Comparison of Whole-Genome Sequence-Based Methods and PCR Ribotyping for Subtyping of Clostridioides difficile.

Clostridioides difficile is the most common cause of antibiotic-associated gastrointestinal infections. Capillary electrophoresis (CE)-PCR ribotyping is currently the gold standard for C. difficile typing but lacks the discriminatory power to study transmission and outbreaks in detail. New molecular methods have the capacity to differentiate better and provide standardized and interlaboratory exchangeable data. Using a well-characterized collection of diverse strains (N = 630; 100 unique ribotypes [RTs]), we compared the discriminatory power of core genome multilocus sequence typing (cgMLST) (SeqSphere and EnteroBase), whole-genome MLST (wgMLST) (EnteroBase), and single-nucleotide polymorphism (SNP) analysis. A unique cgMLST profile (more than six allele differences) was observed in 82 of 100 RTs, indicating that cgMLST could distinguish most, but not all, RTs. Application of cgMLST in two outbreak settings with RT078 and RT181 (known to have low intra-RT allele differences) showed no distinction between outbreak and nonoutbreak strains in contrast to wgMLST and SNP analysis. We conclude that cgMLST has the potential to be an alternative to CE-PCR ribotyping. The method is reproducible, easy to standardize, and offers higher discrimination. However, adjusted cutoff thresholds and epidemiological data are necessary to recognize outbreaks of some specific RTs. We propose to use an allelic threshold of three alleles to identify outbreaks.

A primary drying model-based comparison of conventional batch freeze-drying to continuous spin-freeze-drying for unit doses.

An innovative continuous spin-freeze-drying technology for unit doses was recently developed. For this technology, a mechanistic primary drying model was developed allowing the calculation of the optimal dynamic drying trajectory for spin-frozen formulations. In this work, a model-based and experimentally verified comparison was made between conventional batch freeze-drying and spin-freeze-drying by analyzing the outputs (i.e., primary drying endpoint, optimal shelf temperature/power heater and product temperature profile) of both primary drying models. Input parameters such as dried product layer resistance (Rp) and heat input parameters (Kv,Ptot) were experimentally determined for both freeze-drying methods and compared. In addition, optimal dynamic process parameters were calculated for 3 model formulations by using both mechanistic models. Finally, model predictions were validated by measuring the product temperature and primary drying endpoint. It was observed that, when considering the same layer thickness, Rp was generally lower for continuous spin-frozen formulations compared to vials frozen in a conventional batch freeze-dryer. This observation contributes to the short primary drying times of spin-frozen formulations. In addition, as spin-freezing drastically increases the surface area of the product and lowers the dried layer thickness, drying times can be reduced even further while an excellent cake structure and appearance can still be obtained. The primary drying model for spin-frozen formulations proved to be equally accurate for the prediction of the primary drying endpoint and product temperature compared to the batch freeze-drying model.

The recent emergence of a highly related virulent Clostridium difficile clade with unique characteristics.

OBJECTIVES: Clostridium difficile is a major global human pathogen divided into five clades, of which clade 3 is the least characterized and consists predominantly of PCR ribotype (RT) 023 strains. Our aim was to analyse and characterize this clade. METHODS: In this cohort study the clinical presentation of C. difficile RT023 infections was analysed in comparison with known 'hypervirulent' and non-hypervirulent strains, using data from the Netherlands national C. difficile surveillance programme. European RT023 strains of diverse origin were collected and whole-genome sequenced to determine the genetic similarity between isolates. Distinctive features were investigated and characterized. RESULTS: Clinical presentation of C. difficile RT023 infections show severe infections akin to those seen with 'hypervirulent' strains from clades 2 (RT027) and 5 (RT078) (35%, 29% and 27% severe CDI, respectively), particularly with significantly more bloody diarrhoea than RT078 and non-hypervirulent strains (RT023 8%, other RTs 4%, p 0.036). The full genome sequence of strain CD305 is presented as a robust reference. Phylogenetic comparison of CD305 and a further 79 previously uncharacterized European RT023 strains of diverse origin revealed minor genetic divergence with >99.8% pairwise identity between strains. Analyses revealed distinctive features among clade 3 strains, including conserved pathogenicity locus, binary toxin and phage insertion toxin genotypes, glycosylation of S-layer proteins, presence of the RT078 four-gene trehalose cluster and an esculinase-negative genotype. CONCLUSIONS: Given their recent emergence, virulence and genomic characteristics, the surveillance of clade 3 strains should be more highly prioritized.

