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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. |
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