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

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Baigent S J, Kgosana L B, Gamawa A A, Smith L P, Read A F, Nair V K (2013)

Relationship between levels of very virulent MDV in poultry dust and in feather tips from vaccinated chickens

Avian Diseases 52 (2 supplement 1), 440-447
Publisher’s version: http://dx.doi.org/10.1637/10356-091012-Reg.1

Abstract

To assess the effect of various vaccine strains on replication and shedding of virulent Marek's disease virus from experimentally infected chickens, quantitative PCR (q-PCR) methods were developed to accurately quantify viral DNA in infected chickens and in the environment in which they were housed. Four groups of 10 chickens, kept in poultry isolators, were vaccinated at 1 day old with one of four vaccines covering each of the three vaccine serotypes, then challenged with very virulent MDV strain Md5 at 8 days of age. At regular time-points, feather tips were collected from each chicken and poultry dust was collected from the air-extract prefilter of each isolator. DNA was extracted from feather and dust samples and subjected to real-time q-PCR, targeting the US2 gene of MDV-1, in order to measure Md5 level per 104 feather tip cells or per microgram of dust. Accuracy of DNA extraction from dust and real-time q-PCR were validated by comparing either q-PCR cycle threshold values or the calculated MDV genome level; for use in q-PCR, DNA was extracted from serial dilutions of MDV-infected dust diluted with noninfected dust, or DNA from MDV-infected dust was diluted with DNA from noninfected dust. The results confirmed the accuracy and sensitivity of dust DNA extraction and subsequent q-PCR and showed that differences in virus levels between dust samples truly reflect differences in shedding. Vaccination delayed both replication of Md5 in feather tips and shedding of Md5. First detection of Md5 in feather tips always preceded or coincided with first detection in dust in each group. pCVI988 and HVT+SB-1 were the most efficient vaccines in reducing both replication and shedding of Md5. There was close correlation between mean virus level in feathers of each group and mean virus level in the dust shed by that group. This relationship was similar in each of the vaccinated groups, demonstrating that measurement of the virus in dust can be used to monitor accurately both the infection status of the chickens and environmental contamination by MDV.
Bakshi S, Holzer B, Bridgen A, McMullan G, Quinn D G, Baron M D (2013)

Dugbe virus ovarian tumour domain interferes with ubiquitin/ISG15-regulated innate immune cell signalling

Journal of General Virology 94 (2), 298-307
Publisher’s version: http://dx.doi.org/10.1099/vir.0.048322-0

Abstract

The ovarian tumour (OTU) domain of the nairovirus L protein has been shown to remove ubiquitin and interferon-stimulated gene 15 protein (ISG15) from host cell proteins, which is expected to have multiple effects on cell signalling pathways. We have confirmed that the OTU domain from the L protein of the apathogenic nairovirus Dugbe virus has deubiquitinating and deISGylating activity and shown that, when expressed in cells, it is highly effective at blocking the TNF-alpha/NF-kappaB and interferon/JAK/STAT signalling pathways even at low doses. Point mutations of the catalytic site of the OTU [C40A, H151A and a double mutant] both abolished the ability of the OTU domain to deubiquitinate and deISGylate proteins and greatly reduced its effect on cell signalling pathways, confirming that it is this enzymic activity that is responsible for blocking the two signalling pathways. Expression of the inactive mutants at high levels could still block signalling, suggesting that the viral OTU can still bind to its substrate even when mutated at its catalytic site. The nairovirus L protein is a very large protein that is normally confined to the cytoplasm, where the virus replicates. When the OTU domain was prevented from entering the nucleus by expressing it as part of the N-terminal 205 kDa of the viral L protein, it continued to block type I interferon signalling, but no longer blocked the TNF-alpha-induced activation of NF-kappaB.
Baron J, Baron M (2013)

Creation of a completely helper cell-dependent recombinant morbillivirus

Journal of General Virology 94 (6), 1195-1199
Publisher’s version: http://dx.doi.org/10.1099/vir.0.050872-0

Abstract

We have created a completely helper cell-dependent morbillivirus by modifying the genome to remove the coding sequence of the phosphoprotein (P) and recovering the recombinant virus in a cell line constitutively expressing the P protein. The P protein-deleted virus (P?) grew very inefficiently unless both of the viral accessory proteins (V and C) were also expressed. Growth of the virus was restricted to the P-expressing cell line. The P? virus grew more slowly than the parental virus and expressed much less viral protein in infected cells. The technique could be used to create virus-like particles for use as a vaccine or as antigen in immunological or serological assays.
Barry G, Alberdi P, Schnettler E, Weisheit S, Kohl A, Fazakerley J K, Bell-Sakyi L (2013)

