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Introduction: Mammarenaviruses are negative-sense bisegmented enveloped RNA viruses that are endemic in Africa, the Americas, and Europe. Several are highly virulent, causing acute human diseases associated with high case fatality rates, and are considered to be significant with respect to public health impact or bioterrorism threat., Areas Covered: This review summarizes the status quo of treatment development, starting with drugs that are in advanced stages of evaluation in early clinical trials, followed by promising candidate medical countermeasures emerging from bench analyses and investigational animal research., Expert Opinion: Specific therapeutic treatments for diseases caused by mammarenaviruses remain limited to the off-label use of ribavirin and transfusion of convalescent sera. Progress in identifying novel candidate medical countermeasures against mammarenavirus infection has been slow in part because of the biosafety and biosecurity requirements. However, novel methodologies and tools have enabled increasingly efficient high-throughput molecular screens of regulatory-agency-approved small-molecule drugs and led to the identification of several compounds that could be repurposed for the treatment of infection with several mammarenaviruses. Unfortunately, most of them have not yet been evaluated in vivo . The most promising treatment under development is a monoclonal antibody cocktail that is protective against multiple lineages of the Lassa virus in nonhuman primate disease models.

Five New World mammarenaviruses (NWMs) cause life-threatening hemorrhagic fever (HF). Cellular entry by these viruses is mediated by human transferrin receptor 1 (hTfR1). Here, we demonstrate that an antibody (ch128.1/IgG1) which binds the apical domain of hTfR1, potently inhibits infection of attenuated and pathogenic NWMs in vitro. Computational docking of the antibody Fab crystal structure onto the known structure of hTfR1 shows an overlapping receptor-binding region shared by the Fab and the viral envelope glycoprotein GP1 subunit that binds hTfR1, and we demonstrate competitive inhibition of NWM GP1 binding by ch128.1/IgG1 as the principal mechanism of action. Importantly, ch128.1/IgG1 protects hTfR1-expressing transgenic mice against lethal NWM challenge. Additionally, the antibody is well-tolerated and only partially reduces ferritin uptake. Our findings provide the basis for the development of a novel, host receptor-targeted antibody therapeutic broadly applicable to the treatment of HF of NWM etiology., (© 2022. The Author(s).)

Targeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their proapoptotic properties but rather with their ability to induce cell arrest at the G0/G1 phase. OLX- and ABT-737-mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, small interfering RNA (siRNA)-mediated knockdown of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals. IMPORTANCE Antiapoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors' mediation of cell cycle arrest at the G0/G1 phase, rather than their proapoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate antimammarenavirus activity in vivo and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals.

Mammarenaviruses are prevalent pathogens distributed worldwide, and several strains cause severe cases of human infections with high morbidity and significant mortality. Currently, there is no FDA-approved antiviral drugs and vaccines against mammarenavirus and the potential treatment option is limited to an off-label use of ribavirin that shows only partial protective effect and associates with side effects. For the past few decades, extensive research has reported potential anti-mammarenaviral drugs and their mechanisms of action in host as well as vaccine candidates. This review describes current knowledge about mammarenavirus virology, progress of antiviral drug development, and technical strategies of drug screening.

Mammarenaviruses cause chronic infections in rodents, which are their predominant natural hosts. Human infection with some of these viruses causes high-consequence disease, posing significant issues in public health. Currently, no FDA-licensed mammarenavirus vaccines are available, and anti-mammarenavirus drugs are limited to an off-label use of ribavirin, which is only partially efficacious and associated with severe side effects. Dihydroorotate dehydrogenase (DHODH) inhibitors, which block de novo pyrimidine biosynthesis, have antiviral activity against viruses from different families, including Arenaviridae , the taxonomic home of mammarenaviruses. Here, we evaluate five novel DHODH inhibitors for their antiviral activity against mammarenaviruses. All tested DHODH inhibitors were potently active against lymphocytic choriomeningitis virus (LCMV) (half-maximal effective concentrations [EC 50 ] in the low nanomolar range, selectivity index [SI] > 1000). The tested DHODH inhibitors did not affect virion cell entry or budding, but rather interfered with viral RNA synthesis. This interference resulted in a potent interferon-independent inhibition of mammarenavirus multiplication in vitro, including the highly virulent Lassa and Junín viruses.

