Coronavirus murine hepatitis virus strain 3 induces intestinal injury via caspase 3 dependent apoptosis

Background: Diarrhea was typical symptoms of the coronavirus disease 2019 (COVID-19). However, the underlying mechanism had not been fully understood. Aim(s): The study aimed to explore the mechanism of intestinal injury during COVID-19 in a coronavirus murine hepatitis virus strain 3 (MHV-3) induced acute mouse model. Method(s): MHV-3 induced acute infection Balb/cJ mice model was established. Intestine samples were collected at indicated time points as 0 h, 24 h, 48 h and 60 h post infection. The mRNA and protein expression of IL1b, TNFalpha, IL6, caspase 3 and cleaved caspase 3 were examined by real-time quantitative PCR (qPCR) and western blot respectively. The intestine injury and apoptosis were measured by HE staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Moreover, Z-DEVD-FMK (caspase 3 inhibitor) pre-treated MHV-3 infection mice model were established, in which the apoptosis of intestine was evaluated as well. Meanwhile, the murine intestinal cell MODE-K was infected by MHV-3 in vitro for evaluation of virus induced apoptosis. Result(s): Post MHV-3 infection, the histopathology of intestine tissue showed extraordinary injury with time dependence, as well as high level of TUNEL positivity. The mRNA levels of inflammatory cytokine IL1b, TNFalpha and IL6 were significantly increased. The protein expressions of caspase 3 and cleaved caspase 3 in the intestine was found significantly elevated from 24 to 48 h post MHV-3 infection. Z-DEVD-FMK pretreatment inhibited caspase 3 and cleaved caspase 3 expression and decreased TUNEL positivity. Meanwhile, alleviated gut injury and inhibited TNFalpha expression were observed. In vitro treated by MHV-3, intestinal cell line MODE-K showed nine-fold increase of apoptosis by comparison with saline treated ones. The expressions of apoptosis crucial protein caspase3 and cleaved caspase3 significantly elevated, as well as TNFalpha. Conclusion(s): Coronavirus murine hepatitis virus strain 3 induces intestinal injury via caspase 3 dependent apoptosis, which might shed light on the treatment of intestinal complications in COVID-19.

New Short RNA Motifs Potentially Relevant in the SARS-CoV-2 Genome

Background: The coronavirus disease has led to an exhaustive exploration of the SARS-CoV-2 genome. Despite the amount of information accumulated, the prediction of short RNA motifs encoding peptides mediating protein-protein or protein-drug interactions has received limited attention. Objective(s): The study aims to predict short RNA motifs that are interspersed in the SARS-CoV-2 genome. Method(s): A method in which 14 trinucleotide families, each characterized by being composed of triplets with identical nucleotides in all possible configurations, was used to find short peptides with biological relevance. The novelty of the approach lies in using these families to search how they are distributed across genomes of different CoV genera and then to compare the distributions of these families with each other. Result(s): We identified distributions of trinucleotide families in different CoV genera and also how they are related, using a selection criterion that identified short RNA motifs. The motifs were reported to be conserved in SARS-CoVs;in the remaining CoV genomes analysed, motifs contained, exclusively, different configurations of the trinucleotides A, T, G and A, C, G. Eighty-eight short RNA motifs, ranging in length from 12 to 49 nucleotides, were found: 50 motifs in the 1a polyprotein-encoding orf, 27 in the 1b polyprotein-encoding orf, 5 in the spike-encoding orf, and 6 in the nucleocapsid-encoding orf. Although some motifs (~27%) were found to be intercalated or attached to functional peptides, most of them have not yet been associated with any known functions. Conclusion(s): Some of the trinucleotide family distributions in different CoV genera are not random;they are present in short peptides that, in many cases, are intercalated or attached to functional sites of the proteome.Copyright © 2022 Bentham Science Publishers.

Neutrophils drive pulmonary vascular leakage in MHV-1 infection of susceptible A/J mice.

