Patrick Goubau

Sensitive and rapid assay on MT-4 cells for detection of antiviral compounds against the AIDS virus

Human immunodeficiency virus (HIV) infection of MT-4 cells, an HTLV-I-transformed T-cell line, proved to be a rapid and sensitive assay system for the detection of potential antiviral drugs effective against the acquired immune deficiency syndrome (AIDS). Four days after HIV inoculation of the MT-4 cells, viral antigen expression was monitored in parallel with indirect immunofluorescence microscopy and laser flow cytofluorography. When 3-azido-2,3-dideoxythymidine (AzddThd, AZT) and 2,3-dideoxycytidine (ddCyd) were evaluated under these conditions, they inhibited viral antigen expression at a minimum (33% inhibitory) concentration of 0.0004 and 0.02 microM, respectively. Similar minimum effective concentrations were found for AzddThd and ddCyd in assays where inhibition of viral cytopathogenicity was based on cell survival. While laser flow cytofluorography could be best adapted for quantitative measurements, cell survival and reconstitution of disrupted cell aggregates gave an equally rapid and sensitive endpoint; and the latter may be ideally suited for preliminary drug screening.

Detection of HIV-1 RNA in plasma and serum samples using the NASBA amplification system compared to RNA-PCR

The presence of HIV-1 RNA in the plasma and serum of European and African patients was monitored using RNA-polymerase chain reaction (RNA-PCR) and the new isothermal NASBA nucleic acid amplification system encompassing a gel-based detection assay (ELGA). Identical RNA extraction procedures, provided by the NASBA amplification system, were used for both methods. The detection limit for HIV-1 RNA, measured on a 10-fold dilution series of spiked HIVIIIB in negative plasma, was about 0.05 CCID50 per test for both methods. Both NASBA and RNA-PCR were more sensitive than a p24 assay for the detection of circulating HIV-1 virus in blood: 17 of the 34 (50%) p24 antigen-tested seropositives were p24-positive while 32 (94%) were positive by NASBA and 30 (88%) by RNA-PCR. Among the 45 seropositives, 34 of which were tested for p24 antigen, 43 (96%) were positive by NASBA and 41 (91%) by RNA-PCR. Almost all seropositives had a detectable viral load in 100 microliters plasma. Lower viral loads were only encountered in some healthy seropositives with a higher CD4 count. There was no cross-reactivity with HIV-2 or HIV-I with both the RNA-PCR and NASBA. The extraction method used permitted the detection of HIV-1 RNA equally well in serum and in plasma with heparin or EDTA.

Use of a generic polymerase chain reaction assay detecting human T-lymphotropic virus (HTLV) types I, II and divergent simian strains in the evaluation of individuals with indeterminate HTLV serology

In countries with a low prevalence of human T‐lymphotropic virus (HTLV) infection, indeterminate HTLV serologies are a major problem in blood bank screening because of the uncertainties about infection in these cases. The recent discovery of two new types of simian T‐lymphotropic virus (STLV), which give an HTLV‐indeterminate serology, raises the question whether indeterminate serologies in humans may be linked to new types of HTLV. Starting from a Tax sequence alignment of all available primate T‐cell lymphotropic virus strains (PTLV), including the two new types STLV‐PH969 and STLV‐PP1664, we developed generic and type‐specific nested polymerase chain reactions (PCRs). The generic PCR proved to be highly sensitive and cross‐reactive for all four types of PTLV, while the discriminatory PCRs had a high sensitivity and a specificity of 100%. There was no cross‐reactivity with human immunodeficiency virus (HIV), ensuring correct interpretation of results from coinfected patients. Among the 77 serologically indeterminate samples tested, 6 were found to be HTLV‐IPCR positive and 1 was HTLV‐II PCR positive. Sequencing of one of the HTLV‐I PCR positives excluded PCR contamination, and revealed a divergent type of HTLV‐I. The majority of the seroindeterminate samples (91%) were however HTLV‐PCR negative, and no new types of HTLV were found. This new assay can identify otherwise undetected HTLV‐I or HTLV‐II infections and is a useful tool of screening for new types of HTLV among seroindeterminate samples. J. Med. Virol. 52:1–7, 1997.© 1997 Wiley‐Liss, Inc.

