Vaccine strains of Rift Valley fever virus exhibit attenuation at the maternal-fetal placental interface.

Rift Valley fever virus (RVFV) infection causes abortions in ruminant livestock and is associated with an increased likelihood of miscarriages in women. Using sheep and human placenta explant cultures, we sought to identify tissues at the maternal-fetal interface targeted by RVFV. Sheep villi and fetal membranes were highly permissive to RVFV infection resulting in markedly higher virus titers than human cultures. Sheep cultures were most permissive to wild-type RVFV and ΔNSm infection, while live-attenuated RVFV vaccines (LAVs; MP-12, ΔNSs, and ΔNSs/ΔNSm) exhibited reduced replication. The human fetal membrane restricted wild-type and LAV replication, and when infection occurred, it was prominent on the maternal-facing side. Type I and type III interferons were induced in human villi exposed to LAVs lacking the NSs protein. This study supports the use of sheep and human placenta explants to understand vertical transmission of RVFV in mammals and whether LAVs are attenuated at the maternal-fetal interface.IMPORTANCEA direct comparison of replication of Rift Valley fever virus (RVFV) in sheep and human placental explants reveals comparative efficiencies and permissivity to infection and replication. Vaccine strains of RVFV demonstrated reduced infection and replication capacity in the mammalian placenta. This study represents the first direct cross-host comparison of the vertical transmission capacity of this high-priority emerging mosquito-transmitted virus.

Rift Valley Fever Epizootic, Rwanda, 2022.

A Rift Valley fever epizootic affected livestock in Rwanda during March-October 2022. We confirmed 3,112 infections with the virus, including 1,342 cases, 1,254 abortions, and 516 deaths among cattle, goats, and sheep. We recommend a One Health strategy for investigations and response to protect animal and human health.

Evaluation of the immunogenicity and protective efficacy of an inactivated vaccine candidate for sheep infected with ovine parainfluenza virus type 3.

Respiratory diseases constitute a major health problem for ruminants, resulting in considerable economic losses throughout the world. Parainfluenza type 3 virus (PIV3) is one of the most important respiratory pathogens of ruminants. The pathogenicity and phylogenetic analyses of PIV3 virus have been reported in sheep and goats. However, there are no recent studies of the vaccination of sheep or goats against PIV3. Here, we developed a purified inactivated ovine parainfluenza virus type 3 (OPIV3) vaccine candidate. In addition, we immunized sheep with the inactivated OPIV3 vaccine and evaluated the immune response and pathological outcomes associated with OPIV3 TX01 infection. The vaccinated sheep demonstrated no obvious symptoms of respiratory tract infection, and there were no gross lesions or pathological changes in the lungs. The average body weight gain significantly differed between the vaccinated group and the control group (P < 0.01). The serum neutralization antibody levels rapidly increased in sheep post-vaccination and post-challenge with OPIV3. Furthermore, viral shedding in nasal swabs and viral loads in the lungs were reduced. The results of this study suggest that vaccination with this candidate vaccine induces the production of neutralizing antibodies and provides significant protection against OPIV3 infection. These results may be helpful for further studies on prevention and control strategies for OPIV3 infections.

Ovine adenoviruses infecting sheep and goats in Türkiye: detection and molecular characterization of three different types.

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.

Molecular characterization of Peste des petits ruminants virus (PPRV) in sheep and goats and risk factors associated with it in selected districts of Khyber Pakhtunkhwa-Pakistan.

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.

Prevalence and associated risk factors of peste des petits ruminants in selected districts of the northern border region of Pakistan.

