Natural Immunomodulatory Agents as a Complementary Therapy for Poxviruses.

Poxviruses target innate immunity mediators such as tumor necrosis factors, interleukins, interferons, complement, and chemokines. It also targets adaptive immunity such as CD4+ T cells, CD4+ T cells, and B cells. Emerging of the recent epidemic of monkeypox virus (MPXV), a zoonotic disease native to Central and Western Africa, besides the lack of permitted treatments for poxviruses infections, encouraged researchers to identify effective inhibitors to help in preventing and treating poxviruses infections. Natural bioactive components, particularly polyphenolics, are promising for creating powerful antioxidants, anti-inflammatory, immune-stimulating, and antiviral agents. As a result, they are potentially effective therapies for preventing and treating viral diseases, such as infections caused by poxviruses including the recent pandemic MPXV. Polyphenolics: rosmarinic acid, caffeic acid, resveratrol, quercitrin, myricitrin, gingerol, gallotannin, and propolis-benzofuran A, as well as isoquinoline alkaloids: galanthamine and thalimonine represent prospective antiviral agents against MPXV, they can inhibit MPXV and other poxviruses via targeting different viral elements including DNA Topoisomerase I (TOP1), Thymidine Kinase (TK), serine/threonine protein kinase (Ser/Thr kinase), and protein A48R. The bioactive extracts of different traditional plants including Guiera senegalensis, Larrea tridentata, Sarracenia purpurea, Kalanchoe pinnata (Lam.) Pers., Zingiber officinale Roscoe, Quercus infectoria, Rhus chinensis, Prunella vulgaris L., Salvia rosmarinus, and Origanum vulgare also can inhibit the growth of different poxviruses including MPXV, vaccinia virus (VACV), variola virus, buffalopox virus, fowlpox virus, and cowpox virus. There is an urgent need for additional molecular studies to identify and confirm the anti-poxviruses properties of various natural bioactive components, especially those that showed potent antiviral activity against other viruses.

The Overview of Potential Antiviral Bioactive Compounds in Poxviruses.

Poxviruses belong to the family of double-stranded DNA viruses, and it is pathogenic for humans and spread worldwide. These viruses cause infections and various diseases in human. So, it is required to develop new drugs for the treatment of smallpox or other poxvirus infections. Very few potential compounds for the treatment of poxvirus such as smallpox, chickenpox, and monkeypox have been reported. Most of the compounds has used as vaccines. Cidofovir is most commonly used as a vaccine for the treatment of poxviruses. There are no phytochemicals reported for the treatment of poxviruses. Very few phytochemicals are under investigation for the treatment of poxviruses.

Adamantane derivatives as potential inhibitors of p37 major envelope protein and poxvirus reproduction. Design, synthesis and antiviral activity.

Currently, smallpox, caused by the variola virus belonging to the poxvirus family, has been completely eradicated according to the WHO. However, other representatives of poxviruses, such as vaccinia virus, cowpox virus, ectromelia virus, monkeypox virus, mousepox virus and others, remain in the natural environment and can infect both animals and humans. The pathogens of animal diseases, belonging to the category with a high epidemic risk, have already caused several outbreaks among humans, and can, in an unfavorable combination of circumstances, cause not only an epidemic, but also a pandemic. Despite the fact that there are protocols for the treatment of poxvirus infections, the targeted design of new drugs will increase their availability and expand the arsenal of antiviral chemotherapeutic agents. One of the potential targets of poxviruses is the p37 protein, which is a tecovirimat target. This protein is relatively small, has no homologs among proteins of humans and other mammals and is necessary for the replication of viral particles, which makes it attractive target for virtual screening. Using the I-TASSER modelling and molecular dynamics refinement the p37 orthopox virus protein model was obtained and its was confirmed by ramachandran plot analysis and superimposition of the model with the template protein with similar function. A virtual library of adamantane containing compounds was generated and a number of potential inhibitors were chosen from virtual library using molecular docking. Several compounds bearing adamantane moiety were synthesized and their biological activity was tested in vitro on vaccinia, cowpox and mousepox viruses. The new compounds inhibiting vaccinia virus replication with IC50 concentrations between 0.133 and 0.515 µM were found as a result of the research. The applied approach can be useful in the search of new inhibitors of orthopox reproduction. The proposed approach may be suitable for the design of new poxvirus inhibitors containing cage structural moiety.

