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