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ABSTRACT Mammals have co-evolved with retroviruses, including lentiviruses, over a long period. Evidence supporting this contention is that viral infectivity factor (Vif) encoded by lentiviruses antagonizes the anti-viral action of cellular apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) of the host. To orchestrate E3 ubiquitin ligase complex for APOBEC3 degradation, Vifs utilize mammalian proteins such as core-binding factor beta (CBFB; for primate lentiviruses) or cyclophilin A (CYPA; for Maedi-Visna virus [MVV]). However, the co-evolutionary relationship between lentiviral Vif and the mammalian proteins associated with Vif-mediated APOBEC3 degradation is poorly understood. Moreover, it is unclear whether Vif proteins of small ruminant lentiviruses (SRLVs), including MVV and caprine arthritis encephalitis virus (CAEV), commonly utilize CYPA to degrade the APOBEC3 of their hosts. In this study, molecular phylogenetic and protein homology modeling revealed that Vif co-factors are evolutionarily and structurally conserved. It was also found that not only MVV but also CAEV Vifs degrade APOBEC3 of both sheep and goats and that CAEV Vifs interact with CYPA. These findings suggest that lentiviral Vifs chose evolutionarily and structurally stable proteins as their partners (e.g., CBFB or CYPA) for APOBEC3 degradation and, particularly, that SRLV Vifs evolved to utilize CYPA as their co-factor in degradation of ovine and caprine APOBEC3. [ABSTRACT FROM AUTHOR]

Highlights • Frozen-thawed milk is a suitable alternative to frozen-thawed serum for SRLV cELISA. • Colostral samples caused no interference with agreement between sample types. • Implications of storage conditions and preservative added to milk need evaluation. Abstract Small ruminant lentivirus (SRLV) infection in goats causes progressive inflammatory infiltration of multiple organs including the mammary gland, and has been hypothesized to impact udder health and milk quality on dairy goat operations. The objective of this study was to evaluate frozen-thawed milk as an alternative substrate to serum for detection of antibodies to SRLV by a commercial competitive ELISA. Paired milk and serum samples collected on the same day from 116 goats were compared using a commercial SRLV antibody test kit, cELISA (VMRD, Pullman, WA, USA). Agreement between sample types and sensitivity, specificity, positive and negative predictive values of the cELISA using frozen-thawed milk and serum were calculated using a Bayesian latent class model. Agreement was excellent (κ = 0.97), with only 2/116 pairs yielding discordant results. The sensitivity and specificity of cELISA on milk were 97.0% and 95.9%, respectively. Individual frozen-thawed milk samples were a suitable alternative to frozen-thawed serum samples for the detection of SRLV antibodies in goats using a commercial competitive ELISA. [ABSTRACT FROM AUTHOR]

Small ruminant lentiviruses include viruses with diverse genotypes that frequently cross the species barrier between sheep and goats and that display a great genetic variability. These characteristics stress the need to consider the whole host range and to perform local surveillance of the viruses to opt for optimum diagnostic tests, in order to establish control programes. In the absence of effective vaccines, a comprehensive knowledge of the epidemiology of these infections is of major importance to limit their spread. This article intends to cover these aspects and to summarise information related to characteristics of the viruses, pathogenesis of the infection and description of the various syndromes produced, as well as the diagnostic tools available, the mechanisms involved in transmission of the pathogens and, finally, the control strategies that have been designed until now, with remarks on the drawbacks and the advantages of each one. We conclude that there are many variables influencing the expected cost and benefits of control programs that must be evaluated, in order to put into practice measures that might lead to control of these infections. [ABSTRACT FROM AUTHOR]

The major challenges in diagnosing small ruminant lentivirus (SRLV) infection include early detection and genotyping of strains of epidemiological interest. A longitudinal study was carried out in Rasa Aragonesa sheep experimentally infected with viral strains of genotypes A or B from Spanish neurological and arthritic SRLV outbreaks, respectively. Sera were tested with two commercial ELISAs, three based on specific peptides and a novel combined peptide ELISA. Three different PCR assays were used to further assess infection status. The kinetics of anti-viral antibody responses were variable, with early diagnosis dependent on the type of ELISA used. Peptide epitopes of SRLV genotypes A and B combined in the same ELISA well enhanced the overall detection rate, whereas single peptides were useful for genotyping the infecting strain (A vs. B). The results of the study suggest that a combined peptide ELISA can be used for serological diagnosis of SRLV infection, with single peptide ELISAs useful for subsequent serotyping. [ABSTRACT FROM AUTHOR]

Production and well-being of sheep and goats in many countries are harmfully impacted by small ruminant lentiviruses (SRLV) that cause incurable, progressive diseases. Susceptibility to ovine progressive pneumonia virus (OPPV), the North American form of SRLV, is influenced by variants of the ovine transmembrane protein 154 gene (TMEM154). The experimental objective was to estimate additive and dominance effects of TMEM154 haplotypes 1 and 3 on susceptibility of breeding ewes to infection after natural exposure to OPPV from birth to 39 mo of age. Sires and dams were heterozygous for TMEM154 haplotypes 1 and 3, producing ewe lambs with diplotypes "1 1," "1 3," and "3 3." These lambs were raised by mature, infected dams to ensure natural, maternal exposure to OPPV. Ewe lambs (n = 108) were kept for breeding and joined an infected flock of ewes to guarantee natural, nonmaternal exposure to OPPV. Ewes were bred to lamb at 1, 2, and 3 yr of age. Serum samples were collected at breeding, 1 mo before lambing and shortly after weaning each year to monitor infection status to 39 mo of age. During the experiment, 9 of the 108 ewes died while uninfected and data collected on these ewes were not analyzed. Infection status of the remaining 99 ewes at 39 mo of age was analyzed using logistic regression procedures. Effects of ewe type of birth, ewe type of rearing, and breed type of dam were not detected (P > 0.10), and the estimated sire variance component was nil. Ewe diplotype affected infection status (P < 0.0001), as did additive (P < 0.0001) and dominance (P < 0.0022) effects. Predicted probabilities of infection for ewes with diplotypes "1 1," "1 3," and "3 3" were 0.10, 0.88, and 0.89, respectively, and confidence intervals for diplotypes "1 3" and "3 3" were distinct from "1 1." Haplotype 3 was completely dominant to haplotype 1 at 39 mo of age. The probability of infection for ewes with either diplotype "1 3" or "3 3" averaged 8.5 times that of ewes with diplotype "1 1." Diplotype "1 3" and "3 3" ewes were highly susceptible to nonmaternal transmission of OPPV, in contrast to diplotype "1 1 ewes. Therefore, the distribution of ewes with diplotypes "1 1," "1 3," and "3 3" within a flock will influence the number of infections caused by each route of transmission. Selection and mating strategies can be implemented to produce sheep that are genetically less susceptible to OPPV infection. [ABSTRACT FROM AUTHOR]

