Genome Titers Research Articles

Higher polymerase activity of a human influenza virus enhances activation of the hemagglutinin-induced Raf/MEK/ERK signal cascade

Influenza viruses replicate within the nucleus of infected cells. Viral genomic RNA, three polymerase subunits (PB2, PB1, and PA), and the nucleoprotein (NP) form ribonucleoprotein complexes (RNPs) that are exported from the nucleus late during the infectious cycle. The virus-induced Raf/MEK/ERK (MAPK) signal cascade is crucial for efficient virus replication. Blockade of this pathway retards RNP export and reduces virus titers. Hemagglutinin (HA) accumulation and its tight association with lipid rafts activate ERK and enhance localization of cytoplasmic RNPs. We studied the induction of MAPK signal cascade by two seasonal human influenza A viruses A/HK/218449/06 (H3N2) and A/HK/218847/06 (H1N1) that differed substantially in their replication efficiency in tissue culture. Infection with H3N2 virus, which replicates efficiently, resulted in higher HA expression and its accumulation on the cell membrane, leading to substantially increased activation of MAPK signaling compared to that caused by H1N1 subtype. More H3N2-HAs were expressed and accumulated on the cell membrane than did H1N1-HAs. Viral polymerase genes, particularly H3N2-PB1 and H3N2-PB2, were observed to contribute to increased viral polymerase activity. Applying plasmid-based reverse genetics to analyze the role of PB1 protein in activating HA-induced MAPK cascade showed that recombinant H1N1 virus possessing the H3N2-PB1 (rgH1N1/H3N2-PB1) induced greater ERK activation, resulting in increased nuclear export of the viral genome and higr virus titers. We conclude that enhanced viral polymerase activity promotes the replication and transcription of viral RNA leading to increased accumulation of HA on the cell surface and thereby resulting in an upregulation of the MAPK cascade and more efficient nuclear RNP-export as well as virus production.

Development of a TaqMan assay for the six major genotypes of hepatitis C virus: Comparison with commercial assays

A quantitative real-time PCR assay was developed that detects genomic RNA from reference strains representing the six major genotypes of hepatitis C virus (HCV) with equal sensitivity and accurately measured HCV RNA in JFH1 HCV-infected Huh7.5 cells. The method is indirectly calibrated to the first international (WHO 96/790) HCV standard preparation and has a linear dynamic range of 10(2.6)-10(6.5) IU/ml. In addition, the inter- and intra-assay precision were approximately 3% CV and <2% CV, respectively. Comparison with results obtained by commercially available HCV RNA Nucleic Acid Technology kits (Versant HCV RNA 3.0 b-DNA and Amplicor HCV Monitor), that also employ the WHO standard, allowed validation of the TaqMan assay against all major HCV genotypes. Both commercial methods detected HCV RNA over a wide dynamic range, but showed a consistent difference of about 0.3 log10 when evaluating samples of different HCV genotypes. The genome titers obtained with the three methods correlated with the infectivity titers previously determined for the HCV reference strains. TaqMan assays have become an essential tool to follow viral load in clinical samples and cell culture-based experiments and this technology offers significant advantages in linear dynamic range, sensitivity and customization.

Two Cases of Chronic Active Epstein-Barr Virus Infection (CAEBV) Successfully Treated with Allogeneic Bone Marrow Transplantation and Induction of Specific Cytotoxic T Lymphocyte (CTL).

