scholarly journals Bioaccumulation, Retention, and Depuration of Enteric Viruses by Crassostrea virginica and Crassostrea ariakensis Oysters

2008 ◽  
Vol 74 (22) ◽  
pp. 6825-6831 ◽  
Author(s):  
Sharon P. Nappier ◽  
Thaddeus K. Graczyk ◽  
Kellogg J. Schwab
Keyword(s):  
Crassostrea Virginica ◽  
Hepatitis A Virus ◽  
Proteinase K ◽  
Murine Norovirus ◽  
Viral Pathogens ◽  
Crassostrea Ariakensis ◽  
Reverse Transcription Pcr ◽  
Bacteriophage Ms2 ◽  
Continuous Filtration ◽  
Male Specific

ABSTRACT Crassostrea ariakensis oysters are under review for introduction into the Chesapeake Bay. However, the human health implications of the introduction have not been fully addressed. This study evaluated rates of bioaccumulation, retention, and depuration of viruses by Crassostrea virginica and C. ariakensis when the two oyster species were maintained in separate tanks containing synthetic seawater of various salinities (8, 12, or 20 ppt). Oyster bioaccumulation tanks were seeded with 103 PFU/ml of hepatitis A virus (HAV), poliovirus, male-specific bacteriophage (MS2), and murine norovirus 1 (MNV-1) and 103 PCR units/ml of human norovirus (NoV). After 24 h, depuration commenced as oysters (n = 255) were placed in pathogen-free seawater under continuous filtration. Oysters (n = 6) were sampled weekly for 1 month from each tank. Viral RNA was recovered using a modified proteinase K, guanidine, and glassmilk method and analyzed by quantitative reverse transcription-PCR. The odds of C. ariakensis oysters harboring NoV, MNV-1, or HAV were statistically greater than the odds of C. virginica oysters harboring the same viruses (MNV-1 odds ratio [OR], 4.5; P = 0.01; NoV OR, 8.4; P < 0.001; HAV OR, 11.4; P < 0.001). Unlike C. virginica, C. ariakensis bioaccumulated and retained NoV, MNV-1, and HAV for 1 month at all salinities. Additionally, the odds of an oyster testing positive for NoV was 25.5 times greater (P < 0.001) when the oyster also tested positive for MNV-1. This research helps assess the threat of C. ariakensis as a vehicle for viral pathogens due to the consumption of raw oysters and validates the role for MNV-1 as a surrogate for NoV.

Hollow-Fiber Ultrafiltration for the Concentration and Simultaneous Recovery of Multiple Pathogens in Contaminated Foods

2009 ◽  
Vol 72 (12) ◽  
pp. 2547-2552 ◽  
Author(s):  
HEE-YEON KIM ◽  
HYEUN-JIN PARK ◽  
GWANGPYO KO
Keyword(s):  
Hollow Fiber ◽  
Food Samples ◽  
Murine Norovirus ◽  
Viral Pathogens ◽  
Recovery Rates ◽  
Total Recovery ◽  
E Coli ◽  
Reverse Transcription Pcr ◽  
Elution Buffer ◽  
Multiple Pathogens

We investigated the possibility of using hollow-fiber ultrafiltration (HUF) for the simultaneous recovery of multiple microorganisms in food samples. MS2 bacteriophage, E. coli, Bacillus subtilis spores, and murine norovirus (MNV) were each inoculated into 5 liters of either distilled water (DW) or glycine elution buffer and then concentrated using hollow-fiber polysulfone ultrafilters. The resulting concentrates were further analyzed by either cultivation or TaqMan real-time reverse transcription PCR assay. The overall average recovery rates were 7.1% in DW and 17.1% in glycine elution buffer. When the virus, vegetative bacteria, and bacterial spores were simultaneously inoculated into DW, glycine, or Tris-HCl elution buffers, on average 16.8% of inoculated microorganisms were recovered by HUF. The addition of 3% beef extract blocking buffer to HUF increased the total recovery rate to 46.1%, with incremental recovery rates increasing sharply for B. subtilis spores and MNV. Use of HUF resulted in E. coli recovery rates of 68.0% on lettuce and 66.2% on ham and MNV recovery rates of 1.5% on lettuce and 5.8% on ham. Our study demonstrates that HUF can be effective at simultaneously recovering and concentrating diverse bacterial and viral pathogens from foods.

