Sheeppox Virus Strains Research Articles

Sheeppox virus genome sequences from the European outbreaks in Spain, Bulgaria, and Greece in 2022–2024

In 2022-2024, three outbreaks of sheeppox (SPP) were reported in the European Union. These occurred in Spain, Bulgaria, and Greece and had serious economic consequences due to animal losses and trade restrictions. Five sheeppox virus (SPPV) whole-genome sequences (WGSs) were determined from samples collected during these outbreaks and analyzed in the context of all other published WGSs. Sheeppox virus strains can be divided in two, or possibly three, main groups. The isolates from the recent outbreaks belong to clade A2, which includes strains historically circulating in the Middle East and Northern Africa. Sequence divergence was low among the isolates that caused the recent European outbreaks. These results highlight the need for more regular and dense surveillance in under-sampled areas and the use of WGS to increase the chance of pinpointing the origin of an introduction, identifying potential introduction routes, and providing insights into SPPV evolution.

The Serological Response in Cattle following Administration of a Heterologous Sheep Pox Virus Strain Vaccine for Protection from Lumpy Skin Disease; Current Situation in Armenia

Lumpy skin disease (LSD) is a highly infectious viral disease of cattle caused by LSD virus (LSDV), which was first reported in Armenia in late 2015. It was identified in pasture-raised cattle near the border with Iran. Currently, vaccination plays a key role in preventing further incursion of disease in high-risk areas. The purpose of this work was to assess the quality of vaccination currently used in Armenia by determining the immune response of the heterologous dry culture sheep pox virus-based vaccine against LSD in cattle. Seroprevalence and seroconversion testing was carried out using an ELISA to detect specific antibodies against LSD before and 30 days after vaccination in three adjacent regions of Armenia (Ararat, Armavir, Gegharkunik). Ixodes ticks were also examined for the presence of LSDV via real-time PCR. We found that the heterologous vaccine used in Armenia creates a high level of population immunity of 86.09% (83.83-87.97%) and no adverse side effects were observed in cattle. Of the 6 types of Ixodes ticks identified and tested, we found no evidence of LSDV circulating in these vectors. These results suggest that regular serological monitoring via ELISA and heterologous vaccination should continue in areas of Armenia at high risk for incursion of LSD to reduce the spread of this highly infectious transboundary disease.

Genetic characterization of sheep pox virus strains from outbreaks in Central Russia in 2018-2019.

This study investigates the phylogenomic relatedness between sheep pox viruses (SPPVs) circulating in Central Russia in 2018-2019 with the NISKHI vaccine strain used in the country, based on their complete genome sequences. The sheep pox outbreaks occurred 1 year apart in the adjacent regions of Tula and Moscow. Full genome sequences were generated by sequencing DNA directly obtained from Trizol-extracted scabs, using the DNBSEQ-400 platform (MGI Tech, China). Phylogenetic analysis indicated that the SPPV isolates from Russia clusters with previously published sequences from Srinagar in the Kashmir province of India in 2000 (SPPV-Srinagar strain) as well as SPPV A strain from Kazakhstan in 2000. The aforementioned cluster belonged to a sister clade containing the NISKHI vaccine strain, thus indicating that the recent outbreaks were not genetically linked to the widely used vaccine.

The complete genome sequence of Indian sheeppox vaccine virus and comparative analysis with other capripoxviruses

