Biogenesis and molecular characteristics of serum hepatitis B virus RNA

Shen, Sheng; Xie, Zhanglian; Yu, Xiaoyang; Zhang, Hu; Kim, Elena S.; Zhou, Bin; Hou, Jinlin; Zhang, Xiaoyong; Huang, Qi; Sun, Jian; Guo, Haitao

doi:10.1371/journal.ppat.1008945

Cited by 40 publications

(55 citation statements)

References 51 publications

(96 reference statements)

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“…The primers target a sequence upstream of DR2; the PCR amplicon is located between the 3 splicing site and the RT primer site. This yields a more specific amplicon, with better coverage of the spliced and 3 truncated forms, which are the predominant species of serum HBV pgRNA (Shen et al, 2020). The stability of pgRNA observed in this study might reflect the nature of the HBV RNA itself, and indicates encapsidation of the internal RNA, thus improving its resistance to the environment.…”

Section: Rattanachaisitmentioning

confidence: 78%

“…However, more evidence is needed to confirm this (Liu et al, 2019). In addition, serum HBV RNA is heterogeneous, comprising an intact genome length of 3.5-kb along with spliced and polyA-free pgRNA, and varies depending on the stage of chronic HBV infection, medication administered, and detection methods (Hacker et al, 2004;Liu et al, 2019;Shen et al, 2020;Wang et al, 2016). We used an HBV-specific primer pair as described by Wang et al (2016), which selectively amplified specific pgRNA species.…”

Section: Rattanachaisitmentioning

confidence: 99%

“…There is no evidence for the infectivity of these particles (Lu et al, 2017). HBV RNA-virions do not induce a productive infection in vitro, unlike the HBV DNA-virions (Shen et al, 2020).…”

Section: Introductionmentioning

confidence: 97%

“…Circulating serum HBV RNAs are heterogenous, with the major component being pgRNA species, which are localized either in the unenveloped capsid or virion (Bai et al, 2018). The encapsidated forms are detergent-and ribonuclease-resistant (Shen et al, 2020) and they bind with specific antibodies, forming capsid-antibody complexes (Bai et al, 2018). HBV pgRNA variants are of varying lengths, with some undergoing post-transcriptional splicing at the 5 terminal, while the others are 3 terminally truncated forms (Shen et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

“…The encapsidated forms are detergent-and ribonuclease-resistant (Shen et al, 2020) and they bind with specific antibodies, forming capsid-antibody complexes (Bai et al, 2018). HBV pgRNA variants are of varying lengths, with some undergoing post-transcriptional splicing at the 5 terminal, while the others are 3 terminally truncated forms (Shen et al, 2020). There is no evidence for the infectivity of these particles (Lu et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

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Stability of hepatitis B virus pregenomic RNA in plasma specimens under various temperatures and storage conditions