Chronic active Epstein-Barr virus infection (CAEBV) is a heterogenous EBV-related disorder, characterized by clonal expansion of EBV-infected T or NK cells. Although CAEBV is a high mortality and morbidity disease with life-threatening complications, effective treatment regimens have not yet been established. The pathogenesis and etiology of CAEBV are not well characterized. The clonal expansion of EBV-associated T or NK cells has been suggested to play important roles in the pathogenesis of CAEBV. It is not clear why EBV-associated abnormal cells cannot be excluded, because of a factor on the virus side or a function on the host side. We report two cases of CAEBV in which allogeneic bone marrow transplantation (BMT) was performed soon after diagnosis. After BMT, the EBV genome titer decreased as cytotoxic T lymphocyte (CTL) activity gradually increased.Case 1: A five-year-old boy had exhibited prolonged fever, lymphadenopathy, and severe liver dysfunction for more than 6 months. Clonally proliferating EBV-infected T cells and abnormally high titers of EBV-related antibodies were detected. No EBV-specific CTLs were detected. The patient was given a diagnosis of T-cell type CAEBV and he received BMT (the donor was his 1-locus mismatched mother) 6 months after diagnosis. Severe treatment-related toxicity did not occur. After BMT, the EBV genome titer gradually decreased as the CTL activity increased. At 2 months after BMT, the EBV genome titer of the patient was nearly negative.Case 2: A two-year-old girl had exhibited a persistent fever of unknown etiology and hepatosplenomegaly. Abnormally high titers of EBV-related antibodies were detected and NK cells in her peripheral blood contained a high EBV genome titer. The EBV-specific CTL levels were low. She received BMT from an unrelated donor 3 months after diagnosis. Toxicity other than grade 2 graft-versus-host disease was not detected. After BMT, the EBV genome titers decreased gradually as the CTL activity increased.Conclusion: These cases suggest that not only high-dose chemotherapy as a preconditioning treatment of BMT, but also that increased CTL activity to eliminate virus-infected cells might be effective. Additional cases should be studied to establish effective treatments.

High-titer, serum-free production of adeno-associated virus vectors by polyethyleneimine-mediated plasmid transfection in mammalian suspension cells

Adeno-associated virus (AAV)-based vectors belong to the most promising gene transfer vectors in clinical studies. To provide vector for late-stage clinical trials as well as for a potential commercial phase, a scalable, cGMP-compliant process is required. Nearly all vector production protocols currently approved in Phase I clinical trials rely on AAV production in adherent HEK 293 cells in the presence of serum. In this study, we present a helper- and serum-free production method of AAV vectors in suspension-adapted HEK 293 cells. The method is based on plasmid transfection with 25 kDa linear polyethyleneimine. Compared to existing methods, our system is highly scalable as cells grow in suspension, does not require animal-derived products or the use of an exogenous virus (adenovirus or baculovirus) and yields genomic titers equal to those obtained in adherent HEK 293 cells in the presence of serum. Most importantly, work load and cost could be dramatically reduced in comparison to earlier methods, when comparing the production of equivalent volumes of cell culture media. Thus, our protocol should appeal to both basic research laboratories and cGMP manufacturing units.

Relationship Between Marek's Disease and the Time Course of Viral Genome Proliferation in Feather Tips

A total of 114 male chickens from three sire families of a commercial cross of White Leghorn chickens were infected with RB-1B Marek's disease (MD) virus at 21 days of age by exposing them to chickens previously inoculated with MD virus. The presence of virus in feather tips, feather pulp, and MD viral antibodies indicated all chickens became infected. The first virus-positive chickens were observed at 12 days postexposure (dpe). The frequency reached a maximum at 27 dpe and then decreased. At 80 dpe, when the experiment was terminated, no viral DNA was detected in the feather pulp of the surviving chickens (82%). Death from MD was first observed at 38 dpe and reached 18% by the end of the experiment, with spleen lesions being the major MD lesion. The viral genome titers in spleen extracts of chickens with MD lesions was negatively correlated with the time of death, and, similar to feather pulp, none of the surviving chickens was virus positive at the end of the experiment. Quantization of the viral genome titers in feather tip extracts at 27 and 38 dpe revealed a positive correlation with the presence of MD lesions, but only in the declining phase (38 dpe) and not at the peak (27 dpe) of the viral titer. Sire effects were significant, indicating the presence of genetic factors that affect viral proliferation. Again, significance was only observed at 38 dpe and not at 27 dpe. The results indicate that, in this commercial line, 1) all chickens were susceptible to infection via contact exposure, 2) all surviving chickens recovered from the viral infection, and 3) it is not sufficient to measure viral titers at a single time point when using viral titers as an endpoint for MD susceptibility.

