15 research outputs found
Molecular characterization of hepatitis B virus (HBV) from mono-infected and HBV/human immunodeficiency virus (HIV) co-infected individuals in Sudan
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
Molecular characterization of hepatitis B virus in liver disease patients and asymptomatic carriers of the virus in Sudan
Analysis of ultra-deep pyrosequencing and cloning based sequencing of the basic core promoter/precore/core region of hepatitis B virus using newly developed bioinformatics tools.
AIMS: The aims of this study were to develop bioinformatics tools to explore ultra-deep pyrosequencing (UDPS) data, to test these tools, and to use them to determine the optimum error threshold, and to compare results from UDPS and cloning based sequencing (CBS). METHODS: Four serum samples, infected with either genotype D or E, from HBeAg-positive and HBeAg-negative patients were randomly selected. UDPS and CBS were used to sequence the basic core promoter/precore region of HBV. Two online bioinformatics tools, the "Deep Threshold Tool" and the "Rosetta Tool" (http://hvdr.bioinf.wits.ac.za/tools/), were built to test and analyze the generated data. RESULTS: A total of 10952 reads were generated by UDPS on the 454 GS Junior platform. In the four samples, substitutions, detected at 0.5% threshold or above, were identified at 39 unique positions, 25 of which were non-synonymous mutations. Sample #2 (HBeAg-negative, genotype D) had substitutions in 26 positions, followed by sample #1 (HBeAg-negative, genotype E) in 12 positions, sample #3 (HBeAg-positive, genotype D) in 7 positions and sample #4 (HBeAg-positive, genotype E) in only four positions. The ratio of nucleotide substitutions between isolates from HBeAg-negative and HBeAg-positive patients was 3.5 ∶ 1. Compared to genotype E isolates, genotype D isolates showed greater variation in the X, basic core promoter/precore and core regions. Only 18 of the 39 positions identified by UDPS were detected by CBS, which detected 14 of the 25 non-synonymous mutations detected by UDPS. CONCLUSION: UDPS data should be approached with caution. Appropriate curation of read data is required prior to analysis, in order to clean the data and eliminate artefacts. CBS detected fewer than 50% of the substitutions detected by UDPS. Furthermore it is important that the appropriate consensus (reference) sequence is used in order to identify variants correctly
Overt and occult hepatitis B virus infection in adult Sudanese HIV patients
Objectives: Human immunodeficiency virus (HIV) infection in Sub-Saharan Africa is complicated by co-infection with hepatitis B and C viruses (HBV and HCV), which share similar transmission routes. The aims of this study were to determine the prevalence of hepatitis B surface antigen (HBsAg)-positive and HBsAg-negative HBV infection and of HCV infection among HIV-infected patients.
Methods: A cross-sectional study was conducted among treatment-naïve HIV-positive adults in Khartoum State. HBV, HCV, and HIV infections were detected using immunoassays for HBsAg, hepatitis B core antibodies (anti-HBc), hepatitis C antibodies (anti-HCV), and HIV antibodies (anti-HIV), while real-time PCR was used to measure HBV DNA.
Results: The mean age of the 358 patients was 35.2 ± 9.3 years and the male to female ratio was 1.3:1.0. The mean alanine aminotransferase (ALT) level was 10.9 ± 18.0 U/l. Evidence of 23, current or past HBV infection was detected in 62.8% of the patients. HBV DNA was detected in 96 patients (26.8%), 42 HBsAg-positive (11.7%) and 54 (15.1%) HBsAg-negative, indicating occult hepatitis B infection. Anti-HCV was detected in 1.7%.
Conclusions: Evidence of HBV infection was detected in 26.8% of HIV patients with HBsAg-negative infection, with viraemia detected in 15.1% of the patients. All HIV-infected patients should be screened carefully for HBV infection with HBsAg and anti-HBc IgG antibodies prior to starting antiretroviral therapy
Genotyping and virological characteristics of hepatitis B virus in HIV-infected individuals in Sudan
Objectives: Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) share common routes of blood-borne transmission. In HBV mono-infected Sudanese individuals, genotypes D, E, and A circulate. The objective of this study was to molecularly characterize HBV from HBV/HIV co-infected individuals.
Methods: The polymerase overlapping the S region and the basic core promoter (BCP/PC) of HBV from 32 hepatitis B surface antigen (HBsAg)-positive and 18 HBsAg-negative serum samples were amplified and sequenced.
Results: HBV from 37 samples was successfully genotyped and the genotype distribution was 46.0% D, 21.6% E, 18.9% A, and 13.5% D/E recombinant. Compared to mono-infected individuals, the frequencies of the D/E recombinant and genotype A were higher in HBV/HIV co-infected patients, as was the intra-group divergence of genotype E. BCP/PC mutations affecting hepatitis B e antigen (HBeAg) expression at the transcriptional and translational levels were detected. Two HBsAg-positive individuals had pre-S deletion mutants. The following mutations in the S region could account for the HBsAg negativity: sM133T, sE164G, sV168G, and sS174N. No primary drug resistance mutations were found.
Conclusions: In HBV/HIV co-infected Sudanese patients, the ratio of genotype A to non-A was higher than that in mono-infected patients. The genotype E intra-group divergence in HBV/HIV co-infected individuals was significantly higher than that in HBV mono-infected patients
The second output table of the “Rosetta Tool” (truncated), showing the frequency of each possible codon (triplet) and where it occurs in the alignment.
<p>Frequencies shown with a black background occurred at below-threshold levels and therefore can be disregarded.</p
Flow diagram of the analysis procedure used for the UDPS data.
<p>Flow diagram of the analysis procedure used for the UDPS data.</p
The first output table of the “Rosetta Tool”, showing codon (triplets), followed by single-letter translated amino acids, for each read in the input FASTA alignment.
<p>This alignment can be used to easily locate mutations of interest and to locate synonymous and non-synonymous mutations. The visibility of the nucleotide and/or amino acids columns can be toggled on or off, as required.</p
The input pages of the bioinformatics tools (A) “Deep Threshold Tool”.
<p>The first field specifies the input FASTA file. Fields are available for the user to specify the nucleotide offset mapping of the first position in the input file, the number of nucleotides (length) to process, the starting and ending probabilities of error to examine, and the probability of error increment (step) to use. (<b>B</b>) “Rosetta Tool”. The first field specifies the input FASTA file. Fields are available for the user to specify the nucleotide offset mapping of the first position in the input file, the position of the first in-frame nucleotide of the coding region of interest, the last in-frame nucleotide of the coding region of interest, the amino acid offset of the first amino acid in the coding region of interest, and the probability of error to use.</p