SNPs in APOBEC3 cytosine deaminases and their association with Visna/Maedi disease progression.

The Apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) genes are able to inhibit the replication of a wide range of exogenous retroviruses, as well as endogenous retroviruses and retrotransposons. Three APOBEC3 genes, named APOBEC3Z1, APOBEC3Z2 and APOBEC3Z3, have been described in sheep. In this work the three genes have been screened in order to identify polymorphisms. No polymorphism was detected for the A3Z2 and A3Z3 genes but 16 SNPs and a 3-bp deletion were found in the A3Z1 gene. A thermoestability prediction analysis was applied to the detected amino acidic SNPs by three different programs. This analysis revealed a number of polymorphisms that could affect the protein stability. The SNPs of the 3'UTR were tested to detect alterations on the predicted microRNA target sites. Two new microRNA target sites were discovered for one of the alleles. Two SNPs were selected for association studies in relation with the retroviral disease Visna/Maedi in Latxa and Assaf sheep breeds. Although association analyses resulted unconclusive, probably due to the unsuitability of the SNP allele frequency distribution of the selected polymorphisms in the analyzed breeds, these genes remain good candidates for association studies.

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.

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.

Characterization of ovine Toll-like receptor 9 protein coding region, comparative analysis, detection of mutations and maedi visna infection.

One of the major roles of innate immunity system is the recognition and the determination of the nature of the antigen. This ability is encompassed by specific receptors as Toll-like receptors (TLRs). TLR9 recognizes bacterial and viral CpG motifs, while their potent immunostimulation effect seems to be promising for lentiviral therapies. Recent studies, however, show the presence of a big polymorphism within the TLR genes and the linkage between substitutions and susceptibility to various infections. Moreover, different recognition ability seems to be utilized by different species and possibly breeds. In this study, we characterized the protein coding region of ovine TLR9 gene. By using comparative analysis of two closely related species and humans, we suggest, which characteristics of protein could be responsible for altered recognition. Furthermore, analyzing the presence of the substitutions, we show the intraspecies polymorphism and its possible implications, while attempting to define the association of discovered substitutions with the maedi visna infection.

Characterization of ovine TLR7 and TLR8 protein coding regions, detection of mutations and Maedi Visna virus infection.

Toll-like receptors (TLRs) 2, 3, 4, 7, 8 and 9 play a crucial role in the recognition of viral entities and modulation of the innate immune system. This work presents sequence analysis of ovine TLR7 and TLR8 genes, depicts novel mutations and describes frequencies of mutations in Maedi Visna infected and healthy sheep. Totally 48 samples of the breed Tsigai were analyzed for the presence of mutations. Within 20 mutations, 14 were silent whereas 6 were missense. The frequencies of missense mutations in the Maedi Visna infected compared to non-infected sheep were: Lys115Glu (P-0.766, F-test), Asn117 (P-0.380) and Lys818Arg (P-0.739). These three mutations were localized in extra LRR (lucine rich repeat) region of TLR7, while mutation Ile73Leu (P-0.498) was located within LRR2 motif. Both mutations in TLR8, Asn165Lys (P-1.0) and Tyr349His (P-0.700), were present in extra LRR region. The secondary structure analysis of ovine TLR7 and TLR8 revealed conserved LRR motif structure, however with some irregularities compared to cattle and human. Transmembrane domains of TLR7 and TLR8 showed 100% homology between sheep and cattle wherein no mutations were found. In both TLRs TIR domains were highly conserved with occurrence of 4 silent mutations. Mutations in TLR7 and TLR8 may play an important role as predisposition factor for Maedi Visna infection. Considering the sequence homology among sheep, cattle and human genes encoding TLR7 and TLR8, we predict their similar function, localization and downstream signaling.

Maedi-visna: the meningoencephalitis in naturally occurring cases.

