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.

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.

An Immunodominant Region of the Envelope Glycoprotein of Small Ruminant Lentiviruses May Function as Decoy Antigen.

(1) Background: Small ruminant lentiviruses (SRLV) persist in infected goats that mount a strong humoral immune response characterized by low neutralizing titers. In this study, we characterized the antibody response to SU5, a variable, immunodominant epitope of the envelope glycoprotein of SRLV. We tested the working hypothesis that the variability of SU5 reflects escape from neutralizing antibody. (2) Methods: Affinity purified anti-SU5 antibody were tested for their neutralizing activity to the homologous lentivirus. Virus culture supernatant—in native form or following sonication and filtration—was used to test the ability of free envelope glycoproteins to compete for binding in a SU5-peptide-ELISA. (3) Results: Anti-SU5 antibodies are not neutralizing, strongly suggesting that they do not bind intact viral particles. In contrast, shed envelope glycoproteins efficiently compete for binding in a SU5-ELISA, providing convincing evidence that the SU5 epitope is exposed only on shed envelope glycoproteins. (4) Conclusions: Our results show that the antibody engaging SU5 is not neutralizing and does not appear to bind to SU expressed at the surface of virus particles. We propose that SU5 is a potential decoy epitope exposed on shaded envelope glycoproteins, luring the humoral immune response in committing an original antigenic sin to a functionally irrelevant epitope.

Blood and milk polymorphonuclear leukocyte and monocyte/macrophage functions in naturally caprine arthritis encephalitis virus infection in dairy goats.

The exact influence of caprine arthritis encephalitis virus (CAEV) infection on blood and milk polymorphonuclear leukocytes (PMNLs) and monocyte/macrophages of goats remains unclear. Thus, the present study sought to explore the blood and milk PMNL and monocyte/macrophage functions in naturally CAEV-infected goats. The present study used 18 healthy Saanen goats that were segregated according to sera test outcomes into serologically CAEV negative (n=8; 14 halves) and positive (n=10; 14 halves) groups. All milk samples from mammary halves with milk bacteriologically positive outcomes, somatic cell count ≥2×106cellsmL-1, and abnormal secretions in the strip cup test were excluded. We evaluated the percentage of blood and milk PMNLs and monocyte/macrophages, the viability of PMNLs and monocyte/macrophages, the levels of intracellular reactive oxygen species (ROS) and the nonopsonized phagocytosis of Staphylococcus aureus and Escherichia coli by flow cytometry. In the present study, a higher percentage of milk macrophages (CD14+) and milk polymorphonuclear leukocytes undergoing late apoptosis or necrosis (Annexin-V+/Propidium iodide+) was observed in CAEV-infected goats; we did not find any further alterations in blood and milk PMNL and monocyte/macrophage functions. Thus, regarding our results, the goats naturally infected with CAEV did not reveal pronounced dysfunctions in blood and milk polymorphonuclear leukocytes and monocytes/macrophages.

Impaired Expression of Cytokines as a Result of Viral Infections with an Emphasis on Small Ruminant Lentivirus Infection in Goats.

Knowing about the genes involved in immunity, and being able to identify the factors influencing their expressions, helps in gaining awareness of the immune processes. The qPCR method is a useful gene expression analysis tool, but studies on immune system genes are still limited, especially on the caprine immune system. Caprine arthritis encephalitis, a disease caused by small ruminant lentivirus (SRLV), causes economic losses in goat breeding, and there is no therapy against SRLV. The results of studies on vaccines against other viruses are promising. Moreover, the Marker-Assisted Selection strategy against SRLV is possible, as has been shown in sheep breeding. However, there are still many gaps in our knowledge on the caprine immune response to infection. All types of cytokines play pivotal roles in immunity, and SRLV infection influences the expression of many cytokines in different types of cells. This information encouraged the authors to examine the results of studies conducted on SRLV and other viral infections, with an emphasis on the expression of cytokine genes. This review attempts to summarize the results of studies on the expression of cytokines in the context of the SRLV infection.

Detailed analysis of the promoter activity of an attenuated lentivirus.

