FEN1-aided recombinase polymerase amplification (FARPA) for one-pot and multiplex detection of nucleic acids with an ultra-high specificity and sensitivity.

Recombinase polymerase amplification (RPA) running at 37-42 °C is fast, efficient and less-implemented; however, the existing technologies of nucleic acid testing based on RPA have some limitations in specificity of single-base recognition and multiplexing capability. Herein, we report a highly specific and multiplex RPA-based nucleic acid detection platform by combining flap endonuclease 1 (FEN1)-catalysed invasive reactions with RPA, termed as FEN1-aided RPA (FARPA). The optimal conditions enable RPA and FEN1-based fluorescence detection to occur automatically and sequentially within a 25-min turnaround time and FARPA exhibits sensitivity to 5 target molecules. Due to the ability of invasive reactions in discriminating single-base variation, this one-pot FARPA is much more specific than the Exo probe-based or CRISPR-based RPA methods. Using a universal primer pair derived from tags in reverse transcription primers, multiplex FARPA was successfully demonstrated by the 3-plex assay for the detection of SARS-CoV-2 pathogen (the ORF1ab, the N gene, and the human RNase P gene as the internal control), the 2-plex assay for the discrimination of SARS-CoV-2 wild-type from variants (Alpha, Beta, Epsilon, Delta, or Omicrons), and the 4-plex assay for the screening of arboviruses (zika virus, tick-borne encephalitis virus, yellow fever virus, and chikungunya virus). We have validated multiplex FARPA with 103 nasopharyngeal swabs for SARS-CoV-2 detection. The results showed a 100% agreement with RT-qPCR assays. Moreover, a hand-held FARPA analyser was constructed for the visualized FARPA due to the switch-like endpoint read-out. This FARPA is very suitable for pathogen screening and discrimination of viral variants, greatly facilitating point-of-care diagnostics.

Safety and immunogenicity of aerosolised Ad5-nCoV, intramuscular Ad5-nCoV, or inactivated COVID-19 vaccine CoronaVac given as the second booster following three doses of CoronaVac: a multicentre, open-label, phase 4, randomised trial

Background Aerosolised Ad5-nCoV is the first approved mucosal respiratory COVID-19 vaccine to be used as a booster after the primary immunisation with COVID-19 vaccines. This study aimed to evaluate the safety and immunogenicity of aerosolised Ad5-nCoV, intramuscular Ad5-nCoV, or inactivated COVID-19 vaccine CoronaVac given as the second booster. Methods This is an open-label, parallel-controlled, phase 4 randomised trial enrolling healthy adult participants (≥18 years) who had completed a two-dose primary immunisation and a booster immunisation with inactivated COVID-19 vaccines (CoronaVac only) at least 6 months before, in Lianshui and Donghai counties, Jiangsu Province, China. We recruited eligible participants from previous trials in China (NCT04892459, NCT04952727, and NCT05043259) as cohort 1 (with the serum before and after the first booster dose available), and from eligible volunteers in Lianshui and Donghai counties, Jiangsu Province, as cohort 2. Participants were randomly assigned at a ratio of 1:1:1, using a web-based interactive response randomisation system, to receive the fourth dose (second booster) of aerosolised Ad5-nCoV (0·1 mL of 1·0 × 1011 viral particles per mL), intramuscular Ad5-nCoV (0·5 mL of 1·0 × 1011 viral particles per mL), or inactivated COVID-19 vaccine CoronaVac (0·5 mL), respectively. The co-primary outcomes were safety and immunogenicity of geometric mean titres (GMTs) of serum neutralising antibodies against prototype live SARS-CoV-2 virus 28 days after the vaccination, assessed on a per-protocol basis. Non-inferiority or superiority was achieved when the lower limit of the 95% CI of the GMT ratio (heterologous group vs homologous group) exceeded 0·67 or 1·0, respectively. This study was registered with ClinicalTrials.gov, NCT05303584 and is ongoing. Findings Between April 23 and May 23, 2022, from 367 volunteers screened for eligibility, 356 participants met eligibility criteria and received a dose of aerosolised Ad5-nCoV (n=117), intramuscular Ad5-nCoV (n=120), or CoronaVac (n=119). Within 28 days of booster vaccination, participants in the intramuscular Ad5-nCoV group reported a significantly higher frequency of adverse reactions than those in the aerosolised Ad5-nCoV and intramuscular CoronaVac groups (30% vs 9% and 14%, respectively;p<0·0001). No serious adverse events related to the vaccination were reported. The heterologous boosting with aerosolised Ad5-nCoV triggered a GMT of 672·4 (95% CI 539·7–837·7) and intramuscular Ad5-nCoV triggered a serum neutralising antibody GMT of 582·6 (505·0–672·2) 28 days after the booster dose, both of which were significantly higher than the GMT in the CoronaVac group (58·5 [48·0–71·4];p<0·0001). Interpretation A heterologous fourth dose (second booster) with either aerosolised Ad5-nCoV or intramuscular Ad5-nCoV was safe and highly immunogenic in healthy adults who had been immunised with three doses of CoronaVac. Funding National Natural Science Foundation of China, Jiangsu Provincial Science Fund for Distinguished Young Scholars, and Jiangsu Provincial Key Project of Science and Technology Plan.

