Utility of a Sequence-Independent, Single-Primer-Amplification (SISPA) and Nanopore Sequencing Approach for Detection and Characterization of Tick-Borne Viral Pathogens

Abstract

Currently, next generation sequencing (NGS) is the mainly used approach for identification and monitorization of viruses with a potential public health threat in clinical and environmental samples. To facilitate detection in NGS, the sequence-independent, single-primer-amplification (SISPA) is an effective tool for enriching virus sequences. We performed a preliminary assessment of SISPA-nanopore sequencing as a potential approach for screening tick-borne viruses in six specimens with detectable Crimean-Congo hemorrhagic fever virus (CCHFV) and Jingmen tick virus (JMTV) sequences. A comparison of unbiased NGS and SISPA followed by nanopore sequencing was carried out in 4 specimens with single and pooled ticks. The approach was further used for genome sequencing in culture-grown viruses. Overall, total/virus-specific read counts were significantly elevated in cell culture supernatants in comparison to single or pooled ticks. Virus genomes could be successfully characterized by SISPA with identities over 99%. Genome coverage varied according to the segment and total read count. Base calling errors were mainly observed in tick specimens and more frequent in lower viral loads. Culture-grown viruses were phylogenetically-related to previously-reported local viruses. In conclusion, the SISPA + nanopore sequencing was successful in generating data comparable to NGS and will provide an effective tool for broad-range virus detection in ticks.

An Amplicon-Based Application for the Whole-Genome Sequencing of GI-19 Lineage Infectious Bronchitis Virus Directly from Clinical Samples

“…Whole-genome sequencing, combined with various NGS protocols, is a powerful tool for investigating genome epidemiology and understanding the evolutionary dynamics of viruses [ 21 , 22 , 23 , 24 , 25 ]. Nonspecific amplification of purified nucleic acids in combination with mNGS allows all the genetic material directly recovered from a sample to be sequenced and analyzed, and SISPA represents one such metagenomic approach [ 23 , 24 , 25 ].…”

“…Whole-genome sequencing, combined with various NGS protocols, is a powerful tool for investigating genome epidemiology and understanding the evolutionary dynamics of viruses [ 21 , 22 , 23 , 24 , 25 ]. Nonspecific amplification of purified nucleic acids in combination with mNGS allows all the genetic material directly recovered from a sample to be sequenced and analyzed, and SISPA represents one such metagenomic approach [ 23 , 24 , 25 ]. This method involves random priming and avoids the selection of specific RNA sequences, thus minimizing the biases associated with amplicon-based sequencing and allowing the detection of multiple pathogens [ 22 , 23 ].…”

“…Recently, it was reported that not only the S1 gene but also non-structural proteins may play critical roles in the replication and pathogenicity of IBV [ 19 , 20 ], suggesting that whole-genome sequencing is required to fully characterize IBV viruses and understand their epidemiology, including their antigenicity, tissue tropism, and pathogenicity [ 21 ]. Current advances in metagenomic next-generation sequencing (mNGS) technologies combined with Sequence-Independent, Single-Primer Amplification (SISPA) have provided a useful tool for uncovering the entire genome of IBV [ 22 , 23 , 24 , 25 ]. Generally, these methods have been applied to IBV isolates amplified by serial passage in embryonated chicken eggs [ 26 , 27 , 28 ].…”

Latest Advances in Arbovirus Diagnostics

“…While mNGS is primarily a tool used to monitor the presence of viruses from clinical and environmental samples, which could result in significant public health threats, methods have been developed for the enrichment of specific virus sequences in samples such as vector pools. Viral specific enrichment was successfully used to characterize Crimean-Congo hemorrhagic fever virus (CCHFV) and Jingmen virus (JMTV) in a pooled tick sample [ 51 ] and in an arbovirus surveillance program in Australia that detected unknown Ross River virus circulation [ 52 ].…”

Preparation for the next pandemic: challenges in strengthening surveillance

“…Specimens collected from symptomatic patients, and sick or dead animals should be first screened by multiplex nucleic acid amplification tests for known pathogens or RNA viruses [ 63 , 64 ], and specimens with a negative screen or from clinically suspicious or inconsistent cases should be further tested by unbiased next-generation sequencing to detect novel zoonotic viruses [ 65 , 66 ]. Although the amount of nucleic acid of unknown viruses is usually low in surveillance specimens, the yield can be increased by the use of Sequence-Independent, Single-Primer-Amplification (SISPA) [ 67 , 68 ]. The combination of a comprehensive database and multiple innovative bioinformatics pipelines can overcome the problems of frequent mutations in RNA viruses and the lack of reference genomes.…”