Activation of peroxymonosulfate by sustainable biomass-based carbon nanotubes for controlling the spread of plant viruses in water environments.

With the increasing demand for water in hydroponic systems and agricultural irrigation, viral diseases have seriously affected the yield and quality of crops. By removing plant viruses in water environments, virus transmission can be prevented and agricultural production and ecosystems can be protected. But so far, there have been few reports on the removal of plant viruses in water environments. Herein, in this study, easily recyclable biomass-based carbon nanotubes catalysts were synthesized with varying metal activities to activate peroxymonosulfate (PMS). Among them, the magnetic 0.125Fe@NCNTs-1/PMS system showed the best overall removal performance against pepper mild mottle virus, with a 5.9 log10 removal within 1 min. Notably, the key reactive species in the 0.125Fe@NCNTs-1/PMS system is 1O2, which can maintain good removal effect in real water matrices (river water and tap water). Through RNA fragment analyses and label free analysis, it was found that this system could effectively cleave virus particles, destroy viral proteins and expose their genome. The capsid protein of pepper mild mottle virus was effectively decomposed where serine may be the main attacking sites by 1O2. Long viral RNA fragments (3349 and 1642 nt) were cut into smaller fragments (∼160 nt) and caused their degradation. In summary, this study contributes to controlling the spread of plant viruses in real water environment, which will potentially help protect agricultural production and food safety, and improve the health and sustainability of ecosystems.

The fate of severe acute respiratory syndrome coronavirus-2 and pepper mild mottle virus at various stages of wastewater treatment process.

This study investigated the efficiency of the treatment processes of wastewater treatment plants (WWTPs) to remove severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and pepper mild mottle virus (PMMoV) from the wastewater and sewage sludge, as well as the influence of the mode of operation on the quality of the treated wastewater. SARS-CoV-2 and PMMoV were detected and quantified at different stages of the wastewater and sludge treatment process of three major WWTPs in Stockholm, Sweden. The results showed that primary, biological, and advanced membrane treatment processes are effective in removing SARS-CoV-2 from the wastewater with removal efficiencies of 99-100 % for all WWTPs, while the virus was accumulated in the primary and waste-activated sludges due to higher affinity to biosolids. Operation strategies such as bypass reintroduced the virus into the treated wastewater. The WWTPs achieved relatively low PMMoV removal efficiencies (63-87 %) most probably due to the robust capsid structure of the virus. Anaerobic digestion could not completely remove SARS-CoV-2 and PMMoV from the sludge leading to increased levels of SARS-CoV-2 and PMMoV in dewatered sludge. The study gives an overview of WWTPs' role in tackling pathogen spread in society in the event of a pandemic and disease breakout.

Indicator and pathogenic virus removal in bench scale soil aquifer treatment.

The demonstration of enteric virus removal for indirect potable reuse of advanced purified water is necessary to ensure safe water reclamation practices. This study evaluated the efficacy of soil treatment in reducing concentrations of Pepper Mild Mottle Virus (PMMoV), Hepatitis A (HAV), and Norovirus (NoV) gene markers through bench scale unsaturated soil columns. Three different infiltration rates were evaluated to determine their impact on viral gene marker removal. The concentrations of viral markers in the column influent and effluent samples were measured through RNA extraction and then RT-qPCR, and the log reduction values (LRVs) were calculated to quantify the effectiveness of removal across the columns. The LRVs achieved for PMMoV were 2.80 ± 0.36, 2.91 ± 0.48, and 2.72 ± 0.32 for infiltration rates of 4.9 mm/h, 9.4 mm/h, and 14.0 mm/h, respectively. A one-way ANOVA indicated no statistically significant differences in LRVs among the various infiltration rates (p-value = 0.329). All samples measured for HAV were below the detection limit both in the influent and effluent of the soil columns. While NoV GI and GII markers were measurable in the soil column influent, they were removed to below the detection limit in the effluent. The use of half the Limit-of-Detection (LoD) for effluent values enabled the estimation of log removals, which were calculated as 1.42 ± 0.07, 1.64 ± 0.29, and 1.74 ± 0.18 for NoV GI and 1.14 ± 0.19, 1.58 ± 0.21, and 1.87 ± 0.41 for NoV GII at infiltration rates of 4.9 mm/h, 9.4 mm/h, and 14.0 mm/h. This highlights the efficacy of soil treatment in reducing virus gene marker concentrations at various infiltration rates, and that spreading basins employed for reclaimed water recharge to ground water aquifers are an effective method for reducing the presence of viral contaminants in indirect potable reuse systems.

