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

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Basu S, Aryan A, Overcash J M, Samuel G H, Anderson M A E, Dahlem T J, Myles K M, Adelman Z N (2015)

Silencing of end-joining repair for efficient site-specific gene insertion after TALEN/CRISPR mutagenesis in Aedes aegypti

Proceedings of the National Academy of Sciences of the United States of America 112 (13), 4038-4043
Publisher’s version: http://dx.doi.org/10.1073/pnas.1502370112

Abstract

Conventional control strategies for mosquito-borne pathogens such as malaria and dengue are now being complemented by the development of transgenic mosquito strains reprogrammed to generate beneficial phenotypes such as conditional sterility or pathogen resistance. The widespread success of site-specific nucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in model organisms also suggests that re-programmable gene drive systems based on these nucleases may be capable of spreading such beneficial phenotypes in wild mosquito populations. Using the mosquito Aedes aegypti, we determined that mutations in the FokI domain used in TALENs to generate obligate heterodimeric complexes substantially and significantly reduce gene editing rates. We found that CRISPR/Cas9-based editing in the mosquito Ae. aegypti is also highly variable, with the majority of guide RNAs unable to generate detectable editing. By first evaluating candidate guide RNAs using a transient embryo assay, we were able to rapidly identify highly effective guide RNAs; focusing germ line-based experiments only on this cohort resulted in consistently high editing rates of 24-90%. Microinjection of double-stranded RNAs targeting ku70 or lig4, both essential components of the end-joining response, increased recombination-based repair in early embryos as determined by plasmid-based reporters. RNAi-based suppression of Ku70 concurrent with embryonic microinjection of site-specific nucleases yielded consistent gene insertion frequencies of 2-3%, similar to traditional transposon-or Phi 31-based integration methods but without the requirement for an initial docking step. These studies should greatly accelerate investigations into mosquito biology, streamline development of transgenic strains for field releases, and simplify the evaluation of novel Cas9-based gene drive systems.
Batten C, Frost L, Oura C (2015)

Real-time reverse transcriptase PCR for the detection of bluetongue virus

Veterinary infection biology: Molecular diagnostics and high-throughput strategies (edited by M V Cunha and J Inacio, Humana Press) 1247, 125-131
Publisher’s version: http://dx.doi.org/10.1007/978-1-4939-2004-4_8

Abstract

In recent years, real-time reverse transcription polymerase chain reaction (rRT-PCR) has become one of the most widely used methods for the diagnosis of infectious pathogens. The combined properties of high sensitivity, specificity, and speed, along with a low contamination risk, have made real-time PCR technology a highly attractive alternative to more conventional diagnostic methods. Numerous robust rRT-PCR systems have been developed and validated for important epizootic diseases of livestock, and in this chapter we describe an rRT-PCR protocol for the detection of bluetongue virus. The assay uses oligonucleotide primers to specifically amplify target regions of the viral genome and a dual-labeled fluorogenic (TaqMan()) probe which allows for the assay to be performed in a closed-tube format, thus minimizing the potential for cross-contamination.
Belaganahalli M, Maan S, Maan N, Brownlie J, Tesh R, Attoui H, Mertens P (2015)

Genetic characterization of the tick-borne orbiviruses

Viruses 7 (5), 2185-2209
Publisher’s version: http://dx.doi.org/10.3390/v7052185

Abstract

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.
Bell-Sakyi L, Palomar A M, Bradford E L, Shkap V (2015)

Propagation of the Israeli vaccine strain of Anaplasma centrale in tick cell lines

Veterinary Microbiology 179 (3-4), 270-276
Publisher’s version: http://dx.doi.org/10.1016/j.vetmic.2015.07.008

Abstract

Anaplasma centrale has been used in cattle as a live blood vaccine against the more pathogenic Anaplasma marginale for over 100 years. While A. marginale can be propagated in vitro in tick cell lines, facilitating studies on antigen production, immunisation and vector-pathogen interaction, to date there has been no in vitro culture system for A. centrale. In the present study, 25 cell lines derived from 13 ixodid tick species were inoculated with the Israeli vaccine strain of A. centrale and monitored for at least 12 weeks by microscopic examination of Giemsa-stained cytocentrifuge smears. Infection of 19 tick cell lines was subsequently attempted by transfer of cell-free supernate from vaccine-inoculated tick cells. In two separate experiments, rickettsial inclusions were detected in cultures of the Rhipicephalus appendiculatus cell line RAE25 28-32 days following inoculation with the vaccine. Presence of A. centrale in the RAE25 cells was confirmed by PCR assays targeting the 16S rRNA, groEL and msp4 genes; sequenced PCR products were 100% identical to published sequences of the respective genes in the Israeli vaccine strain of A. centrale. A. centrale was taken through three subcultures in RAE25 cells over a 30 week period. In a single experiment, the Dermacentor variabilis cell line DVE1 was also detectably infected with A. centrale 11 weeks after inoculation with the vaccine. Availability of an in vitro culture system for A. centrale in tick cells opens up the possibility of generating a safer and more ethical vaccine for bovine anaplasmosis.
Bell-Sakyi L, Palomar A M, Kazimirova M (2015)

