Seroprevalence and risk factors of sheep and goat pox virus in selected districts of Wolaita Zone, Southern Ethiopia.

IMPORTANCE: Sheep and goat pox (SGP) virus infection is a highly fatal viral infection of small ruminants that causes major production losses in sheep and goats in Ethiopia while also limiting international trade. OBJECTIVE: This study aimed to estimate the seroprevalence of SGP infection and assess related risk variables. METHODS: A cross-sectional study was conducted from February to August 2023 on 384 serum samples taken from sheep and goats. A serum neutralization test was conducted to detect the presence of antibodies against the SGP virus in Wolaita Sodo Regional Laboratory. RESULTS: The overall seroprevalence rate of SGP was 4.95%. Factors such as sheep (8.26%), female sheep and goats (7.45%), older sheep and goats (8.33%), larger flock size of sheep and goats (10.47%), poorly conditioned sheep and goats (31.58%), sheep and goats with a tick on their skin (10.38%), and animals that had not been vaccinated (5.17%) were found to have higher seroprevalence. Furthermore, the seropositivity in sheep was five times greater than in goats (adjusted odds ratio [AOR], 4.73; 95% confidence interval [CI], 1.39-15.99). Additionally, large-sized flocks of sheep and goats were more likely to be seropositive to pox disease than small-sized flocks (AOR, 6.73; 95% CI, 1.58-28.67). CONCLUSIONS AND RELEVANCE: Thus, the study revealed the prevalence of SGP in the Wolaita zone. Additional research should be conducted to estimate the extent of the disease at the regional level, and management measures should be implemented to reduce the economic losses associated with this condition.

Epidemiological exploration of fleas and molecular identification of flea-borne viruses in Egyptian small ruminants.

The study aimed to investigate molecularly the presence of flea-borne viruses in infested small ruminants with fleas. It was carried out in Egypt's Northern West Coast (NWC) and South Sinai Governorate (SSG). Three specific primers were used targeting genes, ORF103 (for Capripoxvirus and Lumpy skin disease virus), NS3 (for Bluetongue virus), and Rdrp (for Coronavirus), followed by gene sequencing and phylogenetic analyses. The results revealed that 78.94% of sheep and 65.63% of goats were infested in the NWC area, whereas 49.76% of sheep and 77.8% of goats were infested in the SSG region. Sheep were preferable hosts for flea infestations (58.9%) to goats (41.1%) in the two studied areas. Sex and age of the animals had no effects on the infestation rate (p > 0.05). The season and site of infestation on animals were significantly different between the two areas (p < 0.05). Ctenocephalides felis predominated in NWC and Ctenocephalides canis in SSG, and males of both flea species were more prevalent than females. Molecular analysis of flea DNA revealed the presence of Capripoxvirus in all tested samples, while other viral infections were absent. Gene sequencing identified three isolates as sheeppox viruses, and one as goatpox virus. The findings suggest that Capripoxvirus is adapted to fleas and may be transmitted to animals through infestation. This underscores the need for ongoing surveillance of other pathogens in different regions of Egypt.

Lessons Learned from Active Clinical and Laboratory Surveillance during the Sheep Pox Virus Outbreak in Spain, 2022-2023.

In September 2022, more than 50 years after its eradication from Spain, Sheep pox virus was confirmed by laboratory analysis in sheep showing characteristic lesions. This was the start of an outbreak that lasted 9 months and infected 30 farms dispersed over two different areas, Andalusia and Castilla-La Mancha. Early after the initial confirmation, an active surveillance based on clinical inspection with laboratory confirmation of sheep with clinical signs was started in restricted areas. This allowed the confirmation of Sheep pox in 22 out of 28 suspected farms, where limited numbers of sheep with mainly erythema and papules were found, indicative of early detection. Nevertheless, to improve active surveillance and stop the outbreak, clinical inspection was reinforced by laboratory analysis in all inspected farms, even when no clinically diseased sheep were detected. Although more than 35,000 oral swabs from 335 farms were analysed by real-time PCR in pools of five, only two out of six reported outbreaks in this period were detected by laboratory analysis before clinical signs were observed. Furthermore, additional insights were gained from the extensive laboratory surveillance performed on samples collected under field conditions. No evidence of Sheep pox virus infection was found in goats. Oral swabs proved to be the sample of choice for early detection in the absence of scabs and could be tested in pools of five without extensive loss in sensitivity; serology by ELISA was not useful in outbreak detection. Finally, a non-infectious genome of the virus could be detected months after cleaning and disinfection; thus, real-time PCR results should be interpreted with caution in sentinel animals during repopulation. In conclusion, the outbreak of Sheep pox virus in Spain showed that active clinical inspection with laboratory confirmation of clinically diseased sheep via oral swab testing proved a sensitive method for detection of infected farms, providing insights in laboratory surveillance that will be helpful for other countries confronted with Sheep pox outbreaks.

