Quantification of ovine gammaherpesvirus 2 in clinical cases of cattle with sheep-associated malignant catarrhal fever.

Ovine gammaherpesvirus 2 (OvGHV2) produces sheep-associated malignant catarrhal fever (SA-MCF), a frequently lethal, lymphoproliferative disease that is characterized by widespread vascular lesions. Most studies that evaluated the viral load in tissues of animals with SA-MCF were done in the Northern Hemisphere, with scant information from the Southern part of the globe. This study investigated the viral load of OvGHV2 in the tissues of cattle and an underdeveloped fetus with SA-MCF from three distinct biomes of Brazil. All animals had clinical and histopathological manifestations consistent with SA-MCF. Molecular testing identified the OvGHV2 tegument protein and glycoprotein B genes in the tissues of all animals and the fetus. Viral quantification based on the DNA polymerase gene detected elevated loads of OvGHV2 in tissues with histopathological evidence of SA-MCF and organs with unknown histological data, except for the tissues of the fetus, where the viral load was comparatively reduced. The viral loads detected in multiple organs of cattle from this study with SA-MCF are consistent with those identified in different animal species from the USA and Europe. The detection of a low viral load of OvGHV2 in fetal tissue confirmed transplacental dissemination since elevated viral loads were detected in multiple tissues of the cow with SA-MCF. Furthermore, the elevated viral loads detected in the pulmonary tissues of cattle with interstitial pneumonia indicate that OvGHV2 is an inductor of pulmonary disease in cattle.

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.

Occurrence of enteric viruses causing clinical diarrhea in small ruminants in northern Indian plains: a reverse transcription PCR based molecular study.

Fowlpox virus (FPV) infects chickens and turkeys giving rise to pock lesions on various body parts like combs, wattles, legs, shanks, eyes, mouth, etc. The birds, affected with FPV, also show anemia and a ruffled appearance which are clinical symptoms of reticuloendotheliosis. Interestingly, the field strains of FPV are integrated with the provirus of reticuloendotheliosis virus (REV). Due to this integration, the infected birds, upon replication of FPV, give rise to free REV virions, causing severe immunosuppression and anemia. Pox scabs, collected from the infected birds, not only show positive PCR results upon performing FPV-specific 4b core protein gene PCR but also show positive results for the PCR of REV-specific env gene and FPV-REV 5'LTR junction. Homogenized suspension of the pock lesions, upon inoculating to the chorio-allantoic membrane (CAM) of 10-day-old specific pathogen-free embryonated chicken eggs, produces characteristic pock lesions in serial passages. However, the lesions also harbor REV mRNA or free virion, which can be identified by performing REV-specific env gene PCR using REV RNA from FPV-infected CAMs. The study suggests successful replication and availability of REV mRNA and free virion alongside the FPV, although the CAM is an ill-suited medium for any retroviral (like REV) growth and replication.

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.

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.

Proteomic profiles and cytokeratin 13 as a potential biomarker of Ovis aries papillomavirus 3-positive and negative cutaneous squamous cell carcinomas.

Ovis aries papillomavirus 3 (OaPV3) is an epidermotropic PV reported in sheep cutaneous squamous cell carcinoma (SCC). The presence of OaPV3 DNA and its transcriptional activity in cutaneous SCC, as well as its in vitro transforming properties, suggest a viral etiology for this neoplasm. Nevertheless, the reactome associated with viral-host interaction is still unexplored. Here, we investigated and compared the proteomic profiles of OaPV3-positive SCCs, OaPV3-negative SCCs, and non-SCC samples by liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis, bioinformatics tools, and immunohistochemistry (IHC). OaPV3-positive SCCs (n = 3), OaPV3-negative SCCs (n = 3), and non-SCCs samples (n = 3) were subjected to a shotgun proteomic analysis workflow to assess protein abundance differences among the three sample classes. Proteins involved in epithelial cell differentiation, extracellular matrix organization, and apoptotic signaling showed different abundances in OaPV3-positive SCCs tissues (P ≤ 0.05) when compared to the other tissues. Cytokeratin 13 (CK 13) was among the most increased proteins in OaPV3-positive SCC and was validated by immunohistochemistry on 10 samples per class, confirming its potential as a biomarker of OaPV3 infection in SCC. Collectively, results provide a preliminary insight into the reactome associated with viral-host interaction and pave the way to the development of specific biomarkers for viral-induced sheep SCC.

Intracranial squamous cell carcinoma in an Ovis aries.

