Transcriptome Analysis for Genes Associated with Small Ruminant Lentiviruses Infection in Goats of Carpathian Breed.

Small ruminant lentiviruses (SRLV) are economically important viral pathogens of sheep and goats. SRLV infection may interfere in the innate and adaptive immunity of the host, and genes associated with resistance or susceptibility to infection with SRLV have not been fully recognized. The presence of animals with relatively high and low proviral load suggests that some host factors are involved in the control of virus replication. To better understand the role of the genes involved in the host response to SRLV infection, RNA sequencing (RNA-seq) method was used to compare whole gene expression profiles in goats carrying both a high (HPL) and low (LPL) proviral load of SRLV and uninfected animals. Data enabled the identification of 1130 significant differentially expressed genes (DEGs) between control and LPL groups: 411 between control and HPL groups and 1434 DEGs between HPL and LPL groups. DEGs detected between the control group and groups with a proviral load were found to be significantly enriched in several gene ontology (GO) terms, including an integral component of membrane, extracellular region, response to growth factor, inflammatory and innate immune response, transmembrane signaling receptor activity, myeloid differentiation primary response gene 88 (MyD88)-dependent toll-like receptor signaling pathway as well as regulation of cytokine secretion. Our results also demonstrated significant deregulation of selected pathways in response to viral infection. The presence of SRLV proviral load in blood resulted in the modification of gene expression belonging to the toll-like receptor signaling pathway, the tumor necrosis factor (TNF) signaling pathway, the cytokine-cytokine receptor interaction, the phagosome, the Ras signaling pathway, the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) (PI3K-Akt) signaling pathway and rheumatoid arthritis. It is worth mentioning that the most predominant in all pathways were genes represented by toll-like receptors, tubulins, growth factors as well as interferon gamma receptors. DEGs detected between LPL and HPL groups were found to have significantly enriched regulation of signaling receptor activity, the response to toxic substances, nicotinamide adenine dinucleotide (NADH) dehydrogenase complex assembly, cytokine production, vesicle, and vacuole organization. In turn, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway tool classified DEGs that enrich molecular processes such as B and T-cell receptor signaling pathways, natural killer cell-mediated cytotoxicity, Fc gamma R-mediated phagocytosis, toll-like receptor signaling pathways, TNF, mammalian target of rapamycin (mTOR) signaling and forkhead box O (Foxo) signaling pathways, etc. Our data indicate that changes in SRLV proviral load induced altered expression of genes related to different biological processes such as immune response, inflammation, cell locomotion, and cytokine production. These findings provide significant insights into defense mechanisms against SRLV infection. Furthermore, these data can be useful to develop strategies against SRLV infection by selection of animals with reduced SRLV proviral concentration that may lead to a reduction in the spread of the virus.

Serological, Molecular and Culture-Based Diagnosis of Lentiviral Infections in Small Ruminants.

Small ruminant lentiviruses (SRLVs) infections lead to chronic diseases and remarkable economic losses undermining health and welfare of animals and the sustainability of farms. Early and definite diagnosis of SRLVs infections is the cornerstone for any control and eradication efforts; however, a "gold standard" test and/or diagnostic protocols with extensive applicability have yet to be developed. The main challenges preventing the development of a universally accepted diagnostic tool with sufficient sensitivity, specificity, and accuracy to be integrated in SRLVs control programs are the genetic variability of SRLVs associated with mutations, recombination, and cross-species transmission and the peculiarities of small ruminants' humoral immune response regarding late seroconversion, as well as intermittent and epitope-specific antibody production. The objectives of this review paper were to summarize the available serological and molecular assays for the diagnosis of SRLVs, to highlight their diagnostic performance emphasizing on advantages and drawbacks of their application, and to discuss current and future perspectives, challenges, limitations and impacts regarding the development of reliable and efficient tools for the diagnosis of SRLVs infections.

Caprine Arthritis Encephalitis Virus Is Associated with Renal Lesions.

Caprine arthritis encephalitis virus (CAEV) is a monocyte/macrophage-tropic lentivirus that primarily infects goats resulting in a well-recognized set of chronic inflammatory syndromes focused on the joint synovium, tissues of the central nervous system, pulmonary interstitium and mammary gland. Clinically affected animals generally manifest with one or more of these classic CAEV-associated tissue lesions; however, CAEV-associated renal inflammation in goats has not been reported in the peer-reviewed literature. Here we describe six goats with chronic, multisystemic CAEV infections in conjunction with CAEV-associated renal lesions. One of the animals had CAEV antigen-associated thrombotic arteritis resulting in infarction of both the kidney and heart. These goats had microscopic evidence of inflammatory renal injury (interstitial nephritis) with detectable renal immunolabeling for CAEV antigen in three of six animals and amplifiable proviral sequences consistent with CAEV in all six animals. Cardiac lesions (vascular, myocardial or endocardial) were also identified in four of six animals. Within the viral promoter (U3) region, known transcription factor binding sites (TFBSs) were generally conserved, although one viral isolate had a duplication of the U3 A region encoding a second gamma-activated site (GAS). Despite the TFBS conservation, the isolates demonstrated a degree of phylogenetic diversity. At present, the clinical consequence of CAEV-associated renal injury is not clear.

