Epidemiological history of patients
The age of the four patients were 49, 46, 55, and 41 years old, respectively. All of these four cases worked in the same factory in Irkutsk, Russia before returning to China. Each of the four patients had completed two doses of COVID-19 vaccination in Russia on March 2 and March 30, 2021. On June 17, they returned from Moscow, Russia by the same flight. They said they wore masks during the entire flight, and the nasopharyngeal swabs tested negative for the new coronavirus nucleic acid after landing. After three days of health observation in Shanghai, they were transferred from Shanghai to the centralized health observation point in Guangde, Anhui Provence by special vehicles on June 21. There was no activity outside the room during the isolation period (Figure 1).
Investigation and management of close contacts
There were four staff on the transfer vehicle (2 drivers, 1 police officer, and 1 doctor), with secondary protection during the entire transfer period, and the vehicle-mounted staff were strictly separated from the transfer subjects. There were 14 health observation subjects who returned to Anhui in the same vehicle, all of whom reported wearing masks throughout the journey. As of June 29, the nucleic acid test results of all close contacts were negative, and no abnormalities were found in the health monitoring.
Laboratory testing and imaging diagnosis
Among the four patients, there were one case of SARS-CoV-2 mild type and three cases of SARS-CoV-2 normal type. One case had a history of diabetes, and one case had anemia. The shortest experience from admission to discharge was 26 days, and the longest experience was 28 days, with an average of 27 days. Through the analysis of clinical test data, it was found that the absolute value of lymphocyte of the four patients decreased in the early stage after diagnosis (0.73×109/L to 1.01×109/L), and returned to normal levels within 3 to 9 days, and there was no abnormal increase in the absolute value of neutrophil (1.65×109/L to 5.80×109/L), the value of high-sensitivity C-reactive protein (hs-CRP) increased rapidly after diagnosis. The absolute count of white blood cell (WBC) (Liu) decreased to 2.65×109/L, and returned to normal levels on the 15th day. As the course of the disease progression, the value of C-reactive protein (CRP) in three patients began to increase rapidly from the 5th to 7th days, and the levels of CRP reached its peaks at 23.85mg/dL to 54.05mg/dL. Although the levels of CRP increased sharply, other types of examinations did not reveal that there was a bacterial infection. The four patients had transient lactic dehydrogenase (LDH) elevation (253.9U/L-372.1U/L) on the 1th to 6th days, of which two patients were accompanied with an elevation of α-HBDH (200U/L-221U /L). In the four patients, the levels of D-dimer were increased from 0.53mg/L to 19.91mg/L on the 1th to 5th days, no changes of prothrombin time (PT) and activated partial thromboplastin time (APTT) were found. Moreover, the normal platelet count (PLT) and basically stable coagulation function were observed, indicating that there was no risk of disseminated intravascular coagulation (DIC).
Hs-CRP and CRP were used to evaluate the inflammation level of the body. We combined the chest CT to evaluate the progress of the patients. Due to the partial lack of Chen’s data, we could not judge the specific change trend of the detection index while the remaining data of the other three patients were relatively complete. We observed four patients whose hs-CRP exceeded 5 mg/dL for the first 3 to 5 days after admission. In addition to Chen, the value of CRP exceeded 10 mg/dL for the first 4 to 7 days after admission, and multiple patchy shadows appeared for the first time on chest CT at 3 to 6 days, and the time differences between the first time multiple patchy shadows appeared on chest CT and the first time multiple patchy shadows appeared on hs-CRP at 5 mg/dL was no more than 3 days, the time with CRP above 10 mg/dL for the first time was not more than 1 day (Table 1) .
Except for Chen, the levels of CRP of other three patients reached its peak at day 7, Liu’s chest CT showed the increased number/size and density of lung lesions, Pan’s and Wei’s chest CT showed consolidations of lungs. In addition to Chen, the concentrations of hs-CRP were decreased (less than 5 mg/dL) at 15 to 20 days, and the value of CRP also decreased to less than 10 mg/dL, and the first-time chest CT showed lower lesions size and some lesions that showed partial absorption at 15-19 days. The differences between the first occurrence of lesions size decreased / partial absorption of some lesions and the change of hs-CRP/CRP was no more than 5 days (Table 1). Interestingly, we observed three patients other than Chen on day 17th (Supplemental Table 2) with two reagents for at least single-target negative nucleic acid test results, this time was within two days of the first appearance of chest CT of lesions size decreased and partial absorption of some lesions (Figure 2).
Novel coronavirus antibody IgM/IgG and Nab in patients were then tested to assess their humoral immune response. The results showed that IgM and IgG change from negative to positive in all 4 patients at 12-19 days after admission (Figure 3). Nab levels in four patients increased from admission to pre-discharge and exceeded 30 AU/mL at 12-19 days after admission (Figure 4).
Phylogenetic tree analysis of four SARS-COV-2 strains
Representative sequences of different lineages were selected from GISAID database and SARS-COV-2 genome sequences of four cases sequenced this time to construct maximum likelihood phylogenetic tree. Phylogenetic tree analysis showed that 4 cases of SARS-COV-2 in this study were in the Delta-B.1.617.2 lineage, with Bootstrap support values >95% (Figure 5).
Amino acid mutation analysis
Compared with the Wuhan-HU-1 sequence, the four Anhui cases in this study had the same mutation sites, and 36 nucleotide mutation sites were found in all of them (Table 2), where 33 nucleotide mutation sites are located in the coding region, and the sequence of nucleotide mutation sites from more to less is: There are 12 ORF1a regions, 7 S regions, 4 N regions, 4 ORF1b regions, 3 ORF7a regions, 2 5 'UTR regions, 1 M region, 1 ORF3a region, 1 ORF7b region and 1 3' UTR region. There are 6 nucleotide synonymous mutations (G210T, C241T, C3037T, C8986T, A11332G, G29742T) and 30 nucleotide missense mutations. There are 30 sites of amino acid variation and no insertion mutation was found. There are 9 ORF1a proteins, 7 S proteins, 4 N proteins, 4 ORF1b proteins, 3 ORF7a proteins, 1 M proteins, 1 ORF3a proteins, and 1 ORF7b proteins.
At the same time, we compared 28 strains of delta-B.1.617.2 sequences uploaded from China and Russia from April to June, and found a total of 90 mutation sites, where there were only two mutation sites appeared in Anhui cases, nine mutation sites in cases of other provinces in China, and 44 mutation sites observed in Russia’s cases. In addition, there were 11 mutations unique to Anhui cases and Russia cases, and had not been discovered in Chinese other provinces’ input cases, one of the 11 mutations in the N gene, ten of them are on ORF genes. The other 18 mutations occurred simultaneously in the Anhui cases, the Russian cases and the other provinces of China, but there were no common mutations in the Anhui cases and the imported cases in other provinces of China (Figure 6). Therefore, the mutation characteristics of the Anhui cases and the Russian cases are more similar, and it is speculated that the Anhui cases may have been infected in Russia and then imported to Anhui province.