Viral hemorrhagic fevers (VHFs) cause thousands of fatalities every year, but the treatment options for their management remain very limited. In particular, the development of therapeutic interventions is restricted by the lack of commercial viability of drugs targeting individual VHF agents. This makes approaches like drug repurposing and/or the identification of broad range therapies (i.e. those directed at host responses or common proviral factors) highly attractive. However, the identification of candidates for such antiviral repurposing or of host factors/pathways important for the virus life cycle is reliant on high-throughput screening (HTS). Recently, such screening work has been increasingly facilitated by the availability of reverse genetics-based approaches, including tools such as full-length clone (FLC) systems to generate reporter-expressing viruses or various life cycle modelling (LCM) systems, many of which have been developed and/or greatly improved during the last years. In particular, since LCM systems are capable of modelling specific steps in the life cycle, they are a valuable tool for both targeted screening (i.e. for inhibitors of a specific pathway) and mechanism of action studies. This review seeks to summarize the currently available reverse genetics systems for negative-sense VHF causing viruses (i.e. arenaviruses, bunyaviruses and filoviruses), and to highlight the recent advancements made in applying these systems for HTS to identify either antivirals or new virus-host interactions that might hold promise for the development of future treatments for the infections caused by these deadly but neglected virus groups., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The potent antiviral properties of 3-deazaneplanocin, 3-deaza-isoneplanocins (1) and recently discovered l-like carbocyclic nucleosides (2, 3 and 4) prompted us to pursue rationally conceived l-like 3-deazaneplanocin analogues. The synthesis of those analogues including l-like 3-deazaneplanocin (5), l-like 3-bromo-3-deazaneplanocin (6), and l-like 5'-fluoro-5'-deoxy-3-deazaneplanocin (7), was accomplished from a common intermediate, (-)-cyclopentenone (8). Antiviral analysis found 5 and 6 to display favorable activity against the Ebola virus, as expected for 3-deazaadenine carbocyclic nucleosides. Compound 5 also showed activity against arenaviruses, including Pinchinde and Tacaribe., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Several mammarenaviruses, chiefly Lassa virus (LASV) in Western Africa and Junín virus (JUNV) in the Argentine Pampas, cause severe disease in humans and pose important public health problems in their endemic regions. Moreover, mounting evidence indicates that the worldwide-distributed mammarenavirus lymphocytic choriomeningitis virus (LCMV) is a neglected human pathogen of clinical significance. The lack of licensed mammarenavirus vaccines and partial efficacy of current anti-mammarenavirus therapy limited to an off-label use of the nucleoside analog ribavirin underscore an unmet need for novel therapeutics to combat human pathogenic mammarenavirus infections. This task can be facilitated by the implementation of "drug repurposing" strategies to reduce the time and resources required to advance identified antiviral drug candidates into the clinic. We screened a drug repurposing library of 11,968 compounds (Repurposing, Focused Rescue and Accelerated Medchem [ReFRAME]) and identified several potent inhibitors of LCMV multiplication that had also strong anti-viral activity against LASV and JUNV. Our findings indicate that enzymes of the rate-limiting steps of pyrimidine and purine biosynthesis, the pro-viral MCL1 apoptosis regulator, BCL2 family member protein and the mitochondrial electron transport complex III, play critical roles in the completion of the mammarenavirus life cycle, suggesting they represent potential druggable targets to counter human pathogenic mammarenavirus infections., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Lassa virus (LASV) belongs to the Mammarenavirus genus (family Arenaviridae ) and causes severe hemorrhagic fever in humans. At present, there are no Food and Drug Administration (FDA)-approved drugs or vaccines specific for LASV. Here, high-throughput screening of an FDA-approved drug library was performed against LASV entry by using pseudotype virus bearing LASV envelope glycoprotein (GPC). Two hit compounds, lacidipine and phenothrin, were identified as LASV entry inhibitors in the micromolar range. A mechanistic study revealed that both compounds inhibited LASV entry by blocking low-pH-induced membrane fusion. Accordingly, lacidipine showed virucidal effects on the pseudotype virus of LASV. Adaptive mutant analyses demonstrated that replacement of T40, located in the ectodomain of the stable-signal peptide (SSP), with lysine (K) conferred LASV resistance to lacidipine. Furthermore, lacidipine showed antiviral activity against LASV, the closely related Mopeia virus (MOPV), and the New World arenavirus Guanarito virus (GTOV). Drug-resistant variants indicated that V36M in the ectodomain of the SSP mutant and V436A in the transmembrane domain of the GP2 mutant conferred GTOV resistance to lacidipine, suggesting the interface between SSP and GP2 is the target of lacidipine. This study shows that lacidipine is a candidate for LASV therapy, reinforcing the notion that the SSP-GP2 interface provides an entry-targeted platform for arenavirus inhibitor design. IMPORTANCE Currently, there is no approved therapy to treat Lassa fever; therefore, repurposing of approved drugs will accelerate the development of a therapeutic stratagem. In this study, we screened an FDA-approved library of drugs and identified two compounds, lacidipine and phenothrin, which inhibited Lassa virus entry by blocking low-pH-induced membrane fusion. Additionally, both compounds extended their inhibition against the entry of Guanarito virus, and the viral targets were identified as the SSP-GP2 interface., (Copyright © 2018 Wang et al.)