Lesions were examined at different levels of the central nervous system (CNS) in 64 sheep with natural maedi-visna (MV) meningoencephalitis. All animals showed lesions in more than one of the CNS locations examined; the lesions in the cranial regions were periventricular, while those in the spinal cord affected the white matter funicles. Lesions were found particularly in the cerebellar peduncles (non-suppurative meningoencephalitis), followed by the corpus callosum, hippocampus and thoracic spinal cord. Vascular, infiltrative and malacic histopathological patterns were recognized. One pattern predominated in each section examined, although mixed forms occurred. Vascular lesions occurred with similar frequency at all CNS levels, but infiltrative and malacic lesions predominated at rostral and caudal levels, respectively. Cells consistent with macrophages and shown immunohistochemically to be associated with MV virus were seen in malacic and infiltrative lesions, at the periphery of damaged areas.

Mapping and characterization of visna/maedi virus cytotoxic T-lymphocyte epitopes.

CD8(+) cytotoxic T-lymphocyte (CTL) responses have been shown to be important in the control of human and simian immunodeficiency virus infections. Infection of sheep with visna/maedi virus (VISNA), a related lentivirus, induces specific CD8(+) CTL in vivo, but the specific viral proteins recognized are not known. To determine which VISNA antigens were recognized by sheep CTL, we used recombinant vaccinia viruses expressing the different genes of VISNA: in six sheep (Finnish LandracexDorset crosses, Friesland and Lleyn breeds) all VISNA proteins were recognized except TAT. Two sheep, shown to share major histocompatibility complex (MHC) class I alleles, recognized POL and were used to map the epitope. The pol gene is 3267 bp long encoding 1088 aa. By using recombinant vaccinia viruses a central portion (nt 1609-2176, aa 537-725) was found to contain the CTL epitope and this was mapped with synthetic peptides to a 25 aa region (aa 612-636). When smaller peptides were used, a cluster of epitopes was detected: at least three epitopes were present, at positions 612-623: DSRYAFEFMIRN; 620-631: MIRNWDEEVIKN; and 625-635: EEVIKNPIQAR. A DNA-prime-modified vaccinia virus Ankara (MVA)-boost strategy was employed to immunize four sheep shown to share MHC class I allele(s) with the sheep above. Specific CTL activity developed in all the immunized sheep within 3 weeks of the final MVA boost although half the sheep showed evidence of specific reactivity after the DNA-prime immunizations. This is the first report, to our knowledge, of induction of CTL by a DNA-prime-boost method in VISNA infection.

Immune response to maedi-visna virus.

The ovine maedi-visna virus (MVV) was the first lentivirus to be isolated and characterized 1957 in Iceland. MVV leads to a life-long, persistent infection with slow development of lesions in the lung and the central nervous system (CNS). The main target cells of MVV are of the monocyte/macrophage lineage and it does not infect T-lymphocytes or cause immune suppression like human immune deficiency virus (HIV). In spite of a fairly good immune response, including both neutralizing antibodies and cytotoxic T lymphocytes, the virus persists in the host and establishes a life-long infection. There are strong indications that the pathological lesions are immune-mediated and vaccination attempts have not only failed to induce sterile immunity but have occasionally caused increased viremia and more severe disease.

Immune response to individual maedi-visna virus gag antigens.

The lesions caused by maedi-visna virus (MVV) are known to be immune mediated with a presumed contribution by the response to viral antigens. However, very little is known about the T-cell response to individual viral proteins. We have therefore expressed the three individual gag antigens of MVV strain EV1 (p16, p25, and p14) in a bacterial expression system and used the purified recombinant proteins to analyze the antibody and CD4+ T-cell response to MVV. Plasma samples were taken from sheep after 1 year of infection with MVV. The titers for antibodies in these samples were determined by indirect enzyme-linked immunosorbent assays and were as follows: anti-p25 antibody, 1:400 to >1:3,200; anti-p16 antibody, 1:400 to 1:3,200; and anti-p14 antibody, 1:<100 to 1:3,200. When the induction of antibodies was followed over time postinfection (p.i.), samples positive for anti-p25 were seen by day 24 p.i., followed by anti-p16 by day 45 p.i., and lastly anti-p14 by day 100 p.i. T-cell proliferative responses to all three gag antigens were detected in persistently infected sheep peripheral blood lymphocytes. The antigens were therefore used to raise T-cell lines from persistently infected sheep. These T-cell lines were shown to be specific for the recombinant gag antigens and for viral antigen expressed on infected macrophages. The proliferative response was restricted to major histocompatibility complex class II HLA-DR and so was due to CD4+ T lymphocytes. All three gag antigens may therefore play a role in immune-mediated lesion formation in MVV disease by presentation on infected macrophages in lesions.

