Journal of Virological Methods

Published by Elsevier
Online ISSN: 0166-0934
Publications
Article
The objective of this study was to assess the effect of menstrual phase on the ability to quantitate HIV-1 in vaginocervical secretions (VCS) through reconstruction experiments with HIV seronegative VCS collected throughout the menstrual cycle. Measurement of HIV-1 inoculated into both fresh and frozen VCS was undertaken by quantitative micro co-culture, p24 antigen assay and polymerase chain reaction (PCR) for both HIV-1 RNA and pro-viral DNA. Two laboratories carried out these assays over a range of viral concentrations. The study involved a randomized factorial design and the factors were: (1) diluents (phases of the menstrual cycle and controls); (2) laboratories; (3) stock concentrations; and (4) frozen versus fresh VCS samples. Each assay was assessed independently using a random effects analysis of variance (ANOVA) model. No statistical differences due to menstrual cycle were seen in the assay results of p24 antigen (P = 0.08), PBMC culture (P = 0.74), plasma culture (P = 0.13), cell-free RNA (P = 0.44), cell-associated RNA (P = 0.58) and cell-associated DNA (P = 0.43). Inter-laboratory differences were statistically significant for cell-free RNA (P < 0.001), cell-associated DNA (P < 0.001) and p24 (P < 0.001). It is concluded that VCS obtained throughout the menstrual cycle from HIV-uninfected women lacks intrinsic inhibitory factors which could limit detection and quantification by antigen, culture or nucleic acid-based technologies for HIV-1 in VCS throughout the menstrual cycle. Using a standardized collection procedure, we suggest that variation in HIV quantity over time, when reported in VCS of infected women, should be attributed to HIV-associated biologic factors, rather than non-specific or other technical factors.
 
Article
Dengue viruses (DENV) cause the most common arboviral disease afflicting men. Clinical manifestations range from asymptomatic to dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The mechanisms involved in the disease pathogenesis are not fully understood. The severity of the disease seems to be influenced by both viral and host factors. Subgenomic replicons of DENV can be used to study viral replication mechanisms and evaluate the effects of antiviral drugs on viral replication. The objective was to generate and characterize biologically a replicon from a clinical isolate of DENV-3, as part of our studies to understand how this new isolate interacts with cells. To obtain this replicon several RT-PCR fragments encoding the non-structural proteins genes were cloned in high-copy vectors, and used to assemble the replicon in a BAC plasmid vector containing a synthetic DNA molecule encoding the 5' and 3' ends of a viral cDNA with a T7 DNA-dependent RNA polymerase promoter and a ribozyme. In vitro transcribed RNA recovered from this BAC plasmid was transfected into C6/36 mosquito cells, and dengue virus protein expression was assessed by indirect immunofluorescence using polyclonal antibodies. The results showed that the replicon was replicated efficiently in cells, demonstrating successful assembly of a DENV-3 replicon.
 
Article
A recombinant baculovirus construct expressing glycoprotein E (gE) of the Egyptian BoHV-1.1 Abu-Hammad strain (rBac/gE-AbuH) was generated and characterized. The recombinant gE (rgE) secreting protein in culture medium of infected insect cells was used as a coating antigen in an indirect enzyme-linked immunosorbent assay (ELISA) to test its utility for detection of antibody against gE of BoHV-1. Indirect gE-ELISA was compared to standard virus neutralization test and commercial blocking gE-ELISA for detection of anti-gE antibody in a panel of bovine sera. Antibody titers estimated by both ELISAs were closely correlated with those determined by virus neutralization test. In conclusion, the developed indirect gE-ELISA was a reliable candidate for inexpensive detection of anti-gE antibody in control and experimental bovine sera with high specificity and sensitivity. Moreover, it emphasized the diagnostic utility of gE based ELISAs to distinguish cattle infected with BoHV-1 from those vaccinated with the gE negative mutants.
 
Article
A novel method to fractionate phage into its subtypes while fully retaining biological function is reported. Corynebacterium pekinense AS 1.299 phage samples, purified by either conventional ultracentrifugation or gel chromatography on a Superose(R) 6 Prep column (0.78 x 30 cm), were fractionated further into four fractions by anion-exchange chromatography on a Toyopearl SuperQ 650C column (0.5 x 20 cm) with a linear gradient of NaCl concentration from 0.2 to 1.0 M in 0.02 M carbonate-biocarbonate buffer, pH 10.0. Two peaks were identified to be C. pekinense AS 1.299 phages by their ability to infect the host bacteria when inoculated into the culture media, and when examined by electron microscopy. These two types of the phage were found to be morphologically the same except for the difference in the length of their non-contractile tails. Both possessed an isometric head with a diameter of 50 +/- 3 nm, while their tails were 170 +/- 10 and 210 +/- 10 nm, respectively. This simple technique provides a convenient method for phage isolation not only to its species homogeneity, but also to determine its subtype or variant homogeneity.
 
Article
In the absence of a robust infectable cell culture system, assays related to replication of clinical HBV isolates are based on the transfection of replication-competent HBV DNA into hepatoma cell lines that are able to replicate and secrete HBV virions. Current methods for constructing HBV 1.1 genomes work well for drug susceptibility assays, but are not very suitable for research on HBV replication capacity or regulation since a heterogeneous promoter is required to drive pgRNA transcription. A new strategy for constructing HBV 1.3 genomes that contain HBV intrinsic promoter necessary for pgRNA transcription is reported in this paper. Using this strategy, three HBV 1.3 genomes from isolates of three patients were constructed. When the three HBV 1.3 genomes were transfected into the HepG2 cell line, replicative intermediates were detectable by Southern blotting with digoxigenin-labeled DNA probe in two of the three constructs. Using overlap extension PCR and avoiding as much as possible the digestion-and-ligation process, this strategy could be applied to constructing longer-than-genome units for most genotypes of HBV strains.
 
