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Molecular characterization of occult hepatitis B virus infection in patients with end-stage liver disease in Colombia

  • Julio Cesar Rendon,

    Affiliation Grupo de Gastrohepatologia, Facultad de Medicina, Universidad de Antioquia, UdeA, Medellin, Colombia

  • Fabian Cortes-Mancera,

    Affiliation Grupo de Investigación e Innovacion Biomédica GI2B, Facultad de Ciencias Exactas y Aplicadas, Instituto Tecnologico Metropolitano (ITM), Medellin, Colombia

  • Juan Carlos Restrepo-Gutierrez,

    Affiliations Grupo de Gastrohepatologia, Facultad de Medicina, Universidad de Antioquia, UdeA, Medellin, Colombia, Unidad de Hepatologia y Trasplante Hepatico, Hospital Pablo Tobon Uribe, Medellin, Colombia

  • Sergio Hoyos,

    Affiliations Grupo de Gastrohepatologia, Facultad de Medicina, Universidad de Antioquia, UdeA, Medellin, Colombia, Unidad de Hepatologia y Trasplante Hepatico, Hospital Pablo Tobon Uribe, Medellin, Colombia

  • Maria-Cristina Navas

    maria.navas@udea.edu.co

    Affiliation Grupo de Gastrohepatologia, Facultad de Medicina, Universidad de Antioquia, UdeA, Medellin, Colombia

Molecular characterization of occult hepatitis B virus infection in patients with end-stage liver disease in Colombia

  • Julio Cesar Rendon, 
  • Fabian Cortes-Mancera, 
  • Juan Carlos Restrepo-Gutierrez, 
  • Sergio Hoyos, 
  • Maria-Cristina Navas
PLOS
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Abstract

Background

Hepatitis B virus (HBV) occult infection (OBI) is a risk factor to be taken into account in transfusion, hemodialysis and organ transplantation. The aim of this study was to identify and characterize at the molecular level OBI cases in patients with end-stage liver disease.

Methods

Sixty-six liver samples were obtained from patients with diagnosis of end-stage liver disease submitted to liver transplantation in Medellin (North West, Colombia). Samples obtained from patients who were negative for the surface antigen of HBV (n = 50) were tested for viral DNA detection by nested PCR for ORFs S, C, and X and confirmed by Southern-Blot. OBI cases were analyzed by sequencing the viral genome to determine the genotype and mutations; additionally, viral genome integration events were examined by the Alu-PCR technique.

Results

In five cases out of 50 patients (10%) the criteria for OBI was confirmed. HBV genotype F (subgenotypes F1 and F3), genotype A and genotype D were characterized in liver samples. Three integration events in chromosomes 5q14.1, 16p13 and 20q12 affecting Receptor-type tyrosine-protein phosphatase T, Ras Protein Specific Guanine Nucleotide Releasing Factor 2, and the zinc finger 263 genes were identified in two OBI cases. Sequence analysis of the viral genome of the 5 OBI cases showed several punctual missense and nonsense mutations affecting ORFs S, P, Core and X.

Conclusions

This is the first characterization of OBI in patients with end-stage liver disease in Colombia. The OBI cases were identified in patients with HCV infection or cryptogenic cirrhosis. The integration events (5q14.1, 16p13 and 20q12) described in this study have not been previously reported. Further studies are required to validate the role of mutations and integration events in OBI pathogenesis.

Introduction

Hepatitis B virus (HBV) is responsible for significant morbidity and mortality worldwide. It is estimated that one-third of the world population has been infected by HBV and at least 240 million individuals are chronic carriers of the virus. Additionally, more than 780.000 people die each year due to liver diseases associated with HBV infection including cirrhosis and hepatocellular carcinoma (HCC) [1, 2].

HBV infection is diagnosed by detecting the HBV surface antigen (HBsAg) in serum samples using immunoassays; although other serological markers are necessary to identify the type and/or stage of HBV infection including the E antigen (HBeAg), anti-Core protein (anti-HBc), anti-HBsAg (anti-HBs) and anti-HBeAg (anti-HBe) antibodies and viral load [3, 4].

In 1979 evidence of a particular type of HBV infection was described [5], that was subsequently recognized as occult HBV infection (OBI) [6]. OBI is a persistent infection characterized by the absence or undetected HBsAg in the serum, viral load <1000 copies/mL, and with or without detection of other serological markers [7]. Patients with OBI could develop HBV reactivation, cirrhosis and HCC [8, 9].

OBI is a multifactorial pathology in which are involved mutations affecting viral proteins and promoters, Hepatitis C Virus (HCV) and/or Human Immunodeficiency Virus (HIV) coinfection, host immune response, and cellular defense mechanisms such as epigenetic silencing of the viral genome [1012].

OBI has been reported in populations with risk factors including hemodialysis [13, 14], HIV infection [1518], HCV infection [1921], HCC [2224], cryptogenic liver disease [25, 26], and end-liver disease [27, 28]. A large variation in OBI frequency has been reported in the different studies depending on the HBV prevalence in the general population, the risk factor and the criteria for OBI (13–28).

Recently, 8 studies of OBI were carried out in Colombia and have shown a prevalence of OBI from 1% to 8.3% in HIV patients [2931], 0% to 8.75% in blood donors [3234], 0% in undergraduate student populations [35] and 13.3% in patients with diagnosis of cirrhosis and/or HCC [36]. However, there are some limitations in these studies, such as size sample, HBV genome detection techniques and OBI criteria. Thus, additional studies are necessary to establish the prevalence of OBI in the Colombian population.

OBI in recipients of liver transplants is a risk factor to be taken into account, considering de novo infections that occur in 25–95% of transplants from HBsAg negative, anti-HBc positive donors [3739]. According to the Department of Health and Human Resources, in the USA around 79 persons receive organ transplant per day, liver transplantation being approximately 21% of all procedures [40]; meanwhile in Colombia in 2014, a total of 1046 transplant procedures were made, of which 211 (20%) were liver transplants [41].

Evidence has shown that in solid organ transplantations such as kidney [42], bone marrow [43], and heart [44], HBV transmission is usually lower than 5%; while for recipients of liver transplants, a variable rate of de novo HBV infection occurs between 17% and 95% depending on the HBV serological markers of the donor [27, 37, 38, 45, 46]. However, the infection status of the recipient is also highly relevant, considering that among 4.4% and 17.6% of OBI has been reported in liver transplant recipients [28, 4749].

The aim of this study was to determine the frequency of OBI in a cohort of patients with end-stage liver disease, submitted to liver transplantation, attending a hospital in Medellin, and to characterize mutations in the viral genome and viral integration events that could be related with OBI.

Material and methods

Study population

Specimen collection.

This study included 66 liver tissue samples, obtained between August 2002 and February 2008, from patients with diagnosis of end-stage liver disease submitted to liver transplantation at the Pablo Tobon Uribe Hospital, one of the most important centers of liver transplantation in Colombia. The samples were collected in dry sterile 50 mL tubes at the time of surgery, conserved at 4°C and transported at 4°C to the laboratory within 12 hours to 24 hours after collection, where samples were aliquoted in independent containers and conserved at -70°C. In few cases, the liver sample was sent to the laboratory 24–48 hours after surgery.

Cirrhosis and HCC were diagnosed based on the criteria of the European Association for the Study of the Liver (EASL) and the American Association for the Study of Liver Diseases (AASLD) [50, 51].

The OBI cases were defined, based on criteria described by Brechot et al., as HBsAg negative cases in which amplification of at least two different HBV genome regions was confirmed [52].

Hepatic and viral markers

Quantification of serological markers was performed using the platform ARCHITECT ci8200 (Abbott, USA). Serological markers of liver function such as Alanine Aminotransferase (ALT), and Aspartate Aminotransferase (AST) were quantified using the Activated alanine or aspartate aminotransferase assay (Abbott, USA) while serological HBV infection markers were quantified using the chemiluminescent microparticle immunoassay (Abbott, USA).

HBV genome detection

From cases negative for HBsAg (n = 50), total DNA was extracted from liver tissue using the QIAamp DNA Mini Kit (QIAGEN, Germany) according to the manufacturer’s protocol. DNA was quantified using the NanoDrop ND 1000 Spectrophotometer (Thermo Fisher Scientific, USA), and quality tested by amplification of GAPDH gene [53]. DNA (60–80 ηg) was used to amplify three regions of the HBV genome (X, S and Core-X regions) by nested or hemi-nested PCR strategies as described previously (S1 Table).

Amplification of the ORF S was performed using 1 unit of Biolase DNA polymerase (Bioline, USA), 5 mM of MgCl2, and 10 pmol of primers YS1 and YS2 [54]. The second round was performed using 1.25 U of Biolase DNA polymerase (Bioline, USA), 1 mM of MgCl2 and 10 pmol of primers S3s and S3as [55]. Cycling of the first round included an initial denaturation step at 95°C for 3 min and 35 cycles at 94°C for 45 s, 53°C for 60 s and 72°C for 90 s; the second round included an initial denaturation step at 95°C for 3 min and 35 cycles at 94°C for 45 s, 50°C for 60 s, and 72°C for 60 s and one cycle at 72°C for 2 min.

PCR reaction of ORF X was performed using 1.5 unit of Biolase DNA polymerase, 1.5 mM of MgCl2 and 10 pmol of primers X1F and X3R. The second round was carried out in the same conditions using primers X1F and X1R [56, 57]. Cycling of both rounds included an initial denaturation step at 95°C for 3 min and 30 cycles at 94°C for 45 s, 53°C for 30 s, and 72°C for 40 s, and one cycle at 72°C for 2 min.

