O.115 HBsAg polymorphisms: Implications for assay design

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Journal of Clinical Virology 2006, Vol 36 (suppl 2)

acid template and the primer. We have reported one single amino acid substitution in the RT thumb domain (residue rt306 proline substituted by serine) resulted in marked decrease of replicative competency. We aimed to study the function of the "turn" of helix clamp in RT of HBV and the mechanisms involved for decreased replicative competency of mutants in the "turn". Methods: Site-directed mutation was used to generate substitutions at the "turn" (rt 304-308) of a 1.3 copies of HBV genome. Mutants were used to transfect HepG2 cells. Replication of variants, trsncomplemntation between variants and a non-replicative mutant were studied by cell transfection. RNA packaging was monitored after cell transfection and assayed by immunoblotting with core antibody and southern blotting.Endogenous polymerase reaction was used to assay for polymerase activity. Results: When the wild-type rt306P was separately substituted with eight different amino acids (rtP306G, P306A, P306S, P306V, P306D, P306L, P306E, and P306F), all mutants showed decreased replication in HepG2 cells.Mutants of other residues predicted to locate in the "turn (rtG304A, G304F, A307F and L308F) also displayed impaired replication efficiency. Mutations of residues outside of the "turn" did not affect replication competency. Different mechanisms were involved in decreased replicative competency, including decreased enzyme activity, or defect in encapsidation. Conclusion: The conserved amino acid residue sequence in the "turn" of the helix clamp is crucial for intact HBV polymerase function. Virus replicative competency was markedly decreased when the conserved residues (rt304-308) in the predicted "turn" were substituted by alternative amino acids. The diminished replication was residue-site~lependent and amino acid type-dependent. The mechanisms for the decreased replicative competency were mediated by altering the flexibility of the "turn", by hampering the encapsidation of viral particles, and by diminishing RT activity.

Abstracts, 12th ISHVLD therapies, vaccines and the host immune system and is responsible for creating genotypes, subgenotypes and subtypes. At steady state, HCV produces 1012 new virions per day and also has the potential for genetic diversity. However, among chronically infected patients, false-negative anti-HCV test results are highly unusual except in severely immunocompromised individuals. Conversely, at least 7% of patients who spontaneously resolve their infection have no residual anti-HCV markers when tested 25 years later presumably because specific antibody levels decline over time. More recently, a seronegative window period spanning a time interval of 1-4 months has been observed between infection and seroconversion that can only be elucidated by nucleic acid technology or by HCV antigen testing. This does not represent a defect in the antibody test, but is the result of the evolutionary process of infection. Unfortunately, there are no commercial tests for HCV envelope proteins comparable to the HBsAg test in HBV disease. Nevertheless, significant genetic heterogeneity exists within various segments of the genome such as the hypervariable regions (HVR) of E2 and NS5A. Such variability may be associated with immune escape mechanisms designed to evade recognition leading to chronic infection and resistance to antiviral therapy. Indeed, the degree of viral diversity observed among circulating quasispecies of HCV in an individual is associated with persistence of infection when quasispecies composition is most diverse. Conversely, clearance of the virus appears to occur when the number of viral variants is reduced. It is hypothesized that these changes may not be the result of reduced fidelity during HCV replication but may be secondary to viral interference, to random sampling, or to a selection process whereby only a few fit variants are efficiently generated. Thus, the interplay of viral and host factors may be involved in the eventual outcome of disease in HCV infected patients.

