Walter Betancourt

Interests

Teaching

Water Pollution Microbiology, Virology, Environmental Virology, Parasitology, Environmental Parasitology

Research

Research interests are focused on areas of relevance to human and environmental health including: (i) pathogen exposure assessment as part of the microbial risk assessment approach for qualitative and/or quantitative evaluation of the likely intake of water- and foodborne microbial contaminants (viruses, bacteria and protozoan parasites); (ii) reduction of viruses, viral surrogates and protozoan parasites by advanced wastewater treatment processes (integrated membrane processes, advanced oxidation processes, ultraviolet disinfection) for potable and non-potable reuse applications; (iii) microbial and chemical source tracking (MST) to determine the source(s) of fecal contamination in waters for supply, agricultural irrigation, and recreation in combination with pathogen exposure assessment for evaluating health risks; (iv) metagenomic virome analysis for unbiased detection and characterization of all viruses in environmental matrices as a mean to understand virus diversity, frequency, fate, and distribution in the environment, (v) innovative approaches such as flow cytometry and inductively coupled plasma mass spectrometry (ICP-MS) for rapid and sensitive detection of microbial pathogens in low-level contamination environmental scenarios including agricultural waters and highly treated wastewater or underground sources of drinking water supplemented or not with recycled water.

Courses

Scholarly Contributions

Chapters

• Betancourt, W. Q. (2022). Waterborne Plant Viruses ofImportance in Agriculture. In Encyclopedia of Sustainability Science and Technology.
• Betancourt, W. Q. (2019). Cryptosporidium spp.. In Global Waterborne Pathogen.

Journals/Publications

• Betancourt, W. Q. (2022). Extending the life of water reuse reverse osmosis membranes using chlorination. Journal of Membrane Science, 642, 119897. doi:https://doi.org/10.1016/j.memsci.2021.119897
• Betancourt, W. Q. (2022). SARS-CoV-2 variant detection at a university dormitory using wastewater genomic tools. Science of The Total Environment, 805, 149930. doi:https://doi.org/10.1016/j.scitotenv.2021.149930
• Chowdhari, S., Rana, S., Rana, S., Morrison, C. M., Abney, S. E., Singh, R., Gurian, P. L., Kumar, A., Kumar, A., Betancourt, W. Q., & Vivekanandan, P. (2022). Quantitative Assessment of Microbial Pathogens and Indicators of Wastewater Treatment Performance for Safe and Sustainable Water Reuse in India. Microbiology spectrum, 10(6), e0172022.
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  Currently, there is no data on the molecular quantification of microbial indicators of recycled water quality in India. In this study, multiple microbial pathogens and indicators of water quality were evaluated at three wastewater treatment plants located in two Indian cities (New Delhi and Jaipur) to determine the treatment performance and suitability of recycled water for safe and sustainable reuse applications. Real-time polymerase chain reaction (PCR) was used for the rapid evaluation of six human pathogens and six microbial indicators of fecal contamination. Among the microbial indicators, pepper mild mottle virus (PMMoV), FRNA-GII bacteriophage, Bacteroides thetaiotamicron, and four human pathogens (Norovirus genogroups I & II, Giardia, and Campylobacter coli) were detected in all of the influent samples analyzed. This work suggests that the raw influents contain lower levels of noroviruses and adenoviruses and higher levels of Giardia compared to those reported from other geographic regions. Overall, the efficacy of the removal of microbial targets was over 93% in the final effluent samples, which is consistent with reports from across the world. PMMoV and Giardia were identified as the best microbial targets, from the microbial indicators spanning across bacteria, bacteriophages, DNA/RNA viruses, and protozoan parasites, by which to evaluate treatment performance and recycled water quality in Indian settings, as they were consistently present at high concentrations in untreated wastewater both within and across the sites. Also, they showed a strong correlation with other microbial agents in both the raw influent and in the final effluent. These findings provide valuable insights into the use of culture-independent molecular indicators that can be used to assess the microbial quality of recycled water in Indian settings. Wastewater treatment plants (WWTPs) have rapidly increased in India during the last decade. Nonetheless, there are only a few labs in India that can perform culture-based screening for microbial quality. In the last 2 years of the pandemic, India has witnessed a sharp increase in molecular biology labs. Therefore, it is evident that culture-independent real-time PCR will be increasingly used for the assessment of microbial indicators/pathogens in wastewater, especially in resource-limited settings. There is no data available on the molecular quantitation of microbial indicators from India. There is an urgent need to understand and evaluate the performance of widely used microbial indicators via molecular quantitation in Indian WWTPs. Our findings lay the groundwork for the molecular quantitation of microbial indicators in WWTPs in India. We have screened for 12 microbial targets (indicators and human pathogens) and have identified pepper mild mottle virus (PMMoV) and Giardia as the best molecular microbiological indicators in Indian settings.
