Omicron COVID-19 Immune Correlates Analysis of a Third Dose of mRNA-1273 in the COVE Trial (preprint)

In the coronavirus efficacy (COVE) phase 3 efficacy trial of the mRNA-1273 vaccine, IgG binding antibody (bAb) concentration against Spike (BA.1 strain) and neutralizing antibody (nAb) titer against Spike (BA.1 strain) pseudovirus were assessed as correlates of risk of Omicron COVID-19 and as correlates of relative boost efficacy in per-protocol recipients of a third (booster) dose. Markers were measured on the day of the boost (BD1) and 28 days later (BD29). For SARS-CoV-2 naive individuals, BD29 Spike IgG-BA.1 strain bAbs and BD29 BA.1-strain nAbs inversely correlated with Omicron COVID-19: hazard ratio (HR) per 10-fold marker increase [95% confidence interval (CI)] = 0.16 (0.03, 0.79); P=0.024 and 0.31 (0.10, 0.96); P = 0.042, respectively. These markers also inversely correlated with Omicron COVID-19 in non-naive individuals: HR = 0.15 (0.04, 0.63); P = 0.009 and 0.28 (0.07, 1.08); P = 0.06, trend. Fold-rise in markers from BD1 to BD29 had similarly strong inverse correlations. For SARS-CoV-2 naive individuals, overall booster relative (three-dose vs two-dose) efficacy was 46% (95% CI: 20%, 64%) and correlated with BA.1 strain nAb titer at exposure. At 56, 251, and 891 arbitrary units (AU)/ml (10th, 50th, and 90th percentile), the booster relative efficacies were -8% (95% CI: -126%, 48%), 50% (25%, 67%), and 74% (49%, 87%), respectively. Similar relationships were observed for Spike IgG-BA.1 strain bAbs and for the markers measured at BD29. The performance of bAb and nAb markers as correlates of protection against Omicron COVID-19 supports their continued use as surrogate endpoints for mRNA vaccination against Omicron COVID-19.

Early arrival did not ensure the early acquisition of intravenous thrombosis for acute ischemic stroke during the COVID-19 pandemic.

BACKGROUND: Intravenous thrombolysis (IVT) in acute ischemic stroke (AIS) is a time-dependent treatment with a narrow therapeutic time window, in which the time delay could result from the deadline effect. METHODS: One hospital-based cohort was recruited to detect the factors contributing to the deadline effect, where patients with the deadline effect were defined as those who were presented with the onset-to-door time (ODT) in the first 50%, while the door-to-needle time (DNT) was in the last quartile. DNT (in-hospital delay) was further subdivided into several time intervals [door-to-examination time (DET), door-to-imaging time (DIT), door-to-laboratory time (DLT), and decision-making time (DMT) of the patients or their proxies. RESULTS: A total of 186 IVT cases were enrolled, of which 17.2% (32/186) suffered a delay of the deadline effect. The median age was 66 years, and 35.5% were female. Baseline characteristics were similar between the two groups (all p > .05). For the comparisons of the time intervals, DIT (26 versus 15 min, p = .001) was significantly longer in the group with deadline effect, while the differences of DET, DLT, DMT, and ONT did not reach statistical significance (all p > .05). Upon multivariable adjustment in the binary logistic regression model, longer DIT [odds ratio (OR), 1.076; 95% confidence interval (CI), 1.036-1.118; p < .001], and history of coronary heart disease (OR, 3.898; 95%CI, 1.415-10.735; p = .008) were independently associated with deadline effect in the binary logistic regression model, while admitted in the working day (OR, 0.674; 95%CI, 0.096-0.907; p = .033), and having medical insurance (OR, 0.350; 95% CI, 0.132-0.931; p = .035) were negatively associated with the deadline effect. CONCLUSIONS: A speed-safety tradeoff phenomenon from the deadline effect was observed in 17.2% of IVT cases during the COVID-19 pandemic, where longer DIT contributed a lot to this time delay. Patients without medical insurance, or admitted in official holidays were more likely to experience a delay of the deadline effect.

