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.

Abstract

The African swine fever virus (ASFV) DP71L protein is present in all isolates as either a short form of 70 to 72 amino acids or a long form of about 184 amino acids, and both of these share sequence similarity to the C-terminal domain of the herpes simplex virus ICP34.5 protein and cellular protein GADD34. In the present study we expressed DP71L in different mammalian cells and demonstrated that DP71L causes dephosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2?) in resting cells and during chemical-induced endoplasmic reticulum stress and acts to enhance expression of cotransfected reporter genes. We showed that DP71L binds to all the three isoforms (?, ?, and ?) of the protein phosphatase 1 catalytic subunit (PP1c) and acts by recruiting PP1c to eIF2?. We also showed that DP71L inhibits the induction of ATF4 and its downstream target, CHOP. We investigated the eIF2? phosphorylation status and induction of CHOP in porcine macrophages infected by two ASFV field isolates, Malawi Lil20/1 and Benin 97/1, and two DP71L deletion mutants, Malawi?NL and E70?NL. Our results showed that deletion of the DP71L gene did not cause an increase in the level of eIF2? phosphorylation or induction of CHOP, indicating that DP71L is not the only factor required by the virus to control the phosphorylation level of eIF2? during infection. We therefore hypothesize that ASFV has other mechanisms to prevent the eIF2? phosphorylation and the subsequent protein synthesis inhibition.

Abstract

A quarantine period for potentially contaminated personnel can be used to reduce the risk of transfer of foot-and-mouth disease virus (FMDV) from infected to susceptible premises. This is set at 72 hours in the UK, on the basis of results from laboratory studies and field observations. Previous analysis of FMDV carriage within human nasal cavities has relied upon virus isolation by culture in susceptible cells. This study, involving 51 people, evaluated a PCR method, which detected viral genomic material within 35 nasal swabs taken from personnel after up to eight hours exposure to infected animals. Only one of 23 people who was PCR-positive immediately after exposure to FMDV-infected animals remained positive the following day, indicating a low risk of prolonged carriage of virus in the nasal cavities.

Abstract

Background: Immunization of BALB/c mice with a recombinant adenovirus expressing Mycobacterium tuberculosis (M. tuberculosis) antigen 85A (Ad85A) protects against aerosol challenge with M. tuberculosis only when it is administered intra-nasally (i.n.). Immunization with Ad85A induces a lung-resident population of activated CD8 T cells that is antigen dependent, highly activated and mediates protection by early inhibition of M. tuberculosis growth. In order to determine why the i.n. route is so effective compared to parenteral immunization, we used microarray analysis to compare gene expression profiles of pulmonary and splenic CD8 T cells after i.n. or intradermal (i.d.) immunization. Method: Total RNA from CD8 T cells was isolated from lungs or spleens of mice immunized with Ad85A by the i.n. or i.d. route. The gene profiles generated from each condition were compared. Statistically significant (p

Abstract

Streptococcus uberis, strain 0140J, contains a single copy sortase A (srtA), encoding a transamidase capable of covalently anchoring specific proteins to peptidoglycan. Unlike the wild-type, an isogenic mutant carrying an inactivating ISS1 insertion within srtA was only able to infect the bovine mammary gland in a transient fashion. For the first 24 h post challenge, the srtA mutant colonised at a similar rate and number to the wild type strain, but unlike the wild type did not subsequently colonise in higher numbers. Similar levels of host cell infiltration were detected in response to infection with both strains, but only in those mammary quarters infected with the wild type strain were clinical signs of disease evident. Mutants that failed to express individual sortase substrate proteins (sub0135, sub0145, sub0207, sub0241, sub0826, sub0888, sub1095, sub1154, sub1370, and sub1730) were isolated and their virulence determined in the same challenge model. This revealed that mutants lacking sub0145, sub1095 and sub1154 were attenuated in cattle. These data demonstrate that a number of sortase anchored proteins each play a distinct, non-redundant and important role in pathogenesis of S. uberis infection within the lactating bovine mammary gland.