Identification and validation of two peptide markers for the recognition of Clostridioides difficile MLST-1 and MLST-11 by MALDI-MS.

OBJECTIVES: Clostridioides difficile infection (CDI) has become the main cause of nosocomial infective diarrhoea. To survey and control the spread of different C. difficile strains, there is a need for suitable rapid tests. The aim of this study was to identify peptide/protein markers for the rapid recognition of C. difficile strains by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). METHODS: We analysed 44 well-characterized strains, belonging to eight different multi-locus sequence types (MLST), using ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS. The amino acid sequence of two peptide markers specific for MLST-1 and MLST-11 strains was elucidated by MALDI-TOF-MS/MS. The investigation of 2689 C. difficile genomes allowed the determination of the sensitivity and specificity of these markers. C18-solid-phased extraction was used to enrich the MLST-1 marker. RESULTS: Two peptide markers (m/z 4927.81 and m/z 5001.84) were identified and characterized for MLST-1 and MLST-11 strains, respectively. The MLST-1 marker was found in 786 genomes of which three did not belong to MLST-1. The MLST-11 marker was found in 319 genomes, of which 14 did not belong to MLST-11. Importantly, all MLST-1 and MLST-11 genomes were positive for their respective marker. Furthermore, a peptide marker (m/z 5015.86) specific for MLST-15 was found in 59 genomes. We translated our findings into a fast and simple method that allowed the unambiguous identification of the MLST-1 marker on a MALDI-TOF-MS platform. CONCLUSIONS: MALDI-FTICR MS-based peptide profiling resulted in the identification of peptide markers for C. difficile MLST-1 and MLST-11.

Zoonotic Transfer of Clostridium difficile Harboring Antimicrobial Resistance between Farm Animals and Humans.

The emergence of Clostridium difficile as a significant human diarrheal pathogen is associated with the production of highly transmissible spores and the acquisition of antimicrobial resistance genes (ARGs) and virulence factors. Unlike the hospital-associated C. difficile RT027 lineage, the community-associated C. difficile RT078 lineage is isolated from both humans and farm animals; however, the geographical population structure and transmission networks remain unknown. Here, we applied whole-genome phylogenetic analysis of 248 C. difficile RT078 strains from 22 countries. Our results demonstrate limited geographical clustering for C. difficile RT078 and extensive coclustering of human and animal strains, thereby revealing a highly linked intercontinental transmission network between humans and animals. Comparative whole-genome analysis reveals indistinguishable accessory genomes between human and animal strains and a variety of antimicrobial resistance genes in the pangenome of C. difficile RT078. Thus, bidirectional spread of C. difficile RT078 between farm animals and humans may represent an unappreciated route disseminating antimicrobial resistance genes between humans and animals. These results highlight the importance of the "One Health" concept to monitor infectious disease emergence and the dissemination of antimicrobial resistance genes.

Evaluation of spin freezing versus conventional freezing as part of a continuous pharmaceutical freeze-drying concept for unit doses.

Spin-freezing as alternative freezing approach was evaluated as part of an innovative continuous pharmaceutical freeze-drying concept for unit doses. The aim of this paper was to compare the sublimation rate of spin-frozen vials versus traditionally frozen vials in a batch freeze-dryer, and its impact on total drying time. Five different formulations, each having a different dry cake resistance, were tested. After freezing, the traditionally frozen vials were placed on the shelves while the spin-frozen vials were placed in aluminum vial holders providing radial energy supply during drying. Different primary drying conditions and chamber pressures were evaluated. After 2h of primary drying, the amount of sublimed ice was determined in each vial. Each formulation was monitored in-line using NIR spectroscopy during drying to determine the sublimation endpoint and the influence of drying conditions upon total drying time. For all tested formulations and applied freeze-drying conditions, there was a significant higher sublimation rate in the spin-frozen vials. This can be explained by the larger product surface and the lower importance of product resistance because of the much thinner product layers in the spin frozen vials. The in-line NIR measurements allowed evaluating the influence of applied drying conditions on the drying trajectories.

Whole genome sequencing reveals potential spread of Clostridium difficile between humans and farm animals in the Netherlands, 2002 to 2011.