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PS18 -Genetic diversity and diagnostic testing in HBV and HCV infections

(Monday, July 3, 2006, 14:00-15:30)

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An overview of genetic diversity and its impact on diagnostic assays

F.B. Hollinger*. Eugene B. Casey Hepatitis Research Center, Diagnostic Laboratory, Baylor College of Medicine, Houston, TX, USA HBV and HCV circulate in blood as closely-related, but geneticallydiverse molecules called quasispecies. During replication, HBV production may approach 1011 molecules/day, although during peak activity this rate may increase 100-1000 times. Ordinarily, DNA polymerases have excellent fidelity in reading DNA templates because they are associated with an exonuclease which removes incorrectly added nucleotides. However, the HBV DNA polymerase lacks fidelity and proofreading function partly because exonuclease activity is either absent or deficient. Thus, the HBV genome, and especially the envelope gene expressing HBsAg, is mutated with unusually high frequency. These mutations can affect more than one ORF because of overlapping genes. HBsAg preparations consist of three polypeptides: small (S), middle (M) and large (L). These are not distributed uniformly between the various circulating particles. The more numerous 20-nm particles (by a factor of 104 to 106) are composed primarily of the S protein and essentially no L chains. Conversely, the virion contains relatively large amounts of the L chains that include the recognition site for binding to hepatocytes and are important for viral assembly and infectivity. The S gene contains an exposed major hydrophilic region spanning residues 102-169 which encompasses the _a determinant that is important for inducing immunity. Nucleotide substitutions in this region are common and result in reduced binding or failure to detect HBsAg in diagnostic assays. Adaptive immunity also depends on the recognition of HBsAg by specific antibody, and variants pose a threat if they interfere with binding to antibody. Finally, genomic hypervariability allows HBV to escape selection pressures imposed by antiviral

HBsAg polymorphisms: implications for assay design

A. Roque-Afonso 1 *, T.D. Ly2, M.R Ferey 1, E. Dussaix 1. 1Virologie, AP-HR hopital Paul Brousse, Villejuif; 2Serology, L CL laboratory, Ivry, France

Background and Objectives: The serological diagnosis of hepatitis B (HBV) infections is based on the detection of the small surface antigen (HBsAg). Commercially available immunoassays use capture and detection antibodies directed against the "a" determinant, a conformational epitope within HBsAg. Most antibodies found in patients' sera are also directed to this determinant that is thus submitted to selection pressure and exhibits a high degree of polymorphism. Most assays detect as few as 0.5 ng/ml of AgHBs. However, point mutations are susceptible to alter HBsAg antigenicity and impair diagnostic assays. To estimate the prevalence of HBsAg variants, we compared HBsAg polymorphisms of chronic carriers, newly diagnosed patients in two clinical settings and a set of GenBank registered sequences. Methods: One hundred and sixty eight full-length sequences imported from GenBank were used to construct a consensus sequence of the Major Hydrophilic Region (MHR, amino-acid 102 to 169); 55 sequences from chronic carriers, 70 and 66 sequences from patients with a first positive HBsAg detection at the virological laboratory of the Paul Brousse Hospital, and a the private LCL laboratory the HBsAg-positive respectively, were analyzed. HBV genotype was determined by phylogenetic analysis. The frequency of specific changes, the identification of polymorphic sites and antigenic changes (BLOSUM score) were determined with the Mutation Master software. Average dN/dS ratios were computed with MEGA software. Results: Fifteen invariant positions among the 68 under analysis (22%) are common to the 4 populations and correspond to residues thought to be buried according to MHR conformation models: 107111,136-139, and 147-157. Average d N/dS ratio of M H R was 0.327 while that of "a" determinant (124-149) was 0.772. Though chronic carriers strains exhibit the highest number of polymorphic sites (64.7 OYo), variant amino-acids are observed roughly in the half of the 68 MHR amino-acids. The number of different amino acids per site is over 3 in more than 20% of MHR amino-acids. Positions were variant amino-acids are found in more than 10% of the strains, represent about 25% of MHR amino-acids. Negative BLOSUM scores suggest a non-conservative nature of observed substitutions. Substitutions

Oral presentations: PS18: Genetic diversity and diagnostic testing in HBV and HCV infections with BLOSUM scores below -3 (including the G145R substitution) are found respectively in 12 of 168 reference sequences (7%), 6 of 66 community strains (9%), 10 of 70 hospital-diagnosed HBsAg carriers (14.3%) and 13 of 55 chronic carriers (23.6%). Conclusion: Polymorphisms of MHR region are frequent and located in antigenic sites. Thus careful attention has to be paid in the choice and validation of antibodies used in HBsAg detection assays.

