Publications by authors named "Peter P C Mertens"

78 Publications

Continuous Cell Lines from the European Biting Midge (Meigen, 1830).

Microorganisms 2020 May 30;8(6). Epub 2020 May 30.

School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, LE12 5RD, UK.

biting midges (Diptera: Ceratopogonidae) transmit arboviruses of veterinary or medical importance, including bluetongue virus (BTV) and Schmallenberg virus, as well as causing severe irritation to livestock and humans. Arthropod cell lines are essential laboratory research tools for the isolation and propagation of vector-borne pathogens and the investigation of host-vector-pathogen interactions. Here we report the establishment of two continuous cell lines, CNE/LULS44 and CNE/LULS47, from embryos of , a midge distributed throughout the Western Palearctic region. Species origin of the cultured cells was confirmed by polymerase chain reaction (PCR) amplification and sequencing of a fragment of the gene, and the absence of bacterial contamination was confirmed by bacterial 16S rRNA PCR. Both lines have been successfully cryopreserved and resuscitated. The majority of cells examined in both lines had the expected diploid chromosome number of 2 = 6. Transmission electron microscopy of CNE/LULS44 cells revealed the presence of large mitochondria within cells of a diverse population, while arrays of virus-like particles were not seen. CNE/LULS44 cells supported replication of a strain of BTV serotype 1, but not of a strain of serotype 26 which is not known to be insect-transmitted. These new cell lines will expand the scope of research on -borne pathogens.
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http://dx.doi.org/10.3390/microorganisms8060825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356041PMC
May 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

Bluetongue virus outer-capsid protein VP2 expressed in raises neutralising antibodies and a protective immune response in IFNAR mice.

Vaccine X 2019 Aug 22;2:100026. Epub 2019 Jun 22.

School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Leicestershire LE12 5RD, UK.

Bluetongue is a severe, economically important disease of ruminants that is widely distributed in tropical and temperate regions around the world. It is associated with major production losses, restrictions of animal movements and trade, as well as costs associated with developing and implementing effective surveillance and control measures. Mammalian hosts infected with bluetongue virus (BTV) generate a protective neutralising antibody response targeting the major BTV outer-capsid protein and serotype-specific antigen, VP2. BTV VP2 proteins that have been expressed in plants are soluble, with a native conformation displaying neutralising epitopes and can assemble with other BTV structural proteins to form virus-like particles (VLPs). His-tagged VP2 proteins of BTV serotypes 4 and 8 were transiently expressed in then purified by immobilised metal affinity chromatography (IMAC). Antisera from IFNAR mice prime/boost vaccinated with the purified proteins, were shown to contain VP2-specific antibodies by Indirect ELISA (I-ELISA), western blotting and serum neutralisation tests (SNT). Vaccinated mice, subsequently challenged with either the homologous or heterologous BTV serotype, developed viraemia by day 3 post-infection. However, no clinical signs were observed in mice challenged with the homologous serotype (either prime-boost or single-shot vaccinated), all of which survived for the duration of the study. In contrast, all of the vaccinated mice challenged with a heterologous serotype, died, showing no evidence of cross-protection or suppression of viraemia, as detected by real-time RT-qPCR or virus isolation. The induction of protective, serotype-specific neutralising antibodies in IFNAR mice, indicates potential for the use of plant-expressed BTV VP2s as subunit vaccine components, or as a basis for serotype-specific serological assays.
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http://dx.doi.org/10.1016/j.jvacx.2019.100026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6668234PMC
August 2019

A low-passage insect-cell isolate of bluetongue virus uses a macropinocytosis-like entry pathway to infect natural target cells derived from the bovine host.

J Gen Virol 2019 04 7;100(4):568-582. Epub 2019 Mar 7.

2​University of Surrey, Guildford, Surrey, GU2 7XH, UK.

Bluetongue virus (BTV) causes an economically important disease in domestic and wildlife ruminants and is transmitted by Culicoides biting midges. In ruminants, BTV has a wide cell tropism that includes endothelial cells of vascular and lymphatic vessels as important cell targets for virus replication, and several cell types of the immune system including monocytes, macrophages and dendritic cells. Thus, cell-entry represents a particular challenge for BTV as it infects many different cell types in widely diverse vertebrate and invertebrate hosts. Improved understanding of BTV cell-entry could lead to novel antiviral approaches that can block virus transmission from cell to cell between its invertebrate and vertebrate hosts. Here, we have investigated BTV cell-entry using endothelial cells derived from the natural bovine host (BFA cells) and purified whole virus particles of a low-passage, insect-cell isolate of a virulent strain of BTV-1. Our results show that the main entry pathway for infection of BFA cells is dependent on actin and dynamin, and shares certain characteristics with macropinocytosis. The ability to use a macropinocytosis-like entry route could explain the diverse cell tropism of BTV and contribute to the efficiency of transmission between vertebrate and invertebrate hosts.
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http://dx.doi.org/10.1099/jgv.0.001240DOI Listing
April 2019

Quantitative RT-PCR assays for identification and typing of the Equine encephalosis virus.

Braz J Microbiol 2019 Jan 10;50(1):287-296. Epub 2018 Dec 10.

The Pirbright Institute, Ash Road, Pirbright, GU24 0NF, UK.

