Insect-specific viruses and their potential impact on arbovirus transmission

Abstract

Arthropod-borne viruses (arboviruses) are the causative agents of significant morbidity and mortality among humans and animals globally. In the last few years, the widespread adoption of next generation sequencing and metagenomics has led to a new era of virus discovery, where many novel viruses have been documented, exhibiting a restricted host-range in mosquitoes. They represent a wide-range of insect-specific viruses within the families of Bunyaviridae, Flaviviridae, Mesoniviridae, Reoviridae, Rhabdoviridae, Togaviridae, and the newly recognized taxon of Negeviruses. Collectively, their discovery has opened new vistas about the extent of viral diversity and evolution, their influence on vector competence and ability of their insect hosts to transmit human pathogens (e.g. arboviruses), and their potential development as biological control agents or novel vaccine platforms.

Introduction

During the past decade, advances in sequencing technology (next generation sequencing and metagenomics) and phylogenetics have identified a wide range of novel RNA viruses associated with hematophagous insects [1–6]. It is now apparent that biting insects harbor a diverse range of RNA viruses, many of which are not commonly known vertebrate pathogens. The RNA viruses of hematophagous insects can be conveniently divided into two broad groups, based on their host range: (1) the insect viruses that infect vertebrates and (2) the insect viruses that do not. RNA viruses that infect both biting insects and vertebrates are generally classified as arthropod-borne viruses (arboviruses) and include agents such as dengue (DENV), yellow fever (YFV), chikungunya (CHIKV) and West Nile (WNV) viruses. These dual-host viruses typically undergo replicative cycles in both their insect and vertebrate hosts and include many important human and veterinary pathogens. The second group of RNA insect viruses are the insect-specific viruses (ISV). ISVs infect insects and insect cells, but they do not replicate in vertebrates or vertebrate cells. The focus of this chapter will be on the ISVs, and their interaction with their insect hosts.

Most of the known ISVs that have been described from hematophagous insects are associated with mosquitoes. The predominance of mosquito-associated viruses may simply be a reflection of sampling bias, since most of the known ISVs were initially detected during arbovirus surveillance studies; and these types of studies have generally focused on mosquitoes and mosquito-borne viruses of public health and veterinary importance. A second reason for bias is the availability of a stable and highly susceptible mosquito cell line (Aedes albopictus C6/36) for virus isolation. The C6/36 clone has a defective RNAi response after infection to arboviruses infection [7, 8]; and this defect could account for its increased susceptibility to ISVs. There are no comparable cell lines available for culicoid midges, phlebotomine sandflies or other biting flies. It seems likely that some of these latter biting insects also harbor ISVs, but they have been under sampled and relatively few have been tested. Consequently, this review will focus on the ISVs of mosquitoes identified in the last 4 years.

Bunyaviridae

The family Bunyaviridae currently encompasses 5 genera (Hantavirus, Nairovirus, Orthobunyavirus, Phelebovirus and Tospovirus), a classification based on structural, genetic and antigenic characteristics [9]. The first four genera include viruses that infect vertebrates, including humans, whereas the fifth genus contains viruses that are known to cause disease in plants, not animals [9]. Exemplifying the great diversity of species classified within this family, the discovery of Gouléako (GOLV) and Cumuto (CUMV) viruses, during arbovirus surveillance study in Côte d’Ivoire [10] and Trinidad and Tobago [11], suggests a new novel genus evolutionarily related to but distinct from the genus phlebovirus. More recently, a unique clade defined by the restricted host range Herbert (HEBV), Tai (TAIV) and Kibale (KIBV) viruses, forms a phylogenetically sister group to the genus Orthobunyavirus [6], whereas Jonchet (JONV) and Ferak (FERV) viruses are likely two equally novel sister genera [5].

