Development of Methods To Detect “Norwalk-Like Viruses” (NLVs) and Hepatitis A Virus in Delicatessen Foods: Application to a Food-Borne NLV Outbreak

ABSTRACT “Norwalk-like viruses” (NLVs) and hepatitis A virus (HAV) are the most common causes of virus-mediated food-borne illness. Epidemiological investigations of outbreaks associated with these viruses have been hindered by the lack of available methods for the detection of NLVs and HAV in foodstuffs. Although reverse transcription (RT)-PCR methods have been useful in detecting NLVs and HAV in bivalve mollusks implicated in outbreaks, to date such methods have not been available for other foods. To address this need, we developed a method to detect NLVs and HAV recovered from food samples. The method involves washing of food samples with a guanidinium-phenol-based reagent, extraction with chloroform, and precipitation in isopropanol. Recovered viral RNA is amplified with HAV- or NLV-specific primers in RT-PCRs, using a viral RNA internal standard control to identify potential sample inhibition. By this method, 10 to 100 PCR units (estimated to be equivalent to 102 to 103 viral genome copies) of HAV and Norwalk virus seeded onto ham, turkey, and roast beef were detected. The method was applied to food samples implicated in an NLV-associated outbreak at a university cafeteria. Sliced deli ham was positive for a genogroup II NLV as determined by using both polymerase- and capsid-specific primers and probes. Sequence analysis of the PCR-amplified capsid region of the genome indicated that the sequence was identical to the sequence from virus detected in the stools of ill students. The developed method is rapid, simple, and efficient.

Download Full-text

Update: A food-borne outbreak of hepatitis A in the Netherlands related to semi-dried tomatoes in oil, January-February 2010

Eurosurveillance ◽

10.2807/ese.15.20.19572-en ◽

2010 ◽

Vol 15 (20) ◽

Author(s):

M Petrignani ◽

M Harms ◽

L Verhoef ◽

R van Hunen ◽

C Swaan ◽

...

Keyword(s):

The Netherlands ◽

Odds Ratio ◽

Hepatitis A ◽

Case Control Study ◽

Hepatitis A Virus ◽

Case Control ◽

Food Samples ◽

Trace Back ◽

Food Borne ◽

Control Study

Between 31 December 2009 and 10 February 2010, 13 patients were infected by an identical hepatitis A virus strain not previously detected in the Netherlands. They had not been abroad and were widely distributed over the Netherlands. A case-control study including 12 cases and 44 controls identified semi-dried tomatoes in oil as the source of the outbreak (odds ratio: 20.0; 95% confidence interval: 1.5-274). The virus was not detected in any of 81 tested food samples. International trace-back is still ongoing.

Download Full-text

Contamination of Foods by Food Handlers: Experiments on Hepatitis A Virus Transfer to Food and Its Interruption

Applied and Environmental Microbiology ◽

10.1128/aem.66.7.2759-2763.2000 ◽

2000 ◽

Vol 66 (7) ◽

pp. 2759-2763 ◽

Cited By ~ 138

Author(s):

S. Bidawid ◽

J. M. Farber ◽

S. A. Sattar

Keyword(s):

Hepatitis A ◽

Infectious Virus ◽

Hepatitis A Virus ◽

Infectious Hepatitis ◽

Topical Agent ◽

Food Handlers ◽

Virus Removal ◽

Virus Transfer ◽

Food Borne ◽

Water Rinsing

ABSTRACT Hepatitis A virus (HAV) is an important pathogen which has been responsible for many food-borne outbreaks. HAV-excreting food handlers, especially those with poor hygienic practices, can contaminate the foods which they handle. Consumption of such foods without further processing has been known to result in cases of infectious hepatitis. Since quantitative data on virus transfer during contact of hands with foods is not available, we investigated the transfer of HAV from artificially contaminated fingerpads of adult volunteers to pieces of fresh lettuce. Touching the lettuce with artificially contaminated fingerpads for 10 s at a pressure of 0.2 to 0.4 kg/cm2resulted in transfer of 9.2% Ã¯Â¿Â½ 0.9% of the infectious virus. The pretreatments tested to interrupt virus transfer from contaminated fingerpads included (i) hard-water rinsing and towel drying, (ii) application of a domestic or commercial topical agent followed by water rinsing and towel drying, and (iii) exposure to a hand gel containing 62% ethanol or 75% liquid ethanol without water rinsing or towel drying. When the fingerpads were treated with the topical agents or alcohol before the lettuce was touched, the amount of infectious virus transferred to lettuce was reduced from 9.2% to between 0.3 and 0.6% (depending on the topical agent used), which was a reduction in virus transfer of up to 30-fold. Surprisingly, no virus transfer to lettuce was detected when the fingerpads were rinsed with water alone before the lettuce was touched. However, additional experiments with water rinsing in which smaller volumes of water were used (1 ml instead of 15 ml) showed that the rate of virus transfer to lettuce was 0.3% Ã¯Â¿Â½ 0.1%. The variability in virus transfer rates following water rinsing may indicate that the volume of water at least in part influences virus removal from the fingerpads differently, a possibility which should be investigated further. This study provided novel information concerning the rate of virus transfer to foods and a model for investigating the transfer of viral and other food-borne pathogens from contaminated hands to foods, as well as techniques for interrupting such transfer to improve food safety.

