Identification and genetic characterization of a novel Orthobunyavirus species by a straightforward high-throughput sequencing-based approach

Ohad Shifman,Ofir Israeli,Marina Dorozko,Eyal Epstein,Anat Zvi,Dana Stein,Sharon Melamed,Dana Wolf,Shmuel C Shapira,Adi Beth-Din,Shmuel Yitzhaki,Nir Paran,Inbar Cohen-Gihon,Orly Laskar

doi:10.1038/s41598-019-40036-4

Abstract

Identification and characterization of novel unknown viruses is of great importance. The introduction of high-throughput sequencing (HTS)-based methods has paved the way for genomics-based detection of pathogens without any prior assumptions about the characteristics of the organisms. However, the use of HTS for the characterization of viral pathogens from clinical samples remains limited. Here, we report the identification of a novel Orthobunyavirus species isolated from horse plasma. The identification was based on a straightforward HTS approach. Following enrichment in cell culture, RNA was extracted from the growth medium and rapid library preparation, HTS and primary bioinformatic analyses were performed in less than 12 hours. Taxonomical profiling of the sequencing reads did not reveal sequence similarities to any known virus. Subsequent application of de novo assembly tools to the sequencing reads produced contigs, of which three showed some similarity to the L, M, and S segments of viruses belonging to the Orthobunyavirus genus. Further refinement of these contigs resulted in high-quality, full-length genomic sequences of the three genomic segments (L, M and S) of a novel Orthobunyavirus. Characterization of the genomic sequence, including the prediction of open reading frames and the inspection of consensus genomic termini and phylogenetic analysis, further confirmed that the novel virus is indeed a new species, which we named Ness Ziona virus.

Highlights

Viruses are a global source of significant health and economic burdens
Traditional methods rely on morphological characteristics observed by light microscopy or transmission electron microscopy (TEM) in various specimens such as cell cultures and fertilized eggs
Viral safety of biological products derived from equine plasma is verified by testing the starting material of the process for viral contamination[22] and by assessing the efficacy of the entire production process to inactivate infectious viruses[23]. As part of such a process, batches of plasma obtained from a hyperimmune horse, producing anti-botulinum antibodies, were routinely inspected for viruses by incubating the plasma in Vero and BHK cell lines and monitoring them for cytopathic effect (CPE)

Summary

Introduction

Viruses are a global source of significant health and economic burdens. Interactions between humans and animals are the paramount driving force for the ever-growing distribution and emergence of novel viruses. As well as antibody-based diagnostics, allow to identify the virus and in some cases even at the species level These methods provide only morphological clues, depend on the availability of an antiserum and, for the most part, are not strain specific[5]. High-throughput sequencing (HTS) technologies, developed in recent years, have substantially improved the capability of comprehensive detection of pathogens without any prior assumptions about the characteristics of the organisms (i.e., “unknown” samples). These massive parallel sequencing platforms can sequence mixtures of genetic materials from heterogeneous samples with high sensitivity and speed and at a lower cost per base than traditional Sanger sequencing[8,9]. Among these benefits is the ability to detect nonculturable organisms as well as coinfection, drug resistance or response to therapy[10,11]

Methods

Results

Conclusion