| 1. |
- Ban, L., et al.
(författare)
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An improved detection method for the Rhopalosiphum padi virus (RhPV) allows monitoring of its presence in aphids and movement within plants
- 2007
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 142:1-2, s. 136-142
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Tidskriftsartikel (refereegranskat)abstract
- Rhopalosiphumpadi virus (RhPV) is an insect RNA virus that infects aphids, reducing their lifespan and fecundity. It can be transmitted vertically between aphids and horizontally via the plant. An improved detection method for the virus in aphids and plants using RT-PCR was developed; this allowed individual aphids to be tested for RhPV. Testing of R. padi aphids collected from different sites in Sweden revealed the presence of RhPV in wild aphid populations for the first time in Europe. Virus could be detected in several life stages of R. padi, including sexual individuals and eggs, establishing an over-wintering route for the virus. Using RT-PCR, systemic transport of the virus in plants was tracked. Virus spread from the aphid feeding site to all parts of the plant, including roots, within 7 days, and could be acquired by virus-free aphids feeding on the same plant.
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| 2. |
- Banér, Johan, et al.
(författare)
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Microarray-based molecular detection of foot-and-mouth disease, vesicular stomatitis and swine vesicular disease viruses, using padlock probes
- 2007
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 143:2, s. 200-206
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Tidskriftsartikel (refereegranskat)abstract
- The World Organization for Animal Health (Office International des Epizooties, OIE) includes the diseases caused by foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and vesicular stomatitis virus (VSV), as "Diseases Notifiable to the OIE". Foot-and-mouth disease (FMD) outbreaks have severe economical as well as social effects and cannot be differentiated from the diseases caused by the other two viruses on the basis of clinical symptoms. Efficient laboratory techniques are therefore required for detection and identification of the viruses causing similar vesicular symptoms in swine. A rapid method is described using padlock probes and microarrays to detect simultaneously and differentiate the three viruses in a single reaction, as well as providing serotype information in cases of VSV infection. The padlock probe/microarray assay detected successfully and identified 39 cDNA samples of different origin representing the three viruses. The results were in complete agreement with identities and serotypes determined previously. This novel virus detection method is discussed in terms of usefulness and further development.
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| 3. |
- Belak, Sandor
(författare)
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Evaluation of automated nucleic acid extraction methods for virus detection in a multicenter comparative trial
- 2009
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 155, s. 87-90
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Tidskriftsartikel (refereegranskat)abstract
- Five European veterinary laboratories participated in an exercise to compare the performance of nucleic acid extraction robots. Identical sets of coded samples were prepared using serial dilutions of bovine viral diarrhoea virus (BVDV) from serum and cell culture propagated material. Each laboratory extracted nucleic acid from this panel using available robotic equipment (12 separate instruments, comprising 8 different models), after which the processed samples were frozen and sent to a single laboratory for subsequent testing by real-time RT-PCR. In general, there was good concordance between the results obtained for the different automated extraction platforms. In particular, the limit of detection was identical for 9/12 and 8/12 best performing robots (using dilutions of BVDV infected-serum and cell culture material, respectively), which was similar to a manual extraction method used for comparison. The remaining equipment and protocols used were less sensitive, in an extreme case for serum, by a factor of 1000. There was no evidence for cross-contamination of RNA template in any of the negative samples included in these panels. These results are not intended to replace local optimisation and validation, but provide reassurance to laboratories to indicate that the best performing optimised nucleic acid extraction systems can have similar performance. (c) 2008 Elsevier B.V. All rights reserved.
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| 4. |
- Formiga-Cruz, Meritxell, et al.
(författare)
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Nested multiplex PCR assay for detection of human enteric viruses in shellfish and sewage.
- 2005
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 125:2, s. 111-8
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Forsman, Anna, et al.
