Integrated Cell Culture/PCR for Detection of Enteric Viruses in Environmental Samples

2004 ◽  
pp. 069-078 ◽  
Author(s):  
Kelly A. Reynolds
Keyword(s):  
Cell Culture ◽  
Environmental Samples ◽  
Enteric Viruses

Removal of PCR inhibiting substances in sewage sludge amended soil

1995 ◽  
Vol 31 (5-6) ◽  
pp. 311-315 ◽  
Author(s):  
Timothy M. Straub ◽  
Ian L. Pepper ◽  
Charles P. Gerba
Keyword(s):  
Cell Culture ◽  
Enteric Viruses ◽  
Treatment Method ◽  
Cytopathic Effects ◽  
Chelex 100 ◽  
Beef Extract ◽  
Polymerase Chain ◽  
Seminested Pcr ◽  
Soil Colloids ◽  
Amended Soil

Current methods for the detection of enteric viruses in soil involve elution of viruses from soil colloids using beef extract or other proteins. These eluates are then assayed in cell culture and observed daily for cytopathic effects (CPE). While this method is suitable for detection of enteric viruses by cell culture, these eluates contain humic acids and heavy metals that interfere with polymerase chain reaction (PCR) detection. Using beef extract eluates prepared from sludge amended soil, 10 different methods of eluate purification were evaluated for their ability to remove PCR inhibition and maximize sensitivity. The treatment method providing the greatest sensitivity of poliovirus detection by PCR was the combination of Sephadex G-50 and Chelex-100. Using this method 2 plaque forming units (PFU) could be detected after reverse transcription and 30 cycles of PCR. Thirty (30) cycles of seminested PCR were performed on these samples to verify nucleic acid sequences and increase sensitivity after the first 30 cycles of PCR. Using seminested PCR, sensitivity of detection using the Sephadex G-50 and Chelex-100 treatment method to 0.2 PFU. In addition to providing excellent sensitivity for viruses in sludge amended soils, this treatment method is relatively simple compared to other methods.

scholarly journals Assessing the Occurrence of Waterborne Viruses in Reuse Systems: Analytical Limits and Needs

Pathogens ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 107 ◽  
Author(s):  
Charles P. Gerba ◽  
Walter Q. Betancourt
Keyword(s):  
Cell Culture ◽  
Virus Detection ◽  
Enteric Viruses ◽  
Enteric Virus ◽  
Culture Cell ◽  
Virus Infectivity ◽  
Recycled Water ◽  
Chemical Additives ◽  
Potable Reuse ◽  
Passage Number

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.

scholarly journals Rapid and Sensitive Direct Detection and Identification of Poliovirus from Stool and Environmental Surveillance Samples by Use of Nanopore Sequencing

2020 ◽  
Vol 58 (9) ◽  
Author(s):  
Alexander G. Shaw ◽  
Manasi Majumdar ◽  
Catherine Troman ◽  
Áine O’Toole ◽  
Blossom Benny ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Environmental Samples ◽  
Direct Detection ◽  
Nanopore Sequencing ◽  
Wild Type ◽  
Consensus Sequences ◽  
Real Time Analysis ◽  
Detection And Identification ◽  
Culture Algorithm

ABSTRACT Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR, and sequencing of the VP1 region to distinguish vaccine (Sabin), vaccine-derived, and wild-type polioviruses and to ensure an appropriate response. This cell culture algorithm takes 2 to 3 weeks on average between sample receipt and sequencing. Direct detection of viral RNA using PCR allows faster detection but has traditionally faced challenges related to poor sensitivity and difficulties in sequencing common samples containing poliovirus and enterovirus mixtures. We present a nested PCR and nanopore sequencing protocol that allows rapid (<3 days) and sensitive direct detection and sequencing of polioviruses in stool and environmental samples. We developed barcoded primers and a real-time analysis platform that generate accurate VP1 consensus sequences from multiplexed samples. The sensitivity and specificity of our protocol compared with those of cell culture were 90.9% (95% confidence interval, 75.7% to 98.1%) and 99.2% (95.5% to 100.0%) for wild-type 1 poliovirus, 92.5% (79.6% to 98.4%) and 98.7% (95.4% to 99.8%) for vaccine and vaccine-derived serotype 2 poliovirus, and 88.3% (81.2% to 93.5%) and 93.2% (88.6% to 96.3%) for Sabin 1 and 3 poliovirus alone or in mixtures when tested on 155 stool samples in Pakistan. Variant analysis of sequencing reads also allowed the identification of polioviruses and enteroviruses in artificial mixtures and was able to distinguish complex mixtures of polioviruses in environmental samples. The median identity of consensus nanopore sequences with Sanger or Illumina sequences from the same samples was >99.9%. This novel method shows promise as a faster and safer alternative to cell culture for the detection and real-time sequencing of polioviruses in stool and environmental samples.

scholarly journals Adenovirus Infection of Human Enteroids Reveals Interferon Sensitivity and Preferential Infection of Goblet Cells

