scholarly journals Hepatitis A Virus PCR Analysis and <i>E. coli</i> Detection in Oysters at Oualidia Lagoon and Their Correlation

2020 ◽  
Vol 11 (07) ◽  
pp. 884-894
Author(s):  
Naima El Moqri ◽  
Najwa Hassou ◽  
Fatiha El Mellouli ◽  
Hasnae Zekhnini ◽  
Nabil Abouchoaib ◽  
...  
Keyword(s):  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Pcr Analysis ◽  
E Coli ◽  
Oualidia Lagoon

A Polymerase Chain Reaction–Based Method for the Detection of Hepatitis A Virus in Produce and Shellfish

2002 ◽  
Vol 65 (2) ◽  
pp. 393-402 ◽  
Author(s):  
BISWENDU B. GOSWAMI ◽  
MICHAEL KULKA ◽  
DIANA NGO ◽  
PHILLIP ISTAFANOS ◽  
THOMAS A. CEBULA
Keyword(s):  
Polymerase Chain Reaction ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
General Procedure ◽  
Pcr Analysis ◽  
Genomic Rna ◽  
Chain Reaction ◽  
Viral Origin ◽  
Polymerase Chain ◽  
Test Virus

Outbreaks of gastroenteritis that are suspected to be of viral origin are on the rise. Thus, there is a need for regulatory agencies entrusted with food safety to develop adequate techniques for the detection of viruses in foods. We have established a general procedure for the detection of hepatitis A virus (HAV) in shellfish that, with minor modifications, is also applicable to fresh produce such as cilantro. Total RNA was isolated from shellfish or cilantro, followed by isolation of poly(A)-containing RNA. Because HAV genomic RNA contains a poly(A) tail, the isolation of poly(A)-containing RNA also enriches HAV genomic RNA. Reverse transcription was used to convert the RNA to cDNA, and then amplification was carried out by polymerase chain reaction (PCR). Reamplification with internal primers was used to improve the quality and the quantity of amplified DNA, allowing for post-PCR analysis such as sequence identification of the viral strain. With this procedure, multiple samples could be analyzed in four working days by a single trained individual. The nominal sensitivity of detection of the procedure was 0.15 TCID50 (50% tissue culture infective dose) per 0.62 g of tissue with a test virus. The direct RNA isolation protocol avoided pitfalls associated with whole-virus purification procedures by replacing virus precipitation steps involving polyethylene glycol and Procipitate with phenol extraction. The method is straightforward and reliable. We successfully used this procedure to detect naturally occurring HAV in clams involved in a gastroenteritis outbreak, as well as in cilantro artificially contaminated with a test virus.

scholarly journals Occurrence of norovirus and other enteric viruses in untreated groundwaters of Korea

2011 ◽  
Vol 9 (3) ◽  
pp. 544-555 ◽  
Author(s):  
Ji Hee Jung ◽  
Chang Hoon Yoo ◽  
Eung Seo Koo ◽  
Hak Min Kim ◽  
Youjin Na ◽  
...  
Keyword(s):  
Hepatitis A ◽  
Heterotrophic Bacteria ◽  
Hepatitis A Virus ◽  
Enteric Virus ◽  
Additional Treatment ◽  
Total Coliforms ◽  
E Coli ◽  
Virus Contamination ◽  
Three Samples ◽  
Groundwater Wells

A total of 39 water samples from 23 different groundwater wells in Korea were collected and analyzed in order to monitor the occurrence of norovirus (NoV) and other indicator microbes as the first part of a national survey of groundwater. More than 500 L of untreated groundwater were filtered through 1MDS filters. Following elution and concentration by organic flocculation, PCR and sequence analysis were employed to detect and identify NoV, enterovirus, rotavirus, hepatitis A virus and adenovirus (Adv). Somatic and F-specific phages, heterotrophic bacteria, total coliforms and Escherichia coli were also analyzed to infer possible fecal contamination. NoVs were detected in 18% of the 39 samples. Five out of seven NoV-positive samples (71%) were identified as GI while the other two (29%) were GII. Enteroviruses and Advs were detected in two and three samples, respectively. Rotavirus and hepatitis A virus were not detected. Total coliforms, E. coli and coliphages were detected in 49, 15 and 13% of the samples, respectively, but did not appear to be suitable indicators of enteric virus contamination in groundwater. These results suggest that additional treatment may be needed for a significant number of groundwaters prior to use as drinking water.