Farm animals are a potential reservoir for human Clostridium difficile infection (CDI), particularly PCR ribotype 078 which is frequently found in animals and humans. Here, whole genome single-nucleotide polymorphism (SNP) analysis was used to study the evolutionary relatedness of C. difficile 078 isolated from humans and animals on Dutch pig farms. All sequenced genomes were surveyed for potential antimicrobial resistance determinants and linked to an antimicrobial resistance phenotype. We sequenced the whole genome of 65 C. difficile 078 isolates collected between 2002 and 2011 from pigs (n = 19), asymptomatic farmers (n = 15) and hospitalised patients (n = 31) in the Netherlands. The collection included 12 pairs of human and pig isolates from 2011 collected at 12 different pig farms. A mutation rate of 1.1 SNPs per genome per year was determined for C. difficile 078. Importantly, we demonstrate that farmers and pigs were colonised with identical (no SNP differences) and nearly identical (less than two SNP differences) C. difficile clones. Identical tetracycline and streptomycin resistance determinants were present in human and animal C. difficile 078 isolates. Our observation that farmers and pigs share identical C. difficile strains suggests transmission between these populations, although we cannot exclude the possibility of transmission from a common environmental source.

Current application and future perspectives of molecular typing methods to study Clostridium difficile infections.

Molecular typing is an essential tool to monitor Clostridium difficile infections and outbreaks within healthcare facilities. Molecular typing also plays a key role in defining the regional and global changes in circulating C. difficile types. The patterns of C. difficile types circulating within Europe (and globally) remain poorly understood, although international efforts are under way to understand the spatial and temporal patterns of C. difficile types. A complete picture is essential to properly investigate type-specific risk factors for C. difficile infections (CDI) and track long-range transmission. Currently, conventional agarose gel-based polymerase chain reaction (PCR) ribotyping is the most common typing method used in Europe to type C. difficile. Although this method has proved to be useful to study epidemiology on local, national and European level, efforts are made to replace it with capillary electrophoresis PCR ribotyping to increase pattern recognition, reproducibility and interpretation. However, this method lacks sufficient discriminatory power to study outbreaks and therefore multilocus variable-number tandem repeat analysis (MLVA) has been developed to study transmission between humans, animals and food. Sequence-based methods are increasingly being used for C. difficile fingerprinting/typing because of their ability to discriminate between highly related strains, the ease of data interpretation and transferability of data. The first studies using whole-genome single nucleotide polymorphism typing of healthcare-associated C. difficile within a clinically relevant timeframe are very promising and, although limited to select facilities because of complex data interpretation and high costs, these approaches will likely become commonly used over the coming years.

Comparison of real-time PCR techniques to cytotoxigenic culture methods for diagnosing Clostridium difficile infection.

In the past decade, the incidence of Clostridium difficile infections (CDI) with a more severe course has increased in Europe and North America. Assays that are capable of rapidly diagnosing CDI are essential. Two real-time PCRs (LUMC and LvI) targeting C. difficile toxin genes (tcdB, and tcdA and tcdB, respectively) were compared with the BD GeneOhm PCR (targeting the tcdB gene), using cytotoxigenic culture as a gold standard. In addition, a real-time PCR targeting the tcdC frameshift mutation at position 117 (Δ117 PCR) was evaluated for detecting toxigenic C. difficile and the presence of PCR ribotype 027 in stool samples. In total, 526 diarrheal samples were prospectively collected and included in the study. Compared with those for cytotoxigenic culture, sensitivity, specificity, positive predicted value (PPV), and negative predicted value (NPV) were for PCR LUMC 96.0%, 88.0%, 66.0%, and 98.9%, for PCR LvI 100.0%, 89.4%, 69.7%, and 100.0%, for PCR Δ117 98.0%, 90.7%, 71.9%, and 99.5%, and for PCR BD GeneOhm 88.3%, 96.9%, 86.5%, and 97.4%. Compared to those with feces samples cultured positive for C. difficile type 027, the sensitivity, specificity, PPV, and NPV of the Δ117 PCR were 95.2%, 96.2%, 87.0%, and 98.7%. We conclude that all real-time PCRs can be applied as a first screening test in an algorithm for diagnosing CDI. However, the low PPVs hinder the use of the assays as stand-alone tests. Furthermore, the Δ117 PCR may provide valuable information for minimizing the spread of the epidemic C. difficile PCR ribotype 027.

Relatedness of human and animal Clostridium difficile PCR ribotype 078 isolates determined on the basis of multilocus variable-number tandem-repeat analysis and tetracycline resistance.

