Indan Journal of Medical Research Indan Journal of Medical Research Indan Journal of Medical Research Indan Journal of Medical Research
  Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login  
  Home Print this page Email this page Small font sizeDefault font sizeIncrease font size Users Online: 4405       

   Table of Contents      
ORIGINAL ARTICLE
Year : 2020  |  Volume : 151  |  Issue : 4  |  Page : 375-379

Molecular characterization of hepatitis A virus circulating in Uttar Pradesh, India: A hospital-based study


Department of Microbiology, King George's Medical University, Lucknow, Uttar Pradesh, India

Date of Submission01-Mar-2018
Date of Web Publication12-May-2020

Correspondence Address:
Dr Amita Jain
Department of Microbiology, King George's Medical University, Lucknow 226 003, Uttar Pradesh
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmr.IJMR_429_18

Rights and Permissions
   Abstract 

Background & objectives: Hepatitis A is prevalent worldwide and is among the leading cause of acute viral hepatitis in India. Major geographical differences in endemicity of hepatitis A are closely related to hygienic and sanitary conditions and other indicators of the level of socio-economic development. The present study was aimed to know the seropositivity prevalence and predominant circulating strain of HAV in a north India.
Methods: Patients with acute viral hepatitis were enrolled. Blood samples were collected over a period of one year from June 2016 to May 2017. Serum samples were tested for anti-immunoglobulin M (IgM) HAV antibodies. The seropositive samples were analyzed for HAV-RNA by real-time reverse transcription-polymerase chain reaction (RT-PCR). Samples detected on molecular assay were subjected to conventional semi-nested RT-PCR for VP1 gene. Further sequencing of amplified RT-PCR products was done, and data were analyzed.
Results: A total of 1615 patients were enrolled, and serum samples were collected and tested. The male:female ratio was 1.3:1 with a mean age of 24.31±17.02 yr (range 0-83 yr). Among these, 128 (7.93%) were positive for anti-HAV IgM antibodies; 41.63 per cent of seropositive patients were in their childhood or early adolescent age group. Of all seropositive samples, 59 (46.09%) were positive for HAV RNA. Genotyping sequencing of 10 representative strains was carried out, and the circulating genotype was found to be IIIA. The nucleotide sequences showed homology among the strains.
Interpretation & conclusions: Our results showed that hepatitis A was a common disease in children with IIIA as a circulating genotype in this region. In approximately 50 per cent of cases, HAV RNA could be detected. Higher number of HAV IgM-seropositive cases was observed during monsoon period.

Keywords: AVH - genotype - HAV - hepatitis A - endemic - India


How to cite this article:
Prakash S, Shukla S, Shukla R, Bhagat A, Srivastava SS, Jain A. Molecular characterization of hepatitis A virus circulating in Uttar Pradesh, India: A hospital-based study. Indian J Med Res 2020;151:375-9

How to cite this URL:
Prakash S, Shukla S, Shukla R, Bhagat A, Srivastava SS, Jain A. Molecular characterization of hepatitis A virus circulating in Uttar Pradesh, India: A hospital-based study. Indian J Med Res [serial online] 2020 [cited 2020 Oct 21];151:375-9. Available from: https://www.ijmr.org.in/text.asp?2020/151/4/375/284217

Shantanu Prakash & Suruchi Shukla contributed equally


Hepatitis A virus (HAV) is transmitted through faeco-oral route by contaminated water and food. Approximately 1.5 million clinical cases of hepatitis A occur worldwide annually[1]. The incidence rate is strongly related to socio-economic indicators and access to safe drinking water. Approximately 85 per cent of individuals who are infected with HAV recover fully clinically and biochemically within three months, and nearly, all have complete recovery by six months[2]. It has been seen that severe manifestations are more common in young adults requiring hospitalization with overall case fatality rate of 0.3 per cent[3],[4]. HAV is a non-enveloped 27 nm, heat, acid- and ether-resistant RNA virus in the hepatovirus genus of the Picornaviridae family[5]. Its virion contains four capsid polypeptides, designated as VP1 to VP4, which are cleaved post-translationally from the polyprotein product of approximately 7500 nucleotide genome[6]. It has single serotype with six genotypes. Three HAV genotypes, I, II and III, divided into subtypes A and B, infect humans. Genotype I is prevalent in Europe and North and South America, and genotype III is endemic in Asia[7].