Identification of environmental and methodological factors driving variability of Pepper Mild Mottle Virus (PMMoV) across three wastewater treatment plants in the City of Toronto.

PMMoV has been widely used to normalize the concentration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA, influenza, and respiratory syncytial virus (RSV) to account for variations in the fecal content of wastewater. PMMoV is also used as an internal RNA recovery control for wastewater-based epidemiology (WBE) tests. While potentially useful for the interpretation of WBE data, previous studies have suggested that PMMoV concentration can be affected by various physico-chemical characteristics of wastewater. There is also the possibility that laboratory methods, particularly the variability in centrifugation steps to remove supernatant from pellets can cause PMMoV variability. The goal of this study is to improve our understanding of the main drivers of PMMoV variability by assessing the relationship between PMMoV concentration, the physico-chemical characteristics of wastewater, and the methodological approach for concentrating wastewater samples. We analyzed 24-hour composite wastewater samples collected from the influent stream of three wastewater treatment plants (WWTPs) located in the City of Toronto, Ontario, Canada. Samples were collected 3 to 5 times per week starting from the beginning of March 2021 to mid-July 2023. The influent flow rate was used to partition the data into wet and dry weather conditions. Physico-chemical characteristics (e.g., total suspended solids (TSS), biological oxygen demand (BOD), alkalinity, electrical conductivity (EC), and ammonia (NH3)) of the raw wastewater were measured, and PMMoV was quantified. Spatial and temporal variability of PMMoV was observed throughout the study period. PMMoV concentration was significantly higher during dry weather conditions. Multiple linear regression analysis demonstrates that the number and type of physico-chemical parameters that drive PMMoV variability are site-specific, but overall BOD and alkalinity were the most important predictors. Differences in PMMoV concentration for a single WWTP between two different laboratory methods, along with a weak correlation between pellet mass and TSS using one method may indicate that differences in sample concentration and subjective subsampling bias could alter viral recovery and introduce variability to the data.

Campus source to sink wastewater surveillance of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

Wastewater-based surveillance (WBS) offers an aggregate, and cost-effective approach for tracking infectious disease outbreak prevalence within communities, that provides data on community health complementary to individual clinical testing. This study reports on a 16-month WBS initiative on a university campus in England, UK, assessing the presence of SARS-CoV-2 in sewers from large buildings, downstream sewer locations, raw wastewater, partially treated and treated effluents. Key findings include the detection of the Alpha (B.1.1.7) variant in wastewater, with 70 % of confirmed campus cases correlating with positive wastewater samples. Notably, ammonium nitrogen (NH4-N) levels showed a positive correlation (ρ = 0.543, p < 0.01) with virus levels at the large building scale, a relationship not observed at the sewer or wastewater treatment works (WWTW) levels due to dilution. The WWTW was compliant to wastewater standards, but the secondary treatment processes were not efficient for virus removal as SARS-CoV-2 was consistently detected in treated discharges. Tools developed through WBS can also be used to enhance traditional environmental monitoring of aquatic systems. This study provides a detailed source-to-sink evaluation, emphasizing the critical need for the widespread application and improvement of WBS. It showcases WBS utility and reinforces the ongoing challenges posed by viruses to receiving water quality.

Capsicum chinense Jacq.-derived glutaredoxin (CcGRXS12) alters redox status of the cells to confer resistance against pepper mild mottle virus (PMMoV-I).