Characterization of Signal Sequences Determining the Nuclear/Nucleolar Import and Nuclear Export of the Caprine Arthritis-Encephalitis Virus Rev Protein.

Caprine arthritis-encephalitis virus (CAEV), a lentivirus, relies on the action of the Rev protein for its replication. The CAEV Rev fulfills its function by allowing the nuclear exportation of partially spliced or unspliced viral mRNAs. In this study, we characterized the nuclear and nucleolar localization signals (NLS and NoLS, respectively) and the nuclear export signal (NES) of the CAEV Rev protein. These signals are key actors in the nucleocytoplasmic shuttling of a lentiviral Rev protein. Several deletion and alanine substitution mutants were generated from a plasmid encoding the CAEV Rev wild-type protein that was fused to the enhanced green fluorescent protein (EGFP). Following cell transfection, images were captured by confocal microscopy and the fluorescence was quantified in the different cell compartments. The results showed that the NLS region is localized between amino acids (aa) 59 to 75, has a monopartite-like structure and is exclusively composed of arginine residues. The NoLS was found to be partially associated with the NLS. Finally, the CAEV Rev protein's NES mapped between aa 89 to 101, with an aa spacing between the hydrophobic residues that was found to be unconventional as compared to that of other retroviral Rev/Rev-like proteins.

The Vif protein of caprine arthritis encephalitis virus inhibits interferon production.

Caprine arthritis-encephalitis (CAE) is a chronic progressive infectious disease caused by caprine arthritis-encephalitis virus (CAEV) that seriously threatens the goat industry. Chronic infection and life-long multi-tissue inflammation are the typical features of the disease. Innate antiviral immunity is essential for the host defense system that rapidly recognizes and eliminates invading viruses. Interferon ß (IFN-ß) is important for innate immunity and regulates immunity against a broad spectrum of viruses. To investigate the details of the IFN-ß response to CAEV infection, the effects of six viral proteins and the molecular mechanisms by which they affect IFN-ß production were analyzed. Overexpression of DU and Vif promote virus proliferation and inhibit the production of IFN-ß. qRT-PCR and luciferase reporter assays showed that overexpression of Vif inhibits the expression of luciferase under the control of the ISRE, NF-κB or IFN-ß promoter but does not affect the expression of IFN-ß activated by IRF3, indicating that Vif negatively regulates IFN-ß production by affecting upstream signal transduction of IRF3. Amino acids 149-164 of Vif were found to be necessary for the inhibitory effect of IFN-ß production. Our results indicate that CAEV evades surveillance and clearance by intracellular innate immunity by downregulating IFN-ß production.

Development of a recombinase polymerase amplification lateral flow dipstick (RPA-LFD) for the field diagnosis of caprine arthritis-encephalitis virus (CAEV) infection.

Caprine arthritis-encephalitis (CAE) in goats is a complex disease syndrome caused by a lentivirus. This persistent viral infection results in arthritis in adult goats and encephalitis in lambs. The prognosis for the encephalitic form is normally poor, and this form of the disease has caused substantial economic losses for goat farmers. Hence, a more efficient detection platform based on recombinase polymerase amplification (RPA) and a lateral flow dipstick (LFD) was developed in the present study for detecting the proviral DNA of caprine arthritis-encephalitis virus (CAEV). Under the optimal incubation conditions, specifically, 30min at 37°C for RPA followed by 5min at room temperature for LFD, the assay was found to be sensitive to a lower limit of 80pg of total DNA and 10 copies of plasmid DNA. Furthermore, there was no cross-reaction with other tested viruses, including goat pox virus and bovine leukemia virus. Given its simplicity and portability, this RPA-LFD protocol can serve as an alternative tool to ELISA for the primary screening of CAEV, one that is suitable for both laboratory and field application. When the RPA-LFD was applied in parallel with serological ELISA for the detection of CAEV in field samples, the RPA-LFD assay exhibited a higher sensitivity than the traditional method, and 82% of the 200 samples collected in Taiwan were found to be positive. To our knowledge, this is the first report providing evidence to support the use of an RPA-LFD assay as a specific and sensitive platform for detecting CAEV proviral DNA in goats in a faster manner, one that is also applicable for on-site utilization at farms and that should be useful in both eradication programs and epidemiological studies.

