Lumpy skin disease virus (LSDV) has recently undergone rapid spread, now being reported from more than 80 countries, affecting predominantly cattle and to a lesser extent, water buffalo. This poxvirus was previously considered to be highly host-range restricted. However, there is an increasing number of published reports on the detection of the virus from different game animal species. The virus has not only been shown to infect a wide range of game species under experimental conditions, but has also been naturally detected in oryx, giraffe, camels and gazelle. In addition, clinical lumpy skin disease has previously been described in springbok (Antidorcas marsupialis), an African antelope species, in South Africa. This report describes the characterization of lumpy skin disease virus belonging to cluster 1.2, from field samples from springbok, impala (Aepyceros melampus) and a giraffe (Giraffa camelopardalis) in South Africa using PCR, Sanger and whole genome sequencing. Most of these samples were submitted from wild animals in nature reserves or game parks, indicating that the disease is not restricted to captive-bred animals on game farms or zoological gardens. The potential role of wildlife species in the transmission and maintenance of LSDV is further discussed and requires continuing investigation, as the virus and disease may pose a serious threat to endangered species.

Capripoxviruses are the causative agents of sheeppox, goatpox, and lumpy skin disease (LSD) in cattle, which cause economic losses to the livestock industry in Africa and Asia. Capripoxviruses are currently controlled using several live attenuated vaccines. It was previously demonstrated that a lumpy skin disease virus (LSDV) field isolate from Warmbaths (WB) South Africa, ORF 005 (IL-10) gene-deleted virus (LSDV WB005KO), was able to protect sheep and goats against sheeppox and goatpox. Subsequently, genes encoding the protective antigens for peste des petits ruminants (PPR) and Rift Valley fever (RVF) viruses have been inserted in the LSDV WB005KO construct in three different antigen forms (native, secreted, and fusion). These three multivalent vaccine candidates were evaluated for protection against PPR using a single immunization of 104 TCID50 in sheep. The vaccine candidates with the native and secreted antigens protected sheep against PPR clinical disease and decreased viral shedding, as detected using real-time RT-PCR in oral and nasal swabs. An anamnestic antibody response, measured using PPR virus-neutralizing antibody response production, was observed in sheep following infection. The vaccine candidates with the antigens expressed in their native form were evaluated for protection against RVF using a single immunization with doses of 104 or 105 TCID50 in sheep and goats. Following RVF virus infection, sheep and goats were protected against clinical disease and no viremia was detected in serum compared to control animals, where viremia was detected one day following infection. Sheep and goats developed RVFV-neutralizing antibodies prior to infection, and the antibody responses increased following infection. These results demonstrate that an LSD virus-vectored vaccine candidate can be used in sheep and goats to protect against multiple viral infections.

Lumpy skin disease is recognized as a transboundary and emerging disease of cattle, buffaloes and other wild ruminants. Being initially restricted to Africa, and since 1989 the Middle East, the unprecedented recent spread across Eurasia demonstrates how underestimated and neglected this disease is. The initial identification of the causative agent of LSD as a poxvirus called LSD virus, was well as findings on LSDV transmission and epidemiology were pioneered at Onderstepoort, South Africa, from as early as the 1940s by researchers such as Weiss, Haig and Alexander. As more data emerges from an ever-increasing number of epidemiological studies, previously emphasized research gaps are being revisited and discussed. The currently available knowledge is in agreement with the previously described South African research experience that LSDV transmission can occur by multiple routes, including indirect contact, shared water sources and arthropods. The virus population is prone to molecular evolution, generating novel phylogenetically distinct variants resulting from a diverse range of selective pressures, including recombination between field and homologous vaccine strains in cell culture that produce virulent recombinants which pose diagnostic challenges. Host restriction is not limited to livestock, with certain wild ruminants being susceptible, with unknown consequences for the epidemiology of the disease.