Gene silencing in tick cell lines using small interfering or long double-stranded RNA

Experimental and Applied Acarology 59 (3), 319-338
Publisher’s version: http://dx.doi.org/10.1007/s10493-012-9598-x

Abstract

Gene silencing by RNA interference (RNAi) is an important research tool in many areas of biology. To effectively harness the power of this technique in order to explore tick functional genomics and tick-microorganism interactions, optimised parameters for RNAi-mediated gene silencing in tick cells need to be established. Ten cell lines from four economically important ixodid tick genera (Amblyomma, Hyalomma, Ixodes and Rhipicephalus including the sub-species Boophilus) were used to examine key parameters including small interfering RNA (siRNA), double stranded RNA (dsRNA), transfection reagent and incubation time for silencing virus reporter and endogenous tick genes. Transfection reagents were essential for the uptake of siRNA whereas long dsRNA alone was taken up by most tick cell lines. Significant virus reporter protein knockdown was achieved using either siRNA or dsRNA in all the cell lines tested. Optimum conditions varied according to the cell line. Consistency between replicates and duration of incubation with dsRNA were addressed for two Ixodes scapularis cell lines; IDE8 supported more consistent and effective silencing of the endogenous gene subolesin than ISE6, and highly significant knockdown of the endogenous gene 2I1F6 in IDE8 cells was achieved within 48 h incubation with dsRNA. In summary, this study shows that gene silencing by RNAi in tick cell lines is generally more efficient with dsRNA than with siRNA but results vary between cell lines and optimal parameters need to be determined for each experimental system.
Batten C A, Edwards L, Oura C A L (2013)

Evaluation of the humoral immune responses in adult cattle and sheep, 4 and 2.5 years post-vaccination with a bluetongue serotype 8 inactivated vaccine

Vaccine 31 (37), 3783-3785
Publisher’s version: http://dx.doi.org/10.1016/j.vaccine.2013.06.033

Abstract

One of the big surprises about the devastating outbreak of bluetongue serotype-8 that spread across Northern and Western Europe between 2006 and 2008 was how relatively quickly the virus was controlled and eradicated from affected countries. This was at least in part attributed to the high levels of vaccine coverage achieved in affected countries. A previous study revealed that neutralising antibodies persisted in the majority of vaccinated cattle for at least 3 years post-vaccination, indicating that cattle are likely to be protected for this time period. The current study revealed that neutralising antibodies persisted in the same group of cattle for up to 4 years post-vaccination, and that neutralising antibodies persisted for up to 2.5 years in sheep that had been vaccinated on two occasions one year apart. These results have implications for future bluetongue surveillance programmes and vaccine control strategies.
Batten C A, Henstock M R, Steedman H M, Waddington S, Edwards L, Oura C A L (2013)

Bluetongue virus serotype 26: Infection kinetics, pathogenesis and possible contact transmission in goats

Veterinary Microbiology 162 (1), 62-67
Publisher’s version: http://dx.doi.org/10.1016/j.vetmic.2012.08.014

Abstract

The aim of this study was to assess the pathogenicity and infection kinetics of Bluetongue virus serotype 26 (BTV-26) in goats. Out of a group of six goats housed in insect free accommodation, five were experimentally infected with BTV-26 and one was kept uninfected as an in-contact control. Samples taken throughout the study were used to determine the kinetics of infection using a pan specific BTV real time RT-PCR assay and a group specific ELISA. The five infected goats did not show clinical signs of BTV, however high levels of viral RNA were detected and virus was isolated from the blood of all 5 goats. Antibodies against BTV were first detected between 7 and 11 dpi in all 5 experimentally infected goats. Interestingly at 21 dpi viral RNA was detected in, and virus was isolated from, the blood of the in-contact control goat, which also seroconverted. These results suggest that BTV-26 replicates to high levels in goats, causing no obvious clinical disease, suggesting that goats may be the natural host for this virus. Preliminary evidence also indicates that BTV-26 may be spread by contact transmission between goats, however a more detailed study is required in order to confirm this observation.
Belaganahalli M N, Maan S, Maan N S, Nomikou K, Guimera M, Brownlie J, Tesh R, Attoui H, Mertens P P C (2013)

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

PLoS ONE 8 (8), e70779
Publisher’s version: http://dx.doi.org/10.1371/journal.pone.0070779

Abstract

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.
Bell-Sakyi L, Attoui H (2013)

Endogenous tick viruses and modulation of tick-borne pathogen growth

Frontiers in Cellular and Infection Microbiology 3, 25
Publisher’s version: http://dx.doi.org/10.3389/fcimb.2013.00025

