Etiological and Epidemiological of Viral Diarrhea Among Children Under the Age of Five in Shenyang From 2018 to 2020

Diarrhea is one of the leading causes of death among children, especially in the age under ve, but little data are available on the etiology of viral diarrhea in Shenyang. To understand the infection status and the relevant epidemiological characteristics of viral diarrhea and to ll gaps of how the distribution of viruses change across Shenyang in children under the age of ve with diarrhea, stool specimens of children with diarrhea aged 0-59 months and surveillance data was collected from Sentinel Hospital of Shenyang. Rotavirus, calicivirus, adenovirus, and astrovirus 4 viruses were then analyzed, and the proportion of children who tested positive for each pathogen was calculated and seasonal and spatial patterns for major organisms were determined. Viruses were identied in 47.9% of the 897 samples from children with diarrhea. The main viruses of stool samples were rotavirus (16.9%, predominant type G9P[8]), calicivirus (14.7%, norovirus, predominant type GII ), adenovirus (11.8%), and astrovirus (4.5%). Viral infections were mainly detected in the age of 0-12 months. In the area of Shenyang, Huanggu has the most cases (198, 22.1%), followed by Dadong (137, 15.3%) and Hunnan (135, 15.1%). The positive rate of viruses in patients of different ages, seasons, and regions was not same. Public health entities and the government should develop corresponding measures for different age groups, seasons, and regions.


Introduction
Diarrhea is one of the leading causes of death among children under the age of ve and 525,00 children deaths annually from all over the world [1]. In China, infectious diarrhea is a category C legal infectious disease, with an incidence of 2.50-3.38 episodes per person-year in children younger than ve [2]. Diarrhea can be caused by bacterial, viral, and parasitic pathogens [3]. And the previous study has shown that even many pathogens can cause diarrhea, viruses account for over 75% [4]. Viral diarrhea is mainly caused by rotavirus, calicivirus including norovirus and sapovirus, adenovirus and astrovirus [5,6]. Also, the etiology of viral diarrhea differs between regions due to their local economic level [7], sanitary conditions[8], meteorological factors [9], and geography [10]. A better understanding of the epidemiology, etiology, and seasonality of viral diarrhea would be valuable for planning and adopting targeted preventive measures, as well as a clinical therapy. Shenyang,as one of the capital cities of Northeast China, few studies have evaluated viral agents known to be related to diarrhea and none explored the spatiotemporal patterns. Therefore, it is necessary to research viral diarrhea in Shenyang.
In this study, we were not only described the epidemiological features of Enteric viruses but also characterized rotavirus and norovirus strains circulating. We also lled gaps in how the distribution of viruses changes across Shenyang. Scienti c pieces of evidence for early intervention and prevention can be provided by our results.

Study population and fecal specimens
From 2018 to 2020, ongoing surveillance of diarrhea among children under the age of ve was conducted in Sentinel hospitals of Shenyang. Inpatients of sentinel hospitals were registered, and a standard case de nition was used to determine eligibility. Diarrhea [12] was de ned as ≥3 passages of watery, loose, mucus-, or bloody stools within a 24-h period. Stool specimens were collected by a sampler and quickly placed into a sterile sampling tube and stored in a biosafety transport box at 4-8℃. They were sent to the laboratory of Shenyang Center for Disease Control and Prevention within 2 hours, frozen at -80℃, and tested in batches. Also, the relevant demographic and epidemiological data were collected.

Virus detection
Four enteric viruses including rotavirus, calicivirus, adenovirus, and astrovirus were detected. The isolation and identi cation of enteric virus were performed using the uni ed standards operation laid down by the Chinese Center for Disease Control and Prevention [12]. The methods are brie y described below.

Data analysis
The SPSS 24.0 was used to perform statistical analysis on the collected data. The Chi-square test and Fisher's exact test were used to test for statistical signi cance. The P-value of less than 0.05 was considered signi cant. ArcGIS 10.3 was used to explore the spatiotemporal patterns of viral diarrhea.
The isolation rates of rotavirus, calicivirus, adenovirus and astrovirus four enteric viral pathogens showed a similar distinct seasonal variation with a higher rate in winter and autumn (Fig. 2a) In our study, a strong regional pattern in rotavirus, calicivirus, adenovirus, and astrovirus four viruses was also observed (Fig. 3).