An Efficient Face Mask Wearing Detection Algorithm Based on Improved YOLOv3

In recent years, the Corona Virus Disease 2019 (Covid-19) epidemic has raged around the world, with more than 500 million people diagnosed. Relevant medical research and analysis results on Covid-19 indicate that wearing masks is an effective method to prevent and restrain virus transmission. Mask detection stations have been set up in hospitals, railway stations, schools, where there is large crowd flow, but results are not as good as expected. In order to ameliorate pandemic preventing and control measures, a mask wearing detection algorithm YOLOv3-M3 was designed and proposed in this paper. The algorithm can effectively detect people without mask, while consequently reminding them. Firstly, we substituted the feature extraction network of YOLOv3 with MobileNetv3, a lightweight convolutional neural network. Secondly, we utilized K-Means++ to substitute the original ground truth clustering algorithm to improve prediction precision. In addition, the bounding box regression loss function was revised as CIoU loss function. This loss function solves the issues of overlapping between the ground truth and the anchor box, which has increased the training speed. After experiments, the precision of YOLOv3 algorithm on mAP 0.5 and mAP 0.75 is 93.5% and 71.9%, respectively. Elevating 3.1% and 2.6%, respectively, higher than that of YOLOv3 algorithm, and it was superior to SSD, SSD Lite, YOLOv3-Tiny and other one-stage object detection algorithms. The detection speed can reach 13.6 frame/s, which has met the requirements of pandemic prevention and control in most places and can be deployed on terminal devices for object detection. [ FROM AUTHOR]

Multi-period portfolio selection based on uncertainty theory with bankruptcy control and liquidity

Due to the impact of uncertain events, such as the 2008 financial crisis and the outburst of COVID-19 pandemic, the experts’ evaluations information is becoming increasingly important. This paper considers a multi-period portfolio optimization problem under uncertain circumstance, and the return rates of risky securities are regarded as uncertain variables, where the uncertainty theory is used to deal with experts’ evaluations. In light of the complexity of financial markets, we formulate an uncertain multi-period mean-entropy-variance model, where bankruptcy, liquidity, diversification and self-financing are considered as realistic constraints. Furthermore, the maximum return and the minimum risk are both achieved in a single-objective model through the normalization method. Then the equivalent deterministic forms of two secondary models for main model are provided. In addition, we develop a modified root system growth algorithm, which is more suitable for the proposed model. Finally, the effectiveness of the proposed model and designed algorithm is confirmed by numerical simulations.

PF-D-Trimer, a broadly protective SARS-CoV-2 subunit vaccine: immunogenicity and application (preprint)

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has had and still has a considerable impact on global public health. One of the characteristics of SARS-CoV-2 is a surface homotrimeric spike protein, the primary responsible for the host immune response upon infection. Here we show the preclinical studies of a broad protective SARS-CoV-2 subunit vaccine developed from our Trimer Domain platform using the Delta spike protein, from antigen design to purification, vaccine evaluation and manufacturability. The prefusion trimerized Delta spike protein, PF-D-Trimer, was highly expressed in Chinese hamster ovary (CHO) cells, purified by a rapid one-step anti-Trimer Domain monoclonal antibody immunoaffinity process and prepared as a vaccine formulation with an adjuvant. The immunogenicity studies demonstrated that this vaccine candidate induces robust immune responses in mouse, rat and Syrian hamster models. It also protects K18-hACE2 transgenic mice in a homologous virus challenge. The neutralizing antibodies induced by this vaccine display a cross-reactive capacity against the ancestral WA1 and Delta variants as well as different Omicron, including BA.5.2. The Trimer Domain platform was proven to be a key technology in the rapid production of the PF-D-Trimer vaccine and may be crucial to accelerate the development of updated versions of SARS-CoV-2 vaccines.

