Research Article Quantification of Pregenomic RNA and...

Hindawi Publishing CorporationInternational Journal of HepatologyVolume 2013 Article ID 849290 9 pageshttpdxdoiorg1011552013849290

Research ArticleQuantification of Pregenomic RNA andCovalently Closed Circular DNA in Hepatitis B Virus-RelatedHepatocellular Carcinoma

Fugui Bai1 Yoshihiko Yano12 Takumi Fukumoto3 Atsushi Takebe3

Motofumi Tanaka3 Kaori Kuramitsu3 Nungki Anggorowati1 Hanggoro Tri Rinonce1

Dewiyani Indah Widasari1 Masaya Saito2 Hirotaka Hirano2 Takanobu Hayakumo2

Yasushi Seo2 Takeshi Azuma2 Yonson Ku3 and Yoshitake Hayashi1

1 Center for Infectious Diseases Kobe University Graduate School of Medicine Kobe 650-0017 Japan2Department of Gastroenterology Kobe University Graduate School of Medicine Kobe 650-0017 Japan3Department of Hepato-Biliary-Pancreatic Surgery Kobe University Graduate School of Medicine Kobe 650-0017 Japan

Correspondence should be addressed to Yoshihiko Yano yanoyomedkobe-uacjp

Received 6 May 2013 Revised 7 November 2013 Accepted 8 November 2013

Academic Editor Daisuke Morioka

Copyright copy 2013 Fugui Bai et alThis is an open access article distributed under theCreative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Pregenomic RNA (pgRNA) is generated from covalently closed circular DNA (cccDNA) and plays important roles in viral genomeamplification and replication Hepatic pgRNA and cccDNA expression levels indicate viral persistence and replication activityThisstudy was aimed to measure hepatic pgRNA and cccDNA expression levels in various states of hepatitis B virus (HBV) infectionThirty-eight hepatocellular carcinoma (HCC) patients including 14 positive for hepatitis B surface antigen (HBsAg) and 24 negativefor HBsAg but positive for anti-hepatitis B core (anti-HBc) antibody were enrolled in this study In HBsAg-negative but anti-HBc-positive group HBV-DNA was detected in 20 of 24 (83) noncancerous liver tissues for at least two genomic regions based onpolymerase chain reaction (PCR) analysis pgRNA and cccDNA expression levels in occult HBV-infected patients were significantlylower than those in HBsAg-positive patients (119875 lt 0001) pgRNA and cccDNA in cancerous tissues were also detected withoutsignificant difference from those in noncancerous tissues In conclusion cccDNA and pgRNA are detected and represented HBVreplication not only in noncancerous but also in cancerous liver tissues In addition the replication is shown in not only patientswith HBsAg-positive but also occult HBV-infected patients suggesting the contribution to HCC development

1 Introduction

Hepatitis B virus (HBV) infection remains a major healthproblem even though methods for preventing vertical trans-mission and treatment guidelines have been introducedApproximately 2 billion people worldwide are infected withHBVThree hundred fiftymillion of them have chronic infec-tion HBV is particularly endemic in sub-Saharan Africa thePacific and Asia [1] It frequently causes chronic hepatitiswhich subsequently progresses to liver cirrhosis and hepa-tocellular carcinoma (HCC) HBV is responsible for most ofthe worldwide attributable risk of liver cirrhosis and HCCThe risk is even higher in endemic regions [2]

HBV is an incomplete double-stranded circular DNAvirus with a DNA length of approximately 3200 bp AfterHBV infects the liver cells the relaxed-circular DNA is trans-ferred into the nucleus and is repaired to covalently closedcircular DNA (cccDNA) by DNA polymerase The cccDNAplays a key role in the life cycle of the virus It acts as a templatefor the generation of mRNAs including pregenomic RNA(pgRNA) [3ndash5] The transcription of cccDNA generates fourkinds of mRNA PreCC (35 kb) PreS1 (24 kb) S (21 kb)and X (07 kb) The transcription of cccDNA is a critical stepfor viral genomic amplification and replication PgRNA isalso generated during the transcription from cccDNA andworks as a template of viral genomic DNA

2 International Journal of Hepatology

The cccDNA is detected in various phases of chronichepatitis and its levels reflect viral replication in the infectedcells HBV-DNA is sometimes detected in patients negativefor hepatitis B surface antigen (HBsAg) but positive for anti-hepatitis B core (anti-HBc) antibody andor anti-hepatitisB surface (anti-HBs) antibody which is often called occultHBV infection [4] Recent studies revealed that cccDNAwas expressed in HCC without HBs antigen [5] Howeverit is still debated whether HBV-DNA is responsible for thedevelopment of HCC in patients with occult HBV infection

In this study we measured the levels of cccDNA andpgRNA in cancerous and contiguous noncancerous livertissues of HCC patients with overt and occult HBV infectionto examine the viral existence and replication We also deter-mined the nucleotide variations and amino acid mutations inpatients with occult HBV infection

2 Materials and Methods

21 Patients Cancerous and contiguous noncancerous livertissues were obtained from 38 HCC patients (mean plusmn SD6337 plusmn 1201 years male-female ratio 344) who under-went surgical resection at Kobe University Hospital Japanbetween April 2010 and December 2011 HBsAg-positivecarriers (119899 = 14) and HBsAg-negativeHBcAb-positivepatients (119899 = 24) were enrolled in this study Hepatitis Cvirus (HCV) infected patients who were detected by anti-HCV positivity were excluded Tissue samples were rapidlyfrozen in liquid nitrogen and stored at ndash80∘C after resection

The demographic and clinical data including age sexbody mass index (BMI) results of standard test for HBV-related HCC and stage of the disease were collected frommedical record after institutional review board approval wasobtained

Written informed consentwas obtained fromeach patientbefore all the procedures in this study were done This studywas reviewed and approved by the Ethics Committee at KobeUniversity

22 Detection of HBV-DNA cccDNA and pgRNA in TissueSamples Total DNA and RNA from cancerous and contigu-ous noncancerous tissues were extracted from about 20mgof liver tissues using a QIAamp DNA Mini Kit (QIAGENSciences Germantown MD) and Isogen (Nippon GeneTokyo Japan)The DNA and RNA concentrations were mea-sured spectrophotometrically The cDNA was synthesizedfrom five 120583g of total RNA using the Oligo (dT) 15 primer(Promega Madison WI) and M-MLV Reverse Transcriptase(Invitrogen Carlsbad CA) according to the manufacturersrsquoinstructions

To detect intrahepatic HBV-DNA a nested polymerasechain reaction (PCR) was carried out using the specificprimers for amplifying S precore-core polymerase and Xregion The primers used are listed in Table 1 The firstand second round PCRs were performed under the samecondition described previously [5] Appropriate controlswere included in each PCRThe PCR products were resolvedon 2 agarose gels and visualized under ultraviolet illumi-nation with ethidium bromide staining Patients who were

serologically negative for HBsAg but positive for intrahepaticHBV-DNA at least in two regions of HBV genome based onPCR assays were defined as having occult infections

