key: cord-0964753-b3r96swe authors: chen, Yinghu; Yao, Hangping; Fu, Junfen; Shu, Qiang; Chen, Zhimin; Wu, Nanping; Ye, Sheng; Wang, Wei; Ni, Yan; Shang, Shiqiang; Li, Wei; Zheng, Jishan; Li, Shibo; Hong, Liang; Zhang, Qi; Xu, Weize; Chen, Junsong; Fan, Lingyan; Cang, Xiaohui; Wang, Jianbing; Lu, Xiangyun; Cao, Qingyi title: The low contagiousness and new A958D mutation of SARS-CoV-2 in children: An observational cohort study date: 2021-08-25 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2021.08.036 sha: 28656f13d156d50e32b3971e106ba0299f2348e5 doc_id: 964753 cord_uid: b3r96swe AIMS: To explore the contagiousness and new SARS-CoV-2 mutations in pediatric COVID-19. METHODS: This cohort study enrolled all pediatric patients admitted to 8 hospitals in Zhejiang Province of China between 21 January and 29 February 2020, their family members and close-contact classmates. Epidemiological, demographic, clinical and laboratory data were collected. Bioinformatics was used to analyze the features of SARS-CoV-2. Individuals were divided into 3 groups by the first-generation case: Groups 1 (unclear), 2 (adult), and 3 (child). The secondary attack rate (SAR) and R(0) were compared among the groups. RESULTS: The infection rate among 211 individuals was 64%(135/211). The SAR in Groups 2 and 3 was 71%(73/103) and 3%(1/30), respectively; the median R(0) in Groups 2 and 3 was 2 (range: 1-8) and 0 (range: 0-1), respectively. Compared with adult cases, the SAR and R(0) of pediatric cases were significantly lower (p<0.05). We obtained SARS-CoV-2 sequences from the same infant's throat and fecal samples at a two-month interval and found that the new spike protein A958D mutation detected in the stool improved thermostability theoretically. CONCLUSIONS: Children have lower ability to spread SARS-CoV-2. The new A958D mutation is a potential reason for its long residence in the intestine. As the number of pediatric COVID-19 cases increase, exploring the contagiousness of these patients is important for prevention and control, especially in families, kindergartens, and schools. However, data about the contagiousness of pediatric COVID-19 are limited. In Zhejiang Province of China, imported cases from Hubei Province occurred in January 2020 and were followed by second-generation cases in families and communities. The epidemic was essentially controlled by the end of February 2020 by isolating and treating cases and by tracking and screening family members and close-contact persons. As of 7 May 2020, a total of 1268 cases (including adults and children) had been diagnosed in Zhejiang Province, China, and close contacts were traced; 1 adult died . Some children have no digestive tract symptoms with persistent fecal nucleic acid positivity . The virus can be isolated from stool samples at the early stage of the disease, and some mutations were detected (Jin et al., 2020) . The genomic and epidemiological surveillance around the world is highlighted in order to determine the ongoing virus evolution rapidly (Cella et al., 2021) . Despite the abundant SARS-CoV-2 variability (Lokman et al., 2020) , one key question remains with regard to whether these mutations have any functional impact on clinical features. For example, researchers have found that the Europe-prevalent D614G mutation of the S protein may increase the infectivity of SARS-CoV-2 (Korber et al., 2020) . In general, the duration of intestinal excretion of viral nucleic acids varies greatly among patients, and exploring SARS-CoV-2 mutations in the stool of patients who excrete viral nucleic acids for a long period may facilitate the detection of viral mutations with potential clinical impact. We conducted a retrospective cohort study at 8 hospitals (i.e., authors' units) in Zhejiang Province, China, between 21 January and 29 February 2020. The study population included ① all pediatric cases (0-18 years of age) with laboratoryconfirmed COVID-19 admitted to these hospitals and ① family members and closecontact classmates of the pediatric patients. The exclusion criteria were as follows: ① first-generation cases of the family including both adults and children; ① eligible participants exposed to other COVID-19 patients. The study population was divided into 3 groups according to the first-generation case. Group 1, the first-generation case was unclear, and all family members were from the epidemic area; Group 2, the firstgeneration