Hygiene of cell phones of health care workers from NICU of a hospital in Zhejiang Province

OBJECTIVE: To understand the status of pollution of cell phones of health care workers from neonatal intensive care unit(NICU) and explore the effect of frequency of regular disinfection on hygiene of cell phones so as to reduce the pollution of cell phones. METHODS: A questionnaire survey was conducted for the status of use of cell phones among the health care workers, the samples were collected from the cell phones of three groups of health care workers that were disinfected with different frequencies by using sterile sponge sticks. Chi square test was carried out to analyze the impact of use habit of cell phones on the hygiene and the impact of different frequencies of disinfection on the hygiene of cell phones. RESULTS: A total of 46 health care workers were enrolled in the study, all of them used touch-screen cell phones, 97.83% of the health care workers used the cell phone when working, 17.39% used sometimes;13.04% of the health care workers never carried out hand hygiene after they used cell phones, only 1(2.17%) health care worker carried out hand hygiene every time after use;the frequency of regular cleaning of cell phones was less than once per day among 69.57% of the health care workers. The majority of the health care workers used disposable disinfectant wipes for the cleaning of cell phones;15.22% of the health care workers used disposable protective covers. The cell phones of 32 health care workers were sampled, the qualified rate of the samples was 34.38%, and pathogens were not isolated. The cell phones maintained clean by using the disposable protective covers. There was no significant difference in the qualified rate of hygiene among the cell phones with different frequencies of disinfection. CONCLUSION: The regular cleaning and disinfection of the cell phones may effectively reduce the bacterial colonization on the surfaces of cell phones, and the use of disposable protective covers may keep the cell phones relatively clean.

Density Map-based vehicle counting in remote sensing images with limited resolution

Observing traffic flow is of great significance to contemporary urban management. Overhead images, as represented by remote sensing images, provide a major source of information about traffic flow. However, the spatial resolutions of most common high-resolution remote sensing images are often limited to 0.5 m and even below, which makes it unrealistic to count vehicles by means of widely used object detection methods. Therefore, to explore the potential of remote sensing data for studying global urban development and management, this paper introduces a density map-based vehicle counting method for remote sensing imagery with limited resolution. Density map-based models regard the vehicle counting task as estimating the density of vehicle targets in terms of pixel values. We propose an improved CNN-based network, called Congested Scene Recognition Network Minus (CSRNet—), that generates a density map of vehicles from the input remote sensing imagery. A new dataset, RSVC2021, which was generated from the public DOTA and ITCVD datasets, is also introduced for network training and testing. A benchmark on the RSVC2021 dataset is accordingly established and CSRNet— is selected as the baseline model for subsequent experiments. A set of GF-2 time series images with a resolution of 1 m taken before, during and after the COVID-19 epidemic lockdown covering Wuhan city are applied for real-world application testing. The testing results on both the RSVC2021 dataset and real satellite images confirm that, in terms of both the counting values and the visualized density maps, the proposed method achieves good performance and exhibits considerable application potential in this task. The generating codes of RSVC2021 dataset will be publicly available at https://github.com/YinongGuo/RSVC2021-Dataset.

Rapid differentiation of SARS-CoV-2 variants on a nanotechnology enhanced DNA-chip (preprint)

With the persistence of the COVID-19 pandemic caused primarily by constant viral mutations, rapid identification of different lineages of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by large-scale screening at the point-of-care could be key to monitoring and assessing viral evolutions. Herein, we developed a Fluorescence Enhanced Microarray for Multiplex Analysis of Nucleic acids (FEMMAN) for detecting 8 SARS-CoV-2 variants simultaneously in ~ 3 hours without the need of RNA extraction, opening the possibility of point-of-care testing of multiple SARS-CoV-2 variants while reducing the cost significantly to ~ $ 7 per sample from ~ $100 by Next-Generation Sequencing (NGS). Combined with isothermal amplification, the multiplexed RNA assay achieved single-copy detection sensitivity and single nucleotide variant (SNV) distinction owing to the nanotechnology based plasmonic gold (pGOLD) near-infrared fluorescence enhancing platform. Probing 10 targets of three mutational hotspots in S gene, we differentiated 8 viral lineages (Wild type, Alpha, Beta, Gamma, Delta, Lambda, Mu, and Omicron) of SARS-CoV-2, validated using nasopharyngeal swabs obtained from 127 individuals, achieving a 100% sensitivity and 100% specificity in SARS-CoV-2 detection, and a 91.1% concordance with NGS in variant identification. The scalable, multiplexed FEMMAN assay could shift the paradigm of COVID-19 diagnostic and surveillance from positive/negative assessments to simultaneous lineage identification in large-scale screening, greatly facilitating the global monitoring of SARS-CoV-2 variants.