The presence of a divergent T-lymphotropic virus in a wild-caught pygmy chimpanzee (Pan paniscus) supports an African origin for the human T-lymphotropic/simian T-lymphotropic group of viruses

We isolated a divergent simian T-lymphotropic virus (STLV) (strain PP1664) from a wild-caught African bonobo (pygmy chimpanzee, Pan paniscus). Molecular and phylogenetic characterization of this virus show that it reliably separates from the two well-established primate T-lymphotropic virus types, HTLV-I/STLV-I (PTLV-I) and PTLV-II, and from a third type isolated from an African-born Papio hamadryas and designated by us as PTLV-L. Four of eight bonobos kept at the Antwerp Zoo, Belgium, showed an aberrant PTLV serology. We amplified and sequenced a 709 bp PTLV proviral tax/rex fragment from one of the reactive bonobos. It differs by about 25 % from the homologous nucleotide sequences of PTLV-I and PTLV-L and by about 17 % from PTLV-II. This is comparable to the differences among the three known types. Including the most divergent STLV-I strains sequenced to date, for example, strain PHSu1 sequenced here, the divergence in this region within PTLV-I is less than 11 % and within PTLV-II less than 4%. Although very divergent, this new bonobo STLV is the closest well-characterized simian relative of HTLV-II, raising the possibility of very divergent new HTLV strains. Our results show that the number of PTLV types should be considered open and that the variety of indigenous viruses in the PTLV group is greatest in Africa. Thus, as for the other primate retroviruses HIV and SIV, PTLV most probably has its origins in Africa.

Comparative activity of selected antiviral compounds against clinical isolates of human cytomegalovirus

Seventeen fresh clinical isolates of human cytomegalovirus (HCMV) were examined for their in vitro susceptibility to different potential anti-HCMV drugs, including a series of acyclic nucleoside phosphonate analogues as well as the reference compounds ganciclovir, foscarnet and acyclovir. Three sulfated polysaccharides (heparin, dextran sulfate and pentosan polysulfate) known for their ability to inhibit adsorption of enveloped viruses to the cells were also included in these comparative tests. Of the reference compounds, ganciclovir was the most potent. However, it was about five-fold less potent than the phosphonate derivative (s)-1-(3-hydroxy-2-phosphonylmethoxy-propyl)cytosine (HPMPC) and its cyclic form (cHPMPC). All test compounds, including the sulfated polysaccharides, were as active against the clinical isolates as they were against the laboratory strains of HCMV. Furthermore, the choice of the cell line (HEL or MRC-5) did not influence the anti-HCMV activity of the compounds.

Evolutionary rate and genetic heterogeneity of human T-cell lymphotropic virus type II (HTLV-II) using isolates from European injecting drug users

Abstract. Seven new Italian and two new British HTLV-II isolates were obtained from injecting drug users and the entire long terminal repeat (LTR) region was sequenced. Restriction analysis showed that all the Italian isolates are of the IIb subtype, whereas the British isolates are of the IIa subtype. To understand whether the further differentiation of each two principal HTLV-II subtypes in several subgroups could be statistically supported by phylogenetic analysis, the neighbor-joining, parsimony, and maximum likelihood methods were used. The separation between IIa and IIb is very well supported by all three methods. At least two phylogenetic subgroups exist within the HTLV-IIa and at least three within the HTLV-IIb subtype. In the present analysis, no statistical support was obtained for additional phylogroups. Two particular subgroups seem interesting because they include all European and North American injecting drug user strains within the IIa and IIb subtypes, respectively. These data confirm that European HTLV-II infection among drug users is probably derived from North America. They also suggest that though a certain differentiation by restriction analysis in different subgroups is possible, carefully interpreted phylogenetic analyses remain necessary. Using the likelihood ratio test, a molecular clock for the drug user strains was calibrated. A fixation rate between 1.08 × 10−4 and 2.7 × 10−5 nucleotide substitutions per site per year was calculated for the IIa and IIb injecting drug user strains. This is the lowest fixation rate so far reported for RNA viruses, including for HIV, which typically range between 10−2 and 10−4.

