A polytropic caprine arthritis encephalitis virus promoter isolated from multiple tissues from a sheep with multisystemic lentivirus-associated inflammatory disease.
ABSTRACT: Caprine arthritis encephalitis virus (CAEV) is a lentivirus that infects both goats and sheep and is closely related to maedi-visna virus that infects sheep; collectively, these viruses are known as small ruminant lentiviruses (SRLV). Infection of goats and sheep with SRLV typically results in discrete inflammatory diseases which include arthritis, mastitis, pneumonia or encephalomyelitis. SRLV-infected animals concurrently demonstrating lentivirus-associated lesions in tissues of lung, mammary gland, joint synovium and the central nervous system are either very rare or have not been reported. Here we describe a novel CAEV promoter isolated from a sheep with multisystemic lentivirus-associated inflammatory disease including interstitial pneumonia, mastitis, polyarthritis and leukomyelitis. A single, novel SRLV promoter was cloned and sequenced from five different anatomical locations (brain stem, spinal cord, lung, mammary gland and carpal joint synovium), all of which demonstrated lesions characteristic of lentivirus associated inflammation. This SRLV promoter isolate was found to be closely related to CAEV promoters isolated from goats in northern California and other parts of the world. The promoter was denoted CAEV-ovine-MS (multisystemic disease); the stability of the transcription factor binding sites within the U3 promoter sequence are discussed.
Project description:BACKGROUND: Small Ruminant Lentiviruses (SRLV) are widespread in Canadian sheep and goats and represent an important health issue in these animals. There is however no data about the genetic diversity of Caprine Arthritis Encephalitis Virus (CAEV) or Maedi Visna Virus (MVV) in this country. FINDINGS: We performed a molecular and phylogenetic analysis of sheep and goat lentiviruses from a small geographic area in Canada using long sequences from the gag region of 30 infected sheep and 36 infected goats originating from 14 different flocks. Pairwise DNA distance and phylogenetic analyses revealed that all SRLV sequences obtained from sheep clustered tightly with prototypical Maedi visna sequences from America. Similarly, all SRLV strains obtained from goats clustered tightly with prototypical US CAEV-Cork strain. CONCLUSIONS: The data reported in this study suggests that Canadian and US SRLV strains share common origins. In addition, the molecular data failed to bring to light any evidence of past cross species transmission between sheep and goats, which is consistent with the type of farming practiced in this part of the country where single species flocks predominate and where opportunities of cross species transmissions are proportionately low.
Project description:This study was conducted in order to evaluate the transmission of caprine lentivirus to sheep using different experimental groups. The first one (colostrum group) was formed by nine lambs receiving colostrum from goats positive for small ruminant lentiviruses (SRLV). The second group (milk group) was established by nine lambs that received milk of these goats. Third was a control group, consisting of lambs that suckled colostrum and milk of negative mothers. Another experimental group (contact group) was formed by eight adult sheep, confined with two naturally infected goats. The groups were monitored by immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA), agar gel immunodiffusion (AGID) and nested polymerase chain reaction (nPCR). All lambs that suckled colostrum and milk of infected goats and six sheep of the contact group had positive results in the nPCR, although seroconversion was detected only in three of the exposed animals, with no clinical lentiviruses manifestation, in 720 days of observation. There was a close relationship between viral sequences obtained from infected animals and the prototype CAEV-Cork. Thus, it was concluded that SRLV can be transmitted from goats to sheep, however, the degree of adaptation of the virus strain to the host species probably interferes with the infection persistence and seroconversion rate.