The X-Core region was amplified using a hemi-nested PCR. In this strategy both rounds were performed using 1 unit of Biolase DNA polymerase, 1 mM of MgCl2 and 7.5 pmol of primers 1101p and 2440n [58]. The second round was carried out in the same conditions using primers P2 and 1101p. The cycling conditions for the first round included an initial denaturation step at 95°C for 3 min and 35 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 90 s; the second round included an initial denaturation at 95°C for 3 min and 35 cycles at 94°C for 45 s, 55°C for 45 s and 72°C for 60 s and one cycle at 72°C for 2 min.

Southern blot analysis

A full length HBV DNA (3,2 kb) was amplified from the sample TH24 (previously designated FJ589065.1) [59] using the method described by Gunther et al., [60]. The amplification products were purified using the GeneJet Gel Extraction kit (Thermo Fisher Scientific, USA), and cloned into the vector pJET1.2 (Thermo Fisher Scientific, USA) to create the construct pJET-TH24_1,5.

DNA (40–60 μg) obtained from liver samples and 1 μg of the pJET-TH24_1,5 construct were pretreated with EcoRI restriction enzyme (Thermo Fisher Scientific, USA), and separated by electrophoresis on a 1% agarose gel. The gel was blotted onto a nylon membrane (Roche Diagnostics GmbH, Germany) and hybridized with a digoxigenin (DIG)-labeled probe specific for the HBV S region (nucleotides 422–759). The probe was generated using the primers S3s-S3as (S1 Table) and the plasmid pJET-TH24_1,5 with a commercial kit (PCR DIG Probe Synthesis Kit; Roche Diagnostics GmbH, Germany). The hybridization signals were visualized using an enzyme-linked immunoassay (DIG Luminescent Detection Kit; Roche Diagnostics GmbH, Germany) following the protocol recommended by the supplier.

Complete genome sequence

Four overlapping fragments of the HBV genome were amplified using 80 ng of total DNA extracted from the OBI samples, using the HotStart HiFidelity Polymerase kit (Qiagen, Germany) and the following primer sets 58p-1450n, 58n-2440p, P2-1101P and P1-2440n (S1 Table and S1 Fig) [60, 61]. The PCR products were purified using the GeneJet Gel Extraction kit (Thermo Fisher Scientific, USA) and cloned into the cloning vector pJET1.2 (Thermo Fisher Scientific, USA) according to the manufacturer’s indications. Cloned PCR products were sequenced (Macrogen, Inc., Seoul, Korea) and then analyzed using the ContigExpress application of Vector NTI 11.0 (Invitrogen, USA).

The nucleotide sequence data are available in the GeneBank databases (TH3: KX757662, KX757663, KX757664; TH6: KX75766; TH28: KX757666, KX757667; TH75: KX757668; and TH78: KX757670, KX757671, KX757672).

Phylogenetic analysis

HBV genome sequences from the OBI cases were analyzed by phylogenetic reconstruction using 118 HBV sequences including 32 partial and 86 HBV full-length genome sequences (S2 Table) available in GenBank.

The sequences were aligned and edited using BioEdit 7.2 software [62] (available at http://www.mbio.ncsu.edu/bioedit/bioedit.html). The phylogenetic analyses were conducted using the MEGA software version 6.06. The phylogenetic tree was constructed using Neighbor-joining method and GTR model; the bootstrap consensus tree was inferred from 1000 replicates [63]. Pairwise distance test was performed to ensure no contamination between samples.

Pairwise and mutation analysis

Sequences of OBI cases were grouped according to the genotype identified by the phylogenetic analysis, and aligned with 3 HBV full-length genome sequences of genotypes A, D, and F, chosen randomly from the database of reference sequences used for the phylogenetic analysis. None of these sequences were related to OBI (Genotype A: AJ012207, AJ309369 and AF090838; genotype D: AF280817, AB033558 and AB078033; Genotype F: FJ58065, DQ899145 and AY179734, S3 Table).

Based on the DNA sequence, each ORF sequence was aligned, and the deduced amino acid sequence was analyzed. OBI mutations were considered based on previously reported mutations and new mutations identified in OBI sequences compared to the reference sequences of each genotype.

Viral genome integration

HBV DNA integration into the host genome was examined using the Alu-PCR methodology described by Minami et al. and Murakami et al. with some modifications [64, 65]. Briefly, 100 ηg of total DNA extracted from liver samples were amplified in a final volume of 50 μL containing, 1 unit of Biolase DNA polymerase, 1 mM of MgCl2, 50 pmol of Alu primer and 10–100 pmol of primer HB1 (S1 Table). The cycling conditions included an initial denaturation step at 94°C for 3 min and 10 cycles of 94°C for 30 s, 59°C for 30 s, and 70°C for 3 min.

The PCR products were then incubated with 1 U of uracil-DNA glycosylase (Life Technologies, USA) for 30 min at 37°C followed by 10 min at 94°C. Subsequently, 1 unit of Biolase DNA polymerase and 10 mmol of HB2 and Tag5 primers (S1 Table) were added and a Touch Down PCR was carried out. The cycling condition for the first step was 94°C during 3 min, followed by 20 cycles at 94°C for 30 s, 65°C for 30 s decreasing 0.5°C/cycle, 70°C for 3 min, and finally 20 cycles at 94°C for 30 s, 55°C for 30 s, and 70°C for 3 min, followed by one final step at 72°C for 8 min. Finally, 5 μl of the amplified product was used for a second round of amplification using the HB2 and Tag5 primers under the follow conditions: 20 cycles at 94°C for 30 s, 55°C for 30 s, and 70°C for 3 min and one final step at 72°C for 8 min.

The PCR products were analyzed on 1% agarose gels stained with ethidium bromide; the bands were purified using QIAquick gel extraction kit (QIAGEN, Germany) and then sequenced by the Sanger sequencing method (Macrogen, Inc., Seoul, Korea).

The sequences were analyzed using BioEdit software and ContigExpress module from Vector NTI 4.1 (Invitrogen, USA); identification of integration events was made using BLASTn search system.

Statistical analysis

Normality was assessed by the Shapiro-Wiks test and the χ2 test, Student´s t-test and the Mann-Whitney U test were used to identify correlations between parameters. Calculations were performed with the SPSS software package 23. A p <0.05 was considered significant.

Ethics considerations

The study was approved by the Ethics Committees of Universidad de Antioquia (N 007, Facultad de Medicina, UdeA) and the Hospital Pablo Tobon Uribe (No. 13–2012, HPTU). None of the transplant donors were from vulnerable population and all donors provided written informed consent that was freely given.

Results

Study population

The study was carried on liver tissue samples obtained from 66 patients, 39 (39/66, 59.1%) male and 27 (40.9%) female. Forty nine patients (74.2%) had been diagnosed of cirrhosis without HCC; among them 10 (20.4%) had cryptogenic cirrhosis; 9 (18.3%) had cirrhosis associated with HCV infection, 8 (16.3%) cirrhosis associated with cholangiopathies, 7 (14.2%) cirrhosis associated with autoimmune hepatitis, 6 (12.2%) cirrhosis associated with HBV infection, 4 (8.16%) had alcoholic cirrhosis, 2 (4.1%) alcohol and HBV or HCV infection, 1 (2%) HBV/HCV coinfection and 2 (4.1%) related to nonalcoholic steatohepatitis (NASH) or Wilson disease.

Moreover, the 17 (25.8%) cases of cirrhosis and HCC were associated with HBV infection (5/17, 29.4%), HCV infection (5/17, 29.4%), HBV/HCV coinfection (4/17, 23.5%) and autoimmune hepatitis (2/17, 11.8%); In addition, 1 cryptogenic case was identified (1/17, 5.8%) (Table 1). Among all cases, only samples related with autoimmune hepatitis presented a significant correlation with the gender (p<0.003).

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Table 1. Demographical and clinical characteristics of patients with end-stage liver disease submitted to liver transplantation.

https://doi.org/10.1371/journal.pone.0180447.t001

No differences in ALT and AST serum levels were found among the patients with different liver damage etiologies. Meanwhile, AST was significantly higher in patients with cholangiopathies, 90.5 U/L compared with other etiologies 57.5 U/L (p = 0.029). Additionally, AST and ALT serum levels were lower in alcohol consuming patients (42.5 U/L and 25.5 U/L) compared with patients without alcohol consumption (61.5U/L and 44 U/L) (p = 0.039 and p = 0.031, respectively) (Table 2, Fig 1).

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Fig 1. AST and ALT serum levels of the study pupulation.

AST (A) and ALT (B) serum levels before transplant of all patients. Red lines indicates the median value.

https://doi.org/10.1371/journal.pone.0180447.g001

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Table 2. AST and ALT serum levels in patients with end-stage liver disease and submitted to liver transplantation.

https://doi.org/10.1371/journal.pone.0180447.t002

Identification of OBI cases

A total of 50 liver samples obtained from patients negative for serological markers HBsAg and/or anti-HBc antibodies were analyzed for HBV genome amplification. The fragments of ORF S and ORF X were successfully amplified in samples TH3, TH6, TH28, TH75, TH78 (Fig 2A and 2C); while ORF Core was amplified in 3 of them (Fig 2B). OBI cases were identified in samples from patients negative for HBsAg and positive for at least two out of the three PCR strategies used for the HBV genome. The HBV genome was also detected by Southern blot in samples TH3, TH6, TH28, TH75 and TH78 (S2 Fig).

