Despite the availability of effective vaccine, about 1.5 million new hepatitis B virus (HBV) infections are documented annually across the world1; further, it is estimated that around 296 million people are living with chronic hepatitis B infection, leading to 820,000 fatalities in 20191.
HBV belongs to Hepadnaviridae family, and it has ~3.2 kb partially dsDNA that codes for three structural (HBsAg, HBcAg and Pol) and two non-structural proteins (HBeAg and HBxAg)2. Owing to low fidelity rates of reverse transcriptase, high selection pressure, use of antiviral drugs etc. the virus displays high genetic diversity3. Based on nucleotide diversity of eight per cent among HBV genomes, the virus is divided into 10 different genotypes, which are further divided into different sub-genotypes with 4-8 per cent differences4. HBV is also classified into distinct sub-types, based on amino acid positions in surface antigen5.
Distribution of genotypes shows geographical patterns across the globe and within the countries4. Though limited, molecular studies have documented circulation of genotypes D, A and E in northern, genotypes D and A in western, genotype D in eastern and genotypes D, A and C in southern parts of India, with overall dominance of genotype D6. Rarely, genotypes such as B, C, E and I are also reported7. The infecting genotype and the sub-type are known to influence the disease progression, treatment response and clinical outcome4. Moreover, it has been shown that HBV-vaccinated individuals respond unequally to different genotypes3. These facts make it important to monitor circulating genotypes and sub-types in different areas.
In the past, the circulation of genotype D sub-genotype D1, D2, D3 and D5 has been documented from central India6. During the routine molecular surveillance at Jabalpur, central India, a patient infected with HBV genotype A was detected, and the objective of this study was to characterize this genotype detected from central India.
The State-level Virus Research and Diagnostic Laboratory (VRDL) at the ICMR-National Institute of Research in Tribal Health, Jabalpur, India, has mandated to provide diagnosis and monitor viruses of public health importance circulating in central India. Samples referred along with clinical information are tested following syndrome-based approach. To detect hepatitis B infection, the serum samples of patients suspected of viral hepatitis are subjected to HBsAg ELISA using commercially available kit (J. Mitra & Co., New Delhi, India; Cat. No. IR020096). Positive samples having adequate volume and sufficient clinical information are subjected to partial S gene PCR and sequencing for the identification of genotype as described by Gandhe et al8.
In this prospective study (January 2020 to December 2021), during routine surveillance work, one sample sequence showed that it belonged to genotype A. Full genome amplification of this sample by PCR in ABI GeneAmp 9700 PCR-Thermal Cycler, USA, using six sets of overlapping primers. The sequencing and phylogenetic analysis was done as described earlier was carried out by Shivlata et al6. The HBsAg sub-typing was done as described by Purdy et al5. For phylogenetic analysis of HBV full genome sequences, 48 reference sequences of different genotypes from A to J and subgenotypes of A (A1-A7) were retrieved from GenBank database and a phylogenetic tree was constructed by employing the Neighbor Joining method in MEGA (Molecular Evolutionary Genetics Analysis) software version 5.05 by applying 1000 bootstraps to determine genotype/subgenotype. The full genome sequence was submitted to GenBank (Accession Number OK274310).
This study was approved by the Institutional Ethics Committee (No: NIRTH/IEC/2229/2018) and written informed consent was obtained from all the patients.
The Basic Local Alignment Search Tool analysis of the full genome sequence revealed that the HBV detected belonged to genotype A, sub-genotype A1 (Figure); based on the amino acid positions in S protein, the sub-type of the sample was found to be adw2. The sequence showed 99.38 per cent similarity with sequence submitted from Dhaka, Bangladesh (LC519823), 99.32 per cent from Kolkata, India (KT366471.1) followed by sequences from Arunachal Pradesh, India (KF214663.1): 99.10 per cent; New Delhi, India (AY373432.1): 98.60 per cent; Lucknow, India (KT151615.1): 98.67 per cent.
The full length sequence analysis showed several nucleotide and amino acid substitutions of clinical importance (Table). Clinically relevant mutations were observed in the basal core protein/pre-core region of HBV sequences. Mutations occurring in enhancer 1 X promoter region and S1 region were also determined. Interestingly, mutations such as T1041G, A2720G and T3111C which are sub-genotype A2/A39 specific were found in the sequence. On analysis of protein sequence of the open reading frames (ORFs), it was observed that many nucleotide substitutions showed their effect at amino acid level (Table).
