Species-Specific Humoral Immune Responses in Sheep and Goats upon Small Ruminant Lentivirus Infections Inversely Correlate with Protection against Virus Replication and Pathological Lesions.

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

Expression analysis of lung miRNAs responding to ovine VM virus infection by RNA-seq.

BACKGROUND: MicroRNAs (miRNAs) are short endogenous, single-stranded, noncoding small RNA molecules of approximately 22 nucleotides in length. They regulate gene expression posttranscriptionally by silencing mRNA expression, thus orchestrating many physiological processes. The Small Ruminant Lentiviruses (SRLV) group includes the Visna Maedi Virus (VMV) and Caprine Arthritis Encephalitis (CAEV) viruses, which cause a disease in sheep and goats characterized by pneumonia, mastitis, arthritis and encephalitis. Their main target cells are from the monocyte/macrophage lineage. To date, there are no studies on the role of miRNAs in this viral disease. RESULTS: Using RNA-seq technology and bioinformatics analysis, the expression levels of miRNAs during different clinical stages of infection were studied. A total of 212 miRNAs were identified, of which 46 were conserved sequences in other species but found for the first time in sheep, and 12 were completely novel. Differential expression analysis comparing the uninfected and seropositive groups showed changes in several miRNAs; however, no significant differences were detected between seropositive asymptomatic and diseased sheep. The robust increase in the expression level of oar-miR-21 is consistent with its increased expression in other viral diseases. Furthermore, the target prediction of the dysregulated miRNAs revealed that they control genes involved in proliferation-related signalling pathways, such as the PI3K-Akt, AMPK and ErbB pathways. CONCLUSIONS: To the best of our knowledge, this is the first study reporting miRNA profiling in sheep in response to SRLV infection. The known functions of oar-miR-21 as a regulator of inflammation and proliferation appear to be a possible cause of the lesions caused in the sheep's lungs. This miRNA could be an indicator for the severity of the lung lesions, or a putative target for therapeutic intervention.

First survey on association of TMEM154 and CCR5 variants with serological maedi-visna status of sheep in German flocks.

Maedi-visna, a disease caused by small ruminant lentiviruses (SRLVs), is present in sheep from many countries, also including Germany. An amino acid substitution (E/K) at position 35 of the transmembrane protein 154 (TMEM154) as well as a deletion in the chemokine (C-C motif) receptor type 5 gene (CCR5) were reported to be associated with the serological MV status and/or the SRLV provirus concentration in North American sheep populations. The aim of this study was to test if those two gene variants might be useful markers for MV susceptibility in Germany. For this purpose, more than 500 sheep from 17 serologically MV positive German sheep flocks with different breed backgrounds were genotyped applying PCR-based methods. Both, crosstab and non-parametric analyses showed significant associations of the amino acid substitution at position 35 of TMEM154 with the serological MV status (cut-off-based classification) and the median MV ELISA S/P value in all samples and in two of the four analyzed breed subsets. The deletion in the CCR5 promoter did not show a consistent association with serological MV status or median ELISA S/P value. It can be concluded that the amino acid substitution at position 35 of TMEM154 is a promising marker for breeding towards a lower number of serologically MV positive sheep in German flocks, at least in flocks of the Texel breed, while this remains questionable for the deletion in the CCR5 promoter. The findings of this study still need to be verified in additional sheep breeds.

Seroprevalence and risk factors related to small ruminant lentivirus infections in Belgian sheep and goats.

Maedi-Visna virus (MVV) and caprine arthritis encephalitis virus (CAEV) are two prototype members of the group of small ruminant lentiviruses (SRLVs). Both result in progressive and persistent infections of sheep and goats that impact animal health and cause economic losses. In Belgium, the sheep and goat sector is small and consists mostly of hobbyist farmers keeping few animals. A voluntary control program however exists, but less than 2% of the farmers participate to the program. The current lack of SRLV seroprevalence data and knowledge on risk factors related to SRLV seropositivity in this hobbyist sector makes it difficult to evaluate the risk of SRLV transmission from non-certified to SRLV free certified farms. We performed a nationwide SRLV seroprevalence study based on a stratified sampling proportional to the number of sheep and goat holders per province. Randomly selected sheep and goat owners were invited to participate and subject to a short questionnaire to collect information about flock size, animal health condition, age, flock constitution and housing conditions. Samples were collected from maximum 7 animals per farm and tested in a commercial ELISA. In total, we received samples from 87 sheep and 76 goat farms. Sheep flocks showed an overall seroprevalence of 9% (CI 95%: 5-15) and a between-herd seroprevalence of 17% (CI 95%:11-27). Seroprevalence at animal level in goat flocks was 6% (CI 95%: 3-12) and the between-herd seroprevalence was 13% (CI 95%: 7-23). Multiple sheep and goat breeds were found SRLV seropositive. Answers provided during the questionnaire confirmed the mostly hobbyist nature of the sector and showed that more than 65% of sheep and goat farmers had never heard of the disease. The only risk factor found to be related to SRLV seroprevalence was flock size. Herds of more than 10 goats had significantly higher chance to harbor seropositive animals (OR: 4.36; CI: 1.07; 17.73). In conclusion, it was shown that participants to the SRLV free certification program are at risk for reintroduction of the disease in their herds since SRLVs are present on about 15%-20% of non-certified farms. Except from flock size, no clear risk factors were found that are helpfull to identify flocks at risk. Greater effort should be made to inform sheep and goat farmers about the existence and consequences of this disease in order to promote the voluntary control program and further reduce the disease prevalence.

