Molecular Epidemiological Study On Human Caliciviruses Of Acute Diarrhea Patients In Zhejiang Province

[Background and Aims]Human caliciviruses (HuCVs), including norovirus (NV) and sapovirus (SAV), are recognized as the major cause of acute nonbacterial gastroenteritis affecting people worldwide, and also as the common etiologic agents in acute sporadic gastroenteritis in children worldwide, second only to rotavirus (RV).Norovirus, also known as Norwalk-like virus, was originally isolated by Kapikian, etc. in1972in the town of Norwalk, Ohio, from the stools of patients with non-bacterial gastroenteritis outbreaks by immune electron microscopy method. Sapovirus, formerly known as Sapporo-like virus, was named due to its first discovery in Sapporo of Japan in1977, and was one of the major pathogens in acute nonbacterial gastroenteritis in humans and pigs. For a long time, it had been seriously underestimated that calicivirus harmed human health due to the lack of simple, sensitive detection methods on calicivirus.In recent years, with the research on calicivirus molecular epidemiology, its importance and dangers have gradually been comprehensive and systematically understood. In January2005, an outbreak of NV infection was announced in Canada and the United States, which caused widespread concern. In November2006, NV mass infection events took place in Japan, Singapore, Italy and other places one after another. Especially, the most severe infectious gastroenteritis outbreak in Japan in25years had occurred total35.76million people infected with NV in less than two months, causing the world’s attention. In the same period, NV epidemic situation also happened in China including Beijing, Guangdong, Foshan, Fujian and other provinces. According to the monitoring of national monitoring network of viral diarrhea in11provinces diarrhea in children (<5years), NV detection rate was15%. As the virus was highly infectious and often led to sudden public health incidents, the U.S. had listed it as Group B bioterrorism factor. U.S. Centers for Disease Control (CDC) latest estimated that96percent of non-bacterial gastroenteritis outbreaks were due to NV, whereas S AV was the main pathogens of viral diarrhea of infants.Our study on NV started late. In1995, Fang Zhaoyin etc. detected NV firstly in infant diarrhea stool samples in China. Since then, NV was detected in fecal specimens from children with diarrhea in Beijing, Changchun, Hebei Province, Lanzhou, Guangzhou, Fuzhou. Recently, according to Fang Zhaoyin’s studies on HuCVs in infant diarrhea stool from13regions of China in1999-2005, the positive rate of HuCVs was8.3%-38.6%, the detection rate of NV was lower slightly than that of RV, which indicated that NV infection was also quite common in diarrhea cases.Sapovirus have been reported all around the world. Twenty-three persons (including six adults) in a kindergarten of Japan infected with SAV virus in February,2005. HUA Xiu-guo etc.who were from Shanghai Jiao Tong University of china isolated a new calicivirus from people and piglet with unexplained diarrhea in2008, which were proved to be SAV after the identification and animal innoculative experiment. Currently, the international research on the overall level of SAV is still in its initial stage.In spite of the prevalence of HuCVs infections in China, it is short of systematic molecular study of popular features of HuCVs infection in Zhejiang region.Therefore, to investigate the molecular-epidemiologic characteristics and evolution of pandemic strain of HuCVs in acute diarrhea patients in Zhejiang Province from2009to2011can provide the scientific basis to prevent and treat the acute diarrhea caused by HuCVs. It’s also significant to the prevention and control on acute diarrhea in China. [Objects and Methods]1. Experiment ObjectsA total of3153cases of acute diarrhea patients came from five sentinel hospitals intestinal clinic(including Hangzhou, Ningbo, Shaoxing, Huzhou, Quzhou) from January2009to January2011, including children less than5years old and adults.Diarrhea more than3times per day, or significant abnormal stool (watery, mucoid or slimy kind) is defined as cases of diarrhea.2. MethodsCollected more than3153cases of consecutive patients with acute diarrhea, stool specimens, and also to investigate, fill out the questionnaire, telephone follow-up complete the questionnaire during the monitoring period2009-2011. Rotavirus (RV) and HuCVs (NV and SAV) of these specimens were detected by multiplex PCR method and select the part of the amplified positive products were sequenced, the application DNAMAN(5.1.0.0) and MEGA4.0software and related reference strains of calicivirus multiple sequence alignment and phylogenetic analysis.[Results]1. A total of3153acute diarrhea specimens were detected in this study from January2009to January2011in Zhejiang province, the detection rate of HuCVs (18.42%) was lower slightly than that of RV (20.10%).2. A total of265samples were positive for NV (12.36%,265/2144;52positive for NV-G I genotype,213positive for NV-GⅡ genotype), in addition,130were positive for SAV (6.06%,130/2144). The highest positive rate was in<5years old group, followed by15to65age group and5to15age group. Acute diarrhea caused by HuCVs in Zhejiang Province had occurred throughout the year. Infection rates in winter and spring (November to April) was high, in addition, summer (June to August) was relatively higher.3. Random samples of15NV and10SAV were sequenced. Genotypes of HuCVs showed that NV-G Ⅰ subtypes were G Ⅰ-1(3strains) and G Ⅰ-2(1strain). NV-G Ⅱ subtypes were GⅡ-4/2006b variant strains(7strains), GⅡ-2(1strain), GⅡ-7(1strain) and GⅡ-4/2008variant strains(2strains); SAV subtypes were G Ⅰ-2(5strains), G Ⅰ-1(4strains) and GⅡ-1(1strain)[Conclusion]1. HuCVs were one of the major pathogens causing acute diarrhea, the detection rate of which was lower slightly than that of RV. The positive rate of NV was higher than that of SAV. HuCVs mainly infected infants and children less than five years. Prevalent seasons were winter and spring.2. Infection of HuCVs in Zhejiang region showed genetic diversity characteristics. Norovirus included G Ⅰ-1, G Ⅰ-2, GⅡ-2, GⅡ-7, GⅡ-4five genotypes (GⅡ-4/2006b was the predominant strains); Sapovirus included G Ⅰ-2, G Ⅰ-1and G Ⅱ-1three genotypes, and G Ⅰ-2genotype strains was prevalent.