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Bluetongue virus (BT) is a vector-borne virus that causes a disease, called bluetongue, which results in significant economic loss and morbidity in sheep, cattle, goats and wild ungulates across all continents of the world except Antarctica. Despite the geographical breadth of its impact, most BT epidemiological models are informed by parameters derived from the 2006-2009 BTV-8 European outbreak. The aim of this study was to develop a highly adaptable model for BT which could be used elsewhere in the world, as well as to identify the parameters which most influence outbreak dynamics, so that policy makers can be properly informed with the most current information to aid in disease planning. To provide a framework for future outbreak modelling and an updated parameterisation that reflects natural variation in infections, a newly developed and parameterised two-host, two-vector species ordinary differential equation model was formulated and analysed. The model was designed to be adaptable to be implemented in any region of the world and able to model both epidemic and endemic scenarios. It was parameterised using a systematic literature review of host-to-vector and vector-to-host transmission rates, host latent periods, host infectious periods, and vaccine protection factors. The model was demonstrated using the updated parameters, with South Africa as a setting based on the Western Cape's known cattle and sheep populations, local environmental parameters, and Culicoides spp. presence data. The sensitivity analysis identified that the duration of the infectious period for sheep and cows had the greatest impact on the outbreak length and number of animals infected at the peak of the outbreak. Transmission rates from cows and sheep to C. imicola midges greatly influenced the day on which the peak of the outbreak occurred, along with the duration of incubation period, and infectious period for cows. Finally, the protection factor of the vaccine had the greatest influence on the total number of animals infected. This knowledge could aid in the development of control measures. Due to gradual climate and anthropological change resulting in alterations in vector habitat suitability, BT outbreaks are likely to continue to increase in range and frequency. Therefore, this research provides an updated BT modelling framework for future outbreaks around the world to explore transmission, outbreak dynamics and control measures., Competing Interests: Declaration of Competing Interest This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue. The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Peste des Petits Ruminants (PPR) is an economically significant transboundary viral disease of sheep and goats caused by the PPRV virus, affecting annual losses of 1.45-2.10 billion US dollars globally. We designed the current study to evaluate the positive cases, molecular characterization, phylogenetic analysis, and risk factors correlated with the disease in various districts of Khyber Pakhtunkhwa, Pakistan, with the aim of contributing to these strategies., Methods and Results: A total of 384 samples from three selected districts, i.e., Peshawar, Charsadda and Chitral (n = 128 each), were collected, and the virus was investigated by using the sandwich ELISA, while the N gene of the virus was used as a target for molecular detection via RT-PCR. The confirmed samples were then sequenced, and phylogenetic analysis was performed. According to our findings, the highest positive cases was found in district Peshawar (50.87%), followed by Charsadda and Chitral (24.56%), respectively, while risk factor analysis showed that certain categories, such as species, sex, and age less than two years, have higher risk (P < 0.05) in contrast to their respective categories. Furthermore, sequencing and phylogenetic analysis of representative samples showed that the PPRV strains in the current study clustered in lineage IV, which is circulating in the small ruminant population of Asia, the Middle East, and African countries. Comparative residue analysis highlighted the mutation by representing 242 variable sites out of 371 locations., Conclusions: PPRV has foremost importance in Pakistan because the virus was detected in a considerable number of samples, and most of which were sourced from subsidiary areas where veterinary services are not prioritized., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Hepatitis E virus (HEV) is currently recognized as an emerging problem and a growing concern for public health in developed countries, with HEV infections mainly attributable to foodborne transmission of HEV-3. The zoonotic HEV genotype 3 infects a wide range of mammalian hosts, with swine considered as the primary host. This study investigates the occurrence of HEV among small ruminants in Portugal. The primary aim of the present research was to evaluate the circulation and the potential for HEV infection among sheep and goats. A total of 400 bile samples and 493 blood samples were collected from sheep and goats at a slaughterhouse in the center region of Portugal, between January 2022 and March 2023. The HEV RNA detection in bile samples was performed using a nested broad-spectrum RT-PCR targeting the ORF1 region. Serological analysis to detect anti-HEV antibodies was conducted using a commercial double-antigen sandwich multi-species ELISA. The HEV RNA was not detected in any bile samples using the nested broad-spectrum RT-PCR. Serological analysis revealed an overall HEV antibody seroprevalence of 2% (10/493, 95% CI: 0.98-3.70) among the small ruminants, namely 2.2% in goats and 2.0% in sheep. Curiously, no statistically significant association among the factors, age, sex and species and HEV seroprevalence was observed. Although HEV RNA was not detected in the bile of sheep and goats, this study the evidence of seroprevalence in these small ruminant species. Further research could provide additional insights into the factors influencing HEV transmission dynamics in small ruminants in Portugal and its potential implications for public health., (© 2024. The Author(s).)

Adenoviruses (AdVs) have been detected in a wide variety of animals. To date, eight types of AdVs in sheep and two types in goats have been identified, which belong to two distinct genera, Mastadenovirus and Atadenovirus. Typically, the term pneumo-enteritis is used to describe adenovirus-induced disease in small ruminants, which has been associated with both enteric and respiratory symptoms of varying severity. The aim of this study was to detect and identify AdVs of small ruminants belonging to the genera Mastadenovirus and Atadenovirus. For this purpose, diagnostic samples (47 lung, 27 intestine, and two pooled tissue samples including intestine and lung) from 49 small ruminants (39 sheep and 10 goats) were used. Following the viral DNA extraction, PCR was carried out by using the primers targeting the hexon gene in order to detect both mast- and atadenoviruses. Sequencing the amplified fragments revealed the presence of three types of ovine adenovirus (OAdV): OAdV-3, OAdV-4, and OAdV-8. Specifically, OAdV-3 was detected in two sheep and a goat while OAdV-4 and OAdV-8 were found in only one sheep each. There is still limited data on the interaction between the viruses in different adenovirus genera and the detected disease, as well as the genetic diversity of adenoviruses, especially in small ruminants. In conclusion, the detection of AdVs in lung and intestinal tissues of small ruminants in this study suggests that these viruses may have contributed to the disease and/or predisposed to other agents., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The ovine maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) are small ruminant lentiviruses (SRLVs) with striking genetic and structural similarities. The presence of SRLV in Mongolian sheep and goats was serologically demonstrated more than a decade ago; however, the viral genotype remains unknown. In total, 329 blood samples were collected from two sheep breeds (i.e., Khalkha and Sumber) in Tov, Govisumber, Arkhangay, Dornogovi, Zavkhan, and Sukhbaatar provinces, Mongolia. Serological and phylogenetic analyses were performed regardless of any apparent clinical signs, although most of the animals appeared healthy. All sheep in three of the six provinces were seronegative, whereas the seroprevalence in the Tov, Govisumber, and Zavkhan provinces averaged 7.9%. Genomic DNA from seropositive animals was tested using hemi-nested polymerase chain reaction, and sub-genomic SRLV sequences were determined from nine samples. Mongolian SRLV sequences clustered within the divergent subtype A22, which was previously found only in Fertile Crescent regions, including Lebanon, Jordan, and Iran, where the first sheep-domestication (Ovis aries) occurred. According to the phylogenetic analysis, genotype A has two ancestors from the ancient Fertile Crescent: (1) Turkish strains and (2) Iranian, Jordanian, and Lebanese strains. The first ancestor spread westward, whereas the second spread eastward, ultimately reaching Mongolia., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Over the last few decades, several pestiviruses have been discovered in ruminants, pigs, and, more recently, in non-ungulate hosts. Consequently, the nomenclature and taxonomy of pestiviruses have been updated. The Tunisian sheep-like pestivirus (TSV, Pestivirus N ) is an additional ovine pestivirus genetically closely related to classical swine fever virus (CSFV). In this study, during a survey of pestivirus infections in ovine farms in the Lombardy region of Northern Italy, we identified and isolated a pestivirus strain from a sheep that was found to belong to Pestivirus N species based on its genomic nucleotide identity. The sheep itself and its lamb were found to be persistently infected. We performed molecular characterization and phylogenetic analysis of three viral genomic regions (a fragment of 5'-UTR, partial N pro , and the whole E2 region). In conclusion, these results confirmed circulating TSV in Northern Italy after notification in Sicily, Italy, and France. Correlation with Italian, Tunisian, and French strains showed that detection might have resulted from the trading of live animals between countries, which supports the need for health control measures.

