Detection of human herpesvirus 6 DNA in serum by a microplate PCR-hybridization assay.

PCR was performed on DNA extracts derived from clinical serum samples submitted for human herpesvirus 6 (HHV-6) serological examination. To detect amplified HHV-6 products, a hybridization-based microtiter plate assay (PCR ELISA; Boehringer Mannheim) was used. The assay system was found to be rapid, specific, and sensitive. Approximately three copies of a plasmid-based HHV-6 sequence could be detected, and no cross amplification was observed with HHV-7 genomic DNA. There was no correlation found between HHV-6 DNA detection and serological status in clinical serum samples from individuals more than 2 years old. On the other hand, in serum samples from infants less than 2 years old, a high rate of detection of HHV-6 DNA was observed in those who lacked immunoglobulin G and M antibodies to HHV-6 (55%). In this regard, PCR of serum DNA extracts may be used as a sensitive indicator of active HHV-6 infection in infants prior to their seroconversion.

Amplicor HIV monitor, NASBA HIV-1 RNA QT and quantiplex HIV RNA version 2.0 viral load assays: a Canadian evaluation.

BACKGROUND: HIV-1 viral load quantitation is now recognized as a useful tool to monitor the efficiency of antiviral treatment and a powerful predictor of disease outcome. Three HIV-1 viral load quantitation methods have been currently available as commercial kits in Canada since 1996. OBJECTIVE: To evaluate the ability to quantify HIV-1 RNA in plasma of the Amplicor HIV Monitor Test, the NASBA HIV-1 RNA QT Assay and the Quantiplex HIV RNA Assay, version 2.0, at comparable lower detection limits. STUDY DESIGN: Blood was collected from 50 HIV-1-infected patients at various stages of infection and therapy. CD4+ cell count were estimated by flow cytometry. Plasma was isolated and tested in duplicate on four occasions using viral load kits from a single lot. HIV RNA data, performance, sensitivity and intra- and inter-assay variability were compared. RESULTS: RNA could be quantified in 33 patients by each technique. An inverse correlation was observed between viral load level and CD4+ cell counts in patients with counts below 200. Monitor could detect RNA in 94% of patients, but it showed the greatest variability and failure rate. Quantiplex 2.0 could detect HIV-1 RNA in 78%, and NASBA in 88% of the patients at theoretically equivalent lower detection limits, suggesting that the detection limit of Quantiplex 2.0 may be higher than 500 HIV-1 RNA copies per ml. NASBA had the fewest invalid tests and good reproducibility, comparable to that of Quantiplex 2.0. The mean values from NASBA and Monitor were the most similar but the best correlation was observed between Monitor and Quantiplex 2.0 results. CONCLUSIONS: Monitor, NASBA and Quantiplex results were comparable, although those obtained by Quantiplex were significantly lower. Performing this study at comparable detection limits showed that the detection limit of Quantiplex 2.0 may be higher than stated by the manufacturer.

Production of a baculovirus-derived gp50 protein and utilization in a competitive enzyme-linked immunosorbent assay for the serodiagnosis of pseudorabies virus.

The pseudorabies virus (PRV) gp50 envelope glycoprotein gene was cloned and expressed in a recombinant baculovirus. An anti-gp50 Mab (1842) recognized a protein of approximately 40 kDa in immunoblotting assays from infected insect cell lysates, while this product was not present in cells infected with wild-type baculovirus. The recombinant protein was purified by lectin affinity chromatography, utilizing lectins specific for O-linked oligosaccharides (Artocarpus integrifolia and Glycine max). Competitive (c) ELISAs, using either crude or lectin-purified antigen, were devised for the detection of antibodies to PRV in sera, and were capable of monitoring sero-conversion by day 14 post-infection. Furthermore, a specificity of 100% and sensitivity of 98% (crude lysate antigen) or 96% (lectin-purified antigen) was found for a panel of 80 swine sera, using the cELISA, as compared to a serum neutralization (SN) test. These studies demonstrated that recombinant PRV gp50 protein shows promise as a cELISA antigen, for serodetection of PRV.

Bnm1, a Brassica pollen-specific gene.

cDNA and genomic clones of a new pollen-specific gene, Bnm1, have been isolated from Brassica napus cv. Topas. The gene contains an open reading frame of 546 bp and a single intron of 362 bp. A comparison of the deduced amino acid sequence with sequences in data banks did not show similarity with known proteins. Northern blot analysis of developing pollen showed that Bnm1 mRNA was first detected in bicellular pollen and accumulated to higher levels in tricellular pollen. Bnm1 mRNA was not detected in leaves, stems, roots, pistils, seeds or pollen-derived embryos. RNA in situ hybridization of whole flower buds confirmed that Bnm1 was pollen-specific and expressed late in development. A promoter fragment of the Bnm1 gene fused to the gusA reporter gene yielded similar patterns of tissue specificity and developmental regulation in transgenic B. napus cv. Westar plants; however, the promoter was also active during the early stages of pollen development. The Bnm1 gene, cloned in this study, was derived from the A genome of the allotetraploid species B. napus (AACC). Southern blot analysis indicated that sequences similar to the Bnm1 gene were found in both A and C Brassica genomes. Related sequences were found in all 10 members of the Brassiceae tribe examined, but were not present in all tribes of the Brassicaceae family.