Small ruminant lentiviral Vif proteins commonly utilize cyclophilin A, an evolutionarily and structurally conserved protein, to degrade ovine and caprine APOBEC3 proteins.

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.

Generation of a molecular clone of an attenuated lentivirus, a first step in understanding cytopathogenicity and virulence.

Small ruminant lentiviruses infect goats and sheep, inducing clinical disease in a minority of infected animals. Following an eradication campaign, clinical cases may disappear in a population. The complete elimination of these lentiviruses is however difficult to achieve and the spreading of less virulent strains often parallels the elimination of their virulent counterparts. Here, we characterized three such strains isolated from a flock in the post-eradication phase. We completely sequenced their genomes, showing that one of the isolates was most probably the product of a recombination event between the other two viruses. By comparing the sequences of these isolates with those of virulent strains, we found evidence that particular LTR mutations may explain their attenuated phenotype. Finally, we constructed an infectious molecular clone representative of these viruses, analyzing its replication characteristics in different target cells. This clone will permit us to explore the molecular correlates of cytopathogenicity and virulence.

Molecular characterization of the gag gene of caprine arthritis encephalitis virus from goats in the Philippines.

Caprine arthritis encephalitis virus (CAEV) causes caprine arthritis encephalitis syndrome, which is an emerging disease of goats in the Philippines. DNA sequence analysis showed homology of 86-93 % between Philippine CAEV and available CAEV sequences in GenBank. CAEV was detected using nested polymerase chain reaction (PCR), and new sets of primers were designed in order to amplify the gag gene, which is a highly conserved region of the viral genome. In addition, the Philippine CAEV isolate clustered in group B with the prototype caprine lentivirus. Based on amino acid sequence alignments, it is possible that the Philippine CAEV isolate is a new strain of CAEV, but it is also possible that it was already present in the country even before the start of goat importation. Molecular characterization of the CAEV gag gene is important for the development of a detection kit specific for the local strain of CAEV and the establishment of small ruminant lentivirus eradication programs in the Philippines. This study is the first report to describe the molecular characteristics of CAEV circulating in the Philippines.

Small ruminant lentivirus-induced arthritis: clinicopathologic findings in sheep infected by a highly replicative SRLV B2 genotype.

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.

Characterization of small ruminant lentivirus A4 subtype isolates and assessment of their pathogenic potential in naturally infected goats.

BACKGROUND: Small ruminant lentiviruses escaping efficient serological detection are still circulating in Swiss goats in spite of a long eradication campaign that essentially eliminated clinical cases of caprine arthritis encephalitis in the country. This strongly suggests that the circulating viruses are avirulent for goats.To test this hypothesis, we isolated circulating viruses from naturally infected animals and tested the in vitro and in vivo characteristics of these field isolates. METHODS: Viruses were isolated from primary macrophage cultures. The presence of lentiviruses in the culture supernatants was monitored by reverse transcriptase assay. Isolates were passaged in different cells and their cytopathogenic effects monitored by microscopy. Proviral load was quantified by real-time PCR using customized primer and probes. Statistical analysis comprised Analysis of Variance and Bonferroni Multiple Comparison Test. RESULTS: The isolated viruses belonged to the small ruminant lentiviruses A4 subtype that appears to be prominent in Switzerland. The 4 isolates replicated very efficiently in macrophages, displaying heterogeneous phenotypes, with two isolates showing a pronounced cytopathogenicity for these cells. By contrast, all 4 isolates had a poor replication capacity in goat and sheep fibroblasts. The proviral loads in the peripheral blood and, in particular, in the mammary gland were surprisingly high compared to previous observations. Nevertheless, these viruses appear to be of low virulence for goats except for the mammary gland were histopathological changes were observed. CONCLUSIONS: Small ruminant lentiviruses continue to circulate in Switzerland despite a long and expensive caprine arthritis encephalitis virus eradication campaign. We isolated 4 of these lentiviruses and confirmed their phylogenetic association with the prominent A4 subtype. The pathological and histopathological analysis of the infected animals supported the hypothesis that these A4 viruses are of low pathogenicity for goats, with, however, a caveat about the potentially detrimental effects on the mammary gland. Moreover, the high proviral load detected indicates that the immune system of the animals cannot control the infection and this, combined with the phenotypic plasticity observed in vitro, strongly argues in favour of a continuous and precise monitoring of these SRLV to avoid the risk of jeopardizing a long eradication campaign.