Equine encephalosis (EE) is an acute, arthropod-borne, noncontagious, febrile disease of equids. The clinical signs of EE are similar to milder forms of African horse sickness (AHS) and the two diseases can be easily confused. The Equine encephalosis virus (EEV) is a distinct virus species within the genus Orbivirus, family Reoviridae, with ten linear segments of dsRNA genome. Seven distinct serotypes of EEV have been recognised on the basis of sequence analyses of Seg-2. The need for differential diagnosis of similar forms of EE and AHS warranted the development of molecular diagnostic methods for specific detection and identification of EEV. We report the development of quantitative real-time RT-PCR assay for detection of any member of the EEV species targeting the highly conserved EEV Seg-9. Similar serotype-specific qRT-PCR assays were designed for each of the seven EEV serotypes targeting genome Seg-2, encoding the serotype determining VP2 protein. These assays were evaluated using different EEV serotypes and other closely related orbiviruses. They were shown to be EEV virus species-specific, or EEV type-specific capable of detecting 1 to 13 copies of viral RNA in clinical samples. The assays failed to detect RNA from closely related orbiviruses, including AHSV and Peruvian horse sickness virus (PHSV) isolates.
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http://dx.doi.org/10.1007/s42770-018-0034-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6863193PMC
January 2019

How often do mosquitoes bite humans in southern England? A standardised summer trial at four sites reveals spatial, temporal and site-related variation in biting rates.

Parasit Vectors 2017 Sep 15;10(1):420. Epub 2017 Sep 15.

The Pirbright Institute, Ash Road, Woking, Surrey, UK.

Background: This field-based study examined the abundance and species complement of mosquitoes (Diptera: Culicidae) attracted to humans at four sites in the United Kingdom (UK). The study used a systematic approach to directly measure feeding by mosquitoes on humans at multiple sites and using multiple volunteers. Quantifying how frequently humans are bitten in the field by mosquitoes is a fundamental parameter in assessing arthropod-borne virus transmission.

Methods: Human landing catches were conducted using a standardised protocol by multiple volunteers at four rural sites between July and August 2013. Collections commenced two hours prior to sunset and lasted for a total of four hours. To reduce bias occurring due to collection point or to the individual attractiveness of the volunteer to mosquitoes, each collection was divided into eight collection periods, with volunteers rotated by randomised Latin square design between four sampling points per site. While the aim was to collect mosquitoes prior to feeding, the source of blood meals from any engorged specimens was also identified by DNA barcoding.

Results: Three of the four sites yielded human-biting mosquito populations for a total of 915 mosquitoes of fifteen species/species groups. Mosquito species composition and biting rates differed significantly between sites, with individual volunteers collecting between 0 and 89 mosquitoes (over 200 per hour) of up to six species per collection period. Coquillettidia richiardii (Ficalbi, 1889) was responsible for the highest recorded biting rates at any one site, reaching 161 bites per hour, whilst maximum biting rates of 55 bites per hour were recorded for Culex modestus (Ficalbi, 1889). Human-biting by Culex pipiens (L., 1758) form pipiens was also observed at two sites, but at much lower rates when compared to other species.

Conclusions: Several mosquito species are responsible for human nuisance biting pressure in southern England, although human exposure to biting may be largely limited to evening outdoor activities. This study indicates Cx. modestus can be a major human-biting species in the UK whilst Cx. pipiens f. pipiens may show greater opportunistic human-biting than indicated by earlier studies.
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http://dx.doi.org/10.1186/s13071-017-2360-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5602952PMC
September 2017

OIEBTLABNET: the web-based network of the OIE Bluetongue Reference Laboratories.

Vet Ital 2016 Sep;52(3-4):187-193

Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale' in Teramo, Italy.

Bluetongue (BT) is a mild to severe disease of domestic and wild ruminants caused by the Bluetongue virus (BTV) and generally transmitted by Culicoides biting midges. Its occurrence also determines a livestock trade ban in affected countries with severe economic consequences on national and international trade. For this reason, in May 2011, the OIE encouraged the OIE Reference Laboratories to establish and maintain a BT network to provide expertise and training to the OIE and OIE Member Countries for BT diagnosis, surveillance and control. The network is constantly sustained by world leading scientists in the field of virology, epidemiology, serology, entomology and vaccine development. The website, available at http://oiebtnet.izs.it/btlabnet/, hosts an Information System containing data on BTV outbreaks and strains and a WebGIS that distributes maps on BTV occurrence. In this paper we describe the applications and present the benefits derived from the use of the WebGIS in the context of BT international surveillance network.
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http://dx.doi.org/10.12834/VetIt.583.2799.1DOI Listing
September 2016

Development and Evaluation of Real Time RT-PCR Assays for Detection and Typing of Bluetongue Virus.

PLoS One 2016;11(9):e0163014. Epub 2016 Sep 23.

The Pirbright Institute, Pirbright, Surrey, United Kingdom.

