Novel virus discovery in field-collected mosquito larvae using an improved system for rapid determination of viral RNA sequences (RDV ver4.0)

Abstract

In this study, we improved a method for rapid determination of viral RNA sequences (RDV) to overcome the limitations of previous versions. The RDV ver4.0 method can detect RNA sequences with at least 1,000 copies as starting material. A novel virus, which was isolated from field-collected Aedes aegypti larvae in the Phasi Charoen district of Thailand using C6/36 cells, was identified using the RDV ver4.0 protocol. The virus was named Phasi Charoen virus (PhaV). We used a high-throughput pyrosequencing approach to obtain more information about the genome sequence of PhaV. Analysis of a phylogenic tree based on amino acid sequences strongly suggested that PhaV belongs to the family Bunyaviridae.

Sequencing-based Virus Hunting and Virus Detection

“…Yamao et al (2009) used Phi29 DNA polymerase, which exhibits strong strand displacement activity and thus amplifies the DNA sequence in vitro quite efficiently, to amplify a tiny amount of viral nucleic acid. Employing a ligation step with a supplemental oligonucleotide, they successfully improved the analytical sensitivity 32 . As a result, they successfully detected a number of viruses, which had previously been difficult or impossible to detect.…”

“…Although the use of NGS would have eliminated the need for adopter-mediated selective PCR, the Phi29-mediated amplification technique would still be useful. Indeed, Yamao et al (2009) also analyzed their amplification products using NGS to obtain more sequence information for a virus they newly identified 32 ( Table 1).…”

“…Therefore, it is preferable that viral genomes or their replication intermediates be enriched. Amplification using phi29 DNA polymerase was shown to be advantageous in terms of improving the detection sensitivity 32 . Recently, compact models with reduced NGS running costs have been released by a few companies (see the aforementioned manufacturers' web sites) and sequencing-based virus detection in daily diagnosis has now been realized.…”

Development of a Method to Detect Viral RNA Sequences From Cultured Cells by Combining Size Fraction and a Rapid Determination System for Viral RNA Sequences (RDV)

“…Recently, we developed a new method for sequence-independent detection of RNA viruses using direct sequencing, i.e., a rapid determination system for viral RNA sequences (RDV) . Using RDV and improved RDV, several novel viruses have been discovered and identified until now [4,6,[8][9]12,13]. Thus, the RDV method is a powerful tool for identification of unknown viruses.…”

Isolation and characterization of a novel Rhabdovirus from a wild boar (Sus scrofa) in Japan

“…This system does not require a molecular cloning step. We have previously discovered numerous novel viruses, including bat adenovirus 1 (Maeda et al, 2008), bat Hipposideros diadema herpesvirus 1 , bat betaherpesviruses 2, , Ostrich reovirus 1 , mosquito Phasi Charoen bunyavirus (Yamao et al, 2009), mosquito Omono River totivirus (Isawa et al, 2010), Japanese eel endothelial cellsinfecting polyomavirus-like virus , wild boar sapelovirus-like virus 1 (Abe et al, 2011), and goat enterovirus . Information regarding unknown viruses that infect wild boars is valuable for taking measures to prevent virus transmission from wild boars to domestic animals and humans.…”