Evaluation of Sample Pooling for Gene Sequencing of SARS-CoV-2: A Simulation Study (preprint)

COVID-19 continues to pose a significant public health threat, requiring ongoing epidemiological and genomic surveillance. Next-generation sequencing (NGS) is commonly utilized for monitoring viral evolution at high-cost. Real-time reverse transcription polymerase chain reaction(RT-qPCR) pooling test have been have been demonstrated to increase molecular testing throughput effectively during the pandemic, while pooling sequencing shows promise as a cost-effective tool for monitoring virus variants. A simulation study was conducted to evaluate the efficacy of sample pooling for viral gene sequencing of SARS-CoV-2. In this study, 72 original raw data of gene sequencing results with different lineages were collected and combined to create simulated samples based on five pooling strategies. A bioinformatics tool based on Freyja was utilized to analyze the variant composition of these 70 simulated pooled samples. The genetic composition of the pooled samples mostly recovered the variant compositions of the original samples, with discrepancies between the top X results (where X is the number of original samples included in the mixture)and the complete results (P < 0.05).Variations were observed in the calculation method for the top X results (P < 0.05) but not in the complete calculation approach (P > 0.05) across the five pooling strategy groups. Some original samples of suboptimal quality were not accurately identified. The present results indicated that sample pooling coupled with streamlined genotyping offers a promising approach for cost-effective gene sequencing of SARS-CoV-2, which will aid in genomic surveillance efforts against COVID-19.

A Comprehensive Review of Natural Flavonoids with Anti-SARS-CoV-2 Activity.

The COVID-19 pandemic caused by SARS-CoV-2 has majorly impacted public health and economies worldwide. Although several effective vaccines and drugs are now used to prevent and treat COVID-19, natural products, especially flavonoids, showed great therapeutic potential early in the pandemic and thus attracted particular attention. Quercetin, baicalein, baicalin, EGCG (epigallocatechin gallate), and luteolin are among the most studied flavonoids in this field. Flavonoids can directly or indirectly exert antiviral activities, such as the inhibition of virus invasion and the replication and inhibition of viral proteases. In addition, flavonoids can modulate the levels of interferon and proinflammatory factors. We have reviewed the previously reported relevant literature researching the pharmacological anti-SARS-CoV-2 activity of flavonoids where structures, classifications, synthetic pathways, and pharmacological effects are summarized. There is no doubt that flavonoids have great potential in the treatment of COVID-19. However, most of the current research is still in the theoretical stage. More studies are recommended to evaluate the efficacy and safety of flavonoids against SARS-CoV-2.

Genetic characterization of SARS-CoV-2 from cross-border trucks and containers in Kashgar Prefecture, Xinjiang Uygur Autonomous Region, China

Kashgar is a prefecture in Xinjiang Uygur Autonomous Region. China. Kashgar Prefecture (KP) is a land-cargo port connecting China with central Asian countries and Europe. Frequent transportation of cargo has increased the risk of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) introduction into China, which has increased the pressure on coronavirus disease-2019 (COVID-19) prevention and control. In November 2020, an imported virus-induced COVID-19 outbreak occurred in KP. To investigate the genetic characterization of SARS-CoV-2 that contaminated the trucks and containers, and the potential of border rapid logistics system to serve as carriers for SARS-CoV-2 transmission, thirty-five SARS-CoV-2-positive nucleic-acid samples collected from KP cross-border trucks and containers from 6-10 November 2020 were subjected into SARS-CoV-2 genomic sequencing and comparative analyses. The results showed that the median (minimum to maximum) Ct value of ORF1ab was 37.64 (28.91-39.81) . and that of the N gene was 36.50 (26.35-39.30), and the median (minimum to maximum) of the reads mapping ratio to SARS-CoV-2 was 51.95% (0.86%-99.31%), which indicated low viral loads in these environmental samples. Eighteen of 35 samples had genomic coverage >70%. According to the Pango nomenclature, 18 SARS-CoV-2 sequences belonged to six lineages (B.1, B.I.1, B.1.9. B.1.1.220, B.1.153 and B.1.465), three of which (B.I. B.1.1 and 8.1.153) were found in case samples from the same period of four China-neighboring countries. Analyses of nucleotide mutations and phylogenetic trees showed that the genome sequences of SARS-CoV-2 collected from the same location were similar. Four of 18 sequences were in a sub-lineage with the representative strain of COVID-19 outbreak in KP, one of which had 1 or 2 differences in nucleotide mutation sites with the strain that caused the COVID-19 outbreak in KP, which indicated high homology in the viral genome. We showed that cross-border trucks and containers were contaminated by various genotypes of SARS-CoV-2 from other countries during the outbreak in KP. and in which contained the parental virus of the KP cases. These trucks and containers served as carriers for SARS-CoV-2 introduction from other countries to cause local transmission. Our results provide important references for COVID-19 prevention-and-control strategies in border ports and tracing of outbreak sources in China.

