Can HBV Replicate Extrahepatically?
"Hepatitis B virus infection in lymphatic tissues in inactive hepatitis B carriers"
Journal of Hepatitis
Makoto Umedaa, Hiroyuki Marusawaa, Hiroshi Senoa, Akira Katsuradaa, Motoshige Nabeshimaa, Hiroto Egawab, Shinji Uemotoc, Yukihiro Inomatad, Koichi Tanakab, Tsutomu Chibaa
Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, 54 Kawara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan
b Department of Transplantation Immunology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
c First Department of Surgery, Mie University School of Medicine, Mie, Japan
d Department of Transplantation, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
"...Although HBV-DNA could be detected in both the lymph nodes and PBMC of our subjects, we found no evidence of viral replication in tissues of any individual with latent HBV infection. The lack of cccDNA and pregenomic forms of RNA in the lymph nodes and PBMC strongly suggests that HBV can exist in lymphatic tissues without viral replication or proliferation….. our data suggest that lymph nodes and PBMC do not support active replication of HBV in latent HBV carriers who are positive for anti-HBc but negative for HBsAg, in whom the ongoing viral replication occurs in the liver. Instead, HBV may persist as integrated forms in their extrahepatic tissues. Further analysis is needed to determine the molecular status of HBV infection in extrahepatic tissues of highly viremic HBV carriers."
Hepatitis B virus (HBV) infection in extrahepatic tissues is controversial. To clarify whether episomal HBV can infect nonhepatic tissues, we investigated the molecular forms of HBV in the lymphatic cells of inactive HBV carriers who lacked viremia, thus avoiding contamination with HBV genomes originating from the viral particles present in the serum.
We assessed HBV genome, replicative forms, and viral integrants in the liver, serum, peripheral blood mononuclear cells (PBMC), and lymph nodes of 21 inactive HBV carriers who tested positive for antibodies against the HBV core antigen (anti-HBc).
Of the 21 anti-HBc positive individuals, HBV-DNA was detected in liver samples of 15 (71.4%), in the lymph nodes of 11 (52.4%), and in PBMC of three (14.3%). However, none of the detected HBV genomes from lymphatic tissues included the replicative forms of HBV. In one case, integrated HBV was present in the lymphatic tissues and the host–viral junction was present in the intronic sequences of chromosome 17.
These data suggest that human lymphatic tissues cannot support viral replication in anti-HBc positive inactive HBV carriers, while retaining the viral genome as an integrated form.
Hepatitis B virus (HBV) is a partially double-stranded DNA virus belonging to the Hepadnaviridae family [1,2]. Hepadnaviruses are characterized by their hepatotropic features and have a strong preference for infecting hepatocytes, although small amounts of hepadnaviral DNA is found in extrahepatic organs . The existence of extrahepatic replication of HBV is controversial [3–15]. Several previous studies have suggested the presence of replicative intermediate forms of HBV in extrahepatic organs [4–6,14]. For example, viral mRNA and covalently closed circular DNA (cccDNA) were detected in peripheral blood mononuclear cells (PBMC) of highly viremic patients by PCR-based methods . In contrast, others have demonstrated that human PBMC cannot be infected with HBV in vitro and in vivo . Most of those studies tested the PBMC of HBV carriers who were positive for hepatitis B surface antigen (HBsAg) and/or HBV-DNA in the serum. However, in HBV carriers with circulating viral particles, the possibility that the detected viral genomes were attributed to viruses that had only adsorbed to the cells could not be completely excluded .
We recently demonstrated that occult HBV maintains persistent infection in the livers of individuals who have antibody to hepatitis B core antigen (anti-HBc), but not HBsAg without causing any clinical liver dysfunction . Because HBV is frequently transmitted to liver transplant recipients from anti-HBc positive and HBsAg-negative donors, there is growing recognition that most anti-HBc positive healthy individuals have latent HBV infection in their liver tissues [16–21]. Moreover, we and other researchers have shown the reactivation of latent HBV infection in some leukemia patients under newly introduced immunosuppressive therapy or after bone marrow transplantation [22–26]. These findings suggest that most healthy individuals who are positive for anti-HBc have latent infections as the episomal form of HBV. Importantly, active HBV replication was found in the liver tissue of latent HBV carriers without any detectable HBV-DNA in their serum .
The aim of this study was to clarify whether episomal HBV infection can occur in extrahepatic tissues. We investigated the molecular forms of HBV in the lymph nodes and PBMC of latent HBV carriers who lacked viremia. We chose these subjects to exclude the possibility of contamination by HBV genomes originating from the viral particles present in the serum. Our findings showed that the HBV genome could be present in the lymph nodes and PBMC of latent HBV carriers, although these human lymphatic tissues lack the ability to support viral replication.
