Journal of INVERTEBRATE PATHOLOGY Journal of Invertebrate Pathology 86 (2004) 96–103 www.elsevier.com/locate/yjipa

A nucleopolyhedrovirus from sapphirina (Diptera: Culicidae)

Alexandra M. Shapiro,* James J. Becnel, and Susan E. White

Center for Medical, Agricultural and Veterinary Entomology, USDA/ARS, P.O. Box 14565, Gainesville, FL 32604, USA

Received 29 January 2004; accepted 26 April 2004 Available online 9 June 2004

Abstract

In this report we present data on biology, gross pathology, ultrastructure, and host range studies of a naturally occurring nu- cleopolyhedrovirus from the (UrsaNPV). Development of this virus was restricted to nuclei of epithelial cells in posterior midgut and distal gastric caecum. Occlusion bodies contained numerous singly enveloped rod-shaped virions. Early occlusion bodies were irregularly shaped and seemed to subsequently coalesce to form larger polyhedra. Mature occlusion bodies had a unique dumbbell shape, and lacked a polyhedron envelope and crystalline structure. Developmental and structural features of UrsaNPV were generally similar to other mosquito NPVs, with major differences in occlusion body shape and size. Transmission tests showed that only members of Uranotaenia (Ur. sapphirina and Ur. lowii) were susceptible to this virus. Transmission was facilitated by magnesium. Field collected Ur. sapphirina larvae had a relatively high rate of dual infections with UrsaNPV and UrsaCPV (cypovirus). Ó 2004 Elsevier Inc. All rights reserved.

Keywords: Uranotaenia sapphirina; Mosquito viral pathogens; Nuclepolyhedrosis; Cypovirus; Dual viral infections;

1. Introduction white caused by the accumulation of the occlusion bo- dies. Due to the rarity of mosquito NPV isolations from NPVs belong to the genus Nucleopolyhedrovirus the field, the difficulty of transmission in the laboratory (Baculoviridae) and are enveloped, double-stranded and the impossibility to maintain colonies of some DNA viruses with circular genomes that are specific to species, few morphological and transmission studies of . Isolated mostly from Lepidoptera, they are mosquito NPVs have been reported. Most of the de- also known from the Hymenoptera, Diptera, Trichop- tailed ultrastructural studies were conducted with ba- tera, and Crustacea (Federici, 1997). Since the first re- culoviruses from Oc. triseriatus (Federici and Lowe, port of a baculovirus (OcsoNPV) isolated from 1972), Oc. epactius (Stiles et al., 1983), Oc. sollicitans Ochlerotatus sollicitans mosquitoes in Louisiana in 1969 (Federici, 1980) and from Cx. nigripalpus (Moser et al., (Clark et al., 1969), naturally occurring NPVs were 2001). There is only one extensive study on transmission isolated from 13 more mosquito species within the of an NPV in Cx. nigripalpus (Becnel et al., 2001) that genera Aedes, Anopheles, Culex, Ochlerotatus, Psoro- demonstrates the crucial roles of Mg2þ and Ca2þ in this phora, Uranotaenia, and Wyeomyia (Murphy et al., process. A nucleopolyhedrovirus was previously re- 1995). More than 20 mosquito species were found sus- ported in Ur. sapphirina (Clark et al., 1969; Federici, ceptible to NPV infections (field isolates or laboratory 1985), and here we present the first detailed description transmitted infections). Patent infections are detected by of an NPV infection in this mosquito species. A possible, the hypertrophied nuclei of midgut cells that appear but not confirmed by electron microscopy, cypovirus (CPV) infection in Ur. sapphirina was reported by Clark et al. (1969); here we present ultrastructural evidence for * Corresponding author. Fax: +352-374-5966. a CPV infection in this species. This paper describes E-mail address: [email protected]fl.edu (A.M. Shapiro). biology, gross pathology, ultrastructure and host range

