**3.3. Inter- and intra-host genetic diversity**

replicons that cannot be detected using conventional sequencing approaches. Hence, quasis‐ pecies remained unexplored for a considerable time, even though the theoretical concept for quasispecies was put forth by Eigen in 1970 [55]. With the advent of NGS technologies, the generation of large genomic datasets became a reality. Due to the sequencing error issues, it was still tough to demarcate true genetic variations. Circular Sequencing (CirSeq), a novel experimental approach that creates template of tandem repeats of circularized genomic RNA fragments has been developed by Andino's group [56]. CirSeq reduces the sequencing error drastically as the repeats get sequenced in a redundant manner for every genomic fragment. A consensus reduces the theoretical error close to 10−11, which enables capture of the entire mutational spectrum of RNA virus populations. CirSeq was employed to study seven serial passages of *Poliovirus* replicated in HeLa cells. Mutation frequency was computed for every passage and their fitness was determined by mapping onto the 3D structure of proteins. As expected, majority of the mutations detected were neutral substitutions, thus highlighting robustness as driving force for adaptation and evolution [56]. This study clearly delineates the viral mutations responsible for quasispecies structure and highlights the extent of genetic

Microevolution in an evolving quasispecies population is responsible for the sequence diversity in *Porcine reproductive and respiratory syndrome virus* (PRRSV). PRRSV is the causative agent of late-term reproductive failure in sows and respiratory distress in pigs and hence has large economic impact. Genomic complexity of PRRSV due to multiple circulating genotypes results in antigenic diversity, which, in turn, is responsible for lack of effective vaccine development [57]. Sanger sequencing has identified open reading frames ORF5 and ORF7 as the polymorphic regions of the virus genome, encoding major immunogenic epitopes. In order to study the genome-wide polymorphisms, deep sequencing of PRRSV was carried out and amino acid substitutions in ORFs 2–7 in PRRSV strains obtained from pigs that lack B and T cells were studied [58]. By analysing nucleotide substitutions over time followed by compa‐ rative genomics with non-pathogenic variants, the role of mutation and selection in preserving

Low-frequency variants or minority quasispecies are the variants that occur with a frequency of <20–25% in a viral population [59]. Minority quasispecies refers to the memory genomes that were dominant at an earlier phase of quasispecies evolution and can play an important role in conferring drug resistance in viruses such as *Human Immunodeficiency Virus type-1* (HIV-1) and *Influenza virus*. Minority quasispecies of drug-resistant viruses can rapidly reemerge as major populations after the reintroduction of drug pressure. In case of HIV-1, presence of such low-frequency variants has been linked with early failure to the antiretroviral therapy [59, 60]. Emergence of highly pathogenic subtype of *Avian Influenza virus*es (HPAI) has also been explained on the basis of low-frequency variants. Ultra-deep sequencing was used to study the emergence of HPAI from that of less pathogenic (Low Pathogenic Avian

the pathogenesis or fitness of PRRSV was well documented in this study.

variation that can be maintained in a population.

180 Next Generation Sequencing - Advances, Applications and Challenges

**3.2. Detection of low-frequency variants**

Influenza (LPAI)) progenitor viruses [61].

The rate of viral evolution and the effectiveness of its transmission are determined by interand intra-host genetic diversity. Mutation rate and selection pressure ascertain viral diversity. Factors like mixed infections and random processes such as genetic drift and population bottlenecks also contribute to the genetic diversity of viruses both within and among hosts. Transmission fitness influences the effective spread of viruses and is responsible for its stable maintenance in the environment [62].

Intra-host genetic diversity in *Zucchini yellow mosaic virus* (ZYMV), a plant RNA virus known to infect *Cucurbitaceae* plants, has been studied using NGS [63]. Population bottlenecks were investigated for this aphid-borne virus and are thought to occur during both inter-host vector transmission and systemic movement within an individual plant. ZYMV populations infecting cucumbers with and without vector were sequenced followed by *de novo* assembly and variant calling. Analysis revealed that the low-frequency mutants present in the initial population got fixed rapidly in vector-transmitted viruses, whereas the same continued to remain as minor variants in mechanically inoculated viruses. In addition, regions known to be responsible for vector transmission were conserved in all samples. It is interesting to know that previous studies using Sanger sequencing of the coat protein of ZYMV, which is involved in interaction with aphids, could not detect mutations when transmitted between or within plants. However, this study reported six mutations in coat protein with frequency of occurrence as low as ~3%. Such studies provide an insight into the complex dynamics of genetic diversity of an emerging viral infection with implications in disease management.
