Wastewater-based epidemiology (WBE) has been an important tool for population surveillance during the COVID-19 pandemic and continues to play a key role in monitoring SARS-CoV-2 infection levels following reductions in national clinical testing schemes. Studies measuring decay profiles of SARS-CoV-2 in wastewater have underscored the value of WBE, however investigations have been hampered by high biosafety requirements for SARS-CoV-2 infection studies. Therefore, surrogate viruses with lower biosafety standards have been used for SARS-CoV-2 decay studies, such as murine hepatitis virus (MHV), but few studies have directly compared decay rates of both viruses. We compared the persistence of SARS-CoV-2 and MHV in wastewater, using 50 % tissue culture infectious dose (TCID50) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays to assess infectious virus titre and viral gene markers, respectively. Infectious SARS-CoV-2 and MHV indicate similar endpoints, however observed early decay characteristics differed, with infectious SARS-CoV-2 decaying more rapidly than MHV. We find that MHV is an appropriate infectious virus surrogate for viable SARS-CoV-2, however inconsistencies exist in viral RNA decay parameters, indicating MHV may not be a suitable nucleic acid surrogate across certain temperature regimes. This study highlights the importance of sample preparation and the potential for decay rate overestimation in wastewater surveillance for SARS-CoV-2 and other pathogens.

The unprecedented threat of the highly contagious virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes exponentially increased infections of coronavirus disease 2019 (COVID-19), highlights the weak spots of the current diagnostic toolbox. In the midst of catastrophe, nanobiosensors offer a new opportunity as an alternative tool to fill a gap among molecular tests, rapid antigen tests, and serological tests. Nanobiosensors surpass the potential of antigen tests because of their enhanced sensitivity, thus enabling us to see antigens as stable and easy-to-access targets. During the first three years of the COVID-19 pandemic, a substantial number of studies have reported nanobiosensors for the detection of SARS-CoV-2 antigens. The number of articles on nanobiosensors and SARS-CoV-2 exceeds the amount of nanobiosensor research on detecting previous infectious diseases, from influenza to SARS-CoV and MERS-CoV. This unprecedented publishing pace also implies the significance of SARS-CoV-2 and the present pandemic. In this review, 158 studies reporting nanobiosensors for detecting SARS-CoV-2 antigens are collected to discuss the current challenges of nanobiosensors using the criteria of point-of-care (POC) diagnostics along with COVID-specific issues. These advances and lessons during the pandemic pave the way for preparing for the post-COVID era and potential upcoming infectious diseases.

Accurate quantification of infectious contaminants on environmental surfaces, particularly infectious viruses, is essential for contact transmission risk assessment; however, difficulties in recovering viruses from surfaces using swabs complicates this quantification process. Herein, we identified the factors that significantly affected virus recovery rates and developed an ideal swab method that yielded the highest rate of virus recovery. We comprehensively analyzed the effects of swab type (cotton/polyester), swab water content (wet/dry conditions), surface material, and surface area on the rates of viral RNA and infectious virus recovery. The virus recovery rate was significantly lower than the viral RNA recovery rate (P < 0.01), indicating difficulty in the quantification of infectious viruses. The virus recovery rate was significantly higher under wet conditions than that under dry conditions (P < 0.006), and the virus recovery rate obtained using cotton swabs was significantly higher than that using polyester swabs (P < 0.0001). Furthermore, the virus recovery rate had a strong negative correlation (correlation coefficient >0.8) with the target surface area. The maximum surface area where the virus recovery rate was ≥10% (MSA-10%) was identified as the maximum quantifiable area. For influenza virus recovery, MSA-10% on polyvinyl chloride (PVC) sheet, PVC leather, stainless steel, silicone, glass, and polycarbonate surfaces was 66.7, 193, 60.2, 144, 105, and 15.6 cm2, respectively. For feline calicivirus recovery, MSA-10% on PVC sheet, PVC leather, stainless steel, silicone, glass, and polycarbonate surfaces was 210, 111, 2120, 250, 322, and 180 cm2, respectively. The most accurate and ideal method for quantifying infectious viruses on environmental surfaces with the highest recovery rates meets three specifications: \"wet conditions,\" \"the use of cotton swabs,\" and \"a target surface area of approximately 10 cm2.

