Dynamic Zero-Covid strategy curtails mutagenesis and emergence of new variants of the SARS-CoV-2


 Within the local outbreak period of SARS-CoV-2 Delta variant in Nanjing and Yangzhou, China, we analyzed the mutation process of the Delta variants in 520 cases, as well as the production, spread and elimination of new mutant strains under the non-pharmaceutical interventions (NPI) strategy. The investigation on distribution of COVID-19 cases and phylogenetic analysis of SARS-CoV-2 genome sequences attributed to tracking the transmission chains, transmission chains were terminated by the isolation of the COVID-19 patients and quarantine of close-contracts, suggesting the importance of NPI in prompting some mutations to disappear and stopping the transmission of new variants. Dynamic zero-Covid strategy has been implemented successfully to against the second-largest local epidemic caused by an imported COVID-19 case in China.

diseases provide effective and referential ideas for the prevention and control of  NPI (to discover and isolate the source of infection) strategy was implemented during the 86 epidemic prevention and control process in Wuhan, China. Remarkably, this strategy yielded 87 3 zero-covid cases in 76 days. In fact, while China has implemented the zero-Covid strategy 88 successfully, attracting global attention, South Korea,New Zealand,Taiwan,Hong Kong,89 Macao, Australia along with other countries or regions are also working hard to implement the 90 zero-Covid strategy. In the second-largest local epidemic caused by an imported  case in Nanjing and Yangzhou, China, following the first-largest local epidemic in Wuhan, we 92 systematically dissected the mutation process of the Delta variants in different individuals, as 93 well as the production, spread and elimination of new mutant strains under the intervention of 94 the NPI strategy. Moscow, Russia arrived at Nanjing Lukou International Airport (NKG). When tested for 99 COVID-19 at the time of entry using the SARS-CoV-2 RT-PCR test, five COVID-19 positive 100 cases were detected amongst the arrivals. Subsequent genome analysis revealed that the 101 passengers were infected with the Delta variants and one of the cases (Nanjing/Yu) carried 102 very high loadings of the Delta variants(Ct =14.5). Two COVID-19 cases were detected on 103 the 5th and 7th day after arrival of the flight 777-300ER at NKG. However, the two cases are 104 unlikely to be the source of infection that subsequently spread locally because they tested 105 negative in the cabin. According to China's quarantine policy, all the arrivers are lodged in a 106 hotel far from the NKG for quarantine and observed for 14 days immediately after 107 disembarking from the plane. Therefore, the newly detected COVID-19 cases did not directly 108 contact the infected COVID-19 cases among the arrivers. One hour after all the passengers and 109 aircrew left the cabin and after prophylactic disinfection, 16 cleaning staff boarded the cabin 110 to clean. Among them, 6 cases of COVID-19 were found during the weekly nucleic acid testing 111 on July 20; five of them had a history of direct contact with the excreta or pollutants of the 112 case-Nanjing/Yu，including two who cleaned the toilets which case Nanjing/Yu used, two 113 cleaned the aisles where he walked through, and one cleaned up the garbage from Nanjing/Yu's 114 seat. Although the cleaning staffs were wearing masks and gloves, the gloves did not cover the 115 cuffs of the protective clothing. This may have caused their wrists to be exposed. Their hands 116 were not disinfected during the process of cleaning, handling garbage and removing protective 117 clothing after cleaning, and gloves were slipped off, which may have caused hand 118 contamination. Through whole-genome sequencing analysis, it was found that they were 119 infected with the Delta variants. The genomes of the virus isolated from the cleaning staff were 120 highly homologous to those detected in case Nanjing/Yu, showing complete identity, including 121 the spike H146Y mutation of SARS-CoV-2. No other COVID-19 cases from that flight had 122 the spike H146Y mutation and showed low viral loadings. In addition, since July 1 to July 20, 123 a total of six international flights arrived in Nanjing and some passengers have been found 124 infected with SARS-CoV-2, but none of them carried the Delta variants. The outbreak among 125 cleaning staff was not spatially related to the Guangdong and Yunnan outbreak, where local 126 cases of Delta variants have been previously reported. Based on the epidemiological and 127 genomic data, it can be inferred that the cleaning staff probably contracted COVID-19 through 128 indirect contact with the case-Nanjing/Yu. Nanjing is located on the East coast of China, with 9,314,685 permanent residents. From the 132 onset of the outbreak of the epidemic at NKG on July 20th to September 9th, a total of 780 133 COVID-19 cases were reported in Nanjing and Yangzhou. In the Nanjing outbreak, the 10-day 134 period between the cabin exposure and the discovery of the COVID-19 cases, the epidemic ran 135 its natural course and was defined as "natural epidemic period, NE". From July 21st to 26th, 136 nucleic acid tests were carried out for close contacts and high-risk populations. Furthermore, 137 hotel quarantine and treatment in designated hospitals for COVID-19 cases, closure and control 138 of the communities where the cases were located were carried out. These measures could not 139 yet achieve complete hotel quarantine for close contacts, so this 6-day period was termed as 140 "incompletely intervened period, II". Starting from July 27th and 9 days thereafter, nucleic 141 acid tests for close contacts and high-risk populations were performed. Hotel quarantine and 142 treatment in designated hospitals for cases, closure of the communities where the cases were 143 located, and hotel quarantine of all close contacts were achieved. Therefore, this period was 144 called "completely intervened period, CI". The benefits of implementation of vigorous 145 intervention started becoming visible after CI, with the number of COVID-19 cases dropping 146 from over 10 COVID-19 cases per day to less, and the last case was detected on August 11. 147 This phase was called as "epidemic regression period, ER". During the NE, II, CI and ER 148 period, the number of COVID-19 cases or nucleic acid positives found in Nanjing were 41, 149 109, 64 and 7, respectively (Fig. 1A). 150 Yangzhou is located about 100 kms to the Northeast of Nanjing. The epidemic in Yangzhou 151 started from accidental exposure to a COVID-19 case of Nanjing on July 19. The patient 152 travelled to Yangzhou on July 21. She was confirmed as COVID-19 positive by RT-PCR on 153 July 27 in Yangzhou and intervention measures were put in place on July 28. The NE period 154 was 9 days and a total of 48 COVID-19 cases occurred during this time. Hereafter, during the 155 10-day II period, 8-day CI period and 10-day ER period, 358, 141, and 12 COVID-19 cases 156 were detected, respectively (Fig. 1B). 