Serum HBV RNA Correlated with Intrahepatic cccDNA More Strongly than other HBV Markers During Peg-Interferon Treatment

Serum HBV RNA Correlated with Intrahepatic cccDNA More Strongly than other HBV Markers During Peg-Interferon Treatment | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Serum HBV RNA Correlated with Intrahepatic cccDNA More Strongly than other HBV Markers During Peg-Interferon Treatment Xiaomei Wang, Xiumei Chi, Ruihong Wu, Hongqin Xu, Xiuzhu Gao, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-93361/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Jan, 2021 Read the published version in Virology Journal → Version 1 posted 10 You are reading this latest preprint version Abstract Background: Serum hepatitis B virus RNA (HBV RNA) has been reported to be a surrogate marker of intrahepatic cccDNA during nucleos(t)ide analogs therapy. However, whether HBV RNA is superior to other HBV markers reflecting cccDNA profile in HBeAg-positive patients during peg-interferon (peg-IFN) treatment was still unclear. Methods: Serum HBV RNA, HBcrAg, HBV DNA, and HBsAg were longitudinally assessed among 30 HBeAg-positive patients during 48-week peg-IFN treatment. The intrahepatic cccDNA was detected at baseline and week 48, respectively. The individual correlations between HBV RNA, HBcrAg, HBV DNA, HBsAg, and cccDNA were then statistically analyzed. Results: HBV RNA levels in patients with HBeAg seroconversion decreased more rapidly compared with those without HBeAg seroconversion. Among all patients, cccDNA correlated better with HBV RNA than with HBcrAg, HBV DNA, and HBsAg at baseline. After 48 weeks of treatment, cccDNA still correlated more strongly with HBV RNA than other HBV markers. For subsequent analysis, cccDNA positively correlated with HBV RNA and HBcrAg whereas did not correlate with HBV DNA and HBsAg in patients with HBeAg seroconversion. However, cccDNA highly correlated with HBV RNA and HBV DNA, moderately correlated with HBcrAg, while no correlation was observed between cccDNA and HBsAg in patients without HBeAg seroconversion. Conclusion: Serum HBV RNA correlated more strongly than HBcrAg, HBV DNA, and HBsAg with intrahepatic cccDNA levels before and after 48-week peg-IFN treatment. The level of serum HBV RNA may be a superior surrogate marker reflecting the intrahepatic cccDNA profile in HBeAg-positive patients during peg-IFN treatment. Trial registration: ClinicalTrials, NCT03546530. Registered 1 January 2015. https://clinicaltrials.gov/ct2/results?cond=&term=NCT03546530&cntry=&state=&city=&dist= Virology Gastroenterology & Hepatology Hepatitis B virus HBV RNA HBcrAg HBV DNA cccDNA Interferon Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Hepatitis B virus (HBV) infection is a major global health problem. Chronic HBV infection greatly increases the risk of terminal liver diseases, such as cirrhosis, hepatic decompensation, and hepatocellular carcinoma, which contributes to more than 780,000 deaths every year worldwide [ 1 , 2 ]. Currently, the goal for the treatment of HBV is to suppress viral replication to the lowest possible level thus halting disease progression. Nowadays, the approved agents for the treatment of chronic HBV infections mainly belong to two classes: pegylated interferon (peg-IFN) and nucleotide/nucleoside analogs (NAs)[ 3 ]. Among these agents, peg-IFN, which has the effects of suppressing HBV replication and immunomodulation, is still widely used in the clinic as the first-line therapeutic option for patients with HBeAg-positive chronic hepatitis B (CHB) [ 4 ]. Although antiviral agents can effectively reduce the serum HBV DNA level in CHB patients, complete elimination of HBV is difficult due to the existence of intrahepatic covalently closed circular DNA (cccDNA) [ 5 ]. HBV cccDNA, which serves as the template for the transcription of a 3.5-kb pregenomic RNA, can produce progeny viral DNA and proteins even in the absence of detectable HBV DNA or HBsAg in the blood [ 6 – 8 ]. Low levels of intrahepatic cccDNA predict a sustained virologic response after cessation of antiviral treatment. Therefore, quantitation of intrahepatic cccDNA is suggested to be a valuable marker in evaluating the cure of CHB and assessing treatment endpoints. However, the invasive nature of liver biopsy is the major obstacle for quantitation of cccDNA, which greatly limits the use of cccDNA as a marker in real-world clinical practice. Therefore, finding non-invasive surrogate markers of intrahepatic cccDNA is clinically meaningful. Several traditional serum markers, such as quantitative HBsAg and HBV DNA, have been proposed to reflect the intrahepatic cccDNA profile [ 9 , 10 ]. However, the correlations between cccDNA and those traditional markers are too weak to apply them in clinical practice [ 11 ]. Recently, due to being transcribed from cccDNA, serum HBV RNA has also been reported as a potential surrogate marker for reflecting the activity of intrahepatic cccDNA in CHB patients, as well as during NAs therapy [ 12 – 14 ]. Serum HBV RNA was also identified as a good predictor of HBeAg seroconversion in HBeAg-positive patients during therapy with peg-IFN [ 4 ]. In addition, a mouse model experiment revealed that HBV RNA correlated positively with cccDNA before treatment, whereas no correlation between them after 6 weeks of peg-IFN therapy [ 12 ]. HBV core-related antigen (HBcrAg) is another new valuable marker of HBV infection, which consists of three species of related proteins, including hepatitis B core antigen, hepatitis B e antigen, and a truncated 22KDa precore protein[ 15 ]. Serum HBcrAg was also considered to be correlated with cccDNA activity and was a good biomarker in predicting HBeAg seroconversion in patients treated with peg-IFN[ 16 , 17 ]. Despite the findings above, it still remains unclear whether the HBV RNA could offer better predictive performance than HBcrAg, HBV DNA, or HBsAg in reflecting the intrahepatic cccDNA among HBeAg-positive patients during treatment with peg-IFN. Materials And Methods Aim In the present study, the correlations of HBV RNA, HBcrAg, HBV DNA, and HBsAg with cccDNA were evaluated with the aim to find a better surrogate marker of cccDNA in HBeAg-positive patients with peg-IFN treatment. At the same time, the dynamic changes in serum HBV RNA were also investigated. Study participants A total of 30 HBeAg-positive, noncirrhotic CHB patients were recruited from a multicenter, randomized, controlled clinical trial between Mar. 2015 and Dec. 2017 (ClinicalTrials.gov Identifier: NCT03546530). The patients had completed the full course of 48 weeks treatment and had baseline and week 48 liver paired biopsy and serial serum during treatment. The patients were treated with peg-IFN (Kawin Technology, China) at a dosage of 1.5 µg/kg body weight once weekly for 48 weeks. Briefly, the inclusion criteria were as follows: 18 to 60 years old; positive HBsAg for at least 6 months; HBeAg-positive and anti-HBe negative; HBV DNA ≥ 10 5 IU/mL; ALT ≥ 2 and < 10 × the upper limit of normal; no treatment history. The criteria for exclusion were: positivity for antibodies against HCV, HDV, or HIV; other inflammatory diseases such as rheumatoid arthritis, diabetes, or autoimmune hepatitis; hypertension; kidney disease; or a recent history of infectious disease. This study was conducted according to the Helsinki Declaration of 1975 and the research was approved by the Ethics Committee of the First Hospital of Jilin University and all participating institutions. Written informed consent was obtained from all patients. Samples Peripheral serum samples were obtained from all patients at baseline and at week 4, week 12, week 24, week 36, and week 48 during treatment and were stored at -80 °C for biochemical, virological analyses. Paired liver biopsies were collected at baseline and week 48 and snap frozen in liquid nitrogen until cccDNA analyses. Standard laboratory assessments Serum HBV DNA was quantified by quantitative polymerase chain reaction (qPCR) using a Roche COBAS AmpliPrep/COBAS TaqMan system (Roche Diagnostics, Mannheim, Germany) with a lower detection limit of 20 IU/ml. HBV genotypes were determined by real-time PCR with a commercial kit (Shanghai ZJ Bio-Tech, China). HBsAg, HBeAg, anti-HBs, anti-HBe, and anti-HBc were quantitated by chemiluminescence microparticle immunoassays using the Architect i2000SR platform and Abbott Architect reagents (Abbott Laboratories, Chicago, IL) according to manufactures’ instruction. Laboratory assessments were performed at baseline, week 4, week 12, week 24, week 36, and week 48. Serum HBV RNA quantification Serum HBV RNA was quantitatively detected in the period of therapy, at baseline, week 4, week 12, week 24, week 36, and week 48. All samples were measured by previously reported methods [ 18 ]. Briefly, HBV RNA was extracted from 200 µl of serum. HBV DNA was digested with DNase I at 37 °C for 30 min. cDNA was synthesized using a Transcriptor First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany). Then, quantitative