Copyright © 2007 by The American Mosquito Control Association, Inc. M 4229

MOSQUITO PATHOGENIC VIRUSES-THE LAST 20 YEARS James J. Becnel and Susan E. White USDA/ARS, 1600 S.W. 23rd Drive, Gainesville, FL 32608

KEY WORDS Baculovirus, nucleopolyhedrovirus, cytoplasmic polyhedrosis virus, cypovirus, densovirus, brevidensovirus, iridovirus, diagnosis, pathology, transmission ABSTRACT. There are several types of viral pathogens that cause disease in mosquitoes with most belonging to 4 major groups. The most common viruses of mosquitoes are the baculoviruses (NPV5) (Baculoviridae: Nucleopolyhedrovirus) and cytoplasmic polyhedrosis viruses (CPV5) (Reoviridae: Cypo- virus). The other major types of viruses in mosquitoes are represented by the densoviruses (DNVs) (Parvoviridae: Brevidensovirus) and the iridoviruses (MIV5) (lridoviridae: Chloriridovirus). Baculoviruses, densoviruses and iridoviruses are DNA viruses while cypoviruses are the main RNA viruses in mosquitoes. This chapter presents an overview of the recent advancements in the study of mosquito pathogenic viruses and discusses how this new understanding of virus-mosquito interactions can be used to develop novel research and control strategies.

INTRODUCTION OCCLUDED AND NON-OCCLUDED VIRUSES Viruses are extremely small particles (usually ranging from 20-500 nm) composed of a capsid, Viral diseases of insects can be divided into 2 core and genetic material (either DNA or RNA). general groups, those where the virions are These pathogens are some of the smallest and embedded in a protein matrix (occluded viruses) simplest organisms capable of replication. They and those that are not embedded (non-occluded). are obligate intracellular parasites and cause The most common occluded viruses of mosquitoes disease by taking over the host cells machinery are the baculoviruses (NPVs) (Baculoviridae: Nu- to replicate and produce virions that often can cleopolyhedrovirus) and cytoplasmic polhedrosis lead to the death of cells and the host organism. viruses (CPVs) (Reoviridae: Cypovirus). Entomo- The first modern report of a mosquito pathogenic poxviruses are a third group of occluded viruses in virus was made in 1963 with the discovery of mosquitoes but will not be discussed further a putative densovirus from Culex tarsalis in because no new information has been published in California (Kellen et al. 1963). Additional viruses the past 20 years (Federici 1985). The main non- pathogenic to mosquitoes have been reported occluded viruses in mosquitoes are represented by from many different mosquito hosts, primarily by the densoviruses (DNVs) (Parvoviridae: Breviden- researchers in the USA, Europe and Russia sovirus) and the iridoviruses (MIVs) (Iridoviridae: (Federici 1985). Research in this area continued Chloriridovirus). Baculoviruses, densoviruses and through the early 1980s and was thoroughly iridoviruses are DNA viruses and will be discussed reviewed by Federici (1985) but for much of the first followed by a discussion of the cypoviruses intervening 20 years there has been little activity. whose nucleic acid is double stranded RNA. Part of this may be due to the very successful development of Bacillus zhuringien.sis var. israe- DETECTION AND DIAGNOSIS OF lensis and B. sphaericus as mosquito biopesticides MOSQUITO PATHOGENIC VIRUSES (see chapters by Lacey (p. 133) and Federici (p. 164) in this volume). Another possible reason Federici (1985) described methods to examine is that none of the viruses isolated were consid- mosquito larvae for symptoms of viral disease and ered to be suitable for development as biopesti- these techniques are still used today. Infected cides. This was due primarily to the inability to mosquito larvae often exhibit behavioral changes easily manipulate and transmit these viruses and such as body contortions, lethargic motion, or the lack of methods for mass production. Re- hanging near the water surface. Regions of swelling cently, there have been tremendous advances in and/or clearing of the cuticle and unusual pigmen- the ability to transmit some groups of mosquito tation may also be signs of a possible viral infection. pathogenic viruses as well as new molecular tools Field-collected larvae are strained through an i. and capabilities to understand and manipulate appropriate sized sieve, placed on a petri dish and these viruses at the molecular level. This chapter the excess water removed with a pipette or paper. will present an overview of the recent advance- The larvae can be viewed against a dark back- ments in the study of mosquito pathogenic ground with a dissecting microscope. The larval viruses and discuss how this new understanding A recent proposal to revise the family Baculoviridae of virus-mosquito interactions can be used to creates the genus Deltabaculovirus for dipteran specific develop novel research and control strategies. baculoviruses (Jehle et al. 2006).