Totals of 102 and 56 Clostridium difficile type 078 strains of human and porcine origins, respectively, from four European countries were investigated by an optimized multilocus variable-number tandem-repeat analysis (MLVA) and for tetracycline susceptibility. Eighty-five percent of all isolates were genetically related, irrespective of human or porcine origin. Human strains were significantly more resistant to tetracycline than porcine strains. All tetracycline-resistant strains contained the Tn916-like transposon harboring the tet(M) gene. We conclude that strains from human and porcine origins are genetically related, irrespective of the country of origin. This may reflect a lack of diversity and/or common source.

The changing epidemiology of Clostridium difficile infections.

The epidemiology of Clostridium difficile infection (CDI) has changed dramatically during this millennium. Infection rates have increased markedly in most countries with detailed surveillance data. There have been clear changes in the clinical presentation, response to treatment, and outcome of CDI. These changes have been driven to a major degree by the emergence and epidemic spread of a novel strain, known as PCR ribotype 027 (sometimes referred to as BI/NAP1/027). We review the evidence for the changing epidemiology, clinical virulence and outcome of treatment of CDI, and the similarities and differences between data from various countries and continents. Community-acquired CDI has also emerged, although the evidence for this as a distinct new entity is less clear. There are new data on the etiology of and potential risk factors for CDI; controversial issues include specific antimicrobial agents, gastric acid suppressants, potential animal and food sources of C. difficile, and the effect of the use of alcohol-based hand hygiene agents.

Membrane fusion activity of tick-borne encephalitis virus and recombinant subviral particles in a liposomal model system.

We present a kinetic analysis of the membrane fusion activity of tick-borne encephalitis (TBE) virus and TBE-derived recombinant subviral particles (RSPs) in a liposomal model system. Fusion was monitored using a fluorescence assay involving pyrene-labeled phospholipids. Fusion was strictly dependent on low pH, with the optimum being at pH 5.3-5.5 and the threshold at pH 6.8. Fusion did not require a protein or carbohydrate receptor in the target liposomes. Preexposure to low pH of the virus alone resulted in inactivation of its fusion activity. At the optimum pH for fusion and 37 degrees C, the rate and extent of fusion were very high, with more than 50% of the virus fusing within 2 s and the final extent of fusion being 70%. Lowering of the temperature did not result in a significant decrease in the rate and extent of fusion, suggesting that TBE virus fusion is a facile process with a low activation energy, possibly due to the flat orientation of the E glycoprotein on the viral surface facilitating the establishment of direct intermembrane contact. The fusion characteristics of TBE virus and RSPs were similar, indicating that RSPs provide a reliable and convenient model for further study of the membrane fusion properties of TBE virus.

Mutagenesis of the RGD motif in the yellow fever virus 17D envelope protein.

The envelope protein of yellow fever virus 17D (YFV-17D) contains a solvent-exposed RGD motif, which has led to the suggestion that integrins may function as cellular receptors for YFV-17D. We found that mutating the RGD motif to RGE had no effect on viral titers, whereas changing RGD to TGD, TGE, TAD, TAE, or RGS led to reduced titers. Substitution of RGD by RAD or RAE yielded RNA genomes that replicated in mammalian cells but could not spread to neighboring cells at 37 degrees C. These mutants did spread through the cell monolayer at 30 degrees C (both in mosquito cells and in SW13 cells) and viruses grown at this temperature were capable of infecting mammalian cells at 37 degrees C. These results strongly suggest that RGD-mediated integrin binding does not play a major role in YFV-17D entry, since the RGD to RAD mutation, as well as many or all of the other mutations studied, should disrupt all RGD-dependent integrin binding. However, the RGD to RAD or RAE mutations (as well as TAD and TAE) severely destabilized the envelope protein at 37 degrees C, providing an explanation for the observed phenotype. Implications of these findings are discussed in light of the fact that mutations that alter tropism or virulence in different flaviviruses are often found within the loop containing the RGD motif.

Membrane fusion activity of Semliki Forest virus in a liposomal model system: specific inhibition by Zn2+ ions.