The identification of small ruminant lentivirus (SRLV) infected animals is crucial to obtain success through eradication, control and monitoring programs worldwide. Several authors have demonstrated that serology alone is not sufficient to detect SRLV infection. Polymerase chain reaction (PCR) is suggested as the choice test to identify seronegative infected animals. The aim of this study was to evaluate and compare degenerate primer pairs in order to detect a great number of virus variants by a seminested PCR. Based on already described primers and the alignment of gag nucleotide sequences from databases, degenerate primers were designed. Increasing the degree of degeneracy has improved the detection of the provirus in ovine and caprine genomic DNA samples. The number of positive PCR samples was twice higher when using the more degenerate primers in the established experimental conditions. Thus, we propose the use of this seminested PCR as a diagnostic tool, in combination with serology, especially in the situations where the circulating virus variants are unknown. [ABSTRACT FROM AUTHOR]

We describe the clinicopathologic features of an arthritis outbreak in sheep induced by small ruminant lentivirus (SRLV), linked to the presence of a new SRLV isolate phylogenetically assigned to caprine arthritis encephalitis virus–like subgroup B2. Thirteen SRLV seropositive Rasa Aragonesa adult ewes were selected from 5 SRLV highly infected flocks (mean seroprevalence, 90.7%) for presenting uni- or bilateral chronic arthritis in the carpal joint. A complete study was performed, including symptomatology, histopathology, immunocytochemistry, immunohistochemistry, in situ hybridization, and microbiology. The carpus was the joint almost exclusively affected, with 10 sheep (76%) showing a moderate increase in carpal joint size (diameter range, 18–20 cm; normal range, 15–16 cm) without signs of locomotion problems and with 3 ewes (23%) showing severe inflammation with marked increase in diameter (21–24 cm), pain at palpation, and abnormal standing position. Grossly, chronic proliferative arthritis was observed in affected joints characterized by an increased thickness of the synovial capsule and synovial membrane proliferation. Microscopically, synovial membrane inflammation and proliferation and hyperplasia of synoviocytes were observed. More positive cases of SLRV infection were detected by immunocytochemistry of articular fluid than of bronchoalveolar lavage fluid. Immunohistochemistry and in situ hybridization also detected positive cells in the subsynovial connective tissue, lung, mediastinal lymph node, mammary gland, and mammary lymph node. All animals were negative for the presence of Mycoplasma or other bacteria in the articular space. The present outbreak likely represents an adaptation of a caprine virus to sheep. Our results underline the importance of the arthritis induced by SRLV in sheep, a clinical form that might be underestimated. [ABSTRACT FROM PUBLISHER]

Highlights: [•] A model for the genotype B Caprine Arthritis Encephalitis virus disease is presented. [•] Only endemic and disease-free equilibria arise, and a transcritical bifurcation. [•] Eradication is possible only for very small economically unaffordable herds. [•] In absence of vaccination new strategies for fighting the disease are needed. [ABSTRACT FROM AUTHOR]

Small ruminant lentiviruses (SRLV) adversely affect production and well-being of sheep and goats throughout much of the world. The SRLV, including ovine progressive pneumonia virus (OPPV) in North America, cause lifetime infections, and man-agement procedures to eradicate or reduce disease prevalence are costly. Variants of ovine transmembrane protein 154 gene (TMEM154) affect susceptibility to OPPV. The primary experimental objective was to estimate additive and dominance effects of TMEM154 haplotypes 1 and 3 on susceptibility to OPPV infection following natural exposure. A group of 187 trial lambs was born and raised by mature, infected ewes to ensure natural exposure to OPPV. Parents of trial lambs were heterozygous for haplotypes 1 and 3, producing lambs with diplotypes "1 1," "1 3," and "3 3." A group of 20 sentinel lambs was born and raised by mature, unin-fected ewes that were diplotype "1 1." Sentinel lambs had diplotypes "1 1" and "1 3," being sired by the same set of rams as trial lambs. Trial and sentinel lambs were comingled during the experiment. Lambs were weaned at 60 d of age, bled 1 wk after weaning, and thereafter at intervals of 4 or 5 wk until 9 mo of age when OPPV infection status was determined by use of a competitive enzyme-linked immunosorbent assay. Only 1 sentinel lamb became infected. Infection status of trial lambs was analyzed using logistic regression procedures to account for the binary nature of infection status and random effects of sires. Effects of sex, type of birth, type of rearing, age of dam, breed type of dam, and sires were not detected (P > 0.20). Infection status was affected by diplotype of lamb (P = 0.005), with additive (P = 0.002) and dominance (P = 0.052) effects identi-fied. Predicted probabilities of infection for lambs with diplotypes "1 1," "1 3," and "3 3" were 0.094, 0.323, and 0.346, respectively. Confidence intervals for prob-abilities of infection for diplotypes "1 3" and "3 3" were similar, but distinct from diplotype "1 1." These results are consistent with complete dominance of haplotype 3 relative to haplotype 1. The probability of infection at 9 mo of age for lambs with either diplotype "1 3" or "3 3" averaged 3.56 times that of lambs with diplotype "1 1." Genetic susceptibility to OPPV infection can be reduced by selection to increase the frequency of hap-lotype 1, resulting in a greater proportion of lambs with diplotype"! 1." [ABSTRACT FROM AUTHOR]

Multisystemic disease caused by Small Ruminant Lentiviruses (SRLV) in sheep and goats leads to production losses, to the detriment of animal health and welfare. This, together with the lack of treatments, has triggered interest in exploring different strategies of immunization to control the widely spread SRLV infection and, also, to provide a useful model for HIV vaccines. These strategies involve inactivated whole virus, subunit vaccines, DNA encoding viral proteins in the presence or absence of plasmids encoding immunological adjuvants and naturally or artificially attenuated viruses. In this review, we revisit, comprehensively, the immunization strategies against SRLV and analyze this double edged tool individually, as it may contribute to either controlling or enhancing virus replication and/or disease. [ABSTRACT FROM AUTHOR]