Flaviviridae

Biologically, viruses in the genus Flavivirus can be divided into 3 broad groups, based on their arthropod and vertebrate host associations [12, 13]: (i) dual-host viruses which are maintained in an alternating arthropod/vertebrate cycle [14], which includes most of the flavivirus pathogens; and (ii) insect-specific flaviviruses (ISFs) which naturally infect mosquitoes and replicate in insect cells but do not infect vertebrates or vertebrate cell lines [15]; and (iii) vertebrate-only flaviviruses [also known as ‘no known vector’ (NKV) group, which naturally infect and are transmitted between vertebrates but have no known arthropod-association [12]. Some of the bat salivary gland viruses (i.e. Rio Bravo) and rodent-associated flaviviruses (i.e. Modoc) are examples of this group [16]. Viruses in the NKV group infect vertebrates (including sometimes humans), but they do not infect arthropods or insect cells [13]. The ISFs form two distinct phylogenetic and antigenic subgroups or clades: (i) one clade includes viruses, such as the cell fusing agent (CFAV)[17, 18], Culex flavivirus (CxFV)[19–22], Aedes flavivirus (AeFV)[23–27], Kamiti River (KRV)[28, 29], and others (reviewed in [30]), which forms a distinct clade apart from the main flavivirus tree of vertebrate pathogens; and (ii) a second clade, which currently includes Aripo [JA Auguste et al., unpublished], Barkedji (BJV) [31], Chaoyang (CHAOV)[32–34], Donggang (DONV) [N Vasilakis and RB Tesh, unpublished], Ilomantsi (ILOV)[35], Lammi (LAMV)[36], La Tina [N Vasilakis and RB Tesh, unpublished], Marisma (MMV)[37, 38], Nanay (NANV) [39], Nhuhirim (NHUV) [40] and Nounané (NOUV)[41] viruses (and reviewed in [30]), which are antigenically and phylogenetically related to and fall within the dual-host group of flavivirus pathogens in close proximity to WNV, DENV and YFV.

Reoviridae

Members of this family are classified into two subfamilies, Spinareovirinae and Sedoreovirinae, based on their core structures, and both have a wide geographic distribution and wide host range, as they have been isolated from fungi, plants, insects, ticks, arachnids, fish, marine protists, crustaceans, mammals, and birds [42]. Phylogenetic analysis of a single isolate, Aedes pseudoscutellaris reovirus (APRV), a host-restricted virus isolated from the AP61 mosquito cell line in 2005, indicates that it represents a distinct genus (genus Dinovernavirus) within the subfamily Spinareovirinae [43]. A second member of this genus, the host-restricted Fako virus (FAKV), isolated in 2010 during a surveillance study in Cameroon, has the closest genetic and structural relationships are with reoviruses in the genera Cypovirus and Oryzavirus [44]. Cimodo virus (CMDV), is also a unique ISV that belongs to the family Reoviridae. It was isolated in Côte d’Ivoire and likely defines a new genus within the subfamily Spinareovirinae, closely related to myco- and coltiviruses [45].

Rhabdoviridae

The family Rhabdoviridae is comprised of a diverse group of non-segmented, negative-sense RNA viruses that infect a wide range of vertebrates, invertebrates and plants [46], distributed across 17 recognized and proposed genera [47]. Vector-borne rhabdoviruses are present in 12 of the 17 groups, and most prevalent in the dimarhabdovirus supergroup. Two host-restricted rhabdoviruses in mosquitoes, Arboretum (ABTV) and Puerto Almendras (PTAMV) viruses, were isolated from pools of female Ochlerotattus fulvus and Psorophora albigenu mosquitoes, during an arbovirus surveillance in Peru, in 2009 [48]. Experimental infections in vitro failed to produce detectable CPE in vertebrate cell lines, and similarly with in vivo studies, both viruses failed to produce illness and/or death in newborn mice. Since both viruses have only been detected once in mosquito pools, a comprehensive assessment of host range remains to be determined through surveillance and experimental studies.

Togaviridae

The genus Alphavirus within the family Togaviridae, consists of 29 species grouped into 10 complexes based on antigenic and/or genetic similarities [49, 50]. Alphaviruses exhibit a wide host range that includes vertebrates such as fish, equids, birds, amphibians, reptiles, rodents, pigs, humans, and nonhuman primates as well as and insect hosts, including mosquito species encompassing at least six genera and also ticks and lice (reviewed in [51]). While the fish alphaviruses exhibit a restricted host range (replication only in fish), the only known host-restricted alphavirus of mosquitoes is Eilat virus (EILV) [52], which was isolated during a two-year (1982–1984) arbovirus survey of the Negev desert in Israel, from a pool of Anopheles coustani mosquitoes [53].

