Biological Consulting Services of North Central Florida, Inc.

Your source for accurate biological testing.

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Enterovirus Testing Laboratory

 

BCS Laboratory is completely equipped for environmental virology. We utilize innovative methodologies for the detection, enumeration, and identification of enteric viruses. We also provide high titer viral stocks for decontamination/inactivation testing. Our virologists are fully trained in the latest cell culture and molecular techniques to ensure the most reliable and informative results.  We provide routine analysis to numerous private, state and government agencies, and universities.

Water, biosolids, and filter (1MDS, NanoCeram, or Filterite filters) samples are processed according to EPA ICR methodology (US EPA ICR microbial laboratory manual; EPA/600/R-95/178 and the US EPA MANUAL OF METHODS FOR VIROLOGY EPA/600/4-84/013 ), or EPA 1615, or ASTM method D4994-89, or Standard Methods 9510B (Standard Methods for the Examination of Water and Waste Water, 20th Edition, APHA).  BCS holds accreditation for analysis of matrices for the above methods.

Additionally, we provide bacteriophage or Coliphage testing as a surrogate to enterovirus testing.  This analysis provides a low cost prediction of the efficacy of the process of enterovirus transport and/or inactivation.

 

Background Enteric Virus Information

Human enteric viruses are often present in domestic sewage, treated wastewater, recreational surface waters, and biosolids intended for land application. The detection of viable viruses indicates the definite presence of fecal pollution and of human pathogens. Human enteric viruses represent a diverse group.  Most of are non-enveloped RNA viruses; Adenovirus is the sole DNA virus. Viral families represented include picornaviruses, rotavirus, Norwalk and Norwalk-like viruses. Coronaviruses and reoviruses have also been linked to enteric disease.

Symptoms of infection range from gastroenteritis to more life threatening diseases such as myocarditis and aseptic meningitis.  Rotavirus and Norwalk virus are two of the common agents that cause viral gastroenteritis.  Mortality from this condition can be very significant in developing countries.  The mechanisms for their pathogenesis are quite varied, but diarrhea is largely due to the loss of cell lining in the intestinal tract.  High concentrations of virus in excess of 108 per gram of diarrheal stool is often observed.

Transmission is generally considered to occur through the oral/fecal route.  However, other routes such as food, contaminated water, or surface contact can occur.  A potentially important mode of transmission, particularly in developing countries, is by waterborne transmission.  Contaminated vegetables and fruits are also an increasing concern.

The impact of diarrheal disease on a worldwide scale is very significant.  The burden of disease and death falls disproportionately on preschool-age children.  Deaths are attributed to severe dehydration secondary to loss of fluids from diarrhea and vomiting.  Failure to thrive has been reported in children in developing countries where infections may be exacerbated by malnutrition or be synergistic with other enteropathogens.

Relatively little is known about many of the viruses in this group because many are difficult to culture or are not cultureable and produce diseases that are not readily identifiable (i.e. have symptoms that are common with other pathogens).  Molecular methods to detect enteric viruses are becoming more common.

 

Some of the common viruses that we have analyzed for:

Adenovirus - Non-enveloped, double-stranded DNA virus, 60-90 nm. May cause gastroenteritis.

Calicivirus - Non-enveloped, plus-sense RNA virus, 26-32 nm. Major cause of viral gastroenteritis.

Coronavirus - Enveloped, plus-sense RNA virus, 80-200 nm. Primarily causes an upper respiratory disease. However, the virus has been found in the stool of individuals with diarrhea.


Enteroviruses - All in the picornavirus family. Non-enveloped, plus-sense RNA viruses, 22-30 nm. Viruses of human origin within this group include, Poliovirus, Coxsackievirus (type A and B), Echovirus, Enterovirus 68-71. These viruses can be transmitted through the oral-fecal route and many have the ability to target the central nervous system.  Enteroviruses can produce a large number of clinical symptoms depending on the type of virus. These include, myalgia, pancreatitis, diabetes, myocarditis, hand-foot-and-mouth disease, conjunctivitis, and gastroenteritis among others.

Hepatitis A virus - Also in the picornavirus family. Non-enveloped, plus-sense RNA virus. Major cause of viral hepatitis.

Reovirus - Non-enveloped, segmented double-stranded RNA virus, 65-75 nm. Can be found in water or sewage. Possible enteric pathogen.

Rotavirus - Non-enveloped, segmented double-stranded RNA virus, 65-75 nm.  Major cause of viral gastroenteritis.

 

Cell Culture; currently, we use any of following cell strains for the detection of viable enteric viruses:

 

Buffalo Green Monkey (BGM) and RD (ATCC# CCL-136) - for standard pan enteric virus analysis monitoring.

MA104 (ATCC# CRL-2378.1) – enteroviruses, rotaviruses, reoviruses

BS-C-1 (ATCC# CCL-26) – enteroviruses, Hepatitis A

FRhK-4 (ATCC# CRL-1688) – Hepatitis A

MRC-5 (ATCC# CCL-171) – coronaviruses, enteroviruses

Additionally, Caco-2 and PLC/PRF/5 Cell lines are used in conjunction with BGM cell lines in monitoring for enteric viruses in all ASTM and EPA/ICR protocols

 

 

EPA 625/R92/013 Appendix H or Standard Practice for the Recovery of Viruses from Wastewater Sludges; ASTM D4994-89. Elution, concentration and viable enteroviruses enumeration of sample (biosolids) by cell culture analysis on Buffalo Green Monkey cells, Rhabdosarcoma cells, and MA 104 cells.

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Detection of Enteric Viruses in Water and Wastewater (Standard Methods 9510A-G).

Virus recovery, concentration, and enumeration from small volumes of water as per Standard Methods for the Examination of Water and Wastewater, 20th edition, APHA, AWWA, WEF, 1998.

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EPA/600/R-10/181 (2012) Method 1615 Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR

EPA Method 1615 provides culture and molecular procedures for detecting human enteroviruses,human noroviruses, and mammalian orthoreoviruses (culture procedure only) in water. The cell culture procedure detects enterovirus and orthoreovirus species that are capable of infecting and producing cytopathic effects (CPE) in the Buffalo Green Monkey kidney (BGM) cell line. Although this cell
line is considered a "gold standard" for detection of infectious waterborne viruses, noroviruses and a number of enteroviruses do not replicate in BGM cells and thus cannot be detected by cell culture. There is no established cell line for detection of infectious human noroviruses. The molecular procedure incorporated into EPA Method 1615 detects the noroviruses and enteroviruses, including those enteroviruses that do not replicate on BGM cells. However, being a molecular procedure, it does not distiguish infectious from noninfectious viruses and thus does not give any information on viability and thus infectivity of the species detected.

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US EPA ICR EPA/600/R-95/178 or EPA/600/4-84/013 Cultivable enterovirus detection: Recovery of enteric viruses from 1MDS or Filtrite filters and enumeration by cell culture on Buffalo Green Monkey cells, Rhabdosarcoma cells, and MA 104 cells as EPA rule methodology.

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Enteric virus sample collection Kit: sample collection kits can be requested and shipped to customers for sampling. Sampling kit includes container for solid media, or Filter, housing, tubing, and flow meter for aqueous samples. In addition, it also contains cold packs and cooler.

 
 

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