The hepatitis E virus is a serious health concern worldwide, with 20 million cases each year. Growing numbers of autochthonous HEV infections in industrialized nations are brought on via the zoonotic transmission of HEV genotypes 3 and 4. Pigs and wild boars are the main animal reservoirs of HEV and play the primary role in HEV transmission. Consumption of raw or undercooked pork meat and close contact with infected animals are the most common causes of hepatitis E infection in industrialized countries. However, during the past few years, mounting data describing HEV distribution has led experts to believe that additional animals, particularly domestic ruminant species (cow, goat, sheep, deer, buffalo, and yak), may also play a role in the spreading of HEV. Up to now, there have not been enough studies focused on HEV infections associated with animal milk and the impact that they could have on the epidemiology of HEV. This critical analysis discusses the role of domestic ruminants in zoonotic HEV transmissions. More specifically, we focus on concerns related to milk safety, the role of mixed farming in cross-species HEV infections, and what potential consequences these may have on public health.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that has had significant impacts on human health and economies worldwide. SARS-CoV-2 is highly transmissible and the cause of coronavirus disease 2019 in humans. A wide range of animal species have also been shown to be susceptible to SARS-CoV-2 by experimental and/or natural infections. Sheep are a commonly farmed domestic ruminant that have not been thoroughly investigated for their susceptibility to SARS-CoV-2. Therefore, we performed in vitro and in vivo studies which consisted of infection of ruminant-derived cells and experimental challenge of sheep to investigate their susceptibility to SARS-CoV-2. Our results showed that sheep-derived kidney cells support SARS-CoV-2 replication. Furthermore, the experimental challenge of sheep demonstrated limited infection with viral RNA shed in nasal and oral swabs at 1 and 3-days post challenge (DPC); viral RNA was also detected in the respiratory tract and lymphoid tissues at 4 and 8 DPC. Sero-reactivity was observed in some of the principal infected sheep but not the contact sentinels, indicating that transmission to co-mingled naïve sheep was not highly efficient; however, viral RNA was detected in respiratory tract tissues of sentinel animals at 21 DPC. Furthermore, we used a challenge inoculum consisting of a mixture of two SARS-CoV-2 isolates, representatives of the ancestral lineage A and the B.1.1.7-like alpha variant of concern, to study competition of the two virus strains. Our results indicate that sheep show low susceptibility to SARS-CoV-2 infection and that the alpha variant outcompeted the lineage A strain.

The Getah virus (GETV), a mosquito-borne RNA virus, is widely distributed in Oceania and Asia. GETV is not the only pathogenic to horses, pigs, cattle, foxes and boars, but it can also cause fever in humans. Since its first reported case in Chinese mainland in 2017, the number of GETV-affected provinces has increased to seventeen till now. Therefore, we performed an epidemiologic investigation of GETV in the Xinjiang region, located in northwestern China, during the period of 2017-2020. ELISA was used to analyze 3299 serum samples collected from thoroughbred horse, local horse, sheep, goat, cattle, and pigs, with thoroughbred horse (74.8%), local horse (67.3%), goat (11.7%), sheep (10.0%), cattle (25.1%) and pigs (51.1%) being positive for anti-GETV antibodies. Interestingly, the neutralizing antibody titer in horses was much higher than in other species. Four samples from horses and pigs were positive for GETV according to RT-PCR. Furthermore, from the serum of a local horse, we isolated GETV which was designated as strain XJ-2019-07, and determined its complete genome sequence. From the phylogenetic relationships, it belongs to the Group III lineage. This is the first evidence of GETV associated to domestic animals in Xinjiang. Overall, GETV is prevalent in Xinjiang and probably has been for several years. Since no vaccine against GETV is available in China, detection and monitoring strategies should be improved in horses and pigs, especially imported and farmed, in order to prevent economic losses., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2022 The Authors. Publishing services by Elsevier B.V. All rights reserved.)

Maedi-visna (MV) is a lentiviral disease of sheep responsible for severe production losses in affected flocks. There are no vaccination or treatment options with control reliant on test and cull strategies. The most common diagnostic methods used at present are combination ELISAs for Gag and Env proteins with virus variability making PCR diagnostics still largely an experimental tool. To assess variability in viral loads and diagnostic tests results, serology, DNA and RNA viral loads were measured in the blood of 12 naturally infected rams repeatedly blood sampled over 16 months. Six animals tested negative in one or more tests at one or more time points and would have been missed on screening programmes reliant on one test method or a single time point. In addition the one animal homozygous for the 'K' allele of the TMEM154 E35K SNP maintained very low viral loads in all assays and apparently cleared infection to below detectable limits at the final time point it was sampled. This adds crucial data to the strong epidemiological evidence that this locus represents a genuine resistance marker for MV infection and is a strong candidate for selective breeding of sheep for resistance to disease.

Small ruminant lentiviruses (SRLVs) are a group of highly divergent viruses responsible for global infection in sheep and goats. In a previous study we showed that SRLV strains found in mixed flocks in Poland belonged to subtype A13 and A18, but this study was restricted only to the few flocks from Małopolska region. The present work aimed at extending earlier findings with the analysis of SRLVs in mixed flocks including larger numbers of animals and flocks from different part of Poland. On the basis of gag and env sequences, Polish SRLVs were assigned to the subtypes B2, A5, A12, and A17. Furthermore, the existence of a new subtypes, tentatively designed as A23 and A24, were described for the first time. Subtypes A5 and A17 were only found in goats, subtype A24 has been detected only in sheep while subtypes A12, A23, and B2 have been found in both sheep and goats. Co-infection with strains belonging to different subtypes was evidenced in three sheep and two goats originating from two flocks. Furthermore, three putative recombination events were identified within gag and env SRLVs sequences derived from three sheep. Amino acid (aa) sequences of immunodominant epitopes in CA protein were well conserved while Major Homology Region (MHR) had more alteration showing unique mutations in sequences of subtypes A5 and A17. In contrast, aa sequences of surface glycoprotein exhibited higher variability confirming type-specific variation in the SU5 epitope. The number of potential N-linked glycosylation sites (PNGS) ranged from 3 to 6 in respective sequences and were located in different positions. The analysis of LTR sequences revealed that sequences corresponding to the TATA box, AP-4, AML-vis, and polyadenylation signal (poly A) were quite conserved, while considerable alteration was observed in AP-1 sites. Interestingly, our results revealed that all sequences belonging to subtype A17 had unique substitution T to A in the fifth position of TATA box and did not have a 11 nt deletion in the R region which was noted in other sequences from Poland. These data revealed a complex picture of SRLVs population with ovine and caprine strains belonging to group A and B. We present strong and multiple evidence of dually infected sheep and goats in mixed flocks and present evidence that these viruses can recombine in vivo.

