Immunogenic profile of a plant-produced nonavalent African horse sickness viral protein 2 (VP2) vaccine in IFNAR-/- mice.

A safe, highly immunogenic multivalent vaccine to protect against all nine serotypes of African horse sickness virus (AHSV), will revolutionise the AHS vaccine industry in endemic countries and beyond. Plant-produced AHS virus-like particles (VLPs) and soluble viral protein 2 (VP2) vaccine candidates were developed that have the potential to protect against all nine serotypes but can equally well be formulated as mono- and bi-valent formulations for localised outbreaks of specific serotypes. In the first interferon α/ß receptor knock-out (IFNAR-/-) mice trial conducted, a nine-serotype (nonavalent) vaccine administered as two pentavalent (5 µg per serotype) vaccines (VLP/VP2 combination or exclusively VP2), were directly compared to the commercially available AHS live attenuated vaccine. In a follow up trial, mice were vaccinated with an adjuvanted nine-serotype multivalent VP2 vaccine in a prime boost strategy and resulted in the desired neutralising antibody titres of 1:320, previously demonstrated to confer protective immunity in IFNAR-/- mice. In addition, the plant-produced VP2 vaccine performed favourably when compared to the commercial vaccine. Here we provide compelling data for a nonavalent VP2-based vaccine candidate, with the VP2 from each serotype being antigenically distinguishable based on LC-MS/MS and ELISA data. This is the first preclinical trial demonstrating the ability of an adjuvanted nonavalent cocktail of soluble, plant-expressed AHS VP2 proteins administered in a prime-boost strategy eliciting high antibody titres against all 9 AHSV serotypes. Furthermore, elevated T helper cells 2 (Th2) and Th1, indicative of humoral and cell-mediated memory T cell immune responses, respectively, were detected in mouse serum collected 14 days after the multivalent prime-boost vaccination. Both Th2 and Th1 may play a role to confer protective immunity. These preclinical immunogenicity studies paved the way to test the safety and protective efficacy of the plant-produced nonavalent VP2 vaccine candidate in the target animals, horses.

Widespread Reassortment Contributes to Antigenic Shift in Bluetongue Viruses from South Africa.

Bluetongue (BT), a viral disease of ruminants, is endemic throughout South Africa, where outbreaks of different serotypes occur. The predominant serotypes can differ annually due to herd immunity provided by annual vaccinations using a live attenuated vaccine (LAV). This has led to both wild-type and vaccine strains co-circulating in the field, potentially leading to novel viral strains due to reassortment and recombination. Little is known about the molecular evolution of the virus in the field in South Africa. The purpose of this study was to investigate the genetic diversity of field strains of BTV in South Africa and to provide an initial assessment of the evolutionary processes shaping BTV genetic diversity in the field. Complete genomes of 35 field viruses belonging to 11 serotypes, collected from different regions of the country between 2011 and 2017, were sequenced. The sequences were phylogenetically analysed in relation to all the BTV sequences available from GenBank, including the LAVs and reference strains, resulting in the analyses and reassortment detection of 305 BTVs. Phylogenomic analysis indicated a geographical selection of the genome segments, irrespective of the serotype. Based on the initial assessment of the current genomic clades that circulate in South Africa, the selection for specific clades is prevalent in directing genome segment reassortment, which seems to exclude the vaccine strains and in multiple cases involves Segment-2 resulting in antigenic shift.

Potential link of single nucleotide polymorphisms to virulence of vaccine-associated field strains of lumpy skin disease virus in South Africa.

South Africa is endemic for lumpy skin disease and is therefore reliant on various live attenuated vaccines for the control and prevention of the disease. In recent years, widespread outbreaks of vaccine-like strains of lumpy skin disease virus (LSDV) were reported internationally, leading to an increase in the generation of full genome sequences from field isolates. In this study, the complete genomes of six LSDVs submitted during active outbreaks in the 1990s in South Africa were generated. Based on phylogenetic analysis, the six viruses clustered with vaccine strains in LSDV Subgroup 1.1 and are subsequently referred to as vaccine-associated. The genetic differences between the phenotypically distinct vaccine and vaccine-associated strains were 67 single nucleotide polymorphisms (SNPs). This study characterized the location and possible importance of each of these SNPs in their role during virulence and host specificity.

