Complete Genome Sequencing of Field Isolates of Peste des Petits Ruminants Virus from Tanzania Revealed a High Nucleotide Identity with Lineage III PPR Viruses

SIMPLE SUMMARY: Peste des petits ruminants virus (PPRV) causes a highly devastating disease, peste des petits ruminants (PPR), in sheep and goats, which is targeted for global control and eradication. However, in many developing countries, access to expensive sequencing technologies is limited and is compounded by difficulties in transporting clinical samples across international borders. Oxford nanopore MinION is a relatively cheap sequencing technology using portable devices that require minimal supporting laboratory infrastructure or technical expertise for sample preparation and rapid sequencing. In this study, Oxford nanopore MinION sequencing was carried out to generate complete genomes of PPRV from archived PPRV-positive samples collected from PPR outbreaks in goats in Ngorongoro and Momba districts in Tanzania during 2016 and 2018, respectively. Complete genomes of PPRV of 15,948 nucleotides long were generated within four hours of sequencing. The phylogenetic analysis of the complete genomes revealed a high nucleotide identity (96.19–99.24%) with lineage III PPR viruses currently circulating in East Africa, indicating a common origin. The Oxford nanopore MinION sequencer can be deployed to overcome diagnostic and surveillance challenges in developing countries in the PPR Global Control and Eradication program. However, the coverage depth was uneven across the genome and amplicon dropout was observed between the matrix (M) and fusion (F) genes. Thus, larger field studies are needed to allow the collection of sufficient data to assess the robustness of nanopore sequencing technology. ABSTRACT: Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats that threatens food security, small ruminant production and susceptible endangered wild ruminants. With policy directed towards achieving global PPR eradication, the establishment of cost-effective genomic surveillance tools is critical where PPR is endemic. Genomic data can provide sufficient in-depth information to identify the pockets of endemicity responsible for PPRV persistence and viral evolution, and direct an appropriate vaccination response. Yet, access to the required sequencing technology is low in resource-limited settings and is compounded by the difficulty of transporting clinical samples from wildlife across international borders due to the Convention on International Trade in Endangered Species (CITES) of Wild Fauna and Flora, and Nagoya Protocol regulations. Oxford nanopore MinION sequencing technology has recently demonstrated an extraordinary performance in the sequencing of PPRV due to its rapidity, utility in endemic countries and comparatively low cost per sample when compared to other whole-genome (WGS) sequencing platforms. In the present study, Oxford nanopore MinION sequencing was utilised to generate complete genomes of PPRV isolates collected from infected goats in Ngorongoro and Momba districts in the northern and southern highlands of Tanzania during 2016 and 2018, respectively. The tiling multiplex polymerase chain reaction (PCR) was carried out with twenty-five pairs of long-read primers. The resulting PCR amplicons were used for nanopore library preparation and sequencing. The analysis of output data was complete genomes of PPRV, produced within four hours of sequencing (accession numbers: MW960272 and MZ322753). Phylogenetic analysis of the complete genomes revealed a high nucleotide identity, between 96.19 and 99.24% with lineage III PPRV currently circulating in East Africa, indicating a common origin. The Oxford nanopore MinION sequencer can be deployed to overcome diagnostic and surveillance challenges in the PPR Global Control and Eradication program. However, the coverage depth was uneven across the genome and amplicon dropout was observed mainly in the GC-rich region between the matrix (M) and fusion (F) genes of PPRV. Thus, larger field studies are needed to allow the collection of sufficient data to assess the robustness of nanopore sequencing technology.