Molecular Epidemiology Of Viral Diarrhea Among Children In Shanghai

[Background] Global deaths from diarrhea among children aged less than 5 years were estimated at 1.87 million, approximately 19% of total child deaths. WHO reported African and South-East Asia Regions combined contain 78% of all diarrhea deaths occurring among children in the developing world. Before the 1970s of 20th century, Most episodes of diarrhea were attributable to bacteria and parasites. With application of electron microscopy and new diagnostics, such as enzyme immunoassay (EIA)/ELISA and PCR to diarrhea, norovirus, rotavirus, adenovirus, sapovirus and astrovirus were discovered and identified as the major agents causing watery diarrhea. Infectious diarrhea caused by those viruses was defined as viral diarrhea, which accounts for most of childhood diarrhea. However, no specific effective drugs can be used for the treatment of viral diarrhea, hence, vaccine is the best and promising strategy for control and prevention of diarrhea. Before the development and implementation of the effective vaccines, we should have a systemic understanding of the epidemiology of viral diarrhea. Recent years, some studies of the viral diarrhea among children have been carried out in China. However, no systemic study for all known viruses related to childhood diarrhea in Shanghai has been reported. Thus, we conduct a prospective study of acute diarrhea among outpatient children from August of 2008 to July of 2009.This study contains three parts:(1) establishment and improvement of gene diagnosis for viruses causing diarrhea; (2) epidemiology of virus diarrhea among children in Shanghai; (3) genotypes distribution and DNA sequence analysis of viruses causing diarrhea.Part 1 Establishment and improvement of gene diagnosis for viruses causing diarrheaObjective:①To develop a rapid and sensitive diagnostic method for detecting astrovirus.②To improve RT-PCR method for norovirus to facilitate direct sequencing of PCR products. Material and methods:①All capsid and full-length sequences of astrovirus were downloaded from the Genbank and multiple-aligned. According to the conserved domain, the specific primers and probe for detecting astrovirus were determined by the software for primers design. Real-time RT-PCR based on the designed primers and Taqman probe were evaluated by the comparison with the conventional RT-PCR.②The original degenerate primers for norovirus in the literature were modifed, and then they were evaluated by dilution of norovirus-positive specimens. Result:①One-step real-time RT-PCR we developed is 1000 fold sensitive than ordinary RT-PCR with the ideal specificity.②The sensitivity of improved RT-PCR method is the same as the original one in the literature. Conclusion:①One-step real-time RT-PCR we developed was a reliable and sensitive method for detection of astrovirus.②The amplified product of improved RT-PCR method for norovirus can be sequenced directly.Part 2 Epidemiology of virus diarrhea among children in ShanghaiObjective:To give a systemic picture of the epidemiology of virus diarrhea in children. Material and methods:910 stool samples were collected randomly from the outpatient children with acute diarrhea during Aug 2008 to Jul 2009. Stool samples were tested for rotavirus antigen by colloidal gold device. One step real-time RT-PCR was used for screening norovirus genotype I and II, sapovirus, human astrovirus. Real-time PCR was used for screening adenovirus and conventional PCR was used to detect postitve specimens again. Results:Out of 910 stool samples, annual detection rates of rotavirus, norovirus, sapovirus, astrovirus and adenovirus are 29.5%,18.1%,6.5%,3.3% and 6.5%. The high activity of rotavirus began in October and peaked in November, December, then tended to decline from January. Norovirus was prevalent year round with the peak season occurring in July, August and September. Sapovirus, astrovirus and adenovirus diarrhea showed no significant seasonal distribution.91.0% of rotavirus diarrhea episodes occurred in children under 3 years old. And 94.0% of children with norovirus diarrhea are less