Publications

The Pirbright Institute publication directory contains details of selected publications written by our researchers.

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Abstract

Epizootic Haemorrhagic Disease virus serotype 6 (EHDV-6) has recently caused serious outbreaks of Epizootic Haemorrhagic Disease (EHD) on the edges of Europe, in Turkey, Israel and Morocco. The aim of this study was to assess the pathogenicity and infection kinetics of EHD in Holstein-Friesian cattle infected with the two distinct strains of EHDV-6 isolated from the recent Turkish and Moroccan outbreaks. Samples taken throughout the study were used to validate two recently developed diagnostic assays that detect EHDV antibodies and viral genome. Two groups of five Holstein-Friesian cattle were experimentally infected with either the Moroccan or the Turkish isolate of EHDV-6. Cattle in both groups remained clinically unaffected throughout the study, but displayed high levels of viral RNA and virus in their blood, confirming that sub-clinical infection of cattle is likely to play an important role in EHDV transmission. A recently developed and commercialised real-time RT-PCR assay detected viral RNA as early as 2 days post infection (dpi) in both infection studies and viral RNA persisted for the course of the study. Antibodies against EHDV were first detected by 9 dpi using a recently developed EHDV blocking ELISA and antibodies persisted up to the end of the study. All animals developed high levels of neutralising antibodies to EHDV-6, measured by a serum neutralisation test (SNT), with titres (log 10) ranging from 2.20 to 2.38 at the end of the study. Virus was isolated from the blood of infected animals from as early as 2 dpi up to 28 dpi.
Batten C A, Harif B, Henstock M R, Ghizlane S, Edwards L, Loutfi C, Oura C A, El H M (2011)

Experimental infection of camels with bluetongue virus

Research in Veterinary Science 90 (3), 533-535

Abstract

Three camels aged 4-5 years were experimentally infected with Bluetongue virus serotype 1 (BTV-1) and were observed for 75 days. No clinical signs of disease were observed throughout the experiment, however all three animals seroconverted and developed BTV-1 specific neutralising antibodies after challenge. All three camels developed a viraemia from 7 days post infection albeit at a lower level than that usually observed in experimental infections of sheep and cattle. Virus was isolated from the blood of all three animals suggesting that camels may act as a reservoir for BTV and play an important role in its transmission.

Abstract

Development of small footprint, disposable, fast, and inexpensive devices for pathogen detection in the field and clinic would benefit human and veterinary medicine by allowing evidence-based responses to future out breaks. We designed and tested an integrated nucleic acid extraction and amplification device employing a loop-mediated isothermal amplification (LAMP) or reverse transcriptase-LAMP assay. Our system provides a screening tool with polymerase-chain-reaction-level sensitivity and specificity for outbreak detection, response, and recovery. Time to result is similar to 90 min. The device utilizes a swab that collects sample and then transfers it to a disc of cellulose-based nucleic acid binding paper. The disc is positioned within a disposable containment tube with a manual loading port. In order to test for the presence of target pathogens, LAMP reagents are loaded through the tube's port into contact with the sample containing cellulose disc. The reagents then are isothermally heated to 63 degrees C for similar to 1 h to achieve sequence-specific target nucleic acid amplification. Due to the presence of a colorimetric dye, amplification induces visible color change in the reagents from purple to blue. As initial demonstrations, we detected methicillin resistant Staphylococcus aureus genomic DNA, as well as recombinant and live foot-and-mouth disease virus.

Abstract

The genus Orbivirus, family Reoviridae, includes 22 species of viruses with genomes composed of ten segments of linear dsRNA that are transmitted between their vertebrate hosts by insects or ticks, or with no identified vectors. Full-genome sequence data are available for representative isolates of the insect borne mammalian orbiviruses (including bluetongue virus), as well as a tick borne avian orbivirus (Great Island virus). However, no sequence data are as yet available for the mosquito borne avian orbiviruses. We report full-length, whole-genome sequence data for Umatilla virus (UMAV), a mosquito borne avian orbivirus from the USA, which belongs to the species Umatilla virus. Comparisons of conserved genome segments 1, 2 and 8 (Seg-1, Seg-2 and Seg-8) - encoding the polymerase-VP1, sub-core 'T2' protein and core-surface 'T13' protein, respectively, show that UMAV groups with the mosquito transmitted mammalian orbiviruses. The highest levels of sequence identity were detected between UMAV and Stretch Lagoon orbivirus (SLOV) from Australia, showing that they belong to the same virus species (with nt/aa identity of 76.04%/88.07% and 77.96%/95.36% in the polymerase and T2 genes and protein, respectively). The data presented here has assisted in identifying the SLOV as a member of the Umatilla serogroup. This sequence data reported here will also facilitate identification of new isolates, and epidemiological studies of viruses belonging to the species Umatilla virus.
Belhouchet M, Mohd Jaafar F, Firth A E, Grimes J M, Mertens P P C, Attoui H (2011)

