Achieving precise dual detection: One-tube reverse transcription-recombinase aided amplification (RT-RAA) combined with lateral flow strip (LFS) assay for RNA and DNA target genes from pepper mild mottle virus and Colletotrichum species in crude plant samples.

Ensuring the detection sensitivity of both RNA-derived and DNA-derived target genes in a single reaction has posed a significant challenge for on-site detection of plant pathogens. This challenge was addressed by developing a one-tube dual RT-RAA assay combined with LFS for the rapid on-site detection of pepper mild mottle virus (PMMoV) and four Colletotrichum species causing anthracnose in Solanaceous crops. By testing four different combinations of primer groups, two combinations were precisely adjusted within the dual RT-RAA system to balance amplification efficiency and maintain consistent levels of amplification in crude plant samples. Utilizing commercially accessible small-scale equipment and following a streamlined optimization strategy, the assay achieved a limit of detection of 0.32 copies/µL of target genes in the reaction. Importantly, it demonstrated no cross-reactivity with other plant pathogens, thereby affirming the high sensitivity and specificity of the developed dual RT-RAA-LFS detection assay. Moreover, the entire process took only 25 min from sample collection to the visible presentation of results. The assay was validated with 60 field samples and 10 seed samples, producing results consistent with reverse transcription quantitative polymerase chain reaction (RT-qPCR). Notably, it successfully detected PMMoV in systemic leaves without visible symptoms three days post-inoculation, underscoring its effectiveness in early disease detection. This streamlined strategy offers a valuable approach for rapid, low-cost, and highly sensitive on-site simultaneous detection of RNA genome-contained PMMoV and DNA genome-contained Colletotrichum species.

A reverse transcription loop-mediated isothermal amplification assay for quick detection of tomato mosaic virus.

Tomato mosaic virus (ToMV), an economically important virus that affects a wide range of crops, is highly contagious, and its transmission is mediated by mechanical means, and through contaminated seeds or planting materials, making its management challenging. To contain its wide distribution, early and accurate detection of infection is required. A survey was conducted between January and May, 2023 in major tomato growing counties in Kenya, namely, Baringo, Kajiado, Kirinyaga and Laikipia, to establish ToMV disease incidence and to collect samples for optimization of the reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) assay. A RT-LAMP assay, utilizing primers targeting the coat protein, was developed and evaluated for its performance. The method was able to detect ToMV in tomato samples within 4:45 minutes, had a 1,000-fold higher sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR) method and was specific to ToMV. Furthermore, the practical applicability of the assay was assessed using tomato samples and other solanaecous plants. The assay was able to detect the virus in 14 tomato leaf samples collected from the field, compared to 11 samples detected by RT-PCR, further supporting the greater sensitivity of the assay. To make the assay more amenable for on-site ToMV detection, a quick-extraction method based on alkaline polyethylene glycol buffer was evaluated, which permitted the direct detection of the target virus from crude leaf extracts. Due to its high sensitivity, specificity and rapidity, the RT-LAMP method could be valuable for field surveys and quarantine inspections towards a robust management of ToMV infections.

Getting Hold of the Tobamovirus Particle-Why and How? Purification Routes over Time and a New Customizable Approach.

This article develops a multi-perspective view on motivations and methods for tobamovirus purification through the ages and presents a novel, efficient, easy-to-use approach that can be well-adapted to different species of native and functionalized virions. We survey the various driving forces prompting researchers to enrich tobamoviruses, from the search for the causative agents of mosaic diseases in plants to their increasing recognition as versatile nanocarriers in biomedical and engineering applications. The best practices and rarely applied options for the serial processing steps required for successful isolation of tobamoviruses are then reviewed. Adaptations for distinct particle species, pitfalls, and 'forgotten' or underrepresented technologies are considered as well. The article is topped off with our own development of a method for virion preparation, rooted in historical protocols. It combines selective re-solubilization of polyethylene glycol (PEG) virion raw precipitates with density step gradient centrifugation in biocompatible iodixanol formulations, yielding ready-to-use particle suspensions. This newly established protocol and some considerations for perhaps worthwhile further developments could serve as putative stepping stones towards preparation procedures appropriate for routine practical uses of these multivalent soft-matter nanorods.

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.

A soft-sensor approach for predicting an indicator virus removal efficiency of a pilot-scale anaerobic membrane bioreactor (AnMBR).

