G. van der Groen

Filoviridae: a taxonomic home for Marburg and Ebola viruses?

Filoviridae: a Taxonomic Home for Marburg and Ebola Viruses ? M.P. Kiley E.T.W. Bowen G.A. Eddy M. Isaäcson K.M. Johnson J.B. McCormick F.A. Murphy S.R. Pattyn D. Peters O.W. Prozesky R.L. Regnery D.I.H. Simpson W. Slenczka P. Sureau G. van der Groen P.A. Webb H. Wulff Center for Infectious Diseases, Centers for Disease Control, Atlanta, Ga., USA; PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire, UK; US Army Medical Research Institute for Infectious Diseases, Ft. Detrick, Frederick, Md., USA; The South African Institute for Medical Research, Johannesburg, South Africa; Colorado State University, College of Veterinary Medicine and Biomedical Sciences, Ft. Collins, Col., USA;Prins Leopold Instituut voor Tropische Geneeskunde, Antwerpen, Belgium; Bernard-Nocht-Institut für Schiffsund Tropenkrankheiten, Abteilung für Virologie, Hamburg, FRG; National Institute for Virology, Sandringham, Transvaal, South Africa;Hygiene-Institut der Universität, Marburg, FRG; Institut Pasteur, Paris, France

Isolation of HIV-1 RNA from plasma: evaluation of eight different extraction methods.

The efficacy of eight different methods for the extraction of HIV-1 RNA from plasma was compared. The RNA preparation method that gave the best results by RT-PCR was the one described by Chomczynski and Sacchi (1987, Anal. Biochem. 162, 156-159). This method consists of a guanidine thiocyanate treatment followed by three phenol-chloroform-isoamylalcohol extractions and an ethanol precipitation. The disadvantage of this method is that it is time consuming and less suitable for the extraction of large series of samples. Moreover, due to the large number of procedural steps, there is a greater risk of sample mix-up or contamination. Of the single-step RNA purification methods, good results were obtained with the TRIzol method (Gibco Life Technologies, Paisley, UK) and with the extraction method offered by the NASBA kit (Organon Teknika, Turnhout, Belgium). The above single-step methods are recommended since both are sensitive enough to detect low copy numbers of HIV-RNA in the plasma of asymptomatic patients, and require only 2 h for completion. For most of the methods evaluated the inter-test variability was acceptable (mean variation coefficient between duplicate extraction varied between 17.3 and 47.3%). Inter-laboratory reproducibility was evaluated only for the TRIzol-method and found to be low (mean variation coefficient 63.4).

Design and evaluation of an in-house HIV-1 (group M and O), SIVmnd and SIVcpz antigen capture assay.

An enzyme-linked immuno-sorbent assay (ELISA) for the detection of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIVcpz/SIVmnd) antigens was designed using immunoreagents from naturally infected individuals, and compared to the commercially available Vironostika HIV-1 Antigen Microelisa System (Organon Teknika). The in-house assay proved to be specific for HIV-1 isolates belonging to group M (A-H) and group O and for SIVcpz and SIVmnd isolates, but was less sensitive than the Vironostika HIV-1 Antigen Microelisa System, except for SIVmnd. For the strains belonging to HIV-2, SIVmac and SIVagm, the in-house assay could not detect antigen to an appreciable degree. This study shows that a considerably less expensive but sufficiently accurate HIV-1 antigen capture assay can be developed to monitor HIV-1 (group M and O), SIVcpv and SIVmnd antigen in the supernatants of virus cultures.

Reactivity and amplification efficiency of the NASBA HIV-1 RNA amplification system with regard to different HIV-1 subtypes

In view of the genetic diversity of the human immunodeficiency virus Type 1, we assessed the sensitivity and quantification efficiency of the HIV-1 RNA NASBA amplification system with respect to different HIV-1 subtypes and recombinants. Twenty cell culture supernatants representing 17 HIV-1 group M and 3 group O strains were tested, and NASBA RNA loads were compared with results obtained with a RT-PCR based HIV-1 RNA quantitation method, with p24-antigen concentrations and with the infective dose. The current HIV-1 RNA NASBA seemed suitable to quantitate representatives of different HIV-1 M subtypes. Differences between NASBA and RT-PCR loads were observed for certain HIV-1 M strains. Significantly lower RT-PCR loads were measured for most gag A, gag B and gag F strains, whereas NASBA detected lower copy numbers in 1 gag H strain and 1 gag H/env G recombinant. NASBA was not able to quantify 1 HIV-1 group M recombinant. Some of these differences could be explained by the presence and position of mismatches with primers. HIV-1 group O strains were not detectable by both RNA amplification methods. A firm correlation was not observed between the measured RNA loads and either the p24-antigen concentration or the infective dose.

