Eilat Virus Induces Both Homologous and Heterologous Interference
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Virology 484 (2015) 51–58 Contents lists available at ScienceDirect Virology journal homepage: www.elsevier.com/locate/yviro Eilat virus induces both homologous and heterologous interference Farooq Nasar a,b, Jesse H. Erasmus a, Andrew D. Haddow b, Robert B. Tesh a, Scott C. Weaver a,n a Institute for Human Infections and Immunity, Center for Tropical Diseases, and Department of Pathology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555, USA b Virology Division, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA1 article info abstract Article history: Most alphaviruses are mosquito-borne and exhibit a broad host range, infecting many different Received 29 December 2014 vertebrates including birds, rodents, equids, and humans. Occasionally, alphaviruses can spill over into Returned to author for revisions the human population and cause disease characterized by debilitating arthralgia or fatal encephalitis. 10 May 2015 Recently, a unique alphavirus, Eilat virus (EILV), was described that readily infects mosquito but not Accepted 11 May 2015 vertebrate cell lines. Here, we investigated the ability of EILV to induce superinfection exclusion. Prior Available online 10 June 2015 infection of C7/10 (Aedes albopictus) cells with EILV induced homologous and heterologous interference, Keywords: reducing the virus titers of heterologous superinfecting viruses (SINV, VEEV, EEEV, WEEV, and CHIKV) by Alphavirus 10–10,000 fold and delaying replication kinetics by 12–48 h. Similar to in vitro infection, prior in vivo Eilat virus EILV infection of Aedes aegypti mosquitoes delayed dissemination of chikungunya virus for 3 days. This is Superinfection the first evidence of heterologous interference induced by a mosquito-specific alphavirus in vitro and in vivo. & 2015 Elsevier Inc. All rights reserved. Introduction Webb et al., 2008). This ability to infect both mosquitoes and vertebrates enables maintenance of alphaviruses in nature in The genus Alphavirus in the family Togaviridae is comprised of enzootic cycles with occasional spillover into the human popula- small, spherical, enveloped viruses with genomes consisting of a tion. Old World viruses such as chikungunya (CHIKV), o’nyong- single strand, positive-sense RNA approximately 11–12 kb in nyong (ONNV), Sindbis (SINV), and Ross River (RRV) are rarely fatal length (Griffin, 2007). The genus consists of 31 recognized species and cause clinical disease characterized by rash and debilitating classified into ten complexes based on antigenic and/or genetic arthralgia. In contrast, New World viruses such as western similarities (Arrigo et al., 2010; Forrester et al., 2012; Nasar et al., (WEEV), eastern (EEEV), and Venezuelan equine encephalitis 2012; Powers et al., 2001). The two aquatic alphavirus complexes (VEEV) viruses cause fatal encephalitis (Griffin, 2007). Currently, [southern elephant seal virus and salmon pancreas disease virus] there are no licensed vaccines or effective antiviral treatments are not known to utilize arthropods in their transmission cycles, available to prevent or treat human infection by alphaviruses. whereas all the remaining complexes [Barmah Forest, Ndumu, Recently, a host-restricted alphavirus, Eilat virus (EILV), was Middelburg, Semliki Forest, Venezuelan (VEE), eastern (EEE), described that is serologically distinct from other alphaviruses but western equine encephalitis (WEE) and Trocara] consist of arbo- phylogenetically groups within the mosquito-borne clade as a sister viruses that almost exclusively utilize mosquitoes as insect vectors to the WEE complex (Nasar et al., 2012). In contrast to all other (Griffin, 2007; La Linn et al., 2001; Villoing et al., 2000; Weaver mosquito-borne alphaviruses, EILV is unable to infect or replicate in et al., 2012; Weston et al., 1999). Mosquito-borne alphaviruses can vertebrate cells but can readily replicate to high titers (4108 PFU/ infect mosquito species encompassing at least eight genera (Aedes, mL) in mosquito cells (Nasar et al., 2012). The EILV vertebrate host Culex, Anopheles, Culiseta, Haemagogus, Mansonia, Verrallina, and range restriction is present at the attachment/entry as well as Psorophora) and many vertebrate taxa including equids, birds, genomic RNA replication levels (Nasar et al., 2015). EILV is the first amphibians, reptiles, rodents, pigs, as well as human and non- mosquito-specific alphavirus described, and represents a new human primates (Griffin, 2007; Karabatsos, 1985; Jupp et al., 1981; complex within the genus (Nasar et al., 2015). Superinfection exclusion or homologous interference is a phe- nomenon where prior infection with a virus reduces or prevents a n subsequent infection with a closely related virus. This phenomenon Corresponding author. fi E-mail address: [email protected] (S.C. Weaver). was rst described with two closely related genotypes of tobacco 1 Current address. mosaic virus, where plants previously infected with common-mosaic http://dx.doi.org/10.1016/j.virol.2015.05.009 0042-6822/& 2015 Elsevier Inc. All rights reserved. 