Norovirus genotype IIb associated acute gastroenteritis in India

Journal of Clinical Virology 42 (2008) 429–432

Short communication

Norovirus genotype IIb associated acute gastroenteritis in India Preeti Chhabra, Shobha D. Chitambar ∗ Rotavirus Department, National Institute of Virology, 20-A, Dr. Ambedkar Road, Pune 411001, India Received 26 September 2007; received in revised form 11 February 2008; accepted 18 March 2008

Abstract Background: In India the role of noroviruses in causing gastroenteritis is not well defined. Objective: To determine the norovirus prevalence in sporadic cases of acute gastroenteritis in western India. Study design: A total of 236 fecal specimens consisting 192 specimens from hospitalized and 44 from OPD (outpatient department) children ≤5 years of age were tested for the presence of norovirus genogroup (G)I and GII by RT-PCR followed by sequencing. Multiple alignment and phylogenetic analysis were carried out to determine the norovirus genetic types. Results: Norovirus GII infection was detected in 11.9% (28/236) specimens. These included 24 of 192 (12.5%) specimens from hospitalized and 4 of 44 (9.0%) specimens from OPD cases. Nucleotide sequence based analysis of norovirus GII strains indicated circulation of GIIb strains at a significant level in hospitalized (12.5%) and OPD (75%) children. Conclusion: This study demonstrates the predominance of GII/4 (genogroup II/genotype 4) along with co-circulation of GII/1, GII/2, GII/3 and GIIb revealing the possibility of norovirus as the second most common cause of non-bacterial acute gastroenteritis after rotavirus in western India. It also documents for the first time occurrence of norovirus GIIb infections in India. © 2008 Elsevier B.V. All rights reserved. Keywords: Norovirus; Genotype IIb; Caliciviruses; Acute gastroenteritis

1. Introduction Acute gastroenteritis is a major public health problem worldwide reported with a mortality rate of 3.5–4.6 million per year (Synder and Merson, 1982). The disease can be caused by different pathogens including parasites, bacteria and viruses. Etiological viruses include mainly rotavirus group A, enteric adenovirus, astrovirus and human caliciviruses (noro and sapoviruses). Noroviruses are widely recognized as important causative agents of outbreaks of nonbacterial acute gastroenteritis however, sporadic infections are also reported (Buesa et al., 2002; Lindell et al., 2005). The disease is usually mild and self-limiting but the virus is highly infectious (Inouye et al., 2000). Noroviruses belong to family Caliciviridae and are singlestranded RNA viruses. The viral genome encodes three Open Reading Frames (ORFs)—ORF1, ORF2 and ORF3. Noroviruses have been classified genetically into five major groups genogroup I (GI) to genogroup V (GV). GI, GII and ∗

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GIV primarily infect humans; GIII and GV infect bovine and murine species, respectively. GI, GII and GIII have been subdivided in 8, 17 and 2 genetic clusters, respectively whereas GIV and GV have one cluster each (Zheng et al., 2006). Additionally, a fast evolving norovirus genetic type, assumed to play an important role in pediatric gastroenteritis, was detected frequently in European countries in 2000–2001 and was designated as GIIb/Hilversum (Buesa et al., 2002; Reuter et al., 2005; Lindell et al., 2005; Ramirez et al., 2006). This genotype is highly prone to recombination phenomenon (Reuter et al., 2005). Recombinant strains of GIIb have also been reported in Japan during 2003–2005 (Phan et al., 2006, 2007). This is the first report that documents the existence and circulation of GIIb norovirus variants in India.

2. Materials and methods 2.1. Specimens A total of 236 fecal specimens {192 from hospitalized and 44 from outpatient department (OPD) cases} were