The anaerobic membrane bioreactor (AnMBR) is a promising technology for not only water reclamation but also virus removal; however, the virus removal efficiency of AnMBR has not been fully investigated. Additionally, the removal efficiency estimation requires datasets of virus concentration in influent and effluent, but its monitoring is not easy to perform for practical operation because the virus quantification process is generally time-consuming and requires specialized equipment and trained personnel. Therefore, in this study, we aimed to identify the key, monitorable variables in AnMBR and establish the data-driven models using the selected variables to predict virus removal efficiency. We monitored operational and environmental conditions of AnMBR in Sendai, Japan and measured virus concentration once a week for six months. Spearman's rank correlation analysis revealed that the pH values of influent and mixed liquor suspended solids (MLSS) were strongly correlated with the log reduction value of pepper mild mottle virus, indicating that electrostatic interactions played a dominant role in AnMBR virus removal. Among the candidate models, the random forest model using selected variables including influent and MLSS pH outperformed the others. This study has demonstrated the potential of AnMBR as a viable option for municipal wastewater reclamation with high microbial safety.

Pepper mild mottle virus intended for use as a process indicator for drinking water treatment: Present forms and quantitative relations to norovirus and rotavirus in surface water.

Pepper mild mottle virus (PMMoV) has been proposed as a potential indicator of human enteric viruses in environmental water and for viral removal during drinking water treatment. To investigate the occurrence and present forms of PMMoV and quantitative relations to norovirus GII and rotavirus A (RVA) in surface waters, 147 source water samples were collected from 21 drinking water treatment plants (DWTPs) in Japan between January 2018 and January 2021, and the concentrations of viruses in suspended and dissolved fractions were measured using real-time RT-PCR. PMMoV was detected in 81-100 % of samples in each sample month and observed concentrations ranged from 3.0 to 7.0 log10 copies/L. The concentrations of PMMoV were higher in dissolved fraction compared to suspended fractions, while different partitioning was observed for NoV GII depending on seasons. The concentrations of PMMoV were basically higher than those of norovirus GII (1.9-5.3 log10 copies/L) and RVA (1.9-6.6 log10 copies/L), while in 18 samples, RVA presented higher concentrations than PMMoV. Partial regions of VP7, VP4, and VP6 of the RVA in the 18 samples were amplified using nested PCR, and the genotypes were determined using an amplicon-based next-generation sequencing approach. We found that these source water samples included not only human RVA but also various animal RVA and high genetic diversity due to the existence of animal RVA was associated with a higher RVA concentration than PMMoV. Our findings suggest that PMMoV can be used as an indicator of norovirus GII and human RVA in drinking water sources and that the indicator performance should be evaluated by comparing to zoonotic viruses as well as human viruses.

Molecular characterization and host reaction to tomato mottle mosaic virus isolated from sweet pepper seeds in Japan.

Many countries have identified tomato mottle mosaic virus (ToMMV) as a serious threat to tomato production. Here, we constructed and characterized infectious clones of ToMMV isolated from Japanese sweet pepper seeds. The genome of the Japanese isolate is 6399 nucleotides in length and exhibits the highest identity with previously characterized isolates. For example, it is 99.7% identical to that of the Mauritius isolate, which occurs worldwide. Phylogenetic analysis based on complete genome sequences revealed that the Japanese isolates clustered in the same clade as those from other countries. When homozygous tomato cultivars with tobamovirus resistance genes were inoculated with an infectious cDNA clone of ToMMV, the virus systemically infected tomato plants with symptoms typical of Tm-1-carrying tomato cultivars. In contrast, tomato cultivars carrying Tm-2 or Tm-22 showed symptoms only on the inoculated leaves. Furthermore, when commercial cultivars of Tm-22 heterozygous tomato were inoculated with ToMMV, systemic infections were observed in all cultivars, with infection frequencies ranging from 25 to 100%. Inoculation of heterozygous sweet pepper cultivars with tobamovirus resistance genes (L1, L3, and L4) with ToMMV resulted in an infection frequency of about 70%, but most of the infected L1, L3, and L4 cultivars were symptomless, and 10-20% showed symptoms of necrosis and yellowing. Tomato mosaic virus strain L11A, an attenuated virus, did not provide cross-protection against ToMMV and led to systemic infection with typical symptoms. These results suggest that ToMMV might cause extensive damage to existing tomato and sweet pepper cultivars commonly grown in Japan.

Protocol to longitudinally quantify SARS-CoV-2 RNA in wastewater using RT-qPCR and pepper mild mottle virus normalization.

