Variations in the cytomegalovirus DNA polymerase and phosphotransferase genes in relation to foscarnet and ganciclovir sensitivity.

BACKGROUND: Identification of human cytomegalovirus (CMV) genome variation is important for understanding mutations associated with drug resistance. OBJECTIVES: To investigate the CMV resistance to foscarnet (PFA) and ganciclovir (GCV) in patients treated with antiviral drugs and to identify the DNA polymerase (UL54) and phosphotransferase (UL97) gene mutations inducing resistance. STUDY DESIGN: Antiviral susceptibility of CMV strains/isolates for PFA and GCV was compared by plaque reduction assay and in situ ELISA. UL54 and UL97 gene mutations were identified by sequencing. Growth phenotype of two CMV recombinants with mutations in UL54 was studied. RESULTS: Six of seven GCV resistant strains had alterations within the UL97. Five of them also had alterations in the UL54 (F412C, L802M or K513E), previously shown to induce GCV resistance. Seven isolates had no or reduced susceptibility to PFA, which had alterations in the UL54 (D588N, E756K, V781I or L802M). By in vitro mutagenesis, it was shown that a mutation at codon D588N of UL54 conferred 9-fold reduced susceptibility to PFA, while a mutation at codon V781I induced 4-fold reduced susceptibility to PFA and GCV. Both recombinants showed the same kinetics of protein expression (IE, E, and L antigen) and virus yields as the CMV Towne strain. CONCLUSIONS: The recombinants containing alterations within the UL54 (D588N and V781I) showed a reduced susceptibility to antiviral drugs but no change in the replication rate compared to the CMV Towne.

Solid phase ELISA for determination of the virus dose dependent sensitivity of human cytomegalovirus to antiviral drugs in vitro.

The main problems in determining the true in vivo susceptibility of human cytomegalovirus (CMV) to antiviral compounds are the influence of the size of the viral inoculum, the variation in the replication capacity of different CMV strains and the subjective evaluation of the inhibition of viral growth in the plaque assay. In this study, a specific assay was developed which reproducibly determines the sensitivity of primary isolates of CMV. The assay includes simultaneous virus titration and determination of the antiviral sensitivity. When individual virus doses were evaluated, the IC50 was generally dependent on the virus dose, except for resistant isolates, where the IC50 did not change irrespective of the dose of virus. The novel method of IC50 calculation takes into account all values derived from the linear part of the inhibition curve. This may better reflect the in vivo conditions, where the antiviral drug encounters different amounts of virus in different organs. Two human fibroblast-derived cell lines showed similar results.

Elevated antibody levels to Epstein-Bbarr virus antigens in patients with hairy cell leukemia compared to controls in relation to exposure to pesticides, organic solvents, animals, and exhausts.

Epstein-Barr virus (EBV) is a B-lymphotropic human herpes virus infecting B-cells, which has been associated with lymphoid malignancies, above all non-Hodgkin lymphomas (NHL). Severe immunosuppression is the best recognized risk factor for NHL. Many factors in the environment that have been described as risk factors for NHL cause measurable changes in immune functions. Hairy cell leukemia (HCL) is a rare, indolent non-Hodgkin lymphoma, originating from B-lymphocytes. This was a case-control study including 111 male cases with HCL and 400 controls. In a subgroup of 57 cases and 65 controls analysis of antibodies to EBV early antigen, viral capsid antigen, and EBNA-1, measured as P107, was performed. In this study, we confirm other studies describing elevated levels of antibodies to the EBV early antigen (EA) in patients with HCL compared to controls. We found only minor differences in the levels of antibodies to the viral capsid antigen (VCA) and EBNA-1. measured as P107. We found a positive association of a titer to EA IgG > or =40 (OR 4.1; CI 1.9-9.5). The ORs were further elevated when subjects with high levels of EA IgG and exposed to environmental agents such as organic solvents, certain pesticides, impregnating agents, animals, and exhausts were compared to those subjects with low levels that were not exposed. Antibody reactivity against the EBV EBNA 1-alanine-glycine repeat (P107 IgG) above the median gave an increased OR for HCL, which further increased in subjects exposed to organic solvents, certain pesticides. impregnating agents, animals, and exhausts. However, the numbers of exposed cases and controls were small in some of the calculations.

Evaluation of three commercial enzyme-linked immunosorbent assays and two latex agglutination assays for diagnosis of primary Epstein-Barr virus infection.

Three commercially available enzyme-linked immunosorbent assays (ELISAs) from Gull, Biotest, and Behring (Enzygnost) and two latex agglutination tests for heterophile antibodies (Monolatex [Biotest] and Mono-Lex [Trinity Laboratories]) were evaluated for the diagnosis of primary Epstein-Barr virus (EBV) infection and EBV seropositivity. Two hundred fourteen consecutive samples from 197 patients with symptoms of primary EBV infection were analyzed by the five assays at a clinical microbiology laboratory. The samples were also analyzed independently by immunofluorescence methods at a reference laboratory. According to the reference methods, 37 patients (40 serum samples) had primary EBV infections, 120 patients (127 serum samples) had had past EBV infections, 33 patients (36 serum samples) were seronegative, and 7 patients (11 serum samples) exhibited atypical reactions. The respective sensitivities and specificities for the diagnosis of primary EBV infection were 95 and 100% for the Gull assays, 100 and 94% for the Biotest assays, and 100 and 89%, for the Enzygnost assays. The Monolatex and Mono-Lex methods showed similar sensitivities and specificities (78 to 85% and 100 to 99%, respectively) for the diagnosis of primary EBV infection. This study demonstrates the usefulness of commercially available assays for the rapid diagnosis of primary EBV infection, but also the importance of large-scale testing of routine samples before choosing an assay.

Sequence analysis of UL54 and UL97 genes and evaluation of antiviral susceptibility of human cytomegalovirus isolates obtained from kidney allograft recipients before and after treatment.

The frequency of infections caused by drug-resistant cytomegalovirus (CMV) in solid-organ transplant recipients is not known. Only a few resistant strains have been described in transplant recipients. Antiviral susceptibility to ganciclovir (GCV) and foscarnet (PFA) of CMV isolates from 24 renal transplant patients with CMV viremia and CMV disease before and after therapy were investigated by a solid phase ELISA. The CMV DNA polymerase (UL54) and viral phosphotransferase (UL97) genes were also sequenced. Ten patients did not receive antiviral treatment; five and nine patients were treated with PFA and GCV, respectively. No appearance of drug-resistant viruses was observed in the present study, but one isolate showed a reduced sensitivity to PFA after treatment with GCV. This finding could not be explained by the presence or development of mutations that have been associated with drug resistance in UL54. We found no evidence that short-term treatment of CMV with PFA- or GCV-induced resistance.