Since 1989, lumpy skin disease of cattle (LSD) has spread out of Africa via the Middle East northwards and eastwards into Russia, the Far East and South-East Asia. It is now threatening to become a worldwide pandemic, with Australia possibly next in its path. One of the research gaps on the disease concerns its main mode of transmission, most likely via flying insect vectors such as biting flies or mosquitoes. Direct or indirect contact transmission is possible, but appears to be an inefficient route, although there is evidence to support the direct contact route for the newly detected recombinant strains first isolated in Russia. In this study, we used experimental bulls and fed them via virus-inoculated feed to evaluate the indirect contact route. To provide deeper insights, we ran two parallel experiments using the same design to discover differences that involved classical field strain Dagestan/2015 LSDV and recombinant vaccine-like Saratov/2017. Following the attempted indirect contact transmission of the virus from the inoculated feed via the alimentary canal, all bulls in the Dagestan/2015 group remained healthy and did not seroconvert by the end of the experiment, whereas for those in the Saratov/2017 recombinant virus group, of the five bulls fed on virus-inoculated feed, three remained clinically healthy, while two displayed evidence of a mild infection. These results provide support for recombinant virus transmission via the alimentary canal. In addition, of particular note, the negative control in-contact bull in this group exhibited a biphasic fever at days 10 and 20, developed lesions from day 13 onwards, and seroconverted by day 31. Two explanations are feasible here: one is the in-contact animal was somehow able to feed on some of the virus-inoculated bread left over from adjacent animals, but in the case here of the individual troughs being used, that was not likely; the other is the virus was transmitted from the virus-fed animals via an airborne route. Across the infected animals, the virus was detectable in blood from days 18 to 29 and in nasal discharge from days 20 to 42. Post-mortem and histological examinations were also indicative of LSDV infection, supporting further evidence for rapid, in F transmission of this virus. This is the first report of recombinant LSDV strain transmitting via the alimentary mode.

Research into the phylogenetic relationships of lumpy skin disease virus (LSDV) strains was long overlooked, partially due to its original restricted distribution to sub-Saharan Africa. However, recent incursions into northern latitudes, and a rapid spread causing major economic losses worldwide, have intensified additional research on the disease and the causative virus. This study delineates the phylogeny of LSDV in the context of full genome sequences of strains recovered in the field, as well as strains highly passaged in cell culture. We sequenced the oldest known field strain to date (isolate LSDV/Haden/RSA/1954 [South Africa] recovered from an outbreak in 1954), a recent field isolate (LSDV/280-KZN/RSA/2018 [South Africa] sequenced directly from blood during an outbreak in 2018) and strain LSDV/Russia/Dagestan-75 (a high-passaged cell culture strain derived from the field strain, LSDV/Russia/Dagestan/2015 [Russia]). Sequence analysis placed the field strain LSDV/Haden/RSA/1954 in the same cluster (cluster 1.1) with attenuated Neethling-type commercial vaccine viruses, with eight SNP differences, discrediting the previously held hypothesis that cluster 1.1 vaccine strains were derived from cluster 1.2 field viruses via the process of attenuation between them. In contrast, the recent LSDV/280-KZN/RSA/2018 isolate grouped with other recent field isolates in cluster 1.2, providing evidence that cluster 1.1 strains were displaced by cluster 1.2 strains in South Africa. Based on the field isolates between 1954 and 2018, the substitution rate of 7.4 × 10-6 substitutions/site/year was established, with mutations occurring in either synonymous sites or intergenic regions. This is the first evolutionary metric recorded for LSDV. Comparing the genome sequences of high-passage strains of LSDV showed that propagation in vitro without animal host selective pressure generates mainly non-synonymous SNPs in virus-replication genes. These results improve our understanding of LSDV evolution and demonstrate that the population dynamics of circulating isolates is not constant, with LSDV associated with different genetic clusters dominating the landscape during specific periods in time.

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.

