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

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Sweeney T R, Dhote V, Yu Y, Hellen C U (2012)

A distinct class of internal ribosomal entry site in members of the Kobuvirus and proposed Salivirus and Paraturdivirus genera of the Picornaviridae

Journal of Virology 86 (3), 1468-1486
Publisher’s version: https://doi.org/10.1128/jvi.05862-11

Abstract

The 5?-untranslated regions (5' UTRs) of picornavirus genomes contain an internal ribosomal entry site (IRES) that promotes the end-independent initiation of translation. Picornavirus IRESs are classified into four structurally distinct groups, each with different initiation factor requirements. Here, we identify a fifth IRES class in members of Kobuvirus, Salivirus, and Paraturdivirus genera of Picornaviridae: Aichi virus (AV), bovine kobuvirus (BKV), canine kobuvirus (CKoV), mouse kobuvirus (MKoV), sheep kobuvirus (SKV), salivirus A (SV-A), turdivirus 2 (TV2), and TV3. The 410-nucleotide (nt)-long AV IRES comprises four domains (I to L), including a hairpin (L) that overlaps a Yn-Xm-AUG (pyrimidine tract/spacer/initiation codon) motif. SV-A, CKoV, and MKoV also contain these four domains, whereas BKV, SKV, and TV2/TV3 5' UTRs contain domains that are related to domain I and equivalent to domains J and K but lack an AV-like domain L. These IRESs are located at different relative positions between a conserved 5?-terminal origin of replication and divergent coding sequences. Elements in these IRESs also occur elsewhere: domain J's apical subdomain, which contains a GNRA tetraloop, matches an element in type 1 IRESs, and eIF4G-binding motifs in domain K and in type 2 IRESs are identical. Other elements are unique, and their presence leads to unique initiation factor requirements. In vitro reconstitution experiments showed that like AV, but in contrast to other currently characterized IRESs, SV-A requires the DExH-box protein DHX29 during initiation, which likely ensures that the initiation codon sequestered in domain L is properly accommodated in the ribosomal mRNA-binding cleft.

Aguirre S, Maestre A M, Pagni S, Patel J R, Savage T, Gutman D, Maringer K, Bernal-Rubio D, Shabman R S, Simon V, Rodriguez-Madoz J R, Mulder L C, Barber G N, Fernandez-Sesma A (2012)

DENV inhibits type I IFN production in infected cells by cleaving human STING

PLoS Pathogens 8 (10), e1002934
Publisher’s version: https://doi.org/10.1371/journal.ppat.1002934

Abstract

Dengue virus (DENV) is a pathogen with a high impact on human health. It replicates in a wide range of cells involved in the immune response. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response. DENV circumvents the host immune response by expressing proteins that antagonize the cellular innate immunity. We have recently documented the inhibition of type I IFN production by the proteolytic activity of DENV NS2B3 protease complex in human monocyte derived dendritic cells (MDDCs). In the present report we identify the human adaptor molecule STING as a target of the NS2B3 protease complex. We characterize the mechanism of inhibition of type I IFN production in primary human MDDCs by this viral factor. Using different human and mouse primary cells lacking STING, we show enhanced DENV replication. Conversely, mutated versions of STING that cannot be cleaved by the DENV NS2B3 protease induced higher levels of type I IFN after infection with DENV. Additionally, we show that DENV NS2B3 is not able to degrade the mouse version of STING, a phenomenon that severely restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition of DENV infection and spread in mice.

Maringer K, Stylianou J, Elliott G (2012)

A network of protein interactions around the herpes simplex virus tegument protein VP22

Journal of Virology 86 (23), 12971-12982
Publisher’s version: https://doi.org/10.1128/JVI.01913-12

