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J Virol Methods ; 299: 114333, 2022 01.
Article in English | MEDLINE | ID: covidwho-1525873


The increasing prevalence of N501Y variants of SARS-CoV-2 has kindled global concern due to their enhanced transmissibility. Genome sequencing is the gold standard method to identify the emerging variants of concern. But it is time-consuming and expensive, limiting the widespread deployment of genome surveillance in some countries. Health authorities surge the development of alternative assay to expand screening capacity with reduced time and cost. In this study, we developed an in-house TaqMan minor groove binder (MGB) probe-based one-step RT-qPCR assay to detect the presence of N501Y mutation in SARS-CoV-2. A total of 168 SARS-CoV-2 positive respiratory specimens were collected to determine diagnostic accuracy of the RT-qPCR assay. As a reference standard, PANGO lineages and the mutation patterns of all samples were characterised by whole-genome sequencing. The analytical sensitivity and the ability of the assay to detect low frequency of N501Y variants were also evaluated. A total of 31 PANGO lineages were identified from 168 SARS-CoV-2 positive cases, in which 34 samples belonged to N501Y variants, including B.1.1.7 (n = 20), B.1.351 (n = 12) and P.3 (n = 2). The N501Y RT-qPCR correctly identified all 34 samples as N501Y-positive and the other 134 samples as wildtype. The limit-of-detection of the assay consistently achieved 1.5 copies/µL on four different qPCR platforms. N501Y mutation was successfully detected at an allele frequency as low as 10 % in a sample with mixed SARS-CoV-2 lineage. The N501Y RT-qPCR is simple and inexpensive (US$1.6 per sample). It enables robust high-throughput screening for surveillance of SARS-CoV-2 variants of concern harbouring N501Y mutation.

COVID-19 , SARS-CoV-2 , Humans , Real-Time Polymerase Chain Reaction , Whole Genome Sequencing
J Hazard Mater ; 421: 126799, 2022 01 05.
Article in English | MEDLINE | ID: covidwho-1336648


Stack aerosols are generated within vertical building drainage stacks during the discharge of wastewater containing feces and exhaled mucus from toilets and washbasins. Fifteen stack aerosol-related outbreaks of coronavirus disease 2019 (COVID-19) in high-rise buildings have been observed in Hong Kong and Guangzhou. Currently, we investigated two such outbreaks of COVID-19 in Hong Kong, identified the probable role of chimney effect-induced airflow in a building drainage system in the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We injected tracer gas (SF6) into the drainage stacks via the water closet of the index case and monitored tracer gas concentrations in the bathrooms and along the facades of infected and non-infected flats and in roof vents. The air temperature, humidity, and pressure in vertical stacks were also monitored. The measured tracer gas distribution agreed with the observed distribution of the infected cases. Phylogenetic analysis of the SARS-CoV-2 genome sequences demonstrated clonal spread from a point source in cases along the same vertical column. The stack air pressure and temperature distributions suggested that stack aerosols can spread to indoors through pipe leaks which provide direct evidence for the long-range aerosol transmission of SARS-CoV-2 through drainage pipes via the chimney effect.

Aerosols , Air Microbiology , COVID-19 , Housing , COVID-19/transmission , Hong Kong , Humans , Phylogeny , SARS-CoV-2
Cells ; 10(7)2021 07 12.
Article in English | MEDLINE | ID: covidwho-1308302


MicroRNAs (miRNAs) are critical regulators of gene expression that may be used to identify the pathological pathways influenced by disease and cellular interactions. Viral miRNAs (v-miRNAs) encoded by both DNA and RNA viruses induce immune dysregulation, virus production, and disease pathogenesis. Given the absence of effective treatment and the prevalence of highly infective SARS-CoV-2 strains, improved understanding of viral-associated miRNAs could provide novel mechanistic insights into the pathogenesis of COVID-19. In this study, SARS-CoV-2 v-miRNAs were identified by deep sequencing in infected Calu-3 and Vero E6 cell lines. Among the ~0.1% small RNA sequences mapped to the SARS-CoV-2 genome, the top ten SARS-CoV-2 v-miRNAs (including three encoded by the N gene; v-miRNA-N) were selected. After initial screening of conserved v-miRNA-N-28612, which was identified in both SARS-CoV and SARS-CoV-2, its expression was shown to be positively associated with viral load in COVID-19 patients. Further in silico analysis and synthetic-mimic transfection of validated SARS-CoV-2 v-miRNAs revealed novel functional targets and associations with mechanisms of cellular metabolism and biosynthesis. Our findings support the development of v-miRNA-based biomarkers and therapeutic strategies based on improved understanding of the pathophysiology of COVID-19.

COVID-19/metabolism , Coronavirus Nucleocapsid Proteins/genetics , Metabolic Networks and Pathways , MicroRNAs/genetics , RNA, Viral/genetics , SARS-CoV-2/physiology , Animals , COVID-19/virology , Cell Line , Chlorocebus aethiops , Host-Pathogen Interactions , Humans , Phosphoproteins/genetics , SARS-CoV-2/genetics , Vero Cells