Publications

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.

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.

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.

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification(1). Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast(2,3) but more recently has been adapted to use in mammalian cells(4-8). As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E(9,10).The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation(10). The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence(8). To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter. Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.