study shows that BTV virulence is determined by different viral genomic segments. The data obtained will help to characterize thoroughly the pathogenesis of bluetongue. The possibility to determine the pathogenicity of virus isolates on the basis of their genome sequences will help in the design of control strategies that fit the risk posed by new emerging BTV strains.
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http://dx.doi.org/10.1128/JVI.00395-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4442542PMC
May 2015

"Ménage à Trois": the evolutionary interplay between JSRV, enJSRVs and domestic sheep.

Viruses 2014 Dec 9;6(12):4926-45. Epub 2014 Dec 9.

UMR754, Université Claude Bernard Lyon 1, Institut National de la Recherche Agronomique, Ecole Pratique des Hautes Etudes, SFR BioSciences Gerland, 50 avenue Tony Garnier, 69007 Lyon, France.

Sheep betaretroviruses represent a fascinating model to study the complex evolutionary interplay between host and pathogen in natural settings. In infected sheep, the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) coexists with a variety of highly related endogenous JSRVs, referred to as enJSRVs. During evolution, some of them were co-opted by the host as they fulfilled important biological functions, including placental development and protection against related exogenous retroviruses. In particular, two enJSRV loci, enJS56A1 and enJSRV-20, were positively selected during sheep domestication due to their ability to interfere with the replication of related competent retroviruses. Interestingly, viruses escaping these transdominant enJSRVs have recently emerged, probably less than 200 years ago. Overall, these findings suggest that in sheep the process of endogenization is still ongoing and, therefore, the evolutionary interplay between endogenous and exogenous sheep betaretroviruses and their host has not yet reached an equilibrium.
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http://dx.doi.org/10.3390/v6124926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4276937PMC
December 2014