Epidemiological, clinical, and virological characteristics of 465 hospitalized cases of coronavirus disease 2019 (COVID‐19) from Zhejiang province in China

Abstract Background The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and the associated coronavirus disease (COVID‐19) have spread throughout China. Previous studies predominantly focused on its place of origin, Wuhan, causing over estimation of the disease severity due to selection bias. We analyzed 465 confirmed cases in Zhejiang province to determine the epidemiological, clinical, and virological characteristics of COVID‐19. Methods Epidemiological, demographic, clinical, laboratory, and management data from qRT‐PCR confirmed COVID‐19 patients from January 17, 2020, to January 31, 2020, were collected, followed by multivariate logistic regression analysis for independent predictors of severe/critical‐type COVID‐19 and bioinformatic analysis for features of SARS‐CoV‐2 from Zhejiang province. Results Among 465 COVID‐19 patients, median age was 45 years, while hypertension, diabetes, and chronic liver disease were the most common comorbidities. History of exposure to the epidemic area was present in 170 (36.56%) and 185 (39.78%) patients were clustered in 77 families. Severe/critical‐type of COVID‐19 developed in 49 (10.54%) patients. Fever and cough were the most common symptoms, while diarrhea/vomiting was reported in 58 (12.47%) patients. Multivariate analysis revealed eight risk factors for severe/critical COVID‐19. Glucocorticoids and antibiotics were administered to 60 (12.90%) and 218(46.88%) patients, respectively. Bioinformatics showed four single amino acid mutations and one amino acid position loss in SARS‐CoV‐2 from Zhejiang province, with more similarity to humans than to viruses. Conclusions SARS‐CoV‐2 showed virological mutations and more human transmission in Zhejiang province, indicating considerable epidemiological and clinical changes. Caution in glucocorticoid and antibiotics use is advisable.


| INTRODUC TI ON
An increasing number of cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia, known as coronavirus disease , have set off a global alert and put a heavy public health burden on China due to the rapid transmission ability (basic reproductive number = 2.2) and approximately 11% fatality. 1,2 When looking back at its occurrence and dissemination, the original serial cases of pneumonia of unknown etiology were reported from Wuhan, China, on December 8, 2019. 3 On January 7, 2020, a novel coronavirus was identified by the Chinese Center for Disease Control and Prevention (CDC) from the throat swab of a patient and was subsequently named SARS-CoV-2 by the WHO. 4 Though the Chinese government responded rapidly and took drastic measures to stop SARS-CoV-2 dissemination, including quarantining Wuhan city on January 23, it spread inevitably, reaching every province of China and beyond to other countries.
Coronaviruses are named for the crown-like spikes on their surface. People worldwide commonly get infected with the human coronaviruses 229E, NL63, OC43, and HKU1. Two other strains, severe acute respiratory syndrome coronavirus (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV), are zoonotic in origin and have been linked to lethal diseases. 5,6 SARS-CoV-2 is the seventh newly identified coronavirus with the capacity to infect humans by the Chinese authorities. Though the full sequence, homology to other known coronaviruses, and potential invasion mechanism have been gradually revealed, 7 there are still many unresolved questions regarding SARS-CoV-2. Most importantly, the mutations, transmission ability, virulence changes, and associated clinical features of SARS-CoV-2 during dissemination remain unknown.
The first case of COVID-19 in Zhejiang Province was diagnosed on January 17. Zhejiang province is 1000 kilometers from Wuhan. In contrast to the initial cases from Wuhan who had a history of close contact with Huanan seafood market, the majority of patients in Zhejiang province only had a history of working, studying, or travel to Wuhan. Nevertheless, knowledge about the specific epidemiology and clinical characteristics on a large scale still remains incomplete.
In this study, we performed a comprehensive investigation of the epidemiology, clinical, and virological characteristics of 465 patients with laboratory-confirmed COVID-19 in Zhejiang province. To the best of our knowledge, this is by far the largest number of COVID-19 patients reported in Zhejiang province with distinct clinical features compared to Wuhan.

| Data sources and ethics
We performed a retrospective study focusing on the epidemiological, clinical, and virological characteristics of confirmed cases of COVID-19 from January 17, 2020, to January 31, 2020. The data were uniformly collected by the Health Commission of Zhejiang Province. All patients were assigned to specific hospitals for unified treatment according to Zhejiang Province's emergency rule. All patients who were diagnosed with COVID-19 based on the WHO interim guidance were successfully enrolled in this study. 4 All data pertaining to the included cases have been shared with WHO, and the primary analytic results were immediately reported to the authorities of Zhejiang province. Since case collection and analysis were determined by the Health Commission of Zhejiang province under national authorization and considered to be a part of the continuing public health outbreak investigation, our study was regarded as exempt from institutional review board approval.
For in-depth data analysis, the subtype definition of COVID-19 patients was according to the Chinese diagnosis and treatment scheme for SARS-CoV-2 (5th edition) with minor modification based on the WHO standards. Specifically, the severity of COVID-19 was categorized as mild, common, severe, or critical. Mild type was defined as mild symptoms with no pneumonia on imaging. Common type was defined as having respiratory tract symptoms and pneumonia on imaging. Severe type was characterized by dyspnea, respiratory rate ≥30/minute, blood oxygen saturation ≤93%, PaO2/FiO2 ratio <300, and/or lung infiltrates >50% within 24-48 hours. Critical cases were those that exhibited respiratory failure, septic shock, and/or multiple organ dysfunction/failure.

