3.1 Patients with gastroenteritis
During the period 2015-2019, a total of 961 hospitalized children (under 10 years old), including 609 boys and 352 girls, confirmed providing fecal specimens for rotavirus nucleotide detection. RVA was detected in 183 stool samples via qRT-PCR with an overall positivity rate of 19.0 % (Figure 1). Rotavirus B and C were not detected. The maximum number of samples was tested in May 2017, and the largest number of RVA-positive cases occurred in February 2019 (Figure 1). No statistically significant difference was found between two genders and RVA-infection (p>0.05) (Table 3). Types of fecal specimens collected in this study included watery stool, loose stool, pus and blood stool, and mucus stool, no statistically significant difference existed between four sample types positive for RVA (Table 3). Apart from the symptom of diarrhea, some patients were accompanied by other related symptoms such as vomiting, fever, abdominal pain, and dehydration (Table 3). There was a statistically significant difference between RVA-infection and fever (> 38.5 ℃) (p<0.05). Whereas, no significant difference (p>0.05) was found between RVA-infection and the other three symptoms (vomiting, abdominal pain, and dehydration) (Table 3). 20 RVA-positive patients coinfected with other diarrhea associated viruses: 16 with norovirus, 2 with sapovirus, and 1 with astrovirus. One was coinfected with more than two viruses (norovirus and astrovirus). 6 were coinfected with other pathogenic bacteria: 2 with Salmonella, 3 with EAEC, and 1 with ETEC (Table S1).
3.2 Age, geographical and seasonal features
Overall, most cases (292) were found in the age group of 12-23 months which was also the group with the highest average RVA-positive rate (23.7 %) (Figure 2a). On the contrary, the lowest rate (8.5 %) was observed in the group >60 months with a statistically significant difference (P<0.05, Figure 2a). More than a fifth of gastroenteritis cases were found in July and August, but the RVA-positive rate remained low and with a statistically significant difference (P<0.05, Figure 2b). 44.9% of the RVA-positive case was found in December, although gastroenteritis cases were rare in winter (November-January) (Figure 1, Figure 2b). All the patients came from the 7 cities or regions of Shanxi Province, the highest prevalence (29.5%) was observed in Yangquan, followed by 22.4% in Xinzhou, 21.8% in Lvliang, 21.1% in Northern Shanxi, and 16.9% in Taiyuan (Figure 2c). The patients from Southern Shanxi had the lowest average positive rate (11.6%, Figure 2c).
3.3 VP7 and VP4 genotyping
159 samples were genotyped for VP7 and 183 samples were successfully genotyped for VP4. G9 was the prevalent G-genotype, representing 76.0% (139/183) of all samples genotyped, followed by G3 (13/183, 7.1 %), G2 (6/183, 3.3%), and G1(1/183, 0.5 %) (Figure 3a). P (174/183, 95.1 %) was the prevalent P-genotype, followed by P (9/183, 4.9%) (Figure 3b). 159 strains were confirmed both G- and P-genotype. G9P was the most commonly detected genotype (139/183, 76.0 %), followed by G3P (13/183, 7.1 %), G2P (6/183, 3.3 %), G1P (1/183, 0.5 %), and G9P (1/183, 0.5 %) (Figure 3c).
3.4 Geographical distribution of RVA genotypes
Due to the long distance between the patients and the designated hospital, cases from two northern cities (Datong and Shuozhou) and four southern cities (Linfen, Yuncheng, Changzhi, and Jincheng) were less compared with the central cities of Shanxi Province, therefore, the cases from these cities were divided into two independent parts (Northern Shanxi and Southern Shanxi) for next analyzing. G9P was the dominant genotype and occupied for more than 80% of RVA positive cases in most cities or regions of Shanxi Province (Figure 4). The only G1P and G9P were detected in Taiyuan (2016) and Southern Shanxi (2018), respectively (Figure 4). G2P was detected in Taiyuan, Xinzhou, Lvliang, and Yanquan; G3P was detected in Taiyuan, Xinzhou, Lvliang, and Southern Shanxi. Strains that cannot be typed (GntP and GntP) were found in most areas in Shanxi province, except Yangquan (Figure 4).
