Sevskikh, Timofey

Vlasov M., Sindryakova I., Kudryashov D., Morgunov S., Kolbasova O., Lyska V., Zhivoderov S., Pivova E., Balyshev V., Namsrayn S., Sevskikh T., Sereda A., Kolbasov D.

African swine fever (ASF) is a lethal hemorrhagic disease of Suidae, i.e., domestic pigs and wild boars, caused by African swine fever virus (ASFV). The development of cross-protective vaccines against ASF is imperative for effective disease control, particularly in regions where ASF is endemic, potentially featuring multiple circulating ASFV isolates. The investigation of non-hemadsorbing naturally attenuated isolates and laboratory recombinant strains with a deletion in the EP402R gene has attracted interest. Our study aimed to assess the impacts of various administration routes and doses of the naturally attenuated ASFV-PSA-1NH (immunotype IV, genotype I) isolate on the manifestation of clinical signs of ASF and the level of protection against the heterologous ASFV-Stavropol 01/08 strain (seroimmunotype VIII, genotype II). The results demonstrated that the intranasal administration of a low dose of ASFV-PSA-1NH to pigs minimized the clinical signs of ASF and established a high level of protection against the heterologous strain ASFV-Stavropol 01/08. Despite the challenges in standardizing the dosage for intranasal administration, this approach appears as a viable alternative in ASF vaccination.

Vlasov M.E., Kudryashov D.A., Sindryakova I.P., Sevskikh T.A., Pivova E.Y., Lyska V.M., Sereda A.D., Balyshev V.M.

Glazunova A., Krasnova E., Bespalova T., Sevskikh T., Lunina D., Titov I., Sindryakova I., Blokhin A.

Avian influenza (AI) is a global problem impacting birds and mammals, causing economic losses in commercial poultry farms and backyard settings. In 2022, over 8,500 AI cases were reported worldwide, with the H5 subtype being responsible for many outbreaks in wild and domestic birds. In the territory of the Russian Federation, outbreaks of AI have been massively reported since 2020, both among domestic bird species and wild bird species. Wild migratory birds often serve as natural reservoirs for AI viruses, and interactions between bird species can lead to the emergence of new, highly pathogenic variants through genetic recombination between strains. In order to combat the widespread outbreaks of the disease and potential risks of further spread in 2021, monitoring studies were conducted in the Samara Oblast, the southeastern region of European Russian Federation. These studies aimed to diagnose and characterize circulating AI virus variants among wild migratory birds during waterfowl hunting in areas of mass nesting. Among the 98 shot birds, a highly pathogenic A/H5N1 AI virus was detected in a Eurasian Teal from the Bolshechernigovsky district. It was classified into clade 2.3.4.4 based on the cleavage site structure of HA. Phylogenetic analysis showed a high relatedness of the identified strain in the Samara Oblast with field isolates from Russia, Nigeria, Bangladesh, and Benin. The article emphasizes the importance of monitoring AI virus spread in both wild and poultry, highlighting the need for timely information exchange to assess risks. Further comprehensive studies are necessary to understand virus dissemination pathways.

Vlasov M.E., Kudrjashov D.A., Kolbasova O.L., Morgunov S.Y., Pivova E.Y., Repin V.I., Sevskikh T.A., Sindryakova I.P.

This study presents the immunobiological characteristics of the candidate vaccine strain Katanga-350 of African swine fever virus.

Kolbasova O., Sevskikh T., Titov I., Kolbasov D.

This article presents the results of virological and genetic studies of an isolate of caprine arthritis encephalitis (CAE) virus from the republic of Mordovia, Russian Federation. The isolate was found during monitoring studies of goat blood samples for the viral genome, and the presence of antibodies to lentiviruses was detected. According to the recommendation of the OIE, the positive result of PCR was confirmed with nucleotide sequencing. It was found that the obtained nucleotide sequence is identical to the genome of small ruminant lentiviruses presented in the GenBank database. Phylogenetic analysis showed that the isolate “Mordovia-2018” was included in the same cluster with an isolate from the Tver region of the Russian Federation detected in 2008. The sequence of the fragment of the env-gene of the isolate from the republic of Mordovia is available in GenBank under the number MN186380.1. To isolate the virus, a fraction of peripheral blood monocyte cells from the animal’s blood was added to a monolayer of lamb synovial membrane cell culture, and ten passages were carried out. The first manifestations of the cytopathic effect were observed after the third passage on the eighth day of cultivation in the form of single large cells of irregular shape with 5–7 nuclei. At the seventh passage, multiple syncytium with 7–12 nuclei were observed. At subsequent passage levels, the formation of syncytium containing more than 10–14 nuclei was observed.

Pivova E.Y., Vlasov M.E., Sevskikh T.A., Povolyaeva O.S., Zhivoderov S.P.

This article presents the results of a study on the susceptibility of laboratory animals to the lumpy skin disease virus (LSDV). Mice weighing 15–20 g, hamsters weighing 40–60 g, guinea pigs weighing 600–1200 g, and rabbits weighing 2.5–3 kg were used in this study. Nodules were observed on the skin of rabbits and hamsters at the sites of inoculation. The virus was isolated from the affected skin areas in cell culture and examined using real-time PCR, indicating its tropism for animal skin. The production of anticapripoxvirus antibodies was detected using the neutralization reaction, starting from 10 days after infection in mice, 27 days in rabbits, and 14 days in hamsters. Some laboratory animals exhibited multiple skin nodules. This indicates that these animal species may play a role in maintaining the epizootic process.

GLAZUNOVA A.A., SEVSKIKH T.A., LUNINA D.A., KRASNOVA E.A.

African Swine fever (ASF) remains a serious economic threat to pig production around the world, affecting only animals of the Suidae family. The lack of effective treatment and vaccines makes the control of the disease particularly difficult and expensive. Only implementation of strict quarantine measures and a properly chosen eradication strategy remain the only methods to control the disease. ASF also circulates in the wildlife for a long time, persisting in the wild boar carcasses and spreading with infected animals to the new territories. Under natural conditions, the virus spreads slowly with the wild boars, but the intervention of hunters and the extraction of potentially infected meat can significantly accelerate the process. Particular emphasis is placed on the role of dead wild boars killed by ASF and the migration of susceptible wild animals. They are considered as the main source and factor in the maintenance of the ASF virus in the wild. In connection with this problem, the question of the role of hunting in the spread of ASF remains relevant. In order to reveal this issue, the article discusses the behavior of wild susceptible animals, hunting features worldwide, and the impact of hunting on the social behavior of wild boars, the role of hunting and hunters both in the containment and in the spread of ASF. The main attention of the article is focused on the importance of the cohesive work of each stakeholder group in the elimination of ASF. The choice of a strategy to control ASF in the wild and the long term support of joint efforts to implement it is the key to restoring the well-being of the area and preventing the introduction of the virus into farms and households.