scholarly journals Hemocytes Are Sites of Enteric Virus Persistence within Oysters

2011 ◽  
Vol 77 (23) ◽  
pp. 8360-8369 ◽  
Author(s):  
Keleigh Provost ◽  
Brooke A. Dancho ◽  
Gulnihal Ozbay ◽  
Robert S. Anderson ◽  
Gary P. Richards ◽  
...  
Keyword(s):  
Hepatitis A Virus ◽  
Rna Extraction ◽  
Acid Tolerance ◽  
Enteric Virus ◽  
Extraction Methods ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Virus Persistence ◽  
Content Type ◽  
Reverse Transcription Pcr

ABSTRACTThe goal of this study was to determine how enteric viruses persist within shellfish tissues. Several lines of novel evidence show that phagocytic blood cells (hemocytes) of Eastern oysters (Crassostrea virginica) play an important role in the retention of virus particles. Our results demonstrated an association of virus contamination with hemocytes but not with hemolymph. Live oysters contaminated overnight with hepatitis A virus (HAV) and murine norovirus (MNV) had 56% and 80% of extractable virus associated with hemocytes, respectively. Transfer of HAV-contaminated hemocytes to naïve (virus-free) oysters resulted in naïve oyster meat testing HAV positive for up to 3 weeks. Acid tolerance of HAV, MNV, poliovirus (PV), and feline calicivirus (FCV) correlated with the ability of each virus to persist within oysters. Using reverse transcription-PCR (RT-PCR) to evaluate persistence of these viruses in oysters, we showed that HAV persisted the longest (>21 days) and was most acid resistant, MNV and PV were less tolerant of acidic pH, persisting for up to 12 days and 1 day, respectively, and FCV did not persist (<1 day) within oysters and was not acid tolerant. This suggests that the ability of a virus to tolerate the acidic conditions typical of phagolysosomal vesicles within hemocytes plays a role in determining virus persistence in shellfish. Evaluating oyster and hemocyte homogenates and live contaminated oysters as a prelude to developing improved viral RNA extraction methods, we found that viruses were extracted more expediently from hemocytes than from whole shellfish tissues and gave similar RT-PCR detection sensitivities.

scholarly journals Development of an RNA Extraction Protocol for a Norovirus from Raw Oysters and Detection by qRT-PCR and Droplet-Digital RT-PCR

Foods ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1804
Author(s):  
Daniel Plante ◽  
Julio Alexander Bran Barrera ◽  
Maude Lord ◽  
Irène Iugovaz ◽  
Neda Nasheri
Keyword(s):  
Hepatitis A Virus ◽  
Rna Extraction ◽  
Complex Nature ◽  
Murine Norovirus ◽  
Rt Pcr ◽  
Pcr Inhibitors ◽  
Qrt Pcr ◽  
Extraction Protocol ◽  
Viral Particles ◽  
Hands On

Foodborne viruses such as norovirus and hepatitis A virus cause frequent outbreaks associated with the consumption of raw or undercooked oysters. Viral particles are bioaccumulated in the oyster’s digestive glands, making RNA extraction and RT-PCR detection difficult due to the complex nature of the food matrix and the presence of RT-PCR inhibitors. Herein, we have developed a viral RNA extraction protocol from raw oysters using murine norovirus (MNV) as a surrogate for human noroviruses. The method combines lysis in Tri-Reagent reagent, followed by RNA extraction using Direct-Zol purification columns and lithium chloride precipitation. Viral load quantification was performed by both qRT-PCR and droplet-digital RT-PCR. We have demonstrated that this method can efficiently remove RT-PCR inhibitors, and is sensitive enough to reliably detect viral contamination at 25 PFU/0.2 g. We have also compared the efficiency of this method with the ISO 15216-1:2017 method and Method E developed by Quang and colleagues, and observed significantly higher efficiency compared with the ISO 15216-1 method and comparable efficiency with Method E, with less steps, and shorter hands-on time.

scholarly journals Disruption of Type III Interferon (IFN) Genes Ifnl2 and Ifnl3 Recapitulates Loss of the Type III IFN Receptor in the Mucosal Antiviral Response

