|Year : 2019 | Volume
| Issue : 4 | Page : 563-568
Currently circulating genotypes of hepatitis E virus in India, 2014–2018
Fernandes M Mevis, Sasidharanpillai Sabeena, Ramachandran Sanjay, Sudandiradas Robin, Santhosha Devadiga, Varamballi Prasad, Dsa Oliver, Alyusif Ameen, Govindakarnavar Arunkumar
Manipal Institute of Virology, Manipal Academy of Higher Education, Manipal, Karnataka, India
|Date of Submission||25-Nov-2019|
|Date of Acceptance||11-Mar-2020|
|Date of Web Publication||18-May-2020|
Dr. Sasidharanpillai Sabeena
Manipal Institute of Virology, Manipal Academy of Higher Education, Manipal - 576 104, Karnataka
Source of Support: None, Conflict of Interest: None
Purpose: Hepatitis E virus (HEV) is an emerging pathogen causing acute viral hepatitis worldwide. Clinical manifestations often occur in young adults with an increased mortality rate among pregnant women. HEV genotypes 1 and 4 are mainly reported among humans and swines, respectively. Aims: The aim was to study the currently circulating genotypes of HEV in India. Materials and Methods: A retrospective cross-sectional study was carried out at Manipal Institute of Virology to know the circulating genotypes of hepatitis E, spanning over 5 years from August 2014 to September 2018. The serum samples screened serologically positive and confirmed positive for active infection by real-time reverse transcriptase-polymerase chain reaction (PCR) (Real Star® HEV RT-PCR Kit 2.0, Altona Diagnostics, GmbH, Hamburg, Germany) were further subjected to nested conventional PCR targeting the RdRp gene of non-structural ORF1 region. The purified PCR product was sequenced in BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Thermo Fisher Scientific, USA). The chromatograms obtained by sequencing were analysed using Sequencher 5.4.6, and HEV FASTA sequences were compared with reference sequences for HEV in GenBank Nucleotide Blast. Results: During the study period, there were 317 cases of laboratory-confirmed cases of acute viral hepatitis comprising 202, 70, 43 and 2 cases of hepatitis A, E, B and C, respectively. Serum samples of 70 acute hepatitis cases were positive for anti-hepatitis E IgM. According to the clinical case classification, there were 66 cases of acute viral hepatitis and four cases of fulminant hepatic liver failure. The mean age of the patients was 30.3 years (standard deviation = 12.5). The samples from various parts of India were genotyped as 1a. Conclusion: The HEV genotypes 1a was observed to be the currently circulating strain in the regions studied.
Keywords: Genotype, hepatitis E virus, India, viral hepatitis
|How to cite this article:|
Mevis FM, Sabeena S, Sanjay R, Robin S, Devadiga S, Prasad V, Oliver D, Ameen A, Arunkumar G. Currently circulating genotypes of hepatitis E virus in India, 2014–2018. Indian J Med Microbiol 2019;37:563-8
|How to cite this URL:|
Mevis FM, Sabeena S, Sanjay R, Robin S, Devadiga S, Prasad V, Oliver D, Ameen A, Arunkumar G. Currently circulating genotypes of hepatitis E virus in India, 2014–2018. Indian J Med Microbiol [serial online] 2019 [cited 2020 Sep 30];37:563-8. Available from: http://www.ijmm.org/text.asp?2019/37/4/563/284515
| ~ Introduction|| |
Worldwide, hepatitis E virus (HEV) is estimated to infect about 20 million people every year. About 3.3 million cases present with gastrointestinal symptoms or extrahepatic manifestations. This hepatotropic virus was initially referred to as 'enterically transmitted non-A non-B hepatitis'. HEV is a non-enveloped, positive-sense RNA virus with capped and polyadenylated single-stranded genome, belonging to the genus Orthohepevirus under the novel family Hepeviridae. The single-stranded RNA genome consists of three open reading frames of ORF1, ORF2 and ORF3 encoding for non-structural proteins, capsid proteins and regulatory phosphoproteins, respectively.
The main mode of transmission is through the faecal-oral route, leading to outbreaks of viral hepatitis. In developing countries, HEV is one of the major causes of waterborne hepatitis due to inadequate sanitation and hygiene. This is mainly attributed to heavy monsoons, floods, seeping pipes and raw sewage flowing. Amongst the four mammalian lineages, genotypes 1 and 2 infect humans. Meanwhile, genotypes 3 and 4 circulate in animal species including pigs. Consumption of inadequately cooked meat of wild boar, domestic pigs, parboiled flesh, raw livers or eating Corsican Figatelli sausage may lead to outbreaks of foodborne zoonotic hepatitis E. Direct contact with infected animals or animal excreta also can lead to human infection.