Sheeppox virus (SPPV) is responsible for a significant economic loss to sheep husbandry in enzootic regions of Africa, the Middle East, and Asia including the Indian subcontinent. In this study, we present the complete genome sequence of SPPV vaccine strain SPPV-Srin38/00 from India determined by next-generation sequencing (NGS) using Illumina technology. The attenuated Srinagar vaccine strain of SPPV (SPPV-Srin38/00) was developed by serial passaging the virus initially in lamb testes (LT) cells followed by Vero cell line. The SPPV-Srin38/00 virus has a genome size of 150, 103bp, which encodes for 147 functional putative genes and consists of a central coding region flanked by two identical 2353bp inverted terminal repeats (ITRs). Comparative phylogenetic analysis based on complete genome sequences of Capripoxviruses formed three distinct groups each for SPPV, GTPV, and LSDV with clustering of SPPV-Srin38/00 strain with SPPV-A strain. Nine ORFs of SPPV-Srin38/00 namely SPPV-Srin_002/SPPV-Srin_155, SPPV-Srin_004/SPPV-Srin_153, SPPV-Srin_009, SPPV-Srin_013, SPPV-Srin_026, SPPV-Srin_132, and SPPV-Srin_136 were found to be fragmented as compared to LSDV, whereas only one ORF (such as SPPV-Srin_136) was found to be fragmented as compared to GTPV. SPPV genomes, including the SPPV-Srin38/00 strain, shared 99.78-99.98% intraspecies nucleotide identity, indicating that SPPV strains have extremely low genetic diversity. The strain shared 96.80-97.08% and 97.11-97.61% nt identity with GTPV and LSDV strains, respectively. Its ORFs 016, 021, 022, 130 and 138 are the least identical ORFs among three species of the genus Capripoxvirus with 72.5-93% aa identity to GTPV and LSDV strains and may be potentially used for differentiation of CaPV species. This study may contribute to a better understanding of the epidemiology and evolution of capripoxviruses as well as the development of specific detection methods, better expression vectors, and vaccines with improved safety and efficacy.

Selection of a suitable viral DNA extraction method for Sheeppox virus in cell culture

A suitable method for the extraction of nucleic acids should be efficient, sensitive, rapid and simple. Moreover, ideally, good method should yield pure nucleic acid-free from any contaminant inhibitors. Several methods have been reported for viral deoxyribonucleic acid (DNA) isolation but limited information is available on quick and simple isolation of Sheeppox virus (SPPV) genomic DNA in cell culture. In this study, the healthy Vero cells and primary lamb testis cells were infected with SPPV strains such as SPPV-Jaipur, SPPV-Ranipet and SPPV-Roumanian Fanar (RF) and harvested when it exhibited clear cytopathic effect (CPE) in culture. Four different DNA extraction methods i.e., (i) Phenol/chloroform/Isoamyl alcohol method, (ii) Cell lysis buffer method, (iii) Proteinase-k method, and (iv) commercial nucleic acid extraction kit was used to extract optimum yield of viral genomic DNA from clarified culture supernatant of harvested SPPV virus. The DNA sample was characterized using the Nanodrop spectrophotometer and agarose gel electrophoresis. Significantly (p<0.05) higher yield of SPPV genomic DNA was obtained in proteinase-k method which was about 3-5 times more than other methods. Among these methods, proteinase-k protocol was found to be comparatively very effective method in terms of yield of viral genomic DNA, and was free from PCR inhibitors.

Genome-wide expression analysis reveal host genes involved in immediate-early infections of different sheeppox virus strains.

This study explored the transcriptome of lamb testis cells infected with sheeppox virus (SPPV) wild strain (WS) and vaccine strain (VS) at an immediate-early time. Most of the differentially expressed genes (DEGs) and differentially expressed highly connected (DEHC) gene network were found to be involved in SPPV-VS infection compared to SPPV-WS. Further, the signaling pathways were mostly involved in SPPV-VS infection than SPPV-WS. SPPV modulates the expression of several important host proteins such as CD40, FAS, ITGβ1, ITGα1, Pak1, Pak2, CD14, ILK leading to viral attachment and entry; immune-related DEGs such as MAPK, JNK, ERK, NFKB, IKB, PI3K, STAT which provide optimal cellular condition for early viral protein expression; and FOXO3, ATF, CDKNA1, TCF, SRF, BDNF which help in inducing apoptosis and MPTP, BAD and Tp53 inhibits apoptosis or cell death at the immediate-early time. The results captured the specific genes and enabled to understand distinct pathogenic mechanisms employed by VS and WS of SPPV.