than 3 years old. Similarly,88.1% of sapovirus,85.5% of human astrovirus and 98.3% of adenovirus diarrhea episodes occurred in children under 5 years old. Conclusion:Rotavirus and norovirus are the most common viral agent causing childhood diarrhea in Shanghai and sapovirus, human astrovirus and adenovirus are also common causes of acute diarrhea among children in Shanghai. The peak season of rotavirus diarrhea is autumn and winter, and it is summer and autumn for norovirus diarrhea. But no significant seasonal pattern were found in sapovirus, astrovirus and adenovirus diarrhea. Children aged less than five years old are susceptible to diarrhea associated virus after exposure.Part 3 Genotypes distribution and DNA sequence analysis of virus causing diarrheaObjective:To learn more about molecular epidemiological characteristics of viral diarrhea, and molecular characteristics and genotyping of the viruses, in order to provide background information for vaccine development. Material and methods:The specimens tested positive for the five viruses in the second part of our study were used. All rotavirus-positive stool samples were used to further characterize the G and P genotypes by Nested multiplex RT-PCR. Norovirus, sapovirus, human astrovirus and adenovirus were genotyped by constructing molecular phylogenetic tree after direct sequencing.In addition, norovirus and sapovirus sequence two different regions and phylogenetic analysis was done according to the sequences of both domains following recombination analysis. Results:①Of 178 rotavirus-positive strains successful-typed, G1 (33.7%) was the most common G serotype followed by G3 (20.8), Mixed type(20.4%), G2 (14.6%), G9 (6.7%), G4(2.2%); majority of P genotype belonged to P[8] which counted for 77.8% and P[4] comprised 17%.②The capsid domains of 116 strains were successfully sequenced (GⅡ.4 2006b variants:65 (57.5%), GⅡ.3:35 (31.0%) were, GⅡ.6:7 (6.2%), GⅢ.12:4 (3.5%), GⅡ.14:1(0.9%), GⅡ.2:1(0.9%), GⅠ.4:1(0.9%) and GⅠ.5:1(0.9%)).The RdRp domains of 102 strains were successfully sequenced (GⅡ.4 2006b variants: 64(62.7%), other GⅡ.4:33(32.4%), GⅡ.2:2(2.0%), GⅡ.3:2(2.0%), GⅡ.9:2(2.0%) and GⅡ.c:2 (2.0%). Both capsid and RdRp domains of 91 norovirus strains were successfully sequenced.58 (63.7%) were GⅡ.4(capsid)/GⅡ.4(RdRp),25 were(27.5%)GⅡ.4/GⅡ.3, 2 (2.2%) were GⅡ.6/GⅡ.9,2 (2.2%) were GⅢ.6/GⅡ.4,2 (2.2%) were GⅡ.12/GⅡ.3, 1was(1.1%)GⅡ.12/GⅡ.4,1 (1.1%)was GⅡ.2/GⅡ.2 and 1 was(1.1%)GⅠ.4/GⅠ.4. In all, the capsid genotypes of 33 norovirus strain were not consistent to the corresponding RdRp genotypes, which indicated norovirus recombination.③13 strains were were successfully sequenced by the primer pair of SR80/JV33 and 14 strains were were successfully sequenced by the primer pair of SLV5317/SLV5749.8 stain were successfully sequenced by both primer pairs.8 were GⅠ.1,4 were GⅡ.1,3 were GⅣ,1 was GⅠ.2, 1 was GⅠ.3,1 was GⅠ.3 and 1 was GⅠ.8. Strains sequenced successfully in two domain showed the same genotypes.④20 strains of human astrovirus were were successfully sequenced.12 (60%) were G1 and 8 (40%) were G4. And all the G1 strains were subtyped into 3 clusters including cluster 1b, cluster 1d and cluster 1a. Cluster 1b was most common. G4 all belonged to cluster 4a.⑤58 strains were successfully sequenced.32 (55.2%) were serotype 41,2 (3.4%) were serotype 40,9 (15.5%)were serotype 2,5 (8.6%) were serotype 3,4(6.9%) were serotype 1,3 (5.2%) were serotype 31,1 (1.7%) were serotype 15 and 2 (3.4%) was untypable. Conclusion:①The serotypes of rotavirus circulating in Shanghai were globally common serotypes which are integrated into the current rotavirus vaccines, and G1P[8] maybe come to dominate again。②GⅡ.4 2006b norovirus variants were spreading and becoming dominant in Shanghai.And there were many recombinant norovirus spreading in Shanghai.③Coexistence of multiple genotypes of sapovirus were found in Shanghai. So far, no recombination variants were identified.④Human astrovirus Genotype 1 and 4 were commonly found in Shanghai.⑤adenovirus serotype 41 was the dominant serotype in Shanghai. Serotype 31 could be a pathogenic serotype causing diarrhea.