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Fig 2. Identification of OBI cases in liver tissue samples from patients with end-stage liver disease.

Amplification of HBV X, X-Core and S regions in liver tissue samples obtained from patients without serological evidence of HBV infection. A: ORF X (138 nt); B: ORF Core (729 nt), and C: ORF S (310 nt). All samples were tested at a DNA concentration of 60–80 ng (1-3-5-7-9-11-13-14-15) and by a 1:10 dilution (2-4-6-8-10-12-14-16).

https://doi.org/10.1371/journal.pone.0180447.g002

Based on the OBI case criteria described by Brechot et al. [52], 5 cases of OBI (5/50, 10%) were identified in the study population. Two OBI cases corresponding to patients with cryptogenic cirrhosis, two patients with cirrhosis associated with HCV infection and one patient with a diagnosis of cirrhosis and HCC associated with HCV infection (Table 3).

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Table 3. Demographical and clinical characteristics of patients with OBI.

https://doi.org/10.1371/journal.pone.0180447.t003

No differences were observed between OBI cases and HBV infection regarding gender, age and ALT and AST serum level (Table 4)

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Table 4. Demographical and clinical background of patients with OBI and chronic HBV infection.

https://doi.org/10.1371/journal.pone.0180447.t004

Genotyping OBI cases

Using PCR strategies to amplify the viral genome between 55% and 99% of the full-length viral genome sequences were obtained from OBI samples e.g., TH3: 83%, TH6: 97%, TH28: 70%, TH75: 99%, and TH78: 55% based on the NC009937 reference sequence.

The genotyping analysis was performed using partial HBV ORF and complete sequences and evaluated by Maximum likelihood (ML), Neighbor Joining (NJ), and Maximum parsimony (MP). Between the methodologies, the intergenotype branching was conserved and allow us to identify sample TH6 to genotype F, subgenotype F1, while TH75 and TH78 belong to genotype F, subgenotype F3, TH28 to genotype A, subgenotype A2, and TH3 to genotype D, subgenotype D4 (Fig 3).

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Fig 3. Phylogenetic analysis of OBI sequences.

Unrooted phylogenetic tree generated by Neighbor Joining method of partial (part) and full-length HBV sequences. The sequences of this study (TH3, TH6. TH28, TH75, TH78) indicated with black circles, were compared with representative sequences of all HBV genotypes (A-H), denoted with the genotype, GenBank accession number, and country. The genotype of the collapsed blanches is indicated and the bootstrap values from 1000 replicates are indicated in the internal nodes.

https://doi.org/10.1371/journal.pone.0180447.g003

Amino acid substitutions in cases of OBI cases

All amino acid (aa) sequences were deduced and compared with reference sequences of HBV genotypes A, D and F. The changes found in each aa sequence are shown in Table 5 (ORF S), Table 6 (ORF P) and Table 7 (ORF X, ORF Core and DNA changes of preC/C promoter).

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Table 7. ORF X, ORF C and Core promoter mutations identified in OBI cases.

https://doi.org/10.1371/journal.pone.0180447.t007

Regarding the HBsAg sequence, only one missense mutation, N37D, was found in the preS1 domain in samples TH3, TH6, TH75 and TH78 (Table 5). However, this mutation is located in the receptor-binding domain although; there are no reports of this mutation in the literature.

Moreover, 11 amino acid changes were identified in the S domain of HBsAg, seven in the TH3 (Q51E, N52K, C60Y, G70T, R73H, I255M, F259V), two in the TH6 (I195M, W196S), two in TH75 (C76F, C99stop), and one in the TH78 (I195M) liver sample. Interestingly, no mutations were found in this domain in sample TH28 and only three of them I195M, W196S and R73H were previously reported.

None of the HBsAg mutations described in this study modify the “a” determinant and the major hydrophilic region (MHR), although, the overlapping sequence coding for P protein was altered. A single mutation was recognized in both the TP and S domains of P protein, R29N in the TP domain of the TH3 sample and Q219R in the S domain of TH3, TH6, TH75 and TH78 samples (Table 6). These mutations have not been previously reported and its relation with OBI is unknown. Interestingly the mutation Q129R, corresponding to N37D of the preS1 domain was identified in four OBI cases. This finding raises the question concerning the consequences of the A2915G mutation in OBI pathogenesis and the effects on the P (Q219R) and/or HBsAg (N37D) proteins.

Previously reported mutations targeting the P protein were also identified in this study; these mutations are related to resistance to antiviral therapy, and to increase or decrease of polymerase activity (rtL180M, rtM204V, rtP237T, rtN238S/A, rtL217R) (Table 6). Interestingly, although the OBI patients never received antiviral treatment for HBV and/or HIV infection, Lamivudine and Adefovir resistance mutations (rtL180M, rtM204V, rtL217L, and rtP237T) were detected in 4 OBI samples; these mutations are prevalent in patients undergoing antiviral treatment [69, 7375].

The presence of mutations in HBx, HBeAg, HBcAg, and the Core promoter is also remarkable (Table 7). Regarding the Core promoter sequence, the four mutations A1762T, G1764A, C1766T and T1768A were found, that had been previously reported [69, 7375].

HBV integration events in OBI

Integration events were identified in samples TH3 and TH75, being characterized three integration events (Fig 4). Among them, one was detected in the TH3 sample mapped in chromosome 20q12, intron 9 of tyrosine phosphatase receptor type T (PTPRT) gene. While two integration events were detected in TH75 sample: one at chromosome 5q14.1, intron 3 of Ras protein-specific guanine nucleotide-releasing factor 2 (RASGRF2) gene and other on chromosome 16p13, in the Zinc Finger 263 (ZNF263) gene sequence (Fig 4).

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Fig 4. HBV integration events of in cases of OBI cases.

Integration sites are indicated with the approximate location in the ideograms of chromosomes 5, 16 and 20 based on G-banding and the sequence of integration (based on NCBI map viewer).

https://doi.org/10.1371/journal.pone.0180447.g004

Discussion

Colombia is a country with low prevalence for HBV infection considering the average incidence reported (3.11 to 4.68/100.000 habitants); however, in 2015 the incidence range was from 1 to 18.3/100.000 between the states showing a high heterogeneity along the territory. In the state of Antioquia where most patients included in this study are from, the incidence in 2015 was estimated to be 6.6 cases /100.000 habitants [76].

The prevalence of OBI in the Colombian population has been described in some studies carried out on serum samples from HIV patients by Ramirez and Correa (2%, 1/50), by Polo et al. (0.97%, 1/103) and by Bautista et.al (8.7%, 24/275) [2931]; while in serum samples from Colombian blood donor populations, Beltran et al., Arroyave et al., Castellanos et al., and Rios et al., reported 0% (0/129), 3.4% (7/207), 8.75% (14/160) and 2% (6/302) of OBI, respectively [3234, 77]. Recently, we described a frequency of 13.3% of OBI cases in 15 liver samples obtained from patients with diagnosis of cirrhosis and/or HCC [36].

In this study we describe the genetic characteristics of OBI cases in Colombian patients with diagnosis of end-stage liver disease. Reports of HBV genome detection have been published since 1990 with a frequency ranging from 4.8% to 72.7% [52]. The first studies performed in Europe, China and Israel, reported OBI in 55.5% [78], 60.7% [79] and 72.7% [80] of liver samples (frozen or paraffin-embedded tissues) obtained from patients negative for HBsAg.

OBI detection studies using liver tissues have performed in populations from China and Brazil. Wong et al. reported 62% of OBI in a cohort of 48 HCC patients among them 80% of cryptogenic cases, 3% of HCV infection and 17% of alcohol related HCC [26]. On the other hand, Ferrari et al. reported 4.4% of OBI in 62 paraffin-embedded liver tissue samples obtained from patients with diagnosis of liver cirrhosis [47]; Similarly, Barros-Junior et al. reported 17.64% of OBI in 17 liver biopsies obtained from patients diagnosed with chronic hepatitis B [81].

The OBI frequency described in the present study population (10%) is similar to other reports of OBI in a country of the region, Brazil (4.4% to 17.64%) that showed the impact of this infection in patients with diagnosis of end-liver disease [28, 48, 49, 82].

One of the most common risk factor of OBI is HIV and/or HCV infection. The frequency of OBI in patients with HCV infection has been reported to be between 33% and 50%, and between 22% and 73.3% in cases of HCV infection and HCC cases related to HCV [49, 83, 84]. The link between HCV and OBI has been clearly established since the HCV core protein inhibits HBV gene expression, by interaction with HBx, and HBV replication by obstructing encapsidation of the pregenomic RNA [85].

In the present study, 60% (3/5) of the OBI cases were identified in patients with HCV infection (two patients diagnosed with liver cirrhosis and one with HCC); the other 40% (2/5) OBI cases were detected in patients with cryptogenic cirrhosis (Table 3). These results are agree with previous reports of OBI with HCV coinfection and cryptogenic liver disease [21, 48, 49, 82, 86].

Based on data presented in Table 1, OBI was identified in 18.2% of cryptogenic cases and in 21.4% of HCV cases. Taking these results in account, OBI diagnosis should be considered in patients with cryptogenic or HCV associated end-stage liver disease before liver transplantation in order to decide what therapeutic strategy should be applied for these patients [47, 48, 87].