Arenaviruses are a large group of emerging viruses including several causative agents of severe hemorrhagic fevers with high mortality in man. Considering the number of people affected and the currently limited therapeutic options, novel efficacious therapeutics against arenaviruses are urgently needed. Over the past decade, significant advances in knowledge about the basic virology of arenaviruses have been accompanied by the development of novel therapeutics targeting different steps of the arenaviral life cycle. High-throughput, small-molecule screens identified potent and broadly active inhibitors of arenavirus entry that were instrumental for the dissection of unique features of arenavirus fusion. Novel inhibitors of arenavirus replication have been successfully tested in animal models and hold promise for application in humans. Late in the arenavirus life cycle, the proteolytic processing of the arenavirus envelope glycoprotein precursor and cellular factors critically involved virion assembly and budding provide further promising 'druggable' targets for novel therapeutics to combat human arenavirus infection.

Reverse-genetics systems are powerful tools enabling researchers to study the replication cycle of RNA viruses, including filoviruses and other hemorrhagic fever viruses, as well as to discover new antivirals. They include full-length clone systems as well as a number of life cycle modeling systems. Full-length clone systems allow for the generation of infectious, recombinant viruses, and thus are an important tool for studying the virus replication cycle in its entirety. In contrast, life cycle modeling systems such as minigenome and transcription and replication competent virus-like particle systems can be used to simulate and dissect parts of the virus life cycle outside of containment facilities. Minigenome systems are used to model viral genome replication and transcription, whereas transcription and replication competent virus-like particle systems also model morphogenesis and budding as well as infection of target cells. As such, these modeling systems have tremendous potential to further the discovery and screening of new antivirals targeting hemorrhagic fever viruses. This review provides an overview of currently established reverse genetics systems for hemorrhagic fever-causing negative-sense RNA viruses, with a particular emphasis on filoviruses, and the potential application of these systems for antiviral research., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Arenaviruses merit significant attention both as tractable model systems to study acute and persistent viral infections, and as clinically important human pathogens. Evidence indicates that LCMV remains present in the USA and Europe and capable of causing significant morbidity in infected individuals, likely being a neglected human pathogen. Moreover, new arenaviruses are being discovered in the Americas on the average of one every three years, with some of them causing severe hemorrhagic fever. In addition, weaponized forms of these viruses pose a real threat as agents of bioterrorism. Therefore, it is important to develop effective vaccines and better antiviral drugs to combat the dual threats of naturally occurring and intentionally introduced Arenavirus infections. The development of arenavirus reverse genetic systems is allowing investigators to conduct a detailed molecular characterization of the viral cis-acting signals and trans-acting factors that control each of the steps of the Arenavirus life cycle, including RNA synthesis, packaging and budding. We will discuss how this new knowledge is facilitating the establishment of novel assays to identify and characterize compounds capable of interfering with specific steps of the virus life cycle. Likewise, the ability to generate predetermined specific mutations within the arenavirus genome, and analyze their phenotypic expression, would significantly contribute to the elucidation of arenavirus-host interactions, including the bases of their ability to persist, as well as to cause severe HF (hemorrhagic fever) disease in humans. These approaches could also lead to the development of novel potent and safe Arenavirus vaccines.

