Kaposi's sarcoma-associated herpesvirus infection in Chinese patients with chronic hepatitis B†
The authors declare no conflict of interests.
Abstract
The seroprevalence of Kaposi's sarcoma-associated herpesvirus (KSHV) in Chinese patients with chronic hepatitis B, the relationship between KSHV and hepatitis B virus (HBV) infection, and the influence of glycyrrhizic acid on KSHV replication in vivo are undefined. Plasma was collected from 211 patients with chronic hepatitis B. Antibody to KSHV ORF65 was evaluated by ELISA, and real-time PCR was used to quantify KSHV DNA and HBV DNA. The KSHV ORF65 positivity rate in patients with chronic hepatitis B was found to be 28% (59/211): 27.3% (44/161) in males and 30% (15/50) in females (P > 0.05). The seroprevalence of KSHV increased with age until reaching the highest rate (37.1%) in the 31–40 years age group. HBV DNA loads in patients with chronic hepatitis B infected with KSHV were higher than those without KSHV infection (9.2 log (10) IU/ml vs. 7.8 log (10) IU/ml, P < 0.05). The average KSHV DNA loads in patients with HBV genotype B, C, and mixed (B/C) were 409.1, 484.5, and 352 copies/ml, respectively (P > 0.05). Patients treated with glycyrrhizic acid had lower KSHV DNA levels than those without therapy (204.7 copies/ml vs. 533.9 copies/ml, P < 0.05). The KSHV ORF65 positivity rates tended to increase with age, but were not related to gender or HBV genotypes. The data indicated the interaction between KSHV and HBV, and the inhibiting effect of glycyrrhizic acid on KSHV replication in patients with chronic hepatitis B. J. Med. Virol. 83:879–883, 2011. © 2011 Wiley-Liss, Inc.
INTRODUCTION
Although Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), has been recognized for over a decade, various aspects of the epidemiology of KSHV infection need to be defined clearly. For example, transmission routes and risk factors for infection by KSHV have not been elucidated completely. Studies suggest the importance of sexual [Martin et al., 1998; Eltom et al., 2002; Martro et al., 2007], vertical [Brayfield et al., 2003; Lisco et al., 2006], saliva [Duus et al., 2004; Taylor et al., 2004], blood [Dollard et al., 2005; Hladik et al., 2006], and transplant-related [Becuwe et al., 2005; Gentile et al., 2005] transmission of KSHV. In a study among 215 Ugandan children, adolescents, and young adults, KSHV antibodies to the latency-associated nuclear antigen (LANA) and the ORF65 protein were associated independently with hepatitis B infection in children younger than 12 years, and the KSHV infection rate increased with age before puberty [Mayama et al., 1998]. Mayama et al. [1998] suggested that the living conditions which predispose African children to the transmission of hepatitis B also favored infection with KSHV. A study of Jewish population groups in Israel found that the prevalence of KSHV was significantly higher (22.7%) among hepatitis B virus (HBV) seropositive blood donors compared with 14.1% of those who were negative to HBV, and chronic carriers of HBV infection (HBsAg positive) were more likely to have higher KSHV antibody titers [Davidovici et al., 2001]. Davidovici et al. [2001] suggested that both KSHV and HBV are transmitted via similar routes in blood donors. A cross-sectional study in healthy company employees in Greece found HBsAg-positive individuals had an increased risk of being KSHV-seropositive [Zavitsanou et al., 2007]. However, in contrast to the findings of these studies, others indicated that seropositivity for KSHV tended to be inversely related or not associated with HBV markers [Sarmati et al., 2003; Schinaia et al., 2004; Chironna et al., 2006].
Two billion people globally have been infected with HBV, and China has one of the highest rates of HBV endemicity in the world. Among persons older than 3 years in China, the prevalence rates of HBsAg were 9.09% [Liang et al., 2005]. The seroprevalence of KSHV in patients with chronic hepatitis B in China and the relationship between KSHV and HBV infection has not yet been defined.
KSHV is the etiologic agent of Kaposi's sarcoma, and it is also present in lesions in patients with multicentric Castleman disease and primary effusion lymphoma. Both Kaposi's sarcoma and primary effusion lymphoma display minimal response to antiherpesvirus drugs such as ganciclovir, cidofovir, and foscarnet [Robles et al., 1999; Little et al., 2003]. It has been reported that glycyrrhizic acid is effective for destroying primary effusion lymphoma cells that are transformed by KSHV in vitro [Curreli et al., 2005]; however, whether such effects could also occur in humans is not clear.