KEY MESSAGE: The CcGRXS12 gene protects plants from cellular oxidative damage that are caused by both biotic and abiotic stresses. The protein possesses GSH-disulphide oxidoreductase property but lacks Fe-S cluster assembly mechanism. Glutaredoxins (Grxs) are small, ubiquitous and multi-functional proteins. They are present in different compartments of plant cells. A chloroplast targeted Class I GRX (CcGRXS12) gene was isolated from Capsicum chinense during the pepper mild mottle virus (PMMoV) infection. Functional characterization of the gene was performed in Nicotiana benthamiana transgenic plants transformed with native C. chinense GRX (Nb:GRX), GRX-fused with GFP (Nb:GRX-GFP) and GRX-truncated for chloroplast sequences fused with GFP (Nb:Δ2MGRX-GFP). Overexpression of CcGRXS12 inhibited the PMMoV-I accumulation at the later stage of infection, accompanied with the activation of salicylic acid (SA) pathway pathogenesis-related (PR) transcripts and suppression of JA/ET pathway transcripts. Further, the reduced accumulation of auxin-induced Glutathione-S-Transferase (pCNT103) in CcGRXS12 overexpressing lines indicated that the protein could protect the plants from the oxidative stress caused by the virus. PMMoV-I infection increased the accumulation of pyridine nucleotides (PNs) mainly due to the reduced form of PNs (NAD(P)H), and it was high in Nb:GRX-GFP lines compared to other transgenic lines. Apart from biotic stress, CcGRXS12 protects the plants from abiotic stress conditions caused by H2O2 and herbicide paraquat. CcGRXS12 exhibited GSH-disulphide oxidoreductase activity in vitro; however, it was devoid of complementary Fe-S cluster assembly mechanism found in yeast. Overall, this study proves that CcGRXS12 plays a crucial role during biotic and abiotic stress in plants.

Adoption of the 2A Ribosomal Skip Principle to Track Assembled Virions of Pepper Mild Mottle Virus in Nicotiana benthamiana.

The coat protein (CP) is an important structural protein that plays many functional roles during the viral cycle. In this study, the CP of pepper mild mottle virus (PMMoV) was genetically fused to GFP using the foot-and-mouth disease virus peptide 2A linker peptide and the construct (PMMoV-GFP2A) was shown to be infectious. The systemic spread of the virus was monitored by its fluorescence in infected plants. Electron microscopy and immunocolloidal gold labelling confirmed that PMMoV-GFP2A forms rod-shaped particles on which GFP is displayed. Studies of tissue ultrastructure and virion self-assembly confirmed that PMMoV-GFP2A could be used to monitor the real-time dynamic changes of CP location during virus infection. Aggregations of GFP-tagged virions appeared as fluorescent plaques in confocal laser microscopy. Altogether, PMMoV-GFP2A is a useful tool for studying the spatial and temporal changes of PMMoV CP during viral infection.

Evaluation of four human-associated fecal biomarkers in wastewater in Southern Ontario.

Human fecal biomarkers (HFBs) have a longstanding history in the field of microbial source tracking (MST) serving as indicators of human fecal contamination in drinking and recreational water. Further, HFBs have aided in recent efforts to monitor human pathogen transmission within communities. The dilution of wastewater from various sources throughout the sewershed cannot be controlled and human fecal biomarkers (HFBs) can be used to normalize target human pathogen concentrations so that fluctuations in fecal matter in wastewater can be accounted for. In the current study, we monitored the prevalence of four HFBs - including two viruses, Pepper mild mottle virus (PMMoV), cross-assembly phage (crAssphage), as well as two human-associated Bacteroides markers, HF183 and BacHuman - in wastewater samples from ten Southern Ontario wastewater treatment plants and evaluated their temporal and spatial variation in context of environmental factors that may impact the ability of HFB to normalize pathogen concentrations in wastewater. Environmental variables including precipitation, wastewater flow rate, temperature, and concentrated mass were also analyzed for their potential correlation with HFB variation in wastewater. The four HFBs were detected at high concentrations across all 10 sampling locations. The median concentrations across all sampling sites were: PMMoV 3.6 Log gene copies (GC)/mL; crAssphage 5.0 Log GC/mL; HF183 6.8 Log GC/mL and BacHuman 6.9 Log GC/mL. All HFBs were found to be similarly stratified across all 10 sites, and the bacterial markers were consistently found at higher concentration compared to the viral HFBs at all sites. The coefficient of variation (CV) for each HFB was used to characterize the variability of each biomarker at each sewershed. BacHuman and crAssphage were found to have lower CV than PMMoV and HF183, indicating that BacHuman and crAssphage may perform better in reflecting the variations in abundance of human feces in wastewater or MST applications.

