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.

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.

Emergency vaccination of cattle against lumpy skin disease: Evaluation of safety, efficacy, and potency of MEVAC® LSD vaccine containing Neethling strain.

Lumpy skin disease (LSD) is an emerging disease of cattle causing significantly high economic losses. Control of LSD depends on the use of homologous attenuated LSD virus strains isolated originally from South Africa (the Neethling strain). The virus belongs to the genus Capripoxvirus, which includes sheep pox virus and goat pox virus. The present study was conducted to evaluate the safety and efficacy of a new live attenuated LSD vaccine produced by Middle East for Vaccines (MEVAC®) based on the Neethling strain. Tests were performed both in Egypt and Vietnam. Safety was evaluated by inoculation of five cattle with 10 times the recommended dose and observation of the animals for 14 days. Immunogenicity was tested at different periods post-vaccination (PV) in animals receiving the recommended doses of the vaccine using ELISA and virus neutralization test. Five cows were used to determine the protection index (PI) and non-vaccinated control cattle were included. Three calves were challenged by intradermal inoculation of the wild virus (5 × 105 TCID50) 28 days PV. Field or mass vaccination experiments were conducted in Vietnam during national campaigns in the summer of 2021 with 4301 vaccinated animals closely monitored after vaccination. In the field, around 2% (80/4301) of the animals showed hyper-reactivity, and 0.6% (24/4301) showed small skin swellings that disappeared within few hours PV. Abortion was recorded in three animals (0.3% 3/867). Challenged animals were resistant to clinical disease and PI value was 3.5 log10. Meanwhile, antibody levels determined by the ELISA were inconsistent among animals and laboratories during the study period. Overall, the findings point to a new safe and effective LSD vaccine.

LSDV126 gene based molecular assays for specific detection and characterization of emerging Lumpy Skin Disease virus.

Lumpy skin disease (LSD) is a highly infectious and economically important viral disease, which is currently emerging in the Indian subcontinent. LSD is caused by Lumpy Skin Disease Virus (LSDV) under the genus Capripoxvirus and the family Poxviridae. Since its first incursion in India in the year 2019, the virus is rapidly disseminating through different means like direct contact, fomites and mainly by blood-feeding insects. As the disease has never been reported from India or neighbouring countries, there is a lack of planning and preparatory measures in terms of diagnostics and vaccines to control the disease. In the absence of any homologous vaccine, a live attenuated heterologous goat pox vaccine (Uttarkashi strain) is now being widely used in the country for the prevention of LSDV infection. Use of live attenuated goat pox virus vaccine necessitates the availability of an assay which could specifically detect and differentiate LSDV from goat pox virus. In this study, nucleotide sequences of LSDV126 gene encoding extracellular enveloped virus protein of circulating LSDV and goat pox virus were determined and analyzed. Deletion of 27 nt tandem repeats was observed in LSDV in comparison to goat pox and LSDV vaccine viruses. The deletion region was targeted for designing primers specific to LSDV, but not goat pox virus. A novel isothermal polymerase spiral reaction (PSR) was optimized as pen side diagnostic for prompt and sensitive detection of genomic DNA of LSDV. The assay was found to be highly sensitive and specific when compared to the real-time PCR. The assay was found to be specifically detecting only LSDV but not the goat pox virus. The limit of detection was identified as 9 × 10-6 ng of positive DNA. The assay will provide a point of care tool that will be a boon for the successful control of LSD in India.

Design of a multi-epitope vaccine against goatpox virus using an immunoinformatics approach.

