scholarly journals Detection of Hepatitis A Virus by Using a Combined Cell Culture-Molecular Beacon Assay

2008 ◽  
Vol 74 (7) ◽  
pp. 2239-2243 ◽  
Author(s):  
Hsiao-Yun Yeh ◽  
Yu-Chen Hwang ◽  
Marylynn V. Yates ◽  
Ashok Mulchandani ◽  
Wilfred Chen
Keyword(s):  
Cell Culture ◽  
Hepatitis A ◽  
Molecular Beacon ◽  
Hepatitis A Virus ◽  
Mammalian Cell Culture ◽  
Fluorescence Signal ◽  
Cytopathic Effects ◽  
Infected Cells ◽  
Fetal Rhesus Monkey Kidney ◽  
Fluorescent Cells

ABSTRACT Rapid and efficient methods for the detection and quantification of infectious viruses are required for public health risk assessment. Current methods to detect infectious viruses are based on mammalian cell culture and rely on the production of visible cytopathic effects (CPE). For hepatitis A virus (HAV), viral replication in cell culture has been reported to be nonlytic and relatively slow. It may take more than 1 week to reach the maximum production and subsequent visualization of CPE. A molecular beacon (MB), H1, specifically targeting a 20-bp 5′ noncoding region of HAV, was designed and synthesized. MB H1 was introduced into fixed and permeabilized fetal rhesus monkey kidney (FRhK-4) cells infected with HAV strain HM-175. Upon hybridizing with the viral mRNA, fluorescent cells were visualized easily under a fluorescence microscope. Discernible fluorescence was detected only in infected cells by using the specific MB H1. A nonspecific MB, which was not complementary to the viral RNA sequence, produced no visible fluorescence signal. This MB-based fluorescence assay enabled the direct counting of fluorescent cells and could achieve a detection limit of 1 PFU at 6 h postinfection, demonstrating a significant improvement in viral quantification over current infectivity assays.

Rapid Detection Method for Hepatitis A Virus from Lettuce by a Combination of Filtration and Integrated Cell Culture–Real-Time Reverse Transcription PCR

2011 ◽  
Vol 74 (10) ◽  
pp. 1756-1761 ◽  
Author(s):  
JI-YEON HYEON ◽  
JUNG-WHAN CHON ◽  
CHANKYU PARK ◽  
JUNG-BOK LEE ◽  
IN-SOO CHOI ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Rt Pcr ◽  
Cytopathic Effects ◽  
Reverse Transcription Pcr ◽  
Charged Membrane ◽  
Polyethylene Glycol Precipitation ◽  
Positively Charged

We have developed a rapid and simple method for filtration using a positively charged membrane to concentrate hepatitis A virus (HAV) from lettuce and an integrated cell culture–real-time reverse transcription PCR (ICC–real-time RT-PCR) to detect infectious HAV. The most suitable buffer for HAV concentration by filtration was 100 mM Tris-HCl, 50 mM glycine (pH 9.5). Filtration using the NanoCeram matrix was compared with polyethylene glycol precipitation for viral concentration from lettuce inoculated with 6 log RNA copies of HAV. The recovery rate of filtration was statistically higher than that of polyethylene glycol precipitation (47.3 versus 24.9%, respectively). The sensitivity of ICC–real-time RT-PCR for detection of infectious HAV was determined by inoculation of FRhK-4 cells with HAV (4 log to 0 log RNA copies). ICC–real-time RT-PCR detected infectious HAV on average 5 days earlier than cytopathic effects at all inoculation levels. HAV recovered from lettuce (approximately 3 log RNA copies) was also analyzed with ICC–real-time RT-PCR. Infectious HAV was detected within 2 days postinfection by ICC–real-time RT-PCR, whereas cytopathic effects were not observed until 7 days postinfection. Coupled with a virus concentration and purification system using a positively charged membrane, ICC–real-time RT-PCR has the potential to become a novel and rapid method for the detection of infectious HAV in vegetables.

Hepatitis a Virus Concentration from Experimentally Contaminated Distilled, Tap, Waste and Seawater

1989 ◽  
Vol 21 (3) ◽  
pp. 255-258 ◽  
Author(s):  
Evangélos Biziagos ◽  
Jacques Passagot ◽  
Jean-Marc Crance ◽  
Robert Deloince
Keyword(s):  
Cell Culture ◽  
Polyethylene Glycol ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Tap Water ◽  
Virus Concentration ◽  
Beef Extract ◽  
Polyethylene Glycol 6000

The concentration of cell-culture-adapted hepatitis A virus (HAV) from experimentally contaminated distilled, drinking, waste and seawater was performed by using a filter adsorption-elu-tion method in the following conditions: HAV seeded in water was adsorbed at pH 4.0 to two nitrocellulose membranes (1.2 and 0.45 µm porosity for distilled and tap water or 8.0 and 3.0 µm porosity for waste and seawater), then eluted by 3% beef-extract at pH 8.5 and further concentrated by polyethylene glycol 6000 precipitation. Thus, HAV in 5 to 50 liters of seeded waters was concentrated approximately 1,700 to 17,000 fold with greater than 70% recovery of the initial virus added to the samples.