Glazunova A.A., Korogodina E.V., Sevskikh T.A., Krasnova E.A., Kukushkin S.A., Blokhin A.A.

Porcine Reproductive Respiratory Syndrome (PRRS) causes significant economic losses in pig production around the world. The virus is highly contagious and spreads both by aerosol and with all biological secretions, and the disease itself is characterized by fever, weight loss and abortion of fetuses. This swine disease is relatively new, for the first-time clinical signs were described in 1986, and in 1991 the pathogen was isolated. The main problems hindering the fight against the virus currently remain: the insufficient effectiveness of existing vaccines, the high genetic variability of the pathogen, the ability of PRRSV to suppress the host's immune system, and long-term virus carriers in recovered animals. The review on porcine reproductive and respiratory syndrome presents data on the genetic diversity of virus strains that differ in the degree of virulence and pathogenicity, sources of infection and ways of transmission of the virus. The article also contains data on the spread of PRRS in the world and in Russia. It is noted that the disease has been reported in many swine-producing countries, where, after a phase of active spread, the disease became endemic. And in 2021, according to the to the International Epizootic Bureau (OIE), outbreaks of PRRSV were detected in domestic pigs in Ecuador, Uruguay, Nepal, China, Bahrain, Switzerland and Ukraine, as well as in wild boars in Bahrain, Switzerland and Ukraine. According to the published data of monitoring studies conducted in the period from 2012 to 2017, only single territorially limited outbreaks were detected in Russia. Earlier studies indicate a wide distribution of the PRRS pathogen in Russia. The effectiveness of various variants of live and inactivated vaccines is described in detail, and it has been established that live attenuated vaccines are most effective against PRRS. The features of the protection of the territory of a number of states (Norway, Sweden, Finland, Switzerland) from the introduction of the pathogen, which by now remain free from PRRS, as well as the experience of Hungary in the eradication of the disease, are analyzed. All preventive measures in the pig farm in case of detection of the virus should include general, veterinary-sanitary and special measures, for diagnosis it is better to use modern RT-PCR test systems that allow distinguishing PRRS genotypes, in combination with serological tests, and for vaccination choose a registered and approved vaccine based on the genetic characteristics of the pathogen circulating in the area.

GLAZUNOVA A.A., SEVSKIKH T.A., KOROGODINA E.V., LUNINA D.A., KRASNOVA E.A., GOGIN A.E.

The article provides information on the ASF epizootic, and control measures taken. The most likely reason for the introduction of the disease, considering the distance from the nearest foci of the disease, is the anthropogenic factor. The unregulated growth of the wild boar population against the background of poorly controlled movement of pig products from affected areas leads to an increased risk of the virus introduction into the population of the wild susceptible animals. The control strategy adopted by the competent authorities of Italy includes a zonal principle for the selection and implementation of a set of measures. The main control approaches are depopulation of pigs in the unprotected pig sector (PSF), restriction of wild boar migration with the help of fences, as well as active search and removal of carcasses of wild boars, followed by local depopulation of wild boar in the infected zone, reduction of its density in the threatened zone along with measures aimed at early detection of the disease, and enhanced biosecurity measures.

Havas K.A., Gogin A.E., Basalaeva J.V., Sindryakova I.P., Kolbasova O.L., Titov I.A., Lyska V.M., Morgunov S.Y., Vlasov M.E., Sevskikh T.A., Pivova E.Y., Kudrjashov D.A., Doolittle K., Zimmerman S., Witbeck W., et. al.

African swine fever virus causes hemorrhagic disease in swine. Attenuated strains are reported in Africa, Europe, and Asia. Few studies on the diagnostic detection of attenuated ASF viruses are available. Two groups of pigs were inoculated with an attenuated ASFV. Group 2 was also vaccinated with an attenuated porcine reproductive and respiratory syndrome virus vaccine. Commercially available ELISA, as well as extraction and qPCR assays, were used to detect antibodies in serum and oral fluids (OF) and nucleic acid in buccal swabs, tonsillar scrapings, OF, and blood samples collected over 93 days, respectively. After 12 dpi, serum (88.9% to 90.9%) in Group 1 was significantly better for antibody detection than OF (0.7% to 68.4%). Group 1′s overall qPCR detection was highest in blood (48.7%) and OF (44.2%), with the highest detection in blood (85.2%) from 8 to 21 days post inoculation (dpi) and in OF (83.3%) from 1 to 7 dpi. Group 2′s results were not significantly different from Group 1, but detection rates were lower overall. Early detection of attenuated ASFV variants requires active surveillance in apparently healthy animals and is only reliable at the herd level. Likewise, antibody testing will be needed to prove freedom from disease.

Glazunova A.А., Sevskikh T.A., Lunina D.А., Krasnova E.A.

Glazunova A.А., Sevskikh T.A., Lunina D.А., Tkacheva I.S.

Glazunova A.A., Korennoy F.I., Sevskikh T.A., Lunina D.A., Zakharova O.I., Blokhin A.A., Karaulov A.K., Gogin A.E.

African swine fever (ASF) is an incurable viral disease of domestic and wild pigs. A large-scale spread of ASF began in Eurasia in 2007 and has affected territories from Belgium to the Far East, occurring as both local- and regional-level epidemics. In 2020, a massive ASF epidemic emerged in the southeastern region of European Russia in the Samara Oblast and included 41 outbreaks of ASF in domestic pigs and 40 cases in wild boar. The Samara Oblast is characterized by a relatively low density of wild boar (0.04–0.05 head/km2) and domestic pigs (1.1–1.3 head/km2), with a high prevalence of small-scale productions (household farms). This study aims to understand the driving forces of the disease and perform a risk assessment for this region using complex epidemiological analyses. The socioeconomic and environmental factors of the ASF outbreak were explored using Generalized Linear Logistic Regression, where ASF infection status of the Samara Oblast districts was treated as a response variable. Presence of the virus in a district was found to be most significantly (p < 0.05) associated with the importation of live pigs from ASF-affected regions of Russia (OR = 371.52; 95% CI: 1.58–87290.57), less significantly (p < 0.1) associated with the density of smallholder farms (OR = 2.94; 0.82–10.59), volume of pork products' importation from ASF-affected regions of Russia (OR = 1.01; 1.00–1.02), summary pig population (OR = 1.01; 0.99–1.02), and insignificantly (p > 0.1) associated with presence of a common border with an ASF-affected region (OR = 89.2; 0.07–11208.64). No associations were found with the densities of pig and wild boar populations. The colocation analysis revealed no significant concentration of outbreaks in domestic pigs near cases in wild boar or vice versa. These results suggest that outbreaks notified in low biosecurity household farms were mainly associated with the transportation and trade of pigs and pork products from ASF-affected regions of Russia. The findings underline the importance of taking into account animal transportation data while conducting future studies to develop a risk map for the region and the rest of European Russia.