(författare)
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Development of broadly targeted human endogenous gammaretroviralpol-based real time PCRs Quantitation of RNA expression in human tissues
- 2005
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 129:1, s. 16-30
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Tidskriftsartikel (refereegranskat)abstract
- Endogenous retroviral sequences (ERVs) are dynamic genomic components with profound influences on gene expression and genomic structure. Their extent of expression is not well known. Several broadly targeted real-time reverse transcription PCR (QPCRs) systems for surveillance of RNA expression of the major groups of human gammaretroviral ERVs were constructed. The highly conserved reverse transcriptase (RT) and integrase (IN) domains of the pol gene were used as targets for the PCRs, which were both probe-based (TaqMan) and probe-less (SYBR Green). Different levels of primer and probe degeneracy, with or without inosine, were tested. Several of the PCRs had sensitivities of a few HERV nucleic acid copies per PCR reaction. Specificities were approximately as expected from the fit of primers and probes. Gammaretroviral HERV RNA expression was studied in different human tissues. Each HERV group had a specific pattern of expression. HERV-E was highly expressed in testis, HERV-I/T in brain and testis, HERV-H in brain and testis, while HERV-W was highly expressed in placenta. Gammaretroviral RNA was not detected in plasma from 50 blood donors in saliva from 20 persons. In conclusion, a set of tools for investigation of gammaretroviral HERV RNA expression was created.
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| 6. |
- Fridholm, Helena, et al.
(författare)
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Rapid and reproducible infectivity end-point titration of virulent phage in a microplate system
- 2005
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 1879-0984 .- 0166-0934. ; 128:1-2, s. 67-71
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Tidskriftsartikel (refereegranskat)abstract
- The standard method for measuring the it umber of infectious phages in solution has traditionally been the plaque forming assay. An alternative method is described where the number of lytic, infectious phages is determined in an endpoint titration assay adapted for a microplate system. In this model system, susceptible Escherichia coli 136 at a density of 4 x 10(7) cells/ml, were mixed with an equal volume (100 mu l) of Phi X174 diluted serially in a microtest plate. After 3 h of incubation on a microplate shaker the endpoint was determined spectrophotornetrically and calculated according to the method of Reed and Muench. A well was considered positive for infection if the OD630-value was <= 10% compared to the OD630-value of the negative control Of uninfected cells. ID50-titers were 2.5 x higher than the PFU-titers (CV 15%) and the intra assay reproducibility revealed a CV of 9%. The method has several advantages as compared with the conventional PFU-titration. It is less time and material consuming with the possibility to assess several samples at the same time.
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| 7. |
- Gyarmati, Péter, et al.
(författare)
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Universal detection of hepatitis E virus by two real-time PCR assays : TaqMan((R)) and Primer-Probe Energy Transfer
- 2007
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 146:1-2, s. 226-235
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Tidskriftsartikel (refereegranskat)abstract
- Hepatitis E virus (HEV) is a major cause of food- and waterborne diseases in countries with poor sanitation. Furthermore, travellers to such countries are also at risk of contracting the virus. Noteworthily, during the last decade an increasing number of non-travel-related cases were recorded even in countries with high sanitary standards. An alternative, direct route of infection, from animals to humans (zoonotic transmission) is suspected to be the cause of recent cases of hepatitis E. In order to provide rapid and sensitive methods for detecting the virus in various hosts, two real-time PCR methods were developed and compared: a TaqMan (R) and Primer-Probe Energy Transfer (PriProET) assay. These highly sensitive novel methods provide valuable diagnostic tools to investigate zoonotic transmission, to detect the virus in the food chain and in research related to the potential of hepatitis E virus to cross the species barrier. The results show that the two novel PCR assays are robust, highly sensitive and specific for broad range detection of the four genotypes of HEV. Compared to PriProET, the TaqMan (R) assay appears to perform slightly better, with higher fluorescence values for positive samples. However, the PriProET has the benefit of better tolerating the point mutations in the target nucleic acids. Thus, it provides a more powerful tool to detect new virus variants. These new molecular diagnostic assays are practical tools that can be employed in the area of public health, for disease diagnosis and for tracking outbreaks. In basic research the methods provide new tools to study HEV biology, including virus-host interactions and transmission between various host species.
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| 8. |
- Hermening, Stephan, et al.
(författare)
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Improved high-capacity adenoviral vectors for high-level neuron-restricted gene transfer to the CNS
- 2006
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 1879-0984 .- 0166-0934. ; 136:1-2, s. 30-37
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Tidskriftsartikel (refereegranskat)abstract
- Adenovirus-based (Ad) vectors are used widely for experimental gene transfer to the CNS. Ad transduce many cell types including postmitotic neurons. However, their use for, CNS gene transfer is limited due to the host immune response elicited. Furthermore, the extensive distribution of the primary cellular receptor for Ad, the coxsackievirus and adenovirus receptor (CAR), allows adenoviral vectors to infect a broad range of host cells which may be disadvantageous in tissues with various different cell types, like the CNS. The use of tissue-specific promoters allows for neuron-restricted gene expression, even though gene expression driven by these promoters is often very weak. Accordingly, increased transgene expression levels from viral transcription units are needed in order to improve the overall performance of Ad vectors. We designed a high-capacity Ad vector (HC-Ad) that allows for high-level, neuron-restricted transgene expression and shows no obvious signs of immumogenicity or toxicity in the mouse brain. (c) 2006 Elsevier B.V. All rights reserved.