Article
The present study aimed to construct a 1.5X hepatitis B virus (HBV) replication system in vitro that could generate high level of HBV viruses. This system would help compare the replication capacity among the virus strains associated with high and low risk of hepatocellular carcinoma (HCC). Four strains of HBV were isolated from two HCC patients and two HBV carriers. After molecular cloning, four corresponding constructs named as HBV-1.5Xs were generated. Each of them has one and a half copies of HBV 3.2kb genome, a 5'-end redundant sequence of 1.1kb to nt715 and a 3'-end redundant sequence of 500bp to nt2325 that situated after the poly (A) sequence. The HepG2 cells were transfected with the HBV-1.5Xs, and the levels of HBsAg, HBeAg and viral DNA were then detected in both the supernatant and the cells. After 24h and 48h of transfection, a high OD value of HBsAg of 3.5 was observed in the supernatant and also in some of the diluted cell lysate samples. The HBeAg level was relatively low in all strain samples of HBV. The log(10) values of viral loads were also determined with the cell lysate having a higher value (10-11 per ml) than that of the supernatant (6-7 per ml). The results showed that the novel HBV-1.5X system was capable to generate high level of HBV in a consistent manner. However, no significant difference was found among the replication capacities among these strains in vitro. The HBV-1.5X system may be a useful platform that assists the establishment of stable cell lines and transgenic mice for the investigation of viral pathogenesis, particularly for the various strains of HBV.
 
Article
Recent studies have suggested that monitoring the amount of HIV provirus in peripheral blood mononuclear cells (PBMCs) may be a useful end point for HAART where, in combination with plasma viral load, it provides additional information as to the possibility of virus eradication. In the present study, a modified version of the Cobas Amplicor HIV-1 Monitor test (CAHIM), currently used to quantify plasma viremia, have been evaluated to also measure the amount of proviral DNA in PBMCs. The analytical and clinical performance of the modified CAHIM test was assessed by quantifying different amounts of a standard HIV-DNA preparation obtained from the 8E5 cell line and by analysing 165 patients and controls samples. In these experiments, the modified test, that showed a linear dynamic range from 1.7 to 4.7 log10 copies/10(6) cells (r = 0.99) with a maximum CV of 20%, proved able to detect and quantify HIV-DNA in all but one clinical samples, with concentrations varying from 1.3 to 3.8 log10 copies/10(6) cells. During anti-retroviral treatment, the assay revealed different proviral DNA time courses associated with viral load changes and inversely correlated with CD4+ cells count. As expected, HIV-DNA was always detectable even when plasma viremia fell below the CAHIM cut-off. The modified CAHIM test specificity was confirmed by testing 20 HIV-negative samples in triplicates. Taken together, the data showed that the modified CAHIM test can be used to monitor HIV proviral DNA changes during HAART and can help in investigating further the clinical use of this marker.
 
Article
Bovine papilloma virus was purified from crude extracts of bovine warts by gel filtration in Sephacryl® S-1000 Superfine. Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis and electron microscopy shows that the virus preparation is as pure as that obtained by alternative techniques. The yield of virus particles is in the range 55–80%. The main advantage of using the gel filtration technique is that it is much less time-consuming than currently used centrifugation procedures.
 
Article
Barley yellow dwarf virus was purified from infected oats using cellulase to assist virus extraction, clarification of the extract with chloroform-butanol, precipitation of virus by polyethylene glycol and gel filtration of the resulting suspension on Sephacryl S-1000 Superfine. The virus was further purified by sucrose density gradient centrifugation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electron microscopy showed that the virus preparation is of sufficient purity for biochemical investigation. A yield of approximately 0.8 mg virus/kg of infected leaf was obtained. The technique is simple and less time-consuming than conventional centrifugation procedures and can be used routinely for purification of a wide range of spherical plant viruses.
 
Article
A method using flow cytometry (FCM) analysis was developed to quantitate baculovirus total particles produced in insect cell cultures. The method is a direct count of particles and involves staining of the baculovirus DNA with SYBR Green I, a highly fluorescent nucleic acid specific dye. Sample preparation of cell-free supernatant containing budded viral particles involves fixation with paraformaldehyde, freeze-thaw treatment, viral membrane permeabilization with Triton X-100, and sample heating to improve staining efficiency and enhance baculovirus particle green fluorescence intensities. In this study, the effects of the different treatment steps and medium composition on viral particle counts were examined in order to identify optimal preparation conditions. FCM analysis linearity was established over a viral concentration range of two logs with a lower detection limit at 10(5) viral particles per ml. Robustness and reproducibility of the method were assessed using samples from large-scale bioreactor cultures. The events (or virus particle counts) obtained by FCM analysis were usually higher than the titres obtained by end-point dilution assay (EPDA). Results from 16 different viral stocks showed an average ratio of 3.7 total particles (FCM) to infectious particles (EPDA). Essentially, the FCM analysis reported below shortens baculovirus quantitation time to 2 h and provides a good estimation of virus titers. It is believed that these findings will contribute to acceleration of process development in the area of baculovirus expression technology in general and specifically in process where stoichiometric multi-viral infections of cells are critical to the expression of complex products.
 
Article
The susceptibility of a variety of different primary tissues was examined to long-term transduction with recombinant adeno-associated virus type 2 (rAAV-2) and factors influencing the transduction efficiency. In contrast to others using cell lines and animal models, emphasis was placed on the use of primary human cells. Enhanced green fluorescent protein (EGFP) marker gene expression was examined using fluorescence-activated cell sorting analysis. The most effective target cells for rAAV-2-mediated gene transfer were bronchial epithelial, artery endothelial as well as smooth and skeletal muscle cells with mean transduction rates ranging from 34.3 to 81.6%. Lower transduction rates between 4.3 and 19.5% were found in chondrocytes, dermal papilla follicle epithelial cells and fibroblasts. No transduction was observed in melanocytes, granulocyte colony-stimulating factor (G-CSF)-mobilized CD34(+) cells or malignant CD19(+) cells from patients with chronic lymphocytic leukemia. A proportion of EGFP-expressing skeletal muscle and smooth muscle cells was maintained over a period of 6 weeks after transduction (42.7+/-5.4 and 67.1+/-0.9%, respectively). Interestingly, among hair follicle epithelial cells the proportion of transduced cells increased from 8+/-0.5 to 36+/-7.7% in the course of 6 weeks. In contrast, for endothelial cells, bronchial epithelial cells and fibroblasts, a rapid decline in the number of EGFP expressing cells were noted. An inverse relationship between the proportion of cells in G2/M phase of cell cycle and long-term gene expression was observed. All rAAV-2 susceptible primary cells expressed FGFR-1 and the alphaV integrin consistent with their role as co-receptors for AAV-2. In conclusion, AAV-2 is a suitable vector system for transduction and evaluation of functional effects of long-term gene expression in primary human muscle and hair follicle cells.
 