A study was implemented to estimate the pestivirus seroprevalence in sheep and goats in Belgium, to identify circulating species and to check for a potential association between seropositivity of small ruminants and presence of cattle in the same farm. It was based on the testing of serum samples and bulk tank milk samples (BTM) collected in sheep and goat flocks in 2018-2019 all over the country. 7460 serum samples collected from 410 flocks were tested by a commercial ELISA able to detect antibodies (Ab) against Border Disease Virus (BDV), and Bovine Viral Diarrhea Virus (BVDV). BTM samples (n = 144) were collected from dairy flocks in November 2019 and tested with the same Ab ELISA. ELISA positive serum samples were also tested by virus neutralization test (VNT) for neutralizing antibodies against BDV, BVDV-type1 and BVDV-type2. Virological tests (RT-PCR) were performed on pools of serum samples from pestivirus-exposed flocks with at least two seropositive animals and on all Antibody-positive BTM samples. Information about serum and milk samples (identification, test results, farm of origin and location, presence of cattle) were gathered in animal-level and farm-level databases. Based on this study, the apparent animal seroprevalence for pestiviruses in small ruminant flocks in Belgium in 2018 was estimated to be 0.87 % (95 % C.I. [0.68 %-1.11 %]). The prevalence of flocks exposed to pestivirus (i.e. with at least one seropositive animal) was estimated to be 8.5 % (95 % C.I. [6.4 % - 11.6 %]). In exposed flocks, the average within-flock seroprevalence was 9.9 %. In dairy sheep and goats, the estimated proportion of exposed flocks in 2019, based on the detection of pestivirus antibodies in the bulk tank milk, was 9.7 % [5.9 %-15.7 %]. All PCR tests were negative, indicating the likely absence of active pestivirus circulation in these flocks. Although the observed pestivirus seroprevalence was found to be low in Belgian small ruminants, this study also showed, based on VNT results, that they are exposed to several pestivirus species: BDV, BVDV-1 and BVDV-2. 22.4 % of the farms included in the serological survey were holding both a small ruminant flock and a cattle herd, hence with a potential risk of contact between the two species. There was a significant positive association between pestivirus seropositivity in the sheep/goat flocks and the presence of a cattle herd in the same farm (OR = 2.42 (95 %C.I. [1.18-4.94]) but this association was not found for Ab-positive BTM in dairy flocks., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The Morbillivirus peste des petits ruminants virus (PPRV) is the causal agent of a highly contagious disease that mostly affects sheep and goats and produces considerable losses in developing countries. Current PPRV control strategies rely on live-attenuated vaccines, which are not ideal, as they cannot differentiate infected from vaccinated animals (DIVA). Recombinant vector-based vaccines expressing viral subunits can provide an alternative to conventional vaccines, as they can be easily paired with DIVA diagnostic tools. In the present work, we used the bovine herpesvirus-4-based vector (BoHV-4-A) to deliver PPRV hemagglutinin H antigen (BoHV-4-A-PPRV-H-ΔTK). Vaccination with BoHV-4-A-PPRV-H-ΔTK protected sheep from virulent PPRV challenge and prevented virus shedding. Protection correlated with anti-PPRV IgGs, neutralizing antibodies and IFN-γ-producing cells induced by the vaccine. Detection of antibodies exclusively against H-PPRV in animal sera and not against other PPRV viral proteins such as F or N could serve as a DIVA diagnostic test when using BoHV-4-A-PPRV-H-ΔTK as vaccine. Our data indicate that BoHV-4-A-PPRV-H-ΔTK could be a promising new approach for PPRV eradication programs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rodríguez-Martín, Rojas, Macchi, Franceschi, Russo, Sevilla, Donofrío and Martín.)

Maedi-Visna-like genotype A strains and Caprine arthritis encephaltis-like genotype B strains are small ruminant lentiviruses (SRLV) which, for incompletely understood reasons, appear to be more virulent in sheep and goats, respectively. A 9-month in vivo infection experiment using Belgian genotype A and B SRLV strains showed that almost all homologous (genotype A in sheep; genotype B in goats) and heterologous (genotype A in goats; genotype B in sheep) intratracheal inoculations resulted in productive infection. No differences in viremia and time to seroconversion were observed between homologous and heterologous infections. Higher viral loads and more severe lesions in the mammary gland and lung were however detected at 9 months post homologous compared to heterologous infection which coincided with strongly increased IFN-γ mRNA expression levels upon homologous infection. Pepscan analysis revealed a strong antibody response against immune-dominant regions of the capsid and surface proteins upon homologous infection, which was absent after heterologous infection. These results inversely correlated with protection against virus replication in target organs and observed histopathological lesions, and thus require an in-depth evaluation of a potential role of antibody dependent enhancement in SRLV infection. Finally, no horizontal intra- and cross-species SRLV transmission to contact animals was detected.

Phleboviruses (genus P hlebovirus , family Phenuiviridae ) are emerging pathogens of humans and animals. Sand-fly-transmitted phleboviruses are found in Europe, Africa, the Middle East, and the Americas, and are responsible for febrile illness and nervous system infections in humans. Rio Grande virus (RGV) is the only reported phlebovirus in the United States. Isolated in Texas from southern plains woodrats, RGV is not known to be pathogenic to humans or domestic animals, but serologic evidence suggests that sheep ( Ovis aries ) and horses ( Equus caballus ) in this region have been infected. Rift Valley fever virus (RVFV), a phlebovirus of Africa, is an important pathogen of wild and domestic ruminants, and can also infect humans with the potential to cause severe disease. The introduction of RVFV into North America could greatly impact U.S. livestock and human health, and the development of vaccines and countermeasures is a focus of both the CDC and USDA. We investigated the potential for serologic reagents used in RVFV diagnostic assays to also detect cells infected with RGV. Western blots and immunocytochemistry assays were used to compare the antibody detection of RGV, RVFV, and two other New World phlebovirus, Punta Toro virus (South and Central America) and Anhanga virus (Brazil). Antigenic cross-reactions were found using published RVFV diagnostic reagents. These findings will help to inform test interpretation to avoid false positive RVFV diagnoses that could lead to public health concerns and economically costly agriculture regulatory responses, including quarantine and trade restrictions.

