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.

Epidemiology and control of maedi-visna virus: Curing the flock.

Maedi-visna (MV) is a complex lentiviral disease syndrome characterised by long immunological and clinical latencies and chronic progressive inflammatory pathology. Incurable at the individual level, it is widespread in most sheep-keeping countries, and is a cause of lost production and poor animal welfare. Culling seropositive animals is the main means of control, but it might be possible to manage virus transmission effectively if its epidemiology was better quantified. We derive a mathematical epidemiological model of the temporal distributions of seroconversion probabilities and estimate susceptibility, transmission rate and latencies in three serological datasets. We demonstrate the existence of epidemiological latency, which has not explicitly been recognised in the SRLV literaure. This time delay between infection and infectiousness apparently exceeds the delay between infection and seroconversion. Poor body condition was associated with more rapid seroconversion, but not with a higher probability of infection. We estimate transmission rates amongst housed sheep to be at about 1,000 times faster than when sheep were at grass, when transmission was negligible. Maternal transmission has only a small role in transmission, because lambs from infected ewes have a low probability of being infected directly by them, and only a small proportion of lambs need be retained to maintain flock size. Our results show that MV is overwhelmingly a disease of housing, where sheep are kept in close proximity. Prevalence of MV is likely to double each year from an initial low incidence in housed flocks penned in typically-sized groups of sheep (c. 50) for even a few days per year. Ewes kept entirely at grass are unlikely to experience transmission frequently enough for MV to persist, and pre-existing infection should die out as older ewes are replaced, thereby essentially curing the flock.

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.

Prevalence of Maedi-visna in Saskatchewan sheep.

A study was conducted to estimate flock and individual seroprevalence of Maedi-visna in Saskatchewan and evaluate risk factors for seropositive flocks. Thirty-five percent (24/68) of flocks and 4.6% (93/2010) of individual samples were positive. Within-flock prevalence ranged from 3.3% to 96.7%. Significant flock-level predictors of flock prevalence included large flock size, purchasing > 50 sheep and respiratory problems in the previous 5 years.

Prévalence de maedi-visna chez des moutons de la Saskatchewan. Une étude a été réalisée pour estimer la séroprévalence individuelle et dans le troupeau de maedi-visna en Saskatchewan et évaluer les facteurs de risque des troupeaux séropositifs. Trente-cinq pour cent (24/68) des échantillons des troupeaux et 4,6 % (93/2010) des échantillons individuels étaient positifs. La prévalence dans le troupeau variait de 3,3 % à 96,7 %. Les prédicteurs importants au niveau du troupeau incluaient une taille importante du troupeau, l'achat de > 50 moutons et des problèmes respiratoires au cours des cinq années antérieures.(Traduit par Isabelle Vallières).

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.

DNA Vaccines Against Maedi-Visna Virus.

Maedi-visna virus (MVV) is an ovine retrovirus of the Lentivirus genus, responsible for a chronic and progressive disease of sheep with a high prevalence all over the world. Therefore, measures aiming at the control of MVV infection are necessary, and the development of DNA vaccines may be the ideal approach. A DNA vaccine is an antigen-encoding bacterial plasmid designed to mimic infections safely, with ability to generate both humoral and cellular long-lasting immune responses once it is delivered to the host.Here, we describe the development and evaluation of DNA vaccines against ovine maedi-visna virus. The first step is the design of the vaccines, including the choice of the backbone vector and the nucleotide sequences to use as antigen-encoding sequences. Once constructed, the vaccines may be produced with high quality for use in in vitro and in vivo tests. In vitro assays are performed through transfection of animal cells to confirm the expression of the protein, while in vivo tests are carried out by mouse and/or sheep immunization in order to check humoral and cellular responses to the vaccines and conclude about their efficiency. Several approaches may be later performed in order to enhance the effectiveness of the vaccines, such as the introduction of targeting sequences, the use of a prime-boost strategy, the administration of a combined vaccine, and the use of liposomes as delivery vehicle.

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.

Host restriction of lentiviruses and viral countermeasures: APOBEC3 and Vif.

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

Expression analysis of 13 ovine immune response candidate genes in Visna/Maedi disease progression.

