Original article 319
Hepatitis delta virus genotype-1 alone cocirculates with hepatitis B virus genotypes A and D in Pakistan Fatima A. Butta,*, Iram Amina,*, Muhammad Idreesa and Muhammad Iqbalb Introduction Hepatitis delta virus (HDV) and hepatitis B virus (HBV) have been identified as major causes of morbidity and mortality in Pakistan because HDV causes infection only in the presence of HBV. Coinfection with both hepatitis viruses can lead to a more severe acute form of disease and to an increased risk of fulminant hepatitis. HDV infection differs in its distribution and severity depending on the geographical distribution, and several genotypes of HDV have been identified so far. Objectives The aim of the present study was to establish the HDV and HBV genotypes in chronically infected Pakistani patients and to determine whether there is any correlation between HDV and HBV genotypes. Patients and methods We studied samples from a total of 46 chronically infected HBV and HDV patients for HBV and HDV genotype analysis out of a total of 75 chronic HBV carriers enrolled. HBV and HDV genotypes were determined using type-specific PCR, followed by sequencing of PCR amplified products. Results The results of HBV genotyping showed that 33 of 46 (71.7%) patients had genotype D, five (10.9%) had A + D mixed genotypes, whereas eight (17.3) samples were untypable. We could detect only one HDV genotype (HDV-1) prevalent in the Pakistani population. The HDV-1 genotype isolate was associated with HBV genotype D alone or in combination with A (HBV-A + D).
Introduction Hepatitis delta virus (HDV) is a defective virus and is dependent on hepatitis B virus (HBV), which provides the missing function for its infectivity. HDV has become a major public health problem, affecting almost 15–20 million individuals worldwide [1,2]. HDV infection occurs either as a coinfection in individuals with HBV or as a superinfection in individuals who are chronic HBV carriers [3–5]. Individuals with HDV/HBV coinfection can have a more severe form of the disease and an increased risk of fulminant hepatitis [4,6–8]. It has been observed that many individuals with HDV infection develop chronic illness and about 80% of these individuals progress to cirrhosis within 5–10 years [9]. In cases of dual and triple infections, one virus could have an inhibitory effect on the other, such as in HDV/HBV dual infections; the virological levels of HBV have been found to be lower than those in HBV infection alone. HDV infection is found in all age groups, but it varies in its distribution and pattern depending on the prevalence of HBV chronic infection. c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0954-691X
Conclusion The present study concludes that HDV/HBV coinfection is very high in the Pakistani population and was previously underestimated. The most prevalent circulating genotypes of HBV and HDV are HDV-1 and HBV-D, respectively, in the studied area. There is no specific interaction between HBV and HDV genotypes as suggested by HDV-1/HBV-D or HDV-1/HBV-A + D coinfection. Coinfection of HDV-1 and HBV-D simply reflects the most frequent genotypes circulating in this specific geographical region of the world. Eur J c 2014 Wolters Kluwer Gastroenterol Hepatol 26:319–324 Health | Lippincott Williams & Wilkins. European Journal of Gastroenterology & Hepatology 2014, 26:319–324 Keywords: coinfection, genotype, hepatitis B virus, hepatitis delta virus, polymerase chain reaction a Division of Molecular Virology & Molecular Diagnostics, National Centre of Excellence in Molecular Biology (CEMB), University of the Punjab and bCentre for Applied Molecular Biology, Ministry of Science & Technology, Lahore, Pakistan
Correspondence to Muhammad Idrees, PhD, Division of Molecular Virology & Molecular Diagnostics, National Centre of Excellence in Molecular Biology (CEMB), University of the Punjab, 87 West Canal Bank Road, Thokar Niaz Baig, Lahore 53700, Pakistan Tel: + 92 42 529 3141; fax: + 92 42 529 3149; e-mail: [email protected] *Fatima A. Butt and Iram Amin contributed equally to the writing of this article. Received 26 July 2013 Accepted 16 September 2013
HDV is a small defective RNA virus with a negativesense genome, containing single-stranded RNA of 1.7 kb [10,11], a delta antigen, and envelope proteins of hepatitis B surface antigen (HBsAgs) to complete its virion assembly and secretion [12–14] as HBV acts as a covirus for HDV transmission. HDV infection differs in its distribution and severity depending on different geographic areas [15–20], which could be because of possible genetic differences in the prevalence of predominant HDVs [19,21,22]. So far, HDVs have been classified into seven genotypes (HDV-1–7) [23], of which three are major and most widely distributed. HDV-1 is prevalent worldwide and is associated with a huge diversity of diseases, ranging from fulminant hepatitis to asymptomatic chronic liver disease. HDV-2 is prevalent mainly in Asia, Taiwan, Japan, and Siberia. HDV-1 causes more severe disease compared with genotype 2. HDV-3 is mainly found in the north part of South America and causes a severe form of fulminant hepatitis [24]. HDV-4 is found mainly in Japan and Taiwan and HDV-5–7 in DOI: 10.1097/MEG.0000000000000007
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Africa [23–32]. In another study, an extended classification of the delta virus genus to eight clades was proposed [33], which is very similar to the human HBV genetic variability [23,34]. Studies of epidemiology of HDV have shown that the prevalence rate of HDV infection has decreased to 5.7% in 2005 in chronic HBV patients as compared with 18.3% in 1990 [35]. The factor responsible for the control of HDV is the excellent control of HBV achieved by vaccination in the past 15 years in developed countries [36]. However, the prevalence of HDV seems to be increasing in developing countries such as Pakistan because of the lack of preventive strategies and resources [37]. The prevalence of HDV was 16.6% in HBsAgpositive patients from different areas of Pakistan [38]. A few studies are available from different countries that describe the role of the association of different HBV and HDV genotypes with the disease outcome. In Pakistan, especially in the province of Sindh, there are pockets with a high prevalence of concurrent HBV and HDV cases, and few studies on their prevalence, coinfection, disease outcomes, and HBV genotyping are available. However, to the best of our knowledge, no previous study determining the HDV and HBV genotypes circulating in this important region of the world has been available until now. Therefore, this study was carried out with the aim of identifying the HBV and HDV genotypes harbored by chronically infected patients and to further study whether there is any specific interaction between different HBV and HDV genotypes.
