Comparison of Cell Culture and a Poliovirus Gene Probe Assay for the Detection of Enteroviruses in Environmental Water Samples

Nucleic acid hybridization provides a rapid non-cell culture method for the detection of enteric viruses in water. The purpose of this work was to compare the detection of naturally occurring enteroviruses by cell culture with their detection by a poliovirus gene probe in various types of water samples. Samples of activated sludge effluent, tertiary treated wastewater (activated sludge, filtration and passage through reverse osmosis), ground water, surface water and tidal river water were processed through 1 MDS Virozorb filters to concentrate any naturally occurring virus. Viruses were eluted from the filters with pH 9.5 beef extract and reduced in volume by flocculation to 20-30 ml. These concentrates were then assayed in the BGM cell line by the cytopathogenic effects (CPE) method and by a poliovirus cDNA probe (base pairs 115-7440) labeled with 32P. A total of 233 samples were assayed in this manner. In slightly more than 93% of the samples gene probe and cell culture yielded the same results. Of these samples 36 were positive by gene probe and 28 by cell culture assay. Positive samples for gene probe were confirmed by treatment with NaOH or RNAse and then reprobed. Samples demonstrating CPE upon primary passage were confirmed positive by subsequent passage of cell lysate on a new monolayer of BGM cells. Ten samples were positive by gene probe and negative by cell culture, and 4 samples were negative by gene probe and positive by cell culture.

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Comparison of Poliovirus Detection in Water by Cell Culture and Nucleic Acid Hybridization

Water Science & Technology ◽

10.2166/wst.1993.0367 ◽

1993 ◽

Vol 27 (3-4) ◽

pp. 315-319

Author(s):

Carlos E. Enriquez ◽

Morteza Abbaszadegan ◽

Ian L. Pepper ◽

Kenneth J. Richardson ◽

Aaron B. Margolin ◽

...

Keyword(s):

Cell Culture ◽

Tissue Culture ◽

Nucleic Acid ◽

Water Samples ◽

Environmental Water ◽

Nucleic Acid Hybridization ◽

Well Water ◽

Gene Probe ◽

Hybridization Technique ◽

Water Filter

The nucleic acid hybridization technique has been used to detect viral nucleic acid in environmental water samples. This type of assay, in contrast with tissue culture assays, may not distinguish between viable and non-viable viruses. We evaluated, by comparison with tissue culture infectivity assay (plaque forming method), the ability of the gene probe assay to detect viable poliovirus 1 (LSc) in well water, autoclaved well water, filter-sterilized well water and autoclaved phosphate buffered saline kept at 37° C and 15° C for 75 days, and in dechlorinated tapwater held at room temperature. A gradual decline in numbers of poliovirus was observed in all of the samples by cell culture assay. With the exception of autoclaved well water and phosphate buffer samples, a parallel decline in virus detectable by gene probe occurred in all other water samples.

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Aged HCT-8 Cell Monolayers Support Cryptosporidium parvum Infection

Applied and Environmental Microbiology ◽

10.1128/aem.01579-07 ◽

2007 ◽

Vol 73 (23) ◽

pp. 7548-7551 ◽

Cited By ~ 29

Author(s):

Laura Y. Sifuentes ◽

George D. Di Giovanni

Keyword(s):

Water Quality ◽

Cell Culture ◽

Water Sample ◽

Cryptosporidium Parvum ◽

Water Samples ◽

Sample Collection ◽

Sample Processing ◽

Cell Monolayers ◽

Immunofluorescent Assay ◽

Naturally Occurring

ABSTRACT Cell culture assays in various formats have been used to study the infectivity of Cryptosporidium spp. as well as to determine the infectivity of naturally occurring oocysts in water. Currently, cell culture assays for infectious Cryptosporidium spp. in water have largely been limited to practice in research laboratories. One obstacle to the routine use of Cryptosporidium cell culture assays for the analysis of water samples is the coordination of water sample collection and processing with readiness of cell culture monolayers. For most Cryptosporidium cell culture assays, monolayers are allowed to develop for 24 to 48 h to reach 80 to 100% confluence prior to inoculation. In this study, we used immunofluorescent assay microscopy to evaluate freshly confluent (2-day-old) and aged (8- to 67-day-old) HCT-8 cell monolayers for their ability to support Cryptosporidium parvum infection. HCT-8 monolayers as old as 67 days were clearly shown to support infection. In two of three experiments, aged monolayers (8- to 11-day-old and 11- to 22-day-old, respectively) developed the same number of C. parvum clusters of infection as freshly confluent monolayers. Results suggest that it may be possible to use cell monolayers from freshly confluent to 3 weeks old on hand for infectivity assays without having to schedule sample processing to coincide with development of freshly confluent monolayers. This would make Cryptosporidium cell culture assays much more feasible for water quality and utility laboratories.