Background: Lung inflammation, neutrophil infiltration, and pulmonary vascular leakage are pathological hallmarks of acute respiratory distress syndrome (ARDS) which can lethally complicate respiratory viral infections. Despite similar comorbidities, however, infections in some patients may be asymptomatic while others develop ARDS as seen with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections for example. Methods: In this study, we infected resistant C57BL/6 and susceptible A/J strains of mice with pulmonary administration of murine hepatitis virus strain 1 (MHV-1) to determine mechanisms underlying susceptibility to pulmonary vascular leakage in a respiratory coronavirus infection model. Results: A/J animals displayed increased lung injury parameters, pulmonary neutrophil influx, and deficient recruitment of other leukocytes early in the infection. Moreover, under basal conditions, A/J neutrophils overexpressed primary granule protein genes for myeloperoxidase and multiple serine proteases. During infection, myeloperoxidase and elastase protein were released in the bronchoalveolar spaces at higher concentrations compared to C57BL/6 mice. In contrast, genes from other granule types were not differentially expressed between these 2 strains. We found that depletion of neutrophils led to mitigation of lung injury in infected A/J mice while having no effect in the C57BL/6 mice, demonstrating that an altered neutrophil phenotype and recruitment profile is a major driver of lung immunopathology in susceptible mice. Conclusions: These results suggest that host susceptibility to pulmonary coronaviral infections may be governed in part by underlying differences in neutrophil phenotypes, which can vary between mice strains, through mechanisms involving primary granule proteins as mediators of neutrophil-driven lung injury.

Real-time reverse transcription recombinase polymerase amplification for rapid detection of murine hepatitis virus.

Murine hepatitis virus (MHV) is a highly infectious murine coronavirus that has a high potential for causing harm to host animals. This study aimed to develop a real-time reverse transcription recombinase polymerase amplification (RT-RPA) method for rapid detection of MHV in laboratory mice. Methods: Specific primers and probes for RT-RPA assay were designed targeting the conserved region in the M gene of the MHV reference strain (accession no. FJ6647223) according to the TwistDx manual instructions. The specificity, sensitivity, and reproducibility of the RT-RPA method were evaluated and compared with those of the standard RT-qPCR method. The clinical applicability of this assay was evaluated using 68 field samples. Results: Amplification using the newly developed RT-RPA assay was completed within 20 min at 37°C, while that using the RT-qPCR method required nearly 60 min. The RT-RPA method exhibited an obvious time-saving advantage. Both RT-RPA and RT-PCR methods had the same limit of detection, which was 4.45 × 101 copies/µL. The specificity was indicated by a lack of cross-reaction with MHV, pneumonia virus of mice, Sendai virus, hantavirus, minute virus of mice, and reovirus type III. The MHV detection rate of RT-RPA assays was 13.63% (9/66) and RT-qPCR assays was 15.15% (10/66). Cohen's "kappa" (κ) analysis results exhibited a very good agreement between two methods with the value of κ ≥ 0.750(since κ = 0.939) and p < 0.0005 (since p = 0.000). Conclusion: The RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of MHV in laboratory mice and has significant potential for application in laboratories.

SARS-CoV-2 mitochondriopathy in COVID-19 pneumonia exacerbates hypoxemia.