PCR was performed with 2 × RealStar Power Probe Mixture (Gen Star, Beijing, China) with a linear range of 5 × 10 3 copies/ml to 5 × 10 9 copies/ml and a lower detection limit of 1 × 10 3 copies/ml. Intrahepatic HBV cccDNA quantification Intrahepatic HBV cccDNA was quantified by fluorescent probe quantitative PCR assays (SUPBIO Biotechnology, Guangzhou, China). The detailed information for intrahepatic cccDNA measurement is as our previous reported study [ 19 ]. Serum HBcrAg quantification HBcrAg was measured using a fully automated Lumipulse chemiluminescence enzyme immunoassay (CLEIA) analyzer (Fujirebio Inc., Tokyo, Japan), according to the manufacturer’s instructions. Since the general analytic measurement range of this assay is from 1000 U/ml (3 log10 U/ml) to 10,000,000 U/ml (7 log10 U/ml), serial dilutions of the serum samples are required when the serum qHBcrAg level is above the detection limit of the assay. Statistical analysis The characteristics of the study participants are presented as the proportions, means or medians. Comparisons between the patients with HBeAg seroconversion and without HBeAg seroconversion were performed using the chi-squared test for categorical data and the Mann-Whitney U-test for continuous data. Serum HBV RNA, HBcrAg, HBV DNA, HBsAg, and intrahepatic cccDNA were expressed in the logarithm. Differences in mean log-transformed values were calculated using Student’s t-test or one-way ANOVA where it is appropriate. The Pearson’s correlation analysis was applied to the log-transformed values of intrahepatic and serum markers. P values of < 0.05 were considered significant. Analyses were performed using the statistical software SPSS version 18 for Windows PC (SPSS, Chicago, IL, USA). Results Patient characteristics at baseline Our study cohort consisted of 30 patients(18 males and 12 females). The mean age was 26.57 ± 4.17 years. After 48 weeks of peg-IFN treatment, 9 of the 30 patients achieved HBeAg seroconversion. For further comparison, the patients were categorized into two groups: SR group (patients achieving HBeAg seroconversion response, n = 9), NSR group (patients not achieving HBeAg seroconversion response, n = 21). As shown in Table 1 , the comparison of the baseline values between the SR and NSR groups are listed as follows: HBV RNA (7.72 ± 1.42 log 10 copies/mL vs. 7.75 ± 1.09 log 10 copies/mL, P = 0.959), cccDNA (median 30.0 copies/cell vs. median 34.4 copies/cell, P = 0.946), HBcrAg (5.24 ± 1.45 log 10 KU/mL vs. 5.48 ± 1.16 log 10 KU/mL, P = 0.636), HBV DNA (7.52 ± 1.05 log 10 IU/mL vs. 7.59 ± 1.15 log 10 IU/mL, P = 0.878), and HBsAg (3.59 ± 0.49 log 10 IU/mL vs. 3.86 ± 0.69 log 10 IU/mL, P = 0.310). From the above results, it could be seen that there were no significant differences existed in HBV RNA, cccDNA, HBcrAg, HBV DNA, and HBsAg at baseline between the SR and NSR groups. In addition, no significant differences in age, HBV genotype, or ALT (all P > 0.05) were observed between the two groups. Table 1 Baseline clinical characteristics and HBV RNA, HBV cccDNA, HBV DNA, HBsAg, and HBeAg levels in the SR and NSR groups. Characteristic All SR group NSR group P value a (N = 30) (N = 9) (N = 21) (SR vs. NSR) Age, yr (median) 26.57 ± 4.17 27.00 ± 3.08 26.38 ± 4.62 0.216 Gender (male/female) 18/12 8/1 10/11 0.03 HBV genotype (%) 0.614 B 10 (33.3%) 3 (33.3%) 7 (33.3%) C 20 (66.7%) 6 (66.7%) 14 (66.7%) ALT (U/L, median (range)) 120 (66.8–174.0) 131.0 (96.7–181.0) 115.0 (59.0-174.0) 0.380 HBV DNA (log 10 IU/mL) 7.57 ± 1.10 7.52 ± 1.05 7.59 ± 1.15 0.878 HBsAg (log 10 IU/mL) 3.78 ± 0.64 3.59 ± 0.49 3.86 ± 0.69 0.310 HBcrAg (log 10 KU/mL) 5.41 ± 1.23 5.24 ± 1.45 5.48 ± 1.16 0.636 HBV RNA (log 10 copies/mL) 7.73 ± 1.18 7.72 ± 1.42 7.75 ± 1.09 0.959 HBV cccDNA (copies/cell, median (range)) 33.8 (15.4–76.4) 30.0 (13.2-100.8) 34.4 (13.7–66.7) 0.946 ALT, alanine aminotransferase; HBV, hepatitis B virus; HBcrAg, hepatitis B core-related antigen; HBsAg, hepatitis B surface antigen; cccDNA, covalently closed circular DNA. Dynamic changes in HBV RNA, HBV DNA, and cccDNA levels during 48 weeks of peg-IFN treatment Among all patients, all levels of serum HBV RNA, HBV DNA and intrahepatic cccDNA showed a rapid decline from baseline to week 48 (from 7.73 ± 1.18 log 10 copies/mL to 4.66 ± 1.56 log 10 copies/mL for HBV RNA; from 7.57 ± 1.10 log 10 IU/mL to 4.22 ± 2.45 log 10 IU/mL for HBV DNA; from median 33.8 copies/cell to 2.53 copies/cell for cccDNA). In patients with HBeAg seroconversion, the HBV RNA levels decreased more rapidly than those without HBeAg seroconversion. At week 48, serum HBV RNA levels were significantly lower in the SR group than the NSR group (3.56 ± 0.64 log 10 copies/mL vs. 5.00 ± 1.60 log 10 copies/mL, P = 0.002) (Fig. 1 A). In the SR group, serum HBV RNA levels decreased more slowly than serum HBV DNA levels, whereas, both HBV RNA and HBV DNA levels decreased similarly in the NSR group (Fig. 1 A). During the 48-week peg-IFN treatment period, the cccDNA levels decreased significantly from a median value of 30.2 copies/cell to 1.94 copies/cell in the SR group and from a median value of 34.4 copies/cell to 3.23 copies/cell in the NSR group. No difference in cccDNA decline was observed between the SR and NSR groups (Fig. 1 B). Correlations of cccDNA with HBV RNA, HBcrAg, HBV DNA, and HBsAg before and after 48 -week IFN treatment To further evaluate the performance of HBV RNA, HBcrAg, HBV DNA, HBsAg in reflecting cccDNA, the correlation values of each parameter were calculated. Among all patients, intrahepatic cccDNA was positively correlated with serum HBV RNA, HBcrAg, HBV DNA, and HBsAg at baseline. However, cccDNA correlated better with HBV RNA (r = 0.781, P < 0.001) than with HBcrAg (r = 0.741, P < 0.001), HBV DNA (r = 0.664, P < 0.001), and HBsAg (r = 0.484, P = 0.005) (Fig. 2 A- 2 D). At 48 weeks after peg-IFN treatment, intrahepatic cccDNA was still significantly correlated with serum HBV RNA, HBV DNA and HBcrAg ( r = 0.728, P < 0.001; r = 0.655, P < 0.001; r = 0.721, P < 0.001. Figure 3 A, 3 B, 3 C, respectively), while no correlations were observed between cccDNA and HBsAg levels ( r = 0.361, P = 0.064. Figure 3 D). After analyzing the different groups of CHB patients separately at week 48, we found that significant positive correlations of intrahepatic cccDNA with serum HBV RNA ( r = 0.709, P = 0.020. Figure 4 A) and HBcrAg ( r = 0.735, P = 0.014. Figure 4 B) rather than HBV DNA ( r = 0.335, P = 0.102, Fig. 4 C) or HBsAg ( r = 0.354, P = 0.349, Fig. 4 D) were observed in the SR group. Different from the SR group, cccDNA correlated strongly with HBV RNA ( r = 0.766, P < 0.001. Figure 5 A) and HBV DNA ( r = 0.820, P < 0.001. Figure 5 C), moderately with HBcrAg ( r = 0.558, P = 0.008; Fig. 5 B), but not with HBsAg (Fig. 5 D) in the NSR group. In addition, we also analyzed the correlation of HBV RNA with other serum markers among all patients. The results showed that serum HBV RNA levels strongly correlated with HBcrAg ( r = 0.793, P < 0.001. Figure 6 A) and HBV DNA( r = 0.594, P < 0.001, Fig. 6 B) at baseline, and the correlations of HBV RNA with HBcrAg ( r = 0.673, P < 0.001. Figure 6 D) and HBV DNA ( r = 0.907, P < 0.001. Figure 6 E) were still positive after 48 weeks peg-IFN treatment. However, HBV RNA only moderately correlated with HBsAg both at baseline and after 48 weeks of treatment ( r = 0.457, P = 0.010 at baseline; r = 0.314, P = 0.090 at week 48; Fig. 6 C, 6 F). Discussion In previous studies, both serum HBV RNA and HBcrAg were reported to be potential surrogate biomarkers of cccDNA in predicting the outcomes of peg-IFN or NAs treatments [ 4 , 20 , 21 ]. The correlation of cccDNA with HBV RNA has also been studied before and after nucleos(t)ide analog treatment [ 5 , 21 , 22 ]. However, it is still unclear whether serum HBV RNA or HBcrAg is better as the surrogate marker of intrahepatic cccDNA. In the present study, we analyzed the correlations of intrahepatic cccDNA with serum HBV RNA, HBcrAg, HBV DNA, and HBsAg before and after peg-IFN treatment in HBeAg-positive CHB patients, to determine which factor has the strongest correlation with intrahepatic cccDNA and could be a better surrogate marker of intrahepatic cccDNA. Furthermore, the kinetics of HBV RNA and intrahepatic cccDNA among all patients were also analyzed. The results showed that the baseline levels of serum HBV RNA or HBcrAg were similar in patients with and without HBeAg seroconversion (all P > 0.05), which indicated that HBV RNA and HBcrAg at baseline may be not associated with HBeAg-seroconversion in HBeAg-positive patients treated with peg-IFN. During peg-IFN treatment, HBV RNA declined more rapidly in patients with HBeAg seroconversion than those without HBeAg seroconversion. After 48-week peg-IFN treatment, a smaller decrease of serum HBV RNA than serum HBV DNA was observed in the SR group, whereas HBV RNA and HBV DNA decreased parallel in the NSR group. Previous studies have reported that cccDNA reduced rapidly in the first year of antiviral treatment [ 23 , 24 ]. Our study also showed that peg-IFN could profoundly reduce the load of cccDNA after 1 year of treatment. In addition, we found that the reduction of cccDNA in the SR group was greater than that in the NSR group. The possible reason was that Peg-IFN can suppress the activity and transcription of cccDNA, which may result in increased clearance rates of HBV RNA, HBcrAg, and cccDNA [ 25 ]. Our study also showed that intrahepatic cccDNA levels correlated best with serum HBV RNA( r = 0.781) than HBcrAg ( r = 0.741), HBV DNA ( r = 0.664), and HBsAg ( r = 0.484) at baseline, and the correlation between cccDNA and HBV RNA was still strong (r = 0.728, P < 0.001) after 48 weeks of peg-IFN treatment. While HBsAg did not correlate with cccDNA (r = 0.361, P = 0.064). These data indicated that serum HBV RNA might be a superior serological marker for the persistence of active cccDNA molecules during peg-IFN treatment. Our results were different from Gao’s study, which identified that serum HBV RNA only had a weak correlation with cccDNA in a 96-week NAs treatment [ 5 ]. The difference could be attributed to the different antiviral mechanisms of NAs and peg-IFN. Interestingly, an animal experiment in HBV-infected mice found that there was no correlation between serum HBV RNA and intrahepatic cccDNA after 6 weeks of peg-IFN therapy [ 12 ]. The discrepancies might because the mouse model could not fully reflect the real conditions in clinical patients. It has been reported that serum HBcrAg was better than HBV RNA in reflecting intrahepatic cccDNA levels in treatment naive patients [ 17 ]. However, our study showed that although HBcrAg and HBV RNA have a similar correlation with cccDNA at baseline, the correlation of cccDNA with HBcrAg became weaker than with HBV RNA after 48 weeks of peg-IFN therapy. We further compared the correlations of cccDNA with HBV RNA, HBV DNA, and HBsAg between patients with and without HBeAg seroconversion after 48 weeks of peg-IFN treatment. The results showed that HBV RNA and HBcrAg were positively correlated with cccDNA both in the SR and NSR groups at week 48 (HBV RNA: r = 0.709, P = 0.002 in the SR group; r = 0.766, P < 0.001 in the NSR group. HBcrAg: r = 0.735, P = 0.014 in the SR group; r = 0.662, P = 0.008 in the NSR group). HBV DNA was strongly correlated with cccDNA in the NSR group ( r = 0.818, P < 0.001), but no positive correlation was observed in the SR group ( r = 0.335, P = 0.102). The correlation between HBsAg and cccDNA was not found in either the SR group (r = 0.354, P = 0.349) or the NSR group (r = 0.374, P = 0.094). The results indicated that HBV RNA and HBcrAg were better markers than HBV DNA and HBsAg in reflecting cccDNA no matter the results of antiviral treatment. Although previous studies have shown that serum HBsAg quantification may reflect the level of intrahepatic cccDNA both in HBeAg-positive and HBeAg-negative CHB patients [ 26 , 27 ], our study showed that cccDNA weakly correlated with HBsAg before treatment and did not correlate with HBsAg after 48 weeks of peg-IFN therapy. This pattern possibly because HBsAg could be produced not only from cccDNA in infected hepatocytes but also from viral sequences integrated into the host genome [ 28 ]. These data indicated that serum HBsAg level could not accurately reflect the level of intrahepatic cccDNA in the cohort of our study. Our results also showed that serum HBV RNA correlated well with HBcrAg at baseline and at week 48 with peg-IFN treatment, most likely because HBcrAg was translated from preC mRNA which is also transcribed from cccDNA just like HBV RNA [ 17 ]. In contrast, the HBV RNA level decreased significantly, whereas the HBsAg level remained stable after 48 weeks of treatment. As a result, no significant correlation between HBV RNA and HBsAg ( r = 0.314, P = 0.090) was observed after 48 weeks of IFN treatment.. Due to the difficulty of acquiring liver samples both at baseline and after 48 weeks of peg-IFN therapy, the sample size in our study was small and the patients enrolled were mainly those infected with HBV genotype B or C. The selection bias might have affected the internal validity of this study. Future studies are required to recruit more patients and other HBV genotypes. Conclusions Both Serum HBV RNA and HBcrAg correlated significantly with intrahepatic cccDNA before and after 48-week peg-IFN treatment in HBeAg-positive patients. However, serum HBV RNA correlated with cccDNA more strongly than HBcrAg, HBV DNA, and HBsAg, irrespective of treatment results. HBV RNA also correlated well with HBcrAg before and after peg-IFN therapy. The study indicated that serum HBV RNA may be a better surrogate non-invasive marker in reflecting the intrahepatic cccDNA profile during peg-IFN treatment. Abbreviations HBV: Hepatitis B virus; HBV RNA: hepatitis B virus RNA; HBcrAg: HBV core-related antigen; ALT:lanine aminotransferase; cccDNA: Covalently closed circular DNA; CHB: Chronic hepatitis B; HBeAg: Hepatitis B e antigen; HBsAg: Hepatitis B surface antigen; Peg-IFN; Pegylated interferon; NAs: nucleotide/nucleoside analogs. Declarations Ethics approval and consent to participate The study protocol was approved by the Ethics Committee of the First Hospital of Jilin University and all participating institutions. All patients gave written informed consent for their participation in the study. Consent for publication Not applicable. Availability of data and materials The data analyzed in the current study is available from the corresponding author on reasonable request. Conflicts of Interest The authors declare no conflicts of interest. F unding This study was supported by the National Science and Technology Major Project of China (2018ZX10732101, 2014ZX10002002) and the Program for JLU Science and Technology Innovative Research Team (2017TD-08). Author ’s c ontributions Xiaomei Wang performed the research. Xiumei Chi, Xiuzhu Gao, Lei Yu, Longgen Liu, Mingxiang Zhang, and Youwen Tan acquired the data. Ruihong Wu and Hongqin Xu analyzed the data. 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Bowden S, Locarnini S, Chang TT, Chao YC, Han KH, Gish RG, de Man RA, Yu M, Llamoso C, Tang H: Covalently closed-circular hepatitis B virus DNA reduction with entecavir or lamivudine. World J Gastroenterol 2015, 21:4644-4651. Belloni L, Allweiss L, Guerrieri F, Pediconi N, Volz T, Pollicino T, Petersen J, Raimondo G, Dandri M, Levrero M: IFN-alpha inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome. J Clin Invest 2012, 122:529-537. Martinot-Peignoux M, Lapalus M, Asselah T, Marcellin P: HBsAg quantification: useful for monitoring natural history and treatment outcome. Liver Int 2014, 34 Suppl 1:97-107. Chan HL, Wong VW, Tse AM, Tse CH, Chim AM, Chan HY, Wong GL, Sung JJ: Serum hepatitis B surface antigen quantitation can reflect hepatitis B virus in the liver and predict treatment response. Clin Gastroenterol Hepatol 2007, 5:1462-1468. Chuaypen N, Sriprapun M, Praianantathavorn K, Payungporn S, Wisedopas N, Poovorawan Y, Tangkijvanich P: Kinetics of serum HBsAg and intrahepatic cccDNA during pegylated interferon therapy in patients with HBeAg-positive and HBeAg-negative chronic hepatitis B. J Med Virol 2017, 89:130-138. Cite Share Download PDF Status: Published Journal Publication published 06 Jan, 2021 Read the published version in Virology Journal → Version 1 posted Review # 2 received at journal 04 Nov, 2020 Editorial decision: Major revision 04 Nov, 2020 Review # 1 received at journal 28 Oct, 2020 Reviewer # 2 agreed at journal 22 Oct, 2020 Reviewers invited by journal 15 Oct, 2020 Reviewer # 1 agreed at journal 15 Oct, 2020 Editor assigned by journal 14 Oct, 2020 Submission checks completed at journal 13 Oct, 2020 Editor invited by journal 13 Oct, 2020 First submitted to journal 12 Oct, 2020 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiumei","middleName":"","lastName":"Chi","suffix":""},{"id":3584080,"identity":"6669d014-b131-4a4d-be0e-8c283ae3cf52","order_by":2,"name":"Ruihong Wu","email":"","orcid":"","institution":"The First Hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ruihong","middleName":"","lastName":"Wu","suffix":""},{"id":3584081,"identity":"5ef12539-d4f1-4380-b7fd-9aea26b19842","order_by":3,"name":"Hongqin Xu","email":"","orcid":"","institution":"The First Hospital of Jilin University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongqin","middleName":"","lastName":"Xu","suffix":""},{"id":3584082,"identity":"0d0fd972-6131-4316-9d27-c8145749b5a9","order_by":4,"name":"Xiuzhu Gao","email":"","orcid":"","institution":"The First Hospital of Jilin 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Jin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9klEQVRIiWNgGAWjYDACZiB+AMSMzQwMBg8YbHj4+RuI0JIA05LAkCYjOeMAETYlIBiHbQwaEnCrBAG+47yHXyRU3LFrbuc9UJDYdp7HgOEA44ePObi1SB7mS7NIOPMsubGZL8Egse02jzlzA7PkzG24tRgc5jEDqjyczNjMYwDWYtlwgI2ZlwQt53gMDiQQ1GL8AKjFDqrlAGEtkkBbGBLOHE4Aa0k4l8wjOeNgM16/8J0/Y/zhQ8Vhe8P+M2YGH8rs7Pn5mw9++IhHC8MBBjYJIJW4sYGBzQAixNiARz1YC/MHIGUvD0wID/ArHQWjYBSMgpEKANgxVNJafUGBAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-4626-9828","institution":"The