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Optimizing a Suspension Culture Method with a Decreased Cost to Detect Enteroviruses in Water to Increase Surveillance Access

Microbiology Research ◽

10.3390/microbiolres11020008 ◽

2020 ◽

Vol 11 (2) ◽

pp. 35-44

Author(s):

Stephanie Tornberg-Belanger ◽

Jonathan Sreter ◽

Aaron Margolin

Keyword(s):

Cell Culture ◽

Nucleic Acid ◽

Water Samples ◽

Financial Burden ◽

Virus Detection ◽

Culture Method ◽

Virus Concentration ◽

Viral Nucleic Acid ◽

A Cell ◽

Materials Used

Enteroviruses are a public health threat due to the high incidence of infections and potential for serious illness or death. Some laboratories in high-income countries detect enteroviruses in water by integrating cell culture and PCR (ICC/PCR). This combined method carries a high financial burden, due in part to specialized cell culture equipment. Therefore, we expanded upon a pilot study to reduce the cost by using common laboratory polypropylene tubes to create a cell culture in suspension. We optimized the protocol by determining minimal incubation periods post-infection as a function of the initial virus concentration. Cells in suspension and traditional monolayers were inoculated with poliovirus and incubated in 8-hour intervals up to 48 hours prior to extraction. Quantitative PCR (qPCR) was used to detect viral nucleic acid targets. Treated and raw water samples were seeded with virus and the suspension ICC/qPCR protocol used to ascertain whether the protocol performed similar to directly seeding cells. No variation in virus detection occurred using the suspension ICC/qPCR or monolayer ICC/qPCR (p = 0.95). In surface water samples, viral nucleic acid was successfully detected, with no significant increase after 32 h (p > 0.05). Suspension ICC/qPCR is as effective as monolayer ICC/qPCR in detecting enteroviruses in surface waters. Materials used in the suspension ICC/qPCR have a lower monetary cost than traditional cell culture materials without loss of sensitivity. More accessible testing of waters for enterovirus contamination through cost reduction has the potential to reduce human exposure and disease.

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Automated cell counting of astrocytes on patterned substrates containing aliphatic and charged properties

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010014124x ◽

1995 ◽

Vol 53 ◽

pp. 974-975

Author(s):

W. Shain ◽

H. Ancin ◽

H.C. Craighead ◽

M. Isaacson ◽

L. Kam ◽

...

Keyword(s):

Cell Culture ◽

Cell Attachment ◽

Culture Method ◽

Cell Counting ◽

Data Sets ◽

Nuclear Staining ◽

Double Positive ◽

A Cell ◽

Wafer Test ◽

Cell Densities

Neural protheses have potential to restore nervous system functions lost by trauma or disease. Nanofabrication extends this approach to implants for stimulating and recording from single or small groups of neurons in the spinal cord and brain; however, tissue compatibility is a major limitation to their practical application. We are using a cell culture method for quantitatively measuring cell attachment to surfaces designed for nanofabricated neural prostheses.Silicon wafer test surfaces composed of 50-μm bars separated by aliphatic regions were fabricated using methods similar to a procedure described by Kleinfeld et al. Test surfaces contained either a single or double positive charge/residue. Cyanine dyes (diIC18(3)) stained the background and cell membranes (Fig 1); however, identification of individual cells at higher densities was difficult (Fig 2). Nuclear staining with acriflavine allowed discrimination of individual cells and permitted automated counting of nuclei using 3-D data sets from the confocal microscope (Fig 3). For cell attachment assays, LRM5 5 astroglial cells and astrocytes in primary cell culture were plated at increasing cell densities on test substrates, incubated for 24 hr, fixed, stained, mounted on coverslips, and imaged with a 10x objective.

