Background
Norovirus a the nonenveloped, single-stranded positive-sense RNA virus belonging to the Caliciviridae family [
1], is a leading cause for acute gastroenteritis (AGE) in human hosts worldwide, especially among children and the elderly [
2‐
4]. The whole genome of norovirus is 7.7 kb, which can be organized into three open reading frames (ORF 1–3) [
5]. Of these, ORF2 encodes the major capsid protein (VP1). X-ray crystallography of VP1 showed two structural domains, including the N-terminal shell (S) domain and the C-terminal protrusion (P) domain [
6]. The P domain is further divided into the P1 subdomain and the variable P2 subdomain which contains putative neutralization sites and interacts with histo-blood group antigens (HBGAs) [
7].
Norovirus continues to evolve in humans through both mutation and recombination, in particular, recombination appears to be a major force driving virus evolution [
8]. Novel norovirus strains have emerged, and some classified into new tentative genogroups and genotypes [
9‐
11]. In the past years, we also had studied the evolution of norovirus open reading frame 2 (ORF2), utilizing strains derived from population-based surveillance, and detected far more mutations than recombination events in RNA-dependent RNA polymerase (RdRp) region of ORF1 [
12]. These evolutionary characteristics of norovirus are mostly based on symptomatic infections. Yet the fecal excretion of norovirus infection is common in asymptomatic individuals, especially in children [
13]. Asymptomatic infections, constitute a significant reservoir for infection in the community, and may act as a source of endemic and epidemic disease [
14,
15]. Our study characterized norovirus strains in samples collected weekly from symptomatic and asymptomatic infants participating in a birth cohort study. A phylogenetic analysis sequence encoding the norovirus RdRp and ORF2 was performed to classify the strains co-circulating during the surveillance period. The mutation and recombination patterns of norovirus strains were explored by recombination analysis.
Discussion
Norovirus is a leading cause of AGE, especially among children in China [
37]. For a comprehensive understanding of virus evolution and transmission dynamics in symptomatic and asymptomatic infections, a birth cohort study is desirable [
3]. Our previous study estimated that norovirus infection led to an annual incidence rate of 6.0, 15.6 and 5.5 per 100 persons per year in the three age groups a) all ages, b) less than 5 years and c) older than 60 years respectively [
38]. We have studied the evolution of the major capsid protein (VP1) encoded by the ORF2 based on sequences isolated from population-based diarrhea surveillance in Zhengding county spanning between 2001 and 2019, and concluded that antigenic variation was the major mechanism for the emergence of novel VP1s (not including RdRp region), rather than recombination [
12]. However, all of these findings were derived from symptomatic infections caused by norovirus. This study is the first birth cohort study to explore the transmission dynamics of norovirus in Chinese infants. More asymptomatic infections (20%) than symptomatic infections (14%), were detected, which suggested that the asymptomatic infections played an important role in the persistence of transmission, and thus, should be accounted when exploring the evolution of norovirus [
39,
40]. Several factors might be involved in the mechanism for the high occurrence of asymptomatic infection in the community. Firstly, the infants recruited in our study were around 6 months of age when they experienced the epidemic season. Some studies have demonstrated that the maternal antibody decay rapidly in the first months of life, although the measurable maternal antibodies were expected to have waned at 6 months [
41‐
44], and thus could still provide a certain level of protection, and eventually attenuated the severity of infections [
45,
46]. Secondly, the study site is located in rural areas, where infants are generally breastfed up to 12 months of age. It was reported that norovirus-specific immunoglobulin A in breast milk might protect against norovirus associated diarrhea but not norovirus infection [
47]. Similar to some published studies [
48,
49], symptomatic infections were not associated significantly with duration of viral shedding.