Prospective RFID Sensors for the IoT Healthcare System

The outbreak of COVID-19 has attracted people’s attention to our healthcare system, stimulating the advancement of next-generation health monitoring technologies. IoT attracts extensive attention in this advancement for its advantage in ubiquitous communication and sensing. RFID plays a key role in IoT to tackle the challenges in passive communication and identification and is now emerging as a sensing technology which has the ability to reduce the cost and complexity of data collection. It is advantageous to introduce RFID sensor technologies in health-related sensing and monitoring, as there are many sensors used in health monitoring systems with the potential to be integrated with RFID for smart sensing and monitoring. But due to the unique characteristics of the human body, there are challenges in developing effective RFID sensors for human health monitoring in terms of communication and sensing. For example, in a typical IoT health monitoring application, the main challenges are as follows: (1) energy issues, the efficiency of RF front-end energy harvesting and power conversion is measured;(2) communication issues, the basic technology of RFID sensors shows great heterogeneity in terms of antennas, integrated circuit functions, sensing elements, and data protocols;and (3) performance stability and sensitivity issues, the RFID sensors are mainly attached to the object to be measured to carry out identification and parameter sensing. However, in practical applications, these can also be affected by certain environmental factors. This paper presents the recent advancement in RFID sensor technologies and the challenges for the IoT healthcare system. The current sensors used in health monitoring are also reviewed with regard to integrating possibility with RFID and IoT. The future research direction is pointed out for the emergence of the next-generation healthcare and monitoring system.

Surveillance of SARS-CoV-2 in the environment and animal samples of the Huanan Seafood Market (preprint)

Emerging in December 2019, coronavirus disease 2019 (COVID-19) eventually became a pandemic and has posed a tremendous threat to global public health. However, the origins of SARS-CoV-2, the causative agent of COVID-19, remain to be determined. It has reported that a certain number of the early case clusters had a contact history with Huanan Seafood Market. Therefore, surveillance of SARS-CoV-2 within the market is of vital importance. Herein, we presented the SARS-CoV-2 detection results of 1380 samples collected from the environment and the animals within the market in early 2020. By SARS-CoV-2-specific RT-qPCR, 73 environmental samples tested positive for SARS-CoV-2 and three live viruses were successfully isolated. The viruses from the market shared nucleotide identity of 99.980% to 99.993% with the human isolate HCoV/Wuhan/IVDC-HB-01. In contrast, no virus was detected in the animal swabs covering 18 species of animals in the market. The SARS-COV-2 nucleic acids in the positive environmental samples showed significant correlation of abundance of Homo sapiens with SARS-CoV-2. In summary, this study provided convincing evidence of the prevalence of SARS-CoV-2 in the Huanan Seafood Market during the early stage of COVID-19 outbreak.

Potent Neutralizing Antibodies Elicited by RBD-Fc-Based COVID-19 Vaccine Candidate Adjuvanted by the Th2-Skewing iNKT Cell Agonist.

The development of a safe and effective COVID-19 vaccine is of paramount importance to terminate the current pandemic. An adjuvant is crucial for improving the efficacy of the subunit COVID19 vaccine. α-Galactosylceramide (αGC) is a classical iNKT cell agonist which causes the rapid production of Th1- and Th2-associated cytokines; we, therefore, expect that the Th1- or Th2-skewing analogues of αGC can better enhance the immunogenicity of the receptor-binding domain in the spike protein of SARS-CoV-2 fused with the Fc region of human IgG (RBD-Fc). Herein, we developed a universal synthetic route to the Th1-biasing (α-C-GC) and Th2-biasing (OCH and C20:2) analogues. Immunization of mice demonstrated that αGC-adjuvanted RBD-Fc elicited a more potent humoral response than that observed with Alum and enabled the sparing of antigens. Remarkably, at a low dose of the RBD-Fc protein (2 µg), the Th2-biasing agonist C20:2 induced a significantly higher titer of the neutralizing antibody than that of Alum.