The syntheses and the antiviral activities of 2-halo derivatives of neplanocin A (1b,c), (6'R)-6'-C-methylneplanocin A (2b), and dehydroxymethylneplanocin A (3b,c) are described. SN2 reaction of the known cyclopentenyl units 12 and 13 with 2-haloadenines under basic conditions gave the protected carbocyclic nucleosides 14b,c and 15b,c, respectively. Starting from the cyclopentenone derivative 5, the optically active tosyloxycyclopentene derivative 11 was prepared, which was similarly condensed with 2-fluoroadenine to give the protected (6'R)-6'-C-methyl derivative 16b. Deprotection of these compounds afforded the target 2-halo derivatives of neplanocin A. Of these new compounds, 2-fluoroneplanocin A (1b) showed an antiviral potency and a spectrum that was comparable to that of neplanocin A (1a). It was particularly active against vaccinia virus, vesicular stomatitis virus, parainfluenza virus, reovirus, arenaviruses (Junin, Tacaribe), and human cytomegalovirus, i.e., those viruses that fall within the purview of the S-adenosyl-L-homocysteine hydrolase inhibitors.

A novel nucleoside analog, 3'-fluoro-3'-deoxyadenosine (3'F3'dAdo), was evaluated for antiviral activity against several arthropod-borne and arenaviruses in Vero cell culture. The following 50% inhibitory concentrations (EC50) of virus plaque formation were obtained against the test viruses: Semliki Forest (10.3 microM) and Venezuelan equine encephalitis (5.3 microM) alphaviruses, lymphocytic choriomeningitis (7.7 microM) and Pichinde (greater than 32 microM) arenaviruses, Punta Toro (greater than 32 microM) and San Angelo (1.6 microM) bunyaviruses, banzi flavivirus (4.0 microM), and Colorado tick fever orbivirus (0.6 microM). By comparison, the broad-spectrum antiviral agent ribavirin was active against lymphocytic choriomeningitis (18 microM), Pichinde (24 microM), Punta Toro (114 microM), and San Angelo (99 microM) viruses, but was less active against the other 4 viruses (greater than 200 microM). Vero cell proliferation and thymidine and uridine incorporation into replicating Vero cells were inhibited by 50% with 3'F3'dAdo concentrations of 36, 45, and 32 microM, respectively. In virus yield reduction assays, increasing the multiplicity of infections of Semliki Forest and Venezuelan equine encephalitis viruses reduced the inhibitory activity of 3'F3'dAdo. Using the same assay, 3'F3'dAdo was found to enhance Punta Toro virus replication up to 5-fold relative to the untreated control. By adding the nucleoside transport inhibitor nitrobenzylthioinosine (100 microM) to the culture medium, antiviral activity against the two alphaviruses was eliminated, indicating that 3'F3'dAdo uses the nucleoside transport system for cell entry. When actinomycin D (5 microM) was used to greatly suppress cellular RNA synthesis in Semliki Forest virus-infected and uninfected cells, 3'F3'dAdo preferentially inhibited viral RNA synthesis. The results of these studies indicate 3'F3'dAdo is a selective inhibitor of most of the viruses tested and should be a promising candidate for in vivo evaluations.

In the presence of longwave ultraviolet light, psoralen derivatives photoreact with the nucleic acids within intact viruses and cells. This photoreaction can leave protein antigens and other surface components relatively unmodified, while eliminating the infectivity of a wide range of infectious agents. The kinetics of inactivation differ among RNA and DNA viruses photoreacted with different derivatives of psoralen. The inactivation kinetics are nonlinear as a result of photodegradation of psoralens and the unexplained biphasic inactivation of some viruses. In spite of these complexities, the photoreaction is capable of generating broad safety margins in the disinfection of microbial products under gentle, physiologic conditions. The psoralen photoreaction provides a potential method for inactivating both known and unknown viruses in active blood products. Psoralen-inactivated viruses have already proven useful as noninfectious antigens for use in immunoassays and as successful experimental vaccines.

The capacity of BHK-21 cell culture to produce Mopeia virus (Arenaviridae family) to form syncytium upon acidification of the culture medium to pH 5.5 and lower was demonstrated. The cell fusion requires their active virus production: a virus titre in the culture medium must be at least 10(5) PEU/ml. The inhibition of virus multiplication with ammonium chloride as well as treatment of the cells before the medium acidification with immune serum reduced syncytium formation markedly. No cell fusion was observed upon acidification of the medium immediately after virus adsorption to cells. Thus, the observed cell fusion under the influence of the virus is an "internal fusion" and confirms our previous data on the endocytosis mode of arenavirus penetration into the cell.

