Arenavirus chemotherapy—retrospect and prospect

Arenavirus chemotherapy-retrospect and prospect C. J. PFAU1 Two groups of compounds, identifiable by structural similarity, have been found to interfere with the in vitro replication of arenaviruses. All 4 members of the benzimidazole group contain dipolar fused benzene and 5-membered nitrogen-containing rings and share potential chelating ability through the different bidentate structures formed with their side- chains. The biological activity of one of these compounds, metisazone, has been shown to depend on the presence of divalent metals of the first transition series, Cu++ being the most effective. Furthermore, whereas metisazone inactivates cell-free virus, two other members of the group, HBB and 1,2-bis(5-methoxy-JH-benzimidazol-2-yl)-1,2-ethanediol, act intra- cellularly. The site ofaction of the fourth member, SKF 30097, is not known. Using murine lymphocytic choriomeningitis infections as an in vivo model, the bisbenzimidazole derivative has been found to increase life-span without interfering with virus replication. Medication with SKF 30097 or metisazone and copper(2+) sulfate did not significantly or reproducibly change the expected day ofdeath of the animals. The amantadine compounds of the second group have unusual symmetric structures with a 10-carbon cage. The parent compound acts intracellularly, while the site ofaction ofan octachloro derivative is not known. Medication with the parent compound, but not the derivative, shortened the interval between LCM infection and death ofthe mouse. Tissue culture and animal screening of the many available derivatives in these two groups may uncover compounds more efficacious than those already examined. It is difficult to see how antiviral drugs could compete with vaccines in the control of such dis- eases as smallpox, yellow fever, poliomyelitis, and measles. Despite these triumphs, and although I am intrigued by the possibility of using defective inter- fering (DI) viruses in this manner (14), vaccines are not, and probably never will be, the complete answer to the control of arenavirus infections. With the exception of rabies vaccine, all vaccines must be administered prior to infection in order to ensure success. When novel strains of a virus appear, as with influenza, the disease spreads so rapidly that it is impossible to develop, distribute, and administer a vaccine against the new strain soon enough to protect a significant part of a population. Even when it is practical to control a disease by widespread vaccination, this is seldom done. The disease may then smoulder on in a community with occasional isolated cases, outbreaks, and epidemics. Because of the very high case-fatality rates in patients with Lassa fever and with Argentine and 1 Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12181, USA. Bolivian haemorrhagic fevers, and because of the lack of widely accepted vaccines, control through chemotherapy warrants attention. For the fore- seeable future, efforts in arenavirus chemotherapy will be geared to the evaluation of compounds already found to be effective against other types of virus. The reason for this is that the facilities for running large-scale screening programmes are ex- pensive, so that the search for antiviral drugs is almost exclusively the province of pharmaceutical manufacturers. In order to be commercially viable a drug must sell in sufficient quantities to pay for its development and manufacture as well as for future research. Thus, diseases of low incidence or low economic importance, no matter how serious the outcome may be for the individual, will not be primary targets for drug development. This is espe- cially so when the disease occurs in countries where methods of synthesis or drug use are not patentable. This review will be limited to those compounds that are known to interfere with arenavirus-cell interactions. The alkylating agents, the purine and pyrimidine analogues, and the folic acid analogues will not be considered here, even though they are 3406 - 737 - BULL. WORLD HEALTH ORGAN., Vol. 52, 1975 C. J. PFAU known to protect mice from otherwise lethal lym- phocytic choriomeningitis (LCM) virus challenge (5, 7). These antiproliferative agents block the cell- mediated immune