Article
An alternative enzyme linked immunosorbent assay (ELISA) system was developed to analyze antibodies to human papillomavirus capsid antigens. The assay uses glutathione crosslinked to casein to capture the major capsid protein L1 from human papillomavirus (HPV) types 6b, 16 and 18 fused to glutathione S-transferase (GST) as antigen. The method allows efficient one-step purification of L1 fusion protein from crude bacterial lysates on ELISA plates coated with glutathione casein. The GST-L1 capture ELISA detected HPV 16 antibodies with high type specificity. Comparison with the current "gold-standard" for L1-serology that uses virus-like particles (VLP) as antigen demonstrated similar assay sensitivity. Pairwise comparison of the absorbance values of 105 human sera obtained in the two ELISA formats for HPV 16 showed a R(2) value of linear regression of 0.68. Conformity of the two ELISAs in classification of sera as HPV 16 L1 antibody-positive or -negative was verified with Cohen's kappa test, yielding a value of 0.62. These data indicate that the GST-L1 capture ELISA is similar in performance to the VLP ELISA. The ease of antigen production and purification in the GST-based ELISA will be advantageous to screen large sample numbers in vaccine trials or epidemiological studies examining immune responses to many HPV types in parallel.
 
Article
During the European project 'Bacteriophages in bathing waters' (January 1996-June 1999), research was carried out to optimise the method for detection and enumeration of F-specific (RNA) phages in water. It was evaluated whether further optimisation would be possible/needed for the procedure as described in the standard method of the International Organisation for Standardisation (ISO) 10705-1. The research focused mainly on optimisation of the different steps for culturing the host strain WG49 Salmonella Typhimurium. It was concluded that all steps described in ISO 10705-1 are necessary and, if followed carefully, using a culture of host strain WG49 Salmonella Typhimurium of good quality, reliable results could be obtained for the enumeration of F-specific RNA phages.
 
Article
One-step immunocapture enzyme-immunoassay (EIA) was compared with time-resolved fluoroimmunoassay (TR-FIA) for rapid diagnosis of influenza A infection by antigen detection. The high-affinity monoclonal antibodies (MAbs) recognising two independent epitopes on the conservative nucleoprotein were used for capture (MAb 44) and detection (MAb 107L) of antigen by both assays. The detection limit for purified recombinant influenza A virus nucleoprotein was approximately 10 pg by EIA and 5 pg by TR-FIA. The performance of the methods was evaluated by testing 43 known positive and 50 negative clinical specimens (nasopharyngeal washes and aspirates). The sensitivity and specificity was 93% and 92% for EIA and 100% and 98% for TR-FIA, respectively, in comparison to the reference A3/A1 TR-FIA. The relationship of 44/107L immunoassays has been evaluated: in comparison to 44/107L TR-FIA (100%), EIA confirmed 93% of positive and 94% of negative samples. In conclusion, the capture-detector pair of MAbs 44 and 107L can be used for the sensitive detection of influenza A viral antigen in clinical samples by both immunocapture methods. Despite the slightly lower accuracy of the EIA, widespread availability and economy of the EIA methodology makes it an advantageous alternative for the laboratory diagnosis of influenza A virus infections.
 
Article
Murine cytomegalovirus (MCMV) can only be propagated effectively in mouse embryo fibroblast (MEF) cells. We demonstrate that MCMV replicates significantly better in M2-10B4 cells, a continuous line of murine bone marrow stromal cells. M2-10B4 cells were also comparable to MEF cells for detection of small amounts of MCMV reactivating from latently infected spleen explants. M2-10B4 cells will be very useful for studies of MCMV pathogenesis.
 
Comparing HPV detection using two generic primers in single and multiple infections.
Comparison of HPV detection using the GP5+/6+ and MY09/11 primers regarding cytological findings.
Advantages and disadvantages of different HPV detection techniques in clinical samples.
Article
The aims of this study were to determine the prevalence of HPV infection and evaluate the concordance and performance of two primer sets for detecting single and multiple viral infections. A total of 1810 Colombian women were enrolled in the study, and molecular, cytological and epidemiological analyses were performed. Both concordance and performance of two different PCR amplification primer sets (GP5+/6+ and MY09/11) were assessed. The results showed that 60.2% of females with positive HPV DNA were infected by more than one viral type. The OR for multiple infections was 18.2 when using the MY09/11 primer set and 6.52 with the GP5+/6+ primer set. The results also showed an association between GP5+/6+ positivity and the severity of the disease regarding the cytological findings. It was also found that using a single primer set led to underestimating the prevalence for HPV infection. The simultaneous use of these primer sets is an important tool for the detection of HPV DNA, being equally relevant for identifying multiple infections and low viral DNA copies. This study highlights the importance of suitable assessment of HPV epidemiological profiles; screening programs must also be strengthened to broaden the coverage of the most vulnerable populations.
 
Article
A novel PCR-restriction fragment length polymorphism assay (PCR-RFLP) was developed for sensitive detection and reliable differentiation of five low-risk human papillomavirus (lr-HPV) genotypes: HPV 6, HPV 11, HPV 42, HPV 43 and HPV 44, as well as differentiation of prototypic and non-prototypic HPV 6 genomic variants. The assay is based on the amplification of a 320-bp fragment of the HPV E1 gene and subsequent analysis of PCR-products with BsaJI and HinFI. Testing on plasmid standards showed that PCR-RFLP enabled simple and reliable identification and differentiation of five targeted lr-HPV genotypes and could detect reproducibly down to 10 copies of viral genome equivalents per PCR. The PCR-RFLP showed almost complete agreement with previously obtained genotyping results on 42 HPV-DNA negative samples and 223 HPV-DNA positive samples (45 HPV 6, 34 HPV 11, 35 HPV 42, 10 HPV 43, 24 HPV 44 positive samples and 75 samples containing 28 non-targeted HPV genotypes). The novel assay is simple and robust, does not require any sophisticated equipment and can be of great value for epidemiological studies, particularly in settings in which financial resources are limited.
 