We further performed quantitative real-time PCR usingan Applied Biosystems 7500 RT-PCR System (AppliedBiosystems Foster City CA) Briefly 2120583L of HBV-cccDNAor pgRNA was amplified using TaqMan Gene ExpressionMaster Mix (Applied Biosystems Foster City CA) primersfor cccDNA (CCC and BC1) or pgRNA (PGP and BC1) andFRET hybridization probes (hbvLC and hbvFL) as listed inTable 1 The amplification was performed under conditionsdescribed previously [8]

To avoid cross-contamination between samples standardprecautions were used for all procedures Reagents samplesand amplified products were stored in separate areas

23 Determination of HBV Genotypes and SubgenotypesHBV genotypes and subgenotypes were determined byphylogenetic analysis based on the S region Part of Sregion was amplified in PCR using HB2F and HB2Rprimer (Table 1) with condition described previously [7]The amplified fragments were purified using ExoSAP-IT(USB Cleveland OH) and directly sequenced using BigDyeTerminator v31 Cycle Sequencing Kit (Applied BiosystemsFoster City CA) The sequencing was performed in an ABIPRISM 3100-Avant Genetic Analyzer (Applied BiosystemsFoster City CA) The sequences were edited manually andsubsequently aligned using Clustal X version 2012 soft-ware (httpwwwclustalorg) A phylogenetic tree was con-structed by the neighbor-joiningmethod using theMolecularEvolutionary Genetic Analysis (MEGA) version 402 soft-ware (httpmegasoftwarenet) [9] To show the reliability ofthis analysis bootstrap resampling and reconstruction wereperformed 1000 times [10]

24 Detection of Amino Acid Substitutions in the ldquoardquo Deter-minant Region and Mutations in the Core Promoter andPrecore Region Some amino acid substitutions in the ldquoardquodeterminant region are thought to result in detectable HBV-DNA in HBsAg-negative patients Therefore the sequenceswithin this region were aligned and analyzed to identifyamino acid substitutions among HBV strains obtained frompatients positive for HBV-DNA We used the previouslydescribed primer pairs as shown in Table 1 [7] to amplify theldquoardquo determinant region Core promoter and precore regionwere also amplified using primers and condition describedpreviously (Table 1) [7]

Those amplified fragments were directly sequenced andthe obtained sequences from each region were aligned withreference sequences retrieved from GenBank databases toidentify the substitutions or mutations

25 Statistical Analyses Statistical analyses were performedusing IBM SPSS Statistics version 210 (IBM Armonk NY)The categorical variables were analyzed using the 1205942 testor Fisherrsquos exact test whereas continuous variables wereanalyzed using independent t-tests or the Mann-Whitneytest Differences were considered statistically significant at119875 lt 005

International Journal of Hepatology 3

Table 1 Sequences and positions of primers and probes used in this study

Primer and probe Region Position Polarity Sequence (51015840 to 31015840) ReferenceOccult case determination

HBV 1 PreS-S 2815ndash2834 Sense GGTCACCATATTCTTGGGAA [6]HBV 2 PreS-S 690ndash671 Antisense AATGGCACTAGTAAACTGAG [6]HBV 17 PreS-S 3037ndash3057 Sense AATCCAGATTGGGACTTCAA [6]HBV 4 PreS-S 459ndash440 Antisense CCTTGATAGTCCAGAAGAAC [6]HBV 5 Precore-core 2021ndash2040 Sense GCCTTAGAGTCTCCTGAGCA [6]HBV 6 Precore-core 2464ndash2448 Antisense GTCCAAGGAATACTAAC [6]HBV 7 Precore-core 2048ndash2066 Sense CCTCACCATACTGCACTCA [6]HBV 8 Precore-core 2385ndash2366 Antisense GAGGGAGTTCTTCTTCTAGG [6]Pol 1 S Polymerase 2412ndash2430 Sense CGCGTCGCAGAAGATCTCA [5]Pol 1 AS Polymerase 256ndash237 Antisense CGAGTCTAGACTCTGTGGTA [5]Pol 2 S Polymerase 2452ndash2476 Sense GTATYCCTTGGACTCATAAGGTGGG [5]Pol 2 AS Polymerase 2838ndash2814 Antisense CTTGTTCCCAAGAATATGGTGACCC [5]X 1 S X 1100ndash1121 Sense CGCCAACTTACAAGGCCTTTCT [5]HBV 19 X 1550ndash1529 Antisense CGTTCACGGTGGTCTCCAT [6]HBV 15 X 1380ndash1400 Sense GCTAGGCTGTGCTGCCAACTG [6]HBV 21 X 1518ndash1497 Antisense GGTCGGTCGGAACGGCAGACGG [6]

Genotyping andamplification of ldquoardquodeterminant region

HB2F S 414ndash433 Sense TGCTGCTATGCCTCATCTTC [7]HB2R S 989ndash970 Antisense CATACTTTCCAATCAATAGG [7]

Amplification of corepromoter and precoreregions

HB7F C 1611ndash1630 Sense GAGACCACCGTGAACGCCCA [7]HB7R C 2072ndash2048 Antisense CCTGAGTGCTGTATGGTGAGG [7]

cccDNA and pgRNAquantification

CCC XC 1555ndash1573 Sense GTGCCTTCTCATCTGCCGG [8]PGP XC 1826ndash1843 Sense CACCTCTGCCTAATCATC [8]BC1 XC 1974ndash1955 Antisense GGAAAGAAGTCAGAAGGCAA [8]hbvLC C 1874ndash1848 Antisense GGAGGCTTGAACAGTAGGACATGAAC [8]hbvFL C 1897ndash1876 Antisense CYAAAGCCACCCAAGGCACAGC [8]

3 Results

31 Clinical Characteristics of the Patients The clinical char-acteristics of HBsAg-negativeHBcAb-positive patients werecompared with those of HBsAg-positive carriers (Table 2)The HBsAg-positive carriers were significantly younger thanHBsAg-negativeHBcAb-positive patients (557plusmn120 versus687 plusmn 93 years 119875 = 0002) The complication of diabetesmellitus is significantly higher in HBsAg-negativeHBcAb-positive patients It was indicated by higher level of HbA1cin this group (596 plusmn 087 versus 504 plusmn 033 119875 lt 0001) Tenof them (42) were treated by oral medication andor insulininjection therapy for diabetes In addition four patients hadmassive alcohol consumption and had been diagnosed asalcoholic cirrhosis The average body mass index (BMI) inHBsAg-negativeHBcAb-positive patients tended to be high

compared with those in HBsAg-positive carriers (231 plusmn 34versus 205 plusmn 37 119875 = 0079) although not significantstatistically Four HBsAg-positive patients were treated bynucleotide analogue for more than one year and HBV-DNAwas undetectable in their sera A significantly greater propor-tion of HBsAg-positive carriers had an HBV viral load of gt4log copiesmL as compared with HBsAg-negativeHBcAb-positive patients (429 versus 0 119875 = 0012) Thirty-twoHBV strains could be genotyped Almost all (969) patientswere infected by genotype C2 Only one patient (31) wasinfected by genotype B1 (Table 2 and Figure 2)

