Clinical and Virological Features of the First Occurrence of COVID-19 Delta Variant in Anhui Province, China (preprint)

Objectives Since the outbreak of coronavirus disease 2019 (COVID-19), it has caused serious casualties worldwide. In recent months, the virus has mutated into an increasingly infectious form (Delta variant) and spread rapidly.Methods In the current study, we analyzed the clinical, epidemiological and viral genetic characteristics of the first four imported Delta cases in Anhui Province, China.Results The four imported Delta cases developed chest inflammation, tissue damage and recovered after admission, the serum high-sensitivity C-reactive protein (hs-CRP) and CRP levels showed a first increasing and then decreasing trend. The changes of hs-CRP /CRP and serum neutralizing antibodies (Nab) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) levels were associated with the regression of chest lesions. The combination of genetic sequencing and epidemiological analysis suggested that the SARS-CoV-2 delta variant infection of these four patients may originate from Russia.Conclusions Our study found the certain correlations of serum hs-CRP/CRP and Nab levels with the occurrence, development and outcome of COVID-19 delta variant, suggesting that monitoring hs-CRP/CRP and Nab levels of COVID-19 delta variant patients at hospital admission may be useful for understanding the severity of patients’ current conditions.

Spike mutations in SARS-CoV-2 variants confer resistance to antibody neutralization (preprint)

New SARS-CoV-2 variants continue to emerge from the current global pandemic, some of which can replicate faster and with greater transmissibility and pathogenicity. In particular, UK501Y.V1 identified in UK, SA501Y.V2 in South Africa, and BR501Y.V3 in Brazil are raising serious concerns as they spread quickly and contain spike protein mutations that may facilitate escape from current antibody therapies and vaccine protection. Here, we constructed a panel of 28 SARS CoV 2 pseudoviruses bearing single or combined mutations found in the spike protein of these three variants, as well as additional nine mutations that within or close by the major antigenic sites in the spike protein identified in the GISAID database. These pseudoviruses were tested against a panel of monoclonal antibodies (mAbs), including some approved for emergency use to treat SARS CoV 2 infection, and convalescent patient plasma collected early in the pandemic. SA501Y.V2 pseudovirus was the most resistant, in magnitude and breadth, against mAbs and convalescent plasma, followed by BR501Y.V3, and then UK501Y.V1. This resistance hierarchy corresponds with Y144del and 242-244del mutations in the N-terminal domain as well as K417N/T, E484K and N501Y mutations in the receptor binding domain (RBD). Crystal structural analysis of RBD carrying triple K417N E484K N501Y mutations found in SA501Y.V2 bound with mAb P2C-1F11 revealed a molecular basis for antibody neutralization and escape. SA501Y.V2 and BR501Y.V3 also acquired substantial ability to use mouse and mink ACE2 for entry. Taken together, our results clearly demonstrate major antigenic shifts and potentially broadening the host range of SA501Y.V2 and BR501Y.V3, which pose serious challenges to our current antibody therapies and vaccine protection.

Mutation and Virulence of SARS-CoV-2 Strains Circulating in Anhui Province, China (preprint)

Background: The genome of SARS-CoV-2 has shown considerable variation during its spreading. Monitoring variations in the virus genome to understand the evolution and spread of the virus is extremely important. Methods: Seven SARS-CoV-2 strains (BB127, BB183, HB030, MAS525, HF3028, FY1494, and SZ005) circulating in Anhui Province, China were isolated and sequenced for evolutionary analysis. Five strains were further cultured in vitro and were subjected to viral growth assay, TCID50 assay, and detection of spike protein expression. Next generation sequence (NGS) analysis were applied to investigate the mutation frequencies throughout the whole genome at different time gradients in vitro. Findings: Our observations revealed that in vitro cultured SARS-CoV-2 virus had much higher mutation frequency (up to ~20 times) than that in infected patients, and the mutation in nonstructural protein 14 (nsp14) might increase the genomic mutation frequency. Different strains had various amount of spike protein which may positively correlated with the virus replication capacity but may be influenced by other viral factors. Interpretation: Our study suggested that SARS-CoV-2 has the potential to diversify under favorable conditions. Monitoring viral mutations is not only helpful for better understanding of virus evolution and virulence change, but also the key to prevent virus transmission and disease progression. SARS-CoV-2 genomic variation analysis may also provide potential ideas for more efficient vaccine development and clinical treatment. Funding: This work is funded by Special Project for Emergency Scientific and Technological Research on New Coronavirus Infection (YG, No. YD9110002001), Emergency Research Project of Novel Coronavirus Infection of Anhui Province (Grant numbers 202004a07020002; 202004a07020004), Postdoctoral Research Foundation of China (2020M670084ZX) and the Fundamental Research Funds for the Central Universities (WK9110000166; WK9110000167).Declaration of Interests: We declare no competing interests.Ethics Approval Statement: The study was conformed to the principles of the Declaration ofHelsinki and approved by the Ethics Committee of the First Affiliated Hospital of USTC..