scholarly journals Alteration of Hepatitis A Virus (HAV) Particles by a Soluble Form of HAV Cellular Receptor 1 Containing the Immunoglobulin- and Mucin-Like Regions

2003 ◽  
Vol 77 (16) ◽  
pp. 8765-8774 ◽  
Author(s):  
Erica Silberstein ◽  
Li Xing ◽  
Willem van de Beek ◽  
Jinhua Lu ◽  
Holland Cheng ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Soluble Form ◽  
Pcr Analysis ◽  
Cellular Receptor ◽  
African Green Monkey Kidney ◽  
Negative Stain ◽  
Electron Microscopy Analysis ◽  
Human Igg1

ABSTRACT Hepatitis A virus (HAV) infects African green monkey kidney cells via HAV cellular receptor 1 (havcr-1). The ectodomain of havcr-1 contains an N-terminal cysteine-rich immunoglobulin-like region (D1), followed by a mucin-like region that extends D1 well above the cell surface. D1 is required for binding of HAV, and a soluble construct containing D1 fused to the hinge and Fc portions of human immunoglobulin G1 (IgG1), D1-Fc, bound and neutralized HAV inefficiently. However, D1-Fc did not alter the virions. To determine whether additional regions of havcr-1 are required to trigger uncoating of HAV, we constructed D1muc-Fc containing D1 and two-thirds of the mucin-like region fused to the Fc and hinge portions of human IgG1. D1muc-Fc neutralized 10 times more HAV than did D1-Fc. Sedimentation analysis in sucrose gradients showed that treatment of HAV with 20 to 200 nM D1muc-Fc disrupted the majority of the virions, whereas treatment with 2 nM D1muc-Fc had no effect on the sedimentation of the particles. Treatment of HAV with 100 nM D1muc-Fc resulted in low-level accumulation of 100- to 125S particles. Negative-stain electron microscopy analysis revealed that the 100- to 125S particles had the characteristics of disrupted virions, such as internal staining and diffuse edges. Quantitative PCR analysis showed that the 100- to 125S particles contained viral RNA. These results indicate that D1 and the mucin-like region of havcr-1 are required to induce conformational changes leading to HAV uncoating.

scholarly journals Hepatitis A virus polyprotein processing by Escherichia coli proteases

2002 ◽  
Vol 83 (2) ◽  
pp. 359-368 ◽  
Author(s):  
Rosa M. Pintó ◽  
Susana Guix ◽  
Juan F. González-Dankaart ◽  
Santiago Caballero ◽  
Gloria Sánchez ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Study Data ◽  
E Coli ◽  
Polyprotein Processing ◽  
Capsid Formation ◽  
Bacterial Protease ◽  
Low Efficiency ◽  
Virus Polyprotein

Hepatitis A virus (HAV) encodes a single polyprotein, which is post-translationally processed. This processing represents an essential step in capsid formation. The virus possesses only one protease, 3C, responsible for all cleavages, except for that at the VP1/2A junction region, which is processed by cellular proteases. In this study, data demonstrates that HAV polyprotein processing by Escherichia coli protease(s) leads to the formation of particulate structures. P3 polyprotein processing in E. coli is not dependent on an active 3C protease: the same processing pattern is observed with wild-type 3C or with several 3C mutants. However, this processing pattern is temperature-dependant, since it differs at 37 or 42 °C. The bacterial protease(s) cleave scissile bonds other than those of HAV; this contributes to the low efficiency of particle formation.

scholarly journals A Strategy for Detection of Viruses in Groundwater by PCR

1999 ◽  
Vol 65 (2) ◽  
pp. 444-449 ◽  
Author(s):  
Morteza Abbaszadegan ◽  
Peter Stewart ◽  
Mark LeChevallier
Keyword(s):  
Cell Culture ◽  
Large Volume ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Processing Technique ◽  
Molecular Techniques ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Sample Processing ◽  
Specific Primers