Pepper mild mottle virus can infect and traffick within Nicotiana benthamiana plants in non-virion forms.

Virions are responsible for the long-distance transport of many viruses, such as Pepper mild mottle virus (PMMoV). Emerging evidence indicates viral traffic in the form of ribonucleoprotein complexes (RNP), yet comprehensive analysis is scarce. In this study, we inoculated plants with PMMoV-GFP, both with and without the coding sequence for the coat protein (CP). PMMoV-GFP was detected in systemic leaves, even in the absence of the CP, despite the presence of much smaller infection areas. Moreover, using leaf extracts from PMMoV-infected plants to perform a root-irrigation experiment, we confirmed that PMMoV can infect plants through root transmission. Diluting the leaf extracts significantly diminished infectivity, and attempts to compensate for the dilution of other components by adding virions above the original level proved ineffective. Our findings strongly indicate that PMMoV can infect and traffick within plants in non-virion forms. Future studies should aim to identify the specific forms involved.

Inactivating Activities and Mechanism of Imidazo[1,2-c]pyrimidin-5(6H)-one Nucleoside Derivatives Incorporating a Sulfonamide Scaffold.

Twenty-eight imidazo[1,2-c]pyrimidin-5(6H)-one nucleoside derivatives incorporating a sulfonamide scaffold with preferable inactivating activities on pepper mild mottle virus (PMMoV) were designed and synthesized. Then, compound B29 with illustrious inactivating activity against PMMoV was received on the basis of the three-dimensional quantitative structure-activity relationship (3D-QSAR) model, with the EC50 of 11.4 µg/mL, which was superior to ningnanmycin (65.8 µg/mL) and template molecule B16 (15.3 µg/mL). Furthermore, (1) transmission electron microscopy (TEM) indicated that B29 could cause severe fracture of virions; (2) microscale thermophoresis (MST) and molecular docking further demonstrated that B29 had faintish binding affinities with PMMoV CPR62A (Kd = 202.84 µM), PMMoV CPL144A (Kd = 141.57 µM), and PMMoV CPR62A,L144A (Kd = 332.06 µM) compared to PMMoV CP (Kd = 4.76 µM); and (3) western blot and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results of pCB-GFP-PMMoV CPR62A, pCB-GFP-PMMoV CPL144A, and pCB-GFP-PMMoV CPR62A,L144A were consistent with MST and confocal. In brief, the above results indicated that the amino acids at positions 62 and 144 of PMMoV CP might be the key amino acid sites of B29 acted on.

Pepper Mild Mottle Virus as a Potential Indicator of Fecal Contamination in Influents of Wastewater Treatment Plants in Riyadh, Saudi Arabia.

Several indicators of fecal pollution in water resources are continuously monitored for their reliability and, of particular interest, their correlation to human enteric viruses-not justified by traditional bacterial indicators. Pepper mild mottle virus (PMMoV) has recently been proposed as a successful viral surrogate of human waterborne viruses; however, in Saudi Arabia there are no available data in terms of its prevalence and concentration in water bodies. The concentration of PMMoV in three different wastewater treatment plants (King Saud University (KSU), Manfoha (MN), and Embassy (EMB) wastewater treatment plants (WWTP)) was measured using qRT-PCR during a one-year period and compared to the human adenovirus (HAdV), which is highly persistent and considered an indicator for viral-mediated fecal contamination. PMMoV was found in ~94% of the entire wastewater samples (91.6-100%), with concentrations ranging from 62 to 3.5 × 107 genome copies/l (GC/l). However, HAdV was detected in 75% of raw water samples (~67-83%). The HAdV concentration ranged between 1.29 × 103 GC/L and 1.26 × 107 GC/L. Higher positive correlation between PMMoV and HAdV concentrations was detected at MN-WWTP (r = 0.6148) than at EMB-WWTP (r = 0.207). Despite the lack of PMMoV and HAdV seasonality, a higher positive correlation (r = 0.918) of PMMoV to HAdV was recorded at KSU-WWTP in comparison to EMB-WWTP (r = 0.6401) around the different seasons. Furthermore, meteorological factors showed no significant influence on PMMoV concentrations (p > 0.05), thus supporting the use of PMMoV as a possible fecal indicator of wastewater contamination and associated public health issues, particularly at MN-WWTP. However, a continuous monitoring of the PMMoV distribution pattern and concentration in other aquatic environments, as well as its correlation to other significant human enteric viruses, is essential for ensuring its reliability and reproducibility as a fecal pollution indicator.