Small ruminant lentiviruses (SRLVs) infections lead to chronic diseases and remarkable economic losses undermining health and welfare of animals and the sustainability of farms. Early and definite diagnosis of SRLVs infections is the cornerstone for any control and eradication efforts; however, a "gold standard" test and/or diagnostic protocols with extensive applicability have yet to be developed. The main challenges preventing the development of a universally accepted diagnostic tool with sufficient sensitivity, specificity, and accuracy to be integrated in SRLVs control programs are the genetic variability of SRLVs associated with mutations, recombination, and cross-species transmission and the peculiarities of small ruminants' humoral immune response regarding late seroconversion, as well as intermittent and epitope-specific antibody production. The objectives of this review paper were to summarize the available serological and molecular assays for the diagnosis of SRLVs, to highlight their diagnostic performance emphasizing on advantages and drawbacks of their application, and to discuss current and future perspectives, challenges, limitations and impacts regarding the development of reliable and efficient tools for the diagnosis of SRLVs infections.

Phleboviruses are arboviruses transmitted by sand flies, mosquitoes and ticks. Some sand fly-borne phleboviruses cause illnesses in humans, such as the summer fevers caused by the Sicilian and Naples viruses or meningitis caused by the Toscana virus. Indeed, traces of several phleboviral infections have been serologically detected in domestic animals, but their potential pathogenic role in vertebrates other than humans is still unclear, as is the role of vertebrates as potential reservoirs of these viruses. In this study, we report the results of a serological survey performed on domestic animals sampled in Northern Italy, against four phleboviruses isolated from sand flies in the same area. The sera of 23 dogs, 165 sheep and 23 goats were tested with a virus neutralization assay for Toscana virus, Fermo virus, Ponticelli I virus and Ponticelli III virus. Neutralizing antibodies against one or more phleboviruses were detected in four out of 23 dogs, 31 out of 165 sheep and 12 out of 23 goats. This study shows preliminary evidence for the distribution pattern of phleboviral infections in different animal species, highlighting the potential infection of the Toscana virus in dogs and the Fermo virus in goats.

The smallest polycistronic dsRNA segment-10 (S10) of bluetongue virus (BTV) encodes NS3/3A and putative NS5. The S10 sequence data of 46 Indian BTV field isolates obtained between 1985 and 2011 were determined and compared with the cognate sequences of global BTV strains. The largest ORF on S10 encodes NS3 (229 aa) and an amino-terminal truncated form of the protein (NS3A) and a putative NS5 (50-59 aa) due to alternate translation initiation site. The overall mean distance of the global NS3 was 0.1106 and 0.0269 at nt and deduced aa sequence, respectively. The global BTV strains formed four major clusters. The major cluster of Indian BTV strains was closely related to the viruses reported from Australia and China. A minor sub-cluster of Indian BTV strains were closely related to the USA strains and a few of the Indian strains were similar to the South African reference and vaccine strains. The global trait association of phylogenetic structure indicates the evolution of the global BTV S10 was not homogenous but rather represents a moderate level of geographical divergence. There was no evidence of an association between the virus and the host species, suggesting a random spread of the viruses. Conflicting selection pressure on the alternate coding sequences of the S10 was evident where NS3/3A might have evolved through strong purifying (negative) selection and NS5 through a positive selection. The presence of multiple positively selected codons on the putative NS5 may be advantageous for adaptation of the virus though their precise role is unknown.

Nairobi sheep disease orthonairovirus (NSDV) is a zoonotic tick-borne arbovirus, which causes severe gastroenteritis in small ruminants. To date, the virus is prevalent in East Africa and Asia. However, due to climate change, including the spread of transmitting tick vectors and increased animal movements, it is likely that the distribution range of NSDV is enlarging. In this project, sheep and cattle (hitherto classified as resistant to NSDV) were experimentally infected with NSDV for a comparative study of the species-specific pathogenesis. For this purpose, several new diagnostic assays (RT-qPCR, ELISA, iIFA, mVNT, PRNT) were developed, which will also be useful for future epidemiological investigations. All challenged sheep (three different doses groups) developed characteristic clinical signs, transient viremia and virus shedding-almost independent on the applied virus dose. Half of the sheep had to be euthanized due to severe clinical signs, including hemorrhagic diarrhea. In contrast, the course of infection in cattle was only subclinical. However, all ruminants showed seroconversion-implying that, indeed, both species are susceptible for NSDV. Hence, not only sheep but also cattle sera can be included in serological monitoring programs for the surveillance of NSDV occurrence and spread in the future.

The parapoxvirus orf virus (ORFV) encodes several immunomodulatory proteins (IMPs) that modulate host innate and pro-inflammatory responses to infection. Using the ORFV IA82 strain as the parental virus, recombinant viruses with individual deletions in the genes encoding the IMPs chemokine binding protein (CBP; ORFV112), inhibitor of granulocyte-monocyte colony-stimulating factor and IL-2 (GIF, ORFV117) and interleukin 10 homologue (vIL-10; ORFV127) were generated and characterized in vitro and in vivo. The replication properties of the individual gene deletion viruses in cell culture was not affected comparing with the parental virus. To investigate the effect of the individual gene deletions in ORFV infection and pathogenesis, groups of four lambs were inoculated with each virus and were monitored thereafter. Lambs inoculated with either recombinant or with the parental ORFV developed characteristic lesions of contagious ecthyma. The onset, nature and severity of the lesions in the oral commissure were similar in all inoculated groups from the onset (3 days post-inoculation [pi]) to the peak of clinical lesions (days 11-13 pi). Nonetheless, from days 11-13 pi onwards, the oral lesions in lambs inoculated with the recombinant viruses regressed faster than the lesions produced by the parental virus. Similarly, the amount of virus shed in the lesions were equivalent among lambs of all groups up to day 15 pi, yet they were significantly higher in the parental virus group from day 16-21 pi. In conclusion, individual deletion of these IMP genes from the ORFV genome resulted in slight reduction in virulence in vivo, as evidenced by a reduction in the duration of the clinical disease and virus shedding., (Copyright © 2021. Published by Elsevier B.V.)

Highly pathogenic bovine papillomaviruses (BPVs) were detected and quantified for the first time using digital droplet polymerase chain reaction (ddPCR) by liquid biopsy in 103 clinically healthy sheep. Overall, ddPCR detected BPVs in 68 blood samples (66%). BPV infection by a single genotype was revealed in 61.8% of the blood samples, and BPV coinfection by double, triple or quadruple genotypes was observed in 38.2% of liquid biopsies. The BPV-2 genotype was most frequently seen in sheep, whereas BPV-1 was the least common. Furthermore, ddPCR was very useful for detection and quantification; the BPV-14 genotype was observed for the first time in ovine species, displaying the highest prevalence in some geographical areas (Apulia). In 42 of the positive samples (61.8%), a single BPV infection was observed, 26 of which were caused by BPV-2 (61.9%) and 7 by BPV-13 (16.7%). BPV-14 was responsible for 7 single infections (16.7%) and BPV-1 for 2 single infections (4.7%). Multiple BPV coinfections were observed in the remaining 26 positive samples (38.2%), with dual BPV-2/BPV-13 infection being the most prevalent (84.6%). BPV infection by triple and quadruple genotypes was also observed in 11.5% and 3.8% of cases, respectively. The present study showed that ddPCR, a biotechnological refinement of conventional PCR, is by far the most sensitive and accurate assay for BPV detection compared to conventional qPCR. Therefore, ddPCR displayed an essential diagnostic and epidemiological value very useful for the identification of otherwise undetectable BPV genotypes as well as their geographical distributions and suggesting that animal husbandry practices contribute to cross-species transmission of BPVs.