response that causes death in the LCM-murine system, and with the possible excep- tion (15) of N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) they have no specific activity against the arenaviruses. What follows, then, are data gathered mainly in the author's laboratory. Our initial encounter with antiviral agents effec- tive against arenaviruses was fortuitious. In 1966, while searching for a reproducible plaque assay for LCM, we received a strain that caused rapid cyto- pathic effects (CPE) in HeLa cells. Before using this strain routinely, we began what was planned to be a thorough examination of its properties. It was quickly established that this strain was ether-re- sistant and would not kill weanling or adult mice. Another unusual characteristic of the strain, which we confirmed, was its sensitivity to the benzimidazole derivative HBB (I), a drug known at that time to inhibit only the in vitro replication of certain pic- ornaviruses. Doubting the authenticity of the strain we had received, we sent it to the Virus Reference Unit at the Center for Disease Control (CDC), Atlanta, and at the same time determined the effect of HBB on the replication of three well-known strains of LCM (Table 1). Before Dr M. H. Hatch of CDC had identified the strain sent to her as polio- virus type 1, we discovered that true LCM strains were indeed inhibited by HBB. At 55 ,umol/litre, it inhibited HeLa extracellular virus yields by 70-90% (no inhibition was observed with L cells). At 220 ,umol/litre, HBB did not affect the multiplica- tion rate of noninfected cells, but did noticeably inhibit multiplication 2 days after infection. Further- more, HBB changed neither the in vitro inactivation rate of virus, nor its adsorption to cells. Interest- ingly, unlike all other HBB-sensitive viruses, the multiplication of LCM was not inhibited by guani- dine hydrochloride at a concentration of 700 jumol/litre (12). This latter result has been confirmed using a precise plaque assay technique (J. C. Logan and C. J. Pfau, unpublished observations, 1974) instead of the LD30 titration method used in all the above experiments. No in vivo experiments were attempted. In 1972 we decided to reinvestigate the benzimid- azoles. The reason for this was twofold: LCM was now regarded as the prototype of the arenaviruses and the need for chemotherapeutic agents against the highly pathogenic members of the group became apparent; and a very potent derivative of HBB had been discovered. Although many HBB derivatives had been synthesized since the late 1950s, none had been found to be much more effective than the parent compound. However, in 1968 the derivat- ive II [1,2-bis(5-methoxy-1H-benzimidazol-2-yl)- 3 3N CH30ay2CH-CH3 N OH OH N H H 1,2-ethanediol] was reported to be 1000 times as effective as HBB in its ability to inhibit the in vitro replication of poliovirus. In 1972, it was established that, unlike HBB, this compound appeared to inhibit all viruses of the Picorna family, specifically the rhinoviruses. Furthermore, rhinovirus shedding in chimpanzees could be prevented by medication with this compound. Our tissue culture studies revealed that II inhibited the replication of LCM, Parana, and Pichinde vi- ruses in L cells (Table 1) but not in HeLa cells. This made direct comparison with the efficacy of HBB impossible. However, the amount of substance con- centration of HBB (mol/litre) was at least a hundred times that needed with compound II to achieve similar inhibition in LCM synthesis. Like HBB, this compound had no direct inactivating effect on LCM virus nor did it affect the adsorption rate of the virus to L cells. Whereas II exhibited its antiviral activity at concentrations that had little or no effect on L cell division rate, a marked change was noted in the sensitivity of the cells to lysis by standard trypsin dispersal procedures. Because of our positive tissue culture results and the data of others indicating that effective anti-rhinoviral levels of II in mice could easily be attained, evaluation of the compound against murine LCM infection was begun. Indeed, 70% of the mice receiving II lived at least 4 times as long as control mice injected with the neurotropic UBC strain of LCM (Table 2). However, most of 738 I 739CHEMOTHERAPY: RETROSPECT