First Hospital of Jilin University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Qinglong","middleName":"","lastName":"Jin","suffix":""}],"badges":[],"createdAt":"2020-10-15 18:45:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-93361/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-93361/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12985-020-01471-2","type":"published","date":"2021-01-06T15:03:19+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":3064092,"identity":"305774cb-dcf4-4975-b40d-5c906a021582","added_by":"auto","created_at":"2020-10-19 16:11:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":26778,"visible":true,"origin":"","legend":"Dynamic changes in serum HBV RNA and HBV DNA (A), and cccDNA (B) during the 48 weeks of treatment with peg-IFN in the SR group and the NSR group. ","description":"","filename":"OnlineFigure1.Png","url":"https://assets-eu.researchsquare.com/files/rs-93361/v1/6bb4f81dfda6136e4a2cf8a3.Png"},{"id":3064093,"identity":"296ce3e9-a53e-4bff-85ef-2a410780d5ac","added_by":"auto","created_at":"2020-10-19 16:11:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":38957,"visible":true,"origin":"","legend":"Correlations between the level of intrahepatic cccDNA and the level of serum HBV RNA (A), HBcrAg (B), HBV DNA (C), and HBsAg (D) among all patients at baseline. Solid line: linear growth tread; r: correlation coefficient.","description":"","filename":"OnlineFigure2.Png","url":"https://assets-eu.researchsquare.com/files/rs-93361/v1/4eb470f6c1b80648225cd5d1.Png"},{"id":3064094,"identity":"985e0560-4cf0-4c22-afea-64b4da9b9fbb","added_by":"auto","created_at":"2020-10-19 16:11:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":40595,"visible":true,"origin":"","legend":"Correlations between the level of intrahepatic cccDNA and the level of serum HBV RNA (A), HBcrAg (B), HBV DNA (C), and HBsAg (D) among all patients at week 48. Solid line: linear growth tread; r: correlation coefficient.","description":"","filename":"OnlineFigure3.Png","url":"https://assets-eu.researchsquare.com/files/rs-93361/v1/0401930f4966174478207263.Png"},{"id":3064095,"identity":"70d2bdd7-089f-41b1-982b-c6b1050eccdb","added_by":"auto","created_at":"2020-10-19 16:11:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":36097,"visible":true,"origin":"","legend":"Correlations between the level of intrahepatic cccDNA and the level of serum HBV RNA (A), HBcrAg (B), HBV DNA (C), and HBsAg (D) in the SR group at week 48. Solid line: linear growth tread; r: correlation coefficient.","description":"","filename":"OnlineFigure4.Png","url":"https://assets-eu.researchsquare.com/files/rs-93361/v1/f1f387ccad8cfdcffbb36d56.Png"},{"id":3064096,"identity":"392f0c5d-98ac-4bd3-9a3d-e1f8f74eea0b","added_by":"auto","created_at":"2020-10-19 16:11:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":41463,"visible":true,"origin":"","legend":"Correlations between the level of intrahepatic cccDNA and the level of serum HBV RNA (A), HBcrAg (B), HBV DNA (C), and HBsAg (D) in the NSR group at week 48. Solid line: linear growth tread; r: correlation coefficient.","description":"","filename":"OnlineFigure5.Png","url":"https://assets-eu.researchsquare.com/files/rs-93361/v1/79b6b3c1278c5f8dcdd5ff7d.Png"},{"id":3064097,"identity":"7c84f0f8-b4cb-4440-8b42-0b621b640214","added_by":"auto","created_at":"2020-10-19 16:11:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":58842,"visible":true,"origin":"","legend":"Correlation of serum the level of HBV RNA with HBcrAg (A), HBV DNA (B), and HBsAg (C) among all patients at baseline and with HBcrAg (D), HBV DNA (E), and HBsAg (F) at week 48. Solid line: linear growth tread; r: correlation coefficient.","description":"","filename":"OnlineFigure6.Png","url":"https://assets-eu.researchsquare.com/files/rs-93361/v1/cdca83c5088c11b78e7f84a9.Png"},{"id":13604197,"identity":"7c1cd875-9ad7-4799-be14-a48c510f7153","added_by":"auto","created_at":"2021-09-17 05:58:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":874897,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-93361/v1/92793b6e-fe2c-4607-8fb2-86401d5d7397.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eSerum HBV RNA Correlated with Intrahepatic cccDNA More Strongly than other HBV Markers During Peg-Interferon Treatment\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eHepatitis B virus (HBV) infection is a major global health problem. Chronic HBV infection greatly increases the risk of terminal liver diseases, such as cirrhosis, hepatic decompensation, and hepatocellular carcinoma, which contributes to more than 780,000 deaths every year worldwide [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Currently, the goal for the treatment of HBV is to suppress viral replication to the lowest possible level thus halting disease progression. Nowadays, the approved agents for the treatment of chronic HBV infections mainly belong to two classes: pegylated interferon (peg-IFN) and nucleotide/nucleoside analogs (NAs)[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among these agents, peg-IFN, which has the effects of suppressing HBV replication and immunomodulation, is still widely used in the clinic as the first-line therapeutic option for patients with HBeAg-positive chronic hepatitis B (CHB) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough antiviral agents can effectively reduce the serum HBV DNA level in CHB patients, complete elimination of HBV is difficult due to the existence of intrahepatic covalently closed circular DNA (cccDNA) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. HBV cccDNA, which serves as the template for the transcription of a 3.5-kb pregenomic RNA, can produce progeny viral DNA and proteins even in the absence of detectable HBV DNA or HBsAg in the blood [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Low levels of intrahepatic cccDNA predict a sustained virologic response after cessation of antiviral treatment. Therefore, quantitation of intrahepatic cccDNA is suggested to be a valuable marker in evaluating the cure of CHB and assessing treatment endpoints. However, the invasive nature of liver biopsy is the major obstacle for quantitation of cccDNA, which greatly limits the use of cccDNA as a marker in real-world clinical practice. Therefore, finding non-invasive surrogate markers of intrahepatic cccDNA is clinically meaningful.\u003c/p\u003e \u003cp\u003eSeveral traditional serum markers, such as quantitative HBsAg and HBV DNA, have been proposed to reflect the intrahepatic cccDNA profile [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. However, the correlations between cccDNA and those traditional markers are too weak to apply them in clinical practice [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecently, due to being transcribed from cccDNA, serum HBV RNA has also been reported as a potential surrogate marker for reflecting the activity of intrahepatic cccDNA in CHB patients, as well as during NAs therapy [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Serum HBV RNA was also identified as a good predictor of HBeAg seroconversion in HBeAg-positive patients during therapy with peg-IFN [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition, a mouse model experiment revealed that HBV RNA correlated positively with cccDNA before treatment, whereas no correlation between them after 6 weeks of peg-IFN therapy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHBV core-related antigen (HBcrAg) is another new valuable marker of HBV infection, which consists of three species of related proteins, including hepatitis B core antigen, hepatitis B e antigen, and a truncated 22KDa precore protein[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Serum HBcrAg was also considered to be correlated with cccDNA activity and was a good biomarker in predicting HBeAg seroconversion in patients treated with peg-IFN[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Despite the findings above, it still remains unclear whether the HBV RNA could offer better predictive performance than HBcrAg, HBV DNA, or HBsAg in reflecting the intrahepatic cccDNA among HBeAg-positive patients during treatment with peg-IFN.