It is becoming increasingly clear that organisms have developed a variety of mechanisms to fight against viral infection. The viruses have developed means of counteracting these defences in various ways. The APOBEC3 proteins are a mammalian-specific family of nucleic acid cytidine deaminases that block retroviral infection. These inhibitors are counteracted by the Vif proteins encoded by most lentiviruses. In this paper, we will review the interaction of the lentiviral Vif proteins with the APOBEC3 proteins, with an emphasis on sheep APOBEC3 and maedi-visna virus (MVV) Vif. [ABSTRACT FROM AUTHOR]

Small ruminant lentiviruses include members that infect sheep (ovine lentivirus [OvLV]; also known as ovine progressive pneumonia virus/maedi-visna virus) and goats (caprine arthritis encephalitis virus [CAEV]). Breed differences in seroprevalence and proviral concentration of OvLV had suggested a strong genetic component in susceptibility to infection by OvLV in sheep. A genetic marker test for susceptibility to OvLV has been developed recently based on the TMEM154 gene with validation data from over 2,800 sheep representing nine cohorts. While no single genotype has been shown to have complete resistance to OvLV, consistent association in thousands of sheep from multiple breeds and management conditions highlight a new strategy for intervention by selective breeding. This genetic marker-assisted selection (MAS) has the potential to be a useful addition to existing viral control measures. Further, the discovery of multiple additional genomic regions associated with susceptibility to or control of OvLV suggests that additional genetic marker tests may be developed to extend the reach of MAS in the future. This review will cover the strengths and limitations of existing data from host genetics as an intervention and outline additional questions for future genetic research in sheep, goats, small ruminant lentiviruses, and their host-pathogen interactions. [ABSTRACT FROM AUTHOR]

Abstract: Live attenuated vaccines provide the most consistent protective immunity in experimental models of lentivirus infections. In this study we tested the hypothesis that animals infected with a naturally attenuated small ruminant lentivirus field strain of genotype E may control a challenge infection with a virulent strain of the caprine arthritis encephalitis virus (CAEV-CO). Within genotype E, Roccaverano strain has been described as attenuated since decreased arthritic pathological indexes were recorded in Roccaverano-infected animals compared to animals of the same breed infected with genotype B strains. Moreover, under natural conditions, animals double-infected with genotypes B and E appear less prone to develop SRLV-related disease, leading to a putative protective role of Roccaverano strain. Here we present evidence that goats experimentally infected with the avirulent genotype E SRLV-Roccaverano strain control the proviral load of a pathogenic challenge virus (CAEV-CO strain) more efficiently than naïve animals and appear to limit the spread of histological lesions to the contralateral joints. [Copyright &y& Elsevier]

Abstract: Three field isolates of small ruminant lentiviruses (SRLVs) were derived from a mixed flock of goats and sheep certified for many years as free of caprine arthritis encephalitis virus (CAEV). The phylogenetic analysis of pol sequences permitted to classify these isolates as A4 subtype. None of the animals showed clinical signs of SRLV infection, confirming previous observations which had suggested that this particular subtype is highly attenuated, at least for goats. A quantitative real time PCR strategy based on primers and probes derived from a highly variable env region permitted us to classify the animals as uninfected, singly or doubly infected. The performance of different serological tools based on this classification revealed their profound inadequacy in monitoring animals infected with this particular SRLV subtype. In vitro, the isolates showed differences in their cytopathicity and a tendency to replicate more efficiently in goat than sheep cells, especially in goat macrophages. By contrast, in vivo, these viruses reached significantly higher viral loads in sheep than in goats. Both env subtypes infected goats and sheep with equal efficiency. One of these, however, reached significantly higher viral loads in both species. In conclusion, we characterized three isolates of the SRLV subtype A4 that efficiently circulate in a mixed herd of goats and sheep in spite of their apparent attenuation and a strict physical separation between goats and sheep. The poor performance of the serological tools applied indicates that, to support an SRLV eradication campaign, it is imperative to develop novel, subtype specific tools. [Copyright &y& Elsevier]

Abstract: Small Ruminant Lentivirus is transmitted vertically by ingestion of contaminated milk/colostrum and horizontally by the respiratory route through close contact with infected animals. In order to detect presence of virus in a shed environment, we sampled and tested drinking water, pen air and air exhaled from infected sheep in both commercial flocks and groups of culled sheep. Nucleic acids were extracted and analysed by retrotranscription, Pol PCR amplification and sequencing. Virus sequences were detected in 9% and 11% of samples of water and air, respectively, collected from pens, and in 35% of sheep whose expelled air was tested. Our results are in concordance with previous evidences of air and waterborne transmission of the infection and might have implications for the design of control strategies. [Copyright &y& Elsevier]

Abstract: A compulsory control program to control-eradicate Brucella infections was initiated 1985 in the Basque Country in Northern Spain. The program included both Brucella melitensis infection (ovine/caprine brucellosis), which were later included in a national plan, and Brucella ovis infection (ovine epididymitis), which was promoted by the local animal health authorities. Prevalences of these infections in small ruminant farms at that time were 10% and 60% of flocks, respectively. Since then, the flocks/herds have been tested annually and animals found to be infected by means of the tests employed were slaughtered. At the same time, a voluntary Small Ruminant Lentivirus infection control program also started in some sheep flocks of the region. Currently, prevalences of brucellosis have dropped to 0% for B. melitensis infection and <0.1% for B. ovis. This implies an overall rate reduction of 100% and 99.9%, respectively. The results for Small Ruminant Lentivirus infection have not been so good and only three of the eight flocks into the scheme have reached eradication. Good results have been obtained by local and national animal health authorities and farmers during the past 25 years for brucelosis control. Use of simple serological testing and culling has allowed reduction-eradication of the infection. Once advanced control is reached, an economic balance will be necessary, in order to optimize best use of resources, including allocating them to control other small ruminant problems. [Copyright &y& Elsevier]

