Persistence of Fecal Source Tracking Markers Human Fecal-Associated Bacteroidales (HF183), Cross-Assembly Phage-like Marker (crAssphage), Pepper Mild Mottle Virus (PMMoV), and Tomato Brown Rugose Fruit Virus (ToBRFV) in Coastal Waters

This study aimed to compare the performance of polyethylene glycol (PEG) precipitation, and Nanotrap® Microbiome magnetic particle capture workflows for recovering novel fecal marker, pBI143 from 12 wastewater samples collected across six treatment plants in Maryland, USA. Quantitative PCR (qPCR) was used to quantify marker abundance. The Nanotrap workflow yielded significantly higher concentration of pBI143 compared to PEG precipitation workflow (p < 0.05). The Nanotrap workflow used in the study utilized both magnetic nanoparticles A and B, rather than magnetic nanoparticle A alone, highlighting the necessity of optimization based on the intended targets for enhanced recovery. The extracted total nucleic acids by the Nanotrap workflow, were further analyzed to quantify other fecal markers, crAssphage, tomato brown rugose fruit virus (ToBRFV), and pepper mild mottle virus (PMMoV). No significant differences in the concentrations of pBI143, crAssphage, and ToBRFV (p > 0.05) were observed, whereas the concentration of PMMoV was significantly lower than that of the three fecal markers (p < 0.05). Based on the concentration alone, pBI143, ToBRFV, and crAssphage were found to be a better alternative to PMMoV as an endogenous fecal marker.

Pepper (Capsicum annuum L.) is one of the major vegetable crops in Jordan. During the winter growing seasons of 2015 and 2016, virus symptoms including stunting of young plants, puckering, and yellow mottling of leaves in sweet pepper plants grown under plastic houses in Jordan Valley were observed. The most obvious symptoms were the misshapen fruits that affected the market value of the crop, which resembled recent descriptions of tomato brown rugose fruit virus (ToBRFV) on tomato fruits (Salem et al. 2016). Symptoms on mechanically inoculated indicator hosts including Chenopodium quinoa, Datura stramonium, D. metel, Nicotiana glutinosa, and N. tabacum, and serological testing using double-antibody sandwich ELISA (antiserum A128 of the PLAVIT collection at IPSP-CNR, Italy) suggested the presence of a tobamovirus. Total RNA was extracted from fruits and leaves of 39 symptomatic pepper plants, using an SV-Total RNA Extraction kit (Promega, U.S.A.). Samples were tested by reverse transcription polymerase chain reaction (RT-PCR) for the most common tobamoviruses infecting pepper, including tomato mosaic virus, tobacco mosaic virus, tobacco mild green mosaic virus (TMGMV), and pepper mild mottle virus (Takeuchi et al. 2005). In addition, generic primers for detection of tobamoviruses were also used (Dovas et al. 2004). In RT-PCR, amplicons of the expected product size (400 and 728 bp) using the generic tobamovirus and TMGMV-specific primers, respectively, in all symptomatic plant samples were obtained, but such amplicons were not obtained from healthy plant extracts or water negative controls. RT-PCR products of the RdRp (400 bp) and CP (728 bp) partial regions were purified and ligated into pGEM T-Easy Vector (Promega), and two clones for each PCR product were sequenced and deposited in NCBI GenBank (accession nos. MK816313 to 16). BLASTN analysis showed that these nucleotide sequences had 96 to 99% identity to TMGMV genome sequences (JX534224 and MH730962) in NCBI GenBank. Furthermore, total RNA was extracted with TRIzol reagent (Invitrogen, U.S.A.) according to the manufacturer’s specifications. After ribosomal RNA depletion, the cDNA library was constructed using a TruSeq RNA Sample Prep kit (Illumina, U.S.A.) and sequenced by Illumina HiSeq X-ten platform (Biomarker, China). Raw sequencing data were analyzed using CLC Genomics Workbench 9.5 (Qiagen, Denmark). After raw reads were processed, a total of 80,818,734 paired-end reads of 150 bp were obtained, generating 266,412 contigs (>200 nt) with de novo assembly by CLC Genomics Workbench 9.5. BLASTN analysis of the assembled contigs revealed the presence of two virus-derived contigs: TMGMV (6,414 nt, MK648158) and ToBRFV (6,388 nt, MK648157), which represented a nearly full-length genome. To confirm the ToBRFV identity, all samples were tested by RT-PCR using two pairs of primers: ToBRFV F1 (5′-GTATTTTTGTTTTACAACATATACCAAC-3′) and ToBRFV R1 (5′-AGTGCGAATGTGATTTAAAACTGTGAA-3′), and ToBRFV F7 (5′-GGAAGAAGTCCCGATGTCTGTAAGGCTT-3′) and ToBRFV R7 (5′-GATGCAGGTGCAGAGGACCATTGTAAAC-3′), designed on ToBRFV genome (KT383474; Salem et al. 2016). Twenty-two out of 39 tested samples originated specific amplicons (1,300 and 697 bp for RdRp and CP, respectively). RT-PCR products of two samples were purified and sent for direct sequencing, and results were deposited in NCBI GenBank (MK834288, MK834289, MK834294, and MK834295), revealing nucleotide identity of 99 to 100% to ToBRFV for both RdRp and CP (KT383474 and KX619418). To our knowledge, this is the first report of TMGMV and ToBRFV infecting pepper in Jordan. The occurrence of these tobamoviruses that are transmitted through seeds, in Jordan Valley, the main area for pepper production, may represent a potential threat to other susceptible vegetables and requires careful monitoring to avoid future outbreaks and significant yield losses.