Investigation of removal and inactivation efficiencies of human sapovirus in drinking water treatment processes by applying an in vitro cell-culture system.

Here, we examined the efficiencies of drinking water treatment processes for the removal and inactivation of human sapovirus (HuSaV). We applied a recently developed in vitro cell-culture system to produce purified solutions of HuSaV containing virus concentrations high enough to conduct virus-spiking experiments, to develop an integrated cell culture-polymerase chain reaction (ICC-PCR) assay to quantify the infectivity of HuSaV, and to conduct virus-spiking experiments. In virus-spiking coagulation-sedimentation-rapid sand filtration (CS-RSF) and coagulation-microfiltration (C-MF) experiments, HuSaV removals of 1.6-3.7-log10 and 1.2->4.3-log10, respectively, were observed. The removal ratios observed with CS-RSF were comparable and correlated with those of murine norovirus (MNV, a widely used surrogate for human noroviruses) and pepper mild mottle virus (PMMoV, a potential surrogate for human enteric viruses in physical and physicochemical drinking water treatment processes), and those observed with C-MF were higher than but still correlated with those of MNV and PMMoV, indicating that MNV and PMMoV are both potential surrogates for HuSaV in CS-RSF and C-MF. For astrovirus (AstV, a representative human enteric virus), removal ratios of 1.8-3.3-log10 and 1.1->4.0-log10 were observed with CS-RSF and C-MF, respectively. The removal ratios of AstV observed with CS-RSF were comparable and correlated with those of PMMoV, and those observed with C-MF were higher than but still correlated with those of PMMoV, indicating that PMMoV is a potential surrogate for AstV in CS-RSF and C-MF. When the efficacy of chlorine treatment was examined by using the developed ICC-PCR assay, 3.8-4.0-log10 inactivation of HuSaV was observed at a CT value (free-chlorine concentration [C] multiplied by contact time [T]) of 0.02 mg-Cl2·min/L. The infectivity reduction ratios of HuSaV were comparable with those of MNV. For AstV, 1.3-1.7-log10 and >3.4-log10 inactivation, as evaluated by ICC-PCR, was observed at CT values of 0.02 and 0.09 mg-Cl2·min/L, respectively. These results indicate that HuSaV and AstV are both highly sensitive to chlorine treatment and more sensitive than a chlorine-resistant virus, coxsackievirus B5 (1.3-log10 inactivation at a CT value of 0.4 mg-Cl2·min/L, as evaluated by the ICC-PCR assay).

RT-PCR based detection of Pepper mild mottle virus from capsicum seeds and seed transmission assay.

Pepper mild mottle virus (PMMoV), a Tobamovirus from Virgaviridae family, is highly contagious and transmitted by seeds as well as soil in nature. PMMoV has become a greater threat to capsicum cultivation worldwide. To develop an indigenous, rapid, and sensitive protocol for routine detection of PMMoV from seeds, the sensitivity of DAS-ELISA and RT-PCR was compared in the present study. The infected seeds of California Wonder were included in the study. Through DAS-ELISA the virus was successfully detected from 20 mg of seeds. However, using RT-PCR, we were able to detect the virus even from one infected seed with reproducibility. In the present study, vertical seed transmission of the test virus was investigated by employing a grow-out test under greenhouse conditions as well as directly through RT-PCR omitting the grow-out test in three capsicum cultivars. Based on symptoms observations in grow out test, seed transmission was observed in the 3 capsicum cultivars viz., California Wonder (63.04%), Yolo Wonder (33.80%) and Doux des LAndes (33.30%). Through RT-PCR it was estimated to be 55.56% (California Wonder), 28.96% (Yolo Wonder), and 40.64% (Doux des Landes), respectively. Thus, indicating 100% seed-to-seedling PMMoV transmission and reliability of RT-PCR in direct PMMoV detection from seeds. Even a small percentage of infected seed has the potential to greatly increase the PMMoV inoculum in the field and result in 100% plant infection. Therefore, we suggest using the established procedure for PMMoV detection right from the seed. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00807-0.

