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A total of 964 (17.44%, 964/5529) RSV positive samples were identified from 5529 enrolled patients between January 2015 and December 2019 from 14 hospitals located in Beijing, Shanghai, Chongqing, Jilin, Hebei, Ningxia, Zhejiang, Guizhou, Liaoning, Heilongjiang and Guangdong provinces. Over the total research period, RSV-A was predominant, except in 2016 (Fig. 1). The ages of these patients ranged from 1 month to 13 years with a median age of 3.5 years. The male-to-female ratio was 1.92:1 (634 boys and 330 girls).
Figure 1. The phylogenetic dendrogram based on the Chinese RSV sequences from 2015 to 2019 and reference sequences with genotype information worldwide. A, B Detailed phylogenetic trees of the RSV-A and RSV-B taxa were analyzed, respectively. The neighbour-joining method was used to infer the topology by the MGEA 6.06 software. The percentage of bootstrap values (percentage of 1000 pseudoreplicate datasets) over 70% are shown at the nodes of the trees. Filled circles with different colors indicate the all the Chinese sequences from different years (red 2015, yellow 2016, purple 2017, green 2018, blue 2019). The scale bars represent substitutions per base pair per indicated horizontal distance.
Of these 964 RSV positive samples, 524 (54.36%) samples were identified as RSV-A, 183 (18.98%) samples were identified as RSV-B, 23 (2.39%) samples were identified as both RSV-A and B, and 234 (24.27%) samples were not tested due to insufficient specimen volume. Some other respiratory viruses were also detected in 311 (32.26%) RSV positive samples, while the co-detection rates were 37.60%, 29.51% and 34.78% in RSV-A, RSV-B and RSV-A & -B, respectively. The main respiratory viruses co-detected with RSV were Enterovirus/Rhinovirus (EV/HRV) (149, 15.46%), Bocavirus (HBoV) (72, 7.47%), Adenovirus (AdV) (49, 5.08%), Parainfluenza (PIV) (41, 4.25%), Human Metapneumovirus (hMPV) (40, 4.15%) and Influenza Virus (Flu) (40, 4.15%). The co-detection rates were 30.40% (176/579), 36.25% (95/262), 27.55% (27/98), and 52.00% (13/25) in the ages of ≤ 1 year, 1-3 years, 3-6 years, and > 6 years, respectively (P < 0.05).
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A total of 535 RSV HVR-2 sequences of G gene was obtained in this study, including 353 RSV-A and 182 RSV-B. Combined with the 182 sequences (168 RSV-A and 14 RSV-B) retrieved from GenBank between 2015 and 2019, 717 HVR-2 sequences of G gene were performed molecular phylogenetic analysis (Table 1 and 2).
Province/Municipality Beijing Chongqing Jilin Hebei Ningxia Zhejiang Guizhou Liaoning Guangdong Fujian Gansu Total 2015 23 (23) 1 (1) 66 (0) 23 (23) 11 (11) 116 (116) 66 (28) 10 (0) 316 (202) 2016 2 (2) 5 (5) 9 (9) 16 (16) 2017 1 (1) 1 (1) 3 (3) 5 (5) 3 (3) 16 (16) 1 (0) 30 (29) 2018 16 (16) 1 (1) 31 (31) 29 (29) 1 (1) 53 (10) 131 (88) 2019 18 (18) 10 (0) 28 (18) Total 60 (60) 1 (1) 66 (0) 28 (28) 13 (13) 159 (159) 34 (34) 4 (4) 145 (54) 10 (0) 2 (0) 521 (353) Numbers shown are the sum of sequences collected in this study (shown in parentheses) and retrieved from GenBank database. Table 1. Distribution of RSV-A G gene HVR-2 sequences collected in the mainland of China from 2015 to 2019.
Province/Municipality Beijing Shanghai Chongqing Jilin Hebei Zhejiang Guizhou Liaoning Heilongjiang Guangdong Fujian Total 2015 7 (7) 3 (3) 14 (6) 43 (43) 27 (27) 2(2) 4 (4) 5 (0) 105 (92) 2016 5 (4) 1 (1) 3 (3) 20 (20) 21 (21) 50 (49) 2017 2 (2) 1 (1) 3 (3) 2018 6 (6) 2 (2) 13 (13) 1 (1) 22 (22) 2019 3 (3) 13 (13) 16 (16) Total 21 (20) 1 (1) 6 (6) 14 (6) 63 (63) 52 (52) 14 (14) 1 (1) 2 (2) 17 (17) 5 (0) 196 (182) Numbers shown are the sum of sequences collected in this study (shown in parentheses) and retrieved from GenBank database. Table 2. Distribution of RSV-B G gene HVR-2 sequences collected in the mainland of China from 2015 to 2019.
Phylogenetic analysis of 521 RSV-A sequences showed that 512 (98.27%) fell within genotype ON1 with the mean genetic distance of 0.029, 6 (1.15%) within genotype NA1 with the mean genetic distance of 0.052, and 3 (0.58%) within genotype GA5 with the mean genetic distance of 0.040. Almost all the RSV-B sequences clustered into BA9 genotype (193, 98.47%) with a mean genetic distance of 0.025; except three sequences were clustered into genotype SAB4 (mean genetic distance of 0.020). Only genotypes ON1 and BA9 were detected after December 2015 in this study.
For the genotype ON1, all the sequences after 2015 formed 10 lineages presenting temporal distribution. Most of the sequences from 2015-2016 were clustered together and formed lineage 1, 3-6, and 10; while most of the sequences from 2017-2019 formed lineage 2 and 7. Consistent with genotype ON1, the majority of BA9 sequences from 2017-2019 formed a new lineage 7, while these sequences from 2015-2016 separated into other 6 lineages (Fig. 1).