104. Role of the HI Loop in the Adeno- Associated Virus (AAV) Life Cycle

The fivefold axis of symmetry associated with pore region in icosahedral AAV capsids has been extensively studied in regard to its role in deploying the phospholipase A2 domain and packaging of the 4.7kb AAV genome. However, the role of the HI loops surrounding the fivefold pore, which appear to vary significantly in amino acid sequence between AAV serotypes has not been defined thus far. The HI loop is a surface-displayed peptide motif of 10-15 amino acid residues connecting the H and I beta-strands of the VP3 subunit of AAV and extends to interact with a fivefold-related neighboring subunit. In order to understand the role of this capsid region on the AAV life cycle, we substituted the HI loop in the VP3 subunit of the AAV2 capsid with corresponding regions from the VP3 subunits of AAV1, AAV4, AAV5 or AAV8. In addition, we created a deletion mutant lacking the HI loop and an AAV2 mutant substituted with a loop containing ten glycine residues. Determination of the mutant virus titers by dot blot analysis revealed that vector genome titers of AAV2HI1 (AAV1 HI loop substituted in AAV2) and AAV2HI8 displayed a two-fold decrease in titer, while AAV2HI4 displayed a ten-fold decrease in titer when compared to parental AAV2. On the other end of this spectrum, AAV2HI5, AAV2HIdel (deletion mutant), and AAV2HIpolyG (poly-glycine loop) were unable to package vector genomes. Further western and dot blot western analysis revealed that AAV2HI5 and AAV2HIdel mutants were able to synthesize capsid subunits, but unable to assemble into capsids. Interestingly, the AAV2HIpolyG mutant assembled into empty capsids highlighting the plasticity of this loop region. Further characterization of AAV HI loop mutants using transduction assays, heparin binding assays and electron microscopy are currently in progress. Our studies implicate a potential role for the HI loop in AAV genome packaging and capsid assembly.

506. Production of Recombinant Adeno- Associated Virus Type 1 Vector Using a Herpes Simplex Virus Based System

Top of pageAbstract Recombinant adeno-associated virus (rAAV) is an attractive vector for use in gene therapy treatment of various diseases. However, attaining high-level of rAAV has so far been proven problematic and the usefulness of rAAV vectors has been limited by the low production yield with current production methods. Previously we reported a highly scalable method for rAAV serotype 2 (AAV-2) production using two recombinant herpes simplex viruses (rHSV). One rHSV (rHSV-rep2cap2) expresses rep and cap of AAV-2 and provides helper function for AAV replication and packaging, and the other rHSV (rHSV-TR-GFP) contains the reporter gene green fluorescent protein (GFP) flanked by the inverted terminal repeats (ITR) of AAV-2. Both rHSV vectors are replication defective in non complementing cell lines because of the deletion of an essential gene. This rHSV-based system markedly increased the yield of rAAV production when compared to other traditional AAV production methods. It is well known that different serotypes of AAV have distinct host ranges and cell type preferences, and AAV vectors pseudo- typed with serotype one capsid have been shown to be particularly useful in transducing muscle cells. Here we report the development and initial characterization of an rHSV based production system for rAAV-1. Using a replication defective rHSV as the backbone, the AAV-2 Rep and AAV-1 Cap genes were engineered into rHSV by homologous recombination to generate the helper virus rHSV- rep2cap1. The ability of this virus to produce rAAV-1 was tested by co-infecting HEK-293 cells with rHSV-rep2cap1 and rHSV-TR- GFP. AAV genome titers determined by real time PCR showed that rAAV-1-GFP was packaged with a similar efficiency as the positive control rAAV-2-GFP. The infectivity of the produced rAAV-1-GFP was also examined and the results showed that rAAV-1-GFP was able to transduce C12 cells at an expected efficiency when compared to that of AAV-2-GFP. Studies are currently underway to evaluate these rAAV-1 vectors for gene delivery in vivo. In conclusion, this study suggests that the rHSV-based rAAV production system has a great potential for large scale rAAV production of various serotypes and this system may be able to generate sufficient quantities of rAAV for preclinical and clinical applications.

Gene therapy for colon cancer by adeno-associated viral vector-mediated transfer of survivin Cys84Ala mutant