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Abstract: The small ruminant lentiviruses include the prototype for the genus, visna-maedi virus (VMV) as well as caprine arthritis encephalitis virus (CAEV). Infection of sheep or goats with these viruses causes slow, progressive, inflammatory pathology in many tissues, but the most common clinical signs result from pathology in the lung, mammary gland, central nervous system and joints. This review examines replication, immunity to and pathogenesis of these viruses and highlights major differences from and similarities to some of the other lentiviruses. [Copyright &y& Elsevier]

Abstract: An extensive outbreak characterized by the appearance of neurological symptoms in small ruminant lentivirus (SRLV) infected sheep has been identified in Spain, but the genetic characteristics of the strain involved and differential diagnostic tools for this outbreak remain unexplored. In this work, 23 Visna-affected naturally infected animals from the outbreak, 11 arthritic animals (both groups presenting anti-Visna/Maedi virus serum antibodies), and 100 seronegative animals were used. Eight of the Visna-affected animals were further studied post-mortem by immunohistochemistry. All had lesions in spinal cord, being the most affected part of the central nervous system in six of them. A representative strain of the outbreak was isolated. Together with other proviral sequences from the outbreak the virus was assigned to genotype A2/A3. In vitro culture of the isolate revealed that viral production was slow/low in fibroblast-like cells but it was high in blood monocyte-derived macrophages. The long terminal repeat (LTR) of the viral genome of this isolate lacked an U3-duplication, but its promoter activity in fibroblast-like cells was normal compared to other strains. Thus, viral production could not be inferred from the LTR promoter activity in this isolate. Analysis of the viral immunodominant epitopes among SRLV sequences of the outbreak and other known sequences allowed the design of a synthetic SU peptide ELISA that detected the Visna affected animals, representing a tool of epidemiological interest to control viral spread of this highly pathogenic strain. [Copyright &y& Elsevier]

Nucleotide sequences of small ruminant lentiviruses (SRLVs) were determined in sheep and goats, including progeny of imported animals, on a farm in Mexico. On the basis of gag-pol, pol, env and LTR sequences, SRLVs were assigned to the BI subgroup, which comprises caprine arthritis-encephalitis virus (CAEV)-like prototype sequences mainly from goats. In comparison with CAEV-like env sequences of American and French origin, two putative recombination events were identified within the V3-V4 and V4-V5 regions of the env gene of a full length SRLV sequence (FESC-752) derived from a goat on the farm. [ABSTRACT FROM AUTHOR]

Abstract: Genotype E of small ruminant lentivirus has been recently described in goats from different breeds in Italy. Genotype E infection may differ from known genotypes since deletions of dUTPase and VPR proteins have been confirmed in different independent areas and goat breed, and play a key role on virus replication and pathogenesis. In particular, genotype E Roccaverano strain has been described as low pathogenic since does not lead to clinical symptoms in goats. In contrast, classical CAEV infected goats of the same area and breed presented arthritis. In this study, we have used intratracheal and intra-bone marrow routes to establish genotype E persistent infections. Humoral and cellular immune responses elicited in the host against genotype E and genotype B derived antigens were evaluated until 200 days post-inoculation. Compared to genotype B antigen, seroconversion against genotype E GAG P16-25 antigen was detected at 2–3 weeks after inoculation, significantly earlier and at higher titres. Interestingly, antibody avidity did not increase in the course of the experiment neither against P16-25 nor against SU5, both derived from genotype E. T cell proliferation against P25-GST fusion protein antigens derived from genotype E was firstly detected at 15 days post-inoculation and was maintained throughout time until week 20 post-infection, while T cell proliferation against the genotype B P25 was not produced by the end of the experiment at 20 weeks post-inoculation. The strength of reaction was also higher when using P25 E as stimulator antigen. In contrast with antibody and T cell proliferation, cytotoxic-T-lymphocyte (CTL) activity in the circulating lymphocytes (effector cells) using blood-derived macrophages (BDM) as target cells, was not strain specific being surprisingly higher against genotype B infected antigen presenting cells (APCs). This is the first study reporting experimentally induced immunological changes in SRLV genotype E infection and indicates that CTL activity may be the adaptive immune response able to induce protection against heterologous infection. [Copyright &y& Elsevier]

Abstract: Small ruminant lentivirus genotype E lacks the dUTPase subunit and vpr-like gene. Two strains (Roccaverano and Seui) with identical genetic organization have been described, with the env HV1–HV2 domains being the most divergent. Although dUTPase and vpr-like deletions have been involved in the RT fidelity in non dividing cells, both strains were able to replicate efficiently in blood derived macrophages (BDM), while virus production of E1 subtype was reduced or abrogated in replicating fibroblastic-like cells. The transcriptional activity of genotype E was similar in these two cellular populations. When viral pseudotypes were generated with the env of both viruses, Roccaverano pseudotype displayed a paranuclear localization on BDM, suggesting a different mechanism of entry. Polymorphic GAS and TAS sites in the U3 region, further suggest that a population different from classically activated macrophages can be infected by these viruses, opening new insights into lentiviruses with low or null pathogenic potential. [ABSTRACT FROM AUTHOR]

Abstract: Lactogenic transmission plays an important role in the biology of lentiviruses such as HIV and SIV or the small ruminant lentiviruses (SRLV). In this work we analyzed the characteristics of viruses that goats, naturally infected with two strains of SRLV, transmitted to their kids. The spectrum of viral genotypes transmitted was broader and the efficiency of transmission greater compared to their human and simian counterparts. The newly described A10 subgroup of SRLV was more efficiently transmitted than the B1 genotype. The analysis of a particular stretch of the envelope glycoprotein encompassing a potential neutralizing epitope revealed that, as in SIV, the transmitted viruses were positively charged in this region, but, in contrast to SIV, they tended to lack a glycosylation site that might protect against antibody neutralization. We conclude that the physiology of the ruminant neonatal intestine, which permits the adsorption of infected maternal cells, shaped the evolution of these particular lentiviruses that represent a valid model of lactogenic lentivirus transmission. [ABSTRACT FROM AUTHOR]

The article explores the role of serologic diagnostic tests in reducing small ruminant lentivirus (SRLV) infections. Factors affecting the performance of the serologic and molecular diagnostic tests include the format of the assay and the percentage of identity between the viral nucleotide sequences in a flock of a certain geographic region. It was concluded that small ruminant genomics may contribute to the establishment of genetic markers of SRLV infection and disease.