Maedi-visna virus persistence: Antigenic variation and latency.

Maedi-visna virus (MVV), a lentivirus of sheep, shares with other lentiviruses the ability to establish a lifelong infection. In this study five sheep were infected intravenously with MVV and housed together with a number of uninfected sheep for natural transmission. All virus isolates from ten sheep that had been infected naturally had multiple mutations in the principal neutralization domain in Env and were antigenic variants, while three of four isolates from the carrier sheep had identical sequences to the infecting strain and were not antigenic variants. There was evidence of positive selection in the gene, particularly in amino acids comprising the neutralization epitope and some adjacent glycosylation sites. Together these results suggest that virus persistence is acquired by a reservoir of latent viruses, and that there is selection for antigenic variants of virus that is transmitted naturally.

Preparation of a cell line persistently infected with maedi/visna virus and production of viral antigens.

We attempted to prepare a cell line that produces maedi/visna virus (MVV) and is free of contamination by other viruses and mycoplasmas. Three cell lines, which originated from a sheep, goat and bat, were infected with MVV and passaged approximately every 5 days. The cultured cells were then subjected to polymerase chain reaction analysis for MVV provirus. As a result, a cell line persistently infected with MVV was established from ZZ-R cells, which originated from the fetal goat tongue. The 50-fold concentrated culture fluid formed a precipitation line against reference antiserum.

Risk factors associated with small ruminant lentivirus infection in eastern Poland sheep flocks.

An analysis of the risk factors for ovine lentivirus infection was performed in sheep flocks located throughout the central-eastern region of Poland. Here, we report the infection details for 98 flocks with a total of 6470 ewes, 15 sheep breeds. The identification of infected animals and a review of the epidemiological status of each flock were based on an evaluation of serological tests performed on collected blood serum samples. Blood for examination was obtained from 2925 ewes of the 98 flocks under observation. Specific antibodies for Maedi Visna Virus (MVV) were detected via ELISA. Data illustrating the conditions at each sheep farm were obtained through questionnaires completed by farmers, as well as observations, measurements, and breeding records that were available. These observations were used to assess risk factors contributing to small ruminant lentivirus (SRLV) infection in sheep flocks. It was found that both sheep flock size and the type of management system had a significant effect on the increased risk of lentiviral infection. In addition, we demonstrate that there is a significant (p<0.0001) relationship between the occurrence of mastitis (OR 2.01, CI: 1.55-2.61) and diarrhea (OR 4.22, CI: 3.30-5.39) with SRLV infection in the observed sheep. Additionally, the infection rate of the animals translated directly to an impaired physical condition. Notably, the risk of infection could potentially be reduced if sheep producers are further acquainted with SRLV detection and invoke a control program based on diagnostic tests. Moreover, marketing approval should be granted for solely SRLV-seronegative animals.

Genome-Wide Search for Host Association Factors during Ovine Progressive Pneumonia Virus Infection.

Ovine progressive pneumonia virus (OPPV) is an important virus that causes serious diseases in sheep and goats with a prevalence of 36% in the USA. Although OPPV was discovered more than half of a century ago, little is known about the infection and pathogenesis of this virus. In this report, we used RNA-seq technology to conduct a genome-wide probe for cellular factors that are associated with OPPV infection. A total of approximately 22,000 goat host genes were detected of which 657 were found to have been significantly up-regulated and 889 down-regulated at 12 hours post-infection. In addition to previously known restriction factors from other viral infections, a number of factors which may be specific for OPPV infection were uncovered. The data from this RNA-seq study will be helpful in our understanding of OPPV infection, and also for further study in the prevention and intervention of this viral disease.

Two mutations in the vif gene of maedi-visna virus have different phenotypes, indicating more than one function of Vif.

