Replication of Small Ruminant Lentiviruses in Aluminum Hydroxide-Induced Granulomas in Sheep: a Potential New Factor for Viral Dissemination.

Aluminum (Al)-based salts are widely used adjuvants in ruminants and other species to strengthen the immune response elicited against vaccine antigen(s). However, they can lead to the formation of long-lasting granulomas composed of abundant activated macrophages. Small ruminant lentiviruses (SRLV) are widely distributed macrophage-tropic retroviruses that cause persistent infections in sheep and goats. Infected monocytes/macrophages and dendritic cells establish an inflammatory microenvironment that eventually leads to clinical manifestations. The aim of this work was to study the effect of Al-induced granulomas in the replication and pathogenesis of SRLV. Eleven adult, naturally SRLV-infected sheep showing clinical arthritis were distributed in vaccine (n = 6), adjuvant-only (n = 3), and control (n = 2) groups and inoculated with commercial Al-based vaccines, Al hydroxide adjuvant alone, or phosphate-buffered saline, respectively. In vitro studies demonstrated viral replication in Al-induced granulomas in 5 out of 10 sheep. Immunohistochemistry (IHC) evinced granular, intracytoplasmic SRLV presence in macrophages within granulomas. Viral sequences obtained from granulomas, blood monocytes, and other tissues were highly similar in most animals, suggesting virus circulation among body compartments. However, notable differences between isolated strains in granulomas and other tissues in specific animals were also noted. Interestingly, the B2 subtype was the most commonly found SRLV genotype, reaching a wider body distribution than previously described. Recombination events between genotypes B2 and A3 along the gag region were identified in two sheep. Our results indicate that Al-hydroxide-derived granulomas may represent an ideal compartment for SRLV replication, perhaps altering natural SRLV infection by providing a new, suitable target tissue.IMPORTANCE Granulomas are inflammation-derived structures elicited by foreign bodies or certain infections. Aluminum adjuvants included in vaccines induce granulomas in many species. In sheep, these are persistent and consist of activated macrophages. Small ruminant lentiviruses (SRLV), which are macrophage-tropic lentiviruses, cause a chronic wasting disease affecting animal welfare and production. Here, we studied the occurrence of SRLV in postvaccination granulomas retrieved from naturally infected ewes after vaccination or inoculation with aluminum only. SRLV infection was confirmed in granulomas by identification of viral proteins, genomic fragments, and enzymatic activity. The infecting SRLV strain, previously found exclusively in carpal joints, reached the central nervous system, suggesting that occurrence of SRLV in postvaccination granulomas may broaden tissue tropism. SRLV recombination was detected in inoculated animals, a rare event in sheep lentiviruses. Potentially, virus-host interactions within granulomas may modify viral pathogenesis and lead to more widespread infection.

Localization of a TNF-activated transcription site and interactions with the gamma activated site within the CAEV U3 70 base pair repeat.

The cytokines TNFalpha and IFNgamma have previously been shown to activate caprine arthritis encephalitis virus (CAEV) transcription. Increased viral titers correlate with increased lesion severity. Therefore, TNFalpha and IFNgamma may augment the caprine arthritis lesion by increasing viral titers. CAEV transcription is under the control of the viral promoter within the U3 region of the long terminal repeat. A set of U3 deletion mutants was generated and used to establish stably integrated, U937-based cell lines. These cell lines were utilized to define the required promoter sequences for cytokine-induced transcriptional activation. Here we have identified a novel 17 nucleotide TNF-activated site within the U3 region 70 bp repeat which is both required and sufficient in a minimal construct for TNFalpha-induced CAEV transcriptional activation. In contrast to the results of previous studies with IFNgamma, we found that multiple sequences within the U3 region 70 bp repeat were required for IFNgamma-activation of the CAEV promoter. The results identify previously unrecognized complexity in the CAEV promoter that may be relevant to viral replication and disease.

Phosphorothioate oligonucleotides inhibit the replication of lentiviruses and type D retroviruses, but not that of type C retroviruses.

Phosphorothioate analogs of oligodeoxynucleotides at a concentration of 2 microM protected Himalayan tahr cells from infection by caprine arthritis encephalitis virus (CAEV) and equine dermis cells from infection by equine infectious anemia virus (EIAV). The characteristics of this inhibition against these lentiviruses are similar to those previously described for the inhibition of HIV-1 in ATH8 cells [17]. Thus, the 28-mer homo-oligomer of cytidine [S-(dC)28] was at least as effective as three anti-sense sequences targeted to the LTR, gag, and env regions of CAEV. The effectiveness of homo-oligomers of equal length was in the order C >> A > T, and a random 28-copolymer with a composition of 2C:1G was as effective as S-(dC)28. Shorter oligonucleotides were less effective (28 > 14 > 5 mers) for all base compositions tested. While replication of a simian type D retrovirus was inhibited by S-(dC)28, this compound did not inhibit the cytopathogenicity of two type C retroviruses, amphotropic murine leukemia virus (MuLV), and baboon endogenous virus, when they were tested in the same cell lines used to support the replication of lentiviruses. Southern blot analysis of the high molecular weight DNA of drug-treated CAEV-infected cells showed that S-(dC)28 was acting at or before the reverse transcription step. Our present data and the earlier finding that S-(dC)28 is a potent in vitro inhibitor of the MuLV reverse transcriptase [15] suggest that S-(dC)28 is acting very early in the replication cycle of these lentiviruses. Since MuLV reverse transcriptase is inhibited in vitro, but its replication is not blocked in permissive cells, our data suggest that the phosphorothioate oligonucleotides are preventing virus attachment.