Zakharova O.I., Titov I.A., Gogin A.E., Sevskikh T.A., Korennoy F.I., Kolbasov D.V., Abrahamyan L., Blokhin A.A.

African swine fever (ASF) is an emerging viral contagious disease affecting domestic pigs (DP) and wild boar (WB). ASF causes significant economic damage to the pig industry worldwide due to nearly 100% mortality and the absence of medical treatments. Since 2019, an intensive spread of ASF has been observed in the Russian Far East region. This spread raises concerns for epidemiologists and ecologists given the potential threat to the WB population, which is an essential member of the region's wild ungulates and provides a notable share of food resources for predatory species. This study aims to determine the genotype of ASF virus circulating in the region, reveal the spatio-temporal patterns of the ASF outbreaks' emergence, and assess the potential reduction of the regional fauna because of expected depopulation of WB. The first historical case of ASF in the study region was caused by an African swine fever virus (ASFV) isolated from DPs and belonging to Genotype 2, CVR1; IGR-2 (TRS +). Sequencing results showed no significant differences among ASFV strains currently circulating in the Russian Federation, Europe, and China. The spatiotemporal analysis with the space-time permutations model demonstrated the presence of six statistically significant clusters of ASF outbreaks with three clusters in DPs and one cluster in WBs. DP outbreaks prevail in the north-west regions of the study area, while northern regions demonstrate a mixture of DP and WB outbreaks. Colocation analysis did not reveal a statistically significant pattern of grouping of one category of outbreaks around the others. The possible damage to the region's fauna was assessed by modeling the total body mass of wild ungulates before and after the wild boars' depopulation, considering a threshold density of WB population of 0.025 head/km2, according to the currently in force National Plan on the ASF Eradication in Russia. The results suggest the total mass of ungulates of the entire study region will likely decrease by 8.4% (95% CI: 4.1–13.0%), while it may decrease by 33.6% (19.3–46.1%) in the Primorsky Krai, thereby posing an undeniable threat to the predatory species of the region.

Glazunova A.A., Sevskikh T.A., Kustikova O.V., Dresvjannikova S.G., Usadov T.R., Dzhailidi G.A., Debeljak Z., Lunina D.A.

Abstract The article provides assessment of field and laboratory methods for the collection and evaluation of potential vectors of lumpy skin disease virus (LSDV) in one of the districts of Krasnodarskiy Kray in southern Russia. In this study, we tested several methods of vector collection and a PCR protocol for the detection of the LSDV genome in insects. Descriptive data on samples were collected using a free web-based application Epicollect5. Potential LSDV vectors are quite widely spread insects in this region. We identified 15 insect species, including Musca domestica, Musca autumnalis and Stomoxys calcitrans. Analysis of the insect population showed an increase in species diversity and a decrease in abundance of the insect population by the end of the flight season. PCR tests did not detect LSDV genome in the collected samples. All the methods tested were found suitable for large-scale monitoring of lumpy skin disease (LSD). Further studies on potential risk factors of LSD spread are necessary to improve measures on preventing and eliminating the disease.

Mikhaleva T.V., Konnova S.S.

Introduction. Pig farming, as a fast-growing branch of animal husbandry, is capable of prompt achieving a significant increase in the production of pork with high nutritional properties and biological value. One of the acute problems of pig farming is respiratory diseases of viral and bacterial etiology. In the current economic conditions, reducing the dependence of the Russian pig farmingon technological imports is of particular significance. Production of domestically manufactured feeds and veterinary drugs should be considered as the most important condition for achieving the technological sovereignty of the Russian Federation.Objective. To analyze the provision of pig farming with domestic vaccines against such significant porcine respiratory diseases as swine influenza, porcine enzootic (mycoplasmal pneumonia), porcine reproductive and respiratory syndrome and circovirus infection as well as to identify factors that hinder the development of immunobiological drugs against these diseases.Materials and methods. The information base of the research included data from pig-breeding organizations of the Russian Federation, the Rosselkhoznadzor’s state register of veterinary medicinal products, reference andspecial literature, publications of research institutions.Results. Agents of swine influenza, porcine enzootic (mycoplasmal) pneumonia, porcine reproductive and respiratory syndrome, porcine circovirus infection are the most prevalent pathogens that cause respiratory diseases in pig son the pig farms. Over the past few years, Russian biofactories havebeen developing import substitution programs for the necessary immunobiological drugs. By the end of 2023, the domestic establishments manufactured 19.3 billion doses of veterinary vaccines, which is 3 billion doses more than in 2022.Conclusion. Vaccination is the most efficient and cost-effective way to prevent viral infections. However, domestic immunological drugs against swine influenza have not yet been developed in our country, and vaccines against porcine enzootic (mycoplasmal) pneumonia, porcine reproductive and respiratory syndrome, porcine circovirus infection require modification due to high variability of the agents.

González-García G., Gallardo C., Montón M., Barroso-Arévalo S., Casado N., Barasona J.Á., Sánchez-Vizcaíno J.M., Venteo Á., Sastre P., Rueda P.

Background/Objectives: African Swine Fever (ASF) is one of the most significant infectious diseases affecting both domestic pig and wild boar populations, leading to substantial economic and biosanitary consequences. In Europe, disease management relies on stringent biosecurity measures and surveillance through diagnosis, highlighting the urgent need for an effective and safe vaccine for ASF control. In this context, the VACDIVA project has generated several promising vaccine candidates, including those with the EP153R gene deleted and replaced by the eGFP reporter gene. Methods: In this study, pEP153R and eGFP proteins were produced using recombinant technology and demonstrated their antigenicity and DIVA capability through indirect ELISA. Additionally, a prototype serological DIVA test was designed and developed. The assay is based on the detection of antibodies against both DIVA antigens and the well-established immunogenic p72 protein. Results: This preliminary DIVA diagnostic assay complements vaccine candidates based on a genotype II ASFV strain, featuring the deletion of the EP153R gene and/or the insertion of the eGFP reporter gene, exemplified by the Lv17/WB/Rie1-∆CD vaccine candidate. Conclusions: This approach could potentially improve surveillance during prospective vaccination campaigns.

Bespalova T.Y., Glazunova A.A.

