In process metagenomic analysis of LSDV harvest for quality control
In process metagenomic analysis of LSDV harvest for quality control
Hadeer M. Mossa, Ausama A. Yousif, Emad A. Aboelsoud, Mahmoud El Gamal, Ahmed El-
Sanousi
ABSTRACT
Background: Vaccination against lumpy skin disease is carried out by heterologous sheep pox vaccine
(SPPV) giving un-satisfiable results and incomplete protection in vaccinated cattle, since that it was a
must to produce a homologues vaccine for LSDV.
Methods: A local isolate of LSDV strain was propagated on MDBK cell line and CPE was appeared
after 72 hours as cell rounding, cell aggregation and cell degeneration with titer of 105.5TCID50/ml,
DNA extraction and PCR for the candidate strain was performed using universal and specific primers
for virus identification and confirmation. The size of the produced amplicons was 192 pb for the
universal primer and 502pb, 1452pb for the specific primers respectively. Metagenomics is done to
evaluate a virus harvest for standardization by applying a suite of genomic technologies and
bioinformatics tools to directly represent the genetic content of entire communities of organisms in the
given harvest.
Results: In this study a 3rd generation sequencing (TGS) was applied using a Nanopore technology
(MinION) based on identification of DNA bases by measuring the changes in electrical conductivity
generated as DNA strands pass through a biological pore (nanopore). Its portability, affordability, and
speed in data production makes it suitable for real-time applications, the release of the long read
sequencer MinION has thus generated much excitement by producing a high microbial diversity in
biological samples that was limited by using second generation sequencing as Illumina . DNA
concentration and sequencing had been performed in demands for faster and more accurate methods to
analyze, characterize and qualify the virus as a seed for future vaccine production. Analysis workflow
starts on a high performance laptop device (GENUS), sequence data is generated for bacteria, viruses,
fungi and archaea that present in the sample it was then classified to subspecies and strain level in a
quantitative manner in approximately 48min and 23 second.
Conclusion: This method allows screening all the virus harvest content in non-selective, non- targeted
manner. The reasons to choose this technology in qualification of the TC propagated LSDV as a onestep
quality control for in process LSDV indicating all what in the sample as well as an indication for
virus purity. The presence of LSDV with very high degree of confidence and the presence of other
similar species in the capripox genus as they share a lot of conserved areas all over their genomes
remain to be addressed to fully exploit the potential of the nanopore technology, together with some
other conventional testing before judging the sample.
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January 2020
Vol. 7, Pages 1-96
