Prevalence of GII.4 Sydney Norovirus Strains and Associated Factors of Acute Gastroenteritis in Children: Winter 2019-2020 in Guangzhou


 BackgroundNorovirus, the leading cause of non-bacterial acute gastroenteritis (AGE) worldwide, is constantly mutating. Continuous monitoring of the evolution of epidemic genotypes and emergence of novel genotypes is therefore necessary. This study determined the prevalence and clinical characteristics of norovirus strains in AGE in Guangzhou, China in 2019-2020 winter.MethodsThis study included children aged 2-60 months diagnosed with AGE in Guangzhou Women and Children Hospital, from August 2019 to January 2020. Norovirus was detected by real-time polymerase chain reaction and clinical data were obtained. Genotyping and phylogenetic analyses were performed with partial gene sequence fragments located at the open reading frames 1-2 junctions.ResultsDuring the study period, 168 children (61.3% males) were confirmed as norovirus infectious AGE. The main symptoms were diarrhoea and vomiting; and 38 patients (22.6%) had seizures. Norovirus was mainly prevalent in October and November, and GII.P31/GII.4 Sydney was the major genotype circulating in Guangzhou. The phylogenetic tree showed that the Guangzhou strains had high homology with the strains circulating in 2017-2019 worldwide. ConclusionsGII.4 Sydney was the main prevalent norovirus genotype in Guangzhou from August 2019 to January 2020, which had more severe diarrhoea than those of other genotypes. These findings provide a valuable reference for the prevention, control, and treatment of norovirus in the future.


Introduction
Norovirus is the leading cause of non-bacterial acute gastroenteritis (AGE) worldwide, causing approximately 18% of AGE and 212,000 deaths every year [1,2]. Norovirus causes considerable yearly losses to society, and more than half of the expenditure on norovirus, is due to the childhood infections [3]. Norovirus is mainly prevalent in winter, and often causes severe AGE outbreaks. The symptoms of patients with norovirus AGE mainly include vomiting, diarrhoea, and nausea. Most of these symptoms are mild and self-limiting, and generally last for 2-3 days. However, norovirus can cause serious clinical symptoms in some groups, especially children, the elderly, and immunocompromised patients [4,5]. Once transmitted, the virus can remain in the patient for weeks or even years. Due to the lack of speci c antiviral drugs and vaccines, supportive care and symptomatic treatment are the main therapeutic methods for norovirus infections. Moreover, norovirus has a strong ability to spread, and usually causes AGE outbreaks in semi-enclosed areas, such as hospitals, schools, kindergartens, cruise ships, nursing homes, and prisons [1,6].
Norovirus is a single-stranded positive-sense RNA virus belonging to the family Caliciviridae. The norovirus gene is approximately 7.5-7.7 kb long, and includes three open reading frames (ORFs). ORF1 encodes a polyprotein and is involved in viral replication. ORF2 and ORF3 encode the norovirus structural proteins VP1 and VP2, respectively. Norovirus is divided into 7 genogroups (GI to G , but only GI, GII, and GIV can infect humans ) based on the ORF2 gene, and further divided into more than 40 genotypes [7].
Because the mutation and recombination of norovirus mainly occur at the ORF1 and ORF2 junctions, the double genotype classi cation standard, based on ORF1 and ORF2, can well describe the epidemic potential of the virus, and has been widely accepted and applied in recent years [8].
The rst norovirus outbreak occurred in Norwalk, USA, in 1968 [9]. Since 1995, GII.4 has become the most popular genotype worldwide, accounting for more than 90% of all the epidemics [7,10]. In China, norovirus is also a major cause of non-bacterial AGE. It causes more than 20% AGE nationwide, affects about 800,000 people yearly [11]. However, previous studies have shown that non-GII.4 noroviruses(such as GII. 17 and GII. 2) have gradually replaced GII.4 to become the predominant genotypes in recent years [12,13], and that norovirus is constantly mutating. Continuous monitoring of the evolution of epidemic genotypes and the emergence of novel genotypes is necessary to effectively control norovirus transmission.
In this study, we aimed to analyse the epidemiological and clinical characteristics of norovirus infection in children in Guangzhou in the winter of 2019-2020. Furthermore, we performed a phylogenetic analysis of the epidemic strains prevalent in Guangzhou to understand the trend of norovirus. These ndings could provide important references for the control and treatment of norovirus, and for vaccine development, in the future.
As shown in Figure 2, the number of norovirus-infected children showed a monthly increase from 5 in August to 72 in October; in November, there were 55 infected children. The gradually decreasing trend started in November, with only four positive norovirus samples in January. GII.P31/GII.4 Sydney norovirus was the dominant genotype in Guangzhou, Guangdong Province, in the winter of 2019. In October and November, GII.P16/GII.4 Sydney and GII.P16/GII.2 also increased slightly.