Although several previous studies have reported the presence of HBV genomes in PBMC, it is still controversial whether HBV exists as an episomal form and replicates in extrahepatic tissues [3–15]. In this respect, it should be noted that most previous studies examined the PBMC of HBV carriers positive for HBsAg, who normally have HBV-DNA in their serum. Thus, it is possible that the detected viral genomes in PBMC were derived from viruses circulating in the serum or adsorbed to the PBMC. For example, Kock et al. reported that HBV particles can bind tightly to various types of cells so that attached viruses cannot be washed away and remain detectable in culture for many days . To exclude the possibility of contamination of the circulating HBV-DNA, we examined PBMC and lymph nodes from LDLT donors positive for anti-HBc but negative for HBsAg. We have previously shown that HBV-DNA is rarely detected in the serum of these donors, despite their latent infection with the episomal form of HBV which is associated with ongoing viral replication in the liver [16,21]. Therefore, we first confirmed by PCR that HBV-DNA could not be detected in the serum in the anti-HBc positive donors to exclude the possible contamination by serum HBV-DNA in extrahepatic tissues. Our data demonstrated clearly that HBV-DNA is present in lymphatic tissues and PBMC of anti-HBc positive latent HBV carriers. Our data agree with those from a previous report showing identification of HBV-DNA sequences in PBMC of liver transplant recipients who were serum HBV-DNA negative by PCR analysis .
Although HBV-DNA could be detected in both the lymph nodes and PBMC of our subjects, we found no evidence of viral replication in tissues of any individual with latent HBV infection. The lack of cccDNA and pregenomic forms of RNA in the lymph nodes and PBMC strongly suggests that HBV can exist in lymphatic tissues without viral replication or proliferation. These findings are consistent with previously reported clinical outcomes in liver transplant patients, in which none of the recipients who were positive for anti-HBc but negative for HBsAg and who had HBV genomes in their liver tissues had acquired HBV reactivation after total removal of the infected liver through liver transplantation . It may be emphasized that these recipients generally receive intense immunosuppressive therapy; nevertheless, none of them developed HBV reactivation after LDLT. Our results contrast with other previous studies [4–6,14]. For example, cccDNA and pregenomic forms of RNA were detected in PBMC of patients negative for HBsAg but positive for HBV-DNA in the serum . As discussed above, the discrepancies between our results and those from other reports may be related to the presence or absence of circulating viral particles in the patient's serum. A considerable number of viral particles circulating in the serum was shown to contain viral RNA rather than DNA, suggesting that previous observations of the presence of HBV infection in PBMC could be explained by adsorbed virus . Since Soussan et al. showed recently that a singly spliced HBV-RNA encodes a novel HBV protein in vivo , there is room for further investigation to clarify whether HBV-spliced mRNA can be present in extrahepatic tissues.
Our findings also suggest the presence of integrated HBV-DNA in lymph node samples of anti-HBc positive carriers. To obtain direct evidence of HBV integration into host genomes, we used an inverse-PCR-based method using two pairs of inverse primers around the S region of HBV-DNA. It has been reported that many of the viral-host junctions cluster near the DR1 region of HBV sequences [39,40]. However, we found that amplification of the S region was the most sensitive method to detect the minimum amount of HBV genome present in lymphatic tissues of anti-HBc positive individuals. We targeted this region in the inverse-PCR analyses, and identified HBV integrants and flanking host sequences located in chromosome 17q22 in the lymph node of one case with anti-HBc. Our data agree with those in recent reports that observed viral integration in PBMC samples from patients with HBV-related acute and chronic liver disease [41,42]. However, detection of integrated viral DNA by inverse-PCR does not imply the clonal expansion of HBV in lymph nodes, and the question of whether the viral integration reflects the prior infection of proliferating bone marrow cells or the stimulation of PBMC expansion is still unanswered [3,42].
In conclusion, our data suggest that lymph nodes and PBMC do not support active replication of HBV in latent HBV carriers who are positive for anti-HBc but negative for HBsAg, in whom the ongoing viral replication occurs in the liver. Instead, HBV may persist as integrated forms in their extrahepatic tissues. Further analysis is needed to determine the molecular status of HBV infection in extrahepatic tissues of highly viremic HBV carriers.
