Venezuelan equine encephalitis (VEE) complex alphaviruses are important re-emerging arboviruses that cause life-threatening disease in equids during epizootics as well as spillover human infections. We conducted a comprehensive analysis of VEE complex alphaviruses by sequencing the genomes of 94 strains and performing phylogenetic analyses of 130 isolates using complete open reading frames for the nonstructural and structural polyproteins. Our analyses confirmed purifying selection as a major mechanism influencing the evolution of these viruses as well as a confounding factor in molecular clock dating of ancestors. Times to most recent common ancestors (tMRCAs) could be robustly estimated only for the more recently diverged subtypes; the tMRCA of the ID/IAB/IC/II and IE clades of VEE virus (VEEV) were estimated at ca. 149–973 years ago. Evolution of the IE subtype has been characterized by a significant evolutionary shift from the rest of the VEEV complex, with an increase in structural protein substitutions that are unique to this group, possibly reflecting adaptation to its unique enzootic mosquito vector Culex (Melanoconion) taeniopus. Our inferred tree topologies suggest that VEEV is maintained primarily in situ, with only occasional spread to neighboring countries, probably reflecting the limited mobility of rodent hosts and mosquito vectors.

The greatest risk from live-attenuated vaccines is reversion to virulence. Particular concerns arise for RNA viruses, which exhibit high mutation frequencies. We examined the stability of 3 attenuation strategies for the alphavirus, Venezuelan equine encephalitis virus (VEEV): a traditional, point mutation-dependent attenuation approach exemplified by TC-83; a rationally designed, targeted-mutation approach represented by V3526; and a chimeric vaccine, SIN/TC/ ZPC. Our findings suggest that the chimeric strain combines the initial attenuation of TC-83 with the greater phenotypic stability of V3526, highlighting the importance of the both initial attenuation and stability for live-attenuated vaccines.

To develop an effective vaccine against eastern equine encephalitis (EEE), we engineered a recombinant EEE virus (EEEV) that was attenuated and capable of replicating only in vertebrate cells, an important safety feature for live vaccines against mosquito-borne viruses. The subgenomic promoter was inactivated with 13 synonymous mutations and expression of the EEEV structural proteins was placed under the control of an internal ribosomal entry site (IRES) derived from encephalomyocarditis virus (EMCV). We tested this vaccine candidate for virulence, viremia and efficacy in the murine model. A single subcutaneous immunization with 104 infectious units protected 100% of mice against intraperitoneal challenge with a highly virulent North American EEEV strain. None of the mice developed any signs of disease or viremia after immunization or following challenge. Our findings suggest that the IRES-based attenuation approach can be used to develop a safe and effective vaccine against EEE and other alphaviral diseases.

This study examined the effect of naturally occurring Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) gene sequence variation on the LMP-1 half-life in epithelial cells. The LMP-1 half-life was not influenced by sequence variation in amino acids 250 to 307 or amino acids 343 to 352. The LMP-1 half-life was short when the amino acid encoded at position 129 was methionine, the initiation codon product of lytic LMP-1 (lyLMP-1). The mutation of amino acid 129 to isoleucine greatly increased the LMP-1 half-life. Expression of lyLMP-1 in trans down-regulated the LMP-1 half-life in a dose-dependent manner and restored a short-half-life phenotype to the mutated LMP-1 construct lacking the cis ability to express lyLMP-1. This observed dominant negative effect of lyLMP-1 expression on the LMP-1 half-life in epithelial cells in vitro may have implications for EBV epithelial oncogenesis in vivo.Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus that is associated with numerous malignancies, especially nasopharyngeal carcinoma (NPC). Oncogenic EBV latent membrane protein 1 (LMP-1) is a 63-kDa protein of 386 amino acids (strain B958) encoded by the BNLF1 gene ( Fig. 1). LMP-1 inserts into the plasma membrane and functions as a ligand-independent, constitutively active growth factor receptor, similar to CD40 of the tumor necrosis factor receptor (TNFR) family. After oligomerization, LMP-1 binds TNFRassociated factors (TRAFs) and the TNFR-associated death domain protein (26, 38) to activate intracellular signaling through the NF-B, cJun N-terminal kinase, and p38 mitogenactivated protein kinase pathways (10,11,42). LMP-1 also activates the JAK-STAT signaling pathway, possibly through interaction with Janus kinase 3 (JAK3) (19,22). Cumulatively, these signals generate many effects on host cell growth, differentiation, apoptosis, and immune response.The LMP-1 gene manifests remarkable natural sequence heterogeneity (9,35,48). Although a specific LMP-1 genotypedisease phenotype relationship has not yet been identified (48), NPC-derived LMP-1 is both structurally and functionally different from the LMP-1 derived from the laboratory EBV strain B958 in the following ways: (i) NPC LMP-1 activates NF-B signaling and AP-1 transactivation better than B958 LMP-1 (5,7,15,16,27,33,34), (ii) NPC LMP-1 down-regulates cell immune markers, blocks cell apoptosis, and up-regulates the epidermal growth factor receptor (EGFR) better than B958 LMP-1 (7,8,15,27,33,34,52), and (iii) NPC LMP-1 transforms cells and forms tumors in mice more efficiently than B958 LMP-1 (8,24,28,33,52). Chimeric studies of LMP-1 have mapped some of these functional differences to the carboxy-terminal 30-nucleotide domain encoding amino acids 343 to 352 (28) and transmembrane domain amino acids 85 to 129 (5, 34).The intracellular quantity of expressed LMP-1 and the cell type background together influence the strength of LMP-1 signaling and its ultimate phenotypic effect on the cell (4,16,17,20,24,28,49,50). The intracellular half-life of LMP-1 is g...