HAV is considered to be endemic in India. According to National Centre for Disease Control, India, HAV is responsible for about 10-30 per cent of acute hepatitis cases in individuals with acute liver failure in India[8]. The Indian population has shown an upward shift in the average age at the first HAV infection, among the socio-economically developed population resulting in pockets of susceptible populations[8]. Outbreaks of HAV have been reported, mainly affecting young adults from different parts of the country, e.g., in Delhi, Kerala and Shimla[9],[10],[11]. Genotypes I and III of HAV are the predominant strains circulating in India[8]. The present hospital-based study was aimed to know the seropositivity and predominant circulating strain of HAV in Uttar Pradesh (UP), north India.


   Material & Methods Top


Consecutively, all cases with clinical presentation of acute viral hepatitis (AVH) (WHO case definition)[12], referred to the Virology Laboratory, department of Microbiology, King George's Medical University, Lucknow, UP, India, during June 2016-May 2017, were enrolled in this observational study. The protocol was approved by the Institutional Ethics Committee and written informed consent was obtained from each participant. Patients of all age groups and both sexes were included. From each patient, blood sample (5 ml) was collected. Blood samples were centrifuged; serum was collected and tested for anti-HAV immunoglobulin M (IgM) using ELISA kit (DIA Pro Diagnostic Bioprobes Srl., Italy). The remaining sample was stored at −70°C. All anti-HAV IgM positive samples were subjected to HAV real-time reverse transcription-polymerase chain reaction (RT-PCR) assay using a protocol described by Costafreda et al[13]. For molecular profiling, HAV RNA-positive serum samples were subjected to conventional semi-nested RT-PCR targeting 518 base pair (bp) fragment encompassing the VP1 region which was further subjected to sequencing using a protocol described by Tallo et al[14]. As our study was focussed on phylogenetic analysis of HAV only, the other faeco-orally transmitted viruses were not tested.

All the amplified products were purified and sequenced using BigDye Terminator Cycle-Sequencing Kit (Applied Biosystems, USA) on ABI 3130 genetic Analyzer (Applied Biosystems, USA). Nucleotide sequences were edited and subjected to GenBank using Basic Local Alignment Search Tool (BLAST) programme (www.ncbi.nlm.nih.gov/BLAST) for comparing with all the available similar sequences. Further, phylogenetic analysis was carried out, and tree was constructed by maximum likelihood method using MEGA7 software with the neighbour-joining method from a Kimura 2-parameter distance matrix[15]. Genotype was determined using reference sequences belonging to different HAV genotypes.


   Results & Discussion Top


A total of 1615 AVH cases were enrolled over a one-year period. The mean age of the participants was 24.31±17.02 yr (range 0-83 yr) and male:female ratio was 1.3:1. Anti-HAV IgM antibodies seropositivity was 7.9 (128/1615) per cent [Table 1]. The mean age of seropositive cases was 9.3±9.4 yr. About 41 per cent of seropositive patients were in their childhood or early adolescent age group. Of the 128 seropositive samples, HAV RNA was detected in 59 (46.09%) samples. The IgM HAV antibodies percentage positivity in AVH cases referred from Sarawasti (22.2%), Unnao (17%) and Gorakhpur (12.5%) districts was high. The seasonal distribution of enrolled cases showed that AVH occurred throughout the year, though both the number of AVH cases and the HAV IgM seropositive cases increased during monsoon period between June and August [Figure 1]. Of the 59 HAV RNA-positive samples, only 10 high viraemia samples could be amplified for VP1 gene by conventional PCR. [Figure 2] shows phylogenetic tree of 10 representative HAV strains. The HAV strains with genotype IIIA were circulating in this region showing 92-100 per cent of nucleotide identity in a stretch of 408 bp. Estimates of genetic diversity were conducted on MEGA7[15] and the analysis among 10 strains showed a total of 36 variable sites among 408 bp. The NCBI BLAST analysis showed 2-4 per cent (8-15 bp) variation from its closest match of Indian (accession no. FJ360733.1) and Norwegian strains (accession no. AJ299464.3). There was no nonsense mutation observed in the sequenced region. The three sequences ID HAV-2, 3 and 5 from Saraswati and Lucknow districts were minimally deviated without any significant difference. The NCBI accession numbers of the submitted HAV sequences were MH929445-MH929454.
Table 1: Age- and gender-wise distribution of the suspected and confirmed hepatitis A virus (HAV) cases

Click here to view
Figure 1: Seasonal distribution of the total sample tested and confirmed hepatitis A virus case.