Article
Competition binding studies between viruses are usually performed with radiolabelled probes. In this report, a cytofluorimetric method using biotinylated echovirus (EV) 11 is described for the study of competition of enteroviruses for a common cell receptor site. An N-hydroxysuccinimide ester biotin spacer arm was used for biotinylation of CsSO4-purified EV 11. Biotinylation did not change the infectivity of the virus (attachment to and replication in susceptible cells). With the exception of EV 22 and EV 23, all the echovirus serotypes and also coxsackievirus A9 (CA 9) were able to inhibit the absorption of biotinylated EV 11 onto cells. The taxonomic implications of these findings are discussed.
 
Article
Individual types of human papillomaviruses (HPV) which infect mucosal surfaces have been implicated as the causative agents for carcinomas of the cervix, anus, penis, larynx and the buccal cavity, occasional periungal carcinomas, as well as benign anogenital warts. The identification of particular HPV types is thus important for: identifying patients with premalignant lesions who are at risk of progression to malignancy; epidemiological studies; studies of the natural history of these viruses; and even medico-legal cases of suspected sexual abuse of children. In this protocol we describe PCR assays for: the identification of DNA from the mucosal HPVs types -6, -11, -16, -18, -31 and -33; a consensus HPV PCR for detecting DNA from 20 characterised mucosal HPVs, as well as more than 25 novel HPVs; and, for a control PCR for beta-globin.
 
Article
The method of Perrin and Gilliland (1990) was modified to create site-specific mutants. The polymerase chain reaction and a single mutant primer are needed to carry out site-specific mutagenesis. Using this method, removal of the excess primers and nucleotides from the initial amplification is not necessary. This method provides a simpler way to generate site-specific mutants.
 
Article
An indirect solid-phase enzyme-linked immunosorbent assay (ELISA) for the determination of specific IgM and IgG antibodies to echovirus type 11 in a single dilution of serum was developed using partially purified echovirus type 11 bound to microplates. Whole serum was used for IgG antibody but prior to assaying for IgM antibody interfering IgG was removed by ion exchange chromatography. The ELISA for echovirus type 11 IgG antibody was a more sensitive, rapid, technically easier and less costly alternative to the neutralisation test. With the IgG ELISA 12 of 132 sera (10.6%) known to contain enterovirus antibodies other than echovirus type 11 were positive but it could not be determined to what extent this was due to the greater sensitivity of the ELISA or cross-reactions. The IgM ELISA was even more sensitive than the IgG ELISA with acute sera, and showed a reactivity in 4 of 36 sera (11.1%) with no detectable echovirus type 11 neutralising antibodies. Echovirus type 11 IgM antibody was detected in all sera collected after the first week of infection and up to 30 days after infection. However, it was only detected in 58% of sera collected during the first week after onset thus limiting its use for rapid diagnosis. The echovirus type 11 IgM ELISA appears to have considerable laboratory diagnostic potential when a rising antibody level cannot be demonstrated in paired sera or when virus is not cultured.
 
Article
Recurrent respiratory papillomatosis (RRP) is a potentially life-threatening disease caused by human papillomavirus (HPV), usually HPV types 6 and 11. The conventional method used for detection and typing the RRP isolates in our laboratory is the polymerase chain reaction (PCR) and DNA sequencing method. A real-time PCR assay based on fluorescence resonance energy transfer (FRET) probe technology was developed for the detection and rapid genotyping of HPV-6 and-11 isolates from biopsy material. The primers and probes were designed using multiple alignments of HPV-6 and HPV-11 partial E6 and E7 sequences that included prototypic and non-prototypic variants. Real-time PCR followed by probe-specific melting-curve analysis allowed differentiation of HPV-6 and HPV-11. HPV-6 and HPV-11 amplicons were used to determine detection limits and inter- and intra-assay variability. The detection limit of the assay was 12.8 DNA copies for HPV-6 and 22.5 DNA copies for HPV-11. A total of 60 isolates were genotyped using the FRET real-time PCR assay and a 100% concordance was obtained when results were compared with genotyping based on conventional DNA sequencing. The real-time PCR assay based on FRET technology was able to detect and rapidly genotype HPV from tissue biopsy obtained from patients with RRP. The assay reduces the time required for genotyping from three working days to less than a day.
 
Article
We developed a non-radioisotopic (non-RI) reverse transcriptase assay (RTA). The reverse transcriptase (RT) incorporates biotin-11-deoxyuridine-triphosphate (bio-dUTP) using a poly(rA) template hybridized with oligo(dT) primer that is immobilized on the surface of a 96-well microtiter plate. This assay is thus semi-automated by adapting it to an ELISA testing format. The incorporation of bio-dUTP was enhanced by adding cold dTTP to the reaction mixture, optimally in a molar ratio 4:1 (dTTP:bio-dUTP). This non-RI RTA is more sensitive than the conventional RI assay for the detection of purified Rous-associated virus 2 (RAV-2) and of human immunodeficiency virus type 1 (HIV-1) lysate. Because of its simple procedure, higher sensitivity and non-use of RI materials, the assay can be utilized not only for virological studies but also for routine safety screening of biological products for retroviral contamination.
 
Article
A method described previously for determining the concentration of influenza virus antihemagglutinin antibody molecules, the number of epitopes per virus particle and the equilibrium constant of virus antibody interaction was adapted to the use with escape variants (EVs), produced by multiplication of influenza virus A/Brazil (H1N1) in presence of monoclonal antibody directed to each of the four hemagglutinin sites (Sa, Sb, Ca and Cb). The EVs were found to possess an altered antigenic site, which was both antigenic and immunogenic. By use of selected EVs and antibody preparations, the number of epitopes per antigenic site was determined and it was found that each of the four sites was represented by about 390 epitopes per virus particle, suggesting that each of the about 400 hemagglutinin spikes per virion possessed one epitope of the specificity Sa, Sb, Ca and Cb. Alteration of site Sa but not of site Ca increased the avidity of antibody to react with the unchanged sites.
 