Rattanachaisit

Suksawatamnuay

Sriphoosanaphan

et al. 2021

PeerJ

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Background Hepatitis B virus (HBV) pregenomic RNA (pgRNA) has gained increasing attention owing to its role in replication of covalently closed circular DNA (cccDNA) in HBV. This marker has the potential to be used in clinical programs aimed to manage HBV infections. However, several reports on HBV pgRNA levels in clinical cases have conflicting results. RNA is easily degraded when exposed to heat and other environmental stressors. However, the stability of HBV pgRNA, during blood sample collection before the standard automated quantification, has never been estimated. This study aimed to demonstrate the effect of two different temperature conditions and storage durations on the stability of HBV pgRNA. Method Blood from forty patients with chronic hepatitis B infection, who also showed evidence of active HBV DNA replication, was collected and processed within 2 h of collection. Plasma from each patient was divided and stored at 4 °C and 25 °C (room temperature) for six different storage durations (0, 2, 6, 12, 24, and 48 h) and subsequently transferred to −80 °C for storage. The effect of multiple cycles of freezing and thawing of plasma at −20 °C or −80 °C was evaluated using samples from ten patients. Quantification of pgRNA from the samples was performed simultaneously, using the digital polymerase chain reaction (dPCR) method. The differences in pgRNA levels at baseline and each time point were compared using generalized estimating equation (GEE). A change greater than 0.5 log10 copies/mL of pgRNA is considered clinically significant. Statistical analyses were conducted using Stata 16.0. Results The mean HBV pgRNA level in the initially collected plasma samples was 5.58 log10copies/mL (ranging from 3.08 to 8.04 log10 copies/mL). The mean pgRNA levels in samples stored for different time periods compared with the initial reference sample (time 0) significantly decreased. The levels of pgRNA for 6, 12, 24, and 48 h of storage reduced by −0.05 log10 copies/mL (95% confidence interval (CI) −0.095 to −0.005, p = 0.03), −0.075 log10 copies/mL (95% CI [−0.12 to −0.03], p = 0.001), −0.084 log10 copies/mL (95% CI [−0.13 to −0.039], p = < 0.001), and −0.120 log10 copies/mL (95% CI [−0.17 to −0.076], p = < 0.001), respectively. However, these changes were below 0.5 log10 copies/mL and thus were not clinically significant. Compared with the samples stored at 4 °C, there were no significant differences in pgRNA levels in samples stored at 25 °C for any of the storage durations (−0.01 log10 copies/mL; 95% CI [−0.708 to 0.689], p = 0.98). No significant difference in the levels of pgRNA was observed in the plasma samples, following four freeze-thaw cycles at −20 °C and −80 °C. Conclusion The plasma HBV pgRNA level was stable at 4 °C and at room temperature for at least 48 h and under multiple freeze-thaw cycles. Our results suggest that pgRNA is stable during the process of blood collection, and therefore results of pgRNA quantification are reliable.

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Section: Rattanachaisitmentioning

confidence: 78%

Section: Rattanachaisitmentioning

confidence: 99%

“…There is no evidence for the infectivity of these particles (Lu et al, 2017). HBV RNA-virions do not induce a productive infection in vitro, unlike the HBV DNA-virions (Shen et al, 2020).…”

Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Stability of hepatitis B virus pregenomic RNA in plasma specimens under various temperatures and storage conditions

Rattanachaisit

Suksawatamnuay

Sriphoosanaphan

et al. 2021

PeerJ

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Letter to the Editor: Can the Ratio of Serum HBV RNA to DNA Reflect the Reverse‐Transcription Efficiency of Viral pgRNA?

Deng

Shi

et al. 2021

Hepatology

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B virus virions produced under nucleos(t)ide analogue treatment are mainly not infectious because of irreversible DNA chain termination. Hepatology 2020;71:463-476. 4) liu S, liu Z, li W, Zhou B, Liang X, Fan R, et al. Factors associated with the biphasic kinetics of serum HBV RNA in patients with HBeAg-positive chronic hepatitis B treated with nucleos(t)ide analogues. Aliment Pharmacol Ther 2020;52: 692-700. Author names in bold designate shared co-first authorship.

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Circulating HBV RNA: From biology to clinical applications

et al. 2022

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Chronic HBV infection can hardly be cured due to the persistence of an intrahepatic pool of viral covalently closed circular DNA (cccDNA) transcription template, which is refractory to current antivirals. The direct analyses of cccDNA quantity and transcriptional activity require an invasive biopsy. Recently, circulating HBV RNA has been identified as a promising noninvasive surrogate marker of cccDNA and can be used for monitoring disease progression and predicting prognosis of patients with chronic HBV infection. To better understand this surrogate biomarker of cccDNA, we reviewed the current knowledge about the molecular characteristics and potential clinical applications of circulating HBV RNA. Specifically, we summarized the reported species and existing forms of circulating HBV RNA and discussed their biogenesis and the capacity of de novo infection by RNA virions. Moreover, we described the potential applications of circulating HBV RNA in different clinical scenarios, such as classifying the phases of chronic HBV infection, analyzing sustained on‐treatment and off‐treatment outcomes of treated patients, as well as predicting HCC development. Perspectives on future research of circulating HBV RNA were also proposed in this review.

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Biogenesis and molecular characteristics of serum hepatitis B virus RNA

Cited by 40 publications

References 51 publications

Stability of hepatitis B virus pregenomic RNA in plasma specimens under various temperatures and storage conditions

Stability of hepatitis B virus pregenomic RNA in plasma specimens under various temperatures and storage conditions

Letter to the Editor: Can the Ratio of Serum HBV RNA to DNA Reflect the Reverse‐Transcription Efficiency of Viral pgRNA?

Circulating HBV RNA: From biology to clinical applications

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