Proteolytic processing of foot-and-mouth disease virus polyproteins expressed in a cell-free system from clone-derived transcripts

All picornaviral genes are expressed as a single, large polyprotein, which is proteolytically processed into the system produces functional proteins, including viral protease 3C, which plays a major role in processing the precursor proteins. To study the function of the two putative proteases 3C and leader (L) in processing, we constructed several cDNA plasmids encoding various regions of the FMDV type A12 genome. These plasmids, containing FMDV cDNA segments under the control of the T7 promoter, were transcribed in vitro by using T7 RNA polymerase and then translated in rabbit reticulocyte lysates. The expressed FMDV gene products were identified by immunoprecipitation with specific antisera and analyzed by gel electrophoresis. The results demonstrate the following: (i) the leader protein, L, is processed from the structural protein precursor, P1, in the absence of any P2 or P3 region proteins; (ii) protein 2A remains associated with the structural protein precursor, P1, rather than the precursor, P2; (iii) the processing of the P1-2A/P2 junction is not catalyzed by 3C or L; (iv) the proteolytic processing of polyproteins from the structural P1 region (except VP4/VP2) and the nonstructural P2 and P3 region is catalyzed by 3C.     

Leader protein of foot-and-mouth disease virus is required for cleavage of the p220 component of the cap-binding protein complex.

Suppression of host protein synthesis in cells infected by poliovirus and certain other picornaviruses involves inactivation of the cap-binding protein complex. Inactivation of this complex has been correlated with the proteolytic cleavage of p220, a component of the cap-binding protein complex. Since picornaviral RNA is not capped, it continues to be translated as the cap-binding protein complex is inactivated. The cleavage of p220 can be induced to occur in vitro, catalyzed by extracts from infected cells or by reticulocyte lysates translating viral RNA. Expression of polioviral protease 2A is sufficient to induce p220 cleavage, and the presence in 2A of an 18-amino-acid sequence representing a putative cysteine protease active site correlates with the ability of different picornaviruses to induce p220 cleavage. Foot-and-mouth disease virus (FMDV) infection induces complete cleavage of p220, yet the FMDV genome codes for a 2A protein of only 16 amino acids, which does not include the putative cysteine protease active site. Using cDNA plasmids encoding various regions of the FMDV genome, we have determined that the leader protein is required to initiate p220 cleavage. This is the first report of a function for the leader protein, other than that of autocatalytic cleavage from the FMDV polyprotein.     

Pathogenesis of wild-type and leaderless foot-and-mouth disease virus in cattle.

Four calves were experimentally infected via aerosol with foot-and-mouth disease virus. Two were infected with a wild-type virus derived from a full-length infectious clone (A12-IC), and two were infected with a clone-derived virus lacking the leader gene (A12-LLV2), with euthanasia and tissue collection at 24 and 72 h postexposure (hpe). Clinical disease was apparent only in the animal given A12-IC and euthanized at 72 hpe. In situ hybridization revealed that the animal infected with A12-IC and euthanized at 24 hpe had abundant viral nucleic acid in the lung, present in clusters of positive cells in the respiratory bronchiolar epithelium and associated subepithelial regions. At 72 hpe in the A12-IC-infected calf, viral nucleic acid in the lung was present in interstitial areas, and in addition, viral nucleic acid was detectable in epithelial tissues around histologically apparent vesicles. In animals infected with A12-LLV2, viral nucleic acid was detectable in the lung at both 24 and 72 hpe, but staining revealed a more localized distribution with less nucleic acid than was found in animals given A12-IC. Therefore, it appears that after aerosol exposure to A12-IC, early replication is in the region of the lung, with subsequent dissemination to distal sites. In comparison, the A12-LLV2 virus is much less widely disseminated in the lung at 24 hpe, with no lesions or virus detectable in secondary sites at 72 hpe. The greatly reduced pathogenicity of A12-LLV2 may make it an excellent candidate for a modified live viral vaccine.     

Antiviral effects of a thiol protease inhibitor on foot-and-mouth disease virus.

The thiol protease inhibitor E-64 specifically blocks autocatalytic activity of the leader protease of foot-and-mouth disease virus (FMDV) and interferes with cleavage of the structural protein precursor in an in vitro translation assay programmed with virion RNA. Experiments with FMDV-infected cells and E-64 or a membrane-permeable analog, E-64d, have confirmed these results and demonstrated interference in virus assembly, causing a reduction in virus yield. In addition, there is a lag in the appearance of virus-induced cellular morphologic alterations, a delay in cleavage of host cell protein p220 and in shutoff of host protein synthesis, and a decrease in viral protein and RNA synthesis. The implications of using E-64-based compounds as potential antiviral agents for FMDV are discussed.     