Different Neutralizing Antibody Responses of Heterologous Sera on Sheeppox and Lumpy Skin Disease Viruses.

Sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV) are the three members of the genus Capripoxvirus within the Poxviridae family and are the etiologic agents of sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD), respectively. LSD, GTP, and SPP are endemic in Africa and Asia, causing severe disease outbreaks with significant economic losses in livestock. Incursions of SPP and LSD have occurred in Europe. Vaccination with live attenuated homologous and heterologous viruses are routinely implemented to control these diseases. Using the gold standard virus neutralization test, we studied the ability of homologous and heterologous sera to neutralize the SPPV and LSDV. We found that LSD and SPP sera effectively neutralize their homologous viruses, and GTP sera can neutralize SPPV. However, while LSD sera effectively neutralizes SPPV, SPP and GTP sera cannot neutralize the LSDV to the same extent. We discuss the implications of these observations in disease assay methodology and heterologous vaccine efficacy.

Description of Sheep Pox Outbreak in Spain in 2022-2023: Challenges Found and Lessons Learnt in Relation with Control and Eradication of This Disease.

Sheep pox and goat pox are infectious viral diseases that affect ovine and caprine animals and are caused by two viruses of the family Poxviridae, genus Capripoxvirus. Sheep pox has been traditionally endemic in Africa, the Middle East, and several Southeast Asian countries, but it is considered a transboundary disease capable of affecting previously free countries epidemically. It is a disease of compulsory immediate notification to the World Organization for Animal Health (WOAH) and the European Union (EU). On 19 September 2022, the disease reemerged in Spain, which had been free of it since 1968, causing a total of 30 outbreaks until 17 May 2023, when the last outbreak of the disease was reported. The control and eradication measures implemented were those laid down in EU legislation, based on the total stamping out of positive herds, zoning and restriction of movement, and strengthening of biosecurity and passive surveillance. This manuscript describes the outbreak, as well as assesses the challenges and lessons learned in relation to its management, with the aim of helping in the effective management of future outbreaks of this disease.

Structural and sequence analysis of the RPO30 gene of sheeppox and goatpox viruses from India.

Sheeppox and goatpox are transboundary viral diseases of sheep and goats that cause significant economic losses to small and marginal farmers worldwide, including India. Members of the genus Capripoxvirus (CaPV), namely Sheeppox virus (SPPV), Goatpox virus (GTPV), and Lumpy skin disease virus (LSDV), are antigenically similar, and species differentiation can only be accomplished using molecular approaches. The present study aimed to understand the molecular epidemiology and host specificity of SPPV and GTPV circulating in India through sequencing and structural analysis of the RNA polymerase subunit-30 kDa (RPO30) gene. A total of 29 field isolates from sheep (n = 19) and goats (n = 10) belonging to different geographical regions of India during the period: Year 2015 to 2023, were analyzed based on the sequence and structure of the full-length RPO30 gene/protein. Phylogenetically, all the CaPV isolates were separated into three major clusters: SPPV, GTPV, and LSDV. Multiple sequence alignment revealed a highly conserved RPO30 gene, with a stretch of 21 nucleotide deletion in all SPPV isolates. Additionally, the RPO30 gene of the Indian SPPV and GTPV isolates possessed several species-specific conserved signature residues/motifs that could act as genotyping markers. Secondary structure analysis of the RPO30 protein showed four α-helices, two loops, and three turns, similar to that of the E4L protein of vaccinia virus (VACV). All the isolates in the present study exhibited host preferences across different states of India. Therefore, in order to protect vulnerable small ruminants from poxviral infections, it is recommended to take into consideration a homologous vaccination strategy.