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The deduced protein lengths of the RSV-A HVR-2 were 86 (genotypes NA1), 87 (genotypes GA5) and 110 (genotype ON1) amino acids, respectively. All the RSV-A sequences from China between 2015 and 2019 were analyzed to determine the amino acid variations (Fig. 2). Seventy-three amino acid changes were detected in the 512 sequences of HVR-2 of genotype ON1 compared with the reference (ON67-1210, GenBank accession number: JN257693). For the frequency of the amino acid changes, 35 sites were more than 1%, however, other 38 sites with frequency less than 1% were not shown. Only 13 sites more than 5% were identified [V225A (8.20%), T245A (5.27%), T249I (30.47%), H258Q (23.83%), E262K (38.28%), H266L (23.83%), E271K (6.25%), L274P (17.97%), E286K (6.45%), Y297H (7.42%), L298P (18.36%), Y304H (8.59%), L310P (5.86%)], which contained 6 sites more than 10% in them (Fig. 2). All the 196 sequences with amino acid change E262K clustered into lineage 10, including all the 156 sequences with amino acid changes T249I. For the 196 sequences, 161, 12, 6 and 17 of them were from 2015, 2016, 2017 and 2018 respectively. Almost all the sequences with amino acid changes H258Q and H266L were from Beijing, Zhejiang, Ningxia, Guangdong and Jilin municipality/provinces between 2017 and 2019, except 5 from Zhejiang and Fujian provinces in 2015. The two substitutions were co-occurrences, while all sequences with these two substitutions clustered into lineage 7 (Fig. 1).
Figure 2. Summary of amino acid changes and frequencies in HVR-2 of the G gene of genotype GA5 (n = 3), NA1 (n = 6), ON1 (n = 512), SAB4 (n = 3), and BA9 (n = 193) comparing with their reference sequences. The earliest sequence of each genotype was used as the reference (GA5: Mon/1/01, AF516135; NA1: NG-016-04, AB470478; ON1: ON67-1210, JN257693; SAB4: Cam2009-0351, JN119976; BA9: BA/100/04, DQ227395). Only sites with a substitution frequency of more than 2% are indicated. For the RSV-A ON1 and RSV-B BA9 genotypes, these substitution frequency more than 5% or 10% were marked with orange and red label, respectively.
For the genotype NA1, there were 10 specific amino acid changes compared with the reference sequence (NG-016-04, GenBank accession number: AB470478) from Japan in 2004. Specific amino acid substitutions were P222S (66.67%), D237N (100%), N251Y (50.00%), N260S (100%), T269S (33.33%), N273Y (83.33%) and L274P (33.33%). Compared with the reference sequence (Mon/1/01, GenBank accession number: AF516135) from Uruguay in 2001, there were 7 amino acid changes in the genotype GA5, including E224D (66.67%), T227I (66.67%), E232D (66.67%), T243I (100%), S267L (33.33%) (Fig. 2).
The amino acid variations analyses were also performed on the HVR-2 regions of RSV-B protein G between 2015 and 2019 (Fig. 2). There were 5 and 44 amino acid substitutions in SAB4 and BA9, compared with their respective reference sequences (Cam2009-0351, GenBank accession number: JN119976 and BA/100/04, GenBank accession number: DQ227395). For the amino acid changes frequency of the genotype BA9, 14 sites were more than 5% and 9 sites were more than 10% (Fig. 2). Compared with the reference (DQ227395), the deduced 20 amino acid duplicated regions of all genotype BA9 sequences have 10 amino acid substitutions, containing R262G (2.07%), P264T/A (98.45%, 1.55%), L267S/P (98.45%, 1.55%), I270T (3.63%), A271V (16.06%), T275A (2.07%), T276A (5.18%), S269F (1.04%), L272P (1.55%) and D273N (1.55%) (Fig. 2). All the sequences with amino acid changes T290I and T312I were from 2018 and 2019, which clustered into lineage 7 (Fig. 1). The amino acid change P237H (8.81%) was only observed in 17 sequences from Hebei Province between 2015 and 2016, which belonged to the lineage 1 (Fig. 1). For the genotype SAB4, specific amino acid substitutions were found at P216Q (33.33%), T229A (66.67%), E258K (66.67%), I280T (100%) and F287S (33.33%).
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N-X-S/T, where X was not Pro, was identified as the N-glycosylation site. Three predicted N-glycosylation sites (237, 250 and 294) were relatively conserved among the genotype GA5 between 2015 and 2019. For the genotype NA1, three predicted potential N-glycosylation sites were located at 237, 251 and 294, while N251Y amino acid substitution was identified in 50% of the sequences, respectively. In contrast, only one N-glycosylation site 237 was identified among the genotype ON1 sequences. For the RSV-B, two N-glycosylation sites were identified and conserved among all the sequences of genotypes SAB4 and BA9 in this study. The sites were 276 and 290 for the genotype SAB4, while for the genotype BA9 sequences the sites were 296 and 310.
The protein G also contains numbers of serine and threonine residues which are potential O-glycosylation sites (Wertz et al. 1985). Analysis of the HVR-2 predicted 2-10 potential O-glycosylation sites among genotype ON1 sequences, and 12-24 potential O-glycosylation sites among genotype BA9 sequences between 2015 and 2019 (G-scores: 0.5-0.9) (Supplementary Table S3 and S4). The amino acid positions most likely to have O-glycosylation are T219, T220, T227, T228, T231, S270, S275, S277, T281, S283 for genotype ON1 (refer to GYYF-57), and T218, T222, T227, T228, T232, T236, T240, T244, S245, T246, S249, T250, T255, S257, T260, S265, T280, T281, S285, T294, S297, T298, T300, S304 for genotype BA9 (refer to ZJ-293).