Mucosal vaccination with an attenuated maedi-visna virus clone.

Four sheep were infected intratracheally with an attenuated molecular clone of maedi-visna virus (MVV). All four became infected. Ten months later these sheep were challenged intratracheally with a genetically similar but pathogenic clone of MVV. Four unvaccinated sheep were infected simultaneously. All sheep became infected by the challenge virus. The vaccinated sheep were not protected against superinfection with the challenge clone. However, virus was isolated more frequently from the blood of the unvaccinated controls than of the vaccinated animals and ten times more frequently from lungs of unvaccinated sheep than from lungs of vaccinated sheep at sacrifice, indicating partial protection.

Ovine lentivirus-associated leucomyelitis in naturally infected North American sheep.

Leucomyelitis was the predominant feature in four North American adult sheep (cases 1-4) with ovine lentivirus (OvLV) infection. All four animals were OvLV-seropositive and a syncytogenic virus consistent with OvLV was isolated from the brain of case 3 and the lungs of case 4. Clinically, the sheep had dyspnoea and neurologic signs of varying severity. Changes in the central nervous system included asymmetrical meningoleucomyelitis with white matter degeneration in all four sheep and scattered foci of leucoencephalitis in periventricular, subependymal and other white matter areas of the brain of the three animals (cases 1, 2 and 4) for which the brain was examined. In the lungs of two sheep (cases 3 and 4), there was lymphoid interstitial pneumonia with marked lymphoid hyperplasia. The viral capsid antigen (p25) was detected by immunohistochemistry (IHC) in sections of lung, brain and spinal cord of the four sheep and OvLV RNA was detected by in-situ hybridization (ISH) in lung and spinal cord samples. The results confirm the usefulness of the IHC and ISH for differential diagnosis of visna.

Maedi-visna virus and its relationship to human immunodeficiency virus.

Maedi-visna is a slow virus infection of sheep leading to a progressing lymphoproliferative disease which is invariably fatal. It affects multiple organs, but primarily the lungs where it causes interstitial pneumonia (maedi). Infection of the central nervous system was commonly observed in Icelandic sheep (visna), infection of mammary glands (hard udder) in sheep in Europe and the USA, and infection of the joints in sheep in the USA. The name ovine progressive pneumonia (OPP) is commonly used in the USA and ovine lentivirus (OvLV) infection is also a name used for maedi-visna. A related infection of goats, caprine arthritis-encephalitis (CAE), is common in Europe and the USA. The natural transmission of maedi-visna is mostly by the respiratory route, but also to newborn lambs by colostrum and milk. Intrauterine transmission seems to be rare and venereal transmission is not well documented. Macrophages are the major target cells of maedi-visna virus (MVV), but viral replication is greatly restricted in the animal host, apparently due to a posttranscriptional block. The low-grade viral production in infected tissues can explain the slow course of the disease in sheep. The lesions in maedi-visna consist of infiltrates of lymphocytes, plasma cells, and macrophages, and are detectable shortly after experimental transmission. Several studies indicate that the lesions are immune mediated and that cytotoxic T-lymphocytes may be important effector cells. The persistence of the MVV infection is explained by a reservoir of latently infected blood and bone marrow monocytes, which migrate into the target organs and mature into macrophages with proviral DNA transcription, but limited replication of virus. The MVV particles are morphologically similar to those of other retroviruses and the mode of replication follows the same general pattern. The genome organization and gene regulation resembles that of other lentiviruses. In addition to gag, pol and env, MVV has three auxiliary genes (tat, rev and vif), which seem to have similar functions as in other lentiviruses, with a possible exception of the tat gene. A determination of the 9200 nucleotide sequence of the MVV genome shows a close relationship to CAE virus, but limited sequence homology with other lentiviruses, and only in certain conserved domains of the reverse transcriptase and possibly in the surface protein. MVV infection in sheep and HIV-1 infection in humans have a number of features in common such as a long preclinical period following transmission, and a slow development of multiorgan disease with fatal outcome. A brief early acute phase, which is terminated by the immune response, is also an interesting common feature. Like HIV-1, MVV is macrophage tropic and the early stages of the HIV-1 infection which affect the central nervous system and the lungs are in many ways comparable to maedi-visna. In contrast to HIV-1, MVV does not infect T-lymphocytes and does not cause T-cell depletion and immunodeficiency. This is responsible for the difference in the late stages of the HIV-1 and MVV infections and the final clinical outcome. Despite limited sequence homology, certain proteins of MVV and HIV-1 show structural and functional similarities. Studies of MVV may therefore help in the search for new drugs against lentiviruses, including HIV-1.

