Elsevier

Antiviral Research

Volume 40, Issues 1–2, December 1998, Pages 73-84
Antiviral Research

Comparison of antiviral compounds against human herpesvirus 6 and 7

https://doi.org/10.1016/S0166-3542(98)00049-7Get rights and content

Abstract

Four classes of antiviral compounds were evaluated for inhibitory activity against two variants of human herpesvirus 6 (HHV-6A and -6B) and human herpesvirus 7 (HHV-7). These included: (1) a pyrophosphate analog, phosphonoformic acid (PFA); (2) beta-guanine analogs, 9-(2-hydroxyethoxymethyl)guanine (acyclovir or ACV), 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (ganciclovir or GCV) and 9-(4-hydroxy-3-hydroxy-3-hydroxymethylbutylyl)guanine (penciclovir or PCV); (3) acyclic nucleoside phosphonates, (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine [cidofovir or (S)-HPMPC] and its cyclic derivative (S)-cyclic-HPMPC (cHPMPC), 9-[[2-hydroxy-1-phosphonomethoxy)ethoxy]methyl]guanine (HPMEMG) and 9-[(2-phosphonylmethoxy)ethyl]-2,6-diaminopurine (PMEDAP), and the seven other related compounds; and (4) a series of benzimidazole ribonucleosides, including 2-bromo-5,6-dichloro-1-(beta-d-ribofuranosyl)benzimidazole (BDCRB). End-point inhibitory concentration (EPC) and 50% effective inhibitory concentration (EC50) values were determined by a dot-blot antigen detection method in cord blood mononuclear cells infected with HHV-6A, HHV-6B or HHV-7 at a multiplicity of infection of 0.004 CCID50/cell. (S)-HPMPC and cHPMPC had an EC50 value of approximately 0.3 μg/ml for HHV-6A, 1.2 μg/ml for HHV-6B and 3.0 μg/ml for HHV-7. These compounds were the most active of those tested against each virus. The EC50 value of GCV for HHV-6A was 0.65 μg/ml, 1.33 μg/ml for HHV-6B, and >7 μg/ml for HHV-7. The EC50 values of ACV and PCV were approximately 6–8 μg/ml for HHV-6A, 16–24 μg/ml for HHV-6B and 121–128 μg/ml for HHV-7. These drugs were the least active. The sensitivity of HHV-7 to the guanine analogs was different from HHV-6, suggesting a difference in selectivity of specific viral enzymes.

Introduction

In recent years, several emerging viruses have been discovered, including human herpesvirus 6 (HHV-6) (Salahuddin et al., 1986, Lopez et al., 1988, Yamanishi et al., 1988, Pellett and Black, 1996) and human herpesvirus 7 (HHV-7) (Frenkel et al., 1990). HHV-6 was first isolated from peripheral blood lymphocytes of patients with acquired immune deficiency syndrome (AIDS) (Salahuddin et al., 1986), from patients with lymphoproliferative disorders and children with exanthema subitum (Lopez et al., 1988, Yamanishi et al., 1988). HHV-7 was first isolated from peripheral blood lymphocytes (Frenkel et al., 1990) and saliva of healthy adults (Wyatt and Frenkel, 1992, Black et al., 1993, Frenkel and Roffman, 1996, Takahashi et al., 1997b). HHV-6 and HHV-7 have been classified as beta herpesviruses.

HHV-6 has been divided into two variants, HHV-6A and HHV-6B, following molecular and epidemiological characterization (Ablashi et al., 1991, Aubin et al., 1991, Inoue et al., 1993, Pellett and Black, 1996). Clinically, HHV-6B is the most common etiologic agent of exanthema subitum (roseola). Diseases caused by HHV-6A are less apparent. Recently, HHV-7 has been reported as another cause of exanthema subitum (Tanaka et al., 1994). While diseases caused by HHV-6 and HHV-7 in childhood are usually not fatal, complications of exanthema subitum (Asano et al., 1990, Kondo et al., 1993, Torigoe et al., 1996, Torigoe et al., 1997) and life-threatening diseases (e.g. organ transplant, pneumonitis, multiple sclerosis, encephalitis) (Okuno et al., 1990, Asano et al., 1991, Challoner et al., 1995, Knox et al., 1995, McCullers et al., 1995) have been reported.