from 100 ml serum sample using a Nucleospin Viral RNA Extraction Kit (Macherey-Nagel, Duren, Germany) according to the manufacturer’s instructions provided in the kit protocol. The extracted RNA (B20 ng) was reverse transcribed into cDNA with the Moloney Murine Leukemia Virus reverse transcriptase (M-MLV) enzyme according to the manufacturer’s instructions (Invitrogen Biotechnologies, Carlsbad, California, USA) along with the enzyme. HDV-specific antisense primer 50 -ACAAG GAGAGGCAGGATCACCGAC-30 (10 pmol/ml) was used in this reverse transcription reaction. The reaction mixture was incubated at 371C for 50 min, followed by at 951C for 3 min in the thermal cycler to heat inactivate the M-MLV enzyme. The first-round amplification was performed with 4 ml of cDNA using the outer sense primer (50 -GCCCAGGTCGGACCGCGAGGA-30 ) and the outer antisense primer (50 -ACAAGGAGAGGCAG GATCACCGAC-30 ) in a total of 35 cycles of denaturation at 941C for 40 s, annealing at 551C for 40 s, and extension at 721C for 1 min. Final extension was performed at 721C for 7 min. Nested/second-round PCR amplification was performed with 2 ml of the first-round product using inner sense (50 -GAGATGCCATGCCGACCCGAAGAG-30 ) and inner antisense primers (50 -GAAGGAAGGCCCTCGA GAACAAGA-30 ) using the same cycling profile on the thermal cycler as mentioned above, except for annealing temperature, which was 501C instead of 551C, with a final extension step of 10 min at 721C. The PCR products were electrophoresed on a 2% TAE agarose gel and stained with ethidium bromide as DNA stain. Genotyping of hepatitis delta virus
Patients and methods Patients and initial clinical tests
Initially, a total of 80 chronically HBV-infected patients were enrolled for this study. Serum samples from these patients were tested to evaluate the prevalence of HDV/ HBV coinfection at the Genome Centre for Molecular Diagnostics and Research (GCMD), Lahore. PCR was performed at GCMD for the presence of HBV DNA using SmartCycler II (Cepheid, Sunnyvale, California, USA) real-time PCR with a kit from Sacace Biotechnology (Como, Italy). Samples reactive for HBsAg and detected by HBV DNA were further tested for the presence of HDV RNA, HDV genotyping, and HBV genotyping in the Division of Molecular Virology, Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan. For all PCR reactions, strict procedures proposed by Kwok and Higuchi [39] for nucleic acid amplification diagnostic techniques were followed to avoid false-positive results. Detection of hepatitis delta virus
A total of four primers were designed for the amplification of the conserved region of the HDAg gene representing all the available HDV genotypes. HDV RNA was extracted
For HDV genotyping, second-round PCR products were gel eluted using the GF-1 Nucleic Acid Extraction Kit (Vivantis Technologies, Selangor Darul Ehsan, Malaysia) following the manufacturer’s protocol. Eluted DNA was subjected to cycle sequencing with the ABI Prism BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems Inc., Foster City, California, USA) using the inner sense and inner antisense primers (utilized in the nested PCR) in separate sequencing reactions according to the manufacturer’s instructions and run on a 3100 Automated Genetic Analyzer (Applied Biosystems Inc.). Phylogenetic analysis and sequence alignment
Consensus sequences were generated by alignment of both sequenced strands with forward/sense and reverse/antisense primers after validation using the Phred–Phrap Software (Washington, Seattle, USA). These sequences were submitted to the GenBank database (National Center for Biotechnology Information, Bethesda, Maryland, USA). Nucleotide sequences of HDV isolates sequenced in the present study were aligned together using CLUSTAL Omega. These nucleotide sequences were also compared with previously reported sequences (accession numbers of the reference sequences used were retrieved from GenBank). MEGA (version 2.1) (http://www.megasoftware.net/) was
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Hepatitis delta virus genotype-1, Pakistan Butt et al. 321
used to analyze amino acid sequences. The accession numbers of the reference sequences used were as follows: the phylogenetic trees were constructed in MEGA (version 5.05) using the neighbor-joining method [40]. The robustness of phylogenetic analysis and significance of the branch order were determined by bootstrap analysis with 1000 replicates. Genotyping of hepatitis B virus
HBV DNA was extracted using the method and reagents as described for HDV RNA extraction and rehydrated in 50 ml sterile distilled water. A volume of 10 ml (20–50 ng) each of HBV DNA was amplified in different HBV genotyping PCRs: HBV genotypes A through G (HBV-A– G) were determined using HBV genotype-specific primers for the surface gene of HBV as described previously by Naito et al. [41] and HBV-H was determined using the method described previously [34]. The primer names, their sequences, and the resulting PCR product sizes of genotyping analysis are shown in Table 1. Statistical analysis
The data were analyzed and the summary statistic was calculated using the statistical package, SPSS version 17.0 (SPSS Inc., Chicago, Illinois, USA) for Windows. The results for all variables were described as rates (%). w2 and Fisher’s exact test were used to evaluate the association among categorical variables. All data are presented as mean±SD or number of patients. P-values of less than 0.05 were considered significant.