Project description:BACKGROUND: Small ruminant lentiviruses (SRLV) are members of the Retroviridae family and infect goats and sheep worldwide. Detection of specific antibodies using AGID and ELISA is the most commonly used means of diagnosing SRLV infection. The most frequent molecular method for detecting the provirus genome is PCR, using peripheral blood leucocytes as target cells. Real time PCR has also recently been used. The aim of this study was to develop a real time PCR for detection of SRLV in order to improve molecular diagnostics of SRLV infections in sheep and goats. RESULTS: Two new real time PCR assays using TaqMan probes for the specific detection of genotype A (MVV assay) and genoptype B (CAEV assay) SRLV strains and differentiation between them were developed and validated at both analytical and diagnostic levels following MIQE guidelines. The validation results showed that the new real time PCR is 100% specific, with a reliable limit of detection of 26 (CAEV assay) and 72 (MVV assay) plasmid DNA copies, while compared to ELISA the diagnostic sensitivity of both assays was 79% when tested with Slovenian SRLV field samples. Intra-assay and inter-assay coefficients of variation showed overall good repeatability and reproducibility of the new real time PCR assays, except for the highest dilutions. CONCLUSIONS: Two new TaqMan probe based real time PCR assays for the specific detection of genotype A and B SRLV strains and differentiation between them were developed and validated. They can serve as an additional tool for confirming infection with SRLV and may also be useful for early detection of infected animals prior to seroconversion.
Project description:The risk of classical scrapie transmission in small ruminants is highest during the neonatal period with the placenta recognized as a significant source of infection. Milk has also been identified as a source of scrapie with sheep-to-sheep transmission occurring after neonatal consumption of as little as 1-2 liters of milk; concurrent mastitis due to small ruminant lentivirus (SRLV) infection may be associated with increased scrapie transmission via milk in sheep. In contrast, goat-to-sheep transmission has been documented only after prolonged consumption of >30 liters of milk. The goal of the current study was to assess transmission of scrapie to goat kids and lambs following low volume, short duration consumption of milk from infected goats. Milk from two does (female goats) with pre-clinical scrapie was fed to four goat kids (?4.5 L each) and four lambs (~3.7 L each) beginning ~24 hours after birth. Scrapie transmission was detected in three sheep as early as 18 months post inoculation; transmission was also detected in two goats but not until postmortem analyses at 33 months post inoculation. Each milk donor goat also had naturally-acquired infection with SRLV. Different degrees of lymphohistiocytic inflammation and PrPSc accumulation were observed in mammary gland tissues of the donors, which appeared to associate with transmission of scrapie via milk. Thus, similar to the risks of milk transmission of scrapie from sheep, even limited exposure to milk from goats can pose significant risk for scrapie transmission to both goat kids and lambs.
Project description:Small ruminant lentivirus (SRLV), also called ovine progressive pneumonia virus or maedi-visna, is present in 24% of US sheep. Like human immunodeficiency virus, SRLV is a macrophage-tropic lentivirus that causes lifelong infection. The production impacts from SRLV are due to a range of disease symptoms, including pneumonia, arthritis, mastitis, body condition wasting and encephalitis. There is no cure and no effective vaccine for preventing SRLV infection. However, breed differences in prevalence and proviral concentration indicate a genetic basis for susceptibility to SRLV. Animals with high blood proviral concentration show increased tissue lesion severity, so proviral concentration represents a live animal test for control post-infection in terms of proviral replication and disease severity. Recently, it was found that sheep with two copies of TMEM154 haplotype 1 (encoding lysine at position 35) had lower odds of SRLV infection. In this study, we examined the relationship between SRLV control post-infection and variants in two genes, TMEM154 and CCR5, in four flocks containing 1403 SRLV-positive sheep. We found two copies of TMEM154 haplotype 1 were associated with lower SRLV proviral concentration in one flock (P < 0.02). This identified the same favorable diplotype for SRLV control post-infection as for odds of infection. However, frequencies of haplotypes 2 and 3 were too low in the other three flocks to test. The CCR5 promoter deletion did not have consistent association with SRLV proviral concentration. Future work in flocks with more balanced allele frequencies is needed to confirm or refute TMEM154 association with control of SRLV post-infection.