Demographic characteristics and clinical manifestations
The demographic characteristics and clinical manifestations of the 168 children with norovirus infection in this study are shown in Table. 1. Among these children, 103 (61.3%) were male and 45 (38.7%) were female. The median age was 19 (IQR 13.0-28.8) months, and 148 (88.1%) patients were under 3 years of age. A total of 80 (47.6%) children were hospitalised for a median of 5 (IQR 3.0-7.5) days. There were 56 (33.3%) children with fever, and the median body temperature during infection was 37 °C (IQR 37.0-37.9°C). Eighty-two (48.8%) patients had anorexia and 38 (22.6%) had seizures. Clinical data showed that the main symptoms shown by the patients with norovirus infection were diarrhoea (79%) and vomiting (83.9%), and no signi cant differences were found between the genotype groups. However, the duration and the maximum number of diarrhoea cases with GII.4 ( GII.P31/GII.4 Sydney and GII.P16/GII.4 Sydney) were signi cantly higher than those in the other genotypes.  Laboratory examinations showed that the white blood cell (WBC), red blood cell (RBC), aspartate aminotransferase (AST), hydroxybutyrate dehydrogenase (HBDH), and creatine kinase isoenzyme (CK-MB) levels in the blood of patients with norovirus infection increased, but no signi cant differences were found between the genotype groups ( Table 2). The levels of sodium (Na) (P=0.002) and chloride (Cl) (P=0.024) in the blood of patients infected with GII.4(GII.P31/GII.4 Sydney and GII.P16/GII.4 Sydney) were signi cantly higher than those of patients infected with other genotypes.    We performed phylogenetic analysis based on the partial gene sequence fragments (ORF1-2 junction), to investigate the genetic diversity of norovirus strains prevalent in Guangzhou in the winter of 2019. In Figure 3, the sequence alignment of 131 strains of GII.P31/GII.4 Sydney norovirus shows the identities of the nucleotides and amino acids as 94.87%-100.00% and 96.75%-100.00%, respectively. The nucleotide and amino acid identities between our strains and the GII.P31/GII. Norovirus mutates and evolves rapidly, and it is classi ed into 7 genogroups and further divided into more than 40 genotypes based on the ORF2 gene [7]. These different norovirus genotypes are diverse in infectivity and virulence [14,15]. More than 90% of non-bacterial AGE cases are caused by GII, with GII.4 as the major epidemic genotype in many regions of the world during the past two decades [10,16] [13,26]. Our study is the rst to report that the main prevalent strains changed to GII.P31/GII.4 Sydney and GII.P16/GII.4 Sydney in Guangzhou in the winter of 2019-2020. The phylogenetic tree showed that the strains we obtained in Guangzhou had high homology with the strains circulating in 2017-2019 worldwide. This suggests that local strains may have become prevalent after the in ux of the foreign strains. Overall, the epidemic strains of norovirus changes rapidly, and it is necessary to pay more attention to the monitoring of the norovirus outbreaks, which will help reduce the losses caused by norovirus infection.
Our study revealed October and November as the months when the norovirus infections were most prevalent in Guangzhou in the winter 2019-2020, which is different from the previous research that November to March of the following year is the prevalent month [27]. Research by Marshall et al. showed that the prevalence is positively related to the humidity and the amount of rainfall [28]. However, the weather in Guangzhou was characterised by continuous sunshine and drought from mid-October in 2019 [29], which was quite different from that of the previous years. The lack of rainfall in the winter of 2019 might be the reason why norovirus infections began to decrease in November. The molecular epidemiological data of this study demonstrated that norovirus circulating in Guangzhou in 2019 had, to a certain extent, sequence variations from the previous circulating strains, which may also change the prevalent characteristics; however, this needs further study. In addition, the strict control, wearing of face masks, and the increased awareness of hygiene by the citizens, associated with the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) at the beginning of 2020 [30], not only effectively stopped the spread of SARS-COV-2, but may also have effectively reduced the opportunities of spread of norovirus.
In this study, male patients were more among the infected than females (103:65), which is consistent with previous studies [26,27]. That the innate and acquired immunity in females are inherently stronger than in males may be the reason [31]. Children with norovirus infection are more likely to develop AGE, especially children under 5 years. In our study, more than 88% of the patients were aged <36 months, with a median age of 19 (IQR 13-28.75) months. Fifty percent of infected patients were aged 12 to 23 months, which is consistent with previous reports [32][33][34]. These results suggest that children under 3 years of age are more susceptible to norovirus infection. Therefore, in the prevention and control of norovirus, more attention should be paid to children in this age group.
Vomiting and diarrhoea were the main symptoms of norovirus infection in our study. Compared with patients infected with non-GII.4 norovirus, patients with GII.4 were more likely to present with diarrhoea [20,27]. In this study, we found that the duration of diarrhoea was signi cantly longer and the maximum frequency per day in patients with GII.4 infection was more (P<0.05) than that of other genotypes. In addition, laboratory examination results showed that the levels of Na and Cl in the blood of patients with GII.4 infection were lower than those with non-GII.4. These features also indicate that the GII.4 genotype infection is more serious, and effective measures, such as rehydration, are needed to prevent more serious complications. Furthermore, our study showed no signi cant differences in clinical symptoms and laboratory examination parameters between patients with GII.P31/GII.4 Sydney and GII.P16/GII.4 Sydney. The sequence alignment analysis demonstrated that these two strains were only different in the RdRp genes, while the VP1 genes were the same for the GII.4 Sydney. Previous studies have also proved that the VP1 gene did not differ between GII.P31/GII.4 Sydney and GII.P16/GII.4 Sydney [18]. The VP1 gene encodes a viral structural protein with the histo-blood group antigens (HBGAs)-binding sites, which can mediate the cellular interactions [35,36]. The RdRp gene encodes a non-structural protein that participates in the transcription of norovirus, which can in uence viral transmission [21,24]. Perhaps the virulence of norovirus might correlate positively with its receptor-binding ability and be less correlated with its transmissibility. Further research is needed to prove this.
Our research showed nausea, abdominal pain, and dehydration as the complications of norovirus infection. In addition, we recorded that 38 (22.6%) patients had seizures, which was the main reason why some patients were admitted. Previous studies have also shown that the probability of seizures in norovirus infections is signi cantly higher than that in rotavirus infections [37][38][39]. Seizures, in this study, were similar to benign convulsions associated with mild gastroenteritis reported previously by Boman et al. [40]. The mechanism of norovirus-induced seizures in children is still unclear, and may be associated with the children's immature immune system [37]. Recently, some studies found that norovirus infection can cause encephalitis/encephalopathy [37,41]. However, no case of encephalitis/encephalopathy was found in our study. In addition, the laboratory data showed that the levels of cardiac biomarkers, such as HBDH and CK, were elevated in most children. This suggests the need for clinical attention to myocardial damage in patients with norovirus infection.
There were several limitations to our study. For example, this study was not a multicentre or multi-year study; only cases occurring during the norovirus prevalent season (winter) were included in our study. In addition, we only analysed the epidemiological data of patients who visited the hospital, and patients who did not seek for medical advice at the hospital were not included in this study. In addition, we used partial gene sequencing for the phylogenetic analysis; therefore, further analysis using whole-genome analysis is needed.