ABSTRACTMicrobial source tracking (MST) identifies sources of fecal contamination in the environment using host-associated fecal markers. While there are numerous bacterial MST markers that can be used herein, there are few such viral markers. Here, we designed and tested novel viral MST markers based on tomato brown rugose fruit virus (ToBRFV) genomes. We assembled eight nearly complete genomes of ToBRFV from wastewater and stool samples from the San Francisco Bay Area in the United States. Next, we developed two novel probe-based reverse transcription-PCR (RT-PCR) assays based on conserved regions of the ToBRFV genome and tested the markers’ sensitivities and specificities using human and non-human animal stool as well as wastewater. The ToBRFV markers are sensitive and specific; in human stool and wastewater, they are more prevalent and abundant than a commonly used viral marker, the pepper mild mottle virus (PMMoV) coat protein (CP) gene. We used the assays to detect fecal contamination in urban stormwater samples and found that the ToBRFV markers matched cross-assembly phage (crAssphage), an established viral MST marker, in prevalence across samples. Taken together, these results indicate that ToBRFV is a promising viral human-associated MST marker.IMPORTANCE Human exposure to fecal contamination in the environment can cause transmission of infectious diseases. Microbial source tracking (MST) can identify sources of fecal contamination so that contamination can be remediated and human exposures can be reduced. MST requires the use of host-associated MST markers. Here, we designed and tested novel MST markers from genomes of tomato brown rugose fruit virus (ToBRFV). The markers are sensitive and specific to human stool and highly abundant in human stool and wastewater samples.

Pepper mild mottle virus (PMMoV) is a plant virus belonging to the Virgaviridae family; it significantly reduces pepper yield production worldwide. The PMMoV is spread by contaminated seeds and there is no chemical treatment available. Therefore, resistant pepper varieties containing the L4 gene are recommended for the management of PMMoV. A considerable amount of evidence suggests that the L4 gene confers resistance to PMMoV in pepper. The aim of the project is to confirm the status of the L4 gene for resistance to PMMoV in pepper varieties, several inoculations were performed on pepper plants containing L3, L4 resistant genes and susceptible pepper plants without the resistance genes. The L4 resistant plants produced mottling, mosaic, leaf curl, stem necrosis symptoms in the tested pepper plants but there was no amplicon observed with specific primers of PMMoV in RT-PCR analyses. To determine if the L3 and L4 genes are controlling resistance to PMMoV, RT-PCR analyzes were conducted using PMMoV and Tomato brown rugose fruit virus (ToBRFV) where both viruses belong to the same family. The molecular studies revealed that the L4 gene controls resistance mechanisms to PMMoV but it is not able to govern Tobamovirus, ToBRFV. We showed that pepper plants harboring the L3 and L4 gene have the ability to precisely control the mechanism of resistance to PMMoV compared to pepper plants carrying only the L3 gene. A complete genome sequence of PMMoV was obtained and submitted to Genbank with MW523006 accessıon number in the NCBI system.