Like most other lentiviruses, maedi-visna virus (MVV) requires Vif for replication in natural target cells and in vivo. Here, we show that Vif-deficient MVV accumulates G-A mutations in the sequence context characteristic of ovine APOBEC3, consistent with a role of MVV Vif in neutralizing APOBEC3. We studied two point mutations in the vif gene of MVV. One was a tryptophan to arginine mutation that affects the interaction with APOBEC3 and caused G-A hypermutation. The other mutation was a proline to serine mutation that together with a mutation in the capsid protein caused attenuated replication in fetal ovine synovial (FOS) cells but not in sheep choroid plexus (SCP) cells. There was no hypermutation associated with this mutation. These results suggest that MVV Vif exerts more than one function and that there may be interaction between Vif and the capsid. The results also suggest the involvement of an unknown host factor in MVV Vif function.

Role of cullin-elonginB-elonginC E3 complex in bovine immunodeficiency virus and maedi-visna virus Vif-mediated degradation of host A3Z2-Z3 proteins.

BACKGROUND: All lentiviruses except equine infectious anemia virus (EIVA) antagonize antiviral family APOBEC3 (A3) proteins of the host through viral Vif proteins. The mechanism by which Vif of human, simian or feline immunodeficiency viruses (HIV/SIV/FIV) suppresses the corresponding host A3s has been studied extensively. RESULTS: Here, we determined that bovine immunodeficiency virus (BIV) and maedi-visna virus (MVV) Vif proteins utilize the Cullin (Cul)-ElonginB (EloB)-ElonginC (EloC) complex (BIV Vif recruits Cul2, while MVV Vif recruits Cul5) to degrade Bos taurus (bt)A3Z2-Z3 and Ovis aries (oa)A3Z2-Z3, respectively, via a proteasome-dependent but a CBF-ß-independent pathway. Mutation of the BC box in BIV and MVV Vif, C-terminal hydrophilic replacement of btEloC and oaEloC and dominant-negative mutants of btCul2 and oaCul5 could disrupt the activity of BIV and MVV Vif, respectively. While the membrane-permeable zinc chelator TPEN could block BIV Vif-mediated degradation of btA3Z2-Z3, it had minimal effects on oaA3Z2-Z3 degradation induced by MVV Vif, indicating that Zn is important for the activity of BIV Vif but not MVV Vif. Furthermore, we identified a previously unreported zinc binding loop [C-x1-C-x1-H-x19-C] in the BIV Vif upstream BC box which is critical for its degradation activity. CONCLUSIONS: A novel zinc binding loop was identified in the BIV Vif protein that is important for the E3 ubiquination activity, suggesting that the degradation of btA3Z2-Z3 by BIV and that of oaA3Z2-Z3 by MVV Vif may need host factors other than CBF-ß.

Immunogenetics of small ruminant lentiviral infections.

The small ruminant lentiviruses (SRLV) include the caprine arthritis encephalitis virus (CAEV) and the Maedi-Visna virus (MVV). Both of these viruses limit production and can be a major source of economic loss to producers. Little is known about how the immune system recognizes and responds to SRLVs, but due to similarities with the human immunodeficiency virus (HIV), HIV research can shed light on the possible immune mechanisms that control or lead to disease progression. This review will focus on the host immune response to HIV-1 and SRLV, and will discuss the possibility of breeding for enhanced SRLV disease resistance.

Successful Visna/maedi control in a highly infected ovine dairy flock using serologic segregation and management strategies.

A control system for Visna/maedi virus (VMV) infection based on serologic segregation and management strategies was applied in an infected Spanish dairy Manchega breed sheep flock (n=670) that was affected by a severe respiratory process associated to VMV. The control started in 2004 and consisted on the serological study of animals, segregation in two different flocks (seropositive and seronegative), separate management of flocks, selection of young female lambs for replacement only from seronegative ewes offspring, immediate removal of seropositive animals detected in the seronegative flock and a management tending toward the reduction and final culling of the seropositive flock. The serological control was repeated yearly or twice a year, approximately. Initial VMV seroprevalence of the undivided flock was 66.4% (January 2004) that descended to 47.3%, 12.8%, 2.2% and 0.2% between July 2004 and May 2006. Residual seroprevalence fluctuated slightly thereafter with a peak of 2.2% in April 2008. After segregation, number of animals in the seronegative flock was 378 that descended to 323 in October 2005. Since then, this number has increased steadily reaching 650 sheep in December 2011. The seropositive flock was progressively reduced by culling until its total disappearance in June 2010. This work presents the detailed results obtained in the control strategy against VMV in a single dairy sheep flock by implementing a segregation system based on serologic testing. The system is highly successful, as it reduces to residual levels VMV infection in about two years without the need of culling a high number of animals, as required by other methods. Moreover, the original size flock was been recovered within 8 years and has led to a subjective improvement of animal health and welfare in the flock. The residual seroprevalence could be eliminated at this stage by applying more sensitive molecular or other serological techniques to reach eradication.

