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Molecular characterization of hepatitis A virus strains in a tertiary care health set up in north western India.

Singh MP, Majumdar M, Thapa BR, Gupta PK, Khurana J, Budhathoki B, Ratho RK - Indian J. Med. Res. (2015)

Bottom Line: Sequencing of 15 representative strains was carried out and the circulating genotype was found to be III A.The cumulative substitution in AVH strains Vs ALF strains as compared to GBM, Indian and prototype strain in the 200-500 region of 5' NTR was comparable.Our results showed hepatitis A still a disease of children with III A as a circulating genotype in this region.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Postgraduate Institute of Medical Education & Research, Chandigarh, India.

ABSTRACT

Background & objectives: Hepatitis A virus usually causes acute viral hepatitis (AVH) in the paediatric age group with a recent shift in age distribution and disease manifestations like acute liver failure (ALF). This has been attributed to mutations in 5'non-translated region (5'NTR) which affects the viral multiplication. The present study was aimed to carry out the molecular detection and phylogenetic analysis of hepatitis A virus strains circulating in north western India.

Methods: Serum samples from in patients and those attending out patient department of Pediatric Gastroenterology in a tertiary care hospital in north India during 2007-2011 with clinically suspected AVH were tested for anti-hepatitis A virus (HAV) IgM antibodies. Acute phase serum samples were subjected to nested PCR targeting the 5'NTR region followed by sequencing of the representative strains.

Results: A total of 1334 samples were tested, 290 (21.7%) were positive for anti-HAV IgM antibody. Of these, 78 serum samples (< 7 days old) were subjected to PCR and 47.4% (37/78) samples showed the presence of HAV RNA. Children < 15 yr of age accounted for majority (94%) of cases with highest seropositivity during rainy season. Sequencing of 15 representative strains was carried out and the circulating genotype was found to be III A. The nucleotide sequences showed high homology among the strains with a variation ranging from 0.1-1 per cent over the years. An important substitution of G to A at 324 position was shown by both AVH and ALF strains. The cumulative substitution in AVH strains Vs ALF strains as compared to GBM, Indian and prototype strain in the 200-500 region of 5' NTR was comparable.

Interpretation & conclusion: Our results showed hepatitis A still a disease of children with III A as a circulating genotype in this region. The mutations at 5'NTR region warrant further analysis as these affect the structure of internal ribosomal entry site which is important for viral replication.

No MeSH data available.


Related in: MedlinePlus

Disease severity and nucleotide substitutions in HAV 5’NTR region (200-500 nt) of standard strains were compared with HAV sequences found circulating in north India reported in the study (a) Wild type strain of HAV genotype IA: GBM/WT (X75215), (b) Whole genome reported from Pune, India genotype IIIA: FJ360734.1, (c) Prototype strain of genotype IIIA: PA21: M63026.1).
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Figure 3: Disease severity and nucleotide substitutions in HAV 5’NTR region (200-500 nt) of standard strains were compared with HAV sequences found circulating in north India reported in the study (a) Wild type strain of HAV genotype IA: GBM/WT (X75215), (b) Whole genome reported from Pune, India genotype IIIA: FJ360734.1, (c) Prototype strain of genotype IIIA: PA21: M63026.1).

Mentions: Substitutions between nucleotides 200 and 500 of the 5’NTR region: Interestingly all our strains showed an important substitution of G to A at nucleotide 324 position when compared with wild-type HAV GBM/WT RNA (X75215). As proposed by Fujiwara et al20, this nucleotide is positioned at the beginning of the stem structure of domain IV, and the change increases the free energy resulting in more stable structure. However, no difference was found in distribution of this substitution related to disease severity. On comparison of strains isolated from our region with wild-type HAV GBM/WT RNA (X75215), prototype strain for genotype IIIA (M63026.1) and complete genome sequence of an Indian strain reported from Pune, Maharashtra (FJ360734.1 IND-HAV-99F) nucleotide identities were found to range from 90 to 92 per cent, 97 to 98 per cent and 98 to 99 per cent, respectively. HAV GBM strain was recovered from a human faecal sample collected in the pre-icteric phase of infection in Germany and was named GBM wild type as was adapted for rapid growth and release in HFS (human lung fibroblast cells) and, FRhk-4 cells (Foetal Rhesus monkey kidney-derived cells)21. Genotype circulating in north west India was found to be IIIA. Therefore, the strains were next compared with PA21 which is the prototype strain of HAV genotype IIIA and was originally isolated from Panamanian owl monkey, but has subsequently been found associated with human cases of HAV from Nepal and Northern India during 1989 and 199012. Further, we tried comparing the strain reported in the present study from north west with an indigenous strain. Whole genome sequence of HAV genotype IIIA was available only from western India and, therefore, IND-HAV-99F was selected, which was isolated from stool sample of an icteric patient from western India in 199922. The overall nucleotide substitution in 200-500 nt region as compared to the above mentioned standard strains are represented in Fig. 3a, b and c. Further comparison of our strains with IND-HAV-99F revealed a 161A to G substitution in 5 of 15 (33.3%) cases, 207C to T in 8 of 15 (53.3%) cases and 559A to G in 3 of 15(20%) cases. On comparison with prototype strain of genotype IIIA (PA21) five substitutions were observed in domain III of IRES 105C to T in15 of 15 (100%) cases, 148 T to C in 6 of 15 (40%), 187C to T in 15 of 15(100%), 213G to A in 15 of 15 (100%), 266G to A in 15 of 15 (100%). In domain IV two substitution and one insertion were observed 490G to A in 11 of 15 (73.3%), 517T to C in 15 of 15 (100%) and at position of 431 insertion of G was observed in all the strains.


