Abstract：
:Quantification of the viral burden and identification of drug resistant mutations are important laboratory tools in the management of HIV-1 infected patients. However, widespread use of assays for viral load determination and genotyping is still hampered by the high cost. Here, an in-house RT-PCR-sequencing assay for HIV-1 drug resistance monitoring with the potential to be used both as a qualitative assay to detect the virus in plasma and as a genotyping system is described. A total of 377 clinical samples, collected from 374 HIV-infected patients of diverse geographic origin, were tested. The nested RT-PCR for amplification of the protease reverse transcriptase gene was found positive for 350 (92.8%) and 346 (91.8%) of 377 samples, respectively. All amplification-failures were due to viral loads of below 500 copies/ml. However, low viral load does not exclude amplification since 80.2 and 76% of 121 samples with viral loads of less than 500 copies/ml were amplified successfully for protease and reverse transcriptase, respectively. The high sensitivity of the assay was independent of the HIV-subtype, with a broad range of different HIV-1 subtypes tested. In conclusion the RT-PCR-direct sequencing method is convenient for the sensitive detection and subsequent genotyping of plasma RNA from a broad range of different HIV-1 subtypes. The assay enables the accurate follow-up of patients under treatment at a significantly reduced cost compared to the currently available commercial assays for viral load assessment and genotyping.
journal_name
J Virol Methodsjournal_title
Journal of virological methodsauthors
Steegen K,Demecheleer E,De Cabooter N,Nges D,Temmerman M,Ndumbe P,Mandaliya K,Plum J,Verhofstede Cdoi
10.1016/j.jviromet.2005.11.004subject
Has Abstractpub_date
2006-05-01 00:00:00pages
137-45issue
2eissn
0166-0934issn
1879-0984pii
S0166-0934(05)00373-3journal_volume
133pub_type
杂志文章abstract：:A method for coupling cytomegalovirus (CMV) antigens to aldehyde-fixed erythrocytes for use in a passive hemagglutination assay for antibody to CMV is described. This method uses acid buffer instead of tannic acid for coupling CMV-complement fixation (CF) antigen to the stabilized cells. The coated cells are stable fo...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(83)90002-2
更新日期：1983-09-01 00:00:00
abstract：:The Bacille Calmette Guerin (BCG), long valued for its role as a live vaccine for the prevention of tuberculosis, is being used as a recombinant delivery vehicle for foreign antigens, principally, for inducing long-lived specific humoral and cellular immunity. Hepatitis B and its sequelae are major public health probl...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2004.11.026
更新日期：2005-04-01 00:00:00
abstract：:Epstein-Barr virus (EBV) has been implicated in the pathogenesis of several human malignancies including B lymphomas and nasopharyngeal carcinoma. The EBV R transactivator (Rta) has been found to play essential roles in stimulating a lytic cycle and viral gene expression. Recently, it was shown that ELISA detecting se...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2007.03.011
更新日期：2007-09-01 00:00:00
abstract：:The polycation protamine sulfate was compared to polybrene, the usual agent employed, for its ability to increase the efficiency of retroviral infection. The murine retroviral vector SAX, which contains the neoR gene and the human adenosine deaminase (ADA) cDNA, was used as a marker of cell infection. SAX viral supern...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(89)90132-8
更新日期：1989-02-01 00:00:00
abstract：:The disruption of the viral coat of human immunodeficiency virus by Triton X-100, a nonionic detergent, is a time-dependent process which requires incubation times of 30 min or longer. Conditions for the production of a noninfectious sample from a viral pellet that can be used to measure reverse transcriptase activity...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/s0166-0934(97)00169-9
更新日期：1998-01-01 00:00:00
abstract：:Avian influenza viruses (AIVs) of the H9N2 subtype are a major economic problem in the poultry industry in Israel. Most field isolates from the last decade differ significantly from H9N2 isolates from Europe and the USA, rendering published detection methods inadequate. This study aimed to develop a real-time TaqMan((...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2010.04.019
更新日期：2010-09-01 00:00:00
abstract：:The reverse transcriptase of the sheep lentivirus visna/maedi virus has been characterised. Optima for magnesium ion concentration (5-10 mM), potassium ion concentration (150 mM) and pH (8.25) for this enzyme are very similar to those previously described for the human immunodeficiency viruses. The assay used for this...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(92)90144-3
更新日期：1992-02-01 00:00:00