Bluetongue is an insect borne (Culicoides) viral disease of small ruminants. The virus blankets the globe with a wide serotypic variation, numbered from 1 to 28. In India 21 different serotypes have been reported to be circulating across the various agro-climatic zones of the country. Non-structural proteins (NSPs) of bluetongue virus have always remained ideal target for differentiation of infected from vaccinated animals. The current study is an extrapolation of our previous work where a novel fusion construct comprising of bluetongue viral segment NS1 and NS3 was successfully cloned, expressed, purified with an efficient strategy for its suitable implementation as a diagnostic antigen. In this study, the applicability of the fusion construct has been further evaluated and optimised for field applicability. The fusion construct used in an ELISA platform projected a relative diagnostic sensitivity and specificity of 98.1% and 95.5% respectively against a pre-established test panel. The rNS1-NS3 ELISA showed substantially good agreement with the commercial BTV antibody detection kit. Finally, the study brings together the diagnostic capability of two NSPs, which can be a handy tool for sero-surveillance of bluetongue., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Wad Medani virus (WMV) belongs to the genus Orbivirus and is a poorly studied arbovirus with unclear medical significance. Presently, a limited number of WMV strains are characterized and available in NCBI GenBank, some isolated many years ago. A new WMV strain was isolated in 2012 from Dermacentor nuttalli ticks collected from sheep in the Tuva Republic, Russia, and sequenced using high-throughput methods. Complete coding sequences were obtained revealing signs of multiple intersegment reassortments. These point to a high variability potential in WMV that may lead to the formation of strains with novel properties. These new data on WMV can promote better understanding of: ecological features of its circulation; relationships within the genus Orbivirus; and the medical significance of the virus., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Arthropod-borne Batai virus (BATV) is an Orthobunyavirus widely distributed throughout European livestock and has, in the past, been linked to febrile diseases in humans. In Germany, BATV was found in mosquitoes and in one captive harbor seal, and antibodies were recently detected in various ruminant species. We have, therefore, conducted a follow-up study in ruminants from Saxony-Anhalt, the most affected region in Eastern Germany. A total of 325 blood samples from apparently healthy sheep, goats, and cattle were tested using a BATV-specific qRT-PCR and SNT. Even though viral RNA was not detected, the presence of antibodies was confirmed in the sera of all three species: sheep (16.5%), goats (18.3%), and cattle (41.4%). Sera were further analyzed by a glycoprotein Gc-based indirect ELISA to evaluate Gc-derived antibodies as a basis for a new serological test for BATV infections. Interestingly, the presence of neutralizing antibodies was not directly linked to the presence of BATV Gc antibodies. Overall, our results illustrate the high frequency of BATV infections in ruminants in Eastern Germany.

Background: Orf virus (ORFV) is a member of the genus Parapoxvirus and family Poxviridae. The virus has a worldwide distribution and infects sheep, goats, humans, and wild animals. However, due to the complex structure of the poxvirus, the underlying mechanism of the entry and infection by ORFV remains largely unknown. ORFV ORF047 encodes a protein named L1R. Poxviral L1R serves as the receptor-binding protein and blocks virus binding and entry independently of glycosaminoglycans (GAGs). The study aimed to identify the host interaction partners of ORFV ORF047., Methods: Yeast two-hybrid cDNA library of sheep testicular cells was applied to screen the host targets with ORF047 as the bait. ORF047 was cloned into a pBT3-N vector and expressed in the NMY51 yeast strain. Then, the expression of bait proteins was validated by Western blot analysis., Results: Sheep SERP1and PABPC4 were identified as host target proteins of ORFV ORF047, and a Co-IP assay further verified their interaction., Conclusions: New host cell proteins SERP1and PABPC4 were found to interact with ORFV ORF047 and might involve viral mRNA translation and replication.

Most picornaviruses of the family Picornaviridae are relatively well known, but there are certain "neglected" genera like Bopivirus , containing a single uncharacterised sequence (bopivirus A1, KM589358) with very limited background information. In this study, three novel picornaviruses provisionally called ovipi-, gopi- and bopivirus/Hun (MW298057-MW298059) from enteric samples of asymptomatic ovine, caprine and bovine respectively, were determined using RT-PCR and dye-terminator sequencing techniques. These monophyletic viruses share the same type II-like IRES, NPGP-type 2A, similar genome layout (4-3-4) and cre -localisations. Culture attempts of the study viruses, using six different cell lines, yielded no evidence of viral growth in vitro. Genomic and phylogenetic analyses show that bopivirus/Hun of bovine belongs to the species Bopivirus A , while the closely related ovine-origin ovipi- and caprine-origin gopivirus could belong to a novel species "Bopivirus B" in the genus Bopivirus . Epidemiological investigation of N = 269 faecal samples of livestock (ovine, caprine, bovine, swine and rabbit) from different farms in Hungary showed that bopiviruses were most prevalent among <12-month-old ovine, caprine and bovine, but undetectable in swine and rabbit. VP1 capsid-based phylogenetic analyses revealed the presence of multiple lineages/genotypes, including closely related ovine/caprine strains, suggesting the possibility of ovine-caprine interspecies transmission of certain bopiviruses.

There is strong evidence that severe acute respiratory syndrome 2 virus (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, originated from an animal reservoir. However, the exact mechanisms of emergence, the host species involved, and the risk to domestic and agricultural animals are largely unknown. Some domestic animal species, including cats, ferrets, and minks, have been demonstrated to be susceptible to SARS-CoV-2 infection, while others, such as pigs and chickens, are not. Importantly, the susceptibility of ruminants to SARS-CoV-2 is unknown, even though they often live in close proximity to humans. We investigated the replication and tissue tropism of two different SARS-CoV-2 isolates in the respiratory tract of three farm animal species - cattle, sheep, and pigs - using respiratory ex vivo organ cultures (EVOCs). We demonstrate that the respiratory tissues of cattle and sheep, but not of pigs, sustain viral replication in vitro of both isolates and that SARS-CoV-2 is associated to ACE2-expressing cells of the respiratory tract of both ruminant species. Intriguingly, a SARS-CoV-2 isolate containing an amino acid substitution at site 614 of the spike protein (mutation D614G) replicated at higher magnitude in ex vivo tissues of both ruminant species, supporting previous results obtained using human cells. These results suggest that additional in vivo experiments involving several ruminant species are warranted to determine their potential role in the epidemiology of this virus., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Hepatitis E virus (HEV) is the etiological agent behind hepatitis E infection. Domestic pigs and wild boars are the main animal reservoirs of HEV. Very few papers describe HEV infection in goats and sheep. As the data pertaining to the presence of HEV virus in the milk of small ruminants in Europe are lacking, the aim of this paper was to examine a representative number of milk samples from these animals. The detection of HEV genome (HEV RNA) was performed using reverse transcriptase real-time polymerase chain reaction (RT-qPCR). HEV RNA was found in 2.8% of the examined samples. Positivity ranged from 10 1 to 10 3 genome equivalents/mL (GE/mL) with a median of 9.99 × 10 2 GE/mL. On the basis of these results, the milk of small ruminants could represent a source of HEV infection to consumers.