157 Phylogenetic analysis revealed that all the genomes of viruses isolated from COVID-19 158 patients of Nanjing and Yangzhou fell into one clade. The viruses from Yangzhou COVID-19 159 cases belonged to the branch of those from Nanjing COVID-19 cases, which was consistent 160 with the fact that the COVID-19 local outbreak in Yangzhou originated from the old lady case 161 (YZ001) who travelled from Nanjing to Yangzhou. None of the genomes from Yangzhou 162 COVID-19 cases fell into the majority of genomes from Nanjing COVID-19 cases may due to 163 the timely lockdown measure of Yangzhou. The genomes from Nanjing and Yangzhou  169 Compared with Wuhan-Hu-1 strain, the Delta variants isolated from case Nanjing/Yu and the 170 early infected local cases in Nanjing had 35 mutations in the nucleotide sequence and three 171 deletion sites (Extended data Table 1). The mutations seem to have changed many amino acids. 172 Among them, there were four amino acid (aa) substitutions in the nucleocapsid protein (N), 173 namely D63G, R203M, G215C and D377Y. There was one amino acid substitution, I82T, in 174 the membrane protein (M). There were 16 amino acid substitutions in other ORF regions, 175 including 11 substitutions in ORF1ab (K261N, A1306S, P2046L, P2287S, V2930L, T3255I, 176 5 T3646A, P4715L, G5063S, P5401L, A6319V), one substitution in ORF3a (S26L), three 177 substitutions in ORF7a (P45L, V82A, T120I) and one substitution in ORF8 (ERF119-120DF) 178 ( Fig. 2A). G5063S(nucleotide G15451A)substitution in RdRp resulted in the mismatch of 179 Charite primer, which may affect its detection efficiency because it mapped to the 3＇primer 180 region. Another R203M(nucleotide acid G28881T)  and ER periods were 15, 44, 28, and 2, respectively. Of the 89 SNV combinations, 53 were 246 terminated in complete hotel quarantine, and 36 were self-terminated. The self-terminated 247 cases had the chance to infect others. The mutations terminated due to hotel quarantine 248 accounted for 59.55% (53/89) (Fig. 3D) of the total cases, which was lower than that of Nanjing. 249 This discrepancy could be attributed to the longer II period. Specifically, 2, 21 and 30 SNV 250 combinations were terminated with NPIs in NE, II and CI periods, while 6, 15, 12 and 3 were 251 self-terminated in NE, II, CI and ER periods, respectively. Several SNV combinations were 252 also found in single cases in Yangzhou (Fig.3D), 7, 28, 22 and 2 SNV combinations terminated 253 in NE, II, CI, and ER periods, of which 2, 16, 13 and 0 were terminated by hotel quarantine 254 measures, and others were self-terminated in the four periods, respectively. 255 In this epidemic, the elimination of the Delta variants included two aspects. On one hand, the 256 self-elimination of the Delta variants could terminate the transmission chain in the absence of 257 NPI, the percentage of mutants eliminated in self-termination accounted for 32.85% of the total 258 SNV combinations. On the other hand, the number of the SNV combinations decreased 259 gradually from II to ER periods, and eventually the transmission chains were fully terminated, 260 suggesting that hotel quarantine could effectively prompt some mutations to disappear and was 261 important to stop the SNV combination of Delta variants in local epidemic. 262 Respiratory samples were collected from three COVID-19 cases at different time points to 263 analyze the dynamics of the allele frequency in intra-host single nucleotide variations (iSNVs). 264 Changes in the profiles of alternative allele frequencies ( in the S protein like L5F, P384S, N542K, D574Y, A647S, H655Y, G769V, V951L, Q1005H,   314   T1117I, and Q1208H are worthy of attention. Among these, L5F has been found in several 315 variants around the world, especially in B.1.526, which may be related to the increased 316 transmissibility of the variant strain. This probably resulted in the second wave of COVID-19 317 cases to extend its duration in New York in early 2021 41 . H655Y mutation emerging in 318 B.1.1.529 variants and P.1 variants has been proven to evade neutralization by human 319 monoclonal antibodies, and can spread rapidly in cats and persists at intermediate frequencies 320 4,42 . In relation to the local epidemic, H655Y mutation was found among 6 sporadic cases in 321 Yangzhou during the II and CI periods (from Aug 2nd-10th) and none subsequently cases, 322 suggesting that the transmission of variants with this mutation may be terminated by the NPI 323 measures. The G769V mutation was found in the R.1 lineage of SARS-CoV-2 in Japan. This 324 mutation is not considered to be related to immune escape variants like the simultaneous 325 E484K and W152L mutations, and its role needs to be further investigated 43 . T1117I mutation 326 in the spike was found during the pandemic wave beginning in May 2020 in Costa Rica, whose 327 frequency reached 29.2% in full genome analyses. Structural modeling of the T1117I mutation 328 suggests a potential effect on the viral oligomerization needed for cell infection, but no 329 differences with other genomes on transmissibility, severity nor vaccine effectiveness are 330 predicted for this mutation 44 . In terms of the other 7 mutations, which were firstly identified 331 and their information uploaded in GISAID database, the information of mutated locus and the 332 corresponding biological changes in the function have not been reported. As can be analyzed 333 from the chemical structure, the Q1005H and Q1208H substitution may abrogate intra-334 protomer hydrogen bond (H-bond) between the T716 side-chain and main-chain carbonyls of 335 Q1005 and Q1208, but could form a symmetric histidine triad near the base of the spike. In 336 addition, water-mediated interactions could promote the formation of hydrogen bonding 337 between the histidines 45 . In terms of A647S mutation, after the hydrophobic and non-polar 338 alanine converted to hydrophilic and polar serine, the hydroxyl group contained in serine may 339 bind to the oligosaccharide chain of T645 and would lead to the changes of spatial structure 340 between antigens and cells. Furthermore, the D574Y mutation would also be affected by 341 glycan chains of T572 and T573 and may mediate immune escape, although its specific effects 342 have not been confirmed in predicted models and the real world. glycosylation between hosts are the limitations of the method itself. Therefore, the mechanism 366 of these mutation sites is planned to be investigated in our future researches and will be shown 367 in another complete report. 368 As long as the virus continues to spread, it will replicate and produce new mutation sites. Only    Ethical approval for this study was approved by ethics committee of the Jiangsu Provincial 30