Investigation of an outbreak of sheeppox among native sheep breeds in the Western Himalayas of India.

An outbreak of sheeppox was investigated in a cluster of villages situated in Western Himalayan ranges of a Northern Indian state. Non-migratory sheep (n = 80) of native breeds namely Gaddi and Rampur Bushair were infected and 15 have died. The outbreak started after a few animals contracted the disease during the summer grazing period at the highland pastures from migrating flocks of sheep. This initial outbreak resulted in a further spreading of the disease into the valley. Clinical examination revealed varying degree of cutaneous papular lesions and respiratory distress. Upon necropsy, visceral lesions in the lungs, trachea and kidneys were also found. Clinical and morbid samples were found positive for sheeppox virus using group specific P32 gene and I3L gene based multiplex PCR differentiating sheeppox and goatpox viruses. Histopathological, hematological and blood biochemical analysis also supported the pathology of an acute viral infection. The causative sheeppox virus strain was isolated using lamb testicular cell culture and phylogenetic analysis, based upon P32 and RPO30 genes, showed its clustering with other Indian strains reported from neighboring states. This study demonstrated the spread of sheeppox virus to new niches by migratory sheep flocks leading to establishment of endemic infections in many new pockets of higher Western Himalayas.

Establishment of a Challenge Model for Sheeppox Virus Infection.

Sheeppox virus (SPPV) together with goatpox virus and lumpy skin disease virus form the genus Capripoxvirus of the Poxviridae family. Due to their great economic importance and major impact on livelihood of small-scale farmers, OIE guidelines classify capripox viruses as notifiable diseases. In the present study, we examined pathogenesis of an Indian SPPV isolate and an Egyptian SPPV isolate in sheep. Three different infection routes were tested: (i) intravenous infection, (ii) intranasal infection and (iii) contact transmission between infected and naïve sheep. Clinical course, viremia and viral shedding as well as seroconversion were analyzed in order to establish a challenge model for SPPV infections that can be used in future vaccine studies. Next to in vivo characterization, both SPPV strains underwent next- and third-generation sequencing to obtain high quality full-length genomes for genetic characterization and comparison to already published SPPV sequences.

Experimental Infection and Genetic Characterization of Two Different Capripox Virus Isolates in Small Ruminants.

Capripox viruses, with their members “lumpy skin disease virus (LSDV)”, “goatpox virus (GTPV)” and “sheeppox virus (SPPV)”, are described as the most serious pox diseases of production animals. A GTPV isolate and a SPPV isolate were sequenced in a combined approach using nanopore MinION sequencing to obtain long reads and Illumina high throughput sequencing for short precise reads to gain full-length high-quality genome sequences. Concomitantly, sheep and goats were inoculated with SPPV and GTPV strains, respectively. During the animal trial, varying infection routes were compared: a combined intravenous and subcutaneous infection, an only intranasal infection, and the contact infection between naïve and inoculated animals. Sheep inoculated with SPPV showed no clinical signs, only a very small number of genome-positive samples and a low-level antibody reaction. In contrast, all GTPV inoculated or in-contact goats developed severe clinical signs with high viral genome loads observed in all tested matrices. Furthermore, seroconversion was detected in nearly all goats and no differences concerning the severity of the disease depending on the inoculation route were observed. Conclusively, the employed SPPV strain has the properties of an attenuated vaccine strain, consistent with the genetic data, whereas the GTPV strain represents a highly virulent field strain.

