Demographic characteristics of cases
The first cluster outbreaks caused by asymptomatic cases comprised 12 confirmed cases (B–M) and three asymptomatic cases (A1–A3). Patient C first developed a fever, cough and muscle soreness on January 25, 2020, and the last case, M, developed a fever on February 7, 2020. The epidemic occurred in three generations of 15 cases. An symptomatic case, A1, was used as the index patient, while the second generation comprised patients B and C, and the third generation comprised asymptomatic cases A2, A3, and patients D–M. Three patients (Patients B, L and M) were single exposure cases, while the rest were multiple exposure cases. According to the analysis of the onset time in single exposure cases, the incubation period ranged from 4 to 11 days (Patient B, 4 days; Patient L, 4 days, and Patient M, 11 days; Fig. 1a).
The second cluster outbreaks caused by asymptomatic case consisted of six confirmed cases, Patients B*–G*and nine asymptomatic cases, A1*–A9*. Patient B* first developed fever and weakness on 18 January, 2020, and the last case, G*, developed a cough on 12 February, 2020. Four generations of cases of transmission occurred in this outbreak. Patient B* and asymptomatic case A1* were the first generation and the second-generation was patient C*. In the third generation, there were five asymptomatic cases, A2*–A6*, and three patients D*–F*. The fourth generation comprised patient G* and three asymptomatic cases, A7*–A9*. Patient C*and D* were single exposure; the incubation periods of patients C* and D* were 6 days and 1 day, respectively (Fig. 1b) A summary of clinical features and microbiological results from clinical specimens collected from the two clusters of patients infected with SARS-CoV-2 at presentation are shown in Table 2.
Genetic characterization of the virus
The rRT-PCR results of the original clinical samples obtained from the 30 patients in this study are shown in Table 2Twelve complete SARS-CoV-2 spike gene sequences were obtained; these data have been deposited in the NCBI database (accession number: MT415366–MT415377)
We constructed phylogenetic trees based on the nucleotide sequences of these spike gene. The twelve complete spike gene sequences were nearly identical across the whole spike, with sequence identity exceeding 99.9%, and 99.8%–100% similarity between the Anhui and Wuhan strains. We then performed phylogenetic analysis of the collection of coronavirus sequences from NCBI and GS. The phylogenetic tree showed that 12 complete spike gene sequences clustered with seven other spike sequences of viruses belonging to the Betacoronavirus genera from different countries and regions. In terms of phylogeny, RaTG13, Pangolin-CoV and SARS-CoV-2 were clustered into a well-supported group. SARS-CoV-2 and RaTG13 were grouped together within this group, with Pangolin-CoV as their closest common ancestor.
The spike gene cluster was situated with the groups of SARS coronaviruses, and its inner joint neighbors were bat coronavirus RaTG13 or pangolin coronaviruses, with human bat coronavirus and MERS coronaviruses as the outgroup (Fig. 1a). Compared to the spike gene of SARS-CoV and MERS-CoV, the SARS-CoV-2 strains were less genetically similar to SARS-CoV (79.8%–80.3% similarity) and MERS-CoV (53.7% similarity). The similarity plot suggested that RaTG13 was the most closely related sequence to SARS-CoV-2 throughout the spike sequences. SARS-CoV-2 and RaTG13 showed 94.6%–94.7% sequence similarity. The strains in this study were similar to the pangolin Guangdong strain and the Guangxi strain (82.6%–86.9% similarity) .
Sequencing of throat swab and sputum samples collected after the onset of the illness revealed 16 single nucleotide variations within the spike gene (Table 3). The protein structures were predicted based on spike proteins (PDB_ID:6VSB) in the National Genomics Data Center database (https://bigd.big.ac.cn/) using PyMOL. We evaluated amino acid variations in the spike proteins among the Sarbecovirus coronaviruses. Five variations of these sequences occurred in only one isolate. In addition, 1–3 single nucleotide variations were identified in seven samples. In the first cluster, amino acid mutations were detected in the following strains: HN011: E298K, K300N, T302L, L752R, and P812T; HN015: G485R, HN020: A67S, and F1103L; HN023: Y200S, I818S, and V1010L. In the second cluster, amino acid mutations were detected in the following strains: MAS005: S750R, and L752R; M368: G838S; and M525: W152R; M635: S750I. Most of these mutations were located in non-RBD regions, except for one sample, HN015, in which a G485R mutation was detected in the RBD (Fig. 2).
Epidemiological investigation of the two sources of the cluster
In the epidemiological traceability investigation of the first cluster comprising patients B and C, it was found that asymptomatic case A1 had eaten a meal with patient B and C in an enclosed space (10 m2) on January 22, 2020. When asymptomatic case A1 was questioned initially, he deliberately concealed his history, and only admitted traveling from Wuhan after tracking information was obtained from public security authorities. The same pattern was identified in the second cluster, in which asymptomatic case A1* admitted that she and her husband had traveled from Wuhan only after several public security investigations.
In the first clustered case, the second-generation case patient B had not visited Wuhan during the 14 days before the onset of symptoms. On January 27, 2020, she developed a productive cough, fever and muscle soreness. On February 2, 2020, she went to the local hospital for treatment. The viral nucleic acid test was positive on February 3, and she was confirmed as COVID-19 on February 4.
The mode of transmission in the first cluster was close contact and droplet-spreading in a family gathering. After the asymptomatic case A1 returned from Wuhan on January 19, and had meals with patients B and C, it was inferred that asymptomatic case A1 was the source of infection of patients B and C; the rest of the cases were relatives of patients B and C. This cluster outbreak was caused by close contact during multiple meals within a family setting.
In the second cluster case, asymptomatic case A1* and her husband patient B* began selling tofu in a vegetable market in Wuhan from November 2019. Patient B* developed a fever and weakness on January 18, 2020 and was treated in a small local clinic. The couple traveled from Wuhan on January 20. On January 22, asymptomatic case A1* went to a banquet at the same time her husband, patient B*; he was reported to have traveled from Wuhan by his neighbor and had a fever. Patient B* was immediately sent to a local hospital for isolation and treatment and was diagnosed as COVID-19 on 24 January.
Patient C* owned a snack business with her husband in Zhejiang Province. She and her husband returned to Anhui with two neighbors on January 18. Patient C* attended a banquet on January 22 and played cards with the asymptomatic case A1*. She developed a fever, productive cough, and backpain on January 29 and went to the local hospital on February 1. Her chest CT showed pneumonia in both lungs and she tested positive for viral nucleic acids by rRT-PCR on February 9 when she was diagnosed as COVID-19. Prior to the onset illness, patient C* lived with her daughter-in-law, patient E*, and her mother, asymptomatic case A4*. Patient E* developed a fever on February 3. Throat swabs collected from her mother A4* on February 17 tested positive by rRT-PCR, although she did not have any symptoms such as fever or discomfort. Patient F* was accompanied by her husband A5* and treated by fluid infusion in a small clinic on February 5–6, together with patient C*. She had symptoms of discomfort on February 7 and tested positive by rRT-PCR on February 11. She lived with her husband patient A5*, mother-in-law patient A8*, and her 4-year-old son patient A7*, but all were asymptomatic cases.
The main mode of transmission in the second cluster was social activities - playing cards, social gatherings and close contact. In the early phase of the outbreaks, the population had no perception of the risk of the disease.