The average 50% inhibitory concentration (IC 50 ) values for AD169 were 0.22 ؎ 0.09 M 1263W94 and 5.36 ؎ 0.12 M ganciclovir. For 35 human cytomegalovirus (HCMV) clinical isolates the average IC 50 was 0.42 ؎ 0.09 M 1263W94, and for 26 ganciclovir-susceptible HCMV clinical isolates the average IC 50 was 3.78 ؎ 1.62 M ganciclovir. Nine HCMV clinical isolates that were resistant to ganciclovir were completely susceptible to 1263W94.Human cytomegalovirus (HCMV) causes considerable morbidity and mortality in the immunocompromised host (18,19). Organ transplant recipients suffer from retinitis, gastrointestinal disease, hepatitis, and pneumonia caused by HCMV infections, whereas AIDS patients suffer from HCMV-induced retinitis and other complications (1). The current Food and Drug Administration-approved chemotherapies for HCMV infections consist of ganciclovir, foscarnet, cidofovir, and fomivirsen (5,16,17,20). These antiviral drugs are effective against infections caused by HCMV; however, they are not ideal because of their toxicity and poor bioavailability. Furthermore, longterm treatment with these drugs often leads to the selection of drug-resistant mutants (6,8,9). Due to the problems associated with the currently used antiviral compounds for HCMV infection, there is an active search for more useful compounds to combat infections with HCMV.The benzimidazole ribonucleosides represent a new class of antiviral compounds that inhibit HCMV replication by blocking the processing of progeny viral DNA (4,10,22). In an attempt to make a more stable derivative of benzimidazole riboside 2-bromo-5,6-dichloro-1-␤-D-ribofuranosyl benzimidazole (BDCRB), the L form of the compound, was synthesized (4). The L-riboside benzimidazole analogue of BDCRB, 1263W94, has potent activity against HCMV laboratory strains and clinical isolates as well as Epstein-Barr virus (4, 23). Preliminary studies suggest that 1263W94 inhibits HCMV replication by blocking viral DNA synthesis, but not by an effect on the viral DNA polymerase or the phosphotransferase encoded by the UL97 gene (4).In this report, we show that 1263W94 inhibits the replication of the AD169 laboratory strain of HCMV and 35 HCMV clinical isolates at drug concentrations that are approximately 10-fold less than those required by ganciclovir. Nine of the 35 HCMV clinical isolates are resistant to ganciclovir, and several are also resistant to foscarnet and cidofovir (2,3,7,8,9,11).All of these drug-resistant HCMV clinical isolates are susceptible to 1263W94. These results show that 1263W94 inhibits the replication of both ganciclovir-susceptible and single-and multiple-drug-resistant HCMV clinical isolates, confirming reports that 1263W94 has a mode of action different from that of ganciclovir, foscarnet, and cidofovir (4). These results also suggest that this drug is potentially useful for treating patients infected with HCMV clinical isolates that are resistant to the currently used antiviral drugs.Determination of IC 50 values of 1263W94 and ganciclovir for HCMV laboratory str...

BAY38-4766 and BAY43-9695 are nonnucleosidic compounds with activities against human cytomegalovirus (HCMV). Two phenotypic assays were used to determine the drug susceptibilities of 36 HCMV clinical isolates to the BAY compounds and ganciclovir. Using either assay, both BAY compounds at a concentration of approximately 1 M inhibited the replication of all 36 HCMV clinical isolates, including 11 ganciclovirresistant clinical isolates, by 50%.Human cytomegalovirus (HCMV) causes considerable morbidity and mortality in immunocompromised hosts (13). Organ transplant patients suffer from hepatitis and pneumonia caused by HCMV infections, whereas AIDS patients suffer from HCMV-induced retinitis and other complications (1). The current Food and Drug Administration-approved therapies for retinitis due to infection with HCMV include ganciclovir, foscarnet, cidofovir, and fomivirsen (2,11,12,14). These antiviral drugs are active against infections caused by HCMV; however, they are not ideal because of their toxicity and poor bioavailability. Furthermore, long-term treatment with these antiviral agents often leads to the selection of viral mutants that are resistant to one or more of these drugs (3-5). Current research has led to the discovery of several novel compounds with in vitro and in vivo activities against HCMV (6, 7, 16). One such compound, BAY38-4766, is a nonnucleosidic inhibitor of HCMV replication (17). Two phenotypic drug susceptibility assays, a flow cytometric fluorescence-activated cell sorter (FACS) assay (8-10) and a plaque reduction assay (PRA) (15), were used to compare the effects of BAY38-4766, its main metabolite, BAY43-9695, and ganciclovir on the in vitro replication of ganciclovir-susceptible and ganciclovir-resistant HCMV clinical isolates. BAY38-4766 and BAY43-9695 inhibited the replication of ganciclovir-sensitive and ganciclovirresistant HCMV clinical isolates at concentrations less than or equal to 1 M. These results suggest that these compounds are potentially useful for treating patients infected with ganciclovir-sensitive or ganciclovir-resistant HCMV.The use of the FACS assay and the PRA for determining 50% inhibitory concentrations (IC 50 s) for HCMV clinical isolates have been described in detail previously (8-10). These two phenotypic assays were used to determine the susceptibilities of the AD169 laboratory strain of HCMV and 36 HCMV clinical isolates to BAY38-4766, BAY43-9695, and ganciclovir. The FACS assay yielded average IC 50 s of BAY38-4766, BAY43-9695, and ganciclovir for the AD169 laboratory strain of 0.95 Ϯ 0.17 (mean Ϯ standard deviation), 0.70 Ϯ 0.30, and 3.05 Ϯ 0.21 M, respectively. The PRA yielded average IC 50 s of these three drugs for AD169 of 0.64 Ϯ 0.14, 0.55 Ϯ 0.06, and 3.50 Ϯ 0.21 M, respectively. The average IC 50 s of the two BAY compounds and ganciclovir for 36 HCMV clinical isolates are presented in Table 1. Both BAY compounds inhibited the replication of all of the HCMV clinical isolates by 50% at essentially the same concentrations. Of the 36 HCMV clinical i...