Conclusions
In conclusion, this study reported the epidemiological characteristics of norovirus from August 2019 to January 2020 in Guangzhou. The main genotypes of norovirus were GII.P31/GII.4 Sydney and GII.P16/GII.4 Sydney. Most of their strains had high homology with the epidemic strains of 2017-2019. We determined the clinical information of infected patients. Vomiting and diarrhoea were the main symptoms. These ndings help us to understand the prevalence of norovirus strains in Guangzhou, and provide a valuable reference for the prevention, control, and treatment of norovirus in the future.

Study population and specimen collection
A total of 168 children (age range: 2-60 months) diagnosed with AGE following norovirus infection at Guangdong Women and Children Hospital, from August 2019 to January 2020, were included in this study. AGE was de ned as defaecation ≥3 times within 24 hours with trait changes (diluted watery stools), and/or vomiting ≥2 times within 24 hours [42]. All norovirus infection diagnoses were con rmed with molecular detection (described below).

Collection of clinical information
Clinical information (demographic characteristics, clinical symptoms, laboratory testing.) were collected on the participants.This study did not record any data about the patient's personal identity information since the collection process started and all patient names and other relative information were replaced with identifying numbers, therefore, the informed consent was not needed. Genotyping and phylogenetic analyses Reverse transcription was performed using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Beijing, China) to obtain norovirus cDNA. Norovirus belonging to genotypes GI and GII were ampli ed using primers MON432 and GISKF, MON431 and GIISKR, respectively [18]. The length of the PCR-ampli ed products was expected to be 543bp for GI and 557bp for GII, including the partial RdRp gene located in ORF1 as well as the partial VP1 gene located in ORF2 (ORF1-2 junction) [8]. The ampli ed products were sent to Sangon Biotech (Sangon Biotech, Shanghai, China) for sequencing. To con rm the genotype of norovirus and to analyse the norovirus sequences at the ORF1-2 junction, we used the online Norovirus Typing Tool Version 2.0 (http://www.rivm.nl/mpf/norovirus/typingtool). The sequences were uploaded to GenBank (accession numbers: MT856488-MT856646) and are shown in the Additional le 1.
Multiple sequence alignment was performed using ClustalW in MEGA X, and the phylogenetic tree was constructed using the neighbour-joining method, with a bootstrap value of 1000 repetitions. All reference sequences were downloaded from GenBank.

Statistical analysis
The patient's clinical data were collated, imported, and analysed in IBM SPSS Statistics for Windows, version 26.0 (IBM Corp., Armonk, N.Y., USA). Qualitative data were expressed as frequency (percentage), and the chi-square test was used for analysis. Quantitative data were expressed as median and interquartile range (IQR), and t-test or one-way analysis of variance (ANOVA) was used to analyse data that conformed to the normal distribution, while the nonparametric rank-sum test was used for data that did not conform. Availability of data and materials Phylogenetic analysis of the 445bp norovirus nucleotide sequence within the partial RdRp and VP1 genes. The norovirus strains detected in this study (MT856488-MT856646) and reference sequence retrieved from GenBank were constructed using the neighbour-joining method with bootstrap analysis of 1000 replicates using MEGA X. Bootstrap values >80% are shown in red. The nucleotide sequence in this study is presented as GenBank accession number/country/year of isolation/genotype. The sequences