Further characterization of a protein kinase from foot-and-mouth disease virus.

Acid disruption of foot-and-mouth disease virus released a protein kinase activity that sedimented at less than 7S. This enzyme was separated into three peaks of activity by ion-exchange and hydroxylapatite chromatography. Analysis of the various enzyme fractions by polyacrylamide gel electrophoresis and silver staining revealed that one of the fractions lacked the major virion structural proteins, but still contained two or three other polypeptides. This enzyme phosphorylated mainly one protein (P17) in an in vitro assay.     

The foot-and-mouth disease virus leader proteinase gene is not required for viral replication.

The foot-and-mouth disease virus (FMDV) leader (L) proteinase has only two known functions: (i) autocatalytic removal from the N terminus of the viral polyprotein and (ii) cleavage of the p220 subunit of the eukaryotic initiation factor 4F complex, which helps to shut off host protein synthesis. Cleavage of p220 appears to be important for picornavirus replication, since rhinoviruses and enteroviruses utilize a different proteinase (2A) to cleave p220. To explore the role of L in FMDV replication, we generated synthetic FMDV genomes lacking the L gene and tested their viability in cells. Genomes were constructed with the N-terminal Gly codon of VP4 positioned directly following either the first (Lab) or second (Lb) Met codon of the L protein. Cells transfected with synthetic RNAs lacking L and initiating with the Lab Met codon failed to produce viable virus, but cells transfected with RNAs that utilized the second AUG to drive translation of the viral polyprotein produced viable viruses. These leader-deleted viruses produced plaques on BHK cells that were slightly smaller than those produced by wild-type (WT) virus, grew to slightly lower titers than WT virus in BHK cells, shut off host protein synthesis more slowly than WT virus, and were slightly attenuated in mice. These studies indicate that the L proteinase is not essential for FMDV replication and show that in the cells and animals tested the L gene has a limited effect on virus replication.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

The role of snail aestivation in transmission of schistosomiasis in changing climatic conditions

Schistosomiasis vector snails are subjected to extreme seasonal changes, particularly in ephemeral rivers and lentic waterbodies. In the tropics, aestivation is one of the adaptive strategies for survival and is used by snails in times of extremely high temperatures and desiccation. Aestivation therefore plays an important role in maintaining the transmission of schistosomiasis. This review assesses the possible impacts of climate change on the temporal and spatial distribution of schistosomiasis-transmitting snails with special emphasis on aestivation, and discusses the effect of schistosome infection on aestivation ability. The impacts of parasite development on snails, as well as physiological changes, are discussed with reference to schistosomiasis transmission. This review shows that schistosome-infected snails have lower survival rates during aestivation, and that those that survive manage to get rid of the infection. In general, snail aestivation ability is poor and survival chances diminish with time. Longer dry periods result in fewer, as well as uninfected, snails. However, the ability of the surviving snails to repopulate the habitats is high.     

Antibiotic dosing in the 'at risk' critically ill patient: Linking pathophysiology with pharmacokinetics/pharmacodynamics in sepsis and trauma patients

Background

Critical illness, mediated by trauma or sepsis, can lead to physiological changes that alter the pharmacokinetics of antibiotics and may result in sub-therapeutic concentrations at the sites of infection. The first aim of this project is to identify the clinical characteristics of critically ill patients with significant trauma that have been recently admitted to ICU that may predict the dosing requirements for the antibiotic, cefazolin. The second aim of this is to identify the clinical characteristics of critically ill patients with sepsis that may predict the dosing requirements for the combination antibiotic, piperacillin-tazobactam.

Methods/Design

This is an observational pharmacokinetic study of patients with trauma (cefazolin) or with sepsis (piperacillin-tazobactam). Participants will have samples from blood and urine, collected at different intervals. Patients will also have a microdialysis catheter inserted into subcutaneous tissue to measure interstitial fluid penetration of the antibiotic. Participants will be administered sinistrin, indocyanine green and sodium bromide as well as have cardiac output monitoring performed and tetrapolar bioimpedance to determine physiological changes resulting from pathology. Analysis of samples will be performed using validated liquid chromatography tandem mass-spectrometry. Pharmacokinetic analysis will be performed using non-linear mixed effects modeling to determine individual and population pharmacokinetic parameters of antibiotics.

Discussion

The study will describe cefazolin and piperacillin-tazobactam concentrations in plasma and the interstitial fluid of tissues in trauma and sepsis patients respectively. The results of this study will guide clinicians to effectively dose these antibiotics in order to maximize the concentration of antibiotics in the interstitial fluid of tissues.