RNA viruses, such as influenza virus, have a high rate of mutation. Some of these mutations lead to viruses that are resistant to the currently used antiviral drugs and can be selected in the presence of antiviral drugs. If the drug-resistant viruses are biofit, their replication can lead to serious disease that cannot be treated effectively with the previously used antiviral compounds. This scenario has occurred frequently. When amantadine hydrochloride was used to treat influenza virus type A infections, 30% of the virus isolates obtained from treated patients were found to be resistant (9,11,22). With the licensing of the neuraminidase (NA) inhibitors, the selection of influenza viruses resistant to these inhibitors was of concern (32, 39, 43, 52, 61). In vitro resistance associated with amino acid substitutions in the hemagglutinin (HA) or NA antigens or both has been reported for the NA inhibitors (4,14,15,32,40,49,55). Despite these concerns, recent reports have demonstrated that there is little or no natural resistance to oseltamivir or zanamivir (5, 33). To determine if mutations to zanamavir occurred in vivo, the drug susceptibilities of clinical isolates obtained during a phase II clinical trial of zanamivir were determined by the plaque reduction assay (PRA), the NA inhibition (NAI) assay, and an in vivo assay using ferrets (3,17). A comparison of 41 paired isolates obtained before and during therapy with zanamivir showed no shifts in susceptibility to zanamivir when measured by the NAI assay, but the PRA using MDCK cells showed variable susceptibility to zanamivir. The susceptibilities of the clinical isolates determined by the PRA did not correlate with in vivo susceptibility studies in humans and ferrets, whereas the NAI assay did correlate with the in vivo susceptibility assays. In a study of 54 isolates obtained after treatment with oseltamivir, 2 clinical isolates were resistant in the NAI assay and an additional 8 were resistant in the PRA (16). These discrepancies between the PRA and the NAI assay could be due to the isolation of viruses with mutations in the HA gene that lead to in vitro resistance. NA inhibitor-resistant viruses with mutations in the HA gene would be scored in the PRA, but not in the NAI assay. The close relationship between the drug susceptibilities obtained with the NAI assay and the in vivo assays suggests that for these clinical isolates the NAI assay correlates better with the in vivo assay than the PRA for the NA inhibitors. The present evidence suggests that only mutations in the NA gene that lead to resistance to the NA inhibitors are clinically relevant.The currently used in vitro drug susceptibility assays, such as the PRA, the virus yield reduction assay, and the neutral red dye uptake assay, are cumbersome, time-consuming, and subjective (21, 45). A PCR-based drug susceptibility assay has recently been published, but its usefulness in clinical trials has not been evaluated (54). Previously, we demonstrated that the susceptibilities of herpes simplex viruses and h...

This report describes a flow cytometry drug susceptibility assay that uses a single fluorochrome-labeled monoclonal antibody to determine the acyclovir susceptibilities of herpes simplex virus (HSV) type 1 or type 2 clinical isolates. This assay yields 50% effective doses (drug concentrations that reduce the number of antigen-positive cells by 50%) for HSV clinical isolates that are equivalent to those obtained with the plaque reduction assay.Herpes simplex virus (HSV) infections are ubiquitous, with approximately 80% of the adult population infected with HSV type 1 and approximately 20% of the adult population also infected with HSV type 2 (1, 22, 25). Current therapy for primary and recurrent HSV infections involves the use of acyclovir or one of its more bioavailable prodrugs, valacyclovir or famciclovir (2). Long-term use of these antiviral drugs in HSVinfected neonates and immunocompromised patients can lead to the selection of viral mutants that are resistant to these drugs (7,8,17). Less than 1% of the clinical isolates obtained from immunocompetent patients treated with acyclovir are resistant to acyclovir (3). However, 5 to 10% of the clinical isolates obtained from immunocompromised patients subjected to long-term treatment or multiple treatments with acyclovir are resistant to the drug due to mutations in the thymidine kinase (TK) gene and/or DNA polymerase genes (5, 7). Patients with acyclovir-resistant HSV clinical isolates caused by mutations in the TK gene, but not those infected with viruses with mutations in the DNA polymerase gene, can be successfully treated with the HSV DNA polymerase inhibitors foscarnet and cidofovir (9, 10).There are no universally accepted methods for determining the drug susceptibilities of HSV clinical isolates. The most accurate assay for HSV is the plaque reduction assay (PRA) (19-21). The National Committee for Clinical Laboratory Standards (NCCLS) has established a standardized drug susceptibility assay for HSV based on the PRA, but it has not been validated and is seldom used because it is time-consuming, expensive to perform, and subjective. Other drug susceptibility assays are faster than the PRA, and some of the endpoints can be read automatically, but these assays are less sensitive than the PRA (6,12,23,24). With the increased use of acyclovir and its derivatives among HSV-infected neonates and immunocompromised patients leading to the increased selection of drug-resistant HSV clinical isolates, there is a urgent need for a standardized drug susceptibility assay for HSV clinical isolates.HSV-specific fluorochrome-labeled monoclonal antibodies and flow cytometry have been used to detect and quantify HSV-infected cells and to perform drug susceptibility testing of HSV clinical isolates (13,18). These studies used a high multiplicity of infection and monitored the effect of antiviral drugs on HSV replication by measuring the effects of drugs on the synthesis of late antigens. In this report, we show that a single monoclonal antibody to an HSV antigen that is shar...