Epidemiological characteristics of NoV outbreaks
There were 203 NoV outbreaks from September 2015 to August 2018 in Shenzhen. Of 203 outbreaks, 62 (30.5%) outbreaks were from Nanshan district, 42 (20.7%) outbreaks were from Futian district, 32 (15.8%) outbreaks were from Longgang district, 29 (14.3%) outbreaks were from Luohu district, 23 (11.3%) outbreaks were from Longhua district, 5 (2.5%) outbreaks were from Baoan district, 4 (3.9%) outbreaks were from Pinshan district,4 (3.9%) outbreaks were from Dapeng district and 2 (1.0%) were from Guangming district (Figure1A). Information on the outbreak size was reported for 197 outbreaks (97.0%), ranging from 5 to 115 cases per outbreak(Table1). Of the 203 outbreaks, 91.6% of outbreaks occurred in school settings including child care center (143,70.4%), primary school (23, 11.3%), middle schools (7,3.4%), university (4, 2.0%), nine-year school (8,4.0%) and fifteen-year school (1, 0.5%). 8.4% of outbreaks occurred in non-school settings, including company (5, 2.5%), hotel(4, 2.0%), institution(3,1.5%), hospital(2, 1.0%),restaurant(2, 1.0%) and community (1, 0.5%) (Table 2). The reported outbreaks peaked in cold season, especially during November to March (Figure 1B).
Distribution Characteristics of NoV genotypes
Of 203 outbreaks detected as NoV positive by Real-time RT-PCR from September 2015 to August 2018, 150 outbreaks were genotyped successfully. Of these 150 outbreaks with genotype information, 137 (91.3%,137/150) were classified into GII genogroup and 12 (8.0%,12/150) were classified into GI genogroup. There was 1 (0.6%,1/150) outbreak comprised both GI- and GII-positive samples. A total of 15 capsid genotypes and 15 polymerase genotypes were detected. Among the 150 genotyped outbreaks, the capsid types of GI genogroup outbreaks were GI.2 (3,2.0%), GI.3 (3,2.0%), GI.6 (3,2.0%), GI.5 (2,1.3%), GI.1 (1,0.7%) and the polymerase types were GI.Pb (3,2.0%), GI.P2 (3,2.0%), GI.P5 (2,1.3%), GI.Pd (1,0.7%), GI.P1 (1,0.7%). The predominant capsid types of GII genogroup outbreaks were GII.2 (92,61.3%), followed by GII.3 (13,8.7%), GII.6 (8,5.3%), GII.17 (8,5.3%) and GII.4 Sydney 2012 (7,4.7%). The other GII capsid types including GII.4, GII.8, GII.13 and GII.21 were detected in 3 (2.0%,3/150), 2 (1.3%,2/150), 1 (0.7%,1/150) and 2 (1.3%,2/150), respectively. The predominant polymerase types of GII outbreaks were GII.P16 (92,61.3%), followed by GII.P12 (8,5.3%), GII.P7 (6,4.0%), GII.P17 (3,2.0%), GII.Pe (2,1.3%), GII.P8 (1,0.7%) and GII.P21 (1,0.7%), In addition, we found an novel recombination genotype GII.Pe/GII.17 that had not been fund in Shenzhen before(Table 3).
Genotype distribution and outbreak characteristics
For outbreaks caused by GII.2 strain, 73 (79.3%,73/92) outbreaks occurred in child care center, 7 (7.6%,7/92) outbreaks in primary school, 4 (4.3%,4/92) outbreaks in middle school, 4 (4.3%,4/92) outbreaks in nine-year school, 1 (1.1%,1/92) outbreak in university, 1 (1.1%,1/92) outbreak in hospital, 1(1.1%,1/92) outbreak in company and 1 (1.1%,1/92) outbreak in fifteen-year school. The dominant setting distribution (child care center, primary school, middle school) of GII.2 infection have showed no significantly different (Fisher's Exact Test=3.595, p=0.177). The 13 GII.3 strain outbreaks, 11 (84.6%,11/13) outbreaks occurred in child care center, followed by 1 (7.7%,1/13) outbreak in primary school and 1 (7.7%,1/13) outbreak in nine-year school.