Cracking the Code of Lumpy Skin Disease: Identifying Causes, Symptoms and Treatment Options for Livestock Farmers.

The novel bovine viral infection known as lumpy skin disease is common in most African and Middle Eastern countries, with a significant likelihood of disease transfer to Asia and Europe. Recent rapid disease spread in formerly disease-free zones highlights the need of understanding disease limits and distribution mechanisms. Capripox virus, the causal agent, may also cause sheeppox and Goatpox. Even though the virus is expelled through several bodily fluids and excretions, the most common causes of infection include sperm and skin sores. Thus, vulnerable hosts are mostly infected mechanically by hematophagous arthropods such as biting flies, mosquitoes, and ticks. As a result, milk production lowers, abortions, permanent or temporary sterility, hide damage, and mortality occur, contributing to a massive financial loss for countries that raise cattle. These illnesses are economically significant because they affect international trade. The spread of Capripox viruses appears to be spreading because to a lack of effectual vaccinations and poverty in rural areas. Lumpy skin disease has reached historic levels; as a consequence, vaccination remains the only viable option to keep the illness from spreading in endemic as well as newly impacted areas. This study is intended to offer a full update on existing knowledge of the disease's pathological characteristics, mechanisms of spread, transmission, control measures, and available vaccinations.

Targeted Whole Genome Sequencing of the Capripoxvirus Genome from Clinical Tissue Samples and Lyophilized Vaccine Batches.

Diseases caused by Capripoxviruses (CaPVs) are of great economic importance in sheep, goats, and cattle. Since CaPV strains are serologically indistinguishable and genetically highly homologous, typing of closely related strains can only be achieved by whole-genome sequencing. In this chapter, we describe a robust, cost-effective, and widely applicable protocol for reconstructing (nearly) complete CaPV genomes directly from clinical samples or commercial vaccine batches in less than a week. Taking advantage of the genetic similarity of CaPVs, a set of pan-CaPVs long-range PCRs was developed that covers the entire genome with only a limited number of tiled amplicons. The resulting amplicons can be sequenced on all currently available high-throughput sequencing platforms. As an example, we have included a detailed protocol for performing nanopore sequencing and a pipeline for assembling the resulting tiled amplicon data.

Harnessing Attenuation-Related Mutations of Viral Genomes: Development of a Serological Assay to Differentiate between Capripoxvirus-Infected and -Vaccinated Animals.

Sheeppox, goatpox, and lumpy skin disease caused by the sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively, are diseases that affect millions of ruminants and many low-income households in endemic countries, leading to great economic losses for the ruminant industry. The three viruses are members of the Capripoxvirus genus of the Poxviridae family. Live attenuated vaccines remain the only efficient means for controlling capripox diseases. However, serological tools have not been available to differentiate infected from vaccinated animals (DIVA), though crucial for proper disease surveillance, control, and eradication efforts. We analysed the sequences of variola virus B22R homologue gene for SPPV, GTPV, and LSDV and observed significant differences between field and vaccine strains in all three capripoxvirus species, resulting in the truncation and absence of the B22R protein in major vaccines within each of the viral species. We selected and expressed a protein fragment present in wildtype viruses but absent in selected vaccine strains of all three species, taking advantage of these alterations in the B22R gene. An indirect ELISA (iELISA) developed using this protein fragment was evaluated on well-characterized sera from vaccinated, naturally and experimentally infected, and negative cattle and sheep. The developed wildtype-specific capripox DIVA iELISA showed >99% sensitivity and specificity for serum collected from animals infected with the wildtype virus. To the best of our knowledge, this is the first wildtype-specific, DIVA-capable iELISA for poxvirus diseases exploiting changes in nucleotide sequence alterations in vaccine strains.

Cytokine profile in lambs naturally infected with sheeppox virus.