Reactivation of survivin expression is involved in carcinogenesis and angiogenesis in colon cancer. Previous in vitro studies showed that mutation of the cysteine residue at position 84 (Cys84Ala) of survivin generates a dominant-negative mutant that triggers mitotic catastrophe and apoptosis. We investigated the therapeutic effect of the adeno-associated virus (AAV)-mediated survivin mutant (Cys84Ala) on colon cancer. Survivin mutant (Cys84Ala) (Sur-Mut(Cys84Ala)) was cloned into the AAV expression vector pAM/CAG-WPRE.poly(A) to generate recombinant AAV-Sur-Mut(Cys84Ala) virus. Cell proliferation, apoptosis, mitotic catastrophe, and tumor growth were measured in vitro and in vivo. Transduction of colon cancer cells with rAAV-Sur-Mut(Cys84Ala) inhibited cell proliferation and induced apoptosis and mitotic catastrophe in vitro. rAAV-Sur-Mut(Cys84Ala) sensitized colon cancer cells to chemotherapeutic drugs. Furthermore, expression of survivin mutant mediated by AAV inhibited tumorigenesis in colon cancer cells. Intratumoral injection of rAAV-Sur-Mut(Cys84Ala) significantly induced apoptosis and mitotic catastrophe and inhibited angiogenesis and tumor growth in a colon cancer xenograft model in vivo. No obvious cytotoxicity to other tissues was observed. More importantly, rAAV-Sur-Mut(Cys84Ala) expression strongly enhanced the antitumor activity of 5-Fluorouracil (5-FU), resulting in regression of established tumors. Our results showed that rAAV-Sur-Mut(Cys84Ala) induced apoptosis and mitotic catastrophe and inhibited tumor angiogenesis and tumor growth. Thus, use of AAV-mediated survivin mutant (Cys84Ala) is a promising strategy in colon cancer gene therapy.

Serial detection of Epstein–Barr virus DNA in sera and peripheral blood leukocyte samples of pediatric renal allograft recipients with persistent mononucleosis‐like symptoms defines patients at risk to develop post‐transplant lymphoproliferative disease

We tested blood samples of 25 pediatric renal transplant recipients for Epstein-Barr virus (EBV) DNA load by quantitative polymerase chain reaction (PCR). Eleven of these transplant recipients showed clinical persistent mononucleosis-like symptoms years after transplantation (Tx). A quantitation of EBV DNA by PCR in peripheral blood lymphocyte (PBL) and serum samples revealed variable EBV DNA titers. The majority of EBV PCR results in samples of the 14 asymptomatic transplant recipients was repeatedly below detection limit. In contrast, patients with mononucleosis-like symptoms showed persistent EBV genome titers over a period of 6 months, ranging from 75 to 18 750 copies/10 000 PBL and from 680 to 335 000 copies/mL serum, respectively. One child suffering from this mononucleosis-like condition developed an EBV-associated Burkitt-like lymphoma 29 months after Tx. Whereas clinical and histological investigations did not indicate a post-transplant lymphoproliferative disorder (PTLD) until tumor detection, EBV titers in PBL and serum had been high for at least 8 months. We propose that pediatric transplant recipients who show both, recurrent mononucleosis-like symptoms and a sustained high EBV genome load, are at increased risk for severe EBV-related post-transplant complications.

Quantification of adeno-associated virus particles and empty capsids by optical density measurement.

We show here that UV absorbance of denatured adeno-associated virus (AAV) vector provides a simple, rapid, and direct method for quantifying vector genomes and capsid proteins in solution. We determined the molar extinction coefficients of capsid protein to be 3.72 x 10(6) M(-1) cm(-1) at 260 nm and 6.61 x 10(6) M(-1) cm(-1) at 280 nm. For recombinant AAV vectors, extinction coefficients can be calculated by including the predicted absorbance of the vector DNA. Since the amount of empty capsids in purified vector preparations lowers the A(260)/A(280) ratio in a predictable manner, the vector genome (vg) and capsid particle (cp) titers in purified AAV vector preparations can be calculated from the absorbance at 260 nm and the A(260)/A(280) ratio. To validate this method, the vg and cp titers calculated by UV absorbance were compared with titers determined by quantitative (Q)-PCR and capsid ELISA. The vg titers determined by absorbance agreed well with titers determined by Q-PCR. The cp/vg ratio determined by the A(260)/A(280) method also correlated well with those determined by AAV capsid ELISA in conjunction with Q-PCR. This new method provides a simple and rapid means to determine AAV vg titers and the ratio of empty to full particles in purified virus preparations.

