Comprehensive full genome analysis of norovirus strains from eastern India, 2017-2021

Abstract

Background: Worldwide, noroviruses are the leading cause of acute gastroenteritis (AGE) in people of all age groups. In India, norovirus rates between 1.4 to 44.4% have been reported. Only a very few complete norovirus genome sequences from India have been reported.

Objective: To perform full genome sequencing of noroviruses circulating in India during 2017-2021, identify circulating genotypes, assess evolution including detection of recombination events.

Methodology: Forty-five archived norovirus-positive samples collected between October 2017 to July 2021 from patients with AGE from two hospitals in Kolkata, India were processed for full genome sequencing. Phylogenetic analysis, recombination breakpoint analysis and comprehensive mutation analysis were also performed.

Results: Full genome analysis of norovirus sequences revealed that strains belonging to genogroup (G)I were genotyped as GI.3[P13]. Among the different norovirus capsid-polymerase combinations, GII.3[P16], GII.4 Sydney[P16], GII.4 Sydney[P31], GII.13[P16], GII.16[P16] and GII.17 were identified. Phylogenetic analysis confirmed phylogenetic relatedness with previously reported norovirus strains and all viruses were analyzed by Simplot. GII[P16] viruses with multiple residue mutations within the non-structural region were detected among circulating GII.4 and GII.3 strains. Comprehensive mutation analysis and selection pressure analysis of GII[P16] viruses showed positive as well as negative selection sites. A GII.17 strain (NICED-BCH-11889) had an untypeable polymerase type, closely related to GII[P38].

Conclusion: This study highlights the circulation of diverse norovirus strains in eastern India. These findings are important for understanding norovirus epidemiology in India and may have implications for future vaccine development.

Fig. 1

Maximum likelihood phylogenetic analysis of full genome sequences obtained from circulating GII noroviruses in eastern India. GII.4[P16] strains are marked in maroon square, GII.3[P16] are marked in sky blue circle, GII.13[P16] are marked in orange diamond, GII.16[P16] are in blue triangle, GII.4[P31] are in green square and GII.17[P-ut] are shown in red triangle. Bootstrap values < 70% are not shown. (*Partial sequences of genomic regions of noroviruses were reported previously and available in the Genbank database)

Fig. 2

Maximum likelihood phylogenetic analysis of nearly complete genome sequences obtained from circulating GI noroviruses in eastern India. GI.3[P13] strains are marked in black circle. Bootstrap values < 70% are not shown

Fig. 3

Simplot analysis of eastern Indian recombinant noroviruses. a SimPlot analysis of GII.4 Sydney[P16] strains were performed using reference parental strains GII.4[P4]/KC013592.1 (green line), GII.16[P16]/AY772730.1 (blue line) and reference recombinant strain GII.4[P16]/LC175468.1 (red line) was used as reference strain. b Simplot analysis of GII.3[P16] strains were performed using reference parental strain GII.16[P16]/AY772730.1 (blue line), GII.3[P3]/LC122831.1 (green line) and reference recombinant strain GII.3[P16]/KY887597.1 (red line). c SimPlot analysis of GII.4 Sydney[P31] strains performed using GII.4[P4]/KC013592.1 (green line) as reference parental strains for GII.4 and GII.2[P31]/LC209439.1 (blue line) was used as reference parental strains for GII[P31] genotype. GII.4[P31]/LC122827.1 (red line) was used as similar recombinant reference strain. d Simplot analysis of GII.13[P16] strain was performed using reference parental strain GII.16[P16]/AY772730.1 (blue line), GII.13[P12]/KJ196276.1 (green line) and reference recombinant strain GII.13[P16]/MG892908.1 (red line). Recombination breakpoints are marked in vertical red line (dashed)

Fig. 4

Analysis of sub-genomic transcription start region at ORF1/ORF2 junction of different norovirus strains circulated in eastern India; (a) GII.3[P16], (b) GII.13[P16], (c) GII.4 Sydney[P16] and (d) GII.4 Sydney[P31]. Predicted RNA secondary structure with least ΔG are shown in the diagram. For simplicity only one representative strain is shown in the diagram and other strains showing similar structure are listed below the diagram. The predicted secondary structures are shown in antisense orientation, sub-genomic RNA initiation sites are indicated by an arrow and the start anticodons are indicated in black box

Fig. 5

Analysis of variation in amino acid sequence of antigenic epitope regions of VP1 capsid among GII.4 Sydney strains detected in Kolkata, 2017–2021

Fig. 6

Analysis of informative amino acid substitutions in non-structural proteins of GII[P16] genotype associated with circulating GII.4 and GII.3 noroviruses. Amino acid substitution in circulating GII[P16] strains were compared with reference GII[P16] strain (NC_039477.1). Amino acid substitution with more than 5% frequencies were marked by green triangle and complete substitution sites were marked by blue star. Pervasive positive selection sites were indicated in red asterisk. (Details of this analysis are attached as Additional file 3: Fig. S3a–f)

Fig. 7

Phylogenetic analysis VP1 capsid and polymerase genes of a rare GII.17 norovirus strain NICED-BCH-11889. The closest similar polymerase strain Beijing53931/GII.25[P38] (GQ856469.1) was marked in black circle and the study strain was indicated in black triangle

Fig. 8

Sequence similarity of GII.17 NICED-BCH-11889 strain. The horizontal axis represents the nucleotide positions and the vertical axis represents the sequence similarity compared to the GII.17 NICED-BCH-11889 (LC769700.1) strain. The relative position of different ORFs of norovirus and polymerase-capsid junction was shown above the similarity plot

Fig. 9

Amino acid sequence alignment of P domain from different GII.17 clusters. HBGA surface binding loops (A-, B-, P-, S-, T-, U- and N-) are heighted by different colours. The consecutive four amino acid deletions in the P- loop are marked by black arrows