As wastewater-based epidemiology (WBE) broadens its focus to include prevalent diseases with significant global health impact, existing surveillance systems concentrate on sewer-based infrastructure, which excludes the 2.7 billion people using non-sewered systems. To address this gap, our study explores the potential of fecal sludge treatment plants (FSTPs) for WBE, emphasizing the stability of virus RNA targets within pooled fecal sludge. We screened fecal sludge from a centralized treatment facility in Dakar, Senegal for SARS-CoV-2, human norovirus (HuNoV), and microbial source trackers (MSTs) pepper mild mottle virus (PMMoV) and tomato brown rugose fruit virus (ToBRFV). Decay kinetics of genomic RNA markers from these viruses were examined at 4 °C, 15 °C, and 30 °C over 70 days. Results indicate high persistence of viral targets in fecal sludge (T90 value of 3.3 months for exogenous SARS-CoV-2 N1 and N2, 6.2 months for ToBRFV), with all targets detected throughout the 70-day experiment under various temperatures with limited decay (&lt;1 log10 reduction). This study addresses a crucial gap in understanding virus persistence in on-site sanitation systems by providing essential decay rate constants for effective target detection. Our results indicate that sampling at centralized facilities treating fecal sludge from on-site sanitation could facilitate localized pathogen surveillance in low-income settings.HighlightsInvestigation of the persistence of SARS-CoV-2, HuNoV, PMMoV, and ToBRFV genomic RNA in pooled fecal sludge derived from on-site sanitation systems.Novel microbial source tracker (MST), ToBRFV, exhibited comparable abundance to PMMoV, a well-established MST, in fecal sludge.No significant decay observed for HuNoV and PMMoV over 70 days at all temperature conditions (4, 15, and 30 °C).SARS-CoV-2 N1 and N2 showed T90values of 3.3 months at 30 °C.Fecal sludge treatment plants offer a centralized sampling location for wastewater-based epidemiology, providing a strategic approach for monitoring public health.Graphical Abstract

Microbial source tracking (MST) identifies sources of fecal contamination in the environment using fecal host-associated markers. While there are numerous bacterial MST markers, there are few viral markers. Here we design and test novel viral MST markers based on tomato brown rugose fruit virus (ToBRFV) genomes. We assembled eight nearly complete genomes of ToBRFV from wastewater and stool samples from the San Francisco Bay Area in the United States of America. Next, we developed two novel probe-based RT-PCR assays based on conserved regions of the ToBRFV genome, and tested the markers’ sensitivities and specificities using human and non-human animal stool as well as wastewater. TheToBRFV markers are sensitive and specific; in human stool and wastewater, they are more prevalent and abundant than a currently used marker, the pepper mild mottle virus (PMMoV) coat protein (CP) gene. We applied the assays to detect fecal contamination in urban stormwater samples and found that the ToBRFV markers matched cross-assembly phage (crAssphage), an established viral MST marker, in prevalence across samples. Taken together, ToBRFV is a promising viral human-associated MST marker.

This study was initiated to examine the tomato-infecting viruses belonging to the Tobamovirus and Potexvirus genera in Iraq. Field observations and surveys were carried out for three successive cropping seasons (2020/21 to 2022/23) in selected tomato production areas. The purpose was to identify the main viruses associated with tomato epidemics and assess the impact of different tomato cultivars on disease occurrence. A total of 700 tomato leaf samples were collected from seven governorates (Baghdad, Diyala, Babylon, Najaf, Kerbala, Nasiriya, and Basrah) and tested using pathogen-specific immunostrip kits. The survey showed a presence of Tomato brown rugose fruit virus (ToBRFV), Tobacco mosaic virus (TMV), Pepper mild mottle virus (PMMoV), Cymbidium mosaic virus (CymMV), Odontoglossum ringspot virus (ORSV), Cucumber green mottle mosaic virus (CGMMV), Pepino mosaic virus (PepMV) and Potato virus X (PVX) in tomato fields in Iraq. ToBRFV secured the highest relative incidence in tomato fields (7 governorates) followed by PepMV and CymMV and PMMoV (6 out of 7 governorates) and CGMMV, TMV (5 governorates), and PVX (3 governorates). The least was ORSV (only in Basrah). To our knowledge, this is the first comprehensive survey investigating Tobamovirus and Potexvirus on tomato fields in Iraq and the first report of ToBRFV, PMMoV, CymMV, ORSV, CGMMV and PepMV infecting tomato crops in Iraq.