| Procedures
We obtained epidemiological, demographic, clinical, laboratory, management, and outcome data from the patients' medical records.
Clinical outcomes were followed up to January 31, 2020. If data were missing from the records or clarification was needed, we obtained data by direct communication with the attending doctors and other healthcare providers. All data were checked by at least two doctors.
Laboratory confirmation of SARS-CoV-2 was done in the following institutions: the Zhejiang province CDC, the first affiliated hospital, School of Medicine, Zhejiang University, and the local CDC at the city Conclusions: SARS-CoV-2 showed virological mutations and more human transmission in Zhejiang province, indicating considerable epidemiological and clinical changes. Caution in glucocorticoid and antibiotics use is advisable.

K E Y W O R D S
coronavirus disease 2019 , risk factors, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Zhejiang province Science Foundation of China (81770574); and National Nature Science Foundation of China (81700549). level. Throat swab specimens from the upper respiratory tract and sputum samples that were obtained from all patients at admission were maintained in a viral-transport medium. SARS-CoV-2 infection was confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) using the same protocol described previously. 8 Other respiratory viruses including influenza A virus (H1N1, H3N2, H7N9), influenza B virus, respiratory syncytial virus, parainfluenza virus, adenovirus, SARS-CoV, and MERS-CoV were also examined with routine real-time RT-PCR. Possible causative infection with bacteria or fungi in sputum or endotracheal aspirates was also investigated at admission in P3 level laboratories. All patients underwent chest X-rays or chest computed tomography (CT) at admission.

| Outcomes
We collected and described the epidemiological data (ie, living in Wuhan and returning to Hangzhou, traveling back from Wuhan, contact with people from Wuhan and Hubei province, exposure to Wuhan seafood market within 14 days before illness onset); anthropometrics; demographics; symptoms and signs on admission; laboratory and chest X-ray/CT results; comorbidity; coinfection with other respiratory pathogens; treatment (including drugs, intensive care, and mechanical ventilation); and clinical outcomes.

| Viral sequencing, phylogenetic analysis, and synonymous codon usage analysis
All published sample sequences (n = 23) were obtained from the NCBI viral genome database and NGDC (https://bigd.big.ac.cn/ncov/), except for ZJ01 which was the name of the SARS-CoV-2 isolated separately from Zhejiang patients. The coronavirus samples came from all over the world, including Wuhan (n = 4), Guangdong (n = 3), USA (n = 2), and Australia (n = 1). There were also SARS specimens (n = 3) and other animal coronaviruses (n = 10). Multalin (http://multa lin. toulo use.inra.fr/multa lin/multalin. html) was used to compare the differences between these sequences. Further SWISS-MODEL online server (https://swiss model.expasy.org/) was used to reconstruct the three-dimensional structure of protein according to gene or amino acid sequence. The reconstructed protein model was put in PyMol to explore molecular mechanic and protein function. Phylogenetic tree establishment, evolutionary analysis, and other bioinformatic analyses were performed by MEGA7.0. The heat map of relative synonymous codon usage (RSCU) was drawn with MeV 4.9.0, where Codon W1.4.2 was used to estimate the RSCU of these coronaviruses. Hierarchical clustering was used to analyze RSCU of these strains, with utilization of Euclidean distance selection and average linkage clustering.

| Statistical analysis
For continuous variables, mean (SD) and median (IQR) were used for normally and abnormally distributed data. Categorical variables were expressed as numbers (%). For laboratory results, it was determined whether the measurement was outside the normal range.
Univariate logistic regression analysis was used to identify the risk factors of severe/critical-type patients. All significant variables in univariate analysis were included in a multivariate logistic regression model with Forward:Wald method to identify independent predictors of severe/critical-type patients. No adjustment for multiple

| Demographic and epidemiologic characteristics
This study analyzed 465 patients with confirmed SARS-CoV-2infected pneumonia from January 17, 2020, to January 31, 2020, in Zhejiang province. As shown in   (severe type) and ARDS, in the lung window, demonstrates peripheral and multiple ground-glass opacities (GGO). The pulmonary architecture, including vasculature and bronchi, can be still seen. (C-D) Non-contrast axial CT of a 40-year-old man diagnosed with COVID-19 pneumonia (severe type), in the lung window, demonstrates bilateral subpleural consolidation and opacities in the two lower lobes, more densely consolidated on the right. CT lung of the same patient one week after treatment, in the lung window demonstrates that the lung lesions were significantly absorbed critical type, showing lower tendency severity when compared to data from Wuhan.

