Genetic Diversity and Epidemiology of Norovirus in Children with acute sporadic gastroenteritis in Shanghai, China, 2012-2017

Noroviruses are considered the important causes of acute gastroenteritis (AGE) across all age groups especially in children under five years. We investigated the prevalence and molecular epidemiology of norovirus in outpatient children from Children’s Hospital of Fudan University in Shanghai, China. A total of 1433 stool specimens were collected from children under ﬁve years with acute gastroenteritis between January 2012 and December 2017. All the samples were analyzed by conventional reverse transcription-polymerase chain reaction (RT-PCR) for genogroup II targeting both the RNA-dependent RNA polymerase (RdRp) and partial capsid genes. Norovirus Genotyping Tool v.2.0 (https://www.rivm.nl/mpf/typingtool/norovirus/) was used for genotyping strains, and phylogenetic analyses were conducted by MEGA 6.0.


Conclusions
The present study shows high detection rates and genetic diversity of circulating NoVs genotypes in paediatric AGE samples from Shanghai. The findings emphasize the importance of continuous molecular surveillance of emerging NoVs strains.

Background
Despite substantial decreases in recent decades, gastroenteritis remains the second most common cause of morbidity and the fourth most common cause of mortality worldwide in children under the age of 5 years [1]. Noroviruses have been considered as the second most common virus cause of sporadic gastroenteritis after rotavirus in children [2][3][4][5].
Noroviruses (NoVs) have a non-segmented positive-strand RNA genome of approximately 7.6 kb that contains three ORFs [6]. ORF1 encodes a polyprotein that is cleaved by the virus-encoded protease into six nonstructural proteins, including the norovirus protease and RNA-dependent RNA polymerase (RdRp). ORF2 encodes the major structural protein (VP1), and ORF3 encodes a minor structural protein (VP2) [7]. NoVs are highly diverse and divided into seven genogroups (GI-GVII) of which GI, GII and GIV have been found among human, and GII is the most popular genogroup in children with acute diarrhea. GII NoVs have been subdivided into 29 genotypes and 23 genotypes based on the genetic diversity of ORF1 and ORF2, respectively [8,9].
It is well known that natural recombination at the ORF1/ORF2 junction region is considered a common event between NoVs strains, and more and more combination or recombination norovirus strains are currently appearing in many areas, such as GII.Pe/GII.4, GII.P16/GII.4, GII.P17/GII.17 and GII.P16/GII.2 [10][11][12][13][14][15][16][17]. Consequently, a dual nomenclature system of NoVs have been proposed, taking the phylogenetic relationships of both partial ORF1 and ORF2 into account. This dual typing approach is correct to identify genetically different NoVs genotypes. In addition, combinant genotypes are better to understand the molecular epidemiology of NoVs.
Previous studies in Shanghai mainly adopted either ORF1 or ORF2 nomenclature to understand the epidemiology of NoVs in children under five years [18][19][20][21][22]. Here, we conducted the current study to investigate the diversity of NoVs genotypes adopting a dual nomenclature system, based on both ORF1 and ORF2, in children suffering from acute gastroenteritis who visited Children's Hospital of Fudan University in Shanghai from January 2012 to December 2017. Furthermore, we also assessed the overall frequency of NoVs, seasonal distribution of NoVs, and NoVs genotypes distribution by age group.

Study design
Fecal specimens were collected from 1433 children up to 5   Sequences generated from the PCR products of each strain were analyzed by using the Norovirus Genotyping Tool v.2.0 (https://www.rivm.nl/mpf/typingtool/norovirus/), where each sequence was used to assign to a NoVs genotype. Phylogenetic analysis on the nucleotide sequences obtained in our study and sequences data from GenBank, was also performed using the Maximum-Likelihood method (Kimura two-parameter model, 1000 bootstrap replications for branch support) in MEGA 6.0.

Statistical analysis
The difference between NoVs detection rates in boys and girls was compared using a twosided chi-square test in SPSS Statistics Version 19.0 (IBM Corp., Armonk, NY, USA) and the P-value less than 0.05 was considered statistically significant.   Table   2).

Discussion
This study was a long-term monitoring of the epidemiology and molecular characteristics of the NoVs in children under 5 years with acute sporadic gastroenteritis in Shanghai.
Herein, the total detection rate of norovirus positive cases (15.4%) among outpatients was lower than that in our previous monitoring data of NoVs infections in both outpatients and inpatients [20,22]. Although the percentage of NoVs infection in 2012 (25.0%) was similar to our previous data during 2006 to 2011, annual detection rate of NoVs was in fluctuating reduction from 2012 to 2017 [22]. This may be associated with the increased propaganda on how to prevent NoVs infection among people because of the increasing outbreaks of NoVs infection in many areas in recent years.
During 2012 to 2017, children with NoVs infection were mainly aged less than 2 years which is concert with our previous study and other studies [20,[22][23][24][25]. No significant difference of NoVs detection rates was found in girls and boys. This may imply that gender is not a predisposing factor in NoVs infection in children less than 5 years. In accordance with previous studies, which concluded that norovirus mainly peaked in cold seasons [20,22,[26][27][28], our study demonstrated that the highest detection rate was found in September and over 70% of NoVs infection were detected in autumn and winter. However, in some other areas, NoVs-associated diarrhea has a summer peak or no apparent seasonal peak which may be connected with the increased contaminated water and food or other unknown reasons [21,29]. in children was also reported in Huzhou and Shanghai [30,36]. However, adults were more susceptible to GII.P17/GII.17 than children [30,36]. The reason for this infection difference in diverse age groups was still ambiguous and further studies need to be conducted to explore the mechanism.
Combined genotypes of NoVs conducted in this study demonstrated that 19 kinds of RdRp/Capsid genotypes were determined, and 12 of them were distinct in RdRp genotypes and capsid genotypes. All of those discordant RdRp/Capsid genotypes were suspected as recombinant strains and most of them have been reported elsewhere [14,16,17,23,37,41]. Although it needs more analysis on the junction of ORF1 and ORF2 to confirm the recombination site in our study, it obviously suggest that this phenomenon is very common in NoVs as observed elsewhere. Furthermore, we also observed a change in the circulation pattern of the RdRp/Capsid strains, with GII.P4-2006b/GII. 4-2006b predominant  China [17]. Interestingly, this type was mainly observed in Asia-Pacific region, implying that the pandemic of GII.P12/GII.3 may have regional characteristics [17,47,48].
In epidemiological investigations carried out in different age of children, the distribution of NoVs Capsid/RdRp genotypes varied in different age groups. In our study, GII.Pe/GII.4-Sydney_2012 was detected in all age groups while other genotypes were not. Some genotypes were only detected in one age group. Those results may imply that the infection of some NoVs Capsid/RdRp genotypes was age-specific. However, more and longtime surveillance of the epidemiology of NoVs in different age of children should be conducted to illustrate this phenomenon.

Conclusions
In conclusion, our study has demonstrated the epidemiology and great genetic diversity of

Consent for publication
Not applicable.

Availability of data and material
The datasets used in the current study are available from the corresponding author on reasonable request.

Competing interests
The authors have no competing interest.  Tables   Table 1 Primers used for Noroviruses genotyping in this study   Table 3 NoV