Detection of a fourth orbivirus non-structural protein

PLoS One 6, e10

Abstract

The genus Orbivirus includes both insect and tick-borne viruses. The orbivirus genome, composed of 10 segments of dsRNA, encodes 7 structural proteins (VP1-VP7) and 3 non-structural proteins (NS1-NS3). An open reading frame (ORF) that spans almost the entire length of genome segment-9 (Seg-9) encodes VP6 (the viral helicase). However, bioinformatic analysis recently identified an overlapping ORF (ORFX) in Seg-9. We show that ORFX encodes a new non-structural protein, identified here as NS4. Western blotting and confocal fluorescence microscopy, using antibodies raised against recombinant NS4 from Bluetongue virus (BTV, which is insect-borne), or Great Island virus (GIV, which is tick-borne), demonstrate that these proteins are synthesised in BTV or GIV infected mammalian cells, respectively. BTV NS4 is also expressed in Culicoides insect cells. NS4 forms aggregates throughout the cytoplasm as well as in the nucleus, consistent with identification of nuclear localisation signals within the NS4 sequence. Bioinformatic analyses indicate that NS4 contains coiled-coils, is related to proteins that bind nucleic acids, or are associated with membranes and shows similarities to nucleolar protein UTP20 (a processome subunit). Recombinant NS4 of GIV protects dsRNA from degradation by endoribonucleases of the RNAse III family, indicating that it interacts with dsRNA. However, BTV NS4, which is only half the putative size of the GIV NS4, did not protect dsRNA from RNAse III cleavage. NS4 of both GIV and BTV protect DNA from degradation by DNAse. NS4 was found to associate with lipid droplets in cells infected with BTV or GIV or transfected with a plasmid expressing NS4.
Blake D P, Billington K J, Copestake S L, Oakes R D, Quail M A, Wan K L, Shirley M W, Smith A L (2011)

Genetic mapping identifies novel highly protective antigens for an apicomplexan parasite

PLoS Pathogens 7 (2), e1001279

Abstract

Apicomplexan parasites are responsible for a myriad of diseases in humans and livestock; yet despite intensive effort, development of effective sub-unit vaccines remains a long-term goal. Antigenic complexity and our inability to identify protective antigens from the pool that induce response are serious challenges in the development of new vaccines. Using a combination of parasite genetics and selective barriers with population-based genetic fingerprinting, we have identified that immunity against the most important apicomplexan parasite of livestock (Eimeria spp.) was targeted against a few discrete regions of the genome. Herein we report the identification of six genomic regions and, within two of those loci, the identification of true protective antigens that confer immunity as sub-unit vaccines. The first of these is an Eimeria maxima homologue of apical membrane antigen-1 (AMA-1) and the second is a previously uncharacterised gene that we have termed 'immune mapped protein-1' (IMP-1). Significantly, homologues of the AMA-1 antigen are protective with a range of apicomplexan parasites including Plasmodium spp., which suggest that there may be some characteristic(s) of protective antigens shared across this diverse group of parasites. Interestingly, homologues of the IMP-1 antigen, which is protective against E. maxima infection, can be identified in Toxoplasma gondii and Neospora caninum. Overall, this study documents the discovery of novel protective antigens using a population-based genetic mapping approach allied with a protection-based screen of candidate genes. The identification of AMA-1 and IMP-1 represents a substantial step towards development of an effective anti-eimerian sub-unit vaccine and raises the possibility of identification of novel antigens for other apicomplexan parasites. Moreover, validation of the parasite genetics approach to identify effective antigens supports its adoption in other parasite systems where legitimate protective antigen identification is difficult.

