Point mutations in the varicella-zoster virus DNA polymerase gene confers resistance to foscarnet and slow growth phenotype.

Seven independent laboratory mutants were derived from seven distinct wild-type varicella-zoster virus (VZV) isolates after exposure to increasing concentrations of foscarnet (PFA) and were found to be resistant to this drug. Single base changes resulting in amino acid substitutions were observed in the nucleotide sequence of the DNA polymerase gene of each PFA-resistant mutant. The mutations were found to occur within the domain II (Arg-665 --> Gly for strains vrMOR and vrVER; Val-666 --> Leu for vrLEB; Gln-692 --> Arg for vrOLI) and domain III (Arg-806 --> Ser for vrABD; Leu-809 --> Ser for vrALI and vrCHA) of DNA polymerase gene. In addition, the PFA-resistant mutants exhibited a phenotype characterized by slow growth, the strains showing a marked delay in immediate-early antigen plaque formation compared with the wild-type VZV from which they were derived. These results may have implications for successful isolation and characterization of PFA-resistant strains from clinical samples containing mixed viral populations.

Single amino acid change in DNA polymerase is associated with foscarnet resistance in a varicella-zoster virus strain recovered from a patient with AIDS.

The genetic characterization of a foscarnet-resistant strain of varicella-zoster virus (VZV) that was isolated from a patient with AIDS is reported. Compared with the sequence of the Dumas reference strain, this strain, which was isolated from a patient in whom foscarnet treatment failed, had two point mutations. The emergence of one of the mutations, which includes a change from a glutamic acid to a lysine at position 512 in the DNA polymerase, suggests that this mutation is implicated in the VZV foscarnet resistance. The other mutation, which replaces serine 863 by a glycine, is also present in 2 susceptible strains--Oka and a wild-type isolate.

Acyclovir-resistant varicella-zoster virus: phenotypic and genetic characterization.

A man with acquired immunodeficiency syndrome (AIDS) developed zoster of the right arm which was resistant clinically to acyclovir. Varicella-zoster virus (VZV) was cultured from a skin biopsy performed at the beginning of acyclovir therapy (isolate 1) and after its failure (isolate 2). The emergence of acyclovir resistance during treatment was investigated by developing a simple and rapid drug sensitivity assay based on the plaque reduction reference method. This late-antigen synthesis reduction assay involved serial dilutions of cell-associated virus. The 50% inhibitory concentration (IC50) of acyclovir was 16 +/- 7.5 microM for the susceptible reference strain OKA, in agreement with published data. The acyclovir IC50 increased from 6.5 microM for isolate 1 to 100 microM for isolate 2. In comparison with the sequence of isolate 1, isolate 2 had a single mutation consisting of a C to T change at position 907 of the thymidine kinase gene, which changed a glutamine codon into a stop codon at position 303 of the thymidine kinase protein. These results show the emergence of acyclovir resistance through a single previously undescribed mutation in the thymidine kinase gene, and confirm the heterogeneity of mutations inducing acyclovir resistance.

Difference in permissiveness of human fibroblast cells to variants A and B of human herpesvirus-6.

Human herpesvirus-6 (HHV6) is a lymphotropic virus genetically related to human cytomegalovirus (CMV) and for which two variants, A and B, have been distinguished. Human CMV is usually cultivated with human fibroblasts (HF). The lack of cell lines useful for HHV6 isolation and propagation led us to investigate whether HHV6 variants A and B could infect HFs as CMV does. Isolates of HHV6 variants A and B were used to infect MRC-5 HFs. HHV6 infection was detected by means of immunoperoxidase assay using three specific monoclonal antibodies. HHV6-specific antigens were detected in 88 and 38% of cases after infection with variants A and B, respectively. The highest number of HHV6-antigen-positive cells was obtained at 4-5 days p.i. The titre of HHV6 stocks was determined in parallel by immunoperoxidase assay on HFs and by observation of cytopathic effect using serial dilutions on peripheral blood mononuclear cells (PBMC). The number of infectious particles inducing the appearance of antigen-positive HF cells was consistently lower than the titre of virus stocks, expressed as TCID50. The amount of HF-associated HHV6 DNA was measured using limiting dilution PCR assay; it was significantly increased during 4-day infection in the case of variant A but not variant B. The yield of virus from infected HFs was demonstrated only for variant A by the serial propagation of virus from HFs to PBMCs and by the increase in cell-free HHV6 DNA in HF culture supernatant. Our results show that HHV6 can reproducibly infect HFs, albeit at a low level, and that HFs are more permissive to variant A than to variant B, as reported previously for PBMCs and human T-cell lines.

