In process metagenomic analysis of LSDV harvest for quality control

Hadeer mostafa mossa| Ausama Abd El Raouf Yousif| Emad abdelsalam Aboelsoud| Mahmoud El Gamal| Ahmed El-Sanousi

Abstract
Vaccination against lumpy skin disease is carried out by heterologous sheep pox vaccine (SPPV) giving un satsifiable results and incomplete protection in vaccinated cattle, since that it was a must to produce a homologues vaccine for LSDV. A local strain isolate of LSDV was propagated on MDBK cell line and appearance of CPE within 72 hours then tittered till reaching the highest titre105.5TCID50/ml, DNA extraction and PCR using universal and specific primers for virus identification and confirmation producing a192 pb amplicon 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. In this study a 3rd generation sequencer (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 was preformed 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 . Thus allow us to screen all the virus harvest content in non-selective ,non- targeted manner. The reasons to choose such technology in qualification of the TC propagated LSDV as a one-step quality control for in process LSDV indicating all what in the sample as well as an indication for virus purity . it reveals the presence of lumpy in 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 therefore a number of technical challenges remain to be addressed to fully exploit the potential of the nanopore technology, together with some other conventional testing before judging the sample.

Key words: LSDV ,Metagenomic analysis ,Third generation sequencer

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