Abstract: The highly divergent SRLV genotype E has recently been characterized in Italy as a low pathogenic caprine lentivirus in the Roccaverano breed. The availability of a genotype specific diagnostic test based on a comparative assay, using a combination of genotype specific recombinant antigens allows a wide serosurvey in other goat populations. The island of Sardinia still has the highest small ruminant population of any Italian region and crossbreeding has been limited to goats, mainly with the Maltese breed. A serological survey was carried out on sheep flocks and goat herds, using individual sera as well as a bulk milk-adapted procedure. Genotype E was identified in more than 50% of goat herds and none of the sheep flocks thus supporting the idea that this genotype is specifically associated with the goat species. The full-length proviral sequence of a Sardinian isolate revealed and confirmed the deletion of dUTPase subunit and the absence of both vpr gene and the 71bp repeat of the LTR. Genetic similarity of this isolate with the prototype strain Roccaverano was not more than 84%, supporting the designation of two subtypes within genotype E. Nevertheless, in vitro properties of the Sardinian strain were different from those of the Roccaverano strain in terms of ability to infect synovial membrane and produce syncitia. Remarkable differences in the HV1 and HV2 of the env gene were recorded, with the Sardinian isolate displaying sequence motif more similar to arthritic strains. Data presented suggest diffusion of genotype E is wider than previously thought. [Copyright &y& Elsevier]

The highly divergent, small ruminant lentivirus (SRLV) genotype E Roccaverano strain has a full genome consisting of 8,418 nucleotides, which lack the entire dUTPase subunit of the pol gene, the vpr-like accessory gene, and the 71-bp repeat of the U3 region within the long terminal repeat (LTR). These deletions affect in reverse transcriptase fidelity in non-dividing cells (dUTPase and vpr-like) and in the regulation of viral replication. Surprisingly, this SRLV strain was able to replicate efficiently in non-dividing cells (i.e., blood-derived macrophages), while replication in fibroblastic-like cells was somewhat restricted. To evaluate whether this observation was due to the presence/absence of specific transcription factors within these fibroblasts, U3 transcriptional activity was measured in the different cell types and revealed that both fibroblasts and macrophages were able to activate the viral promoter in the same manner. Among the transcription factor-binding sites present within the U3 region, the highly conserved Ap4 tandem repeat was shown to be sufficient for LTR promoter activity. [ABSTRACT FROM AUTHOR]

Abstract: Antibody-based diagnosis of small ruminant lentiviruses (SRLVs) has been efficiently achieved using serum and milk, but not semen, for which polymerase chain reaction (PCR) has been proposed as a confirmatory technique. This work, involving 296 ovine (Ovis aries) and caprine (Capra hircus) semen donors, investigates whether seminal fluid (SF) can be reliably used in antibody-based SRLV diagnosis. First, a gold standard was established to assess the infection status and determine the sensitivity and specificity of three commercial enzyme-linked immunosorbent assays (ELISAs) in serum testing using Western blot and PCR as confirmatory tests. For SF testing, both gold standard and serum testing results were used as reference. The performance of SF testing was affected not only by the ELISA assay sensitivity (related to antigen spectrum) compared with that of the gold standard (as it occurred in serum testing) but also by SF sample quality and SF working dilution. Nonturbid SF samples, commonly collected in artificial insemination centers (AICs), were required. Compared with serum, SF testing had a decreased sensitivity in two of the ELISA assays (with original serum working dilutions ≤1/20 in serum testing) but reached a similar sensitivity (and specificity) in the assay designed to work at the highest serum dilution (1/500). A SF concentration of about 1/2 (250-fold that used in serum testing) was found optimal in this assay, yielding highly repeatable results that were in almost perfect agreement with those of serum testing (κ ± SE, 0.91 ± 0.81). Thus, SF ELISA can be reliably applied in antibody-based SRLV diagnosis. This information may be useful to control infection in AICs and animal and semen trade programs requiring health-certified quality of semen donors. [Copyright &y& Elsevier]

Abstract: Small ruminant lentivirus (SRLV) belonging to the highly divergent genotype E has recently been identified in the Italian goat breed Roccaverano. In this report we have developed a specific serological test based on recombinant matrix/capsid antigen fusion protein. Performance has been evaluated and compared with a similar test based on genotype B antigen. Herds under study were selected according to the infectious status characterized by blood PCR and sequencing. Results clearly showed that B and E based recombinant ELISA only detected homologous infection and an apparent cross-reactivity was recorded in a herd in which co-infection was present. Three commercially available ELISAs showed different abilities in detecting genotype E infection, being the whole virus-based immunoassay the best choice. Genotype E-recombinant antigen was not detected in ELISA by three commercially available Mabs known to be cross-reactive among CAEV and MVV capsid antigens, further supporting the high divergence of the E genotype from others. Finally, a SRLV-free herd according to commercial ELISA testing, was analysed in the same area where genotype E was identified and few animals belonging to Roccaverano breed were found slightly reactive with the E antigens. Our results suggest that the prevalence of genotype E in other small ruminant populations may be conveniently estimated using a comparative assay based on a combination of genotype specific recombinant antigens and may highlight a wider space in which SRLVs evolve. [Copyright &y& Elsevier]

Maedi visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are a heterogeneous group of infectious agents affecting sheep and goats. Due to their natural cross-species infection they are referred to as small-ruminant lentiviruses (SRLV). Recently a new genetic cluster, highly divergent from MVV and CAEV was identified in the north-west part of Italy. A panel of genotype E specific antigens was developed and evaluated in flocks infected with B1 and E strains. The results clearly indicate that a strain specific antigen is required to correctly identify animals infected with different genotypes. [ABSTRACT FROM AUTHOR]