Field study on the use of vaccination to control the occurrence of lumpy skin disease in Ethiopian cattle

The current study was carried out in central and North-western parts of Ethiopia to assess the efficacy of Kenyan sheep pox virus strain vaccine (KS1 O-180) against natural lumpy skin disease (LSD) infection under field conditions by estimating its effect on the transmission and severity of the disease. For this study, an LSD outbreak was defined as the occurrence of at least one LSD case in a specified geographical area. An observational study was conducted on a total of 2053 (1304 vaccinated and 749 unvaccinated) cattle in 339 infected herds located in 10 sub-kebeles and a questionnaire survey was administered to 224 herd owners. Over 60% of the herd owners reported that the vaccine has a low to very low effect in protecting animals against clinical LSD; almost all of them indicated that the vaccine did not induce any adverse reactions. In the unvaccinated group of animals 31.1% were diagnosed with LSD while this was 22.5% in the vaccinated group (P<0.001). Severity of the disease was significantly reduced in vaccinated compared to unvaccinated animals (OR=0.68, 95% CI: 0.49; 0.96). Unvaccinated infected animals were more likely (predicted fraction=0.89) to develop moderate and severe disease than vaccinated infected animals (predicted fraction=0.84).LSD vaccine efficacy for susceptibility was estimated to be 0.46 (i.e. a susceptibility effect of 0.54) while the infectiousness effect of the vaccine was 1.83. In other words, the vaccine reduces the susceptibility by a factor of two and increases infectiousness by approximately the same amount. LSD transmission occurred in both vaccinated and unvaccinated animals, the estimated reproduction ratio (R) was 1.21 in unvaccinated animals compared to 1.19 in vaccinated ones, and not significantly different. In conclusion, KS1 O-180 vaccination, as applied currently in Ethiopia, has poor efficacy in protecting cattle populations against LSD, neither by direct clinical protection nor by reducing transmission, and this signifies the urgent need to either improve the quality of the vaccine or to develop potent alternative vaccines that will confer good protection against LSD.

Investigation of a Possible Link Between Vaccination and the 2010 Sheep Pox Epizootic in Morocco

Sheep pox is endemic in most parts of Northern Africa and has the potential to cause severe economic problems. Live attenuated vaccines are used in Morocco, and in many other countries, to control the disease. Sheep pox virus (SPPV) re-appeared in 2010 causing a nodular clinical form previously not observed in Morocco. The severe clinical signs observed during the course of this outbreak and initial reports citing similarity in nucleotide sequence between the Moroccan vaccine strain and field isolates warranted a more in depth analysis of this epizootic. In this study, sequence analysis showed that isolates obtained from four provinces of eastern Morocco were identical, demonstrating that a single SPPV strain was responsible for the 2010 epizootic. In addition, the genome fragments sequenced and phylogenetic analyses undertaken as part of this study showed significant differences between field isolates and the Moroccan vaccine strain. New PCR methods were developed to differentiate between wild-type isolates and vaccine strains of SPPV. Using these methods, no trace of wild-type SPPV was found in the vaccine and no evidence was found to suggest that the vaccine strain was causing clinical disease.

Process intensification for an enhanced replication of a newly adapted RM-65 sheep pox virus strain in Vero cells grown in stirred bioreactor

Sheep pox virus initially adapted to replicate in primary lamb kidney cells was adapted to Vero cells by serial passages in monolayer cultures. After nine passages the virus was able to correctly replicate in Vero cells, virus titer achieved was 105.875 TCID50 (median tissue culture infective dose)ml−1.To optimize the production process, the effects of MOI (multiplicity of infection), TOI (time of infection) and the culture medium were investigated. Cell infection at a MOI of 0.005 concurrently with cell seeding showed the best results in terms of specific virus productivity. The effect of MEM enrichment with several components was investigated using the experimental design approach. 67 experiments were performed in 6-well plates to select the best combination. The highest titer was achieved when MEM was supplemented with 5mM glucose, 5mM fructose and 25mM sucrose. Spinner culture confirms these data; virus titer was 107.375 TCID50ml−1.In addition Vero cells were cultivated in a 7-l bioreactor in batch mode on 3gl−1 Cytodex1, and infected at cell seeding at a MOI of 0.005. Maximal virus titer was 107.275 TCID50ml−1. This corresponds to 44-fold factor enhancement compared to spinner cultures conducted in MEM+2% FCS.