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| 9. |
- Karlsson, Malin, et al.
(författare)
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A real-time PCR assay for the monitoring of influenza a virus in wild birds
- 2007
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 144:1-2, s. 27-31
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Tidskriftsartikel (refereegranskat)abstract
- A screening system including a new real-time PCR assay for the monitoring of influenza A virus in wild birds was developed. The real-time PCR assay uses SYBR green chemistry and the primers are targeting the matrix gene of influenza A virus. The performance of the assay was compared with two other assays, one assay also using SYBR green chemistry and one assay using TaqMan chemistry, i.e. a specific probe. A total of 45 fecal bird samples were analysed for influenza A virus in three different PCR reactions. Overall, 26 samples were positive in at least one of the three real-time PCR assays. Of the 26 samples, 18 were positive by all three reactions. Eight samples were found positive exclusively by the two SYBR green reactions, six of which were detected by both SYBR green reactions. Of the 26 positive samples, 15 samples were verified as positive either by virus isolation or influenza A M2-gene PCR. The results showed that the two SYBR green systems had a higher performance regarding the detection of influenza A as compared to the PCR reaction using a specific probe.
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| 10. |
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| 11. |
- Käller, Max, et al.
(författare)
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Tag-array based HPV genotyping by competitive hybridization and extension
- 2005
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 129:2, s. 102-112
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Tidskriftsartikel (refereegranskat)abstract
- A method is described for HPV genotyping based on multiplex competitive hybridization (MUCH) combined with apyrase mediated allele-specific extension (AMASE). Two type-specific oligonucleotides were designed for each of the 23 investigated HPV types and directed towards two highly inter-type heterogeneous regions. The type-specific oligonucleotides were allowed to compete in the hybridization to an immobilized template resulting in a highly specific hybridization process. To increase further the specificity, a second step of type discrimination was used in which specific extension of 3'-termini matched oligonucleotides was performed. The 46 type-specific oligonucleotides each had a unique tag sequence to allow detection via an array of oligonucleotides complementary to the tags. To evaluate the genotyping assay, a total of 92 HPV positive samples were tested in this study. Twelve had double infections and five had three to five coexisting HPV types. The results show that MUCH-AMASE can readily detect multiple infections, whereas conventional dideoxy sequencing resulted in ambiguous sequence. Four samples with three to five genotypes detected were cloned and individual clones were sequenced. The cloning procedure verified the MUCH-AMASE results with indications that we can find minor infections (< 2% relative amounts). We can thus conclude that the developed assay is highly sensitive, with improved throughput and with excellent possibility to detect multiple infections.
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| 12. |
- Leblanc, Neil, et al.
(författare)
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Development of a magnetic bead microarray for simultaneous and simple detection of four pestiviruses
- 2009
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 155:1, s. 1-9
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Tidskriftsartikel (refereegranskat)abstract
- This study reports a novel method for the rapid detection and identification of the four recognized species in the pestivirus genus of the Flaviviridae family, i.e. classical swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV1) and type 2 (BVDV2). The analysis of pestivirus PCR products was performed on microarrays by means of magnetic bead detection. The process utilizes an oligonucleotide array, onto which 5' biotinylated PCR products were hybridized, followed by visualization with streptavidin-coated magnetic particles by the naked eye, microscope or biochip reader. The assay was tested on a collection of pestiviruses that included all four species and allowed a specific and sensitive detection. Sensitivity was compared with other post-PCR detection methods, namely gel electrophoresis and suspension microarray. The results indicate that due to its high sensitivity, specificity and simple detection procedure, the magnetic bead assay provides a powerful tool for detection and identification of viral pathogens. Considering the simplicity of the assay, the protocols for hybridization and magnetic bead detection offer an emerging application for molecular diagnoses in virology that is amenable for use in a modestly equipped laboratory.