Abstract

Assembly of the herpesvirus tegument is poorly understood but is believed to involve interactions between outer tegument proteins and the cytoplasmic domains of envelope glycoproteins. Here, we present the detailed characterization of a multicomponent glycoprotein-tegument complex found in herpes simplex virus 1 (HSV-1)-infected cells. We demonstrate that the tegument protein VP22 bridges a complex between glycoprotein E (gE) and glycoprotein M (gM). Glycoprotein I (gI), the known binding partner of gE, is also recruited into this gE-VP22-gM complex but is not required for its formation. Exclusion of the glycoproteins gB and gD and VP22's major binding partner VP16 demonstrates that recruitment of virion components into this complex is highly selective. The immediate-early protein ICP0, which requires VP22 for packaging into the virion, is also assembled into this gE-VP22-gM-gI complex in a VP22-dependent fashion. Although subcomplexes containing VP22 and ICP0 can be formed when either gE or gM are absent, optimal complex formation requires both glycoproteins. Furthermore, and in line with complex formation, neither of these glycoproteins is individually required for VP22 or ICP0 packaging into the virion, but deletion of gE and gM greatly reduces assembly of both VP22 and ICP0. Double deletion of gE and gM also results in small plaque size, reduced virus yield, and defective secondary envelopment, similar to the phenotype previously shown for pseudorabies virus. Hence, we suggest that optimal gE-VP22-gM-gI-ICP0 complex formation correlates with efficient virus morphogenesis and spread. These data give novel insights into the poorly understood process of tegument acquisition.

Benlahrech A, Yasmin A, Westrop S J, Coleman A, Herasimtschuk A, Page E, Kelleher P, Gotch F, Imami N, and Patterson S (2012)

Dysregulated immunophenotypic attributes of plasmacytoid but not myeloid dendritic cells in HIV-1 infected individuals in the absence of highly active anti-retroviral therapy

Clinical and Experimental Immunology 170 (2), 212-221
Publisher’s version: https://doi.org/10.1111/j.1365-2249.2012.04647.x

Abstract

Dendritic cells (DC) in HIV-1-infected individuals are decreased and their dysfunction has been implicated in HIV-1 immunopathogenesis. The mechanism of their dysfunction remains unclear, thus we analysed the expression of membrane molecules associated with immune regulation and DC activation in myeloid (mDC) and plasmacytoid DC (pDC) in therapy-naive and highly active anti-retroviral therapy (HAART)-treated HIV-1+ patients. DC from healthy controls, untreated HIV-1+ and HAART-treated patients were assessed by flow cytometry for expression of: anergy and apoptosis inducing molecules [programmed death (PD)-1 and its ligands PD-L1 and PD-L2], inhibitory and regulatory T cell-inducing molecules [immunoglobulin-like transcript (ILT)-3 and ILT-4], interferon (IFN)-α inhibitory receptor (ILT-7) and co-stimulatory molecules (CD80, CD83, and CD86). pDC from untreated HIV-1+ patients expressed significantly lower levels of ILT-7 compared to healthy controls, while HAART-treated patients showed normal expression. pDC were also found to express moderately higher levels of PD-L1 and ILT-3 and lower levels of PD-L2 receptors in untreated patients compared to controls and HAART-treated patients. No significant changes were observed in mDC. There were no associations between the percentages and levels of expression of these molecules by pDC and viral load or CD4 T cell count. In conclusion, pDC but not mDC from HIV-1+patients with active viraemia display higher levels of apoptosis and T regulatory-inducing molecules and may be predisposed to chronically produce IFN-α through down-regulation of ILT-7. HAART restored normal expression levels of these receptors.

Bao J, Wang Q, Parida S, Liu C, Zhang L, Zhao W, Wang Z (2012)

Complete genome sequence of a peste des petits ruminants virus recovered from wild bharal in Tibet, China

Journal of Virology 86, 10885-10886
Publisher’s version: http://dx.doi.org/10.1128/jvi.01503-12

Abstract

For the first time, here we announce the complete genome sequence of a field isolate of Peste des petits ruminants virus (PPRV) derived from macerated rectal tissue of a free living bharal (Pseudois nayaur) that displayed clinical disease consistent with severe infection with PPRV. Further, we compare the full genome of this isolate, termed PPRV Tibet/Bharal/2008, with previously available PPRV genomes, including those of virus isolates from domestic small ruminants local to the area where the reported isolate was collected. The current sequence is phylogenetically classified as a lineage IV virus, sharing high levels of sequence identity with previously described Tibetan PPRV isolates. Indeed, across the entire genome, only 26 nucleotide differences (0.16% nucleotide variation) and, consequently, 9 amino acid changes were present compared to sequences of locally derived viruses. 