Evaluation of the occurrence of psychotropic drugs in urban sewage and the abuse during the COVID-19 epidemic period in a city of Guangdong

Using the method of sewage epidemiology, the occurrence characteristics and changes of two typical psychoactive drugs methamphetamine and ketamine in the domestic sewage of a city in Guangdong Province during and before and after the new crown epidemic in 2020 were studied, and the mobile phone signaling data was used to analyze The total population, structural characteristics and their change laws were combined to analyze the changes in the consumption of the two drugs during the epidemic and before and after the epidemic. The results show that the actual population of the city during the epidemic decreased by about 45% compared with before the epidemic. The total consumption of various drugs has dropped by about 75% compared to before the epidemic. The significant decline in total consumption was largely caused by the actual population decline during the epidemic. In fact, the per capita consumption levels of methamphetamine and ketamine in the city during the epidemic Compared with the pre-epidemic drop of less than 50%. Taking into account the strict control measures during the epidemic, this decline shows that the abuse of these two psychotropic substances is extremely stubborn;after the epidemic, the consumption of the two drugs and their concentrations in the environment have increased significantly. It has basically returned to the level before the epidemic, which further proves the stubbornness of psychotropic substance abuse.

Novel pectin from crude polysaccharide of Syzygium aromaticum against SARS-CoV-2 activities by targeting 3CLpro (preprint)

To date, COVID-19 is still a severe threat to public health, hence specific effective therapeutic drugs development against SARS-CoV-2 is urgent needed. 3CLpro and PLpro and RdRp are the enzymes required for the SARS-CoV-2 RNA synthesis. Therefore, binding to the enzyme may interfere the enzyme function. Before, we found that sulfated polysaccharide binding to 3CLpro might block the virus replication. Hence, we hypothesize that negative charged pectin glycan may also impede the virus replication. Here we show that 922 crude polysaccharide from Syzygium aromaticum may near completely block SARS-CoV-2 replication. The inhibition rate was 99.9% (EC50 : 0.90 muM). Interestingly, 922 can associates with 3CLpro, PLpro and RdRp. We further show that the homogeneous glycan 922211 from 922 may specifically attenuate 3CL protease activity. The IC50s of 922 and 922211 against 3CLpro are 4.73 plusmn 1.05 muM and 0.18 plusmn 0.01 muM, respectively. Monosaccharide composition analysis reveals that 922211 with molecular weight of 78.7 kDa is composed of rhamnose, galacturonic acid, galactose and arabinose in the molar ratio of 8.21 : 37.81 : 3.58 : 4.49. The structure characterization demonstrated that 922211 is a homogalacturonan linked to RG-I pectin polysaccharide. The linear homogalacturonan part in the backbone may be partly methyl esterified while RG-I type part bearing 1, 4-linked alpha-GalpA, 1, 4-linked alpha-GalpAOMe and 1, 2, 4-linked alpha-Rhap. There are four branches attached to C-1 or C4 position of Rhamnose glycosyl residues on the backbone. The branches are composed of 1, 3-linked beta-Galp, terminal (T)-linked beta-Galp, 1, 5-linked alpha-Araf, T-linked alpha-Araf, 4-linked alpha-GalpA and/or 4-linked beta-GalpA. The above results suggest that 922 and 922211 might be a potential novel leading compound for anti-SARS-CoV-2 new drug development.

