Applications of Next-Generation Sequencing Technologies to Diagnostic Virology

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

Among RNA viruses, HIV quasispecies have been extensively investigated because of their relevance for vaccine design and response to antiviral drug therapy [87] . Within infected individuals, HIV is highly heterogeneous owing to rapid turnover rates, high viral load, and a replication mediated by the error-prone reverse transcriptase enzyme that lacks proofreading activity. High variability is also the consequence of recombination, which can shuttle mutations between viral genomes and lead to major antigenic shifts or alterations in virulence [88] . An example of application of NGS for analysis of HIV quasispecies is the use of massive parallel 454 pyrosequencing with the shotgun approach to characterize the full length genome of an HIV-1 BF recombinant and its quasispecies heterogeneity in a patient who died from multiorgan failure during seroconversion [89] . Another fascinating application of deep sequencing in HIV research is the use of the 454 pyrosequencing methods to analyze the variable regions of heavy and light chains of neutralizing antibodies against HIV in the blood obtained from HIV-1-infected individuals, in order to understand how broadly neutralizing antibodies develop [90] . But the most relevant application of NGS in HIV diagnostics is the detection of anti-viral drug resistant minor variants, which will be discussed in the next section.