In this study, in order to reveal the immune response against the disease in naturally infected sheep with SPPV, the expressions of various pro- or anti-inflammatory cytokines such as tumour necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-1beta (IL-1ß), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and interleukin-12 (IL-12) were evaluated immunohistochemically. The material of this study consisted of tissue samples taken from 24 sheep, which were brought as dead for routine histopathological examination to the Department of Pathology. Avidin-biotin-peroxidase method was used for immunohistochemistry. Characteristic pox lesions were observed in the skin, lungs and kidneys. In histopathological examinations, pox cells, which are very characteristic for the diagnosis of the disease, were observed in all three tissues. Capripoxvirus nucleic acid was detected in 8 of the 24 tissues. Samples were sequenced, and a phylogenetic tree was constructed with reference strains from GenBank. Strains from the study clustered with sheeppox virus references. In conclusion, the levels of pro-inflammatory cytokines such as TNF-α, IFN-γ, IL-1ß, IL-2, IL-8 and IL12 (Th1) were much more dominant compared to the levels of anti-inflammatory cytokines: IL-10 and IL-6 (Th2). This supported the fact that the cellular immune response is much more effective than the humoral immune response in sheeppox.

Spatio-temporal analysis of sheep and goat pox outbreaks in Uganda during 2011-2022.

BACKGROUND: Sheep and goat pox (SGP) caused by sheep poxvirus (SPV) and goat poxvirus (GPV) respectively; are transboundary and World Organisation for Animal Health (WOAH)-notifiable viral diseases. There is barely any coherent information about the distribution and prevalence of SGP for Uganda. We therefore conducted this study to describe the temporal and spatial distribution of SGP suspected outbreaks in Uganda for the period 2011-2020 as well as serologically confirm presence of SGP antibodies in suspected SGP outbreaks reported in 2021-2022. RESULTS: Thirty-seven [37] SGP outbreaks were reported across the country during the study period. North-eastern region [that comprises of Karamoja region] had the highest number of outbreaks [n = 17, 45%]; followed by Central [n = 9, 2.4%], Northern [n = 8, 2.2%] and Western region [n = 3, 0.08%]. Reports from district veterinary personnel indicate that the prevalence of; and mortality rate and case fatality rate associated with SGP were 0.06%, 0.02% and 32% respectively. There was a steady increase in the number of reported SGP outbreaks [x̄ = 4] over the study period. Seropositivity of SGPV antibodies in outbreak sheep and goats that were investigated during the study period [2021-2022] was [n = 41, 27%, 95 CI;] CONCLUSION: Our analyses of SGPV passive and active reports indicate that SGP is present in Uganda with a decade long average of four outbreaks per annum. During this period, about a third of all SGPV-clinically infected animals died. SPG is therefore a major constraint to small ruminant health and productivity in Uganda. Introduction of animals from infected herds and breach in farm biosecurity were the most important predictors of SGP outbreaks. In addition to the already existing SGP commercial vaccines, small ruminant screening for SGPV before introducing them to naïve herds and ensuring on farm biosecurity should be part of the SGP control tool pack for Ugandan small ruminant farmers.

Poxvirus sensitivity of a novel diploid sheep embryonic heart cell line.

Lumpy skin disease virus (LSDV), camelpox virus (CPV), and orf virus (ORFV) are members of the family Poxviridae. These viruses are usually isolated or produced in embryonated eggs or primary cells because continuous cell lines are less sensitive to infection. Disadvantages of the use of eggs or primary cells include limited availability, potential endogenous contaminants, and a limited ability to perform multiple passages. In this study, we developed a diploid cell culture from sheep embryonic hearts (EHs) and demonstrated its high proliferative and long-term storage capacities. In addition, we demonstrated its sensitivity to representatives of three genera of the family Poxviridae: Capripoxvirus (LSDV), Orthopoxvirus (CPV), and Parapoxvirus (ORFV). The cell culture had a doubling time of 24 h and reached 40 passages with satisfactory yield. This is comparable to that observed in primary lamb testis (LT) cells at passage 5 (P5). After infection, each poxvirus titer was 7.0-7.6 log TCID50/mL for up to five passages and approximately 6.8, 6.4, and 5.6 for the three viruses at P6-P25, P30, and P40, respectively. The sensitivity of sheep EH cells to poxvirus infection did not decrease after long-term storage in liquid nitrogen and was higher than that of primary LT cells, which are used for capripoxvirus and parapoxvirus detection and growth, and Vero cells, which are used for orthopoxvirus detection and growth. Thus, EH diploid cells are useful for poxvirus isolation and production without embryonated eggs or primary cells.