36

Biorational Control of Mosquitoes 37

midgut is best seen from the ventral side and can be slide placed on top of a small petri dish tilled with seen by turning the petri dish over to examine the water which makes contact with the bottom of larvae pressed to the glass surface. the slide. Water is placed in-between the black Larvae infected with iridoviruses appear uni- plate on the dissecting scope and the petri dish. formly light in color, appearing brownish-orange, This gives an appearance of dark infinity and turquoise-blue or white in color (Fig. 1E). The reduces the interference of the background. Live larvae might look swollen and larger in size when specimens can he slowed by placing the slide on compared to healthy larvae of the same age. a frozen ice pack for a few minutes prior to These are easily identified without the aid of photographing. Midguts can be dissected in microscopes when examined against a dark back- a fixative of choice (glutaraldehyde, formalin) so ground. Black pans or tightly woven black cloth that they retain their shape during photography. make satisfactory backgrounds with sufficient water for the larvae to float. When fresh smears DNA VIRUSES IN MOSQUITOES are examined, the infected fat body quickly disintegrates into whitish fluid with submicro- BACULOVIRIDAE scopic particles causing Brownian motion. There are currently 2 recognized genera within Larvae infected with baculoviruses or cypo- the family Baculovi ridae, viruses have whitish cysts or nodules in the Nuc/eopo/vhedro virus and Granulo virus. Members of the genus Nucleo- midgut (Figs. 1C, ID). These can be found polyhedro virus have occlusion bodies that contain throughout the midgut and gastric caeca or many virions while members of the genus isolated in one or two regions of the gut. in Granu/o virus have occlusion bodies that contain baculovirus infections (Fig. IC) the cysts are one, or rarely two virions. The baculoviruses hypertrophied nuclei, appear round, and when found in mosquitoes thus far have been restricted the gut is examined under a phase microscope, to the Nucleopolyliedro virus group (NPV). Since the occlusion bodies can be seen in the nuclei. the first report of a baculovirus (OcsoNPV) These sometimes break apart into smaller parti- isolated from Och/erotatus sollicitans (formerly cles as the nuclei disintegrate. Cypoviruses are the Aedes so/licitans) in Louisiana (Clark et al. 1969), reverse (Fig. ID): the inclusion bodies are in the naturally occurring NPVs have been isolated cytoplasm that appears blue to white and the from 13 additional mosquito species in the genera nuclei are compressed, almost not detectable. The A edes, Anopheles, Culex, Ochierotatus, Psoro- infected cells will look irregular; they usually do and Wyeoniyia (Murphy et not disintegrate in water and retain their cell phora, (]ranotaenia, al. 1995). More than 20 mosquito species have membrane when smeared in water. Some healthy been found to he susceptible to NPV infections larvae have normal physiological states that (Federici 1985, Becnel etal. 2001, Andreadis et al. appear similar to cypoviruses. These larvae 2003, Shapiro et al. 2004). frequently have a large quantity of glycogen that gives the gut a milky-white appearance (Fig. I B). Distinguishing Characteristics In this case, however, the nuclei of the midgut Mosquito NPVs are specific for midgut tissues cells are not compressed but are readily discerned. (primarily midgut epithelium) of mainly larval gastric caeca are not involved and if held over- mosquitoes (Federici 1985, Becnel et al. 2001, night, the gut returns to normal transparency. Shapiro et al. 2004) but infections have been Another diagnostic technique is to purify found in adult midguts (Becnel et al. 2003). a suspected virus on a continuous density Patent infections are detected by the hypertro- gradient made with sucrose, cesium or IIS-40 phied nuclei of midgut cells that appear white due Ludox®. Occluded viruses have a density of 1.12- to the accumulation of occlusion bodies 1.18 (Moser et al. 2001, Shapiro et al. 2005) and (Fig. IC). Occlusion bodies can be relatively large will form a band below host tissue and bacteria. (5 20 j.t) and polyhedral in shape or small (0.5 ) Some occlusion bodies may he sensitive to pH and globular in shape (Federici 1985, Becnel et al. and will be lost if not held at an alkaline p1-1. HS- 2001, Shapiro et al. 2004). The virions are 40 Ludox® has a density of 1 .3 and can he used to approximately 60 x 250 nm, rod-shaped and purify occluded and nonoccluded viruses. assemble in the nuclei of midgut cells. Mosquito NPVs are highly virulent with infected larvae PHOTOGRAPHY OF GROSS SIGNS usually dying 72 96 h post-exposure. Prior to 2001, the only detailed studies on development An essential part of examining mosquitoes for and transmission of mosquito NPVs were con- viral infection is to document the gross signs. ducted with the isolate OcsoNPV from Louisiana Black is the best background to visualize the in several different mosquito hosts (Federici and diagnostic characteristics of many viral diseases Lowe 1972, Federici 1980, Stiles et al. 1983) and in mosquitoes. The photographs presented here summarized by Federici (1985). Since 2001, (Figs. IA—lE) were made with a specimen posi- considerable new information has been reported tioned on a glass slide with minimal water and the for a mosquito NPV from Cx. nigripalpu,s AM(A Bulletin No. 7 Voi. 2, Supplement to No. 2

1 ig. 1 . Signs of mosquito \ 1 II I U ii .cs. (A) Healthy ( alex qiti,iqtwla.veiaiu.v larvalgut. ( B) Flealt hy Codex alniwius larval gut with cloudy cells in the posterior midgut, gi ing a milky appearance. Arrow points to milky cell a ith clear nucleus. (C) Colex qtwzque/as!carus larval midgut infected with the baculovirus CuniNPV. Arrow points to hvpertrophied nuclei with clear c ytoplasm. ( D) ( ,anotaenia .vapphiriira larval midgut infected with the CpOviruS I t( PV. (F) Ohf,otooi. itt /l jorht,it has larvae: (toll) health y. (bottom) infected a itli the rido irus R-M IV.

1)1 oration al Control 01 M osq in tocs 39

Basement Membrane Virus / Replication 41 Ocduded Virus

MidgutCell irogenic

Virus ( flflN r.I. M icrovi Iii I — - Peritrophic //// Matrix Mg 2+ Mg Midgut Lumens Mg 2+ Mg2+ 2+ Fig. 2. Diagram of the life cycle of the baculovirus CuniNPV in larval Cule.v mosquitoes.