Visna/Maedi virus (VMV) is a lentivirus that infects cells of the monocyte/macrophage lineage in sheep. Infection with VMV may lead to Visna/Maedi (VM) disease, which causes a multisystemic inflammatory disorder causing pneumonia, encephalitis, mastitis and arthritis. The role of ovine immune response genes in the development of VM disease is not fully understood. In this work, sheep of the Rasa Aragonesa breed were divided into two groups depending on the presence/absence of VM-characteristic clinical lesions in the aforementioned organs and the relative levels of candidate gene expression, including cytokines and innate immunity loci were measured by qPCR in the lung and udder. Sheep with lung lesions showed differential expression in five target genes: CCR5, TLR7, and TLR8 were up regulated and IL2 and TNFα down regulated. TNFα up regulation was detected in the udder.

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.

Vaccination delays Maedi-Visna lentivirus infection in a naturally-infected sheep flock.

BACKGROUND: The Maedi-Visna (MV) lentivirus causes two slowly progressive eventually fatal diseases of sheep, Maedi, a progressive interstitial pneumonia, and Visna, a progressive demyelinating disease of the central nervous system. Other lentiviruses also cause fatal slow infections in their natural hosts, e.g. the HIV virus in humans. Results of experimental vaccination against any lentivirus where vaccinees are challenged by natural routes, may therefore be of general interest. From 1991-1998 experiments with formalin-inactivated whole Maedi-Visna virus vaccine were carried out in the Department of Microbiology at the University of Iceland. Western Blot tests showed good immune response to all major proteins of the virus. When aluminium hydroxide was added to the vaccine all vaccinees developed neutralizing antibodies to the vaccine strain at titers 1/8 - 1/256. After housing 5 twin pairs, one twin in each pair vaccinated, the other unvaccinated, with infected sheep for 4 years, all the unvaccinated twins became infected, but only 2 of their vaccinated siblings as confirmed by virus cultivation experiments on tissues from their lungs spleens lymph nodes and choroid plexuses. RESULTS: One twin in each of 40 female twin pairs, born into a Maedi-Visna-infected sheep flock and kept under natural farming conditions in Cyprus, was vaccinated at birth, 3 weeks and 3 months, with formalin-inactivated whole Maedi-Visna lentivirus vaccine adjuvanted with aluminium hydroxide. 17 mothers of the twins were seronegative, 13 seroconverting and 10 had old infection. Of 17 vaccinees born to seronegative mothers 9 were uninfected at 28 months, but only 2 of their unvaccinated siblings. Of 13 unvaccinated twins born to seroconverting mothers, 12 caught infection during their first 10 weeks, but only 4 of their vaccinated siblings. Vaccination had no effects on 10 vaccinees born to mothers with long-standing Maedi-Visna infections and broad andibody response at birth of their lambs. CONCLUSION: Compared with their unvaccinated siblings, natural infection was delayed in significant number of vaccinated twins born by seronegative and seroconverting mothers and vaccinated at birth, 3 weeks and 3 months with formalin inactivated whole MV vaccine adjuvanted with aluminium hydroxide. Maternal antibodies interfered with vaccination so early in life if the mother had old infection.

First molecular characterization of visna/maedi viruses from naturally infected sheep in Turkey.

Recent worldwide serological and genetic studies of small ruminant lentiviruses (SRLV) have led to the description of new genotypes and the development of new diagnostic tests. This study investigated the detection and molecular characterization of visna/maedi virus (VMV) infection in serum and blood samples from pure and mixed sheep breeds acquired from different regions in Turkey using ELISA and PCR techniques. The prevalence of VMV was 67.8 % by ELISA and/or LTR-PCR with both assays showing a medium level of agreement (kappa: 0.26; ± 0.038 CI). Positivity of VMV in sheep increased according to the age of the animal, although PCR positivity was higher than ELISA in young individuals. Phylogenetic analysis of 33 LTR sequences identified two distinct clades that were closely related to American and Greek LTR sequences. Phylogenetic analysis of 10 partial gag gene sequences identified A2, A3, A5, A9, A11 subtypes of genotype A SRLVs. In vitro culture of all isolates in fetal sheep lung cells (FSLC) showed a slow/low phenotype causing less or no lytic infection compared with infection with the WLC-1 American strain characterized by a rapid/highly lytic phenotype. Phylogenetic analysis revealed that Turkish VMV sequences preceded the establishment of American or Greek strains that were associated with the migration of sheep from the Middle East to Western Europe several centuries ago. This is the first study that describes Turkish VMV sequences with the molecular characterization of LTR and gag genes, and it strongly suggests that SRLV-genotype A originated in Turkey.

