Concentration and detection of hepatitis A virus and rotavirus from shellfish by hybridization tests.

A modified polyethylene glycol precipitation method for concentration of virus followed by a new method to recover nucleic acid was used to detect hepatitis A virus (HAV) and rotavirus (SA11) in shellfish (oysters and hard-shell clams) by hybridization tests. Infectious virus, seeded into relatively large quantities of shellfish, was recovered consistently, with greater than 90% efficiency as measured by either in situ hybridization (HAV) or plaque assay (rotavirus SA11). Viral nucleic acid for dot blot hybridization assays was extracted and purified from virus-containing polyethylene glycol concentrates. Separation of shellfish polysaccharides from nucleic acid was necessary before viral RNA could be detected by dot blot hybridization. Removal of shellfish polysaccharides was accomplished by using the cationic detergent cetyltrimethylammonium bromide (CTAB). Use of CTAB reduced background interference with hybridization signals, which resulted in increased hybridization test sensitivity. After polysaccharide removal, dot blot hybridization assays could detect approximately 10(6) physical particles (corresponding to approximately 10(3) infectious particles) of HAV and 10(4) PFU of SA11 rotavirus present in 20-g samples of oyster and clam meats. These studies show continuing promise for the development of uniform methods to directly detect human viral pathogens in different types of shellfish. However, practical applications of such methods to detect noncultivatable human viral pathogens of public health interest will require additional improvements in test sensitivity.     

Comparison of immunological and molecular hybridization detection methods for the detection of hepatitis A virus in sewage

Immune electron microscopy (IEM), radioimmunoassay (RIA) and molecular hybridization with a digoxigenin-labelled cDNA probe were compared for the detection of wild-type human hepatitis A virus (HAV) in raw and treated sewage. In the same experiments, classic tests for culturable enteroviruses were carried out. With the hybridization probes, HAV was detected in three of the 13 amuent samples (23%) and in eight out of 13 effluent samples (61%). For four of the emuent samples, positivity revealed by IEM was confirmed by the cDNA probe. In contrast, two of the samples shown as positive by IEM were negative with the probes. Detection of HAV by RIA was negative in all cases. Demonstration of HAV was higher in emuent than in affluent. No particular relationship was established between demonstration of HAV, on the one hand, and the various concentrations of enteroviruses observed in the same samples on the other. Overall, if all the results, irrespective of the type of water (affluent or effluent), are taken together, 50% of the sewage samples tested were found to contain HAV by one or another method of detection.     

The detection of rotavirus RNA in nasopharyngeal smears by molecular hybridization]

Rotavirus RNA was detected in nasopharyngeal washings, obtained from patients with rotavirus gastroenteritis (76.3% of cases) and gastroenteritis of unknown etiology (34.7% of cases), by the method of molecular hybridization with the use of RNA of monkey virus SA11, labeled with 32P in the process of T4-ligase reaction. In 100% of cases this detection correlated with the presence of lesions of the upper respiratory ways and was probably indicative of the rotavirus nature of the catarrhal syndrome in rotavirus gastroenteritis.     

Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes.

The development and trials of a nucleic acid hybridization test for the detection of hepatitis A virus (HAV) in estuarine samples within 48 h are described. Approximately 10(4) physical particles of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hybridization stringency, 32P energy level, probe concentration, and nucleic acid binding to filters. Test specificity was shown by a lack of cross-hybridization with other enteroviruses and unrelated nucleic acids. Potential false-positive reactions between bacterial DNA in samples and residual vector DNA contamination of purified nucleotide sequences in probes were eliminated by DNase treatment of samples. Humic acid at concentrations of up to 100 mg/liter caused only insignificant decreases in test sensitivity. Interference with hybridization by organic components of virus-containing eluates was removed by proteinase K digestion followed by phenol extraction and ethanol precipitation. The test is suitable for detecting naturally occurring HAV in samples from polluted estuarine environments.     

A bacteriophage M13 based sandwich hybridization assay for the detection of hepatitis B virus in human blood.

A two step hybridization procedure was developed to detect the presence of hepatitis B virus in blood samples using bacteriophage M13 radiolabelled DNA as probe. During the first step of hybridization, single-stranded bacteriophage M13 tg 130 DNA, with 3.2 kb HBV DNA cloned into it, was hybridized to target HBV DNA immobilized on nitrocellulose membrane filter. In the second step of hybridization, M13 DNA annealed to HBV target is detected with the help of double stranded form of M13 DNA. The assay offers minimum 4- to 6-fold higher sensitivity in comparison to single-step conventional hybridization assays. Additionally M13 DNA offers itself as universal probe.     

A comparison of enzyme-immunoassay and radioimmunoassay for detection of hepatitis A virus and antibodies against hepatitis A virus

A direct comparison has been made of tracers labelled with an enzyme and with 125I in solid phase enzyme-immunoassay (EIA) and solid phase radioimmunoassay (RIA) for the detection of hepatitis A virus (HAV) antigen and antibodies to HAV. By comparing the binding capacity of peroxidase-labelled anti-HAV-IgG and anti-HAV-F(ab)2 fragments tracers, anti-HAV-IgG was found to have a higher binding capacity than anti-HAV-F(ab)2 fragments in both EIA and RIA. For EIA 16.25-fold more anti-HAV-IgG was needed for one test probe compared to RIA and 32.5-fold more anti-HAV-F(ab)2 fragments. For the detection of HAV antigen from stool preparations and IgG and IgM antibodies against HAV, there were only minor quantitative differences in titre. EIA was as sensitive as RIA.     