Fast and reliable titration of recombinant adeno-associated virus type-2 using quantitative real-time PCR

In this study, a quantitative real-time PCR (qPCR) was developed to determine genomic rAAV-2 titers using the Light-Cycler technology. Since the CMV promoter is the most commonly used promoter in gene therapeutic approaches, primers were designed which hybridize with the human CMV promoter sequence. PCR products were detected by the addition of SYBR green. qPCR of a 5 log spanning serial dilution of the vector plasmid containing one CMV promoter per plasmid molecule yielded a high amplification efficiency of 1.99 per cycle. To quantify the copy number of viral genomes, the qPCR curves of adeno-associated virus type 2 (AAV-2) samples were related to a standard curve assessed by the 5 log spanning serial vector plasmid dilution (0.01–100 pg DNA). For validation of the method, rAAV-2 preparations were analyzed by a standard method and qPCR in parallel. As standard method, flow cytometry was used for titration of infectious viral particles on HeLa cells using the Enhanced Green Fluorescent Protein as a marker. A significant correlation was found between the results obtained by flow cytometry and the results from the qPCR over a 5 log range ( r=0.85, P<0.0001). The mean ratio between infectious rAAV-2 particles titrated via flow cytometry and genomic copies of rAAV-2 measured by qPCR of the same sample was 1:253. The higher titers found by qPCR might be due to multiple transduction of a single cell or to non-infectious particles generated during rAAV-2 preparation. In conclusion, qPCR is a fast and reliable method for determination of rAAV-2 titers and might be a powerful tool for standardization of rAAV-2 preparations particularly in the context of clinical studies.

Efficient gene transfer of CD40 ligand into primary B-CLL cells using recombinant adeno-associated virus (rAAV) vectors

B cells of chronic lymphocytic leukemia (B-CLL) are resistant to transduction with most currently available vector systems. Using an optimized adenovirus-free packaging system, recombinant adeno-associated virus (rAAV) vectors coding for the enhanced green fluorescent protein (AAV/EGFP) and CD40 ligand (AAV/CD40L) were packaged and highly purified resulting in genomic titers up to 3 x 10(11)/mL. Cells obtained from 24 patients with B-CLL were infected with AAV/EGFP or AAV/CD40L at a multiplicity of infection (MOI) of 100 resulting in transgene expression in up to 97% of cells as detected by flow cytometry 48 hours after infection. Viral transduction could be specifically blocked by heparin. Transduction with AAV/CD40L resulted in up-regulation of the costimulatory molecule CD80 not only on infected CLL cells but also on noninfected bystander leukemia B cells, whereas this effect induced specific proliferation of HLA-matched allogeneic T cells. Vaccination strategies for patients with B-CLL using leukemia cells infected ex vivo by rAAV vectors now seems possible in the near future.

Experimental studies on subclinical hepatitis E virus infection in cynomolgus macaques.

Serial subclinical transmission among susceptible humans may serve as a reservoir of hepatitis E virus (HEV) in areas in which HEV is endemic. This hypothesis was investigated in an experimental primate model. Four groups of 4 cynomolgus macaques each were inoculated intravenously with 10(4)-10(5) (group 1), 10-100 (group 2), and 1-10 (group 3) cynomolgus macaque HEV infectious doses. All 4 animals in group 1 had clinical disease marked by alanine aminotransferase (ALT) elevation, fecal virus excretion, viremia, and seroconversion. Of the animals in groups 2 and 3, only 1 had evidence of biochemical hepatitis, although most had virus excretion and viremia (3 animals each in groups 2 and 3), and evidence of seroconversion (1 animal in group 2 and 3 animals in group 3). Viral genomic titers in stool specimens of animals with or without ALT elevation were similar. Infectivity studies confirmed the viability and transmission potential of the virus excreted by animals without ALT elevation. These data suggest that subclinical HEV infection may represent an HEV reservoir.

Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes

This study compared a range of mammalian CNS expression cassettes in recombinant adeno-associated virus (AAV-2) vectors using strong endogenous promoter sequences, with or without a strong post-regulatory element and polyadenylation signal. Changes in these elements led to transgene expression varying by over three orders of magnitude. In experiments conducted in primary cell culture and in >100 stereotactically injected rats, we observed highly efficient and stable (>15 months) gene expression in neurons and limited expression in glia; the highest expression occurred with endogenous, nonviral promoters such as neuron-specific enolase and beta-actin. The packaging size of AAV-2 was maximized at 5.7 kb without impairing gene expression, as judged by direct comparison with a number of smaller AAV-2 constructs. The genomic insert size and titer were confirmed by Southern blot and quantitative PCR, and infectivity was tested by particle titer using ELISA with a conformation-dependent epitope that requires the full intact capsid. A packaging and purification protocol we describe allows for high-titer, high-capacity AAV-2 vectors that can transduce over 2 x 10(5) neurons in vivo per microliter of vector, using the strongest expression cassette.

