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

Introduction

Chronic hepatitis B virus (HBV) infection is one of the leading causes of morbidity and mortality worldwide (WHO, 2017). Serum levels of HBV DNA and HBV surface antigen (HBsAg) are used to evaluate the efficacy of HBV treatment (Terrault et al., 2018). However, there are some limitations of their use in indicating the extent of HBV DNA replication. Recently, HBV pregenomic RNA (pgRNA) quantification is being used as a diagnostic marker to represent HBV cccDNA replication (Liu et al., 2019).

The HBV genome is a 3.2 kb long, relaxed, circular, and partially double-stranded DNA (Datta et al., 2012). After attaching to and entering the hepatocytes through endocytosis, the viral particles release the relaxed circular DNA (rcDNA) into the nucleus. The rcDNA is repaired to form a covalently closed circular DNA (cccDNA), which becomes the template for transcription of viral RNAs (Ganem & Schneider, 2001; Seeger & Mason, 2000). Viral RNAs transcribed from cccDNA consist of pregenomic RNA and subgenomic RNA. Pregenomic RNA is a template for reverse transcription of viral DNA and itself translates into precore, core, and polymerase proteins (Datta et al., 2012). New viral particles are assembled from pregenomic RNA, using viral polymerases, in the cytoplasm. Viral polymerase reverse-transcribes some of the pgRNA back to relaxed circular DNA. This viral particle is either released from hepatocytes through the endoplasmic reticulum and Golgi complex modification or travels back to the nucleus for amplification of the cccDNA pool (Datta et al., 2012; Rehermann & Nascimbeni, 2005). A recent study showed that pgRNA is also encapsidated and released from infected hepatocytes (HBV RNA virion-like particles) (Jansen et al., 2016; Wang et al., 2016).

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). 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).

Pregenomic RNA level is used as an indicator of HBV cccDNA replication. Theoretically, this novel marker would be superior to HBsAg (HBsAg (q)), since the latter can be synthesized from both cccDNA and integrated DNA, resulting in decreased specificity as marker for viral replication (Cornberg et al., 2017). Clinical applications of quantitation of pgRNA levels include monitoring of the treatment efficacy of nucleot(s)ide analogs (NAs) and interferons and determination of when to terminate NA treatment (Huang et al., 2015; Jansen et al., 2016; Tsuge et al., 2013).

In general, RNA is easily degraded by various factors during quantitation, including temperature, storage time, ribonuclease (RNase), or contamination with inhibitors (Sanders et al., 2018). RNA stability was confirmed before the clinical application of various RNA assays, such as those for human immunodeficiency virus (HIV) and hepatitis C virus (HCV). HIV RNA in plasma can be stored at room temperature (25 °C) and 4 °C for up to 3 days (Sebire et al., 1998), and HCV RNA in blood remains stable at 4 °C for up to 4 days but stability decreases during storage at 23 °C or 37 °C (De Moreaude Gerbehaye et al., 2002; Krajden et al., 1999).

HBV pgRNA stability under different temperatures and storage durations has not been previously estimated. In addition, stability of the encapsidated form of HBV RNA virion-like particles in blood samples during laboratory processes remains unknown. These estimates are crucial for the use of HBV pgRNA in comparative clinical studies and diagnosis. Thus, we aimed to instigate the effect of two different temperature conditions and storage durations on the stability of HBV pgRNA.

Material and Methods

This study was approved by the Institutional Review Board of Faculty of Medicine, Chulalongkorn University (IRB number 765/61).

Results

From the initial forty plasma samples, HBV pgRNA levels ranged from 3.08 to 8.04 log₁₀ copies/mL with a mean of 5.58 log₁₀ copies/mL (Fig. 1). Changes in HBV pgRNA levels for different durations of storage and temperature are expressed, with mean, standard deviation (SD), minimum (min), median (P50), 25th percentile (P25), 75th percentile (P75), and maximum (max) values, in Table 1. Compared to the initial reference sample (time 0), using GEE, the mean changes in pgRNA levels were found to be statistically significant. The pgRNA levels of samples stored for 6, 12, 24, and 48 h decreased from the initial values and are presented in Fig. 2 and Table 2.

Since the differences in pgRNA levels for the different storage durations were within 0.5 log₁₀ copies/mL, these changes are not clinically significant. There were also no significant changes in pgRNA levels between the two storage temperatures (−0.01 log₁₀ copies/mL; 95% CI [−0.708 to 0.689], p = 0.98) (Fig. 2).