• Malayil, L., Ramachandran, P., Chattopadhyay, S., Allard, S. M., Bui, A., Butron, J., Callahan, M. T., Craddock, H. A., Murray, R., East, C., Sharma, M., Kniel, K., Micallef, S., Hashem, F., Gerba, C. P., Ravishankar, S., Parveen, S., May, E., Handy, E., , Kulkarni, P., et al. (2022). Variations in Bacterial Communities and Antibiotic Resistance Genes Across Diverse Recycled and Surface Water Irrigation Sources in the Mid-Atlantic and Southwest United States: A CONSERVE Two-Year Field Study. Environmental science & technology, 56(21), 15019-15033.
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  Reduced availability of agricultural water has spurred increased interest in using recycled irrigation water for U.S. food crop production. However, there are significant knowledge gaps concerning the microbiological quality of these water sources. To address these gaps, we used 16S rRNA gene and metagenomic sequencing to characterize taxonomic and functional variations (e.g., antimicrobial resistance) in bacterial communities across diverse recycled and surface water irrigation sources. We collected 1 L water samples ( = 410) between 2016 and 2018 from the Mid-Atlantic (12 sites) and Southwest (10 sites) U.S. Samples were filtered, and DNA was extracted. The V3-V4 regions of the 16S rRNA gene were then PCR amplified and sequenced. Metagenomic sequencing was also performed to characterize antibiotic, metal, and biocide resistance genes. Bacterial alpha and beta diversities were significantly different ( < 0.001) across water types and seasons. Pathogenic bacteria, such as , , and were observed across sample types. The most common antibiotic resistance genes identified coded against macrolides/lincosamides/streptogramins, aminoglycosides, rifampin and elfamycins, and their read counts fluctuated across seasons. We also observed multi-metal and multi-biocide resistance across all water types. To our knowledge, this is the most comprehensive longitudinal study to date of U.S. recycled water and surface water used for irrigation. Our findings improve understanding of the potential differences in the risk of exposure to bacterial pathogens and antibiotic resistance genes originating from diverse irrigation water sources across seasons and U.S. regions.
• Prasek, S. M., Pepper, I. L., Innes, G. K., Slinski, S., Ruedas, M., Sanchez, A., Brierley, P., Betancourt, W. Q., Stark, E. R., Foster, A. R., Betts-Childress, N. D., & Schmitz, B. W. (2022). Population level SARS-CoV-2 fecal shedding rates determined via wastewater-based epidemiology. The Science of the total environment, 838(Pt 4), 156535.
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  Wastewater-based epidemiology (WBE) has been utilized as an early warning tool to anticipate disease outbreaks, especially during the COVID-19 pandemic. However, COVID-19 disease models built from wastewater-collected data have been limited by the complexities involved in estimating SARS-CoV-2 fecal shedding rates. In this study, wastewater from six municipalities in Arizona and Florida with distinct demographics were monitored for SARS-CoV-2 RNA between September 2020 and December 2021. Virus concentrations with corresponding clinical case counts were utilized to estimate community-wide fecal shedding rates that encompassed all infected individuals. Analyses suggest that average SARS-CoV-2 RNA fecal shedding rates typically occurred within a consistent range (7.53-9.29 log gc/g-feces); and yet, were unique to each community and influenced by population demographics. Age, ethnicity, and socio-economic factors may have influenced shedding rates. Interestingly, populations with median age between 30 and 39 had the greatest fecal shedding rates. Additionally, rates remained relatively constant throughout the pandemic provided conditions related to vaccination and variants were unchanged. Rates significantly increased in some communities when the Delta variant became predominant. Findings in this study suggest that community-specific shedding rates may be appropriate in model development relating wastewater virus concentrations to clinical case counts.
