To prevent further spread of the disease and find the source of infection, Tianjin Municipal CDC organized relevant professionals to conduct epidemiological investigations and laboratory tests to identify the source of infection as rapidly as possible. Persons included in this investigation were the 137th confirmed COVID-19 patient in Tianjin, his family members and colleagues, residents of his apartment building, and hotel guests including non-Chinese nationals at his place of employment.
The patient was investigated with the “Epidemiological Questionnaire for COVID-19 Cases,” and others were investigated with a checklist that included basic personal information, clinical conditions, travel history, contact with others, and suspected exposures in the previous 14 days (1-3). Type and frequency of contact with the patient were determined.
Throat swabs and blood samples were obtained from all investigated subjects. Environmental samples from the patient’s residence and workplace were obtained including samples from door handles, faucets, kitchenware, refrigerators, garbage cans, air conditioners, sewers, clothes, stoves, sealed meat products, and aquatic equipment in the hotel. Samples from the environment, food, and food packaging from hotel suppliers were also obtained for testing.
Nucleic acid samples were extracted from throat, nasopharyngeal, and anal swabs using viral DNA/RNA extraction kits (Xi’an Tianlong Science and Technology, China), and initially stored at –70 °C. COVID-19 virus (also known as SARS-CoV-2 and 2019-nCoV) detection was conducted via real-time reverse transcriptase polymerase chain reaction (RT-PCR) using nucleic acid detection kits (Shanghai BioGerm Medical Technology, China) according to the manufacturer instructions. ULSEN® SARS-CoV-2 whole genome capture kits (MicroFuture, Beijing, China) were used for COVID-19 virus genome enrichment as described by the manufacturer. Amplified products were purified by Agencourt AMPure XPKit (Beckman Coulter, Brea, CA, USA) and then subjected to library preparation by Nextera® XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA). Whole genome sequencing was carried out using a MiniSeq™ Sequencing System (Illumina, San Diego, CA, USA).
Resulting sequences were assembled with CLC Genomics Workbench 20.0.3 (Qiagen, Hilden, Germany). Sequence alignments and phylogenetic analysis were performed with MEGA X software, and a neighbor-joining tree was assembled with bootstrap values determined from 1,000 replicates. All comparison sequences, including the most recent Beijing outbreak sequences (i.e., ESP_ISL_469254, ESP_ISL_469255, and ESP_ISL_469256), were downloaded from the EpiFlu database of the Global Initiative on Sharing All Influenza Data (GISAID; gisaid.org) and from GenBank (ncbi.nlm.nih.gov/genbank/).
A total of 246 environmental and food specimens were collected during the investigation. This total included 144 environmental samples of 18 types of sources (e.g., door handles, kitchenware items, etc.) and 102 food samples (i.e., food and food packaging for frozen and fresh seafood, beef, mutton, and vegetables). All 246 specimens had negative viral nucleic acid test results.
A total of 1,137 contacts were investigated for suspected exposure (e.g., family members, colleagues, neighbors, hotel guests, etc.). No individuals had travel history to Xinfadi Agricultural Product Wholesale Market in Beijing Municipality or other high-risk areas or contact with persons with travel history to mid- to high-risk areas in Beijing. No individual had travel history overseas or contact with foreign travel returnees. In addition, no individual had contact with confirmed COVID-19 cases or persons experiencing COVID-19 or related respiratory symptoms.
On June 17, a total of 750 throat swab specimens and 365 serum specimens from persons with suspected exposures were collected and tested for both viral nucleic acids and virus-specific antibodies. All test results were negative.
On June 19, 55 employees in the dining room of the hotel were tested a second time. All viral nucleic acid test results were negative. All antibody test results were also negative, except one. A chef who worked in the western dining room of the hotel tested positive for IgM antibody specific to COVID-19 virus. After three consecutive tests, the IgM antibody result remained positive.
As a result, the chef who tested positive for anti-COVID-19-virus IgM antibodies was then further investigated as a possible source, and he reported having had no symptoms and was therefore preliminarily designated as a confirmed asymptomatic COVID-19 patient. He had visited his girlfriend in Beijing many times in the past month. While in Beijing on June 8, he took the subway and spent time in public places including restaurants, bars, and a theater. While in the workplace in the hotel in Tianjin, the chef and the patient had several exposures while in the smoking room on the ground floor. In addition, there was also an instance of close (less than 0.5 meters) communication between the chef and the patient on June 10 where the patient wore a mask but the chef did not. The patient was also responsible for cleaning the tableware in the employee’s dining room and had resulting contact with the chef during the daily lunch. Therefore, it was hypothesized that the source may have been infected in public places in Beijing, returned to work normally due to his asymptomatic status while in Tianjin, and then infected the case through frequent close contact at work.
Furthermore, COVID-19 virus RT-PCR test results on throat, nasopharyngeal, and anal swab samples were all positive with cycle threshold (Ct) values of 16.0/18.0 (ORF1ab/N), 23.0/24.4, and 35.0/35.0, respectively. As the Ct values of the anal swab sample was too high to acquire whole genome sequences, we chose throat (FH-124-Y) and nasopharyngeal (FH-124-B) swab samples for genome sequencing. The whole genome of FH-124-Y was 29,868 nt, and FH-124-B was 29,803 nt, with a shortage in 5’-NTR and 3’-polyA tail. The genome sequences of FH-124-Y and FH-124-B were found to be 100% identical and were both named Tianjin/137/2020.
Phylogenetic analysis has revealed that all known COVID-19 virus genomes fall into two major clades, or lineages, named S and L (Figure 1). We found that Tianjin/137/2020 belonged to the Europe Branch of the L-Lineage, and that it was most closely related to the Beijing strain ESP_ISL_469255 (4), to which it was 100% identical at the nucleotide level.
Phylogenetic tree based on the whole genome sequences of COVID-19 virus. The COVID-19 virus genome of the cases in Beijing Municipality and Tianjin City were highlighted in shades of red and blue, respectively. The reference sequence Wuhan-Hu-1 (NC_045512) were highlighted in orange. S- or L-clade of COVID-19 virus were marked and colored on the right.