Wastewater surveillance allows severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection levels to be tracked in a community. Here, we present a protocol to longitudinally quantify SARS-CoV-2 RNA in wastewater using quantitative reverse-transcription PCR (RT-qPCR) and pepper mild mottle virus (PMMoV) normalization. We describe steps for the pasteurization of wastewater samples, solids separation, supernatant filtration, viral precipitation and concentration, and RNA extraction. We then detail procedures for RT-qPCR, viral concentration extrapolation, PMMoV normalization, and longitudinal analysis. This protocol has the potential to be used for surveillance of other microorganisms. For complete details on the use and execution of this protocol, please refer to Sanchez Jimenez et al.1.

New Resources for the Specific and Sensitive Detection of the Emerging Tomato Brown Rugose Fruit Virus.

Plant viruses can evolve towards new pathogenic entities that may eventually cause outbreaks and become epidemics or even pandemics. Seven years ago, tomato brown rugose fruit virus (ToBRFV) emerged, overcoming the genetic resistance that had been employed for more than sixty years against tobamoviruses in tomato. Since then, ToBRFV has spread worldwide, producing significant losses in tomato crops. While new resistances are deployed, the only means of control is the implementation of effective prevention and eradication strategies. For this purpose, in this work, we have designed, assessed, and compared an array of tests for the specific and sensitive detection of the ToBRFV in leaf samples. First, two monoclonal antibodies were generated against a singular peptide of the ToBRFV coat protein; antibodies were utilized to devise a double-antibody-sandwich enzyme-linked immunosorbent assay (DAS-ELISA) test that sensitively detects this virus and has no cross-reactivity with other related tobamoviruses. Second, a real-time quantitative PCR (RT-qPCR) test targeting the RNA-dependent replicase open reading frame (ORF) was designed, and its performance and specificity validated in comparison with the CaTa28 and CSP1325 tests recommended by plant protection authorities in Europe. Third, in line with the tendency to use field-deployable diagnostic techniques, we developed and tested two sets of loop-mediated isothermal amplification (LAMP) primers to double-check the detection of the movement protein ORF of ToBRFV, and one set that works as an internal control. Finally, we compared all of these methods by employing a collection of samples with different ToBRFV loads to evaluate the overall performance of each test.

Synthesis and Characterization of a Full-Length Infectious cDNA Clone of Tomato Mottle Mosaic Virus.

Tomato mottle mosaic virus (ToMMV) is a noteworthy virus which belongs to the Virgaviridae family and causes serious economic losses in tomato. Here, we isolated and cloned the full-length genome of a ToMMV Chinese isolate (ToMMV-LN) from a naturally infected tomato (Solanum lycopersicum L.). Sequence analysis showed that ToMMV-LN contains 6399 nucleotides (nts) and is most closely related to a ToMMV Mexican isolate with a sequence identity of 99.48%. Next, an infectious cDNA clone of ToMMV was constructed by a homologous recombination approach. Both the model host N. benthamiana and the natural hosts tomato and pepper developed severe symptoms upon agroinfiltration with pToMMV, which had a strong infectivity. Electron micrographs indicated that a large number of rigid rod-shaped ToMMV virions were observed from the agroinfiltrated N. benthamiana leaves. Finally, our results also confirmed that tomato plants inoculated with pToMMV led to a high infection rate of 100% in 4-5 weeks post-infiltration (wpi), while pepper plants inoculated with pToMMV led to an infection rate of 40-47% in 4-5 wpi. This is the first report of the development of a full-length infectious cDNA clone of ToMMV. We believe that this infectious clone will enable further studies of ToMMV genes function, pathogenicity and virus-host interaction.

Enteric Viruses and Pepper Mild Mottle Virus Show Significant Correlation in Select Mid-Atlantic Agricultural Waters.

