Safety and efficacy of inactivated African horse sickness (AHS) vaccine formulated with different adjuvants.

African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae causing African Horse Sickness (AHS) in equids with a mortality of about 95% in naïve horses. AHS causes serious losses in developing countries where horses play a central role in draft power and transportation. There are nine AHSV serotypes inducing no or low cross-neutralizing antibodies. AHSV is spread by biting Culicoides midges. AHS is endemic in sub-Saharan Africa, and a serious threat outside Africa, since Culicoides species in moderate climate conditions are spreading the closely related bluetongue virus. AHS outbreaks will be devastating for the equestrian industry in developed countries. Live-attenuated vaccines (LAVs) are licensed, marketed and in use in Africa. Their application is controversial with regard to safety issues. LAVs are not allowed in AHS-free countries. We here studied inactivated AHSV with different adjuvants in guinea pigs and horses. Subcutaneous and intramuscular vaccination were studied in horses. Local reactions were observed after prime and boost vaccination. In general, neutralizing antibodies (nAbs) titres were very low after prime vaccination, whereas boost vaccination resulted in high nAb titres for some adjuvants. Vaccinated horses were selected based on local reactions and nAb titres to study efficacy. Unfortunately, not all vaccinated horses survived virulent AHSV infection. Further, most survivors temporarily developed clinical signs and viremia. Further, the current prototype inactivated AHS vaccine is not suitable as emergency vaccine, because onset of protection is slow and requires boost vaccinations. On the other hand, inactivated AHS vaccine is completely safe with respect to virus spread, and incorporation of the DIVA principle based on NS3/NS3a serology and exploring a vaccine production platform for other serotypes is feasible. A superior adjuvant increasing the protective response without causing local reactions will be required to develop payable and acceptable inactivated AHS vaccines.

Development and Evaluation of Real Time RT-PCR Assays for Detection and Typing of Bluetongue Virus.

Bluetongue virus is the type species of the genus Orbivirus, family Reoviridae. Bluetongue viruses (BTV) are transmitted between their vertebrate hosts primarily by biting midges (Culicoides spp.) in which they also replicate. Consequently BTV distribution is dependent on the activity, geographic distribution, and seasonal abundance of Culicoides spp. The virus can also be transmitted vertically in vertebrate hosts, and some strains/serotypes can be transmitted horizontally in the absence of insect vectors. The BTV genome is composed of ten linear segments of double-stranded (ds) RNA, numbered in order of decreasing size (Seg-1 to Seg-10). Genome segment 2 (Seg-2) encodes outer-capsid protein VP2, the most variable BTV protein and the primary target for neutralising antibodies. Consequently VP2 (and Seg-2) determine the identity of the twenty seven serotypes and two additional putative BTV serotypes that have been recognised so far. Current BTV vaccines are serotype specific and typing of outbreak strains is required in order to deploy appropriate vaccines. We report development and evaluation of multiple 'TaqMan' fluorescence-probe based quantitative real-time type-specific RT-PCR assays targeting Seg-2 of the 27+1 BTV types. The assays were evaluated using orbivirus isolates from the 'Orbivirus Reference Collection' (ORC) held at The Pirbright Institute. The assays are BTV-type specific and can be used for rapid, sensitive and reliable detection / identification (typing) of BTV RNA from samples of infected blood, tissues, homogenised Culicoides, or tissue culture supernatants. None of the assays amplified cDNAs from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures.

VP2 Exchange and NS3/NS3a Deletion in African Horse Sickness Virus (AHSV) in Development of Disabled Infectious Single Animal Vaccine Candidates for AHSV.