Waterborne pathogenic viruses present a critical public health challenge, particularly in potable water reuse systems where stringent safety standards must be met. Achieving the necessary Log Reduction Values (LRVs) for viruses in water treatment remains difficult due to their low prevalence and high detection limits. This study investigated tomato brown rugose fruit virus (ToBRFV) as a novel viral indicator to assess advanced treatment efficacy of advanced treatment systems in achieving target Log10 Reduction Values (LRVs) for pathogenic viruses. Over 13 months, wastewater samples from the Reno-Sparks Metropolitan Region (Nevada, USA) were analyzed using metagenomics and RT-qPCR to compare ToBRFV, pepper mild mottle virus (PMMoV), and MS2 bacteriophage. Results demonstrated that ToBRFV was consistently detected at high concentrations across all samples, exhibiting minimal seasonal variability. In contrast, PMMoV showed moderate fluctuations, while MS2 was detected at lower levels. The robustness and stability of ToBRFV suggest it could serve as a reliable indicator for verifying LRVs in potable reuse systems, complementing existing methods. Additionally, its plant-based origin reduces human health risks during handling. These findings support ToBRFV’s potential to enhance treatment monitoring and public health safeguards. Further research should validate its applicability across diverse geographic and climatic conditions, as well as its correlation with enteric virus removal, to optimize water reuse frameworks.

Rapid and visual field diagnosis of tomato brown rugose fruit virus using reverse transcription recombinase aided amplification (RT RAA) combined with lateral flow strips (LFS)

Flow virometry (FVM) offers a promising approach for monitoring viruses and virus-like particles (VLPs) in environmental samples. This study compares levels of non-specific VLPs across a wastewater treatment plant (WWTP) with levels of somatic coliphage, (F+) specific coliphage, Pepper Mild Mottle Virus (PMMoV), CrAssphage (CrAss), and Tomato Brown Rugose Fruit Virus (ToBRFV). All targets were quantified in influent, secondary-treated effluent, and tertiary-treated effluent at the University of California, Davis Wastewater Treatment Plant (UCDWWTP) over 11 weeks. We established an FVM-gating boundary for VLPs using bacteriophages T4 and ϕ6 as well as four phages isolated from wastewater. We then utilize T4 alongside three submicron beads as quality controls in the FVM assay. Coliphage was measured by standard plaque assays, and genome copies of PMMoV, CrAss, and ToBRFV were measured by digital droplet (dd)PCR. FVM results for wastewater revealed distinct microbial profiles at each treatment stage. However, correlations between VLPs and targeted viruses were poor. Trends for virus inactivation and removal, observed for targeted viruses during wastewater treatment, were consistent with expectations. Conversely, VLP counts were elevated in the WWTP effluent relative to the influent. Additional sampling revealed a decrease in VLP counts during the filtration treatment step following secondary treatment but a substantial increase in VLPs following ultraviolet disinfection. Defining application boundaries remain crucial to ensuring meaningful data interpretation as flow cytometry and virometry take on greater significance in water quality monitoring.

Optimizing ozone treatment for pathogen removal and disinfection by-product control for potable reuse at pilot-scale

Comparative analysis of column experiments highlights the suitability and challenges of using common plant-based viruses as surrogates for human adenovirus in saturated porous media.

Flow diagram showing sampling in Thailand, nucleic acid extraction, PMMoV qPCR detection, and comparison with ToBRFV and crAssphage across wastewater and animal feces. Pepper mild mottle virus (PMMoV) is one of the most abundant ribonucleic acid viruses in human feces and is widely used in wastewater-based epidemiology (WBE) and microbial source tracking. Its performance has not been evaluated in Thailand. This study analyzed archived nucleic acid extracts from sewage (n = 16), wastewater treatment plant (WWTP) influents (n = 16), and pooled animal feces (n = 35). PMMoV was detected in all sewage and WWTP samples, with concentrations of 4.26–5.83 log10 copies/100 mL in sewage (median 5.49) and 4.51–5.67 log10 copies/100 mL in WWTP influents (median 5.25). Only one pooled pig sample was positive (3.02 log10 copies/100 mg). PMMoV concentrations were higher than Tomato brown rugose fruit virus ToBRFV in sewage (p &amp;lt; 0.001) and WWTP influents (p &amp;lt; 0.05), and greater than crAssphage in WWTP influents (p &amp;lt; 0.001). Correlation analysis showed positive associations with ToBRFV (Spearman′s rho = 0.57–0.62), crAssphage (rho = 0.29–0.59), and Escherichia coli (rho = 0.25–0.52). These findings demonstrate that PMMoV is abundant in Thai wastewater, rarely detected in pooled animal feces, and is suitable as a human-specific marker and WBE normalizer.

Enteric virus and pharmaceutical and personal care product attenuation in a bench-scale ozone-soil aquifer treatment process.

Evaluation of tomato brown rugose fruit virus as a microbial source tracking marker for human sewage in Thailand.