Community based case-control study of rotavirus gastroenteritis among young children during 2008-2010 reveals vast genetic diversity and increased prevalence of G9 strains in Kolkata

Abstract

Background: Group A Rotaviruses are a major etiologic agent of gastroenteritis in infants and young children (<5 years) worldwide. Although rotavirus vaccines have been successfully administered in many countries, in India the introduction of rotavirus vaccine in national immunization program was approved in 2014. Since high disease burden and large number of genetic variants have been reported from low income countries including India, monitoring of rotavirus was initiated prior to implementation of the vaccine in the region.

Methods: A total number of 3,582 stool samples were collected from an urban slum community in Kolkata, among which 1,568 samples were obtained from children of ≤ 5 years of age, with moderate to severe diarrhoea and 2,014 samples were collected from age-sex matched healthy neighbourhood controls. Rotavirus positive samples were typed by multiplex semi-nested PCR and nucleotide sequencing. Circulating strains were phylogenetically analyzed.

Results: Among 1,568 children with diarrhoea, 395 (25.2%), and among 2,014 asymptomatic children, 42 (2%) were rotavirus positive. G1P[8] was identified as the most common strain (32%) followed by G9P[8] (16.9%), G2P[4] (13.5%) and G9P[4] (10.75%). G12 strains with combinations of P[4], P[6] and P[8] comprised 11.9% of total positive strains. The rest (<10%) were rare and uncommon strains like G1P[4], G1P[6], G2P[8] and animal-like strains G4P[6], G6P[14] and G11P[25]. The 42 rotavirus positive samples from asymptomatic children revealed common genotypes like G1, G2 and G9.

Conclusion: This community based case-control study showed increased predominance of genotype G9 in Kolkata. It also confirmed co-circulation of a large number of genetic variants in the community. Asymptomatic rotavirus positive children though low in number can also be a source of dispersal of infection in the community. This study provides background information to the policy makers for implementation of rotavirus vaccines in this region.

Figure 1. Age distribution.

Age –wise distribution of rotavirus-positive Case and Control children (0–5 years) against total collected samples during January 2008 through December 2010.

Figure 2. Seasonality of rotavirus in Kolkata.

Seasonal distribution of the rotavirus positivity against total collected samples in Case and Control children in an urban slum community in Kolkata, India, during January 2008 through December 2010.

Figure 3. A and B. Phylogenetic trees of the G1 and G2 strains of Kolkata.

Phylogenetic trees constructed from the nucleotide sequences of VP7 genes of A. G1; B. G2 strains of Kolkata, isolated during January 2008 through December 2010, with other representative G1 and G2 strains respectively. A. One Kolkata G1 strain showed its relatedness to lineage II G1 strains and the rest G1 strains clustered with lineage I G1 strains. B. All Kolkata G2 strains clustered with lineage IV G2 strains. Scale bar, 0.05 substitutions per nucleotide. Bootstrap values less than 70% are not shown.

Figure 4. A and B. Phylogenetic trees of the G9 and G12 strains of Kolkata.

Phylogenetic trees constructed from the nucleotide sequences of VP7 genes of A. G9; B. G12 strains of Kolkata, isolated during January 2008 through December 2010, with other representative G9 and G12 strains respectively. A. All Kolkata G9 strains showed relatedness to lineage III G9 strains. (**Sub cluster of Japanese and Chinese rotavirus strains). B. All Kolkata G12 strains clustered with lineage III G12 strains. Scale bar, 0.05 substitutions per nucleotide. Bootstrap values less than 70% are not shown.

Figure 5. A and B. Phylogenetic trees of the G4 and P strains of Kolkata.

Phylogenetic trees constructed from the nucleotide sequences of VP7 genes of A. G4; and VP4 genes of B. P strains of Kolkata, isolated during January 2008 through December 2010, with other representative G4 and P strains respectively. All G4 strains indicated its genetic relatedness with the porcine or porcine-like human G4 strains. Scale bar, 0.05 substitutions per nucleotide. Bootstrap values less than 70% are not shown.

Figure 6. A and B. Phylogenetic trees of the P and P strains of Kolkata.

Phylogenetic trees constructed from the nucleotide sequences of VP4 genes of A. P; and B. P strains of Kolkata, isolated during January 2008 through December 2010, with other representative P and P strains respectively. Scale bar, 0.05 substitutions per nucleotide. Bootstrap values less than 70% are not shown.