abstract：:Rapid and accurate detection of measles virus is important for case diagnosis and public health management. This study compared the performance of two monoplex RT-PCR reactions targeting the H and N genes to a duplex RT-PCR targeting both genes simultaneously. The duplex simplified processing without compromising assa...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2016.11.003
更新日期：2017-01-01 00:00:00
abstract：:Emerging life threatening pathogens such as severe acute aspiratory syndrome-coronavirus (SARS-CoV), avian-origin influenzas H7N9, and the Middle East respiratory syndrome coronavirus (MERS-CoV) have caused a high case-fatality rate and psychological effects on society and the economy. Therefore, a simple, rapid, and ...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2014.01.022
更新日期：2014-06-01 00:00:00
abstract：:Green fluorescent protein (GFP) was targeted into bacteriophage T4 heads and proheads as a probe of the internal environment. Targeting was accomplished with internal protein III (IPIII) fusion proteins or capsid targeting sequence (CTS)-tagged proteins, where CTS is the 10-amino acid residue CTS of IPIII. Recombinant...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/s0166-0934(00)00166-x
更新日期：2000-07-01 00:00:00
abstract：:The presence of competent vectors in some countries currently free of Rift Valley fever (RVF) and global changes in climate, travel and trade have increased the risk of RVF spreading to new regions and have emphasised the need for accurate and reliable diagnostic tools for early diagnosis during RVF outbreaks. Highly ...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2011.07.011
更新日期：2011-11-01 00:00:00
abstract：:An epitope-blocking ELISA (EB-ELISA) was developed to distinguish animals infected with foot-and-mouth-disease (FMDV) from those immunized with commercial vaccines. The assay used monoclonal antibodies to target the 3B core repeat motif (QKPLK) and purified recombinant 3AB proteins from the major B cell line epitopes ...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2007.01.025
更新日期：2007-06-01 00:00:00
abstract：:A liquid phase blocking sandwich ELISA has been compared with virus neutralisation for testing pig sera for antibodies against swine vesicular disease (SVD) virus. Highest infectivity titre of SVD virus was obtained using a multiplicity of infection of 30 pfu/cell and harvesting after 21 h. Titres obtained for 300 cli...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(89)90101-8
更新日期：1989-07-01 00:00:00
abstract：:Grapevine shoot cultures infected by Grapevine vitivirus A (GVA) were grown on Quorin-Lepoivre basic medium and submitted to in vitro chemotherapy and thermotherapy sanitation techniques. Ribavirin (Rb) at 20gml(-1), dihydroxypropyladenine (DHPA) at 60gml(-1) and their combination (RbDH) were added to the proliferatin...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2007.06.008
更新日期：2007-12-01 00:00:00
abstract：:The genes encoding the Erns and E2 antigen epitopes of classical swine fever virus (CSFV) were expressed as a chimeric protein in Escherichia coli BL21 by pET expression system. The antigenicity of the expressed protein CnC2 was identified by indirect enzyme-linked immunoabsorbant assay (ELISA) and immunoblot with ant...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2011.12.006
更新日期：2012-03-01 00:00:00
abstract：:The Cucumber mosaic virus (CMV) is a three-component isodiametric plant virus with an extremely wide host range, present worldwide. A pseudorecombinant form has been described, deriving from the RNA3 component of the CMV-S strain, carrying the coat protein (CP) gene, and the RNA 1, 2 components of the CMV-D strain. Th...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2008.10.005
更新日期：2009-02-01 00:00:00
abstract：:A system which allows the reconstitution of measles virus (MV) from cloned cDNA is described. The severely host cell restricted vaccinia vector MVA-T7 expressing bacteriophage T7 RNA polymerase was used to generate full-length antigenomic MV RNA and simultaneously the mRNAs encoding the viral N, P and L proteins in or...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/s0166-0934(96)02137-4
更新日期：1997-02-01 00:00:00
abstract：:The development of a rapid and sensitive system for detecting influenza viruses is a high priority for controlling future epidemics and pandemics. Quantitative real-time PCR is often used for detecting various kinds of viruses; however, it requires more than 2h per run. Detection assays were performed with super high-...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2011.08.015
更新日期：2011-12-01 00:00:00
abstract：:An automated blotter was evaluated for detection of the HPV SPF10-LiPA25 RHA for HPV genotyping with 63 FFPE tissue and 45 WHO proficiency panel samples. The results showed that 10-20% more types were detected and bands were of significantly stronger intensity (p<0.0001) compared to manual processing. Therefore it was...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2014.01.012