case involved an adult (>18 y); Group 3, the first-generation case was a child (①18 y). the guardians of the patients. The procedures for sample collection, RT-PCR analysis, and interpretation of results have been previously described (Huang et al., 2020 Germany), following the manufacturer's recommendations. Data analysis was conducted at BGI according to a published protocol . To perform phylogenetic tree construction and mutation analysis, 2019 SARS-CoV-2 genomic sequences were retrieved from the following databases : Multiple-sequence alignment of the SARS-CoV-2 and reference sequences was performed in Muscle (Edgar RC., 2004) . The phylogenetic tree was constructed using the maximum likelihood method with MEGA X software (Kumar et al., 2018) . Variations in this isolate (SARS-CoV-2-CHZJU) were called using MN908947 as the 15/36 reference sequence. The function of the variation series was annotated manually. The cryo-EM structure of the spike protein of SARS-CoV-2 (PDB entry: 6 VXX) (Walls et al., 2020) was viewed with PyMOL (Janson et al., 2017) ; mutagenesis was also performed and visualized with PyMOL. Continuous variables are expressed as medians (ranges). Kruskal-Wallis H tests were used for comparisons among 3 groups, and Wilcoxon rank-sum tests were used for comparisons between 2 groups. Count data are expressed as numbers (%), and Pearson's chi-squared test was applied for comparisons among groups. We judged a two-sided p value less than 0.05 as statistically significant. All data analyses were carried out using SPSS version 20. This study enrolled all 46 pediatric COVID-19 cases admitted to 8 hospitals between 21 January and 29 February 2020, including 45 pediatric cases in 40 families with 202 family members and 1 pediatric case with 8 close contacts (4 close-contact classmates and 4 roommates). No other students or staff at a residential school became infected 16/36 due to contact with the pediatric patient ( Figure 1 ). The study enrolled a total of 211 individuals from among 215 eligible individuals, and 4 were excluded according to the exclusion criteria ( Figure 1 ). Among the 211 individuals, the total infection rate was 64% (135/211), and the total secondary attack rate (SAR) was 60% (112/188) ( Table 1 ). SARs in Group 1, 2 and 3 were 69% (38/55), 71% (73/103) and 3% (1/30), respectively. The median R 0 in Group 2 and 3 was 2 (range: 1-8) and 0 (range: 0-1), respectively (details on R 0 in each family are listed in appendix pages 1-30). Statistical analysis revealed significant differences (p<0.05) in the SAR and R 0 between Group 2 and 3, suggesting that the ability of children to spread disease is significantly lower than that of adults (Table 1) . Moreover, the children in our study were often the last family members to develop symptoms. Between Group 1 and 2, there was no significant difference (p>0.05) in SAR between children and adults ( (Table 2) . Whole-genome sequencing of SARS-CoV-2 using throat swab and fecal samples from the same infant with a two-month interval. After the throat swab nucleic acid test became negative in pediatric cases, virus cultures based on stool samples were negative, even though 55% ( April; SARS-CoV-2 cultures were carried out. Only the virus culture based on the early pharyngeal swab was positive; it was sent to BGI for whole-genome sequencing. The stool virus cultures were all negative. The Ct value for the stool sample obtained on 3 April was 31.5. However, as the stool culture did not show a cytopathic effect on the third day, the culture supernatant was sent for whole-gene sequencing, and fortunately, we obtained the whole-genome sequence. The evolutionary tree between SARS-CoV-2 sequences was highly similar. The phylogenetic evolutionary tree showed high similarity between the SARS-CoV-2 sequences isolated (Figure 2 .A). The average difference between the sequences is 0.019%, and the sequence difference between this isolate (SARS-CoV-2-CHZJU) and the remaining 50 strains was found to be 0.08-0.10%. Compared to the outgroup, all SARS-CoV-2 strains clustered into one specific group. Among the two reference sequences used as outgroups, the bat-SL-CoVZC45 strain isolated from bats was closest to SARS-CoV-2, with an average sequence difference of 14%; the SARS 19/36 coronavirus was slightly separated, with a sequence difference of 27.8%. The new A958D mutation improves theoretically viral