Applications of Next-Generation Sequencing Technologies to Diagnostic Virology

Barzon, Luisa; Lavezzo, Enrico; Militello, Valentina; Toppo, Stefano; Palù, Giorgio

The human population is exposed to an increasing burden of infectious diseases caused by the emergence of new previously unrecognized viruses. Climate changes, globalization, settlements near animal and livestock habitats, and the increased number of immunocompromised people probably contribute to the emergence and spread of new infections [7] . In addition, several clinical syndromes are suspected to be of viral etiology, but the causing agent cannot be isolated and recognized by traditional culture and molecular methods. Thus, there is the need to improve methods for the identification of unsuspected viral pathogens or new viruses. Subtractive techniques, such as representational difference analysis or random sequencing of plasmid libraries of nuclease resistant fragments of viral genomes, have led in the past to the discovery of several viruses, including human herpesvirus type 8 [8] , human GB virus [9] , Torque Teno Virus [10] , bocavirus [11] , human parvovirus 4 [12] , WU polyomavirus [13] and KI polyomavirus [14] . These techniques are poorly sensitive and time-consuming, and thus are unsuitable for large scale analysis. For these purposes, NGS-based methods have been developed. However, traditional cloning and sequencing methods can be relatively simple and sensitive for the discovery of new viruses when used for the analysis of otherwise sterile samples, and may represent an alternative to NGS. One of these methods is termed VIDISCA (Virus Discovery cDNA Amplified Fragment Length Polymorphism Analysis) and may be applied to sterile specimens, such as cell culture supernatants [15] . In this method, samples are ultracentrifuged for viral particle enrichment and treated by DNase and RNase to digest away cellular nucleic acids. Capsid-protected viral nucleic acids are then purified, converted to double stranded DNA, digested with restriction enzymes and ligated to oligonucleotide adaptors, which are used as primer binding sites for comparative PCR [15] . This method was described originally in the context of the discovery of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2004 [16] . Microarray-based diagnostic assays have also been used to characterize previously unknown viruses, such as SARS-CoVs [17] , but require information on the genome of the virus or closely related viruses that are under investigation [18] .