Results Of the total of 80 HBsAg-reactive patients enrolled, 75 were found to be positive by HBV DNA PCR and were further analyzed for HBV and HDV genotypes. The five HBsAg-reactive but HBV DNA PCR-negative samples were excluded from further analysis as these could be false positive by the ELISA method. HDV RNA was Primer names, sequences, and the resulting polymerase chain reaction product sizes of genotyping analysis
detected in 46 (61.3%) of these 75 HBV-positive patients and were therefore coinfected by these two viruses at the same time. Of the HBV/HDV-coinfected patients, 30 (65.21%) were men and 16 (34.78%) were women. The mean age of the patients was 29±11 years (range 15–59 years). Figure 1 shows the HDV-specific PCR amplified product band of 400 bp (HDAg gene) on an agarose gel. These HDV/HBV coinfected samples were further selected for HBV genotyping using genotype-specific PCR methods. Hepatitis delta virus genotype analysis
A total of 21 samples were selected randomly for HDV genotype analysis using sequencing of the HDAg geneamplified product after gel elusion as shown in Fig. 1. All the sequences were submitted to the GenBank database and the accession numbers allotted are from JN187431 through JN187438 and JN400336 through JN400348. Nucleotide sequences of the local HDAg gene of HDV in Pakistan showed 91% homology with the HDV isolate from Turkey, the dTk38 LHD gene for large HDV antigen (accession no. AM779590), using Blastn (http://www.ncbi. nlm.nih.gov/BLAST). Phylogenetic analysis was carried out using Mega 4 software (Tokyo Metropolitan University, Tokyo, Japan) among the sequenced regions as well as with different reported sequences. Eight different sequences were retrieved from GenBank, representing eight different clades from different geographical regions of the world. A 406-bp region (nt 882–1288) was chosen. The accession numbers and countries of origin for these reference sequences are presented in Table 2. The phylogenetic constructed tree showed that all HDV sequences clustered with HDV-1 as all of the sequences were in the category of genotype-1 (Fig. 2). Hepatitis B virus genotyping analysis
Of the 46 tested HBV PCR-positive samples by genotyping assay, 38 (82.6%) were genotyped successfully, of
Table 1
Primer names Universal sense (P1) Universal antisense Mix A B2 BA1R BB1R BC1R Mix B B2R BD1 BE1 BF1 Mix C HBHKF1 HBHKR2 HBHKF2
Sequences 0
0
5 -TCACCATATTCTTGGGAACAAGA-3 50 -CGAACCACTGAACAAATGGC-30
M 1 2 3 4 5 6 7 8 9 10 M
1063
50 -GGCTCMAGTTCMGGAACAGT-30 50 -CTCGCGGAGATTGACGAGATGT-30 50 -CAGGTTGGTGAGTGACTGGAGA-30 50 -GGTCCTAGGAATCCTGATGTTG-30
68(A) 281(B) 122(C)
50 -GGAGGCGGATYTGCTGGCAA-30 50 -GCCAACAAGGTAGGAGCT-30 50 -CACCAGAAATCCAGATTGGGACCA-30 50 -GYTACGGTCCAGGGTTACCA-30
119(D) 167(E) 97(F)
50 -ACGGGGCGCACCTCTCTTTAC-30 50 -AGCCAAAAAGGCCATATGGCA-30 50 -GCACTTCGTTTCACCTCTGCA-30
Fig. 1
Product size (bp)
357(G&H)
400 bp
500 bp 250 bp
PCR amplification of hepatitis delta virus (HDV) HDAg gene. Lanes 1–9, HDV-positive samples; lane 10, negative sample; M, marker of 50 bp.
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Table 2 Accession numbers and countries of origin of reported geographical sequences of genotypes 1–8 Serial numbers 1 2 3 4 5 6 7 8
Accession number U81989.1 X60193.1 AB037948.1 AF309420.1 AM183328.1 AX741164.1 AM183333.1 AM183327.1
Country of origin Ethiopia Japan Venezuela Japan France Paris France France
Represented in the phylogenetic tree HDV-1 HDV-2 HDV-3 HDV-4 HDV-5 HDV-6 HDV-7 HDV-8
HDV, hepatitis delta virus.
which 33 (71.7%) had genotype D and five (10.9%) had mixed infections of HBV-A + D. A total of eight (17.3%) samples were untypable as the genotyping method could not confirm a type even when these were positive and could thus be genotyped. Figure 3 shows the HBV-D specific band of 119 bp.
