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

Abstract

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.

Figure 1

Virus isolation. RT activity in the supernatant of macrophage cultures monitored over 13 days. Results are expressed as - CT values + 40 according to the described PERT assay.

Figure 2

Cytopathic effect on macrophages. a-b: Cytopathic effect on macrophages. a) Macrophage culture 14 days p.iso. These cells were mock infected at day 7 p.iso. b) Macrophage culture from the same animal as in Fig.  2a 14 days p.iso. These cells were infected at day 7 p.iso. with the SRLV A4 isolate obtained from goat #2, passaged twice in primary macrophage cultures.

Figure 3

Phylogenetic analysis. Phylogenetic analysis of the isolated viruses based on the highly variable SU portion of env encompassing the SU4 and SU5 regions [16,17]. The tree was constructed by the neighbor-joining method, and bootstrap values (1000 replicates) above 70% are considered to be significant. * designates the two SRLV A4 isolates described previously [14].

Figure 4

Monitoring of the proviral load in PBMC. PBMC associated proviral load was quantified by real time PCR for all 5 goats at 4 time points. Viral loads are indicated as means ± SD.

Figure 5

Quantification of the proviral load in different organs and cells. a-e: Quantification of the proviral load in different organs and cells. Viral loads of goats #1 to #5 (Figure  5a to 5e) quantified in PBMC, left and right synovial membrane (SMl and SMr), choroid plexus (PCh), left and right mammary glands (MGl and MGr), lung tissue (L) and alveolar macrophages (alMØ). Bars indicate means ± SD and the respective mean value is shown inside the bar. *indicates statistically significant differences according to the Bonferroni (All-Pairwise) Multiple Comparison Test (p ≤ 0.05).

Figure 6

Histopathology of the mammary gland. a-b: Histopathology of the mammary gland. a: representative histopathological picture of a grade 1 inflammatory reaction in goat #2 with small periductular lymphoid follicles (asterisks) and mild periacinar lymphocytic infiltrates (arrows). b: representative histopathological picture of a grade 4 inflammatory reaction in goat #5 with many large lymphoid follicles (asterisks) and moderate lymphocytic periacinar infiltrates (arrow). H&E stain, scale bar = 100 μm.