The panzootic of African swine fever (ASF) in Europe and Asia caused by viruses of genotypes I and II has led to a comprehensive scientific study of the course of this infection. Researchers have noted that a certain proportion of diseased animals survive. Detection of seropositive (antibody-positive) animals in ASF-affected countries of Europe was especially noticeable in wild boar populations. The role of seropositive animals ASF survivors in the persistence and spread of infection in the population has been long and controversially debated, as they may potentially become persistently infected, acting as virus carriers. The aim of this review was to summarize the current scientific and experimental results on the chronic course of ASF in Eurasian wild boar, virus carriage and spread of infection from surviving seropositive animals. In Eurasia different forms of ASF disease are currently observed in susceptible animals: peracute, acute, subacute, less frequently chronic and asymptomatic; the latter are found to be caused by circulating ASF viruses (ASFV) of reduced virulence. Two types of survivors animals are distinguished: 1) animals that develop persistent infection with periodic viremia and signs of subacute to chronic course; 2) animals that recover completely and clear of infection. Long-term persistence and complete elimination of the virus have been observed in survivors: in the last ten years, the time of virus excretion has been experimentally determined to generally range from 35 to 99 days. Survivors of the 1st type may play a role in the spread of the ASFV due to periodic viremia. Seropositive survived animals of the 2nd type due to their small numbers are not considered by some researchers to play a significant epidemiological role in the persistence of ASFV in wild boar populations. Experimental transmission of ASFV from surviving wild boars has been studied to date to a limited extent, such research should be continued. The knowledge gained in these areas will improve the understanding of the current situation of ASF in wildlife.

Koltsov A., Sukher M., Krutko S., Belov S., Korotin A., Rudakova S., Morgunov S., Koltsova G.

Background/Objectives: The spread of African swine fever virus (ASFV) has led to major economic losses to pork worldwide. In Russia, there are no developed or registered vaccines against ASFV genotype II, which is associated with numerous ASFV outbreaks in populations of domestic pigs and wild boars in the country. Methods: We introduced deletions of the six MGF360 and MGF505 genes of the ASFV virulent Stavropol_01/08 strain, isolated in Russia in 2008. Results: We show here that this deletion did lead to full attenuation of the ASFV virulent Stavropol_01/08 strain. Animals intramuscularly inoculated with 104 HAD50 of ΔMGF360/505_Stav developed a strong immune response and short period of viremia (at 3–7 days post-inoculation). Recombinant ΔMGF360/505_Stav strain provides complete protection of pigs against the ASFV parental Stavropol_01/08 strain (103 HAD50). Therefore, in our experiment, we did not detect the genome of both the virulent and the recombinant strains in the blood and organs post-challenge with the Stavropol_01/08. In contrast, we found only partial protection (40%) of the ΔMGF360/505_Stav-immunized pigs against challenge with the ASFV heterologous Rhodesia strain. Additionally, the surviving animals had a prolonged fever, and their condition was depressed for most of the experiment. Conclusions: Thus, the ASFV recombinant ΔMGF360/505_Stav strain is the first live attenuated vaccine (LAV) in Russia that induces complete protection in pigs challenged with the highly virulent, epidemiologically relevant strains genotype II and serotype 8. However, this ASF LAV is not able to provide a high level of protection against other variants of serotype 8.

Vlasov M.E., Balyshev V.M., Sereda A.D.

African swine fever (ASF) is a hemorrhagic viral disease that brings serious implications for animal health and economy due to high mortality rate, quarantine measures and restrictions on international trade in pig products. Only domestic and wild species of the Suidae family of all breeds and ages are susceptible to infection with ASF virus. To date, no safe and effective ASF vaccines have been developed, but in recent years some progress has been made in development of ASF modified live virus first-generation vaccine candidates, which have been used only in some countries of Southeast Asia. The expansion of their use is hindered, among other things, due to the lack of international and state recommendations (requirements) for the evaluation of purity, activity, safety and effectiveness of ASF vaccine candidates. Clinical signs of the disease are one of the main indicators of safety and effectiveness of ASF modified live virus vaccine candidates. The purpose of this work was to develop a clinical symptom-based scoring system to be used for characterizing of newly recovered ASFV isolates causing various forms of the disease, as well as for the determination of safety and effectiveness of ASF modified live virus vaccine candidates. It is proposed to take into account 7 major clinical manifestations: an increase in body temperature, reduced liveliness, loss of appetite, skin lesions, joint swelling, laboured breathing, neurological disorders, each scored from 0 to 3 or 4. The study of twelve ASFV strains of various virulence revealed that acute and subacute ASF produce the maximum clinical scores ranged from 13 to 22, chronic form gives 6–18 points, subclinical form is scored 0–8.

Oganesyan A.S., Shibayev M.A., Petrova O.N., Baskakova N.Y., Karaulov A.K.

The analysis of the situation on management of main epizootic threats significant for pig industry in the Russian Federation was carried out. The features and effectiveness of various biosecurity measures used in pig holdings and possible ways for their improvement were examined. Options for improvement of biosecurity measures in Russian pig holdings and development of the systems in the Russian Federation having favorable effect on the population protection were discussed focusing on four target aspects related to infection introduction and spread routes: isolation of the population from external sources of threats (elimination of probable environmental impact scenarios); isolation of the population from internal sources of threats (elimination of probable impact scenarios during production process); isolation of the pig population or pig farming system from the human factor impact (elimination of possible scenarios of human factor impact on the population); isolation of the population from the conditions under which the threat potential is manifested (changing of conditions). The analysis results were presented graphically in the form of a schematic diagram“Sources of threats to biosecurity systems and measures aimed at these sources for the protected population creation in pig industry”. Based on the discussion results, automation and digitalization of all processes in pig industry, generation of genetically modified pigs not susceptible to the most significant pathogens such as African swine fever, classical swine fever, porcine reproductive and respiratory syndrome viruses and their use for production purposes, further research and implementation of integrated technological solutions for feed sanitation have been concluded to be the evolutionarily significant ways for effective pig farming intensification in the Russian Federation.

Gobbi P., Pavone S., Orso M., Passamonti F., Righi C., Beato M.S., Feliziani F., Giammarioli M.

Small ruminant lentiviruses (SRLVs) are responsible for chronic and progressive multisystemic clinical forms, which significantly reduce flocks’ productivity and have a considerable economic impact on the small ruminant industry. Due to the increase in genetic analysis studies and the potential for misclassification of certain strains, owing to the high genetic variability of these viruses, a systematic review was deemed necessary. This review explores the types of matrices used for molecular detection and phylogenetic studies, the genomic regions selected as targets, and the software utilized for phylogenetic analysis, assessing the geographical distribution of identified genotypes and subgenotypes over time. A thorough comparison of the diagnostic approaches highlights the strengths and limitations of each method, identifying gaps that need to be addressed. Additionally, recombination events and compartmentalization are examined to provide an updated, detailed, and comprehensive overview of SRLV phylogenesis.