The studies of HBV genotyping in different populations revealed that in the Andean region of Colombia, genotype F, subgenotype F3, is the most prevalent, while genotype F1b is the most frequent in Amazonas state (South East) and genotype A is the most common in the Pacific region of the country; even though genotypes D, C, G and E have also been described in the Colombian populations [32, 61, 8891]. In the present study, the genetic analysis of the viral genome revealed the predominance of genotype F (three cases); subgenotype F1 was identified in one case while F3 was identified in two cases. On the other hand, less common genotypes in the Colombia population, genotypes A and D were also identified in one case each. Currently, there is no evidence of higher OBI risk related with a particular HBV genotype; although variability in CpG islands between genotypes could have an impact on OBI.

Several mutations have been related to OBI; among them, are those affecting the “a” determinant and pre-S domain, that reduce HBsAg immunogenicity and expression, respectively [9295]. Although other mutations could be related to the outcome of OBI including those affecting the RT domain of P protein (affecting the efficiency of viral replication), inducing the expression of the truncated HBx protein (affecting the normal function of HBx), and altering the promoter activity (downregulation of mRNA expression) [66, 96].

Despite the fact that mutations are well described in OBI cases [66, 85], as in this study (Tables 57), the relation between specific mutations and OBI pathogenesis is still controversial [74]. One hypothesis attempting to explain the role of mutations in OBI is the selection of variants as a consequence of host-virus interaction; that is not the case of primary occult infection described in the woodchuck hepatitis virus model that depends on some quasispecies present in the inoculum [97, 98]. Therefore deeper analyses in vitro and in vivo are required to validate the role of mutations in OBI pathogenesis.

In our study, 25 mutations along the viral genome were detected in the OBI samples. Interestingly, not a single mutation has been identified in all OBI cases or in OBI cases previously described in Colombian blood donors [32]. This finding is in agreement of a number of different mutations described in OBI cases [8, 32, 85, 99], and the mutation rate of HBV (2x10-5 nt/generation) [100].

In preS1, N37D, a mutation not previously reported and located in the recognition site for the viral receptor sodium taurocholate cotransporting polypeptide (NTCP) was detected [101]. This mutation may potentially impair the interaction between HBsAg and this receptor by a change of a polar uncharged side chain for a negatively charged chain.

In HBx two mutations were identified, F151L and F132Y, that correspond to changes between two hydrophobic amino acids. The mutations characterized in PreS1 and HBx have not been previously reported, and their role in OBI pathogenesis has to be addressed.

Interestingly, the amino acid sequence of HBc in the TH3 sample shows a combination of mutations in the C-terminus of the protein; this domain is characterized by an arginine-rich region with multiple SPRR motifs, that are important for the interaction with nucleic acids, pgRNA encapsidation and synthesis of the positive strand DNA [102, 103]. As is shown in Table 7, nine mutations were found in sample TH3 (R127L R150I, R151L, R154W, P156R, R157Q, R158S, R166Q, R167K), and one in sample TH78 (R175G) that directly affect the SPRR motif of this region, with potential implications on encapsidation and viral DNA synthesis [104].

Interestingly, the point mutations 1762, 1764, 1766 and 1768 located in the DNA-protein interacting region of the Basal Core promoter region and related with the HBx mutations K130M, V131I and F132Y were detected in sample TH75 (Table 7). These mutations have been reported in both chronic and OBI cases and are known to downregulate the expression of HBeAg and upregulate that of pgRNA by creating an additional HNF1 binding site [7375, 105]; in addition, these mutations have been related to higher risk of HCC development [106, 107].

However, the characterization of the A1762T, G1764A, C1766T, T1768A mutations in OBI cases could seem contradictory considering the low viral load in this infection; further studies are necessary to demonstrate the role of these mutations in OBI. Even more, the overlapping of Basal Core Promoter and the ORF HBx, could affect the activity of this protein by modifying its nuclear transactivation domain [108110].

In this study, most of the missense mutations are located between nt 350 and nt 750, comprising the HBsAg S and the RT domain of P protein, that are important regions for escape mutants and antiviral resistance [111]. We identified 10 mutations, 5 of them previously reported and related to drug resistance, replication rescue, downregulation of HBeAg expression and modification of HBsAg antigenicity (Table 6) [6773, 94].

Ten mutations were characterized on the P protein sequence. The mutation rtL180M, related to adefovir and lamivudine resistance, was found in two cases of OBI (samples TH6 and TH78); while rtM204V related to adefovir, lamivudine, entecavir, and telbivudine resistance [112], was found in one of these two OBI cases, along with rtL180M (sample TH6). Other point mutations previously reported and related to adefovir resistance were rtL217R in TH28, while rtP237T and rtN238S/A were found in the TH6 sample; although none of these cases reported HBV antiviral treatment.

Although P mutations have been described in OBI cases [66], the role of these mutations is still under investigation; moreover, polymerase mutation is usually related to low polymerase activity [113, 114], which could be related to low viral load, one of the hallmarks of OBI.

Considering that the ORF S and ORF P overlap, mutations in one ORF may produce nonsynonymous mutations in the other ORF [115]. Indeed, mutations rtL180M and rtM204L in ORF P produce consecutive mutations I195M and W196S in ORF S; these mutations reduce the recognition of HBsAg by antibodies and therefore their detection by immunoassay [116]; the same effect is also described for R73H, although this change does not correspond to a change in the P protein [66, 117].

Integration of the viral genome is an event of HBV infection, although it plays no role in viral replication. Indeed, integrated viral sequences are detected in tumor tissue of HBV-related HCC and in adjacent non tumor tissue, but the molecular mechanisms of HBV integration is unknown [65, 118, 119]. Integration of viral sequences results in the disruption of one or more ORFs, in deletions, cis/trans activation and expression of truncated protein with novel activity [107, 120, 121].

In this study, three integration events were identified in two liver samples. In sample TH3, the break point of the HBV genome occurs in position nt1682, and was integrated in chromosome 20q12 (Fig 4), intron 9 of PTPRT gene. PTPRT is a member of the protein tyrosine phosphatase (PTP) family. The PTP proteins regulate cell growth, differentiation and mitotic cycle and were identified as potential tumor suppressors genes in colorectal cancer; however, sample TH3 was obtained from a patient without HCC diagnosis. Interestingly, the integration events described in the present study have not been previously reported [122, 123].

In sample TH75, two integrations events were characterized; the HBV genome break point was located in nt 1513 and was integrated in chromosome 5q14, intron 3 of the Ras protein-specific guanine nucleotide-releasing factor 2 (RASGRF2) gene. The second integration event was located in nt 1680 of the HBV genome and was integrated in chromosome 16p13, in the zinc finger 263 (ZNF263) gene (Fig 4).

The RASGRF2 protein is a calcium-regulated nucleotide exchange factor activating RAS and RAS-related protein, that coordinates the signaling of distinct mitogen-activated protein kinase pathways [124]. Interestingly, RASGRF2 expression is usually suppressed in various human cancers [125]. Finally, ZNF263 is a C2H2 zinc-finger protein, containing nine fingers and one KRAB domain [126].

All the integration events occurred in exonic sequence of the genes, potentially impairing the correct mRNA maturation. Beyond the predicted effect of the integration events, functional studies should be made to identify the consequences of these integration events.

Integration of complete or truncated sequences of HBsAg could alter the ratio of ORF S transcripts, affecting the assembly and secretion of virions and subviral particles. Indeed, increased expression of HBsAg L could induce accumulation, and, subsequent reduction of HBsAg secretion [127, 128]. These events may explain the low viral load and lack of detection of HBsAg in occult cases.

Conclusions

This study is the first characterization study of OBI in patients with end-stage liver disease in Colombia to provide data on HBV genotype, mutations and integration events in OBI cases. We could detect the HBV genomes in 10% of liver samples obtained from transplant recipients. The HBV genotypes F (F3, F1), A (A2) and D (D4) were characterized. In addition, a considerable number of mutations were identified in the OBI cases; among them, the mutation A2915G affecting both the P and HBsAg sequences was identified in 4/5 OBI cases. Interestingly, we also identified antiviral resistance-related mutations and preC/Core promoter mutations; however, none could be associated with low fitness viral replication or impaired expression of HBsAg. In addition, three integration events were characterized in two OBI samples; those events were identified in PTPRT, RASGRF2, and ZNF263 genes.

The limitations of this study are the sample size, the detection limit of PCR strategies for the detection and sequencing of the viral genome and the methodological approach to detect integration only downstream of nt 1130 of the HBV genome; indeed, it has been reported that region nt 1513 to nt 1680 is a common break point for integration [119].

Further studies are needed to determine the prevalence of OBI in different populations in Colombia, as well whether the mutants and integration events described in the present study might participate in the pathogenesis of OBI.

Supporting information

S1 Fig. Complete genome sequencing strategies.

Overlapping primers of four PCR strategies were used to amplify the complete genome of HBV.

https://doi.org/10.1371/journal.pone.0180447.s001

(DOCX)

S2 Fig. HBV DNA detection by southern blot.

