Journal of Medical Virology 87:1218–1224 (2015)
Molecular Epidemiology of Hepatitis A Virus Infection in Northeast India Moumita Bose,1 Sujoy Bose,1 Anjan Saikia,2 Subhash Medhi,3 and Manab Deka3* 1
Biotechnology, Gauhati University, Guwahati, India N.F. Railway, Central Hospital, Guwahati, India 3 Biological Science, Gauhati University, Guwahati, India 2
The present study was undertaken to screen the molecular epidemiology of Hepatitis A virus (HAV) in Northeast India (NEI) who are ethnically distinct, tribal dominated and of lower socioeconomic status with almost no information available from NEI on these aspects. Briefly, 3 ml blood was collected from 324 random liver disease cases with jaundice, receiving care at Central Hospital, N.F. Railway, Guwahati, Assam with informed consent. The patients detected with HAV-IgM positive status were included and were stratified as acute viral hepatitis (AVH) and fulminant hepatitis (FHF) based on clinical profile. Viral RNA was isolated and HAV-RNA was detected by Real-time PCR using primers for the VP3–VP1 region. HAV genotyping was studied by PCR-direct sequencingphylogenetic analysis approach using the VP1/ 2A region of HAV isolates. Statistical analysis was performed using SPSS13.0 software. A total of 69 cases were HAV infected with two HBV co-infected cases (n ¼ 69 þ 2 ¼ 71), 62 cases and two co-infected cases were AVH and others were FHF cases. HAV infection was predominant in especially in the young and adult age group. HAV-RNA was detected in 28 cases, out of which 19 cases could be genotyped (12 AVH, 7 FHF); which showed the prevalence of genotype IIIA or IA only. Although HAV genotype IIIA was the major genotype in both the AVH (10/12, 83.33%) and FHF (5/7, 71.43%) group, but the difference in distribution of genotypes in AVH and FHF cases was statistically non-significant (P ¼ 0.550). HAV genotype IIIA is associated with the majority of HAV infected cases and severity in NEI. J. Med. Virol. 87:1218– 1224, 2015. # 2015 Wiley Periodicals, Inc. KEY WORDS:
hepatitis A virus; molecular epidemiology; genotype; liver disease; Northeast India
C 2015 WILEY PERIODICALS, INC.
INTRODUCTION Viral hepatitis is a global health problem that affects hundreds of millions of children and adults. Hepatitis A is an acute, usually self-limiting disease of the liver caused by hepatitis A virus (HAV), which is a small, unenveloped RNA virus within the genus Hepatovirus, a member of the family Picornaviridae. The incidence of hepatitis A is closely related to socio-economic conditions, and sero-epidemiological studies show that prevalence of anti-hepatitis A antibodies varies from 15% to close to 100% in different parts of the world [WHO, 2010]. An estimated 1.5 million clinical cases of hepatitis A occur worldwide each year. The disease is endemic in many lowincome countries where food and water hygiene may be of a low standard. Northeast India has an ethnically distinct population which is mainly tribal dominated compared to other parts of India, and has a diverse culture, rituals, socio-economic conditions; and most importantly poor hygiene which predispose the native population to various diseases of different pathology including hepatitis infection. Clinically Hepatitis A is presented as an acute infection with generalized symptoms accompanied by jaundice and is more common in children. Unlike hepatitis B and C, hepatitis A infection does not cause chronic liver disease and is rarely fatal, but it can cause debilitating symptoms and fulminant hepatitis (acute liver failure) which is observed more in nonimmune adolescents and adults, and is associated with high mortality [Mathiesen et al., 1980; Evangelos et al., 1989; Yotsuyanagi et al., 1996; Chadha et al., 1999; Schwarz and Balistreri, 2002; Acharya et al., 2003]. The incubation period of hepatitis A is usually Correspondence to: Manab Deka, HOD & Professor, Department of Biological Science, GUIST, Gauhati University, Guwahati781014, Assam, India. E-mail: [email protected] Accepted 24 January 2015
DOI 10.1002/jmv.24168 Published online 16 April 2015 in Wiley Online Library (wileyonlinelibrary.com).
Hepatitis A Virus Infection in Northeast India
14–28 days. Symptoms of hepatitis A range from mild to severe, and can include fever, malaise, loss of appetite, diarrhea, nausea, abdominal discomfort, dark-colored urine and jaundice. HAV genome is of positive polarity, i.e., viral RNA can directly serve as messenger RNA [Fujiwara et al., 2003]. The large open reading frame present in HAV genome can be divided into three (P1–P3) functional regions. The P1 region encodes capsid polypeptides VP1–VP3 and the putative VP4. [Kanda et al., 2003]. The nucleotide sequence comparison based on the VP1/2A region of the genome is used to define seven different genotypes [Robertson et al., 1992]. The seven genotypes are differentiated into four human clusters (I–III and VII) and three simian strains (IV–VI). The seven genotypes recovered from human and non-human primates differ from each other at approximately 15 to 25% of base position in the VP1/2A region. The nucleotide diversity within sub-genotypes of HAV (IA, IB, IIIA, and IIIB) is less than 7.5% [Robertson et al., 1992]. Most of the human strains cluster in genotype I. Sub-genotype IIIA had been reported to be the major HAV genotype in India [Hussain et al., 2005]. However, recent report contradicts earlier findings that revealed subgenotype IB in western India from samples of sewage treatment plant [Vaidya et al., 2002]. Even though there is limited data available on viral hepatitis and liver disease spectrum from Northeast India [Deka et al., 2010] and almost no information on the molecular epidemiology of HAV infection, the present work tries to address. MATERIALS AND METHODS Patient Enrolment, Demographical, Biochemical, and Serological Profile Random cases of patients suffering from liver disease with clinical presented jaundice (n ¼ 324) receiving care at Central Hospital, N.F. Railway, Guwahati, Assam were enrolled in the study with informed consent. About 3 ml of blood was collected from each patient with their informed consent with the help of a registered practitioner. The plasma or serum was separated and stored at 20 ˚C. Initial diagnosis of the patients was made on the basis of biochemical test such as liver function test, and serological markers for liver diseases, i.e., HAV-IgM, anti-HCV, HEV-IgM, HBs-Ag and the patients were stratified accordingly. The patients who were diagnosed with HAV-IgM positive status were clinically co-related and stratified under two groups: HAV-related acute viral hepatitis. The clinical onset of an acute viral hepatitis A is defined as the beginning of early symptoms including fever, general malaise, fatigue, nausea, vomiting, anorexia and right upper quadrant discomfort. It is mainly characterized by the onset of jaundice and positive serological test for IgM anti-HAV.