[Selection for disease and epidemic resistance in domestic ruminants and swine by indicator traits, marker and causal genes--a review. Part 2: Special immunogenetics of sheep and goats with particular regard for endoparasitoses, scrapie, foot rot and maedi-visna virus infection]. / Selektion beim domestizierten Wiederkäuer und Schwein auf Krankheits- und Seuchenresistenz mit Hilfe von Indikatormerkmalen, Marker- und Kausalgenen--ein Uberblick. 2. Mitteilung: Spezielle Immungenetik bei Schaf und Ziege unter besonderer Berücksichtigung der Endoparasitosen, Scrapie, Moderhinke und der Maedi-Visna-Virusinfektion.

The introduction of the first part deals with immunogenetic investigations on the field of life-stock. The main chapter is outlined as a tabular overview of current opportunities of the application of indicator traits as well as marker and causal genes in breeding for disease resistance in cattle, sheep, goats and swine. In the discussion of the second part, emphasis was laied on diseases of small ruminants in central and western Europe with special respect of endoparasitoses, scrapie, foot-rot and maedi-visna virus infection. Indicator traits are discussed with respect of their advantages and disadvantages. The rigorous selection on specific traits is connected with an increase of the number of homozygotes. In contrary, pathogens do undergo mutations, thus escaping the host's immune system. Out of this point of view it is advisable, to set on selection very cautiously. The role of technologies of modern immunogenetics is pointed out in respect of constructing disease resistant animals.

How to succeed as a virus: strategies for dealing with the immune system.

Viruses may be viewed as genetic information whose success depends on avoiding elimination from individual hosts, or, if this is not possible, in persisting in the population of their hosts. The immune system represents the crucial defense mechanism responsible for the elimination of viruses from individual hosts and for the establishment of immunity that prevents a recurring infection by the same virus. Herd immunity, i.e., immunity of the population against infection resulting from the immunity of a certain fraction of the individuals of the population, represents an important concept in the interaction of viruses with their hosts. Thus, if the number of susceptible hosts decreases below a critical threshold, viruses may risk extinction because they literally run out of substrate. This possibility is increased due to the viruses' low resistance to inactivation outside their hosts by physical influences, such as heat and ultraviolet radiation. Some viruses have adopted a strategy of dual host tropism, i.e., they may reside in reservoir hosts that permit them to survive for extended periods of times. Examples of such viruses are the large and taxonomically diverse group of arboviruses. Moreover, although not normally discussed under this aspect, influenza viruses can also be said to have adopted this strategy, in view of water fowl representing reservoir hosts from which complete viruses may directly cross over to mammals, as was the case with the equine Jilin (Guo et al., 1995) or, more recently, the H5 subtype of influenza virus in humans (Shortridge et al., 1998). In addition, influenza viruses of birds may be transmitted, albeit only partially, through genetic reassortment (Shu et al., 1996).

CD4(+) T-cells are required for the establishment of maedi-visna virus infection in macrophages but not dendritic cells in vivo.