Discussion
In this study, the updated results on the viral etiology as well as epidemiological characteristics of children under the age of ve with viral diarrhea were obtained by studying their stool samples in sentinel hospitals in Shenyang from 2018 to 2020. These etiologies differ in terms of the demographic characteristics of the infected children, the season and year of prevalence, and the region.
During the period of this study, 897 children aged 0-59 months who were hospitalized in Shenyang Sentinel Hospital provided stool samples, of which 47.9% were virus-positive. Rotavirus and calicivirus were the predominant viral agents. This result was similar to previous studies [9,13]. It's worth noting that the number of cases of rotavirus had a downtrend but the calicivirus had an upward trend. Although no data were available on rotavirus vaccination rates among children in Shenyang, with the coverage of the rotavirus vaccine, the number of diarrhea deaths attributable to rotavirus had already declined and a subsequent increase in calicivirus (norovirus)-associated viral diarrhea was observed [14]. Suggesting that we should pay more attention to the impact of calicivirus on infants and young children. The prevention and control measures of calicivirus (norovirus) should be developed. Now there are some antiviral medicines, disinfectants, and vaccines [15]. And the inclusion of a rotavirus vaccine in the national immunization program is still strongly recommended. Adenovirus and astrovirus infections were 11.8% and 4.5%, respectively. Although the roles of these two viruses in viral diarrhea were minor, they should not be neglected because they still affect the health of children and the global burden of disease [1,16].
Epidemic strains were varied over the place in both developed and developing countries [17]. In our study, we also found a similar conclusion. The predominant strain of rotavirus was G9P[8], increasing from 83% to 100% from 2018 to 2020. For norovirus, the genotype of norovirus includes only GII among 2018 and 2019, but in 2020 included GI, GII, and GI+GII three types. In addition, the G9P[8] and GII were almost concentrated in urban such as Huanggu, Hunnan, and Yuhong. Suggesting that it is essential to conduct continuous surveillance in different areas of Shenyang, especially in urban areas, to understand the prevalence of this viral diarrhea caused by G9P[8] and GII strains.
The infection rate of the male was higher than female. This may be because boys had a higher chance of being exposed to unsanitary conditions. Although there was no statistically signi cant difference in gender, it's also suggested that boys should pay more attention to personal hygiene. Also, the distribution of the virus in different age groups was explored. In general, the frequency and types of viral infections were higher in young children, especially in 0-12 and 13-24 months. With the increase in age, the number of cases decreased gradually. This difference in age distribution may be in uenced by the host immunity, eating habits, hygiene, and environment [18,19]. In China, kindergarten education is at the age of 3-5 years, possibly leading to the establishment of the host protective immunity and the ensuing decreased infection after this age. In addition, most of the children over 2 years old have been vaccinated or have been infected or their immune systems are well developed. Protective antibodies are produced in the body and are not prone to infectious diarrhea. Above all, under the age of two groups are the most vulnerable group to viral diarrhea, they should be taken as primary active surveillance targets.
The analysis of the epidemiological trends of viral diarrhea revealed that the infection rate of viral diarrhea decreased year by year from 2018 to 2020. This change may be related to the number of patients consulted, the hygiene habits of children, and their nutritional status. In terms of seasonal distribution, viral diarrhea can occur throughout the year, and its peak epidemic is mainly concentrated in autumn and winter, which is consistent with the results reported in related studies [20]. We hypothesized that the potential reason for this not only includes the characteristics of the virus (suitable for cold and dry environments) but also in spring and winter, infants are more preference for indoor activities. With doors and windows closed, children can easily inhale viruses from contaminated environments. Shenyang is located in the northern part of China, with cold weather and long winters, suggesting that we should pay attention to seasonal protection.
Due to local economic conditions, sanitary conditions, and other factors, the incidence of viral diarrhea may be different between different cities [1]. In our study, we also found that even in the same city, infection rates may be varied in different regions. An obvious spatial distribution in viral diarrhea could be seen in Shenyang. In particular, it was more prevalent in Huanggu, Dadong, Hunnan, Yuhong, Shenhe, and Tiexi ve districts. Other regions were relatively lower. These ve districts are the urban areas of Shenyang. The result was similar to a previous study in Beijing [17]. These districts are the economic, cultural, transportation, and commercial centers [11]. Relatively high population density and population mobility, which may lead to poor sanitation and make children more vulnerable to exposure to viruses. Or the results may be due to parents of these kids have different medical awareness in other regions, so mild infectious diarrhea may not seek medical care and remain unreported, leading to biases in the incidence of the disease.
In conclusion, we studied the viral pathogens spectrum and relevant epidemiology of viral diarrhea in Sentinel Hospital of Shenyang, to provide evidence for the prevention and treatment of viral diarrhea. We demonstrate that there is a high incidence of viral diarrhea, particularly due to rotavirus infections in Shenyang. And the etiologies differ in terms of the demographic characteristics of the infected children, the season and year of prevalence, and region. This suggests that we should take targeted measures for different situations, the relevant departments should pay more attention to viral diarrhea in infants and young children in both basic research and clinical diagnosis and treatment, conduct long-term systematic surveillance, and carry out relevant health education activities. Providing a scienti c basis for the prevention, control, and immunization of viral diarrhea in infants and young children in China. Besides, the rotavirus vaccine in the national immunization program is strongly necessary.

Limitations
This study had several limitations. First, the number of cases was not as many as others reported in multicenter studies due to restrictions on time and resources. Second, children with mild infectious diarrhea may not seek medical care and remain unreported, leading to biases in the incidence of the disease. Nevertheless, this study will enrich the epidemiology database of China and will be of great importance for preventing and control viral diarrhea. Figure 1 Distribution of different viral infections by age groups in different regions Distribution of rotavirus(a-b) and norovirus (c-d) genotypes from 2018 to 2020 Figure 5