Ovine TRIM5α can restrict visna/maedi virus.

The restrictive properties of tripartite motif-containing 5 alpha (TRIM5α) from small ruminant species have not been explored. Here, we identify highly similar TRIM5α sequences in sheep and goats. Cells transduced with ovine TRIM5α effectively restricted the lentivirus visna/maedi virus DNA synthesis. Proteasome inhibition in cells transduced with ovine TRIM5α restored restricted viral DNA synthesis, suggesting a conserved mechanism of restriction. Identification of TRIM5α active molecular species may open new prophylactic strategies against lentiviral infections.

Small ruminant lentiviruses: immunopathogenesis of visna-maedi and caprine arthritis and encephalitis virus.

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.

Seroprevalence and risk factors associated with Visna/Maedi virus in semi-intensive lamb-producing flocks in northwestern Spain.

A cross-sectional study was carried out to determine Visna/Maedi virus (VMV) seroprevalence and risk factors in semi-intensive lamb-producing flocks as a prelude to establishing a monitoring program in northwestern (NW) Spain. A total of 15,155 serum samples were taken from 78 commercial flocks and were submitted to an indirect VMV ELISA. Association between potential risk factors and seroprevalence at the flock level was assessed using a multivariable logistic regression model. A Generalized Estimating Equations (GEE) model and Exhaustive Chi-squared Automatic Interaction Detector (CHAID) were used to determine the seropositivity against VMV at the individual animal level. Individual seropositivity was 24.8% while 52.6% of the flocks examined had a true seroprevalence ≥1%. Flock size and introduction of new animals in the flock were significantly associated with seropositivity at the flock level. Flock size, sheep-goat contact, type of housing of lambs prior to weaning and age were significantly associated with individual VMV seropositivity. Confinement of lambs in preweaning lamb groups and high sheep-goat contact, regardless of the low number of goats per flock, were risk factors associated with individual VMV seropositivity, suggesting that these two factors are crucial for VMV control in semi-intensive lamb-producing flocks. These factors should be considered for developing more efficient strategies that will reduce the rate of VMV transmission.

Propagating and detecting an infectious molecular clone of maedi-visna virus that expresses green fluorescent protein.

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis. The primary target cells of MVV in vivo are considered to be of the monocyte lineage. Certain strains of MVV can replicate in other cell types, however. The green fluorescent protein is a commonly used marker for studying lentiviruses in living cells. We have nserted the egfp gene into the gene for dUTPase of MVV. The dUTPase gene is well conserved in most lentivirus strains of sheep and goats and has been shown to be important in replication of CAEV. However, dUTPase has been shown to be dispensable for replication of the molecular clone of MVV used in this study both in vitro and in vivo. MVV replication is strictly confined to cells of sheep or goat origin. We use a primary cell line from the choroid plexus of sheep (SCP cells) for transfection and propagation of the virus. The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages. There is good correlation between measurements of TCID50 and EGFP. This virus should therefore be useful for rapid detection of infected cells in studies of cell tropism and pathogenicity in vitro and in vivo.

Identification of the ovine mannose receptor and its possible role in Visna/Maedi virus infection.

This study aims to characterize the mannose receptor (MR) gene in sheep and its role in ovine visna/maedi virus (VMV) infection. The deduced amino acid sequence of ovine MR was compatible with a transmembrane protein having a cysteine-rich ricin-type amino-terminal region, a fibronectin type II repeat, eight tandem C-type lectin carbohydrate-recognition domains (CRD), a transmembrane region, and a cytoplasmic carboxy-terminal tail. The ovine and bovine MR sequences were closer to each other compared to human or swine MR. Concanavalin A (ConA) inhibited VMV productive infection, which was restored by mannan totally in ovine skin fibroblasts (OSF) and partially in blood monocyte-derived macrophages (BMDM), suggesting the involvement of mannosylated residues of the VMV ENV protein in the process. ConA impaired also syncytium formation in OSF transfected with an ENV-encoding pN3-plasmid. MR transcripts were found in two common SRLV targets, BMDM and synovial membrane (GSM) cells, but not in OSF. Viral infection of BMDM and especially GSM cells was inhibited by mannan, strongly suggesting that in these cells the MR is an important route of infection involving VMV Env mannosylated residues. Thus, at least three patterns of viral entry into SRLV-target cells can be proposed, involving mainly MR in GSM cells (target in SRLV-induced arthritis), MR in addition to an alternative route in BMDM (target in SRLV infections), and an alternative route excluding MR in OSF (target in cell culture). Different routes of SRLV infection may thus coexist related to the involvement of MR differential expression.