Differential Efficacy of Novel Antiviral Substances in 3D and Monolayer Cell Culture.

Repurposing of approved drugs that target host functions also important for virus replication promises to overcome the shortage of antiviral therapeutics. Mostly, virus biology including initial screening of antivirals is studied in conventional monolayer cells. The biology of these cells differs considerably from infected tissues. 3D culture models with characteristics of human tissues may reflect more realistically the in vivo events during infection. We screened first, second, and third generation epidermal growth factor receptor (EGFR)-inhibitors with different modes of action and the EGFR-blocking monoclonal antibody cetuximab in a 3D cell culture infection model with primary human keratinocytes and cowpox virus (CPXV) for antiviral activity. Antiviral activity of erlotinib and osimertinib was nearly unaffected by the cultivation method similar to the virus-directed antivirals tecovirimat and cidofovir. In contrast, the host-directed inhibitors afatinib and cetuximab were approx. 100-fold more efficient against CPXV in the 3D infection model, similar to previous results with gefitinib. In summary, inhibition of EGFR-signaling downregulates virus replication comparable to established virus-directed antivirals. However, in contrast to virus-directed inhibitors, in vitro efficacy of host-directed antivirals might be seriously affected by cell cultivation. Results obtained for afatinib and cetuximab suggest that screening of such drugs in standard monolayer culture might underestimate their potential as antivirals.

Antivirals in medical biodefense.

The viruses historically implicated or currently considered as candidates for misuse in bioterrorist events are poxviruses, filoviruses, bunyaviruses, orthomyxoviruses, paramyxoviruses and a number of arboviruses causing encephalitis, including alpha- and flaviviruses. All these viruses are of concern for public health services when they occur in natural outbreaks or emerge in unvaccinated populations. Recent events and intelligence reports point to a growing risk of dangerous biological agents being used for nefarious purposes. Public health responses effective in natural outbreaks of infectious disease may not be sufficient to deal with the severe consequences of a deliberate release of such agents. One important aspect of countermeasures against viral biothreat agents are the antiviral treatment options available for use in post-exposure prophylaxis. These issues were adressed by the organizers of the 16th Medical Biodefense Conference, held in Munich in 2018, in a special session on the development of drugs to treat infections with viruses currently perceived as a threat to societies or associated with a potential for misuse as biothreat agents. This review will outline the state-of-the-art methods in antivirals research discussed and provide an overview of antiviral compounds in the pipeline that are already approved for use or still under development.

Bioluminescence Imaging as a Tool for Poxvirus Biology.

Bioluminescence imaging, with luciferase as a reporter-encoding gene, has been successfully and widely used for studies to follow viral infection in an organism and to measure therapeutic efficacy of antiviral agents in small animal models. Bioluminescence is produced by the reaction of a luciferase enzyme stably inserted into the viral genome with a defined substrate systemically delivered into the animal. The light emitted is captured allowing the detection of viral infection sites and the quantification of viral replication in the context of tissues of a living animal. The goal of this chapter is to provide a technical background for the evaluation of poxvirus infection in cells and animals through bioluminescence imaging technology using luciferase-expressing recombinant poxviruses.

Structural basis for the inhibition of poxvirus assembly by the antibiotic rifampicin.

Poxviruses are large DNA viruses that cause disease in animals and humans. They differ from classical enveloped viruses, because their membrane is acquired from cytoplasmic membrane precursors assembled onto a viral protein scaffold formed by the D13 protein rather than budding through cellular compartments. It was found three decades ago that the antibiotic rifampicin blocks this process and prevents scaffold formation. To elucidate the mechanism of action of rifampicin, we have determined the crystal structures of six D13-rifamycin complexes. These structures reveal that rifamycin compounds bind to a phenylalanine-rich region, or F-ring, at the membrane-proximal opening of the central channel of the D13 trimer. We show by NMR, surface plasmon resonance (SPR), and site-directed mutagenesis that A17, a membrane-associated viral protein, mediates the recruitment of the D13 scaffold by also binding to the F-ring. This interaction is the target of rifampicin, which prevents A17 binding, explaining the inhibition of viral morphogenesis. The F-ring of D13 is both conserved in sequence in mammalian poxviruses and essential for interaction with A17, defining a target for the development of assembly inhibitors. The model of the A17-D13 interaction describes a two-component system for remodeling nascent membranes that may be conserved in other large and giant DNA viruses.

Flavobacteria as secondary pathogens in carp suffering from koi sleepy disease.