Discussion The aim of this study was to determine the sequencebased genotyping of HDV and to correlate this genotype with the HBV genotype in coinfected patients from
Fig. 2
HDV-6 HDV-7 HDV-5 HDV-3 HDV-2 HDV-4 HDV-8 PK-HDV-3 PK-HDV-5 PK-HDV-10 PK-HDV-1 PK-HDV-4 PK-HDV-17 PK-HDV-14 PK-HDV-20 PK-HDV-18 PK-HDV-8 PK-HDV-16 PK-HDV-2 PK-HDV-21 PK-HDV-13 PK-HDV-19 PK-HDV-6 PK-HDV-9 PK-HDV-12 PK-HDV-15 PK-HDV-11 HDV-1 PK-HDV-7
0.2
Phylogenetic tree of studied sequences with geographical strains of genotypes 1–8 generated by the neighbor-joining method with a bootstrap test of 1000 replicates. Each geographical strain is abbreviated by the name shown in Table 2.
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Hepatitis delta virus genotype-1, Pakistan Butt et al. 323
Fig. 3
M
1
2
3
4
5
6
7
119 bp
Genotyping results of hepatitis B virus-positive samples showing genotype D with a band of 119 bp. Lanes 1–7, samples showing genotype D; M, 50 bp ladder.
Pakistan. Although the sample size of our study is not very large as compared with that of earlier studies, the results are very interesting and important as this is the first study of its nature from Pakistan on the subject, wherein a high prevalence rate of HDV/HBV coinfection has been observed and the genotypes of HBV and HDV have been determined in coinfected individuals. The epidemiology of HDV is changing worldwide, consistent with successful efforts to control HBV infection in the developed countries, whereas the infection remains endemic in south Asian countries such as Pakistan. The first and important finding of the current study is the observation that a very high number (61.33%) of HBV-positive patients were coinfected with HDV. This rate is very high as compared with previous studies from this region [37,38] and other parts of the world. Mumtaz et al. [38] reported a prevalence rate of HDV of only 16.6%. This huge difference in the rates of HDV/HBV coinfection may be because of the sensitivity of the detection systems used. We have used the most sensitive and specific molecular diagnostic methods such as nested PCR (with a sensitivity of 10 copies/ml) and real-time PCR (50 copies/ml). In our study, the majority of HBV/ HDV-coinfected patients were men. The second and interesting finding of this study was the detection of only one HDV genotype (HDV-1) that cocirculates with HBV in Pakistan. To the best of our knowledge, this is the first reported HDV genotype from this region of the world using a sequencing method. Our finding is in agreement with the predominant genotype of HDV in Pakistan’s neighboring country Iran and in the middle-east regions such as Lebanon, Egypt, and Turkey [42–45], where HDV-1 is the most prevalent genotype. The third most important finding of this study was the observation that in all cases, mixed infection of HDV-1
coexists with HBV-D. Although it has been reported previously that genotype D is the most common genotype in this country along with other genotypes [46], no study is available that had correlated HDV genotypes with HBV genotypes. In our study, HBV-D was observed in 71.7% of the total isolates. This HBV-D is also considered to be common in south Europe, the Middle-East, and previously in India. In the current study, we also found that 10.9% patients had mixed HBVA + D along with coexistence of HDV. The clinical significance of such mixed HBV genotype infections particularly if coinfected with HDV is unclear and needs further study. Eight samples were HBV DNA and HDV RNA PCR positive; however, no genotypespecific bands were found for HBV. This may be because of some mutations in the HBV genome that cause specific primer annealing and amplification [47]. In this study, we could not isolate HBV genotypes other than A and D, which means that HBV-E–H do not exist here. These genotypes have been reported from Africa, South America, USA, Vietnam, and Japan, respectively [34,48,49]. The high rate of coinfection of HBV and HDV observed in this study indicates that HBV replication is not suppressed by HDV as reported in previous studies that in most cases of HDV infection, HBV replication is suppressed to very low levels by HDV [50,51]. It seems that HBV replication is not suppressed by the presence of HDV RNA in a subset of individuals. A more severe progression toward liver decompensation has been documented in patients with active HBV and HDV replication, with each virus contributing to liver damage, thereby resulting in more severe liver disease [52,53]; however, we did not observe this type of complication in our patients.
Conclusion
The present study concludes that HDV/HBV coinfection is very high and was previously underestimated. All of the HDV/HBV coinfected patients had HDV-1 and HBV-D was the most common. An association exists between HDV-1 and HBV-D.
Acknowledgements F.A.B. and I.A. collected epidemiological data; M. Idrees critically reviewed the manuscript. F.A.B., I.A., and M. Idrees analyzed and compiled the data. M. Iqbal helped with sequence analysis and data interpretations. All authors read and approved the final manuscript. The authors acknowledge and extend their heartfelt gratitude to the Genome Centre for Molecular Based Diagnostics & Research (GCMBDR) for providing the HBV and HDV samples and all the clinicians and patients (involved in this study) for their cooperation.
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The study was financially supported by the Genome Centre for Molecular Diagnostics and Research (GCMD).