In vivo analysis of the 3' untranslated region of the hepatitis C virus after in vitro mutagenesis of an infectious cDNA clone.

Large sections of the 3' untranslated region (UTR) of hepatitis C virus (HCV) were deleted from an infectious cDNA clone, and the RNA transcripts from seven deletion mutants were tested sequentially for infectivity in a chimpanzee. Mutants lacking all or part of the 3' terminal conserved region or the poly(U-UC) region were unable to infect the chimpanzee, indicating that both regions are critical for infectivity in vivo. However, the third region, the variable region, was able to tolerate a deletion that destroyed the two putative stem-loop structures within this region. Mutant VR-24 containing a deletion of the proximal 24 nt of the variable region of the 3' UTR was viable in the chimpanzee and seemed to replicate as well as the undeleted parent virus. The chimpanzee became viremic 1 week after inoculation with mutant VR-24, and the HCV genome titer increased over time during the early acute infection. Therefore, the poly(U-UC) region and the conserved region, but not the variable region, of the 3' UTR seem to be critical for in vivo infectivity of HCV.

Hepatitis C virus in pelvic lymph nodes and female reproductive organs.

Based on epidemiological evidence, hepatitis C virus (HCV) is thought to be involved in the etiology of not only hepatocellular carcinoma, but also lymphoproliferative diseases. In addition, our previous studies using recently developed cell culture systems that support HCV replication have indicated that HCV possesses both hepatotropism and lympholropism. To determine whether HCV is present in extrahepatic organs, we conducted semi‐quantitative reverse transcription‐polymerase chain reaction for the 5 non‐coding region of the HCV genome in surgical specimens (lymph nodes, ovary, uterus, peripheral blood mononuclear cells [PBMCs] and serum) from three patients with gynecological cancer. We found relatively high HCV genome titers in the lymph nodes, not in the sera, irrespective of various titers in PBMCs. These results suggest that lymph nodes may play an important role in the carrier state and the persistence of HCV infection. Moreover, contrary to expectation, high titers of the HCV genome were observed in the ovaries and the uteri, suggesting the feasibility of mother‐toinfant and spouse‐to‐spouse transmissions of HCV.

Characterization of long-term cultures of hepatitis C virus.

The human T- and B-cell lines HPBMa10-2 and Daudi produced infectious hepatitis C virus (HCV) for more than 1 year after infection. The infectivity titer of the cell culture-grown HCV and its genome titer were comparable. The virion density in sucrose was around 1.12 g/ml. Among the 13 variants detected in the inoculum, 7 were adsorbed by the cells and one particular HCV sequence which was present in minor quantities in the inoculum persisted.

Assay of hepatitis B virus genome titers in sera of infected subjects.

A method for quantitative standardization of the DNA hybridization assay for hepatitis B virus (HBV) DNA protein complex in serum is described. This method was used to determine the titer of HBV DNA in various groups of subjects with HB surface antigen (HBsAg) in order to ascertain its accuracy as an index of infectivity. The method's detection limit was 10(5) genome equivalents or 0.3 pg DNA per ml. Titers of 5 X 10(7) to 5 X 10(8) genome equivalents per ml were found to be typical for persistent massive viremia, which occurred more frequently in symptomatic (30 of 48) than in asymptomatic (24 of 72) carriers positive for HBe antigen (HBeAg). Moderate viremia (10(5)-5 X 10(7)) was usually found in patients eliminating the virus from the blood. Patients with resolving acute hepatitis B were frequently positive at the onset (18 of 26) with moderate titers, but became negative within several weeks. In 11 patients who developed chronic hepatitis B, titers increased until typical massive viremia was evident. Whereas healthy HBsAg carriers with anti-HBe always had negative genome titers (144 of 144), symptomatic carriers with anti-HBe often had moderate genome titers (9 of 30). It is recommended that genome titers be monitored in HBeAg-positive and in symptomatic anti-HBe positive virus carriers in order to distinguish between virus carriers with high (greater than 5 X 10(7)), moderate (10(5) -5 X 10(7)) and low (less than 10(5)) infectivity.