Quantification of Epstein-Barr virus load in peripheral blood of human immunodeficiency virus-infected patients using real-time PCR.

Epstein-Barr virus (EBV) reactivation is more likely to occur in immunocompromised patients with subsequent higher susceptibility to EBV-associated lymphoproliferations. In contrast to transplant recipients, limited data are available concerning the EBV load in HIV-infected patients, with or without AIDS-related non-Hodgkin's lymphomas. We developed a TaqMan real-time PCR assay, allowing both the EBV genome and a cellular gene to be quantified in order to obtain a reliable normalized measurement of the EBV load in peripheral blood mononuclear cells (PBMCs). With a wide 6-log(10) quantification range and inter-assay variations of less than 24%, this quantitative PCR was sufficiently accurate and reproducible for routine follow-up. The EBV load was determined in PBMCs from 113 HIV-infected patients, 11 patients with primary HIV infection and 24 HIV-seronegative healthy controls. The rates of EBV detection were similar in the three groups. However, EBV loads were higher in the HIV-infected group (P < 0.00001) except for the patients with primary HIV infection. Unexpectedly, EBV loads were not correlated with the clinical stages of HIV infection or HIV replication, and did not depend on the degree of immunodepression, as judged by CD4+ counts. This study contributes towards the definition of the baseline EBV load during HIV infection and stresses the broad inter-individual variability of the EBV load in HIV-infected patients. Real-time PCR provides a useful tool that can be used in further longitudinal studies to assess the relevance of the EBV load to identify HIV-infected patients with a high risk of EBV-associated lymphoproliferations.

Interstrain variations in the cytomegalovirus (CMV) glycoprotein B gene sequence among CMV-infected children attending six day care centers.

Using the shell vial assay and sequence analysis of a variable region of the glycoprotein B (gB) gene, cytomegalovirus (CMV) excretion rates in urine and virus transmission were studied among 93 children from six day care centers (DCCs). During a 6-month period, excretion rates differed significantly between DCCs (P < .001). The 6 gB gene sequences, obtained from 24 CMV-infected children, were classified in four previously defined groups. In five DCCs, 2 or 3 strains cocirculated, and none was dominant. Infection could have been acquired outside the DCC for 2 children and inside it for 9. Two children from the same DCC had mixed infections. No differences in hygiene, child care practices, or experience and level of qualification of the staff could explain this wide variety of excretion rates between DCCs. The distribution of gB gene patterns observed does not suggest that 1 type was dominant or more efficiently transmitted.

Cross-resistances to antiviral drugs of IUdR-resistant HSV1 in rabbit keratitis and in vitro.

The emergence of cross-resistance to various antiviral drugs was investigated both in vivo and in vitro for herpes simplex virus type 1 (HSV1) resistant to idoxuridine (IUdR 0.24%) obtained by seven successive passages (from P0 to P7) in rabbit keratitis treated by IUdR. The viral population obtained at the seventh IUdR passage (P7) showed an activity of the thymidine kinase (TK) reduced to 5.6% of the parental strain (PO); moreover, most of the clones of P7 showed an altered TK phenotype determined by the [125I]iododeoxycytidine (IDC) procedure. In rabbit keratitis, IUdR-resistant viral population P7 showed cross-resistance to bromovinyl desoxyuridine (BVDU) (0.5%) and to acyclovir (ACV) (3%). Under trifluorothymidine (1%) treatment, P7 showed an intermediate sensitivity. HSV1 at P7 remained sensitive to adenine arabinoside (Ara A) (3%) and to dihydroxy-propoxymethylguanine used at high concentration (3%). The in vitro sensitivity determination to various antiviral drugs was investigated by dye-uptake assay for the initial viral population PO and for HSV1 collected under IUdR treatment at the third (P3) and the seventh (P7) passages. Cross-resistance to TK-dependent drugs, such as IDC, BVDU, and ACV were found at P7. P7 remained sensitive to Ara A and to phosphonoformic acids antiviral drugs known not to be dependent on viral TK.

[Reversibility of IUdR resistance to sensitivity to an HSV1 strain in experimental keratitis in rabbits]. / Réversibilité de la résistance à l'IUdR vers la sensibilité d'une souche HSV1 au cours de la kératite du lapin.

During 7 serial passages of Herpes Simplex Virus (HSV1) in rabbit cornea treated with idoxuridine (IUdR) (P1 to P7), the emergence of resistance had been obtained from P3. The reversion towards IUdR sensitivity has been investigated from either viral population P3 or P6 by 6 serial passages in rabbit cornea treated by Vaseline (V1 to V6). From viral population P3, the reversion to IUdR sensitivity has been obtained at V4. In contrast, from viral population P6, the IUdR resistance was conserved from V1 to V6. In vitro, on Vero cells, the effective doses 50% (ED50) and 90% (ED90), determined by dye uptake assay and plaque reduction assay, confirmed the reversibility towards IUdR sensitivity obtained from P3 and the stability of IUdR resistance from P6.

Heparin fragments regulate collagen phenotype and fibronectin synthesis in the skin of genetically diabetic mice.

The biosyntheses of interstitial collagens of type I and III and of fibronectin were studied in genetically diabetic KK mice, as compared to control C57 Black mice, as well as the effect of low-Mr heparin fragments (CY 222) on these biosyntheses. An increased production of type III collagen, as compared to type I collagen, could be demonstrated in explant cultures of KK mice skins. Fibronectin biosynthesis was also increased. In vivo treatment of KK mice with 1 mg/kg of CY 222 decreased the biosyntheses of type III collagen and of fibronectin to normal levels. These experiments suggest that low-Mr heparin fragments can modulate the expression of extracellular matrix macromolecules.