The differences in biological properties of HHV-6 and HHV-7 (Frenkel and Roffman, 1996, Pellett and Black, 1996, Isomura et al., 1997) assess their sensitivity to antiviral drugs. The antiviral activity of various drugs against these agents has been reported (Streicher et al., 1988Agut et al., 1989Åkesson-Johansson et al., 1990 Burns and Sandford, 1990Williams, 1992Reymen et al., 1995Black et al., 1997). Takahashi et al. (1997a)compared the antiviral activity of several drugs against these viruses with an ELISA method. We standardized the assay methodology in order to define the sensitivity of HHV-6 and HHV-7 to several classes of antiviral compounds.

In order to evaluate differences in the sensitivities of HHV-6 and HHV-7, four classes of antiviral compounds were studied. The four classes included:

(1) Phosphonoformic acid (PFA), a non-nucleoside pyrophosphate analog, is a non-competitive inhibitor of viral DNA replication, acting at the pyrophosphate binding site of DNA polymerase (Crumpacker, 1992, Hirsch et al., 1996).

(2) 9-(2-Hydroxyethoxymethyl)guanine (ACV), 9-(4-hydroxy-3-hydroxy-3-hydroxymethylbutylyl)guanine (PCV) and 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (GCV) are representative guanine analogs which require virus-specific enzymes (i.e. thymidine kinase and UL97 phosphotransferase) for activation (Vere Hodge and Cheng, 1993, Crumpacker, 1996, Hirsch et al., 1996).

(3) (S)-1-[(3-Hydroxy-2-phosphonylmethoxy)propyl]cytosine [(S)-HPMPC] is a representative acyclic nucleoside phoshphonate which has a phosphonate group on an alkyl side-chain (De Clercq, 1993).

(4) 2-Bromo-5,6-dichloro-1-(beta-d-ribofuranosyl)benzimidazole (BDCRB) is one of benzimidazole ribofuranosides that do not require phosphorylation for antiviral activity (Townsend et al., 1995, Saluja et al., 1996).

Some of these compounds are potent inhibitors of herpes simplex virus (HSV), human cytomegalovirus (HCMV) and varicella zoster virus (VZV) (Faulds and Heel, 1990, Whitley and Gnann, 1992, Wagstaff et al., 1994, Townsend et al., 1995, Hirsch et al., 1996, Saluja et al., 1996).

Section snippets

Host cells

Fresh cord blood mononuclear cells (CBMCs) were prepared by centrifugation through Histopaque-1077 (Sigma Chemical Co., St. Louis, MO) and cultured for 3 days in RPMI-1640 medium containing 10% heat inactivated fetal bovine serum, 0.1 U/ml of recombinant human interleukin 2 (GIBCO BRL Life Technology Inc., Grand Island, NY) and 5 μg/ml of phytohemagglutinin (PHA; Sigma Chemical Co., St. Louis, MO) at 37°C in a 5% CO2 incubator. After 3 days, CBMCs were infected with virus and cultured in

Antiviral screening for acyclic nucleoside analogs

The EPC, CC50 and selectivity index (SI) of the acyclic nucleoside phosphonates against the Z29 strain of HHV-6B are summarized in Table 1. The EPC values were first determined by 10-fold dilutions of compounds and, more precisely, by 2-fold dilutions for selected compounds. The EPC values of (R)-PMPA and D4API were 100 μg/ml, being the least effective of the tested compounds. The EPC for PMMMG was >10 μg/ml. (S)-HPMPA and PMEG had EPC values of 1 μg/ml, but the respective cytotoxicities were

Discussion

We developed a dot-blot assay to assess the sensitivity of HHV-6 and HHV-7 to several classes of antiviral compounds. Our results are reproducible and objective by standardizing the MOI and using CBMCs as host cells. The CBMCs allow in vitro propagation of T-lymphotropic viruses, such as HHV-6 and HHV-7. This method, especially visual evaluation of the EPC, is simple and will be applicable to the study of other T-lymphotropic viruses, as well. Recently, Takahashi et al. (1997a)compared the

Acknowledgements

We thank Dr T. Yamagata and his co-workers for their support. Work performed and reported by the authors was supported by Contracts NOI-AI-15113, NOI-AI-12667 from the Virology Branch, DMID, the National Institute of Allergy and Infectious Diseases, a grant from the Division of Research Resources (RR-032) from the National Institutes of Health, Bristol Myers Squibb Unrestricted Infectious Disease Award, a grant from the state of Alabama, USA, and a grant-in-aid from the Ministry of Health and

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