In India, no zoonotic transmission between humans and swine was reported as in developed countries. HEV genotype 4 isolated from swines was not observed to be infecting humans.
Sporadic human cases attributed to genotypes 3 and 4 were reported from developed European countries. HEV infection is widespread among domestic pigs, sheep, goats and buffalo in India. China has perceived alteration in the epidemiology of hepatitis E infection, and genotype 4 has become the dominant circulating genotype, replacing HEV genotype 1 among humans as well as animal hosts.
India is one of the hyperendemic areas for HEV genotype 1, with subtypes a and c reported to cause human infections. HEV genome mutates at an evolutionary rate of 0.8 × 103 nucleotide substitution per base per year, which contributes to occupational exposure to swine handlers, slaughterhouse workers and pig farmers. There is a substantial advancement in the development of recombinant subunit vaccines against HEV. At present, Hecolin® is the only licensed HEV vaccine (Xiamen Innovax Biotech Co., Ltd, Xiamen, Fujian, China) which is available in China to prevent human disease. There are major research gaps pertaining to currently circulating HEV strains among humans as well as various animal hosts in different parts of India.
| ~ Materials and Methods|| |
Based on the hospital-based acute febrile illness (AFI) surveillance at Manipal Institute of Virology (MIV), a retrospective cross-sectional study representing the time period spanning over 5 years from August 2014 to September 2018 was carried out. In this multicentric acute fever surveillance project, there were 33 rural study centres from ten states of India such as Karnataka, Kerala, Tamil Nadu, Maharashtra, Goa, Jharkhand, Odisha, Assam, Gujarat and Tripura, which were linked to community health centres and Taluk hospitals. As per the WHO case definition, serum samples from acute hepatitis cases with malaise, anorexia, nausea, jaundice, dark urine, right upper quadrant tenderness and serum alanine aminotransferase level more than ten times the upper limit of normal (400 IU/L) were included by purposive sampling from different parts of India.
In the present study, viral hepatitis cases <6-month duration with total bilirubin >2 mg/dl were classified as acute viral hepatitis. Patients with altered sensorium and coma within 8 weeks of the onset of hepatitis with no previous history of liver disease were classified as acute fulminant hepatic failure. During the study period, 5 ml of blood was collected in red vacutainer from all the AFI cases after taking written informed consent. Samples were transported at 4°C–8°C to MIV and stored at −80°C. Repeated freeze thaw of samples was avoided by making aliquots separate for serology and molecular diagnosis.
The samples were screened serologically for anti-HEV IgM antibodies using anti-HEV IgM ELISA 3.0 (MP Biomedicals Germany GmbH, Germany). Viral RNA extraction with 150 μl of serum sample was done using Qiagen QIAamp Viral RNA Extraction Kit (Qiagen, Hilden, Germany) as per manufacturer's instructions. Serologically positive samples were further tested by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) (Real Star® HEV RT-PCR Kit 2.0, Altona Diagnostics, GmbH, Hamburg, Germany). For PCR, serum samples tested negative and positive for HEV RNA by real-time PCR (Real Star® HEV RT-PCR Kit 2.0, Altona Diagnostics, GmbH, Hamburg, Germany) were used as negative and positive controls, respectively. Positive samples were further subjected to nested conventional PCR targeting the RdRp gene of non-structural ORF1 region. The extracted RNA from PCR-positive samples was subjected to genotyping by conventional nested PCR using primers (Sigma-Aldrich Chemicals, Mumbai, India) targeting the non-structural ORF1 region gene (RdRp) (Gene Bank accession no. M-32400). For the first-round PCR, forward primer (5'-CCGGATCCA CACACATCTGAGCTACATTCGTGAGCT-3') and reverse primer (5'-CCGAATTCAAAGGCA TCCATGGTGTTTGAGAATGAC-3')with product size 591 bp were used. For the second-round PCR, forward(5'-GGAATTCGACTCCACCCAGAATTACTT-3') and reverse primers(5'-GGAATTCACAGCCGGCGATCAGGACAG-3') with product size 343 bp were employed.
The cycling conditions for the first-round PCR were set at 10 min at 95°C for initial denaturation, followed by 35 cycles of denaturation for 30 s at 94°C, annealing at 61°C for 15 s and extension for 45 s at 72°C, and final extension at 72°C for 5 min. The reaction mixture was prepared using AgPath-ID™ One-step RT-PCR Kit (Applied Biosystems by Thermo fisher Scientific, Bedford, MA, USA). The extracted nucleic acid (5-μl RNA) was added to the 20-μl first-round reaction mixture containing 12.5-μl buffer mix(Ambion, Life technologies, Woodward St, Austin, TX 78744), 1 μl each of primers(HEVF and HEVR), 1 μl of enzyme mix (Ambion® Life Technologies, USA) and 4.5-μl nuclease-free water. The amplification was carried out in ProFlex™ PCR system (Life Technologies, USA).