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAim\u003c/h2\u003e \u003cp\u003eIn the present study, the correlations of HBV RNA, HBcrAg, HBV DNA, and HBsAg with cccDNA were evaluated with the aim to find a better surrogate marker of cccDNA in HBeAg-positive patients with peg-IFN treatment. At the same time, the dynamic changes in serum HBV RNA were also investigated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eStudy participants\u003c/h2\u003e \u003cp\u003eA total of 30 HBeAg-positive, noncirrhotic CHB patients were recruited from a multicenter, randomized, controlled clinical trial between Mar. 2015 and Dec. 2017 (ClinicalTrials.gov Identifier: NCT03546530). The patients had completed the full course of 48 weeks treatment and had baseline and week 48 liver paired biopsy and serial serum during treatment. The patients were treated with peg-IFN (Kawin Technology, China) at a dosage of 1.5\u0026nbsp;\u0026micro;g/kg body weight once weekly for 48 weeks. Briefly, the inclusion criteria were as follows: 18 to 60\u0026nbsp;years old; positive HBsAg for at least 6 months; HBeAg-positive and anti-HBe negative; HBV DNA\u0026thinsp;\u0026ge;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e IU/mL; ALT\u0026thinsp;\u0026ge;\u0026thinsp;2 and \u0026lt;\u0026thinsp;10\u0026thinsp;\u0026times;\u0026thinsp;the upper limit of normal; no treatment history. The criteria for exclusion were: positivity for antibodies against HCV, HDV, or HIV; other inflammatory diseases such as rheumatoid arthritis, diabetes, or autoimmune hepatitis; hypertension; kidney disease; or a recent history of infectious disease.\u003c/p\u003e \u003cp\u003eThis study was conducted according to the Helsinki Declaration of 1975 and the research was approved by the Ethics Committee of the First Hospital of Jilin University and all participating institutions. Written informed consent was obtained from all patients.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSamples\u003c/h2\u003e \u003cp\u003ePeripheral serum samples were obtained from all patients at baseline and at week 4, week 12, week 24, week 36, and week 48 during treatment and were stored at -80\u0026nbsp;\u0026deg;C for biochemical, virological analyses. Paired liver biopsies were collected at baseline and week 48 and snap frozen in liquid nitrogen until cccDNA analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eStandard laboratory assessments\u003c/h2\u003e \u003cp\u003eSerum HBV DNA was quantified by quantitative polymerase chain reaction (qPCR) using a Roche COBAS AmpliPrep/COBAS TaqMan system (Roche Diagnostics, Mannheim, Germany) with a lower detection limit of 20\u0026nbsp;IU/ml. HBV genotypes were determined by real-time PCR with a commercial kit (Shanghai ZJ Bio-Tech, China). HBsAg, HBeAg, anti-HBs, anti-HBe, and anti-HBc were quantitated by chemiluminescence microparticle immunoassays using the Architect i2000SR platform and Abbott Architect reagents (Abbott Laboratories, Chicago, IL) according to manufactures\u0026rsquo; instruction. Laboratory assessments were performed at baseline, week 4, week 12, week 24, week 36, and week 48.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eSerum HBV RNA quantification\u003c/h2\u003e \u003cp\u003eSerum HBV RNA was quantitatively detected in the period of therapy, at baseline, week 4, week 12, week 24, week 36, and week 48. All samples were measured by previously reported methods [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Briefly, HBV RNA was extracted from 200\u0026nbsp;\u0026micro;l of serum. HBV DNA was digested with DNase I at 37\u0026nbsp;\u0026deg;C for 30\u0026nbsp;min. cDNA was synthesized using a Transcriptor First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany). Then, quantitative PCR was performed with 2\u0026thinsp;\u0026times;\u0026thinsp;RealStar Power Probe Mixture (Gen Star, Beijing, China) with a linear range of 5\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e copies/ml to 5\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e9\u003c/sup\u003e copies/ml and a lower detection limit of 1\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e copies/ml.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIntrahepatic HBV cccDNA quantification\u003c/h2\u003e \u003cp\u003eIntrahepatic HBV cccDNA was quantified by fluorescent probe quantitative PCR assays (SUPBIO Biotechnology, Guangzhou, China). The detailed information for intrahepatic cccDNA measurement is as our previous reported study [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSerum HBcrAg quantification\u003c/h2\u003e \u003cp\u003eHBcrAg was measured using a fully automated Lumipulse chemiluminescence enzyme immunoassay (CLEIA) analyzer (Fujirebio Inc., Tokyo, Japan), according to the manufacturer\u0026rsquo;s instructions. Since the general analytic measurement range of this assay is from 1000\u0026nbsp;U/ml (3 log10 U/ml) to 10,000,000\u0026nbsp;U/ml (7 log10 U/ml), serial dilutions of the serum samples are required when the serum qHBcrAg level is above the detection limit of the assay.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe characteristics of the study participants are presented as the proportions, means or medians. Comparisons between the patients with HBeAg seroconversion and without HBeAg seroconversion were performed using the chi-squared test for categorical data and the Mann-Whitney U-test for continuous data. Serum HBV RNA, HBcrAg, HBV DNA, HBsAg, and intrahepatic cccDNA were expressed in the logarithm. Differences in mean log-transformed values were calculated using Student\u0026rsquo;s t-test or one-way ANOVA where it is appropriate. The Pearson\u0026rsquo;s correlation analysis was applied to the log-transformed values of intrahepatic and serum markers. \u003cem\u003eP\u003c/em\u003e values of \u0026lt;\u0026thinsp;0.05 were considered significant. Analyses were performed using the statistical software SPSS version 18 for Windows PC (SPSS, Chicago, IL, USA).\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003ePatient characteristics at baseline\u003c/h2\u003e\n\u003cp\u003eOur study cohort consisted of 30 patients(18 males and 12 females). The mean age was 26.57\u0026thinsp;\u0026plusmn;\u0026thinsp;4.17\u0026nbsp;years. After 48 weeks of peg-IFN treatment, 9 of the 30 patients achieved HBeAg seroconversion. For further comparison, the patients were categorized into two groups: SR group (patients achieving HBeAg seroconversion response, n\u0026thinsp;=\u0026thinsp;9), NSR group (patients not achieving HBeAg seroconversion response, n\u0026thinsp;=\u0026thinsp;21).\u003c/p\u003e\n\u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, the comparison of the baseline values between the SR and NSR groups are listed as follows: HBV RNA (7.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42 log\u003csub\u003e10\u003c/sub\u003e copies/mL \u003cem\u003evs.\u003c/em\u003e 7.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09 log\u003csub\u003e10\u003c/sub\u003e copies/mL, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.959), cccDNA (median 30.0 copies/cell \u003cem\u003evs.\u003c/em\u003e median 34.4 copies/cell, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.946), HBcrAg (5.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45 log\u003csub\u003e10\u003c/sub\u003e KU/mL \u003cem\u003evs.\u003c/em\u003e 5.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16 log\u003csub\u003e10\u003c/sub\u003e KU/mL, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.636), HBV DNA (7.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05 log\u003csub\u003e10\u003c/sub\u003e IU/mL \u003cem\u003evs.\u003c/em\u003e 7.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 log\u003csub\u003e10\u003c/sub\u003e IU/mL, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.878), and HBsAg (3.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 log\u003csub\u003e10\u003c/sub\u003e IU/mL \u003cem\u003evs.\u003c/em\u003e 3.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69 log\u003csub\u003e10\u003c/sub\u003e IU/mL, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.310). From the above results, it could be seen that there were no significant differences existed in HBV RNA, cccDNA, HBcrAg, HBV DNA, and HBsAg at baseline between the SR and NSR groups. In addition, no significant differences in age, HBV genotype, or ALT (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) were observed between the two groups.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eBaseline clinical characteristics and HBV RNA, HBV cccDNA, HBV DNA, HBsAg, and HBeAg levels in the SR and NSR groups.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth rowspan=\"2\" align=\"left\"\u003e\n\u003cp\u003eCharacteristic\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eAll\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSR group\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eNSR group\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e(N\u0026thinsp;=\u0026thinsp;30)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e(N\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e(N\u0026thinsp;=\u0026thinsp;21)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e(SR \u003cem\u003evs.