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Conflicts of interest
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There are no conflicts of interest. 31
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Hepatitis delta virus genotype-1 alone cocirculates with hepatitis B virus genotypes A and D in Pakistan Fatima A. Butta,*, Iram Amina,*, Muhammad Idreesa and Muhammad Iqbalb Introduction Hepatitis delta virus (HDV) and hepatitis B virus (HBV) have been identified as major causes of morbidity and mortality in Pakistan because HDV causes infection only in the presence of HBV. Coinfection with both hepatitis viruses can lead to a more severe acute form of disease and to an increased risk of fulminant hepatitis. HDV infection differs in its distribution and severity depending on the geographical distribution, and several genotypes of HDV have been identified so far. Objectives The aim of the present study was to establish the HDV and HBV genotypes in chronically infected Pakistani patients and to determine whether there is any correlation between HDV and HBV genotypes. Patients and methods We studied samples from a total of 46 chronically infected HBV and HDV patients for HBV and HDV genotype analysis out of a total of 75 chronic HBV carriers enrolled. HBV and HDV genotypes were determined using type-specific PCR, followed by sequencing of PCR amplified products. Results The results of HBV genotyping showed that 33 of 46 (71.7%) patients had genotype D, five (10.9%) had A + D mixed genotypes, whereas eight (17.3) samples were untypable. We could detect only one HDV genotype (HDV-1) prevalent in the Pakistani population. The HDV-1 genotype isolate was associated with HBV genotype D alone or in combination with A (HBV-A + D).
Introduction Hepatitis delta virus (HDV) is a defective virus and is dependent on hepatitis B virus (HBV), which provides the missing function for its infectivity. HDV has become a major public health problem, affecting almost 15–20 million individuals worldwide [1,2]. HDV infection occurs either as a coinfection in individuals with HBV or as a superinfection in individuals who are chronic HBV carriers [3–5]. Individuals with HDV/HBV coinfection can have a more severe form of the disease and an increased risk of fulminant hepatitis [4,6–8]. It has been observed that many individuals with HDV infection develop chronic illness and about 80% of these individuals progress to cirrhosis within 5–10 years [9]. In cases of dual and triple infections, one virus could have an inhibitory effect on the other, such as in HDV/HBV dual infections; the virological levels of HBV have been found to be lower than those in HBV infection alone. HDV infection is found in all age groups, but it varies in its distribution and pattern depending on the prevalence of HBV chronic infection. c 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins 0954-691X
Conclusion The present study concludes that HDV/HBV coinfection is very high in the Pakistani population and was previously underestimated. The most prevalent circulating genotypes of HBV and HDV are HDV-1 and HBV-D, respectively, in the studied area. There is no specific interaction between HBV and HDV genotypes as suggested by HDV-1/HBV-D or HDV-1/HBV-A + D coinfection. Coinfection of HDV-1 and HBV-D simply reflects the most frequent genotypes circulating in this specific geographical region of the world. Eur J c 2014 Wolters Kluwer Gastroenterol Hepatol 26:319–324 Health | Lippincott Williams & Wilkins. European Journal of Gastroenterology & Hepatology 2014, 26:319–324 Keywords: coinfection, genotype, hepatitis B virus, hepatitis delta virus, polymerase chain reaction a Division of Molecular Virology & Molecular Diagnostics, National Centre of Excellence in Molecular Biology (CEMB), University of the Punjab and bCentre for Applied Molecular Biology, Ministry of Science & Technology, Lahore, Pakistan
Correspondence to Muhammad Idrees, PhD, Division of Molecular Virology & Molecular Diagnostics, National Centre of Excellence in Molecular Biology (CEMB), University of the Punjab, 87 West Canal Bank Road, Thokar Niaz Baig, Lahore 53700, Pakistan Tel: + 92 42 529 3141; fax: + 92 42 529 3149; e-mail: [email protected] *Fatima A. Butt and Iram Amin contributed equally to the writing of this article. Received 26 July 2013 Accepted 16 September 2013
HDV is a small defective RNA virus with a negativesense genome, containing single-stranded RNA of 1.7 kb [10,11], a delta antigen, and envelope proteins of hepatitis B surface antigen (HBsAgs) to complete its virion assembly and secretion [12–14] as HBV acts as a covirus for HDV transmission. HDV infection differs in its distribution and severity depending on different geographic areas [15–20], which could be because of possible genetic differences in the prevalence of predominant HDVs [19,21,22]. So far, HDVs have been classified into seven genotypes (HDV-1–7) [23], of which three are major and most widely distributed. HDV-1 is prevalent worldwide and is associated with a huge diversity of diseases, ranging from fulminant hepatitis to asymptomatic chronic liver disease. HDV-2 is prevalent mainly in Asia, Taiwan, Japan, and Siberia. HDV-1 causes more severe disease compared with genotype 2. HDV-3 is mainly found in the north part of South America and causes a severe form of fulminant hepatitis [24]. HDV-4 is found mainly in Japan and Taiwan and HDV-5–7 in DOI: 10.1097/MEG.0000000000000007
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Africa [23–32]. In another study, an extended classification of the delta virus genus to eight clades was proposed [33], which is very similar to the human HBV genetic variability [23,34]. Studies of epidemiology of HDV have shown that the prevalence rate of HDV infection has decreased to 5.7% in 2005 in chronic HBV patients as compared with 18.3% in 1990 [35]. The factor responsible for the control of HDV is the excellent control of HBV achieved by vaccination in the past 15 years in developed countries [36]. However, the prevalence of HDV seems to be increasing in developing countries such as Pakistan because of the lack of preventive strategies and resources [37]. The prevalence of HDV was 16.6% in HBsAgpositive patients from different areas of Pakistan [38]. A few studies are available from different countries that describe the role of the association of different HBV and HDV genotypes with the disease outcome. In Pakistan, especially in the province of Sindh, there are pockets with a high prevalence of concurrent HBV and HDV cases, and few studies on their prevalence, coinfection, disease outcomes, and HBV genotyping are available. However, to the best of our knowledge, no previous study determining the HDV and HBV genotypes circulating in this important region of the world has been available until now. Therefore, this study was carried out with the aim of identifying the HBV and HDV genotypes harbored by chronically infected patients and to further study whether there is any specific interaction between different HBV and HDV genotypes.