| Clinical Features and laboratory abnormalities
The clinical characteristics of the patients are shown in Table 2.
Briefly, fever and cough were the most common symptoms. Concerning infection-related parameters, over 50% patients had increased erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP). Intriguingly, none of the patients had increased procalcitonin (PCT) levels. Since radiography is pivotal for disease identification and diagnosis, 462 patients underwent chest radiography on admission, among which 408 (88.31%) were diagnosed with pneumonia. Bilateral ground-glass opacities and consolidation were the most common radiological findings inEdits our cases ( Figure 1).

| Treatment and complications
All patients were treated in isolation with supportive and empiric medication. As show in Table 3

| Risk factor prediction for severe/critical COVID-19
There were total 49 patients with severe/critical COVID-19 in this study. When compared with mild and common COVID-19, initial univariate analysis of epidemiological, clinical, and laboratory variables identified 25 significant risk factors for severe/critical COVID-19 (Table S1). Based on these variables, further multivariate logistic regression analysis with forward selection method was performed and we found that male sex, any coexisting disease, cough, muscle ache, diarrhea, decreased lymphocytes, increased CRP, and decreased albumin were the independent risk factors for severe/critical COVID-19 (Table 4).

| Multiple sequence alignment and bioinformatics analysis
We confirmed that ZJ01 is a SARS-CoV-2 with 29,381 bases by utilizing biological methods (Appendix S1). However, in the process of sequencing, we failed to perfectly measure its entire genome, resulting in the deletion of a 0.5 KB gene segment encoding ORS 1aboratory ( Figure 2A). The S protein of SARS-CoV-2 is responsible for recognizing angiotensin-converting enzyme 2 (ACE2) receptor and mediating membrane-enveloped fusion for invasion. By sequencing the S protein base of ZJ01, we found that ZJ01 was very similar to MN908947.3 from Wuhan ( Figure 2B). There were only four single amino acid mutations and one amino acid position loss in this sequence, as shown by 3D protein reconstruction ( Figure 2C). Except for one mutation point exposed on the protein surface (blue), the other mutations occurred within the S protein conformation.
The evolutionary tree revealed the evolutionary path of the coronavirus family ( Figure 2D). Though there were variations among the members of the SARS-CoV-2 family, they were not enough to allow them to evolve into additional subspecies. The most surprising result was the RSCU heat map ( Figure 2E). RSCU refers to the relative probability that a specific codon encodes a corresponding amino acid in a synonymous codon. ZJ01's RSCU is more similar to humans than other viruses. This phenomenon is also seen in MN988668 which was found in Wuhan. This suggests that the SARS-CoV-2 is beginning to evolve after a period of human-to-human transmission. There are several limitations of this study that need to be acknowledged. Firstly, the retrospective nature of this study may decrease its credibility and future prospective cohort studies should be considered. Secondly, a complete analysis of COVID-19 on a national level is urgently needed, which might provide more solid data. Thirdly, although we summarized the risk factors for severe/ critical type of COVID-19, there is still lack of a prediction model for disease fatality, since most of the enrolled cases are currently under treatment. Finally, cytokine change is common in coronavirus infections 19 and has been reported in a previous SARS-CoV-2 study. 8 Therefore, it would be better if we could also test cytokine changes in our study.

| D ISCUSS I ON
In summary, Zhejiang province was the first province that exhibited the highest response level for SARS-CoV-2 infection and quarantined every suspected patient, followed by immediate virus detection. All these measures were effective and helped to control virus dissemination. On the basis of our experience with the virus, we revealed the novel epidemiological, clinical, and virological features of SARS-CoV-2 as summarized in this paper. The changes in these features may be due to virus mutation during dissemination, as we observed on comparison of the viral sequence between Zhejiang and Wuhan with focus on the invasion-related S protein.
We have also provided large-scale preliminary data pertaining to our treatment experience, including low and appropriate glucocorticoid usage, early and timely antiviral therapy, avoiding unnecessary antibiotics, and ample oxygen supply. Further in-depth analysis on the therapeutic interventions is under way.

ACK N OWLED G EM ENTS
We thank the Health Commission of Zhejiang Province, China, for coordinating data collection; Prof. Wen-Bo Xiao and Ling-Xiang Ruan for their help in the radiography analysis of X-rays and CT scans. Thanks to all the front-line medical staff of Zhejiang Province for their bravery and efforts in SARS-CoV-2 prevention and control.