Safety and immunogenicity of aerosolised Ad5-nCoV, intramuscular Ad5-nCoV, or inactivated COVID-19 vaccine CoronaVac given as the second booster following three doses of CoronaVac: a multicentre, open-label, phase 4, randomised trial.

BACKGROUND: Aerosolised Ad5-nCoV is the first approved mucosal respiratory COVID-19 vaccine to be used as a booster after the primary immunisation with COVID-19 vaccines. This study aimed to evaluate the safety and immunogenicity of aerosolised Ad5-nCoV, intramuscular Ad5-nCoV, or inactivated COVID-19 vaccine CoronaVac given as the second booster. METHODS: This is an open-label, parallel-controlled, phase 4 randomised trial enrolling healthy adult participants (≥18 years) who had completed a two-dose primary immunisation and a booster immunisation with inactivated COVID-19 vaccines (CoronaVac only) at least 6 months before, in Lianshui and Donghai counties, Jiangsu Province, China. We recruited eligible participants from previous trials in China (NCT04892459, NCT04952727, and NCT05043259) as cohort 1 (with the serum before and after the first booster dose available), and from eligible volunteers in Lianshui and Donghai counties, Jiangsu Province, as cohort 2. Participants were randomly assigned at a ratio of 1:1:1, using a web-based interactive response randomisation system, to receive the fourth dose (second booster) of aerosolised Ad5-nCoV (0·1 mL of 1·0 × 1011 viral particles per mL), intramuscular Ad5-nCoV (0·5 mL of 1·0 × 1011 viral particles per mL), or inactivated COVID-19 vaccine CoronaVac (0·5 mL), respectively. The co-primary outcomes were safety and immunogenicity of geometric mean titres (GMTs) of serum neutralising antibodies against prototype live SARS-CoV-2 virus 28 days after the vaccination, assessed on a per-protocol basis. Non-inferiority or superiority was achieved when the lower limit of the 95% CI of the GMT ratio (heterologous group vs homologous group) exceeded 0·67 or 1·0, respectively. This study was registered with ClinicalTrials.gov, NCT05303584 and is ongoing. FINDINGS: Between April 23 and May 23, 2022, from 367 volunteers screened for eligibility, 356 participants met eligibility criteria and received a dose of aerosolised Ad5-nCoV (n=117), intramuscular Ad5-nCoV (n=120), or CoronaVac (n=119). Within 28 days of booster vaccination, participants in the intramuscular Ad5-nCoV group reported a significantly higher frequency of adverse reactions than those in the aerosolised Ad5-nCoV and intramuscular CoronaVac groups (30% vs 9% and 14%, respectively; p<0·0001). No serious adverse events related to the vaccination were reported. The heterologous boosting with aerosolised Ad5-nCoV triggered a GMT of 672·4 (95% CI 539·7-837·7) and intramuscular Ad5-nCoV triggered a serum neutralising antibody GMT of 582·6 (505·0-672·2) 28 days after the booster dose, both of which were significantly higher than the GMT in the CoronaVac group (58·5 [48·0-71·4]; p<0·0001). INTERPRETATION: A heterologous fourth dose (second booster) with either aerosolised Ad5-nCoV or intramuscular Ad5-nCoV was safe and highly immunogenic in healthy adults who had been immunised with three doses of CoronaVac. FUNDING: National Natural Science Foundation of China, Jiangsu Provincial Science Fund for Distinguished Young Scholars, and Jiangsu Provincial Key Project of Science and Technology Plan.