Semliki Forest virus (SFV) has been shown previously to fuse efficiently with cholesterol- and sphingolipid-containing liposomal model membranes in a low-pH-dependent manner. Several steps can be distinguished in this process, including low-pH-induced irreversible binding of the virus to the liposomes, facilitated by target membrane cholesterol, and subsequent fusion of the viral membrane with the liposomal bilayer, specifically catalyzed by target membrane sphingolipid. Binding and fusion are mediated by the heterodimeric viral envelope glycoprotein E2/E1. At low pH the heterodimer dissociates, and the E1 monomers convert to a homotrimeric structure, the presumed fusion-active conformation of the viral spike. In this paper, we demonstrate that SFV-liposome fusion is specifically inhibited by Zn2+ ions. The inhibition is at the level of the fusion reaction itself, since virus-liposome binding was found to be unaffected. Zn2+ did not inhibit E2/E1 dissociation, but severely inhibited exposure of an acid-specific epitope on E1, E1 homotrimer formation, and acquisition of trypsin-resistance. It is concluded that virus--liposome binding solely requires low-pH-induced E2/E1 heterodimer dissociation, while fusion depends on further rearrangements in the E1 spike protein. As these rearrangements occur subsequent to the binding step, their precise course, including the formation of a fusion complex, may be influenced by interaction of E1 with target membrane lipids.

Inhibition of influenza virus fusion by polyanionic proteins.

Anionic charge-modified human serum albumin (HSA) has previously been shown to exert potent in vitro activity against human immunodeficiency virus type 1 (HIV-1). In these studies, introduction of the additional negative charges was performed by derivatizing the epsilon-amino groups of lysine residues with succinic (Suc-HSA) or cis-aconitic anhydride (Aco-HSA), by which primary amino groups are replaced with carboxylic acids. The anti-HIV-1 activity was related to inhibition of gp41-mediated membrane fusion. Here, we investigated the activity of aconitylated and succinylated proteins on influenza virus membrane fusion, which is mediated by the viral membrane glycoprotein hemagglutinin (HA). Aco-HSA and Suc-HSA markedly inhibited the rates and extents of fusion of fluorescently labeled virosomes bearing influenza HA, with target membranes derived from erythrocytes. The inhibitory activity was dependent on the overall negative-charge density; HSA modified with 36 or less extra negative charges failed to inhibit fusion. The inhibition of fusion showed a certain degree of specificity for the protein carrying the negative charges: polyanionic HSA and beta-lactoglobulin A derivatives had fusion-inhibitory activity, whereas succinylated BSA, lactalbumin, lactoferrin, lysozyme, and transferrin were inactive. Aco60-HSA and Aco-beta-lactoglobulin A inhibited influenza virus membrane fusion in a concentration-dependent manner, IC50 values being about 4 and 10 microg/mL, respectively. HA-mediated membrane fusion is pH dependent. Aco60-HSA did not induce a shift in the pH threshold or in the pH optimum. Fusion with liposomes of another low pH-dependent virus, Semliki Forest virus, was not specifically affected by any of the compounds reported here. In view of some structural and functional similarities between influenza HA and the HIV-1 gp120/gp41 complex, it is tempting to postulate that the current results might have some implications for the anti-HIV-1 mechanism of polyanionic proteins.

Sphingolipids activate membrane fusion of Semliki Forest virus in a stereospecific manner.

The alphavirus Semliki Forest virus (SFV) enters cells through receptor-mediated endocytosis. Subsequently, triggered by the acid pH in endosomes, the viral envelope fuses with the endosomal membrane. Membrane fusion of SFV has been shown previously to be dependent on the presence of cholesterol in the target membrane. Recently, we have demonstrated that fusion of SFV also requires sphingolipids [Nieva, J. L., Bron, R., Corver, J., & Wilschut, J. (1994) EMBO J. 13, 2797-2804]. In the present paper, we show that the activation of low-pH-dependent fusion of SFV by sphingolipids is a stereospecific process. Pyrene-labeled SFV fused rapidly and extensively with liposomes consisting of a mixture of phosphatidylcholine, phosphatidylethanolamine, and cholesterol, supplemented with low concentrations of D-erythro-ceramide, representing the naturally occurring sphingolipid stereoisomer. Fusion was assessed by a decrease in the pyrene excimer fluorescence. L-erythro-, D-threo-, and L-threo-ceramide did not support fusion of the virus. Similar results were obtained with the corresponding sphingomyelin stereoisomers. The stereospecificity of SFV fusion activation was confirmed by using an assay based on degradation of the viral capsid protein by trypsin encapsulated in the target liposomes. Fusion mediated by D-erythro-ceramide was not affected by the additional presence in the target liposomes of ceramide stereoisomers incapable of fusion activation. Binding of the virus to the liposomes, as assessed by flotation on sucrose density gradients, was not dependent on the presence of fusion-competent or fusion-incompetent sphingolipids in the liposomes. The results of this study support the notion that a stereospecific interaction of the viral fusion protein with D-erythro sphingolipids in the target membrane represents an essential step in the activation of the fusion capacity of SFV.