Infectious African swine fever virus (ASFV) can cause the spread and morbidity of African swine fever, while the inactivated virus cannot. When they are not distinguished separately, the detection results will lack authenticity and cause unnecessary panic and detection cost. The detection technology based on cell culture is complex, high-cost, and time-consuming in practice, which is not conducive to the rapid detection of infectious ASFV. In this study, a propidium monoazide (PMA) qPCR detection method for rapid diagnosis of infectious ASFV was constructed. Parameters of PMA concentration, light intensity, and lighting time were under strict safety verification and comparative analysis for optimization. The results determined that the optimal condition for PMA to pretreat ASFV was the final concentration of PMA 100 μM. The light intensity was 40 W, the light duration was 20 min, the target fragment size of the optimal primer probe was 484 bp, and its detection sensitivity for infectious ASFV was 101.28 HAD50/mL. In addition, the method was innovatively applied to the rapid evaluation of disinfection effect. When ASFV concentration was less than 102.28 HAD50/mL, the method could still be effective for the evaluation of thermal inactivation effect, and the evaluation ability of chlorine-containing disinfectants was better, and the applicable concentration could reach 105.28 HAD50/mL. It is worth mentioning that this method can not only reflect whether the virus is inactivated, but also indirectly reflect the degree of damage to viral nucleic acid caused by disinfectants. In conclusion, the PMA-qPCR constructed in this study can be applied to laboratory diagnosis, disinfection effect evaluation, drug development, and other aspects of infectious ASFV and can provide new technical support for effective prevention and control of ASF. KEY POINTS: • A rapid detection method for infectious ASFV was developed • Provide a new scheme for rapid evaluation of disinfection effect of chlorine-containing disinfectants • PMA-qPCR can simultaneously show the survival status of the virus and the damage of nucleic acid.

暂无摘要。

African swine fever (ASF) is a hemorrhagic and often fatal disease occurring in domestic pigs and wild boars. ASF can potentially greatly impact the global trade of pigs and pork products and threaten global food security. Outbreaks of ASF must be notified to the World Organization for Animal Health. In this study, we analyzed the feasibility of applying propidium monoazide (PMA) pretreatment-based infectious virus detection technology to ASF prevention and control and investigated the prospects of applying this technology for epidemic monitoring, disinfection effect evaluation, and drug development. PMA as a nucleic acid dye can enter damaged cells and undergo irreversible covalent crosslinking with nucleic acid under halogen light to prevent its amplification. Although this technology has been widely used for the rapid detection of viable bacteria, its application in viruses is rare. Therefore, we analyzed the theoretical feasibility of applying this technology to the African swine fever virus (ASFV) in terms of gene and cell composition. Rapid infectious ASFV detection technology based on PMA pretreatment would greatly enhance all aspects of ASF prevention and control, such as epidemic monitoring, disinfection treatment, and drug development. The introduction of this technology will also greatly improve the ability to prevent and control ASF.

Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectious virus isolation in outpatients with coronavirus disease 2019 (COVID-19) has been associated with viral RNA levels and symptom duration, little is known about the host, disease, and viral determinants of infectious virus detection.
COVID-19 adult outpatients were enrolled within 7 days of symptom onset. Clinical symptoms were recorded via patient diary. Nasopharyngeal swabs were collected to quantitate SARS-CoV-2 RNA by reverse transcriptase polymerase chain reaction and for infectious virus isolation in Vero E6-cells. SARS-CoV-2 antibodies were measured in serum using a validated ELISA assay.
Among 204 participants with mild-to-moderate symptomatic COVID-19, the median nasopharyngeal viral RNA was 6.5 (interquartile range [IQR] 4.7-7.6 log10 copies/mL), and 26% had detectable SARS-CoV-2 antibodies (immunoglobulin (Ig)A, IgM, IgG, and/or total Ig) at baseline. Infectious virus was recovered in 7% of participants with SARS-CoV-2 antibodies compared to 58% of participants without antibodies (prevalence ratio [PR] = 0.12, 95% confidence interval [CI]: .04, .36; P = .00016). Infectious virus isolation was also associated with higher levels of viral RNA (mean RNA difference +2.6 log10, 95% CI: 2.2, 3.0; P < .0001) and fewer days since symptom onset (PR = 0.79, 95% CI: .71, .88 per day; P < .0001).
The presence of SARS-CoV-2 antibodies is strongly associated with clearance of infectious virus. Seropositivity and viral RNA levels are likely more reliable markers of infectious virus clearance than subjective measure of COVID-19 symptom duration. Virus-targeted treatment and prevention strategies should be administered as early as possible and ideally before seroconversion.
NCT04405570.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) and is capable of human-to-human transmission and rapid global spread. Thus, the establishment of high-quality viral detection and quantification methods, and the development of anti-SARS-CoV-2 agents are critical.
METHODS: Here, we present the rapid detection of infectious SARS-CoV-2 particles using a plaque assay with 0.5% agarose-ME (Medium Electroosmosis) as an overlay medium.
RESULTS: The plaques were capable of detecting the virus within 36-40 h post-infection. In addition, we showed that a monogalactosyl diacylglyceride isolated from a microalga (Coccomyxa sp. KJ) could inactivate the clinical isolates of SARS-CoV-2 in a time- and concentration-dependent manner.
CONCLUSIONS: These results would allow rapid quantification of the infectious virus titers and help develop more potent virucidal agents against SARS-CoV-2.

The infectivity of severe acute respiratory syndrome coronavirus 2 in deceased persons and organisms remains unclear. We studied transgenic K18 hACE2 mice to determine the kinetics of virus infectivity after host death. Five days after death, virus infectivity in the lung declined by >96% and RNA copies declined by 48.2%.

BACKGROUND: While SARS-CoV-2 infectious virus isolation in outpatients with COVID-19 has been associated with viral RNA levels and symptom duration, little is known about the host, disease and viral determinants of infectious virus detection.
METHODS: COVID-19 adult outpatients were enrolled within 7 days of symptom onset. Clinical symptoms were recorded via patient diary. Nasopharyngeal swabs were collected to quantitate SARS-CoV-2 RNA by reverse transcriptase polymerase chain reaction and for infectious virus isolation in Vero E6-cells. SARS-CoV-2 antibodies were measured in serum using a validated ELISA assay.
RESULTS: Among 204 participants with mild-to-moderate symptomatic COVID19, the median nasopharyngeal viral RNA was 6.5 (IQR 4.7-7.6 log10 copies/mL), and 26% had detectable SARS-CoV-2 antibodies (IgA, IgM, IgG, and/or total Ig) at baseline. Infectious virus was recovered in 7% of participants with SARS-CoV-2 antibodies compared to 58% of participants without antibodies (probability ratio (PR)=0.12, 95% CI: 0.04, 0.36; p=0.00016). Infectious virus isolation was also associated with higher levels of viral RNA (mean RNA difference +2.6 log10, 95% CI: 2.2, 3.0; p<0.0001) and fewer days since symptom onset (PR=0.79, 95% CI: 0.71, 0.88 per day; p<0.0001).
CONCLUSIONS: The presence of SARS-CoV-2 antibodies is strongly associated with clearance of infectious virus isolation. Seropositivity and viral RNA levels are likely more reliable markers of infectious virus clearance than subjective measure of COVID-19 symptom duration. Virus-targeted treatment and prevention strategies should be administered as early as possible and ideally before seroconversion.
BACKGROUND: NCT04405570.