Large-scale single-cell analysis reveals critical immune characteristics of COVID-19 patients (preprint)

Dysfunctional immune response in the COVID-19 patients is a recurrent theme impacting symptoms and mortality, yet the detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 205 COVID-19 patients and controls to create a comprehensive immune landscape. Lymphopenia and active T and B cell responses were found to coexist and associated with age, sex and their interactions with COVID-19. Diverse epithelial and immune cell types were observed to be virus-positive and showed dramatic transcriptomic changes. Elevation of ANXA1 and S100A9 in virus-positive squamous epithelial cells may enable the initiation of neutrophil and macrophage responses via the ANXA1-FPR1 and S100A8/9-TLR4 axes. Systemic up-regulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis and designing effective therapeutic strategies for COVID-19.

Using the Kano model to associate the number of confirmed cases of COVID-19 in a population of 100,000 with case fatality rates: an observational study (preprint)

Background: One of the key factors to understand the spread of COVID-19 is the case fatality rate(CFR) rates for counties/regions. However, many merely reported those CFRs on the basis of total confirmed cases(TCC) instead of per population of 100, 000. As such, the disparate definitions of CFR yield inconsistent results in COVID-19 situations. Whether both incident rate and the CFR can be indicators to identify a country/region hit by COVID-19 under(or out of) control is still unclear. This study aims to (1) develop a diagram to disperse both TCC and CFR on a population base of 100,000(namely TCC100 and CFR100, respectively) using the Kano model, (2)discuss selected countries/regions with success on preventative measures to keep COVID-19 under control(i.e., relatively lower TCC100 and CFR100 or CFR), and (3) design an app to display both TCC100 and CFR100 or CFR for all infected countries/regions.  Methods: We downloaded the COVID-19 data of confirmed cases and deaths in countries/regions on a daily basis from the Github website. Three values, TCC100, CFR100, and CFR, were computed for each country/region and displayed on the Kano model. The lower TCC100 and CFR values were deemed as having the COVID-19 situation more under control. An app was developed to display both TCC100 and CFR100 on Google Maps. Results: The correlation coefficient(CC) between TCC100 and CFR100 is 0.92(t=37.87) based on 285 count data (i.e., 12, 225, and 48 dispersed on the Kano diagram) obtained in comparison to the pair of TCC100 and CFR with the CC=-0.02(t=-0.25) and counts(i.e., 56, 145, and 84 on the Kano diagram). The state of Washington, USA, was found having the highest TCC100(=3498.15), CFR100 (=16548.96) and CFR(=5%) in comparison to (1)Taiwan[TCC100(=1.65), CFR100 (=2.17), CFR(=1%)] and (2)South Korea[TCC100(=20.34), CFR100 (=39.8), CFR(=2%)] on April 12,2020. Conclusion: We illustrated both TCC100 and CFR along with CFR100 to compare countries/regions on a Kano diagram to understand the COVID-19 situations better. A larger TCC100 value was strongly associated with a larger CFR100 value that needs further verifications in the future. An app was developed to display both TCC100 and CFR for countries/regions on a dashboard laid over Google Maps. 

Serological diagnostic kit of SARS-CoV-2 antibodies using CHO-expressed full-length SARS-CoV-2 S1 proteins (preprint)

WHO has declared COVID-19 a pandemic with more than 300,000 confirmed cases and more than 14,000 deaths. There is urgent need for accurate and rapid diagnostic kits. Here we report the development and validation of a COVID-19/SARS-CoV-2 S1 serology ELISA kit for the detection of total anti-virus antibody (IgG+IgM) titers in sera from either the general population or patients suspected to be infected. For indirect ELISA, CHO-expressed recombinant full length SARS-CoV-2-S1 protein with 6*His tag was used as the coating antigen to capture the SARS-CoV-2-S1 antibodies specifically. The specificity of the ELISA kit was determined to be 97.5%, as examined against total 412 normal human sera including 257 samples collected prior to the outbreak and 155 collected during the outbreak. The sensitivity of the ELISA kit was determined to be 97.5% by testing against 69 samples from hospitalized and/or recovered COVID-19 patients. The overall accuracy rate reached 97.3%. Most importantly, in one case study, the ELISA test kit was able to identify an infected person who had previously been quarantined for 14 days after coming into contact with a confirmed COVID-19 patient, and discharged after testing negative twice by nucleic acid test. With the assays developed here, we can screen millions of medical staffs in the hospitals and people in residential complex, schools, public transportations, and business parks in the epidemic centers of the outbreaks to fish out the "innocent viral spreaders", and help to stop the further spreading of the virus.