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HAV-related fulminant hepatitis. Fulminant hepatitis A patients were categorized to have clinical syndrome developed as a result of severe impairment of hepatic functions or massive necrosis of hepatocytes in the absence of pre-existing liver disease. Fulminant hepatic failure patients were diagnosed by the presence of encephalopathy within 4 weeks of onset of illness with a prothrombin time of less than 40% that of the standardized value. Viral RNA Extraction Viral RNA extraction was done from serum samples obtained from HAV-IgM positive cases by using the Qiagen viral RNA extraction kit following the manufacturer’s protocol. cDNA was prepared from the extracted RNA using the cDNA synthesis kit (Applied Biosystems, Life Technologies, Singapore), and stored at 20˚C till further use. Viral RNA Detection Detection of HAV-RNA in the HAV-IgM positive samples was done by Real-time PCR using primers specific for the VP3–VP1 region of HAV genome (F: 50 -CCACA CAAGTTGGGGATGAT-30 ; R: 50 TGCTTGCACTCCTGAAACAT-30 ), and the cDNA prepared from the isolated viral RNA of IgM-Anti-HAV positive cases as templates. The amplification conditions comprised of initial denaturation at 94˚C for 5 mins, followed by 35 cycles of 94˚C for 15 sec, 55˚C for 15 sec and 72˚C for 15 sec followed by a single final extension cycle of 72˚C for 7 min. HAV Genotyping HAV genotyping was performed by PCR-direct sequencing-phylogenetic analysis approach using the VP1/2A region of the HAV isolates. The initial PCR was performed with the outer primers F: 50 TTGTCTGTCACAGAACAATCAG-30 and R: 50 -AGTCACACCTCTCCAGGAAAACTT-30 which amplified a product of 361 bp; and after suitable dilution if needed the second PCR amplification was done using the primers F: 50 -TCCCAGAGCTCCATTGAA-30 and R: 50 -AGGAGGTGGAAGCACTTCATTTGA-30 which finally yielded a final product of 234 bp, which was subjected to direct sequencing analysis followed by phylogenetic analysis using the MEGA4.0 software; and the statistical evaluation for distribution of HAV genotype in acute viral hepatitis vis-a`-vis fulminant hepatic failure cases was performed by using the SPSS13.0 software. RESULTS Hepatitis A Virus Infected Cases The diagnosis of HAV infection was screened on the basis of IgM Anti-HAV positivity and was categorized into acute viral hepatitis and fulminant cases based on clinical details and status as discussed J. Med. Virol. DOI 10.1002/jmv
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earlier. Overall, 69 cases and two HBV co-infected cases (N ¼ 69 þ 2 ¼ 71) were found to be HAV infected. Among all the cases, 62 cases along with 2 HBV co-infected cases were clinically diagnosed as acute viral hepatitis with mean SGOT and SGPT levels of 228 148 IU/ml and 323 166 IU/ml, respectively. Rest of the seven cases were categorized as fulminant hepatitis with encephalopathy. Two fulminant cases and one acute viral hepatitis cases were associated with acute liver failure. The mean SGOT and SGPT levels in fulminant hepatic failure cases were 526 328 IU/ml and 741 416 IU/ml, respectively. The details of the demographical and biochemical profile are tabulated in Table I. HAV infection was predominant in the younger age group between the age group of 5–20 years as shown in Table II. Hepatitis A Virus Infected Cases Detection for HAV viral mRNA was performed by Real-time PCR using primers specific for the VP3– VP1 region. Out of the 71 cases, HAV mRNA could be detected in only 28 cases, which may be due to the fact that viremia terminates shortly after hepatitis develops (Fig. 1). HAV Genotyping Since literature suggests that the VP1/2A region is more conserved than the VP3–VP1 region of HAV and thus better for genotyping, we performed the HAV genotyping for the cases enrolled in our study using PCR-direct sequencing-phylogenetic analysis approach using the VP1/2A region of the HAV isolates. The second round semi-nested PCR product of 234 bp of NE Indian isolates was gel extracted and subjected to direct sequencing followed by alignmentbased comparative analysis and phylogenetic analysis using standard Genbank based sequences of HAV isolated and reported from different corners of the world and India representing genotype IA, IB, II, VI, IIIA, and IIIB (Figs. 2–4). The analysis data show that the HAV isolates from NE India belong to either genotype IIIA or IA only. Among the 28 cases which was positive for HAV mRNA, we could amplify, sequence, and genotype for 19 cases only. This included for the 7 fulminant hepatic failure cases and 12 acute viral hepatitis cases. In HAV cases where genotyping was possible, HAV genotype IIIA was the major genotype in both the acute viral hepatitis (10/ 12, 83.33%) and fulminant hepatic failure (5/7, 71.43%) group. The only other genotype found in our studied cohort was HAV genotype IA.
TABLE II. Age- and Gender-Based Distribution of HAVIgM Positive Cases HAV þ HBV
HAV Patients age group 5–10 years 11–20 years 21–30 years 31–40 years 41–50 years 50–60 years >60 years
Male
Female
21 [72.4] 5 [50] 2 [33.3] 4 [36.4] 9 [81.8] 4 [100] 0 [0.0]
8 [27.6] 5 [50] 4 [66.7] 6 [54.5] 1 [9.0] 0 [0.0] 0 [0.0]
Male 0 0 0 1 1 0 0
[0.0] [0.0] [0.0] [9.0] [9.0] [0.0] [0.0]
Female 0 0 0 0 0 0 0
[0.0] [0.0] [0.0] [0.0] [0.0] [0.0] [0.0]
Data represented as number [percentage%].
Although our results showed predominance of HAV genotype IIIA in both acute viral hepatitis and fulminant hepatic failure cases (P ¼ 0.046), the difference in distribution of genotypes in acute viral hepatitis and fulminant hepatic failure cases was statistically non-significant (P ¼ 0.550). All the three patients who suffered from acute liver failure were infected with HAV genotype IIIA, and therefore it can be inferred that genotype IIIA is the predominant genotype in NE India and is associated with liver disease severity. Phylogenetic analysis also revealed that NE Indian isolates belonging to HAV genotype IIIA have close similarity with genotype IIIA reported earlier from India as well as some similarity with the Korean and Mozambique IIIA strain. DISCUSSION The highest prevalence of fecal-oral infection occurs in regions where low standards of sanitation promote virus transmission [Ceyhan et al., 2008]. In most industrialized nations, where hepatitis A is no longer considered a childhood disease, infections with HAV are increasingly contracted by adults [Mathur and Arora, 2008]. Present data on HAV epidemiology is available primarily from either Northern India [Acharya et al., 2003; Hussain et al., 2005], Western [Gadgil et al., 2008; Chadha et al., 2009], or Southern India [John et al., 2009; Rath et al., 2011], with limited data from Northeast India [Deka et al., 2010]. In our study, most of the infected individuals were children and young people. Our data also supports earlier global data that majority of the HAV related cases are from the pediatric age group [Hollinger and Ticehurst, 1990; Hussain et al., 2006]. But a good fraction of patients are of young and adult age group as reflected by a mean age group of 23 16 years;
TABLE I. Demographical and Biochemical Profile of the HAV-IgM Positive Cases Cases
No.