Abstract: RNA transcripts of the B7 family molecule (CD80) are diminished in blood leukocytes from animals clinically affected with Visna/Maedi virus (VMV) infection. This work investigates whether the use of B7 genes enhances immune responses and protection in immunization-challenge approaches. Sheep were primed by particle-mediated epidermal bombardment with VMV gag and env gene recombinant plasmids together with plasmids encoding both CD80 and CD86 or CD80 alone, boosted with gag and env gene recombinant modified vaccinia Ankara virus and challenged intratracheally with VMV. Immunization in the presence of one or both of the B7 genes resulted in CD4+ T cell activation and antibody production (before and after challenge, respectively), but only immunization with CD80 and CD86 genes together, and not CD80 alone, resulted in a reduced number of infected animals and increased early transient cytotoxic T lymphocytes (CTL) responses. Post-mortem analysis showed an immune activation of lymphoid tissue in challenge-target organs in those animals that had received B7 genes compared to unvaccinated animals. Thus, the inclusion of B7 genes helped to enhance early cellular responses and protection (diminished proportion of infected animals) against VMV infection. [Copyright &y& Elsevier]

Abstract: To extend and complete the epitope mapping of gag-encoded structural proteins, the immunodiagnostic potential of nucleoprotein (NP) of two different small ruminant lentivirus (SRLV) genotypes were antigenically characterized. Respective recombinant counterparts were generated and used in an enzyme-linked immunosorbent assay (ELISA) format to test a panel of sera from infected flocks. Results clearly indicate that a single linear epitope located within the C-terminal is partially cross-reactive among different SRLV genotypes and may complement multiple epitope ELISA for serological diagnosis of infection. However, in contrast to matrix and capsid antigen epitopes, which drive a genotype-specific immunoresponse, a moderate degree of variation was identified in NP independently of the genotype to which it belongs. [Copyright &y& Elsevier]

Abstract: Retroviruses are RNA viruses widely distributed in most vertebrate and in many non-vertebrate species. Among the retrovirus family, small ruminant lentiviruses (SRLV), Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) infect small ruminants. JSRV and ENTV are oncogenic retroviruses responsible for pulmonary and nasal adenocarcinomas, respectively, which can lead to the death of the animals in few weeks or months. SRLV are non-oncogenic and replication-competent lentiviruses, related to human immunodeficiency virus (HIV). SRLV are responsible for slowly evolving progressive degenerative diseases leading to the death of the host after few months or years. These viruses are transmitted through the colostrum or milk and aerosolization of infectious particles. This review focuses on the biology of the small ruminant retrovirus and on the main mechanisms of disease induction. [Copyright &y& Elsevier]

Abstract: To be able to predict sexual transmissibility of small ruminant lenti viruses (SRLV), it is necessary to know whether or not the virus is excreted in the semen, and under what circumstances. Thus, this research focussed on establishing the presence of proviral DNA of SRLV in semen and in the male genital tract of small ruminants. After initial results established the presence of SRLV in serum, the emergence of proviral DNA of SRLV in semen and presence in blood in a group of naturally SRLV-infected individuals (13 rams and 4 bucks), was followed temporally using real-time polymerase chain reaction (PCR). The same animals were also systematically serologically monitored by enzyme-linked immuno sorbent assay (ELISA) during the breeding season (August–February). A triple monocyte–macrophage count was performed on both blood and semen using a specific monoclonal antibody in conjunction with flow cytometry. The finding that epididymal semen and tissue samples of the testes, epididymides, ampullary, vesicular, prostate and bulbo-urethral glands all tested positive for the presence of proviral DNA indicates that various male sexual organs may contribute directly to shedding of proviral SRLV DNA in ejaculated semen. Our results suggest that small ruminants show intermittent shedding of proviral SRLV DNA into epididymal as well as ejaculated semen. They also demonstrate that a single PCR-negative semen sample cannot be used as a diagnostic tool to predict that subsequent ejaculates will be SRLV-free. No significant relationship was found between numbers of monocytes and/or macrophages in blood or semen and the detection of proviral SRLV in ejaculates. [Copyright &y& Elsevier]

Abstract: The compartmentalization of small ruminant lentivirus (SRLV) subtype A (Maedi-Visna virus) and B (caprine arthritis–encephalitis virus) variants was analyzed in colostrum and peripheral blood mononuclear cells of four naturally infected goats. Sequence analysis of DNA and RNA encompassing the V4-V5 env regions showed a differential distribution of SRLV variants between the two compartments. Tissue-specific compartmentalization was demonstrated by phylogenetic analysis in three of the four cases. In these animals colostrum proviral sequences were clustered relative to the blood viral sequences. In one goat, the blood and colostrum-derived provirus sequences were intermingled, suggesting trafficking of virus between the two tissues or mirroring a recent infection. Surprisingly, the pattern of free virus variants in the colostrum of all animals corresponded only partially to that of the proviral form, suggesting that free viruses might not derive from infected colostral cells. The compartmentalization of SRLV between peripheral blood and colostrum indicates that lactogenic transmission may involve specific viruses not present in the proviral populations circulating in the blood. [Copyright &y& Elsevier]

Abstract: DNA vaccine candidates against Maedi-Visna virus (MVV) infection in ovines were developed as an alternative to conventional vaccines. Candidates were constructed by cloning genes encoding the MVV gag polyprotein and gag proteins p16 and p25 fused to a β-galactosidase reporter in a plasmid backbone. Transfection of different ovine cells showed a higher protein expression with plasmid lacZp16, which was hence further optimised by (i) removing a putative inhibitory sequence via reduction of the AU-content in the p16 gene or by (ii) introducing a secretory signal (Sc) to promote antigen secretion and increase its presentation to APCs. Unexpectedly, plasmids constructed on the basis of the first strategy by mutagenesis of lacZp16 (lacZp16mut24), led to a reduction in the expression of the antigen/reporter fusion in cultured ovine cells. This indicates that the high AU content in MVV does not inhibit protein expression. However, mice primed with lacZp16mut24 and boosted with MVV protein displayed higher humoral response when compared with control lacZp16. The addition of the Sc signal (Sc-p16) led to lower amounts of intracellular antigen/reporter fusion in transfected ovine cells, thus confirming secretion. These findings correlate with in vivo experiments, which showed that mice primed with Sc-p16 and boosted with MVV exhibited stronger antibody responses when compared with control mice primed with lacZp16 and boosted with MVV. Stronger humoral responses were recorded by immunising mice with (i) Sc-p16 and lacZp16mut24 plasmids together or with (ii) one plasmid containing both the mutations and the Sc signal. [Copyright &y& Elsevier]