Maan S, Maan N S, Singh K P, Belaganahalli M N, Guimera M, Pullinger G, Nomikou K, Mertens P P C (2012)

Complete genome sequence analysis of a reference strain of bluetongue virus Serotype 16

Journal of Virology 86 (18), 10255-10256
Publisher’s version: http://dx.doi.org/10.1128/jvi.01672-12

Abstract

The entire genome of the reference strain of bluetongue virus (BTV) serotype 16 (strain RSArrrr/16) was sequenced (a total of 23,518 base pairs). The virus was obtained from the Orbivirus Reference Collection (ORC) at IAH, Pirbright, United Kingdom. The virus strain, which was previously provided by the Onderstepoort Veterinary Research Institute in South Africa, was originally isolated from the Indian subcontinent (Hazara, West Pakistan) in 1960. Previous phylogenetic comparisons show that BTV RNA sequences cluster according to the geographic origins of the virus isolate/lineage, identifying distinct BTV topotypes. Sequence comparisons of segments Seg-1 to Seg-10 show that RSArrrr/16 belongs to the major eastern topotype of BTV (BTV-16e) and can be regarded as a reference strain of BTV-16e for phylogenetic and molecular epidemiology studies. All 10 genome segments of RSArrrr/16 group closely with the vaccine strain of BTV-16 (RSAvvvv/16) that was derived from it, as well as those recently published for a Chinese isolate of BTV-16 (>99% nucleotide identity), suggesting a very recent common ancestry for all three viruses.

Sampels V, Hartmann A, Dietrich I, Coppens I, Sheiner L, Striepen B, Herrmann A, Lucius R, Gupta N (2012)

Conditional mutagenesis of a novel choline kinase demonstrates plasticity of phosphatidylcholine biogenesis and gene expression in Toxoplasma gondii

Journal of Biological Chemistry 287 (20), 16289-16299
Publisher’s version: https://doi.org/10.1074/jbc.M112.347138

Abstract

The obligate intracellular and promiscuous protozoan parasite Toxoplasma gondii needs an extensive membrane biogenesis that must be satisfied irrespective of its host-cell milieu. We show that the synthesis of the major lipid in T. gondii, phosphatidylcholine (PtdCho), is initiated by a novel choline kinase (TgCK). Full-length ( approximately 70-kDa) TgCK displayed a low affinity for choline (K(m) approximately 0.77 mM) and harbors a unique N-terminal hydrophobic peptide that is required for the formation of enzyme oligomers in the parasite cytosol but not for activity. Conditional mutagenesis of the TgCK gene in T. gondii attenuated the protein level by approximately 60%, which was abolished in the off state of the mutant (Deltatgck(i)). Unexpectedly, the mutant was not impaired in its growth and exhibited a normal PtdCho biogenesis. The parasite compensated for the loss of full-length TgCK by two potential 53- and 44-kDa isoforms expressed through a cryptic promoter identified within exon 1. TgCK-Exon1 alone was sufficient in driving the expression of GFP in E. coli. The presence of a cryptic promoter correlated with the persistent enzyme activity, PtdCho synthesis, and susceptibility of T. gondii to a choline analog, dimethylethanolamine. Quite notably, the mutant displayed a regular growth in the off state despite a 35% decline in PtdCho content and lipid synthesis, suggesting a compositional flexibility in the membranes of the parasite. The observed plasticity of gene expression and membrane biogenesis can ensure a faithful replication and adaptation of T. gondii in disparate host or nutrient environments.

Schnettler E, Ratinier M, Watson M, Shaw A E, McFarlane M, Varela M, Elliott R M, Palmarini M, Kohl A (2012)

RNA interference targets arbovirus replication in Culicoides cells

Journal of Virology 87 (5), 2441-2454
Publisher’s version: https://dx.doi.org/10.1128/jvi.02848-12