2018 ◽  
Vol 92 (9) ◽  
Author(s):  
Mayumi K. Holly ◽  
Jason G. Smith
Keyword(s):  
Cell Culture ◽  
Culture System ◽  
Enteric Viruses ◽  
Goblet Cells ◽  
Clinical Isolates ◽  
Intestinal Cell ◽  
Intestinal Epithelial ◽  
Cell Culture System ◽  
Human Adenoviruses ◽  
Enteric Adenoviruses

ABSTRACTHuman adenoviruses (HAdV) are significant human pathogens. Although only a subset of HAdV serotypes commonly cause gastroenteritis in humans, most HAdV species replicate in the gastrointestinal tract. Knowledge of the complex interaction between HAdVs and the human intestinal epithelium has been limited by the lack of a suitable cell culture system containing relevant cell types. Recently, this need has been met by the stable and prolonged cultivation of primary intestinal epithelial cells as enteroids. Human enteroids have been used to reveal novel and interesting aspects of rotavirus, norovirus, and enterovirus replication, prompting us to explore their suitability for HAdV culture. We found that both prototype strains and clinical isolates of enteric and nonenteric HAdVs productively replicate in human enteroids. HAdV-5p, a respiratory pathogen, and HAdV-41p, an enteric pathogen, are both sensitive to type I and III interferons in human enteroid monolayers but not A549 cells. Interestingly, HAdV-5p, but not HAdV-41p, preferentially infected goblet cells. And, HAdV-5p but not HAdV-41p was potently neutralized by the enteric human alpha-defensin HD5. These studies highlight new facets of HAdV biology that are uniquely revealed by primary intestinal epithelial cell culture.IMPORTANCEEnteric adenoviruses are a significant cause of childhood gastroenteritis worldwide, yet our understanding of their unique biology is limited. Here we report robust replication of both prototype and clinical isolates of enteric and respiratory human adenoviruses in enteroids, a primary intestinal cell culture system. Recent studies have shown that other fastidious enteric viruses replicate in human enteroids. Therefore, human enteroids may provide a unified platform for culturing enteric viruses, potentially enabling isolation of a greater diversity of viruses from patients. Moreover, both the ability of interferon to restrict respiratory and enteric adenoviruses and a surprising preference of a respiratory serotype for goblet cells demonstrate the power of this culture system to uncover aspects of adenovirus biology that were previously unattainable with standard cell lines.

Improvements in the RT-PCR detection of enteric viruses in environmental samples

1998 ◽  
Vol 38 (12) ◽  
pp. 83-86 ◽  
Author(s):  
K. J. Schwab ◽  
F. H. Neill ◽  
M. K. Estes ◽  
R. L. Atmar
Keyword(s):  
Nucleic Acid ◽  
Reverse Transcriptase ◽  
Environmental Samples ◽  
Internal Standard ◽  
Pcr Amplification ◽  
Enteric Viruses ◽  
New Method ◽  
Rt Pcr ◽  
Pcr Product ◽  
Enzyme Method

Current methods for the detection of nucleic acid from enteric viruses in environmental samples usually involve extensive concentration and purification of target viruses followed by RT-PCR amplification using two enzymes, reverse transcriptase and Taq polymerase. We have developed a modified method that improves RT-PCR assays by: (i) the use of an RT-PCR internal standard control RNA to identify potential false negative results caused by inhibition of RT-PCR enzymes; (ii) the use of rTth (Perkin-Elmer, Foster City, CA), a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase in a single-tube, single-buffer, elevated-temperature reaction; and (iii) the use of thermolabile uracil N-glycosylase (HK-UNG) (Epicentre Technologies, Madison, WI) to prevent PCR product carryover contamination. The new method was compared to the traditional two-enzyme, RT-PCR method for detection of Norwalk virus (NV) and hepatitis A virus (HAV) in buffer, stool, clam and oyster samples. The new method was at least as sensitive in NV and HAV detection compared to the traditional two-enzyme method. The internal standard control successfully detected inhibitors to RT-PCR amplification. NV and HAV PCR products generated with dUTP replacing dTTP during amplification were seeded into subsequent samples to test the prevention of PCR product carryover contamination by HK-UNG. The new method successfully eliminated PCR product carryover contamination in contrast to the traditional two-enzyme method. These improvements to viral nucleic acid detection have the potential to improve sensitivity, specificity and confidence in RT-PCR results.

scholarly journals Detection of human enteric viruses in shellfish, vegetables, waters and environmental samples: a preliminary study

2010 ◽  
Vol 14 ◽  
pp. e63-e64
Author(s):  
V. Cannella ◽  
G. Purpari ◽  
A. Ferrari ◽  
A. Migliazzo ◽  
P. Di Marco ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Enteric Viruses ◽  
Preliminary Study ◽  
Human Enteric Viruses

scholarly journals ENTERIC VIRUSES AS CONTAMINANTS AND BIOINDICATORS IN ENVIRONMENTAL SAMPLES: A REVIEW

2015 ◽  
Vol 20 (2) ◽  
Author(s):  
Gislaine Fongaro ◽  
Hugo Deleon Silva ◽  
El Mahdy Mohamed El Mahdy ◽  
Maria Elisa Magri ◽  
Camila Daminelli Schissi ◽  
...  
Keyword(s):  
Environmental Samples ◽  
Enteric Viruses