UNLABELLED: African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae. There are nine serotypes of AHSV showing different levels of cross neutralization. AHSV is transmitted by species of Culicoides biting midges and causes African horse sickness (AHS) in equids, with a mortality rate of up to 95% in naive horses. AHS has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates appear to be competent vectors for the related bluetongue virus (BTV). To control AHS, live-attenuated vaccines (LAVs) are used in Africa. We used reverse genetics to generate "synthetic" reassortants of AHSV for all nine serotypes by exchange of genome segment 2 (Seg-2). This segment encodes VP2, which is the serotype-determining protein and the dominant target for neutralizing antibodies. Single Seg-2 AHSV reassortants showed similar cytopathogenic effects in mammalian cells but displayed different growth kinetics. Reverse genetics for AHSV was also used to study Seg-10 expressing NS3/NS3a proteins. We demonstrated that NS3/NS3a proteins are not essential for AHSV replication in vitro. NS3/NS3a of AHSV is, however, involved in the cytopathogenic effect in mammalian cells and is very important for virus release from cultured insect cells in particular. Similar to the concept of the bluetongue disabled infectious single animal (BT DISA) vaccine platform, an AHS DISA vaccine platform lacking NS3/NS3a expression was developed. Using exchange of genome segment 2 encoding VP2 protein (Seg-2[VP2]), we will be able to develop AHS DISA vaccine candidates for all current AHSV serotypes. IMPORTANCE: African horse sickness virus is transmitted by species of Culicoides biting midges and causes African horse sickness in equids, with a mortality rate of up to 95% in naive horses. African horse sickness has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates are supposed to be competent vectors. By using reverse genetics, viruses of all nine serotypes were constructed by the exchange of Seg-2 expressing the serotype-determining VP2 protein. Furthermore, we demonstrated that the nonstructural protein NS3/NS3a is not essential for virus replication in vitro. However, the potential spread of the virus by biting midges is supposed to be blocked, since the in vitro release of the virus was strongly reduced due to this deletion. VP2 exchange and NS3/NS3a deletion in African horse sickness virus were combined in the concept of a disabled infectious single animal vaccine for all nine serotypes.

Endoluminal thoracic aortic repair in the octogenarian and nonagenarian: the New Zealand experience.

INTRODUCTION: The aim of this study was to review the New Zealand-wide experience of thoracic endovascular aortic repair to determine effect of age on outcome. METHODS: This was an observational, retrospective analytic study comparing two age groups. The New Zealand Thoracic Aortic Stent (NZTAS) registry was reviewed for patient demographics, indications for repair, risk factors, technical success, complications, length of hospital stay and in-hospital mortality. RESULTS: The 264 patients were divided into two groups: <80 (Group I, n = 245) and >80 years on the day of the procedure (Group II, n = 19). Group II comprised 11 males and 8 females with a mean age (range) of 84 (80-90) years. Using the Society of Thoracic Surgeons's scoring system for risk factors, there was no significant difference between the groups. Technical success was 84% (n = 16) in Group II. The mean hospital stay in Group II was 20 (2-90) days and the in-hospital mortality 16% (n = 3). None of these outcomes was significantly different to those in the under 80 group (Group I). CONCLUSIONS: Data from NZTAS registry show outcomes and risk factors do not significantly differ between the two age groups.

Serotype- and serogroup-specific detection of African horsesickness virus using phage displayed chicken scFvs for indirect double antibody sandwich ELISAs.

There is an ongoing need for standardized, easily renewable immunoreagents for detecting African horsesickness virus (AHSV). Two phage displayed single-chain variable fragment (scFv) antibodies, selected from a semi-synthetic chicken antibody library, were used to develop double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to detect AHSV. In the DAS-ELISAs, the scFv previously selected with directly immobilized AHSV-3 functioned as a serotype-specific reagent that recognized only AHSV-3. In contrast, the one selected with AHSV-8 captured by IgG against AHSV-3 recognized all nine AHSV serotypes but not the Bryanston strain of equine encephalosis virus. Serving as evidence for its serogroup-specificity. These two scFvs can help to rapidly confirm the presence of AHSV while additional serotype-specific scFvs may simplify AHSV serotyping.

Rescue of recent virulent and avirulent field strains of bluetongue virus by reverse genetics.

