A novel fast hybrid capture sequencing method for high-efficiency common human coronavirus whole-genome acquisition.

Rapid and accurate sequencing of the entire viral genome, coupled with continuous monitoring of genetic changes, is crucial for understanding the epidemiology of coronaviruses. We designed a novel method called micro target hybrid capture system (MT-Capture) to enable whole-genome sequencing in a timely manner. The novel design of probes used in target binding exhibits a unique and synergistic "hand-in-hand" conjugation effect. The entire hybrid capture process is within 2.5 hours, overcoming the time-consuming and complex operation characteristics of the traditional liquid-phase hybrid capture (T-Capture) system. By designing specific probes for these coronaviruses, MT-Capture effectively enriched isolated strains and 112 clinical samples of coronaviruses with cycle threshold values below 37. Compared to multiplex PCR sequencing, it does not require frequent primer updates and has higher compatibility. MT-Capture is highly sensitive and capable of tracking variants.IMPORTANCEMT-Capture is meticulously designed to enable the efficient acquisition of the target genome of the common human coronavirus. Coronavirus is a kind of virus that people are generally susceptible to and is epidemic and infectious, and it is the virus with the longest genome among known RNA viruses. Therefore, common human coronavirus samples are selected to evaluate the accuracy and sensitivity of MT-Capture. This method utilizes innovative probe designs optimized through probe conjugation techniques, greatly shortening the time and simplifying the handwork compared with traditional hybridization capture processes. Our results demonstrate that MT-Capture surpasses multiplex PCR in terms of sensitivity, exhibiting a thousandfold increase. Moreover, MT-Capture excels in the identification of mutation sites. This method not only is used to target the coronaviruses but also may be used to diagnose other diseases, including various infectious diseases, genetic diseases, or tumors.

Generation of SARS-CoV-2 spike receptor binding domain mutants and functional screening for immune evaders using a novel lentivirus-based system.

The emergence of rapid and continuous mutations of severe acute respiratory syndrome 2 (SARS-CoV-2) spike glycoprotein that increased with the Omicron variant points out the necessity to anticipate such mutations for conceiving specific and adaptable therapies to avoid another pandemic. The crucial target for the antibody treatment and vaccine design is the receptor binding domain (RBD) of the SARS-CoV-2 spike. It is also the site where the virus has shown its high ability to mutate and consequently escape immune response. We developed a robust and simple method for generating a large number of functional SARS-CoV-2 spike RBD mutants by error-prone PCR and a novel nonreplicative lentivirus-based system. We prepared anti-RBD wild type (WT) polyclonal antibodies and used them to screen and select for mutant libraries that escape inhibition of virion entry into recipient cells expressing human angiotensin-converting enzyme 2 and transmembrane serine protease 2. We isolated, cloned, and sequenced six mutants totally bearing nine mutation sites. Eight mutations were found in successive WT variants, including Omicron and other recombinants, whereas one is novel. These results, together with the detailed functional analyses of two mutants provided the proof of concept for our approach.

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.

Case report: Clinical characteristics of two cases of pneumonia caused with different strains of Chlamydia psittaci.

Background: With the development of metagenomic sequencing technologies, more and more cases of pneumonia caused with Chlamydia psittaci (C. psittaci) have been reported. However, it remains unknown about the characteristics of patients with pneumonia caused by different strains of C. psittaci. Here, we shared the clinical characteristics of two cases of pneumonia caused with C. psittaci strains SZ18-2 and SZ15 which were rarely identified in humans. Case presentation: Case 1: A 69-year-old male farmer who fed ducks presented to hospital for cough, diarrhea and lethargy with the temperature of 39.8°C. Case 2: A 48-year-old male worker who slaughtered ducks was transferred to hospital for high fever, cough, myalgia, diarrhea and loss of appetite. Both patients did not take any protective measures (wearing face masks or gloves) while processing ducks. C. psittaci pneumonia was diagnosed by metagenomic next-generation sequencing and polymerase chain reaction. After treatment with doxycycline and azithromycin individually, they recovered well and discharged from hospital. Through OmpA sequencing, two different strains of SZ18-2 and SZ15 were identified in case 1 and case 2, respectively. Conclusions: Patients infected with different strains of C. psittaci may own different clinical manifestations. C. psittaci infection should be suspected when pneumonia appears, accompanied by digestive symptoms and multiple organ dysfunction, especially under the exposure of specific birds.

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.

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.

Exosomal microRNA expression profiles derived from A549 human lung cells in response to influenza A/H1N1pdm09 infection.

Exosomes participate in intercellular communication by shuttling various small molecules from donor to recipient cells. We aimed to examine the role of exosomes and exosomal miRNAs in influenza virus infection. The results showed that influenza A/H1N1pdm09 infection could promote A549 cells to secrete exosomes, while blocking the generation of exosomes reduced viral RNA production. A total of 97 exosomal miRNAs with significantly altered expression were identified during influenza infection. Of 12 candidate miRNAs chosen for further validation, ten were confirmed by qRT-PCR. Among 5978 predicted target genes，we found 37 interferon pathway-related genes to be the potential targets of 29 differentially expressed miRNAs. Many target genes were annotated to various KEGG signaling pathways, some of which played important roles in influenza infection. These data will help to further understand the mechanism of influenza virus-host interactions, which is important for the development of preventative and therapeutic strategies against influenza virus.

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.

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.

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.

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

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

Rapid and Sensitive Detection of Salmonella spp. Using CRISPR-Cas13a Combined With Recombinase Polymerase Amplification.

Salmonella spp. is one of the most common foodborne disease-causing pathogens that can cause severe diseases in very low infectious doses. Rapid and sensitive detecting Salmonella spp. is advantageous to the control of its spread. In this study, a conserved short fragment of the Salmonella invA gene was selected and used to design primers and specific crRNA (CRISPR RNA) for establishing a one-tube and two-step reaction system for Salmonella spp. detection, by combining recombinase polymerase amplification (RPA) with CRISPR-Cas13a (Clustered Regularly Interspaced Short Palindromic Repeats associated protein 13a) cleavage. The established one-tube RPA-Cas13a method can complete the detection within 20 min and the two-step RPA-Cas13a method detection time within 45 min. The designed primers were highly specific to Salmonella spp. and had no cross-reaction with the other nine diarrheal bacteria. The one-tube RPA-Cas13a could detect the Salmonella genome with the limit of 102 copies, which was the same as real-time polymerase chain reaction (PCR), but less sensitive than two-step RPA-Cas13a (100 copies). The detection results of one-tube or two-step RPA-Cas13a and real-time PCR were highly consistent in clinical samples. One-tube RPA-Cas13a developed in this study provides a simple, rapid, and specific detection method for Salmonella spp. While two-step assay was more sensitive and suitable for samples at low abundance.

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.

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.