Sphingolipid-dependent fusion of Semliki Forest virus with cholesterol-containing liposomes requires both the 3-hydroxyl group and the double bond of the sphingolipid backbone.

Low-pH-induced membrane fusion of Semliki Forest virus (SFV) in a model system is mediated by sphingolipids in the target membrane; ceramide is the sphingolipid minimally required (J. L. Nieva, R. Bron, J. Corver, and J. Wilschut, EMBO J. 13:2797-2804, 1994). Here, using various ceramide analogs, we demonstrate that sphingolipid-dependent fusion of SFV with cholesterol-containing liposomes exhibits remarkable molecular specificity, the 3-hydroxyl group and the 4,5-trans carbon-carbon double bond of the sphingosine backbone being critical for the sphingolipid to mediate the process. This observation supports the notion that sphingolipids act as a cofactor in SFV fusion, interacting directly with the viral fusion protein to induce its ultimate fusion-active conformation.

Fusion of Semliki Forest virus with cholesterol-containing liposomes at low pH: a specific requirement for sphingolipids.

Semliki Forest virus (SFV) utilizes a membrane fusion strategy to introduce its genome into the host cell. After binding to cell-surface receptors, virus particles are internalized through receptor-mediated endocytosis and directed to the endosomal cell compartment. Subsequently, triggered by the acid pH in the lumen of the endosomes, the viral envelope fuses with the endosomal membrane. As a result of this fusion reaction the viral RNA gains access to the cell cytosol. Low-pH-induced fusion of SFV, in model systems as well as in cells, has been demonstrated previously to be strictly dependent on the presence of cholesterol in the target membrane. In this paper, we show that fusion of SFV with cholesterol-containing liposomes depends on sphingomyelin (SM) or other sphingolipids in the target membrane, ceramide representing the sphingolipid minimally required for mediating the process. The action of the sphingolipid is confined to the actual fusion event, cholesterol being necessary and sufficient for low-pH-dependent binding of the virus to target membranes. The 3-hydroxyl group on the sphingosine backbone plays a key role in the SFV fusion reaction, since 3-deoxy-sphingomyelin does not support the process. This, and the remarkably low levels of sphingolipid required for half-maximal fusion (1-2 mol%), suggest that the sphingolipid does not play a structural role in SFV fusion, but rather acts as a cofactor, possibly through activation of the viral fusion protein. Domain formation between cholesterol and sphingolipid, although it may facilitate SFV fusion, is unlikely to play a crucial role in the process.

Membrane fusion of Semliki Forest virus requires sphingolipids in the target membrane.

Enveloped animal viruses, such as Semliki Forest virus (SFV), utilize a membrane fusion strategy to deposit their genome into the cytosol of the host cell. SFV enters cells through receptor-mediated endocytosis, fusion of the viral envelope occurring subsequently from within acidic endosomes. Fusion of SFV has been demonstrated before to be strictly dependent on the presence of cholesterol in the target membrane. Here, utilizing a variety of membrane fusion assays, including an on-line fluorescence assay involving pyrene-labeled virus, we demonstrate that low-pH-induced fusion of SFV with cholesterol-containing liposomal model membranes requires the presence of sphingomyelin or other sphingolipids in the target membrane. The minimal molecular characteristics essential for supporting SFV fusion are encompassed by a ceramide. The action of the sphingolipids is confined to the actual fusion event, cholesterol being necessary and sufficient for low-pH-dependent binding of the virus to target membranes. Complex formation of the sphingolipids with cholesterol is unlikely to be important for the induction of SFV--liposome fusion, as sphingolipids that do not interact appreciably with cholesterol, such as galactosylceramide, effectively support the process. The remarkably low levels of sphingomyelin required for half-maximal fusion (1-2 mole%) suggest that sphingolipids do not play a structural role in the SFV fusion process, but rather act as a cofactor, possibly activating the viral fusion protein in a specific manner.