3.5 Phylogenetic analyses of VP7 genes
The G1-G2-G3 VP7 tree was constructed based on 18 Shanxi strains sequenced in this study and 13 representative members (Figure 5, Table 2). The G1 lineage contained the only Shanxi strain (SX/2016/073) and two representative strains (WZ202 and Kerala-RV01) detected in China and Indonesia with 95.9% and 99.8% nucleotide similarity, respectively. SX/2016/073 shared 90.8% nucleotide similarity and 92.8% amino acid similarity with the RotaTeq G1 strain RotaTeq-WI79-9 (Figure 5, G1). 5 Shanxi G2 strains (SX/2015/136, SX/2017/267, SX/2019/270, SX/2014/140, and SX/2016/011) were closely related to a Japanese strain (Tokyo 17-10) detected in Tokyo in 2017, with 99.8% – 99.9% nucleotide similarities. The other Shanxi G2 strain (SX/2018/393) was related to a South Korea strain (Seoul-710) with a nucleotide similarity of 94.9%. All the Shanxi G2 strains shared 92.7% - 92.9% nucleotide similarity and 95.1% - 95.5% amino acid similarity with the RotaTeq G2 strain RotaTeq-SC2-9 (Figure 5, G2). 10 Shanxi G3 strains (SX/2017/001, SX/2017/021, SX/2017/080, SX/2017/172, SX/2017/020, SX/2017/473, SX/2017/082, SX/2017/047, SX/2018/089, and SX/2018/146) were closely related to the representative strains (E2432 and Tokyo 17-08) with high nucleotide similarities (99.3% - 99.6%). The other Shanxi G3 strain (SX/2017/271) was related to 1CR7 detected in South Korea in 2017, with 99.3 % nucleotide similarity. All the Shanxi G3 strains shared 81.8% - 94.1% nucleotide similarity and 92.5% - 97.7% amino acid similarity with the RotaTeq vaccine G3 strain RotaTeq-SC2-9 (Figure 5, G3).
63 Shanxi G9 strains and 10 representative members were selected for the G9 phylogenetic analyses (Figure 6, Table 2). Except for SX/2015/069, 62 Shanxi G9 strains fell into 2 minor lineages (I and II). 55 Shanxi G9 strains (belong to lineage 1) were closely related to each other with 99.2% – 99.9% nucleotide similarity, clustering with other strains isolated in Japan (Tokyo 18-43), USA (VU12-13-101), Thailand (T152), and China (SC6, Hu/JS2013, BJ-Q794) isolated in previous studies during the years 2011 and 2018 (Figure 6, lineage I). 7 Shanxi G9 strains (SX/2015/061, SX/2015/065, SX/2015/081, SX/2015/059, SX/2015/132, SX/2015/156, and SX/2016/002) belong to lineage 2 were closely related to each other with 99.4% - 99.9% nucleotide similarity, clustering with a Chinese strain (km15119) isolated in Yunnan Province in 2016 (Figure 6, lineage II). The SX/2015/069 was separate from other Shanxi G9 strains, but still has 99.2% nucleotide similarity with the reference strain HU/JS2013 (Figure 6).
3.6 Phylogenetic Analyses of VP4 genes.
The P-VP4 tree was constructed based on 9 Shanxi strains and 2 representative members (Figure 7, Table 2). 8 Shanxi strains (SX/2015/136, SX/2019/271, SX/2019/284, SX/2018/400, SX/2019/270, SX/2016/041, SX/2017/267, and SX/2015/140) were closely related to WZ189 detected in Zhejiang Province (China) in 2017, with high nucleotide similarities (99.7 % - 99.9 %). The other Shanxi strain SX/2018/393 was closely related to the Hu/13-146 detected in Shanghai (China), with 98.0% nucleotide similarity (Figure 7).
The P-VP4 tree was constructed based on 93 Shanxi strains and 6 representative members (Figure 8, Table 2). The Shanxi G9 strains fell into 2 minor lineages (I and II, Figure 8). 87 Shanxi P strains (belong to lineage 1) were closely related to each other with 96.9% - 99.9% nucleotide similarity, clustering with a Japanese strain (Tokyo 18-50) and 3 Chinese strains (Z1602, km15119, SC1) isolated in previous studies during the years 2011 and 2018. The Shanxi P strains belong to lineage 1 shared 91.9 % - 93.0 % nucleotide similarity and 93.1% - 94.0% amino acid similarity with the RotaTeq vaccine P1a strain RotaTeq-WI79-4 (Figure 8, lineage I). The other 6 Shanxi P strains (SX/2016/124, SX/2016/172, SX/2016/112, SX/2015/144, SX/2015/117, and SX/2015/069) belong to lineage 2 were closely related to a Chinese strain Hu/JS-2012 detected in Jiangsu Province in 2012, with 98.9 % - 99.3% nucleotide similarities (Figure 8, lineage II). The Shanxi P strains belong to lineage 2 shared 87.6% - 87.7% nucleotide similarity and 88.4% - 88.8% amino acid similarity with the RotaTeq vaccine P1a strain RotaTeq-WI79-4 (Figure 8).