Currently, norovirus is the main cause of non-bacterial AGE, and it often causes AGE outbreaks. In this study, we reported the detailed clinical and epidemiological characteristics of young children with norovirus AGE in Guangzhou City in the winter of 2019-2020. Our results revealed that norovirus was prevalent mainly in October and November during our study period, and GII.P31/GII.4 Sydney was the major genotype circulating in Guangzhou, followed by GII.P16/GII.4 Sydney and GII.P16/GII.2. Additionally, diarrhoea and vomiting were the main symptoms of norovirus AGE and GII.4 Sydney was associated with a more severe AGE presentation than other genotypes. These findings provide a better understanding of the clinical manifestations and aetiology of norovirus AGE, and can be helpful in the prevention, control, and treatment of norovirus AGE in the future.
Norovirus mutates and evolves rapidly, and it is classified 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]. GII.4 norovirus can be further classified into seven strains (GII.4 US95/96 strain, GII.4 Farmington Hills, GII.4 Hunter, GII.4 Den Haag, GII.4 New Orleans, and GII.4 Sydney) due to its continuous variations. These novel GII.4 epidemic strains that are associated with AGE outbreaks usually mutate every 2-3 years [17, 18]. This is because recombination frequently occurs between different strains and the breakpoints are usually at the junction of ORF1-2; hence, a dual typing based on both the RdRp of ORF1(P type) and the VP1 of ORF2 (genotype) of norovirus is widely used currently [8]. In our study, GII.P31/GII.4 Sydney was the main epidemic strain (78.0%) in Guangzhou in winter 2019-2020. This strain was first detected in Australia in 2012 and rapidly became the major epidemic strain in Europe, America, and Asia in 2012–2013 [19, 20]. In 2015, a novel recombinant norovirus strain, GII.P16/GII.4 Sydney, appeared and prevailed in Germany, France, USA, and China [21-23]. There were 20 cases (11.9%) of GII.P16/GII.4 Sydney during the study period. Previous studies have found that the RdRp gene of this novel strain is highly homologous to GII.P16/GII.2, and the VP1 gene is highly homologous to GII.P31/GII.4 Sydney [24, 25]. In addition, few strains of GII.P16/GII.2, GII.P7/GII.6, and GII.P21/GII.13 were also found in our study. GII.P16/GII.2 was the major epidemic strain in Guangdong Province in August 2016 and continued to circulate in 2016 and 2017 [13, 26]. Our study is the first 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 influx 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[32-34]. 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 significantly 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 significant 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 influence 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 significantly higher than that in rotavirus infections [37-39]. 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.