Male: female
Mean age
Mean SGOT (IU/ml)
Mean SGPT (IU/ml)
Acute viral hepatitis Fulminant hepatic failure
62 7
40:22 5:2
22 16 29 13
228 148 526 328
323 166 741 416
J. Med. Virol. DOI 10.1002/jmv
Hepatitis A Virus Infection in Northeast India
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Fig. 1. Representative amplification and raw data plot showing the real-time based detection of HAV mRNA in the studied HAV-IgM positive cases.
and the prevalence in higher age groups is also supported by recent reports from India [Hussain et al., 2005]. Documented literature from India also shows that the age of acquiring HAV infection is shifting from early childhood to adolescence and young adulthood. Such epidemiological shift leads to an increased incidence of symptomatic HAV infection, including heightened risk of liver failure [Mathur and Arora, 2008].
Fig. 2. Representative agarose gel showing post PCR amplification of 234 bp after second round of semi-nested PCR of the VP1/2A region.
Seven HAV genotypes have been defined. Genetic heterogeneity of hepatitis A has been revealed by sequencing different genome regions, including VP3 carboxyl terminus, the VP1 amino terminus and the VP1/2A junction [Cohen et al., 1987; Arauz-ruiz et al., 2001; Costa-mattioli et al., 2002] (Fig. 2). The VP3 C-terminal region is relatively conserved, the VP1 amino acid terminus presents an intermediate variability, while VP1/2A junction is more variable and is used to distinguish one strain from another [Costa-mattioli et al., 2002]. Global genotype distribution shows that genotypes I and III comprise the vast majority of human strains. Sub-genotype IA comprises the majority of the human strains studied and constitutes major virus population in North and South America, China, Japan, Russia and Thailand. The sub-genotype IB contains strains from Jordan, North Africa, Australia, Europe, Japan and South America. Most of the remaining human HAV strains segregate into genotype III that is further divided into two sub-genotypes, IIIA, and IIIB [Cohen et al., 1987, Jansen et al., 1990, Robertson et al., 1992]. The sub-genotype IIIA have been subsequently identified in specimens collected from humans with hepatitis A J. Med. Virol. DOI 10.1002/jmv
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Fig. 3. A: Multiple sequence alignment of representative HAV isolates MouHAV1, 4, 9, 14, 18, 24, 53, 37, and 39 from NE India compared to sequences available from different global pockets at the nucleotide levels using MEGA4.0 software showing the variations amongst the isolates. B: Sequence alignment and phylogenetic analysis using the VP1/2A region of HAV isolates isolated from patients from NE India (MouHAV1, 4, 9, 14, 18, 24, 53, 37, and 39) showing the presence of genotype IIIA and IA only.
Fig. 4. Phylogenetic tree of the compared standard global isolates and representative isolates from NE India using the VP1/2A region showing that, the representative NE Indian isolates belong to HAV genotype IIIA and have close similarity with genotype IIIA reported earlier from India as well as some similarity with the Korean and Mozambique IIIA strain. The evolutionary history was inferred using the neighbour joining method. The bootstrap consensus tree was inferred from 500 replicates using the MEGA4.0 software.
J. Med. Virol. DOI 10.1002/jmv
Hepatitis A Virus Infection in Northeast India
in India, Sri Lanka, Nepal, Malaysia, Sweden, and the United States [Khanna et al., 1992; Hussain et al., 2005]. The IIIB sub-genotype is responsible for cases of HAV infection in Japan and Denmark. Majority of the HAV isolates reported from India were found to be of genotype IIIA [Robertson et al., 1992; Hussain et al., 2005]. Similar to the majority of the studies from Indian population, in our study cohort the majority of the HAV related cases belong to genotype IIIA, and a very few belong to genotype IA, which is distinctly different from what has been reported from western India [Vaidya et al., 2002]. Although, HAV causes an acute self-limited illness but rarely, it is also involved in a severe course such as fulminant hepatitis. The role of HAV genotype in disease severity is equivocal. Although few studies highlights the HAV genotype in disease severity [Fujiwara et al., 2002; Kanda et al., 2010], while several other studies reported no association between genotype or nucleotide changes with disease severity [Fujiwara et al., 2003; Rezende et al., 2003; Normann et al., 2004; Tjon et al., 2006]. Recently, a comparative analysis of disease severity between genotype 1A and IIIA revealed that the patients with genotype IIIA were older and had high alanine aminotransferase (ALT) levels, prolonged prothrombin times and lower serum albumin level [Yoon et al., 2011]. Another study indicated that co-infection of two subgenotypes (1A and 1B) in a patient with acute hepatitis accounted for the prolonged and severe course of illness [Coppola et al., 2007]. Moreover, in our study genotype IIIA was found to be associated with both acute and fulminant HAV infection. Therefore, additional studies are needed to define the precise role of viral genotypes in the severity of hepatitis A. To conclude, HAV infection is a risk factor for liver disease susceptibility and severity in northeast India especially in the young and adult age group. Molecular epidemiology-based analysis shows that genotype IIIA is the major circulating genotype in northeast India, and it shows similarity with the isolates reported from other parts of India, Korea, and Mozambique. Vaccination rates for HAV are low in clinical practice in northeast India; and overall improvement in living standards, environmental hygiene, and identification of NE India as susceptible pocket requiring planned vaccination program needs attention and implementation. REFERENCES Acharya SK, Batra Y, Bhatkal B, Ojha B, Kaur K, Hazari S, Saraya A, Panda SK. 2003. Seroepidemiology of hepatitis a virus infection among school children in Delhi and north Indian patients with chronic liver disease: Implications for HAV vaccination. J Gastroenterol Hepatol 18:822–827. Arauz-ruiz P, Sundqvist L, Garcia Z, Taylor L, Visona K, Norder H, Magnius LO. 2001. Presumed common source outbreaks of hepatitis A in an endemic area confirmed by limited sequencing within the VP1 region. J Med Virol 65:449–456. Ceyhan M, Yildirim I, Kurt N, Uysal G, Dikici B, Ecevit C, Aydogan A, Koc A, Yasa O, Koseoglu M, Onal K, Hacimustafaoglu M,
