Background: Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV.

Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus genus and several species of this family are pathogenic to humans. The viruses are classified as potential agents of biological warfare and terrorism and sensitive detection as well as effective prophylaxis and antiviral therapies are required.In this work, we describe the isolation of the anti-VEEV single chain Fragment variable (scFv), ToR67-3B4, from a non-human primate (NHP) antibody gene library. We report its recloning into the bivalent scFv-Fc format and further immunological and biochemical characterisation.The scFv-Fc ToR67-3B4 recognised viable as well as formalin and ß-propionolactone (ß-Pl) inactivated virus particles and could be applied for immunoblot analysis of VEEV proteins and immuno-histochemistry of VEEV infected cells. It detected specifically the viral E1 envelope protein of VEEV but did not react with reduced viral glycoprotein preparations suggesting that recognition depends upon conformational epitopes. The recombinant antibody was able to detect multiple VEEV subtypes and displayed only marginal cross-reactivity to other Alphavirus species except for EEEV. In addition, the scFv-Fc fusion described here might be of therapeutic use since it successfully inactivated VEEV in a murine disease model. When the recombinant antibody was administered 6 hours post challenge, 80% to 100% of mice survived lethal VEEV IA/B or IE infection. Forty to sixty percent of mice survived when scFv-Fc ToR67-3B4 was applied 6 hours post challenge with VEEV subtypes II and former IIIA. In combination with E2-neutralising antibodies the NHP antibody isolated here could significantly improve passive protection as well as generic therapy of VEE.

The high risk associated with biological threat agents dictates that any suspicious sample be handled under strict surety and safety controls and processed under high-level containment in specialized laboratories. This study attempted to find a rapid, reliable, and simple method for the complete inactivation of a wide range of pathogens, including spores, vegetative bacteria, and viruses, while preserving microbial nucleic acid fragments suitable for PCRs and proteinaceous epitopes for detection by immunoassays. Formaldehyde, hydrogen peroxide, and guanidium thiocyanate did not completely inactivate high titers of bacterial spores or viruses after 30 min at 21°C. Glutaraldehyde and sodium hypochlorite showed high microbicidal activity but obliterated the PCR or enzyme-linked immunosorbent assay (ELISA) detection of bacterial spores or viruses. High-level inactivation (more than 6 log 10 ) of bacterial spores (Bacillus atrophaeus), vegetative bacteria (Pseudomonas aeruginosa), an RNA virus (the alphavirus Pixuna virus), or a DNA virus (the orthopoxvirus vaccinia virus) was attained within 30 min at 21°C by treatment with either peracetic acid or cupric ascorbate with minimal hindrance of subsequent PCR tests and immunoassays. The data described here should provide the basis for quickly rendering field samples noninfectious for further analysis under lower-level containment and considerably lower cost.The high risk associated with biological threats dictates that any suspicious samples be handled under strict surety and safety controls and processed under high-level containment in specialized laboratories. These laboratories are complex, very expensive to operate, and need to be staffed by personnel from an extremely limited pool of experts. In addition, safe means of transporting samples suspected of containing highly virulent agents to specialized high-level containment laboratories for analysis also is expensive, requiring in many countries the custody of armed personnel. It can be estimated that several million dollars are spent annually worldwide to secure and safely transport an ever-increasing stream of suspicious biological samples, which are collected in theaters of war as well as in domestic environments.The objective of this study was to identify liquid chemical reagents with high efficiency to inactivate microbial organisms and viruses that, at the same time, would preserve the high sensitivity of subsequent diagnostics performed at a lower level of containment and cost.Considerable knowledge has been accumulated regarding the efficiency of microbicidal reagents and methods, particularly on inactivating bacterial spores in liquids, on surfaces, or in aerosols (4, 19), but the subsequent effect of these disinfectants on the performance of either nucleic acidbased or immunologically based diagnostics have received relatively little attention. We selected for study two aldehydes (formaldehyde and glutaraldehyde), a halogenating agent (hypochlorite), two peroxides (hydrogen peroxide and peracetic acid), an...

The cloning of a renal Na-Pi contransport in system from winter flounder (P eudopleuronectes americanus) has recently been reported. We used this information to answer the questions 1) what is the distribution of the transport protein along the nephron? and 2) how are renal and intestinal transporters related? The distribution of the flounder NaPi-II protein was tested using two antisera raised against partial sequences (amino acids 1-14 and 388-441) of the transporter. Antibody-specific fluorescence was detected at the basolateral membrane of epithelial cells in the proximal tubular segment PII. Two clones corresponding to the renal Na-Pi cotransporter were isolated from a flounder intestinal cDNA library. Their functional properties were determined using Xenopus laevis oocytes. The apparent affinities for Pi [Michaelis constant (K(m)) = 0.063 mM] and Na (K(m) = 45.3 mM), as well as the pH dependency (increasing transport activity with increasing pH), showed the same characteristics in both intestinal and the renal systems. Sequence analysis revealed that the two intestinal clones were 100% homologous to the renal cDNA, Flounder NaPi-II-specific immunofluorescence was observed predominantly at the apical membrane on intestinal cross sections. We report the cloning and expression of the first intestinal Na-Pi cotransport system. This transporter belongs to the small group of proteins that exhibit the same function in the apical and the basolateral membranes of different cells.

This study describes the development of the first neutralizing antibodies against Western equine encephalitis virus (WEEV), a member of the genus Alphavirus. WEEV is transmitted by mosquitoes and can spread to the human central nervous system, causing symptoms ranging from mild febrile reactions to life-threatening encephalitis. WEEV has been classified as a biological warfare agent by the US Centers for Disease Control and Prevention. No anti-WEEV drugs are currently commercially available. Neutralizing antibodies are useful for the pre- and post-exposure treatment of WEEV infections. In this study, two immune antibody gene libraries were constructed from two macaques immunized with inactivated WEEV. Four antibodies were selected from these libraries and recloned as scFv-Fc, with a human Fc part. These antibodies bound WEEV specifically in ELISA with little or no cross-reaction with other alphaviruses. They were further analyzed by immunohistochemistry. All binders were suitable for the intracellular detection of WEEV particles. Neutralizing activity was determined in vitro. Three of the four antibodies were found to be neutralizing; about 1 ng/mL of the best antibody (ToR69–3A2) neutralized 50% of 5x104 TCID50/mL. Due to its human-like nature with a germinality index of 89% (VH) and 91% (VL), the ToR69–3A2 antibody is a promising candidate for future passive vaccine development.