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collected during December 2005–February 2007 from children, ≤5 years of age (median age 2.3 years), suffering from acute gastroenteritis in Pune, western India. All patients were examined for fever, number of episodes and duration of vomiting and diarrhea, extent of dehydration and treatment for the assessment of severity of disease (Ruuska and Vesikari, 1990). The samples were collected within 24 h of hospitalization with prior informed consent from parents/guardians and stored at −20 ◦ C until analyzed. 2.2. Detection of noroviruses by RT-PCR All specimens were tested for the presence of norovirus GI and GII by RT-PCR using primers from RNA-dependent RNA polymerase (RdRp) gene. The samples were also tested for presence of group A rotavirus by ELISA (DakoCytomation, UK). Briefly, thirty percent fecal suspensions were prepared in 0.01 M phosphate buffered saline (PBS) pH 7.2. Viral RNA was extracted using TRIzol® LS reagent (Invitrogen, USA) as per manufacturer’s protocol. RT-PCR was performed using primers specific for GI (GR12, SR48, SR50 and SR52) and GII (GR12 and SR46) (Girish et al., 2002; Ando et al., 1995) for amplification of 126 bp genomic fragment. To amplify a larger region (685 bp) from RdRp gene, the positive specimens were subjected to another RT-PCR with primers NV4611 and NV5296 specific for genogroup II (Yuen et al., 2001). The products were electrophoresed in 2% agarose gels containing ethidium bromide and visualized under UV transilluminator. 2.3. Nucleotide sequencing All RT-PCR products were excised from the gel for purification (QIAquick gel extraction kit, QIAGEN, UK) and cycle sequencing was carried out using BigDye® Terminator v3.1 cycle sequencing kit (Applied Biosystems, USA). The sequences were collected from automated DNA sequencer (ABI 3130XL, Applied Biosystems). The nucleotide sequences reported in this study have been deposited in GenBank under accession numbers EU019228–EU019230 and EU137728–EU137730.

2.4. Phylogentic analysis Sequence identity was determined through BLAST (www.ncbi.nlm.nih.gov/blast) and multiple sequence alignment was carried out with CLUSTAL W program (Thompson et al., 1994). Phylogenetic analysis of aligned sequences was carried out using MEGA 3.1 (Kumar et al., 2004). The phylogenetic tree was generated with neighbor-joining algorithm and Kimura 2-parameter distance model. The reliability of phylogenetic tree was tested by applying bootstrap test with 1000 bootstrap replications.

3. Results Of the 236 fecal specimens tested 28 (11.9%) showed amplification in RT-PCR for norovirus GII. These included 24 of 192 (12.5%) from hospitalized and 4 of 44 (9%) from OPD cases. Contrary to this, rotavirus positivity was 44.7% and 36.3%, respectively. GI norovirus was not detected in any of the specimens. In two of 24 (8.3%) hospitalized patients there was rotavirus co-infection while all four OPD cases were rotavirus negative. Clinical severity score of the patients with norovirus infection indicated severe disease in majority (70.8%) and moderate disease in 29.2%. Highest number of norovirus infections was detected in the month of March while the lowest was in May. Phylogenetic analysis of nucleotide sequences of all 24 strains from hospitalized patients placed 15 strains in GII/4 (genogroup II/genotype 4) (62.5%), 4 strains in GII/2 (16.6%), 1 strain in GII/3 (4.1%), 1 strain in GII/1 (4.1%) and 3 strains in new norovirus genetic type GIIb (12.5%). In comparison, 3 (75%) of the 4 strains from OPD cases clustered with GIIb while remaining one was placed in GII/2 (25%). All six GIIb strains—three from hospitalized (Pune/PC01/ 05, Pune/PC02/06 and Ahm/PC03/06) and three from OPD (Pune/OPD01/07, Pune/OPD02/07 and Pune/OPD03/07) cases were subjected to RT-PCR for the amplification of a larger genomic fragment (685 bp) from RdRp region to authenticate the presence of GIIb. Multiple alignment of partial nucleotide sequences from RdRp region of three

Table 1 Percent nucleotide identity (in lower triangle) and percent amino acid identity (in upper triangle) between GIIb norovirus variants from India and reference strains in RdRp region PC01 PC01 PC02 PC03 OPD01 OPD02 OPD03 Suria/312 Pont de Roide 673 Hokkaido/134 Yamaguchi17