Controlled study of human herpesvirus 6 detection in acquired immunodeficiency syndrome-associated non-Hodgkin's lymphoma. The French Study Group for HIV-Associated Tumors.

Human herpesvirus 6 (HHV-6) is a recently identified lymphotropic herpesvirus, which has been isolated from patients with acquired immunodeficiency syndrome (AIDS) or lymphoproliferative diseases. Two variants A and B of HHV-6 have been described, variant B being more common in children with exanthema subitum. HHV-6 infection was studied in cases of AIDS-associated non-Hodgkin's lymphoma (NHL), and in three control populations in order to evaluate the possible etiological role of HHV-6 in this lymphoproliferative disease. Tumor specimens from various organs were obtained from 27 patients with AIDS-associated NHL and 20 human immunodeficiency virus (HIV)-seronegative patients with NHL. Lymph node specimens were obtained from four HIV-seropositive and nine HIV-seronegative patients with lymph node follicular hyperplasia. A specific polymerase chain reaction (PCR) was used to detect HHV-6 DNA. Subsequently HHV-6 variant was identified by using variant-specific PCR. Human cytomegalovirus (CMV) infection was detected in parallel by means of specific PCR. HHV-6 DNA was detected in 12 of 27 tumor tissues (44%), including 8 of 15 lymph node specimens (53%) from patients with AIDS-associated NHL. The corresponding values in HIV-seronegative patients with NHL were 35% (7/20) and 36% (5/14), respectively. Lymph node specimens were positive for HHV-6 in two of four (50%) HIV-seropositive and five of nine (55%) HIV-seronegative patients with follicular hyperplasia. Variant A was detected in two cases of AIDS-associated NHL, variant B in one case, and both variants in six cases. The distribution of HHV-6 variants exhibited a similar pattern in the three control groups.(ABSTRACT TRUNCATED AT 250 WORDS)

A nylon membrane enzyme immunoassay for rapid diagnosis of influenza A infection.

A new membrane-enzyme immunofiltration assay (MIFA) was developed for rapid diagnosis of influenza A infection. The pretreated specimens were dispensed into a 1.2 micron Biodyne B nylon membrane-bottomed microplate and vacuum filtration was applied. Blocking solution, peroxidase-conjugated anti-influenza A nucleoprotein monoclonal antibody, washing buffer and substrate were added in that order. The assay was completed within 30 min. Out of 103 nasopharyngeal swabs collected in transport medium, 31 isolates of influenza A virus were obtained and 22 specimens were detected directly by the MIFA technique. The 9 isolation-positive MIFA-negative specimens required 6 days or more for viral detection in cell culture, and probably contained a very low quantity of virus. The 72 cell culture negative specimens were also negative by MIFA. Comparison with a classical immunocapture assay (ICA) gave a better sensitivity for MIFA, as only 15/103 specimens were positive by ICA. MIFA is a rapid test with 71% sensitivity and 100% specificity. It was also very useful to test the cell culture supernatants, as a sensitivity of 100% was obtained with MIFA when the immunofluorescence technique was positive. The same technique could be readily carried out on the same plate for other respiratory viruses since capture antibody is not used.