Several compounds, belonging to different classes of nucleoside analogues and sulfated polysaccharides, were evaluated for their inhibitory effects on the replication of the arenaviruses Junin and Tacaribe in VERO cells. S-Adenosylhomocysteine (AdoHcy) hydrolase inhibitors [i.e. adenosine dialdehyde, carbocyclic 3-deazaadenosine (C-c3 Ado), neplanocin A, 3-deazaneplanocin A, 9-(2,3-dihydroxypropyl)adenine [(S)-DHPA], (RS)-3-adenin-9-yl-hydroxypropanoic acid isobutyl ester [(RS)-AHPA], the 2',3'-dihydroxycyclopentenyl derivatives of adenine (DHCA) and 3-deazaadenine (DHCDA)] inhibited arenavirus replication within the concentration range of 1-10 micrograms/ml, while not being toxic for cell morphology or cellular DNA synthesis at a concentration of 100-400 micrograms/ml. Based on the ratio of the concentrations required to inhibit cell proliferation and virus replication, only (S)-DHPA, DHCA, C-c3 Ado and adenosine dialdehyde could be considered as truly selective inhibitors. Tubercidin, cyclopentenyl cytosine, pyrazofurin and ribavirin also inhibited viral cytopathogenicity at concentrations that were well below the cytotoxic threshold. Carbodine (cyclopentyl cytosine) also proved to be a potent inhibitor of arenavirus replication, but it was not as selective as cyclopentenyl cytosine. Very potent and selective inhibitors were the sulfated polysaccharides dextran sulfate, lambda-carrageenan, fucoidan, heparin and pentosan polysulfate: they inhibited virus replication at a concentration of 0.1-2.8 micrograms/ml, whereas the compounds were not inhibitory to cell growth even at a concentration of 200 micrograms/ml.

Lysosomotropic agents (NH4Cl, amantadine, chloroquine, monensin) which prevent acidification of intracellular vacuoles, when introduced into the culture medium before or during inoculation of cells (BHK-21, Vero) with arenaviruses inhibit reproduction of these viruses completely or significantly. Mozambique virus proved to be 10 times more sensitive to the effect of lysosomotropic agents than Pichinde and Lassa viruses. Thus, arenaviruses have a pH-dependent stage at the beginning of the reproduction cycle which is indirectly indicative of their penetration into cells by receptor endocytosis.

Administration of cyclophosphamide (CY) to guinea pigs infected with a lethal strain of Junin virus (JV) delayed the time of death, with survival of a small number of animals. Virological studies showed a temporary decrease of virus concentration in blood and viscera shortly after the CY injection. In the pathological study no differences were found in the organic lesions present in CY-treated and nontreated animals, with the exception of the pulmonary alterations. In CY-treated guinea pigs the lungs appeared almost normal, but in the control, nontreated animals severe alterations with the pattern of the "respiratory distress syndrome of the adult" were consistently present. In in vitro experiments, incorporation of serum collected from guinea pigs injected 30 minutes before exsanguination with CY to cell cultures, infected with JV, prevented virus replication. On the basis of these results it is suggested that the delay of time of death and eventual survival of CY-treated guinea pigs after JV infection depends on a direct antiviral effect of the drug rather than on its known immunosuppressive action. In addition, the absence of pulmonary alterations in CY-treated animals was tentatively considered to be dependent on the marked polymorphonuclear leukocyte depletion induced by the drug.

Binary combinations of the N-nucleoside ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) and the C-nucleoside analog selenazofurin (2-beta-D-ribofuranosylselenazole-4-carboxamide) or tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) were tested in vitro for activity against Venezuelan equine encephalomyelitis, Japanese encephalitis, yellow fever, Rift Valley fever, Korean hemorrhagic fever, and Pichinde viruses. The 50% effective dose for each compound alone or in a series of combinations was determined with a plaque reduction assay. Combinations of ribavirin and selenazofurin were synergistic against Venezuelan equine encephalomyelitis, Japanese encephalitis, yellow fever, and Pichinde viruses, with fractional inhibitory concentrations of 0.1, 0.2, 0.4, 0.4, respectively, but showed additive effects against Korean hemorrhagic fever and Rift Valley fever viruses. Combinations of ribavirin and tiazofurin were synergistic against yellow fever and Japanese encephalitis (fractional inhibitory concentrations, 0.41 and 0.48, respectively) but showed additive effects against Korean hemorrhagic fever virus. Combinations of selenazofurin and tiazofurin had additive effects against Japanese encephalitis, yellow fever, and Korean hemorrhagic fever viruses. The effect of combinations on cell toxicity was additive, both in monolayers of nondividing cells incubated under agar for the same period as the plaque assay and for rapidly dividing cells given short-term exposure (4 h), followed by determination of the proportion of surviving cells with a colony forming assay.