Isolation and propagation of a Spiroplasma sp. from Slovakian Ixodes ricinus ticks in Ixodes spp. cell lines

Ticks and Tick-borne Diseases 6 (5), 601-606
Publisher’s version: http://dx.doi.org/10.1016/j.ttbdis.2015.05.002

Abstract

Ixodes spp. ticks are known to occasionally harbour spiroplasmas – helical mycoplasmas in the class Mollicutes; a previous study in Slovakia reported an overall prevalence of Spiroplasma ixodetis of 3% in Ixodes ricinus. In the present study, extracts of unfed adult I. ricinus ticks collected from vegetation in south-western Slovakia were added to a panel of cell lines derived from I. ricinus and Ixodes scapularis embryos. The cultures were monitored by preparation and examination of Giemsa-stained cytocentrifuge smears at intervals over the subsequent 16–18 months. Spiroplasma-like microorganisms were detected in cultures of both tick species after 2–3 months and subcultured onto fresh, uninfected cells of the appropriate cell line up to seven times. Molecular analysis using PCR assays targeting fragments of the 16S rRNA, ITS and rpoB genes confirmed the identity of the microorganisms as a Spiroplasma sp., with between 98.9% and 99.5% similarity to S. ixodetis. The sequences of the spiroplasmas isolated from three different pools of ticks collected on two different occasions were identical for all three genes tested.
Blodorn K, Hagglund S, Gavier-Widen D, Eleouet J F, Riffault S, Pringle J, Taylor G, Valarcher J F (2015)

A bovine respiratory syncytial virus model with high clinical expression in calves with specific passive immunity

BMC Veterinary Research 11, 76
Publisher’s version: http://dx.doi.org/10.1186/s12917-015-0389-6

Abstract

Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in cattle worldwide. Calves are particularly affected, even with low to moderate levels of BRSV-specific maternally derived antibodies (MDA). Available BRSV vaccines have suboptimal efficacy in calves with MDA, and published infection models in this target group are lacking in clinical expression. Here, we refine and characterize such a model. In a first experiment, 2 groups of 3 calves with low levels of MDA were experimentally inoculated by inhalation of aerosolized BRSV, either: the Snook strain, passaged in gnotobiotic calves (BRSV-Snk), or isolate no. 9402022 Denmark, passaged in cell culture (BRSV-Dk). All calves developed clinical signs of respiratory disease and shed high titers of virus, but BRSV-Snk induced more severe disease, which was then reproduced in a second experiment in 5 calves with moderate levels of MDA. These 5 calves shed high titers of virus and developed severe clinical signs of disease and extensive macroscopic lung lesions (mean+/?SD, 48.3+/?12.0% of lung), with a pulmonary influx of inflammatory cells, characterized by interferon gamma secretion and a marked effect on lung function. We present a BRSV-infection model, with consistently high clinical expression in young calves with low to moderate levels of BRSV-specific MDA, that may prove useful in studies into disease pathogenesis, or evaluations of vaccines and antivirals. Additionally, refined tools to assess the outcome of BRSV infection are described, including passive measurement of lung function and a refined system to score clinical signs of disease. Using this cognate host calf model might also provide answers to elusive questions about human RSV (HRSV), a major cause of morbidity in children worldwide.
Boyce M, McCrae M A (2015)

Rapid mapping of functional cis-acting RNA elements by recovery of virus from a degenerate RNA population: application to genome segment 10 of bluetongue virus

Journal of General Virology 96 (10), 3072-3082
Publisher’s version: http://dx.doi.org/10.1099/jgv.0.000259

Abstract

The regulatory elements which control the processes of virus replication and gene expression in the Orbivirus genus are uncharacterised both in terms of their locations within genome segments and their specific functions. The reverse genetics system for the type species, Bluetongue virus, has been used in combination with RNA secondary structure prediction to identify and map the positions of cis-acting regions within genome segment 10. Through the simultaneous introduction of variability at multiple nucleotide positions in the rescue RNA population, the functional contribution of these positions was used to map regions containing cis-acting elements essential for virus viability. Nucleotides that were individually lethal when varied mapped within a region of predicted secondary structure involving base pairing between the 5' and 3' end of the transcript. An extended region of predicted perfect base pairing located within the 3' untranslated region (UTR) of the genome segment was also found to be required for virus viability. In contrast to the identification of individually lethal mutations, gross alteration of the composition of this predicted stem region was possible, providing the base pairing potential between the two strands was maintained, identifying a structural feature, predicted to be conserved throughout the Orbivirus genus. The approach of identifying cis-acting sequences through sequencing the recovered virus following the rescue of a degenerate RNA population is broadly applicable to viruses where reverse genetics is available.
Breard E, Belbis G, Viarouge C, Nomikou K, Haegeman A, De Clercq K, Hudelet P, Hamers C, Moreau F, Lilin T, Durand B, Mertens P, Vitour D, Sailleau C, Zientara S (2015)

Evaluation of adaptive immune responses and heterologous protection induced by inactivated bluetongue virus vaccines