BACKGROUND: Peste des Petits Ruminants (PPR) is a world organization for animal health (WOAH) notifiable and economically important transboundary, highly communicable viral disease of small ruminants. PPR virus (PPRV) belongs to the genus Morbillivirus of the family Paramyxoviridae. AIM: The present cross-sectional epidemiological investigation was accomplished to estimate the apparent prevalence and identify the risk factors linked with peste des petits ruminants (PPR) in the previously neglected northern border regions of Pakistan. METHOD: A total of 1300 samples (serum = 328; swabs = 972) from 150 flocks/herds were compiled from sheep (n = 324), goats (n = 328), cattle (n = 324), and buffaloes (n = 324) during 2020-2021 and tested using ELISA for detection of viral antibody in sera or antigen in swabs. RESULTS: An overall apparent prevalence of 38.7% (504 samples) and an estimated true prevalence (calculated by the Rogan and Gladen estimator) of 41.0% (95% CI, 38.0-44 were recorded in the target regions. The highest apparent prevalence of 53.4% (85 samples) and the true prevalence of 57.0%, 95% Confidence Interval (CI) were documented in the Gilgit district and the lowest apparent prevalence of 53 (25.1%) and the true prevalence of 26.0%, 95% Confidence Interval (CI), 19.0-33.0) was reported in the Swat district. A questionnaire was designed to collect data about associated risk factors that were put into a univariable logistic regression to decrease the non-essential assumed risk dynamics with a P-value of 0.25. ArcGIS, 10.8.1 was used to design hotspot maps and MedCalc's online statistical software was used to calculate Odds Ratio (OR). Some of the risk factors significantly different (P < 0.05) in the multivariable logistic regression were flock/herd size, farming methods, nomadic animal movement, and outbreaks of PPR. The odds of large-sized flocks/herds were 1.7 (OR = 1.79; 95% Confidence Interval (CI) = 0.034-91.80%) times more likely to be positive than small-sized. The odds of transhumance and nomadic systems were 1.1 (OR = 1.15; 95% Confidence Interval (CI) = 0.022-58.64%) and 1.0 (OR = 1.02; 95% Confidence Interval (CI) = 0.020-51.97%) times more associated to be positive than sedentary and mixed farming systems, respectively. The odds of nomadic animal movement in the area was 0.7 (OR = 0.57; 95% Confidence Interval (CI) = 0.014-38.06%) times more associated to be positive than in areas where no nomadic movement was observed. In addition, the odds of an outbreak of PPR in the area were 1.0 (OR = 1.00; 95% Confidence Interval (CI) = 0.018-46.73%) times more associated to be positive than in areas where no outbreak of PPR was observed. CONCLUSIONS: It was concluded that many northern regions considered endemic for PPR, large and small ruminants are kept and reared together making numerous chances for virus transmission dynamic, so a big threats of disease spread exist in the region. The results of the present study would contribute to the global goal of controlling and eradicating PPR by 2030.

Genome characterization of Rift Valley fever virus isolated from cattle, goats and sheep during interepidemic periods in Kenya.

Rift Valley fever virus (RVFV) is a mosquito-borne RNA virus of the Phlebovirus genus in the phenuviridae family. Its genome is trisegmented with small (S), medium (M) and large (L) fragments. In nature, the virus exists as a single serotype that is responsible for outbreaks of Rift Valley fever (RVF), a zoonotic disease that often occurs in Africa and the Middle East. RVFV genomes are thought to undergo both recombination and reassortment and investigations of these events is important for monitoring the emergence of virulent strains and understanding the evolutionary characteristics of this virus. The aim of this study was to characterize the genomes of RVFV isolates from cattle, sheep, and goats collected during an interepidemic period in Kenya between June 2016 and November 2021. A total of 691 serum samples from cattle (n = 144), goats (n = 185) and sheep (n = 362) were analysed at the Central Veterinary Laboratories. The competitive IgM-capture ELISA, was used to screen the samples; 205 samples (29.67%) tested positive for RVFV. Of the 205 positive samples, 42 (20.5%) were from cattle, 57 (27.8%) from goats, and 106 (51.7%) from sheep. All the IgM-positive samples were further analyzed by qPCR, and 24 (11.71%) tested positive with Ct values ranging from 14.788 to 38.286. Two samples, 201808HABDVS from sheep and 201810CML3DVS from cattle, had Ct values of less than 20.0 and yielded whole genome sequences with 96.8 and 96.4 coverage, respectively. There was no statistically significant evidence of recombination in any of the three segments and also phylogenetic analysis showed no evidence of reassortment in the two isolated RVFV segments when compared with other isolates of different lineages from previous outbreaks whose genomes are deposited in the GenBank. No evidence of reassortment leaves room for other factors to be the most probable contributors of change in virulence, pathogenicity and emergence of highly virulent strains of the RVFV.

