Publications

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

There were a total of 2599 results for your search.
Johns H L, Bensaude E, La R S A, Seago J, Charleston B, Steinbach F, Drew T W, Crooke H, Everett H (2010)

Classical swine fever virus infection protects aortic endothelial cells from plpC-mediated apoptosis

Journal of General Virology 91 (4), 1038-1046

Abstract

Classical swine fever virus (CSFV) causes severe disease in pigs associated with leukopenia, haemorrhage and fever. We show that CSFV infection protects endothelial cells from apoptosis induced by the dsRNA mimic, pIpC, but not from other apoptotic stimuli, FasL or staurosporine. CSFV infection inhibits pIpC-induced caspase activation, mitochondrial membrane potential loss and cytochrome c release as well as the pro-apoptotic effects of truncated Bid (tBid) overexpression. The CSFV proteins Npro and Erns both contribute to CSFV inhibition of apoptosis. We conclude that CSFV infection can inhibit apoptotic signalling at multiple levels, including at the caspase-8 and the mitochondrial checkpoints. By supporting viral replication, endothelial cells may promote CSFV pathogenesis.

Abstract

The positive-stranded RNA genome of classical swine fever virus (CSFV) encodes 12 known proteins. The first protein to be translated is the N-terminal protease (Npro). Npro helps evade the innate interferon response by targeting interferon regulatory factor-3 for proteasomal degradation and also participates in the evasion of dsRNA-induced apoptosis. To elucidate the mechanisms by which Npro functions, we performed a yeast two-hybrid screen in which the anti-apoptotic protein HAX-1 was identified. The Npro–HAX-1 interaction was confirmed using co-precipitation assays. A dramatic redistribution of both Npro and HAX-1 was observed in co-transfected cells, as well as in transfected cells infected with wild-type CSFV, but not in cells infected with an Npro-deleted CSFV strain.

Abstract

In order to develop novel solutions to avian disease problems, including novel vaccines and/or vaccine adjuvants, and the identification of disease resistance genes which can feed into conventional breeding programmes, it is necessary to gain a more thorough understanding of the avian immune response and how pathogens can subvert that response. Birds occupy the same habitats as mammals, have similar ranges of longevity and body mass, and face similar pathogen challenges, yet birds have a different repertoire of organs, cells, molecules and genes of the immune system compared to mammals. This review summarises the current state of knowledge of the chicken's immune response, highlighting differences in the bird compared to mammals, and discusses how the availability of the chicken genome sequence and the associated postgenomics technologies are contributing to theses studies and also to the development of novel intervention strategies againts avian and zoonotic disease.
Kedmi M, Herziger Y, Galon N, Cohen R M, Perel M, Batten C, Braverman Y, Gottlieb Y, Shpigel N, Klement E (2010)

The association of winds with the spread of EHDV in dairy cattle in Israel during an outbreak in 2006

Preventive Veterinary Medicine 96 (3-4), 152-160

Abstract

Winds may play a major role in spread of arthropod-borne viruses (arboviruses). Arboviruses like epizootic hemorrhagic disease virus (EHDV), bluetongue virus and bovine ephemeral fever virus (BEFV) frequently cause major outbreaks in Israel with a unique pattern of spread. Most of these outbreaks begin in the Jordan valley, near the Sea of Galilee and then spread to the north, south and west through the major valleys of Israel. The aim of this study was to describe the spread pattern in such an outbreak and to find if this pattern can be explained by winds. Herein, we compared the spread rate to each direction and used Cox proportional hazards model to test factors associated with the spread of EHDV, which emerged in diary cattle in Israel during the summer of 2006. Documented, clinical and serological data on spread of the outbreak were then compared with wind data collected by meteorological stations along the trail of virus spread and with modeled winds at high altitude (>500 m). The analysis revealed that both the hazard and the rate of outbreak spread to the south and to the north were significantly higher than to the west. Average rate of outbreak spread during periods in which at least 3 h of winds to spread direction were recorded was 20,880 m/week (SD = 13,230) vs. 7486 m/week (SD = 4936) in periods during which no such winds were recorded. Serological evidence demonstrated exposure to the virus up to 166 km away from the location of the initial outbreak center. Modeled wind data showed that this spread may be explained by winds at high altitudes. Animal movements due to shipments of feedlot calves and slaughters could not explain the spread pattern observed during the outbreak. This study therefore shows that winds are probably a major contributory factor for long and medium distance spread of Culicoides borne viruses in this region.