AND PROSPECT Table 1. Compounds with in vitro efficacy against arenaviruses Inhibitor Site of action Virus compound I (H BB) intracellular LCM Strain Usual working concentration Cell type Reference CA 1371 55iAmol/litre UBC 55/Amol/litre Traub 55,umol/litre compound 11 intracellular LCM UBC 5Mg/ml Parana 12056 5pg/ml Pichinde An 3739 5gg/ml metisazone extracellular LCM UBC Traub CA 1371 Parana 12056 Pichinde An 3739 compound IV (SKF 30097) 20iAmol/litre+20/Amol/litre CuS04 20/umol/litre+20/Amol/litre CuS04 2Oimol/litre+40umol/litre CuS04 20,mol/litre+1 60,Amol/litre CuS04 20,umol/litre+1 60,umol/litre CuS04 ? Pichinde An 3739 30gg/mI amantadine intracellular LCM CA 1371 WE UBC Amapari BEAm 70563 Junin XJ XJ-C13 Parana 12056 Tacaribe TRVL 11573 Tamiami W 10777 octachloro- amantadine ? LCM UBC 50pg/ml 50jug/ml 50MAg/mI 50Ag/ml 3OAg/ml 30Mg/ml 1 00,ug/ml 25gAg/ml 50,4g/ml 20,ug/ml HeLa 1 2 HeLa 1 2 HeLa 1 2 L L L 16 16 16 9 9 9 9 9 HeLa L. & p. a L, BHK 20 L, BHK 20 L, BHK 20 Vero 13 Vero 2 Vero 3 BHK 13 Vero 13 Vero 13 L W. &p.b a J. C. Logan & C. J. Pfau, unpublished observations, 1975. b E. A. Wright & C. J. Pfau, unpublished observations, 1972. these 30-day postinfection survivors died with LCM- like symptoms over the next 4 months. Furthermore, the virus content of organs was identical in drug- treated and control mice during the first 7 days after infection. Thus, we concluded that II had no specific antiviral activity in vivo and probably spared mice by temporal depression of the immune system. A search was then begun for a compound struc- turally related to II that might have both in vitro and in vivo antiviral activity. There is some similarity in Table 2. Evaluation of benzimidazole-like compounds in LCM-infected mice Medication Virus challenge Refer- Substance Outcome ence route a dose strain route b dose time c compound ll drinking 330mg/kg per UBC i.c. 10-100 a.m. lengthened 17 water day for 14 days LDso life-span WCP i.p. 10-100 a.m. no change LD5o in life-span 17 metisazone s.c. 10mg/kg in UBC i.c. 3OLD5o 24 h p.m. slight L. & p.d 1 2h for 7 days lengthen- CuS04 drinking 10mg/kg per ing of life- water day for 7 days span SKF 30097 s.c. 25mg/kg in UBC i.c. 30LD5o 24 h p.m. no change L. & p.d 1 2h for 10 days in life-span a s.c. = subcutaneous. b i.c. = intracutaneous; i.p. = intraperitoneal c a.m. = at initiation of medication; p.m. = after initiation of medication. d J. C. Logan & C. J. Pfau, unpublished observations, 1975. 740 C. J. PFAU structure between II and the compound known as IBT.a Both consist of fused benzene and 5-mem- bered nitrogen-containing rings with a single side- chain attached to the heterocyclic ring. The hetero- cyclic systems, however, are unlike in properties and the characteristics of the side-chains are quite differ- ent. An extensive literature exists on the ability of IBT (and related compounds) to inhibit the replica- tion of vaccinia virus in tissue culture, as well as to protect mice against intracerebral infection with various poxviruses (1, 8). IBT and its cogeners have also been reported to inhibit, in tissue culture, other DNA viruses in the adenovirus and herpesvirus groups, as well as certain RNA-containing reo-, arbo-, myxo-, paramyxo-, and picornaviruses (1). Contact inactivation of cell-free virus was also de- scribed as the mode of action of these compounds against Rous sarcoma virus (8), three viruses caus- ing slow infections of sheep (visna, maedi, and progressive pneumonia), herpesviruses, murine sarcoma and leukaemia viruses, and feline sarcoma virus. Indeed, it was found that the methyl derivative of IBT (metisazone, III) inactivated cell- s NNHCNH2 2=0 N CH3 free Parana and Pichinde viruses as well as three strains of LCM. The rate of inactivation by meti- sazone was greatly enhanced and controlled by addition of copper(2+) sulfate. A difference existed in the copper requirement for fast inactivation of LCM and Parana and Pichinde viruses. In the presence of metisazone at a concentration of 20 ,umol/litre, LCM and Pichinde viruses lost over 90% of their infectivity within 15 minutes (at 37°C) if copper(2+) sulfate was added (20 jumol/litre for LCM virus and 160 ,umol/litre for Pichinde virus) (Table 1). There was little detectable change in inactivation rates when