Molecular characterization of hepatitis B virus (HBV) from mono-infected and HBV/human immunodeficiency virus (HIV) co-infected individuals in Sudan
Date
2014-09-09
Authors
Yousif, Mukhlid
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Abstract
Hepatitis B virus (HBV), the prototype member of the family Hepadnaviridae, is
hepatotropic and replicates by reverse transcription. HBV is responsible for the chronic
infection of more than 240 million people worldwide, of which 65 million reside in Africa.
The nine HBV genotypes (A to I) identified to date, are geographically distributed and exhibit
different clinical manifestations and treatment responses. The term occult HBV infection
(OBI) refers to a HBV infection in which HBV surface antigen (HBsAg) cannot be detected
by conventional serological assays as has been defined by the Taormina expert panel. . HBV
and human immune deficiency virus (HIV) are both endemic in many parts of the world and
share common transmission routes. Worldwide, 10% of those infected with HIV are also
chronically infected with HBV. HIV co-infection has been shown to be a risk factor for the
development of OBI in individuals infected with HBV.
The aim of this study was to characterize, at the molecular level, HBV from mono-infected
and HBV/HIV co-infected individuals in Sudan
The objectives of this study were the systematic and comparative analysis of HBV genotype
D sequences, available in the public databases; the molecular characterization of HBV from
mono-infected Sudanese liver disease patients and from HBV/HIV co-infected Sudanese
patients; and the development and testing of bioinformatics tools to explore HBV sequence
data generated using ultradeep pyrosequencing (UDPS) and comparison of UDPS results
with those obtained from cloning based sequencing (CBS).
All available complete genomes of genotype D of HBV from the GenBank database were
analyzed. The intra-group divergence of the subgenotypes ranged from 0.8% + 0.5 for
subgenotype D6 to 3.0% + 0.3 for subgenotype D8. Phylogenetic analysis of genotype D
showed separation into six distinct clusters (subgenotypes D1, D2, D3/D6, D4, D5 and
D7/D8), with good bootstrap support. The mean intergroup divergence between subgenotype
D3 and subgenotype D6 was 2.6%, falling below the accepted threshold of 4% required to
define a subgenotype. This suggests that subgenotypes D3 and D6 are the same subgenotype
because they also share signature amino acids. Furthermore, subgenotype D8 is a genotype
D/E recombinant, which clusters with subgenotype D7. This analysis provided an update on
the classification of the subgenotypes of genotype D of HBV.
Although HBsAg seroprevalence in Sudan, a central-African country, is greater than 8%, the
only sequencing data for HBV, available prior to our study, was from asymptomatic blood
donors, where genotype E predominates, followed by genotype D and subgenotype A2.
Ninety-nine HBV-positive liver disease patients were enrolled in our study, including: 15
with hepatocellular carcinoma (HCC), 42 with cirrhosis, 30 asymptomatic carriers, 7 with
acute hepatitis and 5 with chronic hepatitis. The surface and basic core promoter/precore
(BCP/PC) regions, and the complete genome of HBV were sequenced. Eighty-two percent of
the samples from HBV mono-infected liver disease patients were genotyped. Fifty-nine
percent were infected with genotype D (74% D1, 10% D2, 3% D3 and 13% D6), 30% with
genotype E, 8.5% with genotype A and 2.5% with a genotype D/E recombinant. Patients
infected with genotype E had a higher frequency of HBeAg-positivity (29.2%) and higher
viral loads compared to patients infected with genotype D. BCP/PC region mutations,
including the G1896A mutation, seen in 37% of the HBeAg-negative individuals, could
account for the HBeAg-negativity.
A total of 358 Sudanese HIV-positive patients were enrolled. HBsAg was detected in 11.7%
of the participants, indicating chronic HBV infection. HBV DNA was detected in 26.8% of
the participants: 11.7% were HBsAg positive (overt infection) and the remaining 15.1% were
HBsAg-negative (OBI). Fifty serum samples from the HBV/HIV DNA-positive co-infected
participants were selected for genomic analysis of HBV. Of these, the HBV genotype of 37
was determined. The genotype distribution of HBV isolates from the HBV/HIV co-infected
participants did not differ significantly from those from the HBV mono-infected patients:
genotype D (46%), E (21.6%), A (18.9%) and a D/E recombinant (13.5%). Compared to the
HBV isolates from mono-infected liver disease patients, the frequency of the D/E
recombinant and genotype A was higher in HBV/HIV co-infected patients, as was the intragroup
divergence of genotype E. No difference in BCP/PC mutations affecting HBeAg
expression at the transcriptional and translational levels between genotype D and E was
observed. The following mutations could account for the HBsAg-negativity: sM133T,
sE164G, sV168G and sS174N. No primary drug resistance mutations were found.
Two online bioinformatics tools, the ―Deep Threshold Tool (DDT)‖ and the ―Rosetta Tool‖,
were built to analyze data generated from UDPS and CBS of the BCP/PC region of four
Sudanese serum samples, infected with either genotype D or E of HBV, from HBeAgpositive
and HBeAg negative patients. A total of 10952 reads were generated by UDPS on the
454 GS Junior platform. The Threshold was calculated using DDT based on probability of
error of 0.5%. In total, 39 unique mutations were identified by UDPS, of which 25 were nonsynonymous.
The ratio of nucleotide substitutions between isolates from HBeAg-negative
and HBeAg-positive patients was 3.5:1. From the sequences analyzed, compared to genotype
E isolates, genotype D isolates showed greater variation in the X, BCP/PC/C regions. Only
18 of the 39 positions identified by UDPS were detected by CBS.
Using the specific criteria, that have been suggested previously, to define
genotypes/subgenotypes of HBV, we determined that genotype D has six and not eight
subgenotypes. The importance of HBV genotypes in clinical consequences of infection and
response to antiviral treatment has led us to characterize HBV genotypes circulating in
Sudan. HBV mono-infected patients and HBV/HIV co-infected individuals, were mainly
infected with genotype D or E. HBV mono-infected patients, infected with genotype E, had
higher HBeAg-positivity and higher viral loads than those infected with genotype D. The
ratio of genotype A to non- A, as well as the genotype E intra-group divergence were higher
in HBV/HIV co-infected individuals compared to HBV mono-infected individuals. OBI was
found in 15.1% HBV/HIV co-infected patients and its clinical relevance remains to be
determined. In order to overcome the limitations of Sanger sequencing, which include its
high cost and inability to detect minor populations in quasispecies, next generation
sequencing techniques have been developed. It was demonstrated that correct analysis of
UDPS data required appropriate curation of read data, in order to clean the data and eliminate
artefacts and that the appropriate consensus (reference) sequence should be used in order to
identify variants correctly. CBS detected fewer than 50% of the substitutions detected by
UDPS. This new technology may allow the detection of minor variants between the different
genotypes of HBV and provide biomarkers for the prediction of clinical manifestation of
HBV and response to antiviral therapy.