32 Detection of HBV-DNA cccDNA and pgRNA in theTissue Based on the nested PCR analysis HBV-DNA wasdetected in all cancerous andnoncancerous tissues ofHBsAg-positive carriers On the other hand at least two HBV


Table 2 Clinical characteristics of patients

HBsAg-positive carriers (119899 = 14) HBsAgminusHBcAb+ patients (119899 = 24) 119875 valueMalefemale 131 213 NSAge (years) 557 plusmn 120 687 plusmn 93 0002BMI 205 plusmn 37 231 plusmn 34 NSPrevalence of BMI gt22 314 (21) 1424 (58) 003lowast

Platelet count (mm3) 181 plusmn 40 193 plusmn 84 NSAlbumin (gdL) 42 plusmn 06 39 plusmn 06 NSAST (IUL) 303 plusmn 59 740 plusmn 596 NSHbA1c 504 plusmn 033 596 plusmn 087 lt0001ICG-R 15 () 954 plusmn 276 1115 plusmn 522 NSChild-Pugh (ABC) 1310 1830 NSHBV-DNA gt 4 log copiesmL 6 (429) 0 (00) 0012Genotype BC 113 017 NSAFP gt 200 (ngmL) 7 (50) 8 (333) NSPIVKA-II gt 400 (mAUmL) 9 (643) 14 (583) NSStage IIIIIIIV 1423 1629 NSAST aspartate aminotransferase AFP 120572-fetoprotein PIVKA-II protein induced by vitamin K absence or antagonist II NS not significantlowastMann-Whitneyrsquos 119880 test

Table 3 Detection of HBV-DNA in 24HCC patients with HBsAg-negative and HBcAb-positive patients

Canceroustissue

Noncanceroustissue 119875 value

S region 9 (38) 17 (71) 0020Precore-core region 4 (17) 4 (17) NSPolymerase region 14 (58) 10 (42) NSX region 12 (50) 21 (88) 0005Positive in ge2 regions 10 (42) 20 (83) 0003NS not significant

genomic regions were detected in 20 of 24 (83) noncancer-ous tissues from HBsAg-negativeHBcAb-positive patients(119875 = 0003) Those patients were classified as having occultHBV infection The S (71 versus 38 119875 = 002) and X(88 versus 50 119875 = 0005) regions were detected morefrequent in noncancerous tissues than in cancerous tissues(Table 3) Twenty patients with occult HBV infection werecomparedwith 14HBsAg-positive carriersThemean pgRNAand cccDNA expression levels in noncancerous tissues weresignificantly lower in occult HBV-infected patients (572 plusmn122 log copies120583g of extracted RNA and 752 plusmn 148 logcopies120583g of extracted DNA resp) than in HBsAg-positivecarriers (755 plusmn 150 log copies120583g of extracted RNA and1037 plusmn 115 log copies120583g of extracted DNA resp both 119875 lt0001) In addition pgRNA and cccDNA expression levels incancerous tissues were lower in occult HBV-infected patients(611plusmn108 log copies120583g of extracted RNA and 784plusmn191 logcopies120583g of extracted DNA resp) than in HBsAg-positivepatients (810 plusmn 177 log copies120583g of extracted RNA and1002 plusmn 428 log copies120583g of extracted DNA resp 119875 = 0004and 119875 = 0201 resp Figure 1) The pgRNA and cccDNAin HBsAg-positive patients were also compared in relationwith nucleotide analogue therapy However no significant

difference was detected between undertreated patients andnaıve patients (pgRNA 758 plusmn 148 versus 745 plusmn 162 logcopies120583g of extracted RNA cccDNA 1013 plusmn 098 versus1015 plusmn 134 log copies120583g of extracted DNA resp) Theratio of pgRNAcccDNA in noncancerous tissues was alsoexamined but no difference was detected between HBsAg-positive patients and occult HBV-infected patients (075 plusmn018 versus 085 plusmn 019 resp)

33 Amino Acid Substitutions in the ldquoardquo Determinant Regionand Mutations in the Core Promoter and Precore RegionTable 4 shows the distribution of amino acid substitutionsin the ldquoardquo determinant region in HBsAg-positive patientsand occult HBV-infected patients Two specific substitutions(I126T and I126V) were detected in the occult HBV infectiongroup However all of the occult infected patients had theviral loads less than 4 log copiesmL Substitutions in I126Tand T131P were also detected in HBsAg-positive patients

The A1762TG1764A T1846A C1858 and G1896Amuta-tions were detected in HBsAg-positive carriers and in occultHBV-infected patients without significant differences intheir prevalence rates However the T1753C mutation wasonly found in HBsAg-positive carriers (29) and it wasstatistically significant (119875 = 0033 Table 5)

4 Discussion

HBV is a major cause of HCC in most Asian countriesChronic HCV infection is the leading cause and HBVinfection is the second most common cause of HCC inJapan [11] However approximately 10 of HCC patientswere serologically negative for HBsAg and HCV-Ab [12]Chronic forms of hepatitis particularly alcoholic hepatitissteatohepatitis and autoimmune hepatitis may induce HCCin the absence of hepatitis viral infection The number ofcases of HCC without hepatitis viral infection has increased


0

1

2

3

4

5

6

7

8

9

10

Occult

pgRNA in noncancerous tissues

P lt 0001

HBsAg(+)

Log

copi

es120583

g of

extr

acte

d RN

A

(a)

0

2

4

6

8

10

12

14

cccDNA in noncancerous tissues

Occult

P lt 0001

HBsAg(+)

Log

copi

es120583

g of

extr

acte

d D

NA

(b)

0

2

4

6

8

10

12

pgRNA in cancerous tissues

P = 0004

OccultHBsAg(+)

Log

copi

es120583

g of

extr

acte

d RN

A

(c)

0

2

4

6

8

10

12

14

16

cccDNA in cancerous tissues

P = 0201

Log

copi

es120583

g of

extr

acte

d D

NA

OccultHBsAg(+)

(d)