Epidemiology of peste des petits ruminants in selected districts of Borena zone, Ethiopia.

BACKGROUND: Peste des petits ruminant (PPR) is a contagious disease caused by the peste des petits ruminants virus (PPRV). The disease poses a significant economic threat to small ruminant production in Ethiopia, particularly to the striving pastoral production system. A cross-sectional study was conducted to estimate the seroprevalence and associated risk factors of PPR in the small ruminants of the Borena Zone. A total of 384 serum samples were collected randomly from sheep and goats and examined for the presence of PPRV antibodies using competition enzyme-linked immune sorbent assay (c-ELISA). Additionally, a retrospective analysis of five years of outbreak data was performed to provide insight into the spatial and temporal distribution of the disease. RESULTS: The seroprevalence of PPRV antibodies in nonvaccinated, vaccinated, and unknown vaccination status of small ruminants in this study was found to be 32.1%, 68.8%, and 45.5%, respectively. Multivariable logistic analysis revealed a statistically significant association between PPRV seropositivity and several factors, including age, animal origin, flock size, and veterinary services status. A retrospective analysis revealed 53 PPR outbreaks in the Borena Zone from 2018 to 2022, exacerbated by low vaccination coverage relative to the at-risk animal population. CONCLUSION: The study revealed significant gaps in current vaccination efforts, with herd immunity levels falling below the FAO-WOAH recommended threshold of 80%. Despite Ethiopia's ambitious goal to eradicate PPR by 2027, the frequent outbreaks and insufficient herd immunity highlight the inadequacy of the existing strategies. To effectively move toward eradication, Ethiopia must align its approach with the global PPR eradication framework, which emphasizes a comprehensive strategy that includes diagnostics, surveillance, prevention, and the establishment of a robust veterinary regulatory system, rather than relying solely on vaccination. Overcoming logistical challenges, improving vaccination coverage, and optimizing the timing of vaccination campaigns, especially in hard-to-reach areas, will be crucial for reducing outbreaks and making progress toward eradication.

ORF virus causes tumor-promoting inflammation in sheep and goats.

ORF virus (ORFV) causes contagious ecthyma ("ORF"), a disease of sheep and goats characterized by lesions ranging from vesicles and pustules to atypical papilloma-like and angiomatous lesions in the skin and mucosae. The authors investigated the molecular factors leading to the ORF-associated atypical tumor-like changes. Fifteen lambs, 15 kids, and an adult ram clinically affected by natural ORFV infection were enrolled in the study and examined by several methods. ORFV was detected by viral culture or real-time polymerase chain reaction (RT-PCR) in the lesioned tissues and in the blood of the clinically affected sheep and goats. Surprisingly, ORFV was also detected in the blood of healthy goats from an affected herd. Microscopically, they found a pseudo-papillomatous proliferation of the epithelium, while the dermis and lamina propria were expanded by a proliferating neovascular component that highly expressed the viral vascular endothelial growth factor (vVEGF) and its host receptor vascular endothelial growth factor receptor 2 (VEGFR2). Immunohistochemistry, immunofluorescence, and in situ hybridization for mRNA showed that epidermal growth factor receptor (EGFR) was expressed in the fibrovascular component, in the infiltrating CD163+ macrophages, and in the basal stratum of the epidermis. Confocal immunofluorescence microscopy demonstrated that CD163+ macrophages were associated with VEGF and VEGFR2. Finally, they found by quantitative RT-PCR the overexpression of the interleukin-6 and VEGFR2 genes in the lesioned tissues. These findings suggest that ORFV activates an inflammatory reaction characterized by CD163+ macrophages expressing EGFR and VEGFR2, which might play an oncogenic role through synergistic action with vVEGF signaling.

Sero-epidemiology of bluetongue in ruminants raised on private holdings in North-Western Pakistan.