Pichinde or LCM virus was grown in a variety of different cell lines. The in vivo efficacy of metisazone was then ex- plored. Although IBT had been reported in 1955 to have no effect on LCM-infected mice, the virus that a From the former chemical name isatin beta-thiosemi- carbazone. was actually used was pseudo lymphocytic chorio- meningitis (ectromelia). Later, an authentic strain was used, still with negative results (D. J. Bauer, personal communication, 1974). However, the syn- ergism between copper (2+) sulfate and metisazone seen in vitro warranted further in vivo investiga- tion. Initially, metisazone alone was tried, using the injection schedule outlined in Table 2, except that the individual injections were with 100 mg of drug per kg of body weight (a concentration found not to affect the growth rate of mice during the medication period). No protection against LCM infection was observed. The subcutaneous injection of the metisa- zone-copper(2+) sulfate complex was found to be very toxic. Within less than 24 hours after the initial injection at the level of 5 mg/kg of each compound, over half the mice had died and the rest were rapidly losing weight (this is about the same toxic dose as observed with the injection of sodium cyanide). Even at 0.05 mg/kg, all mice ceased to gain weight during the injection period. Although there were no toxic effects at 0.005 mg/kg, there was no protection against LCM infection. Since it has been reported that high serum levels of copper can be obtained in mice rather quickly after oral intake, our regimen of medication was switched to subcutaneous injection of metisazone combined with nontoxic levels of copper(2+) sulfate in the drinking-water (Table 2). With many dose combinations we have yet to see a consistently repeatable pattern of protection. Attention was then focused on the 3-substituted triazinoindole IV (SKF 30097). This is an analogue 8 2 1 H3 6 X>4 NHCH CH -OHN 2CH2C. C'H3 CH3 IV of metisazone and, interestingly, shares structural similarities with the benzimidazoles as well as broad spectrum in vivo activity against the picornaviruses (11). At 30 ,ug/ml, a concentration not found to inhibit HeLa cell replication during the experimental period, SKF 30097 led to a 90 %Y drop in the extra- cellular Pichinde virus titre 48 hours after infection (Table 1). A comparable decrease in the Pichinde virus titre was noted with metisazone at 4.7 ,ug/ml (20 Hmol/litre) for the same time period (M. P. Fox & C. J. Pfau, unpublished olbservations, 1975). The mode of action of SKF 30097 against the arena- CHEMOTHERAPY: RETROSPECT AND PROSPECT viruses is unknown but it has been reported not to contact inactivate picornaviruses (11). No protection in LCM lethally-infected mice was afforded by this compound (Table 2). The 2 remaining compounds to be discussed, the amantadines (V and VI), have unusual 10-carbon- oL V VI cage symmetric structures, and are quite unlike the first 4 compounds. The reasons for testing amanta- dine were, again, centered in a plaque assay for LCM. Shortly after our initial success with plaque formation by cell-free LCM virus, the procedure was modified for the detection of virus-infected cells (infective centres). Initial observations were that no matter how high the " input multiplicity of infec- tion" no more than 10% of the cells scored as infective centres after the standard 1-hour adsorp- tion period (14). A by-product of further experi- ments showing that the problem was in the induc- tion, not in the expression, of infective centre forma- tion, was the finding of an apparently preferential intercellular spread of infection (an increasing num- ber of infective centres in the virtual absence of extracellular virus). As proof that virus infection from cell to cell via the overlying fluid is not necessary, the spread of infective centres is usually shown to be unimpeded in the presence of virus-specific neutralizing antibody in the medium. We had no source of potent anti- body, so a literature search was begun for a com- pound that would block some early event in the virus-cell interaction. Amantadine was known to prevent myxovirus growth by inhibiting either pene- tration or uncoating of the virion. It had also been reported to interfere with the replication of