Detection and discrimination of WU/KI polyomaviruses by real-time PCR with melting curve analysis

WU and KI polyomaviruses are novel viruses of the Polyomaviridae family, which have been identified recently in respiratory secretions from patients with acute respiratory tract infection. Their potential role in respiratory disease is still unclear and requires additional investigation. To facilitate further studies and diagnosis, a real-time PCR with melting curve analysis was optimized and evaluated to detect WU and KI polyomaviruses. Primers specific for the VP1 gene were designed from regions conserved among WU and KI polyomaviruses which provided amplification products of 198 and 231bp corresponding to WU and KI, respectively and thus yielded a difference in melting temperature (T(m)) between WU and KI polyomaviruses. The assay proved highly specific for WU and KI polyomaviruses as no cross amplification was detected with other respiratory viruses or human genomic DNA. The assay was also highly sensitive with a detection limit as low as 10copies/muL for both WU and KI polyomaviruses. The performance of the real-time PCR assay was evaluated in terms of amplification efficiency (92%). Finally, the assay was validated using DNA extracted from clinical respiratory specimens for WU and KI polyomaviruses and the results were confirmed by direct nucleotide sequencing. The results obtained by melting curve analysis were in perfect agreement with nucleotide sequencing. In conclusion, this method is advantageous because it is rapid, specific, sensitive, reproducible, accurate, cost-effective and thus, would be feasible and attractive for large-scale analysis aimed at investigating the clinical role of WU and KI polyomaviruses.     

Low pretreatment serum HBsAg level and viral mutations as predictors of response to PEG-interferon alpha-2b therapy in chronic hepatitis B

BackgroundViral genomic mutations have become increasingly recognized as being associated with the outcome of chronic HBV infection. However, the role of viral mutations as a predictor of response to pegylated-interferon (PEG-IFN) therapy has so far remained unclear.Study designViral mutations in the enhancer II/basal core promoter (BCP)/precore and the pre-S regions were characterized by direct sequencing in pretreatment serum samples of 50 patients with chronic hepatitis B (33 HBeAg-positive and 17 HBeAg-negative), who were treated for 48 weeks with PEG-IFN alpha-2b.ResultsSustained virological response at 48 weeks post treatment, defined as HBeAg seroconversion and HBV DNA < 2000 IU/mL for HBeAg-positive patients, and HBV DNA < 200 IU/mL for HBeAg-negative patients, was achieved in 12 (36.4%) and 6 (35.3%) of HBeAg-positive and HBeAg-negative patients, respectively. Response to PEG-IFN therapy correlated to low pretreatment HBsAg level but did not correlate with HBV genotype, pretreatment alanine transaminase and HBV DNA levels. In HBeAg-positive hepatitis, PEG-IFN response correlated with the appearance of double BCP mutations (A1762T/G1764A) at baseline (P = 0.041). In the HBeAg-negative group, response to PEG-IFN therapy was associated with the presence of pre-S mutation/deletions (P = 0.028). Multivariate analysis identified low pretreatment HBsAg level as an independent factor associated with SVR in both groups.ConclusionsPretreatment quantitative HBsAg determination is useful for predicting response to PEG-IFN therapy. The presence of double BCP and pre-S mutation/deletions at entry may be associated with a high rate of antiviral response in HBeAg-positive and HBeAg-negative hepatitis, respectively.     

Long-term antibody persistence in children primed and boosted with a DTPw-HBV vaccine at 2, 4, 6, 18, months of age

This paper presents 7- and 10-year follow-up immunogenicity data from two studies, which explored long-term persistence induced by combined tetravalent DTPw-HBV vaccines. The 10-year follow-up study compared two identical antigen-content, but different formulations of DTPw-HBV vaccine (adsorbed on either Al(OH)(3) or Al(PO(4)), whilst the 7-year follow-up study compared two different formulations of DTPw-HBV vaccines containing 5 and 10 microg of hepatitis B surface antigen (HBsAg), following a birth dose of HBV. Primary vaccination took place at 2, 4, 6 months and booster dosing at 18 months. A booster dose of local DTPw vaccine was given at 4 years of age to all returning subjects. At the end of the 7-year study, 90.9% subjects receiving 10 microg HBsAg had anti-HBs antibody concentrations > or =10mIU/ml; the majority of subjects remained seroprotected against diphtheria and tetanus (> or =90.9 and 100%, respectively) and 100% had seropositive levels for pertussis antigens. At the end of the 10-year follow-up study, > or =60.9% subjects had seroprotective concentrations of anti-HBs antibodies; > or =95.2 and 100% subjects were seroprotected against diphtheria and tetanus, respectively; > or =78.3% subjects were seropositive for pertussis. The results demonstrate long-term antibody persistence induced by combined DTPw-HBV vaccine.     

The distribution of hepatitis B virus genotypes in Thailand

Phylogenetic analysis was performed on hepatitis B virus (HBV) strains obtained from 86 hepatitis B surface antigen (HBsAg) positive donors from Thailand originating throughout the country. Based on the S gene, 87.5% of strains were of genotype C while 10.5% were of genotype B, with all genotype B strains obtained from patients originating from the central or the south Thailand. No genotype B strains were found in the north of Thailand. Surprisingly, one patient was infected with a genotype H strain while another patient was infected with a genotype G strain. Complete genome sequencing and recombination analysis identified the latter as being a genotype G and C2 recombinant with the breakpoint around nucleotide position 700. The origin of the genotype G fragment was not identifiable while the genotype C2 fragment most likely came from strains circulating in Laos or Malaysia. The performance of different HBsAg diagnostic kits and HBV nucleic acid amplification technology (NAT) was evaluated. The genotype H and G/C2 recombination did not interfere with HBV detection. J. Med. Virol. 84:1541–1547, 2012. © 2012 Wiley Periodicals, Inc.     

Molecular analysis of hepatitis B virus associated with vaccine failure in infants and mothers: a case-control study in Thailand

Perinatal transmission of hepatitis B virus (HBV) has been controlled incompletely despite adequate immunoprophylaxis in infants. The aim of this study was to characterize virological factors of HBV associated with vaccine failure in Thailand. Sera of 14 infected infants (13 HBeAg‐positive and one HBeAg‐negative) with vaccine failure and their respective mothers (group M1) were tested quantitatively for HBV DNA by real‐time PCR, HBV genotypes and mutations were characterized by direct sequencing. Sera collected from 15 HBeAg‐positive (group M2) and 15 HBeAg‐negative (group M3) mothers whose infants had been vaccinated successfully served as controls. The results showed that group M1 and group M2 mothers had equal titers of HBV DNA but higher titers than group M3. All infected infants and their respective mothers had the same HBeAg status and HBV genotypes. DNA analysis in a pair of HBeAg‐negative infant and mother revealed that both were infected with an HBV precore mutant (G1896A). Escape mutants in the “a” determinant region (residues 144 and 145) were detected in two (14%) infected infants. The prevalence of BCP mutations/deletions in groups M2 and M3 was higher significantly than in group M1 (P = 0.022 and P < 0.001, respectively). In conclusion, instead of the HBeAg status, a high titer of HBV DNA in mothers was the major contributor to perinatal transmission of HBV. Escape mutants might be associated with vaccine failure in some infants. BCP mutations/deletions in mothers might contribute to the prevention of mother‐to‐infant transmission of HBV. J. Med. Virol. 84: 1177–1185, 2012. © 2012 Wiley Periodicals, Inc.