Taking advantage of numerous patients with chronic hepatitis B in our infectious disease center, the KSHV infection characteristics were studied in patients with chronic hepatitis B, the interaction between HBV and KSHV, and the effect of glycyrrhizic acid on KSHV replication in patients with chronic hepatitis B.
MATERIALS AND METHODS
Study Population and Samples
Anticoagulated blood was collected from 211 patients with chronic hepatitis B who were outpatients or inpatients (161 males and 50 females) recruited from the Infectious Disease Department of the First Affiliated Hospital of Zhejiang University Medical College. There were 19 patients aged 0–20 years, 53 aged 21–30, 62 aged 31–40, 36 aged 41–50, 15 aged 51–60, and 26 aged ≥61. Among them, 91 patients accepted anti-HBV treatment with interferon or nucleoside drugs while the others did not. HBV genotyping was performed as described previously [Pan et al., 2007]. Among these patients, 2 (0.95%) had HBV genotype A, 97 (45.9%) had genotype B, 88 (41.7%) had genotype C, 21 (9.95%) had mixed(B/C), and 3 (1.42%) had genotype D. Patients co-infected with the hepatitis C virus or HIV were excluded from this study. Informed consent was obtained from all adult patients and children's parents where applicable. The protocol was approved by the local ethics committee and performed in accordance with the Helsinki Declaration. Peripheral blood mononuclear cells (PBMCs) and plasma were separated immediately from samples by Ficoll density gradient centrifugation after the collection of blood. Plasma and PBMCs remained frozen at −70°C until use.
Serologic Testing for KSHV
Detection of KSHV-specific antibodies ORF65 in plasma was performed by an enzyme-linked immunosorbent assay (ELISA) as previously described [Zhu et al., 2008]. Briefly, the KSHV ORF65 recombinant protein was diluted in a 1 µg/ml, and a conventional ELISA protocol was performed. Test samples were diluted 1:100, and 100 µl of each sample was added to an ELISA plate for 2 hr at 37°C. A peroxidase-conjugated goat antihuman IgG (Sigma, Shanghai, China) (diluted, 1:10,000) was incubated for 30 min at 37°C to detect KSHV IgG antibodies. The color reaction was developed for 30 min at 37°C using tetramethylbenzidine (Sigma). The reactions were stopped with 50 µl of 1 M H2SO4. The mean OD450 of five KSHV-negative controls plus 3 SD was taken as the cut-off value. Positive and negative control sera were run in parallel to the studied samples in every test, and all plasma samples were tested blindly in duplicate. Samples with values between the cut-off ±0.05 were re-analyzed to confirm the results.
Quantitative PCR for KSHV
KSHV viral loads in plasma were measured by TaqMan real-time polymerase chain reaction (PCR) using a Lightcycler detection system (Roche, Basel, Switzerland) with primers and a probe that targeted the ORF26 region. Primers and the TaqMan probe were designed using Primer 3 online Software and synthesized by Sangon (Shanghai, China). The upstream and downstream primer sequences were 5′-GCTCGAGTCCAACGGATTG-3′ and 5′-AATGGCGTGCCCCAGTTGC-3′, respectively. The fluorogenic TaqMan probe was FAM-5′-TTCCCCATGGTCGTGCCGC-3′-TAMRA. To prepare the KSHV DNA standards, a 172 bp of KSHV ORF26 fragment was amplified using a nested PCR as described previously [Zhu et al., 2008]. The purified PCR products were cloned into competent E. coli cells using a PCR 2.1 TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA), and the inserts were sequenced using a BigDye sequencing kit and an ABI 377 automatic sequencer. The resulting plasmid was used as a standard to quantify the KSHV gene copy number. Standard curves (with duplicate serial 10-fold dilutions of plasmid DNA from 103 to 107 copies) were run in parallel with each analysis. The 40 µl amplification mix consisted 4 µl of sample, 4 µl 10× buffer, 4 µl MgCl2, 4 µl dNTP, 1.5 U Taq DNA polymerase (DRR100A, Takara, Dalian, China), 0.6 µl forward primer (300 nM), 1.8 µl reverse primer (900 nM), and 0.4 µl TaqMan probe (200 nM). Controls containing no template were also performed in triplicate. The reaction conditions were 50°C for 2 min, 95°C for 10 min, 95°C for 15 sec, and 60°C for 1 min for 50 cycles.