Crimean Congo Hemorrhagic Fever (CCHF) is an important disease. The objective of this study was to investigate the presence / prevalence of CCHFV in tick and milk and blood samples of domestic ruminant (cattle, sheep and goat) in Resadiye town of Tokat province, where the disease is endemic. Although no virus RNA was found from whole blood and milk samples, it was detected in 10 of 78 (12.8%) tick pools. Viral loads ranged from 4.8x104 copies/ml to 2.66x109 copies/ml. Out of 171 serum samples examined, 113 (66.1%) were found to be positive for CCHFV. In conclusion, it was revealed that the prevalence of CCHFV was more common in small ruminants than in cattle. It is an important result in terms of public health that virus cannot be detected. The detection of virus RNA in tick samples shows that CCHFV is still endemic in domestic animals., (Copyright© by the Polish Academy of Sciences.)

This study aimed to evaluate by means of Nested Polymerase Chain Reaction (nPCR), co-cultivation and sequencing, with genetic comparison between strains (mother/newborn), the occurrence of vertical transmission of Small Ruminant Lentiviruses (SRLV) from naturally occurring nannies infected for their offspring. For the detection of SRLV seropositive progenitors, blood was collected from 42 nannies in the final third of gestation in tubes with and without anticoagulant. The diagnostic tests used were Western Blot (WB) and nPCR. During the period of birth, the same blood collection procedure was performed on 73 newborns at zero hours of birth, with the same diagnostic tests. Seventeen blood samples from seven-day-old kids, proven positive for SRLV by nPCR, chosen at random, were subjected to coculture in goat synovial membrane (GSM) cells for 105 days. The pro-viral DNA extracted from the cell supernatant from the coculture was subjected to nPCR. For DNA sequencing from the nPCR products, nine positive samples were chosen at random, four nannies with their respective offspring, also positive. Each sample was performed in triplicate, thus generating 27 nPCR products of which only 19 were suitable for analysis. Among the 42 pregnant goats, in 50% (21/42) pro-viral DNA was detected by nPCR, while in the WB, only 7.14% (3/42) presented antibodies against SRLV. Regarding neonates, of the 73 kids, 34 (46.57%) were positive for the virus, using the nPCR technique, while in the serological test (WB), three positive animals (4.10%) were observed. The coculture of the 17 samples with a positive result in the nPCR was confirmed in viral isolation by amplification of the SRLV pro-viral DNA. When aligned, the pro-viral DNA sequences (nannies and their respective offspring) presented homology in relation to the standard strain CAEV Co. It was concluded that the transmission of SRLV through intrauterine route was potentially the source of infection in the newborn goats., Competing Interests: The authors have declared that no competing interests exist.

Sheeppox and goatpox are highly contagious viral diseases of small ruminants causing severe economic losses to the livestock farmers. The disease is enzootic in Asia including India, Middle East and African countries. In the present study, a total of 28 isolates from twenty five sheeppox and goatpox disease outbreaks were phylogenetically analyzed based on P32 gene/protein along with homology modeling and docking using heparan sulfate and UDP-glucose. Three distinct lineage-specific clusters as per their host origin were recorded. Multiple sequence analysis of P32 gene revealed that genetically similar sheeppox virus (SPPV) and goatpox virus (GTPV) strains are circulating in India. Phylogenetically, Lumpy skin disease (LSDV) and SPPV had a closer genetic relationship than GTPV. Comparative sequence alignment indicated conservation of various motifs such as glycosaminoglycan (GAG), chemokine like motif (CX3C) and Asp-Glu-any other residue-Asp (D/ExD), as well as viral specific signature residues in SPPV and GTPV isolates. Structurally, P32 protein of SPPV and GTPV with mixed α helices and β sheets resembled with crystal structure of homologue vaccinia virus H3L protein. Docking studies in P32 protein of SPPV and GTPV revealed conserved binding pattern with heparan sulfate which is involved in the virus attachment and varied glycosyltransferase fold with UDP-glucose. These findings may help in development of suitable vaccines/diagnostics and therapeutics against capripoxviruses., (Copyright © 2020. Published by Elsevier B.V.)

Border disease virus (BDV) envelope glycoprotein E2 is required for entry into cells and is a determinant of host tropism for sheep and pig cells. Here, we describe adaptive changes in the BDV E2 protein that modify virus replication in pig cells. To achieve this, two BDV isolates, initially collected from a pig and a sheep on the same farm, were passaged in primary sheep and pig cells in parallel with a rescued variant of the pig virus derived from a cloned full-length BDV cDNA. The pig isolate and the rescued virus shared the same amino acid sequence, but the sheep isolate differed at ten residues, including two substitutions in E2 (K771E and Y925H). During serial passage in cells, the viruses displayed clear selectivity for growth in sheep cells; only the cDNA-derived virus adapted to grow in pig cells. Sequencing revealed an amino acid substitution (Q739R) in the E2 domain DA of this rescued virus. Adaptation at the same residue (Q739K/Q739R) was also observed after passaging of the pig isolate in sheep cells. Use of reverse genetics confirmed that changing residue Q739 to R or K (each positively charged) was sufficient to achieve adaptation to pig cells. Furthermore, this change in host tropism was suppressed if Q739R was combined with K771E. Another substitution (Q728R), conferring an additional positive charge, acquired during passaging, restored the growth of the Q739R/K771E variant. Overall, this study provided evidence that specific, positively charged, residues in the E2 domain DA are crucial for pig-cell tropism of BDV.

Native animal breeds constitute an invaluable pool of genetic resources in a changing environment. Discovering native breeds and safeguarding their genetic diversity through specific conservation programs is therefore of high importance. Endogenous retroviruses have proved to be a reliable genetic marker for studying the demographic history of sheep (Ovis aries). Previous research has revealed two migratory episodes of domesticated sheep from the Middle East to Europe. The first episode included predominantly 'primitive populations', while the second and most recent is hypothesised to have included sheep with markedly improved wool production. To examine whether the recently discovered Kihnu native sheep in Estonia have historically been part of the first migratory episode and to what extent they have preserved primitive genetic characters, we analysed retroviral insertions in 80 modern Kihnu sheep and 83 ancient sheep from the Bronze Age to Modern Period (850 BCE-1950 CE). We identified that the Kihnu sheep have preserved 'primitive', 'Nordic', and other 'ancient' retrotypes that were present both in archaeological and modern samples, confirming their shared ancestry and suggesting that contemporary Kihnu native sheep originate from the first migratory episode. However, over the course of history, there has been a gradual decrease in the frequency of primitive retrotypes. Furthermore, Kihnu sheep possessed several 'novel' retrotypes that were absent in archaeological individuals, but were shared with improvement breeds, suggesting recent crossing within the last two centuries. To preserve these ancient lineages, our results are being applied in the conservation program of the Kihnu Native Sheep Society.

Bluetongue virus (BTV) and African horse sickness virus (AHSV) are vector-borne viruses belonging to the Orbivirus genus, which are transmitted between hosts primarily by biting midges of the genus Culicoides . With recent BTV and AHSV outbreaks causing epidemics and important economy losses, there is a pressing need for efficacious drugs to treat and control the spread of these infections. The polyanionic aromatic compound aurintricarboxylic acid (ATA) has been shown to have a broad-spectrum antiviral activity. Here, we evaluated ATA as a potential antiviral compound against Orbivirus infections in both mammalian and insect cells. Notably, ATA was able to prevent the replication of BTV and AHSV in both cell types in a time- and concentration-dependent manner. In addition, we evaluated the effect of ATA in vivo using a mouse model of infection. ATA did not protect mice against a lethal challenge with BTV or AHSV, most probably due to the in vivo effect of ATA on immune system regulation. Overall, these results demonstrate that ATA has inhibitory activity against Orbivirus replication in vitro, but further in vivo analysis will be required before considering it as a potential therapy for future clinical evaluation.