Abstract

Eimeria maxima is one of the seven Eimeria spp. that infect the chicken and cause the disease coccidiosis. The well characterised immunogenicity and genetic diversity associated with E. maxima promote its use in genetics-led studies on avian coccidiosis. The development of a genetic map for E. maxima, presented here based upon 647 amplified fragment length polymorphism markers typed from 22 clonal hybrid lines and assembled into 13 major linkage groups, is a major new resource for work with this parasite. Comparison with genetic maps produced for other coccidial parasites indicates relatively high levels of genetic recombination. Conversion of 14% of the markers representing the major linkage groups to sequence characterised amplified region markers can provide a scaffold for the assembly of future genomic sequences as well as providing a foundation for more detailed genetic maps. Comparison with the Eimeria tenella genetic map produced 10 years ago has revealed a less biased marker distribution, with no more than nine markers mapped within any unresolved heritable unit. Nonetheless, preliminary bioinformatic characterisation of the three largest publicly available genomic E. maxima sequences suggest that the feature-poor/feature-rich structure which has previously been found to define the first sequenced E. tenella chromosome also defines the E. maxima genome. The significance of such a segmented genome and the apparent potential for variation in genetic recombination will be relevant to haplotype stability and the longevity of future anticoccidial strategies based upon multiple loci targeted by novel chemotherapeutic drugs or recombinant subunit vaccines.

Abstract

African swine fever (ASF) is an important disease of pigs and outbreaks of ASF have occurred in Europe on multiple occasions. To explore the period for which the European soft tick species Ornithodoros erraticus (Acari: Argasidae) is able to act as a reservoir of African swine fever virus (ASFV) after infected hosts are removed, we collected specimens from farms in the provinces of Alentejo and Algarve in Portugal during the endemic period and tested them subsequently using cell culture and experimental infection. We show that ticks from previously infected farms may contain infectious virus for at least five years and three months after the removal of infectious hosts. Furthermore, in two cases infectious virus was successfully isolated from ticks on restocked farms that had not yet suffered a re-emergence of disease. Experimental transmission to pigs was demonstrated in batches tested up to 380 days after an outbreak. These results clarify the epidemiological role of O. erraticus ticks in the persistence of ASFV in the field, provide additional evidence to support its role in the re-emergence of a sporadic outbreak of ASF in Portugal in 1999 and suggest that the current quarantine legislation and restocking advice when these ticks are present on the pig farm premises is appropriate.

Abstract

CD4+ T cells expressing the latent form of transforming growth factor-beta [latency-associated peptide (LAP) (TGF-beta(1))] play an important role in the modulation of immune responses. Here, we identified a novel peptide ligand (GPC(81-95)) with an intrinsic ability to induce membrane-bound LAP (TGF-beta(1)) expression on a subpopulation of human CD4+ T cells (using flow cytometry; ranging from 0 center dot 8% to 2 center dot 6%) and stimulate peripheral blood mononuclear cells to release LAP (TGF-beta(1)) (using ELISPOT assay; ranging from 0 center dot 03% to 0 center dot 16%). In spite of this low percentage of responding cells, GPC(81-95) significantly reduced Toll-like receptor 4 ligand-induced tumour necrosis factor-alpha production in a TGF-beta(1)- and CD4+ T-cell-dependent manner. The results demonstrate that GPC(81-95) is a useful tool to study the functional properties of a subpopulation of LAP (TGF-beta(1))+ CD4+ T cells and suggest a pathway that can be exploited to suppress inflammatory response.

Abstract

Chimeric foot-and-mouth disease viruses (FMDVs) have been generated from plasmids containing full-length FMDV cDNAs and characterized. The parental virus cDNA was derived from the cell-culture-adapted O1Kaufbeuren B64 (O1K B64) strain. Chimeric viruses, containing capsid coding sequences derived from the O/UKG/34/2001 or A/Turkey 2/2006 field viruses, were constructed using the backbone from the O1K B64 cDNA, and viable viruses (O1K/O-UKG and O1K/A-Tur, respectively) were successfully rescued in each case. These viruses grew well in primary bovine thyroid cells but grew less efficiently in BHK cells than the rescued parental O1K B64 virus. The two chimeric viruses displayed the expected antigenicity in serotype-specific antigen ELISAs. Following inoculation of each virus into cattle, the rescued O1K B64 strain proved to be attenuated whereas, with each chimeric virus, typical clinical signs of foot-and-mouth disease were observed, which then spread to in-contact animals. Thus, the surface-exposed capsid proteins of the O1K B64 strain are responsible for its attenuation in cattle. Consequently, there is no evidence for any adaptation, acquired during cell culture, outside the capsid coding region within the O1K B64 strain that inhibits replication in cattle. These chimeric infectious cDNA plasmids provide a basis for the analysis of FMDV pathogenicity and characterization of receptor utilization in vivo.

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