(CuniNPV) (Becnel et al. 2001. Moser et al. 2001, 72-96 It p.i. at which time most nuclei in the Afonso et al. 200!) and to a lesser extent for an posterior stomach and gastric caeca are infected. N PV from Uranotaenia .vapp/urma ( Ursa N PV) (Shapiro et al. 2004). Information on Life Cycle CuniNPV will be described in detail to illustrate A cascade of events is required for the invasion, the current understanding of the biology, mor- replication and spread of CuniNPV in the phology and genomics of mosquito baculo- mosquito host (Fig. 2). Infections of CuniNPV viruses. are initiated when larval mosquitoes ingest occlu- sion bodies together with the appropriate divalent Gross Nlorphologv cation, usually magnesium (see transmission sec- CuniNPV affects the development, behavior and tion below). Occlusion derived virions (ODVs) are appearance of infected Cy. quinque/a,rciatus and released from occlusion bodies due to the alkaline Cx. nigripaljnis larvae (Becnel et al. 2001. Moser conditions of the midgut together with other et al. 2001). Within 24 Ii post infection (p.i.), possible factors. The released ODVs attach and infected larvae are typically stunted in size when pass through the peritrophic matrix (PM) followed compared to unexposed individuals, indicating by attachment to the membrane of the microvilli a failure to molt or problems with nutrient and entry into the cytoplasm of midgut epithelial uptake. Larvae actively feed through about 48 h cells. Invasion of the nucleus occurs when p.i., but by 72 It p.i. larvae are lethargic and often nucleocapsids attach to the nuclear envelope where remain suspended at the water surface. The larvae DNA is released into the nucleoplasm to initiate of Cx. quinquct usc/al us and Cy. nigripalpus have viral replication. This process of ingestion of a relatively clear cuticle allowing the infected cells infectious particles to the invasion of the nucleus of the midgut to be detected with a dissecting occurs within 2-4 h post exposure to occlusion microscope (Fig. IC). By 48 h p.i., nuclei of most bodies. The next phase of the process involves the cells in the gastric caeca and posterior stomach rapid spread of CuniNPV to other midgut cells. are opaque to white in color due to the pro- The initial viral replication produces nucleocapsids liferation of occlusion bodies (OBs). Cells in the that are released from the nucleus that form anterior stomach rarely support viral develop- extracellular virions (the budded virion, BV) that rnent. Death of the larvae usually occurs within are released out of the midgut cells into the

41) AMCA Bulletin No. 7 VOL. 23. Supplement to No. 2

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Fig. 3. Electron micrographs of OCL ud d ii in mosquito midg ut, (A) Cu/es qiiuu/ii to nu/l/is larval midgut cell infected with the baculovirus CuniNPV. Inset. Occlusion bodies. (B) Li 000/aenia sapp/miruia larval midgut nuclei with the baculovirus UrsaNPV. (C) Uranotaenia .sapp/urina larval midgut cells with the cypovirus UsCPV. Insert: inclusion bodies. (D) Cu/ex ,cstuans larval midgut cells infected with the cyposirus CrCPV.

ectoperitrophic space. The BVs can then attach to Ultrastructure microvilli of other midgut cells and make their way Mature OBs are globular in shape and uniform to the nucleus to initiate another round of in size with a diameter of approximately 400 nm replication. At some point, nucleocapsids acquire (Fig. 3A). The OBs lack an envelope (the poly- de novo envelopes in the nucleus and become hedron envelope of NPVs) that is a characteristic occluded by the occlusion body protein. The nuclei feature of most other baculoviruses. Each OB become packed with occlusion bodies (Fig. 3A) typically contains 4 rod-shaped virions but some- that are released upon death of the host and cause times contains up to 8 virions. Nucleocapsids are infection when ingested by a susceptible mosquito singly enveloped and the resulting virions are larva. The spread within the mosquito midgut and approximately 200 X 40 nm. Each virion consists production of occlusion bodies occurs within 14- of the nucleocapsid, intermediate layer and an 48 h post exposure. outer envelope. The OBs of CuniNPV do not

Biorational Control of Mosquitoes 41 coalesce into larger occlusion bodies as found for (Becnel et al. 2001, Andreadis et al. 2003). These OcsoNPV (Federici and Anthony 1972) and include Cx. nigripalpus, Cx. quinquefasciatus, Cx. IJrsaNPV (Shapiro et al. 2004). Early occlusion .salinarius, Cx. pipiens. Cx. pipiens f. ,nolestus, and bodies of UrsaNPV are irregularly shaped and (7x. rest uans. Cx. territans (subgenus Neoculex) is seem to subsequently coalesce to form large the only O.1e.v species tested that is not suscep- polyhedra (Shapiro et al., 2004). Mature occlusion tible to CuniNPV (Andreadis et al. 2003). No bodies have a dumbbell shape, lack the poly- infections have been found with any species of hedron envelope, and measure up to 10-15 pm in Aede,r (Ac. aegypti, Ac. albopietus). Anopheles length and 2-3 tm in diameter (Fig. 313). (An. alhi,nanus, An. quadri,naculatus), Ochierota- tus (Oc. triseriatu.s, Oc. taeniorhynchus), Culiseta Transmission melanura or Toxorhynch lies amhionen,cis. Purified Prior to work with CuniNPV, transmission suspensions of CuniNPV ODVs administered studies had only been conducted with the NPV either per os or by injection to larvae of from Oc. sollicitans (OcsoNPV). Transmission Ilelicoverpa zea failed to establish infections tests using a viral suspension of OcsoNPV to the (unpublished data). The very restricted host range natural host generally yield low infection levels of CuniNPV to only Culex spp. differs from that average only about 14% (Clark and Fukuda previous transmission studies with OcsoNPV 1971). While infection levels for OcsoNPV vary showing that Aedes, Ochierotatus, and Psoro- with the host and age of mosquitoes, the virus is phora spp. are susceptible but not Cu/ex and generally difficult to consistently transmit (Stiles Anopheles spp. (Clark and Fukuda 1971). and Paschke 1980). Transmission of CuniNPV to larval mosquitoes in the laboratory in deionized Field Epizootiology water usually results in less than 1% infection Natural epizootics of CuniNPV have been levels (Becnel et al. 2001). This situation changed studied at 2 field sites in Florida (Becnel et al. dramatically when divalent cations were found to 2001). One of these was a swine wastewater site play a crucial role in the transmission of with Cx. nigripalpus dominant during the warmer CuniNPV. Deletion analysis of the principal months and Cx. quinquefasciatus dominate dur- cations present in the field water found the ing the cooler months. Regular and extended water-borne factors critical for transmission of epizootics of CuniNPV in (7x. nigripalpus are CuniNPV. The addition of salts to deionized documented at this site with an average infection water significantly improve infection levels in rate of 20.1% and a maximum rate of 60% larvae. Salt mixtures without Mg` result in less (Becnel et al. 2001). Culex quinqueflisciatus larvae than 1.0% infections. Salt mixtures with Mg` or have an average CuniNPV infection rate of 8.6% without Ca- increase infection levels (Becnel et al. and a maximum infection rate of 20%. The 2001). Further investigations found that trans- second site is a dairy wastewater facility where mission is mediated by divalent cations where Cx. quinquefiisciatu.c is the dominant species. magnesium is essential for transmission while the Although high larval populations of Cx. quinque- presence of calcium inhibits the activity of jàsciatu.s are present CuniNPV infected larvae magnesium to mediate transmission. Transmis- have been collected on only 5 occasions and never sion of UrsaNPV is also driven by cations similar at epizootic levels (0.08% infection). The Mg"/ to that found for CuniNPV (Shapiro et al. 2004). Ca" ratios at the swine wastewater site are 1.9 / Transmission of CuniNPV is also enhanced by the ().8 mM while these ratios at the dairy wastewater divalent cations barium, cobalt, nickel, and site were (3.7 / 3.0 mM). It appears that the strontium or inhibited by copper, iron, and zinc favorable Mg2 /Ca2 ratios at the swine wastewa- (Becnel et al. 2001, Andreadis et al. 2003). ter site mediated transmission of CuniNPV while To produce high levels of infection in mosqui- the high calcium levels at the dairy wastewater toes with CuniNPV, Mg2 must be present only site are unfavorable for transmission. during the 1st hours of CuniNPV exposure Molecular Characteristics indicating that the activity of Mg2 occurs early in the infectivity process. Electron microscopic The CuniNPV genome is a circular dsDNA observations of occlusion bodies in the midgut molecule of 108,252 base pairs (50.9% CG lumen revealed that Mg2 is probably not required composition), which encodes 109 putative genes for dissolution (Moser et al. 2001). Other possible (Afonso et al. 2001, Moser et al. 2001). There are targets requiring Mg2 are proteases that specifi- 36 genes in the CuniNPV genome with homo- cally attack the PM allowing virions to cross this logues to other baculoviruses; however its lack of barrier to infection or to facilitate the attachment conservation in gene order, and the absence of of virions to receptors on the PM or microvilli of homologues of numerous genes present in known midgut epithelium to facilitate entry into the cells. lepidopteran and hymenopteran baculoviruses, suggest significant evolutionary distance between Host Range Dipteran and other baculoviruses (Afonso et al. CuniNPV has only been transmitted to mos- 2001, Lauzon et al. 2004, Garcia-Maruniak et al. quitoes within the genus Culex, subgenus Culex 2004). Phylogenetic analysis of 13 baculoviruses