RATIONALE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 pneumonia. We hypothesize that SARS-CoV-2 causes alveolar injury and hypoxemia by damaging mitochondria in airway epithelial cells (AEC) and pulmonary artery smooth muscle cells (PASMC), triggering apoptosis and bioenergetic impairment, and impairing hypoxic pulmonary vasoconstriction (HPV), respectively. OBJECTIVES: We examined the effects of: A) human betacoronaviruses, SARS-CoV-2 and HCoV-OC43, and individual SARS-CoV-2 proteins on apoptosis, mitochondrial fission, and bioenergetics in AEC; and B) SARS-CoV-2 proteins and mouse hepatitis virus (MHV-1) infection on HPV. METHODS: We used transcriptomic data to identify temporal changes in mitochondrial-relevant gene ontology (GO) pathways post-SARS-CoV-2 infection. We also transduced AECs with SARS-CoV-2 proteins (M, Nsp7 or Nsp9) and determined effects on mitochondrial permeability transition pore (mPTP) activity, relative membrane potential, apoptosis, mitochondrial fission, and oxygen consumption rates (OCR). In human PASMC, we assessed the effects of SARS-CoV-2 proteins on hypoxic increases in cytosolic calcium, an HPV proxy. In MHV-1 pneumonia, we assessed HPV via cardiac catheterization and apoptosis using the TUNEL assay. RESULTS: SARS-CoV-2 regulated mitochondrial apoptosis, mitochondrial membrane permeabilization and electron transport chain (ETC) GO pathways within 2 hours of infection. SARS-CoV-2 downregulated ETC Complex I and ATP synthase genes, and upregulated apoptosis-inducing genes. SARS-CoV-2 and HCoV-OC43 upregulated and activated dynamin-related protein 1 (Drp1) and increased mitochondrial fission. SARS-CoV-2 and transduced SARS-CoV-2 proteins increased apoptosis inducing factor (AIF) expression and activated caspase 7, resulting in apoptosis. Coronaviruses also reduced OCR, decreased ETC Complex I activity and lowered ATP levels in AEC. M protein transduction also increased mPTP opening. In human PASMC, M and Nsp9 proteins inhibited HPV. In MHV-1 pneumonia, infected AEC displayed apoptosis and HPV was suppressed. BAY K8644, a calcium channel agonist, increased HPV and improved SpO2. CONCLUSIONS: Coronaviruses, including SARS-CoV-2, cause AEC apoptosis, mitochondrial fission, and bioenergetic impairment. SARS-CoV-2 also suppresses HPV by targeting mitochondria. This mitochondriopathy is replicated by transduction with SARS-CoV-2 proteins, indicating a mechanistic role for viral-host mitochondrial protein interactions. Mitochondriopathy is a conserved feature of coronaviral pneumonia that may exacerbate hypoxemia and constitutes a therapeutic target.

Toxicity and virucidal activity of a neon-driven micro plasma jet on eukaryotic cells and a coronavirus.

Plasma medicine is a developing field that utilizes the effects of cold physical plasma on biological substrates for therapeutic purposes. Approved plasma technology is frequently used in clinics to treat chronic wounds and skin infections. One mode of action responsible for beneficial effects in patients is the potent antimicrobial activity of cold plasma systems, which is linked to their unique generation of a plethora of reactive oxygen and nitrogen species (ROS). During the SARS-CoV-2 pandemic, it became increasingly clear that societies need novel ways of passive and active protection from viral airway infections. Plasma technology may be suitable for superficial virus inactivation. Employing an optimized neon-driven micro plasma jet, treatment time-dependent ROS production and cytotoxic effects to different degrees were found in four different human cell lines with respect to their metabolic activity and viability. Using the murine hepatitis virus (MHV), a taxonomic relative of human coronaviruses, plasma exposure drastically reduced the number of infected murine fibroblasts by up to 3000-fold. Direct plasma contact (conductive) with the target maximized ROS production, cytotoxicity, and antiviral activity compared to non-conductive treatment with the remote gas phase only. Strikingly, antioxidant pretreatment reduced but not abrogated conductive plasma exposure effects, pointing to potential non-ROS-related mechanisms of antiviral activity. In summary, an optimized micro plasma jet showed antiviral activity and cytotoxicity in human cells, which was in part ROS-dependent. Further studies using more complex tissue models are needed to identify a safe dose-effect window of antiviral activity at modest toxicity.