Amantadine susceptibility in influenza A virus isolates: determination methods and lack of resistance in a Canadian sample, 1991-94.

BACKGROUND: Influenza A infections are an important cause of morbidity and mortality in the elderly and patients affected by chronic diseases or immunodeficiencies. Treatment and prevention of infection in hospitals and nursing homes often involve the use of amantadine, but resistant viruses may arise. OBJECTIVES: To assess the effectiveness of specific and sensitive methods for rapid screening and sequence confirmation of amantadine resistance, and the occurrence of amantadine resistance in recent influenza A virus isolates in Canada. STUDY DESIGN: A chicken antiserum-based enzyme linked immunoassay (ELISA) was developed and used to screen fifty influenza A isolates for amantadine resistance. Drug sensitivity was expressed as a percentage of virus growth inhibition. The efficiency of the assay was compared to that of a monoclonal antibody (mab)-based ELISA using influenza A strains from 1968 to 1994. Specific PCR primers, generated to amplify the M2 gene region where amantadine resistance mutations occur, were tested over a wide range of strains. Direct sequencing of the PCR fragments was performed to confirm the presence of resistance mutations. RESULTS: The polyclonal antiserum-based ELISA detected antigens from all recent H1N1 strains and H3N2 strains tested at an inoculum dilution ten-fold lower than the mab-based ELISA. Primers for the detection of amantadine resistance mutations consistently amplified a wide range of strains. The direct sequencing of the RT-PCR amplicons generated, detected resistance mutations in reassortant and control viruses, the only strains found resistant by ELISA. All influenza A isolates (H3N2, H1N1) tested, except resistant controls and two reassortant viruses, were amantadine-sensitive as indicated by greater than 50% virus growth inhibition. CONCLUSIONS: Influenza A virus susceptibility to amantadine could be detected by using an antiserum-based ELISA, offering a simple and more sensitive alternative to the mab-based assay. Coupled with direct sequencing of the M2 gene, it provides a reliable way to detect and confirm resistance in influenza isolates. However, no resistant clinical isolates were detected in the sample.

Specificity and nucleotyping studies of a gp50-based polymerase chain reaction assay for detection of pseudorabies virus.

PCR primers that amplify a region of the gp50 envelope glycoprotein gene of a number of vaccinal and field strains of pseudorabies virus (PRV) have been previously described (Galeota-Wheeler and Osorio, Am J Vet Res 1991: 52; 1799-1803). This gp50-based PCR assay was tested for its diagnostic applicability, utilizing a panel of nine PRV isolates and 13 related herpesviruses, originating from domestic animal species and man. Slight modifications to the original PRV PCR protocol ensured that false positive PCR products from avian herpesviruses were not evident in agarose gel electrophoresis analysis. Nucleotide sequence data derived from the PCR product revealed that the region of the genome amplified was markedly conserved and allowed only for virus subgrouping, rather than definitive isolate characterization.

Evolution of the hemagglutinin gene of influenza B virus was driven by both positive and negative selection pressures.

Nucleotide sequences of the hemagglutinin (HA) gene of twelve influenza B strains and their deduced amino acid sequences were extracted from the GenBank and compared to those of early isolates. Separate analyses of nonsynonymous and synonymous substitutions for the HA1 and HA2 regions individually indicate that the percentage of nonsynonymous substitutions in the HA1 ranges from 44.4-56.0% but in the HA2 from 0.0-6.5%. The results suggest that positive selection as well as negative selection played a role in the evolution of the influenza B HA gene with the former acting on the HA1 and the latter on the HA2.

Genetic characterization of the hemagglutinin gene of influenza B virus which predominated in the 1985/86 Canadian influenza season.