This study investigated the sero-epidemiology of bluetongue in ruminants in North-Western Pakistan. A total of 3,173 serum samples were collected from small (n = 1,651) and large (n = 1,522) ruminants being reared by farmers in 14 districts. Antibodies to bluetongue virus (BTV) were detected using competitive ELISA. The overall prevalence of BTV antibodies was 65%. A significant association (P < 0.05) between the prevalence of BTV antibodies and the risk factors including sex, species, age, area, husbandry practices and breed was shown by univariate analysis. In multivariate analysis, the seroprevalence was 6.5 (95% CL = 3.7-11.4), 5.9 (95% CL = 3.8-9.4) and 2.4 (95% CL = 1.5-3.7) times higher in buffaloes, cattle and goats than sheep, respectively. The seroprevalence was 1.4 (95% CL = 1.1-1.7) times higher in local breeds than in cross/exotic breeds. The seroprevalence was 1.6 (95% CL = 1.1 to 2.3) times higher in sedentary animals than in nomadic animals. The seroprevalence was significantly associated with age. Further work is required to determine the BTV serotypes prevalent in the study area for effective control of the disease.

Seroprevalence and contributing factors of transboundary animal diseases in sheep and goats: a study in Peninsular Malaysia.

Diseases caused by small ruminant lentiviruses, Mycobacterium avium ssp. paratuberculosis (MAP), Schmallenberg virus, and peste des petits ruminants virus (PPR) is globally recognised as serious threats to the ruminant industry due to their potential to spread rapidly across boundaries. Despite their global distribution and negative impacts on ruminant production, there is a gap in knowledge of the current trends in their epidemiology among sheep and goat populations in Peninsular Malaysia. This study was therefore designed to fill the gap of knowledge concerning the seroprevalence and contributing factors of CAEV, paratuberculosis, SBV, and PPRV among small ruminants from selected flocks in Selangor, Negeri Sembilan, and Pahang states in Peninsular Malaysia. A cross-sectional study design was used to collect animal data and blood samples for serological assays simultaneously. The ID Screen (ID.VET, France) indirect ELISA screening tests were used to detect serum antibodies directed against CAEV/MVV (VISNAS Ver 0922), paratuberculosis (PARAS Ver 0516), SBV (SBVC Ver 1114) and PPRV (PPRC Ver 0821). There was 45.4% (95% CI = 40.74-50.74), 6.8% (95% CI = 4.66-9.69), 27.8% (95% CI = 23.35-32.77), and 2.6% (95% CI = 1.11-0.51) true seroprevalence for CAEV, paratuberculosis, SBV, and PPR, respectively. Geographical location and species were the risk factors for CAEV and paratuberculosis, while the management system and age of small ruminants were the risk factors for SBV. The present study is the first to document a large-scale seroprevalence of MAP and PPR infection among sheep and goat flocks in Peninsular Malaysia. The presence of PPRV and MAP antibodies among small ruminant flocks is signalling current or previous exposure to the pathogens or cross reactions with similar antigens. This finding further suggests the potential for future outbreaks of these devastating diseases among sheep and goats in Malaysia. The high seroprevalence of CAEV and SBV among small ruminants indicates high levels of exposure to the viruses in the environment, which is a potential threat to production.

Space-time cluster analysis and maximum entropy modeling of Peste des petits ruminants (PPR) in Türkiye.

Peste des petits ruminants (PPR) is an economically important highly serious transboundary disease that mainly occurs in small ruminants such as sheep and goats. The aim of this study was to identify the probability of risk and and space-time clusters of Peste des Petits Ruminants (PPR) in Türkiye. The occurrence of PPR in Türkiye from 2017 to 2019 was investigated in this study using spatial analysis based on geographic information system (GIS). Between these dates, it was determined that 337 outbreaks and 18,467 cases. The highest number of outbreaks were detected in the Central Anatolia region. It was determined that PPR is seen more intensely in sheep compared to goats in Türkiye. In this study, 34 environmental variables (19 bioclimatic, 12 precipitation, altitude and small livestock density variables) were used to explore the environmental influences on PPR outbreak by maximum entropy modeling (Maxent). The clusters of PPR in Türkiye were identified using the retrospective space-time scan data that were computed using the space-time permutation model. A PPR prediction model was created using data on PPR outbreaks combination with environmental variables. Nineteen significant (p < 0.001) space-time clusters were determined. It was discovered that the variables altitude, sheep density, precipitation in june, and average temperature in the warmest season made important contributions to the model and the PPR outbreak may be strongly related with these variables. In this study, PPR in Türkiye has been characterized significantly spatio-temporal and enviromental factors. In this context, the disease pattern and obtained these findings will contribute to policymakers in the prevention and control of the disease.