There was no significant difference in the levels of pgRNA in the samples, following the freeze-thaw cycles (Fig. 3). Four cycles of freezing and thawing did not result in significant ratio changes in HBV pgRNA levels in any of the samples tested ( p = 0.45 and 0.59 for −20 °C and −80 °C, respectively) (Table 3).

Discussion

HBV pgRNA levels are increasingly being used as a biomarker of intrahepatic cccDNA replication, and have been studied for their role in HBV pathogenesis and treatment. However, there exists no standard for collection, processing, and storage to ensure the accuracy and reproducibility of quantification tests (Liu et al., 2019).

Based on our results, we conclude that blood samples collected in EDTA, and processed at 4 °C within 2 h of collection can be stored at 4 °C and 25 °C for up to 48 h without significantly affecting pgRNA levels. In addition, there was no significant difference in the levels of HBV pgRNA following four freeze-thaw cycles. The aforementioned time duration for blood collection and processing can be applied to real-world situations, in which the time from blood collection to quantification is within 48 h before being shipped to a central laboratory for further processing. The two temperature points investigated in our study are frequently used for storage and transport of blood samples. In addition, multiple freeze-thaw cycles at −20 °C and −80 °C are frequently used in clinical research.

For accuracy and consistency of the quantification process, the automated system for RNA extraction used in this study allowed for less human interference and RNase contamination. The co-extracted DNA that remained after DNase reaction was tested using HBV DNA-specific and housekeeping DNA primers. In the 8 selected samples, DNA did not remain after the DNase reaction.

However, there was a slight decrease in the HBV pgRNA level in samples 29 and 33 (Figs. S1 and S2, and Table S1). One explanation for this decrease might stem from an oxidation reaction (Relova et al., 2018). Thus, shorter period of handling before storage and controlled low-temperature transfer would reduce this variation.

Some of the mean changes in HBV pgRNA levels, from the baseline levels, for storage duration and temperature were found to be significantly different (Table 1). A generalized estimating equation (GEE), adjusting for repeated measurements in each sample, was applied to build a statistical model for repeated measurements. The GEE findings indicated significant changes in pgRNA levels in samples with a storage duration beyond 6 h (Table 2). However, these findings were only an illustration of the statistical reliability. In terms of clinical application, these changes are acceptable and not significant, because clinical differences are considered significant only when the change in pgRNA levels is more than 0.5 log₁₀ copies/mL (Baleriola et al., 2011; Pawlotsky, 1997).

Currently, there is no standard method for serum HBV pgRNA detection (Liu et al., 2019). Digital PCR (dPCR), in addition to its excellent sensitivity and quantification of the target molecule, does not need a standard controlled curve, as those required in real-time or other quantitative PCR assays (qPCR) (Cao et al., 2017). It also sensitive to very low levels of circulating nucleic acids in blood (Kuypers & Jerome, 2017). A study comparing HBV DNA detection using qPCR and dPCR showed lower copy number detection and better reproducibility of the dPCR assay, which improves sensitivity and specificity for serum HBV DNA measurements (Tang et al., 2016). In this study, we performed pgRNA quantification by dPCR to ensure that samples with very low titers of HBV pgRNA could be detected.

Species and forms of serum HBV RNA have not yet been clearly identified. Wang et al. reported that HBV RNA detected in the supernatant is pgRNA contained in the enveloped particles (Wang et al., 2016). 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. 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.

This study had several limitations. In future investigations, only the robust reproduction of specimen handling, processing, and storage techniques, described in our study, can result in pgRNA stabilities similar to that reported in this study. Other storage times and temperatures cannot be applied. Second, we measured only pgRNA specific to the HBV pgRNA-specific primer. There are no data on variation in the levels of other HBV RNA species. Our method, however, can be applied as a future reference. Finally, this stability was observed in patients under the current approved HBV treatment. Patients recruited in this study were either NA naïve or under NA treatment at the time of the study. NA treatment does not interfere with the encapsidation of HBV virions. However, some novel anti-HBV drugs that target pgRNA, such as (Z)-2-(allylamino)-4-amino- N′-cyanothiazole-5-carboximidamide (AACC), inhibit pgRNA and polymerase action, resulting in capsid assembly inhibition and encapsidated pgRNA reduction (Jo et al., 2020). There is still a lack of data on HBV pgRNA levels in patients using these new drugs.

Conclusions

We have demonstrated the stability of the plasma level of HBV pgRNA at 4 °C and room temperature for up to 48 h and following multiple freeze-thaw cycles before it is quantified. Our results confirmed the stability of pgRNA using the established strategy of blood collection and storage, and thus, quantitation of pgRNA is reliable.

Supplemental Information