Molecular Epidemiology Of Norovirus Associated With Acute Gastroenteritis In Shanghai And The Preliminary Investigation Of Evolutionary Mechanisms Of The GII.4 Epidemic Strains

Objectives1. To describe the epidemiological characteristics of norovirus （NoV）-associated acute gastroenteritis in Shanghai from 2012.1 to 2013.12 and characterize the evolution pattern of circulating strains;2. To establish the molecular evolution model of NoV GII.4 epidemic strains in vitro to assess the evolutionary tendency and to screen the key informative site in the major capsid protein;3. To determine the receptor binding patterns of the recent GII.4 epidemic strains: Den_Haag₂006b, New_Orleans₂009 and Sydney₂012 and to evaluate their antigenic variation, which aims to investigate the role of receptor switch and antigenic drift in the persistence of GII.4 epidemic strains.Methods1. Molecular epidemiology of norovirus in adults in Shanghai From 2012.1 to 2013.12, stool specimens were collected from adult （≥16 years） outpatients who visited either of the two sentinel hospitals in Shanghai for acute gastroenteritis. Molecular detection and genotyping of NoV were performed and the phylogenetic relationship of the circulating strains has also been comprehensively analyzed.2. Molecular evolution analysis of GII.4 epidemic strains in vitro The full-length of capsid sequences of GII.4 epidemic strains since 1995 （US9596, Farmington_Hills₂002, Hunter₂004, Den_Haag₂006b, New_Orleans₂009 and Sydney₂012） were downloaded from GenBank and Camberwell₁994 was referred to as the prototype. Site variation was analyzed through multiple sequences aligned analysis and the evolutionary tendency was assessed by phylogenetic analysis. And positive selection analysis was employed for screening the key informative sites.3. The preliminary investigation of the molecular evolutionary mechanisms of the GⅡ.4 epidemic strainsThe pET-42a（+）/VP1 expression vector were constructed for the three recent GⅡ.4 epidemic strains （Den_Haag₂006b, New_Orleans₂009 and Sydney₂012）. The VP1 proteins were expressed in Rosetta （DE3） bacterial strain, purified and verified through western-blot analysis. Receptor binding patterns of these three epidemic strains were determined. Serum from health control and convalescent serum from patients infected with GII.4 epidemic strains were employed to investigate the role of receptor switch and antigenic drift in the persistence of GⅡ.4 epidemic strains through cross-reactivity analysis and receptor-binding blockade assays.Results1. Molecular epidemiology of norovirus in adults in Shanghai A total of 846 stool samples from adults （≥16 years） with acute gastroenteritis were collected from 2012.1 to 2013.12. The epidemics level of GI NoV was low throughout the surveillance period, with the positive rate of 2.60%（22 cases）, and no seasonality of GI NoV infection could be distinguished. For GⅡ genogroup, higher epidemics in adults in Shanghai, with the detection rate of 13.83%（117 cases）, were observed. And relatively high epidemics of GⅡ NoV infection were spotted between October and December in 2012. The frequency of NoV-associated acute gastroenteritis in older people is significantly higher than that in young individuals （p<0.05）, while no significant age distribution was spotted for GI NoV.Sequencing and genotyping analysis revealed that the majority of GI NoV circulated in Shanghai turned out to be GI.3 and GI.4, while GII.4 accounted for 93.40% of GIl NoV. Besides, the high epidemics of GII NoV infection between October and December in 2012 is associated with the emergence of a novel GII.4 norovirus strain, termed Sydney₂012. Sequence analysis also demonstrated that this was a recombinant virus between a GlI.e polymerase and GII.4 capsid, with the recombination break point between 5037bp-5062bp, which has also been the dominant circulating strain in Shanghai.2. Molecular evolution analysis of GII.4 epidemic strains in vitro57 varied amino acid sites were detected through multiple sequences aligned analysis for the 188 full-length of capsid sequences of GII.4 epidemic strains, while 40 of the 57 varied sites were located in the P2 region. Interestingly, the same amino acid residues were repeatedly employed by GII.4 epidemic strains from different times in the same varied sites. Phylogenetic analysis of the full-length of the nucleotide sequences through different methods identified similar but not identical trees in which all six identified clusters were represented. And all extant clusters were supposed to arise from the Camberwell cluster. Phylogenetic analysis of each domain of the capsid revealed that only the P2 region has been evolving in a pattern similar to the full-length of the nucleotide sequences.13 informative sites were determined through positive selection analysis, and 9 of them were located in the P2 region.3. The preliminary investigation of the molecular evolutionary mechanisms of the GII.4 epidemic strainsThe receptor binding analysis revealed that the three recent GII.4 epidemic strains, Den_Haag₂006b, New_Orleans₂009 and Sydney₂012, all showed a high affinity to all kinds of ABO histo-blood group antigens. Sero-prevalence of anti-NoV IgG was above 80%in health adults. An intense cross-reactivity of the convalescent serum from patients infected with Den_Haag₂006b or Sydney₂012 with GII.4 epidemic strains in different times were observed, while the convalescent serum can specifically block the interaction of the VP1 protein of the GII.4 epidemic strain at the same time with ABO histo-blood group antigens. Serum samples from health adults didn’t exert any tremendous effects on the blockade of receptor binding.Conclusions1. Periodic epidemics were demonstrated by NoV GII.4 strains, and Sydeny₂012 has replaced Den_Haag₂006b and New_Orleans₂009 and has been the dominant circulating strain in Shanghai.2. P2 region is the key domain for molecular evolution of VP1, and only this region has been evolving in a pattern similar to the full-length of the nucleotide sequences. Due to the limitation of the small genome size, varied sites in VP1 are mainly spotted in P2 domain and the single variation site may exert tremendous influence on receptor binding and antigenicity of VP1.3. The three recent GII.4 strains all have a high affinity with ABO histo-blood group antigens and the receptor binding pattern didn’t change dramatically, while antigenic drift continually to develop, suggesting that antigenic drift may play a key role in the recent GII.4 strains’persistence in human population.