Epidemiological And Genetic Features Of Gastrointestinal Viruses In Pediatric Outpatients With Acute Diarrhea In Chongqing

BackgroundWith a high morbidity and mortality, acute diarrhea cause seriouseconomic and healthy burden, especially in undeveloped countries. It is reported that more than700million children younger than5years are affected by acute diarrhea in the world per year,and more than2million deaths in children are related with the disease in developing countriesannually.With improvements in hygiene and sanitation,the incidence of diarrhea associated with bacterial and parasitic pathogen has decreased, but viral diarrhea has not been controlled effectively due to lack of specifictreatments. Rotavirus, norovirus, sapovirus, adenovirus and astrovirus are the most common etiologic agents of viral diarrhea. As the second most common cause of pediatric mortality, acute diarrhea has become a worldwide public health problem.ObjectiveThe current surveillance was performed to identify the spectrum of viral pathogens causing diarrhea and their epidemiological characteristics. The norovirus was further investigated for the prevalent genotypes, epidemiological trend and genetic features. We also aimed to find out whether there existed new recombinant strains in Chongqing.Material and MethodsDuring2009.8to2013.11, a sentinel surveillance focus on the detection of diarrhea virus was performedat the outpatient department of Children’s Hospital of Chongqing Medical University, China. The children with acute diarrheawere recruited in accordance with uniform standards. A standard questionnaire was established to collect demographic information and clinical symptoms of children.The stool specimens were collected for the detection of the presence of eight kinds of common diarrhea virusby using ELISA, Real-time PCR and other molecular technologies. These gastrointestinal virus included Group A Rotavirus, Group B rotavirus, Group C Rotavirus, type Ⅰ Norovirus （GⅠ）, type Ⅱ Norovirus （GⅡ）, adenovirus, astrovirus and sapovirus. Positive Norwalk-like virus samples were further determined for the sequences of ORF1and ORF2genes. Homology analysis of nucleotide sequence and recombination analysis were performed. Using automated genotyping tools, the nucleotide sequences were uploaded to www.Noronet.nl to determine the genotype of ORF1and ORF2respectively. Phylogenetic trees of partial ORF1and ORF2were performed and recombinant strainswere determined by inconsistentgenotyping results ofORFland ORF2segment. Recombinant analysis was performed to identifythe recombination sites using RDP4and SimPlot analysis. Real-time PCR and nested PCR were performed to determine the presence of HBoV and its genotype.Results1. DiarrheaPathogens and its epidemiological characteristics（1）During the study period,2416outpatients with acute diarrhea were includedwith70.58%of the patients infected with at least one of the viral pathogens. Norovirus was the most commonly detected virus, with detection rate of50.45%, followed by38.54%for the rotavirus,4.60%for adenovirus,3.74%for sapovirus,2.67%for Astroviruses. Totally586cases were infected withmultiple viruses, from which the mixed infection by norovirus and rotavirus was the most commontype.（2）Children aged between7-12month were more susceptible to norovirus infection thanchildren of other age group（chi-square test, P<0.0001）. Children aged between7-24months are more vulnerable to rotavirus single infection and mixed infection than other age group （rotavirus infection:chi-square test, P<0.0001; mixed infection: chi-square test, P<0.0001）. Virus detection rates did not differ between patients of different gender.（3） The detection rate of GⅡ and RVA virus had obvious seasonal characters, but the epidemic seasons was different. The detection rate of GⅡ peaked in summer and autumn, and reached bottom in spring. However, the detection rate of RVA presented a bimodal distribution, with two popular peaks in fall and winter and one bottom in summer. There was an abnormal peak during2012.4-2012.6, which reminded us of a possible outbreak.