Available filtration methods to concentrate waterborne viruses are either too costly for studies requiring large numbers of samples, limited to small sample volumes, or not very portable for routine field applications. Sodocalcic glass wool filtration is a cost-effective and easy-to-use method to retain viruses, but its efficiency and reliability are not adequately understood. This study evaluated glass wool filter performance to concentrate the four viruses on the U.S. Environmental Protection Agency contaminant candidate list, i.e., coxsackievirus, echovirus, norovirus, and adenovirus, as well as poliovirus. Total virus numbers recovered were measured by quantitative reverse transcription-PCR (qRT-PCR); infectious polioviruses were quantified by integrated cell culture (ICC)-qRT-PCR. Recovery efficiencies averaged 70% for poliovirus, 14% for coxsackievirus B5, 19% for echovirus 18, 21% for adenovirus 41, and 29% for norovirus. Virus strain and water matrix affected recovery, with significant interaction between the two variables. Optimal recovery was obtained at pH 6.5. No evidence was found that water volume, filtration rate, and number of viruses seeded influenced recovery. The method was successful in detecting indigenous viruses in municipal wells in Wisconsin. Long-term continuous filtration retained viruses sufficiently for their detection for up to 16 days after seeding for qRT-PCR and up to 30 days for ICC-qRT-PCR. Glass wool filtration is suitable for large-volume samples (1,000 liters) collected at high filtration rates (4 liters min ؊1 ), and its low cost makes it advantageous for studies requiring large numbers of samples.Waterborne viruses are an important cause of disease, being responsible for 14% of outbreaks (9 of 64 cases) and 38% of illnesses (1,153 of 3,008 cases) associated with drinking water in the United States from 1999 to 2002 (21, 49). During the same period, noroviruses were responsible for 6% (8 of 66 cases) of outbreaks and 17% (348 of 2,093 cases) of illnesses associated with recreational water. If waterborne illnesses of unknown etiology during the period 1999-2002 are included in the above statistics, as these are believed to be of viral origin, up to 56% and 28% of illness cases associated with drinking water and recreational water, respectively, may be attributed to viruses.To detect and quantify waterborne viruses from environmental samples, the first step in the protocol usually requires concentration from a large water volume. Several concentration methods have been developed and applied successfully in the past two decades (see reviews by Wyn-Jones and Sellwood [48] and Grabow [13]). These include adsorption onto (and subsequent elution from) electropositive cartridges and membranes (3,25,27,29,33,35), gauze pads and glass powder (2, 9, 34), electronegative membranes, and microporous materials (1,8,12,16,20,27) and concentration by ultrafiltration (15, 17, 36, 37) and ultracentrifugation (26). Adsorption onto electropositive cartridges, for example, the CUNO 1-MDS Viroso...

Background: Groundwater supplies for drinking water are frequently contaminated with low levels of human enteric virus genomes, yet evidence for waterborne disease transmission is lacking.Objectives: We related quantitative polymerase chain reaction (qPCR)–measured enteric viruses in the tap water of 14 Wisconsin communities supplied by nondisinfected groundwater to acute gastrointestinal illness (AGI) incidence.Methods: AGI incidence was estimated from health diaries completed weekly by households within each study community during four 12-week periods. Water samples were collected monthly from five to eight households per community. Viruses were measured by qPCR, and infectivity assessed by cell culture. AGI incidence was related to virus measures using Poisson regression with random effects.Results: Communities and time periods with the highest virus measures had correspondingly high AGI incidence. This association was particularly strong for norovirus genogroup I (NoV-GI) and between adult AGI and enteroviruses when echovirus serotypes predominated. At mean concentrations of 1 and 0.8 genomic copies/L of NoV-GI and enteroviruses, respectively, the AGI incidence rate ratios (i.e., relative risk) increased by 30%. Adenoviruses were common, but tap-water concentrations were low and not positively associated with AGI. The estimated fraction of AGI attributable to tap-water–borne viruses was between 6% and 22%, depending on the virus exposure–AGI incidence model selected, and could have been as high as 63% among children < 5 years of age during the period when NoV-GI was abundant in drinking water.Conclusions: The majority of groundwater-source public water systems in the United States produce water without disinfection, and our findings suggest that populations served by such systems may be exposed to waterborne viruses and consequent health risks.

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There is a growing demand for the development of experimental strategies for efficient articular cartilage repair. Current tissue engineering-based regenerative strategies make use of human mesenchymal stromal cells (hMSCs). However, when implanted in a cartilage defect, control of hMSCs differentiation toward the chondrogenic lineage remains a significant challenge. We have recently demonstrated that silencing the antichondrogenic regulator microRNA-221 (miR-221) was highly effective in promoting in vitro chondrogenesis of monolayered hMSCs in the absence of the chondrogenic induction factor TGF-b. Here we investigated the feasibility of this approach first in conventional 3D pellet culture and then in an in vivo model. In pellet cultures, we observed that miR-221 silencing was sufficient to drive hMSCs toward chondrogenic differentiation in the absence of TGF-b. In vivo, the potential of miR-221 silenced hMSCs was investigated by first encapsulating the cells in alginate and then by filling a cartilage defect in an osteochondral biopsy. After implanting the biopsy subcutaneously in nude mice, we found that silencing of miR-221 strongly enhanced in vivo cartilage repair compared to the control conditions (untreated hMSCs or alginate-only). Notably, miR-221 silenced hMSCs generated in vivo a cartilaginous tissue with no sign of collagen type X deposition, a marker of undesired hypertrophic maturation. Altogether our data indicate that silencing miR-221 has a prochondrogenic role in vivo, opening new possibilities for the use of hMSCs in cartilage tissue engineering. STEM CELLS 2016;34:1801-1811 SIGNIFICANCE STATEMENTWe demonstrated here the effectiveness of an innovative approach based on transient transfection of inhibitor of antichondrogenic miR-221 to direct human mesenchymal stromal cells (hMSCs) toward chondrogenesis both in vitro and in vivo, without exposure to the chondrogenic inducer TGF-b. miR-221 knockdown was sufficient to repair an osteochondral defect subcutaneously implanted in mice, promoting the production of newly formed tissue expressing Collagen type II but not Collagen type X. It is well known that the expression of Collagen type X, a marker for chondrocyte hypertrophy, is an undesired outcome and remains an issue to be solved in the cell-based approach for cartilage regeneration. Therefore, the ability of miR-221 depleted hMSCs to downregulate Collagen type X, both in vitro and in vivo, represents a crucial event on formation of cartilaginous repair tissue and a promising approach to translate into the clinic.

The description of a microencapsulation procedure for Wharton's jelly mesenchymal stem cells (WJMSCs) is reported. The applied method is based on the generation of monodisperse droplets by a vibrational nozzle. An ionic alginate encapsulation procedure was utilized for the microbeads hardening. Different experimental parameters were analyzed, including frequency and amplitude of vibration, polymer pumping rate, and distance between the nozzle and the gelling bath. The produced barium-alginate microbeads were characterized by excellent morphological characteristics as well as a very narrow size distribution. The microencapsulation procedure did not alter the morphology and viability of the encapsulated WJMSCs. In addition, the current paper reports the functional properties in terms of secretive profiles of both free and encapsulated WJMSCs. The analyzed factors were members of the family of interleukins, chemokines, growth factors, and soluble forms of adhesion molecules. These experiments showed that despite encapsulation, most of the proteins analyzed were secreted both by the free and encapsulated cells, even if in a different extent. In conclusion, the described encapsulation procedure represents a promising strategy to utilize WJMSCs for possible in vivo applications in tissue engineering and biomedicine.