• Anderson-Coughlin, B. L., Craighead, S., Kelly, A., Gartley, S., Vanore, A., Johnson, G., Jiang, C., Haymaker, J., White, C., Foust, D., Duncan, R., East, C., Handy, E. T., Bradshaw, R., Murray, R., Kulkarni, P., Callahan, M. T., Solaiman, S., Betancourt, W., , Gerba, C., et al. (2021). Enteric Viruses and Pepper Mild Mottle Virus Show Significant Correlation in Select Mid-Atlantic Agricultural Waters. Applied and environmental microbiology, 87(13), e0021121.
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  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% [ = 22]) than in surface waters (4% [ = 49]), similar to PMMoV detection frequency in surface (33% [ = 42]) and reclaimed (67% [ = 21]) waters. Our data show a significant correlation between EV and PMMoV ( = 0.628, 0.05) detection levels in reclaimed water samples but not in surface water samples ( = 0.476, 0.78). Water salinity significantly affected the detection of both EVs and PMMoV (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. 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.
• Betancourt, W. Q. (2021). COVID-19 containment on a college campus via wastewater-based epidemiology, targeted clinical testing and an intervention. Science of The Total Environment, 779, 146408. doi:https://doi.org/10.1016/j.scitotenv.2021.146408
• Betancourt, W. Q. (2021). Enteric Viruses and Pepper Mild Mottle Virus Show Significant Correlation in Select Mid-Atlantic Agricultural Waters. Appl Environ Microbiol, 87(13), e0021121. doi:10.1128/aem.00211-21
• Betancourt, W. Q. (2021). Enumerating asymptomatic COVID-19 cases and estimating SARS-CoV-2 fecal shedding rates via wastewater-based epidemiology. Science of The Total Environment, 801, 149794. doi:https://doi.org/10.1016/j.scitotenv.2021.149794
• Betancourt, W. Q. (2021). Environmental Surveillance of SARS-CoV-2 RNA in Wastewater and Groundwater in Quintana Roo, Mexico. Food and Environmental Virology, 13(4), 457-469. doi:10.1007/s12560-021-09492-y
• Betancourt, W. Q. (2021). Norovirus detection in water samples at the level of single virus copies per microliter using a smartphone-based fluorescence microscope. Nature Protocols, 16(3), 1452-1475. doi:10.1038/s41596-020-00460-7
• Betancourt, W. Q. (2020). Potential indicators of virus transport and removal during soil aquifer treatment of treated wastewater effluen. Water Research, 177, 115812. doi:https://doi.org/10.1016/j.watres.2020.115812
• Betancourt, W. Q. (2019). Novel Circular Rep-Encoding Single-Stranded DNA Viruses Detected in Treated Wastewater. Microbiology Resource Announcements, 8(18), e00318-19. doi:10.1128/mra.00318-19
• Betancourt, W. Q. (2019). Variable non-linear removal of viruses during transport through a saturated soil column. Journal of Contaminant Hydrology, 103479. doi:https://doi.org/10.1016/j.jconhyd.2019.04.002
• Betancourt, W. Q., Schijven, J., Regnery, J., Wing, A., Morrison, C. M., Drewes, J. E., & Gerba, C. P. (2019). Variable non-linear removal of viruses during transport through a saturated soil column. Journal of contaminant hydrology, 223, 103479.
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  Reduction of viral surrogates (bacteriophage MS2 and murine norovirus-1 [MNV-1]) and viruses naturally present in wastewater (enteroviruses, adenoviruses, Aichi viruses, reovirus, pepper mild mottle virus) was studied in a long-term experiment simulating soil-aquifer treatment of a non-disinfected secondary treated wastewater effluent blend using a 4.4 m deep saturated soil column (95% sand, 4% silt, 1% clay) with a hydraulic residence time of 15.4 days under predominantly anoxic redox conditions. Water samples were collected over a four-week period from the column inflow and outflow as well as from seven intermediate sampling ports at different depths. Removal of MS2 was 3.5 log over 4.4 m and removal of MNV-1 was 3 log over 0.3 m. Notably, MNV-1 was removed to below detection limit within 0.3 m of soil passage. In secondary treated wastewater effluent, MNV-1 RNA and MS2 RNA degraded at a first-order rate of 0.59 day and 0.12 day, respectively. In 15.4 days, the time to pass the soil column, the RNA-degradation of MS2 would amount to 0.8 log and in one day that of MNV-1 0.3 log implying that attachment of MNV-1 and MS2 to the sandy soil took place. Among the indigenous viruses, genome copies reductions were observed for Aichi virus (4.9 log) and for pepper mild mottle virus (4.4 log). This study demonstrated that under saturated flow and predominantly anoxic redox conditions MS2 removal was non-linear and could be described well by a power-law relation. Pepper mild mottle virus was removed less than all of the other viruses studied, which substantiates field studies at managed aquifer recharge sites, suggesting it may be a conservative model/tracer for enteric virus transport through soil.
