Characterization of Hepatitis C Virus (HCV) Envelope Diversification from Acute to Chronic Infection within a Sexually Transmitted HCV Cluster by Using Single-Molecule, Real-Time Sequencing

Abstract

In contrast to other available next-generation sequencing platforms, PacBio single-molecule, real-time (SMRT) sequencing has the advantage of generating long reads albeit with a relatively higher error rate in unprocessed data. Using this platform, we longitudinally sampled and sequenced the hepatitis C virus (HCV) envelope genome region (1,680 nucleotides [nt]) from individuals belonging to a cluster of sexually transmitted cases. All five subjects were coinfected with HIV-1 and a closely related strain of HCV genotype 4d. In total, 50 samples were analyzed by using SMRT sequencing. By using 7 passes of circular consensus sequencing, the error rate was reduced to 0.37%, and the median number of sequences was 612 per sample. A further reduction of insertions was achieved by alignment against a sample-specific reference sequence. However, in vitro recombination during PCR amplification could not be excluded. Phylogenetic analysis supported close relationships among HCV sequences from the four male subjects and subsequent transmission from one subject to his female partner. Transmission was characterized by a strong genetic bottleneck. Viral genetic diversity was low during acute infection and increased upon progression to chronicity but subsequently fluctuated during chronic infection, caused by the alternate detection of distinct coexisting lineages. SMRT sequencing combines long reads with sufficient depth for many phylogenetic analyses and can therefore provide insights into within-host HCV evolutionary dynamics without the need for haplotype reconstruction using statistical algorithms.IMPORTANCE Next-generation sequencing has revolutionized the study of genetically variable RNA virus populations, but for phylogenetic and evolutionary analyses, longer sequences than those generated by most available platforms, while minimizing the intrinsic error rate, are desired. Here, we demonstrate for the first time that PacBio SMRT sequencing technology can be used to generate full-length HCV envelope sequences at the single-molecule level, providing a data set with large sequencing depth for the characterization of intrahost viral dynamics. The selection of consensus reads derived from at least 7 full circular consensus sequencing rounds significantly reduced the intrinsic high error rate of this method. We used this method to genetically characterize a unique transmission cluster of sexually transmitted HCV infections, providing insight into the distinct evolutionary pathways in each patient over time and identifying the transmission-associated genetic bottle-

Hepacivirus A Infection in Horses Defines Distinct Envelope Hypervariable Regions and Elucidates Potential Roles of Viral Strain and Adaptive Immune Status in Determining Envelope Diversity and Infection Outcome

“…PacBio single-molecule, real-time (SMRT) sequencing was performed on one to two samples before and after seroconversion in order to define the founder and evolving E1/E2 sequence variants. SMRT sequencing technology is coupled with circular consensus sequencing and was ideal for this analysis because it allowed the full-length envelope genes to be sequenced in a single read with minimal sequencing errors (23,24). The genetic diversity of E1/E2 quasispecies in each sample was determined from the predicted amino acid sequences by calculating the mean Hamming distance.…”

High resolution evolutionary analysis of within-host hepatitis C virus infection

“…The sequence data were generated by PacBio SMRT sequencing using stringent multiple passes to infer the circular consensus sequence (CCS) reads. This approach reduced the consensus sequence error rate from 1.6% to 0.4% and resulted in approximately 500 and 740 CCS reads per sample (11). Further information about the sequencing and individual samples can be found in C. K. Ho, et al (11).…”

“…(7)), but comparable studies for HCV are more challenging due to the high viral diversity observed during HCV infection, which makes it difficult to link infected individuals phylogenetically. In particular, there is a growing body of work indicating that within-host HCV diversity is maintained by multiple viral subpopulations with independent replication behaviour, where distinct long-lived lineages are intermittently detected in blood plasma over time (8)(9)(10)(11)(12). This observation implies that transmitted viruses are unlikely to be representative of the donor within-host HCV population sampled at a different point in time, and suggests there is potential for divergent viral strains to be transmitted from the same source individual.…”

“…to detect low-frequency viral variants and estimate transmission bottlenecks) such data are less suitable for reconstructing infection dynamics from sequences due to the large phylogenetic uncertainty associated with short reads. To address these challenges, we undertook an evolutionary analysis of full-length HCV envelope (E1/E2) gene sequences serially sampled from four patients, which were generated previously using Pacbio SMRT sequencing (12). The high resolution provided by long-read, high-throughput, serially-sampled virus sequences is ideal for studying the within-host evolution of chronic viruses, such as HIV and HCV, which are characterised by complex and genetically diverse viral populations.…”

Inconsistent temporal patterns of genetic variation of HCV among high-risk subjects may impact inference of transmission networks

“…A multitude of observational studies suggest that a substantial proportion of PWID carry mixed infections [18]. Furthermore, within-host evolutionary dynamics are complex [19][20][21][22][23][24][25][26]. Multiple co-existing lineages persist over time and are not necessarily detected at every sampling time [19,20,26], even using highthroughput sequencing [20,26], likely resulting from viral population structure [19,20,26,27].…”