Petkevičius S., Klibavičė P., Šalomskas A., Kupčinskas T., Moroz-Fik A., Biernacka K., Mickiewicz M., Nowek Z., Ózsvári L., Bárdos K., Stuen S., Abril C.E., Bertoni G., Kaba J., Czopowicz M.

Wang J., Wan S., Liu S., Wang Z., Ding X., Wu Q., Liu X., Chen Z., Chen L., Wang H., Jia K., Li S.

Oganesyan A.S., Shibayev M.A., Petrova O.N., Baskakova N.Y., Karaulov A.K.

The results of the situational analysis on porcine diseases in the Russian Federation and the expert assessment prioritizing the list of porcine pathogens significant for the pig industry of the country are presented. The method applied to analyse the expert estimates in the situational analysis allows for rapid assessment and interpretation of the situation with identification of priority diseases to be further addressed. The calculations demonstrated the sufficient degree of agreement among the experts (coefficient of concordance W = 0.61), and Pearson’s chi-squared test statistic χ2 = 51.33 (≥ 21.02607) indicated that the concordance is not random and the results can be used in subsequent studies. The specific features of epizootiology of the agents of African swine fever, classical swine fever, porcine reproductive and respiratory syndrome that can impact the effectiveness of biosecurity systems of pig establishments, as well as further ways for improving biosecurity management measures are discussed. The overall risk for the pig industry in the Russian Federation that is associated with external sources is currently characterized as permanently high, requiring maintaining risk management measures at the pig establishments by both the managerial staff of the establishments and the State Veterinary Service. It is recommended that biosecurity measures against external threats should focus on diseases such as African swine fever (weight λ = 0.52), porcine reproductive and respiratory syndrome (λ = 0.071), classical swine fever (λ = 0.068) and infections considered emerging for the Russian Federation (λ = 0.05) according to the weights based on the expert estimation results. The biosecurity systems of the establishments should equally address other threats significant for the pig industry of the country: swine enzootic pneumonia, porcine pleuropneumonia (Actinobacillus pleuropneumoniae), Aujeszky’s disease, streptococcosis (Streptococcus suis), porcine circovirus infection, foot-and-mouth disease, leptospirosis, transmissible gastroenteritis, cysticercosis (λ = 0.02…0.05). The improvement of the governmental policy for eradication of African swine fever, porcine reproductive and respiratory syndrome, classical swine fever (including the substantial modification of the existing official pig turnover control, zoning, diagnosis and prevention quality, as well as the implementation of biosecurity standards) is the most significant factor, without which the disease eradication perspective is questionable.

Koltsov A., Sukher M., Krutko S., Belov S., Korotin A., Rudakova S., Morgunov S., Koltsova G.

African swine fever (ASF) is an emerging disease caused by the African swine fever virus (ASFV), which is a great threat to the swine industry worldwide. Currently registered vaccines that have demonstrated protection against the homologous ASFV strains are live attenuated vaccines based on recombinant ASFV strains with the deletions of virulence-associated genes. In this study, we evaluated the deletion of the A137R gene in the ASFV virulent Stavropol_01/08 strain isolated in Russia in 2008. Our animal experiment results demonstrated that the deletion of the A137R gene did not lead to the full attenuation of this strain, and increasing the dose of the A137R-deletion mutant during infection led to the death of 87.5% of the infected animals. In this report, we also demonstrated that immunofluorescence (IFA) and Western blotting assays based on the recombinant p11.5 protein can be used to detect antibodies in animals infected with the attenuated ASFV variants of several genotypes/serotypes. Both assays were specific to ASFV p11.5 protein and showed negative results when examining the sera of the non-infected animals or those infected with the A137R-deletion mutant. Therefore, we propose to use the p11.5 protein along with other previously proposed ASFV proteins, such as CD2v, as negative antigenic DIVA markers for an attenuated ASF vaccine.

Suwankitwat N., Deemagarn T., Bhakha K., Songkasupa T., Vitoonpong R., Trakunjaroonkit P., Rodphol S., Nuansrichay B., Chintapitaksakul L., Wongsarattanasin K., Kwon O., Kang H., Shin Y.

Lumpy skin disease (LSD) is a viral infection that impacts the cattle industry. The most efficient approach to prevent disease involves the utilization of live-attenuated LSD vaccines (LAVs), which stands out as the most successful method. However, LAVs might be subjected to changes to their genomes during replication that increase viral infectivity or virulence. The objective of this study was to monitor alterations in the genetic characteristics of the lumpy skin disease virus (LSDV) in beef cattle following the administration of LAVs in Lopburi Province of Central Thailand. A total of four skin samples from LSD cases were collected from non-vaccinated animals that exhibited LSD clinical symptoms from two distinct districts, spanning three subdistricts within the region. The samples of cattle were analyzed using real-time PCR targeting the LSDV074 p32 gene, the virus was isolated, and the entire genome sequences were evaluated through a single nucleotide polymorphisms (SNPs) analysis, and phylogenetic trees were assembled. The investigations revealed that LSDVs from two isolates from Chai Badan district exhibited significant mutations in the open reading frame (ORF) 023 putative protein, while another two isolates from Lam Sonthi district had a change in the untranslated region (UTR). For a result, the most proficient disease diagnosis and control should be evaluated on viral genetics on a regular basis.

Rathakrishnan A., Reis A.L., Petrovan V., Goatley L.C., Moffat K., Lui Y., Vuong M., Ikemizu S., Davis S., Dixon L.K.

Sereda A.D., Namsrayn S., Balyshev V.M., Vlasov M.E., Sindryakova I.P., Koltsova G., Kolbasov D.V.

The extreme genetic and immunobiological heterogeneity exhibited by the African swine fever virus (ASFV) has been a significant impediment in the development of an efficacious vaccine against this disease. Consequently, the lack of internationally accepted protocols for the laboratory evaluation of candidate vaccines has become a major concern within the scientific community. The formulation of such protocols necessitates the establishment of a consensus at the international level on methods for the determination of homologous and heterologous isolates/strains of ASFV. The present article provides a comprehensive description of biological techniques employed in the classification of ASFV by seroimmunotypes. These techniques involve a holistic evaluation of ASFV isolates/strains based on their antigenic properties as determined by the hemadsorption inhibiting test (HAdI) using type-specific sera and an immunological test (IT) conducted on pigs inoculated with attenuated strains. The article outlines the methods for setting up the HAdI test, an IT on pigs, and the processes involved in the acquisition of type-specific serums for the HAdI test. It is pertinent to note that the definitive classification of seroimmunotype can only be ascertained after conducting an IT on pigs. The findings from the HAdI test or the phylogenetic analysis of the EP402R gene should be considered preliminary in nature.