Download Full-text

Outbreaks of Shellfish-Associated Enteric Virus Illness in the United States: Requisite for Development of Viral Guidelines

Journal of Food Protection ◽

10.4315/0362-028x-48.9.815 ◽

1985 ◽

Vol 48 (9) ◽

pp. 815-823 ◽

Cited By ~ 79

Author(s):

GARY P. RICHARDS

Keyword(s):

United States ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Enteric Virus ◽

The United States ◽

Viral Gastroenteritis ◽

Norwalk Virus ◽

Virus Contamination ◽

Pathogenic Viruses

Outbreaks of hepatitis A, Norwalk illness, and nonspecific viral gastroenteritis are associated with consumption of sewage-contaminated shellfish. Over 100 outbreaks have been reported in the United States during the past 50 years. Reported cases of shellfish-associated enteric virus illness are on the increase, whereas bacterial illness from shellfish is on the decline. As yet, there are no procedures for detecting hepatitis A virus, Norwalk virus and numerous other pathogenic viruses in environmental samples, but virus extraction and assay procedures for water and shellfish are available for the more easily cultivated enteric viruses. Current standards rely on bacterial indicators as a means to evaluate the sanitary quality of shellfish and their growing waters, but the adequacy of using bacteria as indicators of possible virus contamination is questionable. The feasibility of employing enteroviruses or rotaviruses as possible viral indiators is discussed. It is proposed that easily cultivated enteroviruses, such as poliovirus, be used as an interim indicator for the possible presence of human pathogenic viruses in seafoods, with the subsequent formulation of guidelines to limit the levels of virus contamination in shellfish.

Download Full-text

Concentration and purification of beef extract mock eluates from water samples for the detection of enteroviruses, hepatitis A virus, and Norwalk virus by reverse transcription-PCR.

Applied and Environmental Microbiology ◽

10.1128/aem.61.2.531-537.1995 ◽

1995 ◽

Vol 61 (2) ◽

pp. 531-537 ◽

Cited By ~ 106

Author(s):

K J Schwab ◽

R De Leon ◽

M D Sobsey

Keyword(s):

Reverse Transcription ◽

Water Samples ◽

Hepatitis A ◽

Hepatitis A Virus ◽

Norwalk Virus ◽

Reverse Transcription Pcr ◽

Beef Extract

Download Full-text

Three-Year Study To Assess Human Enteric Viruses in Shellfish

Applied and Environmental Microbiology ◽

10.1128/aem.66.8.3241-3248.2000 ◽

2000 ◽

Vol 66 (8) ◽

pp. 3241-3248 ◽

Cited By ~ 193

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.

Download Full-text

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

Applied and Environmental Microbiology ◽

10.1128/aem.65.1.322-326.1999 ◽

1999 ◽

Vol 65 (1) ◽

pp. 322-326 ◽

Cited By ~ 19

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.

Download Full-text

Hepatitis A virus proteinase 3C binding to viral RNA: correlation with substrate binding and enzyme dimerization

Biochemical Journal ◽

10.1042/bj20041153 ◽

2005 ◽

Vol 385 (2) ◽

pp. 363-370 ◽

Cited By ~ 19

Author(s):

Hannelore PETERS ◽

Yuri Y. KUSOV ◽

Sonja MEYER ◽

Andrew J. BENIE ◽

Englbert BÄUML ◽

...

Keyword(s):

Life Cycle ◽

Proteolytic Activity ◽

Hepatitis A ◽

Kinetic Resolution ◽

Rna Binding ◽

Hepatitis A Virus ◽

Viral Life Cycle ◽

Binding Kinetics ◽

Viral Rna ◽

Binding Studies

Proteinase 3C of hepatitis A virus (HAV) plays a key role in the viral life cycle by generating mature viral proteins from the precursor polyprotein. In addition to its proteolytic activity, 3C binds to viral RNA, and thus influences viral genome replication. In order to investigate the interplay between proteolytic activity and RNA binding at the molecular level, we subjected HAV 3C and three variants carrying mutations of the cysteine residues [C24S (Cys-24→Ser), C172A and C24S/C172A] to proteolysis assays with peptide substrates, and to surface plasmon resonance binding studies with peptides and viral RNA. We report that the enzyme readily forms dimers via disulphide bridges involving Cys-24. Dissociation constants (KD) for peptides were in the millimolar range. The binding kinetics for the peptides were characterized by kon and koff values of the order of 102 M−1·s−1 and 10−2 to 10−1 s−1 respectively. In contrast, 3C binding to immobilized viral RNA, representing the structure of the 5′-terminal domain, followed fast binding kinetics with kon and koff values beyond the limits of the kinetic resolution of the technique. The affinity of viral RNA depended strongly on the dimerization status of 3C. Whereas monomeric 3C bound to the viral RNA with a KD in the millimolar range, dimeric 3C had a significantly increased binding affinity with KD values in the micromolar range. A model of the 3C dimer suggests that spatial proximity of the presumed RNA-binding motifs KFRDI is possible. 3C binding to RNA was also promoted in the presence of substrate peptides, indicating co-operativity between RNA binding and protease activity. The data imply that the dual functions of 3C are mutually dependent, and regulate protein and RNA synthesis during the viral life cycle.

Download Full-text