ABSTRACT We evaluated the use of the PCR for detection of enteric viruses in groundwater. To do this, we used an improved sample-processing technique and a large-volume amplification protocol. The objective of this study was to use advanced molecular techniques to develop a rapid and simple method which can be used by the water industry for detection of viral contamination in a variety of water samples. The strategy described here fulfills the water industry’s need for a rapid, reliable, easily performed method for analyzing groundwater for virus contamination. Viruses were detected after concentration from at least 400 gallons (1,512 liters) of water by a filter adsorption and elution method, which resulted in a concentrate containing viruses. A total of 150 samples were analyzed by performing cell culture assays for enteroviruses and by performing reverse transcription PCR (RT-PCR) analyses for enteroviruses, hepatitis A virus, and rotavirus. Thirteen samples (8.7%) produced cellular cytopathic effects when the Buffalo green monkey cell line was used. When primers specific for enteroviruses were used in RT-PCR, 40 of 133 samples (30.1%) tested positive for the presence of enterovirus RNA. When hepatitis A virus-specific primers were used, 12 of 139 samples (8.6%) were considered positive for the presence of hepatitis A viral RNA. The RT-PCR analysis performed with rotavirus-specific primers identified 18 of 130 samples (13.8%) that were positive for rotavirus RNA sequences. Our sample-processing technique and large-volume PCR protocol (reaction volume, 300 μl) resulted in sufficient removal or dilution of inhibitors so that more than 95% of the samples could be assayed by PCR. Because of its sensitivity for detecting viral nucleic acid sequences, PCR analysis should produce more positive results than cell culture analysis. Since either cell culture analysis or PCR can reveal only a “snapshot” of the quality of the groundwater being sampled, PCR seems to be a desirable rapid initial screening tool.

Application of solid-phase immune electron microscopy to study physical and stability characteristics of enterically transmitted non-a, non-b hepatitis virus

1988 ◽  
Vol 46 ◽  
pp. 80-81
Author(s):  
Charles D. Humphrey ◽  
E. H. Cook ◽  
Karen A. McCaustland ◽  
Daniel W. Bradley
Keyword(s):  
Electron Microscopy ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Solid Phase ◽  
Etiologic Agent ◽  
World Health ◽  
Health Concern ◽  
Morphological Identification ◽  
Immune Electron Microscopy

Enterically transmitted non-A, non-B hepatitis (ET-NANBH) is a type of hepatitis which is increasingly becoming a significant world health concern. As with hepatitis A virus (HAV), spread is by the fecal-oral mode of transmission. Until recently, the etiologic agent had not been isolated and identified. We have succeeded in the isolation and preliminary characterization of this virus and demonstrating that this agent can cause hepatic disease and seroconversion in experimental primates. Our characterization of this virus was facilitated by immune (IEM) and solid phase immune electron microscopic (SPIEM) methodologies.Many immune electron microscopy methodologies have been used for morphological identification and characterization of viruses. We have previously reported a highly effective solid phase immune electron microscopy procedure which facilitated identification of hepatitis A virus (HAV) in crude cell culture extracts. More recently we have reported utilization of the method for identification of an etiologic agent responsible for (ET-NANBH).

Hepatitis A Antigen in Liver, Bile and Stool

1975 ◽  
Vol 33 ◽  
pp. 340-341
Author(s):  
D.R. Jackson ◽  
J.H. Hoofnagle ◽  
A.N. Schulman ◽  
J.L. Dienstag ◽  
R.H. Purcell ◽  
...  
Keyword(s):  
Hepatitis A ◽  
Liver Enzymes ◽  
Hepatitis A Virus ◽  
Type A ◽  
Initial Study ◽  
Virus Like Particle ◽  
Elevated Liver Enzymes ◽  
Ag Particles ◽  
Ag Particle ◽  
Human Stool

Using immune electron microscopy Feinstone et. al. demonstrated the presence of a 27 nm virus-like particle in acute-phase stools of patients with viral hepatitis, type A, These hepatitis A antigen (HA Ag) particles were aggregated by convalescent serum from patients with type A hepatitis but not by pre-infection serum. Subsequently Dienstag et. al. and Maynard et. al. produced acute hepatitis in chimpanzees by inoculation with human stool containing HA Ag. During the early acute disease, virus like particles antigenically, morphologically and biophysically identical to the human HA Ag particle were found in chimpanzee stool. Recently Hilleman et. al. have described similar particles in liver and serum of marmosets infected with hepatitis A virus (HAV). We have investigated liver, bile and stool from chimpanzees and marmosets experimentally infected with HAV. In an initial study, a chimpanzee (no.785) inoculated with HA Ag-containing stool developed elevated liver enzymes 21 days after exposure.

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