Visualized RNA detection of SARS-CoV-2 in a closed tube by coupling RT-PCR with nested invasive reaction.

A simple, cost-effective and reliable diagnosis of pathogen nucleic acids assay is much required for controlling a pandemic of a virus disease, such as COVID-19. Our previously developed visualized detection of pathogen DNA in a single closed tube is very suitable for POCT. However, virus RNA could not be detected directly and should be reverse-transcribed into cDNA in advance. To enable this visualized assay to detect virus RNA directly, various types of reverse transcriptase were investigated, and finally we found that HiScript II reverse transcriptase could keep active and be well adapted to the one-pot visualized assay in optimized conditions. Reverse transcription, template amplification and amplicon identification by PCR coupled with invasive reaction, as well as visualization by self-assembling of AuNP probes could be automatically and sequentially performed in a closed tube under different temperature conditions, achieving "sample (RNA)-in-result (red color)-out" only by a simple PCR engine plus the naked eye. The visualized RT-PCR is sensitive to unambiguous detection of 5 copies of the N and ORFlab genes of SARS-CoV-2 RNA comparing favourably with qPCR methods (commercialized kit), is specific to genotype 3 variants (Alpha, Beta and Omicron) of SARS-CoV-2, and is very accurate for picking up 0.01% Omicron variant from a large amount of sequence-similar backgrounds. The method is employed in detecting 50 clinical samples, and 10 of them were detected as SARS-CoV-2 positive samples, identical to those by conventional RT-PCR, indicating that the method is cost-effective and labor-saving for pathogen RNA screening in resource-limited regions.

Establishment and evaluation of fluorescent RT-RAA assay for SARS-CoV-2 sgRNAs detection

Objective To establish a reverse-transcription recombinase-aided amplification assay（RT-RAA） to rapidly detect SARS-CoV-2 sub-genomic RNAs（sgRNAs）. Methods The primers and probe for isothermal nucleic acid amplification were designed based on the 5′-leader and 7a and N gene sequence of SARS-CoV-2, and the sgRNAs of SARS-COV-2 were rapidly detected within 30 min at 39 ℃.The sensitivity, specificity and consistency of the assay were evaluated. Results The detection limit of the method was 20 copies/μl and there were no cross-reactions with other respiratory pathogens, showing decent sensitivity and specificity.The results of the assay were concordant with that of real-time PCR, indicating a better consistency of two methods（κ=0.762,P<0.001）. Conclusions The fluorescence RT-RAA assay established in the study can be used for the rapid detection of SARS-CoV-2 sgRNAs, which is of great significance for the rapid diagnosis of COVID 19.

Corrigendum: Systematic benchmarking of nanopore Q20+ kit in SARS-CoV-2 whole genome sequencing.

[This corrects the article DOI: 10.3389/fmicb.2022.973367.].

Antibody persistence and safety after heterologous boosting with orally aerosolised Ad5-nCoV in individuals primed with two-dose CoronaVac previously: 12-month analyses of a randomized controlled trial.

Antibody persistence and safety up to 12 months of heterologous orally administered adenovirus type-5 vector-based COVID-19 vaccine (Ad5-nCoV) in individuals who were primed with two-dose inactivated SARS-CoV-2 vaccine (CoronaVac) previously, has not been reported yet. This randomized, open-label, single-centre trial included Chinese adults who have received two-dose CoronaVac randomized to low-dose or high-dose aerosolised Ad5-nCoV group, or CoronaVac group. In this report, we mainly evaluated the geometric mean titres (GMTs) of neutralizing antibodies (NAbs) against live wild-type SARS-CoV-2 virus and omicron BA.4/5 pseudovirus at 12 months after the booster dose and the incidence of serious adverse events (SAEs) till month 12. Of 419 participants, all were included in the safety analysis and 120 (28.64%) were included in the immunogenicity analysis. Serum NAb GMT against live wild-type SARS-CoV-2 was 204.36 (95% CI 152.91, 273.14) in the low-dose group and 171.38 (95% CI 121.27, 242.19) in the high-dose group at month 12, significantly higher than the GMT in the CoronaVac group (8.00 [95% CI 4.22, 15.17], p < 0.0001). Serum NAb GMT against omicron BA.4/5 pseudovirus was 40.97 (95% CI 30.15, 55.67) in the low-dose group and 35.08 (95% CI 26.31, 46.77) in the high-dose group at month 12, whereas the GMT in the CoronaVac group was below the lower limit of detection. No vaccine-related SAEs were observed. Orally administered aerosolised Ad5-nCoV following two-dose CoronaVac priming has a good safety profile and is persistently more immunogenic than three-dose CoronaVac within 12 months after the booster dose.Trial registration: ClinicalTrials.gov identifier: NCT05043259..

