The predominance of recombinant Norovirus GII.4Sydney[P16] strains in children less than 5 years of age with acute gastroenteritis in Tehran, Iran, 2021–2022

Highlights • The RdRp and VP1-based dual typing by genetic analysis of circulating NoVs in Iran.• GII.4 Sydney_2012[P16] was determined as the most common strain.• GII.8[P8] viruses were also found to be as the second most common.


Introduction
Noroviruses are the most common cause of both sporadic and epidemic non-bacterial acute gastroenteritis (AGE) across all age groups. Moreover, noroviruses are the second leading cause of AGE in children less than 5 years of age, after rotavirus, associated with an estimated 70,000-200,000 deaths annually, mostly in developing countries (Pires et al., 2015;Cannon et al., 2019;Bányai et al., 2018). Noroviruses have also emerged and well recognized as the predominant pediatric viral enteric agent following the implementation of rotavirus vaccine. Regarding the global prevalence of noroviruses, several systematic review and meta-analysis have indicated that norovirus prevalence increased from 12% in 1990 − 2008 (Patel et al., 2008) to 18% in 2015-2021 (Ahmed et al., 2014;Liao et al., 2021;Farahmand et al., 2022), which further highlights the increasing trend of norovirus infections.
In 1979, Kapikian and others were first identified Norwalk virus, the prototype agent of the genus norovirus (previously denoted as "Norwalklike viruses"), as the etiological cause of AGE, during an outbreak of gastroenteritis in students at an elementary school in Norwalk, Ohio, United States (Kapikian et al., 1972). Noroviruses, as a genus of the Caliciviridae family, are non-enveloped, positive sense with a single-stranded RNA genome 7.5-7.7 kb in length (Chhabra et al., 2019). The genome is organized into three open reading frames (ORFs) with ORF1 encoding the nonstructural (NS) viral proteins, ORF2 and ORF3 encoding the major (VP1) and minor (VP2) capsid proteins, respectively (Chhabra et al., 2019). Complete VP1 amino acid sequences and the ORF1 NS7 region (which encodes the RNA-dependent RNA polymerase; RdRp) nucleotide sequences were used for classification based on its genetic variability (Chhabra et al., 2019). Accordingly, based on the complete amino acid sequences of VP1 gene, 10 genogroups (G1-GX) of norovirus have been established so far. Among which, norovirus GI, GII, GIV, GVIII, and GIX are reported to infect humans although GI and GII is by far the most medically important species globally (Chhabra et al., 2019). These genogroups were further segregated into 49 confirmed genotypes, based on amino acids of the complete VP1. Among genotypes, GII genotype 4 (GII.4) is the most predominantly reported circulating strain globally, and can be classified into subtypes or variants. Every 2 to 3 years, new GII.4 variants emerge and replace the previously predominant strains (Chhabra et al., 2019;Kroneman et al., 2013). In 1995, GII.4 pandemic first documented by GII.4 US95_96 variant, followed by the emergence of GII.4 Farmington Hills in 2002, GII.4 Hunter in 2004, GII.4 Yerseke and GII.4 Den Haag in 2006, GII.4 New Orleans in 2009, and GII.4 Sydney in 2012(Kroneman et al., 2013Vinjé, 2015;Tohma et al., 2019) as well as several local epidemics of GII.4 variants such as Grimsby 1995, Henry 2001, Japan 2001, Asia 2003, 2006a, Cairo 2007, Apeldoorn 2008, and Japan 2008(Tohma et al., 2019Tu et al., 2008;Parker et al., 2005;Seto et al., 2005;Belliot et al., 2010;Kamel et al., 2009;Motomura et al., 2010). Recombination, as a major driving force of norovirus diversity, may further increased the emergence of novel strains (Chhabra et al., 2019). The recombination breakpoints are most frequently identified in the ORF1-ORF2 junction region (Chhabra et al., 2019). In this regard, dual typing has been proposed to include diversity at the level of the partial RdRp sequences in strain designations. Based on nucleotide diversity in the RdRp region, noroviruses can be classified into 60 P-types (Chhabra et al., 2019). Based on the last norovirus classification update, it has been proposed the following designations of norovirus strains: GI. genotype [P-type] or GII.genotype[P-type], first listing the capsid genotype followed by the P-type between brackets (Chhabra et al., 2019). In this regard, Kendra et al., (Kendra et al., 2022) (Kendra et al., 2022).
Previous studies in Iran have indicated that positivity rates of norovirus in children less than 5 years of age ranges from 4.14 to 21.3% (Fazeli et al., 2010;Jalilian et al., 2012;Najafi et al., 2013;Romani et al., 2012;Roodsari et al., 2013;Sharifi-Rad et al., 2015;Shoja et al., 2014;Nasab et al., 2016). Although little information is available with regards to the norovirus genotypes in Iran, GII.4 is shown to be the most predominant capsid genotype, followed by GII.3, GII.2, GII.6, GII.7, GII.12, GI.4 (Romani et al., 2012;Farsi et al., 2018). While studies on noroviruses were mainly limited to the prevalence of noroviruses, less data are available regarding RdRp and VP1-based dual typing of noroviruses circulating in Iran. As such, the present study was aimed to identify the genetic variation and dual typing pattern of norovirus strains across the Iranian children on the basis of distinct RdRp and VP1 genes.

Specimen collection
Fecal specimens were collected from children less than 5 years of age with AGE at Children's Hospitals in Tehran from January 2021 to January 2022. Specimens were transported and stored at -20 ̊C in the laboratory of Molecular Virology Division at Pasteur Institute of Iran. The study was conducted according to the Helsinki guidelines and approved by the ethics committee of the Pasteur Institute of Iran.