Micronucleus Production, Activation of DNA Damage Response and cGAS-STING Signaling in Syncytia Induced by SARS-CoV-2 Infection (preprint)

SARS-CoV-2 infection could cause severe acute respiratory syndrome, largely attributed to dysregulated immune activation and extensive lung tissue damage. However, the underlying mechanisms are not fully understood. Here, we reported that viral infection could induce syncytia formation within cells expressing ACE2 and the SARS-CoV-2 spike protein, leading to the production of micronuclei with an average rate of about 4 per syncytium (> 93%). Remarkably, these micronuclei were manifested with a high level of activation of both DNA damage response and cGAS -STING signaling, as indicated by micronucleus translocation of gH2Ax and cGAS, and upregulation of their respective downstream target genes. Since activation of these signaling pathways were known to be associated with cellular catastrophe and aberrant immune activation, these findings help explain the pathological effects of SARS-CoV-2 infection at cellular and molecular levels, and provide novel potential targets for COVID-19 therapy.

The nucleic acid of SARS-CoV-2 was negetive in 775 fur animal samples

In December 2019, an outbreak of a respiratory illness took place in Wuhan, Hubei Province, China, and spread throughout China and other countries. A beta genus coronavirus, SARS-CoV-2 was isolated and identified as a cause of the outbreak. Currently, the main infectious sources(host)of SARS-CoV-2 have not been identified. It has been reported that SARS-CoV-2 was possibly originated from wild animals, for example, bats. However, there are no reports on the traceability of SARS-CoV-2 to fur animals, such as minks, foxes and raccoon dogs, which are widely farmed in Northern China. Therefore, it is necessary to investigate the epidemiology of SARS-CoV-2 in fur animals. In this study, we detected SARS-CoV-2 in 625 tissue specimens collected from farmed minks, foxes and raccoon dogs of unknown death cases in 14 regions of China from 2016 to 2019 and 150 fecal samples collected from farmed minks, foxes and raccoon dogs in Jilin Province, China from August to December in 2019 using the Real-time RT-PCR method recommended by WHO. The results showed that SARS-CoV-2 was not detected in all the 775 samples.

Persistently Reduced Pulmonary Function and Quality of Life in Patients With Mild COVID-19 One Year After Discharge: a Cohort Study (preprint)

Purpose: The epidemiological and clinical features, pathogenesis, and complications of patients with COVID-19 in the acute phase have been well described, but the long-term prognosis and rehabilitation of the patients remain largely unknown. Methods: 39 COVID-19 patients were followed up at 3, 6, and 12 months after discharge. The mental health, pulmonary function, exercise capacity, and quality of life were assessed by Self-Depression Scale and the Self Anxiety Scale, pulmonary function test, 6MWT, 36-Item Short-Form, respectively. Results: Total 33 survivors completed the assessment, 40.8 ± 0.8 years, body mass index= 22.7 ±1.3 kg/m 2 . The length of hospital stays was 19.6 ± 6.6 d. One year after discharge, the mean scores of SDS and SAS showed decreasing trends from 3-months to 12-months post-discharge. 6 patients (18.2%) had FVC <80% of the predicted value, 12 patients (36.4%) had FEV1 <80% of the predicted value. And 9 (27.3%), 3 (9.1%), and 2 (6.1%) of the patients showed reduced FEF25, FEF50, and FEF75 (<70% expected values), respectively. The mean 6MWD values increased significantly from 397±25.4 m at 3-months to 514±40.8 m at 12-months. Conclusions: the impaired pulmonary function in mild COVID-19 survivors was noted after 12 months discharging from hospital. The exercise capacity, mental status, and health status were lower than those of the normal population.