Mesoniviridae

The newly recognized family Mesoniviridae consists of single-stranded RNA, positive-sense RNA viruses belongs within the order Nidovirales [54]. To date, members of this family have only been isolated from various pools of mosquitoes within the family Culicidae [3, 55–60] and appear to have an extensive geographic distribution. The family Mesoniviridae includes a single genus Alphamesonivirus with seven recognized species: Alphamesonivirus-1 includes the two closely related viruses, Nam Dinh (NDiV)[55], Cavally (CavV)[56], and the recently characterized NDiV Ngewotan [3] and NDiV Houston [3]; Alphamesonivirus-2-4 includes the previously described African mesoniviruses, HanaV, MenoV, and NseV, respectively [57]; Alphamesonivirus-5 includes the four Bontag Baru (BBaV) isolates sampled in the early 1980’s in Indonesia [3]; Alphamesonivirus-6 includes the single isolate of Karang Sari (KSaV) virus, sampled in Indonesia [3]; and Alphamesonivirus-7 includes the three isolates of Kamphaeng Phet (KPhV), sampled in Thailand in the mid-1980’s [3]. Moreover, recent phylogenetic and DEmARC-based analyses involving all these viruses using either all replicase non-structural proteins (nsps) or its large subset (from 3CLpro to OMT domains), reassigns the above viruses in five Alphamesonivirus species and two unassigned species (A. Gorbalenya, personal communication).

Negeviruses

The newly recognized taxon of Negeviruses consists of single-stranded RNA of positive polarity viruses consists of six prototype ISVs that have been designated as Negev (NEGV), Ngewotan (NWTV), Piura (PIUV), Loreto (LORV), Dezidougou (DEZV) and Santana (SANV), which were isolated from mosquitoes and phlebotomine sand flies collected in Brazil, Peru, USA, Ivory Coast, Israel and Indonesia [2]. Subsequently, others have reported the isolation and characterization of negeviruses from the Philippines (Tanay virus (TANAV))[61], Trinidad (Wallerfield virus (WALV))[11], Côte d’Ivoire (Goutanap virus (GANV))[62], Brasil (Brajeira and Wallerfield virus (M Nunes, personal communication)), Colombia and the USA (N Vasilakis and RB Tesh, unpublished). Phylogenetic analysis of the prototype negeviruses demonstrated that their closest but still distant relatives are mite-transmitted plant viruses in the genus Cilevirus, which occupied a basal position [2]. Moreover, recent analyses that expanded the sequence dataset to include the more recently discovered viruses suggest that negeviruses may exhibit more complex taxonomic relationship on the genus level [11, 61, 62]. Their close relationship with plant viruses of the genera Cilevirus, Higrevirus and Blunervirus, but heterogenous genome organization and architecture suggests that the Negevirus taxon may eventually be designated as a new virus family [62].

Evolution

To date there is little known about the evolution of ISVs. Confounding this limitation is the lack of available data about their host range, ecology and distribution, which underscores the necessity of further studies to decipher their evolution. What is known is that ISVs belong to viral families where many arboviruses are resident [6, 10, 25, 44, 53] or occupy clades with large phylogenetic distances to established families [2, 55]. A number of recent studies [66, 69, 70] indicate that many of the ISVs in the families Bunyaviridae [1, 5], Flaviviridae [63] and Rhabdoviridae [47] are ancient with highly diverse lineages suggesting that they likely evolved and diversified with their insect hosts. This notion that ISVs have been closely associated with their insect hosts for a long period of time is further supported by the fact that many of these appear to be vertically transmitted, whereas some become integrated within the genomes of their arthropod hosts [64]. Many of the extant arboviruses likely evolved from being ISVs to dual host viruses gaining the ability to infect the vertebrates upon which their insect hosts fed [1], and some may have completely adapted to their vertebrate hosts and lost the need for an insect host [1, 16]. Given the long evolutionary history and genome plasticity of ISVs, it is likely that others will develop the ability to infect vertebrates and thus become new emerging pathogens.

