The Study of Viral RNA Diversity in Bird Samples Using De Novo Designed Multiplex Genus-Specific Primer Panels.

Andrey A Ayginin,Ekaterina V Pimkina,Alina D Matsvay,Ilya V Artyushin,Anna S Speranskaya,Ekaterina A Blinova,Vladimir G Dedkov,German A Shipulin,Kamil Khafizov,Marina V Safonova

doi:10.1155/2018/3248285

Abstract

Advances in the next generation sequencing (NGS) technologies have significantly increased our ability to detect new viral pathogens and systematically determine the spectrum of viruses prevalent in various biological samples. In addition, this approach has also helped in establishing the associations of viromes with many diseases. However, unlike the metagenomic studies using 16S rRNA for the detection of bacteria, it is impossible to create universal oligonucleotides to target all known and novel viruses, owing to their genomic diversity and variability. On the other hand, sequencing the entire genome is still expensive and has relatively low sensitivity for such applications. The existing approaches for the design of oligonucleotides for targeted enrichment are usually involved in the development of primers for the PCR-based detection of particular viral species or genera, but not for families or higher taxonomic orders. In this study, we have developed a computational pipeline for designing the oligonucleotides capable of covering a significant number of known viruses within various taxonomic orders, as well as their novel variants. We have subsequently designed a genus-specific oligonucleotide panel for targeted enrichment of viral nucleic acids in biological material and demonstrated the possibility of its application for virus detection in bird samples. We have tested our panel using a number of collected samples and have observed superior efficiency in the detection and identification of viral pathogens. Since a reliable, bioinformatics-based analytical method for the rapid identification of the sequences was crucial, an NGS-based data analysis module was developed in this study, and its functionality in the detection of novel viruses and analysis of virome diversity was demonstrated.

Highlights

An increase in globalization, climate change, and interaction with livestock animals has resulted in the emergence of novel viral pathogens or zoonoses [1], which pose a serious health problem for birds and animals and for humans
A preliminary evidence of the presence of the certain viruses is required for performing the polymerase chain reaction (PCR), in the absence of which the process of pathogen identification could take a significant amount of time, which can be a major obstacle in the prevention and control of the infection
We have introduced a method for designing oligonucleotide panels for targeted enrichment of viral nucleic acids, where the main objective is to use a minimum number of primer oligonucleotides to cover the maximum number of diverse viral taxa within a single PCR reaction

Summary

Introduction

Climate change, and interaction with livestock animals has resulted in the emergence of novel viral pathogens or zoonoses [1], which pose a serious health problem for birds and animals and for humans. The natural reservoirs of pathogens, such as birds, bats, rodents, and bloodsucking arthropods play a significant role in the sustenance and transmission of zoonotic infections. Migratory birds warrant special attention, as their rich diversity and migratory behavior contribute to the spread of infections to considerable distances. Such migrations are strongly associated with the emergence of the epidemic and enzootic outbreaks as well as the formation and activation of natural sources of viral infections. A preliminary evidence of the presence of the certain viruses is required for performing the PCR, in the absence of which the process of pathogen identification could take a significant amount of time, which can be a major obstacle in the prevention and control of the infection

Methods

Results

Conclusion