Molecular detection and phylogenetic analysis of ovine herpesvirus-2 in subclinical infections of cattle and sheep.

Ovine herpesvirus-2 (OvHV-2) is the causative agent of malignant catarrhal fever (MCF), a serious and often fatal disease that affects cattle and other ruminants. This study aimed to investigate the molecular epidemiology and genetic diversity of OvHV-2 strains circulating in sheep and cattle populations in the Jammu and Kashmir region of India. Screening of 150 sheep and 57 cattle blood samples revealed the presence of the OvHV-2 polymerase (pol) gene in 8.6% of sheep, 10% of apparently healthy cattle, and 29.7% of cattle exhibiting MCF-like symptoms. The full-length glycoprotein B (gB) gene (2800 bp) and an 875 bp internal fragment were successfully amplified, cloned, and sequenced from pol-positive samples. Comparative sequence analysis of the deduced gB amino acid sequences identified seven substitutions at positions 278, 341, 390, 440, 468, 539, and 566 compared to reference strains. Phylogenetic analysis based on the gB nucleotide sequences clustered the OvHV-2 strains from this study within the Indian clade, distinct from strains reported in the UK and US. These findings provide insights into the genetic diversity of OvHV-2 strains circulating in Jammu and Kashmir, with the identified mutations potentially influencing virus-host interactions. Further investigations into the functional implications of these mutations are warranted to understand their role in viral pathogenesis and tropism.

Field-Reassortment of Bluetongue Virus Illustrates Plasticity of Virus Associated Phenotypic Traits in the Arthropod Vector and Mammalian Host In Vivo.

Segmented RNA viruses are a taxonomically diverse group that can infect plant, wildlife, livestock and human hosts. A shared feature of these viruses is the ability to exchange genome segments during coinfection of a host by a process termed "reassortment." Reassortment enables rapid evolutionary change, but where transmission involves a biological arthropod vector, this change is constrained by the selection pressures imposed by the requirement for replication in two evolutionarily distant hosts. In this study, we use an in vivo, host-arbovirus-vector model to investigate the impact of reassortment on two phenotypic traits, virus infection rate in the vector and virulence in the host. Bluetongue virus (BTV) (Reoviridae) is the causative agent of bluetongue (BT), an economically important disease of domestic and wild ruminants and deer. The genome of BTV comprises 10 linear segments of dsRNA, and the virus is transmitted between ruminants by Culicoides biting midges (Diptera: Ceratopogonidae). Five strains of BTV representing three serotypes (BTV-1, BTV-4, and BTV-8) were isolated from naturally infected ruminants in Europe and ancestral/reassortant lineage status assigned through full genome sequencing. Each strain was then assessed in parallel for the ability to replicate in vector Culicoides and to cause BT in sheep. Our results demonstrate that two reassortment strains, which themselves became established in the field, had obtained high replication ability in C. sonorensis from one of the ancestral virus strains, which allowed inferences of the genome segments conferring this phenotypic trait. IMPORTANCE Reassortment between virus strains can lead to major shifts in the transmission parameters and virulence of segmented RNA viruses, with consequences for spread, persistence, and impact. The ability of these pathogens to adapt rapidly to their environment through this mechanism presents a major challenge in defining the conditions under which emergence can occur. Utilizing a representative mammalian host-insect vector infection and transmission model, we provide direct evidence of this phenomenon in closely related ancestral and reassortant strains of BTV. Our results demonstrate that efficient infection of Culicoides observed for one of three ancestral BTV strains was also evident in two reassortant strains that had subsequently emerged in the same ecosystem.

High genetic variability of Schmallenberg virus M-segment leads to efficient immune escape from neutralizing antibodies.