Outstanding increase of oral absorption, bioavailability, and antiviral efficacy of phosphorylated nucleosides and basic antiviral influence of abacavir is the central idea for the development of new series of phosphorylated abacavir (ABC) derivatives. The designed compounds were primarily screened for antiviral nature against HN protein of NDV and VP7 protein of BTV using the molecular environment approach. Out of all the designed compounds, the compounds which are having higher binding energies against these two viral strains were prompted for the synthesis of the target compounds (5A-K). Among the synthesized title compounds (5A-K), the compounds which have exhibited higher dock scores akin to the rest of the compounds were then selected and screened for the antiviral activity against NDV and BTV infected embryonated eggs and BHK 21 cell lines through the in ovo and in vitro approaches. The results revealed that all the designed compounds have formed higher binding energies against both the targets. Among all, the compounds which are selected based on their dock scores such as 5A, 5F, 5G, 5H, 5I, and 5K against NDV and 5J, 5E, 5I, 5C, 5A, and 5K against BTV have shown significant antiviral activity against HN protein of NDV, VP7 protein of Bluetongue virus in both NDV- and BTV-treated embryonated eggs and BHK 21 cell lines. Hence, it is concluded that, the best lead compounds will stand as the potential antiviral agents and prompted them as virtuous therapeutics against NDV and BTV in future.

Small ruminants (e.g., sheep and goats) contribute considerably to the cash income and nutrition of small farmers in most countries in Africa and Asia. Their husbandry is threatened by the highly infectious transboundary viral disease peste des petits ruminants (PPR) caused by peste-des-petits-ruminants virus (PPRV). Given its social and economic impact, PPR is presently being targeted by international organizations for global eradication by 2030. Since its first description in Côte d'Ivoire in 1942, and particularly over the last 10 years, a large amount of molecular epidemiological data on the virus have been generated in Africa. This review aims to consolidate these data in order to have a clearer picture of the current PPR situation in Africa, which will, in turn, assist authorities in global eradication attempts.

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

Maedi-visna (MV) is a complex lentiviral disease syndrome characterised by long immunological and clinical latencies and chronic progressive inflammatory pathology. Incurable at the individual level, it is widespread in most sheep-keeping countries, and is a cause of lost production and poor animal welfare. Culling seropositive animals is the main means of control, but it might be possible to manage virus transmission effectively if its epidemiology was better quantified. We derive a mathematical epidemiological model of the temporal distributions of seroconversion probabilities and estimate susceptibility, transmission rate and latencies in three serological datasets. We demonstrate the existence of epidemiological latency, which has not explicitly been recognised in the SRLV literaure. This time delay between infection and infectiousness apparently exceeds the delay between infection and seroconversion. Poor body condition was associated with more rapid seroconversion, but not with a higher probability of infection. We estimate transmission rates amongst housed sheep to be at about 1,000 times faster than when sheep were at grass, when transmission was negligible. Maternal transmission has only a small role in transmission, because lambs from infected ewes have a low probability of being infected directly by them, and only a small proportion of lambs need be retained to maintain flock size. Our results show that MV is overwhelmingly a disease of housing, where sheep are kept in close proximity. Prevalence of MV is likely to double each year from an initial low incidence in housed flocks penned in typically-sized groups of sheep (c. 50) for even a few days per year. Ewes kept entirely at grass are unlikely to experience transmission frequently enough for MV to persist, and pre-existing infection should die out as older ewes are replaced, thereby essentially curing the flock., Competing Interests: The authors have declared that no competing interests exist.

An oral mass was surgically excised from the gingiva of a pig. As the mass had a similar histological appearance to an equine sarcoid, DNA was extracted and consensus PCR primers used to amplify papillomavirus (PV) DNA. DNA sequences from Ovis aries papillomavirus (OaPV) type 2 were amplified both from a section of the entire mass as well as an area deeper within the mass away from the surface of the lesion. No other PV types were detected within the oral lesion. Ovis aries PV2 is a Delta PV that is closely related to the bovine Delta PVs that cause sarcoids in horses and cats. These results suggest that OaPV2 may be able to infect pigs and this virus could have caused the mesenchymal neoplasm in the mouth of this pig. This is the first evidence that a non-bovine PV can infect a non-host species and the first report of a sarcoid-like mass in pigs. These observations add to the range of species in which PV-associated neoplasia has been reported and suggest that cross-species infection by other Delta PV types may be possible., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study shows the origin and the pathogenic role of a novel ovine pestivirus (OVPV) isolated in 2017 in Italy, as a pathogenic agent causing severe abortions after infection in pregnant ewes and high capacity for virus trans-placental transmission as well as the birth of lambs suffering OVPV-persistent infection. The OVPV infection induced early antibody response detected by the specific ELISA against classical swine fever virus (CSFV), another important virus affecting swine. The neutralizing antibody response were similar against CSFV strains from genotype 2 and the OVPV. These viruses showed high identity in the B/C domain of the E2-glycoprotein. Close molecular diagnostics cross-reactivity between CSFV and OVPV was found and a new OVPV molecular assay was developed. The phylodynamic analysis showed that CSFV seems to have emerged as the result of an inter-species jump of Tunisian sheep virus (TSV) from sheep to pigs. The OVPV and the CSFV share the TSV as a common ancestor, emerging around 300 years ago. This suggests that the differentiation of TSV into two dangerous new viruses for animal health (CSFV and OVPV) was likely favored by human intervention for the close housing of multiple species for intensive livestock production.

Objectives: Hepatitis E virus (HEV) is zoonotic and endemic in several countries. There are no data on the farm level-prevalence and risk factors of HEV in ruminant farms in Jordan or elsewhere. This study aimed to estimate the seroprevalence and risk factors of HEV in ruminant farms in all regions of Jordan., Material and Methods: A total of 460 apparently healthy ruminants from 115 (31 cow, 51 sheep and 33 goat) farms were tested for HEV antibodies using a double antigen sandwich enzyme linked immunosorbent test. A validated questionnaire was used to collect data on animal health and husbandry practices., Results: The results showed that 37.4% of the dairy farms under study (51.6%, 37.2% and 24.2% of dairy cow, sheep and goat farms; respectively) had at least one HEV seropositive animal. At the individual animal level, 12.1% of the tested animals were HEV positive; 14.5% (n = 18), 12.7% (n = 26) and 8.3% (n = 11) of cows, sheep and goats; respectively. Infrequent cleaning of feeders was associated with a significantly greater odds of HEV seropositivity in both large and small dairy ruminant farms (AOR = 16.0, p-val = 0.03, AOR = 3.4, p-val = 0.02, respectively). Farms which reported that small ruminants (sheep and goats) were mixed together had a greater odds of farm-level HEV seroprevalence (AOR = 3.1, p-val = 0.04)., Conclusions: This study shows widespread and high farm-level HEV seroprevalence in dairy farms in Jordan. Husbandry practices and off-abattoir carcass processing in Jordan could amplify emergence and transmission of zoonotic HEV. Future studies should include HEV genotyping in ruminants, their products and humans to better understand HEV epidemiology in Jordan., Competing Interests: Declaration of Competing Interest We declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