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| 13. |
- Liu, Lihong, et al.
(författare)
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Development of a primer-probe energy transfer real-time PCR assay for improved detection of classical swine fever virus
- 2009
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 160, s. 69-73
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Tidskriftsartikel (refereegranskat)abstract
- Classical swine fever (CSF) is a contagious and devastating disease, causing serious losses in the pig industry worldwide. Vaccination of pigs with the conventional C-strain vaccine has been practised in different regions of the world in order to prevent the disease. In the control programmes of CSF, rapid detection and identification of the causing agent, classical swine fever virus (CSFV) is a crucial step. This study describes a novel real-time PCR assay based on primer-probe energy transfer (PriProET) technology for improved detection of CSFV. The assay is able to detect 20 copies of viral cDNA per reaction, showing a high sensitivity. The specificity has been evaluated by testing 57 pestiviruses, representing all species and unclassified pestiviruses. The assay has been found to be highly reproducible. Following PCR amplification, melting curve analysis allows confirmation of specific amplicons, and differentiation between wild-type CSFV and certain C-strain vaccines. This study provides a new tool for the diagnosis of CSF. (C) 2009 Elsevier B.V. All rights reserved.
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| 14. |
- Liu, Lihong, et al.
(författare)
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Two real-time RT-PCR assays of classical swine fever virus, developed for the genetic differentiation of naturally infected from vaccinated wild boars
- 2009
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 159, s. 131-133
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Tidskriftsartikel (refereegranskat)abstract
- Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a disease notifiable to the Office international des Epizooties (OIE). A live marker vaccine would be the ultimate choice for controlling CSF, which enables serological and genetic differentiation of vaccine from wild type CSFV. Recently, a marker vaccine CP7_E2alf has been reported [Koenig, P., Lange, E., Reimann, L, Beer, M., 2007. CP7_E2alf. a safe and efficient marker vaccine strain for oral immunisation of wild boar against classical swine fever virus (CSFV). Vaccine 25, 3391-3399]. A vaccine-specific TaqMan real-time RT-PCR assay was developed and evaluated, and a second, wild type-specific assay was modified from an established one in such a way that both can be performed in two wells side-by-side in a microplate in a single run. The detection limit is 50 viral RNA copies per reaction for the vaccine-specific assay, and 20 copies per reaction for the wild type assay. The two assays have been shown to be highly specific and reproducible, with potential application for genetic differentiation of wild type CSFV from the marker vaccine CP7_E2alf in wild boar vaccination programs. (C) 2009 Elsevier B.V. All rights reserved.
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| 15. |
- Mantke, Oliver Donoso, et al.
(författare)
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A new quantitative real-time reverse transcriptase PCR assay and melting curve analysis for detection and genotyping of Ljungan virus strains
- 2007
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 141:1, s. 71-77
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Tidskriftsartikel (refereegranskat)abstract
- Ljungan virus (LV), a new member of the Picornaviridoe, recently isolated from vole species in both Sweden and the USA, is suspected to be pathogenic for humans as an actiological agent in myocarditis, diabetes, neurological disease and perinatal disease. This study describes for the first time an RT-PCR assay that can identify and quantify LV infection in various tissue sample types using primers and two different minor-groove-binder probes targeting the 5'-untranslated region of the LV genome. The assay, evaluated using control samples derived from various virus cultures and rodent tissues, allows precise quantification of viral load over six orders of magnitude (101 to 106 viral copies per assay) for all known strains of LV with high sensitivity and specificity. Furthermore, a melting curve analysis (MCA) was developed using two amplicon-specific hybridisation probes that allows rapid genotyping of different LV strains. These new methods provide useful tools to investigate the putative role of LV as a pathogen in both rodents and humans.
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| 16. |
- Muradrasoli, Shaman, et al.