Lumpy skin disease and Rift Valley fever are two high-priority livestock diseases which have the potential to spread into previously free regions through animal movement and/or vectors, as well as intentional release by bioterrorists. Since the distribution range of both diseases is similar in Africa, it makes sense to use a bivalent vaccine to control them. This may lead to the more consistent and sustainable use of vaccination against Rift Valley fever through a more cost-effective vaccine. In this study, a recombinant lumpy skin disease virus was constructed in which the thymidine kinase gene was used as the insertion site for the Gn and Gc protective glycoprotein genes of Rift Valley fever virus using homologous recombination. Selection markers, the enhanced green fluorescent protein and Escherichia coli guanidine phosphoribosyl transferase (gpt), were used for selection of recombinant virus and in a manner enabling a second recombination event to occur upon removal of the gpt selection-pressure allowing the removal of both marker genes in the final product. This recombinant virus, LSD-RVF.mf, was selected to homogeneity, characterized and evaluated in cattle as a vaccine to show protection against both lumpy skin disease and Rift Valley fever in cattle. The results demonstrate that the LSD-RVF.mf is safe, immunogenic and can protect cattle against both diseases.

The transmission of “lumpy skin disease virus” (LSDV) has prompted intensive research efforts due to the rapid spread and high impact of the disease in recent years, especially in Eastern Europe and Balkan countries. In this study, we experimentally evaluate the vaccine-derived virulent recombinant LSDV strain (Saratov/2017) and provide solid evidence on the capacity of the virus for transmission in a vector-proof environment. In the 60-day long experiment, we used inoculated bulls (IN group) and two groups of in-contact animals (C1 and C2), with the former (C1) being in contact with the inoculated animals at the onset of the trial and the latter (C2) being introduced at day 33 of the experiment. The infection in both groups of contact animals was confirmed clinically, serologically and virologically, and viremia was demonstrated in blood, nasal and ocular excretions, using molecular tools. Further studies into LSDV biology are a priority to gain insights into whether the hypothesized indirect contact mode evidenced in this study is a de novo-created feature, absent from both parental stains of the novel (recombinant) LSDV isolate used, or whether it was dormant, but then unlocked by the process of genetic recombination. Author summary: In global terms, LSD has been termed a “neglected disease” due to its historic natural occurrence of being restricted to Africa and, occasionally, Israel. However, after its slow spread throughout the Middle East, the disease is now experiencing a resurgence of research interest following a recent and rapid spread into more northern latitudes. Given the dearth of solid findings on potential transmission mechanisms, no efficient or reliable control program currently exists, which does not involve the use of live attenuated vaccines or stamping out policies – both of which are controversial for implementation in non-endemic regions or countries. The vector-borne mode is the only working concept currently available, but with scarce evidence to support the aggressive spread northwards – except for human-assisted spread, including legal or illegal animal transportation. The emergence of outbreaks is not consistently linked to weather conditions, with the potential for new outbreaks to occur and spread rapidly. Here, for the first time, we provide evidence for indirect contact-mode transmission for a naturally-occurring recombinant LSDV isolated from the field. In an insect-proof facility, we obtained solid evidence that the novel LSDV strain can pass to in-contact animals. Given the recombinant nature of the virus utilised, its genetic background relating to the observed transmission pattern within the study needs to be delineated.

Wide spread incidences of vaccine-like strains of lumpy skin disease virus (LSDV) have recently been reported in a Russian region with a neighboring country that actively vaccinate with a live attenuated LSD vaccine. The use of live-attenuated viruses (LAVs) as vaccines during an active outbreak, creates potential ground for coinfection of hosts and emergence of a strain combining genetic fragments of both parental vaccine and field strains. In this study, we analyse the vaccine-like strain LSDV RUSSIA/Saratov/2017 detected in Saratovskaya oblast, a region sharing border with Kazakhstan. To gain insight into possible recombination signals, a full-genome next-generation sequencing of the viral genome was performed using the Illumina platform. The genome contains the backbone of a live-attenuated vaccine with a patchwork of wild-type field virus DNA fragments located throughout. A total of 27 recombination events were identified. The average distance between the recombination sites was 3400 base pairs (bp). The impact of the recombination events on the virulence and transmission capacity of the identified virus remains to be clarified. These findings provide evidence for the first time of genetic exchanges between closely related strains of capripoxviruses in the field and a vaccine strain, and prompt a revisiting of the vaccination issue for a safe and efficacious prevention and control strategy of LSD.