An unprecedented outbreak of rabies occurred in Rio Grande do Sul state (RS) from 2012 onward, resulting in thousands of bovine deaths, important economic losses, and posing risk to human health. This article describes a genetic analysis of 145 rabies viruses (RABV) recovered from herbivorous from RS between 2012 and 2017, based on partial sequence analysis of the nucleoprotein (N) gene. High nucleotide (nt) identity (95.5 to 100%) and amino acid (aa) similarity (96.7 to 100%) were observed among the analyzed sequences. These sequences displayed a high sequence nt identity/aa similarity with bovine RABV sequences (96.4-97.9%; 98.1-100%, respectively) and vampire bat RABV sequences (96.3-97.5%; 97.8-99.5%). Phylogenetic analyzes based on the N sequence allowed for the segregation of viruses into two distinct clusters. Cluster 1 comprised RABV sequences covering the whole studied period, whereas cluster 2 grouped a lower number of viruses from 2013, 2014, 2015, to 2017. In some cases, viruses obtained from the same region within a short period of time grouped to distinct clusters or sub-clusters, indicating the co-circulation of distinct virus lineages in these outbreaks. The segregation into sub-clusters was also observed for viral sequences obtained from the same region at different times, indicating the involvement of distinct viruses. In summary, partial sequence analyses revealed a high conservation of N protein and the circulation of two lineages and different sublineages of RABV in the region. In addition, our results confirm the suitability of N gene to study the genetic relationships among RABV isolates.

Sheeppox and goatpox are important transboundary animal viral diseases of sheep and goats caused by sheeppox virus (SPPV) and goatpox virus (GTPV), respectively, of the genus Capripoxvirus, family Poxviridae. Among the proteins encoded by the capripoxvirus (CaPV) genome, ORF095 (vaccinia virus A4L homolog) is an immunodominant virion core protein that plays a pivotal role in virus assembly and morphogenesis. In the present study, sequence analysis of the ORF095 genes of 27 SPPV and GTPV isolates or field samples from different geographical regions of India was performed, and structure was prediction was done by homology modeling. A multiple sequence alignment of different CaPV isolates revealed that CaPV-A4L is highly conserved, with several species-specific signature residues, namely A93, A216, A315, G136 and G146 in GTPV, G47, A63, A168 and A276 in SPPV, and G48 and C98 in lumpy skin disease virus (LSDV). Phylogenetically, the CaPV isolates were separated into three major clusters, GTPV, SPPV and LSDV, based on the complete coding sequence of the CaPV-A4L gene. Genus-specific clustering of poxviruses was observed in phylogenetic analysis based on A4L protein homologs of chordopoxviruses. A secondary structure prediction showed the presence of six α-helices and one β-sheet as well as some coils. The signature residues identified here are potentially useful for genotyping, and the predicted characteristics of the CaPV-A4L protein make it an ideal candidate for use as an immunogenic or diagnostic antigen for the development of immunoassays in  the sero-evaluation of CaPV in target hosts.

Although members of rotavirus group A (RVA) are major enteric pathogens of humans and animals of many species, their impact on the health of small ruminants is not well documented. In this study, we conducted a molecular analysis of VP4, VP7, VP6 and NSP4 genes of RVAs detected using a commercial antigen ELISA in small ruminants with or without diarrhea in Turkey. Of the RVAs detected in sheep, one strain (Kutahya) was characterized as genotype G8P[1]-I2-E2. Two others (Ankara-1 and Ankara-2) were identified as NSP4 E2 and VP6 I2 genotypes, although they were untyped for the VP4 and VP7 genes. The RVAs from two goats were characterized as genotype G6P [1]-I2-E2. This is the first detection of in goats RVA genotypes G6P [1], which had previously only been found in cattle in Turkey, and of RVA in sheep. The study extends our current knowledge about the circulation of two RVA G genotypes, G6 and G8, in goat herds, and the detection of the G8 genotype in sheep in Turkey. This provides further information about the molecular epidemiology of RVAs in different animal species and indicates that additional surveillance programs are needed to determine the epidemiology of RVA in small ruminants and other species.

In September 2016, clinical signs, indicative of bluetongue, were observed in sheep in Cyprus. Bluetongue virus serotype 8 (BTV-8) was detected in sheep, indicating the first incursion of this serotype into Cyprus. Following virus propagation, Nextera XT DNA libraries were sequenced on the MiSeq instrument. Full-genome sequences were obtained for five isolates CYP2016/01-05 and the percent of nucleotide sequence (% nt) identity between them ranged from 99.92% to 99.95%, which corresponded to a few (2-5) amino acid changes. Based on the complete coding sequence, the Israeli ISR2008/13 (98.42-98.45%) was recognised as the closest relative to CYP2016/01-05. However, the phylogenetic reconstruction of CYP2016/01-05 revealed that the possibility of reassortment in several segments: 4, 7, 9 and 10. Based on the available sequencing data, the incursion BTV-8 into Cyprus most likely occurred from the neighbouring countries (e.g., Israel, Lebanon, Syria, or Jordan), where multiple BTV serotypes were co-circulating rather than from Europe (e.g., France) where a single BTV-8 serotype was dominant. Supporting this hypothesis, atmospheric dispersion modelling identified wind-transport events during July-September that could have allowed the introduction of BTV-8 infected midges from Lebanon, Syria or Israel coastlines into the Larnaca region of Cyprus., Competing Interests: The authors declare no conflict of interest.

Foot-and-mouth disease virus (FMDV) is capable of infecting all cloven-hoofed domestic livestock species, including cattle, pigs, goats, and sheep. However, in contrast to cattle and pigs, the pathogenesis of FMDV in small ruminants has been incompletely elucidated. The objective of the current investigation was to characterize tissue- and cellular tropism of early and late stages of FMDV infection in sheep following three different routes of simulated natural virus exposure. Extensive post-mortem harvest of tissue samples at pre-determined time points during early infection (24 and 48 hours post infection) demonstrated that tissues specifically susceptible to primary FMDV infection included the paraepiglottic- and palatine tonsils, as well as the nasopharyngeal mucosa. Additionally, experimental aerosol inoculation of sheep led to substantial virus replication in the lungs at 24-48 hours post-inoculation. During persistent infection (35 days post infection), the paraepiglottic- and palatine tonsils were the only tissues from which infectious FMDV was recovered. This is strikingly different from cattle, in which persistent FMDV infection has consistently been located to the nasopharyngeal mucosa. Analysis of tissue sections by immunomicroscopy revealed a strict epithelial tropism during both early and late phases of infection as FMDV was consistently localized to cytokeratin-expressing epithelial cells. This study expands upon previous knowledge of FMDV pathogenesis in sheep by providing detailed information on the temporo-anatomic distribution of FMDV in ovine tissues. Findings are discussed in relation to similar investigations previously performed in cattle and pigs, highlighting similarities and differences in FMDV pathogenesis across natural host species., Competing Interests: The authors have declared that no competing interests exist.