Bluetongue virus is the type species of the genus Orbivirus, family Reoviridae. Bluetongue viruses (BTV) are transmitted between their vertebrate hosts primarily by biting midges (Culicoides spp.) in which they also replicate. Consequently BTV distribution is dependent on the activity, geographic distribution, and seasonal abundance of Culicoides spp. The virus can also be transmitted vertically in vertebrate hosts, and some strains/serotypes can be transmitted horizontally in the absence of insect vectors. The BTV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in order of decreasing size (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable BTV protein and the primary target for neutralising antibodies. Consequently VP2 (and Seg-2) determine the identity of the twenty seven serotypes and two additional putative BTV serotypes that have been recognised so far. Current BTV vaccines are serotype specific and typing of outbreak strains is required in order to deploy appropriate vaccines. We report development and evaluation of multiple 'TaqMan' fluorescence-probe based quantitative real-time type-specific RT-PCR assays targeting Seg-2 of the 27+1 BTV types. The assays were evaluated using orbivirus isolates from the 'Orbivirus Reference Collection' (ORC) held at The Pirbright Institute. The assays are BTV-type specific and can be used for rapid, sensitive and reliable detection / identification (typing) of BTV RNA from samples of infected blood, tissues, homogenised Culicoides, or tissue culture supernatants. None of the assays amplified cDNAs from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5035095PMC
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0163014PLOS
September 2016

Generation of virus like particles for epizootic hemorrhagic disease virus.

Res Vet Sci 2016 Aug 31;107:116-122. Epub 2016 May 31.

Dipartimento di Scienze Veterinarie, Università di Pisa, Italy.

Epizootic hemorrhagic disease virus (EHDV) is a distinct species within the genus Orbivirus, within the family Reoviridae. The epizootic hemorrhagic disease virus genome comprises ten segments of linear, double stranded (ds) RNA, which are packaged within each virus particle. The EHDV virion has a three layered capsid-structure, generated by four major viral proteins: VP2 and VP5 (outer capsid layer); VP7 (intermediate, core-surface layer) and VP3 (innermost, sub-core layer). Although EHDV infects cattle sporadically, several outbreaks have recently occurred in this species in five Mediterranean countries, indicating a potential threat to the European cattle industry. EHDV is transmitted by biting midges of the genus Culicoides, which can travel long distances through wind-born movements (particularly over water), increasing the potential for viral spread in new areas/countries. Expression systems to generate self-assembled virus like particles (VLPs) by simultaneous expression of the major capsid-proteins, have been established for several viruses (including bluetongue virus). This study has developed expression systems for production of EHDV VLPs, for use as non-infectious antigens in both vaccinology and serology studies, avoiding the risk of genetic reassortment between vaccine and field strains and facilitating large scale antigen production. Genes encoding the four major-capsid proteins of a field strain of EHDV-6, were isolated and cloned into transfer vectors, to generate two recombinant baculoviruses. The expression of these viral genes was assessed in insect cells by monitoring the presence of specific viral mRNAs and by western blotting. Electron microscopy studies confirmed the formation and purification of assembled VLPs.
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http://dx.doi.org/10.1016/j.rvsc.2016.05.013DOI Listing
August 2016

Using Combined Diagnostic Test Results to Hindcast Trends of Infection from Cross-Sectional Data.

PLoS Comput Biol 2016 07 6;12(7):e1004901. Epub 2016 Jul 6.

SRUC, Edinburgh, United Kingdom.

Infectious disease surveillance is key to limiting the consequences from infectious pathogens and maintaining animal and public health. Following the detection of a disease outbreak, a response in proportion to the severity of the outbreak is required. It is thus critical to obtain accurate information concerning the origin of the outbreak and its forward trajectory. However, there is often a lack of situational awareness that may lead to over- or under-reaction. There is a widening range of tests available for detecting pathogens, with typically different temporal characteristics, e.g. in terms of when peak test response occurs relative to time of exposure. We have developed a statistical framework that combines response level data from multiple diagnostic tests and is able to 'hindcast' (infer the historical trend of) an infectious disease epidemic. Assuming diagnostic test data from a cross-sectional sample of individuals infected with a pathogen during an outbreak, we use a Bayesian Markov Chain Monte Carlo (MCMC) approach to estimate time of exposure, and the overall epidemic trend in the population prior to the time of sampling. We evaluate the performance of this statistical framework on simulated data from epidemic trend curves and show that we can recover the parameter values of those trends. We also apply the framework to epidemic trend curves taken from two historical outbreaks: a bluetongue outbreak in cattle, and a whooping cough outbreak in humans. Together, these results show that hindcasting can estimate the time since infection for individuals and provide accurate estimates of epidemic trends, and can be used to distinguish whether an outbreak is increasing or past its peak. We conclude that if temporal characteristics of diagnostics are known, it is possible to recover epidemic trends of both human and animal pathogens from cross-sectional data collected at a single point in time.
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http://dx.doi.org/10.1371/journal.pcbi.1004901DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4934910PMC
July 2016

Using shared needles for subcutaneous inoculation can transmit bluetongue virus mechanically between ruminant hosts.

Sci Rep 2016 Feb 8;6:20627. Epub 2016 Feb 8.

The Pirbright Institute, Woking, Surrey, United Kingdom.