Complete Genome Sequences of Virus Strains Isolated from Bottle A of the South African Live Attenuated Bluetongue Virus Vaccine.

This is a report of the complete genome sequences of plaque-selected isolates of five virus strains included in bottle A of the South African Onderstepoort Biological Products commercial live attenuated bluetongue virus vaccine.

Phylogenetic Characterization of the Palyam Serogroup Orbiviruses.

The Palyam serogroup orbiviruses are associated with abortion and teratogenesis in cattle and other ruminants. Of the 13 different serotypes that have been identified, the full genome sequence of only one, Kasba, has been published. We undertook to perform Next Generation Sequencing (NGS) and phylogenetic analysis on 12 Palyam serotypes plus field isolates of the African serotypes in our possession. The Palyam serogroup was found to be most closely related to the African horse sickness virus group and showed the most distant evolutionary relationship to the equine encephalosis viruses (EEV). Amino acid sequence analysis revealed that the gene encoding VP7 was the most conserved within serotypes and VP2 and VP5 showed the highest degree of variation. A high degree of sequence identity was found for isolates from the same geographical region. The phylogenetic analysis revealed two clades where the African serotypes were all very closely related in one clade and the other clade contained the Australian and Asian serotypes and one African serotype, Petevo. It was evident from the sequence data that the geographical origin of Palyam serogroup viruses played an important role in the development of the different serotypes.

Effect of using frozen-thawed bovine semen contaminated with lumpy skin disease virus on in vitro embryo production.

Lumpy skin disease (LSD) is an important transboundary animal disease of cattle with significant economic impact because of the implications for international trade in live animals and animal products. LSD is caused by a Capripoxvirus, LSD virus (LSDV), and results in extensive hide and udder damage, fever and pneumonia. LSDV can be shed in semen of infected bulls for prolonged periods and transmitted venereally to cows at high doses. This study examined the effects of LSDV in frozen-thawed semen on in vitro embryo production parameters, including viral status of media and resulting embryos. Bovine oocytes were harvested from abattoir-collected ovaries and split into three experimental groups. After maturation, the oocytes were fertilized in vitro with frozen-thawed semen spiked with a high (HD) or a lower (LD) dose of LSDV, or with LSDV-free semen (control). Following day 7 and day 8 blastocyst evaluation, PCR and virus isolation were performed on all embryonic structures. After completing sufficient replicates to reach 1,000 inseminated oocytes, further in vitro fertilization (IVF) runs were performed to provide material for electron microscopy (EM) and embryo washing procedures. Overall, in vitro embryo yield was significantly reduced by the presence of LSDV in frozen-thawed semen, irrespective of viral dose. When semen with a lower viral dose was used, significantly lower oocyte cleavage rates were observed. LSDV could be detected in fertilization media and all embryo structures, when higher doses of LSDV were present in the frozen-thawed semen used for IVF. Electron microscopy demonstrated LSDV virions inside blastocysts. Following the International Embryo Transfer Society washing procedure resulted in embryos free of viral DNA; however, this may be attributable to a sampling dilution effect and should be interpreted with caution. Further research is required to better quantify the risk of LSDV transmission via assisted reproductive procedures.

Effect of semen processing methods on lumpy skin disease virus status in cryopreserved bull semen.