1223 Celebi S. 2008. Differences in hepatitis A seroprevalence among geographical regions in Turkey: A need for regional vaccination recommendations. J Viral Hepat 15:69–72. Chadha MS, Chtambar SD, Shaikh NJ, Arankalle VA. 1999. Exposure of Indian children to hepatitis A virus and vaccination age. Ind J Med Res 9:11–15. Chadha MS, Lole KS, Bora MH, Arankalle VA. 2009. Outbreaks of hepatitis A among children in western India. Trans R Soc Trop Med Hyg 103:911–916. Cohen JI, Ticehurst JR, Purcell RH, Buckler-white A, Baroudy BM. 1987. Complete nucleotide sequence of wild type hepatitis A virus: Comparison with different strains of hepatitis A virus and other picornaviruses. J Virol 61:50–59. Coppola N, Genovese D, Pisaturo M, Taffon S, Argentini C, Pasquale G, Sagnelli C, Piccinino F, Rapicetta M, Sagnelli E. 2007. Acute hepatitis with severe cholestasis and prolonged clinical course due to hepatitis A virus Ia and Ib coinfection. Clin Infect Dis 44:e73–e77. Costa-mattioli M, Cristina J, Romero H, Perez-Bercof R, Casane D, Colina R, Garcia L, Vega I, Glikman G, Romanowsky V, Castello A, Nicand E, Gassin M, Billaudel S, Ferre´ V. 2002. Molecular evolution of hepatitis A virus: A new classification based on the complete VP1 protein. J Virol 76:9516–9525. Deka M, Bose M, Baruah B, Bose PD, Medhi S, Bose S, Saikia A, Kar P. 2010. Oct Role of CYP2E1 gene polymorphisms association with hepatitis risk in Northeast India World J Gastroenterol 14:4800–4808. Evangelos A, Akriviadis EA, Redker AG. 1989. Fulminant hepatitis A in intravenous drug users with chronic liver disease. Ann Intern Med 110:838. Fujiwara K, Yokosuka O, Ehata T, Saisho H, Saotome N, Suzuki K, Okita K, Kiyosawa K, Omata M. 2002. Association between severity of type A hepatitis and nucleotide variations in the 50 non-translated region of hepatitis A virus RNA: strains from fulminant hepatitis have fewer nucleotide substitutions. Gut 5:82–888. Fujiwara K, Yokosuka O, Imazeki F, Saisho H, Saotome N, Suzuki K, Okita K, Tanaka E, Omata M. 2003. Analysis of the genotype determining region of hepatitis A viral RNA in relation to disease severities. Hepatol Res 25:124–134. Gadgil PS, Fadnis RS, Joshi MS, Rao PS, Chitambar SD. 2008. Mar. Seroepidemiology of hepatitis A in voluntary blood donors from Pune, western India (2002 and 2004–2005) Epidemiol Infect 136:406–409. Hollinger FB, Ticehurst JR. 1990. Hepatitis A virus. In: Fields BN, Knipe DM, Chanock RM, Hirsch MS, Melnick JL, Monath TP, Roizman B, editors. Virology. 2nd edition. New York: Raven Press, pp 631–667. Hussain Z, Das BC, Husain SA, Asim M, Chattopadhyay S, Malik A, Poovorawan Y, Theamboonlers A, Kar P. 2005. May. Hepatitis A viral genotypes and clinical relevance: Clinical and molecular characterization of hepatitis A virus isolates from northern India Hepatol Res 32:16–24. Hussain Z, Das BC, Husain SA, Polipalli SK, Ahmed T, Begum N, Medhi S, Verghese A, Raish M, Theamboonlers A, Poovorawan Y, Kar P. 2006. Virological course of hepatitis A virus as determined by real time RT-PCR: Correlation with biochemical, immunological and genotypic profiles. World J Gastroenterol 12:4683–4688. Jansen RW, Siegl G, Lemon SM. 1990. Molecular epidemiology of human hepatitis A virus defined by an antigen-capture polymerase chain reaction method. Proc Natl Acad Sci U S A 87:2867– 2871. John A, Chatni S, Narayanan VA, Balakrishnan V, Nair P. 2009. Seroprevalence of hepatitis A virus in patients with chronic liver disease from Kerala: Impact on vaccination policy J Indian Med Assoc 107:859–861. Kanda T, Jeong SH, Imazeki F, Fujiwara K, Yokosuka O. 2010. Analysis of 50 non-translated region of hepatitis A viral RNA genotype I from South Korea: Comparison with disease severities. PLoS ONE 5:e15139. Kanda T, Yokosuka O, Kato N, Imazeki F, Fujiwara K, Kawai S, Saisho H, Omata M. 2003. Hepatitis A virus VP3 may activate serum response element associated transcription. Scand J Gastroenterol 38:307–313. Khanna B, Spelbring JE, Innis BL, Robertson BH. 1992. Characterization of a genetic variant of human hepatitis A virus. J Med Virol 36:118–124.
J. Med. Virol. DOI 10.1002/jmv
1224 Mathiesen LR, Skinoj P, Nielsen JO, Parcell RH, Wong D, Ranek L. 1980. Hepatitis type A, B and non A non B in fulminant hepatitis. Gut 21:72–77. Mathur P, Arora NK. 2008. Epidemiological transition of hepatitis A in India: Issues for vaccination in developing countries Indian J Med Res 128:699–704. Normann A, Jung C, Vallbracht A, Flehmig B. 2004. Time course of hepatitis A viremia and viral load in the blood of human hepatitis A patients. J Med Virol 72:10–16. Rath CP, Akki A, Patil SV, Kalyanshettar SS. 2011. Jan. Seroprevalence of hepatitis A virus antibody in Bijapur, Karnataka Indian Pediatr 48:71–73. Rezende G, Roque-afonso AM, Samuel D, Gigou M, Nicand E, Ferre V, Dussaix E, Bismuth H, Fe´ray C. 2003. Viral and clinical factors associated with the fulminant course of hepatitis A infection. Hepatology 38:613–618. Robertson BH, Jansen RW, Khanna B, Totsuka A, Nainan OV, Siegl G, Widell A, Margolis HS, Isomura S, Ito K, Ishizo T, Moritsugu Y, Lemon SM. 1992. Genetic relatedness of hepatitis A virus strains recovered from different geographical regions. J Gen Virol 73:1365–1377.
J. Med. Virol. DOI 10.1002/jmv
Bose et al. Schwarz KS, Balistreri WF. 2002. Viral hepatitis. J Pediatr.Gastroenterol Nutr 35:29–32. Tjon GM, Coutinho RA, van den Hoek A, Esman S, Wijkmans CJ, Hoebe CJ, Wolters B, Swaan C, Geskus RB, Dukers N, Bruisten SM. 2006. High and persistent excretion of hepatitis A virus in immunocompetent patients. J Med Virol 78:1398–1405. Vaidya SR, Chitambar SD, Arankalle VA. 2002. Polymerase chain reaction base prevalence of hepatitis A, hepatitis E endemic area. J Hepatol 37:131–136. World Health Organization 2010. The global prevalence of hepatitis A virus infection and susceptibility: A systematic review. WHO/ IVB/10.01 (available at: http://whqlibdoc.who.int/hq/2010/ WHO_IVB_10.01_eng.pdf). Yoon YK, Yeon JE, Kim JH, Park D, Sohn JW, Chun BC, Kim M. 2011. Comparative analysis of disease severity between genotypes IA and IIIA of hepatitis A virus. J Med Virol 83:1308– 1314. Yotsuyanagi H, Koike K, Yasuda K, Moriya K, Shintani Y, Fujie H, Kurokawa K, Iino S. 1996. Prolonged fecal excretion of hepatitis A virus in adult patients with hepatitis A as determined by polymerase chain reaction. Hepatology 24:10–13.