Total DNA from OBI identified samples was tested for HBV detection using DIG-labeled probe of 319bp targeting S gene was used to detect viral DNA. Line 1: pJET-TH24-1,5, 2: TH3, 3: TH6, 4: TH28, 5: TH78, 6: TH75. Plasmid pJET-TH24-1,5 (10μg) and total DNA (40–50μg) were used.

https://doi.org/10.1371/journal.pone.0180447.s002

(DOCX)

S1 Table. Primers used for HBV genome detection and sequencing.

https://doi.org/10.1371/journal.pone.0180447.s003

(DOCX)

S2 Table. List of sequences used for phylogenetic analysis.

https://doi.org/10.1371/journal.pone.0180447.s004

(DOCX)

S3 Table. List of sequences used for pairwise mutation analysis.

https://doi.org/10.1371/journal.pone.0180447.s005

(DOCX)

Acknowledgments

We thank Dr. Anne Lise Haenni, from the Institut Jacques Monod, for revision of the manuscript.

Author Contributions

  1. Conceptualization: MCN.
  2. Data curation: JCR.
  3. Formal analysis: JCR SH.
  4. Funding acquisition: MCN FCM.
  5. Investigation: JCR.
  6. Project administration: MCN.
  7. Resources: MCN JCRG SH FCM.
  8. Supervision: MCN.
  9. Validation: MCN FCM.
  10. Visualization: JCR MCN.
  11. Writing – original draft: JCR.
  12. Writing – review & editing: MCN FCM.