Click here to view
Figure 2: Molecular phylogenetic analysis of 10 representative hepatitis A virus (HAV) strains by maximum likelihood method. Each strain is labelled by GeneBank accession number followed by country. The strains from this study are marked with (▴). The respective genotypes are mentioned with accession number and country. The evolutionary distances were computed using the Maximum Composite Likelihood method and the tree was drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA7.

Click here to view


An earlier study from our centre in 2013 observed high HAV seroprevalence of 26.9 per cent[16]. The HAV positivity has declined over the time, and this may be due to the continuous effort by the government in improving sanitation and hygiene; however, the need for continuous surveillance can be emphasized looking at the changing pattern. High HAV seropositivity in the early age group depicted HAV endemicity in UP, India. Other studies from Delhi[17], Madhya Pradesh[18] and Chandigarh[19] reported >90 per cent seropositivity in children <15 yr.

HAV RNA positivity among seropositive cases was found to be 46.09 per cent in our study. A similar finding of HAV RNA positivity (47.7%) was reported from Chandigarh in a hospital-based study on 1334 AVH participants[19]. The molecular detection of HAV RNA ranged from 39.7 per cent in a hospital-based study to 63.2 per cent in outbreak investigations[11],[17],[19].

Peaks in total AVH cases admitted and IgM HAV seropositivity occurred during the monsoon season (June-August). This pattern of upsurge of HAV cases in monsoon has been reported in earlier studies[4] and suggests a possibility related to contamination of drinking water during periods of heavy rain. Since the study was done on referred samples, it could not represent true picture as far as district-wise positivity was concerned. More studies on larger sample sizes may be conducted. Moreover, HAV was not detected in several districts; this may be because of less number/adult samples.

Molecular epidemiology of HAV is important to understand the strains circulating in various geographical regions and tracing the source of contamination in an outbreak situation[20]. On molecular profiling genotype IIIA was found to be the prevalent circulating genotype in UP. Genotype III has been reported as the predominant genotype (70%) followed by genotype IA (30%) from Delhi, north India[17]. Co-circulation and co-infections with subgenotypes IIIA and IB have been reported from Pune, western India[21]. HAV genotype IIIA has been reported as an aetiological agent of various other waterborne outbreaks from northern, southern and western India[8]. The genetic diversity in VP1 gene of this region (2-4%) suggests diversity in circulating genotype IIIA in India and requires further analysis of other gene targets. This study of the mutational analysis of HAV RNA from different parts of the country would determine the characteristics and source of infection and also provide information on trends and transmission pattern.

In conclusion, HAV infection was found common in children in the region. Genotype IIIA was found to be circulating genotype. The mutations at VP1 region warrant further analysis.

Financial support & sponsorship: The financial support received from the Indian Council of Medical Research, New Delhi (Grant 83rd ECM IIA/P9) is acknowledged.

Conflict of Interest: None.



 
   References Top

1.
Previsani N, Lavanchy D. Hepatitis A (WHO/CDS/CSR/EDC/2000.7). World Health Organization; 2000.  Back to cited text no. 1
    
2.
Hollinger FB, Emerson SU. Hepatitis A virus. In: Knipe D, Howley P, editors. Fields virology. 5th ed. Philadelphia, USA: Lippincott Williams Wilkins, 2007. p. 911-47.  Back to cited text no. 2
    
3.
Strickland GT, El-Kamary S. Viral hepatitis. In: McGill A, Ryan E, Hill D, Solomon T, editors. Hunter's tropical medicine and emerging infectious diseases. 9th ed. New York: Saunders Elsevier, 2013. p. 290-305.  Back to cited text no. 3
    
4.
Wasley A, Grytdal S, Gallagher K; Centers for Disease Control and Prevention (CDC). Surveillance for acute viral hepatitis – United States, 2006. MMWR Surveill Summ 2008; 57 : 1-24.  Back to cited text no. 4
    
5.
Marvil P, Knowles NJ, Mockett AP, Britton P, Brown TD, Cavanagh D. Avian encephalomyelitis virus is a picornavirus and is most closely related to hepatitis A virus. J Gen Virol 1999; 80 (Pt 3) : 653-62.  Back to cited text no. 5
    