更新日期：2014-04-01 00:00:00
abstract：:Tissue culture ID50 and plaque assays for the Autographa californica nuclear polyhedrosis virus, using the Spodoptera littoralis cell line HPB-SL, were developed. Direct comparison using these assay methods showed that these cells were as susceptible to infection as the more commonly used Spodoptera frugiperda cell li...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(85)90082-5
更新日期：1985-01-01 00:00:00
abstract：:The Cucumber mosaic virus (CMV) is an isodiametric plant virus with an extremely wide host range, present worldwide. CMV chimeric particles (R9-CMV), engineered to express a 27-aa synthetic peptide derived from Hepatitis C virus (HCV), were demonstrated to be stable under simulated gastric and intestinal conditions. T...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2010.01.021
更新日期：2010-05-01 00:00:00
abstract：:In this study, we generated recombinant virus-like particles (VLPs) against family Filoviridae, genus Ebolavirus, species Zaire ebolavirus, strain Makona (EBOV) in Drosophila melanogaster Schneider 2 (S2) cells using the EBOV Makona. S2 cells were cotransfected with four viral plasmids encoding EBOV Makona proteins an...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2018.08.016
更新日期：2018-11-01 00:00:00
abstract：:The mosaic disease caused by fig mosaic virus (FMV) is considered the plague of fig worldwide. A naïve phage display library, raised against the recombinant nucleocapsid protein of FMV (FMV-Np) was screened to obtain specific monoclonal recombinant antibodies in the form of single chain variable fragments (scFvs). Aft...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2019.113796
更新日期：2020-02-01 00:00:00
abstract：:Rapid diagnosis of herpes simplex encephalitis (HSE) can only be achieved by the polymerase chain reaction (PCR). In order to carry out PCR under routine conditions, it is of great importance to establish an easy DNA extraction protocol and especially a rapid and sensitive DNA detection method. In the present study, t...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(94)90174-0
更新日期：1994-12-01 00:00:00
abstract：:A one-step centrifugal ultrafiltration method was developed to enhance rapid detection of human enteric viruses and co-occurring viruses in wastewater. Samples were collected pre- and post-UV treatment at two full-scale tertiary municipal wastewater treatment plants in Calgary, Canada. Viruses were concentrated from 1...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2016.09.010
更新日期：2016-11-01 00:00:00
abstract：:A sensitive and specific blocking enzyme-linked immunosorbent assay (ELISA) was developed to distinguish infectious bovine rhinotracheitis virus (IBRV)-infected animals from those immunized with a glycoprotein gIII deletion mutant, IBRV(NG)dltkdlgIII. For this ELISA, undiluted test sera are used to block the binding o...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(92)90006-y
更新日期：1992-10-01 00:00:00
abstract：:Rapid characterization of herpesvirus isolates exemplified by equine herpesviruses is described. Total DNAs were isolated from virus infected small scale cell cultures. The DNA fragments obtained after restriction enzyme digestion were separated on agarose gels, transferred and immobilized on filter membranes. A radio...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(86)90030-3
更新日期：1986-11-01 00:00:00
abstract：:A rapid and accurate detection and identification system was developed for flaviviruses that makes use of reverse transcription-polymerase chain reaction (RT-PCR). A primer pair (YF-1 and YF-3), which corresponds to the highly conserved sequence at 3' noncoding region among flaviviruses, was useful for identification ...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(93)90020-r
更新日期：1993-03-01 00:00:00
abstract：:The plaque assay is essential for virion quantitation but the classic protocol requires considerable efforts. A simplified dengue 96-well plaque assay with automated quantitation program is an alternative to access the level of infectious virus. Dengue plaque assay was simplified using LLC/MK2 cells and virus mixing s...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/j.jviromet.2016.08.009
更新日期：2016-11-01 00:00:00
abstract：:Ten plasmids containing viral cDNA inserts of portions of the dengue virus type 2 (DEN-2) or Kunjin virus (KUN) genomes were biotinylated using photobiotin acetate and used as probes for the detection of flavivirus RNA in infected Vero cells. The viral cDNA inserts ranged in length from 0.19 to 2.7 kilobase pairs, and...
journal_title：Journal of virological methods
pub_type： 杂志文章
doi：10.1016/0166-0934(87)90055-3
更新日期：1987-02-01 00:00:00