In spite of an eradication campaign that eliminated clinical cases of caprine arthritis encephalitis virus-induced arthritis in the Swiss goat population, seroconversions are still observed. In the affected flocks, viruses belonging mainly to the small ruminant lentivirus A4 subtype are regularly isolated. These viruses are considered attenuated, except in the mammary gland, where high viral loads and histopathological lesions have been observed. We previously characterized and sequenced such field isolates, detecting several potentially attenuating mutations in their LTR. Here we present a detailed analysis of the promoter activity of these genetic elements, which was comparable to those of virulent isolates. An AP-1 binding site was shown to be crucial for promoter activity in reporter gene assays and also in the context of a replicating molecular clone. Other sites, such as AML(vis) and a conserved E-box, appeared to be less crucial. Analysis of a unique AP-4 site showed a clear discrepancy between results obtained with reporter gene assays and those with mutated viruses. Within the limits of this in vitro study, we did not find evidence pointing to the LTR as the genetic correlate of attenuation for these viruses. Finally, the limited replication of SRLV A4 in mammary cell culture could not explain the suggested mammary tropism. In contrast, and in view of the abundance of macrophages in the mammary gland, it is the striking replication capacity of SRLV A4 in these cells, unaffected by all LTR mutations tested, which may explain the apparent mammary tropism of these viruses.

Use of serological diagnostic techniques in the control and eradication of caprine arthritis encephalitis: an update / Uso de técnicas sorológicas de diagnóstico no controle e erradicação da artrite encefalite caprina: atualização

Caprine arthritis encephalitis (CAE) is a chronic disease caused by a small ruminant lentivirus (SRLV), which causes significant losses in goat breeding. The actual state of animal infection with SRLV is difficult to determine due to a complex pathogenesis of the virus, including factors such as delayed or intermittent seroconversion in serological tests. Several serological techniques are available for disease diagnosis, such as screening or confirmation tests, which are different in sensitivity and specificity. Regarding the choice of the test to be applied, availability of commercial immunoreagents, team training, antigen used, and cost of techniques must be considered. This review presents the serological methods available for use in different stages of CAE control and eradication programs, and management measures to be adopted in conjunction with serological diagnosis of the disease...

A artrite encefalite caprina (CAE) é uma enfermidade crônica causada por um lentivírus de pequenos ruminantes (LVPR), que ocasiona perdas significativas na caprinocultura. O estado real da infecção animal pelo LVPR é de difícil determinação em virtude da complexa patogenia do vírus, incluindo fatores como soroconversão tardia ou intermitente em testes sorológicos. Para o diagnóstico da enfermidade, diversas técnicas sorológicas estão disponíveis, como testes de triagem ou confirmatórios, com variações na sensibilidade e especificidade. Para escolha do teste a ser usado, a disponibilidade de imunorreagentes comerciais, o treinamento da equipe, o antígeno utilizado, e o custo das técnicas devem ser considerados. Esta revisão apresenta os métodos sorológicos disponíveis para uso em diferentes fases dos programas de controle e erradicação da CAE e as medidas de manejo que devem ser adotadas em conjunto com o diagnóstico sorológico da enfermidade...

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.

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.

Monoclonal antibodies against Caprine arthritis-encephalitis virus epitopes in the p28 and p55(gag) viral proteins.

The genome of the Caprine Arthritis-Encephalitis Virus (CAEV) encodes the polycistronic precursor protein p55(gag). Proteolytic cleavage of p55(gag) generates the viral core proteins. Some studies suggest that the CAEV p55(gag) protein contains epitopes or antigenic determinants for these core proteins. This work reinforces this hypothesis and demonstrates that monoclonal antibodies (MAbs) that are directed against the capsid protein (p28) of CAEV are also reactive against the precursor p55(gag) protein and the intermediate cleavage products, p44, p36 and p22. The major activity of the MAbs was directed against p28. The MAbF12 binding site in p28 was found to be a linear epitope with a structure that is stable after SDS treatment and remains unaltered after ß-mercaptoethanol (ß-ME) treatment. The MAbF12 binding site in the p55(gag), p36 and p22 proteins was found to be a linear epitope with cross-linked sulphide bonds. In conclusion, these findings suggest that the p28 epitope is presented differently from the epitope in the polycistronic precursor protein p55(gag). The highly immunogenic p28 contains a linear epitope that is detergent-stable and is not altered by ß-ME treatment, whereas the p55(gag) epitope contains a linear epitope susceptible to denaturing agents.

Large-scale serological survey of caprine arthritis-encephalitis virus (CAEV) in Korean black goats (Capra hircus aegagrus).