Bluetongue virus (BTV) is an economically important arbovirus of ruminants that is transmitted by Culicoides spp. biting midges. BTV infection of ruminants results in a high viraemia, suggesting that repeated sharing of needles between animals could result in its iatrogenic transmission. Studies defining the risk of iatrogenic transmission of blood-borne pathogens by less invasive routes, such as subcutaneous or intradermal inoculations are rare, even though the sharing of needles is common practice for these inoculation routes in the veterinary sector. Here we demonstrate that BTV can be transmitted by needle sharing during subcutaneous inoculation, despite the absence of visible blood contamination of the needles. The incubation period, measured from sharing of needles, to detection of BTV in the recipient sheep or cattle, was substantially longer than has previously been reported after experimental infection of ruminants by either direct inoculation of virus, or through blood feeding by infected Culicoides. Although such mechanical transmission is most likely rare under field condition, these results are likely to influence future advice given in relation to sharing needles during veterinary vaccination campaigns and will also be of interest for the public health sector considering the risk of pathogen transmission during subcutaneous inoculations with re-used needles.
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http://dx.doi.org/10.1038/srep20627DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4745043PMC
February 2016

Full-Genome Sequencing as a Basis for Molecular Epidemiology Studies of Bluetongue Virus in India.

PLoS One 2015 29;10(6):e0131257. Epub 2015 Jun 29.

Vector-borne Viral Diseases Programme, The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey, GU24 0NF, United Kingdom.

Since 1998 there have been significant changes in the global distribution of bluetongue virus (BTV). Ten previously exotic BTV serotypes have been detected in Europe, causing severe disease outbreaks in naïve ruminant populations. Previously exotic BTV serotypes were also identified in the USA, Israel, Australia and India. BTV is transmitted by biting midges (Culicoides spp.) and changes in the distribution of vector species, climate change, increased international travel and trade are thought to have contributed to these events. Thirteen BTV serotypes have been isolated in India since first reports of the disease in the country during 1964. Efficient methods for preparation of viral dsRNA and cDNA synthesis, have facilitated full-genome sequencing of BTV strains from the region. These studies introduce a new approach for BTV characterization, based on full-genome sequencing and phylogenetic analyses, facilitating the identification of BTV serotype, topotype and reassortant strains. Phylogenetic analyses show that most of the equivalent genome-segments of Indian BTV strains are closely related, clustering within a major eastern BTV 'topotype'. However, genome-segment 5 (Seg-5) encoding NS1, from multiple post 1982 Indian isolates, originated from a western BTV topotype. All ten genome-segments of BTV-2 isolates (IND2003/01, IND2003/02 and IND2003/03) are closely related (>99% identity) to a South African BTV-2 vaccine-strain (western topotype). Similarly BTV-10 isolates (IND2003/06; IND2005/04) show >99% identity in all genome segments, to the prototype BTV-10 (CA-8) strain from the USA. These data suggest repeated introductions of western BTV field and/or vaccine-strains into India, potentially linked to animal or vector-insect movements, or unauthorised use of 'live' South African or American BTV-vaccines in the country. The data presented will help improve nucleic acid based diagnostics for Indian serotypes/topotypes, as part of control strategies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0131257PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4488075PMC
March 2016

Genetic characterization of the tick-borne orbiviruses.

Viruses 2015 Apr 28;7(5):2185-209. Epub 2015 Apr 28.

Vector-borne Viral Diseases Programme, The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK.

The International Committee for Taxonomy of Viruses (ICTV) recognizes four species of tick-borne orbiviruses (TBOs): Chenuda virus, Chobar Gorge virus, Wad Medani virus and Great Island virus (genus Orbivirus, family Reoviridae). Nucleotide (nt) and amino acid (aa) sequence comparisons provide a basis for orbivirus detection and classification, however full genome sequence data were only available for the Great Island virus species. We report representative genome-sequences for the three other TBO species (virus isolates: Chenuda virus (CNUV); Chobar Gorge virus (CGV) and Wad Medani virus (WMV)). Phylogenetic comparisons show that TBOs cluster separately from insect-borne orbiviruses (IBOs). CNUV, CGV, WMV and GIV share low level aa/nt identities with other orbiviruses, in 'conserved' Pol, T2 and T13 proteins/genes, identifying them as four distinct virus-species. The TBO genome segment encoding cell attachment, outer capsid protein 1 (OC1), is approximately half the size of the equivalent segment from insect-borne orbiviruses, helping to explain why tick-borne orbiviruses have a ~1 kb smaller genome.
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http://dx.doi.org/10.3390/v7052185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4452902PMC
April 2015

Genome Sequence of Bluetongue Virus Type 2 from India: Evidence for Reassortment between Outer Capsid Protein Genes.

Genome Announc 2015 Apr 9;3(2). Epub 2015 Apr 9.

Vector-borne Diseases Programme, The Pirbright Institute, Pirbright, Woking, Surrey, United Kingdom

Southern Indian isolate IND1994/01 of bluetongue virus serotype 2 (BTV-2), from the Orbivirus Reference Collection at the Pirbright Institute (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-2.htm#IND1994/01), was sequenced. Its genome segment 6 (Seg-6) [encoding VP5(OCP2)] is identical to that of the Indian BTV-1 isolate (IND2003/05), while Seg-5 and Seg-9 are closely related to isolates from South Africa and the United States, respectively.
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http://dx.doi.org/10.1128/genomeA.00045-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4392135PMC
April 2015

A quantitative real-time reverse transcription PCR (qRT-PCR) assay to detect genome segment 9 of all 26 bluetongue virus serotypes.

J Virol Methods 2015 Mar 5;213:118-26. Epub 2014 Dec 5.