Lumpy skin disease virus (LSDV) is responsible for causing severe economic losses to cattle farmers throughout Africa, the Middle East, and more recently, South-Eastern Europe and Russia. It belongs to the Capripoxvirus genus of the Poxviridae family, with closely related sheeppox and goatpox viruses. Like other poxviruses, the viral genome codes for a number of genes with putative immunomodulatory capabilities. Current vaccines for protecting cattle against lumpy skin disease (LSD) based on live-attenuated strains of field isolates passaged by cell culture, resulting in random mutations. Although generally effective, these vaccines can have drawbacks, including injection site reactions and/or limited immunogenicity. A pilot study was conducted using a more targeted approach where two putative immunomodulatory genes were deleted separately from the genome of a virulent LSDV field isolate. These were open reading frame (ORF) 005 and ORF008, coding for homologues of an interleukin 10-like and interferon-gamma receptor-like gene, respectively. The resulting knockout constructs were evaluated in cattle for safety, immunogenicity and protection. Severe post-vaccinal reactions and febrile responses were observed for both constructs. Two calves inoculated with the ORF008 knockout construct developed multiple lesions and were euthanised. Following challenge, none of the animals inoculated with the knockout constructs showed any external clinical signs of LSD, compared to the negative controls. Improved cellular and humoral immune responses were recorded in both of these groups compared to the positive control. The results indicate that at the high inoculation doses used, the degree of attenuation achieved was insufficient for further use in cattle due to the adverse reactions observed.

Sheep and goat pox continue to be important livestock diseases that pose a major threat to the livestock industry in many regions in Africa and Asia. Currently, several live attenuated vaccines are available and used in endemic countries to control these diseases. One of these is a partially attenuated strain of lumpy skin disease virus (LSDV), KS-1, which provides cross-protection against both sheep pox and goat pox. However, when used in highly stressed dairy cattle to protect against lumpy skin disease (LSD) the vaccine can cause clinical disease. In order to develop safer vaccines effective against all three diseases, a pathogenic strain of LSDV (Warmbaths [WB], South Africa) was attenuated by removing a putative virulence factor gene (IL-10-like) using gene knockout (KO) technology. This construct (LSDV WB005KO) was then evaluated as a vaccine for sheep and goats against virulent capripoxvirus challenge. Sheep and goats were vaccinated with the construct and the animals were observed for 21days. The vaccine appeared to be safe, and did not cause disease, although it induced minor inflammation at the injection site similar to that caused by other attenuated sheep and goat pox vaccines. In addition, no virus replication was detected in blood, oral or nasal swabs using real-time PCR following vaccination and low levels of neutralising antibodies were detected in both sheep and goats. Leukocytes isolated from vaccinated animals following vaccination elicited capripoxvirus-specific IFN-γ secretion, suggesting that immunity was also T-cell mediated. Following challenge with virulent capripoxvirus, vaccinated sheep and goats were found to be completely protected and exhibited no clinical disease. Furthermore, real-time PCR of blood samples at various time points suggested that viremia was absent in both groups of vaccinated animals, as opposed to capripoxvirus-related clinical disease and viremia observed in the unvaccinated animals. These findings suggest that this novel knockout strain of LSDV has potential as a vaccine to protect livestock against sheep pox and goat pox.

Lumpy skin disease (LSD) is a highly infectious disease of cattle caused by a virus belonging to the Capripoxvirus genus of the family Poxviridae. The purpose of this study is to place on record the first confirmation of LSD in the Sultanate. The disease was diagnosed and confirmed using polymerase chain reaction, histopathology, transmission electron microscopy and serum neutralization testing. The epizootic occurred in 2009 involving a large number of animals and covering a wide area including Nezwa, Alqabel, Sohar, Saham and Burimi. Morbidity and mortality rates of 29.7 and 26.3 %, and 13.6 and 15.4 % were observed at Nezwa and Sohar, respectively. The clinical signs were much more severe in Holstein–Friesian cattle compared to indigenous breeds and were characterized by multiple skin nodules covering the neck, back, perineum, tail, limbs and genital organs. Affected animals also exhibited lameness, emaciation and cessation of milk production. Oedema of limbs and brisket, and superficial lymph node enlargement were highly prominent. It is not known from where the virus originated, or how it spread to the Sultanate. The disease has become endemic in the country and is liable to extend to other Gulf Cooperation Council Countries and cause a pandemic. It is of major concern to the Omani dairy industry. Due to the widespread presence of screw worm, serious economic losses can follow outbreaks.