Monitoring Enteroviruses and SARS-CoV-2 in Wastewater Using the Polio Environmental Surveillance System in Japan.

In the global strategy for polio eradication, environmental surveillance (ES) has been established worldwide to monitor polioviruses. In addition, nonpolio enteroviruses are simultaneously isolated from wastewater under this ES program. Hence, ES can be used to monitor enteroviruses in sewage to supplement clinical surveillance. In response to the coronavirus disease 2019 (COVID-19) pandemic, we also monitored severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in sewage using the polio ES system in Japan. Enterovirus and SARS-CoV-2 were detected in sewage from January 2019 to December 2021 and from August 2020 to November 2021, respectively. Enterovirus species such as echoviruses and coxsackieviruses were frequently detected by ES in 2019, indicating the circulation of these viruses. After the onset of the COVID-19 pandemic, sewage enterovirus detection and related patient reports were notably reduced in 2020 to 2021, suggesting changes in the hygiene behaviors of the human population in response to the pandemic. Our comparative experiment with a total of 520 reverse transcription-quantitative PCR (RT-qPCR) assays for SARS-CoV-2 detection demonstrated that the solid-based method had a significantly higher detection rate than that of the liquid-based method (24.6% and 15.9%, respectively). Moreover, the resulting RNA concentrations were correlated with the number of new COVID-19 cases (Spearman's r = 0.61). These findings indicate that the existing polio ES system can be effectively used for enterovirus and SARS-CoV-2 sewage monitoring using different procedures such as virus isolation and molecular-based detection. IMPORTANCE Long-term efforts are required to implement surveillance programs for the ongoing COVID-19 pandemic, and they will be required even in the postpandemic era. We adopted the existing polio environmental surveillance (ES) system for SARS-CoV-2 sewage monitoring in Japan as a practical and cost-effective approach. Moreover, the ES system routinely detects enteroviruses from wastewater and, therefore, can be used for enterovirus monitoring. The liquid fraction of the sewage sample is used for poliovirus and enterovirus detection, and the solid fraction can be used for SARS-CoV-2 RNA detection. The present study demonstrates how the existing ES system can be used for monitoring enteroviruses and SARS-CoV-2 in sewage.

Monitoring of SARS-CoV-2 concentration and circulation of variants of concern in wastewater of Leuven, Belgium.

Wastewater surveillance plays an important role in the management of the coronavirus disease 2019 (COVID-19) pandemic all over the world. Using different wastewater collection points in Leuven, we wanted to investigate the use of wastewater surveillance as an early warning system for an uprise of infections and as a tool to follow the circulation of specific variants of concern (VOCs) in particular geographic areas. Wastewater samples were collected from local neighborhood sewers and from a large regional wastewater treatment plant (WWTP) in the area of Leuven, Belgium. After virus concentration, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was quantified by real-time quantitative polymerase chain reaction (RT-qPCR) and normalized with the human fecal indicator pepper mild mottle virus (PMMoV). A combination of multiplex RT-qPCR assays was used to detect signature mutations of circulating VOCs. Fecal virus shedding of SARS-CoV-2 variants was measured in feces samples of hospitalized patients. In two residential sampling sites, a rise in wastewater SARS-CoV-2 concentration preceded peaks in positive cases. In the WWTP, viral load peaks were seen concomitant with the consecutive waves of positive cases caused by the original Wuhan SARS-CoV-2 strain and subsequent VOCs. During the Omicron BA.1 wave, the wastewater viral load increased to a lesser degree, even after normalization of SARS-CoV-2 concentration using PMMoV. This might be attributable to a lower level of fecal excretion of this variant. Circulation of SARS-CoV-2 VOCs Alpha, Delta, Omicron BA1/BA.2, and BA.4/BA.5 could be detected based on the presence of specific key mutations. The shift in variants was noticeable in the wastewater, with key mutations of two different variants being present simultaneously during the transition period. Wastewater-based surveillance is a sensitive tool to monitor SARS-CoV-2 circulation levels and VOCs in larger regions. In times of reduced test capacity, this can prove to be highly valuable. Differences in excretion levels of various SARS-CoV-2 variants should however be taken into account when using wastewater surveillance to monitor SARS-CoV-2 circulation levels in the population.

