Arenaviridae is a family of enveloped viruses some of which are capable of causing hemorrhagic fever syndromes in humans. In this report, we demonstrate that treatment of host cells with the tyrosine kinase inhibitor genistein inhibits infection of cells with the New World arenavirus Pichindé (PICV). The greatest degree of inhibition was observed in pre-treated target cells, but modest inhibition of infection was also seen when drug was added to cultures up to 48h after infection. We show that PICV-induced phosphorylation of the activating transcription factor-2 protein (ATF-2) and cyclic adenosine monophosphate response element binding protein (CREB) is inhibited following genistein treatment. Lastly, genistein treatment also inhibited transduction of cells with pseudotyped retrovirus particles expressing envelope proteins of the Old World arenavirus Lassa virus. These results demonstrate that kinase activity is required for arenavirus infection and that therapeutics designed to inhibit kinase activity should be explored.

The chemical interaction of (i) HCl, (ii) NH 3 , and (iii) HCl + NH 3 with crystalline ice has been examined as a function of temperature (T ) 80-150 K) utilizing FTIR spectroscopy. The infrared spectra reveal the formation of ionic hydrates for HCl and molecular hydrates for NH 3 . The acid-base reaction leads to the formation of the ammonium ion, NH 4 + . While it is produced to a limited extent between 80 and 140 K, NH 4 + is a dominant species above 140 K. All species appear as lower amorphous hydrates for T ) 80-140 K. Dramatic changes in the spectra occur above 140 K as higher crystalline hydrates are formed. This temperature coincides with the amorphous to crystalline phase transformation of ice. This change appears to be due to a greater number of water molecules on the surface that are available for hydration and crystallization.

Arenaviruses are important causes of viral hemorrhagic fevers in humans. Arenavirus infection of cells occurs via a pH-dependent endocytic route, but detailed studies of entry pathways have not been done. We investigated the role of cell membrane cholesterol, caveolae, and clathrin coated pits in infection by Lassa virus (LASV), which utilizes alpha-dystroglycan (alpha-DG) as a receptor, and Pichindé virus (PICV), which does not. Depletion of cellular cholesterol by treatment with methyl betacyclodextrin (MbetaCD) or nystatin/progesterone inhibited PICV replication and transfer of packaged marker gene by LASV or PICV pseudotyped retroviral particles. In cells lacking caveolae due to silencing of the caveolin-1 gene, no inhibition of PICV infection or LASV pseudotype transduction was observed. However, PICV infection and LASV and PICV pseudotype transduction was inhibited when an Eps15 dominant negative mutant was used to inhibit clathrin-mediated endocytosis. Altogether, the results indicate that diverse arenaviruses have a common requirement for cell membrane cholesterol and clathrin mediated endocytosis in establishing infection.

A dependence of poliovirus on an unorthodox translation initiation mode can be targeted selectively to drive viral protein synthesis and cytotoxicity in malignant cells. Transformed cells are naturally susceptible to poliovirus, due to widespread ectopic upregulation of the poliovirus receptor, Necl-5, in ectodermal/neuroectodermal cancers. Viral tumor cell killing and the host immunologic response it engenders produce potent, lasting antineoplastic effects in animal tumor models. Clinical application of this principle depends on unequivocal demonstration of safety in primate models for paralytic poliomyelitis. We conducted extensive dose-range-finding, toxicity, biodistribution, shedding, and neutralizing antibody studies of the prototype oncolytic poliovirus recombinant, PVS-RIPO, after intrathalamic inoculation in Macaca fascicularis. These studies suggest that intracerebral PVS-RIPO inoculation does not lead to viral propagation in the central nervous system (CNS), does not cause histopathological CNS lesions or neurological symptoms that can be attributed to the virus, is not associated with extraneural virus dissemination or replication and does not induce shedding of virus with stool. Intrathalamic PVS-RIPO inoculation induced neutralizing antibody responses against poliovirus serotype 1 in all animals studied. Several years ago, we proposed consideration of recombinant, nonpathogenic poliovirus (PV) for the treatment of glioblastoma (GBM) (17). This proposal is based on widespread ectopic expression of the PV receptor, nectin-like molecule-5 (Necl-5), in such cancers (26). Necl-5, an onco-fetal cell adhesion molecule of the nectin family, is broadly associated with ectodermal/neuroectodermal cancers (reviewed in reference 42). Necl-5 expression is abundant in GBM cells, "stem cell-like" GBM cells, and tumorassociated vasculature (6) and is implicated in GBM cell dispersion and invasion (39,40). Due to Necl-5 expression, GBM cells are naturally susceptible to infection with and rapid destruction by PV (17). Direct cytocidal effects of PV elicit host immunogenic responses directed against tumors in vivo (43).Any clinical application of engineered PVs must include a rigorous demonstration of safety in established nonhuman primate models for paralytic poliomyelitis. Such safety studies are modeled after standard neurovirulence assays for the live-attenuated (Sabin) PV vaccines (41). The three Sabin vaccine serotypes (PV1-S, PV2-S, and PV3-S), some stemming from serial passage in diverse simian tissue culture systems, exhibit substantially reduced primate neurovirulence (36). While the genetic base for attenuation is different for each Sabin strain (29), they have key sequence variables in common.PV plus-strand RNA genomes are not equipped with a 7-methyl-guanidine cap (31) and thus are unable to recruit ribosomal subunits via the cap-binding eukaryotic initiation factor (eIF) 4E. Instead, PV RNAs rely on an internal ribosomal entry site (IRES) within their 5= untranslated region (UTR) to recruit 40S ribosoma...

Influenza A viruses continue to pose a threat to human health; thus, various vaccines and prophylaxis continue to be developed. Testing of these products requires various animal models including mice, guinea pigs, and ferrets. However, because ferrets are naturally susceptible to infection with human influenza viruses and because the disease state resembles that of human influenza, these animals have been widely used as a model to study influenza virus pathogenesis. In this report, a statistical analysis was performed to evaluate data involving 269 ferrets infected with seasonal influenza, swine influenza, and highly pathogenic avian influenza (HPAI) from 16 different studies over a five year period. The aim of the analyses was to better qualify the ferret model by identifying relationships among important animal model parameters (endpoints) and variables of interest, which include survival, time-to-death, changes in body temperature and weight, and nasal wash samples containing virus, in addition to significant changes from baseline in selected hematology and clinical chemistry parameters. The results demonstrate that a disease clinical profile, consisting of various changes in the biological parameters tested, is associated with various influenza A infections in ferrets. Additionally, the analysis yielded correlates of protection associated with HPAI disease in ferrets. In all, the results from this study further validate the use of the ferret as a model to study influenza A pathology and to evaluate product efficacy.