Restricted replication of human hepatitis A virus in cell culture: intracellular biochemical studies.

1981 ◽  
Vol 37 (1) ◽  
pp. 216-225 ◽  
Author(s):  
S A Locarnini ◽  
A G Coulepis ◽  
E G Westaway ◽  
I D Gust
Keyword(s):  
Cell Culture ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Human Hepatitis ◽  
Biochemical Studies

Hepatitis A virus cDNA and its RNA transcripts are infectious in cell culture.

1987 ◽  
Vol 61 (10) ◽  
pp. 3035-3039 ◽  
Author(s):  
J I Cohen ◽  
J R Ticehurst ◽  
S M Feinstone ◽  
B Rosenblum ◽  
R H Purcell
Keyword(s):  
Cell Culture ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Rna Transcripts ◽  
Virus Cdna

UV Light Inactivation of Hepatitis A Virus, Aichi Virus, and Feline Calicivirus on Strawberries, Green Onions, and Lettuce

2008 ◽  
Vol 71 (5) ◽  
pp. 908-913 ◽  
Author(s):  
VIVIANA R. FINO ◽  
KALMIA E. KNIEL
Keyword(s):  
Foodborne Pathogens ◽  
Hepatitis A ◽  
Fruits And Vegetables ◽  
Hepatitis A Virus ◽  
Uv Light ◽  
Mammalian Cell Culture ◽  
Aichi Virus ◽  
Feline Calicivirus ◽  
Virus Inactivation ◽  
Need To Evaluate

A majority of illnesses caused by foodborne viruses are associated with fresh produce. Fruits and vegetables may be considered high-risk foods, as they are often consumed raw without a specific inactivation step. Therefore, there is a need to evaluate nonthermal treatments for the inactivation of foodborne pathogens. This study investigates the UV inactivation of three viruses: feline calicivirus (a surrogate for norovirus), and two picornaviruses, hepatitis A virus and Aichi virus. Three produce types were selected for their different surface topographies and association with outbreaks. Green onions, lettuce, and strawberries were individually spot inoculated with 107 to 109 50% tissue culture infective doses (TCID50) of each virus per ml and exposed to UV light at various doses (≤240 mW s/cm2), and viruses were eluted using an optimized recovery strategy. Virus infection was quantified by TCID50 in mammalian cell culture and compared with untreated recovered virus. UV light applied to contaminated lettuce resulted in inactivation of 4.5 to 4.6 log TCID50/ml; for contaminated green onions, inactivation ranged from 2.5 to 5.6 log TCID50/ml; and for contaminated strawberries, inactivation ranged from 1.9 to 2.6 log TCID50/ml for the three viruses tested. UV light inactivation on the surface of lettuce is more effective than inactivation on the other two produce items. Consistently, the lowest results were observed in the inactivation of viruses on strawberries. No significant differences (P > 0.05) for virus inactivation were observed among the three doses applied (40, 120, and 240 mW s/cm2)on the produce, with the exception of hepatitis A virus and Aichi virus inactivation on green onions, where inactivation continued at 120 mW s/cm2 (P < 0.05).

scholarly journals Real-Time Nucleic Acid Sequence-Based Amplification Assay for Detection of Hepatitis A Virus

2005 ◽  
Vol 71 (11) ◽  
pp. 7113-7116 ◽  
Author(s):  
Khaled H. Abd El Galil ◽  
M. A. El Sokkary ◽  
S. M. Kheira ◽  
Andre M. Salazar ◽  
Marylynn V. Yates ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Hepatitis A ◽  
Molecular Beacon ◽  
Hepatitis A Virus ◽  
Immunomagnetic Separation ◽  
Nucleic Acid Sequence ◽  
The Real ◽  
Nasba Assay ◽  
Environmental Pathogens

ABSTRACT A nucleic acid sequence-based amplification (NASBA) assay in combination with a molecular beacon was developed for the real-time detection and quantification of hepatitis A virus (HAV). A 202-bp, highly conserved 5′ noncoding region of HAV was targeted. The sensitivity of the real-time NASBA assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demonstrated by testing with other environmental pathogens and indicator microorganisms, with only HAV positively identified. When combined with immunomagnetic separation, the NASBA assay successfully detected as few as 10 PFU from seeded lake water samples. Due to its isothermal nature, its speed, and its similar sensitivity compared to the real-time RT-PCR assay, this newly reported real-time NASBA method will have broad applications for the rapid detection of HAV in contaminated food or water.

Mutational Events in Consecutive Passages of Hepatitis A Virus Strain GBM during Cell Culture Adaptation

Virology ◽  
1994 ◽  
Vol 204 (1) ◽  
pp. 60-68 ◽  
Author(s):  
Judith Graff ◽  
Christa Kasang ◽  
Andrea Normann ◽  
Mechtild Pfisterer-Hunt ◽  
Stephen M. Feinstone ◽  
...  
Keyword(s):  
Cell Culture ◽  
Virus Strain ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Culture Adaptation
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