Vlasov M.E., Sindryakova I.P., Kudrjashov D.A., Morgunov S.Y., Kolbasova O.L., Lyska V.M., Zhivoderov S.P., Pivova E.Y., Balyshev V.M., Sereda A.D., Kolbasov D.V.

African swine fever virus (ASFV) is an extremely genetically and phenotypically heterogeneous pathogen. Previously, we have demonstrated that experimental inoculation of pigs with an attenuated strain, Katanga-350 (genotype I, seroimmunotype I) (ASFV-Katanga-350), can induce protective immunity in 80% of European domestic pigs against the homologous virulent European strain Lisbon-57. At least 50% of the surviving pigs received protection from subsequent intramuscular infection with a heterologous virulent strain, Stavropol 01/08 (genotype II, seroimmunotype VIII) (ASFV-Stavropol 01/08). In this study, we assessed clinical signs, the levels of viremia, viral DNA, anti-ASFV antibodies and post-mortem changes caused by subsequent intramuscular injection with ASFV-Katanga-350 and heterologous ASFV-Stavropol 01/08. Inoculation of pigs with the ASFV-Katanga-350 did not protect animals from the disease in the case of the subsequent challenged ASFV-Stavropol 01/08. However, 40% of pigs were protected from death. Moreover, the surviving animals showed no pathomorphological changes or the presence of an infectious virus in the organs after euthanasia at 35 days post challenging. The ability/inability of attenuated strains to form a certain level of protection against heterologous isolates needs a theoretical background and experimental confirmation.

Liu H., Wang A., Yang W., Liang C., Zhou J., Chen Y., Liu Y., Zhou Y., Zhang G.

African swine fever virus (ASFV), a highly pathogenic large DNA virus, is the cause of African swine fever worldwide. The ASFV virulence gene EP402R encodes CD2v, a structural protein that plays an important role in the ASFV infection process. In this study, a CHO-S cell line stably expressing the extracellular region of CD2v was generated and secretory CD2v(sCD2v)was purified from the cell culture supernatant. The purified glycosylated sCD2v protein possessed high immunoreactivity and immunogenicity. In addition, we found that glycosylation had a decisive effect on the immune reactivity of CD2v. Then sCD2v was used to generate five CD2v-specific monoclonal antibodies. The reactivity of all monoclonal antibodies with CD2v protein was confirmed by Western blot and indirect immunofluorescence assay (IFA). Interestingly, mAb 8D5 reactivity with sCD2v depended on sCD2v glycosylation status. Subsequent B cell epitope mapping experiments conducted using a series of overlapping synthetic peptides of the CD2v extracellular domain led to identification of mAb B cell epitopes of 128TCKKNNGTNT137 for mAb 4B11 and 148VKYTNESILE157 for mAbs 5H4 and 5F7. Due to their well-defined epitopes, these three mAbs will likely serve as valuable tools for use in ASFV CD2v structure-function studies, diagnostic assays, and prophylactic methodologies to control ASFV transmission.

Carrozza M., Niewiadomska A., Mazzei M., Abi-Said M.R., Hué S., Hughes J., Gatseva A., Gifford R.J.

Abstract Small ruminant lentiviruses (SRLVs) cause chronic, persistent infections in populations of domestic sheep (Ovis aries) and goats (Capra hircus) worldwide. The vast majority of SRLV infections involve two genotypes (A and B) that spread in association with the emergence of global livestock trade. However, SRLVs have likely been present in Eurasian ruminant populations since at least the early Neolithic period. Here, we use phylogenetic and phylogeographic approaches to reconstruct the origin of pandemic SRLV strains and infer their historical pattern of global spread. We constructed an open computational resource (‘Lentivirus-GLUE’) via which an up-to-date database of published SRLV sequences, multiple sequence alignments (MSAs), and sequence-associated metadata can be maintained. We used data collated in Lentivirus-GLUE to perform a comprehensive phylogenetic investigation of global SRLV diversity. Phylogenies reconstructed from genome-length alignments reveal that the deep divisions in the SRLV phylogeny are consistent with an ancient split into Eastern (A-like) and Western (B-like) lineages as agricultural systems disseminated out of domestication centres during the Neolithic period. These findings are also consistent with historical and phylogeographic evidence linking the early 20th century emergence of SRLV-A to the international export of Central Asian Karakul sheep. Investigating the global diversity of SRLVs can help reveal how anthropogenic factors have impacted the ecology and evolution of livestock diseases. The open resources generated in our study can expedite these studies and can also serve more broadly to facilitate the use of genomic data in SRLV diagnostics and research.

Olech M., Kuźmak J., Kycko A., Junkuszew A.

Abstract Introduction Previous gag and env sequence studies placed Polish small ruminant lentiviruses (SRLVs) isolated from sheep and goats in subtypes B1, B2, A1, A5, A12, A13, A16–A18, A23, A24 and A27. This study extended the genetic/phylogenetic analysis of previously identified Polish SRLV strains by contributing long terminal repeat (LTR) sequences. Material and Methods A total of 112 samples were analysed. Phylogenetic analyses were carried out on the LTR fragment using the neighbour-joining, maximum likelihood, and unweighted pair group method with arithmetic mean methods. Results Polish caprine and ovine LTR sequences clustered within group A and grouped in at least 10 clusters (subtypes A1, A5, A12, A13, A16–A18, A23, A24 and A27). Most of the Polish strains (78%) belonged to the same subtype by the indication of the gag, env and LTR genomic regions. Discrepancies in affiliation depending on the particular sequence were observed in 24 (21%) strains, most of which came from mixed-species flocks where more than one SRLV genotype circulated. Sequences of the LTR reflected subtype-specific patterns. Several subtype-specific markers were identified, e.g. a unique substitution of T to A in the fifth position of the TATA box in A17, A27, A20 and B3. Conclusion This study provides valuable insights into the genetic diversity of SRLV field strains in Poland, their phylogenetic relationships and their position in the recently established SRLV classification. Our results confirmed the existence of the ten subtypes listed and the readier emergence of new SRLV variants in mixed-species flocks.

Wang J., Xu Z., Wang Z., Li Q., Liang X., Ye S., Cheng K., Xu L., Mao J., Wang Z., Meng W., Sun Y., Jia K., Li S.