Figure 1 Quantitative analysis of pgRNA (a c) and cccDNA (b d) in noncancerous and cancerous liver tissues The pgRNA and cccDNAexpression levels were significantly higher in HBsAg-positive patients than in occult HBV-infected patients

in recent years because of the introduction of vaccinationprogrammes aimed at preventing vertical transmission ofHBV and screening systems to prevent HCV infection fromtransfusion and transmission [13]

Occult HBV infection is diagnosed when serum is neg-ative for HBsAg but serum or liver is positive for HBV-DNA regardless of anti-HBc status [14] Several studies haveassessed HBV-DNA status in HBsAg-negative HCC patients[6 15] However it is still controversial whether occultHBV infection is associated with liver damage In particularalthough it was reported that occult HBV infection did notprogress to severe liver disease [16] other studies showed

that occult HBV infection caused liver disease progression inpatients with chronic HCV infection [17]

Earlier studies suggested that individuals could recoverfrom self-limited acute hepatitis because of the role of cyto-toxic or memory T cells However these patients might carrythe HBV genome for a long time without showing clinicalsigns of hepatic injury [18] Furthermore histopathologicalexamination of such patients showed mild necroinflamma-tion after decades of recovery [19] In the immunocompetentstate occult HBV is not destructive but progressive liverdysfunctionmay occur if other deleterious factors are present[20] In this study the HBsAg-negativeHBcAb-positive


OBI 5OBI 14

HBSAG 3OBI 4

OBI 1OBI 3

OBI 15HBSAG 13

HBSAG 1HBSAG 4

HBSAG 7OBI 10

OBI 11HBSAG 14

HBSAG 5OBI 13

HBSAG 11OBI 6

OBI 8OBI 2

HBSAG 8HBSAG 9

OBI 7HBSAG 6

AY641558-genotype C2HBSAG 10

OBI 9X75665-genotype C3

AB048704-genotype C4X75657-genotype E

AF160501-genotype GX02496-genotype D

AB116083-genotype AAB033554-genotype B3

AB073839-genotype B2HBSAG 12D23679-genotype B1

AY090457-genotype HX75658-genotype F

Genotype C

Genotype B

Figure 2 Phylogenetic tree analysis of the S gene sequences of strains isolated in this studyTheHBV strains fromdifferent genotypes (A toH)are obtained from GenBankThe GenBank HBV sequences are indicated with their accession number followed by genotypes The sequencesdetermined in this study are indicated by isolate number starting with HBSAG or OBI and are labelled with black circles and triangles forHBsAg-positive patients and patients with occult HBV infection respectively

patients were significantly older than the HBsAg-positivecarriers suggesting that the hepatocarcinogenesis betweenthese two groups was different

The association of occult HBV infection with HCC hasbeen extensively reviewed [21] It was also reported thatcryptic HBV infection had a pro-oncogenic role in chronicHCV carriers and in cryptogenic liver diseases [6] In animalstudies for woodchucks and ground squirrels infected with

woodchuck or ground-squirrel hepatitis virus liver cancerprogression was apparent even after elimination of theseviruses [22] A previous study showed that occult HBV wasstrongly associated with liver cancer independently of agesex cirrhosis and contemporary HCV infection Aside fromindirect oncogenic mechanisms (eg mild necroinflamma-tion) direct oncogenic mechanisms including viral integra-tion into the host genome andmaintenance of transcriptional


Table 4 The alignment of ldquoardquo determinant amino acid sequences

Sample number or GenBank accession numbers Genotype Amino acids alignment (124ndash147) in the ldquoardquo determinant regionConsensus sequences

D23684 C2 CTIPAQGTSMFPSCCCTKPSDGNC

D23678 B1 -------------------T----

Occult HBV infectionOBI-1 C2 ------------------------

OBI-2 C2 --V---------------------

OBI-4 C2 ------------------------

OBI-5 C2 ------------------------

OBI-6 C2 ------------------------

OBI-7 C2 ------------------------

OBI-8 C2 ------------------------

OBI-9 C2 ------------------------

OBI-10 C2 ------------------------

OBI-11 C2 ------------------------

OBI-12 C2 --T---------------------

OBI-13 C2 ------------------------

OBI-14 C2 ------------------------

OBI-15 C2 ------------------------

OBI-16 C2 ------------------------

OBI-17 C2 ------------------------

OBI-18 C2 ------------------------

HBsAg-positive patientsHBSAG-1 C2 -------P----------------

HBSAG-2 C2 ------------------------

HBSAG-3 C2 ------------------------

HBSAG-4 C2 ------------------------

HBSAG-5 C2 ------------------------

HBSAG-6 C2 --T---------------------

HBSAG-7 C2 ------------------------

HBSAG-8 C2 ------------------------

HBSAG-9 C2 ------------------------

HBSAG-10 C2 ------------------------

HBSAG-11 C2 ------------------------

HBSAG-12 B1 ------------------------

HBSAG-13 C2 ------------------------

HBSAG-14 C2 ------------------------

activity allow the production of potential pro-oncogenicfactors such as X protein which may contribute to thedevelopment of cirrhosis and liver cancer [6]

In the present study cccDNA and pgRNA in canceroustissues were higher than those in noncancerous tissues andthis was supported by previous data [5] However severalreports showed that cccDNA levels in cancerous tissueswere lower than those in noncancerous tissues [23] Thecontroversial result might be dependent on the differenceof the necroinflammation and liver fibrosis [24] It was alsoreported that cccDNA in the liver was correlated with serumHBV-DNA viral load and decreased during antiviral therapy[25] In the present study four HBsAg-positive patients weretreated by nucleotide analogue therapy more than one yearHowever cccDNA was not different regardless of therapy It

might be partly caused by sample number and therapeuticduration

This study revealed that intrahepatic cccDNAandpgRNAlevels in cancerous and noncancerous tissues were low inoccult HBV-infected patients and were lower than those inHBsAg-positive carriersThese results suggest that transcrip-tional activity was low in occult HBV-infected HCC patientsEven though transcriptional activity and replication werelower in HCC patients with occult HBV infection it mightstill be sufficient to induce HCC [5]

Other studies have detected cccDNA and pgRNA incancerous and noncancerous tissues in patients with occultHBV infection [5 6] HBV cccDNA is formed from relaxedcircular-DNA and enters the nuclei of the hostrsquos cells Thisstable nonintegrated minichromosome is used as a template


Table 5 Mutations in the core promoter and precore regions inpatients divided by serological status

HBsAg-positivecarriers (119899 = 14)

OccultHBV-infectedpatients (119899 = 9)

119875 value

T1753C 4 (29) 0 (0) 0033A1762TG1764A 11 (79) 5 (56) NST1846A 3 (21) 3 (33) NSG1896A 8 (57) 4 (44) NSNS not significant

for the transcription of viral mRNAs and pgRNA In turnviral mRNA is used to generate viral proteins and replica-tion factors The pgRNA is then encapsidated and reverse-transcribed into HBV-DNA Animal experiments revealedthat 1ndash50 cccDNA molecules gather in the nucleus but theviral and host factors that regulate cccDNA are still poorlydefined [26]