Characteristics of mutations of the virus causing local epidemic
Center for Disease Control and Prevention. Written informed consent was waived in light of the 31 urgent need to collect data. 32 33 Epidemiological investigation of populations 34 COVID-19 cases in Nanjing and Yangzhou were included in this study, whose demographic 35 characteristics were described in Extended data Table 8. Similar distribution of sex, CT values and 36 clinical type were observed in Nanjing and Yangzhou, as well as age, except for the subgroup of 37 '[40-50)' and '≥60'. Cases in Nanjing and Yangzhou were comparable regarding cluster 38 distribution; 450/559 (80.50%) cases were classified into clustered group in Yangzhou, while in 39 Nanjing, this proportion was 32/221 (14.50%). The differences are probably caused by differences 40 in daily activities of the cases in Yangzhou. In addition, a significantly higher proportion of non-41 vaccination subgroup existed in Yangzhou when compared to Nanjing, may be for the reason that 42 most of the cases in Yangzhou were the elderly or children who have not been immunized. 43 Verification of diagnosis and epidemiological investigation were conducted for each newly 44 detected COVID-19 nucleic acid positive case. The verification of diagnosis included rechecking 45 the first positive specimen and re-collecting the patient's nasal/pharyngeal swab specimens within 46 24 hours for nucleic acid testing. Patients with positive nucleic acid test at least twice were 47 included in this study. 48 Epidemiological investigation includes the following four parts.
(1) Investigation of clinical 49 manifestations (whether there are symptoms, first symptoms and onset time) and the sampling 50 time of the first positive nucleic acid test of COVID-19; (2) Activities of the infected person within 51 14 days before the onset time or the sampling time of first positive test, focus on investigating 52 whether the contacts have been identified as infected, and investigating the date, duration, 53 frequency, and protection of contact methods with the infected persons. In addition, exposure to 54 objects or environment contaminated by infected persons, including exposure to foreign aircraft, 55 exposure to environments in which known cases have been active or the contact with contaminated 56 items (duration, frequency, exposure methods, protection conditions, and environmental 57 characteristics). These activities help to determine the source of infection, assist in inferring the 58 possible time of infection, and thus analyze the transmission relationship.
(3) Close contacts, those 59 who were in contact before symptom onset or 4 days before the first positive test, or those who 60 were exposed to the same small confined space together with them, were defined as close contacts. 61 (4) Sub-close contacts, a person who has been in contact with a close contact from contacting with 62 an infected person to hotel quarantine was defined as a sub-close contact. 63 Through epidemiological investigation, clarify the transmission chain of the cluster epidemic 64 and determine whether each infected person has continued transmission. If it is judged that there 65 is no further spreader, it is determined as the last case of a transmission chain, and the infection 66 period can be inferred based on the detection time or onset time of the last infected person. If a 67 case has been quarantined before the infectious period, the termination of the variant transmission 68 is considered to be due to non-drug intervention (NPI) measures. If a case has not been fully 69 controlled during the infectious period but no transmission occurs, the termination of the variant 70 transmission is considered to be due to self-termination of the virus. 71 72 Non-pharmaceutical interventions (NPI) 73

