A nucleic acid detection assay combining reverse transcription recombinase-aided amplification with a lateral flow dipstick for the rapid visual detection of porcine deltacoronavirus.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen causing severe diarrhoea, dehydration, and death in nursing piglets and enormous economic losses for the global swine industry. Furthermore, it can infect multiple animal species including humans. Therefore, a rapid, definitive diagnostic assay is required for the effective control of this zoonotic pathogen. To identify PDCoV, we developed a nucleic acid detection assay combining reverse transcription recombinase-aided amplification (RT-RAA) with a lateral flow dipstick (LFD) targeting the highly conserved genomic region in the ORF1b gene. The RT-RAA-LFD assay exhibited good PDCoV detection reproducibility and repeatability and could be completed within 11 min. Ten minutes at 40 °C was required for nucleic acid amplification and 1 min at room temperature was needed for the visual LFD readout. The assay specifically detected PDCoV and did not cross-react with any other major swine pathogens. The 95% limit of detection (LOD) was 3.97 median tissue culture infectious dose PDCoV RNA per reaction. This performance was comparable to that of a reference TaqMan-based real-time RT-PCR (trRT-PCR) assay for PDCoV. Of 149 swine small intestine, rectal swab, and serum samples, 71 and 75 tested positive for PDCoV according to RT-RAA-LFD and trRT-PCR, respectively. The diagnostic coincidence rate for both assays was 97.32% (145/149) and the kappa value was 0.946 (p < 0.001). Overall, the RT-RAA-LFD assay is a user-friendly diagnostic tool that can rapidly and visually detect PDCoV.

Development of a Nucleocapsid Protein-Based Blocking ELISA for the Detection of Porcine Deltacoronavirus Antibodies.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogen which mainly causes diarrhea, dehydration and death in nursing piglets, threatening the global swine industry. Moreover, it can infect multiple animal species and humans. Hence, reliable diagnostic assays are needed to better control this zoonotic pathogen. Here, a blocking ELISA was developed using a recombinant nucleocapsid (N) protein as the coating antigen paired with an N-specific monoclonal antibody (mAb) as the detection antibody. The percent inhibition (PI) of the ELISA was determined using 384 swine serum samples, with an indirect immunofluorescence assay (IFA) as the reference method. Through receiver operating characteristic analysis in conjunction with Youden's index, the optimal PI cut-off value was determined to be 51.65%, which corresponded to a diagnostic sensitivity of 98.79% and a diagnostic specificity of 100%. Of the 330 serum samples tested positive via IFA, 326 and 4 were tested positive and negative via the ELISA, respectively, while the 54 serum samples tested negative via IFA were all negative via the ELISA. The overall coincidence rate between the two assays was 98.96% (380/384). The ELISA exhibited good repeatability and did not cross-react with antisera against other swine pathogens. Overall, this is the first report on developing a blocking ELISA for PDCoV serodiagnosis.

Chlorination of isothiazolinone biocides: kinetics, reactive species, pathway, and toxicity evolution.

Due to the Covid-19 pandemic, the worldwide biocides application has been increased, which will eventually result in enhanced residuals in treated wastewater. At the same time, chlorine disinfection of secondary effluents and hospital wastewaters has been intensified. With respect to predicted elevated exposure in wastewater, the chlorination kinetics, transformation pathways and toxicity evolution were investigated in this study for two typical isothiazolinone biocides, methyl-isothiazolinone (MIT) and chloro-methyl-isothiazolinone (CMIT). Second-order rate constants of 0.13 M-1·s-1, 1.95 × 105 M-1·s-1 and 5.14 × 105 M-1·s-1 were determined for the reaction of MIT with HOCl, Cl2O and Cl2, respectively, while reactivity of CMIT was around 1-2 orders of magnitude lower. While chlorination of isothiazolinone biocides at pH 7.1 was dominated by Cl2O-oxidation, acidic pH and elevated Cl- concentration favored free active chlorine (FAC) speciation into Cl2 and increased overall isothiazolinone removal. Regardless of the dominant FAC species, the elimination of MIT and CMIT resulted in an immediate loss of acute toxicity under all experimental conditions, which was attributed to a preferential attack at the S-atom resulting in subsequent formation of sulfoxides and sulfones and eventually an S-elimination. However, chlorination of isothiazolinone biocides in secondary effluent only achieved <10% elimination at typical disinfection chlorine exposure 200 mg·L-1·min, but was predicted to be remarkably increased by acidizing solution to pH 5.5. Alternative measures might be needed to minimize the discharge of these toxic chemicals into the aquatic environment.