Summary of global surveillance data of infectious diseases in April 2021

In April 2021, a total of 66 infectious diseases were reported globally, affecting 225 countries and regions. Except for influenza, the top five infectious diseases affecting greatest number of countries and regions were Corona virus disease 2019 (COVID-19, 225), dengue fever (33), measles (26), poliomyelitis (10) and pneumococcal disease (7). The top five infectious diseases with highest case fatality rates were Ebola virus disease (51.4%), yellow fever (33.3%), lassa fever (20.5%), diphtheria (7.0%) and meningitis (5.4%). The top five infectious diseases with greatest number of deaths were COVID-19, cholera, measles, dengue fever and lassa fever. The prevalent infectious diseases in Asia were COVID-19 and dengue fever, the prevalent infectious diseases in Africa were COVID-19, Ebola virus disease, cholera, yellow fever, lassa fever and Rift valley fever, the prevalent infectious diseases in America were COVID-19 and dengue fever, the prevalent infectious disease in Europe was COVID-19.

COVID-ONE-humoral immune: The One-stop Database for COVID-19-specific Antibody Responses and Clinical Parameters (preprint)

Coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2, varies with regard to symptoms and mortality rates among populations. Humoral immunity plays critical roles in SARS-CoV-2 infection and recovery from COVID-19. However, differences in immune responses and clinical features among COVID-19 patients remain largely unknown. Here, we report a database for COVID-19-specific IgG/IgM immune responses and clinical parameters (COVID-ONE humoral immune). COVID-ONE humoral immunity is based on a dataset that contains the IgG/IgM responses to 21 of 28 known SARS-CoV-2 proteins and 197 spike protein peptides against 2,360 COVID-19 samples collected from 783 patients. In addition, 96 clinical parameters for the 2,360 samples and information for the 783 patients are integrated into the database. Furthermore, COVID-ONE humoral immune provides a dashboard for defining samples and a one-click analysis pipeline for a single group or paired groups. A set of samples of interest is easily defined by adjusting the scale bars of a variety of parameters. After the "START" button is clicked, one can readily obtain a comprehensive analysis report for further interpretation. COVID-ONE-humoral immune is freely available at www.COVID-ONE.cn.

COVID-ONE-humoral immune: The One-stop Database for COVID-19-specific Antibody Responses and Clinical Parameters (preprint)

Coronavirus disease 2019 (COVID-19), which is caused by SARS-CoV-2, varies with regard to symptoms and mortality rates among populations. Humoral immunity plays critical roles in SARS-CoV-2 infection and recovery from COVID-19. However, differences in immune responses and clinical features among COVID-19 patients remain largely unknown. Here, we report a database for COVID-19-specific IgG/IgM immune responses and clinical parameters (COVID-ONE humoral immune). COVID-ONE humoral immunity is based on a dataset that contains the IgG/IgM responses to 21 of 28 known SARS-CoV-2 proteins and 197 spike protein peptides against 2,360 COVID-19 samples collected from 783 patients. In addition, 96 clinical parameters for the 2,360 samples and information for the 783 patients are integrated into the database. Furthermore, COVID-ONE humoral immune provides a dashboard for defining samples and a one-click analysis pipeline for a single group or paired groups. A set of samples of interest is easily defined by adjusting the scale bars of a variety of parameters. After the START button is clicked, one can readily obtain a comprehensive analysis report for further interpretation. COVID-ONE-humoral immune is freely available at www.COVID-ONE.cn.

Human Mesenchymal Stem Cells Treatment for Severe COVID-19: 1-Year Follow-Up Results of a Randomized, Double-Blind, Placebo-Controlled Trial (preprint)