A national serological survey of caprine arthritis-encephalitis virus (CAEV) infection was conducted using an enzyme-linked immunosorbent assay (ELISA) and an agar gel immunodiffusion (AGID) test. A total of 658 black goats of various breeds were sampled from 59 farms in three regions of Korea. The CAEV-positive goats were predominantly detected in the Southern region (n=17) as compared with the Northern (n=1) and Central regions (n=0) (χ(2)=6.26, P=0.044). Among 658 goats tested, 18 were positive in both ELISA and AGID, indicating a CAEV prevalence of 2.73% (95% confidence interval: 1.74-4.28). These results indicate that CAEV is present in Korean black goats.

Twelve-year cohort study on the influence of caprine arthritis-encephalitis virus infection on milk yield and composition.

This long-term observational cohort study was carried out to evaluate the effect of caprine arthritis-encephalitis virus (CAEV) infection on the quantitative and qualitative characteristics of milk production in dairy goats. For this purpose, a dairy herd comprising both CAEV-infected and uninfected female goats was observed for 12 consecutive years. Records on daily milk yield, somatic cell count (SCC), and contents of the major milk components (fat, protein and lactose) were collected every month. In total, 3,042 records (1,114 from CAEV-positive and 1,928 from CAEV-negative animals) from 177 female goats were used for statistical analysis. The multi-trait repeatability test-day animal model using the derivative-free multivariate analysis package with the average information-REML method was applied to eliminate the influence of factors other than CAEV infection on milk production in goats. The statistical significance of the differences between estimates for seropositive and seronegative goats was evaluated using Student's t-test. The effect of age of goats (parity) on their serological status was also estimated with the one-trait repeatability test-day model. The serological status of goats was linked to parity: the higher the parity, the greater the probability of CAEV infection. No significant differences between infected and uninfected goats with respect to daily milk yield and SCC were found. On the other hand, the milk of uninfected goats contained more total protein (3.40% vs. 3.35%), fat (3.69% vs. 3.54%), and lactose (4.30% vs. 4.25%) than the milk of infected goats. Even though these differences were highly significant, they were small when expressed numerically.

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.

Vaccinia and other viruses with available vaccines show marked homology with the HIV-1 envelope glycoprotein: the prospect of using existing vaccines to stem the AIDS pandemic.

Cross-reactive immunity occurs when infection with or vaccination against one virus protects against another related family member. A search for homologues of the HIV-1 envelope glycoprotein revealed that it is composed of thousands of intercalating and overlapping viral matches of pentapeptide or longer gapped consensi, belonging to over 70% of the currently sequenced virome, infecting all kingdoms from bacteria to man. It was also highly homologous to proteins from the Visna/Maedi and other ovine viruses, while other proteins (nef/tat/gag/pol) were homologous to proteins from the equine infectious anaemia virus and HTLV-2/HTLV-3 viruses. This phenomenon suggests that horizontal gene transfer from coinfecting RNA and DNA viruses to retroviruses is extensive, providing a route for the subsequent insertion of non-retroviral genes into human and other genomes via retroviral integration. This homology includes all viruses for which vaccines already exist. Cross-reactive immunity may be operative in AIDS, as Vaccinia vaccination decreases viral replication in HIV-1 infected patients' cells, for the CCR5 tropic form. Measles, Dengue virus, or GB virus C infections also decrease the HIV-1 viral load. A resumption of Vaccinia/smallpox vaccination might be expected to have a significant effect on the AIDS pandemic, and a careful study of the potential uses of other existing viral and bacterial vaccines merits close attention. This phenomenon may also be relevant to other recalcitrant viruses, bacteria, and parasites for which no vaccine exists and the armory of existing vaccines may have a role to play in diseases other than those for which they were designed.

Diagnostic performance of PCR and ELISA on blood and milk samples and serological survey for small ruminant lentiviruses in central Spain.

The diagnostic performance of an ELISA for the detection of antibodies to the small ruminant lentiviruses (SRLVs) maedi-visna virus and caprine arthritis-encephalitis virus in milk and corresponding blood samples was evaluated in 50 sheep. The agreement between ELISA results in blood and milk was 90 per cent, and the κ value was 0.79. In addition, a serological survey in the central zone of Spain was performed using milk samples from 413 animals (250 sheep and 163 goats) from 12 flocks/herds. All flocks/herds had some animals that were positive for SRLV. Among the animals, 60.0 per cent of the sheep and 8.0 per cent of the goats tested were seropositive. Each sample was also tested using a PCR technique, which increased the percentage of positive animals detected. Using a combination of ELISA and PCR gave a total of 72.2 per cent of sheep and 28.8 per cent of goats positive for SRLV.