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Reasons for the lack of anti-HBC antibody detection in hepatitis B virus chronically infected patients

V. Avettand-Fenoel 1 *, D. Thabut 2, C. Katlama 3, T. Poynard 2, V. Thibault 1. 1Virology - CERVI; 2Hepato-Gastro-Enterology,

3Infectious Diseases, GH Piti#-Salp#triere, Paris, France Background and Objectives: Chronic hepatitis B (CHB) carriage serologic profile is usually characterized by concomitant detection of at least HBs Antigen (Ag) and anti-HBc antibodies (Ab). However, in few cases an absence of anti-HBc Ab despite persistence of HBs Ag is observed. The aim of our work was to study the possible reasons for the lack of anti-HBc Ab detection in some chronic hepatitis B carriers. Methods: Thirty nine CHB patients who had a confirmed serological profile without anti-HBc Ab were selected among 2169 CHB patients. First line screening assays were based on Axsym (Abbott Diagnostics) serological assays for HBs and HBe Ag and anti-HBc and anti-HBe Ab. The lack of anti-HBc Ab was confirmed using a direct sandwich ELISA (Monolisa Anti-HBc Plus, Biorad). The precore-core HBV encoding gene of CHB patients lacking anti-HBc Ab was analyzed for specific mutations by direct sequencing. Results: Among 2169 infected patients who had a chronic confirmed positive HBs Ag detection, 39 (1.79%) lacked anti-HBc Ab, searched for with the specific Axsym assay. All patients had detectable HBV genome using a PCR assay (Monitor, Cobas, Roche) and were HBe Ag positive. The absence of anti-HBc Ab was confirmed using a second assay in 13/19 (68.4%) patients. Genotype distribution was representative of all CHB patients followed in our unit. Direct sequencing of the preC-C gene did not reveal any particular mutations for 14 out of 16 patients. However, 1 patient presented with an unusual pre-core mutation (S11T) and 1 patient with both the classical PC (W28stop) codon change and an E64stop in the C ORE Noteworthy, all but 1 HCV coinfected patient were immunocompromised due to either HIV coinfection or specific treatments for kidney, heart or bone marrow transplantation or systemic inflammatory disease. Conclusion: Lack of anti-HBc Ab in CHB patients is rarely seen and is largely explained both by lower sensitivity of some serological assays and by decreased antibody production in immunocomproraised patients. The hypothesis of mutated HBc epitopes or particular HBV strains was not evidenced in our study. The lack of anti-HBc Ab is not unusual in immunocompromised patients and therefore should be interpreted cautiously to avoid misinterpretation of a hepatitis B infection. I-0-_~

Genetic diversity and complexity changes correlate with early virological response during chronic hepatitis C therapy

R. Kaczanowski 1 *, J. Sta~czak 2, H. Berdak 2, T. Dyda 2, K. Kucharczyk 1. 1Molecular Diagnostic, Kucharczyk TE. Ltd.,

2Laboratory of Genetics, Hospital for Infectious Diseases, Warszawa, Poland Background and Objectives: The predictions concerning the efficacy of PeglFN plus ribavirin therapy are based on the early virological response (EVR) defined as at least 2-log decrease from base-line HCV RNA viral load or undetectable RNA by week 12 of the therapy. Among patients who do not have EVR (approx. 14% of treated patients) 97% do not achieve sustained virological response (SVR). In the presented study we demonstrate that the changes of genetic diversity and complexity within the HCV HVR1 region correlate with the EVR to PeglFN therapy. We propose and assay which can be possibly used as a diagnostic method.