Abstract

Rapid and accurate diagnostic tests make an important contribution to programmes to monitor and eradicate infectious diseases that impact animal and plant health. Using foot-and-mouth disease (FMD) and sudden oak death as examples, this review outlines recent progress to develop new field tools for detection of the infectious agents that cause high-impact livestock and plant diseases. The principal driver for this work is to develop tools that can be used locally to assist in decision making. Advances in this area have developed simple-to-use lateral-flow devices for the detection of FMD virus and the genus Phytophthora (including Phytophthora ramorum, the causal agent of sudden oak death and the related pathogen P. kernoviae), as well as new hardware platforms to allow PCR testing for these agents by non-specialists in the field. Although developed for different diseases, the user requirements for rapid diagnostic tools for FMD and sudden oak death share many similarities. Using generic solutions to these challenging problems, it is now possible to imagine a new paradigm for how the collection and testing of samples to monitor the spread of important livestock and plant diseases might be achieved.

Abstract

Tumour necrosis factor alpha (TNF-?) is an innate pro-inflammatory cytokine involved in protection against intracellular pathogens. Existing methods for measuring TNF-? production and function in ruminants are limited to ELISA and many rely on polyclonal antisera. With a view to developing improved detection methods for bovine (bov) TNF-?, monoclonal antibodies (mAb) were produced by immunising mice with a plasmid encoding bov TNF-?. Two of the resulting mAb, termed CC327 and CC328, were used to develop a sandwich ELISA capable of detecting both native and recombinant bov TNF-?. This ELISA did not detect recombinant ovine (ov) TNF-?. A luminometric method was applied to the ELISA to improve sensitivity for detection of native bov TNF-? in culture supernatants derived from bovine monocyte-derived dendritic cells (DC) infected with Mycobacterium bovis. Both CC327 and CC328 detected intracytoplasmic expression of TNF-? in mitogen-activated bovine T lymphocytes. However, only CC328 detected intracytoplasmic ovine TNF-? in transfected cells, explaining the failure of the sandwich ELISA to detect recombinant ov TNF-?. These mAbs have generated the capability to study the role of TNF-? in host immune protection and disease pathogenesis in ruminants.

Abstract

Background: The Asian tiger mosquito, Aedes albopictus (Skuse), is a vector of several arboviruses including dengue and chikungunya. This highly invasive species originating from Southeast Asia has travelled the world in the last 30 years and is now established in Europe, North and South America, Africa, the Middle East and the Caribbean. In the absence of vaccine or antiviral drugs, efficient mosquito control strategies are crucial. Conventional control methods have so far failed to control Ae. albopictus adequately. Methodology/Principal Findings: Germline transformation of Aedes albopictus was achieved by micro-injection of embryos with a piggyBac-based transgene carrying a 3xP3-ECFP marker and an attP site, combined with piggyBac transposase mRNA and piggyBac helper plasmid. Five independent transgenic lines were established, corresponding to an estimated transformation efficiency of 2-3%. Three lines were re-injected with a second-phase plasmid carrying an attB site and a 3xP3-DsRed2 marker, combined with PhiC31 integrase mRNA. Successful site-specific integration was observed in all three lines with an estimated transformation efficiency of 2-6%. Conclusions/Significance: Both piggybac-and site-specific PhiC31-mediated germline transformation of Aedes albopictus were successfully achieved. This is the first report of Ae. albopictus germline transformation and engineering, a key step towards studying and controlling this species using novel molecular techniques and genetic control strategies.
Lallinger G, Zweygarth E, Bell-Sakyi L, Passos L M F (2010)