Caprine arthritis encephalitis virus detection in blood by loop-mediated isothermal amplification (LAMP) assay targeting the proviral gag region.

Caprine arthritis encephalitis virus (CAEV), of the genus Lentivirus of the Retroviridae family, causes persistent disease, which is characterized by polyarthritis and mastitis in adult goats and progressive paresis (leukoencephalomyelitis) in kids. A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of CAEV in blood samples. Species-specific primers amplifying the gag gene region in the provirus were used for the detection of CAEV. The LAMP assay result was obtained 30 min after incubation on a constant temperature at 63 °C in a heat block. Resulting amplicons were visualized by addition of SYBR green dye after the reaction and checked by agarose gel electrophoresis. The sensitivity of LAMP assay was evaluated by comparing the result with the nested polymerase chain reaction. Based on the experiments, the result of the assay indicated a rapid and sensitive test for the detection of CAEV.

Evolution of specific antibodies and proviral DNA in milk of small ruminants infected by small ruminant lentivirus.

The diagnosis of Small Ruminant Lentivirus (SRLV) is based on clinical signs, pathological lesions and laboratory testing. No standard reference test for the diagnosis of maedi visna has been validated up to the present, and it is puzzling that tests which detect antibodies against the virus and tests which detect the proviral genome may render opposite results. The aim of this study was to evaluate the presence in milk throughout a lactation period of specific antibodies by ELISA and of SRLV proviral DNA by a PCR of the highly conserved pol region. A six-month study was conducted with the milk of 28 ewes and 31 goats intensively reared. The percentage of animals with antibodies against SRLV increased throughout the study period. Seroprevalence in sheep was 28% at the beginning of the study and by the end it had increased up to 52.4%. In goats, initial seroprevalence of 5.6% increased to 16%. The percentage of PCR positive ewes was stable throughout the study period. Of the positive sheep, 21.4% were PCR-positive before antibodies could be detected and most of them became PCR-negative shortly after the first detection of antibodies. This might suggest that antibodies have a neutralizing effect. In addition, an equal percentage of sheep were always PCR-negative but either became ELISA-positive or was always ELISA-positive, which might support this hypothesis. On the other hand, the PCR results in goats did not follow any pattern and oscillated between 35.3% and 55.6% depending on the month. Most goats positive by PCR failed to develop antibodies in the 6 months tested. We may conclude that the infection and the antibody response to it follow a different trend in sheep and goats.

A polytropic caprine arthritis encephalitis virus promoter isolated from multiple tissues from a sheep with multisystemic lentivirus-associated inflammatory disease.

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.

Small ruminant lentiviruses (SRLVs) break the species barrier to acquire new host range.

Zoonotic events of simian immunodeficiency virus (SIV) from non-human primates to humans have generated the acquired immunodeficiency syndrome (AIDS), one of the most devastating infectious disease of the last century with more than 30 million people dead and about 40.3 million people currently infected worldwide. Human immunodeficiency virus (HIV-1 and HIV-2), the two major viruses that cause AIDS in humans are retroviruses of the lentivirus genus. The genus includes arthritis-encephalitis virus (CAEV) and Maedi-Visna virus (MVV), and a heterogeneous group of viruses known as small ruminant lentiviruses (SRLVs), affecting goat and sheep. Lentivirus genome integrates into the host DNA, causing persistent infection associated with a remarkable diversity during viral replication. Direct evidence of mixed infections with these two closely related SRLVs was found in both sheep and goats. The evidence of a genetic continuum with caprine and ovine field isolates demonstrates the absence of an efficient species barrier preventing cross-species transmission. In dual-infected animals, persistent infections with both CAEV and MVV have been described, and viral chimeras have been detected. This not only complicates animal trade between countries but favors the risk that highly pathogenic variants may emerge as has already been observed in the past in Iceland and, more recently, in outbreaks with virulent strains in Spain. SRLVs affecting wildlife have already been identified, demonstrating the existence of emergent viruses adapted to new hosts. Viruses adapted to wildlife ruminants may acquire novel biopathological properties which may endanger not only the new host species but also domestic ruminants and humans. SRLVs infecting sheep and goats follow a genomic evolution similar to that observed in HIV or in other lentiviruses. Lentivirus genetic diversity and host factors leading to the establishment of naturally occurring virulent versus avirulent infections, in addition to the emergence of new strains, challenge every aspect of SRLV control measures for providing efficient tools to prevent the transmission of diseases between wild ungulates and livestock.