Schmallenberg virus (SBV) is the cause of severe fetal malformations when immunologically naïve pregnant ruminants are infected. In those malformed fetuses, a "hot-spot"-region of high genetic variability within the N-terminal region of the viral envelope protein Gc has been observed previously, and this region co-localizes with a known key immunogenic domain. We studied a series of M-segments of those SBV variants from malformed fetuses with point mutations, insertions or large in-frame deletions of up to 612 nucleotides. Furthermore, a unique cell-culture isolate from a malformed fetus with large in-frame deletions within the M-segment was analyzed. Each Gc-protein with amino acid deletions within the "hot spot" of mutations failed to react with any neutralizing anti-SBV monoclonal antibodies or a domain specific antiserum. In addition, in vitro virus replication of the natural deletion variant could not be markedly reduced by neutralizing monoclonal antibodies or antisera from the field. The large-deletion variant of SBV that could be isolated in cell culture was highly attenuated with an impaired in vivo replication following the inoculation of sheep. In conclusion, the observed amino acid sequence mutations within the N-terminal main immunogenic domain of glycoprotein Gc result in an efficient immune evasion from neutralizing antibodies in the special environment of a developing fetus. These SBV-variants were never detected as circulating viruses, and therefore should be considered to be dead-end virus variants, which are not able to spread further. The observations described here may be transferred to other orthobunyaviruses, particularly those of the Simbu serogroup that have been shown to infect fetuses. Importantly, such mutant strains should not be included in attempts to trace the spatial-temporal evolution of orthobunyaviruses in molecular-epidemiolocal approaches during outbreak investigations.

Ovine Herpesvirus 2 Encodes a Previously Unrecognized Protein, pOv8.25, That Targets Mitochondria and Triggers Apoptotic Cell Death.

Malignant catarrhal fever (MCF) is a rare but frequently lethal disease of certain cloven-hoofed animals. At least 10 different viruses, all members of the Macavirus genus in the subfamily Gammaherpesvirinae, are known as causative agents of MCF. Among these, ovine herpesvirus 2 (OvHV-2) is the most frequent and economically most important MCF agent. Phenotypically, MCF is characterized by severe lymphocytic arteritis-periarteritis, which leads to the accumulation of activated lymphocytes accompanied by apoptosis and necrosis in a broad range of tissues. However, a viral factor that might be responsible for tissue damage has not yet been identified. We have studied a seemingly intergenic locus on the OvHV-2 genome, which was previously shown to be transcriptionally highly active in MCF-affected tissue. We identified by 5' and 3' rapid amplification of cDNA ends (RACE) a conserved, double-spliced transcript that encoded a 9.9-kDa hydrophobic protein. The newly detected gene, Ov8.25, and its splicing pattern were conserved among OvHV-2 strains of different origins. Upon transient expression of synthetic variants of this gene in various cell types, including bovine lymphocytes, the protein (pOv8.25) was shown to target mitochondria, followed by caspase-dependent apoptosis and necrosis. Notably, a deletion mutant of the same protein lost these abilities. Finally, we detected pOv8.25 in brain-infiltrating lymphocytes of cattle with MCF. Thus, the cell death-causing properties of pOv8.25 in affected cells may be involved in the emergence of typical MCF-associated apoptosis and necrosis. Thus, we have identified a novel OvHV-2 protein, which might contribute to the phenotype of MCF-related lesions.IMPORTANCE Ovine herpesvirus 2 (OvHV-2) circulates among sheep without causing disease. However, upon transmission to cattle, the same virus instigates a frequently lethal disease, malignant catarrhal fever (MCF). While the cause of death and pathogenesis of tissue lesions are still poorly understood, MCF is characterized by the accumulation of lymphocytes in various tissues, associated with vasculitis and cell death. As infectious virus is hardly present in these lesions, the cause of cell death cannot be explained simply by viral replication. The significance of our research is in identifying and characterizing a previously overlooked gene of OvHV-2 (Ov8.25), which is highly expressed in animals with MCF. Its encoded protein targets mitochondria, causing apoptosis and necrosis, thus contributing to an understanding of the source and nature of cell death. As the corresponding genetic locus is also active in the context of MCF due to a different macavirus, we may have detected a common denominator of the disease phenotype.