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Methods: Eight patients infected with HCV genotype lb, undergoing Peginterferon-alpha-2b (PEG-intron, Shering-Plough, USA) + ribavirin (Rebetol, US. Pharmacia, USA) therapy were chosen from a large group. Four of them demonstrated EVR and four did not achieve 2 log viremia reduction at week 12 of treatment. The genetic heterogeneity of HCV's HVR1 region was analyzed with the Multitemperature SSCP (MSSCP) method followed by the isolation of ssDNA bands from poliacrylamide gel, and sequencing. This technique (we call it PMES), gives more information about HCV quasispecies and is less time consuming when compared with molecular cloning and sequencing of quasispecies. Results: Patients achieving EVR had significant decrease in genetic complexity within the HVR1 region, after 2 weeks of treatment (p = 0.001). The level of genetic diversity was also reduced, however this difference was not significant (p= 0.15). When the genetic diversity reduction was measured as reduction by per cent, decrease of genetic diversity within HVR1 was significant (p=0.007) after 2 weeks of treatment. At that time in most patients with EVR the genetic diversity decreased to highly homogenous population before the subsequent clearance of HCV RNA at week 12 occurred. In contrast in the group of patients not reaching the 2log viremia reduction at the 12-th week of treatment, no decrease in the genetic diversity and the number of viral strains within the HVR1 region after two weeks of treatment was observed. No significant differences in reduction of viral loads between these groups of patients in week 2rid and 12th of treatment were seen. Conclusion: Changes of genetic diversity and complexity between week "0" and week "2" of the HCV treatment correlate with EVR. Obtained results suggest that early evaluation of diversity and complexity changes in the viral quasispecies population by the PMES method during anti-viral treatment, could be a useful tool for early identification of patients not likely to respond for the therapy. I 0 ~

Combination hepatitis C virus antigen and antibody immunoassay as a new tool for early diagnosis of infection

F. Ansaldi*, B. Bruzzone, M. Frabetti, F. Cardinale, S. Calderisi, D. Demetri, G. Icardi. Dept of Health Sciences, University of Genoa,

Genoa, Italy Background and Objectives: Reduction of the window period of HCV infection represents an important goal in the diagnostic and transfusional settings. A prototype assay designed to simultaneously detect circulating HCV antigen and anti-HCV has been developed by Biorad (Biorad Laboratories Limited,France). Aim of the present study was (i) to define the cut-off (CO) value of the assay, (ii) to evaluate the performance of this new assay in the detection both of antibody and antigen comparing its efficacy with commercial assays and (iii) to perform an on-field evaluation of surveillance of a high risk group, such as haemodialysis patients. Methods: In order to establish the CO value and to evaluate the specificity of the assay, we tested 500 anti-HCV negative sera. The CO value was considered the 99.9 percentile of negative sample distribution. To assess sensitivity, the new test was used on 76 PCRpositive and anti-HCV negative sera, 7 seroconversion panels that included 17 RNA-positive and anti-HCV negative sera and 31 antiHCV positive sera, 20 weak anti-HCV positive sera, 80 viraemic and anti-HCV-positive sera from patients infected with different subtypes and 10 sera from patients with HBV-HCV or HIV-HCV coinfections. A six-month-surveillance of the haemodialysis patients was performed testing every sero-negative subjects monthly with the new assay, anti-HCV and PCR. Results: The mean and the 99.9 percentile OD values of 500 HCV-negative samples were 0.047 (95% CI 0.043-0.05)±0.032 and 0.309, respectively. Of 500 anti-HCV negative samples, 499 (99.8%) were negative with a cut-off index <0.5, while one sample was within the grey zone. Of the 93 HCV-RNA positive and anti-HCV negative sera from patients and panels, 85 (91.4%) resulted positive, and 1 had the cut-off index in the grey zone. The reduction in the diagnostic window period observed with the new test and HCVRNA assays were equal, on average, to 24 days and 34.4 days, respectively. All anti-HCV positive sera from the seroconversion panels, patients infected with different HCV subtypes or patients co-infected with HBV or HIV, were positive with the new assay with