Abstract

Ticks transmit a wide range of viral, bacterial and protozoan pathogens, many of which can establish persistent infections of lifelong duration in the vector tick and in some cases are transmitted transovarially to the next generation. In addition many ixodid and argasid tick cell lines and, by inference the parent ticks from which they were derived, harbor endogenous viruses (ETV) of which almost nothing is known. In general, low level persistent infections with viral pathogens (arboviruses) are not known to have a deleterious effect on tick survival and fitness, suggesting that they can strike a balance with the tick innate immune response. This tolerance of arbovirus infection may be modulated by the permanent presence of ETV in the host cell. In mosquito cells, temporary or permanent silencing of the genes of an endogenous virus by RNA interference can result in changes in replication rate of a co-infecting arbovirus. We propose that tick cell lines offer a useful model system for in vitro investigation of the modulatory effect of ETV on superinfecting pathogen survival and replication in ticks, using the molecular manipulation techniques applied to insect cells.
Kirunda H, Kabi F, Muwereza N, Kabuuka T, Magona J W, Lukwago G (2012)

Knowledge and perceptions of smallholder dairy farmers of cattle disease burdens in selected agro-ecological zones of Uganda

Uganda Journal of Agricultural Sciences 13 (2), 107-116
Publisher’s version: https://www.ajol.info/index.php/ujas/article/view/126213

Abstract

Diseases have continued to affect production and productivity in smallholder dairy farming systems in Uganda. This study sought to establish farmers knowledge and perceptions of disease burden in the Eastern Semi-Arid Zone (ESAZ), Lake Victoria Basin (LVB) and Western Rangelands (WR) agro-ecological zones (AEZs). A structured questionnaire was administered to 150 farm household heads or cattle attendants. Data analysis by cross tabulations was done using SPSS Statistical Software and descriptive statistics generated in XLSTAT. Linear Discriminant analysis and Multivariate Analysis of Variance were computed to establish significant relationships (P<0.05) between variables. East Coast fever (ECF), calf scours, foot rot, mastitis and fascioliasis were reported of high prevalence (>50%), while brucellosis and eye infection had low prevalence (<16.7%). Season, age and breed of cattle were significantly associated with severity, morbidity, mortality and treatment costs in all AEZs. Morbidity and mortality were higher during the long (4.5; 0.7) than short rains (3.1; 0.6), respectively. Comparatively, average treatment costs were higher during the short rains (US$ 22) compared to long rains (US$ 17) each rains lasting three months. Results of our study show that some diseases were common to all AEZs, yet ESAZ had a higher disease burden than WR and LVB.

Dhote V, Sweeney T R, Kim N, Hellen C U, Pestova T V (2012)

Roles of individual domains in the function of DHX29, an essential factor required for translation of structured mammalian mRNAs

Proceedings of the National Academy of Sciences U S A 109 (46), E3150-3159
Publisher’s version: https://doi.org/10.1073/pnas.1208014109

Abstract

On most eukaryotic mRNAs, initiation codon selection involves base-by-base inspection of 5′ UTRs by scanning ribosomal complexes. Although the eukaryotic initiation factors 4A/4B/4G can mediate scanning through medium-stability hairpins, scanning through more stable structures additionally requires DHX29, a member of the superfamily 2 DEAH/RNA helicase A (RHA) helicase family that binds to 40S subunits and possesses 40S-stimulated nucleoside triphosphatase (NTPase) activity. Here, sequence alignment and structural modeling indicated that DHX29 comprises a unique 534-aa–long N-terminal region (NTR), central catalytic RecA1/RecA2 domains containing a large insert in the RecA2 domain, and the C-terminal part, which includes winged-helix, ratchet, and oligonucleotide/oligosaccharide-binding (OB) domains that are characteristic of DEAH/RHA helicases. Functional characterization revealed that specific ribosomal targeting is required for DHX29’s activity in initiation and is determined by elements that map to the NTR and to the N-terminal half of the winged-helix domain. The ribosome-binding determinant located in the NTR was identified as a putative double-stranded RNA-binding domain. Mutational analyses of RecA1/RecA2 domains confirmed the essential role of NTP hydrolysis for DHX29’s function in initiation and validated the significance of a β-hairpin protruding from RecA2. The large RecA2 insert played an autoinhibitory role in suppressing DHX29’s intrinsic NTPase activity but was not essential for its 40S-stimulated NTPase activity and function in initiation. Deletion of the OB domain also increased DHX29's basal NTPase activity, but more importantly, abrogated the responsiveness of the NTPase activity to stimulation, which abolished DHX29's function in initiation. This finding suggests that the OB domain, which is specific for DEAH/RHA helicases, plays an important role in their NTPase cycle.

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