COVID-19 cases from the first local outbreak of SARS-CoV-2 B.1.1.7 variant in China presented more serious clinical features: a prospective, comparative cohort study (preprint)

Background: The SARS-CoV-2 B.1.1.7 variant which was first identified in the United Kingdom (U.K.) has increased sharply in numbers worldwide and was reported to be more contagious. On January 17, 2021, a COVID-19 clustered outbreak caused by B.1.1.7 variant occurred in a community in Daxing District, Beijing, China. Three weeks prior, another non-variant (lineage B.1.470) COVID-19 outbreak occurred in Shunyi District, Beijing. This study aimed to investigate the clinical features of B.1.1.7 variant infection. Methods: A prospective cohort study was conducted on COVID-19 cases admitted to Ditan hospital since January 2020. Data of 74 COVID-19 cases from two independent COVID-19 outbreaks in Beijing were extracted as study subjects from a Cloud Database established in Ditan hospital, which included 41 Shunyi cases (Shunyi B.1.470 group) and 33 Daxing cases (Daxing B.1.1.7 group) that have been hospitalized since December 25, 2020 and January 17, 2021, respectively. We conducted a comparison of the clinical characteristics, RT-qPCR results and genomic features between the two groups. Findings: Cases from Daxing B.1.1.7 group (15 [45.5%] male; median age, 39 years [range, 30.5, 62.5]) and cases from Shunyi B.1.470 group (25 [61.0%] male; median age, 31 years [range, 27.5, 41.0]) had a statistically significant difference in median age (P =0.014). Seven clinical indicators of Daxing B.1.1.7 group were significantly higher than Shunyi B.1.470 group including patients having fever over 38 (14/33 [46.43%] in Daxing B.1.1.7 group vs. 9/41 (21.95%) in Shunyi B.1.470 group [P = 0 .015]), C-reactive protein ([CRP, mg/L], 4.30 [2.45, 12.1] vs. 1.80, [0.85, 4.95], [P = 0.005]), Serum amyloid A ([SAA, mg/L], 21.50 [12.50, 50.70] vs. 12.00 [5.20, 26.95], [P = 0.003]), Creatine Kinase ([CK, U/L]), 110.50 [53.15,152.40] vs. 70.40 [54.35,103.05], [P = 0.040]), D-dimer ([DD, mg/L], 0.31 [0.20, 0.48] vs. 0.24 [0.17,0.31], [P = 0.038]), CD4+ T lymphocyte ([CD4+ T, mg/L], [P = 0.003]) , and Ground-glass opacity (GGO) in lung (15/33 [45.45%] vs. 5/41 [12.20%], [P =0.001]). After adjusting for the age factor, B.1.1.7 variant infection was the risk factor for CRP (P = 0.045, Odds ratio [OR] 2.791, CI [1.025, 0.8610]), SAA (0.011, 5.031, [1.459, 17.354]), CK (0.034, 4.34, [0.05, 0.91]), CD4+ T ( 0.029, 3.31, [1.13, 9.71]), and GGO (0.005, 5.418, [1.656, 17.729]) of patients. The median Ct value of RT-qPCR tests of the N-gene target in the Daxing B.1.1.7 group was significantly lower than the Shunyi B.1.470 group (P=0.036). The phylogenetic analysis showed that only 2 amino acid mutations in spike protein were detected in B.1.470 strains while B.1.1.7 strains had 3 deletions and 7 mutations. Interpretation: Clinical features including a more serious inflammatory response, pneumonia and a possible higher viral load were detected in the cases infected with B.1.1.7 SARS-CoV-2 variant. It could therefore be inferred that the B.1.1.7 variant may have increased pathogenicity.

Safety and immunogenicity of a recombinant tandem-repeat dimeric RBD protein vaccine against COVID-19 in adults: pooled analysis of two randomized, double-blind, placebo-controlled, phase 1 and 2 trials (preprint)

BackgroundA safe and effective coronavirus disease 2019 (COVID-19) vaccine is urgently needed to control the ongoing pandemic. Although progress has been made recently with several candidates reporting positive efficacy results, COVID-19 vaccines developed so far cannot meet the global vaccine demand. We developed a protein subunit vaccine against COVID-19, using dimeric form of receptor-binding domain (RBD) as the antigen. We aimed to assess the safety and immunogenicity of this vaccine in humans and determine the appropriate dose and schedule for an efficacy study. MethodsWe did two randomized, double-blind, placebo-controlled, phase 1 and 2 trials for an RBD-based protein subunit vaccine, ZF2001. In phase 1 study, 50 healthy adults aged 18-59 years were enrolled and randomly allocated to three groups to receive three doses of vaccine (25 g or 50 g RBD-dimer, with adjuvant) or placebo (adjuvant-only) intramuscularly, 30 days apart. In phase 2 study, 900 healthy adults aged 18-59 years were enrolled and randomly allocated to six groups to receive vaccine (25 g or 50 g RBD-dimer, with adjuvant) or placebo (adjuvant-only) intramuscularly, with the former 3 groups given two doses and the latter 3 groups given three doses, 30 days apart. For phase 1 trial, the primary outcome was safety, as measured by the occurrence of adverse events and serious adverse events. The secondary outcome was immunogenicity as measured by the seroconversion rate and magnitude of antigen-binding antibodies, neutralizing antibodies and T-cell cytokine production. For phase 2 trial, the primary outcome included both safety and immunogenicity. These trials are registered with ClinicaTrials.gov, NCT04445194 and NCT04466085. FindingsBetween June 22 and September 15, 2020, 50 participants were enrolled to the phase 1 study (mean age 32.6 years) and 900 participants were enrolled to phase 2 study (mean age 43.5 years), to receive vaccine or placebo with a two-dose or three-dose schedule. For both trials, local and systemic adverse reactions were absent or mild in most participants. There were no serious adverse events related to vaccine in either trial. After three doses, neutralizing antibodies were detected in all participants receiving either 25 g or 50 g dose of vaccine in phase 1 study, and in 97% (the 25 g group) and 93% (the 50 g group) of participants, respectively, in phase 2 study. The SARS-CoV-2-neutralizing geometric mean titres (GMTs) were 94.5 for the 25 g group and 117.8 for the 50 g group in phase 1, and 102.5 for the 25 g group and 69.1 for the 50 g group in phase 2, exceeding the level of a panel of COVID-19 convalescent samples (GMT, 51). Vaccine induced balanced TH1 and TH2 responses. The 50 g group did not show enhanced immunogenicity compared with the 25 g group. InterpretationThe protein subunit vaccine ZF2001 is well-tolerated and immunogenic. The safety and immunogenicity data from phase 1 and 2 trials for ZF2001 support the use of 25 g vaccine dose with three-dose schedule to an ongoing phase 3 large-scale evaluation for safety and efficacy. FundingNational Program on Key Research Project of China, National Science and Technology Major Projects of Drug Discovery, Strategic Priority Research Program of the Chinese Academy of Sciences, and Anhui Zhifei Longcom Biopharmaceutical.