Compared to FiO2 21[percnt], Exposure to FiO2 Levels of 30 or 60[percnt] Produce Dose-Related Reductions in Survival in a Lethal Murine MHV-1 ß-Coronavirus Pneumonia Model

Rationale: While oxygen therapy is standard for patients with pneumonia, a potential for increased oxidant damage exists. Understanding how oxygen therapy impacts inflammatory lung injury with SARS-CoV-2 infection (COVID-19) and related viruses will inform patient management. We investigated the effects of fractional inspired oxygen concentrations (FiO2s) of 30 or 60% in a mouse hepatitis virus-1 (MHV-1) model of acute lung injury we developed in A/J mice. Methods: MHV-1, a ß-coronavirus like SARS-CoV-2, can be studied at Biosafety Level-2. Intratracheal installation of MHV-1 in our model produces inflammatory lung injury, progressive arterial desaturation, and lethality over 14d, similar to COVID-19. Using this model, we compared outcomes in animals exposed in sealed chambers to atmospheric FiO2s of 21, 30 or 60% beginning 2h after of MHV-1 challenge and continuing for up to 14d. In each of three experiments, MHV-1 challenged animals were randomized to receive FiO2s of 21, 30 or 60% (10 animals per FiO2 group per experiment, 90 animals total). In another experiment, 30 animals challenged with noninfected viral culture medium were randomized to the same three FiO2s. Animals were observed for up to 14d. Results: Compared to FiO2 21%, chambers with FiO2 30 and 60% had similar humidities and temperatures but slightly lower carbon dioxide levels (CO2, p≤0.05) but all chamber CO2s were in the range of 400-2000 ppm. Compared to animals surviving with FiO2 21% in each of the three experiments [#survivors/#total animals (%)] [1/10 (10%);5/10 (50%);4/10 (40%)], and their survival times (Figure-1), survival was reduced in respective experiments with FiO2 30% [1/10 (10%);2/10 (20%);0/10 (0%)] and FiO2 60% [0/10 (0%);0/10 (0%);0/10 (0%)]. Patterns of survival were similar comparing the three experiments for each FiO2 and when combined, there was a significant dose-related difference in survival across the three FiO2's (p<0.0001) (Figure-1). Compared to FiO2 21%, survival decreased with FiO2 30% (p=0.06) and more so with FiO2 60% (p<0.0001) (log-rank test with Dunnett-Hsu adjustment). All animals challenged with noninfected viral culture medium and exposed similarly to FiO2s 21, 30 or 60% (n=10 per group) survived except one 30% animal that died at 12d despite appearing well. Conclusions: FiO2s of 30 and 60% that are considered therapeutic and relatively safe clinically, markedly worsened survival in mice with MHV-1 pneumonia, a ß-coronavirus like SARS-CoV-2. These findings emphasize the need to better understand how oxygen therapy impacts the pathogenesis of SARS-CoV-2 in patients.

DIFFERENTIAL TRANSCRIPTOMIC CHANGES IN THE CENTRAL NERVOUS SYSTEM AND NEUROGENIC BLADDERS OF MICE INFECTED WITH A CORONAVIRUS