pseudo- rabies, rubella, Rous and Esh sarcoma, and fowl leukosis (4). We found that in several cell lines amantadine inhibited 4 viruses of the Tacaribe group, as well as three strains of LCM (Table 1). The compound had no direct inactivating effect on LCM infectivity nor did it affect the adsorption of virus to L or BHK cells. By observing the susceptibility of cell-adsorbed virus to neutralizing antibody, aman- tadine was shown to delay penetration of LCM. Furthermore, addition of the drug at any time during ongoing virus replication, with all cells in- fected, inhibited LCM and Parana virus synthesis and release (Table 1). Unknown to us, and two years before full publication of the LCM studies, Coto et al. (3) had reported that amantadine inhibited the replication of Junin virus in Vero cells. In vivo studies with LCM were easily begun because the toxicological and pharmacological pro- perties of amantadine in the mouse were well estab- lished (4). Using drug levels that had no effect on weight gain or humoral antibody response in mice, we found that intracerebral injection of the UBC strain of LCM led to death 1-2 days earlier than in the infected control mice. These drug-treated mice had the typical LCM-induced post mortem convul- sion position. The mouse studies were also done before we became aware of the work of Coto et al. with amantadine and Junin virus. These Argentine workers had tested the compound in vivo and, surprisingly, also found that amantadine medication shortened the life-span of virus-infected guinea-pigs (Table 3). No explanation of these results was offered. My working hypothesis (without any proof) to account for these observations is as follows: Amantadine, especially at low concentrations in tissue culture medium, affects not so much the synthesis of LCM but its release from L cells. If the compound does affect virus synthesis in vivo in the same manner, then virus-infected cells (by retaining more foreign antigen on their surface) might be recognized more quickly, or bound more firmly, by the lymphoid cells responsible for their destruction. We also switched to intraperitoneal injection of the viscerotropic WCP strain of LCM, since it had been reported that amantadine concentrations at- tainable in the mouse brain were rather low. Under these conditions, amantadine neither shortened nor lengthened the life span (Table 3). An octachloro chlorination product (VI) of amantadine was tested. Unlike the parent compound, it had a remarkable curative effect on lethal neurotropic influenza A/WSN murine infections (10). It was felt that, owing to its lipid solubility, pharmacologically active concentrations of the compound could be obtained in nervous tissue. Again, no prophylactic activity against LCM could be observed in the mouse (Table 3) even though, at 20 ,tg/ml, it was able to decrease the extracellular yield of LCM by 60% within 24 hours after infection of L cells (Table 1). As many of the molecular aspects of viral replica- tion have been unravelled within the last 15 years, a groundswell of enthusiasm has developed in anti- 741 C. J. PFAU Table 3. In vivo evaluation of adamantane compounds against arenavirus infections Medication Virus challenge Refer- Animal Outcome ence route dose type route c dose time d guinea-pig oral 50mg/kg daily Junin ? 5OLDso 24 h p.m. shortened 3 intubation for 14 days a Xi life span mouse drinking 300mg/kg daily LCM i.c. 5OLDso 72 h p.m. shortened p e water for 14 days a UBC lifespan LCM i.p. 5OLD5o 72 h p.m. no change p e WCP in life span mouse oral 1 00mg/ks LCM i.c. 1 OLDso 2 h p.m. no change p e intubation for 1 day0 UBC in life span a 1-adamantanamine (compound V). b N-methyl-1 -adamantanecarboxamide octachloro chlorination product (compound VI). C i.c. = intracutaneous; i.p. = intraperitoneal. d p.m. = after initiation of medication. e C. J. Pfau, unpublished observations, 1972. cipation of an era of viral chemotherapy rivalling that experienced with the bacterial diseases. Un- fortunately, the great expenditure of time, effort, and money has thus far yielded a paucity of practical results. Antiviral agents with in vitro activity are widespread, those having prophylactic activity in laboratory animals are not uncommon, but those useful in man are rare. Considering our