OBJECTIVE: To characterize the hemagglutinin (HA) gene of B/Canada/3/85, a prototype strain of influenza B virus variants that emerged in the 1984/85 influenza season and predominated in the 1985/86 season in Canada. DESIGN: Sequencing and comparison of the HA genes of B/Canada/3/85 and the vaccine strains for the 1985/86 season, B/USSR/100/83, and for the 1986/87 season, B/Ann Arbor/1/86. RESULTS: B/Canada/3/85 was similar to B/Ann Arbor/1/86 and significantly different from B/USSR/100/83. Phylogenetic analysis of the HA1-coding sequences indicated that B/Canada/3/85 and several other 1985 strains isolated in distant parts of the world were very closely related and were early variants representing the emergence of a new lineage, the B/Victoria/2/87 lineage. B/Canada/3/85 differed from B/USSR/100/83 in nucleotide sequence by 3.44% and in amino acid sequence by 3.33%. There was also an insertion of two amino acids in the HA1 region of B/Canada/3/85. CONCLUSIONS: B/Canada/3/85 was one of the herald strains for the 1985/1986 influenza B epidemic. The amino acid mutations and the two-codon insertion together may account for the observed antigenic changes in the HA of the influenza B variants.

Evaluation of nucleic acid amplification methods for the detection of hog cholera virus.

A blind panel was tested in a diagnostic evaluation of a reverse transcription (RT) polymerase chain reaction (PCR) method for detecting hog cholera virus (HCV) from pig tissues. The capability of the RT-PCR test to discriminate between HCV and related pestiviruses, bovine viral diarrhea virus (BVDV), and those viruses causing similar diseases in swine, including African swine fever virus (ASFV) and pseudorabies virus (PRV), was also considered. Nucleic acid extraction involved either kit-based or conventional phenol:chloroform:isoamyl alcohol methods. A single-round PCR assay, using primers that hybridize to the conserved p120 nonstructural gene region, was 82.5% sensitive (n = 17) and 100% specific (n = 18) in the detection of the presence of HCV RNA. However, the sensitivity was increased to 100% following a second PCR test. In all, 4 HCV, 7 BVDV, 2 ASFV, and 1 PRV isolates were studied. Novel nucleic acid sequences were generated for 9 HCV strains. Analysis of a portion of the p120 region using these methods was suitable for HCV isolate characterization.

Development of PCR-based tests for the identification of North American isolates of epizootic haemorrhagic disease virus.

A serogroup-specific polymerase chain reaction (PCR) assay and isolate identification strategies (restriction endonuclease analysis (REA) and nucleotide sequencing) were developed for the detection of North American isolates of epizootic haemorrhagic disease virus (EHDV). PCR primers (EHDV-pr4, EHDV-pr5) were designed to hybridize to the L3 gene of a North American isolate of EHDV serotype 1. Total nucleic acid was extracted from preparations of infected tissue culture and PCR was performed using a cDNA-PCR kit, according to the manufacturer's specifications. The PCR assay generated a 459 base pair product from North American isolates of EHDV serotypes 1 and 2, while bluetongue virus (BTV) serotypes 10, 11, 13, and 17, and cell controls, failed to demonstrate PCR products. Slight modifications allowed for the PCR detection of EHDV-1 and -2 in white-tailed deer blood (Odocoileus virginiatus); PCR fragments were not amplified from uninfected deer blood. A number of restriction endonucleases and sequencing primers were evaluated for their utility in isolate identification experiments. Specifically, REA employing HincII and cycle sequencing with an internal primer (EHDV-1-pr3) proved most successful for identifying isolate-specific genome markers. The techniques presented are expected to prove valuable for rapid and specific detection of possible future EHDV incursions in wild and domestic animal species.

Identification of the major North American bluetongue viruses using nucleic acid amplification techniques.

A set of primers (BTV-pr1/2) were selected that hybridized to the VP3 gene of the major North American serotypes of bluetongue virus (BTV). Polymerase chain reaction (PCR) testing yielded positive results from specimens of major North American BTV isolates (serotypes 10, 11, 13 and 17) propagated in Vero cells. In addition, PCR assays were positive from samples of all other BTV serotypes, except BTV-16; however, an alternative primer pair (BTV-prN1/N2) was devised for amplification of this serotype and the major North American BTV serotypes. PCR products were not evident following amplification of related viruses, epizootic haemorrhagic disease virus (EHDV) serotypes 1 or 2, in either PCR test. In addition, slight modification of the nucleic acid extraction method allowed for the amplification of BTV template from ovine and cervine blood, but not from the respective control blood samples. Restriction endonuclease analysis (REA) using AluI and TaqI discriminated the PCR products of BTV serotypes 10, 13 and 11/17. Identification of BTV-11 and -17 was accomplished by PCR product nucleotide sequencing. Thus, using a single gene region (VP3), nucleic acid amplification methods were devised for expeditious serogroup-specific detection of all BTV serotypes and identification of individual North American BTV nucleotypes, which is expected to prove valuable for disease control strategies and retrospective epidemiological analyses.

Reverse transcriptase-PCR assay for detection of hog cholera virus.

A reverse transcriptase-PCR strategy was developed for the detection of hog cholera virus. Hog cholera virus template was amplified from tissue culture fluids and from tissues and blood of infected pigs, but not from samples containing other pestiviruses. Restriction endonuclease analysis identified samples as historic or recent isolates.