Molecular Epidemiology of Hepatitis A Virus Infection in Northeast India Moumita Bose,1 Sujoy Bose,1 Anjan Saikia,2 Subhash Medhi,3 and Manab Deka3* 1
Biotechnology, Gauhati University, Guwahati, India N.F. Railway, Central Hospital, Guwahati, India 3 Biological Science, Gauhati University, Guwahati, India 2
The present study was undertaken to screen the molecular epidemiology of Hepatitis A virus (HAV) in Northeast India (NEI) who are ethnically distinct, tribal dominated and of lower socioeconomic status with almost no information available from NEI on these aspects. Briefly, 3 ml blood was collected from 324 random liver disease cases with jaundice, receiving care at Central Hospital, N.F. Railway, Guwahati, Assam with informed consent. The patients detected with HAV-IgM positive status were included and were stratified as acute viral hepatitis (AVH) and fulminant hepatitis (FHF) based on clinical profile. Viral RNA was isolated and HAV-RNA was detected by Real-time PCR using primers for the VP3–VP1 region. HAV genotyping was studied by PCR-direct sequencingphylogenetic analysis approach using the VP1/ 2A region of HAV isolates. Statistical analysis was performed using SPSS13.0 software. A total of 69 cases were HAV infected with two HBV co-infected cases (n ¼ 69 þ 2 ¼ 71), 62 cases and two co-infected cases were AVH and others were FHF cases. HAV infection was predominant in especially in the young and adult age group. HAV-RNA was detected in 28 cases, out of which 19 cases could be genotyped (12 AVH, 7 FHF); which showed the prevalence of genotype IIIA or IA only. Although HAV genotype IIIA was the major genotype in both the AVH (10/12, 83.33%) and FHF (5/7, 71.43%) group, but the difference in distribution of genotypes in AVH and FHF cases was statistically non-significant (P ¼ 0.550). HAV genotype IIIA is associated with the majority of HAV infected cases and severity in NEI. J. Med. Virol. 87:1218– 1224, 2015. # 2015 Wiley Periodicals, Inc. KEY WORDS:
hepatitis A virus; molecular epidemiology; genotype; liver disease; Northeast India
C 2015 WILEY PERIODICALS, INC.
INTRODUCTION Viral hepatitis is a global health problem that affects hundreds of millions of children and adults. Hepatitis A is an acute, usually self-limiting disease of the liver caused by hepatitis A virus (HAV), which is a small, unenveloped RNA virus within the genus Hepatovirus, a member of the family Picornaviridae. The incidence of hepatitis A is closely related to socio-economic conditions, and sero-epidemiological studies show that prevalence of anti-hepatitis A antibodies varies from 15% to close to 100% in different parts of the world [WHO, 2010]. An estimated 1.5 million clinical cases of hepatitis A occur worldwide each year. The disease is endemic in many lowincome countries where food and water hygiene may be of a low standard. Northeast India has an ethnically distinct population which is mainly tribal dominated compared to other parts of India, and has a diverse culture, rituals, socio-economic conditions; and most importantly poor hygiene which predispose the native population to various diseases of different pathology including hepatitis infection. Clinically Hepatitis A is presented as an acute infection with generalized symptoms accompanied by jaundice and is more common in children. Unlike hepatitis B and C, hepatitis A infection does not cause chronic liver disease and is rarely fatal, but it can cause debilitating symptoms and fulminant hepatitis (acute liver failure) which is observed more in nonimmune adolescents and adults, and is associated with high mortality [Mathiesen et al., 1980; Evangelos et al., 1989; Yotsuyanagi et al., 1996; Chadha et al., 1999; Schwarz and Balistreri, 2002; Acharya et al., 2003]. The incubation period of hepatitis A is usually Correspondence to: Manab Deka, HOD & Professor, Department of Biological Science, GUIST, Gauhati University, Guwahati781014, Assam, India. E-mail: [email protected] Accepted 24 January 2015
DOI 10.1002/jmv.24168 Published online 16 April 2015 in Wiley Online Library (wileyonlinelibrary.com).
Hepatitis A Virus Infection in Northeast India
14–28 days. Symptoms of hepatitis A range from mild to severe, and can include fever, malaise, loss of appetite, diarrhea, nausea, abdominal discomfort, dark-colored urine and jaundice. HAV genome is of positive polarity, i.e., viral RNA can directly serve as messenger RNA [Fujiwara et al., 2003]. The large open reading frame present in HAV genome can be divided into three (P1–P3) functional regions. The P1 region encodes capsid polypeptides VP1–VP3 and the putative VP4. [Kanda et al., 2003]. The nucleotide sequence comparison based on the VP1/2A region of the genome is used to define seven different genotypes [Robertson et al., 1992]. The seven genotypes are differentiated into four human clusters (I–III and VII) and three simian strains (IV–VI). The seven genotypes recovered from human and non-human primates differ from each other at approximately 15 to 25% of base position in the VP1/2A region. The nucleotide diversity within sub-genotypes of HAV (IA, IB, IIIA, and IIIB) is less than 7.5% [Robertson et al., 1992]. Most of the human strains cluster in genotype I. Sub-genotype IIIA had been reported to be the major HAV genotype in India [Hussain et al., 2005]. However, recent report contradicts earlier findings that revealed subgenotype IB in western India from samples of sewage treatment plant [Vaidya et al., 2002]. Even though there is limited data available on viral hepatitis and liver disease spectrum from Northeast India [Deka et al., 2010] and almost no information on the molecular epidemiology of HAV infection, the present work tries to address. MATERIALS AND METHODS Patient Enrolment, Demographical, Biochemical, and Serological Profile Random cases of patients suffering from liver disease with clinical presented jaundice (n ¼ 324) receiving care at Central Hospital, N.F. Railway, Guwahati, Assam were enrolled in the study with informed consent. About 3 ml of blood was collected from each patient with their informed consent with the help of a registered practitioner. The plasma or serum was separated and stored at 20 ˚C. Initial diagnosis of the patients was made on the basis of biochemical test such as liver function test, and serological markers for liver diseases, i.e., HAV-IgM, anti-HCV, HEV-IgM, HBs-Ag and the patients were stratified accordingly. The patients who were diagnosed with HAV-IgM positive status were clinically co-related and stratified under two groups: HAV-related acute viral hepatitis. The clinical onset of an acute viral hepatitis A is defined as the beginning of early symptoms including fever, general malaise, fatigue, nausea, vomiting, anorexia and right upper quadrant discomfort. It is mainly characterized by the onset of jaundice and positive serological test for IgM anti-HAV.