Article
A multiplex polymerase chain reaction (PCR) based on the simultaneous amplification of human papillomavirus (HPV) types 6/11, 16 and 18 in a single-step procedure was developed, using primers chosen in the E6-E7 region. The specificity and sensitivity of this technique have been proved by amplifying mixtures or various amounts of plasmid-containing HPV DNA; it allowed the detection of as few as 5-25 HPV DNA copies. Application of the multiplex PCR to 71 clinical samples showed that HPV DNA was detected in 80% (45/57 cases) of mucosal biopsies and 35% (5/14 cases) of cutaneous specimens. HPV 16 was predominant in high-grade CIN whereas HPV 6 and 11 were detected more frequently in genital condylomas and laryngeal papillomas. In cutaneous Bowen's disease HPV 16, 18 or 6/11 + 16 were detected and in squamous cell carcinomas HPV 6/11 or 16 were found. After sequence amplification with primers of one HPV type, the clinical samples displayed the same HPV types but the frequency of positive and coinfected lesions increased. Thus, multiplex PCR is a valuable technique for typing HPV DNA but coinfections may be underestimated.
 
Article
Environmental samples and contaminated shellfish present frequently low concentrations of more than one viral species. For this reason, a nested multiplex RT-PCR was developed for the detection of adenoviruses, enteroviruses and hepatitis A viruses in different environmental samples such as urban sewage and shellfish. This assay will save time and cost for detection of these enteric viruses with a smaller sample volume, which otherwise can be a limiting factor in routine analysis. The limit of detection was approximately 1 copy for adenovirus and 10 copies for enterovirus and hepatitis A virus per PCR reaction using titrated cell-cultured viruses as template material. In shellfish and environmental samples, this multiplex PCR was optimized to detect all three viruses simultaneously when the concentration of each virus was equal or lower than 1000 copies per PCR reaction. This is the level found predominantly in the environment and in shellfish when the numbers of fecal bacterial and phage indicators are low. The detection of human adenoviruses by PCR has been suggested as a molecular index of fecal contamination of human origin in the environment and food and the multiplex assay developed may be a tool for evaluating the presence of viral contamination in shellfish and water and to expand microbiological control to include viral markers.
 
Article
Hepatitis B surface antigen (HBsAg) is a crucial serum marker for the diagnosis of Hepatitis B virus (HBV) infection. It is imperative to compare test results from different detection methods based on different principles. Four methods, Chemiluminescent microparticle immunoassay (CMIA), Electrochemiluminescent immunoassay (ECLIA), Enzyme- linked immunosorbent assay (ELISA) and Golden immunochromato-graphic assay (GICA) were applied to test the HBsAg level in 250 specimens. According to the EP12-A2 and EP15-A2 documents from Clinical and Laboratory Standards Institute (CLSI), the concentration at which repeated results are 50% positive (C50) of HBsAg detected by CMIA, ECLIA, ELISA and GICA was 0.05, 0.08, 0.15 and 15.0IU/ml, respectively. When the detection concentration of HBsAg was 0.5IU/ml, the imprecision degree of CMIA, ECLIA and ELISA was 8.1%, 5.9% and 14.9% respectively. When detecting high HBsAg level (≥20.0IU/ml) and HBsAg negative specimens, the consistency of the four methods was high, while for the low level (0.05-20.0IU/ml), the consistency was poor (except for the CMIA and ECLIA, P<0.05). When evaluation of the four methods in qualitative diagnosis of HBsAg level in the 116,455 specimens, there was no significant discrepancy among CMIA, CMIA and ECLIA, however, GICA was significantly different from the other 3 methods. Compared with CMIA, the false negative rate of ECLIA, ELISA and GICA was 0.2%, 1.3% and 12.3% respectively. In conclusion, GICA was only suitable for the preliminary screening of HBsAg positive individuals and ELISA can be applied to the qualitative diagnosis of HBsAg. Both CMIA and ECLIA were suitable for the quantitative determination of HBsAg.
 
Article
Two novel enzyme-linked immunoassays (ELISA) for the quantitation of human immunodeficiency virus type 1 (HIV-1) coded glycoprotein with an Mr 120 (gp120) are described. These are based on the highly specific interaction between gp120 and the mannose-specific lectins from Narcissus pseudonarcissus (NPL) and Galanthus nivalis (GNL). Two systems were developed: (1) an HIV-protein ELISA using HIV-protein (also containing HIV-gp120) for the solid phase and NPL as a detector and (2) a lectin-ELISA using the NPL bound to the solid phase and GNL as detector. The HIV-protein ELISA was validated for quantitation of gp120 within the range 3 to 600 ng/ml; the lectin-ELISA for concentrations between 0.6 and 20000 ng gp120/ml. Serum components did not interfere with the binding of gp120 to the lectins. The ELISAs were used for the quantitation of gp120 in HIV-infected CEM cells in vitro. It was found that gp120 appeared in the medium earlier after infection than HIV-p24 and reverse transcriptase, suggesting that gp120 is released as free glycoprotein. Moreover, the ELISAs were also applied successfully for the detection of compounds that bind to gp120 and for the identification of antibodies directed against the highly pathogenic mannan portion of gp120. These ELISAs are considered to be suitable also for the detection of gp120 in the serum of HIV-infected individuals.
 
Article
The goal of the present study was to develop an efficient transient transfection method for large-scale production of high titer lentivirus vector stocks of eight different pseudotypes. The envelope genes used for this purpose were those from VSV-G, Mokola, Rabies, MLV-Ampho, MLV-10A1, LCMV-WE, and LCMV-Arm53b. All envelopes were cloned into phCMV, which yielded lentivirus vector titers one, two, or three orders of magnitude higher than the original plasmids for the Rabies, MLV-10A1, and MLV-Ampho envelopes, respectively. When these newly constructed envelope expression plasmids were used for packaging, treatment with sodium butyrate resulted in almost five-fold increase in titers for some of the pseudotypes, had no effect for others (VSV-G and Rabies), and negatively impacted titers for the LCMV-derived pseudotypes. Production of vectors in serum-free media yielded titers only slightly lower than those obtained in the presence of serum. The efficiency of concentrating vector supernatants by ultracentrifugation or ultrafiltration was compared, with higher recovery efficiencies for the latter method, but the highest titers for most pseudotypes were obtained by ultracentrifugation. The best conditions for each individual pseudotype yielded lentivirus vector stocks with titers above 1 x 10(9) tu/mL for most pseudotypes, and higher than 1 x 10(10) tu/mL for VSV-G.
 