In this study, 14 strains that caused symptomatic infections were identified as GII.3[P12] and GII.4[P31], while 20 strains that caused asymptomatic infections were involving GII.4[P31], GII.2[P16] and GII.4[P16]. Among them, GII.4 Sydney2012 strains were detected in both AGE patients and asymptomatic carriers, which is in agreement with previous studies [
50‐
52]. Since first detected in USA in 2012, GII.4 Sydney[P31] strains have been the predominated strain resulting in global pandemics [
53]. The phylogenetic tree suggested that GII.4[P31] strains isolated from our study have a close genetic distance with previously circulating strains in Shanghai, Beijing, Zhengzhou and Guangzhou in 2016–2020. This finding proves that GII.4[P31] strains have circulated in the mainland China before 2016 [
54,
55]. The GII.4[P16] strain in our study was similar to the viruses circulating in Beijing in 2019–2020, and strains detected in Thailand in 2019–2020. It has been reported that the GII.4 Sydney[P16] strain emerged in 2015 and was soon replaced by the GII.4 Sydney[P31] strain as the primary cause of outbreaks in the United States [
56]. Despite the fact that GII.4 Sydney[P16] strains were rarely detected in our study, it remains necessary to monitor the prevalence of this strain. GII.3[P12] strains in our study clustered with strains from China, Japan, Russia and the United States in 2015–2018, and GII.2[P16] strains were clustered with China, Thailand and Japan in 2011–2013, suggesting that the strains were spread across the globe. We found that the GII.3[P12] strains were only detected in symptomatic infections, and GII.2[P16] strains were only detected in asymptomatic infections. This result may provide new perspective for the investigation of symptomatic and asymptomatic infections, but the results in this study are not sufficient to provide that the infection patterns are specifically distinguishable for those two genotypes. In the future, with the increasing accumulation of molecular epidemiological data, it may be possible for us to verify whether symptomatic and asymptomatic infections are related to the specific genotypes of norovirus.
The emergence and spread of novel norovirus strains is associated with point mutations in ORF1 and ORF2 region, and recombination events that produce chimeric viruses [
57]. Although all norovirus utilized both mechanisms, different genotypes may preferentially emerge and persist in populations. Our study found that the mutation frequency of ORF2 region was higher than RdRp region of the ORF1, especially in the P2 subdomain. The amino acid mutations of the VP1 protein identified in our study (amino acid positions 309, 373, 414) corresponded to the same characteristics identified by another Chinese group, and the remaining amino acid mutation sites were located at or near the amino acids identified by this group [
55]. Most of these mutations are located at or near the epitope region of the P2 sub-domain. It has been reported that residue changes of the epitope region are likely to enable the norovirus to escape the pressure of population immunity and cause global epidemic [
58,
59]. The predicted recombination breakpoints in norovirus strains identified from symptomatic and asymptomatic infants in our study around 5020 to 5105nt, overlapped with the RdRp (4044–5097) and partially overlapped with ORF2 (5085–6707). This is consistent with previously reported strains isolated during gastroenteritis outbreaks [
60]. RdRp of norovirus is a key enzyme responsible for viral transcription and replication and was suggested to be a driving factor in norovirus recombination [
61]. Norovirus recombination typically occurs at the RdRp-ORF2 junction [
62,
63], which is also the transcription initiation site for viral sub-genomic RNA. It has been shown that exchanges in RdRp region caused by genomic recombination result in an increased mutation rate through acquisition of an RdRp with lower fidelity and/or increased replicative ability, improving viral fitness under certain selective pressures and hence drive virus evolution [
64]. Therefore, recombination may be one of the reason for the diversity of norovirus, which not only causing the genomic exchange of RdRp region, but also increasing the mutation rate in other regions such as VP1 [
65]. Meanwhile, mutations in regions other than RdRp, may be caused by antigenic drift or shift that result in point mutations to escape immune response [
57]. Together, the recombination and the point mutation may lead to the generation of new immune escape strains. Thus, large molecular epidemiological cohort studies are required to further verify the causes of norovirus diversity.
Conclusions
In conclusion, during the study period, more episodes of asymptomatic infection were detected in comparison with that of symptomatic infection. Genetic diversity between isolated strains of symptomatic and asymptomatic infection was not observed. The predominant circulating strains were GII.4, followed by GII.3 and GII.2, which origin possibly for recombination include GII.4[P31], GII.4[P16], GII.2[P16], and GII.3[P12] circulating in China, and other Asian countries in recent years. The role of asymptomatic infection in the evolution and transmission of noroviruses needs to be evaluated continuously.
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