from 100 ml serum sample using a Nucleospin Viral RNA Extraction Kit (Macherey-Nagel, Duren, Germany) according to the manufacturer’s instructions provided in the kit protocol. The extracted RNA (B20 ng) was reverse transcribed into cDNA with the Moloney Murine Leukemia Virus reverse transcriptase (M-MLV) enzyme according to the manufacturer’s instructions (Invitrogen Biotechnologies, Carlsbad, California, USA) along with the enzyme. HDV-specific antisense primer 50 -ACAAG GAGAGGCAGGATCACCGAC-30 (10 pmol/ml) was used in this reverse transcription reaction. The reaction mixture was incubated at 371C for 50 min, followed by at 951C for 3 min in the thermal cycler to heat inactivate the M-MLV enzyme. The first-round amplification was performed with 4 ml of cDNA using the outer sense primer (50 -GCCCAGGTCGGACCGCGAGGA-30 ) and the outer antisense primer (50 -ACAAGGAGAGGCAG GATCACCGAC-30 ) in a total of 35 cycles of denaturation at 941C for 40 s, annealing at 551C for 40 s, and extension at 721C for 1 min. Final extension was performed at 721C for 7 min. Nested/second-round PCR amplification was performed with 2 ml of the first-round product using inner sense (50 -GAGATGCCATGCCGACCCGAAGAG-30 ) and inner antisense primers (50 -GAAGGAAGGCCCTCGA GAACAAGA-30 ) using the same cycling profile on the thermal cycler as mentioned above, except for annealing temperature, which was 501C instead of 551C, with a final extension step of 10 min at 721C. The PCR products were electrophoresed on a 2% TAE agarose gel and stained with ethidium bromide as DNA stain. Genotyping of hepatitis delta virus
Patients and methods Patients and initial clinical tests
Initially, a total of 80 chronically HBV-infected patients were enrolled for this study. Serum samples from these patients were tested to evaluate the prevalence of HDV/ HBV coinfection at the Genome Centre for Molecular Diagnostics and Research (GCMD), Lahore. PCR was performed at GCMD for the presence of HBV DNA using SmartCycler II (Cepheid, Sunnyvale, California, USA) real-time PCR with a kit from Sacace Biotechnology (Como, Italy). Samples reactive for HBsAg and detected by HBV DNA were further tested for the presence of HDV RNA, HDV genotyping, and HBV genotyping in the Division of Molecular Virology, Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan. For all PCR reactions, strict procedures proposed by Kwok and Higuchi [39] for nucleic acid amplification diagnostic techniques were followed to avoid false-positive results. Detection of hepatitis delta virus
A total of four primers were designed for the amplification of the conserved region of the HDAg gene representing all the available HDV genotypes. HDV RNA was extracted
For HDV genotyping, second-round PCR products were gel eluted using the GF-1 Nucleic Acid Extraction Kit (Vivantis Technologies, Selangor Darul Ehsan, Malaysia) following the manufacturer’s protocol. Eluted DNA was subjected to cycle sequencing with the ABI Prism BigDye terminator cycle sequencing ready reaction kit (Applied Biosystems Inc., Foster City, California, USA) using the inner sense and inner antisense primers (utilized in the nested PCR) in separate sequencing reactions according to the manufacturer’s instructions and run on a 3100 Automated Genetic Analyzer (Applied Biosystems Inc.). Phylogenetic analysis and sequence alignment
Consensus sequences were generated by alignment of both sequenced strands with forward/sense and reverse/antisense primers after validation using the Phred–Phrap Software (Washington, Seattle, USA). These sequences were submitted to the GenBank database (National Center for Biotechnology Information, Bethesda, Maryland, USA). Nucleotide sequences of HDV isolates sequenced in the present study were aligned together using CLUSTAL Omega. These nucleotide sequences were also compared with previously reported sequences (accession numbers of the reference sequences used were retrieved from GenBank). MEGA (version 2.1) (http://www.megasoftware.net/) was
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Hepatitis delta virus genotype-1, Pakistan Butt et al. 321
used to analyze amino acid sequences. The accession numbers of the reference sequences used were as follows: the phylogenetic trees were constructed in MEGA (version 5.05) using the neighbor-joining method [40]. The robustness of phylogenetic analysis and significance of the branch order were determined by bootstrap analysis with 1000 replicates. Genotyping of hepatitis B virus
HBV DNA was extracted using the method and reagents as described for HDV RNA extraction and rehydrated in 50 ml sterile distilled water. A volume of 10 ml (20–50 ng) each of HBV DNA was amplified in different HBV genotyping PCRs: HBV genotypes A through G (HBV-A– G) were determined using HBV genotype-specific primers for the surface gene of HBV as described previously by Naito et al. [41] and HBV-H was determined using the method described previously [34]. The primer names, their sequences, and the resulting PCR product sizes of genotyping analysis are shown in Table 1. Statistical analysis
The data were analyzed and the summary statistic was calculated using the statistical package, SPSS version 17.0 (SPSS Inc., Chicago, Illinois, USA) for Windows. The results for all variables were described as rates (%). w2 and Fisher’s exact test were used to evaluate the association among categorical variables. All data are presented as mean±SD or number of patients. P-values of less than 0.05 were considered significant.