Molecular characterization of hepatitis A virus strains in a tertiary care health set up in north western India.

Singh MP, Majumdar M, Thapa BR, Gupta PK, Khurana J, Budhathoki B, Ratho RK - Indian J. Med. Res. (2015)

Disease severity and nucleotide substitutions in HAV 5’NTR region (200-500 nt) of standard strains were compared with HAV sequences found circulating in north India reported in the study (a) Wild type strain of HAV genotype IA: GBM/WT (X75215), (b) Whole genome reported from Pune, India genotype IIIA: FJ360734.1, (c) Prototype strain of genotype IIIA: PA21: M63026.1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4418158&req=5

Figure 3: Disease severity and nucleotide substitutions in HAV 5’NTR region (200-500 nt) of standard strains were compared with HAV sequences found circulating in north India reported in the study (a) Wild type strain of HAV genotype IA: GBM/WT (X75215), (b) Whole genome reported from Pune, India genotype IIIA: FJ360734.1, (c) Prototype strain of genotype IIIA: PA21: M63026.1).
Mentions: Substitutions between nucleotides 200 and 500 of the 5’NTR region: Interestingly all our strains showed an important substitution of G to A at nucleotide 324 position when compared with wild-type HAV GBM/WT RNA (X75215). As proposed by Fujiwara et al20, this nucleotide is positioned at the beginning of the stem structure of domain IV, and the change increases the free energy resulting in more stable structure. However, no difference was found in distribution of this substitution related to disease severity. On comparison of strains isolated from our region with wild-type HAV GBM/WT RNA (X75215), prototype strain for genotype IIIA (M63026.1) and complete genome sequence of an Indian strain reported from Pune, Maharashtra (FJ360734.1 IND-HAV-99F) nucleotide identities were found to range from 90 to 92 per cent, 97 to 98 per cent and 98 to 99 per cent, respectively. HAV GBM strain was recovered from a human faecal sample collected in the pre-icteric phase of infection in Germany and was named GBM wild type as was adapted for rapid growth and release in HFS (human lung fibroblast cells) and, FRhk-4 cells (Foetal Rhesus monkey kidney-derived cells)21. Genotype circulating in north west India was found to be IIIA. Therefore, the strains were next compared with PA21 which is the prototype strain of HAV genotype IIIA and was originally isolated from Panamanian owl monkey, but has subsequently been found associated with human cases of HAV from Nepal and Northern India during 1989 and 199012. Further, we tried comparing the strain reported in the present study from north west with an indigenous strain. Whole genome sequence of HAV genotype IIIA was available only from western India and, therefore, IND-HAV-99F was selected, which was isolated from stool sample of an icteric patient from western India in 199922. The overall nucleotide substitution in 200-500 nt region as compared to the above mentioned standard strains are represented in Fig. 3a, b and c. Further comparison of our strains with IND-HAV-99F revealed a 161A to G substitution in 5 of 15 (33.3%) cases, 207C to T in 8 of 15 (53.3%) cases and 559A to G in 3 of 15(20%) cases. On comparison with prototype strain of genotype IIIA (PA21) five substitutions were observed in domain III of IRES 105C to T in15 of 15 (100%) cases, 148 T to C in 6 of 15 (40%), 187C to T in 15 of 15(100%), 213G to A in 15 of 15 (100%), 266G to A in 15 of 15 (100%). In domain IV two substitution and one insertion were observed 490G to A in 11 of 15 (73.3%), 517T to C in 15 of 15 (100%) and at position of 431 insertion of G was observed in all the strains.

Bottom Line: Sequencing of 15 representative strains was carried out and the circulating genotype was found to be III A.The cumulative substitution in AVH strains Vs ALF strains as compared to GBM, Indian and prototype strain in the 200-500 region of 5' NTR was comparable.Our results showed hepatitis A still a disease of children with III A as a circulating genotype in this region.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Postgraduate Institute of Medical Education & Research, Chandigarh, India.

ABSTRACT

Background & objectives: Hepatitis A virus usually causes acute viral hepatitis (AVH) in the paediatric age group with a recent shift in age distribution and disease manifestations like acute liver failure (ALF). This has been attributed to mutations in 5'non-translated region (5'NTR) which affects the viral multiplication. The present study was aimed to carry out the molecular detection and phylogenetic analysis of hepatitis A virus strains circulating in north western India.

Methods: Serum samples from in patients and those attending out patient department of Pediatric Gastroenterology in a tertiary care hospital in north India during 2007-2011 with clinically suspected AVH were tested for anti-hepatitis A virus (HAV) IgM antibodies. Acute phase serum samples were subjected to nested PCR targeting the 5'NTR region followed by sequencing of the representative strains.

Results: A total of 1334 samples were tested, 290 (21.7%) were positive for anti-HAV IgM antibody. Of these, 78 serum samples (< 7 days old) were subjected to PCR and 47.4% (37/78) samples showed the presence of HAV RNA. Children < 15 yr of age accounted for majority (94%) of cases with highest seropositivity during rainy season. Sequencing of 15 representative strains was carried out and the circulating genotype was found to be III A. The nucleotide sequences showed high homology among the strains with a variation ranging from 0.1-1 per cent over the years. An important substitution of G to A at 324 position was shown by both AVH and ALF strains. The cumulative substitution in AVH strains Vs ALF strains as compared to GBM, Indian and prototype strain in the 200-500 region of 5' NTR was comparable.

Interpretation & conclusion: Our results showed hepatitis A still a disease of children with III A as a circulating genotype in this region. The mutations at 5'NTR region warrant further analysis as these affect the structure of internal ribosomal entry site which is important for viral replication.

No MeSH data available.


Related in: MedlinePlus