(författare)
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Broadly targeted multiprobe QPCR for detection of coronaviruses : Coronavirus is common among mallard ducks (Anas platyrhynchos)
- 2009
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 159:2, s. 277-287
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Tidskriftsartikel (refereegranskat)abstract
- Coronaviruses (CoV) can cause trivial or fatal disease in humans and in animals. Detection methods for a wide range of CoVs are needed, to understand viral evolution, host range, transmission and maintenance in reservoirs. A new concept, "Multiprobe QPCR", which uses a balanced mixture of competing discrete non- or moderately degenerated nuclease degradable (TaqMan((R))) probes was employed. It provides a broadly targeted and rational single tube real-time reverse transcription PCR ("NQPCR") for the generic detection and discovery of CoV. Degenerate primers, previously published, and the new probes, were from a conserved stretch of open reading frame 1b, encoding the replicase. This multiprobe design reduced the degree of probe degeneration, which otherwise decreases the sensitivity, and allowed a preliminary classification of the amplified sequence directly from the QPCR trace. The split probe strategy allowed detection of down to 10 viral nucleic acid equivalents of CoV from all known CoV groups. Evaluation was with reference CoV strains, synthetic targets, human respiratory samples and avian fecal samples. Infectious-Bronchitis-Virus (IBV)-related variants were found in seven of 35 sample pools, from 100 wild mallards (Anas platyrhynchos). Ducks may spread and harbour CoVs. NQPCR can detect a wide range of CoVs, as illustrated for humans and ducks.
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| 17. |
- Muradrasoli, Shaman, et al.
(författare)
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Development of real-time PCRs for detection and quantitation of human MMTV-Iike (HML) sequences HML expression in human tissues
- 2006
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 136:1-2, s. 83-92
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Tidskriftsartikel (refereegranskat)abstract
- The human genome contains around 1000 betaretrovirus-like copies, human mouse mammary tumour virus (MMTV)-like (HML) groups 1 - 10, also referred to as human endogenous retroviruts "HERV-K". Despite many efforts, it is not established whether betaretroviruses, exo- or endogenous, are involved in the etiology of breast cancer, or other cancer diseases, in humans. Quantitative real-time PCR (QPCR) TaqMan((R))-based assays for HML groups 1-7, targeting the conserved reverse transcriptase (RT) and integrase (IN) domains of the pol gene were designed. Plasmids containing the entire pol gene of HML 1-7 were used as standards. The RT and IN based QPCRs could detect 10(0)-10(3) copies per PCR reaction of the plasmids. However, not all plasmids gave a signal in both RT and IN QPCRs, probably due to mismatches. Furthermore, RT and IN based HML6 specific QPCRs were developed. They were specific for amplification of transcripts for the whole HML6 group. The methods allow the monitoring in body fluids and tissues of expression of a wide range of betaretrovirus-like sequences. Betaretrovirus-like RNA was studied in normal human tissues and of HML6 in brains of multiple sclerosis (MS) patients. Brain, adrenal gland and testis had a high betaretrovirus-like expression. Multiple sclerosis plaques contained the same HML6 RNA concentration as control tissue. These assays are expected to enhance studies on involvement of betaretroviruses in physiology and disease.
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| 18. |
- Näslund, Jonas, 1979-, et al.
(författare)
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Kinetics of Rift Valley fever virus in experimentally infected mice using quantitative real-time RT-PCR
- 2008
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 151:2, s. 277-282
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Tidskriftsartikel (refereegranskat)abstract
- Rift Valley Fever (RVF) is an important viral zoonosis in Africa affecting animals and humans. Since no protective vaccines or effective treatments are available for human use, accurate and reliable diagnostic methods are essential for surveillance of the disease in order to implement adequate public health actions. To study the kinetics of the RVF Virus (RVFV) infection, a SYBR Green-based quantitative real-time RT-PCR assay was developed. By using primers targeting the S-segment of RVFV, the detection limit of this assay was estimated to 30 RNA templates. Blood and organs of experimentally infected mice were sampled at different time points and RVFV RNA was quantified. High amounts of RVFV RNA were found in blood, brain, and liver samples shortly after infection with a 1-4 days post infection window for viral RNA detection. Mice developed symptoms after the appearance of serum antibodies, indicating that the host response plays an important role in the outcome of the disease. The RVFV quantitative RT-PCR proved to be a valuable diagnostic tool during the first days of infection, before detectable antibody levels and visual symptoms of RVF were observed.
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| 19. |
- Rodriguez, Jesus, 1977-, et al.