Article
A full-length and a truncated gene for the core protein of hepatitis C virus (HCV) were linked to the gene for glutathione S-transferase (GST), and the expression of each GST-HCV core fusion protein was analyzed. The truncated GST-HCV core (1-123) fusion protein was expressed as a mostly soluble and partly insoluble form comprising more than 50% of the total protein in Escherichia coli after induction by isopropylthio-beta-D-galactoside (IPTG), while the full length GST-HCV core (1-191) fusion protein was not expressed, suggesting that the hydrophobic carboxy terminal region in the core protein affects its expression. In addition, the GST-HCV core (1-123) fusion protein purified by GST-agarose chromatography reacted specifically with an anti-HCV serum from a patient.
 
Article
While the majority of dengue infections worldwide are transmitted by the Aedes aegypti mosquito, the majority of research into the interaction between dengue and insect cells is undertaken in the Aedes albopictus derived cell line C6/36. The CCL-125 cell line is a long established A. aegypti derived cell line that was originally characterized as not susceptible to infection by the dengue virus. The present study establishes that CCL-125 is permissive to dengue virus infection and is able to be infected productively as determined by both plaque assay and immunocytochemistry. Infection occurred without observable cytopathic effect. This study demonstrates the utility of the A. aegypti derived cell line CCL-125 as a dengue permissive cell line and suggests that it may be a useful alternative to C6/36 cells in dissecting out the dengue virus-insect cell interaction.
 
Article
The resolution potential of reverse-phase high-performance liquid chromatography (HPLC) for peptide analysis of hydrophobic viral membranes has been investigated, using as model the membrane (M) protein of influenza virus. Proteolytic digests of 125I-labelled M protein and CNBr fragments, extracted from radioiodinated whole virus, have been separated on a uBondapak C18 column with an isopropanol or acetonitrile solvent system. Peptide mapping of trypsin digests of M protein from A/PR/8/34 (H1N1) and A/chicken/Germany/N/49 (H10N7) viruses was identical, whereas Staphylococcus aureus V8 protease digests showed minor differences in at least two peptides. The results also show that HPLC is a powerful tool for the separation of proteolytic digests of viral proteins, since the peptide maps are highly reproducible and recovery was greater than 85%.
 
Article
A rapid and sensitive polymerase chain reaction (PCR) was developed to detect conserved sequences from the immediate early gene of human cytomegalovirus (HCMV). The primers sequences were from EcoRI J fragment of Ad169. The first primer set was selected to amplify a 242 bp fragment and the next primer set was nested within the first and amplified a 146 bp fragment. With the single PCR system it was possible to detect 100 fg HCMV DNA but with double PCR 5-10 fg were detectable. Specific amplification was seen in urines from patients with HCMV infections. 20 urine samples were analysed by single PCR, double PCR and virus cultivation. The double PCR was the most sensitive method. Urines from healthy seropositive persons and cells infected with other members of the herpes virus family were negative with all three methods. This suggests that specific amplification by double PCR is sensitive and can be used for rapid detection of HCMV DNA in cases with activated infection.
 
Article
The further development of Taqman quantitative real-time PCR (qPCR) assays for the absolute quantitation of Marek's disease virus serotype 1 (MDV1) and Herpesvirus of turkeys (HVT) viruses is described and the sensitivity and reproducibility of each assay reported. Using plasmid DNA copies, the lower limit of detection was determined to be 5 copies for the MDV1 assay and 75 copies for the HVT assay. Both assays were found to be highly reproducible for Ct values and calculated copy numbers with mean intra- and inter-assay coefficients of variation being less than 5% for Ct and 20% for calculated copy number. The genome copy number of MDV1 and HVT viruses was quantified in PBL and feather tips from experimentally infected chickens, and field poultry dust samples. Parallelism was demonstrated between the plasmid-based standard curves, and standard curves derived from infected spleen material containing both viral and host DNA, allowing the latter to be used for absolute quantification. These methods should prove useful for the reliable differentiation and absolute quantitation of MDV1 and HVT viruses in a wide range of samples.
 
Article
Human papillomavirus type 13 (HPV-13) is associated with oral focal epithelial hyperplasia (FEH). The purpose of this study was to establish conditions for the application of polymerase chain reaction (PCR) to the specific detection and amplification of HPV-13 DNA. To design primers for HPV-13 a part of the HPV-13 genome was sequenced first: the smallest BamHI fragment (597 bp) of HPV-13 was subcloned and sequenced. The sequence was found to be part of a large open reading frame and had significant homology with the L1 gene of other HPVs. HPV-13 specific primers were designed to amplify a 240 bp fragment from the L1 gene by PCR. Conditions for PCR were standardized for this set of primers.
 
Article
Quantitative multiplex real-time RT-PCR assays utilizing fluorescence resonance energy transfer (FRET) hybridization probes were developed for the detection of 13 respiratory viruses, including well recognized viral causes (respiratory syncytial virus, influenza viruses A and B, parainfluenza viruses types 1, 2, and 3, adenovirus) as well as viruses described recently as causes of acute respiratory tract infections (human coronaviruses NL63, HKU1, 229E, and OC43, human bocavirus, and human metapneumovirus). FRET probes have an improved toleration for single base mismatches than other probe chemistries, reducing the chances of missing highly variable RNA viruses. The assay could detect 2.5-25 DNA/RNA copies/microl (2.5 x 10(3)-2.5 x 10(4) copies/ml). Validation on 91 known positive respiratory specimens indicated similar specificity as commercial direct immunofluorescence assays (IFA) or single-round PCRs used in initial identification. Screening of 270 IFA negative respiratory specimens identified new viruses in 40/270 (14.8%) cases and additional 79/270 (29.3%) well recognized viruses missed by routine diagnostic assays including 6.7% co-infections. All viruses could be detected in the clinical screening panel. The assays demonstrates an improved sensitivity and scope of detecting respiratory viruses relative to routine antigen detection assays while the quantitative utility may facilitate investigation of the role of co-infections and viral load in respiratory virus pathogenesis.
 