Lumpy skin disease is an economically important disease of cattle, caused by the lumpy skin disease virus (LSDV; Capripoxvirus). It has a variable clinical appearance but, in severely affected animals, is associated with extensive skin damage, pneumonia and death. The LSDV can be found in the semen of infected bulls for prolonged periods of time, from where it can be transmitted by mating or artificial insemination and cause clinical disease in heifers and cows. In this study, an ejaculate was collected from a LSDV seronegative bull and confirmed free from LSDV DNA by PCR. The ejaculate was split into a control sample (C), a sample spiked with a 4 log TCID50 dose of an LSDV isolate (HD) and a 103 dilution of the virus suspension (ND) and frozen routinely. Two straws from each of the different semen treatment groups (HD, ND and C) were subsequently thawed and subjected to swim-up, single layer centrifugation, Percoll® density gradient and a Percoll® density gradient with added trypsin. For one set of straws, semen quality variables were recorded, and viral DNA status determined using PCR; the other set was used for positive staining electron microscopy. Samples determined to be positive for LSDV DNA by PCR were then subjected to virus isolation (VI). Complete elimination of LSDV from semen did not occur with use of any of the processing methods. Trypsin did reduce the viral load, and eliminated LSDV from the ND sample, but severely negatively influenced semen quality. The LSDV virions, as assessed by electron microscopy, were associated with the sperm plasma membrane. Further investigation is needed to establish the efficacy of immuno-extenders for rendering semen free from LSDV.

Possible over-wintering of bluetongue virus in Culicoides populations in the Onderstepoort area, Gauteng, South Africa.

Several studies have demonstrated the ability of certain viruses to overwinter in arthropod vectors. The over-wintering mechanism of bluetongue virus (BTV) is unknown. One hypothesis is over-wintering within adult Culicoides midges (Diptera; Ceratopogonidae) that survive mild winters where temperatures seldom drop below 10 °C. The reduced activity of midges and the absence of outbreaks during winter may create the impression that the virus has disappeared from an area. Light traps were used in close association with horses to collect Culicoides midges from July 2010 to September 2011 in the Onderstepoort area, in Gauteng Province, South Africa. More than 500 000 Culicoides midges were collected from 88 collections and sorted to species level, revealing 26 different Culicoides species. Culicoides midges were present throughout the 15 month study. Nine Culicoides species potentially capable of transmitting BTV were present during the winter months. Midges were screened for the presence of BTV ribonucleic acid (RNA) with the aid of a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay. In total 91.2% of midge pools tested positive for BTV RNA. PCR results were compared with previous virus isolation results (VI) that demonstrated the presence of viruses in summer and autumn months. The results indicate that BTV-infected Culicoides vectors are present throughout the year in the study area. Viral RNA-positive midges were also found throughout the year with VI positive midge pools only in summer and early autumn. Midges that survive mild winter temperatures could therefore harbour BTV but with a decreased vector capacity. When the population size, biting rate and viral replication decrease, it could stop BTV transmission. Over-wintering of BTV in the Onderstepoort region could therefore result in re-emergence because of increased vector activity rather than reintroduction from outside the region.

An improved method for determining virucidal efficacy of a chemical disinfectant using an electrical impedance assay.

A major problem with the testing of virucidal efficacy using current protocols is that scoring of virus-induced cytopathic effect (CPE) is dependent on subjective visual interpretation using light microscopy. The current report details the use of an electrical impedance assay (xCELLigence, ACEA Biosciences) for its utility in virucidal efficacy testing. In this study, the xCELLigence system was used in a procedure developed from guidelines given by the Deutsche Vereiniging zur Bekämpfung der Viruskrankheiten (DVV) (German Association for the Control of Virus Diseases) in order to demonstrate the inactivation of infectious bursal disease virus using a commercial virucide. Although the modified DVV assay using the xCELLigence system yielded identical results (i.e. a 5-log10 reduction in viral infectivity) as the traditional DVV assay, the system allows virucidal efficacy and cytotoxicity to be measured in a more precise and reproducible fashion.

Culicoides species abundance and potential over-wintering of African horse sickness virus in the Onderstepoort area, Gauteng, South Africa.