Koi sleepy disease (KSD) is a disease with increasing importance in global common carp aquaculture. Despite the fact that carp edema virus (CEV) is most likely the causative agent of KSD, the disease often presents itself as multifactorial with several parasites and bacteria species present on gills, skin or in internal organs. Therefore, in this study, we analysed and presented initial results on an interaction of flavobacteria and CEV in the development of clinical KSD in carp suffering from proliferative gill disease. We examined selected field samples from Germany and Hungary and confirmed the presence of CEV and flavobacteria co-infections in subset of the samples. In several infection experiments, we studied the transfer and dynamics of both infections. Furthermore, we analysed which Flavobacterium species could be isolated from KSD-affected fish and concluded that Flavobacterium branchiophilum is a possible copathogen. Antibiotic treatment experiments showed that CEV seems to be the primary pathogen causing an insult to the gills of carp and by these enabling other pathogens, including F. branchiophilum, to establish co-infections. Despite the fact that F. branchiophilum co-infection is not required for the development of clinical KSD, it could contribute to the pathological changes recorded during the outbreaks.

OX40:OX40L axis: emerging targets for improving poxvirus-based CD8(+) T-cell vaccines against respiratory viruses.

The human respiratory tract is an entry point for over 200 known viruses that collectively contribute to millions of annual deaths worldwide. Consequently, the World Health Organization has designated respiratory viral infections as a priority for vaccine development. Despite enormous advances in understanding the attributes of a protective mucosal antiviral immune response, current vaccines continue to fail in effectively generating long-lived protective CD8(+) T-cell immunity. To date, the majority of licensed human vaccines afford protection against infectious pathogens through the generation of specific immunoglobulin responses. In recent years, the selective manipulation of specific costimulatory pathways, which are critical in regulating T cell-mediated immune responses, has generated increasing interest. Impressive results in animal models have shown that the tumor necrosis factor receptor (TNFR) family member OX40 (CD134) and its binding partner OX40L (CD252) are key costimulatory molecules involved in the generation of protective CD8(+) T-cell responses at mucosal surfaces, such as the lung. In this review, we highlight these new findings with a particular emphasis on their potential as immunological adjuvants to enhance poxvirus-based CD8(+) T-cell vaccines.

Solution structure of a DNA duplex containing the potent anti-poxvirus agent cidofovir.

Cidofovir (1(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine, CDV) is a potent inhibitor of orthopoxvirus DNA replication. Prior studies have shown that, when CDV is incorporated into a growing primer strand, it can inhibit both the 3'-to-5' exonuclease and the 5'-to-3' chain extension activities of vaccinia virus DNA polymerase. This drug can also be incorporated into DNA, creating a significant impediment to trans-lesion DNA synthesis in a manner resembling DNA damage. CDV and deoxycytidine share a common nucleobase, but CDV lacks the deoxyribose sugar. The acyclic phosphonate bears a hydroxyl moiety that is equivalent to the 3'-hydroxyl of dCMP and permits CDV incorporation into duplex DNA. To study the structural consequences of inserting CDV into DNA, we have used (1)H NMR to solve the solution structures of a dodecamer DNA duplex containing a CDV molecule at position 7 and of a control DNA duplex. The overall structures of both DNA duplexes were found to be very similar. We observed a decrease of intensity (>50%) for the imino protons neighboring the CDV (G6, T8) and the cognate base G18 and a large chemical shift change for G18. This indicates higher proton exchange rates for this region, which were confirmed using NMR-monitored melting experiments. DNA duplex melting experiments monitored by circular dichroism revealed a lower T(m) for the CDV DNA duplex (46 °C) compared to the control (58 °C) in 0.2 M salt. Our results suggest that the CDV drug is well accommodated and stable within the dodecamer DNA duplex, but the stability of the complex is less than that of the control, suggesting increased dynamics around the CDV.

Synthesis of ester prodrugs of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) as anti-poxvirus agents.