(författare)
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Design of a multiplex nested PCR for genotyping of the NSP4 from group A rotavirus
- 2008
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 149:2, s. 240-245
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Tidskriftsartikel (refereegranskat)abstract
- A novel PCR method was developed to discriminate amongst genotypes A-C of the rotavirus non-structural protein 4 (NSP4). Genotype-specific primers were designed that correctly identified the NSP4 genotype when evaluated as a multiplex PCR with cell culture adapted rotavirus strains. Rotavirus strains B223 SGIG6P6[1], NCDV SGIG6P6[1] and SA11 SGIG3P5B[2] were used as control for NSP4 genotype A, A34 SGIG5P14[23], Gottfried SGIIG4P2B[6] and Wa SGIIG1P1A[8] for NSP4 genotype B, RRV SGIG3P5B[3] for NSP4 genotype C. Subsequently, the same set of specific primers was used to genotype a set of 77 Swedish clinical samples. The results showed that all human clinical samples analyzed belong to the NSP4 genotype B and the VP6 subgroup II. © 2008 Elsevier B.V. All rights reserved.
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| 20. |
- Tolf, Conny, et al.
(författare)
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Characterization of polyclonal antibodies against the capsid proteins of Ljungan virus
- 2008
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 150:1-2, s. 34-40
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Tidskriftsartikel (refereegranskat)abstract
- Ljungan virus (LV) is a suspected human pathogen isolated from voles in Sweden and North America. To enable virus detection and studies of localization and activity of virion proteins, polyclonal antibodies were produced against bacterially expressed capsid proteins of the LV strain, 87-012G. Specific detection of proteins corresponding to viral antigens in lysates of LV infected cells was demonstrated by immunoblotting using each one of the generated polyclonal antibodies. In addition, native viral antigens present in cell culture infected with LV strains 87-012G or 145SLG were detected in ELISA and by immunofluorescence using the antibodies against the VP0 and VP1 proteins. The anti-VP3 antibody did not react with native proteins of the LV virion, suggesting that the VP3 is less potent in evoking humoral response and may have a less exposed orientation in the virus capsid. No activity of the antibodies was observed against the closely related human parechovirus type 1. The polyclonal antibody against the VP1 protein was further used for detection of LV infected myocytes in a mouse model of LV-induced myocarditis. Thus, polyclonal antibodies against recombinant viral capsid proteins enabled detection of natural LV virions by several different immunological methods.
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| 21. |
- Yacoub, A., et al.
(författare)
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The rapid molecular subtyping and pathotyping of avian influenza viruses
- 2009
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Ingår i: Journal of Virological Methods. - : Elsevier BV. - 0166-0934 .- 1879-0984. ; 156:1-2, s. 157-161
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Tidskriftsartikel (refereegranskat)abstract
- Highly conserved nucleotide stretches flanking the cleavage site of the haemagglutinin (HA) gene of influenza type A viruses were utilised for generating PCR amplicons from a broad range of avian influenza viruses (AIV) in a one-step real-time SYBR Green RT-PCR assay. The nucleotide sequencing of the amplified PCR products simultaneously reveals both the HA subtype and the pathotype of the AIV isolates, as we demonstrated in case of H5 subtype viruses. The specificity of the assay was confirmed by investigating 66 strains of AIV and nine heterologous pathogens, including influenza B, C and various avian pathogenic viruses. This assay enables a general HA subtype identification and pathotype determination of AIV isolates providing a useful alternative tool for avian influenza diagnosis.
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| 22. |
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| 23. |
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| 24. |
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| 25. |
- Elfaitouri, Amal, et al.
(författare)
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Quantitative real-time PCR assay for detection of human polyomavirus infection.
- 2006
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Ingår i: J Virol Methods. - 0166-0934. ; 135:2, s. 207-13
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Tidskriftsartikel (refereegranskat)abstract
- The overall aim was to study factors that affect behaviour related to CVD (cardiovascular diseases). Study I tested whether gender, education and so-cioeconomic status correlated to knowledge about risk factors, and Study II studied knowledge and risk behaviour from a national perspective (Sweden versus Poland). Furthermore, Study III examined whether obese people dif-fered from people of normal weight regarding knowledge about risk factors, and Study IV examined whether risk behaviour is affected by personal ex-perience of illness and family history of CVD.The studies are population-based with cross-sectional design. Data were obtained by questionnaires and by screening results of risk factors related to CVD. The studies were carried out among 50-year old men and women in Västmanland, Sweden (n=1011) and in Wroclaw, Poland (n=1043).The results show that women are more knowledgeable than men about the risk factors for CVD, and that low education is associated with insufficient knowledge about CVD (Study I). The discrepancy between knowledge and behaviour was greater among the Poles than it was among the Swedes (Study II). Obese individuals did not differ significantly from individuals with a normal weight regarding knowledge of cardiovascular risk factors when education was controlled for (Study III). Individuals with a personal experience of illness may be more inclined to change smoking behaviour than the average person (Study IV).In conclusion, knowledge about risk factors for CVD varies with education, gender and, to a certain degree, nationality. However, knowledge does not only consist of the conditions of behaviour change. The results in the thesis substantiate theories suggesting that change in risk behaviour is a process over time. Predictors of risk behaviours on the individual level as well as national level are of importance, and needs to be considered in the every day practice of health care professionals.