Transmission and clinical characteristics of coronavirus disease 2019 in 104 outside-Wuhan patients, China (preprint)

Background: Cases with coronavirus disease 2019 (COVID-19) emigrated from Wuhan escalated the risk of spreading in other cities. This report focused on the outside-Wuhan patients to assess the transmission and clinical characteristics of this illness. Methods: Contact investigation was conducted on each patient who admitted to the assigned hospitals in Hunan Province (geographically adjacent to Wuhan) from Jan 22, 2020 to Feb 12, 2020. Demographic, clinical, laboratory and radiological characteristics, medication therapy and outcomes were collected and analyzed. Patients were confirmed by PCR test. Results: Of the 104 patients, 48 (46.15%) were imported cases and 56 (53.85%) were indigenous cases; 93 (89.42%) had a definite contact history with infections. Family clusters were the major body of patients. Transmission along the chain of 3 &ldquo:generations" was observed. Mean age was 43 (rang, 8-84) years (including 3 children) and 49 (47.12%) were male. Most patients had typical symptoms, 5 asymptomatic infections were found and 2 of them infected their relatives. The median incubation period was 6 (rang, 1-32) days, of 8 patients ranged from 18 to 32 days. Just 9 of 16 severe patients required ICU care. Until Feb 12, 2020, 40 (38.46%) discharged and 1 (0.96%) died. For the antiviral treatment, 80 (76.92%) patients received traditional Chinese medicine therapy. Conclusions: Family but not community transmission occupied the main body of infections in the two centers. Asymptomatic transmission demonstrated here warned us that it may bring more risk to the spread of COVID-19. The incubation period of 8 patients exceeded 14 days.

Mutations, Recombination and Insertion in the Evolution of 2019-nCoV (preprint)

BackgroundThe 2019 novel coronavirus (2019-nCoV or SARS-CoV-2) has spread more rapidly than any other betacoronavirus including SARS-CoV and MERS-CoV. However, the mechanisms responsible for infection and molecular evolution of this virus remained unclear. MethodsWe collected and analyzed 120 genomic sequences of 2019-nCoV including 11 novel genomes from patients in China. Through comprehensive analysis of the available genome sequences of 2019-nCoV strains, we have tracked multiple inheritable SNPs and determined the evolution of 2019-nCoV relative to other coronaviruses. ResultsSystematic analysis of 120 genomic sequences of 2019-nCoV revealed co-circulation of two genetic subgroups with distinct SNPs markers, which can be used to trace the 2019-nCoV spreading pathways to different regions and countries. Although 2019-nCoV, human and bat SARS-CoV share high homologous in overall genome structures, they evolved into two distinct groups with different receptor entry specificities through potential recombination in the receptor binding regions. In addition, 2019-nCoV has a unique four amino acid insertion between S1 and S2 domains of the spike protein, which created a potential furin or TMPRSS2 cleavage site. ConclusionsOur studies provided comprehensive insights into the evolution and spread of the 2019-nCoV. Our results provided evidence suggesting that 2019-nCoV may increase its infectivity through the receptor binding domain recombination and a cleavage site insertion. One Sentence SummaryNovel 2019-nCoV sequences revealed the evolution and specificity of betacoronavirus with possible mechanisms of enhanced infectivity.