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. 2010 Nov 4:7:302.
doi: 10.1186/1743-422X-7-302.

Molecular analysis of HBV genotypes and subgenotypes in the Central-East region of Tunisia

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Molecular analysis of HBV genotypes and subgenotypes in the Central-East region of Tunisia

Naila Hannachi et al. Virol J. .

Abstract

Background: In Tunisia, country of intermediate endemicity for Hepatitis B virus (HBV) infection, most molecular studies on the virus have been carried out in the North of the country and little is known about other regions. The aim of this study was to determine HBV genotype and subgenotypes in Central-East Tunisia. A total of 217 HBs antigen positive patients were enrolled and determination of genotype was investigated in 130 patients with detectable HBV DNA. HBV genotyping methods were: PCR-RFLP on the pre-S region, a PCR using type-specific primers in the S region (TSP-PCR) and partial sequencing in the pre-S region.

Results: Three genotypes (D, B and A) were detected by the PCR-RFLP method and two (D and A) with the TSP-PCR method, the concordance between the two methods was 93%. Sequencing and phylogenetic analysis of 32 strains, retrieved the same genotype (D and A) for samples with concordant results and genotype D for samples with discordant results. The sequences of discordant genotypes had a restriction site in the pre-S gene which led to erroneous result by the PCR-RFLP method. Thus, prevalence of genotype D and A was 96% and 4%, respectively. Phylogenetic analysis showed the predominance of two subgenotypes D1 (55%) and D7 (41%). Only one strain clustered with D3 subgenotype (3%).

Conclusions: Predominance of subgenotype D7 appears to occur in northern regions of Africa with transition to subgenotype D1 in the East of the continent. HBV genetic variability may lead to wrong results in rapid genotyping methods and sequence analysis is needed to clarify atypical results.

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Figures

Figure 1
Figure 1
Genotyping of HBV by the RFLP-PCR method. Ava II : pre-S region digested with AvaII; DpnII: pre-S region digested with DpnII; Und: undigested pre-S fragment; M: molecular size standards,
Figure 2
Figure 2
Phylogenetic analysis based on 86 sequences of 431 nucleotides within the HBsAg region. The evolutionary history was inferred using the Neighbor-Joining method. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. There were a total of 431 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4. Sequences from patients included in this study are labeled with a red dot. Reference sequences are indicated by their accession number.

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