• Gerba, C. P., & Betancourt, W. Q. (2019). Assessing the Occurrence of Waterborne Viruses in Reuse Systems: Analytical Limits and Needs. Pathogens (Basel, Switzerland), 8(3).
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  Detection of waterborne enteric viruses is an essential tool in assessing the risk of waterborne transmission. Cell culture is considered a gold standard for detection of these viruses. However, it is important to recognize the uncertainty and limitations of enteric virus detection in cell culture. Cell culture cannot support replication of all virus types and strains, and numerous factors control the efficacy of specific virus detection assays, including chemical additives, cell culture passage number, and sequential passage of a sample in cell culture. These factors can result in a 2- to 100-fold underestimation of virus infectivity. Molecular methods reduce the time for detection of viruses and are useful for detection of those that do not produce cytopathogenic effects. The usefulness of polymerase chain reaction (PCR) to access virus infectivity has been demonstrated for only a limited number of enteric viruses and is limited by an understanding of the mechanism of virus inactivation. All of these issues are important to consider when assessing waterborne infectious viruses and expected goals on virus reductions needed for recycled water. The use of safety factors to account for this may be useful to ensure that the risks in drinking water and recycled water for potable reuse are minimized.
• Panthi, S., Sapkota, A. R., Raspanti, G., Allard, S. M., Bui, A., Craddock, H. A., Murray, R., Zhu, L., East, C., Handy, E., Callahan, M. T., Haymaker, J., Kulkarni, P., Anderson, B., Craighead, S., Gartley, S., Vanore, A., Betancourt, W. Q., Duncan, R., , Foust, D., et al. (2019). Pharmaceuticals, herbicides, and disinfectants in agricultural water sources. Environmental research, 174, 1-8.
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  Agricultural water withdrawals account for the largest proportion of global freshwater use. Increasing municipal water demands and droughts are straining agricultural water supplies. Therefore, alternative solutions to agricultural water crises are urgently needed, including the use of nontraditional water sources such as advanced treated wastewater or reclaimed water, brackish water, return flows, and effluent from produce processing facilities. However, it is critical to ensure that such usage does not compromise soil, crop, and public health. Here, we characterized five different nontraditional water types (n = 357 samples) for the presence of pharmaceuticals, herbicides, and disinfectants using ultra-high-pressure liquid chromatography tandem mass spectrometry based method (UPLC-MS/MS). We then evaluated whether the levels of these contaminants were influenced by season. The highest level of herbicides (atrazine) was detected in untreated pond water (median concentration 135.9 ng/L). Reclaimed water had the highest levels of antibiotics and stimulants including azithromycin (215 ng/L), sulfamethoxazole (232.1 ng/L), and caffeine (89.4 ng/L). Produce processing plant water also tended to have high levels of atrazine (102.7 ng/L) and ciprofloxacin (80.1 ng/L). In addition, we observed seasonal variability across water types, with the highest atrazine concentrations observed during summer months, while the highest median azithromycin concentrations were observed in reclaimed water during the winter season. Further studies are needed to evaluate if economically feasible on-farm water treatment technologies can effectively remove such contaminants from nontraditional irrigation water sources.
• Rashes-Gonzalez, G., Avila-Torres, G., Moreno-Valenzuela, O. A., Chaidez-Quiroz, C., Hernandez-Flores, C. I., Acosta-Gonzalez, G., Brown, J. K., Betancourt, W. Q., Gerba, C. P., & Hernandez-Zepeda, C. (2019). Norovirus and human adenovirus occurrence and diversity in recreational water in a karst aquifer in the Yucatan Peninsula, Mexico. JOURNAL OF APPLIED MICROBIOLOGY, 127(4), 1255-1269.