References

  1. 1. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. Lancet. 2012;380(9859):2095–128. Epub 2012/12/19. pmid:23245604.
  2. 2. www.who.int. World Health Organization, WHO. Hepatitis B [Internet]. Geneva: WHO; 2015 [cited 2015 Oct-10-2015]. http://www.who.int/mediacentre/factsheets/fs204/en/.
  3. 3. Krajden M, McNabb G, Petric M. The laboratory diagnosis of hepatitis B virus. Can J Infect Dis Med Microbiol. 2005;16(2):65–72. Epub 2007/12/27. pmid:18159530;
  4. 4. Deny P, Zoulim F. Hepatitis B virus: from diagnosis to treatment. Pathol Biol (Paris). 2010;58(4):245–53. Epub 2010/06/29. pmid:20580167.
  5. 5. Tabor E, Hoofnagle JH, Smallwood LA, Drucker JA, Pineda-Tamondong GC, Ni LY, et al. Studies of donors who transmit posttransfusion hepatitis. Transfusion. 1979;19(6):725–31. Epub 1979/11/01. pmid:230620.
  6. 6. Baginski I, Chemin I, Hantz O, Pichoud C, Jullien AM, Chevre JC, et al. Transmission of serologically silent hepatitis B virus along with hepatitis C virus in two cases of posttransfusion hepatitis. Transfusion. 1992;32(3):215–20. Epub 1992/03/01. pmid:1557801.
  7. 7. Ocana S, Casas ML, Buhigas I, Lledo JL. Diagnostic strategy for occult hepatitis B virus infection. World J Gastroenterol. 2011;17(12):1553–7. Epub 2011/04/08. pmid:21472120;
  8. 8. Raimondo G, Pollicino T, Cacciola I, Squadrito G. Occult hepatitis B virus infection. J Hepatol. 2007;46(1):160–70. Epub 2006/11/23. pmid:17112622.
  9. 9. Hu KQ. Occult hepatitis B virus infection and its clinical implications. J Viral Hepat. 2002;9(4):243–57. Epub 2002/06/26. pmid:12081601.
  10. 10. Zhang Z, Zhang L, Dai Y, Zhang Y, Li J, Li X. Occult hepatitis B virus infection: influence of S protein variants. Virol J. 2016;13:10. Epub 2016/01/21. pmid:26786229;
  11. 11. Vivekanandan P, Kannangai R, Ray SC, Thomas DL, Torbenson M. Comprehensive genetic and epigenetic analysis of occult hepatitis B from liver tissue samples. Clin Infect Dis. 2008;46(8):1227–36. Epub 2008/05/01. pmid:18444860;
  12. 12. Levrero M, Pollicino T, Petersen J, Belloni L, Raimondo G, Dandri M. Control of cccDNA function in hepatitis B virus infection. J Hepatol. 2009;51(3):581–92. Epub 2009/07/21. pmid:19616338.
  13. 13. Di Stefano M, Volpe A, Stallone G, Tartaglia L, Prato R, Martinelli D, et al. Occult HBV infection in hemodialysis setting is marked by presence of isolated antibodies to HBcAg and HCV. J Nephrol. 2009;22(3):381–6. Epub 2009/06/27. pmid:19557715.
  14. 14. Minuk GY, Sun DF, Greenberg R, Zhang M, Hawkins K, Uhanova J, et al. Occult hepatitis B virus infection in a North American adult hemodialysis patient population. Hepatology. 2004;40(5):1072–7. Epub 2004/10/16. pmid:15486926.
  15. 15. Tsui JI, French AL, Seaberg EC, Augenbraun M, Nowicki M, Peters M, et al. Prevalence and long-term effects of occult hepatitis B virus infection in HIV-infected women. Clin Infec Dis. 2007;45(6):736–40. Epub 2007/08/23. pmid:17712758.
  16. 16. Hofer M, Joller-Jemelka HI, Grob PJ, Luthy R, Opravil M. Frequent chronic hepatitis B virus infection in HIV-infected patients positive for antibody to hepatitis B core antigen only. Swiss HIV Cohort Study. Eur J Clin Microbiol Infect Dis. 1998;17(1):6–13. Epub 1998/03/25. pmid:9512175.
  17. 17. Nunez M, Rios P, Perez-Olmeda M, Soriano V. Lack of 'occult' hepatitis B virus infection in HIV-infected patients. Aids. 2002;16(15):2099–101. Epub 2002/10/09. pmid:12370518.
  18. 18. Santos EA, Yoshida CF, Rolla VC, Mendes JM, Vieira IF, Arabe J, et al. Frequent occult hepatitis B virus infection in patients infected with human immunodeficiency virus type 1. Eur J Clin Microbiol Infect Dis. 2003;22(2):92–8. Epub 2003/03/11. pmid:12627282.
  19. 19. Kao JH, Chen PJ, Lai MY, Chen DS. Occult hepatitis B virus infection and clinical outcomes of patients with chronic hepatitis C. J Clin microbiol. 2002;40(11):4068–71. Epub 2002/11/01. pmid:12409376;
  20. 20. Koike K, Kobayashi M, Gondo M, Hayashi I, Osuga T, Takada S. Hepatitis B virus DNA is frequently found in liver biopsy samples from hepatitis C virus-infected chronic hepatitis patients. J Med Virol. 1998;54(4):249–55. Epub 1998/04/29. pmid:9557290.
  21. 21. Cacciola I, Pollicino T, Squadrito G, Cerenzia G, Orlando ME, Raimondo G. Occult hepatitis B virus infection in patients with chronic hepatitis C liver disease. New Engl J Med. 1999;341(1):22–6. Epub 1999/07/01. pmid:10387938.
  22. 22. Hassan ZK, Hafez MM, Mansor TM, Zekri AR. Occult HBV infection among Egyptian hepatocellular carcinoma patients. Virol J. 2011;8:90. Epub 2011/03/05. pmid:21371325;
  23. 23. Squadrito G, Pollicino T, Cacciola I, Caccamo G, Villari D, La Masa T, et al. Occult hepatitis B virus infection is associated with the development of hepatocellular carcinoma in chronic hepatitis C patients. Cancer. 2006;106(6):1326–30. Epub 2006/02/03. pmid:16453330.
  24. 24. Kannangai R, Molmenti E, Arrazola L, Klein A, Choti M, Thomas DL, et al. Occult hepatitis B viral DNA in liver carcinomas from a region with a low prevalence of chronic hepatitis B infection. J Viral Hepat. 2004;11(4):297–301. Epub 2004/07/03. pmid:15230851.
  25. 25. Chan HL, Tsang SW, Leung NW, Tse CH, Hui Y, Tam JS, et al. Occult HBV infection in cryptogenic liver cirrhosis in an area with high prevalence of HBV infection. Am J Gastroenterol. 2002;97(5):1211–5. Epub 2002/05/17. pmid:12014730.
  26. 26. Wong DK, Huang FY, Lai CL, Poon RT, Seto WK, Fung J, et al. Occult hepatitis B infection and HBV replicative activity in patients with cryptogenic cause of hepatocellular carcinoma. Hepatology. 2011;54(3):829–36. Epub 2011/08/03. pmid:21809355.
  27. 27. Chazouilleres O, Mamish D, Kim M, Carey K, Ferrell L, Roberts JP, et al. "Occult" hepatitis B virus as source of infection in liver transplant recipients. Lancet. 1994;343(8890):142–6. Epub 1994/01/15. pmid:7904004.
  28. 28. Ghisetti V, Marzano A, Zamboni F, Barbui A, Franchello A, Gaia S, et al. Occult hepatitis B virus infection in HBsAg negative patients undergoing liver transplantation: clinical significance. Liver Transpl. 2004;10(3):356–62. Epub 2004/03/09. pmid:15004761.
  29. 29. Polo P, Castañeda C, Sierra M, Alvis N. Hepatitis B oculta en pacientes VIH positivos de una institución de salud en Barranquilla Colombia. Infectio. 2009;14(1):39–46.
  30. 30. Bautista-Amorocho H, Castellanos-Dominguez YZ, Rodriguez-Villamizar LA, Velandia-Cruz SA, Becerra-Pena JA, Farfan-Garcia AE. Epidemiology, risk factors and genotypes of HBV in HIV-infected patients in the northeast region of Colombia: high prevalence of occult hepatitis B and F3 subgenotype dominance. PLoS ONE. 2014;9(12):e114272. Epub 2014/12/03. pmid:25462190;
  31. 31. Ramirez IC, J.C. C. Prevalencia de hepatitis B oculta en una cohorte prospectiva de pacientes con VIH. Iatreia. 2008:S10–1.
  32. 32. Rios-Ocampo WA, Cortes-Mancera F, Olarte JC, Soto A, Navas MC. Occult hepatitis B virus infection among blood donors in Colombia. Virol J. 2014;11:206. Epub 2014/12/05. pmid:25471066;
  33. 33. Beltran M, Berrio-Perez M, Bermudez MI, Rey-Benito G, Camacho B, Forero P, et al. [Absence of occult hepatitis B in Colombian blood donors]. Biomedica. 2011;31(4):580–9. Epub 2012/06/08. pmid:22674369.
  34. 34. Arroyave J, Cortes-Mancera F. Caracterizacion molecular de la infeccion por el virus de la hepatitis B en donantes de sangre HBsAg negativo/anti-HBc positivo. Rev Hech Microbiol. 2011:22.
  35. 35. Bautista H, Castellanos YZ, Farfan AE. Marcadores serológicos y moleculares de infección por el virus de la hepatitis B en estudiantes universitarios colombianos. Rev Col Gastroenterol. 2012;27(4).
  36. 36. Duque-Jaramillo A, Rendon JC, Cortes-Mancera F, Correa G, Restrepo JC, Hoyos S, et al. Occult Hepatitis B Virus Infection in Liver Transplant Patients. Rev Col Gastroenterol. 2016;31(4):7.
  37. 37. Dickson RC, Everhart JE, Lake JR, Wei Y, Seaberg EC, Wiesner RH, et al. Transmission of hepatitis B by transplantation of livers from donors positive for antibody to hepatitis B core antigen. The National Institute of Diabetes and Digestive and Kidney Diseases Liver Transplantation Database. Gastroenterology. 1997;113(5):1668–74. Epub 1997/11/14. S0016508597005350 [pii]. pmid:9352871.
  38. 38. Muñoz SJ. Use of hepatitis B core antibody-positive donors for liver transplantation. Liver Transpl. 2002;8(10 S1):S82–7. Epub 2002/10/04. pmid:12362304.
  39. 39. Niu Y, Chen X, Feng L, You H, Ren X, Liu H, et al. Anti-HBc-positive/HBsAg-negative liver donors pose a higher risk of occult HBV infection but do not cause severe histological damage in liver grafts. Clin Res Hepatol Gastroenterol. 2014;38(4):475–80. Epub 2014/05/20. pmid:24835492.
  40. 40. www.srtr.org. Scientific Registry of Transplant Recipients. U.S. Organ Donors by Organ and Donor Type, 2002 to 2011 [Internet]. Minneapolis: SRTR; 2016 [2016-07-07]. http://www.srtr.org/annual_reports/2011/101_dh.aspx.
  41. 41. www.minsalud.gov.co. Minsalud Colombia. Datos Globales de Donación y Trasplantes, 2014 [Internet]. Bogota: Minsalud; 2016 [2016-07-07]. https://www.minsalud.gov.co/sites/rid/Lists/BibliotecaDigital/RIDE/VS/MET/Datos-Globales-Donacion-Trasplantes.pdf.
  42. 42. Mahboobi N, Tabatabaei SV, Blum HE, Alavian SM. Renal grafts from anti-hepatitis B core-positive donors: a quantitative review of the literature. Transpl Infect Dis. 2012;14(5):445–51. Epub 2012/09/14. pmid:22970743.
  43. 43. Strasser SI, McDonald GB. Hepatitis viruses and hematopoietic cell transplantation: A guide to patient and donor management. Blood. 1999;93(4):1127–36. Epub 1999/02/09. pmid:9949154.
  44. 44. De Feo TM, Poli F, Mozzi F, Moretti MP, Scalamogna M. Risk of transmission of hepatitis B virus from anti-HBC positive cadaveric organ donors: a collaborative study. Transplant Proc. 2005;37(2):1238–9. Epub 2005/04/26. pmid:15848681.