9-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) and its cyclic form were selected for further evaluation as potential drug candidates against poxvirus infections. To increase bioavailability of these compounds, synthesis of their structurally diverse ester prodrugs was carried out: alkoxyalkyl (hexadecyloxypropyl, octadecyloxyethyl, hexadecyloxyethyl), pivaloyloxymethyl (POM), 2,2,2-trifluoroethyl, butylsalicylyl, and prodrugs based on peptidomimetics. Most HPMPDAP prodrugs were synthesized in the form of monoesters as well as the corresponding cyclic phosphonate esters. The activity was evaluated not only against vaccinia virus but also against different herpes viruses. The most potent and active prodrugs against vaccinia virus were the alkoxyalkyl ester derivatives of HPMPDAP, with 50% effective concentrations 400-600-fold lower than those of the parent compound. Prodrugs based on peptidomimetics, the 2,2,2-trifluoroethyl, the POM, and the butylsalicylyl derivatives, were able to inhibit vaccinia virus replication at 50% effective concentrations that were equivalent or ∼10-fold lower than those observed for the parent compounds.

Protective effect of partially purified 35 kDa protein from silk worm (Bombyx mori) fecal matter against carbon tetrachloride induced hepatotoxicity and in vitro anti-viral properties.

CONTEXT: It has been found that many proteins from silkworm (Bombyx mori L.) fecal matter have been active against human immunodeficiency virus, Sendai virus, herpes simplex virus type-1, and nuclear polyhedrosis virus. OBJECTIVE: A partially purified 35 kDa protein from silkworm was screened for its hepatoprotective activity, and in vitro antioxidant, and antiviral properties against camelpox and goatpox viruses. MATERIALS AND METHODS: The study investigated the efficiency of the partially purified 35 kDa protein from silk worm fecal matter against CCl4-induced liver damage measured in terms of enzyme levels such as aspartate aminotransferase (AST), alanine amino transferase(ALT), alkaline phosphatase (ALP) and total bilirubin, which maintain liver integrity. In vitro antioxidant potential of this protein was determined based on its ability to scavenge 2, 2-diphenylpicrylhydrazyl (DPPH) and superoxide anions scavenging activity. Further, in vitro cytotoxic effect on Vero cells and antiviral activity against goatpox and camelpox viruses were also studied. RESULTS: The protein had significant hepatoprotection against CCl4-induced liver damage and scavenging of DPPH radical and superoxide anion activity. However, the protein did not inhibit the multiplication of either virus tested at its maximum non-toxic concentration (MNTC) in vitro. DISCUSSION AND CONCLUSION: The partially purified 35 kDa protein from silk worm Bombyx mori L fecal matter possessed protective effect against CCl4-induced oxidative stress in rat model. The protein was found to be ineffective against camelpox and goatpox viruses at its MNTC in vitro.

Efficacy of ST-246 versus lethal poxvirus challenge in immunodeficient mice.

The threat of smallpox as a bioweapon and the emerging threat of human monkeypox, among other poxviral diseases, highlight the need for effective poxvirus countermeasures. ST-246, which targets the F13L protein in vaccinia virus and its homologs in other orthopoxvirus species, provides full protection from lethal poxviral disease in numerous animal models and seems to be safe in humans. All previous evaluations of ST-246 efficacy have been in immunocompetent animals. However, the risk of severe poxviral disease is greater in immunodeficient hosts. Here we report on the efficacy of ST-246 in preventing or treating lethal poxviral disease in immunodeficient mice. After lethal challenge with the Western Reserve strain of vaccinia, Nude, SCID, and J(H) knockout mice additionally depleted of CD4(+) and CD8(+) T cells were not fully protected by ST-246, although survival was significantly extended. However, CD4(+) T cell deficient, CD8(+) T cell deficient, J(H) knockout, and J(H) knockout mice also deficient for CD4(+) or CD8(+) T cells survived lethal challenge when treated with ST-246 starting on the day of challenge. Delaying treatment until 72 h after infection reduced ST-246 efficacy in some models but provided full protection from lethal challenge in most. These findings suggest that ST-246 may be effective in controlling smallpox or other pathogenic orthopoxviruses in some immunodeficient human populations for whom the vaccine is contraindicated.

A new synthesis and an antiviral assessment of the 4'-fluoro derivative of 4'-deoxy-5'-noraristeromycin.

A synthetic route to (1S,2S,3R,5S)-3-(6-amino-9H-purin-9-yl)-5-fluorocyclopentane-1,2-diol (that is, the 4'-fluoro derivative of 4'-deoxy-5'-noraristeromycin, 3) is described via a fluorinated cyclopentanol, which is in contrast to existing schemes where fluorination occurred once the purine ring was present. Compound 3 was assayed versus a number of viruses. A favorable response was observed towards measles (IC(50) of 1.2 microg/mL in the neutral red assay and 14 microg/mL by the visual assay) but this was accompanied by cytotoxicity in the CV-1 host cells (21-36 microg/mL). Among the viruses unaffected by 3 were human cytomegalovirus and the poxviruses (vaccinia and cowpox), which are three viruses that were inhibited by the 4',4'-difluoro analog of 3 (that is, 2).