Enteric viruses (EVs) are the largest contributors to foodborne illnesses and outbreaks globally. Their ability to persist in the environment, coupled with the challenges experienced in environmental monitoring, creates a critical aperture through which agricultural crops may become contaminated. This study involved a 17-month investigation of select human EVs and viral indicators in nontraditional irrigation water sources (surface and reclaimed waters) in the Mid-Atlantic region of the United States. Real-time quantitative PCR was used for detection of Aichi virus, hepatitis A virus, and norovirus genotypes I and II (GI and GII, respectively). Pepper mild mottle virus (PMMoV), a common viral indicator of human fecal contamination, was also evaluated, along with atmospheric (air and water temperature, cloud cover, and precipitation 24 h, 7 days, and 14 days prior to sample collection) and physicochemical (dissolved oxygen, pH, salinity, and turbidity) data, to determine whether there were any associations between EVs and measured parameters. EVs were detected more frequently in reclaimed waters (32% [n = 22]) than in surface waters (4% [n = 49]), similar to PMMoV detection frequency in surface (33% [n = 42]) and reclaimed (67% [n = 21]) waters. Our data show a significant correlation between EV and PMMoV (R2 = 0.628, P < 0.05) detection levels in reclaimed water samples but not in surface water samples (R2 = 0.476, P = 0.78). Water salinity significantly affected the detection of both EVs and PMMoV (P < 0.05), as demonstrated by logistic regression analyses. These results provide relevant insights into the extent and degree of association between human (pathogenic) EVs and water quality data in Mid-Atlantic surface and reclaimed waters, as potential sources for agricultural irrigation. IMPORTANCE Microbiological analysis of agricultural waters is fundamental to ensure microbial food safety. The highly variable nature of nontraditional sources of irrigation water makes them particularly difficult to test for the presence of viruses. Multiple characteristics influence viral persistence in a water source, as well as affecting the recovery and detection methods that are employed. Testing for a suite of viruses in water samples is often too costly and labor-intensive, making identification of suitable indicators for viral pathogen contamination necessary. The results from this study address two critical data gaps, namely, EV prevalence in surface and reclaimed waters of the Mid-Atlantic region of the United States and subsequent evaluation of physicochemical and atmospheric parameters used to inform the potential for the use of indicators of viral contamination.

Pepper Mild Mottle Virus as Indicator of Pollution: Assessment of Prevalence and Concentration in Different Water Environments in Italy.

Pepper mild mottle virus (PMMoV), a plant pathogenic virus belonging to the family Virgoviridae, has been proposed as a potential viral indicator for human faecal pollution in aquatic environments. The present study investigated the occurrence, amount and diversity of PMMoV in water environments in Italy. A total of 254 water samples, collected between 2017 and 2019 from different types of water, were analysed. In detail, 92 raw sewage, 32 treated sewage, 16 river samples, 9 estuarine waters, 20 bathing waters, 67 groundwater samples and 18 drinking waters were tested. PMMoV was detected in 79% and 75% of untreated and treated sewage samples, respectively, 75% of river samples, 67% and 25% of estuarine and bathing waters and 13% of groundwater samples. No positive was detected in drinking water. The geometric mean of viral concentrations (genome copies/L) was ranked as follows: raw sewage (2.2 × 106) > treated sewage (2.9 × 105) > river waters (6.1 × 102) > estuarine waters (4.8 × 102) > bathing waters (8.5 × 101) > groundwater (5.9 × 101). A statistically significant variation of viral loads could be observed between raw and treated sewage and between these and all the other water matrices. PMMoV occurrence and viral loads did not display seasonal variation in raw sewage nor correlation with faecal indicator bacteria in marine waters and groundwater. This study represents the first report on the occurrence and quantification PMMoV in different water environments in Italy. Further studies are required to evaluate the suitability of PMMoV as a viral indicator for human faecal pollution and for viral pathogens in waters.

Real-time tracking of Tomato brown rugose fruit virus (ToBRFV) outbreaks in the Netherlands using Nextstrain.

Tomato brown rugose fruit virus (ToBRFV) is a Tobamovirus that was first observed in 2014 and 2015 on tomato plants in Israel and Jordan respectively. Since the first description, the virus has been reported from all continents except Oceania and Antarctica, and has been found infecting both tomato and pepper crops. In October 2019, the Dutch National Plant Protection Organization received a ToBRFV infected tomato sample as part of a generic survey targeting tomato pests. Presence of the virus was verified using Illumina sequencing. A follow-up survey was initiated to determine the extent of ToBRFV presence in the Dutch tomato horticulture and identify possible linkages between ToBRFV genotypes, companies and epidemiological traits. Nextstrain was used to visualize these potential connections. By November 2019, 68 companies had been visited of which 17 companies were found to be infected. The 50 ToBRFV genomes from these outbreak locations group in three main clusters, which are hypothesized to represent three original sources. No correlation was found between genotypes, companies and epidemiological traits, and the source(s) of the Dutch ToBRFV outbreak remain unknown. This paper describes a Nextstrain build containing ToBRFV genomes up to and including November 2019. Sharing data with this interactive online tool will enable the plant virology field to better understand and communicate the diversity and spread of this new virus. Organizations are invited to share data or materials for inclusion in the Nextstrain build, which can be accessed at https://nextstrain.nrcnvwa.nl/ToBRFV/20191231.