52 F. Nasar et al. / Virology 484 (2015) 51–58 virus could not be subsequently infected with the yellow-mosaic SINV-eGFP or VEEV-TC83-eGFP infection, whereas minimal eGFP tobacco virus (McKinney, 1926, 1929). This phenomenon has been expression was detected in EILV-infected cells at either MOI successfully developed as a tool to prevent or reduce the loss of crops (Fig. 4). At 72 h post-superinfection, increased eGFP expression and is termed “cross protection” (Ziebell and Carr, 2010). Following was observed irrespective of the MOI or virus (Fig. 4). its discovery, superinfection exclusion was also demonstrated with To further explore heterologous interference, superinfection animal and mosquito-only viruses including alphaviruses (Bolling et was also monitored via replication kinetics. Following mock or al., 2012; Bratt and Rubin, 1968a; Claus et al., 2007; Geib et al., 2003; EILV infection, C7/10 cells were superinfected with SINV or VEEV Hobson-Peters et al., 2013; Kenney et al., 2014; Kent et al., 2010; strain TC83 at an MOI of 1 or 0.1 (Fig. 1). EILV-infected cells Laskus et al., 2001; Lee et al., 2005; Steck and Rubin, 1966a; Tscherne displayed a statistically significant reduction in superinfecting et al., 2007; Walters et al., 2004; Whitaker-Dowling et al., 1983; Zou SINV or VEEV-TC83 titers at early time points [6, 12, and 24 (both et al., 2009). The available in vitro data demonstrate that a prior viruses) and 48 (SINV only)] at both MOIs, with p-values o0.02 alphavirus infection can reduce replication of a superinfecting for all time points (Fig. 5). SINV and VEEV-TC83 titers were homologous or heterologous virus (Adams and Brown, 1985; Eaton, reduced by 100 to 10,000-fold between 6 and 24 hpi (Fig. 5). 1979; Karpf et al., 1997; Lennette and Koprowski, 1946; Renz and In addition to the reduction in virus titers, the replication kinetics Brown, 1976; Stollar and Shenk, 1973; Zebovitz and Brown, 1968). of superinfecting viruses were delayed by 12–48 h regardless of These studies raise the intriguing possibility of utilizing alphaviruses virus or MOI (Fig. 5). These data corroborated results obtained via as transmission control measures to limit or eliminate human and/or fluorescent microscopy, and suggested that prior EILV infection animal infection. However, all of the viruses within the mosquito- was able to induce heterologous interference, reducing super- borne clade are capable of replication in both vertebrates and insects, infecting virus production and delaying replication kinetics. and in addition many can cause severe disease in animals and Next, we investigated the heterologous interference of alpha- humans (Griffin, 2007). Consequently, the use of these viruses is viruses associated with severe human disease: EEEV strain FL93, unsuitable for transmission control measures. In contrast, the host- VEEV subtype IC strain 3908, WEEV strain McMillan, and CHIKV restricted and therefore vertebrate apathogenic EILV is potentially strain JKT. C7/10 cells were infected with EILV at an MOI of 10 ideal for this application. Here, we investigated the ability EILV to followed by superinfection with heterologous viruses at an MOI of induce homologous and heterologous superinfection exclusion. 0.1 (Fig. 1). Similar to results obtained with superinfection of SINV and VEEV-TC83, all heterologous viruses displayed a reduction in virus production and delayed replication kinetics at early time Results points in EILV-infected cells (Fig. 6). Virus titers of superinfecting viruses were reduced by 10 to 1,000-fold at 6, 12, 24 and 48 hpi Homologous interference in C7/10 cells with p-values o0.04 for all time points (Fig. 6). VEEV and WEEV displayed the greatest reduction of 1,000-fold at 24 and 48 hpi, To assess superinfection with homologous and heterologous respectively (Fig. 6). Replication kinetics of all superinfecting viruses in vitro in C7/10 cells, an optimal multiplicity of infection viruses were also delayed by at least 12–48 h (Fig. 6). WEEV was (MOI) to establish EILV infection was investigated (Fig. 1). Infection of the only virus that replicated to peak virus titers similar to those in C7/10 cells at an MOI of 10 plaque-forming units (PFU)/mL with EILV- mock-infected cells, whereas the peak titers of other three viruses eRFP established infection in 490% of cells within 24 h and was were reduced by 10 to 52-fold compared to their respective subsequently utilized