results to epitomize what occurs in the field, it seems worth while to restate some generalities. Why use tissue culture screens, when testing for antiviral activity directly in animals would seem to be more valid and useful? It is commonly agreed that, apart from the interferon inducers, unless a compound manifests some activity in vitro it is highly unlikely to do so in vivo. If we assume that a compound should prevent disease in vivo if it inhibits virus replication in tissue culture, why are most of the compounds that are active in vitro ineffective in the laboratory animal? Perhaps in vivo they lack: (a) a reasonably long half- life, (b) good solubility, (c) the capacity to penetrate target cells, and (d) effectiveness against the quanti- ties of virus administered to an animal to produce some measurable effect, which are vastly greater than would ordinarily be encountered by the natural route of infection. An advantage of tissue culture screens that is not to be overlooked is that compounds discovered in this way may be used as probes in studying virus replication at the molecular level. Amantadine, be- cause of its unique ability to inhibit an early as well as a late stage in arenaviral replication, can be used as a classification aid for new isolates. The fact that the rapid contact inactivation of arenaviruses by metisazone requires the addition of copper sulfate has predictive value in choosing viruses that will not be substantially inactivated during purification. Both Parana and Pichinde virus require relatively high concentrations of copper(2+) sulfate for quick in- activation by metisazone and they lose little infectiv- ity during purification (9). However, LCM (9), Latino, and Tacaribe viruses (J. B. Kubis, J. C. Logan, and C. J. Pfau, unpublished observations, 1975) require only one eighth the amount of cop- per(2+) sulfate to achieve comparable inactivation rates. LCM is well known for the large infectivity loss incurred during purification, and Tacaribe virus has also been found to respond similarly (R. R. Rosato, personal communication, 1975). Other areas at present being investigated and developed from the initial metisazone-copper(2+) sulfate studies, include determining the antigenicity of contact-inactivated LCM and using the metisazone-copper(2+) sulfate complex in a selective medium for the isolation of LCM mutants resistant (or relatively resistant) to the complex and thus stable in purification procedures. Future research into the treatment of arenaviral diseases by drugs should be directed towards three general areas: attempts to (a) stimulate the defence mechanisms of the host (interferon inducers), (b) find drugs that directly block some virus-specific process, and (c) palliate symptoms of the disease (the main- stay of medical practice from time immemorial and still the case with most virus diseases). Our long- 742 CHEMOTHERAPY: RETROSPECT AND PROSPECT 743 range interest is to continue to make a selective examination of compounds structurally related to those already proved to have in vitro activity. Since the initial report of the antiviral activity of amanta- dine, a large number of alicyclic compounds have been synthesized, many of which have antiviral activity in tissue culture and experimental animal systems (4). Similarly, large numbers of compounds related to HBB (18) and the thiosemicarbazones (1, 8, 19) are available. Clearly, if arenavirus infections become a global public health problem a substantial effort will be needed to screen these compounds. RNISUMS CHIMIOTHERAPIE DES INFECTIONS A ARENAVIRUS -RIETROSPECTWE ET PERSPECTIVES L'article traite de la decouverte de deux groupes de composes de structure apparent6e, actifs contre les infec- tions i ar6navirus in vitro. La figure 1 donne la formule developpee des composes du premier groupe (composes I A IV). Ces quatre composes presentent tous un systeme heterocyclique condense comportant un cycle azote pen- tagonal et un noyau benzenique accole, et ont les memes possibilites de chelation grace aux differents bidentates qui forment les cha^mes laterales. Jusqu'ici, on n'a encore montre que pour un seul de ces composes - la metisa- zone - que l'activite biologique dependait de la presence de m6taux divalents de la premiere serie de