For second-round PCR, the initial denaturation was set at 95°C for 10 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 45°C for 15 s, extension at 72°C for 45 s and a final extension at 72°C for 5 min. The reaction mixture was prepared using AgPath-ID™ One-step RT-PCR Kit (Applied Biosystems by Thermo Fisher Scientific, USA) and the 0.7-μl template was used. The final PCR products were analysed using 1.8% gel (w/v) agarose Gel (Merck, Kenilworth, NJ 07033, USA) electrophoresis and were visualised using an ultraviolet transilluminator. The band of interest was excised, and gel purification was done using GenElute™ Gel Purification Kit (Sigma Aldrich, St. Louis, MO, USA), and a final purification product of 25 μl was obtained.
The purified PCR product was sequenced in BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies, Thermo Fisher Scientific, USA). The chromatograms obtained by sequencing were analysed using Sequencher 5.4.6. The sequences were aligned, trimmed and saved in FASTA format. The HEV FASTA sequences were compared with reference sequences for HEV in GenBank Nucleotide Blast. The prototype strains for all the HEV genotypes were extracted from The International Committee on Taxonomy of Viruses (ICTV).
The data obtained from the participants regarding sociodemographic characteristics, clinical features and hospital records regarding liver function parameters were recorded using MS Office Excel 2016, and statistical analysis was done using SPSS software (SPSS 15.0 for Windows (SPSS™ Inc., Chicago, IL, USA).
| ~ Results|| |
During the study period, amongst the AFI cases, 344 cases presented as acute hepatitis. Two hundred and forty-seven cases of hepatitis were laboratory confirmed as acute viral hepatitis including 202, 43 and 2 cases of hepatitis A, B and C, respectively. Out of the 97 serum samples negative for hepatitis A, B and C, 70 cases were serologically positive for anti-HEV IgM antibodies. These 70 HEV-positive samples were collected from acute hepatitis cases from Goa (n = 17, 24.2%), Gujarat (n = 11, 15.7%), Odisha (n = 10, 14.9%), Karnataka (n = 8, 11.4%), Kerala (n = 8, 11.4%), Jharkhand (n = 3, 4.2%), Maharashtra (n = 3, 4.2%), Tripura (n = 2, 2.85%), Assam (n = 5, 7.14%) and Tamil Nadu (n = 3, 4.28%). Thirty-one samples serologically screened positive were excluded as the RT-PCR results were negative. Out of the 39 samples positive for HEV RNA, 11 samples were sequenced and genotyped.
Even though there was no distinct seasonality, a greater number of cases were observed during the months of July and November. The mean age of the HEV-infected study participants was 30.3 years (standard deviation = 12.5). The most affected age group was 21–40 years (48.57%), as shown in [Table 1]. Amongst the 70 HEV-positive cases, distinct male gender predisposition was observed with 49 (70%) males and 21 (30%) females. Most of the acute hepatitis cases belonged to low socioeconomic status (68.57%), and the remaining patients were semi-skilled workers, professionals and students. Eleven (15.7%) participants had a history of close contact with clinical cases. The main source of drinking water was well water or piped water stored in tanks, steel pots, drums, syntax, earthen pots and aluminium vessels. In Gujarat, drinking water was observed to be mainly stored at homes in plastic and earthen pots. Travel history to neighbouring districts was given by 22 (31.4%) patients.
All the study participants presented with a fever. Other predominant symptoms were malaise (n = 63, 90%), headache (n = 54, 77%), nausea/vomiting (n = 40, 57%) and abdominal discomfort (n = 44, 63%). Thirty-six (51.4%) participants had a total bilirubin level above 2 mg/dl. According to the clinical case classification, there were 66 cases of acute viral hepatitis and four cases of fulminant hepatic liver failure. There were two pregnant women from Assam and Karnataka enrolled in the present study with acute hepatitis E viral infection. Even though both the women recovered without any complications, the 19-year-old primigravida from Assam had an abortion at 24-week gestational age.