Systematic benchmarking of nanopore Q20+ kit in SARS-CoV-2 whole genome sequencing.

Whole genome sequencing provides rapid insight into key information about the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), such as virus typing and key mutation site, and this information is important for precise prevention, control and tracing of coronavirus disease 2019 (COVID-19) outbreak in conjunction with the epidemiological information of the case. Nanopore sequencing is widely used around the world for its short sample-to-result time, simple experimental operation and long sequencing reads. However, because nanopore sequencing is a relatively new sequencing technology, many researchers still have doubts about its accuracy. The combination of the newly launched nanopore sequencing Q20+ kit (LSK112) and flow cell R10.4 is a qualitative improvement over the accuracy of the previous kits. In this study, we firstly used LSK112 kit with flow cell R10.4 to sequence the SARS-CoV-2 whole genome, and summarized the sequencing results of the combination of LSK112 kit and flow cell R10.4 for the 1200bp amplicons of SARS-CoV-2. We found that the proportion of sequences with an accuracy of more than 99% reached 30.1%, and the average sequence accuracy reached 98.34%, while the results of the original combination of LSK109 kit and flow cell R9.4.1 were 0.61% and 96.52%, respectively. The mutation site analysis showed that it was completely consistent with the final consensus sequence of next generation sequencing (NGS). The results showed that the combination of LSK112 kit and flow cell R10.4 allowed rapid whole-genome sequencing of SARS-CoV-2 without the need for verification of NGS.

Systematic benchmarking of nanopore Q20+ kit in SARS-CoV-2 whole genome sequencing

Whole genome sequencing provides rapid insight into key information about the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), such as virus typing and key mutation site, and this information is important for precise prevention, control and tracing of coronavirus disease 2019 (COVID-19) outbreak in conjunction with the epidemiological information of the case. Nanopore sequencing is widely used around the world for its short sample-to-result time, simple experimental operation and long sequencing reads. However, because nanopore sequencing is a relatively new sequencing technology, many researchers still have doubts about its accuracy. The combination of the newly launched nanopore sequencing Q20+ kit (LSK112) and flow cell R10.4 is a qualitative improvement over the accuracy of the previous kits. In this study, we firstly used LSK112 kit with flow cell R10.4 to sequence the SARS-CoV-2 whole genome, and summarized the sequencing results of the combination of LSK112 kit and flow cell R10.4 for the 1200bp amplicons of SARS-CoV-2. We found that the proportion of sequences with an accuracy of more than 99% reached 30.1%, and the average sequence accuracy reached 98.34%, while the results of the original combination of LSK109 kit and flow cell R9.4.1 were 0.61% and 96.52%, respectively. The mutation site analysis showed that it was completely consistent with the final consensus sequence of next generation sequencing (NGS). The results showed that the combination of LSK112 kit and flow cell R10.4 allowed rapid whole-genome sequencing of SARS-CoV-2 without the need for verification of NGS.

Safety and immunogenicity of heterologous boost immunisation with an orally administered aerosolised Ad5-nCoV after two-dose priming with an inactivated SARS-CoV-2 vaccine in Chinese adults: a randomised, open-label, single-centre trial.