The role of CD4(+) lymphocytes in the establishment of lentivirus infection in macrophages has been studied in an in vivo system of lentivirus infection where CD4(+) lymphocytes are not the targets for infection. Using the non-T-cell-tropic lentivirus, maedi-visna virus (MVV), in CD4-depleted sheep, we have found that CD4(+) T cells were required for MVV infection in macrophages but not dendritic cells. CD4-depleted sheep had significantly lower levels of MVV-infected cells in lymph nodes and efferent lymph after MVV challenge in the drainage area of the lymph node. Due to the absence of virus in combination with the lack of CD4(+) T helper cells, virus-specific immune responses were reduced. There was delayed induction of cytotoxic T cell precursors, a marked reduction in virus-specific in vitro proliferative responses, and a delay in the appearance of MVV-specific antibodies. By contrast, CD4 depletion had no effect on the establishment of MVV infection in afferent lymph dendritic cells migrating from the skin infection site to the lymph node.

Serological survey and epidemiological investigation of maedi-visna in sheep in Finland.

A survey for antibodies to maedi-visna virus (MV) in the Finnish sheep surveillance flocks was conducted in 1994. Examination of a total of 12931 serum samples from animals over 1 year of age from 545 flocks (81% of all flocks) revealed eight seropositive flocks and the subsequent epidemiological investigation yielded one additional seropositive flock, indicating a low prevalence of 1.6%. The infection was very probably imported from Sweden in 1981, but it was not detected until the survey was conducted 13 years later. The entire primary infection flock was slaughtered in 1995. 77% of the sheep were seropositive but the animals were clinically healthy and only one (5%) of the contact flocks of the primary infection flock had contracted the infection. This secondary infection flock, 77% of which was seropositive, was slaughtered in 1994; however, animals in this flock had respiratory problems and the lungs of three sheep showed typical MV lesions. Seven (24%) of its contact flocks had contracted the infection and these each had one or two seropositive animals except for one flock which had seven (18%) seropositive animals. The results show that the initial spread of MV can be insidious and wide before infection is revealed in surveys or any clinical cases are encountered.

A comparative study on the diagnosis of maedi-visna infection in serum and colostrum samples using agar gel immunodiffusion (AGID) technique.

Serum/colostrum pairs were collected from 245 ewes in 6 sheep herds which had been determined previously to be infected with MV virus and were tested against maedi-visna infection using AGID test. Positive rates were detected as 3.8-41.2% in tested flocks. Serum and colostrum samples obtained from 53 sheep were positive for MV virus specific antibodies by AGID test. 16 colostrum samples were negative although serum samples obtained from the same animals were found to be positive for MV antibodies. Of the 245 sera and colostrum pairs tested, there was total agreement of results (+ or -) in 229 and disagreement in the results with the other 16 serum/colostrum pairs. Of the latter, all serum samples were positive and all colostrum samples were negative for MV antibodies. This study compared colostrum and serum samples for the determination of MV antibodies using AGID test under field conditions on naturally infected animals and on healthy animals. The results show that colostrum antibodies can be detected using AGID test and that colostrum is a reliable material to determine anti-MV virus antibodies. The procedure can be used for herd diagnosis.

Constitutive and visna virus induced expression of class I and II major histocompatibility complex antigens in the central nervous system of sheep and their role in the pathogenesis of visna lesions.

Expression of major histocompatibility complex (MHC) antigens was studied in the brains of 10 healthy sheep 2 months to 5 years old and 13 sheep infected with visna virus by intracerebral inoculation and killed one and 6 months post infection (p.i.). In healthy sheep there was prominent expression of class I, mainly on endothelial cells but also detected on ependyma, choroid plexus and in the leptomeninges. Class II expression was sparse. It was observed on perivascular cells, in choroid plexus, leptomeninges and on microglial cells in the white matter. No definite increase with age in the constitutive expression of class I and II was observed, confirming that we are dealing with a true constitutive expression. In visna-infected sheep a considerable induction of MHC antigens on microglia was observed, which correlated with severity of lesions and was mainly found in or adjacent to inflammatory infiltrates of the white matter. Increase in class II antigen expression was detected in all sheep but class I only in sheep with the most severe lesions 6 months p.i., an indication of a higher threshold for induction of class I than class II antigens on microglia. Few cells expressed viral antigens, indicating that direct immune-mediated destruction of infected cells plays a minor role in evolution of lesions. Since the preferential induction of MHC antigens on microglia in the white matter correlated with the lesion pattern, activated microglia may play a considerable role in the pathogenesis of lesions.