Some studies of duck hepatitis B virus revealed a neg-ative feedback mechanism by which large surface proteinsuppresses cccDNA production [27] In the present studythe S and X regions were detected in significantly morenoncancerous tissues than in cancerous tissues A previousstudy reported that a decrease in HBsAg expression in thecancer may contribute to an increase in intrahepatic HBV-DNA in the form of cccDNA in cancerous tissues suggestingthat viral replication is minimal within cancer cells and thatthe majority of intrahepatic HBV-DNA is in the form ofcccDNA [6] In the present study the expression levels ofcccDNA and pgRNA in HCC patients with occult HBVinfection were similar or slightly lower in noncanceroustissues than in cancerous tissues although more studiesare needed to confirm these findings However the presentresults suggest that HBV persisted and was continuouslyreplicated in cancerous and noncancerous cells

Mutations in the S genemay alter HBsAg antigenicity andanti-HBs production It was reported that a single amino acidmutation in the ldquoardquo determinant region (amino acids 124ndash147)of HBsAg could lead to a change in the immunologic epitope[28] and mutations or deletion in the S gene disruptedHBsAg production [29] In the present study two specificsubstitutions (I126T and I126V) were detected in occultHBV-infected patients However the HBV-DNA titre waslt4 log copiesmL in all of the occult HBV-infected patientsTherefore it is still unclear whether these substitutions arerelated to viral replication

In conclusion the present study confirmed that HBVpersisted and was continuously replicated in cancerous andnoncancerous tissues In addition cccDNAandpgRNA levelswere thought to represent HBV replication in the liver andmight contribute to the development of HCC in HBsAgcarriers and occult HBV-infected patients

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgments

This study was supported by a Grant-in-Aid from the JapanInitiative for Global Research Network on Infectious Disease(J-GRID) supported by the Ministry of Education CultureSports Science and Technology Japan and a SATREPSGrant from Japan Science and Technology Agency and JapanInternational Cooperation Agency

References

[1] D Lavanchy ldquoHepatitis B virus epidemiology disease burdentreatment and current and emerging prevention and controlmeasuresrdquo Journal of Viral Hepatitis vol 11 no 2 pp 97ndash1072004

[2] E Tabor ldquoHepatocellular carcinoma global epidemiologyrdquoDigestive and Liver Disease vol 33 no 2 pp 115ndash117 2001

[3] J Beck and M Nassal ldquoHepatitis B virus replicationrdquo WorldJournal of Gastroenterology vol 13 no 1 pp 48ndash64 2007

[4] G Raimondo T Pollicino I Cacciola and G SquadritoldquoOccult hepatitis B virus infectionrdquo Journal of Hepatology vol46 no 1 pp 160ndash170 2007

[5] D K H Wong F Y Huang C L Lai et al ldquoOccult hepatitisB infection and HBV replicative activity in patients withcryptogenic cause of hepatocellular carcinomardquo Hepatologyvol 54 no 3 pp 829ndash836 2011

[6] T Pollicino G Squadrito G Cerenzia et al ldquoHepatitis B virusmaintains its pro-oncogenic properties in the case of occultHBV infectionrdquo Gastroenterology vol 126 no 1 pp 102ndash1102004

[7] F Sugauchi M Mizokami E Orito et al ldquoA novel variantgenotype C of hepatitis B virus identified in isolates fromAustralian Aborigines complete genome sequence and phylo-genetic relatednessrdquo Journal of General Virology vol 82 no 4pp 883ndash892 2001

[8] A Laras J Koskinas E Dimou A Kostamena and S JHadziyannis ldquoIntrahepatic levels and replicative activity ofcovalently closed circular hepatitis B virus DNA in chronicallyinfected patientsrdquoHepatology vol 44 no 3 pp 694ndash702 2006

[9] N Saitou and M Nei ldquoThe neighbor-joining method a newmethod for reconstructing phylogenetic treesrdquo Molecular Biol-ogy and Evolution vol 4 no 4 pp 406ndash425 1987

[10] K Tamura J Dudley M Nei and S Kumar ldquoMEGA4 Molec-ular Evolutionary Genetics Analysis (MEGA) software version40rdquo Molecular Biology and Evolution vol 24 no 8 pp 1596ndash1599 2007

[11] H Nordenstedt D LWhite and H B El-Serag ldquoThe changingpattern of epidemiology in hepatocellular carcinomardquoDigestiveand Liver Disease vol 42 no 3 pp 206ndash214 2010

[12] A Zidan H Scheuerlein S Schule U Settmacher and FRauchfuss ldquoEpidemiological pattern of hepatitis B and hepatitisC as etiological agents for hepatocellular carcinoma in Iran andworldwiderdquo Hepatitis Monthly vol 12 no 10 Article ID e68942012

[13] S G Lim R Mohammed M-F Yuen and J-H Kao ldquoPreven-tion of hepatocellular carcinoma in hepatitis B virus infectionrdquoJournal of Gastroenterology and Hepatology vol 24 no 8 pp1352ndash1357 2009

[14] G Raimondo J-P Allain M R Brunetto et al ldquoStatementsfrom the Taormina expert meeting on occult hepatitis B virus


infectionrdquo Journal of Hepatology vol 49 no 4 pp 652ndash6572008

[15] C Brechot M Hadchouel and J Scotto ldquoState of hepatitis Bvirus DNA in hepatocytes of patients with hepatitis B surfaceantigen-positive and -negative liver diseasesrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 78 no 6 pp 3906ndash3910 1981

[16] J-H Kao P-J Chen M-Y Lai and D-S Chen ldquoOcculthepatitis B virus infection and clinical outcomes of patients withchronic hepatitis Crdquo Journal of ClinicalMicrobiology vol 40 no11 pp 4068ndash4071 2002

[17] A Tamori ldquoPossible contribution to hepatocarcinogenesis of Xtranscript of hepatitis b virus in Japanese patients with hepatitisC virusrdquo Hepatology vol 29 no 5 pp 1429ndash1434 1999

[18] B Rehermann C Ferrari C Pasquinelli and F V Chisari ldquoThehepatitis B virus persists for decades after patientsrsquo recoveryfrom acute viral hepatitis despite active maintenance of acytotoxic T-lymphocyte responserdquo Nature Medicine vol 2 no10 pp 1104ndash1108 1996

[19] N Yuki T Nagaoka M Yamashiro et al ldquoLong-term histologicand virologic outcomes of acute self-limited hepatitis Brdquo Hepa-tology vol 37 no 5 pp 1172ndash1179 2003