Article
F-specific RNA bacteriophages have been classified into four genogroups (GI, GII, GIII and GIV). It was suggested that two of these genogroups are more frequent in human excreta (GII and GIII) and the two other (GI and GIV) are specific for animal excreta. Real-time RT-PCR methods using TaqMan MGB probe were developed to detect the four genogroups. Primers and probes of each specific RT-PCR were designed to target all sequenced bacteriophages belonging to one genogroup, without cross-reactivity with other genogroups. These four methods showed detection limits ranging between 0.01 and 10 PFU/mL and PCR efficiencies ranging between 87 and 95%. The newly methods were tested in urban raw wastewater. Genogroups I and II were detected in all samples (n=7); GIII in six samples and GIV was never detected. GI was predominant in one sample, in which the quantity of Cryptosporidium and Giardia was, respectively, three and eight times higher than the mean values. Because GI is mainly observed in animals, it was hypothesized that this increase was due to an animal input. The use of F-specific RNA phage genotyping to estimate the origin of faecal pollution requires appropriate validation. In this context, real-time RT-PCR will undoubtedly be useful.
 
Article
Oncolytic adenoviruses are exploited as possible anticancer agents in clinical trails. To monitor adenoviral gene expression, a real-time RT-PCR method with a LightCycler was developed that allows the rapid and easy quantification of a number of early and late adenoviral genes in infected tumor cells. Primers were designed that can amplify the spliced forms of the genes encoding E1A13S, DNA polymerase (Pol), pre-terminal protein (pTP), adenoviral death protein (ADP), Hexon (Hex) and Penton (Pent) genes. Standard curves were generated using two-fold serial dilutions of cDNAs derived from non-small cell lung cancer (NSCLC) H460 cells infected for 24h with wild-type adenovirus serotype 5. For all genes correlation coefficients of the standard curves of 0.984 or higher were obtained. The dynamic range of the assay was sufficient to allow the quantitative determination of adenoviral gene expression during a lytic cycle. This RT-PCR assay could be used as a research tool to study the effect of host-cell factors or exogenous treatments on adenoviral gene expression. As example, it is shown that the procedure is suitable to detect changes in adenoviral gene expression in infected H460 cells treated with paclitaxel that is known to enhance the antitumor effect of oncolytic adenoviruses.
 
Article
The influenza A components of live attenuated vaccines used in Russia have been prepared as reassortants of the cold-adapted (ca) H2N2 viruses, A/Leningrad/134/17/57-ca (Len/17) and A/Leningrad/134/47/57-ca (Len/47), and virulent epidemic strains. The lesions responsible for attenuation within the six internal genes of each donor strain have been sequenced and described, but relatively little is known as to their stability before and after passage in susceptible hosts. In the work reported in this paper, RT-PCR restriction analysis and limited sequencing of individual genes were used to evaluate the stability of lesions in stocks of the both donor strains after passage in ferrets, which have been used widely as susceptible hosts for assessment of the virulence of influenza strains. Len/47 was shown to possess expected lesions by RT-PCR and restriction analysis. Substitution at position 1066 of the NP gene, which has been previously reported to be unique to Len/47 [Klimov et al., Virology 186 (1992) 795], was also shown to be present in all clones of Len/17. This change was confirmed by limited sequence analysis and was shown to be retained in progeny viruses isolated from the lungs and turbinates of inoculated ferrets. Two other changes in the PB2 and PB1 genes that were present in Len/47 were detected by limited sequence analysis alone. Further previously unreported minor changes were shown to be present for Len/17 and Len/47, but not both, and their significance is unknown. Limited replication of each donor strain occurred in ferrets and minimal clinical signs and histopathology were present. By contrast, the parental strain Len/57 and the recent epidemic strain A/Sydney/6/97 induced clinical signs and histopathology that were typical of influenza disease.
 
Article
Better, easier and cheaper than polymerase chain reaction (PCR) or antibody detection, rolling circle amplification (RCA) using the bacteriophage varphi 29 DNA polymerase allows for a reliable diagnosis of geminiviruses and presumably all viruses with small single-stranded circular DNA genomes. The results show the efficiency of this technique in characterizing viral DNA components of several geminiviruses from experimental and natural host plant sources. The advantages are: (a) that no expensive devices are necessary, (b) simple handling, (c) detection of all infecting circular DNA components without any knowledge of sequence information in a single step, and (d) low costs per reaction. In addition, RCA-amplified viral DNA can be characterized by restriction fragment length polymorphism analysis and directly sequenced up to 900 bases in a single run circumventing cloning and plasmid purification. This shortcut will considerably accelerate genomics of at least gemini-, nano- and circoviruses in the future.
 
Article
Recombinant AAV vectors are produced by transient transfection of mammalian cells. The virus is usually purified from a combination of lysed cells and spent culture medium by HPLC. We have developed a quantitative, real-time PCR assay for quantifying encapsidated single-stranded viral DNA (i.e. DNA-containing virions) in cell lysates and the spent culture medium. This requires extensive DNaseI digestion to reduce the amount of AAV replicative DNA, as well as plasmid and cellular DNA, to negligible amounts. To demonstrate the utility of this assay, we produced recombinant AAV in HeLa cells and five different types of 293 cells. We used primers to the EGFP transgene to detect the production of a recombinant AAV. We assayed the cell lysates and media by both our quantitative PCR assay and a functional transduction assay. The quantitative PCR assay data correlated well with the transduction assay data. Because this assay only requires standard PCR primers and SYBR Green I dye to detect the amplification of the PCR template, it will readily adapt to any target DNA sequence within the recombinant AAV genome. The recombinant AAV vector does not need to express a reporter gene, such as EGFP or beta-galactosidase in order to assay the amount of virus produced.
 