Development of the isothermal recombinase polymerase amplification assays for rapid detection of the genus Capripoxvirus.

Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) belong to the genus Capripoxvirus (CaPV), and are important pathogens of sheep, goat and cattle, respectively. Rapid and reliable detection of CaPV is critical to prevent its spread and promote its eradication. This study aimed to develop the recombinase polymerase amplification (RPA) assays combined with real-time fluorescence (real-time RPA) and naked-eye visible lateral flow strip (LFS RPA) for rapid detection of CaPV. Both developed RPA assays worked well at 39 °C within 20 min. They were highly specific for the detection of GTPV, SPPV and LSDV, while no cross-reactivity was observed for other non-targeted pathogens and genomic DNA of goat, sheep and cattle. The limit of detection for real-time RPA and LFS RPA were 1.0 × 102 and 1.0 × 101 copies per reaction, respectively. In the artificially contaminated samples with GTPV, the detection results of RPA assays were consistent with those of real-time PCR. For 15 clinical samples, LSDV was detected by real-time RPA, LFS RPA and real-time PCR in 13, 15 and 15 samples, respectively. The developed RPA assays were specific, sensitive, and user-friendly for the rapid detection of CaPV, and could be a better alternative method applied in low-resources settings.

Evidence of Lumpy Skin Disease Virus Transmission from Subclinically Infected Cattle by Stomoxys calcitrans.

Lumpy skin disease virus (LSDV) is a vector-transmitted capripox virus that causes disease in cattle. Stomoxys calcitrans flies are considered to be important vectors as they are able to transmit viruses from cattle with the typical LSDV skin nodules to naive cattle. No conclusive data are, however, available concerning the role of subclinically or preclinically infected cattle in virus transmission. Therefore, an in vivo transmission study with 13 donors, experimentally inoculated with LSDV, and 13 naïve acceptor bulls was performed whereby S. calcitrans flies were fed on either subclinical- or preclinical-infected donor animals. Transmission of LSDV from subclinical donors showing proof of productive virus replication but without formation of skin nodules was demonstrated in two out of five acceptor animals, while no transmission was seen from preclinical donors that developed nodules after Stomoxys calcitrans flies had fed. Interestingly, one of the acceptor animals which became infected developed a subclinical form of the disease. Our results show that subclinical animals can contribute to virus transmission. Therefore, stamping out only clinically diseased LSDV-infected cattle could be insufficient to completely halt the spread and control of the disease.

Poxvirus Infections in Dairy Farms and Transhumance Cattle Herds in Nigeria.

Lumpy Skin disease (LSD) is an economically important disease in cattle caused by the LSD virus (LSDV) of the genus Capripoxvirus, while pseudocowpox (PCP) is a widely distributed zoonotic cattle disease caused by the PCP virus (PCPV) of the genus Parapoxvirus. Though both viral pox infections are reportedly present in Nigeria, similarities in their clinical presentation and limited access to laboratories often lead to misdiagnosis in the field. This study investigated suspected LSD outbreaks in organized and transhumance cattle herds in Nigeria in 2020. A total of 42 scab/skin biopsy samples were collected from 16 outbreaks of suspected LSD in five northern States of Nigeria. The samples were analyzed using a high-resolution multiplex melting (HRM) assay to differentiate poxviruses belonging to Orthopoxvirus, Capripoxvirus, and Parapoxvirus genera. LSDV was characterized using four gene segments, namely the RNA polymerase 30 kDa subunit (RPO30), G-protein-coupled receptor (GPCR), the extracellular enveloped virus (EEV) glycoprotein and CaPV homolog of the variola virus B22R. Likewise, the partial B2L gene of PCPV was also analyzed. Nineteen samples (45.2%) were positive according to the HRM assay for LSDV, and five (11.9%) were co-infected with LSDV and PCPV. The multiple sequence alignments of the GPCR, EEV, and B22R showed 100% similarity among the Nigerian LSDV samples, unlike the RPO30 phylogeny, which showed two clusters. Some of the Nigerian LSDVs clustered within LSDV SG II were with commonly circulating LSDV field isolates in Africa, the Middle East, and Europe, while the remaining Nigerian LSDVs produced a unique sub-group. The B2L sequences of Nigerian PCPVs were 100% identical and clustered within the PCPV group containing cattle/Reindeer isolates, close to PCPVs from Zambia and Botswana. The results show the diversity of Nigerian LSDV strains. This paper also reports the first documented co-infection of LSDV and PCPV in Nigeria.