Comparative activity of various compounds against clinical strains of herpes simplex virus

The following compounds were evaluated for their inhibitory activity against clinical strains of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in both primary rabbit kidney (PRK) and HeLa cell cultures: (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA), 9-(2-phosphonylmethoxyethyl)adenine (PMEA), (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC), (RS)-9-(3-hydroxy-2-phosphonylmethoxypropyl)-2,6-diaminopurine (HPMPDAP), 5-(5-bromothien-2-yl)-2′-deoxyuridine (BTDU), 5-(5-chlorothien-2-yl)-2′-deoxyuridine (CTDU), 9-(2-deoxy-2-hydroxymethyl-β-D-erythro-oxetanosyl)guanine (OXT-G), pentosan polysulfate, heparin, dextran sulfate (MW 10,000), acyclovir, 9-(2-hydroxyethoxymethyl)guanine (ACV), (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU), 1-β-D-arabinofuranosyl-(E)-5-(2-bromovinyl)-uracil (BVaraU), vidarabine (9-β-D-arabinofuranosyladenine) (ara-A) and phosphonoformate (PFA). The most potent inhibitors of HSV-1 were (in order of decreasing activity in PRK cells) BVDU, ACV, BVaraU and OXT-G, their mean 50 % inhibitory concentration (IC50) ranging from 0.02 µg/ml to 0.9 µg/ml. Then followed BTDU and CTDU (IC50 1–2 µg/ml), the sulfated polysaccharides (IC50 1.3–5.8 µg/ml), the phosphonylmethoxyalkyl derivatives (IC50 5.6–25 µg/ml), ara-A (IC50 11 µg/ml) and PFA (IC50 38.5 µg/ml). Except for BVDU, BVaraU, BTDU and CTDU, the compounds did not discriminate between HSV-2 and HSV-1. All the compounds studied could be considered specific anti-HSV agents. Their selectivity indexes varied from 3 (PFA) to 6400 (BVDU).

Comparative evaluation of three ELISA techniques and an indirect immunofluorescence assay for the serological diagnosis of Epstein-Barr virus infection

BACKGROUNDnThe reference method for detecting specific Epstein-Barr virus (EBV) antibodies is indirect immunofluorescence (IF) with EBV-infected cells. The availability of protein purified from infected cells and more recently of recombinant polypeptides designed to contain immunodominant epitopes, has enabled the development of commercial enzyme-linked immunosorbent assays (ELISA) for the specific serodiagnosis of EBV infection.nnnOBJECTIVEnEvaluation of ELISA-based EBV serodiagnosis in comparison with indirect immunofluorescence.nnnSTUDY DESIGNnWe have first compared three commercial ELISA test systems with our in house indirect immunofluorescence assay for classifying correctly a set of serum samples into clinical categories (acute infection, past infection, interfering non-EBV infection, persistent infection). Additionally a prospective analysis with the best performing ELISA test (Enzygnost) was then carried out by running the ELISA test in parallel with the indirect immunofluorescence assay on 324 consecutive clinical samples sent to our laboratory for EBV serodiagnosis.nnnRESULTSnFor the serodiagnosis of past EBV infection and acute EBV infection all three commercial ELISAs performed well in comparison with indirect immunofluorescence. When testing samples positive for cytomegalovirus (CMV), Toxoplasma or herpes simplex IgM, interference in the IgM tests was observed with the three ELISAs. In some instances we could demonstrate that the positive IgM results were due to EBV reactivation. The observed discrepancies between ELISA and IF for the serodiagnosis of chronic EBV infection or EBV reactivation, point to the difficulty for the serodiagnosis of persistent EBV infection on single serum samples. According to our prospective study the EBV IgG determination was accurate. A positive IgM result was not always indicative of an acute infection. Positive IgM results due to EBV reactivation were observed. A positive EBV nuclear antigen (EBNA) IgG result in those samples precluded acute infection.nnnCONCLUSIONSn90-95% of samples could be classified correctly into clinical categories by a two parameter ELISA system detecting IgG and IgM against a standardized mixture of EBV antigens, allowing standardization and automation of EBV-specific serology. The absence of EBNA IgG was useful as a second line confirmatory assay for acute EBV infection.