Pepper Mild Mottle Virus: An Infectious Pathogen in Pepper Production and a Potential Indicator of Domestic Water Quality.

Pepper (Capsicum spp.; Family: Solanaceae; 2n = 24) is an important crop cultivated worldwide for the consumption of its fresh and dried processed fruits. Pepper fruits are used as raw materials in a wide variety of industrial processes. As a multipurpose vegetable crop, there is a need to increase the yield. However, yield productivity of pepper is severely constrained by infectious plant pathogens, including viruses, bacteria, fungi, and oomycetes. The pepper mild mottle virus (PMMoV) is currently one of the most damaging pathogens associated with yield losses in pepper production worldwide. In addition to impacts on pepper productivity, PMMoV has been detected in domestic and aquatic water resources, as well as in the excreta of animals, including humans. Therefore, PMMoV has been suggested as a potential indicator of domestic water quality. These findings present additional concerns and trigger the need to control the infectious pathogen in crop production. This review provides an overview of the distribution, economic impacts, management, and genome sequence variation of some isolates of PMMoV. We also describe genetic resources available for crop breeding against PMMoV.

SARS-CoV-2 concentration in wastewater consistently predicts trends in COVID-19 case counts by at least two days across multiple WWTP scales.

Wastewater surveillance of SARS-CoV-2 has proven instrumental in mitigating the spread of COVID-19 by providing an economical and equitable approach to disease surveillance. Here, we analyze the correlation of SARS-CoV-2 RNA in influents of seven wastewater plants (WWTPs) across the state of South Carolina with corresponding daily case counts to determine whether underlying characteristics of WWTPs and sewershed populations predict stronger correlations. The populations served by these WWTPs have varying social vulnerability and represent 24% of the South Carolina population. The study spanned 15 months from April 19, 2020, to July 1, 2021, which includes the administration of the first COVID-19 vaccines. SARS-CoV-2 RNA concentrations were measured by either reverse transcription quantitative PCR (RT-qPCR) or droplet digital PCR (RT-ddPCR). Although populations served and average flow rate varied across WWTPs, the strongest correlation was identified for six of the seven WWTPs when daily case counts were lagged two days after the measured SARS-CoV-2 RNA concentration in wastewater. The weakest correlation was found for WWTP 6, which had the lowest ratio of population served to average flow rate, indicating that the SARS-CoV-2 signal was too dilute for a robust correlation. Smoothing daily case counts by a 7-day moving average improved correlation strength between case counts and SARS-CoV-2 RNA concentration in wastewater while dampening the effect of lag-time optimization. Correlation strength between cases and SARS-CoV-2 RNA was compared for cases determined at the ZIP-code and sewershed levels. The strength of correlations using ZIP-code-level versus sewershed-level cases were not statistically different across WWTPs. Results indicate that wastewater surveillance, even without normalization to fecal indicators, is a strong predictor of clinical cases by at least two days, especially when SARS-CoV-2 RNA is measured using RT-ddPCR. Furthermore, the ratio of population served to flow rate may be a useful metric to assess whether a WWTP is suitable for a surveillance program.

Novel Cyclized Derivatives of Ferulic Acid as Potential Antiviral Agents through Activation of Photosynthesis.