INTRODUCTION AND OBJECTIVE: Neurodegenerative diseases, such as multiple sclerosis (MS), often lead to the development of neurogenic lower urinary tract symptoms (LUTS). We previously characterized neurogenic bladder dysfunction in a mouse model of MS induced by a coronavirus, mouse hepatitis virus (MHV). The objective of this study was to identify genes and pathways linking neuroinflammation in the central nervous system with urinary bladder dysfunction to enhance our understanding of the mechanisms underlying LUTS in demyelinating diseases. METHODS: Adult C57BL/6 male mice (N=12) received either an intracranial injection of MHV (6,000 PFU) or sterile saline (control). The lumbosacral (L6-S2) spinal cord (SC) segments and urinary bladders were collected during acute infection stage (week 1) and at the first peak of demyelination (week 4) after inoculation with the virus. Total RNA was isolated and analyzed using Nanostring nCounter Neuroinflammation panel. The expression levels of 770 genes associated with neuroinflammation were assessed and compared between the specimens. RESULTS: Transcriptome analysis of SC specimens confirmed a significantly increased expression of 132 genes in MHV mice (tens to hundreds fold change) involved in the regulation of astrocyte, microglia and oligodendrocyte functions, neuroinflammation and immune responses. Out of 132 genes up-regulated in the SC, only 2 genes (siglec1, 46-fold in the SC, 2.6-fold at 1 week and 1.8-fold at 4 weeks in the bladder;and zbp1, 568-fold in the SC, 2.8-fold at 1 week and 2.2-fold at 4 weeks in the bladder) were up-regulated in the urinary bladders of MHV-infected mice. Additionally, two genes were significantly up-regulated (ttr, 2.2-fold at 1week and 1.7-fold at 4 weeks;and ms4a4a, 2.3-fold at 1week and 1.6-fold at 4 weeks), and two were down-regulated (asb2, -1.8-fold at 1 week and -1.6-fold at 4 weeks, and myct1, -1.7-fold at 1week and -1.6-fold at 4 weeks) exclusively in the urinary bladders of MHV mice. CONCLUSIONS: Two genes, siglec1 (encodes type 1 transmembrane protein, expressed in microglia and macrophages, promotes neuroinflammation) and zbp1 (encodes a Z-DNA binding protein, plays role in the innate immune response) link the development of neuroinflammation in the central nervous system with neurogenic changes in the urinary bladders of MHV-infected mice. Further research is needed to establish a functional relationship between expression of these genes and neurogenic LUTS.

Comparative analysis of rapid concentration methods for the recovery of SARS-CoV-2 and quantification of human enteric viruses and a sewage-associated marker gene in untreated wastewater.

To support public-health-related disease surveillance and monitoring, it is crucial to concentrate both enveloped and non-enveloped viruses from domestic wastewater. To date, most concentration methods were developed for non-enveloped viruses, and limited studies have directly compared the recovery efficiency of both types of viruses. In this study, the effectiveness of two different concentration methods (Concentrating pipette (CP) method and an adsorption-extraction (AE) method amended with MgCl2) were evaluated for untreated wastewater matrices using three different viruses (SARS-CoV-2 (seeded), human adenovirus 40/41 (HAdV 40/41), and enterovirus (EV)) and a wastewater-associated bacterial marker gene targeting Lachnospiraceae (Lachno3). For SARS-CoV-2, the estimated mean recovery efficiencies were significantly greater by as much as 5.46 times, using the CP method than the AE method amended with MgCl2. SARS-CoV-2 RNA recovery was greater for samples with higher titer seeds regardless of the method, and the estimated mean recovery efficiencies using the CP method were 25.1 ± 11% across ten WWTPs when wastewater samples were seeded with 5 × 104 gene copies (GC) of SARS-CoV-2. Meanwhile, the AE method yielded significantly greater concentrations of indigenous HAdV 40/41 and Lachno3 from wastewater compared to the CP method. Finally, no significant differences in indigenous EV concentrations were identified in comparing the AE and CP methods. These data indicate that the most effective concentration method varies by microbial analyte and that the priorities of the surveillance or monitoring program should be considered when choosing the concentration method.

UV Inactivation of SARS-CoV-2 across the UVC Spectrum: KrCl* Excimer, Mercury-Vapor, and Light-Emitting-Diode (LED) Sources.