Introduction: Goatpox, a severe infectious disease caused by goatpox virus (GTPV), leads to enormous economic losses in the livestock industry. Traditional live attenuated vaccines cause serious side effects and exist a risk of dispersal. Therefore, it is urgent to develop efficient and safer vaccines to prevent and control of GTPV. Methods: In the present study, we are aimed to design a multi-epitope subunit vaccine against GTPV using an immunoinformatics approach. Various immunodominant cytotoxic T lymphocytes (CTL) epitopes, helper T lymphocytes (HTL) epitopes, and B-cell epitopes from P32, L1R, and 095 proteins of GTPV were screened and liked by the AAY, GPGPG, and KK connectors, respectively. Furthermore, an adjuvant ß-defensin was attached to the vaccine's N-terminal using the EAAAK linker to enhance immunogenicity. Results: The constructed vaccine was soluble, non-allergenic and non-toxic and exhibited high levels of antigenicity and immunogenicity. The vaccine's 3D structure was subsequently predicted, refined and validated, resulting in an optimized model with a Z-value of -3.4. Molecular docking results demonstrated that the vaccine had strong binding affinity with TLR2(-27.25 kcal/mol), TLR3(-39.84 kcal/mol), and TLR4(-59.42 kcal/mol). Molecular dynamics simulation results indicated that docked vaccine-TLR complexes were stable. Immune simulation analysis suggested that the vaccine can induce remarkable increase in antibody titers of IgG and IgM, higher levels of IFN-γ and IL-2. Conclusion: The designed GTPV multi-epitope vaccine is structurally stable and can induce robust humoral and cellular immune responses, which may be a promising vaccine candidate against GTPV.

[Adaptation of the sheep pox virus (Poxviridae: Capripoxvirus: Sheeppox virus) to African green monkey kidney cell line and evaluation of its immunobiological properties].

INTRODUCTION: Outbreaks of infectious diseases seriously hinder the preservation and increase of the number of small ruminants. Such infections include sheep pox virus (SPPV). According to the OIE data of 2021, SPP outbreaks were registered in countries such as Turkey, Israel, China, Maldives, Mongolia, Thailand, Russia, Algeria, Kenya, and in 2019 in Mangistau and Atyrau regions. In Kazakhstan annually conducts routine immunization of sheep at risk with a live attenuated vaccine produced by RIBSP. MATERIALS AND METHODS: The object of the study was the vaccine strain of NISHI and the virulent strain A of the sheep pox virus. The virus was propagated in Vero cells. To determine the harmlessness and immunogenicity, sheep of the Kazakh fine-wool breed aged from 6 to 12 months were used. Virological, serological and immunobiological methods were used in the study. RESULTS: The results of the adaptation of the NISHI strain of SPPV to the Vero cell line are presented. Five passages in Vero cells resulted to the adaptation of the NISHI strain with the manifestation of a cytopathogenic effect specific to SPPV with a titer of 6.50 lg TCD50/ml. Following immunization, the formation of immunity was observed in animals on day 7 with an average protective titer 1.8 log2, which increased by day 21 to 4.33 log2. CONCLUSION: It has been established that the NISHI strain of SPPV retains its virological and immunobiological properties during reproduction in a Vero cell line.

Cellular infiltration, cytokines, and histopathology of skin lesions associated with different clinical forms and stages of naturally occurring lumpy skin disease in cattle.

Lumpy skin disease (LSD) caused by the Capripoxvirus LSD virus which infects cattle, leading to a serious disease characterized by fever and the eruption of skin nodules all over the surface of the body. Our understanding of the pathogenesis of this disease is still incomplete, particularly the immunopathological alterations occurring in the skin nodules of infected animals. Therefore, we collected skin nodules from naturally infected cattle with different forms of the disease, both in the early stage of clinical infection and after disease progression. The skin samples were examined both histopathologically and immunohistochemically using a variety of antibodies targeting immune cellular markers and cytokines. As a result, the dermatohistopathology revealed orthokeratotic hyperkeratosis, vasculitis, epidermal microvesicles, and cellules claveleuses of Borrel in the early stage of infection, with the severity of the lesions correlating with the severity of the clinical disease. Meanwhile, late-stage samples had epidermal hyperkeratosis as well as dermal lymphocytic and histiocytic infiltrations. The predominant cellular infiltrates in the cutaneous lesions of early-stage LSD samples were interferon (IFN)-γ+ cells and CD4+ T lymphocytes with few macrophage lineage cells. However, in the late-stage samples, numerous Iba-1+ macrophages, with few IFN-γ+ cells and CD4+ T lymphocytes, were detected. Our findings indicate that IFN-γ+ cells, CD4+ T lymphocytes, and macrophages play a key role in the immunity against natural LSD virus infection and imply that cutaneous vasculopathy associated with LSD virus infection is an immune-mediated lesion. The current study contributes to our understanding of the pathogenesis of LSD.

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.