Use of rotavirus virus-like particles as surrogates to evaluate virus persistence in shellfish

Rotavirus virus-like particles (VLPs) and MS2 bacteriophages were bioaccumulated in bivalve mollusks to evaluate viral persistence in shellfish during depuration and relaying under natural conditions. Using this nonpathogenic surrogate virus, we were able to demonstrate that about 1 log10 of VLPs was depurated after 1 week in warm seawater (22 degrees C). Phage MS2 was depurated more rapidly (about 2 log10 in 1 week) than were VLPs, as determined using a single-compartment model and linear regression analysis. After being relayed in the estuary under the influence of the tides, VLPs were detected in oysters for up to 82 days following seeding with high levels of VLPs (concentration range between 10(10) and 10(9) particles per g of pancreatic tissue) and for 37 days for lower contamination levels (10(5) particles per g of pancreatic tissue). These data suggest that viral particles may persist in shellfish tissues for several weeks.     

Detection of hepatitis A virus in seeded estuarine samples by hybridization with cDNA probes

The development and trials of a nucleic acid hybridization test for the detection of hepatitis A virus (HAV) in estuarine samples within 48 h are described. Approximately 10(4) physical particles of HAV per dot could be detected. Test sensitivity was optimized by the consideration of hybridization stringency, 32P energy level, probe concentration, and nucleic acid binding to filters. Test specificity was shown by a lack of cross-hybridization with other enteroviruses and unrelated nucleic acids. Potential false-positive reactions between bacterial DNA in samples and residual vector DNA contamination of purified nucleotide sequences in probes were eliminated by DNase treatment of samples. Humic acid at concentrations of up to 100 mg/liter caused only insignificant decreases in test sensitivity. Interference with hybridization by organic components of virus-containing eluates was removed by proteinase K digestion followed by phenol extraction and ethanol precipitation. The test is suitable for detecting naturally occurring HAV in samples from polluted estuarine environments.     

A structural protein of hepatitis C virus expressed in E. coli facilitates accurate detection of hepatitis C virus.

A putative core protein derived from hepatitis C virus was expressed in E. coli. More than 5% of the total protein expressed in the bacteria after induction by isopropylthio-beta-D-galactoside was shown to be the expected protein. Western blotting with this E. coli lysate proved to be more efficient than ELISA with a non-structural viral protein, C100, to detect infection of hepatitis C virus in the sera of patients with non-A, non-B chronic hepatitis, hepatocellular carcinoma as well as in sera from healthy persons.     

Determination of hepatitis B virus DNA in serum by molecular hybridization

Hepatitis B virus (HBV) DNA was detected by direct spotting of alkali-denatured serum on a nitrocellulose filter and molecular hybridization with cloned HBV DNA as the probe. Measurement of the autoradiographic signals as the intensity of hybridization allowed the quantitation of HBV DNA content in serum specimens in reference to cloned HBV DNA. Direct spotting of denatured serum was approximately three times as sensitive as the conventional method in which proteinase-treated serum was extracted with phenol-chloroform. The intensity of hybridization with 25 specimens of HB virion concentrates correlated well with DNA polymerase activity (r = 0.89, P less than 0.01).     

An improved reverse dot hybridization for simple and rapid detection of adefovir dipivoxil-resistant hepatitis B virus

Early detection of adefovir dipivoxil-resistant mutants during long-term treatment of chronic hepatitis B virus (HBV) infection with this drug is of great clinical importance. We developed an improved reverse dot hybridization test for simple and rapid detection of the rtA181V/T and rtN236T mutations associated with adefovir dipivoxil resistance in chronic hepatitis B patients. Probes were designed for genotypes B, C, and D of this resistance characteristic; a total of 70 clinical samples were analyzed with this improved reverse dot hybridization assay. Its usefulness was validated by comparing with sequencing data. Discordant results were confirmed by subclone sequencing. This reverse dot hybridization assay was sufficiently sensitive to detect 10(3) copies/mL; it also detected adefovir dipivoxil-resistant mutant strains when they comprised more than 5% of a mixed virus population. This reverse dot hybridization array correctly identified adefovir dipivoxil-resistant mutants; it had high concordance (98.5%) with direct sequencing data. There was no clear relationship between the HBV genotype and the development of adefovir dipivoxil-resistant mutants. This reverse dot hybridization assay proved to be simple and rapid for detection of rtA181V/T and rtN236T mutations associated with resistance to adefovir dipivoxil.     

Purification of hepatitis A virus from chimpanzee stools.

Hepatitis A virus antigen was purified from early acute-phase chimpanzee stools by a rapid three-step procedure using 7% polyethylene glycol precipitation, CsCl banding, and Sepharose 2B column chromatography. Electron microscopic examination of the hepatitis A virus entigen preparation revealed highly purified hepatitis A virus particles.     

Persistence of mouse hepatitis virus A59 RNA in a slow virus demyelinating infection in mice as detected by in situ hybridization.

Mouse hepatitis virus strain A59 produces chronic central nervous system demyelination in rodents. As late as 6 months after intracerebral inoculation of mice 4 to 6 weeks old, when infectious virus cannot be recovered and viral antigens cannot be detected in the central nervous systems and livers of these animals, primary demyelination is still evident. Using cloned virus-specific DNAs and the highly sensitive and specific technique of in situ hybridization, we have detected low levels of mouse hepatitis virus A59 RNA in the central nervous systems and livers of mice 10 months after inoculation. We suggest that viral persistence may play a role in mouse hepatitis virus A59-induced chronic demyelination.