Abstract: Little sequence information exists on the matrix-protein (MA) encoding region of small ruminant lentiviruses (SRLV). Fifty-two novel sequences were established and permitted a first phylogenetic analysis of this region of the SRLV genome. The variability of the MA encoding region is higher compared to the gag region encoding the capsid protein and surprisingly close to that reported for the env gene. In contrast to primate lentiviruses, the deduced amino acid sequences of the N- and C-terminal domains of MA are variable. This permitted to pinpoint a basic domain in the N-terminal domain that is conserved in all lentiviruses and likely to play an important functional role. Additionally, a seven amino acid insertion was detected in all MVV strains, which may be used to differentiate CAEV and MVV isolates. A molecular epidemiology analysis based on these sequences indicates that the Italian lentivirus strains are closely related to each other and to the CAEV-CO strain, a prototypic strain isolated three decades ago in the US. This suggests a common origin of the SRLV circulating in the monitored flocks, possibly related to the introduction of infected goats in a negative population. Finally, this study shows that the MA region is suitable for phylogenetic studies and may be applied to monitor SRLV eradication programs. [Copyright &y& Elsevier]

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

• The prevalence (PCR) and seroprevalence (ELISA) of SRLV in the goats analyzed ranged between 19 % and 11 %, respectively. • The SRLV identified in this study corresponds to the B1 genotype. • The infection with the Mexican SRLV strain did not show serious lesions or signs in the short period evaluated in kids. Small ruminant lentiviruses (SRLVs) are one of the main viral agents affecting goats worldwide. They cause a systemic, chronic, incurable, degenerative disease. In Mexico, the B1 genotype of SRLV has been detected as endemic in goats and sheep; however, its pathological characteristics have not yet been evaluated. In this study, sampling was performed to identify the percentage of SRLV positivity in different farms in Mexico. Prevalence rates of 19.6 % and 11.6 % were detected through PCR and serology, respectively. Through a real-time PCR differential test, the prevalence of genotype B was identified. From a chosen case, an SRLV strain was isolated (which was called SRLV/B1/Goat/Mx/INIFAP-1/2013). The phylogenetic analysis revealed that the strain SRLV/B1/Goat/Mx/INIFAP-1/2013 shares a great homology with an American and Asiatic strains of the B1 genotype. Virulence of the isolated strain was analyzed during an experimental infection in kids. The kids did not present clinical disease after the experimental infection. The high viral load values persisted throughout the entire experiment (90 days), and no seroconversion was detected. At necropsy, macroscopic alterations were not observed, except in the lungs. The microscopic lesions in the respiratory tract consisted of moderate interstitial pneumonia, peribronchial lymphoid hyperplasia, and bronchial exudate. In the central nervous system, moderate gliosis, meningeal congestion, lymphocyte infiltration, satellitosis, and demyelination were observed. The virus was systemically distributed among organs; the cerebellum and carpal synovial fluid samples were those with the highest viral load, 5.73 copies of virus per ng of total DNA (log 10) and 5.07 log 10 , respectively. In this study, we identified a strain with high homology to Asiatic strains that manifested low virulence in kids during short infection. Understanding the strains that circulate in the goat population will allow implementing better diagnostic methodologies to propose disease control alternatives for this emerging animal production system in Mexico. [ABSTRACT FROM AUTHOR]

• Lay-out of the Dutch SRLV accreditation scheme. • Results showed a close call for the top three ELISAs in specificity and specificity. • Fitness for purpose of five SRLV ELISA's in accreditation schemes. Maedi-visna (MV) in sheep and caprine arthritis encephalitis (CAE) in goats are progressive inflammatory diseases caused by MV virus (MVV) and CAE virus (CAEV), retroviruses that belong to the group of small ruminant lentiviruses (SRLV). In the Netherlands, SRLV accreditation based on screening of goat and sheep sera for specific SRLV antibodies, followed by confirmatory testing of positive samples by a second antibody test, will reach its 40th birthday in 2022. The aim of this study was to evaluate the strategy used within the Dutch SRLV accreditation scheme, based on test characteristics of five different commercially available ELISAs and an AGIDT. The specificity of the ELISAs was determined using Icelandic sheep sera and sera from Dutch SRLV accredited sheep flocks and goat herds. When inconclusive test results are considered negative, specificity did not differ between ELISAs. In the absence of a gold standard test for SRLV infection, goats and sheep from infected herds and flocks, and sheep with clinical maedi-visna were considered positive if three or more out of five ELISAs tested positive. Significant differences in sensitivity were observed between ELISAs, which were most apparent for sheep samples. Based on specificity, sensitivity and the possibility for sample pooling, the ELISA that was already in place as a screening test was still considered to be the most suitable ELISA for accreditation purposes. Another ELISA with very good test characteristics has been selected to replace the AGIDT as a confirmatory test for the test scheme. Two of the ELISAs were considered to be less suitable. To conclude, we here present a SRLV accreditation scheme in the Netherlands that has been updated according to fitness for purpose of commercially available SRLV antibody tests. [ABSTRACT FROM AUTHOR]

Abstract Background A central nervous system (CNS) disease outbreak caused by small ruminant lentiviruses (SRLV) has triggered interest in Spain due to the rapid onset of clinical signs and relevant production losses. In a previous study on this outbreak, the role of LTR in tropism was unclear and env encoded sequences, likely involved in tropism, were not investigated. This study aimed to analyze heterogeneity of SRLV Env regions - TM amino terminal and SU V4, C4 and V5 segments - in order to assess virus compartmentalization in CNS. Results Eight Visna (neurologically) affected sheep of the outbreak were used. Of the 350 clones obtained after PCR amplification, 142 corresponded to CNS samples (spinal cord and choroid plexus) and the remaining to mammary gland, blood cells, bronchoalveolar lavage cells and/or lung. The diversity of the env sequences from CNS was 11.1-16.1% between animals and 0.35-11.6% within each animal, except in one animal presenting two sequence types (30% diversity) in the CNS (one grouping with those of the outbreak), indicative of CNS virus sequence heterogeneity. Outbreak sequences were of genotype A, clustering per animal and compartmentalizing in the animal tissues. No CNS specific signature patterns were found. Conclusions Bayesian approach inferences suggested that proviruses from broncoalveolar lavage cells and peripheral blood mononuclear cells represented the common ancestors (infecting viruses) in the animal and that neuroinvasion in the outbreak involved microevolution after initial infection with an A-type strain. This study demonstrates virus compartmentalization in the CNS and other body tissues in sheep presenting the neurological form of SRLV infection.