The Vector-Borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Woking, Surrey, United Kingdom. Electronic address:

Bluetongue (BT) is an arboviral disease, which can often be fatal in naïve sheep and white tailed deer, but is usually less severe, or unapparent in other ruminants. Twenty-six bluetongue virus (BTV) serotypes have been recognised so far, two of which (BTV-25 and BTV-26) were recently identified by phylogenetic comparisons of genome-segment/outer-capsid protein VP2 (subsequently confirmed by serological 'virus-neutralisation' assays). Rapid, sensitive, reliable and quantitative diagnostic-assays for detection and identification of BTV represent important components of effective surveillance and control strategies. The BTV genome comprises 10 linear segments of dsRNA. We describe a 'TaqMan' fluorescence-probe based quantitative real-time RT-PCR assay, targeting the highly conserved genome-segment-9 (encoding the viral-helicase 'VP6' and NS4). The assay detected Seg-9 from isolates of all 26 BTV types, as well as from clinical samples derived from BTV-6w and BTV-8w outbreaks (in Europe), BTV-25 from Switzerland, BTV-26 from Kuwait, BTV-1w, BTV-4w and BTV-8w from Spain, BTV-4w, BTV-8, BTV-10 and BTV-16 from Brazil. Assay efficiency was evaluated with RNA derived from the reference strain of BTV-1w [RSArrrr/01] and was 99.6%, detecting down to 4 copies per reaction. Samples from uninfected insect or mammalian cell-cultures, hosts-species (uninfected sheep blood) or vector-insects, all gave negative results. The assay failed to detect RNA from heterologous but related Orbivirus species (including the nine African horse sickness virus [AHSV] and seven epizootic haemorrhagic disease virus [EHDV] serotypes).
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http://dx.doi.org/10.1016/j.jviromet.2014.11.012DOI Listing
March 2015

Full genome characterization of the culicoides-borne marsupial orbiviruses: Wallal virus, Mudjinbarry virus and Warrego viruses.

PLoS One 2014 9;9(10):e108379. Epub 2014 Oct 9.

Vector-borne Viral Diseases Programme, Institute for Animal Health, Pirbright, Woking, Surrey, United Kingdom.

Viruses belonging to the species Wallal virus and Warrego virus of the genus Orbivirus were identified as causative agents of blindness in marsupials in Australia during 1994/5. Recent comparisons of nucleotide (nt) and amino acid (aa) sequences have provided a basis for the grouping and classification of orbivirus isolates. However, full-genome sequence data are not available for representatives of all Orbivirus species. We report full-genome sequence data for three additional orbiviruses: Wallal virus (WALV); Mudjinabarry virus (MUDV) and Warrego virus (WARV). Comparisons of conserved polymerase (Pol), sub-core-shell 'T2' and core-surface 'T13' proteins show that these viruses group with other Culicoides borne orbiviruses, clustering with Eubenangee virus (EUBV), another orbivirus infecting marsupials. WARV shares <70% aa identity in all three conserved proteins (Pol, T2 and T13) with other orbiviruses, consistent with its classification within a distinct Orbivirus species. Although WALV and MUDV share <72.86%/67.93% aa/nt identity with other orbiviruses in Pol, T2 and T13, they share >99%/90% aa/nt identities with each other (consistent with membership of the same virus species - Wallal virus). However, WALV and MUDV share <68% aa identity in their larger outer capsid protein VP2(OC1), consistent with membership of different serotypes within the species - WALV-1 and WALV-2 respectively.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108379PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4191977PMC
June 2015

Characterization of sheep pox virus vaccine for cattle against lumpy skin disease virus.

Antiviral Res 2014 Sep 25;109:1-6. Epub 2014 Jun 25.

The Pirbright Institute, Ash Road, Pirbright, Surrey GU24 0NF, UK.

Lumpy skin disease is of significant economic impact for the cattle industry in Africa. The disease is currently spreading aggressively in the Near East, posing a threat of incursion to Europe and Asia. Due to cross-protection within the Capripoxvirus genus, sheep pox virus (SPPV) vaccines have been widely used for cattle against lumpy skin disease virus (LSDV). In the Middle East and the Horn of Africa these vaccines have been associated with incomplete protection and adverse reactions in cattle post-vaccination. The present study confirms that the real identity of the commonly used Kenyan sheep and goat pox vaccine virus (KSGP) O-240 is not SPPV but is actually LSDV. The low level attenuation of this virus is likely to be not sufficient for safe use in cattle, causing clinical disease in vaccinated animals. In addition, Isiolo and Kedong goat pox strains, capable of infecting sheep, goats and cattle are identified for potential use as broad-spectrum vaccine candidates against all capripox diseases.
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http://dx.doi.org/10.1016/j.antiviral.2014.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4149609PMC
September 2014

Immunisation with bacterial expressed VP2 and VP5 of bluetongue virus (BTV) protect α/β interferon-receptor knock-out (IFNAR(-/-)) mice from homologous lethal challenge.

Vaccine 2014 Jul 2;32(32):4059-67. Epub 2014 Jun 2.