PCR-based assay to detect sheeppox virus in ocular, nasal, and rectal swabs from infected Moroccan sheep

Sheeppox is now enzootic in Morocco. The development of a reliable method for rapid diagnosis of the disease is a central part of any control strategy. The aim of this study is to determine the diagnostic value of a variety of clinical samples such as ovine nasal, ocular or rectal swabs for the detection of sheeppox virus (SPPV) by qualitative conventional polymerase chain reaction (PCR), using a single pair of primers targeting the inverted terminal repeats of the SPPV InS-1 strain, a virulent field isolate. Swab and blood samples were collected from forty animals naturally infected with SPPV who had clinical signs of sheeppox. All animals tested PCR-positive for SPPV. Positive results were obtained infrequently with blood samples, whereas swab samples from at least two sites (nasal, ocular, rectal) were positive per evaluated animal. These results indicate that swab samples are suitable for quantitative molecular SPPV diagnosis. PCR product sequences obtained from all types of sheep samples proved to be identical to the corresponding regions of sheeppox virus strain Romania 65.

A description of two outbreaks of capripoxvirus disease in Mongolia

Mongolia had no reported cases of capripoxvirus disease from 1977 until an outbreak of sheeppox in 2006–2007 and then goatpox in 2008. The two outbreaks occurred in geographically distant areas of Mongolia and, most strikingly, were highly species-specific. The 2006–2007 sheeppox outbreak affected no goats and the 2008 goatpox outbreak affected no sheep despite communal herding. The diseases were diagnosed using the polymerase chain reaction and virus neutralisation test. The P32 gene of the Mongolian sheeppox and goatpox viruses from the recent outbreaks were sequenced and compared with an archived 1967 strain of Goatpox virus from Mongolia. The P32 gene of the 2006–2007 Mongolian Sheeppox virus strain was identical to previously published sheeppox strains. The P32 gene of the 2008 Mongolian Goatpox virus strain was identical to the gene from virus isolated from recent goatpox outbreaks in China and Vietnam. The archived Mongolian Goatpox virus strain was unique.

Cytopathic and immunogenic studies of sheep pox virus serially cultivated in cell culture

Lamb kidney (LK) and lamb testis (LT) cell cultures of serially passaged sheep pox virus (SPV) were examined for their cytopathogenicity and immunogenicity. All 3 SPV strains used behaved similarly in both traits. While the CPE was examined at low (4th), mid (18th) and high (40th) passages, the low and mid-passaged LT virus produced cytological changes almost identical to those of the LK virus. With acridine orange staining the cytoplasmic inclusions, seen in infected cells stained by May-Grunwald-Giemsa or HE, gave a greenish-yellow fluorescence suggestive of viral DNA and intracytoplasmic replication. In contrast, the high-passaged LT virus induced unusual intranuclear inclusions without any change in the cytoplasm. With acridine orange these inclusions exhibited a greenish-yellow fluorescence indicative of viral DNA and intranuclear replication. In tests on the immunity conferred by high LK cell culture passaged virus, immunized sheep resisted challenge, whereas sheep given high-LT cell culture passaged virus developed a generalized infection after challenge. It is concluded that the virus used for immunization should possess the property of intracytoplasmic replication.

A new freeze-dried living virus vaccine against sheep-pox

A sheep-pox virus strain has been adapted and multiplied in primary lamb kidney cell cultures. The main characteristics of the strain have been verified in vitro after clones were isolated, and the results confirmed its identity. The safety and the potency of the strain have been investigated in sheep. The inoculation of the strain to sheep was followed by a post-vaccinal reaction materialised by a nodule at the site of inoculation and an increase of temperature by about 1°C. No reactions adversely affecting pregnancy have been noted. Immunisation was demonstrated by an increase in the level of neutralising serous antibodies and protection against the pathogenic virus. The immunity tended to decrease during the second year after primovaccination and a yearly booster vaccination appeared to be necessary. Primovaccination of lambs over 2 months of age produced a better immunity, especially when the lambs were born from vaccinated ewes. This strain forms the active principle of a freeze-dried vaccine containing no adjuvant of the immunity.