2019 ◽  
Vol 93 (22) ◽  
Author(s):  
Stefan T. Peterson ◽  
Elizabeth A. Kennedy ◽  
Pamela H. Brigleb ◽  
Gwen M. Taylor ◽  
Kelly Urbanek ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Viral Infections ◽  
Murine Norovirus ◽  
Viral Pathogens ◽  
Genetic Ablation ◽  
Type Iii ◽  
Mucosal Surfaces ◽  
Receptor Interactions ◽  
Type Iii Ifn ◽  
Type Iii Interferon

ABSTRACT Type III interferon (IFN), or IFN lambda (IFN-λ), is an essential component of the innate immune response to mucosal viral infections. In both the intestine and the lung, signaling via the IFN-λ receptor (IFNLR) controls clinically important viral pathogens, including influenza virus, norovirus, and rotavirus. While it is thought that IFN-λ cytokines are the exclusive ligands for signaling through IFNLR, it is not known whether genetic ablation of these cytokines phenotypically recapitulates disruption of the receptor. Here, we report the serendipitous establishment of Ifnl2−/− Ifnl3−/− mice, which lack all known functional murine IFN-λ cytokines. We demonstrate that, like Ifnlr1−/− mice lacking IFNLR signaling, these mice display defective control of murine norovirus, reovirus, and influenza virus and therefore genocopy Ifnlr1−/− mice. Thus, for regulation of viral infections at mucosal sites of both the intestine and lung, signaling via IFNLR can be fully explained by the activity of known cytokines IFN-λ2 and IFN-λ3. Our results confirm the current understanding of ligand-receptor interactions for type III IFN signaling and highlight the importance of this pathway in regulation of mucosal viral pathogens. IMPORTANCE Type III interferons are potent antiviral cytokines important for regulation of viruses that infect at mucosal surfaces. Studies using mice lacking the Ifnlr1 gene encoding the type III interferon receptor have demonstrated that signaling through this receptor is critical for protection against influenza virus, norovirus, and reovirus. Using a genetic approach to disrupt murine type III interferon cytokine genes Ifnl2 and Ifnl3, we found that mice lacking these cytokines fully recapitulate the impaired control of viruses observed in mice lacking Ifnlr1. Our results support the idea of an exclusive role for known type III interferon cytokines in signaling via IFNLR to mediate antiviral effects at mucosal surfaces. These findings emphasize the importance of type III interferons in regulation of a variety of viral pathogens and provide important genetic evidence to support our understanding of the ligand-receptor interactions in this pathway.

Persistence of Hepatitis A Virus in Oysters†

2003 ◽  
Vol 66 (2) ◽  
pp. 331-334 ◽  
Author(s):  
DAVID H. KINGSLEY ◽  
GARY P. RICHARDS
Keyword(s):  
Polymerase Chain Reaction ◽  
Crassostrea Virginica ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Infectious Virus ◽  
Hepatitis A Virus ◽  
Complete Removal ◽  
Chain Reaction ◽  
Daily Feeding ◽  
Polymerase Chain

We investigated the ability of hepatitis A virus (HAV) to persist for up to 6 weeks in Eastern oysters (Crassostrea virginica). Viral RNA was detected by reverse transcription–polymerase chain reaction 6 weeks after 16 h of exposure to 90,000 PFU (180 PFU/ml of seawater) of HAV. Assaying for infectious virus in oysters that received a daily feeding of phytoplankton recovered 3,800, 650, and 500 PFU of HAV 1, 2, and 3 weeks after contamination with 90,000 PFU of HAV, respectively. However, no infectious HAV was isolated from oysters 4, 5, or 6 weeks after contamination. These results support the position that shellfish depuration is insufficient for the complete removal of infectious viruses. Extended relay times (in excess of 4 weeks) may be required to produce virologically safe shellfish.

scholarly journals Three-Year Study To Assess Human Enteric Viruses in Shellfish

2000 ◽  
Vol 66 (8) ◽  
pp. 3241-3248 ◽  
Author(s):  
F. Le Guyader ◽  
L. Haugarreau ◽  
L. Miossec ◽  
E. Dubois ◽  
M. Pommepuy
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Virus Detection ◽  
Enteric Viruses ◽  
Molecular Technique ◽  
Norwalk Virus ◽  
Viral Contamination ◽  
Long Period ◽  
Reverse Transcription Pcr ◽  
Human Enteric Viruses