Synthesis and Biochemical Evaluation of 8H-Indeno[1,2-d]thiazole Derivatives as Novel SARS-CoV-2 3CL Protease Inhibitors.

The COVID-19 pandemic caused by SARS-CoV-2 is a global burden on human health and economy. The 3-Chymotrypsin-like cysteine protease (3CLpro) becomes an attractive target for SARS-CoV-2 due to its important role in viral replication. We synthesized a series of 8H-indeno[1,2-d]thiazole derivatives and evaluated their biochemical activities against SARS-CoV-2 3CLpro. Among them, the representative compound 7a displayed inhibitory activity with an IC50 of 1.28 ± 0.17 µM against SARS-CoV-2 3CLpro. Molecular docking of 7a against 3CLpro was performed and the binding mode was rationalized. These preliminary results provide a unique prototype for the development of novel inhibitors against SARS-CoV-2 3CLpro.

Synthesis and Biochemical Evaluation of 8H-Indeno[1,2-d]thiazole Derivatives as Novel SARS-CoV-2 3CL Protease Inhibitors

The COVID-19 pandemic caused by SARS-CoV-2 is a global burden on human health and economy. The 3-Chymotrypsin-like cysteine protease (3CLpro) becomes an attractive target for SARS-CoV-2 due to its important role in viral replication. We synthesized a series of 8H-indeno[1,2-d]thiazole derivatives and evaluated their biochemical activities against SARS-CoV-2 3CLpro. Among them, the representative compound 7a displayed inhibitory activity with an IC50 of 1.28 ±0.17 μM against SARS-CoV-2 3CLpro. Molecular docking of 7a against 3CLpro was performed and the binding mode was rationalized. These preliminary results provide a unique prototype for the development of novel inhibitors against SARS-CoV-2 3CLpro.

Ocular manifestations and SARS-CoV-2 detection in tears and conjunctival scrape from non-severe COVID-19 patients.

AIM: To explore the ocular features of corona virus disease (COVID)-19 and severe acute respiratory syndrome coronavirus (SARS-CoV)-2 detection in tears and conjunctival scrapes in non-severe COVID-19 patients. METHODS: This is a multicenter observational clinical study with no intervention conducted from Jan 25th to March 1st, 2020. Clinical data and samples of tears and conjunctival scraping were collected in consecutive laboratory-confirmed, non-severe COVID-19 patients from three hospitals. COVID-19 virus was analyzed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) kits. RESULTS: Totally 255 laboratory-confirmed, non-severe COVID-19 patients were recruited for ocular manifestation investigation. Of them, 54.9% were females, with a mean age of 49.4y. None of the patients has evidence of uveitis; 11 patients (4.3%) complained of mild asthenopia; 2 (0.8%) had mild conjunctival congestion and serous secretion. Twenty-five of them had performed tears and conjunctival scrape for COVID-19 virus detection, with 4 yield possible positive results in the nucleoprotein gene. One of them were asymptomatic with normal chest CT and positive pharyngeal swab result. CONCLUSION: Ocular manifestations are neither common nor specific in non-severe COVID-19 patients. Meanwhile, COVID-19 virus nucleotides can be detected in the tears and conjunctival scrape samples, warranting further research on the transmissibility by the ocular route.