To further develop new antiviral agents, several novel cyclized derivatives of ferulic acid were designed and synthesized. Their antiviral activities were evaluated against the cucumber mosaic virus (CMV), pepper mild mottle virus (PMMoV), and tomato spotted wilt virus (TSWV). The results showed that some ferulic acid derivatives exhibited desirable antiviral activities. Particularly, compound 5e exhibited excellent protective activities against CMV, PMMoV, and TSWV, with EC50 values of 167.2, 102.5, and 145.8 µg mL-1, respectively, which were superior to those obtained for trans-ferulic acid (581.7, 611.2, and 615.4 µg mL-1), dufulin (312.6, 302.5, and 298.2 µg mL-1), and ningnanmycin (264.3, 282.5, and 276.5 µg mL-1). Thereafter, the protective mechanisms of 5e were evaluated through photosynthesis evaluation, transcriptome profiling, and proteomic analysis. The results indicated that 5e significantly activated the expression levels of photosynthesis-related regulatory genes and proteins in tobacco plants and promoted the accumulation of defense molecules to resist viral infection. Thus, the findings of this study indicated that novel cyclized ferulic acid derivatives are potential antiviral agents that act via regulating photosynthesis in the host.

Testing specificity and sensitivity of wastewater-based epidemiology for detecting SARS-CoV-2 in four communities on Vancouver Island, Canada.

We report wastewater surveillance of the spread of SARS-CoV-2 based upon 24-h composite influent samples taken weekly from four wastewater treatment plants (WWTP) on Vancouver Island, BC, Canada between January 3, 2021 and July 10, 2021. Samples were analyzed by reverse transcription quantitative polymerase chain reaction targeting the N1 and N2 gene fragments of SARS-CoV-2 and a region of the replication associate protein of the pepper mottle mosaic virus (PMMoV) serving as endemic control. Only a small proportion of samples had quantifiable levels of N1 or N2. Overall case rates are weakly correlated with the concentration (gene copies/L) and with the flux of viral material influent to the WWTP (gene copies/day); the latter accounts for influent flow variations. Poisson multimodal rank correlation accounts for differences between the four WWTP and shows a significant correlation with a significant positive intercept. Receiver operator characteristics (ROC) analysis confirms a cut-off of cases based on amplified/not-amplified experimental data. At the optimal cut point of 19 (N1) or 17 (N2) cases/week/100,000 the sensitivity and specificity is about 75% for N1 and 67% for N2.

The Efficient and Practical virus Identification System with ENhanced Sensitivity for Solids (EPISENS-S): A rapid and cost-effective SARS-CoV-2 RNA detection method for routine wastewater surveillance.

Wastewater-based epidemiology has attracted attention as a COVID-19 surveillance tool. Here, we developed a practical method for detecting SARS-CoV-2 RNA in wastewater (the EPISENS-S method), which employs direct RNA extraction from wastewater pellets formed via low-speed centrifugation. The subsequent multiplex one-step RT-preamplification reaction with forward and reverse primers for SARS-CoV-2 and a reverse primer only for pepper mild mottle virus (PMMoV) allowed for qPCR quantification of the targets with different abundances in wastewater from the RT-preamplification product. The detection sensitivity of the method was evaluated using wastewater samples seeded with heat-inactivated SARS-CoV-2 in concentrations of 2.11 × 103 to 2.11 × 106 copies/L. The results demonstrated that the sensitivity of the EPISENS-S method was two orders of magnitude higher than that of the conventional method (PEG precipitation, followed by regular RT-qPCR; PEG-QVR-qPCR). A total of 37 untreated wastewater samples collected from two wastewater treatment plants in Sapporo, Japan when 1.6 to 18 new daily reported cases per 100,000 people were reported in the city (March 4 to July 8, 2021), were examined using the EPISENS-S method to confirm its applicability to municipal wastewater. SARS-CoV-2 RNA was quantified in 92 % (34/37) of the samples via the EPISENS-S method, whereas none of the samples (0/37) was quantifiable via the PEG-QVR-qPCR method. The PMMoV concentrations measured by the EPISENS-S method ranged from 2.60 × 106 to 1.90 × 108 copies/L, and the SARS-CoV-2 RNA concentrations normalized by PMMoV ranged from 5.71 × 10-6 to 9.51 × 10-4 . The long-term trend of normalized SARS-CoV-2 RNA concentration in wastewater was consistent with that of confirmed COVID-19 cases in the city. These results demonstrate that the EPISENS-S method is highly sensitive and suitable for routine COVID-19 wastewater surveillance.