BACKGROUND: Due to waning immunity and protection against infection with SARS-CoV-2, a third dose of a homologous or heterologous COVID-19 vaccine has been proposed by health agencies for individuals who were previously primed with two doses of an inactivated COVID-19 vaccine. METHODS: We did a randomised, open-label, controlled trial to evaluate the safety and immunogenicity of heterologous boost immunisation with an orally administered aerosolised adenovirus type-5 vector-based COVID-19 vaccine (Ad5-nCoV) in Chinese adults (≥18 years old) who had previously received two doses of an inactivated SARS-CoV-2 vaccine-Sinovac CoronaVac. Eligible participants were randomly assigned (1:1:1) to receive a heterologous booster vaccination with a low dose (1·0 × 1011 viral particles per mL; 0·1 mL; low dose group), or a high dose (1·0 × 1011 viral particles per mL; 0·2 mL; high dose group) aerosolised Ad5-nCoV, or a homologous intramuscular vaccination with CoronaVac (0·5 mL). Only laboratory staff were masked to group assignment. The primary endpoint for safety was the incidence of adverse reactions within 14 days after the booster dose. The primary endpoint for immunogenicity was the geometric mean titres (GMTs) of serum neutralising antibodies (NAbs) against live SARS-CoV-2 virus 14 days after the booster dose. This study was registered with ClinicalTrials.gov, NCT05043259. FINDINGS: Between Sept 14 and 16, 2021, 420 participants were enrolled: 140 (33%) participants per group. Adverse reactions were reported by 26 (19%) participants in the low dose group and 33 (24%) in the high dose group within 14 days after the booster vaccination, significantly less than the 54 (39%) participants in the CoronaVac group (p<0·0001). The low dose group had a serum NAb GMT of 744·4 (95% CI 520·1-1065·6) and the high dose group had a GMT of 714·1 (479·4-1063·7) 14 days after booster dose, significantly higher than the GMT in the CoronaVac group (78·5 [60·5-101·7]; p<0·0001). INTERPRETATION: We found that a heterologous booster vaccine with an orally administered aerosolised Ad5-nCoV is safe and highly immunogenic in adults who have previously received two doses of CoronaVac as the primary series vaccination. FUNDING: National Natural Science Foundation of China and Jiangsu Provincial Key Research and Development Program.

Safety and immunogenicity of a live-attenuated influenza virus vector-based intranasal SARS-CoV-2 vaccine in adults: randomised, double-blind, placebo-controlled, phase 1 and 2 trials.

BACKGROUND: All currently available SARS-CoV-2 vaccines are administered by intramuscular injection. We aimed to evaluate the safety and immunogenicity of a live-attenuated influenza virus vector-based SARS-CoV-2 vaccine (dNS1-RBD) administered by intranasal spray in healthy adults. METHODS: We did double-blind, randomised, placebo-controlled phase 1 and 2 trials, followed by a phase 2 extension trial, at a single centre in Jiangsu, China. Healthy adults (≥18 years) who had negative serum or fingertip blood total antibody tests for SARS-CoV-2 (in phases 1 and 2), with no prevalent SARS-CoV-2 infection or history of infection and no SARS-CoV-2 vaccination history (in all three trials reported here), were enrolled. Participants were randomly allocated (4:1 in phase 1, 2:1 in phase 2, and 1:1 in the extension trial) to receive two intranasal doses of the dNS1-RBD vaccine or placebo on days 0 and 14 or, for half of the participants in phase 2, on days 0 and 21. To avoid cross-contamination during administration, vaccine and placebo recipients were vaccinated in separate rooms in the extension trial. The phase 1 primary outcome was safety (adverse events recorded on days 0-44; serious adverse events recorded from day 0 until 12 months after the second dose). In the phase 2 and extension trials, the primary immunogenicity outcomes were SARS-CoV-2-specific T-cell response in peripheral blood (measured by IFN-γ ELISpot), proportion of participants with positive conversion for SARS-CoV-2 receptor-binding domain (RBD)-specific IgG and secretory IgA (s-IgA) antibodies, and concentration of SARS-CoV-2 RBD IgG in serum and SARS-CoV-2 RBD s-IgA in the nasopharynx (measured by ELISA) at 1 month after the second dose in the per-protocol set for immunogenicity. χ2 test and Fisher's exact test were used to analyse categorical data, and t test and Wilcoxon rank sum test to compare the measurement data between groups. These trials were registered with the Chinese Clinical Trial Registry (ChiCTR2000037782, ChiCTR2000039715, and ChiCTR2100048316). FINDINGS: Between Sept 1, 2020, and July 4, 2021, 63, 724, and 297 participants without a history of SARS-CoV-2 vaccination were enrolled in the phase 1, phase 2, and extension trials, respectively. At least one adverse reaction after vaccination was reported in 133 (19%) of 684 participants in the vaccine groups. Most adverse reactions were mild. No vaccine-related serious adverse event was noted. Specific T-cell immune responses were observed in 211 (46% [95% CI 42-51]) of 455 vaccine recipients in the phase 2 trial, and in 48 (40% [31-49]) of 120 vaccine recipients compared with one (1% [0-5]) of 111 placebo recipients (p<0·0001) in the extension trial. Seroconversion for RBD-specific IgG was observed in 48 (10% [95% CI 8-13]) of 466 vaccine recipients in the phase 2 trial (geometric mean titre [GMT] 3·8 [95% CI 3·4-4·3] in responders), and in 31 (22% [15-29]) of 143 vaccine recipients (GMT 4·4 [3·3-5·8]) and zero (0% [0-2]) of 147 placebo recipients (p<0·0001) in the extension trial. 57 (12% [95% CI 9-16]) of 466 vaccine recipients had positive conversion for RBD-specific s-IgA (GMT 3·8 [95% CI 3·5-4·1] in responders) in the phase 2 trial, as did 18 (13% [8-19]) of 143 vaccine recipients (GMT 5·2 [4·0-6·8]) and zero (0% [0-2]) of 147 placebo recipients (p<0·0001) in the extension trial. INTERPRETATION: dNS1-RBD was well tolerated in adults. Weak T-cell immunity in peripheral blood, as well as weak humoral and mucosal immune responses against SARS-CoV-2, were detected in vaccine recipients. Further studies are warranted to verify the safety and efficacy of intranasal vaccines as a potential supplement to current intramuscular SARS-CoV-2 vaccine pools. Steps should be taken in future studies to reduce the potential for cross-contamination caused by the vaccine strain aerosol during administration. FUNDING: National Key Research and Development Program of China, National Science, Fujian Provincial Science, CAMS Innovation Fund for Medical Sciences, and Beijing Wantai Biological Pharmacy Enterprise.