In August 2019, lumpy skin disease occurred for the first time in Xinjiang, China, and then quickly spread to many provinces in China. Here, the virus was isolated from the skin scabs of affected cattle during June 2020 in Guangdong, China. Virus isolation, transmission electron microscopy and polymerase chain reaction identified lumpy skin disease virus (LSDV) in the skin crusts of sick cattle. For the isolation of LSDV, the most sensitive cell line is primary cattle testicular (PCT) cells, while Vero cells cannot be used for the isolation of LSDV. In addition, we evaluated the growth characteristics of LSDV in vitro. Compared with MDBK and Vero cells, LSDV produced higher virus titters in PCT cells at 72 h. Phylogenetic analysis based on second-generation sequencing of the LSDV whole genome showed that the isolated virus (LSDV/MZGD/2020) is closely related to Asian strains and formed a new branch. LSDV/MZGD/2020 is also a vaccine recombinant strain that is distinct from the recombinant strain found in Russia. Through Recombination Detection Program (RDP), Simplot and phylogenetic analyses, strong evidence for recombination events was found in Chinese field LSDV strains. The China LSDV/MZGD/2020 strain may be the result of multiple recombination events between the Neethling 2490 and Neethling vaccine LW 1959 strains. This study expands our knowledge of the genetic diversity and evolution of LSDV.

Havas K.A., Gogin A.E., Basalaeva J.V., Sindryakova I.P., Kolbasova O.L., Titov I.A., Lyska V.M., Morgunov S.Y., Vlasov M.E., Sevskikh T.A., Pivova E.Y., Kudrjashov D.A., Doolittle K., Zimmerman S., Witbeck W., et. al.

African swine fever virus causes hemorrhagic disease in swine. Attenuated strains are reported in Africa, Europe, and Asia. Few studies on the diagnostic detection of attenuated ASF viruses are available. Two groups of pigs were inoculated with an attenuated ASFV. Group 2 was also vaccinated with an attenuated porcine reproductive and respiratory syndrome virus vaccine. Commercially available ELISA, as well as extraction and qPCR assays, were used to detect antibodies in serum and oral fluids (OF) and nucleic acid in buccal swabs, tonsillar scrapings, OF, and blood samples collected over 93 days, respectively. After 12 dpi, serum (88.9% to 90.9%) in Group 1 was significantly better for antibody detection than OF (0.7% to 68.4%). Group 1′s overall qPCR detection was highest in blood (48.7%) and OF (44.2%), with the highest detection in blood (85.2%) from 8 to 21 days post inoculation (dpi) and in OF (83.3%) from 1 to 7 dpi. Group 2′s results were not significantly different from Group 1, but detection rates were lower overall. Early detection of attenuated ASFV variants requires active surveillance in apparently healthy animals and is only reliable at the herd level. Likewise, antibody testing will be needed to prove freedom from disease.

Arcangeli C., Torricelli M., Sebastiani C., Lucarelli D., Ciullo M., Passamonti F., Giammarioli M., Biagetti M.

Small ruminant lentiviruses (SRLVs) represent a very heterogeneous group of ss-RNA viruses that infect sheep and goats worldwide. They cause important, deleterious effects on animal production and limit the animal trade. SRLVs show a high genetic variability due to high mutation rate and frequent recombination events. Indeed, five genotypes (A–E) and several subtypes have been detected. The aim of this work was to genetically characterize SRLVs circulating in central Italy. On this basis, a phylogenetic study on the gag-pol genetic region of 133 sheep, collected from 19 naturally infected flocks, was conducted. In addition, to evaluate the frequency of mutation and the selective pressure on this region, a WebLogo 3 analysis was performed, and the dN/dS ratio was computed. The results showed that 26 samples out of 133 were clustered in genotype A and 106 samples belonged to genotype B, as follows: A9 (n = 8), A11 (n = 10), A24 (n = 7), B1 (n = 2), B2 (n = 59), and B3 (n = 45). No recombination events were found. Mutations were localized mainly in the VR-2 region, and the dN/dS ratio of 0.028 indicated the existence of purifying selection. Since the genetic diversity of SRLVs could make serological identification difficult, it is important to perform molecular characterization to ensure a more reliable diagnosis, to maintain flock health status, and for the application of local and national control programs.

Havas K.A., Makau D.N., Shapovalov S., Tolkova E., VanderWaal K., Tkachyk T., Spronk G.D., Heron B., Dee S.A., Perez A.

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating disease of swine in many parts of the world. Porcine reproductive and respiratory syndrome virus (PRRSV) type 1 is endemic in Europe, and prevalence of the subtypes differ spatially. In this study, we investigated a severe PRRS outbreak reported in 30 farms located in eastern Russia that belong to a large swine production company in the region that was also experiencing a pseudorabies outbreak in the system. Data included 28 ORF5 sequences from samples across 18 of the 25 infected sites, reverse transcriptase real-time polymerase chain reaction (RT-qPCR) results from diagnostic testing, reports of clinical signs, and animal movement records. We observed that the outbreak was due to two distinct variants of wildtype PRRSV type 1 subtype 1 with an average genetic distance of 15%. Results suggest that the wildtype PRRSV variants were introduced into the region around 2019, before affecting this production system (i.e., sow farms, nurseries, and finisher farms). Clinical signs did not differ between the variants, but they did differ by stage of pig production. Biosecurity lapses, including movement of animals from infected farms contributed to disease spread.

Schaer J., Cvetnic Z., Sukalic T., Dörig S., Grisiger M., Iscaro C., Feliziani F., Pfeifer F., Origgi F., Zanoni R.G., Abril C.E.

Small ruminant lentiviruses (SRLVs), i.e., CAEV and MVV, cause insidious infections with life-long persistence and a slowly progressive disease, impairing both animal welfare and productivity in affected herds. The complex diagnosis of SRLVs currently combines serological methods including whole-virus and peptide-based ELISAs and Immunoblot. To improve the current diagnostic protocol, we analyzed 290 sera of animals originating from different European countries in parallel with three commercial screening ELISAs, Immunoblot as a confirmatory assay and five SU5 peptide ELISAs for genotype differentiation. A newly developed nested real-time PCR was carried out for the detection and genotype differentiation of the virus. Using a heat-map display of the combined results, the drawbacks of the current techniques were graphically visualized and quantified. The immunoblot and the SU5-ELISAs exhibited either unsatisfactory sensitivity or insufficient reliability in the differentiation of the causative viral genotype, respectively. The new truth standard was the concordance of the results of two out of three screening ELISAs and the PCR results for serologically false negative samples along with genotype differentiation. Whole-virus antigen-based ELISA showed the highest sensitivity (92.2%) and specificity (98.9%) among the screening tests, whereas PCR exhibited a sensitivity of 75%.

Shuralev E.A., Khammadov N.I., Osyanin K.A., Elizarova I.A., Salmanova G.R., Shamaev N.D., Petrov S.V., Whelan C., Saushkin N.Y., Samsonova J.V., Galimzyanov I.G., Efimova M.A., Khaertynov K.S., Faizov T.K., Mukminov M.N., et. al.