Replication of Small Ruminant Lentiviruses in Aluminum Hydroxide-Induced Granulomas in Sheep: a Potential New Factor for Viral Dissemination.

Aluminum (Al)-based salts are widely used adjuvants in ruminants and other species to strengthen the immune response elicited against vaccine antigen(s). However, they can lead to the formation of long-lasting granulomas composed of abundant activated macrophages. Small ruminant lentiviruses (SRLV) are widely distributed macrophage-tropic retroviruses that cause persistent infections in sheep and goats. Infected monocytes/macrophages and dendritic cells establish an inflammatory microenvironment that eventually leads to clinical manifestations. The aim of this work was to study the effect of Al-induced granulomas in the replication and pathogenesis of SRLV. Eleven adult, naturally SRLV-infected sheep showing clinical arthritis were distributed in vaccine (n = 6), adjuvant-only (n = 3), and control (n = 2) groups and inoculated with commercial Al-based vaccines, Al hydroxide adjuvant alone, or phosphate-buffered saline, respectively. In vitro studies demonstrated viral replication in Al-induced granulomas in 5 out of 10 sheep. Immunohistochemistry (IHC) evinced granular, intracytoplasmic SRLV presence in macrophages within granulomas. Viral sequences obtained from granulomas, blood monocytes, and other tissues were highly similar in most animals, suggesting virus circulation among body compartments. However, notable differences between isolated strains in granulomas and other tissues in specific animals were also noted. Interestingly, the B2 subtype was the most commonly found SRLV genotype, reaching a wider body distribution than previously described. Recombination events between genotypes B2 and A3 along the gag region were identified in two sheep. Our results indicate that Al-hydroxide-derived granulomas may represent an ideal compartment for SRLV replication, perhaps altering natural SRLV infection by providing a new, suitable target tissue.IMPORTANCE Granulomas are inflammation-derived structures elicited by foreign bodies or certain infections. Aluminum adjuvants included in vaccines induce granulomas in many species. In sheep, these are persistent and consist of activated macrophages. Small ruminant lentiviruses (SRLV), which are macrophage-tropic lentiviruses, cause a chronic wasting disease affecting animal welfare and production. Here, we studied the occurrence of SRLV in postvaccination granulomas retrieved from naturally infected ewes after vaccination or inoculation with aluminum only. SRLV infection was confirmed in granulomas by identification of viral proteins, genomic fragments, and enzymatic activity. The infecting SRLV strain, previously found exclusively in carpal joints, reached the central nervous system, suggesting that occurrence of SRLV in postvaccination granulomas may broaden tissue tropism. SRLV recombination was detected in inoculated animals, a rare event in sheep lentiviruses. Potentially, virus-host interactions within granulomas may modify viral pathogenesis and lead to more widespread infection.

In vitro and in vivo analyses of co-infections with peste des petits ruminants and capripox vaccine strains.