Development of TaqMan-based qPCR method for detection of caprine arthritis-encephalitis virus (CAEV) infection.

A specific and sensitive two-step TaqMan real-time PCR has been developed for rapid diagnosis of caprine arthritis-encephalitis virus (CAEV) infection by using a set of specific primers and a TaqMan probe targeting a highly conserved region within the gene encoding the viral capsid protein (CA). The assay successfully detected CAEV proviral DNA in total DNA extracts originating from cell culture, whole blood samples and isolated PBMCs, with a lower detection limit of 10(2) copies and a linear dynamic range of 10(5) to 10(10) copies/ml. There was no cross-reaction with other animal viruses (e.g., goat pox virus, bovine leukemia virus, bovine mucosal disease virus, swine influenza virus and Nipah virus). When applied in parallel with serological AGID and conventional PCR for detection of CAEV in field samples, this assay exhibited a higher sensitivity than these traditional methods, and 7.8 % of the 308 specimens collected in the Shanxi and Tianjin regions of China from 1993 to 2011 were found to be positive. Thus, the TaqMan qPCR assay provides a fast, specific and sensitive means for detecting CAEV proviral DNA in goat specimens and should be useful for large-scale detection in eradication programs and epidemiological studies.

Caprine arthritis encephalitis virus (CAEV) replicates productively in cultured epididymal cells from goats.

The transmission of CAEV from male goats has not been well studied and the target cells that support viral replication are not well characterized. Epididymal epithelial cells (EECs) are important and play a key role in the fertility and motility of spermatozoa. During their transit, spermatozoa incorporate several EEC-produced proteins into their plasma membranes to stabilize them and prevent premature acrosomal reaction. This intimate interaction between spermatozoa and EECs may increase the likelihood of the infection of semen with CAEV if epididymal tissue is productively infected and sheds the virus into the duct. The aim of this study was to examine whether goat EECs are susceptible to CAEV infection in tissue culture. Cells were isolated from epididymides obtained from goats that were sampled from a certified-CAEV-free herd. Cultured cells were then inoculated with a molecularly-cloned isolate of CAEV (CAEV-pBSCA). Inoculated cells developed cytopathic effects (CPE), showing numerous multinucleated giant cells (MGC) in cell-culture monolayers. Expression of CAEV proteins was detected by immunofluorescence using an anti-p28, Gag-specific antibody. The culture medium of inoculated cells was shown to contain high titers (10(6) tissue culture infectious doses 50 per ml (TCID50/ml)) of infectious, cytopathic virus when assayed using indicator goat synovial membrane (GSM) cells. Our findings clearly demonstrate that cells of the buck genital tract are targets of CAEV and are thus a potential reservoir that sheds infectious CAEV into the semen of infected animals. These data suggest the use of sperm from CAEV-free goat males for artificial insemination in genetic selection programs to minimize CAEV dissemination.

An insight into a combination of ELISA strategies to diagnose small ruminant lentivirus infections.

A single broadly reactive standard ELISA is commonly applied to control small ruminant lentivirus (SRLV) spread, but type specific ELISA strategies are gaining interest in areas with highly prevalent and heterogeneous SRLV infections. Short (15-residue) synthetic peptides (n=60) were designed in this study using deduced amino acid sequence profiles of SRLV circulating in sheep from North Central Spain and SRLV described previously. The corresponding ELISAs and two standard ELISAs were employed to analyze sera from sheep flocks either controlled or infected with different SRLV genotypes. Two outbreaks, showing SRLV-induced arthritis (genotype B2) and encephalitis (genotype A), were represented among the infected flocks. The ELISA results revealed that none of the assays detected all the infected animals in the global population analyzed, the assay performance varying according to the genetic type of the strain circulating in the area and the test antigen. Five of the six highly reactive (57-62%) single peptide ELISAs were further assessed, revealing that the ELISA based on peptide 98M (type A ENV-SU5, consensus from the neurological outbreak) detected positives in the majority of the type-A specific sera tested (Se: 86%; Sp: 98%) and not in the arthritic type B outbreak. ENV-TM ELISAs based on peptides 126M1 (Se: 82%; Sp: 95%) and 126M2 0,65 0.77 (Se: 68%; Sp: 88%) detected preferentially caprine arthritis encephalitis (CAEV, type B) and visna/maedi (VMV, type A) virus infections respectively, which may help to perform a preliminary CAEV vs. VMV-like typing of the flock. The use of particular peptide ELISAs and standard tests individually or combined may be useful in the different areas under study, to determine disease progression, diagnose/type infection and prevent its spread.