The patient, a 65 yr old male a native of central India, presented to the department of General Medicine, Netaji Subhash Chandra Bose Medical College & Hospital (NSCBMC & H), Jabalpur, with complaints of fever, abdominal distension with abdominal pain, jaundice and melena over one month. He had not travelled to any other States in the last six months. Clinical examination revealed moderate pallor and icterus. His abdomen examination revealed a significantly distended abdomen, with subcutaneous abdominal wall oedema with a positive fluid thrill. On ultrasonographic assessment, liver echotexture was found to be altered suggestive of liver cirrhosis, along with gross ascites and bilateral mild pleural effusion. There was no evidence of hepatocellular carcinoma (HCC) in the patient. Blood investigations revealed an increased ALT (alanine aminotransferase) level of 162.7 U/l with total bilirubin level of 3.87 mg/dl with direct bilirubin of 1.87 mg/dl, and total protein levels were found to be 6.41 g/dl. Persistent viraemia was evident with an increased HBV DNA viral load of 1×108 IU/l (tested commercially using kit from Altona: AltoStar® HBV PCR Kit 1.5). Serologically, the patient was negative for HBeAg but positive for anti-HBe antibody, indicating an immune reactivation phase with no virological remission. Complete blood count revealed decreased haemoglobin levels of 8.7 g/dl with a total leucocyte count of 10,300/µl. Serum creatinine was found to be within normal range (1.15 mg/dl). The patient succumbed to the complications of decompensated liver disease after one month of treatment; therefore, the effects of the detected mutations could not be studied further.
This study documents the detection and molecular characterization of genotype A sub-genotype A1 from central India. Earlier studies from this region have documented detection of various sub-genotypes of genotype D6. Genotypes A-F are reported from different parts of India; however, genotype D is reported as the most dominant genotype18. Genotype A infection is shown to be associated with more severe liver disease than genotype D19; the detection of this genotype from the area thus has clinical and epidemiological importance. The patient had no travel history to HBV A1-endemic areas, suggesting that he acquired the infection in the region. Circulation of subtype adw2 of genotype A1 has been documented from south and north as well as east and west India8,18,20,21, and our findings are in agreement with those studies.
The genotype A/A1 is known to have clinically important characteristic mutations throughout its genome. We detected various nucleotide and amino acid substitutions/mutations of clinical and epidemiological significance in enhancer I X promoter, basal core promoter, pre-core, core, S1 promoter, RT region, X protein, pre-core and core protein. Important ones are given in the Table.
Our sequence showed the presence of sub-genotype A1-specific pre-core kozak sequence 5’-TCAT-3’ at position 1809-1812 when aligned to the reference sequence9. However, we detected guanine at 1041 (in the enhancer I X promoter region) instead of thymine which is generally found in genotype A39. Further, two genotype A2-specific mutations (A2720G and T3111C) were also found in our sequence. We also detected mutations G1467A and C1674T in the enhancer I X promoter region reported to cause low-gene expression and increased apoptosis, respectively10-12, which was observed in this case. The mutation in the basal core region of G1862T (that is a presumed cause resulting in fulminant hepatitis) was detected in the sequence3. The mutation G1888A in pre-core region responsible for decreased expression of HBcAg, was detected.
At amino acid level, the sequence showed a mutation H126Y in HBV pol region which is associated with tenofovir resistance17. Mutations in the X region (S31A) responsible for HCC11 and increased apoptosis (P101S)12 were detected; however, our patient did not have HCC. The amino acid change at position 17 of pre-core region from valine to phenylalanine results in low or no synthesis of HBeAg13, and this mutation was present in the sequence and was the probable reason that the patient was serologically negative for HBeAg and anti-HBeAg antibodies positive.
The sequence reported in the study showed fewer number of mutations compared to the mutations reported from south16, north9 and east10 India, probably because the virus is not under high immunological and antiviral pressure owing to low circulation in the area. Further studies with larger sample size and full genome next generation sequencing will help shed more light on not only on these aspects but will also help understand if there are any quasispecies complex as described from African country22 in genotype A specific to this region.
This study was based on single sequence and this was a major limitation of the study; however, the study reported maiden detection and detailed full genome characterization of HBV genotype A1 from central India and a full genome analysis of HBV genotype A1. Continuous molecular surveillance is required to keep a track of emerging genotypes and the mutations occurring in a region which will help public health programme and treating clinicians.
Financial support & sponsorship: This study was supported under the State VRDL project (Ref No. DHR/VDL/04/2018), Department of Health Research, Ministry of Health & Family Welfare, Government of India, New Delhi.
Conflicts of Interest: None.
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