Poxvirus host range protein CP77 contains an F-box-like domain that is necessary to suppress NF-kappaB activation by tumor necrosis factor alpha but is independent of its host range function.

Tumor necrosis factor alpha (TNF-alpha) activates the nuclear factor kappaB (NF-kappaB) signaling pathway that regulates expression of many cellular factors playing important roles in innate immune responses and inflammation in infected hosts. Poxviruses employ many strategies to inhibit NF-kappaB activation in cells. In this report, we describe a poxvirus host range protein, CP77, which blocked NF-kappaB activation by TNF-alpha. Immunofluorescence analyses revealed that nuclear translocation of NF-kappaB subunit p65 protein in TNF-alpha-treated HeLa cells was blocked by CP77. CP77 did so without blocking IkappaBalpha phosphorylation, suggesting that upstream kinase activation was not affected by CP77. Using GST pull-down, we showed that CP77 bound to the NF-kappaB subunit p65 through the N-terminal six-ankyrin-repeat region in vitro. CP77 also bound to Cullin-1 and Skp1 of the SCF complex through a C-terminal 13-amino-acid F-box-like sequence. Both regions of CP77 are required to block NF-kappaB activation. We thus propose a model in which poxvirus CP77 suppresses NF-kappaB activation by two interactions: the C-terminal F-box of CP77 binding to the SCF complex and the N-terminal six ankyrins binding to the NF-kappaB subunit p65. In this way, CP77 attenuates innate immune response signaling in cells. Finally, we expressed CP77 or a CP77 F-box deletion protein from a vaccinia virus host range mutant (VV-hr-GFP) and showed that either protein was able to rescue the host range defect, illustrating that the F-box region, which is important for NF-kappaB modulation and binding to SCF complex, is not required for CP77's host range function. Consistently, knocking down the protein level of NF-kappaB did not relieve the growth restriction of VV-hr-GFP in HeLa cells.

Mechanism of antiviral drug resistance of vaccinia virus: identification of residues in the viral DNA polymerase conferring differential resistance to antipoxvirus drugs.

The acyclic nucleoside phosphonate (ANP) family of drugs shows promise as therapeutics for treating poxvirus infections. However, it has been questioned whether the utility of these compounds could be compromised through the intentional genetic modification of viral sequences by bioterrorists or the selection of drug resistance viruses during the course of antiviral therapy. To address these concerns, vaccinia virus (strain Lederle) was passaged 40 times in medium containing an escalating dose of (S)-1-[3-hydroxy-2-(phosphonomethoxypropyl)-2,6-diaminopurine [(S)-HPMPDAP], which selected for mutant viruses exhibiting a approximately 15-fold-increased resistance to the drug. (S)-HPMPDAP-resistant viruses were generated because this compound was shown to be one of the most highly selective and effective ANPs for the treatment of poxvirus infections. DNA sequence analysis revealed that these viruses encoded mutations in the E9L (DNA polymerase) gene, and marker rescue studies showed that the phenotype was produced by a combination of two (A684V and S851Y) substitution mutations. The effects of these mutations on drug resistance were tested against various ANPs, both separately and collectively, and compared with E9L A314T and A684V mutations previously isolated using selection for resistance to cidofovir, i.e., (S)-1-[3-hydroxy-2-(phosphonomethoxypropyl)cytosine]. These studies demonstrated a complex pattern of resistance, although as a general rule, the double-mutant viruses exhibited greater resistance to the deoxyadenosine than to deoxycytidine nucleotide analogs. The S851Y mutant virus exhibited a low level of resistance to dCMP analogues but high-level resistance to dAMP analogues and to 6-[3-hydroxy-2-(phosphonomethoxy)propoxy]-2,4-diaminopyrimidine, which is considered to mimic the purine ring system. Notably, (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-3-deazaadenine retained marked activity against most of these mutant viruses. In vitro studies showed that the A684V mutation partially suppressed a virus growth defect and mutator phenotype created by the S851Y mutation, but all of the mutant viruses still exhibited a variable degree of reduced virulence in a mouse intranasal challenge model. Infections caused by these drug-resistant viruses in mice were still treatable with higher concentrations of the ANPs. These studies have identified a novel mechanism for the development of mutator DNA polymerases and provide further evidence that antipoxviral therapeutic strategies would not readily be undermined by selection for resistance to ANP drugs.