SARS-CoV-2 clearance in COVID-19 patients with Novaferon treatment: A randomized, open-label, parallel-group trial.

BACKGROUND: The antiviral effects of Novaferon, a potent antiviral protein drug, on COVID-19 was evaluated in the laboratory, and in a randomized, open-label, parallel-group trial. METHODS: In the laboratory, Novaferon's inhibition of viral replication in cells infected with SARS-CoV-2, and prevention of SARS-CoV-2 entry into healthy cells was determined. Antiviral effects of Novaferon in COVID-19 patients with treatment of Novaferon, Novaferon plus Lopinavir/Ritonavir, or Lopinavir/Ritonavir were evaluated. The primary endpoint was the SARS-CoV-2 clearance rates on day six of treatment, and the secondary endpoint was the time to SARS-CoV-2 clearance. RESULTS: Novaferon inhibited viral replication (EC50=1.02ng/ml), and prevented viral infection (EC50=0.10ng/ml). Results from the 89 enrolled COVID-19 patients showed that both Novaferon and Novaferon plus Lopinavir/Ritonavir groups had significantly higher viral clearance rates on day six than Lopinavir/Ritonavir group (50.0% vs. 24.1%, p=0.0400, and 60.0% vs. 24.1%, p=0.0053). The median time to viral clearance was six days, six days, and nine days for three groups, respectively, a 3-day reduction in both the Novaferon and Novaferon plus Lopinavir/Ritonavir groups compared with the Lopinavir/Ritonavir group. CONCLUSIONS: Novaferon exhibited anti-SARS-CoV-2 effects in vitro and in COVID-19 patients. These data justify further evaluation of Novaferon. TRIAL REGISTRATION NUMBER: Number ChiCTR2000029496 at the Chinese Clinical Trial Registry (http://www.chictr.org.cn/).

The implications of preliminary screening and diagnosis: Clinical characteristics of 33 mild patients with SARS-CoV-2 infection in Hunan, China.

BACKGROUND: In December 2019, coronavirus Disease 2019 (COVID-19) occurred in Wuhan, Hubei Province, China. The disease has rapidly spread from Wuhan to other regions. OBJECTIVES: To describe the clinical manifestations and epidemiological characteristics of patients with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection in Hunan Province in 2020. STUDY DESIGN: From January 19 to February 7, 2020, 33 patients with positive in nucleic acid test of pharyngeal swab were retrospectively collected and analyzed. RESULTS: There are 33 COVID-19 patients (16 male, 17 female), and the median age was 46 years. Nineteen patients (48 %) were associated with a family cluster outbreak. Seventeen patients (52 %) had traveled or lived in Hubei Province. These patients are early mild cases, most common symptoms are fever [23 (70 %)] and cough [13 (39 %)]. Most patients' white blood cell counts are normal, while they manifest as significant reduction in lymphocytes [17/28 (61 %)]. The levels of c-reactive protein and erythrocyte sedimentation rate suggest a typical viral infection. Procalcitonin did not increase and D-dimer increased slightly. Lactate dehydrogenase (LDH) levels have elevated in most patients. CT images of these patients showed bilateral multiple plaques or nodular ground-glass opacities (68.4 %). Fecal nucleic acid results were positive in eight COVID-19 patients accompanied with diarrhea. Tear nucleic acid results were negative in six COVID-19 patients. And four asymptomatic patients were infected with SARS-CoV-2. CONCLUSIONS: The clinical symptoms, laboratory results and imaging reports of patients with COVID-19 in Hunan area are significantly different from those in Wuhan area. For non-Wuhan epidemic areas, more attention should be paid to nucleic acid test results of throat swabs and stools, and it is not easily to diagnose based on clinical symptoms and CT results. Reduced whole blood lymph count can be used as an adjuvant diagnosis of early SARS-CoV-2 infection. Attention should be paid to asymptomatic carriers, which is of great significance for the control of the global epidemic.