New approach for Small Ruminant Lentivirus full genome characterization revealed the circulation of divergent strains

Small Ruminant Lentiviruses (SRLV) are heterogeneous group of viruses that cause chronic, multisystemic infections in sheep and goats. Although historically ascribed to two different viruses, strictly associated with specific clinical features and host, Caprine Arthritis Encephalitis virus (CAEV) and Visna Maedi virus (MVV) are now considered host adapted but not host specific. The group include at least 4 genotypes and several subtypes and shows high variability of both in vivo and in vitro properties. Many studies demonstrated that this viral heterogeneity can influence the viral-host interaction, the cell tropism, the biological properties and, not least, the diagnostic test results. Although different cell types may be infected, the major in vivo tropism of SRLV is for the monocyte/macrophages and dendritic cells while in vitro high virulence viral strains production can be obtained also in fibro-epithelial synovial and choroid plexus cells where they show the classical cytopathic effect of cell fusion (syncytia). Nevertheless several viral strains, characterized by low pathogenic effects, showed a limited ability to replicate in fibroblast cells as well as to produce syncytia, leading to an underestimation of isolation success. Keeping in mind the heterogeneity of in vitro properties of SRLV we developed a fast and effective method for SRLV full genome characterization from spleen explants after cell culture isolation. Blood and spleen samples were collected from 42 adult animals (16 sheep and 26 goats) during regular slaughter in two different slaughterhouse in Northern Italy. Blood samples were tested with a commercial kit for antibody screening and genotyping. Spleen derived macrophage culture was maintained over 5 passages and monitored for the appearance of cytopathic effect and Reverse Transcriptase Activity (RTA). Total RNA was extracted from the concentrated supernatant with the highest value of RTA and reverse transcribed as double stranded cDNA. The samples were sequenced with a Next Generation Sequencing (NGS) approach, using Nextera XT protocol and Illumina MiSeq platform. Data analysis was conducted following two parallel approaches, resequencing and de-novo assembling, with Geneious and Velvet softwares respectively. With this approach 21 spleen explants allowed viral isolation and full genome sequencing. All the samples showed high variability of in vitro behavior. Only 13 RTA positive samples showed CPE on overgrowing fibroblastic-like cells. A positive correlation was observed between the phylogroup A8 and the absence of CPE in culture while all strains belonging to subtype B1 and B2 were fusogenic in vitro, as well as subtype A9 in a sheep and a novel subtype A in a goat. In conclusion the proposed method proved to be a fast and economically feasible approach for viral full genome characterization as well as for biological and in vitro properties study. Despite the limited sampling area it allows to detect both pathogenic subgroup B1 and B2, putative non pathogenic A8 in goats, and 2 additional A isolates (A18 and A19). The success in virus isolation with this method is based on the association of three features: the choice of the spleen as target tissue for virus isolation, the use of RTA as sensitive method to detect viral replication and the use of a high throughput technology for genome sequencing.