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| 26. |
- Johannesson, Per, et al.
(författare)
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A novel and rapid method to quantify cytolytic replication of picornaviruses in cell culture
- 2005
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Ingår i: Journal of virological methods. - : Elsevier BV. - 0166-0934. ; 130, s. 117-123
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Tidskriftsartikel (refereegranskat)abstract
- Determining viral titers is a key issue in a wide variety of studies regarding different aspects of virology. The standard methods used for determining picornavirus titers are endpoint titration assay and plaque assay, both time consuming and laborious. The method described uses the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide) that is reduced to formazane by cellular dehydrogenase, genes shown to be down-regulated during picornavirus infection. The amount formazane produced correlates with the viral titers obtained and can easily be measured using an ELISA plate reader. The colorimetric method has been evaluated using virus types from different genera of the Picornaviridae family. The MTT method reduces the time spent on determining the viral titers and still maintains a reliable accuracy.
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| 27. |
- Kasubi, Mabula Joseph, et al.
(författare)
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A branched, synthetic oligopeptide corresponding to a region of glycoprotein G of HSV-1 reacts sensitively and specifically with HSV-1 antibodies in an ELISA.
- 2005
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Ingår i: Journal of virological methods. - : Elsevier BV. - 0166-0934. ; 125:2, s. 137-43
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Tidskriftsartikel (refereegranskat)abstract
- Herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2), which are common worldwide, are so similar that antibodies directed against one serotype may crossreact with antigens from the other one. Methods for specific detection of antibodies against HSV-1 or HSV-2 are based upon the antigenicities of glycoproteins G. However, due to the cost, the available commercial methods may not readily be used in developing countries. A different enzyme-linked immunosorbent assay (ELISA) method, based upon a synthetic oligopeptide corresponding to an immunogenic region in glycoprotein G of HSV-2, has been used recently and successfully for detection of HSV-2 antibodies. In the present study, the sequences of a newly identified immunogenic and type-specific region in glycoprotein G of HSV-1 was used to synthesize three different, branched oligopeptides. The performances of these peptides in an ELISA were investigated by testing Scandinavian and African sera which were characterized by commercial ELISA and Western blotting methods and divided into four groups either lacking HSV antibodies, containing antibodies against one or the other virus, or against both types. The peptide which corresponded in sequence to the immunodominant region was as specific and sensitive by an ELISA as were the commercial methods. The method is inexpensive and reliable.
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| 28. |
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| 29. |
- Malmström, Sebastian, 1976, et al.
(författare)
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Mutation analysis of lamivudine resistant hepatitis B virus strains by TaqMan PCR.
- 2007
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Ingår i: Journal of virological methods. - : Elsevier BV. - 0166-0934. ; 143:2, s. 147-52
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Tidskriftsartikel (refereegranskat)abstract
- Hepatitis B virus infected patients on long-term lamivudine treatment are exposed to a 15-32% risk compounded annually of developing resistance mutations. Such resistance results in a progression of the liver damage caused by chronic hepatitis B, and may also impair the effect of other antivirals through cross-resistance. At present lamivudine is used frequently as monotherapy because of its relatively low price and negligible side effects. Thus, simple methods for identifying resistance mutations are required. A method based on real-time polymerase chain reaction with TaqMan chemistry is described. The method combines both primer specificity, in order to target wild type and mutant viral strains at codon 180, and a mixture of three minor groove binding probes distinguishing the YMDD wild type and the YVDD and YIDD variants at codon 204. The accuracy of the method was verified by concordance with results of direct sequencing and restriction fragment length polymorphism when examining 27 samples from five patients, in whom lamivudine resistance was known to have developed. This method is rapid, cost effective, and should prove useful for monitoring patients treated with lamivudine.
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| 30. |
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