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Eilat Virus, a Unique Alphavirus with Host Range Restricted to Insects by RNA Replication

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Eilat Virus, a Unique Alphavirus with Host Range Restricted to Insects by RNA Replication Eilat virus, a unique alphavirus with host range restricted to insects by RNA replication Farooq Nasara,1, Gustavo Palaciosb,1,2, Rodion V. Gorchakova, Hilda Guzmana, Amelia P. Travassos Da Rosaa, Nazir Savjib,3, Vsevolod L. Popova, Michael B. Shermanc, W. Ian Lipkinb, Robert B. Tesha, and Scott C. Weavera,c,4 aInstitute for Human Infections and Immunity and Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555; bCenter for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, NY 10032; and cSealy Center for Structural Biology and Molecular Biophysics and Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555 Edited* by Barry J. Beaty, Colorado State University, Fort Collins, CO, and approved July 18, 2012 (received for review March 23, 2012) Most alphaviruses and many other arboviruses are mosquito-borne (2, 3). The western equine encephalitis (WEE) complex contains and exhibit a broad host range, infecting many different verte- both Old World [Whataroa virus (WHATV), Sindbis virus (SINV)] brates including birds, rodents, equids, humans, and nonhuman and New World [Aura virus (AURAV)] viruses as well as recom- primates. Consequently, they can be propagated in most verte- binant viruses [WEE virus (WEEV), Highlands J, Fort Morgan, brate and insect cell cultures. This ability of arboviruses to infect and Buggy Creek] (2–5). The latter are decedents of an ancient arthropods and vertebrates is usually essential for their mainte- recombinant virus that obtained nonstructural and capsid genes nance in nature. However, several flaviviruses have recently been from an EEE-like virus and the remaining genes from a Sindbis- described that infect mosquitoes but not vertebrates, although the like ancestor (4, 5). Last, the aquatic alphaviruses comprise two mechanism of their host restriction has not been determined. Here groups, Southern elephant seal virus and salmon pancreas disease we describe a unique alphavirus, Eilat virus (EILV), isolated from virus (SPDV) (6, 7). SPDV and its subtype sleeping disease virus a pool of Anopheles coustani mosquitoes from the Negev desert are distantly related to all other alphaviruses (7). of Israel. Phylogenetic analyses placed EILV as a sister to the West- Most alphaviruses infect terrestrial vertebrates via mosquito- ern equine encephalitis antigenic complex within the main clade of borne transmission and thereby exhibit a broad host range (8). mosquito-borne alphaviruses. Electron microscopy revealed that, Occasionally, these cycles spill over into humans and domesti- like other alphaviruses, EILV virions were spherical, 70 nm in diameter, cated animals to cause disease. Human infections with Old World MICROBIOLOGY and budded from the plasma membrane of mosquito cells in culture. viruses such as Ross River virus, chikungunya virus, and SINV are EILV readily infected a variety of insect cells with little overt cyto- typically characterized by fever, rash, and polyarthritis, whereas pathic effect. However, in contrast to typical mosquito-borne alpha- infections with the New World viruses VEE virus (VEEV), EEE viruses, EILV could not infect mammalian or avian cell lines, and viral virus (EEEV), and WEEV can cause fatal encephalitis (8). as well as RNA replication could not be detected at 37 °C or 28 °C. Alphaviruses infect a wide range of vertebrate and insect hosts, Evolutionarily, these findings suggest that EILV lost its ability to infect including mosquito species encompassing at least six genera as vertebrate cells. Thus, EILV seems to be mosquito-specific and repre- well as ticks and lice (6, 8–10). Vertebrate hosts include fish, sents a previously undescribed complex within the genus Alphavirus. equids, birds, amphibians, reptiles, rodents, pigs, humans, and Reverse genetic studies of EILV may facilitate the discovery of deter- nonhuman primates (9). Consequently, alphaviruses can be cul- minants of alphavirus host range that mediate disease emergence. tured in many vertebrate and insect cell lines (11–13). In contrast, the distantly related fish alphaviruses, which are not known to evolution | Togavirus have arthropod vectors, exhibit a narrow host range (fish cells only) that is at least partially a result of temperature sensitivity he genus Alphavirus in the family Togaviridae comprises (14–16). However, the viral factor(s) that underlie the varying Tsmall, spherical, enveloped viruses with single strand, posi- host range of alphaviruses are poorly understood. Host-restricted tive-sense, 11- to 12-kb RNA genomes that contains two ORFs alphaviruses that group within the mosquito-borne clade may (1): the 5′ two thirds of the genome encodes four nonstructural provide insights into these factor(s); however, to date, none has proteins (nsPs; nsP1–nsP4); the 3′ third encodes five structural been identified. Here we describe a host-restricted alphavirus proteins (sPs; Capsid, E3, E2, 6K, and E1). Alphaviruses enter of mosquitoes and demonstrate that its inability to infect verte- the host cell via receptor-mediated endocytosis. Following in- brates is caused at least in part by restricted RNA replication. ternalization, low endocytic pH induces a conformational change Results that exposes an E1 fusion peptide resulting in the cytoplasmic release of the nucleocapsid. The genomes of alphaviruses are Virus Isolation. Eilat virus (EILV) was one of 91 virus isolates capped and polyadenylated and serve as mRNA for translation obtained during an arbovirus survey the Negev desert, including of the nsPs. The resulting polyprotein is sequentially cleaved into in the city of Eilat, in Israel, during 1982 to 1984 (17). EILV was four nsPs responsible for RNA replication, modification, and proteolytic cleavage. The nsPs facilitate the synthesis of negative and positive strands as well as the transcription of subgenomic Author contributions: F.N., R.V.G., R.B.T., and S.C.W. designed research; F.N., G.P., R.V.G., A.P.T.D.R., N.S., V.L.P., and M.B.S. performed research; F.N., R.V.G., H.G., R.B.T., and S.C.W. mRNA encoding the sPs. Following translation, glycosylated E1/ contributed new reagents/analytic tools; F.N., R.V.G., A.P.T.D.R., V.L.P., M.B.S., W.I.L., E2 heterodimers are inserted into the plasma membrane. Capsid R.B.T., and S.C.W. analyzed data; and F.N. and S.C.W. wrote the paper. proteins interact with one genomic RNA copy to form nucleo- The authors declare no conflict of interest. capsids, which interact with the cytoplasmic tail of E2 to initiate *This Direct Submission article had a prearranged editor. virion budding from host cell membranes (1). 1F.N. and G.P. contributed equally to this work. The genus Alphavirus currently includes 29 species grouped into 2Present address: US Army Medical Research Institute for Infectious Diseases, Fort Detrick, 10 complexes based on antigenic and/or genetic similarities (2, 3). Frederick, MD 21702. The Barmah Forest, Ndumu, Middelburg, and Semliki Forest 3Present address: School of Medicine, New York University, New York, NY 10016. complexes occur almost exclusively in the Old World, whereas the 4To whom correspondence should be addressed. E-mail: [email protected]. Venezuelan equine encephalitis (VEE), eastern equine encepha- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. litis (EEE), and Trocara complexes comprise New World viruses 1073/pnas.1204787109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1204787109 PNAS Early Edition | 1of6 originally isolated in mosquito cells by Joseph Peleg (Hebrew In Vitro Characterization. An EILV genomic cDNA clone was con- University, Jerusalem) from a pool of Anopheles coustani mos- structed and rescued by electroporation of transcribed RNA. EILV quitoes, and was subsequently sent to one of the authors (R.B.T.) infection did not cause any overt cytopathic effects on C7/10 cells, for further study. Preliminary characterization showed that EILV although they grew at a slower rate than uninfected cells. EILV was unable to infect mammalian cells or to kill infant mice in- formed 3- to 4-mm plaques 3 d after infection of C7/10 cells (Fig. oculated intracerebrally, but could replicate to high titers in a 1B). RNA analysis of EILV-infected C7/10 cells revealed the syn- variety of insect cells. thesis of genomic as well as subgenomic RNA, characteristic of all alphaviruses (Fig. 1C). Genomic Analysis. The complete genomic EILV sequence, de- termined by 454 pyrosequencing, was translated and compared EM. Transmission EM and cryoEM imaging of EILV virions with that of SINV to determine the length of each gene product; showed that they were spherical, 70 nm in diameter, and budded a schematic illustration is shown in Fig. 1A. The lengths of the from the plasma membrane of mosquito cells (Fig. 2 A and B). A UTRs and intergenic regions, as well as of each gene, were similar 20-Å-resolution cryoEM reconstruction (Fig. 2A) revealed an to those of other alphaviruses. Nucleotide and amino acid se- unusual protrusion on the glycoprotein spikes that is absent in quence identity of EILV with other alphaviruses ranged from 57% SINV. The observed volume of this protrusion was consistent to 43% and 58% to 28%, respectively (Dataset S1). In both anal- with the expected volume of the E3 protein. yses, EILV had greater similarity to WHATV, AURAV, SINV, Phylogenetic and Serological Analysis. Neighbor-joining, maximum- and Trocara virus (TROV), and had the lowest sequence identity likelihood, and Bayesian methods were used to determine the to SPDV. The EILV nsPs displayed higher amino acid identity to relationship of EILV within the genus Alphavirus. Trees were those of other alphavirus than did the sPs, with nsP4 exhibiting the generated using full-length as well as nonstructural and structural highest amino acid identity and nsP3 the least (Dataset S2). polyprotein gene nucleotide alignments. All three methods placed Analyses of putative EILV conserved sequence elements (CSEs) EILV within the clade of mosquito-borne alphaviruses (Fig.
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