Squamous cell carcinoma (SCC) is a malignant mucoepithelial tumor that affects pets and farm animals. Common sites are dorsal areas and/or areas of poor skin pigmentation exposed to mutagenic ultraviolet (UV) radiation. Novel ovine papillomavirus (OaPV3) was recently described in SCC lesions in Sardinia breed ovines. In 2017, a 7­year­old half­breed aries was presented with symptoms compatible with a vestibular syndrome. The animal was euthanized 1 month after the onset of clinical signs due to a lack of response to treatment and poor prognosis. A complete postmortem examination was performed. Necropsy revealed only a loss of incisors, associated with alveolar necrotic osteomyelitis, and left unilateral purulent nasal discharge. No other thoracic or abdominal lesions were observed. Opening of the skull revealed a cauliflower­like space­occupying mass. Histological examination showed trabecules and islands of squamous, neoplastic epithelial cells with the formation of concentric keratin layers. This raised the suspicion of SCC, which was confirmed with cytokeratin­positive immunostaining. Simplex PCR on the frozen tissue mass was negative for OaPV1, OaPV2, and OaPV3. This case report suggests that SCC, although rare, should be included in the differential diagnosis of cases of vestibular disorder.

Detection and characterisation of sheep-associated malignant catarrhal fever infection from ruminants by using tegument and gB gene sequences of OvHV-2.

In this study, positive blood and organ samples were obtained from different mixed herds of sheep and cattle against ovine herpesvirus 2 (OvHV-2) infection. Target-positive DNA was sequenced and compared with worldwide distributed OvHV-2 sequences. Tegument gene (422 base pairs) and glycoprotein B (gB) gene (2800 base pairs) amplicons of OvHV-2 genome were used for understanding of epidemiology of malignant catarrhal fever (MCF) infection in Turkey. The results of nucleotide sequencing of polymerase chain reaction (PCR) products indicated presence of sheep-associated form for MCF infection in Turkey. Although the obtained sequences were genetically different from each other, it was found that genetic variations were limited.

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.

Molecular phylogeny of Capripoxviruses based on major immunodominant protein (P32) reveals circulation of host specific sheeppox and goatpox viruses in small ruminants of India.

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 ß 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.

A review of the epidemiological, clinical, and pathological aspects of malignant catarrhal fever in Brazil.

Sheep-associated malignant catarrhal fever (SA-MCF), the form of MCF that occurs in Brazil, is a severe, frequently fatal, infectious disease caused by ovine gammaherpesvirus-2 (OvHV-2), in which sheep are the asymptomatic hosts and cattle and other cloven-hoofed animals are the accidental hosts. This review provides a critical analysis of the historical, epidemiological aspects and the estimated economic impacts associated with SA-MCF in Brazil. Moreover, the clinical manifestations and pathological lesions associated with SA-MCF in cattle are reviewed and discussed and the phylogenetic distribution of OvHV-2 in Brazil is presented. OvHV-2 is the only MCF virus identified in animals from Brazil. It is recommended that a histopathologic diagnosis of SA-MCF be based on all aspects of vascular disease in the affected animal and not only lymphocytic/necrotizing vasculitis and/or fibrinoid change. Conformation of the intralesional participation of OvHV-2 in these alterations can be achieved by immunohistochemistry and/or in situ hybridization assays. Additionally, it is proposed that OvHV-2 should be considered as a possible infectious disease agent associated with the development of bovine respiratory disease in cattle. Furthermore, the possible role of the small intestine in the dissemination of OvHV-2 is discussed.

Signature patterns in region V4 of small ruminant lentivirus surface protein in sheep and goats.

The env gene in Small Ruminant Lentiviruses (SRLV) encodes the surface glycoprotein (SU) that divides into conserved (C1-C4) and variable regions (V1-V5). SRLV region V4 has been found to be homologous to the V3 region of human lentivirus (HIV). HIV V3 is responsible for tropism and the development of nervous clinical patterns when there is a tendency to conserve amino acids in specific "signature pattern" positions. The goal of this study was to identify signature patterns in the V4 region of the SU, which is encoded by the SRLV env gene. Secondarily, to understand how these signature patterns are associated with different clinical status in naturally infected sheep and goats. Starting with 244 samples from seropositive animals from nine Mexican states, we amplified the V4 region using nested PCR and obtained 49 SRLV sequences from peripheral blood leukocytes. Based on phylogenetic analysis results, we identified three groups: asymptomatic genotypes A (Ssx GA) and B (Ssx GB), as well as animals with arthritic presentation, genotype B (A GB). Similarity levels between group sequences ranged from 67.9%-86.7%, with a genetic diversity ranging from 12.7%-29.5% and a dN / dS ratio that indicated negative selection. Analyses using Vespa and Entropy programs identified four residues at positions 54, 78, 79 and 82 in SU region V4 as possible signature patterns, although with variable statistical significance. However, position 54 residues "N" (p = 0.017), "T" (p = 0.001) and "G" (p = 0.024) in groups A GB, Ssx GA and Ssx GB respectively, best characterized the signature patterns. The results obtained identified a signature pattern related to different genotypes and clinical status by SRLV in sheep and goats.