Peste des petits ruminants (PPR) is a viral disease which primarily affects small ruminants, causing significant economic losses for the livestock industry in developing countries. It is endemic in Saharan and sub-Saharan Africa, the Middle East and the Indian sub-continent. The primary hosts for peste des petits ruminants virus (PPRV) are goats and sheep; however recent models studying the pathology, disease progression and viremia of PPRV have focused primarily on goat models. This study evaluates the tissue tropism and pathogenesis of PPR following experimental infection of sheep and goats using a quantitative time-course study. Upon infection with a virulent strain of PPRV, both sheep and goats developed clinical signs and lesions typical of PPR, although sheep displayed milder clinical disease compared to goats. Tissue tropism of PPRV was evaluated by real-time RT-PCR and immunohistochemistry. Lymph nodes, lymphoid tissue and digestive tract organs were the predominant sites of virus replication. The results presented in this study provide models for the comparative evaluation of PPRV pathogenesis and tissue tropism in both sheep and goats. These models are suitable for the establishment of experimental parameters necessary for the evaluation of vaccines, as well as further studies into PPRV-host interactions.

•We have developed a 4 color or 4-plex real-time PCR assay for RVFV RNA.•Two targets are confirmatory for presence of RVFV RNA.•One target allows differentiation of infected from vaccinated animals for NSs deleted vaccines.•One target is an exogenous RNA amplification control.•Assay proven to be specific and robust.

Rift Valley fever virus (RVFV) infects humans and livestock, causing haemorrhaging and abortions in animals. Three major RVF epizootics have occurred in South Africa since the 1950s and the outbreak in 2010 had a mortality rate of 10.7% in humans. Accurate and early detection is therefore essential for management of this zoonotic disease. Enzyme-linked immunosorbent assays (ELISAs) have been developed for the detection of either IgM or IgG antibodies to RVFV in animal sera. In this study, data are presented on the validation of a double-antigen ELISA for the simultaneous detection of both classes of antibodies to RVFV ina single test. ELISA plates were coated with a recombinant nucleoprotein. The nucleoprotein,conjugated to horseradish peroxidase, was used as the detecting reagent. A total of 534 sera from sheep and cattle were used in the validation. The sheep sera were collected during a RVF pathogenesis study at the Agricultural Research Council (ARC) - Onderstepoort Veterinary Institute and the cattle sera were collected during an outbreak of RVF in 2008 at the ARC -Animal Production Institute in Irene, Pretoria. The ELISA had a diagnostic sensitivity of 98.4% and a specificity of 100% when compared to a commercial cELISA. This convenient and fast assay is suitable for use in serological surveys or monitoring immune responses in vaccinated animals.

Arthropod-Borne Animal Diseases Research Unit, Agricultural Research Service, United States Department of Agriculture, Manhattan, KS 66502, USA Biotechnology Building, ARC-Onderstepoort Veterinary Institute, P/Bag X5, Onderstepoort 0110, South Africa National Centre for Foreign Animal Disease, Canadian Food Inspection Agency, 1015 Arlington Street, Winnipeg, Manitoba, Canada Biotechnology Center, Kenya Agricultural Research Institute, Off Waiyaki Way, P.O. Box 57811-00200, City Square, Nairobi, Kenya Department Veterinary Tropical Diseases, Veterinary Faculty, University of Pretoria, P/Bag X4, Onderstepoort 0110, South Africa Special Viral Pathogens Laboratory, Center for Emerging and Zoonotic Diseases, National Institute for Communicable Diseases, A Division of the National ealth Laboratory Service, Private Bag X4, Sandringham 2131, South Africa