Establishment of long-read nanopore sequencing and proficient nanobodies against peste des petits ruminants virus on the road to develop diagnostic and therapeutic tools

Abstract

Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats, peste des petits ruminants (PPR), that threatens food security, animal production and the conservation of wild small ruminants. Growing body of evidence suggests that multiple wildlife and atypical host species can be infected with PPRV, posing a serological diagnostic challenge in multi-host environment. Recent studies confirmed that single- domain antigen binding fragments (nanobodies) derived from heavy-chain-only camelid antibodies and nanopore sequencing have proven to be powerful technologies for the development of cost-effective and robust therapeutic and diagnostic tools, respectively. Therefore, the main objective of this study was to generate PPRV-reactive nanobodies in order to set pace for the development of diagnostic and possible therapeutic nanobodies in the future, alongside with establishment of rapid complete genome nanopore sequencing of PPRV. Firstly, a strategy was developed to generate nanobodies against PPRV, whereby an alpaca (Vicugna pacos) was immunized with live attenuated vaccine strain (PPRV/Nigeria/75/1) to raise an affinity-matured immune response in the heavy-chain- only antibody classes. An immune nanobody library with approximately 64 million independent transformants was engineered, of which 100% contained an insert with the proper size of nanobody gene. Following phage display and in vitro affinity selection (biopanning), nine nanobodies that specifically recognise PPRV were identified on enzyme-linked immunosorbent assay. They showed superb potency in identifying rapidly PPRV, which is likely to open a new perspective in the diagnosis and possible treatment of PPRV infection. Secondly, prior to the full genome sequencing of PPRV, nanopore sequencing protocol was tested for amplification and sequencing of PPRV. With this protocol, there were no DNA fragments and nucleotide sequences in the GC-rich region between matrix (M) and fusion (F) genes at the genome position between 4,444 and 5,526. Thus, a tiling multiplex polymerase chain reaction method was developed to amplify the missing DNA fragments. Following redesigning of three pairs of overlapping long read primers and cascade of optimization, the GC rich region was successfully amplified and sequenced (accession numbers: MW580394, MW580395 and MW580396). These three pairs of primers targeting the GC-rich region were used along with other 22 pairs of primers in tiling multiplex PCR for complete PPRV genome sequencing. The resulting PCR amplicons were used for nanopore library preparation and ultimate sequencing. This method has resulted into complete genomes of PPRV, with 15,948 nucleotides long for both isolates that were 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 PPR viruses currently circulating in East Africa indicating a common origin. The nanopore sequencing platform can be deployed to overcome PPR diagnostic and surveillance challenges, unanticipated variations in virus pathogenicity, circulation of disease in wildlife populations and to service remote and nomadic communities with challenging geographical landscapes. However, further investigations are recommended for PPRV reactive nanobodies especially on diagnostic and therapeutic applications. Once validated, these technologies have great potential for use in the field as rapid and cost-effective tools in context of planned PPR Global Control and Eradication Programme.