Lysosomotropic compounds (ammonium chloride, chloroquine, amantadine, monensin) effectively inhibited the replication of Pichinde, Mopeia, and Lassa viruses in BHK-21 and Vero cells. The inhibitory effect was dependent upon the time of drug addition and was most effective when the drugs were added 1 h before the viral adsorption. The drugs had no direct effect on the infectious viruses nor on adsorption of the arenaviruses. These results suggest that the arenaviruses enter cells by adsorptive endocytosis with the participation of acidic intracellular vesicules.

The antibiotic pyrazofurin, 3-(beta-D-ribofuranosyl)-4-hydroxypyrazole-5-carboxamide, markedly inhibited the in vitro replication of a number of RNA viruses including Rift Valley fever (RVF), Venezuelan equine encephalomyelitis (VEE), Sandfly, Pichinde, Lassa and LCM virus. Plaque formation was reduced by 80% or more with 2-10 micrograms/ml of pyrazofurin while 2 micrograms/ml reduced by 1000-fold the yield of Lassa and LCM virus in a yield reduction assay. In vivo, pyrazofurin failed to protect mice and guinea pigs against a lethal challenge with VEE and Pichinde virus, respectively. On the other hand, pyrazofurin caused a slight increase in the mean time to death of mice infected with RVF virus.

The sensitivity of lymphocytic choriomeningitis virus (LCMV) and Tacaribe virus to several chemical reagents was investigated. The viruses were sensitive to lipid solvents (ether, chloroform) and to detergents (sodium desoxycholate and Triton X-100); they were rapidly inactivated by formalin, beta-propiolactone, hydrogen peroxide and chloramine B. The results obtained contribute to a more complete characterization of the biological features of the arenavirus group and may be useful for scientific research and manufacturing of viral preparations.

In an attempt to define the involvement of host transcription in arenavirus growth, a study was made of the effect of actinomycin D (AMD) on the yields of infectious Pichinde, Tacaribe and Junin viruses. The drug was added either immediately after virus adsorption or later after infection, at the stationary phase of virus growth. The time of exposure of the infected cells to the inhibitor was so chosen that the generation and release of virus into the medium took place in the presence of AMD. A double label technique was used to estimate the generation time of an arenavirus particle. This was found to be 6 h or less for all of the arenaviruses examined. The results indicated that treatment of the host cells with AMD, either immediately after virus adsorption or later after infection, does not affect the yield of infectious Pichinde, Tacaribe or Junin viruses, thus implying that continuous host transcription is not required for the replication cycle of these arenaviruses.

The multiplication of arenaviruses Tacaribe, Pichinde and Junin was inhibited by 16 mM of glucosamine and by 10 mM of 2-deoxi-D-glucose.

The antibiotic tunicamycin, an inhibitor of glycoprotein synthesis, suppressed Junin virus multiplication in Vero cells. No infectious virus was formed if tunicamycin was added, at a concentration of 1 microgram/ml, immediately after virus adsorption and remained up to the end of the replicative cycle. Tunicamycin added at 24,48 or 72 hours p.i. immediately stopped virus synthesis. The effect was irreversible. Virus grown in the presence or absence of tunicamycin and labelled with 3H-uridine was purified and layered on a (20-60%) sucrose gradient. The radioactive profile of both gradients was coincident. The total number of cpm associated to the peak gradient of virus grown with tunicamycin represented 70% of the cpm from control virus. Since viral infectivity was 99.4% inhibited in the presence of TM it can be assumed that most of the radioactive particles formed are not infectious. Therefore, in the presence of tunicamycin Junin virus buds normally. From these results we can conclude that carbohydrates are important for viral infectivity but play no role in the transport of viral proteins through cellular membranes.