\u003c/em\u003e NSR)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAge, yr (median)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e26.57\u0026thinsp;\u0026plusmn;\u0026thinsp;4.17\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e27.00\u0026thinsp;\u0026plusmn;\u0026thinsp;3.08\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e26.38\u0026thinsp;\u0026plusmn;\u0026thinsp;4.62\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.216\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGender (male/female)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e18/12\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8/1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10/11\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.03\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHBV genotype (%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.614\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eB\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10 (33.3%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3 (33.3%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7 (33.3%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eC\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20 (66.7%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6 (66.7%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14 (66.7%)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eALT (U/L, median (range))\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e120 (66.8\u0026ndash;174.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e131.0 (96.7\u0026ndash;181.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e115.0 (59.0-174.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.380\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHBV DNA (log\u003csub\u003e10\u003c/sub\u003e IU/mL)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.52\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.59\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.878\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHBsAg (log\u003csub\u003e10\u003c/sub\u003e IU/mL)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.310\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHBcrAg (log\u003csub\u003e10\u003c/sub\u003e KU/mL)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.41\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.16\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.636\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHBV RNA (log\u003csub\u003e10\u003c/sub\u003e copies/mL)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.42\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.959\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eHBV cccDNA (copies/cell, median (range))\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e33.8 (15.4\u0026ndash;76.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e30.0 (13.2-100.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e34.4 (13.7\u0026ndash;66.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e0.946\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"5\"\u003eALT, alanine aminotransferase; HBV, hepatitis B virus; HBcrAg, hepatitis B core-related antigen; HBsAg, hepatitis B surface antigen; cccDNA, covalently closed circular DNA.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eDynamic changes in HBV RNA, HBV DNA, and cccDNA levels during 48 weeks of peg-IFN treatment\u003c/h2\u003e\n\u003cp\u003eAmong all patients, all levels of serum HBV RNA, HBV DNA and intrahepatic cccDNA showed a rapid decline from baseline to week 48 (from 7.73\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18 log\u003csub\u003e10\u003c/sub\u003e copies/mL to 4.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56 log\u003csub\u003e10\u003c/sub\u003e copies/mL for HBV RNA; from 7.57\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10 log\u003csub\u003e10\u003c/sub\u003e IU/mL to 4.22\u0026thinsp;\u0026plusmn;\u0026thinsp;2.45 log\u003csub\u003e10\u003c/sub\u003e IU/mL for HBV DNA; from median 33.8 copies/cell to 2.53 copies/cell for cccDNA). In patients with HBeAg seroconversion, the HBV RNA levels decreased more rapidly than those without HBeAg seroconversion. At week 48, serum HBV RNA levels were significantly lower in the SR group than the NSR group (3.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64 log\u003csub\u003e10\u003c/sub\u003e copies/mL \u003cem\u003evs.\u003c/em\u003e 5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60 log\u003csub\u003e10\u003c/sub\u003e copies/mL, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). In the SR group, serum HBV RNA levels decreased more slowly than serum HBV DNA levels, whereas, both HBV RNA and HBV DNA levels decreased similarly in the NSR group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). During the 48-week peg-IFN treatment period, the cccDNA levels decreased significantly from a median value of 30.2 copies/cell to 1.94 copies/cell in the SR group and from a median value of 34.4 copies/cell to 3.23 copies/cell in the NSR group. No difference in cccDNA decline was observed between the SR and NSR groups (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n\u003ch2\u003eCorrelations of cccDNA with HBV RNA, HBcrAg, HBV DNA, and HBsAg before and after 48 -week IFN treatment\u003c/h2\u003e\n\u003cp\u003eTo further evaluate the performance of HBV RNA, HBcrAg, HBV DNA, HBsAg in reflecting cccDNA, the correlation values of each parameter were calculated. Among all patients, intrahepatic cccDNA was positively correlated with serum HBV RNA, HBcrAg, HBV DNA, and HBsAg at baseline. However, cccDNA correlated better with HBV RNA (r\u0026thinsp;=\u0026thinsp;0.781, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) than with HBcrAg (r\u0026thinsp;=\u0026thinsp;0.741, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), HBV DNA (r\u0026thinsp;=\u0026thinsp;0.664, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and HBsAg (r\u0026thinsp;=\u0026thinsp;0.484, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA-\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). At 48 weeks after peg-IFN treatment, intrahepatic cccDNA was still significantly correlated with serum HBV RNA, HBV DNA and HBcrAg (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.728, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.655, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.721, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC, respectively), while no correlations were observed between cccDNA and HBsAg levels (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.361, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.064. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e\n\u003cp\u003eAfter analyzing the different groups of CHB patients separately at week 48, we found that significant positive correlations of intrahepatic cccDNA with serum HBV RNA (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.709, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.020. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA) and HBcrAg (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.735, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.014. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB) rather than HBV DNA (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.335, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.102, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC) or HBsAg (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.354, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.349, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD) were observed in the SR group. Different from the SR group, cccDNA correlated strongly with HBV RNA (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.766, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA) and HBV DNA (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.820, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC), moderately with HBcrAg (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.558, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB), but not with HBsAg (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD) in the NSR group.\u003c/p\u003e\n\u003cp\u003eIn addition, we also analyzed the correlation of HBV RNA with other serum markers among all patients. The results showed that serum HBV RNA levels strongly correlated with HBcrAg (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.793, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA) and HBV DNA(\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.594, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB) at baseline, and the correlations of HBV RNA with HBcrAg (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.673, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD) and HBV DNA (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.907, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE) were still positive after 48 weeks peg-IFN treatment. However, HBV RNA only moderately correlated with HBsAg both at baseline and after 48 weeks of treatment (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.457, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.010\u0026nbsp;at baseline; \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.314, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.090\u0026nbsp;at week 48; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":" \u003cp\u003eIn previous studies, both serum HBV RNA and HBcrAg were reported to be potential surrogate biomarkers of cccDNA in predicting the outcomes of peg-IFN or NAs treatments [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The correlation of cccDNA with HBV RNA has also been studied before and after nucleos(t)ide analog treatment [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, it is still unclear whether serum HBV RNA or HBcrAg is better as the surrogate marker of intrahepatic cccDNA.