Antiviral Research 62 (2004) A1–A92

Programs and Abstracts

The Seventeenth International Conference on

Antiviral Research

A Colorimetric Cell Culture Assay for the Identification

of SARS Coronavirus Inhibitors

E. Keyaerts1, L. Vijgen1, J. Neyts1, E. De Clercq1, J.

Balzarini1, M. Van Ranst1_2

1Rega Institute for Medical Research, K.U. Leuven, 3000

Leuven, Belgium; 2U.Z. Leuven, 3000 Leuven, Belgium

Severe acute respiratory syndrome (SARS) has recently

emerged as a new severe human disease, resulting globally

in 774 deaths from 8098 reported probable cases. A novel

member of the Coronaviridae family has been identified as

the causative agent of this pulmonary disease. Although the

initial global outbreak of SARS appears to have been successfully

contained, SARS will remain a serious concern

while there continues to be no suitable vaccine or effective

drug treatment. A colorimetric assay based on the reduction

of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-5-(3-

carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium

(MTS) into a water soluble formazan product which can

be directly quantified using a microtiter ELISA reader,

has been developed for SARS coronavirus strain Frankfurt

1 drugsusceptibility testing. Optimal conditions were

determined and the standard routine assay was calibrated

with a viral input of 100 CCID50 and a density of 10,000

cells per well in a 96-well microtiter plate for an incubation

period of 3 days. Interferon _ was used as a positive

control to validate the assay. The effective IC50 concentration

value obtained with interferon _ in the present assay

was in agreement with interferon _ activity results published

by others. This method presents the advantage of

being rapid, reliable, reproducible, and convenient for high

throughput screening capacity in a stringent P3 biosafety

environment.

120

Antiviral Activity of Glycyrrhizic Acid (GL) Derivatives

Against SARS-Coronavirus (SARS-CoV) and Human

Cytomegalovirus (HCMV)

G. Hoever1, L. Baltina2, R. Kondratenko2, L. Baltina Jr.2,

G. Tolstikov2, H.W. Doerr1, J. Cinatl1

1Institute of Medical Virology, Johann Wolfgang Goethe

University Frankfurt, Paul-Ehrlich Str. 40,60596 Frankfurt,

Germany; 2Institute of Organic Chemistry Ufa Research

Centre of Russian Academy of Sciences, Prospect Oktyabrya,

71, Ufa Glycyrrhizic Acid (GL) is the major bioactive triterpene glycoside

of licorice root (Glycyrrhiza Radix). The antiviral

activity of GL against a broad spectrum of viruses, among

others HIV, HSV1, Influenza Virus, SARS-CoV, HBV and

HCV has been reported.

In the effort to discover GL analogous with strong enhanced

antiviral activity, we tested a number of new synthesized

GL derivatives against HCMV and SARS-CoV. These

compounds were received by introduction of different functional

groups in the Carboxyl and Hydroxyl groups as well

as transformations of the carbohydrate part of the molecule.

Our results show that the GL-amides BL43 and BL49,

the reduced GL-trimethyl ester BL 44, and the Glycopeptide

_-Cys-GL present a more than 10-fold increased antiviral

activity against SARS-CoV compared to Glycyrrhizin,

while the GL-amides BL26, and BL43 presented antiretroviral

activity against HCMV.

121

Antiviral and Virucidal Activities of Oreganol P73-based

Spice Extracts Against Human Coronavirus In Vitro

M_K_ Ijaz1_2_3, Z. Chen1, S.S. Raja1, D.B. Suchmann1, P.W.

Royt2, C. Ingram4, J.K. Gray4, G. Paolilli4

1MICROBIOTEST, INC., Sterling, VA 20164, USA;

2George Mason University, Fairfax, VA 22030, USA;

3University of Ottawa, Ottawa, Ont., Canada; 4North American

Herb & Spice, Buffalo Grove, IL 60085, USA

Human Coronavirus (HCoV) infection is very common, disseminated

by air and occurs worldwide. Recently, a previously

unknown HCoV has been implicated as a causative

agent of severe acute respiratory syndrome (SARS). Finding

a successful antiviral drug for SARS-associated HCoV

is particularly challenging. The virucidal and antiviral activities

of two Oreganol P73-based spice extracts were evaluated

during in vitro HCoV (ATCC VR-740) infection. To

determine, the virucidal potentials of non-cytotoxic dilutions

of Oreganol P73 Extra Strength Formula (0.1%) and Oregacyn

(0.01%), the virus was exposed to each drug dilution,

and samples were collected at various times post-exposure

prior to assay on the host cells. The antiviral ability of

both Oreganol P73 Extra Strength Formula and Oregacyn

was determined by maintaining the non-cytotoxic concentration

of both drugs throughout the viral–host interaction

period. The cell culture plates were examined microscopically

for the presence of HCoV-induced cytopathic effects

produced by viral infection. The virucidal part of the study

indicated that both Oreganol P73 Extra Strength Formula

and Oregacyn at final concentrations of 0.1 and 0.01%, respectively,

proved to be coronavirucidal in direct proportion

to exposure time ranging from 2 to 20min at ambient

temperature.

In contrast, the antiviral studies revealed both

Oreganol P73 Extra Strength Formula and Oregacyn completely

inhibited HCoV infection in vitro. These data indicate

the potential value of these Oreganol P73-based spice

extracts as anti-HCoV compounds and merit further investigation

against other mammalian viruses including HIV,

HBV, HCV, influenza-, parainfluenza-, respiratory syncytial

virus, herpes-, Hanta- and West Nile viruses.