0022-2011/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2004.04.005 A.M. Shapiro et al. / Journal of Invertebrate Pathology 86 (2004) 96–103 97 studies of an NPV, as well as biology and ultrastructure ability of field collected healthy and infected Uranotae- of an NPV/CPV dual infections from Ur. sapphirina nia. Uranotaenia larvae collected from sites which larvae collected in Florida. historically did not have UrsaNPV or UrsaCPV present Uranotaenia sapphirina is a common mosquito in were exposed to virus from field-infected Uranotaenia Florida and may be found throughout most of the year. spp. The healthy Uranotaenia were primarily in the third Larvae occur in permanent pools and ponds, lakes and or fourth instars and the number exposed varied from 20 swamps that contain emergent or floating vegetation to 100 larvae. They were exposed to one to five larval exposed to sunlight. Although Ur. sapphirina feeds pri- equivalents of infected Ur. sapphirina in 10–100 ml of 5 marily on frogs and reptiles, has no known medical or or 10 mM MgCl2, depending on the number of lar- economic importance and the females are not known to vae available, and examined two to three days after bite humans, it is nevertheless on the CDC list of mos- exposure. quitoes testing positive for West Nile Virus. Recent Other mosquito exposures: Second instar larvae of findings (ProMed, 2003) that levels of the virus in alli- Aedes aegypti, Anopheles quadrimaculatus, and Culex gators are as high as what is found in birds (high enough quinquefasciatus, in groups of 100 larvae, were exposed to infect mosquitoes), suggest that alligators might to 3–5 larval equivalents of field infected Uranotaenia transmit the virus and Ur. sapphirina might play a small spp. infected with UrsaNPV and UrsaCPV and exposed role in the transmission cycle of WNV and its circulation in 100 ml of 10–20 mM MgCl2 and examined 2–3 days in the environment. post exposure for signs of infection. It should be noted that the identification of UrsaNPV and UrsaCPV at this point is based on morphological 2.5. Ultrastructural studies criteria alone. Without molecular data on the charac- terization of these viruses its identities are tentative. Guts were dissected from fourth instar larvae and transferred to 2.5% (v/v) glutaraldehyde (EM grade) in 0.1 M cacodylate buffer containing 0.1% CaCl2 for 3 h. 2. Materials and methods Following fixation, samples were washed three times in 0.1 M cacodylate buffer (pH 7.2) and post-fixed in 1% (w/ 2.1. Field collections v) aqueous osmium tetroxide for 1 h at room tempera- ture. Samples were washed three times with water, de- Uranotaenia spp. larvae were collected in the grass hydrated through a standard ethanol series (30–100% surrounding a small, sunlit pond located in Cross Creek, ethanol, 15 min/step) followed by two washes in acetone. Florida. The pond encompassed part of a pasture and a Tissues were infiltrated with Epon-Araldyte resin (Poly- roadside ditch, however, it did go dry during prolonged bed 812, 2 g; Araldyte 502, 1 g; DDSA, 4.5 g; DMP-30 4 drought. Although many predators and crayfish drops) in a stepwise fashion with ratios of acetone to were present, fish were not. Other mosquitoes collected at resin of 3:1, 1:1, and 1:3 and finally several changes of this site included Anopheles crucians and Culex erraticus. pure resin. Resin blocks were cured overnight in a 65 °C oven. Thin gold interference sections (85–100 nm thick) 2.2. Gross pathology were collected on 200-mesh copper grids. The sections were poststained with 2.5% aqueous uranyl acetate for Larvae were separated by genus, placed on a clear 10 min, followed by Standard Reynolds Lead Citrate glass petri dish with minimal water and the ventral side stain for 5 min and were examined with a Hitachi H-600 examined with a dissecting scope against a black back- electron microscope at an accelerating voltage of 75 kV. ground. The larvae had a relatively clear cuticle allowing the infected cells of the midgut to be detected with a dissecting microscope. 3. Results