Since 1998, Bluetongue virus (BTV)-serotypes 1, 2, 4, 9, and 16 have invaded European countries around the Mediterranean Basin. In 2006, a huge BT-outbreak started after incursion of BTV-serotype 8 (BTV8) in North-Western Europe. More recently, BTV6 and BTV11 were reported in North-Western Europe in 2008. These latter strains are closely related to live-attenuated vaccine, whereas BTV8 is virulent and can induce severe disease in ruminants, including cattle. In addition, Toggenburg orbivirus (TOV) was detected in 2008 in Swiss goats, which was recognized as a new serotype of BTV (BTV25). The (re-)emergency of known and unknown BTV-serotypes needs a rapid response to supply effective vaccines, and research to study this phenomenon. Recently, orbivirus research achieved an important breakthrough by the establishment of reverse genetics for BTV1. Here, reverse genetics for two recent BTV strains representing virulent BTV8 and avirulent BTV6 was developed. For this purpose, extensive sequencing of full-genomes was performed, resulting in the consensus sequences of BTV8/net07 and BTV6/net08. The recovery of 'synthetic BTV', respectively rgBTV8 and rgBTV6, completely from T7-derived RNA transcripts was confirmed by silent mutations by which these 'synthetic BTVs' could be genetically distinguished from wild type BTV, respectively wtBTV6 and wtBTV8. The in vitro and in vivo properties of rgBTV6 or rgBTV8 were comparable to the properties of their parent strains. The asymptomatic or avirulent properties of rgBTV6 and the virulence of rgBTV8 were confirmed by experimental infection of sheep. Reverse genetics of the vaccine-related BTV6 provides a perfect start to develop new generations of BT-vaccines. Reverse genetics of the virulent BTV8 will accelerate research on the special features of BTV8, like transmission by species of Culicoides in a moderate climate, transplacental transmission, and pathogenesis in cattle.

An investigation into the possibility of bluetongue virus transmission by transfer of infected ovine embryos.

Bluetongue (BT), a disease that affects mainly sheep, causes economic losses owing to not only its deleterious effects on animals but also its associated impact on the restriction of movement of livestock and livestock germplasm. The causative agent, bluetongue virus (BTV), can occur in the semen of rams and bulls at the time of peak viraemia and be transferred to a developing foetus. The risk of the transmission of BTV by bovine embryos is negligible if the embryos are washed according to the International Embryo Transfer Society (IETS) protocol. Two experiments were undertaken to determine whether this holds for ovine embryos that had been exposed to BTV. Firstly, the oestrus cycles of 12 ewes were synchronised and the 59 embryos that were obtained were exposed in vitro to BTV-2 and BTV-4 at a dilution of 1 x 102.88 and 1 x 10(3.5) respectively. In the second experiment, embryos were recovered from sheep at the peak of viraemia. A total of 96 embryos were collected from BTV-infected sheep 21 days after infection. In both experiments half the embryos were washed and treated with trypsin according to the IETS protocol while the remaining embryos were neither washed nor treated. All were tested for the presence of BTV using cell culture techniques. The virus was detected after three passages in BHK-21 cells only in one wash bath in the first experiment and two unwashed embryos exposed to BTV-4 at a titre of 1 x 103.5. No embryos or uterine flush fluids obtained from viraemic donors used in the second experiment were positive for BTV after the standard washing procedure had been followed. The washing procedure of the IETS protocol can thus clear sheep embryos infected with BTV either in vitro or in vivo.

Full genome characterisation of bluetongue virus serotype 6 from the Netherlands 2008 and comparison to other field and vaccine strains.

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.

Abdominal aortic aneurysm surveillance: application of the UK Small Aneurysm Trial to a New Zealand tertiary hospital.

AIMS: We present 5-year results of an abdominal aortic aneurysm surveillance programme at Christchurch Hospital, based on the UK Small Aneurysm Trial. METHOD: Patients with infrarenal abdominal aortic aneurysms between 30 and 55 mm were placed in an ultrasound-based surveillance programme with an intention to treat when their aneurysms reached the Vascular Service determined threshold, when the AAA became symptomatic, or when rapid AAA growth was demonstrated. Patients were divided into three groups: Group 1, those currently under or those who had completed surveillance (n=198); Group 2, those excluded from surveillance and therefore treatment due to unsuitability for open surgical or endoluminal exclusion who had not completed surveillance (n=18); and Group 3, those who declined surgery on completion of surveillance (n=5). We looked at the number of aneurysm-related deaths in these groups and examined any issues that arose during or upon completion of surveillance. RESULTS: There were five aneurysm-related deaths in Group 1. There was one aneurysm-related death in Group 2, and one in Group 3. CONCLUSION: The data highlights problems related to setting threshold diameters for abdominal aortic aneurysms in women, and to the interval between completion of surveillance and undergoing endoluminal or open surgical repair.