Tobamoviruses can be frequently present in the oropharynx and gut of infants during their first year of life.

Plant viruses have been reported to be common in the gut of human adults, presumably as result of food ingestion. In this work, we report that plant viruses can also be found frequently in the gut and oropharynx of children during their first year of life, even when they are exclusively breast-fed. Fecal and oropharynx samples were collected monthly, from birth to 1 year of age, from three apparently healthy children in a semi-rural community and analyzed by next generation sequencing. In 100% of the fecal samples and 65% of the oropharynx samples at least one plant virus was identified. Tobamoviruses in the Virgaviridae family were by far the most frequently detected, with tropical soda apple mosaic virus, pepper mild mottle virus, and opuntia tobamovirus 2 being the most common species. Seventeen complete virus genomes could be assembled, and phylogenetic analyses showed a large diversity of virus strains circulating in the population. These results suggest that children are continuously exposed to an extensive and highly diverse collection of tobamoviruses. Whether the common presence of plant viruses at an early age influences the infant's immune system, either directly or through interaction with other members of the microbiota, remains to be investigated.

Genome characterization of brugmansia latent virus, a novel tobamovirus.

A novel tobamovirus, brugmansia latent virus (BrLV), was discovered during a study of brugmansia (Brugmansia spp.) in the living collections held at the Royal Botanic Gardens, Kew. Here, we report the complete genome sequence of BrLV, which is 6,397 nucleotides long and contains the four open reading frames (RNA-dependent RNA polymerase, methyltransferase/helicase, movement, and coat proteins) typical of tobamoviruses. The complete genome sequence of BrLV shares 69.7% nucleotide sequence identity with brugmansia mild mottle virus (BrMMV) and 66.7 to 68.7% identity with other tobamoviruses naturally infecting members of the Solanaceae plant family. Phylogenetic analysis of the complete genome nucleotide sequence and the deduced amino acid sequences of the four tobamovirus proteins place BrLV in a subcluster with BrMMV within the Solanaceae-infecting tobamovirus subgroup as a new species.

Reduction of Human Enteric and Indicator Viruses at a Wastewater Treatment Plant in Southern Louisiana, USA.

This study assessed wastewater quality through the quantification of four human enteric viruses and the applicability of pepper mild mottle virus (PMMoV) and tobacco mosaic virus (TMV) as indicators of viral reduction during wastewater treatment. Thirty-three samples were collected from three steps of a wastewater treatment plant in Southern Louisiana, USA for a year between March 2017 and February 2018. Noroviruses of genogroup I were the most prevalent human enteric viruses in influent samples. The concentrations of PMMoV in influent samples (5.9 ± 0.7 log10 copies/L) and biologically treated effluent samples (5.9 ± 0.5 log10 copies/L) were significantly higher than those of TMV (P < 0.05), and the reduction ratio of PMMoV (1.0 ± 0.8 log10) was found comparable to those of TMV and Aichi virus 1. Because of the high prevalence, high correlations with human enteric viruses, and lower reduction ratios, PMMoV was deemed an appropriate indicator of human enteric viral reduction during wastewater treatment process.

Reduction of Pathogenic and Indicator Viruses at a Drinking Water Treatment Plant in Southern Louisiana, USA.

Monthly sampling was conducted at a drinking water treatment plant (DWTP) in Southern Louisiana, USA from March 2017 to February 2018 to determine the prevalence and reduction efficiency of pathogenic and indicator viruses. Water samples were collected from the DWTP at three different treatment stages (raw, secondary-treated, and chlorinated drinking water) and subjected to quantification of seven pathogenic viruses and three indicator viruses [pepper mild mottle virus (PMMoV), tobacco mosaic virus (TMV), and crAssphage] based on quantitative polymerase chain reaction. Among the seven pathogenic viruses tested, only Aichi virus 1 (AiV-1) (7/12, 58%) and noroviruses of genogroup II (NoVs-GII) (2/12, 17%) were detected in the raw water samples. CrAssphage had the highest positive ratio at 78% (28/36), and its concentrations were significantly higher than those of the other indicator viruses for all three water types (P < 0.05). The reduction ratios of AiV-1 (0.7 ± 0.5 log10; n = 7) during the whole treatment process were the lowest among the tested viruses, followed by crAssphage (1.1 ± 1.9 log10; n = 9), TMV (1.3 ± 0.9 log10; n = 8), PMMoV (1.7 ± 0.8 log10; n = 12), and NoVs-GII (3.1 ± 0.1 log10; n = 2). Considering the high abundance and relatively low reduction, crAssphage was judged to be an appropriate process indicator during drinking water treatment. To the best of our knowledge, this is the first study to assess the reduction of crAssphage and TMV during drinking water treatment.