Diagnostic performance of ELISA and PCR in identifying SRLV-infected sheep and goats using serum, plasma and milk samples and in early detection of infection in dairy flocks through bulk milk testing.

The objective of the study was to evaluate the diagnostic performances of the ELITEST-MVV ELISA for detection of antibodies against small ruminant lentiviruses and of two recently published PCRs for the detection of proviral DNA of SRLV in blood and corresponding individual milk samples. In addition, the feasibility of bulk milk testing was investigated by titrating ELISA positive pooled milk samples in negative milk, and by investigating bulk milk samples by ELISA and PCR in relation to the SRLV-status of the flocks. The results show that plasma and milk are suitable replacements for serum. For sheep, both PCRs showed a better diagnostic performance than for goats. ELISA results for bulk milk samples were promising with a putative detection limit of <3% within-herd prevalence using 1/10 pre-diluted samples and even <1% within-herd prevalence when samples were tested undiluted. In a panel of 249 bulk milk samples, all samples from SRLV free flocks (n=138) tested negative in the ELISA, while 50% of the samples from flocks with an unknown SRLV-status (n=111) were positive. For a subset of 59 bulk milk samples, agreement between ELISA results and leader-gag PCR results was almost 100%. These results demonstrate the potential of bulk milk testing as a cost effective tool for early detection of infection in dairy flocks, which is essential for SRLV-monitoring programs.

Ovine progressive pneumonia provirus levels are unaffected by the prion 171R allele in an Idaho sheep flock.

Selective breeding of sheep for arginine (R) at prion gene (PRNP) codon 171 confers resistance to classical scrapie. However, other effects of 171R selection are uncertain. Ovine progressive pneumonia/Maedi-Visna virus (OPPV) may infect up to 66% of a flock thus any affect of 171R selection on OPPV susceptibility or disease progression could have major impact on the sheep industry. Hypotheses that the PRNP 171R allele is 1) associated with the presence of OPPV provirus and 2) associated with higher provirus levels were tested in an Idaho ewe flock. OPPV provirus was found in 226 of 358 ewes by quantitative PCR. The frequency of ewes with detectable provirus did not differ significantly among the 171QQ, 171QR, and 171RR genotypes (p > 0.05). Also, OPPV provirus levels in infected ewes were not significantly different among codon 171 genotypes (p > 0.05). These results show that, in the flock examined, the presence of OPPV provirus and provirus levels are not related to the PRNP 171R allele. Therefore, a genetic approach to scrapie control is not expected to increase or decrease the number of OPPV infected sheep or the progression of disease. This study provides further support to the adoption of PRNP 171R selection as a scrapie control measure.

Role of alveolar macrophages in respiratory transmission of visna/maedi virus.

A major route of transmission of Visna/maedi virus (VMV), an ovine lentivirus, is thought to be via the respiratory tract, by inhalation of either cell-free or cell-associated virus. In previous studies, we have shown that infection via the lower respiratory tract is much more efficient than via upper respiratory tissues (T. N. McNeilly, P. Tennant, L. Lujan, M. Perez, and G. D. Harkiss, J. Gen. Virol. 88:670-679, 2007). Alveolar macrophages (AMs) are prime candidates for the initial uptake of virus in the lower lung, given their in vivo tropism for VMV, abundant numbers, location within the airways, and role in VMV-induced inflammation. Furthermore, AMs are the most likely cell type involved in the transmission of cell-associated virus. In this study, we use an experimental in vivo infection model that allowed the infection of specific segments of the ovine lung. We demonstrate that resident AMs are capable of VMV uptake in vivo and that this infection is associated with a specific up-regulation of AM granulocyte-macrophage colony-stimulating factor mRNA expression (P < 0.05) and an increase in bronchoalveolar lymphocyte numbers (P < 0.05), but not a generalized inflammatory response 7 days postinfection. We also demonstrate that both autologous and heterologous VMV-infected AMs are capable of transmitting virus after lower, but not upper, respiratory tract instillation and that this transfer of virus appears not to involve the direct migration of virus-infected AMs from the airspace. These results suggest that virus is transferred from AMs into the body via an intermediate route. The results also suggest that the inhalation of infected AMs represents an additional mechanism of virus transmission.