Endovascular repair of chronic mesenteric occlusive disease: the role of duplex surveillance.

BACKGROUND: Chronic occlusive mesenteric ischaemia can be treated surgically or endovascularly. Endovascular techniques as elsewhere in the vascular tree are limited by restenosis. The aim of this study was to determine if duplex ultrasound proven restenosis correlates with recurrence of symptoms. METHODS: Our study looks at successful percutaneous revascularization of the mesenteric circulation associated proven restenosis using colour Doppler ultrasound and the relation to recrudescence of symptoms or weight loss. A retrospective review of five patients treated endovascularly at our institution for mesenteric angina secondary to visceral artery stenosis was carried out. RESULTS: Technical success was achieved in four out of the five patients in our study. One patient had a procedure complicated by thrombus in the coeliac axis and superior mesenteric artery (SMA) stents, subsequently showed SMA occlusion and 90% stenosis of the CA and inferior mesenteric artery and required an aorto-mesenteric graft. Three of the four patients with a technically successful procedure had significant (>70%) restenosis of the SMA. All three, including one patient with both SMA restenosis and chronic inferior mesenteric artery occlusion, remain asymptomatic and have maintained their postprocedural weight gain. CONCLUSION: Although ultrasound is a convenient, non-invasive tool for follow up of endovascular treatment of mesenteric stenosis, its use is unclear as in our study restenosis did not correlate with recrudescence of symptoms.

Comparison of duplex ultrasound with digital subtraction angiography in the assessment of infra-inguinal autologous vein bypass grafts.

BACKGROUND: Postoperative surveillance of infra-inguinal vein grafts has arisen because of the high incidence of vein graft stenoses, which frequently progress to vein graft occlusion. The use of duplex ultrasound as the primary imaging method for graft surveillance is well established. This study aims to compare the accuracy of duplex ultrasound with the reference standard of digital subtraction angiography in the assessment of infra-inguinal vein grafts. METHODS: Sixty patients underwent routine postoperative duplex ultrasound as part of the local graft surveillance programme. Angiography was subsequently carried out on 18 grafts. Each lower limb arterial tree was divided into three segments (native arteries proximal to the graft, the graft itself and native arteries distal to the graft) resulting in a total of 42 comparisons. Degree of diameter stenosis on ultrasound was compared with angiography findings to determine concordance. Agreement was also expressed as a kappa value. RESULTS: Overall accuracy of duplex ultrasound was 88% (37/42). A kappa value of 0.80 indicates good agreement. In three of the five discordant cases, ultrasound correctly identified a stenosis, but overestimated the degree of stenosis compared with angiography. In each of the remaining two discordant cases, ultrasound identified a focal stenosis that was not apparent on angiography. In both cases, the area of duplex described abnormality responded to balloon angioplasty. CONCLUSION: Duplex ultrasound as part of the local vein graft surveillance programme is a reliable and accurate method in the detection of failing grafts and in some instances may be more sensitive.

Spectral Doppler characterization of endoleaks following endoluminal abdominal aortic aneurysm repair.

BACKGROUND: Colour Doppler ultrasound of endoluminal abdominal aortic aneurysm repair is becoming an established imaging technique in identifying endoleak. Management and treatment of endoleak is determined in part by the exact nature of the endoleak, namely its type and whether it has single or multiple vessel inflow and outflow. To date, spectral Doppler waveform analysis has provided some information about the propensity for spontaneous seal of isolated type II endoleaks, rather than assisting in their classification. METHODS: We present a collection of three case reports outlining the directionality/phasicity of the Doppler waveform profile associated with endoleaks whose type and subtype (uni- /or multi-conduital) were angiographically determined. RESULTS: In all three cases uniconduital type II endoleak demonstrated a to-and-fro waveform on Doppler ultrasound imaging. CONCLUSIONS: To-and-fro Doppler waveforms may be associated with uniconduital type II endoleaks. If upon investigation of further cases this is found to be the case, this waveform classification may facilitate determination of the subtype (uni- or multi-conduital) of endoleak, thus identifying those cases which may be more amenable to percutaneous repair.