Vaccine 33 (4), 512-518
Publisher’s version: http://dx.doi.org/10.1016/j.vaccine.2014.11.053

Abstract

Eradication of bluetongue virus is possible, as has been shown in several European countries. New serotypes have emerged, however, for which there are no specific commercial vaccines. This study addressed whether heterologous vaccines would help protect against 2 serotypes. Thirty-seven sheep were randomly allocated to 7 groups of 5 or 6 animals. Four groups were vaccinated with commercial vaccines against BTV strains 2, 4, and 9. A fifth positive control group was given a vaccine against BTV-8. The other 2 groups were unvaccinated controls. Sheep were then challenged by subcutaneous injection of either BTV-16 (2 groups) or BTV-8 (5 groups). Taken together, 24/25 sheep from the 4 experimental groups developed detectable antibodies against the vaccinated viruses. Furthermore, sheep that received heterologous vaccines showed significantly reduced viraemia and clinical scores for BTV-16 when compared to unvaccinated controls. Reductions in clinical signs and viraemia among heterologously vaccinated sheep were not as common after challenge with BTV-8. This study shows that heterologous protection can occur, but that it is difficult to predict if partial or complete protection will be achieved following inactivated-BTV vaccination.
Browning H M, Gulland F M D, Hammond J A, Colegrove K M, Hall A J (2015)

Common cancer in a wild animal: the California sea lion (Zalophus californianus) as an emerging model for carcinogenesis

Philosophical Transaction of the Royal Society B-Biological Sciences 370 (1673), 2014.0228
Publisher’s version: http://dx.doi.org/10.1098/rstb.2014.0228

Abstract

Naturally occurring cancers in non-laboratory species have great potential in helping to decipher the often complex causes of neoplasia. Wild animal models could add substantially to our understanding of carcinogenesis, particularly of genetic and environmental interactions, but they are currently underutilized. Studying neoplasia in wild animals is difficult and especially challenging in marine mammals owing to their inaccessibility, lack of exposure history, and ethical, logistical and legal limits on experimentation. Despite this, California sea lions (Zalophus californianus) offer an opportunity to investigate risk factors for neoplasia development that have implications for terrestrial mammals and humans who share much of their environment and diet. A relatively accessible California sea lion population on the west coast of the USA has a high prevalence of urogenital carcinoma and is regularly sampled during veterinary care in wildlife rehabilitation centres. Collaborative studies have revealed that genotype, persistent organic pollutants and a herpesvirus are all associated with this cancer. This paper reviews research to date on the epidemiology and pathogenesis of urogenital carcinoma in this species, and presents the California sea lion as an important and currently underexploited wild animal model of carcinogenesis.
Brugman V A, Hernández-Triana L M, Prosser S W, J., Weland C, Westcott D G, Fooks A R, Johnson N (2015)

Molecular species identification, host preference and detection of myxoma virus in the Anopheles maculipennis complex (Diptera: Culicidae) in southern England, UK

Parasites and Vectors 8 (1), e421
Publisher’s version: http://dx.doi.org/10.1186/s13071-015-1034-8

Abstract

Determining the host feeding patterns of mosquitoes by identifying the origin of their blood-meals is an important part of understanding the role of vector species in current and future disease transmission cycles. Collecting large numbers of blood-fed mosquitoes from the field is difficult, therefore it is important to maximise the information obtained from each specimen. This study aimed to use mosquito genome sequence to identify the species within Anopheles maculipennis sensu lato (An. maculipennis s.l.), identify the vertebrate hosts of field-caught blood-fed An. maculipennis s.l. , and to test for the presence of myxoma virus (Poxviridae, genus Leporipoxvirus) in specimens found to have fed on the European rabbit (Oryctolagus cuniculus). Blood-fed An. maculipennis s.l. were collected from resting sites at Elmley Nature Reserve, Kent, between June and September 2013. Hosts that An. maculipennis s.l. had fed on were determined by a PCR-sequencing approach based on the partial amplification of the mitochondrial cytochrome C oxidase subunit I gene. Mosquitoes were then identified to species by sequencing a region of the internal transcribed spacer-2. DNA extracts from all mosquitoes identified as having fed on rabbits were subsequently screened using PCR for the presence of myxoma virus. A total of 94 blood-fed Anopheles maculipennis s.l. were collected, of which 43 (46 %) provided positive blood-meal identification results. Thirty-six of these specimens were identified as Anopheles atroparvus, which had fed on rabbit (n?=?33, 92 %) and cattle (n?=?3, 8 %). Seven mosquitoes were identified as Anopheles messeae, which had fed on cattle (n?=?6, 86 %) and dog (n?=?1, 14 %). Of the 33 An. atroparvus that contained rabbit blood, nine (27 %) were positive for myxoma virus. Results demonstrate that a single DNA extract from a blood-fed mosquito can be successfully used for molecular identification of members of the An. maculipennis complex, blood-meal identification, and for the targeted detection of a myxoma virus. This study shows that An. atroparvus has a strong feeding preference for both healthy and myxoma-infected rabbits, providing evidence that this species may play a significant role in the transmission of myxomatosis among wild rabbit populations in the United Kingdom.

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