Discovery of small molecule inhibitors of ubiquitin-like poxvirus proteinase I7L using homology modeling and covalent docking approaches.

Essential for viral replication and highly conserved among poxviridae, the vaccinia virus I7L ubiquitin-like proteinase (ULP) is an attractive target for development of smallpox antiviral drugs. At the same time, the I7L proteinase exemplifies several interesting challenges from the rational drug design perspective. In the absence of a published I7L X-ray structure, we have built a detailed 3D model of the I7L ligand binding site (S2-S2' pocket) based on exceptionally high structural conservation of this site in proteases of the ULP family. The accuracy and limitations of this model were assessed through comparative analysis of available X-ray structures of ULPs, as well as energy based conformational modeling. The 3D model of the I7L ligand binding site was used to perform covalent docking and VLS of a comprehensive library of about 230,000 available ketone and aldehyde compounds. Out of 456 predicted ligands, 97 inhibitors of I7L proteinase activity were confirmed in biochemical assays ( approximately 20% overall hit rate). These experimental results both validate our I7L ligand binding model and provide initial leads for rational optimization of poxvirus I7L proteinase inhibitors. Thus, fragments predicted to bind in the prime portion of the active site can be combined with fragments on non-prime side to yield compounds with improved activity and specificity.

Virus distribution of the attenuated MVA and NYVAC poxvirus strains in mice.

Recombinant vaccinia viruses based on the attenuated NYVAC and MVA strains are promising vaccine candidates against a broad spectrum of diseases. Whilst these vectors are safe and immunogenic in animals and humans, little is known about their comparative behaviour in vivo. In this investigation, a head-to-head analysis was carried out of virus dissemination in mice inoculated by the mucosal or systemic route with replication-competent (WRluc) and attenuated recombinant (MVAluc and NYVACluc) viruses expressing the luciferase gene. Bioluminescence imaging showed that, in contrast to WRluc, the attenuated recombinants expressed the reporter gene transiently, with MVAluc expression limited to the first 24 h and NYVACluc giving a longer signal, up to 72 h post-infection, for most of the routes assayed. Moreover, luciferase levels in MVAluc-infected tissues peaked earlier than those in tissues infected by NYVACluc. These findings may be of immunological relevance when these vectors are used as recombinant vaccines.

Purification of 3 monomeric monocot mannose-binding lectins and their evaluation for antipoxviral activity: potential applications in multiple viral diseases caused by enveloped viruses.

Three monomeric monocot lectins from Zephyranthes carinata, Zephyranthes candida, and Gloriosa superba with carbohydrate specificity towards mannose derivatives and (or) oligomannose have been isolated and purified from their storage tissues. The lectins were purified by anion-exchange chromatography on DEAE-Sephacyl and by gel filtration chromatography on Biogel P-200 followed by high-performance liquid chromatography. The purified lectins, Z. carinata, Z. candida, and G. superba had molecular masses of 12, 11.5, and 12.5 kDa, respectively, as determined by gel filtration and SDS-PAGE, indicating that they are monomers. In a hapten inhibition assay, methyl-alpha-D-mannopyranoside inhibited agglutination of both Z. candida and Z. carinata; the latter was also inhibited by Man(alpha1-2)Man and Man(alpha1-3)Man. Gloriosa superba showed inhibition only with Man(alpha1-4)Man of all of the sugars and glycoproteins tested. All purified lectins agglutinated red blood cells from rabbit, whereas G. superba was also reactive towards erythrocytes from guinea pig. All of the lectins were nonglycosylated and did not require metal ions for their activity. They were labile above 60 degrees C and were affected by denaturing agents such as urea, thiourea, and guanidine-HCl. The lectins were virtually nonmitogenic, like other members of Amaryllidaceae and Liliaceae. Of the 3 lectins, G. superba was found to be highly toxic to the BSC-1 cell line (African green monkey kidney epithelial cells), while both of the Zephyranthes species showed significant in vitro inhibition of poxvirus replication in BSC-1 cells without any toxic effects to the cells. In addition, Z. candida also exhibited significant anticancer activity against SNB-78, a CNS human cancer cell line.