3.1. The presence of HBV-DNA in lymph nodes and PBMC of anti-HBc positive latent HBV carriers
We first examined whether HBV-DNA was present in the serum, liver, lymph node, and PBMC samples of individuals who were positive for anti-HBc but negative for HBsAg using primers specific for the S, pre-S, pre-C/C, and X regions. The sensitivity of our nested PCR analysis used in this study has been described previously . As shown in Table 1, amplification of HBV-DNA was not observed in the serum of anti-HBc positive individuals using any primer sets. In contrast, the liver tissues of 15 of the 21 (71.4%) anti-HBc positive donors were positive for HBV-DNA. These data are consistent with previous data indicating that the anti-HBc positive healthy individuals have latent HBV infection in the liver [16–21]. The lymph node samples of 11 of the 21 (52.4%) anti-HBc positive individuals were also HBV-DNA positive in the three repeated assays. Moreover, three individuals positive for anti-HBc but negative for HBsAg also had HBV-DNA in their PBMC. These three were also positive for HBV genomes in their liver, although they lacked viral DNA in their lymph nodes from the hepatoduodenal ligament. HBV genomes were not detected in any of the liver or serum samples of donors without HBV-related serological markers. These findings suggest the presence of HBV genome in lymphatic tissues of individuals who have latent HBV infection in the liver.
3.2. Analyses of the replicative form of HBV in the lymphatic tissues
To clarify whether HBV infection was maintained as an episomal form in the extrahepatic tissues, we examined the presence of HBV replicative forms, including cccDNA and intermediate RNA [35,36], in the lymph nodes of five anti-HBc positive donors who were positive for HBV genome sequences in their lymphatic tissues. For the selective detection of the cccDNA form of HBV, we performed cccDNA-specific PCR amplification accompanied by mung bean nuclease treatment [16,29]. We first confirmed that a faint signal derived from the X region in the serum sample with a high level of HBV-DNA titer had completely disappeared after the nuclease digestion, whereas the S sequences were amplified in the same sample after the treatment with the endonuclease. This effect of endonuclease indicated the specificity of the digestion method for rcDNA in virions and agreed with previous reports [15,29]. As shown in Fig. 2A, both the liver and the lymph node tissues of all five anti-HBc positive donors were positive for HBV-DNA by conventional PCR assay using a primer set for the S region, which is conserved in both cccDNA and rcDNA molecules. Selective amplification of cccDNA detected HBV-derived cccDNA-specific bands of the expected size (658bp) in the liver tissue of all five donors. In contrast, the amplified products originating from HBV-cccDNA were detected in none of the lymph node samples of these five donors in three replicate assays. Similar results were obtained by highly sensitive amplification of cccDNA by PCR accompanied by a single cut of cccDNA by EcoRI treatment. To further examine whether active transcription and replication were present in the lymphatic tissues, we performed RT-PCR followed by Southern blotting assay to detect the pregenomic HBV-RNA. Total RNA extracted from both the lymph node and the liver was available in three anti-HBc positive donors for further analyses. As shown in Fig. 2C, the positive signals at the expected size representing HBV-RNA were detectable in the liver tissues of all three anti-HBc positive donors tested. However, no amplification of the HBV-RNA was observed from the total RNA samples extracted from the lymph nodes of these individuals. These findings suggested that the HBV genomes detected in the lymph nodes of anti-HBc positive individuals did not contain the replicative form of HBV.
3.3. The presence of the integrated form of HBV in the lymphatic tissues
As HBV replicative forms were not detected in lymphatic tissues of anti-HBc positive individuals, we reasoned that the HBV genome might be present as an integrated form in the extrahepatic tissues. To address this question, we separated the DNA from the lymph node and liver tissues of anti-HBc positive individuals into two fractions according to their molecular sizes, as described previously . Human β-actin and p53 gene sequences were amplified from the HMW fractions of all subjects, revealing that this fraction contained host chromosomal DNA (Fig. 3A for β-actin, data not shown for p53). In the liver tissues, two of six cases were positive for HBV-DNA in the HMW host chromosomal DNA fraction. Interestingly, HBV sequences were detected in three of six HMW fractions of DNA samples extracted from lymphatic tissues (lymph nodes of cases #13, #17, and #21), suggesting that the HBV genome in the lymph nodes of anti-HBc positive individuals is most likely present as an integrated form.
To further examine the presence of HBV integration in lymphatic tissues, we performed inverse-PCR-based amplification of HBV-DNA to identify the host-viral junction sequences. We selected the S region of HBV as the target sequences of inverse-PCR analyses because amplification of this region shows the highest sensitivity for detecting HBV sequences in lymphatic tissues of anti-HBc positive individuals. In the lymph nodes of one of three cases (case #21), one visible DNA signal was observed, which showed the different sizes from the common fragments found in all three samples, suggesting a unique HBV integrant in the inversely-amplified PCR product. The nucleotide sequence analysis in this specifically amplified fragment contained both viral and human genome sequences. As shown in Fig. 3B, the human DNA sequence located in chromosome 17q22 was identified as a host flanking sequence, which was connected with the S region of HBV-DNA. Together, these data suggest the presence of HBV integration in host chromosomal DNA of lymph nodes of anti-HBc positive latent HBV carriers.