Heterologous AD5-nCOV plus CoronaVac versus homologous CoronaVac vaccination: a randomized phase 4 trial.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and the waning of vaccine-elicited neutralizing antibodies suggests that additional coronavirus disease 2019 (COVID-19) vaccine doses may be needed for individuals who initially received CoronaVac. We evaluated the safety and immunogenicity of the recombinant adenovirus type 5 (AD5)-vectored COVID-19 vaccine Convidecia as a heterologous booster versus those of CoronaVac as homologous booster in adults previously vaccinated with CoronaVac in an ongoing, randomized, observer-blinded, parallel-controlled phase 4 trial ( NCT04892459 ). Adults who had received two doses of CoronaVac in the past 3-6 months were vaccinated with Convidecia (n = 96) or CoronaVac (n = 102). Adults who had received one dose of CoronaVac in the past 1-3 months were also vaccinated with Convidecia (n = 51) or CoronaVac (n = 50). The co-primary endpoints were the occurrence of adverse reactions within 28 d after vaccination and geometric mean titers (GMTs) of neutralizing antibodies against live wild-type SARS-CoV-2 virus at 14 d after booster vaccination. Adverse reactions after vaccination were significantly more frequent in Convidecia recipients but were generally mild to moderate in all treatment groups. Heterologous boosting with Convidecia elicited significantly increased GMTs of neutralizing antibody against SARS-CoV-2 than homologous boosting with CoronaVac in participants who had previously received one or two doses of CoronaVac. These data suggest that heterologous boosting with Convidecia following initial vaccination with CoronaVac is safe and more immunogenic than homologous boosting.

Effectiveness of Disinfectants Suitable for Inactivating SARS-CoV-2 at Cold-Chain Temperature.

To prevent the spread of SARS-CoV-2 in cold-chain transportation in China, we developed specific cryogenic disinfectants. Carrier tests were performed against SARS-CoV-2 at - 20 °C for the four cryogenic disinfectants developed and qRT-PCR was used to test the virus RNA. Peracetic acid, chlorine disinfectants (two different concentrations), and quaternary ammonium disinfectant with their antifreeze can all inactivate SARS-CoV-2 in 5 min at - 20 °C. However, after 2-3 h of exposure, only chlorine disinfectant could destroy SARS-CoV-2 RNA. The viruses treated with peracetic acid and quaternary disinfectants showed positive Ct values even after 3 h detected with qRT-PCR. The conclusion was that the cold-chain disinfectants we tested could inactivate SARS-CoV-2 quickly and effectively, but only chlorine disinfectants could destroy nucleic acids in 3 h. Our study also illustrated that using qRT-PCR detection of viral nucleic acids to assess disinfection was inappropriate.