Results Of the total of 80 HBsAg-reactive patients enrolled, 75 were found to be positive by HBV DNA PCR and were further analyzed for HBV and HDV genotypes. The five HBsAg-reactive but HBV DNA PCR-negative samples were excluded from further analysis as these could be false positive by the ELISA method. HDV RNA was Primer names, sequences, and the resulting polymerase chain reaction product sizes of genotyping analysis
detected in 46 (61.3%) of these 75 HBV-positive patients and were therefore coinfected by these two viruses at the same time. Of the HBV/HDV-coinfected patients, 30 (65.21%) were men and 16 (34.78%) were women. The mean age of the patients was 29±11 years (range 15–59 years). Figure 1 shows the HDV-specific PCR amplified product band of 400 bp (HDAg gene) on an agarose gel. These HDV/HBV coinfected samples were further selected for HBV genotyping using genotype-specific PCR methods. Hepatitis delta virus genotype analysis
A total of 21 samples were selected randomly for HDV genotype analysis using sequencing of the HDAg geneamplified product after gel elusion as shown in Fig. 1. All the sequences were submitted to the GenBank database and the accession numbers allotted are from JN187431 through JN187438 and JN400336 through JN400348. Nucleotide sequences of the local HDAg gene of HDV in Pakistan showed 91% homology with the HDV isolate from Turkey, the dTk38 LHD gene for large HDV antigen (accession no. AM779590), using Blastn (http://www.ncbi. nlm.nih.gov/BLAST). Phylogenetic analysis was carried out using Mega 4 software (Tokyo Metropolitan University, Tokyo, Japan) among the sequenced regions as well as with different reported sequences. Eight different sequences were retrieved from GenBank, representing eight different clades from different geographical regions of the world. A 406-bp region (nt 882–1288) was chosen. The accession numbers and countries of origin for these reference sequences are presented in Table 2. The phylogenetic constructed tree showed that all HDV sequences clustered with HDV-1 as all of the sequences were in the category of genotype-1 (Fig. 2). Hepatitis B virus genotyping analysis
Of the 46 tested HBV PCR-positive samples by genotyping assay, 38 (82.6%) were genotyped successfully, of
Table 1
Primer names Universal sense (P1) Universal antisense Mix A B2 BA1R BB1R BC1R Mix B B2R BD1 BE1 BF1 Mix C HBHKF1 HBHKR2 HBHKF2
Sequences 0
0
5 -TCACCATATTCTTGGGAACAAGA-3 50 -CGAACCACTGAACAAATGGC-30
M 1 2 3 4 5 6 7 8 9 10 M
1063
50 -GGCTCMAGTTCMGGAACAGT-30 50 -CTCGCGGAGATTGACGAGATGT-30 50 -CAGGTTGGTGAGTGACTGGAGA-30 50 -GGTCCTAGGAATCCTGATGTTG-30
68(A) 281(B) 122(C)
50 -GGAGGCGGATYTGCTGGCAA-30 50 -GCCAACAAGGTAGGAGCT-30 50 -CACCAGAAATCCAGATTGGGACCA-30 50 -GYTACGGTCCAGGGTTACCA-30
119(D) 167(E) 97(F)
50 -ACGGGGCGCACCTCTCTTTAC-30 50 -AGCCAAAAAGGCCATATGGCA-30 50 -GCACTTCGTTTCACCTCTGCA-30
Fig. 1
Product size (bp)
357(G&H)
400 bp
500 bp 250 bp
PCR amplification of hepatitis delta virus (HDV) HDAg gene. Lanes 1–9, HDV-positive samples; lane 10, negative sample; M, marker of 50 bp.
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Table 2 Accession numbers and countries of origin of reported geographical sequences of genotypes 1–8 Serial numbers 1 2 3 4 5 6 7 8
Accession number U81989.1 X60193.1 AB037948.1 AF309420.1 AM183328.1 AX741164.1 AM183333.1 AM183327.1
Country of origin Ethiopia Japan Venezuela Japan France Paris France France
Represented in the phylogenetic tree HDV-1 HDV-2 HDV-3 HDV-4 HDV-5 HDV-6 HDV-7 HDV-8
HDV, hepatitis delta virus.
which 33 (71.7%) had genotype D and five (10.9%) had mixed infections of HBV-A + D. A total of eight (17.3%) samples were untypable as the genotyping method could not confirm a type even when these were positive and could thus be genotyped. Figure 3 shows the HBV-D specific band of 119 bp.