6.
Costa-Mattioli M, Di Napoli A, Ferré V, Billaudel S, Perez-Bercoff R, Cristina J. Genetic variability of hepatitis A virus. J Gen Virol 2003; 84 : 3191-201.  Back to cited text no. 6
    
7.
Robertson BH, Jansen RW, Khanna B, Totsuka A, Nainan OV, Siegl G, et al. Genetic relatedness of hepatitis A virus strains recovered from different geographical regions. J Gen Virol 1992; 73 (Pt 6) : 1365-77.  Back to cited text no. 7
    
8.
National Health Mission. National Action Plan Combating Viral Hepatitis in India. New Delhi: Ministry of Health & Family Welfare, Government of India; 2019.  Back to cited text no. 8
    
9.
Mathur P, Arora NK. Epidemiological transition of hepatitis A in India: Issues for vaccination in developing countries. Indian J Med Res 2008; 128 : 699-704.  Back to cited text no. 9
    
10.
Arankalle VA, Sarada Devi KL, Lole KS, Shenoy KT, Verma V, Haneephabi M. Molecular characterization of hepatitis A virus from a large outbreak from Kerala, India. Indian J Med Res 2006; 123 : 760-9.  Back to cited text no. 10
    
11.
Chobe LP, Arankalle VA. Investigation of a hepatitis A outbreak from Shimla Himachal Pradesh. Indian J Med Res 2009; 130 : 179-84.  Back to cited text no. 11
    
12.
World Health Organization. WHO-recommended surveillance standard of acute viral hepatitis. Available from: https://www.who.int/immunization/monitoring_surveillance/burden/vpd/surveillance_type/passive/hepatitis_standards/en/, accessed on December 18, 2018.  Back to cited text no. 12
    
13.
Costafreda MI, Bosch A, Pintó RM. Development, evaluation, and standardization of a real-time TaqMan reverse transcription-PCR assay for quantification of hepatitis A virus in clinical and shellfish samples. Appl Environ Microbiol 2006; 72 : 3846-55.  Back to cited text no. 13
    
14.
Tallo T, Norder H, Tefanova V, Ott K, Ustina V, Prukk T, et al. Sequential changes in hepatitis A virus genotype distribution in Estonia during 1994 to 2001. J Med Virol 2003; 70 : 187-93.  Back to cited text no. 14
    
15.
Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 2016; 33 : 1870-4.  Back to cited text no. 15
    
16.
Jain P, Prakash S, Gupta S, Singh KP, Shrivastava S, Singh DD, et al. Prevalence of hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus and hepatitis E virus as causes of acute viral hepatitis in North India: A hospital based study. Indian J Med Microbiol 2013; 31 : 261-5.  Back to cited text no. 16
    
17.
Hussain Z, Das BC, Husain SA, Murthy NS, Kar P. Increasing trend of acute hepatitis A in North India: Need for identification of high-risk population for vaccination. J Gastroenterol Hepatol 2006; 21 : 689-93.  Back to cited text no. 17
    
18.
Barde PV, Shukla MK, Pathak R, Kori BK, Bharti PK. Circulation of hepatitis A genotype IIIA virus in paediatric patients in central India. Indian J Med Res 2014; 139 : 940-4.  Back to cited text no. 18
    
19.
Singh MP, Majumdar M, Thapa BR, Gupta PK, Khurana J, Budhathoki B, et al. Molecular characterization of hepatitis A virus strains in a tertiary care health set up in North Western India. Indian J Med Res 2015; 141 : 213-20.  Back to cited text no. 19
    
20.
Chironna M, Lopalco P, Prato R, Germinario C, Barbuti S, Quarto M, et al. Outbreak of infection with hepatitis A virus (HAV) associated with a foodhandler and confirmed by sequence analysis reveals a new HAV genotype IB variant. J Clin Microbiol 2004; 42 : 2825-8.  Back to cited text no. 20
    
21.
Chadha MS, Chitambar SD, Shaikh NJ, Arankalle VA. Exposure of Indian children to hepatitis A virus & vaccination age. Indian J Med Res 1999; 109 : 11-5.  Back to cited text no. 21
    


    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1]



 

Top
 
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
    Abstract
    Material & M...
    Results & Di...
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed266    
    Printed1    
    Emailed0    
    PDF Downloaded136    
    Comments [Add]    

Recommend this journal