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Ultra-deep sequencing of VHSV isolates contributes to understanding the role of viral quasispecies.

ABSTRACT: The high mutation rate of RNA viruses enables the generation of a genetically diverse viral population, termed a quasispecies, within a single infected host. This high in-host genetic diversity enables an RNA virus to adapt to a diverse array of selective pressures such as host immune response and switching between host species. The negative-sense, single-stranded RNA virus, viral haemorrhagic septicaemia virus (VHSV), was originally considered an epidemic virus of cultured rainbow trout in Europe, but was later proved to be endemic among a range of marine fish species in the Northern hemisphere. To better understand the nature of a virus quasispecies related to the evolutionary potential of VHSV, a deep-sequencing protocol specific to VHSV was established and applied to 4 VHSV isolates, 2 originating from rainbow trout and 2 from Atlantic herring. Each isolate was subjected to Illumina paired end shotgun sequencing after PCR amplification and the 11.1 kb genome was successfully sequenced with an average coverage of 0.5-1.9 × 10(6) sequenced copies. Differences in single nucleotide polymorphism (SNP) frequency were detected both within and between isolates, possibly related to their stage of adaptation to host species and host immune reactions. The N, M, P and Nv genes appeared nearly fixed, while genetic variation in the G and L genes demonstrated presence of diverse genetic populations particularly in two isolates. The results demonstrate that deep sequencing and analysis methodologies can be useful for future in vivo host adaption studies of VHSV.

SUBMITTER: Schonherz AA

PROVIDER: S-EPMC4705744 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Project description:Background & aimsWe aimed to investigate how viral quasispecies of the HBV whole genome evolves and diversifies in response to HBeAg seroconversion and viral control utilising next-generation sequencing (NGS).MethodsFifty HBeAg-positive chronic hepatitis B patients, including 18 treatment-naïve and 32 interferon (IFN)-treated individuals, were recruited. Serial HBV whole genomes in serum were analysed by NGS to determine sequence characteristics and viral quasispecies.ResultsHBV quasispecies diversity, measured by nucleotide diversity, was negatively correlated with viral load and hepatitis activity. Spontaneous HBeAg seroconverters exhibited significantly greater viral quasispecies diversity than treatment-naïve non-seroconverters from >1 year before seroconversion (0.0112 vs. 0.0060, p <0.01) to >1 year after seroconversion (0.0103 vs. 0.0068, p <0.01). IFN-induced HBeAg seroconverters tended to have higher viral genetic diversity than non-seroconverters along with treatment. Particularly, the IFN responders, defined as IFN-induced HBeAg seroconversion with low viraemia, exhibited significantly greater genetic diversity of whole HBV genome at 6 months post-IFN treatment than IFN non-responders (0.0148 vs. 0.0106, p = 0.048). Moreover, spontaneous HBeAg seroconverters and IFN responders exhibited significantly higher evolutionary rates and more intra-host single-nucleotide variants. Interestingly, in spontaneous HBeAg seroconverters and IFN responders, there were distinct evolutionary patterns in the HBV genome.ConclusionsHigher HBV quasispecies diversity is associated with spontaneous HBeAg seroconversion and IFN-induced HBeAg seroconversion with low viraemia, conferring a favourable clinical outcome.Lay summaryHBeAg seroconversion is a landmark in the natural history of chronic HBV infection. Using next-generation sequencing, we found that the nucleotide diversity of HBV was negatively correlated with viral load and hepatitis activity. Patients undergoing HBeAg seroconversion had more diverse HBV genomes and a faster viral evolution rate. Our findings suggest HBeAg seroconversion is driven by host selection pressure, likely immune selection pressure.

Project description:BACKGROUND: Ultra-deep pyrosequencing (UDPS) allows identification of rare HIV-1 variants and minority drug resistance mutations, which are not detectable by standard sequencing. PRINCIPAL FINDINGS: Here, UDPS was used to analyze the dynamics of HIV-1 genetic variation in reverse transcriptase (RT) (amino acids 180-220) in six individuals consecutively sampled before, during and after failing 3TC and AZT containing antiretroviral treatment. Optimized UDPS protocols and bioinformatic software were developed to generate, clean and analyze the data. The data cleaning strategy reduced the error rate of UDPS to an average of 0.05%, which is lower than previously reported. Consequently, the cut-off for detection of resistance mutations was very low. A median of 16,016 (range 2,406-35,401) sequence reads were obtained per sample, which allowed detection and quantification of minority resistance mutations at amino acid position 181, 184, 188, 190, 210, 215 and 219 in RT. In four of five pre-treatment samples low levels (0.07-0.09%) of the M184I mutation were observed. Other resistance mutations, except T215A and T215I were below the detection limit. During treatment failure, M184V replaced M184I and dominated the population in combination with T215Y, while wild-type variants were rarely detected. Resistant virus disappeared rapidly after treatment interruption and was undetectable as early as after 3 months. In most patients, drug resistant variants were replaced by wild-type variants identical to those present before treatment, suggesting rebound from latent reservoirs. CONCLUSIONS: With this highly sensitive UDPS protocol preexisting drug resistance was infrequently observed; only M184I, T215A and T215I were detected at very low levels. Similarly, drug resistant variants in plasma quickly decreased to undetectable levels after treatment interruption. The study gives important insights into the dynamics of the HIV-1 quasispecies and is of relevance for future research and clinical use of the UDPS technology.

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Ultra-deep sequencing of VHSV isolates contributes to understanding the role of viral quasispecies.

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