Project description:BACKGROUND:The small ruminant lentiviruses (SRLVs) are a heterogeneous group of viruses that includes caprine arthritis encephalitis virus (CAEV) and Maedi-Visna virus (MVV). SRLVs affect the production and welfare of sheep and goats worldwide. There is currently no effective treatment. Their high mutation rate precludes vaccine development, making innovative control measures necessary. A variant of the chemokine (C-C motif) receptor 5 (CCR5) gene is reportedly involved in resistance to human immunodeficiency (HIV) infection in humans and to SRLV in sheep. The aim of this study was to analyse the genetic structure and variability of the CCR5 gene in goats and to carry out a cross-sectional study to investigate the role of CCR5 genetic variants in controlling susceptibility/resistance to CAEV. RESULTS:The variant g.1059?T located in the promoter region revealed an interesting association with high proviral loads (a 2.8-fold increased risk). A possible explanation could be an alteration of the transcriptional level. Overexpression of the CCR5 receptor on the cell surface may increase virus internalization and proviral load as a consequence. CONCLUSIONS:Our findings could be advantageously used to reduce the susceptibility of goat herds to CAEV by negatively selecting animals carrying the g.1059?T mutation. Eliminating animals predisposed to high proviral loads could also limit the development of clinical signs and the spread of the virus, since these animals are also highly efficient in shedding the virus.
Project description:Small ruminant lentiviruses (SRLV) have high genetic variability which results in different viral strains around the world. This create a challenge to design sensible primers for molecular diagnosis in different regions. This work proposes a protocol of duplex nested-PCR for the precise diagnosis of SRLV. The technique was designed and tested with the control strains CAEV Co and MVV 1514. Then, field strains were submitted to the same protocol of duplex nested-PCR. Blood samples of sheep and goats were tested with AGID and nested PCR with specific primers for pol, gag and LTR. The AGID results showed low detection capacity of positive animals, while the nested PCR demonstrated a greater capacity of virus detection. Results demonstrated that LTR-PCR was more efficient in detecting positive sheep samples, whereas gag-PCR allowed a good detection of samples of positive goats and positive sheep. In addition, pol-PCR was more efficient with goat samples than for sheep. Duplex nested PCR performed with standard virus samples and field strains demonstrated that the technique is more efficient for the detection of multiple pro-viral DNA sequences. This study demonstrated a successful duplex nested PCR assay allowing a more accurate diagnosis of SRLV.
Project description:Small-ruminant lentivirus (SRLV) infections are widespread in Poland, and circulation of subtypes A1, A12, A13, A16, A17, B1 and B2 has been documented. The aim of this study was to characterize the SRLV strains circulating in sheep and goats in mixed flocks in the Malopolska region, where the highest seroprevalence has been detected. Phylogenetic analysis revealed that most of the isolates from sheep belonged to subtype A13, suggesting that this subtype may be predominant in the Malopolska region. Furthermore, the existence of a new subtype, tentatively designated as A18, was described for the first time. This work extends the current knowledge on the distribution of SRLV subtypes in sheep and goats in Poland and provides further information on the genetic diversity of SRLV. The new data are important for both epidemiological studies and eradication programs and provide insight into the evolution of SRLV.
Project description: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.
Project description:Small ruminant lentivirus (SRLV) infections are widespread in Poland and circulation of subtypes A1, A12, A13, B1 and B2 was detected. The present work aimed at extending previous study based on the analysis of a larger number of animals from single-species flocks. Animals were selected for genetic analysis based on serological reactivity towards a range of recombinant antigens derived from Gag and Env viral proteins. Phylogenetic analysis revealed the existence of subtypes B2 and A12 in both goats and sheep and subtypes A1 and B1 in goats only. In addition, two novel subtypes, A16 and A17, were found in goats. Co-infections with strains belonging to different subtypes within A and B groups were detected in 1 sheep and 4 goats originating from four flocks. Although the reactivity of serum samples towards the recombinant antigens confirmed immunological relatedness between Gag epitopes of different subtypes and the cross-reactive nature of Gag antibodies, eleven serum samples failed to react with antigens representing all subtypes detected up-to-date in Poland, highlighting the limitations of the serological diagnosis. These data showed the complex nature of SRLV subtypes circulating in sheep and goats in Poland and the need for improving SRLV-related diagnostic capacity.