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.

Analysis of the transcripts encoding for antigenic proteins of bovine gammaherpesvirus 4.

The major glycoproteins of bovine gammaherpesvirus 4 (BoHV-4) are gB, gH, gM, gL, and gp180 with gB, gH, and gp180 being the most glycosylated. These glycoproteins participate in cell binding while some act as neutralization targets. Glycosylation of these envelope proteins may be involved in virion protection against neutralization by antibodies. In infected cattle, BoHV-4 induces an immune response characterized by low neutralizing antibody levels or an absence of such antibodies. Therefore, virus seroneutralization in vitro cannot always be easily demonstrated. The aim of this study was to evaluate the neutralizing capacity of 2 Argentine BoHV-4 strains and to associate those findings with the gene expression profiles of the major envelope glycoproteins. Expression of genes coding for the envelope glycoproteins occurred earlier in cells infected with isolate 10/154 than in cells infected with strain 07/435, demonstrating a distinct difference between the strains. Differences in serological response can be attributed to differences in the expression of antigenic proteins or to post-translational modifications that mask neutralizing epitopes. Strain 07/435 induced significantly high titers of neutralizing antibodies in several animal species in addition to bovines. The most relevant serological differences were observed in adult animals. This is the first comprehensive analysis of the expression kinetics of genes coding for BoHV-4 glycoproteins in 2 Argentine strains (genotypes 1 and 2). The results further elucidate the BoHV-4 life cycle and may also help determine the genetic variability of the strains circulating in Argentina.

An Immunoperoxidase Monolayer Assay (IPMA) for the detection of lumpy skin disease antibodies.

During this study a new Immunoperoxidase Monolayer Assay (IPMA) was developed for the detection of antibodies against lumpy skin disease virus (LSDV) in an easy and low tech setting. Using two dilutions (1:50 and 1:300) in a duplicate format, the test was shown to be highly sensitive, specific and repeatable. In comparison to the VNT and a commercial ELISA, the LSDV-IPMA was able to detect the LSDV antibodies earlier in infected, vaccinated and vaccinated/infected animals. The assay is very flexible as it can be easily adapted for the detection of sheeppox or goatpox antibodies and it can be scaled-up to handle medium size sample sets by preparing the IPMA plates in advance. These plates are safe and can be handled in low biosafety level labs.

Quantification of ovine herpesvirus 2 by digital PCR in an outbreak of malignant catarrhal fever.

Infection with ovine gammaherpesvirus 2 (OvHV-2) is generally asymptomatic in sheep; however, when it crosses the species barrier, it causes malignant catarrhal fever (MCF) in cattle. In the present study, we developed a real-time PCR assay and a droplet digital PCR assay and use both methods to study an outbreak caused by OvHV-2. Both PCR methods showed high sensitivity and specificity and were able to detect low copy numbers of OvHV-2 in sheep and cattle. The present study describes the first digital PCR quantification of OvHV-2 genome copies in samples collected from sheep and cattle.

Domestic sheep and bighorn sheep carry distinct gammaherpesviruses belonging to the genus Macavirus.

The genus Macavirus of the subfamily Gammaherpesvirinae comprises two genetically distinct lineages of lymphotropic viruses. One of these lineages includes viruses that can cause malignant catarrhal fever (MCF), which are known as MCF viruses (MCFV). All MCFVs are genetically and antigenically related but carried by different hosts. In this study, we report the recognition of new MCFV carried by bighorn sheep. The virus was first identified in a bighorn sheep from Banff National Park, Alberta, Canada. Analysis of a conserved region of the viral DNA polymerase gene of the virus carried by this bighorn sheep showed 85.88% nucleotide identity to the MCFV carried by domestic sheep, ovine herpesvirus 2 (OvHV-2). Further investigation of bighorn samples obtained from animals in the US and Canada showed 98.87-100% identity to the DNA polymerase sequence of the first bighorn in the study. Phylogenetic analysis indicated that the MCFV carried by bighorn sheep is closely related but distinct from OvHV-2. Epidemiological and virulence features of the newly recognized MCFV are still unknown and warrant further investigation. Considering the current nomenclature for MCFVs, we suggest a tentative designation of ovine herpesvirus-3 (OvHV-3) for this newly identified bighorn sheep MCFV.