Abstract

Limitations with current chemotherapeutic and vaccinal control of coccidiosis caused by Eimeria species continue to prompt development of novel controls, including the identification of new drug targets. Glucose-6-phosphate isomerase (G6-PI) has been proposed as a valid drug target for many protozoa, although polymorphism revealed by electrophoretic enzyme mobility has raised doubts for Eimeria. In this study we identified and sequenced the Eimeria tenella G6-PI orthologue (EtG6-PI) from the reference Houghton strain and confirmed its position within the prevailing taxonomic hierarchy, branching with the Apicomplexa and Plantae, distinct from the Animalia including the host, Gallus gallus. Comparison of the deduced 1647 bp EtG6-PI coding sequence with the 9016 bp genomic locus revealed 15 exons, all of which obey the intron-AG-/exon/-GT-intron splicing rule. Comparison with the Weybridge and Wisconsin strains revealed the presence of 33 single nucleotide polymorphisms (SNPs) and 14 insertion/deletion sites. Three SNPs were exonic and all yielded non-synonymous substitutions. Preliminary structural predictions suggest little association between the coding SNPs and key G6-PI catalytic residues or residues thought to be involved in the coordination of the G6-PI's substrate phosphate group. Thus, the significant polymorphism from its host orthologue and minimal intra-specific polymorphism suggest G6-PI remains a valid anti-coccidial drug target.
Lorenzo Romero-Trevejo J, Carlos Gomez-Villamandos J, Pedrera M, Blanco A, Jose Bautista M, Jose Sanchez-Cordon P (2010)

Immunohistochemical study of macrophage and cytokine dynamics in the gut of scrapie-infected mice

Histology and Histopathology 25 (8), 1025-1038

Abstract

To study numerical changes in intestinal macrophages and variations in cytokine production by immune cells in the intestine, conventional C57BL/6J mice were orally infected with the Rocky Mountain Laboratory strain of scrapie. Animals were sacrificed at different timepoints, and samples were taken and processed by routine methods for morphological and immunohistochemical analysis. The results point to a possible role for macrophages in the uptake and transport of the infective agent to Peyer's patches. The observed increase in macrophage numbers in subepithelial sites, taken in conjunction with a drop in tumour necrosis factor-alpha production at these sites, suggests a possible secretory inhibition that could be induced by the disease-associated prion protein (PrPd). On the other hand, cytokine dynamics indicated the presence of an impaired Th1-Th2 cell mediated response, which could facilitate the spread of PrPd to the central nervous system. Further research is required to confirm these hypotheses.

Abstract

Epizootic haemorrhagic disease virus (EHDV) infects wild ruminants, causing a frequently fatal haemorrhagic disease. However, it can also cause bluetongue-like disease in cattle, involving significant levels of morbidity and mortality, highlighting a need for more rapid and reliable diagnostic assays. EHDV outer-capsid protein VP2 (encoded by genome-segment 2 [Seg-2]) is highly variable and represents the primary target for neutralising antibodies generated by the mammalian host. Consequently VP2 is also the primary determinant of virus “serotype”, as identified in virus neutralisation tests (VNT). Although previous reports have indicated eight to ten EHDV serotypes, recent serological comparisons and molecular analyses of Seg-2 indicate only seven EHDV “types”. Oligonucleotide primers were developed targeting Seg-2, for use in conventional RT-PCR assays to detect and identify these seven types. These assays, which are more rapid and sensitive, still show complete agreement with VNT and were used to identify recent EHDV isolates from the Mediterranean region and North America.

Abstract

In mid September 2008, clinical signs of bluetongue (particularly coronitis) were observed in cows on three different farms in eastern Netherlands (Luttenberg, Heeten, and Barchem), two of which had been vaccinated with an inactivated BTV-8 vaccine (during May-June 2008). Bluetongue virus (BTV) infection was also detected on a fourth farm (Oldenzaal) in the same area while testing for export. BTV RNA was subsequently identified by real time RT-PCR targeting genome-segment (Seg-) 10, in blood samples from each farm. The virus was isolated from the Heeten sample (IAH “dsRNA virus reference collection” [dsRNA-VRC] isolate number NET2008/05) and typed as BTV-6 by RT-PCR targeting Seg-2. Sequencing confirmed the virus type, showing an identical Seg-2 sequence to that of the South African BTV-6 live-vaccine-strain. Although most of the other genome segments also showed very high levels of identity to the BTV-6 vaccine (99.7 to 100%), Seg-10 showed greatest identity (98.4%) to the BTV-2 vaccine (RSAvvv2/02), indicating that NET2008/05 had acquired a different Seg-10 by reassortment. Although Seg-7 from NET2008/05 was also most closely related to the BTV-6 vaccine (99.7/100% nt/aa identity), the Seg-7 sequence derived from the blood sample of the same animal (NET2008/06) was identical to that of the Netherlands BTV-8 (NET2006/04 and NET2007/01). This indicates that the blood contained two different Seg-7 sequences, one of which (from the BTV-6 vaccine) was selected during virus isolation in cell-culture. The predominance of the BTV-8 Seg-7 in the blood sample suggests that the virus was in the process of reassorting with the northern field strain of BTV-8. Two genome segments of the virus showed significant differences from the BTV-6 vaccine, indicating that they had been acquired by reassortment event with BTV-8, and another unknown parental-strain. However, the route by which BTV-6 and BTV-8 entered northern Europe was not established.