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.

Simple Summary: Sheep are affected by a viral infection that causes an incurable and difficult to treat lung, joint and brain disease that decreases production efficiency. This small ruminant lentivirus is closely related to human immunodeficiency virus (HIV) that causes AIDS in humans. Differences in breed susceptibility to disease are known in sheep that indicate genetic, or hereditary, resilience factors exist. Our objective was to study the source of this hereditary advantage so that it can eventually be translated into a tool for sheep breeders to improve herd health. Previously, one such hereditary region was detected, but little was known about possible mechanisms or nearby mutations. Here, we report several mutations that may underlie this hereditary mechanism and are in regions of DNA that are known to affect genes by increasing or decreasing gene expression, akin to gene "on/off switches." These mutations were strongly associated with a predictor of disease severity in live animals and had a greater predicted effect on the degree of disease than previously studied mutations. Statistical association (p < 0.05) of disease was demonstrated in two different groups of sheep that were reared in different environments, which indicates increased likelihood that a genetic factor is producing this effect. Small ruminant lentivirus (SRLV) causes Maedi-Visna or Ovine Progressive Pneumonia in sheep and creates insidious livestock production losses. This retrovirus is closely related to human immunodeficiency virus and currently has no vaccines or cure. Genetic marker assisted selection for sheep disease resiliency presents an attractive management solution. Previously, we identified a region containing a cluster of zinc finger genes that had association with ovine SRLV proviral concentration. Trait-association analysis validated a small insertion/deletion variant near ZNF389 (rs397514112) in multiple sheep breeds. In the current study, 543 sheep from two distinct populations were genotyped at 34 additional variants for fine mapping of the regulatory elements within this locus. Variants were selected based on ChIP-seq annotation data from sheep alveolar macrophages that defined active cis-regulatory elements predicted to influence zinc finger gene expression. We present a haplotype block of variants within regulatory elements that have improved associations and larger effect sizes (up to 4.7-fold genotypic difference in proviral concentration) than the previously validated ZNF389 deletion marker. Hypotheses for the underlying causal mutation or mutations are presented based on changes to in silico transcription factor binding sites. These variants offer alternative markers for selective breeding and are targets for future functional mutation assays. [ABSTRACT FROM AUTHOR]

Simple Summary: Caprine arthritis encephalitis, caused by small ruminant lentivirus (SRLV), is a disease that develops with various signs in adult goats, e.g., arthritis, mastitis, and progressive weight loss, while in goat kids, the disease presents with only neuropathy and extremely rarely. The disease results in reduced milk production and economic losses in herds of goats. Previously described changes in single gene expression do not fully explain all the processes occurring in the infected goats. Therefore, the present study describes the first use of a transcriptomic array designed specifically for goats in Poland. Its aim was to investigate the gene expression profiles of peripheral blood nuclear cells from SRLV-seropositive and SRLV-seronegative goats using a custom-made Capra hircus gene expression array. Just four genes out of ~50,000 were found to have differential expression; moreover, changes in their expression suggest an active inflammatory mechanism in SRLV-seropositive goats at the early stage of SRLV infection. The immune response to a viral antigen causes inflammatory cell infiltration to the tissue, which creates a suitable environment for the replication of the virus in macrophages, and the recruitment of more monocytes to the site of infection, or latently infected monocytes. The aim of the study was to analyze the transcriptomic profile of peripheral blood nuclear cells isolated from SRLV-seropositive and SRLV-negative goats at the peak of their first lactation. SRLV-seropositive goats were probably infected via colostrum. Custom transcriptomic microarrays for goats were designed and developed, namely the Capra hircus gene expression array, which features ~50,000 unique transcripts per microarray. Only four genes were differentially expressed, with up-regulated expression of the GIMAP2, SSC5D and SETX genes, and down-regulated expression of the GPR37 gene in SRLV-seropositive vs. SRLV-seronegative goats. However, in an RT-qPCR analysis, the result for the SETX gene was not confirmed. The differences in the expressions of the studied genes indicate an active inflammatory process in the SRLV-seropositive goats at the early stage of infection. [ABSTRACT FROM AUTHOR]

Ovine progressive pneumonia virus (OPPV) is a small ruminant lentivirus that is widespread throughout U.S. sheep flocks. Infections with OPPV are lifelong and effects are multi-systemic with significant implications for animal well-being and productivity. A protein isoform with lysine at position 35 (K35, haplotype "1") encoded by the ovine transmembrane protein 154 (TMEM154) gene has been associated with reduced susceptibility to infection when two copies are present (i.e., diplotype "1,1"). Conversely, the ancestral protein isoform with glutamate at position 35 (E35, haplotype "3") is associated with high susceptibility to infection when at least one copy is present. The beneficial effect of TMEM154 K35 alleles on ewe productivity has not been previously measured in controlled challenge experiments and was a major objective of this study. Ewes with TMEM154 diplotypes "1,1"; "1,3"; and "3,3" (n = 31, 47, and 30, respectively) were born and reared by OPPV-infected dams and managed under continual natural exposure to OPPV. Ewes were tested for serological status at 4-mo intervals for up to 5.5 yr. The incidence of infection in ewes with diplotype "1,1" was 6.5% to 9.7% and significantly lower (P < 0.001) than ewes with diplotype "1,3" (60.5 to 97.3%) or "3,3" (64.0 to 91.4%). Furthermore, the incidence among ewes with diplotype "1,1" did not increase from 10 to 67 mo of age (P > 0.99), whereas the incidence among diplotype "1,3" and "3,3" ewes increased steadily until reaching an asymptote at approximately 52 mo of age. Total number and weight of lamb weaned per ewe exposed through 5.5 yr from ewes with diplotype "1,1" far exceeded (P ≤ 0.05) those with diplotypes "1,3" and "3,3" by, on average, 2.1 lambs and 40 kg, respectively. The present study confirmed that TMEM154 diplotype "1,1" animals have reduced incidence of OPPV infection and, correspondingly, improved productivity. In flocks with a high frequency of TMEM154 haplotype "3," selection for haplotype "1" appears to be a cost-effective approach to mitigate the impact of this economically important disease., (Published by Oxford University Press on behalf of the American Society of Animal Science 2021.)