Discussion The aim of this study was to determine the sequencebased genotyping of HDV and to correlate this genotype with the HBV genotype in coinfected patients from
Fig. 2
HDV-6 HDV-7 HDV-5 HDV-3 HDV-2 HDV-4 HDV-8 PK-HDV-3 PK-HDV-5 PK-HDV-10 PK-HDV-1 PK-HDV-4 PK-HDV-17 PK-HDV-14 PK-HDV-20 PK-HDV-18 PK-HDV-8 PK-HDV-16 PK-HDV-2 PK-HDV-21 PK-HDV-13 PK-HDV-19 PK-HDV-6 PK-HDV-9 PK-HDV-12 PK-HDV-15 PK-HDV-11 HDV-1 PK-HDV-7
0.2
Phylogenetic tree of studied sequences with geographical strains of genotypes 1–8 generated by the neighbor-joining method with a bootstrap test of 1000 replicates. Each geographical strain is abbreviated by the name shown in Table 2.
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Hepatitis delta virus genotype-1, Pakistan Butt et al. 323
Fig. 3
M
1
2
3
4
5
6
7
119 bp
Genotyping results of hepatitis B virus-positive samples showing genotype D with a band of 119 bp. Lanes 1–7, samples showing genotype D; M, 50 bp ladder.
Pakistan. Although the sample size of our study is not very large as compared with that of earlier studies, the results are very interesting and important as this is the first study of its nature from Pakistan on the subject, wherein a high prevalence rate of HDV/HBV coinfection has been observed and the genotypes of HBV and HDV have been determined in coinfected individuals. The epidemiology of HDV is changing worldwide, consistent with successful efforts to control HBV infection in the developed countries, whereas the infection remains endemic in south Asian countries such as Pakistan. The first and important finding of the current study is the observation that a very high number (61.33%) of HBV-positive patients were coinfected with HDV. This rate is very high as compared with previous studies from this region [37,38] and other parts of the world. Mumtaz et al. [38] reported a prevalence rate of HDV of only 16.6%. This huge difference in the rates of HDV/HBV coinfection may be because of the sensitivity of the detection systems used. We have used the most sensitive and specific molecular diagnostic methods such as nested PCR (with a sensitivity of 10 copies/ml) and real-time PCR (50 copies/ml). In our study, the majority of HBV/ HDV-coinfected patients were men. The second and interesting finding of this study was the detection of only one HDV genotype (HDV-1) that cocirculates with HBV in Pakistan. To the best of our knowledge, this is the first reported HDV genotype from this region of the world using a sequencing method. Our finding is in agreement with the predominant genotype of HDV in Pakistan’s neighboring country Iran and in the middle-east regions such as Lebanon, Egypt, and Turkey [42–45], where HDV-1 is the most prevalent genotype. The third most important finding of this study was the observation that in all cases, mixed infection of HDV-1
coexists with HBV-D. Although it has been reported previously that genotype D is the most common genotype in this country along with other genotypes [46], no study is available that had correlated HDV genotypes with HBV genotypes. In our study, HBV-D was observed in 71.7% of the total isolates. This HBV-D is also considered to be common in south Europe, the Middle-East, and previously in India. In the current study, we also found that 10.9% patients had mixed HBVA + D along with coexistence of HDV. The clinical significance of such mixed HBV genotype infections particularly if coinfected with HDV is unclear and needs further study. Eight samples were HBV DNA and HDV RNA PCR positive; however, no genotypespecific bands were found for HBV. This may be because of some mutations in the HBV genome that cause specific primer annealing and amplification [47]. In this study, we could not isolate HBV genotypes other than A and D, which means that HBV-E–H do not exist here. These genotypes have been reported from Africa, South America, USA, Vietnam, and Japan, respectively [34,48,49]. The high rate of coinfection of HBV and HDV observed in this study indicates that HBV replication is not suppressed by HDV as reported in previous studies that in most cases of HDV infection, HBV replication is suppressed to very low levels by HDV [50,51]. It seems that HBV replication is not suppressed by the presence of HDV RNA in a subset of individuals. A more severe progression toward liver decompensation has been documented in patients with active HBV and HDV replication, with each virus contributing to liver damage, thereby resulting in more severe liver disease [52,53]; however, we did not observe this type of complication in our patients.
Conclusion
The present study concludes that HDV/HBV coinfection is very high and was previously underestimated. All of the HDV/HBV coinfected patients had HDV-1 and HBV-D was the most common. An association exists between HDV-1 and HBV-D.
Acknowledgements F.A.B. and I.A. collected epidemiological data; M. Idrees critically reviewed the manuscript. F.A.B., I.A., and M. Idrees analyzed and compiled the data. M. Iqbal helped with sequence analysis and data interpretations. All authors read and approved the final manuscript. The authors acknowledge and extend their heartfelt gratitude to the Genome Centre for Molecular Based Diagnostics & Research (GCMBDR) for providing the HBV and HDV samples and all the clinicians and patients (involved in this study) for their cooperation.
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The study was financially supported by the Genome Centre for Molecular Diagnostics and Research (GCMD).
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Conflicts of interest
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There are no conflicts of interest. 31
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