Eilat Virus Host Range Restriction Is Present at Multiple Levels of the Virus Life Cycle

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 essential 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 pathogenic mosquito-borne viruses (Eastern equine encephalitis virus [EEEV], Western equine encephalitis virus [WEEV], Venezuelan equine encephalitis virus [VEEV], and Chikungunya virus [CHIKV]) that cause severe disease in humans. Our data demonstrate 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 worldwide (1, 2). Arboviruses encompass diverse virus families, including Togaviridae, Bunyaviridae, Flaviviridae, Rhabdoviridae, Orthomyxoviridae, Reoviridae, and Asfarviridae, and are maintained in nature primarily through biological transmission between susceptible 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 persist in nature; however, the viral factors that facilitate their broad host ...

Host Range Restriction of Insect-Specific Flaviviruses Occurs at Several Levels of the Viral Life Cycle

“…However, how ISFs are hindered from infecting vertebrate cells is of special interest in studying virus emergence. The only reverse genetics-based data available in this field have been generated recently from EILV, a novel alphavirus related to the WEEV serocomplex of arbo-alphaviruses, where restriction occurs at the levels of both entry and replication (14, 15). …”

“…EILV is in a sister relationship with vertebrate-infecting alphaviruses of the Western equine encephalitis virus (WEEV) complex (14). Studies of the replication of an EILV replicon, as well as chimerized constructs in which EILV protein domains were replaced by homologous domains of the arbo-alphavirus Sindbis virus (SINV), revealed that EILV is blocked at two independent stages of the replication cycle, namely, RNA replication and viral entry (14, 15). The latter findings were further supported by in vivo infection experiments in mosquitoes and newborn mice.…”

Insect-Specific Viruses

“…As briefly discussed earlier, the Eilat alphavirus is an ISV found to be host restricted at multiple levels (Nasar et al, 2012(Nasar et al, , 2015b. This profound degree of host restriction coupled to the phylogenetic clustering to the WEE complex of alphaviruses, has allowed for the synthesis of Eilat/chikungunya chimeras for use as ELISA antigens with native structure and conformation and without the need for inactivation .…”

Chikungunya Virus as Cause of Febrile Illness Outbreak, Chiapas, Mexico, 2014

“…Standard plaque assays were performed for the samples positive by qRT-PCR with Vero cells. Anti-CHIKV IgM-capture enzyme-linked immunosorbent assays (ELISAs) ( 8 ) were performed by using a chimeric Eilat-CHIKV ( 9 ) that contained the nonstructural proteins from Eilat virus and structural proteins from CHIKV, resulting in a structure indistinguishable from that of CHIKV. Plaque-reduction neutralization tests were used to confirm ELISA results.…”