  45. 45. Cheung CK, Lo CM, Man K, Lau GK. Occult hepatitis B virus infection of donor and recipient origin after liver transplantation despite nucleoside analogue prophylaxis. Liver transplant. 2010;16(11):1314–23. Epub 2010/10/30. pmid:21031547.
  46. 46. Marzano A, Gaia S, Ciardo V, Premoli A, Ghisetti V, Salizzoni M, et al. Prophylaxis of hepatitis B virus infection before liver transplantation, 1990–2001: a single-center experience. Transplant Proc. 2003;35(3):1020–1. Epub 2003/09/02. pmid:12947843.
  47. 47. Ferrari TC, Xavier MA, Vidigal PV, Amaral NS, Diniz PA, Resende AP, et al. Occult hepatitis B virus infection in liver transplant patients in a Brazilian referral center. Braz J Med Biol Res. 2014;47(11):990–4. Epub 2014/10/09. S0100-879X2014001100990 [pii]. pmid:25296362;
  48. 48. Toniutto P, Minisini R, Fabris C, De Feo T, Marangoni F, Burlone M, et al. Occult hepatitis B virus infection in liver transplant recipients with recurrent hepatitis C: relationship with donor age and fibrosis progression. Clin Transplant. 2009;23(2):184–90. Epub 2009/02/13. pmid:19210526.
  49. 49. Shetty K, Hussain M, Nei L, Reddy KR, Lok AS. Prevalence and significance of occult hepatitis B in a liver transplant population with chronic hepatitis C. Liver Transpl. 2008;14(4):534–40. Epub 2008/03/08. pmid:18324677.
  50. 50. www.easl.eu/. European Association for the Study of the liver [Internet]. Geneva: EASL; 2016 [2016-12-09]. http://www.easl.eu/research/our-contributions/clinical-practice-guidelines.
  51. 51. www.aasld.org. American Association for the Study of Liver Diseases [Internet]. Alexandria: AASLD; 2016 [2016-12-09]. http://www.aasld.org/publications/practice-guidelines-0.
  52. 52. Brechot C, Thiers V, Kremsdorf D, Nalpas B, Pol S, Paterlini-Brechot P. Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen: clinically significant or purely "occult"? Hepatology. 2001;34(1):194–203. Epub 2001/06/30. pmid:11431751.
  53. 53. Mazzolla R, Puliti M, Barluzzi R, Neglia R, Bistoni F, Barbolini G, et al. Differential microbial clearance and immunoresponse of Balb/c (Nramp1 susceptible) and DBA2 (Nramp1 resistant) mice intracerebrally infected with Mycobacterium bovis BCG (BCG). FEMS Immunol Med Microbiol. 2002;32(2):149–58. Epub 2002/02/01. pmid:11821237.
  54. 54. Zeng GB, Wen SJ, Wang ZH, Yan L, Sun J, Hou JL. A novel hepatitis B virus genotyping system by using restriction fragment length polymorphism patterns of S gene amplicons. World J Gastroenterol. 2004;10(21):3132–6. Epub 2004/10/01. pmid:15457558.
  55. 55. Schaefer S, Glebe D, Wend UC, Oyunbileg J, Gerlich WH. Universal primers for real-time amplification of DNA from all known Orthohepadnavirus species. J Clin Virol. 2003;27(1):30–7. Epub 2003/05/03. pmid:12727526.
  56. 56. Abedi-Ardekani B, Gouas D, Villar S, Sotoudeh M, Hainaut P. TP53 Mutations and HBX Status Analysis in Hepatocellular Carcinomas from Iran: Evidence for Lack of Association between HBV Genotype D and TP53 R249S Mutations. Hepat Res Treat. 2011;2011:475965. Epub 2011/08/27. pmid:21869931;
  57. 57. Ma NF, Lau SH, Hu L, Xie D, Wu J, Yang J, et al. COOH-terminal truncated HBV X protein plays key role in hepatocarcinogenesis. Clin Cancer Res. 2008;14(16):5061–8. Epub 2008/08/14. pmid:18698024.
  58. 58. Hu X, Margolis HS, Purcell RH, Ebert J, Robertson BH. Identification of hepatitis B virus indigenous to chimpanzees. Proc Natl Acad Sci U S A. 2000;97(4):1661–4. Epub 2000/03/04. pmid:10677515;
  59. 59. Cortes-Mancera F, Loureiro CL, Hoyos S, Restrepo JC, Correa G, Jaramillo S, et al. Etiology and Viral Genotype in Patients with End-Stage Liver Diseases admitted to a Hepatology Unit in Colombia. Hepat Res Treat. 2011;2011:363205. Epub 2011/09/24. pmid:21941645;
  60. 60. Gunther S, Li BC, Miska S, Kruger DH, Meisel H, Will H. A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients. J Virol. 1995;69(9):5437–44. Epub 1995/09/01. pmid:7636989;
  61. 61. Devesa M, Loureiro CL, Rivas Y, Monsalve F, Cardona N, Duarte MC, et al. Subgenotype diversity of hepatitis B virus American genotype F in Amerindians from Venezuela and the general population of Colombia. J Med Virol. 2008;80(1):20–6. Epub 2007/11/28. pmid:18041024.
  62. 62. Hall AT. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. 1999;41:4.
  63. 63. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Bio Evol. 2011;28(10):2731–9. Epub 2011/05/07. pmid:21546353;
  64. 64. Minami M, Poussin K, Brechot C, Paterlini P. A novel PCR technique using Alu-specific primers to identify unknown flanking sequences from the human genome. Genomics. 1995;29(2):403–8. Epub 1995/09/20. pmid:8666388.
  65. 65. Murakami Y, Saigo K, Takashima H, Minami M, Okanoue T, Brechot C, et al. Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV related hepatocellular carcinomas. Gut. 2005;54(8):1162–8. Epub 2005/07/13. pmid:16009689;
  66. 66. Zhu HL, Li X, Li J, Zhang ZH. Genetic variation of occult hepatitis B virus infection. World J Gastroenterol. 2016;22(13):3531–46. Epub 2016/04/08. pmid:27053845;
  67. 67. Lee YS, Chung YH, Kim JA, Jin YJ, Park WH, Kim SE, et al. rtL180M mutation of hepatitis B virus is closely associated with frequent virological resistance to adefovir dipivoxil therapy. J Gastroenterol Hepatol. 2012;27(2):300–5. Epub 2011/07/23. pmid:21777282.
  68. 68. Allen MI, Deslauriers M, Andrews CW, Tipples GA, Walters KA, Tyrrell DL, et al. Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group. Hepatology. 1998;27(6):1670–7. Epub 1998/06/10. pmid:9620341.
  69. 69. Pollicino T, Isgro G, Di Stefano R, Ferraro D, Maimone S, Brancatelli S, et al. Variability of reverse transcriptase and overlapping S gene in hepatitis B virus isolates from untreated and lamivudine-resistant chronic hepatitis B patients. Antivir Ther. 2009;14(5):649–54. Epub 2009/08/26. pmid:19704167.
  70. 70. Michailidis E, Kirby KA, Hachiya A, Yoo W, Hong SP, Kim SO, et al. Antiviral therapies: focus on hepatitis B reverse transcriptase. Int J Biochem Cell Biol. 2012;44(7):1060–71. Epub 2012/04/26. pmid:22531713;
  71. 71. Zhong Y, Lv J, Li J, Xing X, Zhu H, Su H, et al. Prevalence, virology and antiviral drugs susceptibility of hepatitis B virus rtN238H polymerase mutation from 1865 Chinese patients with chronic hepatitis B. Antivir Res. 2012;93(1):185–90. Epub 2011/12/06. pmid:22138714.
  72. 72. Bottecchia M, Madejon A, Sheldon J, Garcia-Samaniego J, Barreiro P, Soriano V. Hepatitis B virus genotype A2 harbours an L217R polymorphism which may account for a lower response to adefovir. J Antimicrob Chemother. 2008;62(3):626–7. Epub 2008/05/10. pmid:18467302.
  73. 73. Parekh S, Zoulim F, Ahn SH, Tsai A, Li J, Kawai S, et al. Genome replication, virion secretion, and e antigen expression of naturally occurring hepatitis B virus core promoter mutants. J Virol. 2003;77(12):6601–12. Epub 2003/05/28. pmid:12767980;
  74. 74. Jammeh S, Tavner F, Watson R, Thomas HC, Karayiannis P. Effect of basal core promoter and pre-core mutations on hepatitis B virus replication. J Gen Virol. 2008;89(Pt 4):901–9. Epub 2008/03/18. pmid:18343830.
  75. 75. Han Z, Liu Y, Pan J, Bi Y, Liu J, Zhou YH. Occult hepatitis B virus infection with positive hepatitis B e antigen. Clin Chim Acta. 2015;438:266–8. Epub 2014/09/15. pmid:25218700.
  76. 76. www.ins.gov.co. 2015 Boletin epidemiologico semana 52. Boletin Epidemiológico Semanal [Internet]. 2015. http://www.ins.gov.co/boletin-epidemiologico/Boletn%20Epidemiolgico/2015%20Boletin%20epidemiologico%20Semana%2052.pdf.
  77. 77. Castellanos YZ, Portilla V, Chacon L, Dias G, Miranda G, Farfan A, et al. Identificación de casos de hepatitis B oculta en donantes de bancos de sangre de Santander. Rev Hech Microbiol. 2013:17.
  78. 78. Paterlini P, Driss F, Nalpas B, Pisi E, Franco D, Berthelot P, et al. Persistence of hepatitis B and hepatitis C viral genomes in primary liver cancers from HBsAg-negative patients: a study of a low-endemic area. Hepatology. 1993;17(1):20–9. Epub 1993/01/01. pmid:8380790.
  79. 79. Paterlini P, Gerken G, Nakajima E, Terre S, D'Errico A, Grigioni W, et al. Polymerase chain reaction to detect hepatitis B virus DNA and RNA sequences in primary liver cancers from patients negative for hepatitis B surface antigen. New Engl J Med. 1990;323(2):80–5. Epub 1990/07/12. pmid:2359427.
  80. 80. Liang TJ, Baruch Y, Ben-Porath E, Enat R, Bassan L, Brown NV, et al. Hepatitis B virus infection in patients with idiopathic liver disease. Hepatology. 1991;13(6):1044–51. Epub 1991/06/01. S027091399100143X [pii]. pmid:2050320.
  81. 81. Barros GM Junior, Braga WS, Oliveira CM, Castilho Mda C, Araujo Jde R. [Occult hepatitis B: prevalence and clinical characteristics in a population with high endemicity of hepatitis B infection in the western Brazilian Amazon region]. Rev Soc Bras Med Trop. 2008;41(6):596–601. Epub 2009/01/15. pmid:19142439.
  82. 82. Alencar RS, Gomes MM, Sitnik R, Pinho JR, Malta FM, Mello IM, et al. Low occurrence of occult hepatitis B virus infection and high frequency of hepatitis C virus genotype 3 in hepatocellular carcinoma in Brazil. Braz J Med Biol Res. 2008;41(3):235–40. Epub 2007/12/22. pmid:18097499.
  83. 83. Raimondo G, Cacciamo G, Saitta C. Hepatitis B virus and hepatitis C virus co-infection: additive players in chronic liver disease? Ann Hepatol. 2005;4(2):100–6. Epub 2005/07/13. 16551 [pii]. pmid:16010242.
  84. 84. Raimondo G, Caccamo G, Filomia R, Pollicino T. Occult HBV infection. Semin Immunopathol. 2013;35(1):39–52. Epub 2012/07/26. pmid:22829332;
  85. 85. Ponde RA. Molecular mechanisms underlying HBsAg negativity in occult HBV infection. Eur J Clin Microbiol Infect Dis. 2015. Epub 2015/06/25. pmid:26105620.
  86. 86. Coppola N, Onorato L, Pisaturo M, Macera M, Sagnelli C, Martini S, et al. Role of occult hepatitis B virus infection in chronic hepatitis C. World J Gastroenterol. 2015;21(42):11931–40. Epub 2015/11/18. pmid:26576082;