Effective disinfection technology to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can help reduce viral transmission during the ongoing COVID-19 global pandemic and in the future. UV devices emitting UVC irradiation (200 to 280 nm) have proven to be effective for virus disinfection, but limited information is available for SARS-CoV-2 due to the safety requirements of testing, which is limited to biosafety level 3 (BSL3) laboratories. In this study, inactivation of SARS-CoV-2 in thin-film buffered aqueous solution (pH 7.4) was determined across UVC irradiation wavelengths of 222 to 282 nm from krypton chloride (KrCl*) excimers, a low-pressure mercury-vapor lamp, and two UVC light-emitting diodes. Our results show that all tested UVC devices can effectively inactivate SARS-CoV-2, among which the KrCl* excimer had the best disinfection performance (i.e., highest inactivation rate). The inactivation rate constants of SARS-CoV-2 across wavelengths are similar to those for murine hepatitis virus (MHV) from our previous investigation, suggesting that MHV can serve as a reliable surrogate of SARS-CoV-2 with a lower BSL requirement (BSL2) during UV disinfection tests. This study provides fundamental information on UVC's action on SARS-CoV-2 and guidance for achieving reliable disinfection performance with UVC devices. IMPORTANCE UV light is an effective tool to help stem the spread of respiratory viruses and protect public health in commercial, public, transportation, and health care settings. For effective use of UV, there is a need to determine the efficiency of different UV wavelengths in killing pathogens, specifically SARS-CoV-2, to support efforts to control the ongoing COVID-19 global pandemic and future coronavirus-caused respiratory virus pandemics. We found that SARS-CoV-2 can be inactivated effectively using a broad range of UVC wavelengths, and 222 nm provided the best disinfection performance. Interestingly, 222-nm irradiation has been found to be safe for human exposure up to thresholds that are beyond those effective for inactivating viruses. Therefore, applying UV light from KrCl* excimers in public spaces can effectively help reduce viral aerosol or surface-based transmissions.

Combination Treatment With Remdesivir and Ivermectin Exerts Highly Synergistic and Potent Antiviral Activity Against Murine Coronavirus Infection.

The recent COVID-19 pandemic has highlighted the urgency to develop effective antiviral therapies against the disease. Murine hepatitis virus (MHV) is a coronavirus that infects mice and shares some sequence identity to SARS-CoV-2. Both viruses belong to the Betacoronavirus genus, and MHV thus serves as a useful and safe surrogate model for SARS-CoV-2 infections. Clinical trials have indicated that remdesivir is a potentially promising antiviral drug against COVID-19. Using an in vitro model of MHV infection of RAW264.7 macrophages, the safety and efficacy of monotherapy of remdesivir, chloroquine, ivermectin, and doxycycline were investigated. Of the four drugs tested, remdesivir monotherapy exerted the strongest inhibition of live virus and viral RNA replication of about 2-log10 and 1-log10, respectively (at 6 µM). Ivermectin treatment showed the highest selectivity index. Combination drug therapy was also evaluated using remdesivir (6 µM) together with chloroquine (15 µM), ivermectin (2 µM) or doxycycline (15 µM) - above their IC50 values and at high macrophage cell viability of over 95%. The combination of remdesivir and ivermectin exhibited highly potent synergism by achieving significant reductions of about 7-log10 of live virus and 2.5-log10 of viral RNA in infected macrophages. This combination also resulted in the lowest cytokine levels of IL-6, TNF-α, and leukemia inhibitory factor. The next best synergistic combination was remdesivir with doxycycline, which decreased levels of live virus by ~3-log10 and viral RNA by ~1.5-log10. These results warrant further studies to explore the mechanisms of action of the combination therapy, as well as future in vivo experiments and clinical trials for the treatment of SARS-CoV-2 infection.

SARS-CoV-2 in environmental perspective: Occurrence, persistence, surveillance, inactivation and challenges.

The unprecedented global spread of the severe acute respiratory syndrome (SARS) caused by SARS-CoV-2 is depicting the distressing pandemic consequence on human health, economy as well as ecosystem services. So far novel coronavirus (CoV) outbreaks were associated with SARS-CoV-2 (2019), middle east respiratory syndrome coronavirus (MERS-CoV, 2012), and SARS-CoV-1 (2003) events. CoV relates to the enveloped family of Betacoronavirus (ßCoV) with positive-sense single-stranded RNA (+ssRNA). Knowing well the persistence, transmission, and spread of SARS-CoV-2 through proximity, the faecal-oral route is now emerging as a major environmental concern to community transmission. The replication and persistence of CoV in the gastrointestinal (GI) tract and shedding through stools is indicating a potential transmission route to the environment settings. Despite of the evidence, based on fewer reports on SARS-CoV-2 occurrence and persistence in wastewater/sewage/water, the transmission of the infective virus to the community is yet to be established. In this realm, this communication attempted to review the possible influx route of the enteric enveloped viral transmission in the environmental settings with reference to its occurrence, persistence, detection, and inactivation based on the published literature so far. The possibilities of airborne transmission through enteric virus-laden aerosols, environmental factors that may influence the viral transmission, and disinfection methods (conventional and emerging) as well as the inactivation mechanism with reference to the enveloped virus were reviewed. The need for wastewater epidemiology (WBE) studies for surveillance as well as for early warning signal was elaborated. This communication will provide a basis to understand the SARS-CoV-2 as well as other viruses in the context of the environmental engineering perspective to design effective strategies to counter the enteric virus transmission and also serves as a working paper for researchers, policy makers and regulators.