HIV-1 group O and group M dual infection in Bénin

HIV-1 is subdivided into group M (major) viruses and group O (outlier) viruses. Group M viruses are worldwide while group O viruses have been reported mainly in Cameroon and among Cameroonians living in France Germany and Belgium. Group O infection has also been reported in Gabon Equatorial Guinea and Nigeria. Double infection with genetically very diverse HIV-1 group M and O viruses may occur. Between 1993 and 1994 sera from 142 HIV-1-seropositive people (128 prostitutes 8 pregnant women and 6 people consulting for STD symptoms) were collected in Benin. 20 of the individuals had antibody reactivity to one or more group O V3-loop peptides in ELISA. One of the 20 reactive samples reacted with the group O-ANT70 V3-loop peptide in LIA and had antibodies to p66 gp41 p33 p24 on group O ANT70 CA9 and VI686 western blot suggesting group O infection. To confirm this result RNA was extracted from the serum of the individual and RT-PCR was conducted with specific primers to distinguish group O and M infection. PCR was positive with both group O-specific and group M-specific primers suggesting a mixed group O/M infection. Sequencing and phylogenetic analysis of the amplified pol fragments confirmed the observation. The HIV-1 group O/M dually infected individual was a woman 19 years old when the sample was collected in 1993. A Fon of Benin origin but born in Cote dIvoire she was married and pregnant with her third child.

Isolation of HIV-1 RNA from plasma : evaluation of seven different methods for extraction. (Part two)

Some new commercial methods for the extraction of viral RNA have been introduced recently. In addition to the study published previously (Verhofstede, C., Reniers, S., Van Wanzeele. F., Plum J., 1996. AIDS 8, 1421-1427), seven different methods (four newly developed and three reference methods) for extraction of HIV-1 RNA from plasma have been evaluated. The RNA preparation method that gave the best results (acceptable reproducibility, highest sensitivity, reasonable price, fast and easy to perform), was the QIAamp Viral RNA kit from QIAgen. The High Pure Viral RNA Kit (Boehringer Mannheim) as well as the non-commercialised extraction kits were also very sensitive. The non-commercial tests seem less suitable for routine use and for the processing of large number of samples. Two methods, RNA Insta-Pure LS (Eurogentec) and PANext RNA extraction kit 1 (NTL, PANsystems GmbH) are not adapted for HIV plasma extraction. The single step methods using glass fibre or silica column are rapid (from 60 to 75 min depending on the number of wash steps) and although the price is high they are cheaper than the Boom extraction methods: High Pure Viral RNA Kit (Boehringer Mannheim) (

HIV antigen detection in circulating immune complexes

3.3/sample), QIAamp Viral RNA Kit (Qiagen) (

Evaluation of a line immunoassay for simultaneous confirmation of antibodies to HIV-1 and HIV-2

3.6/sample), Boom extraction (

Evidence of the presence of two hantaviruses in Slovenia, Yugoslavia

5/sample). The Qiagen kit is the only kit that combines sensitivity with reproducibility, it is commercialised, rapid and affordable in price and can be automated. For most of the methods evaluated the inter-test variability was acceptable (mean variation coefficient between duplicate extractions varied between 26.4 and 48.6%).

Characterization of some ungrouped viruses

Various methods were evaluated for their effectiveness in releasing HIV antigen (Ag) from artificial immune complexes (IC) and from IC present in serum from HIV antibody (Ab) positive subjects. The most effective methods for recovering HIV Ag from IC were those which included a denaturation step to prevent reassociation of Ag with Ab. IC precipitation in 2.5% polyethylene glycol followed by acid treatment with 1 M glycine.HCl (pH 2) for 10 min at 70 degrees C in the presence of 0.05% SDS gave very satisfactory results. With this method, IC were detected in sera from HIV antibody positive Caucasian subjects at all stages of infection. After HIV IC dissociation, HIV Ag was detected in a significant number (8/17 or 47%) of asymptomatic subjects. IC were most prevalent during the late stages of infection. A substantial increase in HIV Ag positivity was also observed in 20 Senegalese HIV Ab positive sera. After HIV IC dissociation HIV antigen detection increased from 2/20 to 12/20. The relevance of IC detection is discussed.