Serum samples with adequate viral load (Ct value <30) from the study sites of east, west and south of India were typed as genotype 1a (GenBank accession numbers MN164692–MN164702). This comprised three samples from Goa, four from Gujarat, two from Assam and one each from Karnataka and Kerala. The 11 strains demonstrated 98%–99% of similarity with HEV isolates from the United Kingdom (Oldham, London) and also strains from Chhattisgarh, East India (KT071749–53). The sequence homology of 97% was observed with strains circulating in Pakistan (AB513496) and Nepal (KY629738). The evolutionary history was deduced using the maximum likelihood method and general time-reversible distance correction. The reliability of the tree was verified by the bootstrap method (1000 replicates). The phylogenetic analyses were done using MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms 9 (version 10.0) (www.megasoftware.net) where all the representative study strains clustered under HEV-1a, as depicted in [Figure 1]. The p-distance calculated between the subtypes a–f of HEV genotype 1 ranged from 0.060 to 0.148 as in [Table 2]. The p-distance between the genotypes 1–8 including the study strains was 0.261–0.563 as shown in [Table 3].
|Figure 1: The phylogenetic tree representing various genotypes of HEV. Labelled sequences with black diamonds illustrate the study samples. Bootstrap value >60% is shown on the nodes. Representative strains: Representative strains for genotype 1 included strains from India (9), Pakistan (6), Nepal (4) and London (2); representative strains for genotype 1b included strains from China (2); representative strains for genotype 1c included strain from India (1); representative strains for genotype 1d included strain from Morocco (1); representative strains for genotype 1e included strains from Chad in Africa (1) and Nigeria (1); representative strains for genotype 2 included strains from Mexico (2); representative strains for genotype 3 included strain from Japan (3), Germany (3), France (1), USA (1), Canada (1), China (1) and Osh in Kyrgyzstan (1); representative strains for genotype 4 included strains from China (6) and Japan (2); representative strains for genotype 5 included strains from Japan (1); representative strains for genotype 6 included strains from Japan (1); representative strains for genotype 7 included strains from UAE (2); representative strains for genotype 8 included strains from China (2). Outgroup: Representative strains for Orthohepevirus B included strains from the USA (2). Representative strains for Orthohepevirus C included strains from the Netherlands (1), Germany (1) and Vietnam (1). Representative strains for Orthohepevirus D included strains from Germany (1)|
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|Table 2: A pair-wise comparison of the nucleotide p-distance over the target gene amongst genotype hepatitis E virus 1 study and reference strains with p-distance values ranging from 0.060 to 0.148 between the subtypes|
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|Table 3: A pair-wise comparison of the nucleotide p-distance over the target gene among genotype 1-8 hepatitis E virus strains, including the strains that obtained in the present study with p-distance values ranging from 0.261 to 0.563|
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| ~ Discussion|| |
HEV infection contributes to 30%–70% of acute sporadic hepatitis cases in developing countries, including India. Molecular confirmation of HEV infection is an epidemiologic tool rather than a diagnostic test. In the present study, the major proportion of infected cases was constituted by the working class and students. Most of the HEV-infected patients were within the age group of 21–40 years. This was in accordance with previous studies from India.,, Symptomatic hepatitis E infection occurs frequently in adolescents as well as young adults, and children below 10 years are often asymptomatic. During the study period of 5 years, we did not observe any distinct seasonality of HEV infection. A study from Central India reported seasonality peaks from March to June. Meanwhile, a study from North India observed peaking of cases during winter months of January and February as well as in the month of August.
Amongst the immunocompromised hosts, there is an important role for molecular tests prior to blood transfusion as well as organ transplantation. Graft hepatitis among the population with post-orthotopic liver transplantation is usually undetected and unreported, which is majorly caused by HEV. Selective HEV RNA screening of blood donors was introduced in many European countries for high-risk patients. Hepatitis E genotypes often show geographical restriction, and our study strains were grouped along with strains from Pakistan. Indian studies since 1967 have reported the circulation of HEV genotype 1 among humans. The population-based survey from Tamil Nadu observed higher seroprevalence among all the age groups from cities compared to rural regions. A change in the circulating genotype from 1 to 4 was reported from HEV hyperendemic countries such as China. Even though HEV genotypes 3 and 4 mainly circulate among various animal hosts worldwide, sporadic human cases were reported from developed countries such as Japan, China, North America and Europe. Further multicentric studies among humans and animals are necessary for early recognition of variation in genotype and disease severity in endemic countries.
In the present study, significant epidemiological information regarding the use of boiled water for drinking purposes; the distance between sewage tank and drinking water source; proximity to slaughterhouses and consumption of partially cooked meat, uncooked vegetables and fruits could not be collected. As there were no study centres from North India, the present study lacks information regarding circulating HEV strains in North India. However, representative serum samples in 5 consecutive years from different parts of India were analysed and genotyped.
| ~ Conclusion|| |
The present study concludes that HEV genotype 1a is the most prevalent and currently circulating strain in most parts of India.
We acknowledge our sincere gratitude to the clinicians from different parts of India.
Financial support and sponsorship
The study was partially supported by the CDC Cooperative Agreement (5U01GH001051) and ICMR grant (file no. 5/8/7/15/2010/ECD-I).
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]