HBV DNA Quantitation
Serum HBV DNA levels were quantified by TaqMan real-time PCR with a Lightcycler detection system (Roche) with a lowest limit of detection of 1,000 virus genome copies/ml. The primer was provided in the kit and the handling procedures were carried out in strict accordance with the reagent kit package insert (PG Biotech, Shenzhen, China). The reaction volume was 40 µl, and the following thermocycling conditions were applied: 37°C for 5 min, 94°C for 1 min, then 40 cycles at 95°C for 5 sec, and 60°C for 30 sec.
Statistical Analysis
Statistical analysis was performed with a χ2 test, a Student's t-test, and one-way ANOVA. Values of P < 0.05 were considered statistically significant. All data were analyzed with SPSS 12.0 (SPSS, Inc., Chicago, IL).
RESULTS
KSHV Seroprevalence in Patients With Chronic Hepatitis B
KSHV seroprevalence in patients with chronic hepatitis B was 28% (59/211). There was no significant difference in KSHV ORF65 antibody prevalence between males (44/161, 27.3%) and females (15/50, 30%) (P > 0.05). The sample size for each age group was calculated to establish the prevalence of KSHV. As shown in Table I, the prevalence of antibodies to KSHV antigens increased rapidly and reached the highest rate (37.1%) in the 31- to 40-year-old age group. However, the rate decreased in patients older than 40 years.
| Age (years) | Total no. | KSHV-positive no. (%) |
|---|---|---|
| 0–20 | 19 | 4 (21.1) |
| 21–30 | 53 | 16 (30.2) |
| 31–40 | 62 | 23 (37.1) |
| 41–50 | 36 | 9 (25.0) |
| 51–60 | 15 | 2 (13.3) |
| ≥61 | 26 | 5 (19.2) |
Influence of KSHV Infection on HBV DNA Levels
Among 120 patients who did not receive nucleoside or interferon treatment, 49 cases were positive for the KSHV ORF65 antibody. The HBV DNA positivity rate in patients with chronic hepatitis B infected with KSHV, and those without KSHV infection, was 73.5% (36/49) and 56.3% (40/71), respectively (P < 0.05). The HBV DNA load was significantly higher in the former group than the latter (9.2 log (10) IU/ml vs. 7.8 log (10) IU/ml, P < 0.05; Table II).
| Total no.a | HBV DNA positive (%) | HBV DNA (log(10) IU/ml) | |
|---|---|---|---|
| KSHV positive | 49 | 36/49 (73.5)b | 9.2 ± 1.8c |
| KSHV negative | 71 | 40/71 (56.3)b | 7.8 ± 1.3c |
- a Cases who had received nucleoside or interferon treatment have been excluded.
- b χ2 = 3.969, P < 0.05.
- c t = −3.915, P < 0.05.
Relationship of HBV Genotypes and KSHV DNA Levels
As seen in Table III, the average KSHV DNA loads in patients with HBV genotype B, C, and mixed (B/C) were 409.1, 484.5, and 352 copies/ml, respectively. No significant difference was observed in the KSHV DNA levels between these HBV genotypes (P > 0.05).
Comparison of KSHV DNA Levels in Patients With Chronic Hepatitis B
The serum KSHV DNA loads of 49 patients with chronic hepatitis B with KSHV ORF65 antibodies were measured, and the results were summarized in Table IV. Among these patients, 16 were treated with glycyrrhizic acid (compound glycyrrhizin injection) for 1–8 weeks; most of the patients were treated with glycyrrhizic acid for 2–4 weeks (120–160 mg, q.d., intravenously guttae). The KSHV DNA load was found to be significantly lower in the patients with glycyrrhizic acid treatment than those without therapy (204.7 copies/ml vs. 533.9 copies/ml, P < 0.05). If KSHV DNA loads >100 copies/ml were considered to be KSHV DNA positive, 50% (8/16) of the former group were KSHV DNA positive, which was also significantly lower than the later positivity rate of 23/33 (69.7%).
| KSHV positive no. | KSHV DNA (copies/ml) | KSHV DNA >100 (copies/ml) no. (%) | |
|---|---|---|---|
| With glycyrrhizic acid treatment | 16 | 204.7 ± 103.9a | 8 (50.0)b |
| Without glycyrrhizic acid treatment | 33 | 533.9 ± 298.5a | 23 (69.7)b |
- a t = −4.267, P < 0.05.
