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.

FIG 1

Schematic diagrams of SINV and EILV chimeras.

FIG 2

Plaque phenotype and replication kinetics of SINV and EILV chimeras in C7/10 cells. (A and C) C7/10 cells were infected with chimeras at 2 dpi (SINV-eGFP and SINV/EILV chimeras) (A) or 3 dpi (EILV-eRFP and EILV/SINV chimeras) (C). (B and D) Replication kinetics of SINV-eGFP and SINV/EILV chimeras (B), and EILV-eRFP and EILV/SINV chimeras (D) were performed at an MOI of 10. Each infection was performed in triplicate. Average titers ± standard deviations (SD) (error bars) are shown.

FIG 3

Infection of C7/10 cells with SINV (A) and EILV (B) chimeras. C7/10 cells were infected at an MOI of 10. Phase-contrast (top row) and fluorescence micrographs (bottom row) were taken at 36 hpi.

FIG 4

Infection of vertebrate and mosquito cell lines with SINV-eGFP with EILV structural ORF. Each cell line was infected at an MOI of 20, and phase-contrast (left) and fluorescence (right) micrographs were taken at 1 and 4 days postinfection (dpi).

FIG 5

Electroporation of vertebrate cell lines with SINV-eGFP with EILV structural ORF. RNA was transcribed in vitro, and each cell line was electroporated with ∼10 μg of RNA, and phase-contrast (left) and fluorescence (right) micrographs were taken at 1 day postelectroporation (dpe).

FIG 6

Replication kinetics of SINV (A) and EILV (B) chimeras in vertebrate cell lines. Infections were performed in triplicate at an MOI of 10. Average titers ± SD are shown.

FIG 7

Infection of vertebrate and mosquito cell lines with EILV-eRFP with SINV structural ORF. Each cell line was infected at an MOI of 10 or electroporated with 10 μg of genomic RNA. Phase-contrast (left) and fluorescence (right) micrographs were taken at 1 dpi (C7/10 cells) or 4 dpi (vertebrate cells).

FIG 8

Predicted secondary structure of EILV (A) and SINV (B) 3′ UTRs. Minimal free energy structures were generated using M-fold at 37°C.

FIG 9

Infection of vertebrate and mosquito cell lines with SINV-eGFP with EILV 3′ UTR (A) and EILV-eRFP with SINV 3′ UTR (B). Each cell line was infected at an MOI of 10 with SINV-eGFP with EILV 3′ UTR or electroporated with 10 μg of genomic EILV-eRFP with SINV 3′ UTR RNA. Phase-contrast (left) and fluorescence (right) micrographs were taken at 1 dpi or 4 dpe.

FIG 10

(A) Schematic diagrams of SINV-eGFP with EILV nsP chimeras. (B) Electroporation or coelectroporation of SINV and/or SINV/EILV nsP chimera genomic RNAs. Fluorescence micrographs were taken at 12 (BHK-21) or 20 (C7/10) hpe.

FIG 11

Detection of EILV negative-strand RNA in vertebrate cell lines. (A) Coelectroporation of SINV and/or EILV-eRFP genomic RNAs into BHK-21, Vero, HEK-293, C7/10 cells and 6 hpe EILV negative-strand RNA was detected via RT-PCR. (B) Phase-contrast (left) and fluorescence (right) micrographs of BHK-21 cells were taken at 12 hpe.