[20] G Raimondo T Pollicino and G Squadrito ldquoWhat is theclinical impact of occult hepatitis B virus infectionrdquoTheLancetvol 365 no 9460 pp 638ndash640 2005

[21] M Torbenson and D L Thomas ldquoOccult hepatitis Brdquo LancetInfectious Diseases vol 2 no 8 pp 479ndash486 2002

[22] B E Korba F V Wells B Baldwin et al ldquoHepatocellularcarcinoma in woodchuck hepatitis virus-infected woodchuckspresence of viral DNA in tumor tissue from chronic carriersand animals serologically recovered from acute infectionsrdquoHepatology vol 9 no 3 pp 461ndash470 1989

[23] L C Faria M Gigou A M Roque-Afonso et al ldquoHepatocel-lular carcinoma is associated with an increased risk of hepatitisB virus recurrence after liver transplantationrdquoGastroenterologyvol 134 no 7 pp 1890ndash1899 2008

[24] Q Wang M I Fiel W Luan et al ldquoImpact of intrahepatichepatitis BDNAand covalently closed circularDNAon survivalafter hepatectomy in HBV-associated hepatocellular carcinomapatientsrdquo Annals Surgical Oncology vol 20 no 12 pp 3761ndash3770 2013

[25] MAbu-Amara and J J Feld ldquoDoes antiviral therapy for chronichepatitis B reduce the risk of hepatocellular carcinomardquo Semi-nars in Liver Disease vol 33 no 2 pp 157ndash166 2013

[26] M Levrero T Pollicino J Petersen L Belloni G Raimondoand M Dandri ldquoControl of cccDNA function in hepatitis Bvirus infectionrdquo Journal of Hepatology vol 51 no 3 pp 581ndash5922009

[27] R J Lenhoff and J Summers ldquoCoordinate regulation of replica-tion and virus assembly by the large envelope protein of an avianhepadnavirusrdquo Journal of Virology vol 68 no 7 pp 4565ndash45711994

[28] K Yamamoto M Horikita F Tsuda et al ldquoNaturally occurringescape mutants of hepatitis B virus with various mutations inthe S gene in carriers seropositive for antibody to hepatitis Bsurface antigenrdquo Journal of Virology vol 68 no 4 pp 2671ndash2676 1994

[29] M Cabrerizo J Bartolome C Caramelo G Barril and VCarreno ldquoMolecular analysis of hepatitis B virus DNA inserum and peripheral blood mononuclear cells from hepatitisB surface antigen-negative casesrdquo Hepatology vol 32 no 1 pp116ndash123 2000

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


The cccDNA is detected in various phases of chronichepatitis and its levels reflect viral replication in the infectedcells HBV-DNA is sometimes detected in patients negativefor hepatitis B surface antigen (HBsAg) but positive for anti-hepatitis B core (anti-HBc) antibody andor anti-hepatitisB surface (anti-HBs) antibody which is often called occultHBV infection [4] Recent studies revealed that cccDNAwas expressed in HCC without HBs antigen [5] Howeverit is still debated whether HBV-DNA is responsible for thedevelopment of HCC in patients with occult HBV infection

In this study we measured the levels of cccDNA andpgRNA in cancerous and contiguous noncancerous livertissues of HCC patients with overt and occult HBV infectionto examine the viral existence and replication We also deter-mined the nucleotide variations and amino acid mutations inpatients with occult HBV infection

2 Materials and Methods

21 Patients Cancerous and contiguous noncancerous livertissues were obtained from 38 HCC patients (mean plusmn SD6337 plusmn 1201 years male-female ratio 344) who under-went surgical resection at Kobe University Hospital Japanbetween April 2010 and December 2011 HBsAg-positivecarriers (119899 = 14) and HBsAg-negativeHBcAb-positivepatients (119899 = 24) were enrolled in this study Hepatitis Cvirus (HCV) infected patients who were detected by anti-HCV positivity were excluded Tissue samples were rapidlyfrozen in liquid nitrogen and stored at ndash80∘C after resection

The demographic and clinical data including age sexbody mass index (BMI) results of standard test for HBV-related HCC and stage of the disease were collected frommedical record after institutional review board approval wasobtained

Written informed consentwas obtained fromeach patientbefore all the procedures in this study were done This studywas reviewed and approved by the Ethics Committee at KobeUniversity

22 Detection of HBV-DNA cccDNA and pgRNA in TissueSamples Total DNA and RNA from cancerous and contigu-ous noncancerous tissues were extracted from about 20mgof liver tissues using a QIAamp DNA Mini Kit (QIAGENSciences Germantown MD) and Isogen (Nippon GeneTokyo Japan)The DNA and RNA concentrations were mea-sured spectrophotometrically The cDNA was synthesizedfrom five 120583g of total RNA using the Oligo (dT) 15 primer(Promega Madison WI) and M-MLV Reverse Transcriptase(Invitrogen Carlsbad CA) according to the manufacturersrsquoinstructions

To detect intrahepatic HBV-DNA a nested polymerasechain reaction (PCR) was carried out using the specificprimers for amplifying S precore-core polymerase and Xregion The primers used are listed in Table 1 The firstand second round PCRs were performed under the samecondition described previously [5] Appropriate controlswere included in each PCRThe PCR products were resolvedon 2 agarose gels and visualized under ultraviolet illumi-nation with ethidium bromide staining Patients who were

serologically negative for HBsAg but positive for intrahepaticHBV-DNA at least in two regions of HBV genome based onPCR assays were defined as having occult infections

We further performed quantitative real-time PCR usingan Applied Biosystems 7500 RT-PCR System (AppliedBiosystems Foster City CA) Briefly 2120583L of HBV-cccDNAor pgRNA was amplified using TaqMan Gene ExpressionMaster Mix (Applied Biosystems Foster City CA) primersfor cccDNA (CCC and BC1) or pgRNA (PGP and BC1) andFRET hybridization probes (hbvLC and hbvFL) as listed inTable 1 The amplification was performed under conditionsdescribed previously [8]

To avoid cross-contamination between samples standardprecautions were used for all procedures Reagents samplesand amplified products were stored in separate areas

23 Determination of HBV Genotypes and SubgenotypesHBV genotypes and subgenotypes were determined byphylogenetic analysis based on the S region Part of Sregion was amplified in PCR using HB2F and HB2Rprimer (Table 1) with condition described previously [7]The amplified fragments were purified using ExoSAP-IT(USB Cleveland OH) and directly sequenced using BigDyeTerminator v31 Cycle Sequencing Kit (Applied BiosystemsFoster City CA) The sequencing was performed in an ABIPRISM 3100-Avant Genetic Analyzer (Applied BiosystemsFoster City CA) The sequences were edited manually andsubsequently aligned using Clustal X version 2012 soft-ware (httpwwwclustalorg) A phylogenetic tree was con-structed by the neighbor-joiningmethod using theMolecularEvolutionary Genetic Analysis (MEGA) version 402 soft-ware (httpmegasoftwarenet) [9] To show the reliability ofthis analysis bootstrap resampling and reconstruction wereperformed 1000 times [10]