Influence on vector competence

The overwhelming evidence on the influence of ISVs on vector competence is derived from in vitro and in vivo experiments with ISFs. Initial in vivo studies with several Culex species suggested that mosquitoes infected with CxFV were less susceptible to secondary WNV infection than control mosquitoes (those without CxFV infection) [65]. Subsequent studies with another ISF, Palm Creek virus (PCV), indicated that it reduced replication of Kunjin (KUNV) and Murray Valley (MVEV) viruses in dually infected C6/36 cells [66]. A recent report indicated that NHUV significantly reduced replication of WNV, Japanese encephalitis (JEV) and St. Louis encephalitis (SLEV) viruses in co-infected C6/36 cells [67]. The mechanism of this reduction is unknown; but competitive inhibition is one possibility. The in vitro studies were performed in C6/36 cells, which have a dysfunctional RNAi response [7, 8] so the obvious next step is to determine if inhibition occurs in vivo (in live mosquitoes). The data suggest that the inhibition does not occur with all arboviruses, since the most pronounced reduction was observed in co-infection with WNV and SLEV (10⁶ - to 10⁴, respectively), whereas JEV co-infection exhibited an 80-fold reduction from peak titer [67]. A recent publication [68] indicated that EILV, an insect-specific alphavirus, also induces heterologous interference with several other alphavirus pathogens.

Possible Modes of Transmission

Currently the mechanism of ISV transmission and maintenance in nature is not known. Experimental [65, 69, 70] and field studies [19, 26, 29] suggest that transmission of insect-specific viruses in the genus Flaviviridae could be maintained by transovarial transmission (from adult female to progeny). Another possible mechanism may be venereal transmission as recent observations such as tissue tropisms of CxFV in C. pipiens [70] and CxFV transmission from naturally infected males to naïve females [65] support this notion. Moreover, the extensive mosquito host-range of mesoniviruses and negeviruses within the family Culicidae (Aedes, Culex, Anopheles, Armigeres, Psorophora, Uranotaenia, Deinocerites, Wyeomyia and Trichoprosopan)(N Vasilakis and RB Tesh, unpublished) and their inability to infect and replicate in laboratory animals and/or various vertebrate cell lines [2, 3] suggest that their hosts do not acquire them from feeding on viremic animals. Based on the negeviruses genomic organization, phylogenetic topology and close relationship to plant viruses of the genera Cilevirus, Higrevirus and Blunervirus [2, 11, 62], it is possible that their adult insect hosts acquire them by feeding on plant juices (e.g. nectar) or feeding on plant material present in the aquatic environment during their early stages of development [71]. Therefore elucidation of the mechanisms of ISV maintenance and transmission in nature, as well as homeostasis effects on their mosquito hosts, is important for understanding how they could potentially modulate arbovirus transmission.

Conclusion and future directions

The discovery of numerous ISVs in recent years within many virus families that harbor arboviruses of extreme public health importance has provided new insights into the complex nature of vector-borne disease systems. Experimental studies have demonstrated their host-restricted range in various arthropod vectors, in contrast to arboviruses, sensu strictu, which exhibit a dual host range in both vertebrate and invertebrate hosts. Phylogenetic analyses have also shown that a large number of these ISVs are closely related to human arboviruses, suggesting they may influence arbovirus transmission. However, many questions remain unanswered. Of highest priority, future studies should explore the ecology and distribution of these viruses in nature as well as their effect on their host homeostasis (e.g. pathogenicity and effect on their vector competence). Understanding microbial diversity in vector hosts is important for control strategies. Given that bacterial endosymbionts have shown promise as control agents in reducing vector competence of mosquitoes to transmit pathogenic arboviruses [72] or modulating their host’s immune response to subsequent arbovirus infection [73, 74], it is likely that ISVs may have a similar effect and limited experimental studies [65, 66, 75] support this notion. Thus, further studies are warranted to investigate the potential for using ISVs as an innovative method for vector-borne disease prevention. Lastly, their potential use as platforms for vaccine or diagnostic development should be further exploited given their restrictive host-range facilitated by their defect for vertebrate cell infection at both the entry and RNA replication levels [76]. This inherent safety characteristic should be exploited to use recombinant DNA technology to generate candidate vaccines against emerging arboviruses of public health importance.

Highlights.

• ISVs are novel RNA viruses associated with hematophagous insects

• Belong in diverse families such as Bunyaviridae, Mesoviviridae, and Rhabdoviridae

• Little is known about the determinants of the ISVs restricted host range

• Little is known about their mode of transmission and maintenance in nature