Applicability of crAssphage, pepper mild mottle virus, and tobacco mosaic virus as indicators of reduction of enteric viruses during wastewater treatment.

This study was conducted to evaluate the applicability of crAssphage, pepper mild mottle virus (PMMoV), and tobacco mosaic virus (TMV) as indicators of the reduction of human enteric viruses during wastewater treatment. Thirty-nine samples were collected from three steps at a wastewater treatment plant (raw sewage, secondary-treated sewage, and final effluent) monthly for a 13-month period. In addition to the three indicator viruses, eight human enteric viruses [human adenoviruses, JC and BK polyomaviruses, Aichi virus 1 (AiV-1), enteroviruses, and noroviruses of genogroups I, II, and IV] were tested by quantitative PCR. Indicator viruses were consistently detected in the tested samples, except for a few final effluents for crAssphage and TMV. The mean concentrations of crAssphage were significantly higher than those of most tested viruses. The concentrations of crAssphage in raw sewage were positively correlated with the concentrations of all tested human enteric viruses (p <0.05), suggesting the applicability of crAssphage as a suitable indicator to estimate the concentrations of human enteric viruses in raw sewage. The reduction ratios of AiV-1 (1.8 ± 0.7 log10) were the lowest among the tested viruses, followed by TMV (2.0 ± 0.3 log10) and PMMoV (2.0 ± 0.4 log10). Our findings suggested that the use of not only AiV-1 and PMMoV but also TMV as indicators of reductions in viral levels can be applicable during wastewater treatment.

Identification and genomic characterization of a novel tobamovirus from prickly pear cactus.

In this work, we describe the complete sequence and genome organization of a novel tobamovirus detected in a prickly pear plant (Opuntia sp.) by high-throughput sequencing, tentatively named "opuntia virus 2". The full genome of opuntia virus 2 is 6,453 nucleotides in length and contains four open reading frames (ORFs) coding for the two subunits of the RNA polymerase, the movement protein, and the coat protein, respectively. Phylogenetic analysis using the complete nucleotide sequence revealed that the virus belongs to the genus Tobamovirus (family Virgaviridae), showing the highest nucleotide sequence identity (49.8%) with cactus mild mottle virus (CMMoV), being indicating that it belongs in the Cactaceae subgroup of tobamoviruses.

Molecular characterization and In Vitro synthesis of infectious RNA of a Turnip vein-clearing virus isolated from Alliaria petiolata in Hungary.

A tobamovirus was isolated from leaves of Alliaria petiolata plants, showing vein-clearing, interveinal chlorosis, and moderate deformation. Host range experiments revealed a high similarity of isolate ApH both to ribgrass mosaic viruses and turnip vein-clearing viruses. The complete nucleotide sequence of the viral genome was determined. The genomic RNA is composed of 6312 nucleotides and contains four open reading frames (ORF). ORF1 is 3324 nt-long and encodes a polypeptide of about 125.3 kDa. The ORF1 encoded putative replication protein contains an Alphavirus-like methyltransferase domain. ORF2 is 4806 nt-long and encodes a polypeptide of about 182 kDa. The ORF2 encoded putative replication protein contains an RNA-dependent RNA polymerase, catalytic domain. ORF3 encodes the putative cell-to-cell movement protein with a molecular weight of 30.1 kDa. ORF4 overlaps with ORF3 and encodes the coat protein with a size of 17.5 kDa. Sequence comparisons revealed that the ApH isolate has the highest similarity to turnip vein-clearing viruses and should be considered an isolate of Turnip vein-clearing virus (TVCV). This is the first report on the occurrence of TVCV in Hungary. In vitro transcripts prepared from the full-length cDNA clone of TVCV-ApH were highly infectious and induced typical symptoms characteristic to the original isolate of the virus. Since infectious clones of TVCV-ApH and crTMV (another isolate of TVCV) markedly differed in respect to recovery phenotype in Arabidopsis thaliana, it is feasible to carry out gene exchange or mutational studies to determine viral factors responsible for the symptom recovery phenotype.