\u003c/p\u003e \u003cp\u003eIn the present study, we analyzed the correlations of intrahepatic cccDNA with serum HBV RNA, HBcrAg, HBV DNA, and HBsAg before and after peg-IFN treatment in HBeAg-positive CHB patients, to determine which factor has the strongest correlation with intrahepatic cccDNA and could be a better surrogate marker of intrahepatic cccDNA. Furthermore, the kinetics of HBV RNA and intrahepatic cccDNA among all patients were also analyzed.\u003c/p\u003e \u003cp\u003eThe results showed that the baseline levels of serum HBV RNA or HBcrAg were similar in patients with and without HBeAg seroconversion (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), which indicated that HBV RNA and HBcrAg at baseline may be not associated with HBeAg-seroconversion in HBeAg-positive patients treated with peg-IFN. During peg-IFN treatment, HBV RNA declined more rapidly in patients with HBeAg seroconversion than those without HBeAg seroconversion. After 48-week peg-IFN treatment, a smaller decrease of serum HBV RNA than serum HBV DNA was observed in the SR group, whereas HBV RNA and HBV DNA decreased parallel in the NSR group. Previous studies have reported that cccDNA reduced rapidly in the first year of antiviral treatment [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Our study also showed that peg-IFN could profoundly reduce the load of cccDNA after 1\u0026nbsp;year of treatment. In addition, we found that the reduction of cccDNA in the SR group was greater than that in the NSR group. The possible reason was that Peg-IFN can suppress the activity and transcription of cccDNA, which may result in increased clearance rates of HBV RNA, HBcrAg, and cccDNA [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study also showed that intrahepatic cccDNA levels correlated best with serum HBV RNA(\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.781) than HBcrAg (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.741), HBV DNA (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.664), and HBsAg (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.484) at baseline, and the correlation between cccDNA and HBV RNA was still strong (r\u0026thinsp;=\u0026thinsp;0.728, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) after 48 weeks of peg-IFN treatment. While HBsAg did not correlate with cccDNA (r\u0026thinsp;=\u0026thinsp;0.361, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.064). These data indicated that serum HBV RNA might be a superior serological marker for the persistence of active cccDNA molecules during peg-IFN treatment. Our results were different from Gao\u0026rsquo;s study, which identified that serum HBV RNA only had a weak correlation with cccDNA in a 96-week NAs treatment [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The difference could be attributed to the different antiviral mechanisms of NAs and peg-IFN. Interestingly, an animal experiment in HBV-infected mice found that there was no correlation between serum HBV RNA and intrahepatic cccDNA after 6 weeks of peg-IFN therapy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The discrepancies might because the mouse model could not fully reflect the real conditions in clinical patients.\u003c/p\u003e \u003cp\u003eIt has been reported that serum HBcrAg was better than HBV RNA in reflecting intrahepatic cccDNA levels in treatment naive patients [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. However, our study showed that although HBcrAg and HBV RNA have a similar correlation with cccDNA at baseline, the correlation of cccDNA with HBcrAg became weaker than with HBV RNA after 48 weeks of peg-IFN therapy.\u003c/p\u003e \u003cp\u003eWe further compared the correlations of cccDNA with HBV RNA, HBV DNA, and HBsAg between patients with and without HBeAg seroconversion after 48 weeks of peg-IFN treatment. The results showed that HBV RNA and HBcrAg were positively correlated with cccDNA both in the SR and NSR groups at week 48 (HBV RNA: \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.709, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002 in the SR group; \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.766, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 in the NSR group. HBcrAg: \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.735, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.014 in the SR group; \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.662, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.008 in the NSR group). HBV DNA was strongly correlated with cccDNA in the NSR group (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.818, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but no positive correlation was observed in the SR group (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.335, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.102). The correlation between HBsAg and cccDNA was not found in either the SR group (r\u0026thinsp;=\u0026thinsp;0.354, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.349) or the NSR group (r\u0026thinsp;=\u0026thinsp;0.374, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.094). The results indicated that HBV RNA and HBcrAg were better markers than HBV DNA and HBsAg in reflecting cccDNA no matter the results of antiviral treatment.\u003c/p\u003e \u003cp\u003eAlthough previous studies have shown that serum HBsAg quantification may reflect the level of intrahepatic cccDNA both in HBeAg-positive and HBeAg-negative CHB patients [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], our study showed that cccDNA weakly correlated with HBsAg before treatment and did not correlate with HBsAg after 48 weeks of peg-IFN therapy. This pattern possibly because HBsAg could be produced not only from cccDNA in infected hepatocytes but also from viral sequences integrated into the host genome [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These data indicated that serum HBsAg level could not accurately reflect the level of intrahepatic cccDNA in the cohort of our study.\u003c/p\u003e \u003cp\u003eOur results also showed that serum HBV RNA correlated well with HBcrAg at baseline and at week 48 with peg-IFN treatment, most likely because HBcrAg was translated from preC mRNA which is also transcribed from cccDNA just like HBV RNA [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In contrast, the HBV RNA level decreased significantly, whereas the HBsAg level remained stable after 48 weeks of treatment. As a result, no significant correlation between HBV RNA and HBsAg (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.314, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.090) was observed after 48 weeks of IFN treatment..\u003c/p\u003e \u003cp\u003eDue to the difficulty of acquiring liver samples both at baseline and after 48 weeks of peg-IFN therapy, the sample size in our study was small and the patients enrolled were mainly those infected with HBV genotype B or C. The selection bias might have affected the internal validity of this study. Future studies are required to recruit more patients and other HBV genotypes.\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eBoth Serum HBV RNA and HBcrAg correlated significantly with intrahepatic cccDNA before and after 48-week peg-IFN treatment in HBeAg-positive patients. However, serum HBV RNA correlated with cccDNA more strongly than HBcrAg, HBV DNA, and HBsAg, irrespective of treatment results. HBV RNA also correlated well with HBcrAg before and after peg-IFN therapy. The study indicated that serum HBV RNA may be a better surrogate non-invasive marker in reflecting the intrahepatic cccDNA profile during peg-IFN treatment.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eHBV: Hepatitis B virus; HBV RNA: hepatitis B virus RNA; HBcrAg: HBV core-related antigen; ALT:lanine aminotransferase; cccDNA: Covalently closed circular DNA; CHB: Chronic hepatitis B; HBeAg: Hepatitis B e antigen; HBsAg: Hepatitis B surface antigen; Peg-IFN; Pegylated interferon; NAs: nucleotide/nucleoside analogs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the Ethics Committee of the First Hospital of Jilin University and all participating institutions. All patients gave written informed consent for their participation in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data analyzed in the current study is available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eF\u003c/strong\u003e\u003cstrong\u003eunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Science and Technology Major Project of China (2018ZX10732101, 2014ZX10002002) and the Program for JLU Science and Technology Innovative Research Team (2017TD-08).