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HAV-related fulminant hepatitis. Fulminant hepatitis A patients were categorized to have clinical syndrome developed as a result of severe impairment of hepatic functions or massive necrosis of hepatocytes in the absence of pre-existing liver disease. Fulminant hepatic failure patients were diagnosed by the presence of encephalopathy within 4 weeks of onset of illness with a prothrombin time of less than 40% that of the standardized value. Viral RNA Extraction Viral RNA extraction was done from serum samples obtained from HAV-IgM positive cases by using the Qiagen viral RNA extraction kit following the manufacturer’s protocol. cDNA was prepared from the extracted RNA using the cDNA synthesis kit (Applied Biosystems, Life Technologies, Singapore), and stored at 20˚C till further use. Viral RNA Detection Detection of HAV-RNA in the HAV-IgM positive samples was done by Real-time PCR using primers specific for the VP3–VP1 region of HAV genome (F: 50 -CCACA CAAGTTGGGGATGAT-30 ; R: 50 TGCTTGCACTCCTGAAACAT-30 ), and the cDNA prepared from the isolated viral RNA of IgM-Anti-HAV positive cases as templates. The amplification conditions comprised of initial denaturation at 94˚C for 5 mins, followed by 35 cycles of 94˚C for 15 sec, 55˚C for 15 sec and 72˚C for 15 sec followed by a single final extension cycle of 72˚C for 7 min. HAV Genotyping HAV genotyping was performed by PCR-direct sequencing-phylogenetic analysis approach using the VP1/2A region of the HAV isolates. The initial PCR was performed with the outer primers F: 50 TTGTCTGTCACAGAACAATCAG-30 and R: 50 -AGTCACACCTCTCCAGGAAAACTT-30 which amplified a product of 361 bp; and after suitable dilution if needed the second PCR amplification was done using the primers F: 50 -TCCCAGAGCTCCATTGAA-30 and R: 50 -AGGAGGTGGAAGCACTTCATTTGA-30 which finally yielded a final product of 234 bp, which was subjected to direct sequencing analysis followed by phylogenetic analysis using the MEGA4.0 software; and the statistical evaluation for distribution of HAV genotype in acute viral hepatitis vis-a`-vis fulminant hepatic failure cases was performed by using the SPSS13.0 software. RESULTS Hepatitis A Virus Infected Cases The diagnosis of HAV infection was screened on the basis of IgM Anti-HAV positivity and was categorized into acute viral hepatitis and fulminant cases based on clinical details and status as discussed J. Med. Virol. DOI 10.1002/jmv
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earlier. Overall, 69 cases and two HBV co-infected cases (N ¼ 69 þ 2 ¼ 71) were found to be HAV infected. Among all the cases, 62 cases along with 2 HBV co-infected cases were clinically diagnosed as acute viral hepatitis with mean SGOT and SGPT levels of 228 148 IU/ml and 323 166 IU/ml, respectively. Rest of the seven cases were categorized as fulminant hepatitis with encephalopathy. Two fulminant cases and one acute viral hepatitis cases were associated with acute liver failure. The mean SGOT and SGPT levels in fulminant hepatic failure cases were 526 328 IU/ml and 741 416 IU/ml, respectively. The details of the demographical and biochemical profile are tabulated in Table I. HAV infection was predominant in the younger age group between the age group of 5–20 years as shown in Table II. Hepatitis A Virus Infected Cases Detection for HAV viral mRNA was performed by Real-time PCR using primers specific for the VP3– VP1 region. Out of the 71 cases, HAV mRNA could be detected in only 28 cases, which may be due to the fact that viremia terminates shortly after hepatitis develops (Fig. 1). HAV Genotyping Since literature suggests that the VP1/2A region is more conserved than the VP3–VP1 region of HAV and thus better for genotyping, we performed the HAV genotyping for the cases enrolled in our study using PCR-direct sequencing-phylogenetic analysis approach using the VP1/2A region of the HAV isolates. The second round semi-nested PCR product of 234 bp of NE Indian isolates was gel extracted and subjected to direct sequencing followed by alignmentbased comparative analysis and phylogenetic analysis using standard Genbank based sequences of HAV isolated and reported from different corners of the world and India representing genotype IA, IB, II, VI, IIIA, and IIIB (Figs. 2–4). The analysis data show that the HAV isolates from NE India belong to either genotype IIIA or IA only. Among the 28 cases which was positive for HAV mRNA, we could amplify, sequence, and genotype for 19 cases only. This included for the 7 fulminant hepatic failure cases and 12 acute viral hepatitis cases. In HAV cases where genotyping was possible, HAV genotype IIIA was the major genotype in both the acute viral hepatitis (10/ 12, 83.33%) and fulminant hepatic failure (5/7, 71.43%) group. The only other genotype found in our studied cohort was HAV genotype IA.
TABLE II. Age- and Gender-Based Distribution of HAVIgM Positive Cases HAV þ HBV
HAV Patients age group 5–10 years 11–20 years 21–30 years 31–40 years 41–50 years 50–60 years >60 years
Male
Female
21 [72.4] 5 [50] 2 [33.3] 4 [36.4] 9 [81.8] 4 [100] 0 [0.0]
8 [27.6] 5 [50] 4 [66.7] 6 [54.5] 1 [9.0] 0 [0.0] 0 [0.0]
Male 0 0 0 1 1 0 0
[0.0] [0.0] [0.0] [9.0] [9.0] [0.0] [0.0]
Female 0 0 0 0 0 0 0
[0.0] [0.0] [0.0] [0.0] [0.0] [0.0] [0.0]
Data represented as number [percentage%].
Although our results showed predominance of HAV genotype IIIA in both acute viral hepatitis and fulminant hepatic failure cases (P ¼ 0.046), the difference in distribution of genotypes in acute viral hepatitis and fulminant hepatic failure cases was statistically non-significant (P ¼ 0.550). All the three patients who suffered from acute liver failure were infected with HAV genotype IIIA, and therefore it can be inferred that genotype IIIA is the predominant genotype in NE India and is associated with liver disease severity. Phylogenetic analysis also revealed that NE Indian isolates belonging to HAV genotype IIIA have close similarity with genotype IIIA reported earlier from India as well as some similarity with the Korean and Mozambique IIIA strain. DISCUSSION The highest prevalence of fecal-oral infection occurs in regions where low standards of sanitation promote virus transmission [Ceyhan et al., 2008]. In most industrialized nations, where hepatitis A is no longer considered a childhood disease, infections with HAV are increasingly contracted by adults [Mathur and Arora, 2008]. Present data on HAV epidemiology is available primarily from either Northern India [Acharya et al., 2003; Hussain et al., 2005], Western [Gadgil et al., 2008; Chadha et al., 2009], or Southern India [John et al., 2009; Rath et al., 2011], with limited data from Northeast India [Deka et al., 2010]. In our study, most of the infected individuals were children and young people. Our data also supports earlier global data that majority of the HAV related cases are from the pediatric age group [Hollinger and Ticehurst, 1990; Hussain et al., 2006]. But a good fraction of patients are of young and adult age group as reflected by a mean age group of 23 16 years;
TABLE I. Demographical and Biochemical Profile of the HAV-IgM Positive Cases Cases
No.