24 Detection of Amino Acid Substitutions in the ldquoardquo Deter-minant Region and Mutations in the Core Promoter andPrecore Region Some amino acid substitutions in the ldquoardquodeterminant region are thought to result in detectable HBV-DNA in HBsAg-negative patients Therefore the sequenceswithin this region were aligned and analyzed to identifyamino acid substitutions among HBV strains obtained frompatients positive for HBV-DNA We used the previouslydescribed primer pairs as shown in Table 1 [7] to amplify theldquoardquo determinant region Core promoter and precore regionwere also amplified using primers and condition describedpreviously (Table 1) [7]

Those amplified fragments were directly sequenced andthe obtained sequences from each region were aligned withreference sequences retrieved from GenBank databases toidentify the substitutions or mutations

25 Statistical Analyses Statistical analyses were performedusing IBM SPSS Statistics version 210 (IBM Armonk NY)The categorical variables were analyzed using the 1205942 testor Fisherrsquos exact test whereas continuous variables wereanalyzed using independent t-tests or the Mann-Whitneytest Differences were considered statistically significant at119875 lt 005











3 Results









Canceroustissue







4 Discussion



0

1

2

3

4

5

6

7

8

9

10

Occult


P lt 0001

HBsAg(+)

Log

copi

es120583

g of

extr

acte

d RN

A

(a)

0

2

4

6

8

10

12

14


Occult

P lt 0001

HBsAg(+)

Log

copi

es120583

g of

extr

acte

d D

NA

(b)

0

2

4

6

8

10

12


P = 0004

OccultHBsAg(+)

Log

copi

es120583

g of

extr

acte

d RN

A

(c)

0

2

4

6

8

10

12

14

16


P = 0201

Log

copi

es120583

g of

extr

acte

d D

NA

OccultHBsAg(+)

(d)







OBI 5OBI 14

HBSAG 3OBI 4

OBI 1OBI 3

OBI 15HBSAG 13

HBSAG 1HBSAG 4

HBSAG 7OBI 10

OBI 11HBSAG 14

HBSAG 5OBI 13

HBSAG 11OBI 6

OBI 8OBI 2

HBSAG 8HBSAG 9

OBI 7HBSAG 6








Genotype C

Genotype B









D23678 B1 -------------------T----


OBI-2 C2 --V---------------------

OBI-4 C2 ------------------------

OBI-5 C2 ------------------------

OBI-6 C2 ------------------------

OBI-7 C2 ------------------------

OBI-8 C2 ------------------------

OBI-9 C2 ------------------------

OBI-10 C2 ------------------------

OBI-11 C2 ------------------------

OBI-12 C2 --T---------------------

OBI-13 C2 ------------------------

OBI-14 C2 ------------------------

OBI-15 C2 ------------------------

OBI-16 C2 ------------------------

OBI-17 C2 ------------------------

OBI-18 C2 ------------------------


HBSAG-2 C2 ------------------------

HBSAG-3 C2 ------------------------

HBSAG-4 C2 ------------------------

HBSAG-5 C2 ------------------------

HBSAG-6 C2 --T---------------------

HBSAG-7 C2 ------------------------

HBSAG-8 C2 ------------------------

HBSAG-9 C2 ------------------------

HBSAG-10 C2 ------------------------

HBSAG-11 C2 ------------------------

HBSAG-12 B1 ------------------------

HBSAG-13 C2 ------------------------

HBSAG-14 C2 ------------------------










119875 value








Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























3 Results









Canceroustissue







4 Discussion



0

1

2

3

4

5

6

7

8

9

10

Occult


P lt 0001

HBsAg(+)

Log

copi

es120583

g of

extr

acte

d RN

A

(a)

0

2

4

6

8

10

12

14


Occult

P lt 0001

HBsAg(+)

Log

copi

es120583

g of

extr

acte

d D

NA

(b)

0

2

4

6

8

10

12


P = 0004

OccultHBsAg(+)

Log

copi

es120583

g of

extr

acte

d RN

A

(c)

0

2

4

6

8

10

12

14

16


P = 0201

Log

copi

es120583

g of

extr

acte

d D

NA

OccultHBsAg(+)

(d)







OBI 5OBI 14

HBSAG 3OBI 4

OBI 1OBI 3

OBI 15HBSAG 13

HBSAG 1HBSAG 4

HBSAG 7OBI 10

OBI 11HBSAG 14

HBSAG 5OBI 13

HBSAG 11OBI 6

OBI 8OBI 2

HBSAG 8HBSAG 9

OBI 7HBSAG 6








Genotype C

Genotype B









D23678 B1 -------------------T----


OBI-2 C2 --V---------------------

OBI-4 C2 ------------------------

OBI-5 C2 ------------------------

OBI-6 C2 ------------------------

OBI-7 C2 ------------------------

OBI-8 C2 ------------------------

OBI-9 C2 ------------------------

OBI-10 C2 ------------------------

OBI-11 C2 ------------------------

OBI-12 C2 --T---------------------

OBI-13 C2 ------------------------

OBI-14 C2 ------------------------

OBI-15 C2 ------------------------

OBI-16 C2 ------------------------

OBI-17 C2 ------------------------

OBI-18 C2 ------------------------


HBSAG-2 C2 ------------------------

HBSAG-3 C2 ------------------------

HBSAG-4 C2 ------------------------

HBSAG-5 C2 ------------------------

HBSAG-6 C2 --T---------------------

HBSAG-7 C2 ------------------------

HBSAG-8 C2 ------------------------

HBSAG-9 C2 ------------------------

HBSAG-10 C2 ------------------------

HBSAG-11 C2 ------------------------

HBSAG-12 B1 ------------------------

HBSAG-13 C2 ------------------------

HBSAG-14 C2 ------------------------










119875 value








Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















Canceroustissue







4 Discussion



0

1

2

3

4

5

6

7

8

9

10

Occult


P lt 0001

HBsAg(+)

Log

copi

es120583

g of

extr

acte

d RN

A

(a)

0

2

4

6

8

10

12

14


Occult

P lt 0001

HBsAg(+)

Log

copi

es120583

g of

extr

acte

d D

NA

(b)

0

2

4

6

8

10

12


P = 0004

OccultHBsAg(+)

Log

copi

es120583

g of

extr

acte

d RN

A

(c)

0

2

4

6

8

10

12

14

16


P = 0201

Log

copi

es120583

g of

extr

acte

d D

NA

OccultHBsAg(+)

(d)