42 AMCA Bulletin No. 7 VOL. 23, Supplement to No. 2

(Herniou et al. 2003) separated the group into 4 Ultrastructure distinct lineages, Group I NPVs, Group 2 NPVs, The virions of MIVs are icoshedral with an Granulosis viruses, and CunINPV. The authors electron dense core of DNA surrounded by an concluded that CunINPV represents a new hacu- intermediate membrane-like lipid layer contained lovirus genus supporting the earlier conclusions within a proteinaceous capsid (Fig. 413). Virions of Moser et al. 2001. range is size from 180- 200 nm in fixed sections. CuniNPV contains genes with potential host The principal site of infection for MIVs is range functions including a p35 homologue larval fat body with the epidermis as the next (Cun75, antiapotosis gene), homologues of per most commonly infected tissue. os infectivity factors (Cun29 and Cun38) that are Transmission important in infectivity of lepidopteran and RMIV is host specific, infecting only hymenopteran baculoviruses (Lauzon et al. Oc. taenwr/iynchus and Or. so//ic/tans (Woodward 2004. Kikhno et al. 2002, Pijlman et al. 2003). and Chapman 1968, Becnel and Fukuda 1989). The presence of novel cellular homologues The virus is horizontally transmitted and usually suggests unique mechanisms of viral-host in- results in infection levels of less that 20% teraction in mosquito baculoviruses and might (Woodard and Chapman 1968, Undeen and have application for the identification of novel genes and gene products. Fukuda 1994). RMIV can also be transmitted transovarially with high levels of infection in progeny (Woodard and Chapman 1968, Linley I RIDOVIRIDAE and Nielsen 1968, Hall and Anthony 1971, Fukuda and Clark 1975). Infected larvae usually Mosquito Iridescent Viruses (MIVs) are mem- die as 4th instars due to destruction of the fat bers of the family lridoviridae that includes body. The natural transmission cycle of RMIV in isolates from both vertebrate and invertebrate Or. taeniorhync/ius populations has been proposed hosts. The first report of an MIV was by Clark et to occur by alternating horizontal and vertical al. (1965) isolated from Or. taeniorhtnc/ius in transmission events (Linley and Nielson 1968). Louisiana. Recent studies have demonstrated that To evaluate the non-susceptibility of most of MTVs are distinct from other known iridescent the mosquito population exposed to RMIV, viruses and they have been placed in a new genus tindeen and Fukuda (1994) examined the possi- Ch/oriridoviru.s (Williams 1996, 1998). Currently, bility that resistance is genetic and that RMIV the genus chloriridoiirus contains only the type has no specific means of entering the host. To test 1V3 from Oc. taenior/iynchu.s (Becnel 2002). for genetic resistance, mean infection rates in Distinguishing Characteristics randomly selected and sibling Or. taeniorhynchus The MIVs are large, icosahedral viruses (ap- larvae were compared. There was no difference in proximately 180 nm in diameter) that replicate infection levels between the 2 groups rendering and assemble in the cytoplasm of the host, genetic resistance unlikely. Injury to larvae by primarily in fat body cells (Figs. 4A. 413). The feeding silicon carbon fibers consistently yielded paracrystalline arrays of virions that accumulate higher infection rates (17%) compared with only in the cytoplasm of host tissues produce iridescent 4% infection rates for virus alone. These authors colors when exposed to light and viewed against concluded that RMLV has no active means of • dark background. The genome is composed of entering the larval host but invades through • single molecule of linear double-stranded DNA. breaks in the cuticle or peritrophic matrix. MIVs have been reported in the USA, Russia and Mosquitoes are also susceptible to invertebrate Europe from Aedes, Psorophora, Cu/ex. and iridescent virus 6 (IIV-6) which was originally Cu/i,ceta spp. There have been few studies on isolated from Chi/o suppressa/is (Lepidoptera: MIVs over the past 20 years. Pyralidae) which is the type for the genus Iridovirus (Williams 1998). Sublethal (covert) Gross Pathology infections in adult Ac. aegypti results in reduced Infections in larvae are usually easily detected longevity, smaller body size and reduced fecun- due to the iridescent color produced by the dity than non-infected conspecifics (Marina et al. paracrystalline arrays of virions in fat body 1999. Marina et al. 2003a, 2003b). The net (Fig. 4A). The color can he yellow-green or reproductive rate for covertly infected females is orange and sometime turquoise (Fig. lE). Matta reduced by 50% (Marina et al. 2003b). and Lowe (1970) designated the originally iso- lated virus in Or. taenwrhvnchus as RMIV Molecular Characteristics (regular mosquito iridovirus) that produces an With the exception of the genus Chioriridovirus, orange iridescence in larvae. This was to separate complete genome DNA sequences have been it from a spontaneously formed laboratory strain determined for viruses representing all IV genera. of the virus designated TMIV (turquoise mos- Recently, the DNA sequence and analysis of the quito iridovirus) that produces a blue-green MIV genome has been completed (Delhon et al. iridescence in Oc. taeniorhvnchus. 2006). The 190,132 bp MIV geriome has a 48% Biorational Control of Mosquitoes 43