Analysis of Clinical Characteristics and Virus Strains Variation of Patients Infected With SARS-CoV-2 in Jiangsu Province—A Retrospective Study

Background: At present, the global sever acute respiratory syndrome coronavirus 2 (SARS-CoV-2) situation is still grim, and the risk of local outbreaks caused by imported viruses is high. Therefore, it is necessary to monitor the genomic variation and genetic evolution characteristics of SARS-CoV-2. The main purpose of this study was to detect the entry of different SARS-CoV-2 variants into Jiangsu Province, China. Methods: First, oropharyngeal swabs were collected from 165 patients (55 locally confirmed cases and 110 imported cases with confirmed and asymptomatic infection) diagnosed with SARS-CoV-2 infection in Jiangsu Province, China between January 2020 and June 2021. Then, whole genome sequencing was used to explore the phylogeny and find potential mutations in genes of the SARS-CoV-2. Last, association analysis among clinical characteristics and SARS-CoV-2 Variant of Concern, pedigree surveillance analysis of SARS-COV-2, and single nucleotide polymorphisms (SNPs) detection in SARS-COV-2 samples was performed. Results: More men were infected with the SARS-CoV-2 when compared with women. The onset of the SARS-CoV-2 showed a trend of younger age. Moreover, the number of asymptomatic infected patients was large, similar to the number of common patients. Patients infected with Alpha (50%) and Beta (90%) variants were predominantly asymptomatic, while patients infected with Delta (17%) variant presented severe clinical features. A total of 935 SNPs were detected in 165 SARS-COV-2 samples. Among which, missense mutation (58%) was the dominant mutation type. About 56% of SNPs changes occurred in the open reading frame 1ab (ORF1ab) gene. Approximately, 20% of SNP changes occurred in spike glycoprotein (S) gene, such as p.Asp501Tyr, p.Pro681His, and p.Pro681Arg. In total, nine SNPs loci in S gene were significantly correlated with the severity of patients. It is worth mentioning that amino acid substitution of p.Asp614Gly was significantly positively correlated with the clinical severity of patients. The amino acid replacements of p.Ser316Thr and p.Lu484Lys were significantly negatively correlated with the course of disease. Conclusion: Sever acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may further undergo a variety of mutations in different hosts, countries, and weather conditions. Detecting the entry of different virus variants of SARS-CoV-2 into Jiangsu Province, China may help to monitor the spread of infection and the diversity of eventual recombination or genomic mutations.

An integrative drug repositioning framework discovered a potential therapeutic agent targeting COVID-19.

The global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires an urgent need to find effective therapeutics for the treatment of coronavirus disease 2019 (COVID-19). In this study, we developed an integrative drug repositioning framework, which fully takes advantage of machine learning and statistical analysis approaches to systematically integrate and mine large-scale knowledge graph, literature and transcriptome data to discover the potential drug candidates against SARS-CoV-2. Our in silico screening followed by wet-lab validation indicated that a poly-ADP-ribose polymerase 1 (PARP1) inhibitor, CVL218, currently in Phase I clinical trial, may be repurposed to treat COVID-19. Our in vitro assays revealed that CVL218 can exhibit effective inhibitory activity against SARS-CoV-2 replication without obvious cytopathic effect. In addition, we showed that CVL218 can interact with the nucleocapsid (N) protein of SARS-CoV-2 and is able to suppress the LPS-induced production of several inflammatory cytokines that are highly relevant to the prevention of immunopathology induced by SARS-CoV-2 infection.

HTCC as a Polymeric Inhibitor of SARS-CoV-2 and MERS-CoV.