• Rosario, K., Morrison, C. M., Mettel, K. A., & Betancourt, W. Q. (2019). Novel Circular Rep-Encoding Single-Stranded DNA Viruses Detected in Treated Wastewater. Microbiology resource announcements, 8(18).
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  Here, we present the complete genome sequences of three circular replication-associated protein (Rep)-encoding single-stranded DNA (CRESS DNA) viruses detected in secondary treated and disinfected wastewater effluent. The discovered viruses, named astewater RESS NA irus (WCDV)-1 to -3, represent novel viral species that seem to persist in wastewater effluent.
• Yoon, J., Reynolds, K. A., Betancourt, W. Q., Morrison, C. M., Perea, S., Breshears, L. E., & Chung, S. (2019). Smartphone-Based Paper Microfluidic Particulometry of Norovirus from Environmental Water Samples at the Single Copy Level. ACS Omega, 4(6), 11180-11188. doi:https://doi.org/10.1021/acsomega.9b00772
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  Human enteric viruses can be highly infectious and thus capable of causing disease upon ingestion of low doses ranging from 10^0 to 10^2 virions. Norovirus is a good example with a minimum infectious dose as low as a few tens of virions, that is, below femtogram scale. Norovirus detection from commonly implicated environmental matrices (water and food) involves complicated concentration of viruses and/or amplification of the norovirus genome, thus rendering detection approaches not feasible for field applications. In this work, norovirus detection was performed on a microfluidic paper analytic device without using any sample concentration or nucleic acid amplification steps by directly imaging and counting on-paper aggregation of antibody-conjugated, fluorescent submicron particles. An in-house developed smartphone-based fluorescence microscope and an image-processing algorithm isolated the particles aggregated by antibody–antigen binding, leading to an extremely low limit of norovirus detection, as low as 1 genome copy/μL in deionized water and 10 genome copies/μL in reclaimed wastewater.
• Zhu, L., Torres, M., Betancourt, W. Q., Sharma, M., Micallef, S. A., Gerba, C., Sapkota, A. R., Sapkota, A., Parveen, S., Hashem, F., May, E., Kniel, K., Pop, M., & Ravishankar, S. (2019). Incidence of fecal indicator and pathogenic bacteria in reclaimed and return flow waters in Arizona, USA. Environmental research, 170, 122-127.
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  The quality of irrigation water used to cultivate produce that is consumed raw is an important issue with regard to food safety. In this study, the microbiological quality of potential irrigation water sources in Arizona was evaluated by testing for the presence of indicator and pathogenic bacteria. Reclaimed water samples were collected from two wastewater treatment plants and return flow samples were collected from two drainage canals and one return flow pond. Standard membrane filtration methods were used for detection of indicator bacteria. Water samples (n = 28) were filtered through cellulose ester membrane filters and bacterial populations were enumerated by placing the filters on selective agar. For detection of pathogens (Salmonella enterica, Listeria monocytogenes and Shiga toxin-producing E. coli (STEC)), water samples were filtered through Modified Moore swabs and enriched in Universal Pre-enrichment Broth, followed by selective enrichment broth for each pathogen. The enriched broth was streaked onto agar media selective for each pathogen. Presumptive colonies were confirmed by PCR/real-time PCR. Among the 14 reclaimed water samples from two sites, the ranges of recovered populations of E. coli, total coliforms, and enterococci were 0-1.3, 0.5-8.3 × 10, and 0-5.5 CFU/100 mL, respectively. No L. monocytogenes, Salmonella or STEC were found. In the 13 return flow water samples from 3 sites, the ranges of recovered populations of E. coli, total coliforms and enterococci were 1.9-5.3 × 10, 6.5 × 10-9.1 × 10, and 2.9-3.7× 10 CFU/100 mL, respectively. All samples were negative for L. monocytogenes. One (7.1%) of the return flow samples was positive for E. coli O145. Nine (64.3%) of the samples were positive for Salmonella. Both real-time PCR and culture-based methods were used for the detection of Salmonella and L. monocytogenes, and the results from the two methods were comparable. The findings of this study provide evidence that irrigation waters in Arizona, including reclaimed water and return flows, could be potential sources of bacterial contamination of produce. Additional work is needed to evaluate whether bacteria present in irrigation water sources transfer to the edible portion of irrigated plants and are capable of persisting through post-harvest activities.
