Molecular and phylogenetic analyses
The total nucleic acids (viral nucleic acids and nucleic acids of the host) were extracted from homogenized tissue samples. The extraction was carried out with an extraction robot KingFisher TM and ID Kit Gene TM Mag Universal Extraction (IDvet), as described by the manufacturer. An RT-PCR was performed using the qScript XLT One-Step RT-PCR Kit (Quantabio, VWR, France) to amplify a 351 base pair (bp) segment of the PPRV N gene with the NP3/NP4 primer pair modified from Couacy-Hymman, et al., (2002). (Forward NP3: 5’-GTC-TCG-GAA-ATC-GCC-TCA-CAG-ACT-3’ and Reverse NP4: 5’-CCT-CCT-CCT-GGT-CCT-CCA-GAA-TCT-3’) at a final concentration of 0.6 μM. PCR was set up under the following programme: 50 °C for 30 min; 95 °C for 15 min and 40 amplification cycles (10 sec at 95 °C, 30 sec at 60 °C and 30 sec at 72 °C) and a final extension step at 72 °C for 5 min. The PCR products were resolved on 1.5% agarose gel to reveal the expected band size.
The clean-up and sequencing of positive PCR products in both forward and reverse directions were carried out by Genewiz (United Kingdom). The sequences were submitted to GenBank (Table 1). Forward and reverse DNA sequences were assembled using BioEdit, and trimmed to remove poor-quality portions of the sequences (final size = 255 bp). Corrected sequences were aligned with different data sets PPRV N gene sequences publicly available in GenBank using MEGA 6. A first dataset contained representatives of the four genetic lineages. Two other datasets contained an increased number of sequences from African lineage II and IV, respectively, to allow for lineage-specific phylogenetic analyses. Phylogenetic trees were constructed using the Maximum Likelihood method as implemented in MEGA 6, with node supports evaluated by bootstrap analyses (1,000 replicates).