Complete genome of a 2014 isolate of peste des petits ruminants virus from Ethiopia

ABSTRACT This report describes the complete genome sequence of a peste des petits ruminants virus (PPRV) isolate from Ethiopia in 2014. The strain (PPRV/Ethiopia/Habru/2014), which showed a normal virulence and relatively low morbidity in the field, belongs to the North African subclade of Lineage IV.

P este des petits ruminants (PPR) is a highly contagious disease affecting sheep and goats, caused by peste des petits ruminants virus (PPRV) of the genus Morbillivirus in the family Paramyxoviridae (1). PPRV is widely distributed through large parts of Africa, the Middle East, and Asia, posing an increasing threat to poor livestock keepers, primarily in developing countries (2)(3)(4)(5). While there is an increasing number of PPRV full-genome sequences in databases, information on the number of cell culture passages prior to sequencing or pathogenicity associated with the viruses sequenced is generally lacking.
Nucleic acid was extracted from 200 µL of cell culture supernatant using the MagMAX Core Nucleic Acid purification kit on a KingFisher Flex automated extraction platform (both ThermoFisher Scientific, Waltham, MA). Libraries were prepared from total RNA using the Trio RNA-Seq kit (Tecan, Männedorf, Switzerland) and underwent paired-end sequencing (2 × 150 bp) on an Illumina MiSeq using v2 reagents (Illumina, San Diego, CA).
Sanger sequencing was used to determine parts of the M-F intergenic region not determined by next-generation sequencing (NGS). This region was amplified in two fragments using primers designed from the Illumina data (Table 1). Double-stranded cDNA was synthesized using SuperScript III First-Strand Synthesis System (ThermoFisher Scientific), followed by NEBNext Ultra II Non-Directional Second Strand Synthesis module A total of 292,050 and 196,212 reads were generated, with 124,730 and 35,158 reads mapping to the reference for the third and fourth passages, respectively. Next-genera tion sequencing (NGS) data sets were analyzed as previously described (9), using bowtie2 (10) (v2.3.4.2) and bwa mem (11) (v0.7.15) for the search, bcftools (12) (v1.6) to call the consensus, and PPRV/Ethiopia/2010 (GenBank accession KJ867541) as the reference genome. Sanger sequencing data were assembled with the NGS-derived consensus using SeqMan Pro, v17.0.2 (DNASTAR, Inc).
The complete genome has a total length of 15,948 nucleotides (GC content 47.9%). The 5′ and 3′ UTRs were determined using the NGS data. There was an average read depth of 939× and 272× per base for the third and fourth passages, respectively. No difference in sequence was found between the two passages.
Phylogenetic comparison of the complete sequence [bar the M-F intergenic region (13)] to those of other PPRV genomes (Fig. 1) showed that the virus belongs to Lineage IV. PPRV/Ethiopia/Habru/2014 clustered with other recent Ethiopia PPRV Funding acquisition, Project administration, Resources, Supervision, Validation, Writingreview and editing | Carrie Batten, Funding acquisition, Project administration, Resources, Writing -review and editing