• Betancourt, W. Q., Abd-Elmaksoud, S., & Gerba, C. P. (2018). Efficiency of Reovirus Concentration from Water with Positively Charged Filters. Food and environmental virology, 10(2), 209-211.
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  This study examined the efficacy of reovirus concentration from large volumes of water using two positively charged filters: Zeta Plus 1MDS and NanoCeram. The results indicated that an average of 61 and 81% of input reoviruses were effectively recovered, respectively, from recycled water and tap water using NanoCeram filtration.
• Gerba, C. P., Betancourt, W. Q., Kitajima, M., & Rock, C. M. (2018). Reducing uncertainty in estimating virus reduction by advanced water treatment processes. Water research, 133, 282-288.
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  Treatment of wastewater for potable reuse requires the reduction of enteric viruses to levels that pose no significant risk to human health. Advanced water treatment trains (e.g., chemical clarification, reverse osmosis, ultrafiltration, advanced oxidation) have been developed to provide reductions of viruses to differing levels of regulatory control depending upon the levels of human exposure and associated health risks. Importance in any assessment is information on the concentration and types of viruses in the untreated wastewater, as well as the degree of removal by each treatment process. However, it is critical that the uncertainty associated with virus concentration and removal or inactivation by wastewater treatment be understood to improve these estimates and identifying research needs. We reviewed the critically literature to assess to identify uncertainty in these estimates. Biological diversity within families and genera of viruses (e.g. enteroviruses, rotaviruses, adenoviruses, reoviruses, noroviruses) and specific virus types (e.g. serotypes or genotypes) creates the greatest uncertainty. These aspects affect the methods for detection and quantification of viruses and anticipated removal efficiency by treatment processes. Approaches to reduce uncertainty may include; 1) inclusion of a virus indicator for assessing efficiency of virus concentration and detection by molecular methods for each sample, 2) use of viruses most resistant to individual treatment processes (e.g. adenoviruses for UV light disinfection and reoviruses for chlorination), 3) data on ratio of virion or genome copies to infectivity in untreated wastewater, and 4) assessment of virus removal at field scale treatment systems to verify laboratory and pilot plant data for virus removal.
• Sassi, H. P., Tuttle, K. D., Betancourt, W. Q., Kitajima, M., & Gerba, C. P. (2018). Persistence of Viruses by qPCR Downstream of Three Effluent-Dominated Rivers in the Western United States. Food and environmental virology, 10(3), 297-304.
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  This study was designed to determine the quantitative polymerase chain reaction (qPCR) signal persistence of viruses in three effluent-dominated streams. Samples were collected from the effluent outfall of three wastewater treatment plants in the Western United States and downstream at different locations. All samples were tested for the presence of pepper mild mottle virus (PMMoV), adenoviruses, norovirus GI and GII, Aichi virus, and enteroviruses using qPCR. PMMoV was detected most frequently in 54/57 (94.7%) samples, followed by adenoviruses which was detected in 21/57 (36.8%) samples. PMMoV was detected at all locations downstream and up to 32 km from the discharge point. This study demonstrated that the detection signal of PMMoV was able to persist in wastewater discharges to a greater degree than human enteric viruses in effluent-dominated rivers.
• Gerba, C. P., & Betancourt, W. Q. (2017). Viral Aggregation: Impact on Virus Behavior in the Environment. Environmental science & technology, 51(13), 7318-7325.
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  Aggregates of viruses can have a significant impact on quantification and behavior of viruses in the environment. Viral aggregates may be formed in numerous ways. Viruses may form crystal like structures and aggregates in the host cell during replication or may form due to changes in environmental conditions after virus particles are released from the host cells. Aggregates tend to form near the isoelectric point of the virus, under the influence of certain salts and salt concentrations in solution, cationic polymers, and suspended organic matter. The given conditions under which aggregates form in the environment are highly dependent on the type of virus, type of salts in solution (cation, anion. monovalent, divalent) and pH. However, virus type greatly influences the conditions when aggregation/disaggregation will occur, making predictions difficult under any given set of water quality conditions. Most studies have shown that viral aggregates increase the survival of viruses in the environment and resistance to disinfectants, especially with more reactive disinfectants. The presence of viral aggregates may also result in overestimation of removal by filtration processes. Virus aggregation-disaggregation is a complex process and predicting the behavior of any individual virus is difficult under a given set of environmental circumstances without actual experimental data.