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Detection of enteric adenoviruses in south African waters using gene probes

Water Science & Technology ◽

10.2166/wst.1995.0638 ◽

1995 ◽

Vol 31 (5-6) ◽

pp. 345-350 ◽

Cited By ~ 2

Author(s):

B. Genthe ◽

M. Gericke ◽

B. Bateman ◽

N. Mjoli ◽

R. Kfir

Keyword(s):

South African ◽

Water Samples ◽

Cellulose Fibre ◽

Gene Probe ◽

Viral Dna ◽

Virus Particles ◽

Gene Probes ◽

Probe Hybridization ◽

Enteric Adenoviruses ◽

Effect Of Chlorine

Gene probes developed locally for both enteric Adenoviruses 40 and 41 were used to determine whether these viruses were present in both raw and treated waters. Approximately sixty water samples were concentrated by ultrafiltration and analysed directly for the presence of enteric adenoviruses. Three pretreatment techniques, namely sephadex columns, cellulose fibre and GenecleanTM were tested for the removal of inhibitory substances from concentrated water samples. The effect of chlorine treatment on viral detection using gene probe hybridization was also examined by exposing adenoviruses to chlorine concentrations of up to 20mg/l for 1 hour. Enteric adenoviruses were detected in up to 59% of both raw and treated waters analysed. Cellulose fibre and GenecleanTM were found to successfully remove inhibitory substances from concentrated raw waters. Viral DNA was detected after exposure to a range of chlorine concentrations indicating that the viruses detected in the treated waters may have been inactivated virus particles.

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Evaluation of flow-cytometry for the monitoring of infectious human rotavirus in water

Water Science & Technology ◽

10.2166/wst.1997.0776 ◽

1997 ◽

Vol 35 (11-12) ◽

pp. 451-453

Author(s):

F. X. Abad ◽

A. Bosch ◽

J. Comas ◽

D. Villalba ◽

R. M. Pintó

Keyword(s):

Flow Cytometry ◽

Water Samples ◽

Wild Type ◽

Cell Monolayers ◽

Naturally Occurring ◽

Human Rotavirus ◽

Faecal Samples

A method has been developed for the detection of infectious human rotavirus (HRV), based on infection of MA104 and CaCo-2 cell monolayers and ulterior flow cytometry. The sensitivity of the flow cytometry procedure for the cell-adapted HRV enabled the detection of 200 and 2 MPNCU in MA104 and CaCo-2 cells, respectively. Flow cytometry performed five days after infection of CaCo-2 enabled the detection of naturally occurring wild-type HRV in faecal samples and concentrated water samples.

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Efficiency and Technological Reliability of Contaminant Removal in Household WWTPs with Activated Sludge

Applied Sciences ◽

10.3390/app11041889 ◽

2021 ◽

Vol 11 (4) ◽

pp. 1889 ◽

Cited By ~ 1

Author(s):

Agnieszka Micek ◽

Krzysztof Jóźwiakowski ◽

Michał Marzec ◽

Agnieszka Listosz ◽

Tadeusz Grabowski

Keyword(s):

Activated Sludge ◽

Wastewater Treatment Plants ◽

Oxygen Demand ◽

Domestic Wastewater ◽

Pollutant Removal ◽

Treated Wastewater ◽

Contaminant Removal ◽

Nitrogen And Phosphorus ◽

Wastewater Samples ◽

Polish Law

The results of research on the efficiency and technological reliability of domestic wastewater purification in two household wastewater treatment plants (WWTPs) with activated sludge are presented in this paper. The studied facilities were located in the territory of the Roztocze National Park (Poland). The mean wastewater flow rate in the WWTPs was 1.0 and 1.6 m3/day. In 2017–2019, 20 series of analyses were done, and 40 wastewater samples were taken. On the basis of the received results, the efficiency of basic pollutant removal was determined. The technological reliability of the tested facilities was specified using the Weibull method. The average removal efficiencies for the biochemical oxygen demand in 5 days (BOD5) and chemical oxygen demand (COD) were 66–83% and 62–65%, respectively. Much lower effects were obtained for total suspended solids (TSS) and amounted to 17–48%, while the efficiency of total phosphorus (TP) and total nitrogen (TN) removal did not exceed 34%. The analyzed systems were characterized by the reliability of TSS, BOD5, and COD removal at the level of 76–96%. However, the reliability of TN and TP elimination was less than 5%. Thus, in the case of biogenic compounds, the analyzed systems did not guarantee that the quality of treated wastewater would meet the requirements of the Polish law during any period of operation. This disqualifies the discussed technological solution in terms of its wide application in protected areas and near lakes, where the requirements for nitrogen and phosphorus removal are high.

