Eilat Virus Host Range Restriction Is Present at Multiple Levels of the Virus Life Cycle Farooq Nasar, Rodion V. Gorchakov,* Robert B. Tesh, Scott C. Weaver Institute for Human Infections and Immunity, Center for Tropical Diseases, and Department of Pathology, University of Texas Medical Branch, Galveston, Texas, USA ABSTRACT Most alphaviruses are mosquito-borne and exhibit a broad host range, infecting many different vertebrates, including birds, rodents, equids, humans, and nonhuman primates. This ability of most alphaviruses to infect arthropods and vertebrates is es- sential for their maintenance in nature. Recently, a new alphavirus, Eilat virus (EILV), was described, and in contrast to all other mosquito-borne viruses, it is unable to replicate in vertebrate cell lines. Investigations into the nature of its host range restriction showed the inability of genomic EILV RNA to replicate in vertebrate cells. Here, we investigated whether the EILV host range restriction is present at the entry level and further explored the viral factors responsible for the lack of genomic RNA replication. Utilizing Sindbis virus (SINV) and EILV chimeras, we show that the EILV vertebrate host range restriction is also manifested at the entry level. Furthermore, the EILV RNA replication restriction is independent of the 3= untranslated genome region (UTR). Complementation experiments with SINV suggested that RNA replication is restricted by the inability of the EILV nonstructural proteins to form functional replicative complexes. These data demonstrate that the EILV host range restriction is multigenic, involving at least one gene from both nonstructural protein (nsP) and structural protein (sP) open reading frames (ORFs). As EILV groups phylogenetically within the mosquito-borne virus clade of pathogenic alphaviruses, our findings have important evolutionary implications for arboviruses. IMPORTANCE Our work explores the nature of host range restriction of the first “mosquito-only alphavirus,” EILV. EILV is related to patho- genic mosquito-borne viruses (Eastern equine encephalitis virus [EEEV], Western equine encephalitis virus [WEEV], Venezue- lan equine encephalitis virus [VEEV], and Chikungunya virus [CHIKV]) that cause severe disease in humans. Our data demon- strate that EILV is restricted both at entry and genomic RNA replication levels in vertebrate cells. These findings have important implications for arbovirus evolution and will help elucidate the viral factors responsible for the broad host range of pathogenic mosquito-borne alphaviruses, facilitate vaccine development, and inform potential strategies to reduce/prevent alphavirus transmission. A rthropod-borne viruses (arboviruses) such as dengue virus, Chikungunya virus, West Nile virus, and Rift Valley fever virus are important causes of human and animal disease world- wide (1, 2). Arboviruses encompass diverse virus families, includ- ing Togaviridae, Bunyaviridae, Flaviviridae, Rhabdoviridae, Ortho- myxoviridae, Reoviridae, and Asfarviridae, and are maintained in nature primarily through biological transmission between suscep- tible vertebrate hosts and hematophagous arthropods such as ticks, mosquitoes, and other biting flies (1, 2). This ability to infect diverse vertebrate and arthropod hosts enables arboviruses to per- sist in nature; however, the viral factors that facilitate their broad host range are poorly understood. Mostly during the past decade, many “insect-only” or arthro- pod viruses have been discovered in several arbovirus families: Sigma and Moussa viruses in Rhabdoviridae; Gouleako virus in Bunyaviridae; cell-fusing agent virus, Kamiti River virus, Aedes flavivirus, Culex flavivirus, Quang Binh, Calbertado, Nounané, Lammi, and Marisma mosquito viruses in Flaviviridae (3–17). Based on phylogenetic findings, these arthropod flaviviruses can be classified into two groups: the first consists of viruses distantly related to human and animal pathogens (Sigma, Moussa, Goule- ako, cell-fusing agent, Kamiti River, Aedes flavivirus, Culex flavi- virus, Quang Binh, and Calbertado viruses), while the second, including Nounané, Lammi, and Marisma mosquito viruses, falls within the mosquito-borne flavivirus clade that includes many human pathogens such as dengue and yellow fever viruses (3–17). The phylogenetic placement of the later arthropod virus group suggests that these arthropod flaviviruses have evolutionarily lost the ability to infect vertebrates. These viruses could therefore yield significant insights into viral factors that facilitate the broad host range of pathogenic flaviviruses. However, the determinants of host range restriction of these flaviviruses remain to be elucidated. Another major taxon of arboviruses is the genus Alphavirus in the family Togaviridae, which also encompasses a very broad host range. The genus is comprised of small, spherical, enveloped vi- ruses with genomes consisting of a single-strand, positive-sense Received 25 July 2014 Accepted 7 November 2014 Accepted manuscript posted online 12 November 2014 Citation Nasar F, Gorchakov RV, Tesh RB, Weaver SC. 2015. Eilat virus host range restriction is present at multiple levels of the virus life cycle. J Virol 89:1404 –1418. doi:10.1128/JVI.01856-14. Editor: A. García-Sastre Address correspondence to Scott C. Weaver, sweaver@utmb.edu. * Present address: Rodion V. Gorchakov, Department of Pediatrics, Section of Tropical Medicine, Baylor College of Medicine, Houston, Texas, USA. Copyright © 2015, American Society for Microbiology. 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