transition, Cu++ etant le plus actif. La metisazone se distingue encore en ceci qu'elle inactive le virus en dehors de la cellule tandis que les composes I et II (HBB et bisbenzimidazole) ont une action intracellulaire; quant au site d'action du compose III, SKF30097, il est encore inconnu. L'exigence en cuivre pour l'inactivation rapide (90% de perte d'acti- vite apres 15 minutes a 370) n'est pas la meme pour l'arenavirus prototype (choriomeningite lymphocytaire) et deux membres du complexe viral Tacaribe (Parana et Pichinde). En presence de 20 nm de metisazone, les virus CML et Pichinde sont inactives a une allure a peu pres egale si l'on ajoute 20 mm de CuS04 au premier et 160 nm de CuS04 au second (tableau 1). Les autres composes du groupe inhibent la reproduction du virus a partir des cellules infectees. Le tableau 1 indique les types cellulaires sensibles et les concentrations non- cytotoxiques de medicament qui sont necessaires pour inhiber le virus de fa$on significative. Mis a part le compose II, aucun des compos6s eprouves n'a modifie le syndrome letal dans l'infection CML chez la souris (tableau 2). Toutefois, on n'a decele chez les souris trai- tees au benzimidazole aucune inhibition de la synthese du virus. Les composes du second groupe ont des struc- tures insolites, symetriques a 10 atomes de carbone en forme de cage (fig. 1 E-F). Le compose initial, I'amanta- dine, retarde la p6netration du virion dans la cellule et inhibe la synthese et la liberation du virus A un stade ulterieur. Le mode d'action in vitro d'un derive octa- chlore d'amantadine est inconnu mais, sur une base molaire, il est beaucoup moins actif que le compos6 initial (voir, dans le tableau 1, les types cellulaires sen- sibles et les concentrations non-cytotoxiques actives). Des cobayes et des souris, respectivement infectes par les virus Junin et CML, et traites a l'amantadine ont suc- combe plus rapidement que les animaux temoins infectes (tableau 3). L'auteur propose 1'hypothese suivante: si I'amantadine agit sur I'animal comme il le fait a de faibles concentrations in vitro (affectant davantage la liberation du virus que sa synthese totale) il se pourrait que la destruction du tissu infect6 par les cellules lym- phoides soit acceleree. Aucun effet de ce genre n'a ete observe avec le derive octachlore de ce compos6 (ta- bleau 3). Peut-etre la culture tissulaire et l'experimenta- tion animale des nombreux derives existants de ces deux groupes permettraient-elles de decouvrir des composes plus efficaces que ceux qui ont ete etudies jusqu'ici. REFERENCES 1. BAUER, D. J. Thiosemicarbazones. In: Bauer, D. J., ed. International Encyclopedia of Pharmacology and Therapeutics, vol. 1: Chemotherapy of virus dis- eases, section 61. London, Pergamon Press, 1972, pp. 35-113. 2. CoTo, C. E. Susceptibility of Junin virus to amanta- dine. Medicina (B. Aires), 32: 23-26 (1972). 3. COTO, C. E. ET AL. Efecto de la Amantadina HC1 sobre la infectividad del virus de Junin (F.H.A.) in vitro e in vivo. Rev. Asoc. argent. Microbiol., 1: 3-8 (1969). 4. HOFFMAN, C. E. Amantadine HC1 and related com- pounds. In: Carter, W. A., ed. Selective inhibitors of viral functions, Cleveland, CRC Press, 1973, pp. 199- 211. 5. HOTCH1N, J. Persistent and slow virus infections. In: Melnick, J. L., ed. Monographs in virology, Basel, Karger, 1971, vol. 3, pp. 44-46. 6. KATZ, E. ET AL. The effect of isatin beta thiosemi- carbazone (IBT)-related compounds on IBT-resistant and on IBT-dependent mutants of vaccinia virus. J. gen. Virol., 25: 239-244 (1974). 744 C. J. PFAU 7. LEHMANN-GRUBE, F. Lymphocytic choriomeningitis virus. In: Gard, S. et al., ed., Virology monographs, Wien/New York, Springer, 1971, vol. 10, pp. 53-56. 8. LEVINSON, W. Inhibition of viruses, tumors, and pathogenic microorganisms by isatin beta thiosemi- carbazone and other thiosemicarbazones. In: Carter, W. A., ed. Selective inhibitors of viral functions. Cleveland, CRC Press, 1973, pp. 213-226. 9. LOGAN, J. C. ET AL. Arenavirus inactivation on contact with N-substituted isatin beta-thiosemi- carbazones and certain cations. J. gen. Virol., 28: 271-283 (1975). 10. McGAHEN, J. W. Curative antiviral activity of N- methyl-l-adamantanecarboxamide octachloro chlo- rination product. Fed. Proc., 25 (abst. 2109): 562 (1966). 