The Epstein-Barr virus (EBV) is an oncogenic human herpesvirus. EBV latent membrane protein 1 (LMP-1) is a viral oncogene that manifests its oncogenic phenotype through activation of cellular signaling pathways involved in cell growth, survival, differentiation, and transformation. Lytic LMP-1 (lyLMP-1) is a related EBV gene without oncogenic properties. The lyLMP-1 gene is found in 60% of the EBV strains circulating in nature, but it is not found in EBV strains associated with nasopharyngeal carcinoma. We recently demonstrated that lyLMP-1 down-regulates the half-life of LMP-1 in epithelial cells. Therefore in this study, we tested the hypothesis that lyLMP-1 concomitantly down-regulates LMP-1 oncogenic activity. The results demonstrated that lyLMP-1 inhibits LMP-1-mediated intracellular signaling activation, epithelial cell growth and survival, and fibroblast cell transformation in a dose-dependent manner. Lytic LMP-1 manifested this effect through the promotion of LMP-1 degradation and a reduction in the expressed quantity of LMP-1. Thus, lyLMP-1 functions as a posttranslational negative regulator of LMP-1 oncogenesis. These results support a model of EBV-associated epithelial oncogenesis in which lyLMP-1 may act in vivo to reduce the risk of LMP-1-mediated transformation and is therefore subjected to negative selection in nasopharyngeal carcinoma pathogenesis. Epstein-Barr virus (EBV)is an oncogenic human herpesvirus associated with a broad spectrum of benign and malignant diseases, including infectious mononucleosis, oral hairy leukoplakia, African Burkitt's lymphoma, Hodgkin's disease lymphoma, lymphoproliferative disorders of immunocompromised hosts, and nasopharyngeal carcinoma. The EBV latent membrane protein 1 (LMP-1) is a viral oncogene (30) that is believed to be important in the pathogenesis of many EBVassociated diseases, including nasopharyngeal carcinoma (25). In the B958 EBV strain, LMP-1 is a 63-kDa protein of 386 amino acids encoded by the BNLF1 gene. LMP-1 localizes to cellular membranes and functions as a constitutively active tumor necrosis factor receptor homologue that propagates intracellular signaling, including the NF-B, cJun N-terminal protein kinase/AP-1, and Janus kinase/STAT pathways (5,12,15,27). Through these signaling pathways, LMP-1 generates a myriad of effects on host cell growth, differentiation, and apoptosis, including growth promotion and survival in epithelial cells (4,11,16,23,32) and transformation of rodent fibroblasts (3, 30). Although the mechanisms by which LMP-1 influences cell biology have been intensively studied, little is known about the mechanisms that regulate LMP-1 oncogenic activity. Some EBV strains also encode an amino-terminally truncated form of LMP-1 called lytic LMP-1 (lyLMP-1). Transcription of lyLMP-1 is driven by the ED-L1A promoter that is located in the first intron of the LMP-1 gene and that is present in all EBV strains (6, 17, 29) (Fig. 1). However, the presence or absence of the lyLMP-1 open reading frame (ORF) is determined by the sequence ...