In South Africa, outbreaks of African horse sickness (AHS) occur in summer; no cases are reported in winter, from July to September. The AHS virus (AHSV) is transmitted almost exclusively by Culicoides midges (Diptera: Ceratopogonidae), of which Culicoides imicola is considered to be the most important vector. The over-wintering mechanism of AHSV is unknown. In this study, more than 500 000 Culicoides midges belonging to at least 26 species were collected in 88 light traps at weekly intervals between July 2010 and September 2011 near horses in the Onderstepoort area of South Africa. The dominant species was C. imicola. Despite relatively low temperatures and frost, at least 17 species, including C. imicola, were collected throughout winter (June-August). Although the mean number of midges per night fell from > 50 000 (March) to < 100 (July and August), no midge-free periods were found. This study, using virus isolation on cell cultures and a reverse transcriptase polymerase chain reaction (RT-PCR) assay, confirmed low infection prevalence in field midges and that the detection of virus correlated to high numbers. Although no virus was detected during this winter period, continuous adult activity indicated that transmission can potentially occur. The absence of AHSV in the midges during winter can be ascribed to the relatively low numbers collected coupled to low infection prevalence, low virus replication rates and low virus titres in the potentially infected midges. Cases of AHS in susceptible animals are likely to start as soon as Culicoides populations reach a critical level.

Black-backed jackal exposure to rabies virus, canine distemper virus, and Bacillus anthracis in Etosha National Park, Namibia.

Canine distemper virus (CDV) and rabies virus (RABV) occur worldwide in wild carnivore and domestic dog populations and pose threats to wildlife conservation and public health. In Etosha National Park (ENP), Namibia, anthrax is endemic and generates carcasses frequently fed on by an unusually dense population of black-backed jackals (Canis mesomelas). Using serology, phylogenetic analyses (on samples obtained from February 2009-July 2010), and historical mortality records (1975-2011), we assessed jackal exposure to Bacillus anthracis (BA; the causal bacterial agent of anthrax), CDV, and RABV. Prevalence of antibodies against BA (95%, n = 86) and CDV (71%, n = 80) was relatively high, while that of antibodies against RABV was low (9%, n = 81). Exposure to BA increased significantly with age, and all animals >6 mo old were antibody-positive. As with BA, prevalence of antibodies against CDV increased significantly with age, with similar age-specific trends during both years of the study. No significant effect of age was found on the prevalence of antibodies against RABV. Three of the seven animals with antibodies against RABV were monitored for more than 1 yr after sampling and showed no signs of active infection. Mortality records revealed that rabid animals are destroyed nearly every year inside the ENP tourist camps. Phylogenetic analyses demonstrated that jackal RABV in ENP is part of the same transmission cycle as other dog-jackal RABV cycles in Namibia.

Molecular characterisation of Newcastle disease virus isolates from different geographical regions in Mozambique in 2005.

Newcastle disease (ND) is regarded as a highly contagious and economically important disease in poultry and has a worldwide distribution. Viral determinants for Newcastle disease virus (NDV) virulence are not completely understood and viruses of different pathotypes can be found at live-bird markets in different geographical areas. The prevalence of Newcastle disease in village poultry in Mozambique is not well documented and strains of NDV involved in yearly outbreaks are unknown. The fusion (F) protein is an important determinant of pathogenicity of the virus and is used commonly for phylogenetic analysis. Newcastle disease viruses from various geographical regions of Mozambique were sequenced and compared genetically to published sequences obtained from GenBank. Samples were collected in three different areas of Mozambique and NDV was isolated by infection of embryonated chicken eggs. Sequence analysis of the F-protein encoding gene was used to classify 28 isolates from Mozambique into genotypes and compare these genotypes phylogenetically with existing genotypes found in GenBank. The isolates obtained from Mozambique grouped mainly into two clades. In the first clade, 12 isolates grouped together with sequences of isolates representing genotypes from Mozambique that were previously described. In the second clade, 16 isolates group together with sequences obtained from GenBank originating from Australia, China, South Africa and the USA. Eleven of these isolates showed a high similarity with sequences from South Africa. The number of samples sequenced (n = 28), as well as the relatively small geographical collection area used in this study, are too small to be a representation of the circulating viruses in Mozambique in 2005. Viruses characterised in this study belonged to lineage 5b, a similar finding of a previous study 10 years ago. From this data, it merely can be concluded that no new introduction of the virus occurred from 1995 to 2005 in Mozambique.