The discovery of two new divergent STLVs has implications for the evolution and epidemiology of HTLVs.

We have isolated and characterised two divergent simian T‐lymphotropic viruses (STLV), not belonging to the established human and simian T‐lymphotropic virus lineages HTLV‐1/STLV‐1 and HTLV‐2. STLV‐L, from an Eritrean sacred baboon (Papio hamadryas), has been typed as a third type of simian T‐lymphotropic virus, distinct from HTLV‐1/STLV‐1 and HTLV‐2. The other virus, isolated from Congolese bonobos (Pan paniscus), is a distinct member of the HTLV‐2 clade and has been designated STLV‐2. The isolation of these two simian viruses shows that the spectrum of HTLVs/STLVs is larger than previously expected. Our data indicate that the two lineages STLV‐L and HTLV‐2/STLV‐2 are of African origin, while the HTLV‐1/STLV‐1 lineage has been shown to be of Asian origin. These data, together with our phylogenetic analyses, suggest an African origin of the HTLV/STLV ancestor, which provides new clues about virus dissemination. Furthermore, the atypical serological profiles exhibited by STLV‐L or STLV‐2 infected animals in western blot, raise questions about the efficiency of current screening methods to type highly divergent HTLVs/STLVs. Considering the growing interest in xenotransplantations, more epidemiological and biological knowledge of simian and human T‐lymphotropic viruses is necessary to estimate the risk of interspecies transmissions. Copyright

Differentiated umbilical cord matrix stem cells as a new in vitro model to study early events during hepatitis B virus infection

The role of cell differentiation state on hepatitis B virus (HBV) replication has been well demonstrated, whereas how it determines cell susceptibility to HBV entry is far less understood. We previously showed that umbilical cord matrix stem cells (UCMSC) can be differentiated towards hepatocyte‐like cells in vitro. In this study we infected undifferentiated (UD‐) and differentiated (D‐) UCMSCs with HBV and studied the infection kinetics, comparing them to primary human hepatocytes (PHHs). UD‐UCMSCs, although permissive to viral binding, had a very limited uptake capacity, whereas D‐UCMSCs showed binding and uptake capabilities similar to PHHs. Likewise, asialoglycoprotein receptor (ASGPR) was up‐regulated in UCMSCs upon differentiation. In D‐UCMSCs, a dose‐dependent inhibition of HBV binding and uptake was observed when ASGPR was saturated with known specific ligands. Subsequent viral replication was shown in D‐UCMSCs but not in UD‐UCMSCs. Susceptibility of UCMSCs to viral replication correlated with the degree of differentiation. Replication efficiency was low compared to PHHs, but was confirmed by (1) a dose‐dependent inhibition by specific antiviral treatment using tenofovir; (2) the increase of viral RNAs along time; (3) de novo synthesis of viral proteins; and (4) secretion of infectious viral progeny. Conclusion: UCMSCs become supportive of the entire HBV life cycle upon in vitro hepatic differentiation. Despite low replication efficiency, D‐UCMSCs proved to be fully capable of HBV uptake. Overall, UCMSCs are a unique human, easily available, nontransformed, in vitro model of HBV infection that could prove useful to study early infection events and the role of the cell differentiation state on such events. (HEPATOLOGY 2013)