Development of enzyme-linked immunosorbent assay for detection of antibody against Caprine arthritis encephalitis virus using recombinant protein of the precursor of the major core protein, p55gag.

An indirect enzyme-linked immunosorbent assay (ELISA) by using recombinant Caprine arthritis encephalitis virus (CAEV) p55gag antigen (rELISA), an indirect ELISA by using whole CAEV (wELISA), and Western blot analysis by using the recombinant p55gag antigen (rWB) were developed for detection of CAEV-specific antibodies in goats. Seven hundred and forty-five sera from goats were tested by rELISA, wELISA, rWB, and agar gel immunodiffusion test (AGID), and the results were compared with those of WB analysis by using the whole CAEV antigen (wWB). The AGID test and rWB had similar sensitivities of 93.3% (95% confidence interval [CI]) and 93% (95% CI), respectively, and similar specificities of 96.0% (95% CI) and 96.3% (95% CI), respectively, compared with wWB. The wELISA had substantially lower sensitivity (80.4%) and specificity (78.0%) compared with wWB, and rELISA had the lowest sensitivity (78.2%) and specificity (61.1%) compared with wWB. The lack of adequate sensitivity and specificity for rELISA and wELISA suggests that these assays need considerable modification. However, the results for rWB show that this assay has excellent agreement with wWB and that it can be used as a confirmatory test for the presence of anti-CAEV antibodies.

Avaliação de antígenos para o diagnóstico de lentivírus em rebanho caprino sob programa de controle / Antigen evaluation for the diagnosis of lentivirus in goat herds under and not under a control program

Este trabalho teve como objetivo comparar os resultados do diagnóstico do Lentivírus Caprino, por Imunodifusão em Gel de ágar - IDGA, utilizando o kit comercial americano e o kit nacional produzido com cepa CAEV Cork. Foram utilizados dois rebanhos, sendo um da Embrapa Caprinos submetido a doze anos de programa de controle e um outro rebanho infectado pelo CAEV, que não teve nenhuma ação prévia de controle. Analisando os resultados dos antígenos (nacional e americano) no rebanho não controlado, verificou-se que o antígeno comercial americano, quando foi utilizado pela primeira vez para o diagnóstico apresentou resultados mais significativos do que o nacional. Já no rebanho controlado, o antígeno nacional detectou um número maior de positivos. Analisando os dados do trabalho verificou-se a importância da alternância de proteínas imunogênicas presentes no antígeno dos kits de diagnóstico usados em programas de controle da Artrite Encefalite Caprina, haja vista a variação das respostas ao diagnóstico segundo a proteína expressa pelo vírus.

Caprine arthritis encephalitis is an infection caused by lentivirus and found on all the continents with a high prevalence in the more technified milk production flocks, causing considerable economic losses for goat production. The aim of this work was the comparison, by Agar gel immunodiffusion (AGID), between the diagnosis using a national test produced with the strain CAEV Cork and an American commercial kit in a controlled flock and in another flock without a control program for goat lentivirosis. The controlled flock had been under control for twelve years by Embrapa Goats, while the other flock was infected by CAEV and had not undergone any previous program of control. Analyzing the results of the antigens (national and American) in the uncontrolled flock, it was verified that when the antigen was used for the first time, the American commercial antigen showed more significative results than the national one. In the controlled flock the national antigen detected a higher number of cases. Analysis of the data revealed the importance of the diagnosis kits in caprine arthritis encephalitis control programs, as seen in the variation of the responses to the diagnosis according to the expressed protein for the virus.

Elimination of small ruminant lentivirus infection from sheep flocks and goat herds aided by health schemes in Great Britain.

Over a period of 11 years, 33 sheep or goat holdings lost their maedi-visna or caprine arthritis-encephalitis accredited status (mean 2.8 per year [0.09 per cent] of the accredited holdings in Great Britain). Of these, 22 sheep flocks and two goat herds eradicated the infection and regained their accredited status. In addition, 10 sheep flocks and two goat herds managed to eradicate infection, having joined the scheme with infected animals. In flocks and herds with a high initial prevalence of infection, the adoption of an indirect ELISA, with greater sensitivity than the agar gel immunodiffusion test, improved success rates. A strategy was devised to interpret the ELISA results depending upon the prevalence of infection at the time. Eighteen of the 33 flocks/herds (54.5 per cent) that had introductions of infection also owned non-accredited stock.