BACKGROUND: Peste des petits ruminants (PPR) and goat pox (GTP) are two devastating animal epidemic diseases that affect small ruminants. Vaccination is one of the most important measures to prevent and control these two severe infectious diseases. METHODS: In this study, we vaccinated sheep with PPR and POX vaccines to compare the changes in the antibody levels between animals vaccinated with PPRV and POX vaccines alone and those co-infected with both vaccines simultaneously. The cell infection model was used to explore the interference mechanism between the vaccines in vitro. The antibody levels were detected with the commercial ELISA kit. The Real-time Quantitative PCR fluorescent quantitative PCR method was employed to detect the viral load changes and cytokines expression after the infection. RESULTS: The concurrent immunization of GTP and PPR vaccine enhanced the PPR vaccine's immune effect but inhibited the immune effect of the GTP vaccine. After the infection, GTP and PPR vaccine strains caused cytopathic effect; co-infection with GTP and PPR vaccine strains inhibited the replication of PPR vaccine strains; co-infection with GTP and PPR vaccine strains enhanced the replication of GTP vaccine strains. Moreover, virus mixed infection enhanced the mRNA expressions of TNF-α, IL-1ß, IL-6, IL-10, IFN-α, and IFN-ß by 2-170 times. GTP vaccine strains infection alone can enhanced the mRNA expression of IL-1ß, TNF-α, IL-6, IL-10, while the expression of IFN-α mRNA is inhibited. PPR vaccine strains alone can enhanced the mRNA expression of IFN-α, IFN-ß, TNF-α, and has little effect the mRNA expression of IL-1ß, IL-6 and IL-10. The results showed that GTP and PPR vaccine used simultaneously in sheep enhanced the PPR vaccine's immune effect but inhibited the immune effect of the GTP vaccine in vivo. Furthermore, an infection of GTP and PPR vaccine strains caused significant cell lesions in vitro; co-infection with GTP + PPR vaccine strains inhibited the replication of PPR vaccine strains, while the co-infection of GTP followed by PPR infection enhanced the replication of GTP vaccine strains. Moreover, virus infection enhanced the expressions of TNF-α, IL-1ß, IL-6, IL-10, IFN-α, and IFN-ß. CONCLUSIONS: Peste des petits ruminants and capripox vaccine strains interfere with each other in vivo and vitro.

A new highly divergent copiparvovirus in sheep.

The subfamily Parvovirinae within the family Parvoviridae consists of viruses that can infect a wide range of vertebrate hosts and cause effects ranging from severe disease to asymptomatic infection. In the present study, high-throughput sequencing (HTS) was utilized to analyze samples obtained from an abortion outbreak in a sheep flock to identify a putative viral etiology. A highly divergent nearly complete parvovirid genome sequence, approximately 4.9 kb in length, was determined. The nonstructural protein (NS1) amino acid (aa) sequence of this virus shared less than 30% identity with those of other copiparvoviruses and less than 22% identity with those of members of other genera in the subfamily Parvovirinae. Phylogenetically, this virus, which we have provisionally named "sheep copiparvovirus 1", formed a cluster with copiparvovirus sequences and should be classified as a member of a new species in the genus Copiparvovirus.

Cross-sectional study and genotyping of rotavirus-A infections in ruminants in Kuwait.

BACKGROUND: Group A rotaviruses (RVA) are zoonotic pathogens responsible for acute enteritis in human and neonatal ruminants. This research aimed to determine the prevalence of RVA in ruminants (cattle, sheep, and goats) and investigate the circulating RVA genotypes in these animals in Kuwait. We conducted a cross-sectional study to detect RVA in ruminants, using an immunochromatography test (IC), direct sandwich ELISA test, and real-time RT-PCR (RT-qPCR) assay using fecal samples. RESULTS: A total of 400 cattle, 334 sheep, and 222 goats were examined. The prevalence of RVA was 5.3, 1.2, and 2.3%, respectively, using IC. The ELISA test detected RVA from 4.3% of cattle, 0.9% of sheep, and 1.8% of goats. There was a significant association between the occurrence of diarrhea and the presence of RVA in bovine fecal samples (p-value = 0.0022), while no statistical association between diarrhea and the presence of RVA in fecal samples of sheep and goats was observed (p-value = 0.7250; p-value = 0.4499, respectively). Twenty-three of the IC-positive samples (17 from cattle, two from sheep, and four from goats) were tested using a RT-qPCR RVA detection assay targeting the NSP3 gene. The results showed that 21 of 23 IC-positive samples tested positive by RT-qPCR. Detection of RVA genotypes revealed that G10P[11] was the predominant strain in cattle (58.8%), followed by G8P[1] (11.7%). One sheep sample was genotyped as G8P[1]. In addition, G6P[1] and G6P[14] were detected in goat samples. CONCLUSION: The present study revealed that the IC was more sensitive in detecting RVA antigen in fecal samples than the ELISA test. A higher occurrence of RVA infection was observed in cattle than in sheep and goats. This study suggests that RVA might be a risk factor of diarrhea in bovine calves less than 2 weeks old. This research also demonstrates the circulation of RVA in sheep and goat populations in Kuwait. Finally, the G10P[11] RVA genotype was the most prevalent genotype identified from cattle samples.