An Improved Barcoded Oligonucleotide Primers-based Next-generation Sequencing Approach for Direct Identification of Viral Pathogens in Clinical Specimens

Objective To provide a feasible and cost-effective next-generation sequencing （NGS） method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples. Methods Viruses from cell culture materials or clinical specimens were identified following an improved NGS procedure： reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide （random hexamer or non-ribosomal hexanucleotide） primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline. Results NGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease （HFMD） and identified other potential viruses or differentiated misdiagnosis events. Conclusion The improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.

Identification of a novel torque teno mini virus in cerebrospinal fluid from a child with encephalitis

Dear Editor, Anelloviruses, which are all single-strand circular DNA viruses, have extremely heterogeneous genetic features. To date, the family Anelloviridae is divided into 1 l genus, and the most dominated species include torque teno virus （TTV）, torque teno mini virus （TTMV） and torque teno midi virus （TTMDV）, belonging to the genus Alphatorquevirus, Betatorquevirus and Gammatorquevirus, respectively. Most of these viruses are detected in the body fluid from the majority of human populations, although their disease associations are still elusive （de Oliveira, 2015; Focosi et al., 2016）.

Tick-Borne Viruses

Ticks are important vectors for the transmission of pathogens including viruses.The viruses carried by ticks also known as tick-borne viruses(TBVs),contain a large group of viruses with diverse genetic properties and are concluded in two orders,nine families,and at least 12 genera.Some members of the TBVs are notorious agents causing severe diseases with high mortality rates in humans and livestock,while some others may pose risks to public health that are still unclear to us.Herein,we review the current knowledge of TBVs with emphases on the history of virus isolation and identification,tick vectors,and potential pathogenicity to humans and animals,including assigned species as well as the recently discovered and unassigned species.All these will promote our understanding of the diversity of TBVs,and will facilitate the further investigation of TBVs in association with both ticks and vertebrate hosts.