Genotyping Based on the LTR Region of Small Ruminant Lentiviruses from Naturally Infected Sheep and Goats from Mexico.

Small ruminant lentiviruses (SRLVs) belong to the genus Lentivirus in the Retroviridae family. There are five genotypes (A, B, C, D, and E), where genotypes A and B have a global distribution and genotypes C, D, and E are limited to Europe. The presence of SRLV has been confirmed in Mexico, with genotype B detected in the central region of the country. We examined the presence of SRLVs and genotype prevalence in 1014 sheep and 1383 goats from 12 Mexican states. Using a commercial competitive ELISA (cELISA) test, we detected SRLV antibodies in 107 sheep (10.55%) and 466 goats (33.69%). We used an endpoint PCR to amplify the LTR region on seropositive animals. A total of 50 sheep and 75 goats tested positive via PCR. Positive amplicons from 11 sheep and 17 goats from ten Mexican States were cloned and sequenced. With the LTR sequence data obtained in this study, a phylogenetic analysis was performed; we also constructed a phylogenetic tree using the obtained sequences and GenBank's available sequences. All studied sequences were associated with genotype B, specifically with the FESC-752 isolate previously identified in Mexico. Highly conserved transcription factor binding sites were observed in analyzed alignments, such as AML (vis), AP-4, and TATA box. However, we identified nucleotide differences at site AP-1 that suggest function loss. Our study found that ovine and caprine genotype B SRLVs are widely distributed in Mexico; a highly conserved LTR region among the sequences evaluated in this study was also found.

Detection of enzootic nasal tumor virus (ENTV) in a sheep flock in southern Brazil.

Enzootic nasal tumor (ENT) is a contagious neoplasm associated with enzootic nasal tumor virus (ENTV), which may induce disease in sheep (ENTV-1) and goats (ENTV-2). This study aimed to describe the occurrence of ENT in two Texel sheep (Ovis aries) from a 75-sheep flock, located in the city of Gravataí, southern Brazil. Animals used to be purchased from different origins, and no specific tests for disease monitoring or quarantine procedure were performed. Affected animals presented respiratory distress, anorexia with severe weight loss, and mucopurulent unilateral nasal discharge. Necropsy was performed in both animals and nasal cavity masses were observed. Histopathological analysis demonstrated an epithelial neoplasm compatible with nasal adenocarcinoma. PCR using a protocol that amplifies a 591 bp sequence of 5'LTR-gag region of ENTV1 was performed followed by DNA sequencing. Both samples were positive, and the sequences obtained presented highest identity (97%) with ENTV strain TN28 (GenBank accession number MH899613) detected in a Texel sheep from Scotland. This is the first report of ENTV-1 leading to enzootic nasal tumor in sheep in Latin America, which confirms the presence of the retrovirus in sheep flocks in the Brazilian territory.

A new approach for Small Ruminant Lentivirus full genome characterization revealed the circulation of divergent strains.

Small Ruminant Lentiviruses (SRLV) include at least 4 viral highly divergent genotypes. Genotypes A and B are widely distributed and genotypes C and E have been recognized in restricted geographic areas. New phylogroups have been identified targeting conserved regions. However, this approach suffers from the potential risk to misamplify highly divergent strains. Pathogenic strains are easily adapted to fibroblastic cells, but non-pathogenic strains isolation may require a different approach. We developed a fast and effective method for SRLV full genome characterization after cell culture isolation. Spleen samples were collected during regular slaughter from sheep and goats in northwestern Italy. Spleen-derived macrophage cultures were monitored for reverse transcriptase activity and RNA was extracted from the supernatant of positive cultures. Using Illumina MiSeq platform 22 new full genome sequences were obtained. The success of this approach is based on the following features: spleen is one of the main target for SRLV persistence; red pulp is a reserve of resident macrophages, the main target for SRLV replication in vivo; RTA is a sensitive assay for any replicating retrovirus; de novo sequencing do not require genetic knowledge in advance.