99.20 98.90 95.20 94.50 94.60 98.20 97.60 96.20 96.00

PC02

PC03

OPD01

OPD02

OPD03

Suria/312

Pont de Roide 673

Hokkaido/134

Yamaguchi17

100.0

100.0 99.50

98.20 98.20 98.20

98.90 99.00 98.40 99.40

98.30 98.40 97.70 99.40 99.50

100.0 100.0 100.0 100.0 100.0 100.0

98.90 99.00 98.40 98.20 99.00 98.50 98.70

98.90 99.00 98.40 99.40 100.0 99.50 100.0 99.00

98.90 99.00 98.40 99.40 100.0 99.50 100.0 99.0 100.0

98.70 94.90 94.30 94.40 97.80 97.60 96.10 95.90

95.50 94.60 94.80 98.30 97.90 96.40 96.20

99.20 99.40 95.60 96.90 96.10 95.90

99.20 95.20 96.00 96.30 96.20

95.50 96.60 96.30 96.10

97.40 97.90 97.40

97.30 97.10

99.80

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Fig. 1. Phylogenetic tree depicting the genetic relatedness of Indian GIIb norovirus variants with other norovirus strains of GII in 685 bp fragment of RdRp region. Indian strains are identified by abbreviations—name of city, sample number and the year of illness. The accession numbers of the reference strains included for the comparison are as follows: GIIb-Suria/312/2001/Sp (AJ487795), Pont de Roide 673/2004/France (AY682549), Hokkaido/134/2003/JP (AB231355), Yamaguchi17/2003/JP (DQ372861), GII/1-Hawai virus/1971/US (U07611), GII/2-Melksham/1995/UK (X81879), GII/3-TV24/1993/CAN (U02030), GII/4-Lordsdale virus/1995/UK (X86557), GII/5-White River/290/1994/US (AF414423), GII/6-Saitama U4/2000/JP (AB039777), GII/7Gwynedd/273/1994/US (AF414409), GII/8-Saitama U25/2000/JP (AB039780), GII/9-VA97207/1997/US (AY038599), GII/11-Sw918/1997/JP (AB074893) and GII/12-Gifu’96/2000/JP (AB045603), GI/1-Norwalk virus/1993/US (M87661), GI/2-Southampton virus/1991/UK (L07418), GI/3-DSV395/1993/US (U04469), GI/4-Chiba407/1987/JP (AB042808). Scale indicates genetic distance.

strains each from hospitalized and OPD cases showed 97.8–98.3% and 95.2–95.6% nucleotide identities with norovirus Suria/312/01 of GIIb from Spain (Table 1). With Pont de Roide 637/04 strain from France, Indian strains showed 96–97.9% nucleotide identity while comparison with two Japanese strains Hokkaido/134/03 and Yamaguchi17/03 nucleotide identities were 96.10–96.40% and 95.90–96.20%, respectively (Table 1). Nucleotide and amino acid differences within the strains from hospitalized cases varied from 0.8% to 1.3% and 0% to 0.5%, respectively while in OPD strains the differences were 0.6–0.8% and 0.5–0.6%, respectively. Within hospitalized and OPD cases the differences varied from 4.5% to 5.7% and 1.0% to 2.3%, respectively. By phylogenetic analysis, strains from hospitalized and OPD cases appeared closer to European strains Suria312/01 and Pont de Roide 637/04, respectively (Fig. 1).

4. Discussion In India, the role of noroviruses in causing gastroenteritis is not very clear on account of limited data till date. Our study conducted to identify the norovirus strains circulating in western India demonstrates the predominance of GII/4 along with co-circulation of GII/1, GII/2, GII/3 and GIIb noroviruses. This is in agreement with previous studies from other parts of the world. Interestingly, GII/4 was not found to be the most prevalent genotype in the hospitalized cases in southern India (Bindhu et al., 2007). Genetic type GIIb was identified in 3 of 24 (12.5%) positive specimens from hospitalized and 3 of 4 (75%) from OPD cases. Thus norovirus GIIb strains are prevailing in both hospitalized and OPD cases of acute gastroenteritis. It was of note that patients who suffered from GIIb infection were residing in different districts suggesting co-

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circulation of diverse norovirus strains in western region of India. To our knowledge, this is the first report on norovirus GIIb infections in India. Although our study has limitations on account of analysis restricted to small size of samples and short period of collection, it is indicative of notable role of noroviruses in causing gastroenteritis and the need for longitudinal studies on surveillance of norovirus disease and strains in India. Earlier reports have described GIIb strains as naturally occurring recombinant norovirus strains with different capsid types (Reuter et al., 2006). This warrants more work towards complete characterization of RNA polymerase and capsid genes for better understanding of mechanism involved in recombination and pathogenicity of noroviruses from India.

Acknowledgments We are grateful to Dr. A.C. Mishra, Director, National Institute of Virology, for his constant support. We also acknowledge the cooperation extended by Drs. R. Dhongade, V. Kalrao, A.N. Pandiat and A.R. Bavdekar for clinical specimens. Thanks are due to Mr. Atul Walimbe for assistance in phylogenetic analysis.

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