11. MATSUMOTO, S. ET AL. The antiviral activity of a triazinoindole (SK&F 30097). Proc. Soc. exp. Biol. Med., 139: 455-460 (1972). 12. PFAU, C. J. & CAMYRE, K. P. Inhibition of lympho- cytic choriomeningitis virus multiplication by 2- (alpha-hydroxybenzyl) benzimidazole. Virology, 35: 375-380 (1968). 13. PFAU, C. J. ET AL. Arenaviruses: Inhibition by amantadine hydrochloride. J. gen. Virol., 14: 209-211 (1972). 14. PFAU, C. J. ET AL. Plaque assays and current concepts of regulation in arenavirus infections. In: Lehmann- Grube, F., ed. Lymphocytic choriomeningitis virus and other arenaviruses. Berlin, Heidelberg, & New York, Springer, 1973, pp. 101-111. 15. SIDWELL, R. W. ET AL. In vitro studies on the antiviral activity of 1,3-bis(2-chloroethyl)-1-nitroso- urea. Appl. Microbiol., 14: 405-410 (1966). 16. STELLA, J. P. ET AL. Characteristics of the in vitro inhibition of arenavirus synthesis by bis-benzimida- zoles. Antimicrob. Agents Chemother., 6: 747-753 (1974). 17. STELLA, J. P. ET AL. Evaluation of bis-benzimidazoles in the treatment of murine lymphocytic choriomenin- gitis virus infections. Antimicrob. Agents Chemo- ther., 6: 754-756 (1974). 18. TAMM, I. & CALIGUnU, L. A. 2-(alpha.-hydroxyben- zyl) benzimidazole and related compounds. In: Bauer, D. J. ed. International Encyclopedia of Pharmacology and Therapeutics, vol. 1: Chemo- therapy of virus diseases, section 61. London, Per- gamon Press, 1972, pp. 115-179. 19. ToNEw, M. ET AL. Antiviral thiosemicarbazones and related compounds. II. Antiviral action of substituted isatinisothiosemicarbazones. Acta Virologica, 18: 17- 24 (1974). 20. WELSH, R. M. ET AL. Amantadine hydrochloride inhibition of early and late stages of lymphocytic choriomeningitis virus-cell interactions. Virology, 45: 679-686 (1971). DISCUSSION WELSH: What is the general feeling regarding the ability of arenaviruses to induce interferon production and their sensitivity to it? The administration of interferon would be one type of chemotherapeutic approach to consider. PADNOS: We have found that LCM (M-P) virus induces 2500 IU of serum interferon in mice on day 3 after infection and that production subsides by day 5. This serum interferon protects mice against lethal challenge with pseudorabies virus, LCM virus, and other arena- viruses. EDDY: We have carried out fairly simple interferon stud- ies, using primate interferon and viruses of the Tacaribe complex. We found that they were remarkably sensitive; if my memory is correct, we found that Pichinde is about 100-fold less sensitive than is VSV, and Machupo virus appears to be about tenfold less sensitive than Pichinde. In addition, we have carried out a very dif- ficult study with Dr Levy using stabilized poly IC, which is an in vivo interferon inducer; we found that the mon- keys became more viraemic and appeared to die earlier when given the drug. HOTCHIN: We have also found interferon in mice in the early days of LCM infection. However, I do not think that this is made by the virus per se. I think it is prob- ably due to some particulate interference with the reticuloendothelial system, causing a temporary outpour- ing of interferon. In vitro we could find no evidence of the slightest amounts of interferon produced by LCM-- infected cells, and in L-cells persistently infected with LCM virus it is possible to grow extremely interferon- sensitive viruses without interference. Mims: We also have found that LCM does not induce interferon in mouse cells in vitro, although it will induce interferon in chick cells. LCM is highly sensitive to inter- feron in vitro, but in an infected animal it is very difficult to produce protection, as has been found with interferoa in other studies. LEHMANN-GRUBE: In a discussion of this kind I think that we should make a clear distinction between infection and disease. Infection does not necessarily lead to disease. Factors limiting infection do not necessarily also limit the clinical signs that follow infection. By the time the clinical signs appear, virus multiplication has probably already finished. So when we look for prophylactic or therapeutic agents, we must consider the effects on infection and the effects on the symptoms or signs. It is. very improbable that we shall find something that wilL have a beneficial effect on both.