ABSTRACT The main pathogenic enteric viruses able to persist in the environment, such as hepatitis A virus (HAV), Norwalk-like virus (NLV), enterovirus (EV), rotavirus (RV), and astrovirus (AV), were detected by reverse transcription-PCR and hybridization in shellfish during a 3-year study. Oyster samples (n = 108), occasionally containing bacteria, were less frequently contaminated, showing positivity for AV (17%), NLV (23%), EV (19%), and RV (27%), whereas mussel samples, collected in areas routinely impacted by human sewage, were more highly contaminated: AV (50%), HAV (13%), NLV (35%), EV (45%), and RV (52%). Sequences obtained from HAV and NLV amplicons showed a great variety of strains, especially for NLV (strains close to Mexico, Snow Mountain Agent, or Norwalk virus). Viral contamination was mainly observed during winter months, although there were some seasonal differences among the viruses. This first study of virus detection over a fairly long period of time suggests that routine analysis of shellfish by a molecular technique is feasible.

scholarly journals Quantification of Hepatitis A Virus in Shellfish by Competitive Reverse Transcription-PCR with Coextraction of Standard RNA

1999 ◽  
Vol 65 (1) ◽  
pp. 322-326 ◽  
Author(s):  
Charlotte Arnal ◽  
Virginie Ferre-Aubineau ◽  
Berangere Mignotte ◽  
Berthe Marie Imbert-Marcille ◽  
Sylviane Billaudel
Keyword(s):  
Tissue Culture ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Internal Standard ◽  
Wild Type ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Reproducible Method ◽  
Dna Enzyme Immunoassay

ABSTRACT To quantify hepatitis A virus (HAV) in experimentally contaminated mussels, we developed an internal standard RNA with a 7-nucleotide deletion for competitive reverse transcription (RT)-PCR. Deposited directly into the sample, this standard was used both as extraction control and as quantification tool. After coextraction and competitive RT-PCR, standard and wild-type products were detected by differential hybridization with specific probes and a DNA enzyme immunoassay. The quantifiable range with this reproducible method was 104 to 107 copies of HAV/gram or 400 to 106 50% tissue culture infective doses/ml.

scholarly journals Capsid Functions of Inactivated Human Picornaviruses and Feline Calicivirus

2003 ◽  
Vol 69 (1) ◽  
pp. 350-357 ◽  
Author(s):  
Suphachai Nuanualsuwan ◽  
Dean O. Cliver
Keyword(s):  
Thermal Inactivation ◽  
Hepatitis A Virus ◽  
The United States ◽  
Proteinase K ◽  
Feline Calicivirus ◽  
Virus Attachment ◽  
Food Borne ◽  
Attachment Sites ◽  
Exceptional Stability

ABSTRACT The exceptional stability of enteric viruses probably resides in their capsids. The capsid functions of inactivated human picornaviruses and feline calicivirus (FCV) were determined. Viruses were inactivated by UV, hypochlorite, high temperature (72°C), and physiological temperature (37°C), all of which are pertinent to transmission via food and water. Poliovirus (PV) and hepatitis A virus (HAV) are transmissible via water and food, and FCV is the best available surrogate for the Norwalk-like viruses, which are leading causes of food-borne and waterborne disease in the United States. The capsids of all 37°C-inactivated viruses still protected the viral RNA against RNase, even in the presence of proteinase K, which contrasted with findings with viruses inactivated at 72°C. The loss of ability of the virus to attach to homologous cell receptors was universal, regardless of virus type and inactivation method, except for UV-inactivated HAV, and so virus inactivation was almost always accompanied by the loss of virus attachment. Inactivated HAV and FCV were captured by homologous antibodies. However, inactivated PV type 1 (PV-1) was not captured by homologous antibody and 37°C-inactivated PV-1 was only partially captured. The epitopes on the capsids of HAV and FCV are evidently discrete from the receptor attachment sites, unlike those of PV-1. These findings indicate that the primary target of UV, hypochlorite, and 72°C inactivation is the capsid and that the target of thermal inactivation (37°C versus 72°C) is temperature dependent.

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