  87. 87. Gerstenkorn C, Cacciola R, Robertson H, Talbot D. Occult hepatitis B infection in chronic hepatitis C liver disease—implications for liver transplantation. Liver. 2000;20(5):425. Epub 2000/11/25. pmid:11092264.
  88. 88. Alvarado Mora MV, Romano CM, Gomes-Gouvea MS, Gutierrez MF, Botelho L, Carrilho FJ, et al. Molecular characterization of the Hepatitis B virus genotypes in Colombia: a Bayesian inference on the genotype F. Infect Genet Evol. 2011;11(1):103–8. Epub 2010/10/19. pmid:20951841.
  89. 89. Alvarado Mora MV, Romano CM, Gomes-Gouvea MS, Gutierrez MF, Carrilho FJ, Pinho JR. Molecular epidemiology and genetic diversity of hepatitis B virus genotype E in an isolated Afro-Colombian community. J Gen Virol. 2010;91(Pt 2):501–8. Epub 2009/10/23. pmid:19846674.
  90. 90. Rios D, di Filippo D, Insuasty M, Rendon JC, Rios WA, Medina C, et al. Hepatitis B Infections in Individuals with Exposure Factors in Quibdo and Apartado, Colombia. Rev Col Gastroenterol. 2015;30(1):11–7.
  91. 91. di Filippo Villa D, Cortes-Mancera F, Payares E, Montes N, de la Hoz F, Arbelaez MP, et al. Hepatitis D virus and hepatitis B virus infection in Amerindian communities of the Amazonas state, Colombia. Virol J. 2015;12:172. Epub 2015/10/27. pmid:26497287;
  92. 92. Kim H, Lee SA, Won YS, Lee H, Kim BJ. Occult infection related hepatitis B surface antigen variants showing lowered secretion capacity. World J Gastroenterol. 2015;21(6):1794–803. Epub 2015/02/17. pmid:25684944;
  93. 93. Fang Y, Teng X, Xu WZ, Li D, Zhao HW, Fu LJ, et al. Molecular characterization and functional analysis of occult hepatitis B virus infection in Chinese patients infected with genotype C. J Med Virol. 2009;81(5):826–35. Epub 2009/03/26. pmid:19319940.
  94. 94. Huang CH, Yuan Q, Chen PJ, Zhang YL, Chen CR, Zheng QB, et al. Influence of mutations in hepatitis B virus surface protein on viral antigenicity and phenotype in occult HBV strains from blood donors. J Hepatol. 2012;57(4):720–9. Epub 2012/05/29. pmid:22634131.
  95. 95. Ireland JH, O'Donnell B, Basuni AA, Kean JD, Wallace LA, Lau GK, et al. Reactivity of 13 in vitro expressed hepatitis B surface antigen variants in 7 commercial diagnostic assays. Hepatology. 2000;31(5):1176–82. Epub 2000/05/05. pmid:10796895.
  96. 96. Zhang Z, Zhang L, Dai Y, Jin L, Sun B, Su Q, et al. Occult hepatitis B virus infection among people with a family history of chronic hepatitis B virus infection. J Med Virol. 2015;87(11):1890–8. Epub 2015/05/13. pmid:25964194.
  97. 97. Gujar SA, Michalak TI. Primary occult hepadnavirus infection induces virus-specific T-cell and aberrant cytokine responses in the absence of antiviral antibody reactivity in the Woodchuck model of hepatitis B virus infection. J Virol. 2009;83(8):3861–76. Epub 2009/02/06. pmid:19193791;
  98. 98. Coffin CS, Pham TN, Mulrooney PM, Churchill ND, Michalak TI. Persistence of isolated antibodies to woodchuck hepatitis virus core antigen is indicative of occult infection. Hepatology. 2004;40(5):1053–61. Epub 2004/09/24. pmid:15382154.
  99. 99. Samal J, Kandpal M, Vivekanandan P. Molecular mechanisms underlying occult hepatitis B virus infection. Clin Microbiol Rev. 2012;25(1):142–63. Epub 2012/01/11. pmid:22232374;
  100. 100. Pult I, Abbott N, Zhang YY, Summers J. Frequency of spontaneous mutations in an avian hepadnavirus infection. J Virol. 2001;75(20):9623–32. Epub 2001/09/18. pmid:11559794;
  101. 101. Zhong G, Yan H, Wang H, He W, Jing Z, Qi Y, et al. Sodium taurocholate cotransporting polypeptide mediates woolly monkey hepatitis B virus infection of Tupaia hepatocytes. J Virol. 2013;87(12):7176–84. Epub 2013/04/19. pmid:23596296;
  102. 102. Nassal M. The arginine-rich domain of the hepatitis B virus core protein is required for pregenome encapsidation and productive viral positive-strand DNA synthesis but not for virus assembly. J Virol. 1992;66(7):4107–16. Epub 1992/07/01. pmid:1602535;
  103. 103. Birnbaum F, Nassal M. Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein. J Virol. 1990;64(7):3319–30. Epub 1990/07/01. pmid:2191149;
  104. 104. Kock J, Nassal M, Deres K, Blum HE, von Weizsacker F. Hepatitis B virus nucleocapsids formed by carboxy-terminally mutated core proteins contain spliced viral genomes but lack full-size DNA. J Virol. 2004;78(24):13812–8. Epub 2004/11/27. pmid:15564489;
  105. 105. Tong S. Mechanism of HBV genome variability and replication of HBV mutants. J Clin Virol. 2005;34(S1):S134–8. Epub 2006/02/08. pmid:16461214.
  106. 106. Dong Q, Chan HL, Liu Z, Chan DP, Zhang B, Chen Y, et al. A1762T/G1764A mutations of hepatitis B virus, associated with the increased risk of hepatocellular carcinoma, reduce basal core promoter activities. Biochem Bioph Res Co. 2008;374(4):773–6. Epub 2008/08/05. pmid:18675784.
  107. 107. Gouas DA, Villar S, Ortiz-Cuaran S, Legros P, Ferro G, Kirk GD, et al. TP53 R249S mutation, genetic variations in HBX and risk of hepatocellular carcinoma in The Gambia. Carcinogenesis. 2012;33(6):1219–24. Epub 2012/07/05. pmid:22759751;
  108. 108. Kumar V, Sarkar DP. Hepatitis B Virus X Protein: Structure—Function Relationships and Role in Viral Pathogenesis. In: Goosen M, Kaufmann J, Triezenberg SJ, editors. Transcription Factors. 166: Springer-Verlag; 2004. p. 377–407.
  109. 109. Belloni L, Pollicino T, De Nicola F, Guerrieri F, Raffa G, Fanciulli M, et al. Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function. Proc Natl Acad Sci U S A. 2009;106(47):19975–9. Epub 2009/11/13. pmid:19906987;
  110. 110. Lucifora J, Arzberger S, Durantel D, Belloni L, Strubin M, Levrero M, et al. Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection. J Hepatol. 2011;55(5):996–1003. Epub 2011/03/08. pmid:21376091.
  111. 111. Zhang ZH, Wu CC, Chen XW, Li X, Li J, Lu MJ. Genetic variation of hepatitis B virus and its significance for pathogenesis. World J Gastroenterol. 2016;22(1):126–44. Epub 2016/01/13. pmid:26755865;
  112. 112. Menendez-Arias L, Alvarez M, Pacheco B. Nucleoside/nucleotide analog inhibitors of hepatitis B virus polymerase: mechanism of action and resistance. Curr Opin Virol. 2014;8:1–9. Epub 2014/05/13. pmid:24814823.
  113. 113. Seigneres B, Aguesse-Germon S, Pichoud C, Vuillermoz I, Jamard C, Trepo C, et al. Duck hepatitis B virus polymerase gene mutants associated with resistance to lamivudine have a decreased replication capacity in vitro and in vivo. J Hepatol. 2001;34(1):114–22. Epub 2001/02/24. pmid:11211887.
  114. 114. Zoulim F. Mechanism of viral persistence and resistance to nucleoside and nucleotide analogs in chronic hepatitis B virus infection. Antivir Res. 2004;64(1):1–15. Epub 2004/09/29. pmid:15451174.
  115. 115. Wakil SM, Kazim SN, Khan LA, Raisuddin S, Parvez MK, Guptan RC, et al. Prevalence and profile of mutations associated with lamivudine therapy in Indian patients with chronic hepatitis B in the surface and polymerase genes of hepatitis B virus. J Med Virol. 2002;68(3):311–8. Epub 2002/09/13. pmid:12226816.
  116. 116. Torresi J, Earnest-Silveira L, Deliyannis G, Edgtton K, Zhuang H, Locarnini SA, et al. Reduced antigenicity of the hepatitis B virus HBsAg protein arising as a consequence of sequence changes in the overlapping polymerase gene that are selected by lamivudine therapy. Virology. 2002;293(2):305–13. Epub 2002/03/12. pmid:11886250.
  117. 117. Kim H, Kim BJ. Association of preS/S Mutations with Occult Hepatitis B Virus (HBV) Infection in South Korea: Transmission Potential of Distinct Occult HBV Variants. Int J Mol Sci. 2015;16(6):13595–609. Epub 2015/06/18. pmid:26084041;
  118. 118. Kimbi GC, Kramvis A, Kew MC. Integration of hepatitis B virus DNA into chromosomal DNA during acute hepatitis B. World J Gastroenterol. 2005;11(41):6416–21. Epub 2006/01/21. pmid:16425409.
  119. 119. Sung WK, Zheng H, Li S, Chen R, Liu X, Li Y, et al. Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma. Nat Genet. 2012;44(7):765–9. Epub 2012/05/29. pmid:22634754.
  120. 120. Takada S, Koike K. Trans-activation function of a 3' truncated X gene-cell fusion product from integrated hepatitis B virus DNA in chronic hepatitis tissues. Proc Natl Acad Sci U S A. 1990;87(15):5628–32. Epub 1990/08/01. pmid:2165598;
  121. 121. Freitas N, Cunha C, Menne S, Gudima SO. Envelope proteins derived from naturally integrated hepatitis B virus DNA support assembly and release of infectious hepatitis delta virus particles. J Virol. 2014;88(10):5742–54. Epub 2014/03/14. pmid:24623409;
  122. 122. www.genecards.or. Weizmann Institute of Science. PTPRT Gene [Internet]. Alameda: Weizmann Institute of Science; 2016 [Jun-27-2016]. http://www.genecards.org/cgi-bin/carddisp.pl?gene=PTPRT.
  123. 123. Tonks NK. Protein tyrosine phosphatases: from genes, to function, to disease. Nat Rev Mol Cell Biol. 2006;7(11):833–46. Epub 2006/10/24. pmid:17057753.
  124. 124. www.genecards.org. Weizmann_Institute_of_Science. RASGRF2 Gene [Internet]. Alameda: Weizmann Institute of Science; 2016 [Jun-27-2016]. http://www.genecards.org/cgi-bin/carddisp.pl?gene=RASGRF2.
  125. 125. Fernandez-Medarde A, Santos E. The RasGrf family of mammalian guanine nucleotide exchange factors. Biochim Biophys Acta. 2011;1815(2):170–88. Epub 2010/11/30. pmid:21111786.
  126. 126. Frietze S, Lan X, Jin VX, Farnham PJ. Genomic targets of the KRAB and SCAN domain-containing zinc finger protein 263. J Biol Chem. 2010;285(2):1393–403. Epub 2009/11/06. pmid:19887448;
  127. 127. Huang ZM, Yen TS. Dysregulated surface gene expression from disrupted hepatitis B virus genomes. J Virol. 1993;67(12):7032–40. Epub 1993/12/01. pmid:8230428;
  128. 128. Chisari FV, Filippi P, McLachlan A, Milich DR, Riggs M, Lee S, et al. Expression of hepatitis B virus large envelope polypeptide inhibits hepatitis B surface antigen secretion in transgenic mice. J Virol. 1986;60(3):880–7. Epub 1986/12/01. pmid:3783819