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).