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Fig. 4. Election microcLipli iiILILlcd \ ii \ I IL zaiiijoihiiii/iiii Lii infected with the iridovirus k-MI V in the c toplasm arranged in crystalline arrays. (B) R-M IV %irlolls. ((. ) C it/c. \ with probable densovirus in the nucleus and the cytoplasm of an imaginal bud cell. (D). Cuboidal inclusion of probable densosirus in the c ytoplasm of an imaginal bud cell.

GC content with 126 predicted MIV genes. Of cies, the Ae. aegt-pti densovirus (AaeDNV. these, 21% are present in all IV genera, including following the terminology of Bergoin and Tijssen enzymes and factors involved in viral transcription 1998) and the .4 e. albopictus densovirus and DNA replication. Genes only present in insect (AaIDNV) with a number of tentative species IVs represented 41% of the MIV genes. Twenty- reported (Büchen-Osmond 2003). The only ex- three percent of MIV genes, mostly of unknown ception to this is a mosquito isolate from function, have no homologues in the other IVs. a laboratory strain of (is-. pipiens (CpDNV) that appears to be most closely related to Junonia coenia densovirus (JcDNV) in the genus Denso- PARVOVIRIDAE virus (Jousset et al. 2000). The first report of Most mosquito densoviruses (M DVs) are a probable MDV was from C.v. iarsaliic larvae in members of the genus Brcvh./en.voviru.s w ithin the California (Kellen et al. 1963. Kellen et at. 1966) family Parvoviridae. The genus contains 2 spe- and subsequently found in Cx. sali,,arius isolated

44 AMCA Bulletin No. 7 VOL. 23, Supplement to No. 2

in Louisiana (Clark and Chapman 1969). The not glycosylated. The capsid consists of 60 First verified Ml)V (AaeDNV) was found in capsomers, each a quadrilateral kite-shaped a laboratory colony of Ac. aegpti in Russia wedge (Büchen-Osmon d 2003). Surface projec- (Lebedeva and Zelenko 1972). The second de- tions are small and the surface appears rough scribed MDV (AaIDNV) was isolated from an (Chen et al. 2004). Replication of AaIDNV Ac. albopictus cell line (Jousset et al. 1993, occurs in the nucleus and is first observed as Boublik et al. 1994a). MDVs have also been multiple dense bodies that appear to fuse isolated from natural populations of Ac. a/hop/c- (Barreau et al. 1996). As development progresses, Ins (Boublik et al. 1994b). Ac. aegvpri (Afanasiev the nuclei become greatly hypertrophied and et al. 1991. Kittayapong et al. 1999 and An. filled with particles arranged in arrays. Virions mininius (Rwegoshora ci a]. 2000), but mosquito are not found in the cytoplasm. Development of colonies and mosquito cell lines from species of AaeDNV is similar to AaIDNV except that A edes. Toxorhvnch ftcs. Haen ?agogus, and Cu/er paracrystaline arrays of virions are formed in have been the most common sources of MDV the cytoplasm without destruction of the nuclear isolations (Jousset et al. 1993, ONeill et al. 1995, envelope (Buchatsky and Raikova 1979). In the Jousset et al. 2000, Rwegoshora et al. 2000). putative MDV from Cr. larsa/is and Cy. sa/maruis, inclusion bodies are formed in the Distinguishing Characteristics cytoplasm of infected cells consisting of para- The M DVs are extremely small, isometric, erystaline arrays of virions. (Fig 3D) either nonenveloped viruses about 20 nm in diameter. cubodial or spherical and range in size from 2- Replication occurs in the nuclei and in some 6 .t (Kellen et al. 1963. 1966, Clark and Chapman species inclusion bodies. consisting of arrays of 1969). virons. are found in the cytoplasm (Figs. 3C, 3D). Most tissues of the host are susceptible but not Transmission midgut epithelium (Lebedeva et al. 1973, Jousset Considerable new information on the trans- et al. 1993). The genome is composed of linear, mission and the portal of entry for MDVs has single-stranded DNA of about 4 kbp in length. been obtained through the use of a recombinant Cross Pathology AaeDNV line expressing the reporter gene GFP Most MDVs have been isolated from colony (Afanasiev et al. 1999. Allen-Miura et al. 1999. mosquitoes or ccli lines because patent infections Ward et a]. 2001). These studies with Ac. aegtpti in larvae are more subtle than other mosquito larvae have confirmed that the virions do not pathogenic viruses. Mosquito larvae infected with enter via the midgut but that the primary site of AaIDNV often became lethargic and hang near viral entry is the anal papillae with bristle cells the the water surface and exhibit body contortions second most common entry point (Ward et al. such as a curved abdomen (Barreau ci al 1996). 2001). The virus then spreads to cells of the fat These infected larvae also lose their pigmentation body within about 2 days p.i. Subsequently, and exhibit a whitish color about I day prior to many other tissues are infected including muscle death. Field collected larvae of Cr. iarsalis and fibers and neurons. These studies also determined that older larvae (72-96 h old) are less susceptible C.Y. sa/inarius infected with a putative MDV are sluggish with abnormally curved abdomens (S- to AaeDNV infection and if infected are more shaped) and pupae often exhibit malformed parts likely to survive to the adult stage. (Kellen ci al. 1963. Clark and Chapman 1969). Horizontal transmission of MDVs is dose and Small dark shiny regions are often present on the age dependent with higher doses resulting in larval thorax (sometimes on abdominal segments) higher rates of larval mortality. AaIDNV results and when these tissues are examined with phase in 90% mortality of Ac. aegtyni larvae exposed as microscopy. large, 2-3 js cuboidal inclusions were 1st instars (Barreau et al. 1996). A Thai-strain of observed. Similar signs of the disease are found densovirus (AThl)NV), isolated from an .4e. for AaeDNV except that the inclusion bodies are aegvpti colony (Kittayapong et al. 1999) kill up spherical rather than cuboidal (Lebedeva et al. to 80% of Ac. a/hop/dos. 50% of Ac. aeglpli and 1973. 1975). Many tissues of the mosquito are 17.5% of An. rn/n/inns larvae (Rwegoshora and susceptible to infection (fat body, hypodermis, Kittayapong 2004). At lower doses, large num- central nervous system, muscular membranes, bers of infected larvae survive to produce adults tracheal cells, imaginal disks, hemocytes) but not that carry the virus (Barreau et al. 1996. midgut epithelium (Lebedeva et al. 1973, Jousset Kittayapong et al. 1999, Ledermann et al. et al. 1993. Ward et al. 2001). 2004). There have been several reports of vertical transmission of MDVs (Buehatsky 1989, ONeil Ultrastructure et al. 1995, Barreau et a]. 1997, Kitta yapong et al. MDVs are extremely small with icosahedral 1999, Rwegoshora and Kittayapong 2004). For virlons of approximately 18-26 nm in diameter example, adult Ac. aegvpti infected as larvae with (Fig. 3D). The particles consist of single stranded AThDNV transmit the virus to 57.5% of their DNA and protein without a lipid layer and are offspring (Kittayapong et al. 1999). The MDVs