Decay of SARS-CoV-2 and surrogate murine hepatitis virus RNA in untreated wastewater to inform application in wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) demonstrates potential for COVID-19 community transmission monitoring; however, data on the stability of SARS-CoV-2 RNA in wastewater are needed to interpret WBE results. The decay rates of RNA from SARS-CoV-2 and a potential surrogate, murine hepatitis virus (MHV), were investigated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in untreated wastewater, autoclaved wastewater, and dechlorinated tap water stored at 4, 15, 25, and 37 °C. Temperature, followed by matrix type, most greatly influenced SARS-CoV-2 RNA first-order decay rates (k). The average T90 (time required for 1-log10 reduction) of SARS-CoV-2 RNA ranged from 8.04 to 27.8 days in untreated wastewater, 5.71 to 43.2 days in autoclaved wastewater, and 9.40 to 58.6 days in tap water. The average T90 for RNA of MHV at 4 to 37 °C ranged from 7.44 to 56.6 days in untreated wastewater, 5.58-43.1 days in autoclaved wastewater, and 10.9 to 43.9 days in tap water. There was no statistically significant difference between RNA decay of SARS-CoV-2 and MHV; thus, MHV is suggested as a suitable persistence surrogate. Decay rate constants for all temperatures were comparable across all matrices for both viral RNAs, except in untreated wastewater for SARS-CoV-2, which showed less sensitivity to elevated temperatures. Therefore, SARS-CoV-2 RNA is likely to persist long enough in untreated wastewater to permit reliable detection for WBE application.

Comparison of virus concentration methods for the RT-qPCR-based recovery of murine hepatitis virus, a surrogate for SARS-CoV-2 from untreated wastewater.

There is currently a clear benefit for many countries to utilize wastewater-based epidemiology (WBE) as part of ongoing measures to manage the coronavirus disease 2019 (COVID-19) global pandemic. Since most wastewater virus concentration methods were developed and validated for nonenveloped viruses, it is imperative to determine the efficiency of the most commonly used methods for the enveloped severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Municipal wastewater seeded with a human coronavirus (CoV) surrogate, murine hepatitis virus (MHV), was used to test the efficiency of seven wastewater virus concentration methods: (A-C) adsorption-extraction with three different pre-treatment options, (D-E) centrifugal filter device methods with two different devices, (F) polyethylene glycol (PEG 8000) precipitation, and (G) ultracentrifugation. MHV was quantified by reverse-transcription quantitative polymerase chain reaction and the recovery efficiency was calculated for each method. The mean MHV recoveries ranged from 26.7 to 65.7%. The most efficient methods were adsorption-extraction methods with MgCl2 pre-treatment (Method C), and without pre-treatment (Method B). The third most efficient method used the Amicon® Ultra-15 centrifugal filter device (Method D) and its recovery efficiency was not statistically different from the most efficient methods. The methods with the worst recovery efficiency included the adsorption-extraction method with acidification (A), followed by PEG precipitation (F). Our results suggest that absorption-extraction methods with minimal or without pre-treatment can provide suitably rapid, cost-effective and relatively straightforward recovery of enveloped viruses in wastewater. The MHV is a promising process control for SARS-CoV-2 surveillance and can be used as a quality control measure to support community-level epidemic mitigation and risk assessment.