Vector-borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Woking, Surrey GU240NF, United Kingdom. Electronic address:

BTV-4 structural proteins VP2 (as two domains: VP2D1 and VP2D2), VP5 (lacking the first 100 amino acids: VP5Δ1-100) and full-length VP7, expressed in bacteria as soluble glutathione S-transferase (GST) fusion-proteins, were used to immunise Balb/c and α/β interferon receptor knock-out (IFNAR(-/-)) mice. Neutralising antibody (NAbs) titres (expressed as log10 of the reciprocal of the last dilution of mouse serum which reduced plaque number by ≥50%) induced by the VP2 domains ranged from 1.806 to 2.408 in Balb/c and IFNAR(-/-) mice. The immunised IFNAR(-/-) mice challenged with a homologous live BTV-4 survived and failed to develop signs of infection (ocular discharge and apathy). Although subsequent attempts to isolate virus were unsuccessful (possibly reflecting presence of neutralising antibodies), a transient/low level viraemia was detected by real time RT-PCR. In contrast, mice immunised with the two VP2 domains with or without VP5Δ1-100 and VP7, then challenged with the heterologous serotype, BTV-8, all died by day 7 post-infection. We conclude that immunisation with bacterially-expressed VP2 domains can induce strong serotype-specific NAb responses. Bacterial expression could represent a cost effective and risk-free alternative to the use of live or inactivated vaccines, particularly if viruses prove to be difficult to propagate in cell culture (like BTV-25). A vaccine based on bacterially expressed VP2 and VP5 of BTV is also DIVA-compatible.
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http://dx.doi.org/10.1016/j.vaccine.2014.05.056DOI Listing
July 2014

Real time RT-PCR assays for detection and typing of African horse sickness virus.

PLoS One 2014 10;9(4):e93758. Epub 2014 Apr 10.

Vector-borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Surrey, United Kingdom.

Although African horse sickness (AHS) can cause up to 95% mortality in horses, naïve animals can be protected by vaccination against the homologous AHSV serotype. Genome segment 2 (Seg-2) encodes outer capsid protein VP2, the most variable of the AHSV proteins. VP2 is also a primary target for AHSV specific neutralising antibodies, and consequently determines the identity of the nine AHSV serotypes. In contrast VP1 (the viral polymerase) and VP3 (the sub-core shell protein), encoded by Seg-1 and Seg-3 respectively, are highly conserved, representing virus species/orbivirus-serogroup-specific antigens. We report development and evaluation of real-time RT-PCR assays targeting AHSV Seg-1 or Seg-3, that can detect any AHSV type (virus species/serogroup-specific assays), as well as type-specific assays targeting Seg-2 of the nine AHSV serotypes. These assays were evaluated using isolates of different AHSV serotypes and other closely related orbiviruses, from the 'Orbivirus Reference Collection' (ORC) at The Pirbright Institute. The assays were shown to be AHSV virus-species-specific, or type-specific (as designed) and can be used for rapid, sensitive and reliable detection and identification (typing) of AHSV RNA in infected blood, tissue samples, homogenised Culicoides, or tissue culture supernatant. None of the assays amplified cDNAs from closely related heterologous orbiviruses, or from uninfected host animals or cell cultures.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0093758PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983086PMC
January 2015

Full-genome characterisation of Orungo, Lebombo and Changuinola viruses provides evidence for co-evolution of orbiviruses with their arthropod vectors.

PLoS One 2014 24;9(1):e86392. Epub 2014 Jan 24.

Department of Vector-Borne Viral Diseases, The Pirbright Institute, Pirbright, United Kingdom.

The complete genomes of Orungo virus (ORUV), Lebombo virus (LEBV) and Changuinola virus (CGLV) were sequenced, confirming that they each encode 11 distinct proteins (VP1-VP7 and NS1-NS4). Phylogenetic analyses of cell-attachment protein 'outer-capsid protein 1' (OC1), show that orbiviruses fall into three large groups, identified as: VP2(OC1), in which OC1 is the 2nd largest protein, including the Culicoides transmitted orbiviruses; VP3(OC1), which includes the mosquito transmitted orbiviruses; and VP4(OC1) which includes the tick transmitted viruses. Differences in the size of OC1 between these groups, places the T2 'subcore-shell protein' as the third largest protein 'VP3(T2)' in the first of these groups, but the second largest protein 'VP3(T2)' in the other two groups. ORUV, LEBV and CGLV all group with the Culicoides-borne VP2(OC1)/VP3(T2) viruses. The G+C content of the ORUV, LEBV and CGLV genomes is also similar to that of the Culicoides-borne, rather than the mosquito-borne, or tick borne orbiviruses. These data suggest that ORUV and LEBV are Culicoides- rather than mosquito-borne. Multiple isolations of CGLV from sand flies suggest that they are its primary vector. OC1 of the insect-borne orbiviruses is approximately twice the size of the equivalent protein of the tick borne viruses. Together with internal sequence similarities, this suggests its origin by duplication (concatermerisation) of a smaller OC1 from an ancestral tick-borne orbivirus. Phylogenetic comparisons showing linear relationships between the dates of evolutionary-separation of their vector species, and genetic-distances between tick-, mosquito- or Culicoides-borne virus-groups, provide evidence for co-evolution of the orbiviruses with their arthropod vectors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0086392PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3901712PMC
February 2015

Bluetongue virus infection induces aberrant mitosis in mammalian cells.

Virol J 2013 Oct 28;10:319. Epub 2013 Oct 28.

The Pirbright Institute, Pirbright, Woking GU24 0NF, UK.