Biorational Control of Mosquitoes 45 are also reported to reduce adult longevity and laboratory animals (white common mice, rats, fertility (Barreau et al. 1996, 1997). It has also guinea pigs. rabbits), chicken embryos and cell been demonstrated that venereal transmission of cultures of warm-blooded animals. Despite the AaIDNV occurs at a low level (2.2%) when promising preliminary results with Viroden, healthy females of Ac. aegypti are allowed to large-scale tests of Viroderi were never conducted. mate with Aal[)NV infected males (Barreau et at. Further field studies of Ml)Vs as potential 1997) biocontrol agents will require production and standardization of large quantities of the virus Host Range that can potentially be achieved by growth in MDVs do not appear to be able to replicate in large-scale mosquito cell culture (Suchman and vertebrates or mammals (Bergoin and Tijssen Carlson 2004). 1998). AaeDNV could he horizontally trans- mitted to Ac. albopictus, Oc. cwltans, Oc. casplus, AaeDNV has also been developed as a gene Oc. dorsalis, Ac. geniculatus, Ae. vexans, Cx. transfer vector that is able to transduce genes into pipiens, and Cu. annulata (Lebedinets and Ze- mosquito larvae that can be infected via horizon- lenko 1975, Buchatsky 1989). AalI)NV is hor- tal transmission in the aquatic environment izontally transmitted to larvae of Ac. aegypti and through the anal papillae (Afanasiev et at. 1994, by injection to adult Ac. aegypti and Ac. 1999, Carlson et at. 2000). Positive aspects of ,nettalicus (Barreau et at. 1994, Barreau et al. MDVs as gene transfer vectors are their relative 1996). AaIDNV inoculated into adult Drosophila stability in the environment and that species such melanogaster and Spodoptera. lit toralis larvae do as AaeDNV can specifically infect mosquito not result in infection (Jousset et al. 1993). The larvae (Buchatsky et al. 1987). Two limitations putative MDV from Cx. salinarius (Clark and to using MDVs as tranducing vectors are the size Chapman 1969) was highly infectious for Cx. of the DNA that can be delivered (4-6 kb) and tarsal/s larvae but does not infect larvae of Cx. the necessity to use transfection to generate peccator, Cx. quinquefasciatus, Cu. inornala, or tranducing particles (Carlson et al. 2000). MDVs Oc. taeniorhync/ius. have considerable potential as tranducing vectors to conduct basic biological research on mosqui- Molecular Characteristics toes and perhaps for new methods of control. The complete genomes of AaeDNV and Introducing genes that increase the virulence of Aa1DNV have been sequenced and are 4009 and the virus or perhaps modify the life cycle or 4126 bp long respectively (Afanasiev et al. 1991 behavior of the mosquito are areas under in- Boublik et at. 1994). These 2 MDV genoines are vestigation (Carlson et al. 2000). similar to mammalian parvoviruses in which all genes are on I strand. The genomes contain 2 sets of generally large open reading frames (ORFs), RNA VIRUSES IN MOSQUITOES one for the nonstructural proteins (NS) and one REOVI RIDAE for the structural (VP) proteins (Bergoin and Tijssen 1998). Two overlapping ORFs occupy The genus (ypo virus within the Reoviridae two-thirds of the genome and code for the contains the insect cytoplasmic polyhedrosis nonstructural proteins designated NSI and NS2 viruses (CPVs) (Belloncik and Mori 1998). CPVs (Carlson et at. 2000). The remainder of the have been isolated from more that 250 insect genome contains a gene that codes for the species collected both in the laboratory and from structural proteins of the virion (VPI and VP2). the field with the great majority of these from lepidopteran hosts (Hukuhara and Bonami 1992). Numerous viruses from the larvae of 20 different PRODUCT DEVELOPMENT species of mosquito (representing nine different The first viral formulation developed for genera), have been identified as cytoplasmic mosquito control was based on AaeI)NV and polyhedrosis viruses based on similarities to called Viroden (Buchatsky et al. 1987). Labora- viruses from Lepidoptera (Federici 1985). These tory studies demonstrated the effectiveness of the similarities included virion structure, the presence Viroden preparation against the immature stages of inclusion bodies (polyhedra) and chronic of Ac. aegypti mosquitoes. The formulation infection in the cytoplasm of larval midgut reduced larval populations of Ac. aegypti in the epithelial cells. There were also 3 reports of CPVs field by about 77% (Buchatsky et al. 1987). The from the midgut epithelium of adult mosquitoes preparation was stable and resistant to unfavor- (Bird et al. 1972, Davies et al. 1971, Federici able environmental conditions such as UV 1973). Since Federici (1985) summarized the radiation, heat and variations in pH without status of CPVs at that time, nearly 20 years have significant loss of activity. Toxicity-pathogenicity passed without any appreciable new knowledge test of Viroden, were carried out on warm- on this common group of viruses in mosquitoes, blooded animals (Vasileva et al. 1990). It was and their taxonomic status has been in limbo. shown that the preparation is not toxic for However, this has recently changed with a new

