Whole genome deep sequencing analysis of viral quasispecies diversity and evolution in HBeAg seroconverters

Su-Ru Lin; Ta-Yu Yang; Cheng-Yuan Peng; You-Yu Lin; Chia-Yen Dai; Hurng-Yi Wang; Tung-Hung Su; Tai-Chung Tseng; I-Jung Liu; Huei-Ru Cheng; Yueh-Chi Shen; Fang-Yi Wu; Chun-Jen Liu; Ding-Shinn Chen; Pei-Jer Chen; Hung-Chih Yang; Jia-Horng Kao

doi:10.1016/j.jhepr.2021.100254

Whole genome deep sequencing analysis of viral quasispecies diversity and evolution in HBeAg seroconverters

JHEP Rep. 2021 Feb 18;3(3):100254. doi: 10.1016/j.jhepr.2021.100254. eCollection 2021 Jun.

Authors

Su-Ru Lin¹, Ta-Yu Yang¹, Cheng-Yuan Peng^{2

3}, You-Yu Lin⁴, Chia-Yen Dai^{5

6}, Hurng-Yi Wang⁴, Tung-Hung Su^{7

8}, Tai-Chung Tseng^{7

8}, I-Jung Liu⁹, Huei-Ru Cheng⁴, Yueh-Chi Shen¹, Fang-Yi Wu¹, Chun-Jen Liu^{4

7

8

10

11}, Ding-Shinn Chen^{4

7

8

10

12}, Pei-Jer Chen^{4

7

8

10

11}, Hung-Chih Yang^{1

4

7

8}, Jia-Horng Kao^{4

7

8

10

11}

Affiliations

¹ Department of Microbiology, National Taiwan University, Taipei, Taiwan.
² School of Medicine, China Medical University, Taichung, Taiwan.
³ Department of Internal Medicine, Center for Digestive Medicine, China Medical University Hospital, Taichung, Taiwan.
⁴ Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan.
⁵ Department of Internal Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
⁶ Hepato-Biliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan.
⁷ Division of Gastroenterology and Hepatology, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan.
⁸ Hepatitis Research Center, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan.
⁹ Cardinal Tien Junior College of Healthcare and Management, New Taipei City, Taiwan.
¹⁰ Department of Internal Medicine, National Taiwan University, Taipei, Taiwan.
¹¹ Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan.
¹² Genomics Research Center, Academia Sinica, Taipei, Taiwan.

Abstract

Background & aims: We 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).

Methods: Fifty 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.

Results: HBV 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.

Conclusions: Higher HBV quasispecies diversity is associated with spontaneous HBeAg seroconversion and IFN-induced HBeAg seroconversion with low viraemia, conferring a favourable clinical outcome.

Lay summary: HBeAg 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.

Keywords: ALT, alanine aminotransferase; AUC, area under curve; BCP, basal core promoter; C, core; CHB, chronic hepatitis B; Chronic hepatitis B; EOT, end of treatment; HBeAg seroconversion; IFN, interferon; IFN-NR, IFN-non-responders; IFN-No-eSC, IFN-treated HBeAg non-seroconverters; IFN-RS, IFN-responders; IFN-eSC, IFN-treated HBeAg seroconverters; Intra-host single nucleotide variants; NGS, next-generation sequencing; ORFs, open reading frames; P, polymerase; S, surface; TN-No-eSC, treatment-naïve non-seroconverters; TN-eSC, treatment-naïve HBeAg seroconverters; dN, nonsynonymous substitution rate; dS, synonymous substitution rate; iSNVs, intra-host single-nucleotide variants.