Eilat virus, a unique alphavirus with host range restricted to insects by RNA replication

Abstract

Most alphaviruses and many other arboviruses are mosquito-borne and exhibit a broad host range, infecting many different vertebrates including birds, rodents, equids, humans, and nonhuman primates. Consequently, they can be propagated in most vertebrate and insect cell cultures. This ability of arboviruses to infect arthropods and vertebrates is usually essential for their maintenance in nature. However, several flaviviruses have recently been described that infect mosquitoes but not vertebrates, although the mechanism of their host restriction has not been determined. Here we describe a unique alphavirus, Eilat virus (EILV), isolated from a pool of Anopheles coustani mosquitoes from the Negev desert of Israel. Phylogenetic analyses placed EILV as a sister to the Western equine encephalitis antigenic complex within the main clade of mosquito-borne alphaviruses. Electron microscopy revealed that, like other alphaviruses, EILV virions were spherical, 70 nm in diameter, and budded from the plasma membrane of mosquito cells in culture. EILV readily infected a variety of insect cells with little overt cytopathic effect. However, in contrast to typical mosquito-borne alphaviruses, EILV could not infect mammalian or avian cell lines, and viral as well as RNA replication could not be detected at 37 °C or 28 °C. Evolutionarily, these findings suggest that EILV lost its ability to infect vertebrate cells. Thus, EILV seems to be mosquito-specific and represents a previously undescribed complex within the genus Alphavirus. Reverse genetic studies of EILV may facilitate the discovery of determinants of alphavirus host range that mediate disease emergence.

Fig. 1.

Schematic diagram of the EILV genome (A). Amino acid size of each protein is denoted below. The intergenic region, 5′ and 3′ UTR nucleotide lengths are above in gray. EILV plaques 3 d after infection on C7/10 cells (B). Synthesis of virus-specific RNAs in C7/10 cells infected with EILV or SINV 7 hpi, analyzed by agarose gel electrophoresis (C). G, genomic RNA; SG, subgenomic RNA.

Fig. 2.

Eilat virion morphology determined by cryoEM and transmission EM. A 20-Å cryoEM reconstruction of EILV glycoprotein spikes on the virion surface (A). The protrusion possibly representing the E3 protein is highlighted in purple. SINV glycoprotein spikes are shown as a comparison (45). EILV virions are shown budding from the surface of C7/10 cells (B).

Fig. 3.

Bayesian phylogenetic tree based on nucleotide sequences of the alphavirus structural ORF. A midpoint rooted tree is shown with all posterior probabilities <1 shown on major branches. Alphavirus complexes are denoted in bold.

Fig. 4.

Complement fixation (A) and Hemagglutination inhibition (B) tests with EILV and other alphavirus antigens and hyperimmune mouse ascitic fluids (MIAF). Asterisk indicates the reciprocal of heterologous titer (Ht)/reciprocal of homologous titer (Ho).

Fig. 5.

Replication kinetics of EILV on representative insect (gray, 28 °C) (A) and vertebrate (black, 37 °C) (B) cell lines. Monolayers were infected at an MOI of 10 (measured in mosquito cells). Supernatants were collected at indicated intervals postinfection and titrated on C7/10 cell monolayers. Each data point represents the mean titer of samples taken from triplicate infections ± SD. A6 cells were incubated at 28 °C.

Fig. 6.

Replication of EILV genomic RNA in vertebrate (37 °C) and insect (28 °C) cell lines. RNA was transcribed in vitro from the cDNA clone and ∼10-μg aliquots of RNA were electroporated into vertebrate and insect cells. Phase-contrast and fluorescent field photographs were taken at day 4 after electroporation.