46 AMCA Bulletin No. 7 VOL. 23. Supplement to No. 2 study on the biology, morphology and molecular ally multiply) by the deposition of a crystalline characteristics of a CPV from (Jranotaenia polyhedron matrix around individual particles. sapphirina (UsCPV) establishing this as a new The polyhedron protein appears to be arranged Cypovirus species (Shapiro et al. 2005). as a face-centered crystalline lattice. Although a similar process for formation of inclusion Distinguishing Characteristics bodies has been reported in An. quadrimaculatus Mosquito Cypoviruses (MCPVs) are charac- (Anthony et al. 1973), it is different from that terized by isometric virions approximately 60 nm observed in other mosquitoes. In Cx. restuan.s in diameter (Federici 1985). Occlusion bodies are (Andreadis 1986) polyhedra are also formed by of 2 types: small cuboidal inclusion bodies deposition of polyhedron around individual (Fig. 3C) that contain only one or a few virions particles, although these subsequently coalesce (Anthony et al. 1973, Shapiro et al. 2005) and to form larger pleomorphic inclusion bodies. In large irregularly shaped inclusion bodies Oc. cantator (Andreadis 1981) and Oc. taenio- (Fig. 3D) that contain many virions (Clark et rhynchus (Federici 1973) relatively large inclusion al. 1969, Clark and Fukuda 1971, Federici 1973, bodies are formed in clusters by deposition of Andreadis 1981, 1986). MCPV development is protein around groups of virions. Thus, the restricted to the cytoplasm of cells of the gastric majority of inclusion bodies in these mosquitoes caeca and posterior stomach (Anthony et al. are large, irregularly shaped and contain multiple 1973, Federici 1973, Andreadis 1981, 1986, virions (Fig. 3D). In contrast, the great majority Shapiro et al. 2005). The first report of a MCPV of UsCPV polyhedra (and the virus reported in was from Cx. salinarius collected in Louisiana An. quadrirnaculatus by Anthony et al. 1973) (Clark et at. 1969). Currently, there are reports of contain only one virion and are cuboidat in shape MCPVs from about 20 mosquito species repre- (Fig. 3C). senting 7 genera. The only new information for MCPVs since these earlier reports is for UsCPV Host Range (Shapiro et al. 2005) and is summarized below. UsCPV has a relatively broad host range including Cx. quinquejásciatus, Ac. aeg ypti, Oc. Gross Pathology triseriatus, Ur. lowii, and An. albinianus, although Patent infections of MCPVs produce a charac- it was not transmitted to An. quadrimaculatus or teristic porcelain-white or iridescent color in the lepidopteran host H. zea. Previous per os midgut cells as a result of the accumulation of transmission studies with mosquito CPVs were occlusion bodies in the cytoplasm (Fig. 1D). successful and also exhibited a certain degree of MCPV infections are localized to the gastric specificity (Andreadis 1986, Clark et at. 1969, caeca and the posterior portion of the stomach Clark and Fukuda 1971). This indicates that there (Federici 1985). A novel cypovirus from Urano- may be a number of distinct mosquito CPVs, taenia sapp/lirina (UsCPV) is characterized by the several of which can infect the same mosquito production of large numbers of virions and host species. Cx. erraticus and An. crucians may inclusion bodies, and their arrangement into also act as hosts for UsCPV since they were paracrystalline arrays gives the gut of infected found together with infected Ur. sapphirina in the insects a distinctive blue iridescence (Shapiro et field and exhibited similar infection character- al. 2005). MCPVs are usually benign with most istics including the iridescent color (Shapiro et at. infected individuals surviving to the adult stage 2005). (Federici 1985, Shapiro et al. 2005). Transmission Ultrastructure Horizontal transmission of several MCPVs Cypovirus virus particle structure differs from has been demonstrated through direct exposure vertebrate, plant and other insect reoviruses by of mosquito larvae to inclusion bodies but the absence of outer capsid layers and by the infection levels were generally low (Clark and production of a viral coded polyhedron protein Fukuda 1971, Federici 1973, Andreadis 1981. that occludes the virions (Belloncik and Mon 1986). Recent studies by Shapiro et al. (2005) 1998). The virions of UsCPV and most other have shown that oral transmission of UsCPV to MCPVs are icosahedral (55-65 nm in diameter) mosquito larvae is enhanced by magnesium and with a central core that is surrounded by a single inhibited by calcium ions. UsCPV exposed to capsid layer (Fig. 3C). They are usually packaged larvae in deionized water was non-infectious for individually within cubic inclusion bodies (poly- Ae. aegypti larvae but the infection rate was hedra —100 nm across), although 2 8 virus significantly increased by the addition of 10 mM particles were sometimes occluded together (Sha- Mg2 ions (Shapiro et at. 2005). However, the piro et al. 2005). In general the development of addition of 10 mM Ca 2, ions to this mixture UsCPV appears to be similar to that of other inhibited infection. Most of the UsCPV infected mosquito cypoviruses (Federici 1985, Andreadis larvae survived to adulthood and retained the 1981, 1986). The virus is assembled within infections in the midgut (Shapiro et at. 2005). virogenic stroma and occluded singly (occasion- This indicates that UsCPV may be vertically