OBI 5OBI 14

HBSAG 3OBI 4

OBI 1OBI 3

OBI 15HBSAG 13

HBSAG 1HBSAG 4

HBSAG 7OBI 10

OBI 11HBSAG 14

HBSAG 5OBI 13

HBSAG 11OBI 6

OBI 8OBI 2

HBSAG 8HBSAG 9

OBI 7HBSAG 6








Genotype C

Genotype B









D23678 B1 -------------------T----


OBI-2 C2 --V---------------------

OBI-4 C2 ------------------------

OBI-5 C2 ------------------------

OBI-6 C2 ------------------------

OBI-7 C2 ------------------------

OBI-8 C2 ------------------------

OBI-9 C2 ------------------------

OBI-10 C2 ------------------------

OBI-11 C2 ------------------------

OBI-12 C2 --T---------------------

OBI-13 C2 ------------------------

OBI-14 C2 ------------------------

OBI-15 C2 ------------------------

OBI-16 C2 ------------------------

OBI-17 C2 ------------------------

OBI-18 C2 ------------------------


HBSAG-2 C2 ------------------------

HBSAG-3 C2 ------------------------

HBSAG-4 C2 ------------------------

HBSAG-5 C2 ------------------------

HBSAG-6 C2 --T---------------------

HBSAG-7 C2 ------------------------

HBSAG-8 C2 ------------------------

HBSAG-9 C2 ------------------------

HBSAG-10 C2 ------------------------

HBSAG-11 C2 ------------------------

HBSAG-12 B1 ------------------------

HBSAG-13 C2 ------------------------

HBSAG-14 C2 ------------------------










119875 value








Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

5

6

7

8

9

10

Occult


P lt 0001

HBsAg(+)

Log

copi

es120583

g of

extr

acte

d RN

A

(a)

0

2

4

6

8

10

12

14


Occult

P lt 0001

HBsAg(+)

Log

copi

es120583

g of

extr

acte

d D

NA

(b)

0

2

4

6

8

10

12


P = 0004

OccultHBsAg(+)

Log

copi

es120583

g of

extr

acte

d RN

A

(c)

0

2

4

6

8

10

12

14

16


P = 0201

Log

copi

es120583

g of

extr

acte

d D

NA

OccultHBsAg(+)

(d)







OBI 5OBI 14

HBSAG 3OBI 4

OBI 1OBI 3

OBI 15HBSAG 13

HBSAG 1HBSAG 4

HBSAG 7OBI 10

OBI 11HBSAG 14

HBSAG 5OBI 13

HBSAG 11OBI 6

OBI 8OBI 2

HBSAG 8HBSAG 9

OBI 7HBSAG 6








Genotype C

Genotype B









D23678 B1 -------------------T----


OBI-2 C2 --V---------------------

OBI-4 C2 ------------------------

OBI-5 C2 ------------------------

OBI-6 C2 ------------------------

OBI-7 C2 ------------------------

OBI-8 C2 ------------------------

OBI-9 C2 ------------------------

OBI-10 C2 ------------------------

OBI-11 C2 ------------------------

OBI-12 C2 --T---------------------

OBI-13 C2 ------------------------

OBI-14 C2 ------------------------

OBI-15 C2 ------------------------

OBI-16 C2 ------------------------

OBI-17 C2 ------------------------

OBI-18 C2 ------------------------


HBSAG-2 C2 ------------------------

HBSAG-3 C2 ------------------------

HBSAG-4 C2 ------------------------

HBSAG-5 C2 ------------------------

HBSAG-6 C2 --T---------------------

HBSAG-7 C2 ------------------------

HBSAG-8 C2 ------------------------

HBSAG-9 C2 ------------------------

HBSAG-10 C2 ------------------------

HBSAG-11 C2 ------------------------

HBSAG-12 B1 ------------------------

HBSAG-13 C2 ------------------------

HBSAG-14 C2 ------------------------










119875 value








Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















OBI 5OBI 14

HBSAG 3OBI 4

OBI 1OBI 3

OBI 15HBSAG 13

HBSAG 1HBSAG 4

HBSAG 7OBI 10

OBI 11HBSAG 14

HBSAG 5OBI 13

HBSAG 11OBI 6

OBI 8OBI 2

HBSAG 8HBSAG 9

OBI 7HBSAG 6








Genotype C

Genotype B









D23678 B1 -------------------T----


OBI-2 C2 --V---------------------

OBI-4 C2 ------------------------

OBI-5 C2 ------------------------

OBI-6 C2 ------------------------

OBI-7 C2 ------------------------

OBI-8 C2 ------------------------

OBI-9 C2 ------------------------

OBI-10 C2 ------------------------

OBI-11 C2 ------------------------

OBI-12 C2 --T---------------------

OBI-13 C2 ------------------------

OBI-14 C2 ------------------------

OBI-15 C2 ------------------------

OBI-16 C2 ------------------------

OBI-17 C2 ------------------------

OBI-18 C2 ------------------------


HBSAG-2 C2 ------------------------

HBSAG-3 C2 ------------------------

HBSAG-4 C2 ------------------------

HBSAG-5 C2 ------------------------

HBSAG-6 C2 --T---------------------

HBSAG-7 C2 ------------------------

HBSAG-8 C2 ------------------------

HBSAG-9 C2 ------------------------

HBSAG-10 C2 ------------------------

HBSAG-11 C2 ------------------------

HBSAG-12 B1 ------------------------

HBSAG-13 C2 ------------------------

HBSAG-14 C2 ------------------------










119875 value








Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















D23678 B1 -------------------T----


OBI-2 C2 --V---------------------

OBI-4 C2 ------------------------

OBI-5 C2 ------------------------

OBI-6 C2 ------------------------

OBI-7 C2 ------------------------

OBI-8 C2 ------------------------

OBI-9 C2 ------------------------

OBI-10 C2 ------------------------

OBI-11 C2 ------------------------

OBI-12 C2 --T---------------------

OBI-13 C2 ------------------------

OBI-14 C2 ------------------------

OBI-15 C2 ------------------------

OBI-16 C2 ------------------------

OBI-17 C2 ------------------------

OBI-18 C2 ------------------------


HBSAG-2 C2 ------------------------

HBSAG-3 C2 ------------------------

HBSAG-4 C2 ------------------------

HBSAG-5 C2 ------------------------

HBSAG-6 C2 --T---------------------

HBSAG-7 C2 ------------------------

HBSAG-8 C2 ------------------------

HBSAG-9 C2 ------------------------

HBSAG-10 C2 ------------------------

HBSAG-11 C2 ------------------------

HBSAG-12 B1 ------------------------

HBSAG-13 C2 ------------------------

HBSAG-14 C2 ------------------------










119875 value








Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















119875 value








Acknowledgments


References





































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Quantification of Pregenomic RNA and...

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