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CHARACTERIZATION OF HEPATITIS B VIRUS GENOTYPE G INFECTION IN CANADA
C Osiowy1, D Gordon1, J Borlang1, E Giles1, JP Villeneuve2
1National Microbiology Laboratory, Winnipeg, Manitoba; 2Hôpital Saint-Luc du Centre Hospitalier Universitaire de Montréal, Montréal, Quebec
Hepatitis B virus (HBV) is grouped into 8 different genotypes, A to H, based on 8% or greater nucleotide divergence within the viral genome. Very little is known regarding the epidemiology, replication, and natural history of genotype G HBV. To investigate the possibility that genotype G requires co-infection with a helper HBV, as suggested from numerous case reports, the viral quasispecies were characterized from the sera of genotype G-infected Canadian patients. Full-length genome sequencing was further performed on several isolates showing unique characteristics.
HBV DNA was isolated from the sera of 13 HBV/G-infected patients from across Canada. Sequencing and phylogenetic analysis of approx. 20-40 clones derived from the amplified HBsAg-coding region was performed to determine the genotype of each quasispecies clone. Genotype-specific nested and real time PCR as well as line probe assay analysis (INNO-LiPA, Innogenetics Inc.) was performed to verify the genotype designation and increase the sensitivity of detecting minority quasispecies. Sequence recombination was investigated by bootscan analysis (SimPlot v3.5.1).
A total of 41 HBV/G sera were analyzed. Consecutive samples taken at 6-month intervals were available for 7 patients. All patients were found to be co-infected with HBV/A and G. Genotype G quasispecies were normally in the minority during co-infection, except in several cases of A/G recombination and putative HBV/G monoinfection. HBV/A and HBV/G quasispecies were observed to fluctuate over time, with HBV/G dominance often associated with stable, high level HBV DNA (=>105 copies/ml). Recombination between genotypes A and G was observed in two patients, with different breakpoints among the two viral genomes.
The majority of genotype G infections in Canada occur in the context of co-infection or recombination with genotype A, suggesting genotype G strains are defective and require a helping function during chronic infection. HBV/A and G quasispecies populations fluctuate over time within individual patients, but the “switch” determinant is presently unknown. Phenotypic analysis is required to investigate the replication competency among the coinfecting strains.