Abstract

T cell receptor (TCR) recognition of peptide-MHC class I (pMHC) complexes is a crucial event in the adaptive immune response to pathogens. Peptide epitopes often display a strong dominance hierarchy, resulting in focusing of the response on a limited number of the most dominant epitopes. Such T cell responses may be additionally restricted by particular MHC alleles in preference to others. We have studied this poorly understood phenomenon using Theileria parva, a protozoan parasite that causes an often fatal lymphoproliferative disease in cattle. Despite its antigenic complexity, CD8(+) T cell responses induced by infection with the parasite show profound immunodominance, as exemplified by the Tp1(214-224) epitope presented by the common and functionally important MHC class I allele N*01301. We present a high-resolution crystal structure of this pMHC complex, demonstrating that the peptide is presented in a distinctive raised conformation. Functional studies using CD8+ T cell clones show that this impacts significantly on TCR recognition. The unconventional structure is generated by a hydrophobic ridge within the MHC peptide binding groove, found in a set of cattle MHC alleles. Extremely rare in all other species, this feature is seen in a small group of mouse MHC class I molecules. The data generated in this analysis contribute to our understanding of the structural basis for T cell-dependent immune responses, providing insight into what determines a highly immunogenic p-MHC complex, and hence can be of value in prediction of antigenic epitopes and vaccine design.
Manjunatha B N, Prasad M, Maan S, Prasad G (2010)

Differentiation of Indian isolates of bluetongue virus serotype 1 from Australian and African isolates based on analysis of vp5 gene

Indian Journal of Biotechnology 9 (2), 117-125

Abstract

Bluetongue virus (BTV), prototype species of genus Orbivirus, belongs to the family Reoviridae. It is a non-enveloped, double shelled virus with ten segmented dsRNA genome. RNA segment 6 encodes an outer capsid serotype specific virus protein VP5. A pair of primers (forward 207-229 bp & reverse 1284-1304) was designed from the published BTV-1 segment 6 sequences to specifically amplify vp5 gene from Indian isolates of BTV. These primers specifically amplified PCR product of 1098 bp from cell culture adapted isolates of BTV-1 (Hisar isolate-BTV-1H, Avikanagar isolate-BTV-1A and Sirsa isolate-BTV-1S(3)), but did not give any amplification with BTV-9 and BTV-23, indicating serotype specificity. vp5 coding sequences amplified from Indian BTV-1 isolates were cloned into pPCR Script (TM) Amp SK (+) vector and transformed into XL10-Gold (R) Kan ultracompetent Escherichia coli cells. The positive clones selected by blue white screening and colony touch PCR were sequenced. The sequence analysis of the vp5 gene (253-1255 bp) revealed that Indian isolates of BTV-1 showed 89-91.1% nucleotide identity with Australian isolates of BTV-1, whereas it showed only 77-79.7% similarity with the BTV-1 African isolates. All three Indian isolates shared 99.4% nucleotide sequence similarity amongst themselves. Comparison of the deduced amino acid sequences revealed that the Indian BTV-1 isolates shared 96.7-98.8% and 94.9-95.8% amino acid similarity with Australian and African BTV-1 isolates, respectively. In silico restriction enzyme (RE) profile analysis of vp5 gene sequences showed that Indian isolates of BTV-1 can be differentiated from other BTV-1 isolates from South Africa and Australia using TaqI and BsmI restriction endonucleases.

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