Ribavirin, a broad-spectrum antiviral drug, is active against hemorrhagic fever viruses (with the exception of Ebola virus) in cell culture systems. In model infections with arenaviruses in guinea pigs and monkeys, ribavirin has demonstrated both prophylactic and therapeutic efficacy. In therapeutic studies it has not prevented late-onset neurologic disease. In human cases of Lassa fever, it significantly reduces mortality when administered before day 7 of illness to persons at high risk. In rodents and monkeys infected with Rift Valley fever virus, ribavirin therapy resulted in reduced mortality; prophylactic administration to volunteers infected with sandfly fever virus, Sicilian strain, prevented development of illness. Ribavirin increased the number of survivors and the mean time to death in suckling mice infected with Crimean-Congo hemorrhagic fever virus and in suckling mice infected with Hantaan virus. In the People's Republic of China, ribavirin significantly reduced mortality in patients with hemorrhagic fever with renal syndrome. Ribavirin has not been effective in animal models of filoviral and flaviviral infections. The only important adverse effect of ribavirin in humans is manageable, reversible anemia.

Machupo virus labeled with radioactive precursors of RNA or protein synthesis was purified by isodensity ultracentrifugation in sucrose concentration gradient. The purified virus was disrupted with NP-40 nonionic detergent in the presence of 1 M KCl, and subvirion fractions were separated by ultracentrifugation in urografin density gradient. This treatment resulted in formation of two subvirion components with a buoyant density 1.25-1.26 and 1.10-1.12 g/cm3 in urografin concentration gradient. The subvirion fraction with the density of 1.25-1.26 g/cm3 contained high-molecular virion RNAs, a major protein of molecular weight of 64 kD, and seemed to be the virion nucleocapsid. The solubilised fraction (1.10-1.12 g/cm3) contained glycosylated protein 37 kD which seems to be a surface glycoprotein.

An immunofluorescent assay (IFA) for Pichinde virus (PCV), a member of the family Arenaviridae, was developed for antiviral drug assays against the virus. The assay was performed by adding fluorescein-labeled anti-PCV monoclonal antibody to virus-infected cells at 24 h after the initial infection and counting the infected cells with an epifluorescence microscope. The average 50% effective dose (ED50) for a series of nucleoside analogues tested against PCV using this IFA was: 2-beta-D-ribofuranosylselenazole-4-carboxamide (selenazofurin), less than 1.0 microgram/ml; 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin), 6.0 micrograms/ml; ammonium 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide- 5'-phosphate hydrate (ribavirin-5'-monophosphate), 15.8 micrograms/ml; ammonium 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide-5'-hemisuccinate (ribavirin-5'-hemisuccinate), 14.7 micrograms/ml; ammonium 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide-5'-(2,3- dimethyl)hemisuccinate [ribavirin-5'-(2,3-dimethyl)hemisuccinate], 213.5 micrograms/ml; 4-hydroxy-1-beta-D-ribofuranosyl-2-pyridone (3-deazauridine), 5.2 micrograms/ml; and (S)-9-(2,3-dihydroxypropyl)adenine, ([S]-DHPA), 471.0 micrograms/ml. In comparison, the ED50 of ribavirin using inhibition of marginal PCV-induced cytopathogenic effect after 12 days was 6.0 micrograms/ml and using plaque reduction after 5 days was 2.5 micrograms/ml, indicating that this IFA was of comparable sensitivity to these other tests.

When Vero cells infected with Junín virus are cultured in the presence of 16 mM glucosamine, no infectious virus is produced and no surface antigens or virions are detected. Cytoplasmic viral antigen, however, is synthesized normally.

The effect of inhibitors of mRNA synthesis (actinomycin D and alpha-amanitin) and DNA replication (mitomycin C and ethidium bromide) on Machupo virus reproduction was studied. Actinomycin D (1.0-4.0 micrograms/ml) and alpha-amanitin (10.0 micrograms/ml) added immediately after adsorption reduced the infectious virus titre by 2 lg or more. Actinomycin D inhibited virus reproduction not only at the early but also at the later stages of infection. Mitomycin C and ethidium bromide did not inhibit Machupo virus reproduction. The cells treated with actinomycin D at early and late stages of infection were found to contain immunoprecipitable virus-specific proteins 78, 64, and 37 KD described previously. The total amount of virus-specific proteins in the inhibitor-treated cells was reduced 3.7 and 2,6-fold after addition of actinomycin at 0 and 12 h postinfection, respectively, as compared with untreated cells. The mechanism of action of actinomycin D on the Machupo virus reproduction is discussed.