thermostability. H), which we expect would dramatically improve the thermostability of the spike protein. SARS-CoV-2 is a highly contagious virus that has caused a global pandemic. To explore the contagiousness of pediatric cases, close-contact persons in families or 20/36 schools were divided into 3 groups according to the first-generation case. The firstgeneration case had close contact with family members from 2 days before the onset of symptoms to 1 day after onset, and this contact period was within the contagious time of the first-generation case. There were no significant differences among the groups in age or sex. In the 21 families of Group 2, the median R 0 was 2 (range: Maltezou et al., 2020). Children are young with less muscle mass, and the ability to produce droplets when coughing is lower in children than in adults. In addition, children tend to swallow respiratory secretions instead of expectorating, which reduces their ability to transmit virus. COVID-19 is similar to tuberculosis in that children are significantly less contagious than adults. Our study shows that COVID-19 in children mainly comprises upper respiratory infections and pneumonia, followed by asymptomatic infection. This result is consistent with Wuhan's and national reports, but the proportion of asymptomatic infections in our study was higher Dong et al., 2020) . Fever, cough and runny nose are the main symptoms. Among pediatric COVID-19 patients, nearly one-third had pneumonia, and a quarter exhibited lung ground-glass opacities, though both rates were lower than those reported in Wuhan . After the respiratory viral nucleic acid test became negative, fecal samples in half of the pediatric cases were still positive for nucleic acid. In a 3-month-old infant, the fecal nucleic acid test continued to be positive for 53 days after the pharyngeal swab became negative. The Ct value was approximately 30, suggesting that SARS-CoV-2 first multiplied in the respiratory tract, causing upper respiratory infection; after SARS-CoV-2 was swallowed, it multiplied in the intestine, resulting in asymptomatic infection of the digestive tract and excretion of viral nucleic acid for a long time. As primary human airway epithelial cells and gut enterocytes are permissive to SARS-CoV-2 infection (Parolin et al., 2021) . Overall, the pediatric COVID-19 patients shed viral nucleic acid in the feces for a long time after the throat swab nucleic acid test became negative, which has also been reported previously . SARS-CoV-2 enters host cells through the receptor angiotensin-converting enzyme-2 (ACE-2), and ACE-2 is highly expressed in lung AT2 cells, esophageal epithelial cells, stratified epithelial cells and absorptive enterocytes in the ileum and colon (Harmer et al., 2002; Lalitha Guruprasad, 2020) . Other research teams have also isolated SARS-CoV-2 from stool samples (Jin et al., 2020) . The intestines of children are susceptible to SARS-CoV-2 infection, which may be related to their immature function and imperfect intestinal flora. The sequence isolated from the infant (SARS-CoV-2-CHZJU) is highly homologous to known strains. The sequence difference between this isolate (SARS-CoV-2-CHZJU) and the 50 known strains is 0.08-0.10%. This isolate (SARS-CoV-2-CHZJU) does not harbor many mutations, except for the orf1ab region (hypervariable region), which is highly variable in all SARS-CoV-2 strains worldwide. Compared to the reference sequence (MN908947), only five missense mutations (S, ORF3a, E, M, and ORF8) were found in the relatively conserved region, and two of these mutations (S and ORF8) occur frequently in multiple SARS-CoV-2 strains. Compared to the sequence isolated from throat swabs, the 24435C>A mutation detected in stool leads to a missense A958D mutation in the S2 domain of the spike protein. Because A958 faces R1014 of the neighboring helix, we predict that the A958D mutation will inevitably lead to salt bridge formation between D958 and 23/36 R1014, which will dramatically improve the thermostability of the spike protein. A958 is located on HR1 of the S2 subunit; therefore, it is possible that this mutation may also affect cell-cell fusion after viral attachment to the host cell receptor. We deduce that the new A958D mutation might have been a potential reason for SARS-CoV-2 long residence in the infant's intestines without apparent symptoms. 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