Background: Bluetongue virus (BTV) is an arbovirus that is responsible for 'bluetongue', an economically important disease of livestock. Although BTV is well characterised at the protein level, less is known regarding its interaction with host cells. During studies of virus inclusion body formation we observed what appeared to be a large proportion of cells in mitosis. Although the modulation of the cell cycle is well established for many viruses, this was a novel observation for BTV. We therefore undertook a study to reveal in more depth the impact of BTV upon cell division.

Methods: We used a confocal microscopy approach to investigate the localisation of BTV proteins in a cellular context with their respective position relative to cellular proteins. In addition, to quantitatively assess the frequency of aberrant mitosis induction by the viral non-structural protein (NS) 2 we utilised live cell imaging to monitor HeLa-mCherry tubulin cells transfected with a plasmid expressing NS2.

Results: Our data showed that these 'aberrant mitoses' can be induced in multiple cell types and by different strains of BTV. Further study confirmed multiplication of the centrosomes, each resulting in a separate mitotic spindle during mitosis. Interestingly, the BTV NS1 protein was strongly localised to the centrosomal regions. In a separate, yet related observation, the BTV NS2 protein was co-localised with the condensed chromosomes to a region suggestive of the kinetochore. Live cell imaging revealed that expression of an EGFP-NS2 fusion protein in HeLa-mCherry tubulin cells also results in mitotic defects.

Conclusions: We hypothesise that NS2 is a microtubule cargo protein that may inadvertently disrupt the interaction of microtubule tips with the kinetochores during mitosis. Furthermore, the BTV NS1 protein was distinctly localised to a region encompassing the centrosome and may therefore be, at least in part, responsible for the disruption of the centrosome as observed in BTV infected mammalian cells.
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http://dx.doi.org/10.1186/1743-422X-10-319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3874736PMC
October 2013

Full genome sequence of a Western reference strain of bluetongue virus serotype 16 from Nigeria.

Genome Announc 2013 Sep 19;1(5). Epub 2013 Sep 19.

Vector-borne Diseases Programme, the Pirbright Institute, Woking, Surrey, United Kingdom.

The genome of NIG1982/10, a Nigerian bluetongue virus serotype 16 (BTV-16) strain, was sequenced (19,193 bp). Comparisons to BTV strains from other areas of the world show that all 10 genome segments of NIG1982/10 are derived from a western lineage (w), indicating that it represents a suitable reference strain of BTV-16w.
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http://dx.doi.org/10.1128/genomeA.00684-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3778194PMC
September 2013

Full genome sequencing of Corriparta virus, identifies California mosquito pool virus as a member of the Corriparta virus species.

PLoS One 2013 27;8(8):e70779. Epub 2013 Aug 27.

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

The species Corriparta virus (CORV), within the genus Orbivirus, family Reoviridae, currently contains six virus strains: corriparta virus MRM1 (CORV-MRM1); CS0109; V654; V370; Acado virus and Jacareacanga virus. However, lack of neutralization assays, or reference genome sequence data has prevented further analysis of their intra-serogroup/species relationships and identification of individual serotypes. We report whole-genome sequence data for CORV-MRM1, which was isolated in 1960 in Australia. Comparisons of the conserved, polymerase (VP1), sub-core-shell 'T2' and core-surface 'T13' proteins encoded by genome segments 1, 2 and 8 (Seg-1, Seg-2 and Seg-8) respectively, show that this virus groups with the other mosquito borne orbiviruses. However, highest levels of nt/aa sequence identity (75.9%/91.6% in Seg-2/T2: 77.6%/91.7% in Seg-8/T13, respectively) were detected between CORV-MRM1 and California mosquito pool virus (CMPV), an orbivirus isolated in the USA in 1974, showing that they belong to the same virus species. The data presented here identify CMPV as a member of the Corriparta virus species and will facilitate identification of additional CORV isolates, diagnostic assay design and epidemiological studies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0070779PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3754974PMC
May 2014

Complete genome sequence analysis of a reference strain of bluetongue virus serotype 16.

J Virol 2012 Sep;86(18):10255-6

Vector-borne Diseases Programme, Institute for Animal Health, Pirbright, Woking, Surrey, United Kingdom.

The entire genome of the reference strain of bluetongue virus (BTV) serotype 16 (strain RSArrrr/16) was sequenced (a total of 23,518 base pairs). The virus was obtained from the Orbivirus Reference Collection (ORC) at IAH, Pirbright, United Kingdom. The virus strain, which was previously provided by the Onderstepoort Veterinary Research Institute in South Africa, was originally isolated from the Indian subcontinent (Hazara, West Pakistan) in 1960. Previous phylogenetic comparisons show that BTV RNA sequences cluster according to the geographic origins of the virus isolate/lineage, identifying distinct BTV topotypes. Sequence comparisons of segments Seg-1 to Seg-10 show that RSArrrr/16 belongs to the major eastern topotype of BTV (BTV-16e) and can be regarded as a reference strain of BTV-16e for phylogenetic and molecular epidemiology studies. All 10 genome segments of RSArrrr/16 group closely with the vaccine strain of BTV-16 (RSAvvvv/16) that was derived from it, as well as those recently published for a Chinese isolate of BTV-16 (>99% nucleotide identity), suggesting a very recent common ancestry for all three viruses.
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http://dx.doi.org/10.1128/JVI.01672-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3446625PMC
September 2012

Isolates of Liao ning virus from wild-caught mosquitoes in the Xinjiang province of China in 2005.