Male: female
Mean age
Mean SGOT (IU/ml)
Mean SGPT (IU/ml)
Acute viral hepatitis Fulminant hepatic failure
62 7
40:22 5:2
22 16 29 13
228 148 526 328
323 166 741 416
J. Med. Virol. DOI 10.1002/jmv
Hepatitis A Virus Infection in Northeast India
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Fig. 1. Representative amplification and raw data plot showing the real-time based detection of HAV mRNA in the studied HAV-IgM positive cases.
and the prevalence in higher age groups is also supported by recent reports from India [Hussain et al., 2005]. Documented literature from India also shows that the age of acquiring HAV infection is shifting from early childhood to adolescence and young adulthood. Such epidemiological shift leads to an increased incidence of symptomatic HAV infection, including heightened risk of liver failure [Mathur and Arora, 2008].
Fig. 2. Representative agarose gel showing post PCR amplification of 234 bp after second round of semi-nested PCR of the VP1/2A region.
Seven HAV genotypes have been defined. Genetic heterogeneity of hepatitis A has been revealed by sequencing different genome regions, including VP3 carboxyl terminus, the VP1 amino terminus and the VP1/2A junction [Cohen et al., 1987; Arauz-ruiz et al., 2001; Costa-mattioli et al., 2002] (Fig. 2). The VP3 C-terminal region is relatively conserved, the VP1 amino acid terminus presents an intermediate variability, while VP1/2A junction is more variable and is used to distinguish one strain from another [Costa-mattioli et al., 2002]. Global genotype distribution shows that genotypes I and III comprise the vast majority of human strains. Sub-genotype IA comprises the majority of the human strains studied and constitutes major virus population in North and South America, China, Japan, Russia and Thailand. The sub-genotype IB contains strains from Jordan, North Africa, Australia, Europe, Japan and South America. Most of the remaining human HAV strains segregate into genotype III that is further divided into two sub-genotypes, IIIA, and IIIB [Cohen et al., 1987, Jansen et al., 1990, Robertson et al., 1992]. The sub-genotype IIIA have been subsequently identified in specimens collected from humans with hepatitis A J. Med. Virol. DOI 10.1002/jmv
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Fig. 3. A: Multiple sequence alignment of representative HAV isolates MouHAV1, 4, 9, 14, 18, 24, 53, 37, and 39 from NE India compared to sequences available from different global pockets at the nucleotide levels using MEGA4.0 software showing the variations amongst the isolates. B: Sequence alignment and phylogenetic analysis using the VP1/2A region of HAV isolates isolated from patients from NE India (MouHAV1, 4, 9, 14, 18, 24, 53, 37, and 39) showing the presence of genotype IIIA and IA only.
Fig. 4. Phylogenetic tree of the compared standard global isolates and representative isolates from NE India using the VP1/2A region showing that, the representative NE Indian isolates belong to HAV genotype IIIA and have close similarity with genotype IIIA reported earlier from India as well as some similarity with the Korean and Mozambique IIIA strain. The evolutionary history was inferred using the neighbour joining method. The bootstrap consensus tree was inferred from 500 replicates using the MEGA4.0 software.
J. Med. Virol. DOI 10.1002/jmv
Hepatitis A Virus Infection in Northeast India
in India, Sri Lanka, Nepal, Malaysia, Sweden, and the United States [Khanna et al., 1992; Hussain et al., 2005]. The IIIB sub-genotype is responsible for cases of HAV infection in Japan and Denmark. Majority of the HAV isolates reported from India were found to be of genotype IIIA [Robertson et al., 1992; Hussain et al., 2005]. Similar to the majority of the studies from Indian population, in our study cohort the majority of the HAV related cases belong to genotype IIIA, and a very few belong to genotype IA, which is distinctly different from what has been reported from western India [Vaidya et al., 2002]. Although, HAV causes an acute self-limited illness but rarely, it is also involved in a severe course such as fulminant hepatitis. The role of HAV genotype in disease severity is equivocal. Although few studies highlights the HAV genotype in disease severity [Fujiwara et al., 2002; Kanda et al., 2010], while several other studies reported no association between genotype or nucleotide changes with disease severity [Fujiwara et al., 2003; Rezende et al., 2003; Normann et al., 2004; Tjon et al., 2006]. Recently, a comparative analysis of disease severity between genotype 1A and IIIA revealed that the patients with genotype IIIA were older and had high alanine aminotransferase (ALT) levels, prolonged prothrombin times and lower serum albumin level [Yoon et al., 2011]. Another study indicated that co-infection of two subgenotypes (1A and 1B) in a patient with acute hepatitis accounted for the prolonged and severe course of illness [Coppola et al., 2007]. Moreover, in our study genotype IIIA was found to be associated with both acute and fulminant HAV infection. Therefore, additional studies are needed to define the precise role of viral genotypes in the severity of hepatitis A. To conclude, HAV infection is a risk factor for liver disease susceptibility and severity in northeast India especially in the young and adult age group. Molecular epidemiology-based analysis shows that genotype IIIA is the major circulating genotype in northeast India, and it shows similarity with the isolates reported from other parts of India, Korea, and Mozambique. Vaccination rates for HAV are low in clinical practice in northeast India; and overall improvement in living standards, environmental hygiene, and identification of NE India as susceptible pocket requiring planned vaccination program needs attention and implementation. REFERENCES Acharya SK, Batra Y, Bhatkal B, Ojha B, Kaur K, Hazari S, Saraya A, Panda SK. 2003. Seroepidemiology of hepatitis a virus infection among school children in Delhi and north Indian patients with chronic liver disease: Implications for HAV vaccination. J Gastroenterol Hepatol 18:822–827. Arauz-ruiz P, Sundqvist L, Garcia Z, Taylor L, Visona K, Norder H, Magnius LO. 2001. Presumed common source outbreaks of hepatitis A in an endemic area confirmed by limited sequencing within the VP1 region. J Med Virol 65:449–456. Ceyhan M, Yildirim I, Kurt N, Uysal G, Dikici B, Ecevit C, Aydogan A, Koc A, Yasa O, Koseoglu M, Onal K, Hacimustafaoglu M,