Among seven coronaviruses that infect humans, three (severe acute respiratory syndrome coronavirus [SARS-CoV], Middle East respiratory syndrome coronavirus [MERS-CoV], and the newly identified severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) are associated with a severe, life-threatening respiratory infection and multiorgan failure. We previously proposed that the cationically modified chitosan N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC) is a potent inhibitor of human coronavirus NL63 (HCoV-NL63). Next, we demonstrated the broad-spectrum antiviral activity of the compound, as it inhibited all low-pathogenicity human coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1). Here, using in vitro and ex vivo models of human airway epithelia, we show that HTCC effectively blocks MERS-CoV and SARS-CoV-2 infection. We also confirmed the mechanism of action for these two viruses, showing that the polymer blocks the virus entry into the host cell by interaction with the S protein.IMPORTANCE The beginning of 2020 brought us information about the novel coronavirus emerging in China. Rapid research resulted in the characterization of the pathogen, which appeared to be a member of the SARS-like cluster, commonly seen in bats. Despite the global and local efforts, the virus escaped the health care measures and rapidly spread in China and later globally, officially causing a pandemic and global crisis in March 2020. At present, different scenarios are being written to contain the virus, but the development of novel anticoronavirals for all highly pathogenic coronaviruses remains the major challenge. Here, we describe the antiviral activity of an HTCC compound, previously developed by us, which may be used as a potential inhibitor of currently circulating highly pathogenic coronaviruses-SARS-CoV-2 and MERS-CoV.

Serum Cytokine and Chemokine Profile in Relation to the Severity of Coronavirus Disease 2019 in China.

Coronavirus disease 2019 (COVID-19) is an emerging infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We investigated the serum cytokine and chemokine levels in asymptomatic, mild, moderate, severe, and convalescent SARS-CoV-2-infected cases. Proinflammatory cytokine and chemokine production induced by SARS-CoV-2 were observed not only in symptomatic patients but also in asymptomatic cases, and returned to normal after recovery. IL-6, IL-7, IL-10, IL-18, G-CSF, M-CSF, MCP-1, MCP-3, IP-10, MIG, and MIP-1α were found to be associated with the severity of COVID-19. Moreover, a set of cytokine and chemokine profiles were significantly higher in SARS-CoV-2-infected male than female patients. The serum levels of MCP-1, G-CSF, and VEGF were weakly and positively correlated with viral titers. We suggest that combinatorial analysis of serum cytokines and chemokines with clinical classification may contribute to evaluation of the severity of COVID-19 and optimize the therapeutic strategies.

Toilets dominate environmental detection of severe acute respiratory syndrome coronavirus 2 in a hospital.

Respiratory and fecal aerosols play confirmed and suspected roles, respectively, in transmitting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). An extensive environmental sampling campaign of both toilet and non-toilet environments was performed in a dedicated hospital building for patients with coronavirus disease 2019 (COVID-19), and the associated environmental factors were analyzed. In total, 107 surface samples, 46 air samples, two exhaled condensate samples, and two expired air samples were collected within and beyond four three-bed isolation rooms. The data of the COVID-19 patients were collected. The building environmental design and the cleaning routines were reviewed. Field measurements of airflow and CO2 concentrations were conducted. The 107 surface samples comprised 37 from toilets, 34 from other surfaces in isolation rooms, and 36 from other surfaces outside the isolation rooms in the hospital. Four of these samples were positive, namely two ward door handles, one bathroom toilet seat cover, and one bathroom door handle. Three were weakly positive, namely one bathroom toilet seat, one bathroom washbasin tap lever, and one bathroom ceiling exhaust louver. Of the 46 air samples, one collected from a corridor was weakly positive. The two exhaled condensate samples and the two expired air samples were negative. The fecal-derived aerosols in patients' toilets contained most of the detected SARS-CoV-2 in the hospital, highlighting the importance of surface and hand hygiene for intervention.

A reverse-transcription recombinase-aided amplification assay for the rapid detection of N gene of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).

The current outbreak of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China firstly. A rapid, highly sensitive, specific, and simple operational method was needed for the detection of SARS-CoV-2. Here, we established a real-time reverse-transcription recombinase-aided amplification assay (RT-RAA) to detect SARS-CoV-2 rapidly. The primers and probe were designed based on the nucleocapsid protein gene (N gene) sequence of SARS-CoV-2. The detection limit was 10 copies per reaction in this assay, which could be conducted within 15 min at a constant temperature (39 °C), without any cross-reactions with other respiratory tract pathogens, such as other coronaviruses. Furthermore, compared with commercial real-time RT-PCR assay, it showed a kappa value of 0.959 (p < 0.001) from 150 clinical specimens. These results indicated that this real-time RT-RAA assay may be a valuable tool for detecting SARS-CoV-2.