Phylogenetic Analysis of RdRp region and VP1 gene
In this study, all the GII.2 NoV were GII.P16/GII.2 strain. To examine the evolution of the strains, 52 full-length RdRp region sequences of GII.P16/GII.2 strain from Shenzhen and 95 reference sequences from GenBank were collected to analyze. The best substitution model was TN93 (Tamura-Nei)+G (Gamma). After MCMC chains were run for 1.0×108 steps for the RdRp region sequences and the first 10% state was buin-in, effective sample sizes greater than 200 were accepted. The MCC tree shown that the evolutionary rate of the RdRp region of Gll.P16/GII.2 strain was estimated as 2.1×10-3 substitutions/site/year (95% HPD interval,1.7×10-3- 2.5×10-3 substitutions/site/year). The common ancestors of GII.P16/GII.2 strain from Shenzhen and GII.P16/GII.4Sydney 2012 diverged during 2011 to 2012, and the prototype of RdRp region of GII.P16/GII.2 strain from Shenzhen were formed during 2012 to 2013. The pholygentic analyses suggested the RdRp region come from GII.P16/GII.4 Sydney2012 from USA (Figure 2A).
Simultaneously, 72 full-length VP1 gene sequences of GII.P16/GII.2 strain retrieved from Shenzhen and 146 reference sequences from GenBank were used to explore evolutionary rate. The best substitution model was TN93 (Tamura-Nei)+G (Gamma)+I (Invariable). After MCMC chains were run for 2.0×108 steps for the VP1 gene and the first 10% state was buin-in, effective sample sizes greater than 200 were accepted. And the MCC tree shown that the evolutionary rate of the VP1 gene of GII.P16/GII.2 strain was estimated as 2.7×10-3 substitutions/site/year (95% HPD interval, 2.4×10-3-3.1×10-3 substitutions/site/year). The common ancestors of GII.P16/GII.2 strain from Shenzhen and previous GII.P16/GII.2 diverged during 2003 to 2004, and the prototype of VP1 gene of GII.P16/GII.2 strain in Shenzhen was formed during 2013 to 2014. The pholygentic analyses suggested the VP1 gene come from GII.P16/GII.2 (2010) from Japan (Figure 2B).
Nonstructural Polymeras Region of GII.P16/GII.2 recombination strain
To explore the aa mutations of nonstructural region within recombination strains, 14 nearly full-length nonstructural polymerase sequences and 22 full-length reference sequences, including GII.P16/GII.17 (2002), GII.P16/GII.2 (2009-2014), GII.P16/GII.2 (2010-2012), GII.P16/GII.13 (2015), GII.P16/GII.3 (2012-2013), GII.P16/GII.4 (2015-2016), GII.P16/GII.17 (2016-2018), from GenBank were aligned. Sequences data indicated there were 102 (6%) parsim-infomative sites revealed, but no aa mutation in nonstructural region of GII.P16/GII.2 recombination strain. Furthermore, 6 aa substitutions (*77E, R750K, P845Q, H1310Y, K1546Q, T1549A) were found only in recent strains (GII.P16/GII.4 Sydney 2012 and GII.P16/GII.2 recombination strain), 2 aa sites (A644P, A1521V) achieved substitution in GII.P16/GII.2 recombination strains and 1 aa site (S/T753T) retured to anterior aa site. RdRp region could be divided into three highly conserved ingredients according to function and structure, including the fingers, thumb, and palm sub-domains ,which could be organized into Motifs A to G [11]. The result shown 1310 aa site (Motifs G) was substituted (Table 4).
HBGA-Binding Profile, Epitopes Predicted and Epitope A to E sites of GII.P16/GII.2 recombination strain
To explore HBGA-Binding profile, epitopes predicted and epitope A to E sites of GII.P16/GII.2 recombination strain, 72 full-length VP1 gene sequences from this study and 65 reference sequences, including GII.Pc/GII.2 (1976-1978), GII.Ph/GII.2 (1997), GII.P2/GII.2 (1987-2015), GII.P12/GII.2 (2004-2006), GII.P21/GII.2 (2010), GII.Pe/GII.2 (2014), GII.P16/GII.2 (2010-2012), GII.P16/GII.2 (2008-2014) and GII.P16/GII.2 (2016-2018), from 1975 to 2018 were collected and aligned. Sequences data shown 29 (5.3%) parsim-infomative sites were revealed but there were no mutations in the amino acids of the HBGA-Binding profile, epitopes predicted and epitope A to E of GII.P16/GII.2 strain (S2 Table)