Background and Aim: Several reports described the detection of specific caprine arthritis-encephalitis virus (CAEV) antibodies in Russian goat populations, which indicates the circulation of CAEV in Russian goat farms. The aim of this study was to use a multi-target approach to testing with both serological tests and an in-house real-time (RT) molecular test to investigate the prevalence of CAEV in goats from three hobbyist farms in the Republic of Tatarstan, Russia. Materials and Methods: We applied a multi-target approach to testing with both enzyme-linked immunosorbent assay (ELISA) and an in-house RT polymerase chain reaction test to investigate the prevalence of CAEV in goats. Animals from the three hobbyist farms were used in this study. The animals from two farms (n=13 for F1 and n=8 for F2) had clinical signs of arthritis and mastitis. In the third farm (n=15 for F3), all goats were home-bred and had no contact with imported animals. Results: CAEV antibodies (ELISA targets TM env and gag genes) were detected in serum samples from two farms (F1 and F2), indicating seroprevalence of 87.50-92.31%. Specific CAEV antibodies were also detected in milk samples. CAEV proviral DNA was detected in 53.85-62.50%. The results from all tests performed in the third farm (F3) were negative, indicating that all tests were 100% specific. Conclusion: The results showed that CAEV is circulating and present in small hobbyist goat farms in Russia. Serological and molecular tests could be important for programs to control and eradicate CAEV in Russia for hobbyist goat farms.

Paul B.T., Hashi H.A., Burhannuddin N.N., Lim T.C., Jesse F.F., Mohd Lila M.A., Haron A.W., Che Amat A., Abba Y., Maqbool A., Bhutto K.U., Mat Isa K., Amira N.A., Odhah M.N., Hambali I.U., et. al.

Caprine arthritis-encephalitis virus (CAEV) is a member of the genus lentivirus causing caprine arthritis-encephalitis (CAE), a chronic inflammatory condition affecting the lungs, joints, udder and central nervous system of small ruminants such as sheep and goats. CAE is distributed worldwide and is recognised as a significant cause of morbidity and decreased milk production in dairy goats. Earlier studies highlighted the clinicopathological features and supplied preliminary serological evidence for the existence of CAE among selected goat herds in Malaysia. Therefore, this study aims to provide further insights into the seroprevalence and contributing factors of CAE among sheep and goat herds in two states of Peninsular Malaysia. The blood samples and biodata were randomly collected from a total of 262 individual sheep (40) and goat (222) in seven smallholder farms. Blood sera were tested for specific anti-CAEV antibodies using Qayee-Bio CAEV sandwich-ELISA test kits according to standard procedures. Our results of the study revealed 21.4% (95% CI: 15.8–28.6) apparent and 20.6% (95% CI: 14.5–27.8) true seroprevalence with significant differences (p < 0.05) in seroconversion rates between the states, farms, production systems and breeds of small ruminants. The prevalence of CAE in the Malaysian Peninsular is a potential threat to the small ruminant industry and developing agricultural economy. Further studies are required to determine the genetic characteristics, distribution and risk factors of CAEV for effective prevention and control in Malaysia.

Sánchez-Cordón P.J., Floyd T., Hicks D., Crooke H.R., McCleary S., McCarthy R.R., Strong R., Dixon L.K., Neimanis A., Wikström-Lassa E., Gavier-Widén D., Núñez A.

The understanding of the pathogenic mechanisms and the clinicopathological forms caused by currently circulating African swine fever virus (ASFV) isolates is incomplete. So far, most of the studies have been focused on isolates classified within genotypes I and II, the only genotypes that have circulated outside of Africa. However, less is known about the clinical presentations and lesions induced by isolates belonging to the other twenty-two genotypes. Therefore, the early clinicopathological identification of disease outbreaks caused by isolates belonging to, as yet, not well-characterised ASFV genotypes may be compromised, which might cause a delay in the implementation of control measures to halt the virus spread. To improve the pathological characterisation of disease caused by diverse isolates, we have refined the macroscopic and histopathological evaluation protocols to standardise the scoring of lesions. Domestic pigs were inoculated intranasally with different doses (high, medium and low) of ASFV isolate Ken05/Tk1 (genotype X). To complement previous studies, the distribution and severity of macroscopic and histopathological lesions, along with the amount and distribution of viral antigen in tissues, were characterised by applying the new scoring protocols. The intranasal inoculation of domestic pigs with high doses of the Ken05/Tk1 isolate induced acute forms of ASF in most of the animals. Inoculation with medium doses mainly induced acute forms of disease. A less severe but longer clinical course, typical of subacute forms, characterised by the presence of more widespread and severe haemorrhages and oedema, was observed in one pig inoculated with the medium dose. The severity of vascular lesions (haemorrhages and oedema) induced by high and medium doses was not associated with the amount of virus antigen detected in tissues, therefore these might be attributed to indirect mechanisms not evaluated in the present study. The absence of clinical signs, lesions and detectable levels of virus genome or antigen in blood from the animals inoculated with the lowest dose ruled out the existence of possible asymptomatic carriers or persistently infected pigs, at least for the 21 days period of the study. The results corroborate the moderate virulence of the Ken05/Tk1 isolate, as well as its capacity to induce both the acute and, occasionally, subacute forms of ASF when high and medium doses were administered intranasally.

Kumar N., Chander Y., Kumar R., Khandelwal N., Riyesh T., Chaudhary K., Shanmugasundaram K., Kumar S., Kumar A., Gupta M.K., Pal Y., Barua S., Tripathi B.N.

Lumpy skin disease (LSD) has devastating economic impact. During the last decade, LSD had spread to climatically new and previously disease-free countries, which also includes its recent emergence in the Indian subcontinent (2019). This study deals with the LSD outbreak(s) from cattle in Ranchi (India). Virus was isolated from the scabs (skin lesions) in the primary goat kidney cells. Phylogenetic analysis based on nucleotide sequencing of LSD virus (LSDV) ORF011, ORF012 and ORF036 suggested that the isolated virus (LSDV/Bos taurus-tc/India/2019/Ranchi) is closely related to Kenyan LSDV strains. Further, we adapted the isolated virus in Vero cells. Infection of the isolated LSDV to Vero cells did not produce cytopathic effect (CPE) until the 4th blind passage, but upon adaptation, it produced high viral titres in the cultured cells. The kinetics of viral DNA synthesis and one-step growth curve analysis suggested that Vero cell-adapted LSDV initiates synthesizing its genome at ~24 hours post-infection (hpi) with a peak level at ~96 hpi whereas evidence of progeny virus particles was observed at 36–48 hours (h) with a peak titre at ~120 h. To the best of our knowledge, this study describes the first successful isolation of LSDV in India, besides providing insights into the life cycle Vero cell-adapted LSDV.