（4）The GⅡ patients had longer duration of diarrhea before going to outpatient department than RVA patients （3.61VS3.23, P=0.0118）. The RVA patients had higher frequency of vomiting than GⅡ patients（56.69VS50.00, P=0.0220）. The patients with virus coinfection are more vulnerable to vomit than those with singe infection（57.17%VS50.08%, P=0.0064）. 2. Genotypes and recombination of norovirus（1） The prevalent norovirus genotypes differed among various years in ChongQing. During2010, GⅡ.42006b and GⅡ.4New Orleans2009were the most prevalent genotypes; during2011, GⅡ.12/GⅡ.3and GⅡ.42006b, were the most prevalent ones, and after April, there were no GⅡ.4New Orleans2009detected in Chongqing; Before March2012, GⅡ.42006b was the predominant strain, but after the detection of GⅡ.4Sydney₂012in October for the first time,it quickly replaced2006b and become the predominant strain; During2013, GⅡ.4Sydney₂012and GⅡ.12/GⅡ.3were the most important epidemic strain, and GⅡ.42006b were no longer popular. The predominant strain in Chongqing outpatients, including GⅡ.42006b, GⅡ.12/GⅡ.3andGⅡ.4Sydney₂012show no difference in terms of age,gender composition and the clinical manifestations.（2） Some sporadic norovirus were detected in Chongqing, including GⅡ.16/GⅡ.2, GⅡ.21/GⅡ.3, GⅡ.15/GⅡ15, GⅡ.6/GⅡ.6and GⅡ.16/GⅡ.13. The GⅡ16/GⅡ.13recombinant strains were detected for the first time in China. SimPlot analysis and RDP.4recombinant analysis confirmed the presence of GⅡ.16/GⅡ.13strains. The GⅡ.16/GⅡ.13in Chongqing has99%identity to the Berlin1195/2012/DE （KC832472）, which cause large-scale outbreak of acute gastroenteritis in German. But the virus did not cause the outbreak but only a few sporadic cases annuallyin the Chongqing region.3. Genotypes and recombination of bocavirusA total of504stool specimens were collected, from which49HBoV positive samples were identified, with a detect rate of9.72%. A seasonal distribution of HBoV was obtained, with two peaks in February and July to August observed respectively. No age or gender specific distribution was obtained for the HBoV positive detection. Based on Blast and mega analysis,25HBoVl,23HBoV2and1HBoV3were determined. All the HBoV2strains detected belonged to HBoV-2B genotype.Conclusion:（1） Most pediatric outpatients with acute diarrhea were caused by virus, among which GⅡ and RVA were the most common ones, followed by the ADV, ASV and SLV. Patients aged between7-24months were most susceptible to norovirus and rotavirus infection. Seasonal pattern of virus positive detection was observed. The detection rate of GⅡ and RVA virus had obvious seasonal characters, but the epidemic seasons were different. The detection rate of GⅡ peaked in summer and autumn, and reached bottom in spring. However, the detection rate of RVA presented a bimodal distribution, with two popular peaks in fall and winter and one bottom in summer. There was a abnormal peak during2012.4-2012.6, which reminded us of a possible outbreak. The GⅡ patients had longer duration of diarrhea before going to outpatient department while RVA patients had more vulnerability to vomiting. The incidence rate of vomiting in mixed infection patients was higher than that of single infection.（2） The predominantnorovirus genotype differed among various years. GⅡ.4Sydney₂012and GⅡ.12/GⅡ.3had replaced GⅡ.42006b and GⅡ.4New Orleans2009tobecame the predominant strains by the year2013. The prevalence of norovirus was typical and closely related to time.On the one hand, the epidemic and disappearance of worldwide epidemic strain-GⅡ.42006b and GⅡ.4New Orleans2009, as well as the epidemic of the latest worldwide epidemic strain GⅡ.4Sydney₂012in Chongqingregion, were consistent with time trend in other regions. On the other hand,the endemic strain GⅡ.12/GⅡ.3, which was mainly prevalent in Asia Pacific region, also had high detection rate during2012-2013in Chongqing region.With the disappearance of GⅡ.42006b, the norovirus detected in Chongqing were all recombinant strains. This epidemic trend model in Chongqing reminded us of the value of recombination of norovirus, especially in ORF1/ORF2overlap regions in the evolution and epidemic of norovirus.（3） The detect rate of HBoV had obvious seasonality, with two peaks in February and July to August respectively. HBoV1and HBoV2were the major genotypes and all the HBoV2strains belonged to HBoV-2B.