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Six novel insect-specific viruses, isolated from mosquitoes and phlebotomine sand flies collected in Brazil, Peru, the United States, Ivory Coast, Israel, and Indonesia, are described. Their genomes consist of single-stranded, positive-sense RNAs with poly(A) tails. By electron microscopy, the virions appear as spherical particles with diameters of ϳ45 to 55 nm. Based on their genome organization and phylogenetic relationship, the six viruses, designated Negev, Ngewotan, Piura, Loreto, Dezidougou, and Santana, appear to form a new taxon, tentatively designated Negevirus. Their closest but still distant relatives are citrus leposis virus C (CiLV-C) and viruses in the genus Cilevirus, which are mite-transmitted plant viruses. The negeviruses replicate rapidly and to high titer (up to 10 10 PFU/ml) in mosquito cells, producing extensive cytopathic effect and plaques, but they do not appear to replicate in mammalian cells or mice. A discussion follows on their possible biological significance and effect on mosquito vector competence for arboviruses.

4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virusThis study uses high-resolution cryo-electron microscopy to provide a complete structural model of the VEEV alphavirus, bridging the gap between incomplete crystal structures and lower resolution electron microscopy analyses.

Replication of alphaviruses in vertebrate cells strongly affects cell physiology and ultimately leads to development of a cytopathic effect (CPE) and cell death. Sindbis virus (SIN) replication causes major changes in cellular macromolecular synthesis, in which the strong downregulation of transcription of cellular mRNAs and rRNAs plays a critical role. SIN nonstructural protein nsP2 was previously proposed as one of the main regulators of virus-host cell interactions, because point mutations in the carboxy-terminal part of nsP2 could make SIN and other alphaviruses and replicons less cytopathic and capable of persisting in some vertebrate cell lines. These mutants were incapable of inhibiting transcription and downregulating a viral stress-induced cell response. In the present work, we demonstrate that (i) SIN nsP2 is critically involved in CPE development, not only during the replication of SIN-specific RNAs, but also when this protein is expressed alone from different expression cassettes; (ii) the cytotoxic effect of SIN nsP2 appears to be at least partially determined by its ability to cause transcriptional shutoff; (iii) these functions of SIN nsP2 are determined by the integrity of the carboxy-terminal peptide of this protein located outside its helicase and protease domains, rather than by its protease activity; and (iv) the cytotoxic activity of SIN nsP2 depends on the presence of this protein in a free form, and alterations in P123 processing abolish the ability of nsP2 to cause CPE.The alphavirus genus of the Togaviridae family contains a number of human and animal pathogens. Nearly 30 members of the genus are widely distributed on all continents. They are transmitted by mosquitoes to vertebrates that serve as amplifying hosts (17,20,38). In infected vertebrates, some of the alphaviruses cause an acute disease, often characterized by high-titer viremia or host death (16,18). Accordingly, these viruses exhibit a highly cytopathic phenotype in cell cultures of mammalian and avian origin (38).Sindbis virus (SIN) is a prototype member of the genus and one of the least pathogenic alphaviruses. SIN is widely used in experimental research, since it can infect a wide variety of commonly used vertebrate cell lines, where it replicates to high titers approaching 10 10 PFU/ml. Moreover, SIN replication leads to the rapid development of a cytopathic effect (CPE) and cell death within 24 to 48 h postinfection (10). The SIN genome is a single-stranded RNA of almost 11.5 kb which has a positive polarity (37) and contains a 5Ј methylguanylate cap and a 3Ј polyadenylate tail. After release from the nucleocapsids (42, 43), the genome is translated into the viral nonstructural proteins nsP1 to nsP4, which are encoded by the 5Ј two-thirds of the genome. Together with host factors these proteins form the replicative enzyme complex (RC) required for viral genome replication and transcription of the subgenomic RNA (38). The latter RNA is encoded by the 3Ј one-third of the genome and translated into the structural proteins t...

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