Application of Gene Probes to Virus Detection in Water

1989 ◽  
Vol 21 (3) ◽  
pp. 147-154 ◽  
Author(s):  
Charles P. Gerba ◽  
Aaron B. Margolin ◽  
Martinez J. Hewlett
Keyword(s):  
Cell Culture ◽  
Nucleic Acid ◽  
Virus Detection ◽  
Enteric Viruses ◽  
Serological Tests ◽  
Nucleic Acid Probes ◽  
Gene Probes ◽  
Detection Capabilities ◽  
Further Development ◽  
A Current

Gene probes offer a rapid and sensitive method for the detection of viruses in water and other environmental samples. Gene probes are small strands of nucleic acid labeled with radioactive or nonradioactive compounds for their detection. The target organism is identified by the hybridization of the probe to the organism's nucleic acid. Nucleic acid probes are at least 1000-fold more sensitive than serological tests such as enzyme-linked-immunoassay and do not first require cultivation of the virus for detection. Gene probes have been developed for organisms that do not grow in cell culture, and probes have been constructed for most of the major groups of enteric viruses. Gene probes have been applied to the detection of enteric viruses in water, marine sediment and shellfish. Radioactively labeled probes can detect as little as 1-10 infectious units of virus within 48 hours. A current disadvantage of probes is that they cannot determine the infectivity of the viruses; however, they can be used to quickly determine the growth of viruses in cell culture. Further development of nonradioactive probes should place virus detection capabilities into the hands of most water quality laboratories.

Virological control of groundwater quality using biomolecular tests

2003 ◽  
Vol 47 (3) ◽  
pp. 261-266 ◽  
Author(s):  
A. Carducci ◽  
B. Casini ◽  
A. Bani ◽  
E. Rovini ◽  
M. Verani ◽  
...  
Keyword(s):  
Cell Culture ◽  
High Efficiency ◽  
Enteric Viruses ◽  
Water Infiltration ◽  
Proteinase K ◽  
Rt Pcr ◽  
Specific Primers ◽  
Deep Groundwater ◽  
Deep Aquifers ◽  
Groundwater Samples

Deep groundwater, even if generally protected, could be contaminated by surface or rain water infiltration through soil fractures, septic tanks, cesspits, land irrigation, disposal of wastewater and disposal of muds from depuration systems. The sanitary importance of such possible contamination is related to the different uses of the water and it is at the maximum level when it is intended for human use. Routine microbiological analyses do not consider viruses, only bacterial parameters, as contamination indicators. However, it is known that enteric viruses can survive a long time in deep aquifers and that they may not always be associated with bacterial indicators. The virological analysis of waters intended for drinking use is provided only as an occasional control exercised at the discretion of the sanitary authority. Technological difficulties with obtaining data about groundwater viral contamination led to a study to devise rapid and efficient methods for their detection and the application of these methods to samples from different sources. Four acid nucleic extraction techniques have been tested (classic proteinase K- phenol/chloroform, QIAamp Viral RNA Kit (Qiagen), SV Total RNA Isolation System (Promega) and NucleoSpin Virus L (Macherey-Nagel). Sensitivity and specificity of RT-PCR protocols for entero- (EV), hepatitis A (HAV) and small round structured (SRSV) viruses have been verified. Deep groundwater samples (100 L) were concentrated (2-step tangential flow ultrafiltration) and the concentrate contaminated with serial 10-fold dilutions of a known titre of poliovirus type 3. Extracted RNA was concentrated (microcon-100) and analysed by RT-PCR using specific EV primers and visualising amplification products by agarose gel electrophoresis. In addition, two different methods of RT-PCR for non-cultivable viruses have been tested: (a) RT-PCR and nested RT-PCR for HAV and (b) RT-PCR with generic primers and RT-PCR with specific primers for SRSV. Different specificity tests have been carried out in the presence of some of the commoner microorganisms. The most efficient, sensitive and specific protocols were used to test 35x100L deep groundwater samples. Sample concentrates were split with one part treated with chloroform and analysed by cell culture (BGM and Frp/3, derived from FrHK/4, cells) and the other tested by RT-PCR for HAV, EV and SRSV. Results demonstrated the high efficiency of the classic and QIAamp methods. Microcon-100 did not increase the sensitivity of the technique used. The highest sensitivity was observed for RT-PCR with specific primers for SRSV and for nested RT-PCR for HAV. One sample showed a cytopathic effect, not confirmed at the third subculture, while the RT-PCR allowed the detection of echovirus 7. Cell culture did not allow detection of the majority of the enteric viruses while PCR gave sensitive, specific and rapid detection of a range of agents in the same samples. Even if it was impossible to fix a virological quality standard, it would be necessary to find a viral indicator in order to achieve a complete preventive check which would be particularly useful in some cases (e.g. water never used before, after pollution accidents, for seasonal checking).

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