Perivascular inflammatory cells in ovine Visna/maedi encephalitis and their possible role in virus infection and lesion progression.

We examined the distribution in the perivascular spaces of Visna/maedi antigen, T cells (CD3+, CD4+ and CD8+), B cells and macrophages by immunohistochemistry in 22 natural cases of Visna/maedi encephalitis. Sheep showed lymphocytic or histiocytic lesions. In mild lymphocytic lesions, the viral antigen was detected in perivascular cuffs where CD8+ T cells predominated, but in severe lymphocytic lesions, sparse antigen was identified, and CD8+/CD4+ T cells appeared in a similar proportion in multilayer perivascular sleeves. In histiocytic lesions, vessels were surrounded by macrophages with abundant viral antigen, with CD8+/CD4+ T cells and B cells in the periphery. These results could reflect different stages of virus neuroinvasion and clarify the neuropathogenesis of Visna/maedi encephalitis.

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.

Visna/Maedi virus genetic characterization and serological diagnosis of infection in sheep from a neurological outbreak.

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

Patterns of lesion and local host cellular immune response in natural cases of ovine maedi-visna.

This study investigates the nervous form of ovine maedi-visna by histological and immunohistochemical techniques. The aim was to study the lesion types and the local cellular immune response related to each lesion type, and the possible relationship between these parameters. Thirty-four Assaf ewes were studied, 29 of which had shown nervous signs. Microscopical lesion patterns were described according to location, extent and predominance of inflammatory cell type. Immunohistochemical labelling of T cells (CD3(+), CD4(+), CD8(+) and cells expressing the γδ form of the T-cell receptor), B cells and macrophages revealed clear differences between the lesion patterns. Two main lesion types were described. Lymphocytic lesions had areas of mild-moderate injury characterized by a predominance of infiltrating T cells. Histiocytic lesions were more severe and had extensive areas of malacia and dominant infiltration by macrophages and B cells. Each animal had a unique lesion pattern and these differences could be due to individual resistance to the progression of infection. The lymphocytic lesions appear to represent initial or latent phases of slow progression, in which the animal presents some natural resistance to the infection. The histiocytic pattern may reflect a poor immune response or a greater virulence of the viral strain.

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.

Maedi-visna virus infection in Karayaka and Amasya Herik breed sheep from provinces in northern Turkey.

Maedi-visna is an important virus infection of sheep having prolonged incubation period (slow disease) and reflecting two distinct forms clinically and pathologically. In this study, the presence of MVV was investigated serologically in 58 Amasya Herik sheep breed and 525 Karayaka sheep breed. Seropositivity rates in Amasya Herik sheep breed and Karayaka sheep breed were detected as 69.0% and 18.5%, respectively. MVV antibodies were found in 137 of 583 serum samples (23.5%). Positivity rates for the provinces varied and were as follows: Samsun 19.4%, Sinop 15.4%, Ordu 25.8%, Trabzon 26.7%, Rize 36.7%, Amasya 69.0% and Tokat 35.0%, however no antibody response was detected in all of the sheep in Giresun province.

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.

Herd risk factors associated with sero-prevalence of Maedi-Visna in the Manitoba sheep population.

Disease associated with Maedi-Visna infection results in substantial economic losses in affected sheep producing areas of the world. A survey was conducted to estimate herd and individual seroprevalence in the province of Manitoba and evaluate risk factors for seropositive herds. Of 2207 sheep sampled from 77 selected sheep flocks, the animal level seroprevalence was 2.47% and herd level seroprevalence was 25.10%. The herd-level factors of presence of clinical skin disease, herd size of > 70, history of musculoskeletal/lameness abnormalities, and the purchase of new stock (> 50) in the last 1 to 5 y, showed significant associations with seropositive herd status. The study documented a remarkable stability of low seroprevalence in the province over a 20-year period in the absence of a systematic disease control program.