Bioraiional Control of Mosquitoes 47 transmitted to the filial generation similar to what characteristics as well as the possibility of has been reported for an MCPV from Or. modifying these viruses to use as tools for .so//icitan.c (Clark and Fukuda 1971) and an laboratory studies or to enhance traits to make MCPV from C.Y. resluans (Andreadis 1986). them more viable as biocontrol agents. There is still a great need to isolate new viruses (or Molecular Characteristics previously described ones) from various geo- Cypoviruses usually have a 10-segmented graphical locations to build a repertoire of viruses dsRNA gcnome and the different types are and characterize their unique biological and initially identified on the basis of differences in molecular properties. Understanding how these the migration patterns of their genome segments pathogens exploit their host provides the oppor- during polyacrylamide gel electrophoresis (Payne tunity to devise novel research and control and Rivers 1976). Analysis of the UsCPV genonie strategies for the development of mosquito by 1% AGE generated 10 RNA bands (Shapiro et pathogenic viruses as components of integrated al. 2005). Electrophoretic analysis of the UsCPV mosquito control programs. genome segments demonstrated a migration pat- tern (elect ropherotype) that is different from those of the 16 cypovirus species already recog- A CKNOWLEDGMENTS nized. The smallest band (segment 10) encodes the viral poyhedrin protein. The UsCPV poly- We acknowledge the technical support of hedron sequence showed no significant nucleotide Heather Furlong (U.S. Department of Agricul- similarity to the corresponding segment of the ture/Agricultural Research Service, Gainesville, other cypoviruses that have previously been FL) and reviewers Dr. James Maruniak and 1)r. analyzed, and it has different conserved termini Terry Green. (Shapiro et al. 2005). It was concluded that UsCPV should be recognized as a member of a new cypoiiru.s species (Cypovirus 17: Strain REFERENCES UsCPV- 17). Afanasiev RN. Kozlov YV, Carlson JO, Beaty BJ. 1994. Densovirus of Acde,r aegipli as an expression vector in mosquito cells, £xp Parasiiol 79:322-39. CONCLUSION .Afonasicv RN. Ward fW. Beaty 13.1. Carlson JO. 1999. Transduction of Aëdes cscgly)It mosquitoes with While progress has been made over the past vectors derived from Arc/es densovirus. Virnloi,i 20 years in understanding the biological and 257:62 72. molecular properties of mosquito pathogenic Afanasiev RN. (Ial yov FF. Ruchatsky LP. Kozlov YV. viruses (particularly baculoviruses and denso- 1991. Nucleotide sequence and genomic organization viruses) only one potential commercial product of Aëdes densonucleosis virus. lj,/ y 185:323 36. containing a densovirus (Viroden) has been Afonso (.;I... Tulman ER. Lii Z. Balinsky CA, Moser evaluated in small scale field studies (Butchatsky BA, Beene] JJ. Rock DL, Kutish G1. 200!. Genonic 1989). These initial evaluations were promising, sequence of a haculovirus pathogenic for Culex but large-scale field evaluations have not been ?ngl7pa/pu.. J Virol 75:11157 11165. Allen-Miura TM. Afanasiev RN. Olson KE. Beaty RJ. conducted due primarily to production con- Carlson JO. 1999. Packaging of Ael)NV-GFP trans- straints. Recent studies have shown that MDVs ducing virus by expression of densovirus structural can he grown in continuous cultures of Arc/es proteins from a Sindhis virus expression sysieni. aI/poicius C6/C36 cells adapted to serum-free froi/ogi 257:54-61, protein- free-media (Suchman and Carlson 2004). Andreadis TG. 1981. A new cytoplasmic polyhedrosis To utilize Viroden. or any other mosquito virus from the salt-marsh mosquito. Aëdes cUfltUlOr pathogenic viruses, as an applied biocontrol agent (Diptera: Culicidue). .1 Inieriehr Pat/wI 37:160 167. will require further advancements in the de- Andrcadis 1G. 1986. Characterization of a cytoplasmic velopment of large-scale mosquito culture meth- polyhedrosis virus affecting the mosquito CuIe.v restuao.s. J hivertebr Patho/ 47:194 202. ods for production. Andreadis T(i, Becnel J.J. White SE. 2003. Infectivity There are still many research and developnten- and pathogenicity of a novel haculovirus. CuniNPV tal obstacles to overcome before viral pathogens froni Culcx o,gripalpu.s (Diptera : Culicidae) for call utilized as part of a control strategy for thirteen species and four genera of mosquitoes. mosquitoes. Genomic information on mosquito J Med Entoniol 40:512 .517. pathogenic viruses and their mosquito hosts is Anthony DW. Hazard El, Crosby SW. 1973. A virus rapidly accumulating and offers exciting possibil- disease in Aoopheles qua(/rinwcceIaIus.i hoerte/,fl ities to understand virus-mosquito interactions at Iat/iol 22:1 5. l3arrcau C, Jousset FX, Bergoin M. 1996. the molecular level. These studies can address Pathogenicity of the Aec/e.s a/l)opIetu.v parvovirus (AaPV). a denso- basic and important properties of these viruses like virus, for Aëdes org ep/i mosquitoes..! J,irertehr such as transmission, host range, safety, virulence Ic,t/u/ 68:299-309. and other biological features. Molecular studies BaiTeau C. Jousset FX, Rergoin M. 1997. 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48 AMCA Bulletin No. 7 Vot,. 23, Supplement to No. 2

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