Cold storage and cryopreservation of tick cell lines

Parasites and Vectors 3, 5

Abstract

Background: Tick cell lines are now available from fifteen ixodid and argasid species of medical and veterinary importance. However, some tick cell lines can be difficult to cryopreserve, and improved protocols for short- and long-term low temperature storage will greatly enhance their use as tools in tick and tick-borne pathogen research. In the present study, different protocols were evaluated for cold storage and cryopreservation of tick cell lines derived from Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, Ixodes ricinus and Ixodes scapularis. For short-term cold storage, cells were kept under refrigeration at 6 C for 15, 30 and 45 days. For cryopreservation in liquid nitrogen, use of a sucrose-phosphate-glutamate freezing buffer (SPG) as cryoprotectant was compared with dimethylsulfoxide (DMSO) supplemented with sucrose. Cell viability was determined by the trypan blue exclusion test and cell morphology was evaluated in Giemsa-stained cytocentrifuge smears. Results: Cold storage at 6 degrees C for up to 30 days was successful in preserving R. (B.) microplus, R. (B.) decoloratus, I. ricinus and I. scapularis cell lines; lines from the latter three species could be easily re-cultivated after 45 days under refrigeration. While cell lines from all four tick species cryopreserved with 6% DMSO were successfully resuscitated, the R. (B.) decoloratus cells did not survive freezing in SPG and of the other three species, only the R. (B.) microplus cells resumed growth during the observation period. Conclusions: This constitutes the first report on successful short-term refrigeration of cells derived from R. (B.) decoloratus, R. (B.) microplus, and I. ricinus, and use of SPG as an alternative to DMSO for cryopreservation, thus making an important contribution to more reliable and convenient tick cell culture maintenance.

Abstract

Background: Combinatorial RNA interference (co-RNAi) is a valuable tool for highly effective gene suppression of single and multiple-genes targets, and can be used to prevent the escape of mutation-prone transcripts. There are currently three main approaches used to achieve co-RNAi in animal cells; multiple promoter/shRNA cassettes, long hairpin RNAs (lhRNA) and miRNA-embedded shRNAs, however, the relative effectiveness of each is not known. The current study directly compares the ability of each co-RNAi method to deliver pre-validated siRNA molecules to the same gene targets. Results: Double-shRNA expression vectors were generated for each co-RNAi platform and their ability to suppress both single and double-gene reporter targets were compared. The most reliable and effective gene silencing was achieved from the multiple promoter/shRNA approach, as this method induced additive suppression of single-gene targets and equally effective knockdown of double-gene targets. Although both lhRNA and microRNA-embedded strategies provided efficient gene knockdown, suppression levels were inconsistent and activity varied greatly for different siRNAs tested. Furthermore, it appeared that not only the position of siRNAs within these multi-shRNA constructs impacted upon silencing activity, but also local properties of each individual molecule. In addition, it was also found that the insertion of up to five promoter/shRNA cassettes into a single construct did not negatively affect the efficacy of each individual shRNA. Conclusions: By directly comparing the ability of shRNAs delivered from different co-RNA platforms to initiate knockdown of the same gene targets, we found that multiple U6/shRNA cassettes offered the most reliable and predictable suppression of both single and multiple-gene targets. These results highlight some important strengths and pitfalls of the currently used methods for multiple shRNA delivery, and provide valuable insights for the design and application of reliable co-RNAi
Lamien C E, Lelenta M, Silber R, Goff C l, Wallace D, Gulyaz V, Tuppurainen E, Luckins A G, Albina E, Diallo A (2010)

Phylogenetic analysis of the capripoxvirus RPO30 gene and its use in a PCR test for differentiating sheep poxvirus from goat poxvirus

Sustainable improvement of animal production and health. FAO/IAEA International Symposium on Sustainable Improvement of Animal Production and Health, Vienna, Austria, 8-11 June 2009., 323-326
Publisher’s version:

Abstract

The Genus Capripoxvirus (CaPV) of the Poxviridae family comprises sheep poxvirus (SPPV), goat poxvirus (GTPV) and lumpy skin disease virus (LSDV) which are responsible for economically important diseases affecting sheep, goats and cattle respectively. To date, there have been no molecular criteria upon which to base strain designation. The complexity of CaPVs host specificity shows the need to develop more reliable tools for CaPVs identification than the current method which is based on the host origin. Previous reports, based on partial or full genome sequencing indicated that CaP viruses are genetically distinct from each other and can be grouped as three different species: SPPV, GTPV and LSDV. In contributing to the creation of more stringent data for genotyping CaPVs, we have analysed the RPO30 gene of several isolates. The phylogenetic reconstructions have shown that the viruses can be segregated into three different lineages according to their host origins: the SPPV, the GTPV and the LSDV lineages. In addition, a 21-nucleotides deletion found in all individuals within only the SPPV group was exploited to design a classical PCR method to differentiate SPPV from GTPV. This test allows the rapid differential diagnosis of diseases caused by either SPPV or GTPV strains.

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