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Interferonogenic activity of the strain BH-3 duck hepatitis virus type I (Picornaviridae: Avihepatovirus: Avihepatovirus A)

Voprosy Virusologii ◽

10.36233/0507-4088-43 ◽

2021 ◽

Vol 66 (2) ◽

pp. 162-166

Author(s):

N. V. Nikitina ◽

I. K. Leonov ◽

L. I. Yavdoshak

Keyword(s):

Cell Culture ◽

Virus Type ◽

Hepatitis Virus ◽

Effective Means ◽

Culture Method ◽

Etiologic Agent ◽

Research Centre ◽

Type I ◽

Duck Hepatitis Virus ◽

Duck Hepatitis

Introduction. Duck viral hepatitis type I (DVH-I) is a poorly studied contagious disease caused by RNA-containing duck (Anatinae) hepatitis virus type I (Picornaviridae: Avihepatovirus: Avihepatovirus A). This infection is widespread in many countries, including Russia, and causes significant damage to industrial duck breeding. The study of interferonogenic activity of its etiologic agent strains is of great importance in solving the problem of developing effective means to control the disease.Material and methods. Strain BH-3 of duck hepatitis virus type I isolated from the liver of sick ducklings was used in the study. The strain was adapted to developing 10–12 day old duck embryos, to the cell culture of chicken and duck fibroblasts and deposited in the State Collection of Viruses of the D.I. Ivanovsky Institute of Virology of FSBI «National Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya» of the Ministry of Health of Russia. Experiments were performed using the standard tissue culture method.Results and discussion. Data on the ability of the viral strain BH-3 to induce interferon (IFN) and its sensitivity to the action of exogenous interferon in the culture of duck fibroblasts are presented. It has been shown that the interferonogenic activity of this strain of the hepatitis virus is in direct proportion to the multiplicity of infection. The maximum induction of IFN (1 : 256 CEPD50) was observed at a dose of 1.0 TCD50/cell in 72–96 hrs after inoculation of the cell culture. Exogenous IFN at a dose of 1 : 128 completely suppressed the cytopathic effect and death of duck embryos infected with hepatitis virus at a dose of 100 TCD50/cell.Conclusion. The data obtained allow us to state that the vaccine strain BH-3 of duck hepatitis virus type I has a pronounced interferonogenic activity and sensitivity to the action of exogenous IFN. This may have implications for the development of effective therapeutic agents against DVH-I.

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Digoxigenin-Labeled Probe-Based Colony Blotting Assay for Rapid Quantification of Salmonella Serovars in Seafood and Water

Journal of AOAC International ◽

10.5740/jaoacint.11-341 ◽

2012 ◽

Vol 95 (6) ◽

pp. 1652-1655 ◽

Cited By ~ 1

Author(s):

Rakesh Kumar ◽

K V Lalitha

Keyword(s):

Real Time ◽

Water Samples ◽

Real Time Pcr ◽

Quantitative Detection ◽

Gene Probe ◽

Pcr Assay ◽

Nylon Membrane ◽

Rapid Enumeration ◽

Salmonella Serovars ◽

Labeled Probe

Abstract A non-radio-labeled probe-based detection method was developed for rapid enumeration of Salmonella in seafood and water samples. A Salmonella-specific invA gene probe was developed using a digoxigenin-based non-radio labeling assay, which was evaluated with naturally contaminated seafood and water samples. The probe-based technique was further compared with the quantitative PCR assay. The method was specific for detection of different Salmonella serovars without any nonspecific hybridization with other Salmonella-related Enterobacteriaceae. The optimum labeling efficiency was determined for the labeled probe, and 10 pg/μL probe concentration was observed to be most efficient for detection of Salmonella colonies on nylon membrane. Quantification of Salmonella in naturally contaminated seafood and water samples (n = 21) was in the range 10–102 CFU/mL. The assay successfully quantified Salmonella in spiked seafood and water samples in the presence of background flora, and the entire assay was completed within 48 h. The probe-based assay was further evaluated with real-time PCR, and results showed that the assay was comparable to real-time PCR assay. Thus, this probe-based assay can be a rapid, useful, and alternative technique for quantitative detection of Salmonella in food, feed, and water samples.

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