Article
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Article
Purification of 14 S subunits from extracts of poliovirus-infected HeLa cells was achieved by a combination of sucrose gradient ultracentrifugation and high-performance size-exclusion chromatography. The particles were free of admixtures of other subviral particles, of nonstructural viral proteins, and of host cell proteins. The purified material retained the physical and antigenic properties of native 14 S subunits fully, as well as their ability to assemble to empty capsids in vitro.
 
Article
The performance of 14 commercially available HIV-1/2 antibody assays were compared using well-characterized serum panels containing in total 1500 1800 sera. The panels included consecutive HIV-negative blood donor sera from Sweden, unselected blood donor and patient sera from Tanzania and unselected sera from outpatient clinics in Guinea-Bissau. Furthermore selected HIV-1 antibody positive sera from Sweden and Tanzania and HIV-2 antibody positive sera from Guinea-Bissau were included in the panels. The HIV-1 antibody positive sera were from individuals at various stages of HIV infection, from primary infection, to asymptomatic phase and late stage disease. 12 of the 14 assays identified correctly all HIV-1 and HIV-2 antibody positive sera. One Tanzanian HIV-1 antibody positive sample with complete banding pattern on Western blot was not detected by two of the ELISAs employing synthetic peptides. There were small differences in sensitivity between the assays when used for analysis of seroconversion panels. The most sensitive assay, Abbott IMx HIV-1/HIV-2 III Plus detected antibodies in all nine samples collected from four individuals during the first week after onset of symptoms of primary HIV-1 infection. Most of the assays became reactive during the second week after onset of symptoms and the least sensitive assays were reactive from the third week. The assays showed a high specificity ranging from 99.2 to 100% when used for analysis of Swedish blood donor sera, while most of the assays showed a significantly lower specificity, 91.9-99.6%, when used for testing African specimens.
 
Article
To evaluate the performance of 22 assays for the detection of antibodies to HIV. Twenty-two assays for the combined detection of antibodies to HIV-1 and HIV-2, were evaluated on the same panel of serum specimens of diverse origin. Eight of the assays were ELISAs and the remaining 14 were simple, assays read visually. The specimen panel consisted of anti-HIV positive and negative samples from Africa (n=192), Europe (n=206), Asia (n=99) and Latin America (n=98). In addition to estimations of sensitivity and specificity, the assays were assessed, using a novel scoring system, for their ease of performance and for their suitability for use in small laboratories and clinics. The sensitivities of the assays in terms of seroconversion were assessed using series of specimens collected from nine individuals undergoing seroconversion. Eight ELISAs and eight of 14 simple assays had sensitivities and specificities of >99 and 95%, respectively. The results of these evaluations will be of assistance to those responsible for the selection of appropriate anti-HIV assays according to laboratory circumstances, the purpose of the testing and the population being tested.
 
Article
A specific and practical method was developed for high throughput 14 human papillomavirus (HPV) genotypes assay in clinical samples by a single PCR. GP5+/6+ polymerase chain reaction (PCR) system was used to amplify HPV DNA in 1127 samples. The PCR product was assayed by AcycloPrime reaction with fluorescence polarization (FP). Fourteen HPV genotypes specific sequence primers designed within GP5+/GP6+ amplification polymorphism regions of L1 genes for corresponding HPV genotypes were annealed with the type specific PCR products and special fluorescent terminator was added to the end of the primer under direction of the PCR products. AcycloPrime-FP analysis showed specific anneal and incorporation without any cross-reaction. The types detected with FP showed an excellent overall agreement with sequence when the individual monotype results were taken into account. The proposed method could detect more than one type of HPV infection, but the sequence method was limited. AcycloPrime-FP could reach the detection level: 100 ag for representative phylogenetically distant HPV genotypes: HPV6, 18, 31, 39, 42, 51 and 58. The results of AcycloPrime-FP showed excellent reproducibility. The proposed method allowed an economical detection of HPV genotypes without any use of labeled probe. It is expected to be an extremely useful tool for HPV genotypes screening.
 
Article
A synthetic peptide corresponding to residues 135-155 (S135-155) of the major protein component of HBsAg was conjugated to beta-galactosidase. This conjugate reacted with monoclonal anti-HBs antibodies having anti-alpha group specificity. The reaction was inhibited by: HBsAg of either subtype ad or ay; by unconjugated S135-155 or a shorter peptide S140-155, but not by unrelated peptides. Modification of lysine residues of either HBsAg or S135-155 reduced this inhibitory effect. These results indicate that Lys 141 is essential for maintaining the antigenicity of one of the epitopes responsible for the common alpha specificity of HBsAg and that studies involving the use of synthetic peptides and modifications of distinct amino acid residues in the native protein or in the peptide may help in characterizing epitopes of viral antigens in general.
 
Article
Attenuated strains of bacteria have been developed as potential live vectors to express homologous or heterologous antigens of many pathogens for inducing protective immune responses. The non-pathogenic and rapidly growing Mycobacterium smegmatis can be transformed effectively by genes for pathogenic antigens, and has been used as a valuable vector for the development of live vaccines. However, little is known on whether M. smegmatis could be transformed with the genes for HBV antigens and could express those genes, and whether vaccination with recombinant M. smegmatis could induce humoral and cellular immune responses in vivo. Both the core protein and preS1 peptide of the hepatitis B virus (HBV) are immunogenic and can induce cellular and humoral immune responses. This made them ideal platform for the development of new vaccines. In the present study, both recombinant M. smegmatis and DNA vaccines were generated to express the CS1 antigen, a fusion protein that comprises truncated core protein (amino acids 1-155) and preS1 peptide (amino acids 1-55) of HBV. Following vaccination of BALB/c mice with the live recombinant M. smegmatis, the CS1-based DNA vaccine, or controls, antigen-specific humoral and cellular immune responses were characterized. Vaccination with live recombinant M. smegmatis induced a stronger cellular immune response and a longer period of humoral immune response than with the DNA vaccination. These results indicate that the recombinant M. smegmatis can express efficiently immunogenic CS1 antigen of HBV in vivo, and may be used for the prevention of HBV infection.
 
Top-cited authors
Jan Balzarini
  • KU Leuven
Piet Herdewijn
Dominique Schols
Robert Snoeck
  • KU Leuven
Scott M Reid
  • Department for Environment, Food and Rural Affairs