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(1) Management of COVID-19 cases. Confirmed cases were transferred to designated 74 infectious disease institutions within 2 hours for treatment, observation or quarantine, until they 75 meet the discharge standards of the "Diagnosis and Treatment Protocol for COVID-19". 76 (2) Management of close contacts. Close contacts were transferred to the hotel quarantine 77 within 12 hours after discovery, and take a single hotel room to quarantine until 14 days after 78 contact with an infected person (people with difficulties in living on their own are allowed to be 79 isolated at home or accompanied by someone), and respiratory samples were collected on the 1, 4, 80 7, 14 days of the quarantine period for testing nucleic acid of SARS-CoV-2. Two nasopharyngeal 81 swabs were collected on the 14th day, and were tested by two reagents separately. Those who 82 tested negative during the quarantine period were released and be monitored at home for 7 days 83 after release. They were not allowed to participate in gathering activities during the period and 84 were suggested to take nucleic acid tests on the 2 nd day and 7 th day respectively, then the control 85 would be lifted after all negative results of nucleic acid tests. 86 (3) Management of sub-close contacts. They were transferred to a centralized quarantine 87 place within 12 hours and quarantine in a single room of a hotel for 7 days. If those close contacts 88 they contact were tested negative within 4 days before quarantine, these sub-close contacts would 89 be released after testing negative on the 1 st , 4 th or 7 th day. If those close contacts were tested 90 positive, these sub-close contacts will be determined as close contacts for further control. 91 (4) Closed management of the community. Communities with community transmission or 92 communities with more than 5 cases were closed for management. People in the communities were 93 not allowed to leave their homes, and supplies were uniformly distributed after secondary 94 protection by community management personnel. Statistical analyses 156 In this real-world study, we included all 520 cases from which genome-wide data were 157 obtained during an outbreak in Nanjing and Yangzhou from July 13 to August 25, 2021. 158 Demographic and laboratory data were obtained from the stream database, and the combined 159 mutation data for each case were summarized and compared. Descriptive analyses of demographic 160 data are presented as mean when continuous and as frequency and ratio (%) when categorical. 161 Univariate and multivariate logistic regression methods were used to investigate the factors 162 influencing whether the SNV combinations could be terminated. Six variables were selected for 163 multivariate analysis based on preliminary statistical findings and clinical limitations. Statistical 164 5 analyses were done using IBM SPSS18.0 and R (Version 9.3). A two-sided α of less than 0.05 was 165 considered statistically significant. 166

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Univariable and multivariable analysis of factors affecting the termination of iSNV mutation combinations.

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Vaccination: No = Unvaccinated persons were defined as those who had not been vaccinated and those who had less than 14 days between the time of last vaccination and the time of illness.

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Yes = Vaccinations were defined as those who were vaccinated and the interval between the last dose of vaccination and the onset of illness was 14 days or more.

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NI †= These factors were not included in the multivariable analysis.

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Results are expressed as median [minimum and maximum values) and n (%).

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