PLoS One 2012 23;7(5):e37732. Epub 2012 May 23.

State Key Laboratory for Infectious Disease Prevention and Control, Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People's Republic of China.

Liao ning virus (LNV) is related to Banna virus, a known human-pathogen present in south-east Asia. Both viruses belong to the genus Seadornavirus, family Reoviridae. LNV causes lethal haemorrhage in experimentally infected mice. Twenty seven isolates of LNV were made from mosquitoes collected in different locations within the Xinjiang province of north-western China during 2005. These mosquitoes were caught in the accommodation of human patients with febrile manifestations, or in animal barns where sheep represent the main livestock species. The regions where LNV was isolated are affected by seasonal encephalitis, but are free of Japanese encephalitis (JE). Genome segment 10 (Seg-10) (encoding cell-attachment and serotype-determining protein VP10) and Seg-12 (encoding non-structural protein VP12) were sequenced for multiple LNV isolates. Phylogenetic analyses showed a less homogenous Seg-10 gene pool, as compared to segment 12. However, all of these isolates appear to belong to LNV type-1. These data suggest a relatively recent introduction of LNV into Xinjiang province, with substitution rates for LNV Seg-10 and Seg-12, respectively, of 2.29×10(-4) and 1.57×10(-4) substitutions/nt/year. These substitution rates are similar to those estimated for other dsRNA viruses. Our data indicate that the history of LNV is characterized by a lack of demographic fluctuations. However, a decline in the LNV population in the late 1980s-early 1990s, was indicated by data for both Seg-10 and Seg-12. Data also suggest a beginning of an expansion in the late 1990s as inferred from Seg-12 skyline plot.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0037732PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3359322PMC
October 2012

Genome sequence of a reassortant strain of bluetongue virus serotype 23 from western India.

J Virol 2012 Jun;86(12):7011-2

Vector-Borne Diseases Programme, Institute for Animal Health, Pirbright, Woking Surrey, United Kingdom.

The full genome sequence (19,177 bp) of an Indian strain (IND1988/02) of bluetongue virus (BTV) serotype 23 was determined. This virus was isolated from a sheep that had been killed during a severe bluetongue outbreak that occurred in Rahuri, Maharashtra State, western India, in 1988. Phylogenetic analyses of these data demonstrate that most of the genome segments from IND1988/02 belong to the major "eastern" BTV topotype. However, genome segment 5 belongs to the major "western" BTV topotype, demonstrating that IND1988/02 is a reassortant. This may help to explain the increased virulence that was seen during this outbreak in 1988. Genome segment 5 of IND1988/02 shows >99% sequence identity with some other BTV isolates from India (e.g., BTV-3 IND2003/08), providing further evidence of the existence and circulation of reassortant strains on the subcontinent.
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http://dx.doi.org/10.1128/JVI.00731-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3393570PMC
June 2012

The genome sequence of a reassortant bluetongue virus serotype 3 from India.

J Virol 2012 Jun;86(11):6375-6

Vector-borne Diseases Programme, Institute for Animal Health, Pirbright, Woking Surrey, United Kingdom.

All 10 genome segments (Seg-1 to 10-a total of 19,188 bp) were sequenced from a strain of bluetongue virus serotype 3 (BTV-3) from India (strain IND2003/08). Sequence comparisons showed that nine of the genome segments from this virus group with other eastern topotype strains. Genome Seg-2 and Seg-6 group with eastern BTV-3 strains from Japan. However, Seg-5 (the NS1 gene) from IND2003/08 belongs to a western lineage, demonstrating that IND2003/08 is a reassortant between eastern and western topotype bluetongue viruses. This confirms that western BTV strains have been imported and are circulating within the subcontinent.
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http://dx.doi.org/10.1128/JVI.00671-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3372206PMC
June 2012

The genome sequence of bluetongue virus type 10 from India: evidence for circulation of a western topotype vaccine strain.

J Virol 2012 May;86(10):5971-2

Vector-borne Diseases Programme, Institute for Animal Health, Pirbright, Surrey, United Kingdom.

Bluetongue virus is the type species of the genus Orbivirus in the family Reoviridae. We report the first complete genome sequence of an isolate (IND2004/01) of bluetongue virus serotype 10 (BTV-10) from Andhra Pradesh, India. This isolate, which is stored in the Orbivirus Reference Collection (ORC) at IAH Pirbright, shows >99% nucleotide identity in all 10 genome segments with a vaccine strain of BTV-10 from the United States.
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http://dx.doi.org/10.1128/JVI.00596-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3347277PMC
May 2012

The genome sequence of bluetongue virus type 2 from India: evidence for reassortment between eastern and western topotype field strains.

J Virol 2012 May;86(10):5967-8

Vector-borne Diseases Programme, Institute for Animal Health, Pirbright, Surrey, United Kingdom.

Bluetongue virus type 2, isolated in India in 1982 (IND1982/01), was obtained from the Orbivirus Reference Collection at IAH Pirbright (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-2.htm#IND1982/01). Full genome sequencing and phylogenetic analyses show that IND1982/01 is a reassortant virus containing genome segments derived from both eastern and western topotypes. These data will help to identify further reassortment events involving this or other virus lineages in the subcontinent.
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http://dx.doi.org/10.1128/JVI.00536-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3347281PMC
May 2012