2. Patients and methods
Between April 5, 1996 and August 22, 2003, 724 patients underwent living-donor liver transplantation (LDLT) at Kyoto University. Before surgery, the liver function of all donors was examined by blood chemistry and serological analyses of HBV markers including HBsAg, antibodies to HBsAg, anti-HBc, hepatitis B e antigen(HBeAg), and antibodies to HBeAg using commercial enzyme immunoassay kits as described previously . Of the original 724 patients, 103 donors (14.2%) were positive for anti-HBc and negative for HBsAg. From these 103 patients, the liver tissues, lymph nodes, PBMC and serum of 21 donors were available for further analyses. These 21 anti-HBc positive individuals included 10 men and 11 women, aged 24 to 63 years (mean age, 43.4 years). None had a history of liver dysfunction, blood transfusion, drug abuse, or family history of HBV infection. From the remaining 621 donors without any HBV markers, 10 were randomly selected as the negative control group (six men and four women). None of the donors enrolled in this study was positive for HBV-DNA in their sera at the time of operation. All subjects provided written informed consent and the study was conducted in accordance with the principles of the Declaration of Helsinki.
2.2. Tissue samples
Liver tissue samples were obtained at the time of transplantation, frozen immediately, and stored at -80°C until use. Blood samples were obtained before surgery, and samples from the lymph node in the hepatoduodenal ligament were taken by biopsy during the operation. DNA was extracted from the liver tissue, lymph node, and serum using procedures as described previously . DNA extraction from the PBMC was performed using the Gene Trapping by Liquid Extraction kit (Takara, Tokyo, Japan) according to the manufacturer's protocol.
2.3. PCR amplification of HBV-DNA
HBV-DNA was amplified by nested or semi-nested polymerase chain reaction (PCR) using AmpliTaq Gold (Perkin–Elmer Cetus, Norwalk, CT) . Primer sets for amplification of the S, pre-S, Core(C) /pre-C, and X regions have been described previously . We defined a sample as HBV-DNA positive when amplification products were detected in two or more of four regions in the same sample in three or more independent experiments. As a positive control, DNA samples were prepared from liver tissues of patients with hepatocellular carcinoma (HCC) who were positive for HBsAg. As negative controls, PCR was performed using DNA samples extracted from liver tissues of healthy donors without any HBV markers, PCR buffer without DNA, or water only.
2.4. Selective detection of cccDNA and pregenomic RNA of HBV
To detect the cccDNA forms of HBV-DNA, PCR amplification was performed using DNA samples treated with mung bean nuclease and primer sets specific for the X region spanning DR1 and DR2 across the gap of the relaxed circular DNA (rcDNA). Mung bean nuclease cleaves a part of the single stranded gap and triple stranded region selectively. Thus, the sequences around the DR region in HBV rcDNA are expected to be digested by this nuclease . In contrast, the digestion with mung bean nuclease prior to PCR amplification does not affect the same region of cccDNA . In addition, to enhance the efficiency of cccDNA amplification, cellular DNA samples were digested with EcoRI (5U) at 37°C for 2h before the PCR analysis . Isolation of total RNA from lymphatic and liver tissues, RT-PCR, and southern blotting analyses were performed as described previously .
2.5. Detection of the integrated form of HBV-DNA
To discriminate the integrated viral DNA from the episomal HBV-DNA forms, the host genomic DNA (high molecular weight fraction; HMW) was separated from the low molecular weight fraction by applying the modified alkali-lysis procedure used to isolate plasmid DNA, as previously described . Inverse-PCR is based on the digestion of DNA with certain restriction enzymes and circularization of cleavage products before amplification using primers synthesized in the opposite orientation to those normally employed for PCR [31–34]. As amplification of the S region of HBV-DNA was found to be the most sensitive among the four sets of primers for the HBV genome, selective digestion of the HBV-S region was performed using the restriction enzyme DdeI or RsaI followed by the amplification of this fragment using inversely designed primer sets specific for the S region. Accordingly, 4μg of extracted DNA was digested with DdeI or RsaI in a total volume of 50μL at 37°C for 4h. After confirmation of complete digestion by agarose gel electrophoresis, the enzyme was heat inactivated at 70°C for 15min. After the purification of the digested DNA using a PCR clean-up system (Promega, Madison, WI), samples were incubated with T4 DNA ligase (New England BioLabs, Beverly, MA) at 16°C for 6h. Finally, circularized DNA was used as the template for PCR amplification. The primer sets used for inverse-PCR amplification were as follows: R-HBS1.5′-GGGTCCTAGGAATCCTGAT-3′: R-HBR1.5′-GTATGTTGCCCGTTTGTCCT-3′: R-HBS2.5′-GTCAACAAGAAAAACCCCGC-3′: R-HBR2.5′-GCCTCATCTTCTTGTTGGTTC-3′: Equal amounts of restriction enzyme-digested but noncircularized DNA were used as a negative control.