• Gerba, C. P., Betancourt, W. Q., & Kitajima, M. (2017). How much reduction of virus is needed for recycled water: A continuous changing need for assessment?. Water research, 108, 25-31.
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  To ensure the safety of wastewater reuse for irrigation of food crops and drinking water pathogenic viruses must be reduced to levels that pose no significant risk. To achieve this goal minimum reduction of viruses by treatment trains have been suggested. For use of edible crops a 6-log reduction and for production of potable drinking water a 12-log reduction has been suggested. These reductions were based on assuming infective virus concentrations of 10 to 10 per liter. Recent application of molecular methods suggests that some pathogenic viruses may be occurring in concentrations of 10 to 10 per liter. Factors influencing these levels include the development of molecular methods for virus detection, emergence of newly recognized viruses, decrease in per capita water use due to conservation measures, and outbreaks. Since neither cell culture nor molecular methods can assess all the potentially infectious virus in wastewater conservative estimates should be used to assess the virus load in untreated wastewater. This review indicates that an additional 2- to 3-log reduction of viruses above current recommendations may be needed to ensure the safety of recycled water. Information is needed on peak loading of viruses. In addition, more virus groups need to be quantified using better methods of virus quantification, including more accurate methods for measuring viral infectivity in order to better quantify risks from viruses in recycled water.
• Rosiles-González, G., Ávila-Torres, G., Moreno-Valenzuela, O. A., Acosta-González, G., Leal-Bautista, R. M., Grimaldo-Hernández, C. D., Brown, J. K., Chaidez-Quiroz, C., Betancourt, W. Q., Gerba, C. P., & Hernández-Zepeda, C. (2017). Occurrence of Pepper Mild Mottle Virus (PMMoV) in Groundwater from a Karst Aquifer System in the Yucatan Peninsula, Mexico. Food and environmental virology, 9(4), 487-497.
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  The Yucatan Peninsula of Mexico hosts a karst aquifer system that is the only source of freshwater for the area; however, it is vulnerable to human-mediated contamination. Pepper mild mottle virus (PMMoV) is one of the most abundant RNA viruses associated with human feces, making it a viable indicator for tracking fecal pollution in aquatic environments, including groundwater. In this study, groundwater samples collected from a karst aquifer from fresh and brackish water locations were analyzed for fecal indicator bacteria, somatic and male F+ specific coliphages, and PMMoV during the rainy and dry seasons. Total coliform bacteria were detected at all sites, whereas Escherichia coli were found at relatively low levels  0.05). Physicochemical and indicator bacteria were not correlated with PMMoV concentrations. The abundance and prevalence of PMMoV in the karst aquifer may reflect its environmental persistence and its potential as a fecal indicator in this karst aquifer system.
• Betancourt, W. Q., & Gerba, C. P. (2016). Rethinking the Significance of Reovirus in Water and Wastewater. Food and environmental virology, 8(3), 161-73.
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  The genus Orthoreovirus contains nonenveloped viruses with double-stranded gene segments encased in a double-layered icosahedral capsid shell. These features constitute major determinants of virion stability in the environment and virion resistance against physical and chemical agents. Reovirus (ReoV) is the general term most commonly used for all virus strains that infect humans and nonhuman animals. Several studies have demonstrated the frequent occurrence of ReoV in wastewaters and natural waters, including surface and ground waters from different geographical areas. Most of these studies have reported higher concentrations of ReoV than any other enteric virus analyzed. They are more commonly isolated in chlorine-disinfected wastewaters than other enteric viruses, and appear to survive longer in water. The ability of ReoV to form large aggregates, even with different types of enteric viruses (e.g., poliovirus) and their ability to undergo mechanisms of gene segment reassortment among different serotypes may also explain their greater stability. Different approaches have been applied for concentration of ReoV from water; however, the recovery efficiency of the filtration methods has not been fully evaluated. Recently, molecular methods for identification of ReoV strains and quantification of virus genome have been developed. Studies have shown that the overall detection sensitivity of ReoV RNA is enhanced through initial replication of infectious virions in cell culture. More studies are needed to specifically address unresolved issues about the fate and distribution of ReoV in the environment since this virus is not commonly included in virological investigations.