Multiple accurate and sensitive arrays for Capripoxvirus (CaPV) differentiation.

Capripoxvirus (CaPV) contains three viruses that have caused massive losses in the livestock and dairy industries. Accurate CaPV differentiation has far-reaching implications for effectively controlling outbreaks. However, it has a great challenge to distinguishing three viruses due to high homology of 97%. Here, we established a sensitive CRISPR/Cas12a array based on Multiple-recombinase polymerase amplification (M-RPA) for CaPV differentiation, which provided a more comprehensive and accurate differentiation mode targeting VARV B22R and RPO30 genes. By sensitive CRISPR/Cas12a and M-RPA, the actual detection limits of three viruses were as low as 50, 40 and 60 copies, respectively. Moreover, Lateral flow dipstick (LFD) array based on CRISPR/Cas12a achieved portable and intuitive detection, making it suitable for point-of-care testing. Therefore, CRISPR/Cas12a array and LFD array paved the way for CaPV differentiation in practice. Additionally, we constructed a real-time quantitative PCR (qPCR) array to fill the qPCR technical gap in differentiation and to facilitate the quarantine departments.

Development and Validation of a New DIVA Real-Time PCR Allowing to Differentiate Wild-Type Lumpy Skin Disease Virus Strains, Including the Asian Recombinant Strains, from Neethling-Based Vaccine Strains.

The current epidemic in Asia, driven by LSDV recombinants, poses difficulties to existing DIVA PCR tests, as these do not differentiate between homologous vaccine strains and the recombinant strains. We, therefore, developed and validated a new duplex real-time PCR capable of differentiating Neethling-based vaccine strains from classical and recombinant wild-type strains that are currently circulating in Asia. The DIVA potential of this new assay, seen in the in silico evaluation, was confirmed on samples from LSDV infected and vaccinated animals and on isolates of LSDV recombinants (n = 12), vaccine (n = 5), and classic wild-type strains (n = 6). No cross-reactivity or a-specificity with other capripox viruses was observed under field conditions in non-capripox viral stocks and negative animals. The high analytical sensitivity is translated into a high diagnostic specificity as more than 70 samples were all correctly detected with Ct values very similar to those of a published first-line pan capripox real-time PCR. Finally, the low inter- and intra-run variability observed shows that the new DIVA PCR is very robust which facilitates its implementation in the lab. All validation parameters that are mentioned above indicate the potential of the newly developed test as a promising diagnostic tool which could help to control the current LSDV epidemic in Asia.

The emergence of novel Iranian variants in sheeppox and goatpox viral envelope proteins with remarkably altered putative binding affinities with the host receptor.

