were dissolved in ethanol, reconstituted with Hanks balanced salt solution, and buffered to pH 7.2 with 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) plus 2% (vol/vol) heat-inactivated fetal bovine serum (4% ethanol, final concentration). Drug solutions (0.3 ml) of appropriate concentration (0 to 2.5 ,ig/ml for didemnin A and 0 to 0.5 ,ug/ml for didemnin B) were added to 24-well tissue culture trays (Falcon). Virus (0.1 ml, 50 to 200 PFU) or medium (for tissue culture toxicity controls) was then added. After incubating the trays for 60 min to allow adsorption of the virus, 0. Didemnins A and B were both found to exhibit significant activity against RVF (median inhibition dose [ID50] for didemnins A and B was 1.37 and 0.04 .Ig/ml, respectively), Venezuelan equine encephalomyelitis virus (ID50, 0.43 and 0.08 gjg/ml, respectively), and yellow fever virus (ID50, 0.4 and 0.08 ,ug/ml, respectively). A concentration of 0.1 ,ug of didemnin B per ml inhibited plaque formation by these three viruses by more than 80%o. Didemnin A, on the other hand, was less efficient, requiring a 25-fold increase in concentration to achieve the same level of virus plaque inhibition. Both didemnins A and B showed mild cytotoxicity in Vero-76 cells as judged by phase-contrast microscopy at concentrations greater than 5 and 0.5 Fg/ml (5.3 and 0.45 M), respectively, consisting of increased vacuolation with moderate cell rounding but no detachment. As compared with the other three test viruses, Pichinde virus (ID50 for didemnins A and B was 2.9 and 0.22 ,ug/ml, respectively), a representative arenavirus, was less sensitive to the in vitro antiviral effect of both didemnins.Studies were also conducted with RVF-infected mice. Female Swiss Webster mice, 3 to 4 weeks of age, were challenged subcutaneously with approximately 250 PFU of RVF per 0.1 ml.

Rhesus monkeys (Macaca mulatta) treated with a newly developed nuclease-resistant complex of polyriboinosinic-polyribocytidylic acid, poly-L-lysine, and carboxymethylcellulose [poly (ICLC)] did not die after challenge with virulent Asibi strain yellow fever (YF) virus. The strain of virus is sensitive to the effects of interferon in vitro and is lethal for rhesus monkeys four to six days after subcutaneous administration of 1,000 plaque-forming units of the virus. The mortality rate was reduced in monkeys initially treated 8 hr before or after inoculation of virus but was unchanged in monkeys initially treated 24 hr after challenge. Treated monkeys developed neutralizing antibody to YF virus. The successful treatment of yellow fever in a primate model with use of poly (ICLC) suggests a meaningful role for the interferon system in the host defense against this viral infection.

No abstract

The antiviral activity of tilorone hydrochloride and three of its analogues (11,002, 11,567, and 11,877) was assessed by oral and intraperitoneal (i.p.) administration to Venezuelan equine encephalitis (VEE) virus-infected mice. Significant increases in the percentage of survival ( P < 0.01) were apparent after oral administration of tilorone and analogue 11,877 at dosages of 250 and 500 mg/kg. Neither tilorone nor 11,877 increased percentage of survival when dosages of 31.25 to 500 mg/kg were given by the i.p. route. Orally administered analogue 11,002 was effective against 100 mouse intracranial median lethal doses (MICLD 50 ) of VEE virus at doses at 250 to 1,000 mg/kg; doses of 31.25 to 250 mg/kg given i.p. were effective against 10 MICLD 50 . Oral dosages of 250 to 1,000 mg of analogue 11,567 per kg were active against 100 MICLD 50 of virus. By the i.p. route, 250 mg of 11,567 per kg protected mice against 1,000 MICLD 50 , and a dose of 125 mg/kg protected against 100 MICLD 50 . Oral treatment of VEE infection with analogue 11,567 24 h after subcutaneous inoculation of VEE virus resulted in no significant increase in the percentage of survivors. All survivors of these studies were susceptible to rechallenge 21 days after the first inoculation of virus.

An indirect mouse model was utilized to evaluate the antiviral activity of several compounds against Venezuelan equine encephalomyelitis (VEE) virus infection in mice. Mice were given various dosages of lysine-stabilized polyriboinosinic acid-polyribocytidylic acid, a tilorone analogue, kethoxal, or mepacrine before and/or shortly after receiving one of several dose levels of attenuated strain TC-83 VEE virus. Twenty-one days later, the same mice were rechallenged intracranially with virulent Trinidad donkey strain VEE virus. Susceptibility to rechallenge was interpreted as evidence of drug effectiveness in completely preventing the initial immunizing virus infection. In contrast, if a drug lacked antiviral effectiveness, the initial attenuated infection stimulated sufficient immunity to protect mice against the virulent rechallenge. Both of the interferon inducers, lysine-stabilized polyriboinosinic acid-polyribocytidylic acid and tilorone analogue 11,567, possessed significant (P < 0.01) antiviral activity based upon this indirect model, whereas mepacrine and kethoxal were inactive. Results using the indirect method were confirmed by using the conventional direct method for evaluating the effectiveness of potentially useful antiviral compounds. The indirect mouse model described should prove useful for studying drug efficacy against certain viruses that are lethal only by intracranial inoculation.