Abstract

Arboviruses are transmitted to vertebrate hosts by biting arthropod vectors such as mosquitoes, ticks and midges. These viruses replicate in both arthropods and vertebrates and are thus exposed to different antiviral responses in these organisms. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism that has been shown to play a major role in the antiviral response against arboviruses in mosquitoes. Culicoides midges are important vectors of arboviruses, known to transmit pathogens of humans and livestock such as bluetongue virus (BTV) (Reoviridae), Oropouche virus (Bunyaviridae), and likely the recently discovered Schmallenberg virus (Bunyaviridae). In this study we investigated whether Culicoides cells possess an antiviral RNAi response and whether this is effective against arboviruses including those with dsRNA genome such as BTV. Using reporter-gene based assays we established the presence of a functional RNAi response in Culicoides sonorensis-derived KC cells, which is effective in inhibiting BTV infection. Sequencing of small RNAs from KC and Aedes aegypti-derived Aag2 cells infected with BTV or the unrelated Schmallenberg virus resulted in the production of virus-derived small interfering RNAs (viRNAs) of 21 nucleotides, similar to the viRNAs produced during arbovirus infections of mosquitoes. In addition, viRNA profiles strongly suggest that the BTV dsRNA genome is accessible to a Dicer-type nuclease. Thus, we show for the first time that midge cells target arbovirus replication by mounting an antiviral RNAi response mainly resembling that of other insect vectors of arboviruses.

Leftwich P T, Edward D A, Alphey L, Gage M J G, Chapman T (2012)

Variation in adult sex ratio alters the association between courtship, mating frequency and paternity in the lek-forming fruitfly Ceratitis capitata

Journal of Evolutionary Biology 25 (9), 1732-1740
Publisher’s version: http://dx.doi.org/10.1111/j.1420-9101.2012.02556.x

Abstract

The intensity with which males deliver courtship and the frequency with which they mate are key components of male reproductive success. However, we expect the strength of the relationship between these traits and a male's overall paternity to be strongly context dependent, for example to be altered significantly by the extent of post-mating competition. We tested this prediction in a lekking insect, Ceratitis capitata (medfly). We examined the effect of manipulating the sex ratio from male- to female-biased (high and low male competition, respectively) on courtship behaviour, mating frequency and paternity of focal males. Under high male competition, focal males delivered significantly more courtship but gained lower paternity than under lower competition. Paternity was positively associated with mating frequency and small residual testes size. However, the association between mating frequency and paternity was significantly stronger under low competition. We conclude that manipulation of sex ratio significantly altered the predictors of mating success and paternity. The relationship between pre- and post-mating success is therefore plastic and alters according to the prevailing level of competition. The results highlight the importance of post-copulatory processes in lekking species and illuminate selection pressures placed on insects such as medflies that are mass reared for pest control.

Limon G, Guitian J, Gregory N G (2012)

A review of the humaneness of puntilla as a slaughter method

Animal Welfare 21 (1), 03-8
Publisher’s version: http://www.ingentaconnect.com/contentone/ufaw/aw/2012/00000021/A00201s2/art00001

Abstract

Puntilla is a traditional slaughter method in which a knife is plunged into the back of the neck to sever the spinal cord. The aim is to produce immediate collapse of the animal. Puntilla is not condoned as a stunning method by the World Animal Health Organisation (OIE) because there is concern that the animal could be conscious during and after the neck stab. Nonetheless, it is still used in some developing countries. The effectiveness and humaneness of puntilla followed by neck sticking was examined at two slaughterhouses in Bolivia. Twenty llamas (Lama glama) and 309 cattle were observed during routine puntilla without stunning. The number of neck stabs was recorded, and then brain and spinal functions (rhythmic breathing, palpebral reflex and eyeball rotation) were assessed. In addition, the presence of specific cognitive responses (such as responses to a threat stimulus and noise, as well as to flavours and odours), were also assessed in cattle. Breed, sex, live weight, body condition score and the slaughterman's experience were recorded. Repeat stabbing was needed to penetrate the foramen ovale in 45% of the llamas and two of them attempted to stand following collapse after the initial stab. All llamas showed rhythmic breathing movements at the flank following puntilla and before sticking, and 95% had a positive palpebral reflex at the same time. Twenty-four percent of the cattle needed repeat stabbing. Repeat stabbing was significantly less frequent with experienced slaughtermen, and more frequent in heavyweight animals (> 380 kg). Brain and spinal responses were present in 91% of the cattle following the stabs. When cattle attempted to stand after a neck stab they were more likely to have rhythmic breathing, positive palpebral response and responsiveness to threat, noise and brief air stimulus applied to the face. These findings indicate that it is difficult in practice to penetrate the spinal cord with a single puntilla stab. Some nerve pathways are often functional after the neck stab and therefore it is highly likely that the animals remain conscious in at least some modalities for the next part of the slaughter procedure. The challenge in developing countries, however, is to find a strategy that encourages use of a method which limits suffering whilst being accessible for routine slaughter practice.

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