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;s c\u003c/strong\u003e\u003cstrong\u003eontributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiaomei Wang performed the research. Xiumei Chi, Xiuzhu Gao, Lei Yu, Longgen Liu, Mingxiang Zhang, and Youwen Tan acquired the data. Ruihong Wu and Hongqin Xu analyzed the data. Junqi Niu and Qinglong Jin edited, reviewed, and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful for all subjects who participated in this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGanem D, Prince AM: Hepatitis B virus infection--natural history and clinical consequences. \u003cem\u003eN Engl J Med \u003c/em\u003e2004, 350:1118-1129.\u003c/li\u003e\n\u003cli\u003eLau GK, Piratvisuth T, Luo KX, Marcellin P, Thongsawat S, Cooksley G, Gane E, Fried MW, Chow WC, Paik SW, et al: Peginterferon Alfa-2a, lamivudine, and the combination for HBeAg-positive chronic hepatitis B. \u003cem\u003eN Engl J Med \u003c/em\u003e2005, 352:2682-2695.\u003c/li\u003e\n\u003cli\u003eLiver EAFTSOT: EASL clinical practice guidelines: Management of chronic hepatitis B virus infection. \u003cem\u003eJ Hepatol \u003c/em\u003e2012, 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\u003cem\u003eJ Viral Hepat \u003c/em\u003e2019, 26:586-595.\u003c/li\u003e\n\u003cli\u003eWang J, Shen T, Huang X, Kumar GR, Chen X, Zeng Z, Zhang R, Chen R, Li T, Zhang T, et al: Serum hepatitis B virus RNA is encapsidated pregenome RNA that may be associated with persistence of viral infection and rebound. \u003cem\u003eJ Hepatol \u003c/em\u003e2016, 65:700-710.\u003c/li\u003e\n\u003cli\u003eWang X, Wang Z, Chi X, Wu R, Jin Q, Xu H, Gao X, Yu L, Chen Y, Shang J, et al: Efficacy of a combination of HBV RNA and HBeAg in predicting HBeAg seroconversion in patients treated with entecavir for 144 weeks. \u003cem\u003eInt J Infect Dis \u003c/em\u003e2020, 99:171-178.\u003c/li\u003e\n\u003cli\u003eZhang M, Li G, Shang J, Pan C, Zhang M, Yin Z, Xie Q, Peng Y, Mao Q, Xiao X, et al: Rapidly decreased HBV RNA predicts responses of pegylated interferons in HBeAg-positive patients: a longitudinal cohort study. \u003cem\u003eHepatol Int \u003c/em\u003e2020, 14:212-224.\u003c/li\u003e\n\u003cli\u003eLuo H, Tan N, Kang Q, Pan J, Chen H, Xi H, Yu M, Xu X: Hepatitis B virus pregenomic RNA status can reveal the long-term prognoses of chronic hepatitis B patients treated with nucleos(t)ide analogues. \u003cem\u003eJ Viral Hepat \u003c/em\u003e2020, 27:323-328.\u003c/li\u003e\n\u003cli\u003eWong DK, Seto WK, Cheung KS, Chong CK, Huang FY, Fung J, Lai CL, Yuen MF: Hepatitis B virus core-related antigen as a surrogate marker for covalently closed circular DNA. \u003cem\u003eLiver Int \u003c/em\u003e2017, 37:995-1001.\u003c/li\u003e\n\u003cli\u003eWong DK, Seto WK, Fung J, Ip P, Huang FY, Lai CL, Yuen MF: Reduction of hepatitis B surface antigen and covalently closed circular DNA by nucleos(t)ide analogues of different potency. \u003cem\u003eClin Gastroenterol Hepatol \u003c/em\u003e2013, 11:1004-1010 e1001.\u003c/li\u003e\n\u003cli\u003eBowden S, Locarnini S, Chang TT, Chao YC, Han KH, Gish RG, de Man RA, Yu M, Llamoso C, Tang H: Covalently closed-circular hepatitis B virus DNA reduction with entecavir or lamivudine. \u003cem\u003eWorld J Gastroenterol \u003c/em\u003e2015, 21:4644-4651.\u003c/li\u003e\n\u003cli\u003eBelloni L, Allweiss L, Guerrieri F, Pediconi N, Volz T, Pollicino T, Petersen J, Raimondo G, Dandri M, Levrero M: IFN-alpha inhibits HBV transcription and replication in cell culture and in humanized mice by targeting the epigenetic regulation of the nuclear cccDNA minichromosome. \u003cem\u003eJ Clin Invest \u003c/em\u003e2012, 122:529-537.\u003c/li\u003e\n\u003cli\u003eMartinot-Peignoux M, Lapalus M, Asselah T, Marcellin P: HBsAg quantification: useful for monitoring natural history and treatment outcome. \u003cem\u003eLiver Int \u003c/em\u003e2014, 34 Suppl 1:97-107.\u003c/li\u003e\n\u003cli\u003eChan HL, Wong VW, Tse AM, Tse CH, Chim AM, Chan HY, Wong GL, Sung JJ: Serum hepatitis B surface antigen quantitation can reflect hepatitis B virus in the liver and predict treatment response. \u003cem\u003eClin Gastroenterol Hepatol \u003c/em\u003e2007, 5:1462-1468.\u003c/li\u003e\n\u003cli\u003eChuaypen N, Sriprapun M, Praianantathavorn K, Payungporn S, Wisedopas N, Poovorawan Y, Tangkijvanich P: Kinetics of serum HBsAg and intrahepatic cccDNA during pegylated interferon therapy in patients with HBeAg-positive and HBeAg-negative chronic hepatitis B. \u003cem\u003eJ Med Virol \u003c/em\u003e2017, 89:130-138.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hepatitis B virus, HBV RNA, HBcrAg, HBV DNA, cccDNA, Interferon","lastPublishedDoi":"10.21203/rs.3.rs-93361/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-93361/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eSerum hepatitis B virus RNA (HBV RNA) has been reported to be a surrogate marker of intrahepatic cccDNA during nucleos(t)ide analogs therapy. However, whether HBV RNA is superior to other HBV markers reflecting cccDNA profile in HBeAg-positive patients during peg-interferon (peg-IFN) treatment was still unclear.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eSerum HBV RNA, HBcrAg, HBV DNA, and HBsAg were longitudinally assessed among 30 HBeAg-positive patients during 48-week peg-IFN treatment. The intrahepatic cccDNA was detected at baseline and week 48, respectively. The individual correlations between HBV RNA, HBcrAg, HBV DNA, HBsAg, and cccDNA were then statistically analyzed.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eHBV RNA levels in patients with HBeAg seroconversion decreased more rapidly compared with those without HBeAg seroconversion. Among all patients, cccDNA correlated better with HBV RNA than with HBcrAg, HBV DNA, and HBsAg at baseline. After 48 weeks of treatment, cccDNA still correlated more strongly with HBV RNA than other HBV markers. For subsequent analysis, cccDNA positively correlated with HBV RNA and HBcrAg whereas did not correlate with HBV DNA and HBsAg in patients with HBeAg seroconversion. However, cccDNA highly correlated with HBV RNA and HBV DNA, moderately correlated with HBcrAg, while no correlation was observed between cccDNA and HBsAg in patients without HBeAg seroconversion.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eSerum HBV RNA correlated more strongly than HBcrAg, HBV DNA, and HBsAg with intrahepatic cccDNA levels before and after 48-week peg-IFN treatment. The level of serum HBV RNA may be a superior surrogate marker reflecting the intrahepatic cccDNA profile in HBeAg-positive patients during peg-IFN treatment.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTrial registration:\u003c/strong\u003e ClinicalTrials, NCT03546530. Registered 1 January 2015. https://clinicaltrials.gov/ct2/results?cond=\u0026amp;term=NCT03546530\u0026amp;cntry=\u0026amp;state=\u0026amp;city=\u0026amp;dist=\u003c/p\u003e","manuscriptTitle":"Serum HBV RNA Correlated with Intrahepatic cccDNA More Strongly than other HBV Markers During Peg-Interferon Treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-10-19 16:11:45","doi":"10.21203/rs.3.rs-93361/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2020-11-05T00:00:00+00:00","index":2,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"decision","content":"Major revision","date":"2020-11-05T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2020-10-28T12:00:00+00:00","index":1,"fulltext":"Recommendation: Reviewer's comments unavailable due to the journal's policy.\n"},{"type":"reviewerAgreed","content":"","date":"2020-10-22T12:00:00+00:00","index":2,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2020-10-15T12:00:00+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2020-10-15T12:00:00+00:00","index":1,"fulltext":""},{"type":"editorAssigned","content":"","date":"2020-10-14T12:00:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2020-10-13T12:00:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2020-10-13T12:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"","date":"2020-10-12T12:00:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"929c87c4-8149-4fad-9b88-d7feb294b263","owner":[],"postedDate":"October 19th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":821566,"name":"Virology"},{"id":821567,"name":"Gastroenterology \u0026 Hepatology"}],"tags":[],"updatedAt":"2021-01-10T15:06:02+00:00","versionOfRecord":{"articleIdentity":"rs-93361","link":"https://doi.org/10.1186/s12985-020-01471-2","journal":{"identity":"virology-journal","isVorOnly":false,"title":"Virology Journal"},"publishedOn":"2021-01-06 15:03:19","publishedOnDateReadable":"January 6th, 2021"},"versionCreatedAt":"2020-10-19 16:11:45","video":"","vorDoi":"10.1186/s12985-020-01471-2","vorDoiUrl":"https://doi.org/10.1186/s12985-020-01471-2","workflowStages":[]},"version":"v1","identity":"rs-93361","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-93361","identity":"rs-93361","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}