Background The long-term consequences of human umbilical cord-derived mesenchymal stem cell (UC-MSC) treatment for COVID-19 patients are yet to be reported. This study assessed the 1-year outcomes in patients with severe COVID-19, who were recruited in our previous UC-MSC clinical trial.Methods: In this prospective, longitudinal, cohort study, 100 patients enrolled in our phase 2 trial were prospectively followed up at 3-month intervals for 1 year to evaluate the long-term safety and effectiveness of UC-MSC treatment. The primary endpoint was an altered proportion of whole-lung lesion volumes measured by high-resolution CT. Other imaging outcomes, 6-minute walking distance (6-MWD), lung function, plasma biomarkers, and adverse events were also recorded and analyzed. This trial was registered with ClinicalTrials.gov (NCT04288102).Findings: Within 3 months, MSC administration exerted numerical improvement in whole-lung lesion volume compared with the placebo, leading to a significant difference of −10.82% (95% CI: −20.69%, −1.46%, P=0.030) on day 10. MSC also reduced the proportion of solid component lesion volume compared with the placebo at each follow-up point, with a significant difference of − 9.02% (95%CI: − 17.44%, − 0.10%, P=0.045) at month 9. More interestingly, 17.86% (10/56) of patients in the MSC group had normal CT images at month 12 ( P= 0.013), but none in the placebo group. The incidence of symptoms was lower in the MSC group than in the placebo group at each follow-up time, particularly sleep difficulties at month 3 (OR 0.19, 95% CI 0.07,0.50; P=0.001), and usual activity at month 12 (OR 0.15, 95% CI 0.03,0.79; P=0.018). Neutralizing antibodies were all positive, with a similar median inhibition rate (61.6% vs. 67.55%) in both groups at month 12. No difference in adverse events at the 1-year follow-up and tumor markers at month 12 were observed between the two groups.Interpretation: UC-MSC administration achieves a long-term benefit in the recovery of lung lesions and symptoms in COVID-19 patients.Trial Registration: This trial was registered with ClinicalTrials.gov (NCT04288102).Funding The National Key R&D Program of China, the Innovation Groups of the National Natural Science Foundation of China, and the National Science and Technology Major Project.Declaration of Interest: None to declare. Ethical Approval: This study was approved by the Ethics Committee of the Fifth Medical Center, Chinese PLA General Hospital (2020-013-D).

A Short Plus Long-Amplicon Based Sequencing Approach Improves Genomic Coverage and Variant Detection In the SARS-CoV-2 Genome (preprint)

High viral transmission in the COVID-19 pandemic has enabled SARS-CoV-2 to acquire new mutations that impact genome sequencing methods. The ARTIC.v3 primer pool that amplifies short amplicons in a multiplex-PCR reaction is one of the most widely used methods for sequencing the SARS-CoV-2 genome. We observed that some genomic intervals are poorly captured with ARTIC primers. To improve the genomic coverage and variant detection across these intervals, we designed long amplicon primers and evaluated the performance of a short (ARTIC) plus long amplicon (MRL) sequencing approach. Sequencing assays were optimized on VR-1986D-ATCC RNA followed by sequencing of nasopharyngeal swab specimens from five COVID-19 positive patients. ARTIC data covered >90% of the virus genome fraction in the positive control and four of the five patient samples. Variant analysis in the ARTIC data detected 67 mutations, including 66 single nucleotide variants (SNVs) and one deletion in ORF10. Of 66 SNVs, five were present in the spike gene, including nt22093 (M177I), nt23042 (S494P), nt23403 (D614G), nt23604 (P681H), and nt23709 (T716I). The D614G mutation is a common variant that has been shown to alter the fitness of SARS-CoV-2. Two spike protein mutations, P681H and T716I, which are represented in the B.1.1.7 lineage of SARS-CoV-2, were also detected in one patient. Long-amplicon data detected 58 variants, of which 70% were concordant with ARTIC data. Combined analysis of ARTIC +MRL data revealed 22 mutations that were either ambiguous (17) or not called at all (5) in ARTIC data due to poor sequencing coverage. For example, a common mutation in the ORF3a gene at nt25907 (G172V) was missed by the ARTIC assay. Hybrid data analysis improved sequencing coverage overall and identified 59 high confidence mutations for phylogenetic analysis. Thus, we show that while the short amplicon (ARTIC) assay provides good genomic coverage with high throughput, complementation of poorly captured intervals with long amplicon data can significantly improve SARS-CoV-2 genomic coverage and variant detection.