Viral RNA extraction and cDNA synthesis
10% (wt/vol) fecal suspension was prepared with phosphatebuffered saline (PBS) and clarified using centrifugation at 1500 x g for 20 min. Viral RNA was then extracted from clarified supernatant using viral RNA/DNA extraction kit (GeneAll, Korea) according to the manufacturer's instructions. Extracted RNA was used for complementary DNA (cDNA) synthesis, which was performed with SinaClon First Strand cDNA synthesis kit (Cat. No. RT5201; SINACLON, Tehran, Iran) using a single cycle in a final volume of 20 μL according to the manufacturer's instructions.

Screening of GI and GII noroviruses
The presence of GI and GII noroviruses in fecal specimens was assessed using real-time PCR with the Corbett Research Rotor-GeneTM (Qiagen, USA). The primer and probe sets COG1F, COG1R, probe Ring 1 and COG2F, COG2R, probe Ring 2 (Table 1) were used to screen GI and GII norovirus strains, respectively (Kageyama et al., 2003;Rolfe et al., 2007). Briefly, PCR reactions were prepared in 20 μl reaction mixtures containing cDNA, 4X CAPITAL™ qPCR Probe Master Mix (Bio-techRabbit, Germany), 500 nM of each primer, and 100 nM of each probe. PCR amplification were performed at 95 • C for 2-3 min, followed by 40 cycles of PCR at 95 • C for 5 s and 60 • C for 30 s.

Amplification of partial RDRP and VP1 of GI and GII noroviruses
The positive specimens by real-time RT-PCR were then genotyped for the RdRp and VP1 regions. Semi-nested PCR was used to amplify RdRp and VP1 regions using primer sets COG2R, JV12Y, JV13I (Vennema et al., 2002) (for GI and GII) and COG1F, G1SKF, G1SKR (Kojima et al., 2002) (for GI), COG2F, G2SKF, G2SKR (Kojima et al., 2002) (for GII) ( Table 1). The PCR amplification reactions for the first round PCR were prepared in 25 μl reaction mixtures containing 2X Hot-Start PCR Master Mix (BiotechRabbit, Germany), 500 nM of each first round primers (JV12Y/COG2R for RdRp; COG1F/G1SKR and COG2F/G2SKR for VP1 of GI and GII, respectively), and cDNA as template. The nested-PCR amplification reactions were also performed in 25 μl reaction mixtures containing 2X Hot-Start PCR Master Mix (BiotechRabbit, Germany), 500 nM of each second primers (JV12Y/JV13I) for RdRp; G1SKF/G1SKR and G2SKF/G2SKR for VP1 of GI and GII, respectively), and 1 μl of the first round PCR product. The following condition was applied for PCR amplification of both rounds: initial activation for 2 min at 95 • C, followed by 35 cycles of denaturation for 30 s at 95 • C, annealing for 30 s at 37 • C, extension for 45 s at 72 • C, and a final extension for 5 min at 72 • C. The PCR products of the expected size were finally visualized in 1.5% agarose gel and sequenced.

Sequence analysis and molecular typing
The nucleotide sequence of the partial RdRp and VP1 was obtained by sequencing with a 3130 Genetic Analyzer Automated Sequencer following Applied Biosystems protocols (Applied BioSystems, Foster City, CA, USA) and edited with the CLC Main Workbench (CLC Bio). Sequences were then genotyped using a web-based Norovirus Typing Tool Version 2 (Tatusov et al., 2021). Multiple sequence alignments of norovirus RdRp and VP1 nucleotide sequences of Iran as well as the reference norovirus strains from GenBank were established using Bio-Edit. The Clustal W method was used to generate phylogenetic tree using the maximum likelihood method (MLM) based on the Kimura 2-parameter model (MEGA 6 software) (Tamura et al., 2013). The trees were statistically supported by bootstrapping with 1000 replicates.

Discussion
Norovirus infections in children with AGE, which has been reported from 2006 to 2014 from several regions of Iran, well indicate the prevalence rates of norovirus infection from 4.14 to 21.3% (Fazeli et al., 2010;Jalilian et al., 2012;Najafi et al., 2013;Romani et al., 2012;Roodsari et al., 2013;Sharifi-Rad et al., 2015;Shoja et al., 2014;Nasab et al., 2016). In our previous study, we have detected genogroup GII of norovirus in 17.1% of specimens taken from children less than 5 years of age with AGE (Farsi et al., 2018). The overall prevalence of norovirus infection in the present study was found to be 20% in children with AGE, which was higher than the previous report from Iran. The GII viruses were significantly predominant, representing 95% of the total norovirus infections. In addition to detection and analysis of norovirus GI and GII prevalence, we also investigated the genetic characterization of noroviruses in the community, which further highlighted the circulation and dominancy of recombinant norovirus GII. and GII.4 Sydney[P31] strains. The genogroup GI of norovirus accounted for 5% of all norovirus infections, which one sample were successfully sequenced for the partial RdRp and VP1 genes and typed as GI.3 [P3]. The GI.3 genotype was reported to be the most frequently detected GI viruses in outbreaks in China, Republic of Korea, Tailand, and Taiwan that mainly occurred in pre-school and school students (Chiu et al., 2020).
In conclusion, the present study indicated yearly diversity in the predominant norovirus strains and/or recombinants compared to our previous study. To our knowledge, this is the first report that highlights the dominancy of recombinant norovirus GII.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
Data will be made available on request.