The outbreak of Sheep and goat pox (SGP) viral infections have increasingly been reported despite vaccinating the majority of sheep populations in Iran. The objective of this study was to predict the impacts of the SGP P32/envelope variations on the binding with host receptors as a candidate tool to assess this outbreak. The targeted gene was amplified in a total of 101 viral samples, and the PCR products were subjected to Sanger sequencing. The polymorphism and phylogenetic interactions of the identified variants were assessed. Molecular docking was performed between the identified P32 variants and the host receptor and the effects of these variants were evaluated. Eighteen variations were identified in the investigated P32 gene with variable silent and missense effects on the envelope protein. Five groups (G1-G5) of amino acid variations were identified. While there were no amino acid variations in the G1 (wild-type) viral protein, G2, G3, G4, and G5 proteins had seven, nine, twelve, and fourteen SNPs, respectively. Based on the observed amino acid substitutions, multiple distinct phylogenetic places were occupied from the identified viral groups. Dramatic alterations were identified between G2, G4, and G5 variants with their proteoglycan receptor, while the highest binding was revealed between goatpox G5 variant with the same receptor. It was suggested that the higher severity of goatpox viral infection originated from its higher affinity to bind with its cognate receptor. This firm binding may be explained by the observed higher severity of the SGP cases from which G5 samples were isolated.

Generation and application of immortalized sertoli cell line from sheep testis.

Primary sheep testicular Sertoli cells (STSCs) are ideal for investigating the molecular and pathogenic processes of capripoxvirus. However, the high cost of isolation and culture of primary STSCs, time-consuming operation, and short lifespan greatly limit their real-world application. In our study, the primary STSCs were isolated and immortalized by transfection of a lentiviral recombinant plasmid containing simian virus 40 (SV40) large T antigen. Androgen-binding protein (ABP) and vimentin (VIM) protein expression, SV40 large T antigen activity, proliferation assays, and apoptosis analysis results showed that immortalized large T antigen STSCs (TSTSCs) still had the same physiological characteristics and biological functions as primary STSCs. Moreover, immortalized TSTSCs had strong anti-apoptosis ability, extended lifespan, and enhanced proliferative activity compared to primary STSCs, which had not transformed in vitro and showed any signs of malignancy phenotype in nude mice. Besides, immortalized TSTSCs were susceptible to goatpox virus (GTPV), lumpy skin disease virus (LSDV), and Orf virus (ORFV). In conclusion, immortalized TSTSCs are useful in vitro models to study GTPV, LSDV, and ORFV in a wide range of ways, suggesting that it can be safely used in virus isolation, vaccine and drug screening studies in future.

A recombinant capripoxvirus expressing the F protein of peste des petits ruminants virus and the P12A3C of foot-and-mouth disease virus.

BACKGROUND: Peste des petits ruminants (PPR), foot-and-mouth disease (FMD) and sheep pox and goat pox are three important infectious diseases that infect goats, sheep and other small ruminants. It is well-known that the prevention of three diseases rely mainly on their individual vaccines. However, the vaccines have a variety of different disadvantages, such as short duration of immunity, increasing the number of vaccinations, and poor thermal stability. The purpose of this study is to construct a recombinant goat pox virus (rGPV) capable of expressing the F gene of PPRV and the P12A3C gene of FMDV as a live vector vaccine. RESULTS: The IRES, FMDV P12A3C and PPRV F genes into the multi-cloning site of the universal transfer plasmid pTKfpgigp to construct a recombinant transfer plasmid pTKfpgigpFiP12A3C, and transfected GPV-infected lamb testis (LT) cells with liposomes and produced by homologous recombination Recombinant GPV (rGPV/PPRVF-FMDVP12A3C, rGPV). The rGPV was screened and purified by green florescence protein (GFP) and xanthine-guanine-phosphoribosyltransferase gene (gpt) of Escherichia coli as selective markers, and the expression of rGPV in LT cells was detected by RT-PCR and immunofluorescence techniques. The results showed that the virus strain rGPV/PPRVF-FMDVP12A3C containing FMDV P12A3C and PPRV F genes was obtained. The exogenous genes FMDV P12A3C and PPRV F contained in rGPV were normally transcribed and translated in LT cells, and the expression products could specifically react with PPRV and FMDV antiserum. Then, the rGPV was intradermally inoculated with goats, the animal experiments showed that rGPV/PPRVF-FMDVP12A3C could induce high levels of specific antibodies against GPV, PPRV and FMDV. CONCLUSIONS: The constructed rGPV induced high levels of specific antibodies against GPV, PPRV and FMDV. The study provides a reference for " one vaccine with multiple uses " of GPV live vector vaccine.