1223 Celebi S. 2008. Differences in hepatitis A seroprevalence among geographical regions in Turkey: A need for regional vaccination recommendations. J Viral Hepat 15:69–72. Chadha MS, Chtambar SD, Shaikh NJ, Arankalle VA. 1999. Exposure of Indian children to hepatitis A virus and vaccination age. Ind J Med Res 9:11–15. Chadha MS, Lole KS, Bora MH, Arankalle VA. 2009. Outbreaks of hepatitis A among children in western India. Trans R Soc Trop Med Hyg 103:911–916. Cohen JI, Ticehurst JR, Purcell RH, Buckler-white A, Baroudy BM. 1987. Complete nucleotide sequence of wild type hepatitis A virus: Comparison with different strains of hepatitis A virus and other picornaviruses. J Virol 61:50–59. Coppola N, Genovese D, Pisaturo M, Taffon S, Argentini C, Pasquale G, Sagnelli C, Piccinino F, Rapicetta M, Sagnelli E. 2007. Acute hepatitis with severe cholestasis and prolonged clinical course due to hepatitis A virus Ia and Ib coinfection. Clin Infect Dis 44:e73–e77. Costa-mattioli M, Cristina J, Romero H, Perez-Bercof R, Casane D, Colina R, Garcia L, Vega I, Glikman G, Romanowsky V, Castello A, Nicand E, Gassin M, Billaudel S, Ferre´ V. 2002. Molecular evolution of hepatitis A virus: A new classification based on the complete VP1 protein. J Virol 76:9516–9525. Deka M, Bose M, Baruah B, Bose PD, Medhi S, Bose S, Saikia A, Kar P. 2010. Oct Role of CYP2E1 gene polymorphisms association with hepatitis risk in Northeast India World J Gastroenterol 14:4800–4808. Evangelos A, Akriviadis EA, Redker AG. 1989. Fulminant hepatitis A in intravenous drug users with chronic liver disease. Ann Intern Med 110:838. Fujiwara K, Yokosuka O, Ehata T, Saisho H, Saotome N, Suzuki K, Okita K, Kiyosawa K, Omata M. 2002. Association between severity of type A hepatitis and nucleotide variations in the 50 non-translated region of hepatitis A virus RNA: strains from fulminant hepatitis have fewer nucleotide substitutions. Gut 5:82–888. Fujiwara K, Yokosuka O, Imazeki F, Saisho H, Saotome N, Suzuki K, Okita K, Tanaka E, Omata M. 2003. Analysis of the genotype determining region of hepatitis A viral RNA in relation to disease severities. Hepatol Res 25:124–134. Gadgil PS, Fadnis RS, Joshi MS, Rao PS, Chitambar SD. 2008. Mar. Seroepidemiology of hepatitis A in voluntary blood donors from Pune, western India (2002 and 2004–2005) Epidemiol Infect 136:406–409. Hollinger FB, Ticehurst JR. 1990. Hepatitis A virus. In: Fields BN, Knipe DM, Chanock RM, Hirsch MS, Melnick JL, Monath TP, Roizman B, editors. Virology. 2nd edition. New York: Raven Press, pp 631–667. Hussain Z, Das BC, Husain SA, Asim M, Chattopadhyay S, Malik A, Poovorawan Y, Theamboonlers A, Kar P. 2005. May. Hepatitis A viral genotypes and clinical relevance: Clinical and molecular characterization of hepatitis A virus isolates from northern India Hepatol Res 32:16–24. Hussain Z, Das BC, Husain SA, Polipalli SK, Ahmed T, Begum N, Medhi S, Verghese A, Raish M, Theamboonlers A, Poovorawan Y, Kar P. 2006. Virological course of hepatitis A virus as determined by real time RT-PCR: Correlation with biochemical, immunological and genotypic profiles. World J Gastroenterol 12:4683–4688. Jansen RW, Siegl G, Lemon SM. 1990. Molecular epidemiology of human hepatitis A virus defined by an antigen-capture polymerase chain reaction method. Proc Natl Acad Sci U S A 87:2867– 2871. John A, Chatni S, Narayanan VA, Balakrishnan V, Nair P. 2009. Seroprevalence of hepatitis A virus in patients with chronic liver disease from Kerala: Impact on vaccination policy J Indian Med Assoc 107:859–861. Kanda T, Jeong SH, Imazeki F, Fujiwara K, Yokosuka O. 2010. Analysis of 50 non-translated region of hepatitis A viral RNA genotype I from South Korea: Comparison with disease severities. PLoS ONE 5:e15139. Kanda T, Yokosuka O, Kato N, Imazeki F, Fujiwara K, Kawai S, Saisho H, Omata M. 2003. Hepatitis A virus VP3 may activate serum response element associated transcription. Scand J Gastroenterol 38:307–313. Khanna B, Spelbring JE, Innis BL, Robertson BH. 1992. Characterization of a genetic variant of human hepatitis A virus. J Med Virol 36:118–124.
J. Med. Virol. DOI 10.1002/jmv
1224 Mathiesen LR, Skinoj P, Nielsen JO, Parcell RH, Wong D, Ranek L. 1980. Hepatitis type A, B and non A non B in fulminant hepatitis. Gut 21:72–77. Mathur P, Arora NK. 2008. Epidemiological transition of hepatitis A in India: Issues for vaccination in developing countries Indian J Med Res 128:699–704. Normann A, Jung C, Vallbracht A, Flehmig B. 2004. Time course of hepatitis A viremia and viral load in the blood of human hepatitis A patients. J Med Virol 72:10–16. Rath CP, Akki A, Patil SV, Kalyanshettar SS. 2011. Jan. Seroprevalence of hepatitis A virus antibody in Bijapur, Karnataka Indian Pediatr 48:71–73. Rezende G, Roque-afonso AM, Samuel D, Gigou M, Nicand E, Ferre V, Dussaix E, Bismuth H, Fe´ray C. 2003. Viral and clinical factors associated with the fulminant course of hepatitis A infection. Hepatology 38:613–618. Robertson BH, Jansen RW, Khanna B, Totsuka A, Nainan OV, Siegl G, Widell A, Margolis HS, Isomura S, Ito K, Ishizo T, Moritsugu Y, Lemon SM. 1992. Genetic relatedness of hepatitis A virus strains recovered from different geographical regions. J Gen Virol 73:1365–1377.
J. Med. Virol. DOI 10.1002/jmv
Bose et al. Schwarz KS, Balistreri WF. 2002. Viral hepatitis. J Pediatr.Gastroenterol Nutr 35:29–32. Tjon GM, Coutinho RA, van den Hoek A, Esman S, Wijkmans CJ, Hoebe CJ, Wolters B, Swaan C, Geskus RB, Dukers N, Bruisten SM. 2006. High and persistent excretion of hepatitis A virus in immunocompetent patients. J Med Virol 78:1398–1405. Vaidya SR, Chitambar SD, Arankalle VA. 2002. Polymerase chain reaction base prevalence of hepatitis A, hepatitis E endemic area. J Hepatol 37:131–136. World Health Organization 2010. The global prevalence of hepatitis A virus infection and susceptibility: A systematic review. WHO/ IVB/10.01 (available at: http://whqlibdoc.who.int/hq/2010/ WHO_IVB_10.01_eng.pdf). Yoon YK, Yeon JE, Kim JH, Park D, Sohn JW, Chun BC, Kim M. 2011. Comparative analysis of disease severity between genotypes IA and IIIA of hepatitis A virus. J Med Virol 83:1308– 1314. Yotsuyanagi H, Koike K, Yasuda K, Moriya K, Shintani Y, Fujie H, Kurokawa K, Iino S. 1996. Prolonged fecal excretion of hepatitis A virus in adult patients with hepatitis A as determined by polymerase chain reaction. Hepatology 24:10–13.