- b χ2 = 4.337, P < 0.05.
DISCUSSION
The seroprevalence rates of KSHV vary due to a combination of geographic and behavioral risk factors. The KSHV prevalence in the general population is lower than 10% in the United States, Asia, and Western Europe, whereas in the Mediterranean countries it is intermediate, and in African countries it is higher than 25% [Belec et al., 1998; Whitby and Boshoff, 1998; Fujii et al., 1999]. Previous studies have detected the KSHV antibody in different Chinese populations, including healthy people, immunodeficient patients, healthy blood donors, intravenous drug users, non-drug users, and non-A-E hepatitis patients [Wang et al., 2000, 2001; Zhu et al., 2008]. However, there have been no reports on KSHV seroprevalence in patients with chronic hepatitis B. For the first time, the current survey provides information about the endemicity of KSHV in Chinese patients with chronic hepatitis B. A KSHV infection rate of 28% (59/211) in patients with chronic hepatitis B is significantly higher than in healthy people, 13% (16/122), which was reported previously [Zhu et al., 2008]. It should be noted that in the current study, antibodies to KSHV LANA or the K8.1 glycoprotein [Biggar et al., 2003; Laney et al., 2004] were not measured, making the seroprevalence not comparable with the data reported in other studies from China. However, this limitation does not affect the comparison of KSHV ORF65 antibody positive and negative patients. In that regard, the KSHV ORF65 positivity rate tended to increase with age until reaching its highest level in the 31–40 years age group. This pattern is consistent with previous studies which showed that the KSHV seroprevalence increased with age [Stein et al., 2004; Zavitsanou et al., 2007]. No significant difference in KSHV positivity status by gender was observed and this lack of association has been previously described [Stein et al., 2004; Zavitsanou et al., 2007].
The HBV DNA positivity rate and median HBV DNA load were significantly higher in patients with chronic hepatitis B infected with KSHV than those without KSHV infection. This strong correlation between HBV replication level and KSHV-positive status supports the hypothesis of an association between these two viral infections. The finding is consistent with higher KSHV antibody titers measured by fluorescent signal intensity in chronic carriers of HBV infection among Jewish population in Israel [Davidovici et al., 2001]. It could be that factors associated with KSHV infection facilitate re-exposure to or reactivation of HBV, resulting in an increased HBV DNA load, compared with KSHV-negative patients with chronic hepatitis B. The association between KSHV and HBV infection, the latter a marker of exposure to blood-borne viruses, also supported the previous conclusion of a blood-borne route as the main route of KSHV transmission in China [Wang et al., 2000]. In order to determine whether HBV genotypes had any impact on KSHV infection, KSHV DNA loads between patients with different HBV genotypes were studied. However, no significant difference was observed in genotype B, C, and mixed (B/C). This suggested that HBV genotypes are not related to KSHV infection in Chinese patients with chronic hepatitis B.
Studies demonstrated that triterpenoid compounds, such as glycyrrhizic acid which is a component of licorice, have a strong antiviral effect, especially on herpesviruses [Pompei et al., 1979; Dargan et al., 1988]. Glycyrrhizic acid and its derivatives were shown to be effective in vitro against KSHV. It is reported that glycyrrhizic acid decreased the expression of LANA and the reduction of LANA led to reactivation of p53 and apoptosis of nearly all of the virally infected cells [Curreli et al., 2005], so it might be useful for eradicating latent KSHV infection and have future therapeutic implications. Glycyrrhizic acid is now routinely used in Japan and China for the treatment and control of chronic viral hepatitis in clinical practice, particularly for patients with chronic hepatitis B [Sato et al., 1996; Arase et al., 1997]. In this study, glycyrrhizic acid may have inhibited KSHV replication in patients with chronic hepatitis B in China.
In conclusion, the seroprevalence of KSHV in Chinese patients with chronic hepatitis B and the interaction between KSHV and HBV were studied. The KSHV seroprevalence rates tended to increase with age, but were not related to gender or HBV genotypes. The association between HBV and KSHV, and glycyrrhizic acid inhibition of KSHV replication in vivo requires further investigation.
