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.

Post-entry blockade of small ruminant lentiviruses by wild ruminants.

Small ruminant lentivirus (SRLV) infection causes losses in the small ruminant industry due to reduced animal production and increased replacement rates. Infection of wild ruminants in close contact with infected domestic animals has been proposed to play a role in SRLV epidemiology, but studies are limited and mostly involve hybrids between wild and domestic animals. In this study, SRLV seropositive red deer, roe deer and mouflon were detected through modified ELISA tests, but virus was not successfully amplified using a set of different PCRs. Apparent restriction of SRLV infection in cervids was not related to the presence of neutralizing antibodies. In vitro cultured skin fibroblastic cells from red deer and fallow deer were permissive to the SRLV entry and integration, but produced low quantities of virus. SRLV got rapidly adapted in vitro to blood-derived macrophages and skin fibroblastic cells from red deer but not from fallow deer. Thus, although direct detection of virus was not successfully achieved in vivo, these findings show the potential susceptibility of wild ruminants to SRLV infection in the case of red deer and, on the other hand, an in vivo SRLV restriction in fallow deer. Altogether these results may highlight the importance of surveilling and controlling SRLV infection in domestic as well as in wild ruminants sharing pasture areas, and may provide new natural tools to control SRLV spread in sheep and goats.

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.

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.

Virological and phylogenetic characterization of attenuated small ruminant lentivirus isolates eluding efficient serological detection.

Three field isolates of small ruminant lentiviruses (SRLVs) were derived from a mixed flock of goats and sheep certified for many years as free of caprine arthritis encephalitis virus (CAEV). The phylogenetic analysis of pol sequences permitted to classify these isolates as A4 subtype. None of the animals showed clinical signs of SRLV infection, confirming previous observations which had suggested that this particular subtype is highly attenuated, at least for goats. A quantitative real time PCR strategy based on primers and probes derived from a highly variable env region permitted us to classify the animals as uninfected, singly or doubly infected. The performance of different serological tools based on this classification revealed their profound inadequacy in monitoring animals infected with this particular SRLV subtype. In vitro, the isolates showed differences in their cytopathicity and a tendency to replicate more efficiently in goat than sheep cells, especially in goat macrophages. By contrast, in vivo, these viruses reached significantly higher viral loads in sheep than in goats. Both env subtypes infected goats and sheep with equal efficiency. One of these, however, reached significantly higher viral loads in both species. In conclusion, we characterized three isolates of the SRLV subtype A4 that efficiently circulate in a mixed herd of goats and sheep in spite of their apparent attenuation and a strict physical separation between goats and sheep. The poor performance of the serological tools applied indicates that, to support an SRLV eradication campaign, it is imperative to develop novel, subtype specific tools.