• Betancourt, W. Q., Duarte, D. C., Vásquez, R. C., & Gurian, P. L. (2014). Cryptosporidium and Giardia in tropical recreational marine waters contaminated with domestic sewage: estimation of bathing-associated disease risks. Marine pollution bulletin, 85(1), 268-73.
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  Sewage is a major contributor to pollution problems involving human pathogens in tropical coastal areas. This study investigated the occurrence of intestinal protozoan parasites (Giardia and Cryptosporidium) in tropical recreational marine waters contaminated with sewage. The potential risks of Cryptosporidium and Giardia infection from recreational water exposure were estimated from the levels of viable (oo) cysts (DIC+, DAPI+, PI-) found in near-shore swimming areas using an exponential dose response model. A Monte Carlo uncertainty analysis was performed in order to determine the probability distribution of risks. Microbial indicators of recreational water quality (enterococci, Clostridium perfringens) and genetic markers of sewage pollution (human-specific Bacteroidales marker [HF183] and Clostridium coccoides) were simultaneously evaluated in order to estimate the extent of water quality deterioration associated with human wastes. The study revealed the potential risk of parasite infections via primary contact with tropical marine waters contaminated with sewage; higher risk estimates for Giardia than for Cryptosporidium were found. Mean risks estimated by Monte Carlo were below the U.S. EPA upper bound on recreational risk of 0.036 for cryptosporidiosis and giardiasis for both children and adults. However, 95th percentile estimates for giardiasis for children exceeded the 0.036 level. Environmental surveillance of microbial pathogens is crucial in order to control and eradicate the effects that increasing anthropogenic impacts have on marine ecosystems and human health.
• Betancourt, W. Q., Kitajima, M., Wing, A. D., Regnery, J., Drewes, J. E., Pepper, I. L., & Gerba, C. P. (2014). Assessment of virus removal by managed aquifer recharge at three full-scale operations. Journal of environmental science and health. Part A, Toxic/hazardous substances & environmental engineering, 49(14), 1685-92.
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  Managed aquifer recharge (MAR) systems such as riverbank filtration and soil-aquifer treatment all involve the use of natural subsurface systems to improve the quality of recharged water (i.e. surface water, stormwater, reclaimed water) before reuse. During MAR, water is either infiltrated via basins, subsurface injected or abstracted from wells adjacent to rivers. The goal of this study was to assess the removal of selected enteric viruses and a potential surrogate for virus removal at three full-scale MAR systems located in different regions of the United States (Arizona, Colorado, and California). Samples of source water (i.e., river water receiving treated wastewater and reclaimed water) before recharge and recovered groundwater at all three sites were tested for adenoviruses, enteroviruses, Aichi viruses and pepper mild mottle virus (PMMoV) by quantitative polymerase chain reaction (qPCR). Samples of groundwater positive for any virus were also tested for the presence of infectious virus by cell culture. PMMoV was the most commonly detected virus in the groundwater samples. Infectious enteric viruses (reovirus) were only detected in one groundwater sample with a subsurface residence time of 5 days. The results suggested that in groundwater with a residence time of greater than 14 days all of the viruses are removed below detection indicating a 1 to greater than 5 log removal depending upon the type of virus. Given its behavior, PMMoV may be suitable to serve as a conservative tracer of enteric virus removal in managed aquifer treatment systems.

Presentations

• Yoon, J., Reynolds, K. A., Betancourt, W. Q., Morrison, C. M., Perea, S., Breshears, L. E., & Chung, S. (2019, Aug.). Smartphone-Based Paper Microfluidic Particulometry of Norovirus from Environmental Water Samples at Single Copy Level. ACS National Meeting & Expo. San Diego, CA: ACS.
• Yoon, J., Reynolds, K. A., Betancourt, W. Q., Morrison, C. M., Breshears, L. E., & Chung, S. (2018, May). Single Particle Level Norovirus Detection Assay Using Smartphone-Based Fluorescence Microscope and a Microfluidic Paper Analytic Device. 2nd KU-UA Joint International Symposium. Seoul, South Korea: Korea University.