2.3. Statistical analysis 3.1. Field collection

Percent infection was estimated as the number of The first UrsaNPV infected larvae were found in infected larvae divided by the number of larvae exam- October 2001 with 8 of 152 (5.3%) Ur. sapphirina larvae ined. This was averaged over the number of collections infected. Several of these infected larvae were dually which had infected larvae. infected with a CPV. During October and November 2001, 14 of the 38 collections had patently infected lar- 2.4. Laboratory bioassay vae with either UrsaNPV or UrsaCPV or both. The maximum infection rate of UrsaNPV was 14.0% in Uranotaenia spp exposures: Horizontal transmission November 2001 with an equal number dually infected. of UrsaNPV studies were limited because of the avail- The site was totally dry by June 2002. Uranotaenia 98 A.M. Shapiro et al. / Journal of Invertebrate Pathology 86 (2004) 96–103 sapphirina were collected again after the pond flooded in 3.2. Gross pathology July 2002, but UrsaNPV and UrsaCPV were not re- covered until October 2002. The highest infection rate of Virus infection affected development, feeding and both viruses was 18.8% in November 2002. During this behavior of the infected larvae. Patent infections devel- time, both Culex erraticus (5.4 0.7%, n ¼ 3) and oped in 2–4 days post inoculum (pi), and death occurred Anopheles crucians (3.7 2.5%, n ¼ 3) were infected on 3–5 days pi. Patently infected UrsaNPV larvae had with a CPV similar to the UrsaCPV, but neither species hypertrophied nuclei which were tightly packed with were infected with UrsaNPV. In 2003, UrsaCPV was occlusion bodies, many exhibiting the unique ‘‘bow-tie’’ recovered in October, but not UrsaNPV. Table 1 gives shape (Figs. 1–3). These nuclei appeared as white nod- the seasonal infection rates for both viruses. ules in the posterior midgut and distal gastric caecum Uranotaenia lowii were also breeding in the same site, (Figs. 1 and 2). We did not observe infection in the but in much lower numbers than Ur. sapphirina. Urano- cardia. taenia lowii infected with UrsaNPV and UrsaCPV (viral Midgut cells infected with UrsaCPV primarily local- identification based on light and electron microscopy) ized to the gastric caecum and posterior stomach. Ur- were collected in 2001. In October 2001, 0.1 0.1% were saCPV infections, were normally characterized by an infected with UrsaNPV and 1.4 0.6% infected with iridescent blue appearance, perhaps due to the size/ar- UrsaCPV. The infection rates in November were rangement of occlusion bodies. Sometimes, larvae 0.6 0.3% for UrsaNPV and 2.0 0.7% for UrsaCPV. thought to have only UrsaNPV would also be infected Since then no infected Ur. lowii have been recovered. with UrsaCPV.

Table 1 Seasonal infection rates of UrsaNPV and UrsaCPV in field collected Uranotaenia sapphirina larvae and the number of collections with infected larvae Year Month Number of col- Total UrsaNPV only Dual UrsaNPV and UrsaCPV only lections made larvae UrsaCPV % Infection N* % Infection N* % Infection N* 2001 October 25 1642 4.2 0.9 9 3.2 1.0 8 10.8 1.7 8 November 13 721 8.1 1.9 5 7.1 2.9 4 9.4 0.8 4 December 3 99 2.0 1 2.0 1 3.0 1 2002 April–September 14 266 0 0 0 October 6 260 14.7 3.5 3 45.6 1 7.5 6.5 2 November 14 970 8.3 4.4 6 8.8 2.6 2 12.5 5.5 4 December 2 7 0 0 0 Totals 77 3665 24 16 19 N ¼ number of collections with infected larvae.

Figs. 1–3. (1) Gross pathology of the ventral side of a fourth-instar UrsaNPV infected Uranotaenia sappirina larva with visible hypertrophied nuclei in the midgut. (2) Dissected midgut from a fourth-instar UrsaNPV infected Uranotaenia sappirina larva showing hypertrophied nuclei in the posterior midgut and distal gastric caecum. (3) Fresh smear of a UrsaNPV infected Ur. sappirina larva with ruptured nuclei viewed with a phase-contrast microscope. Arrows point to numerous dumbbell shaped occlusion bodies. A.M. Shapiro et al. / Journal of Invertebrate Pathology 86 (2004) 96–103 99

3.3. Laboratory bioassays ons budding through the nuclear membranes were en- veloped (Figs. 10 and 11), and among those found in the UrsaNPV was successfully transmitted to Ur. sap- cytoplasm, some were enveloped and some were not phirina and Ur. lowii in the laboratory. In two paired (Figs. 11 and 13). The non-enveloped nucleocapsids tests, Ur. sapphirina exposed to UrsaNPV in deionized found in the cytoplasm were located mostly in the part water or 10 mM MgCl2, the addition of magnesium in- of the cell that faced the basal membrane (Fig. 13) while creased the infection rate by 12.9 3.8%. In additional the enveloped ones did not seem to have any specific tests, UrsaNPV was transmitted to Ur. lowii localization. We did not observe virions crossing plasma (14.5 9.2%, n ¼ 11) in the presence of MgCl2. Control membranes towards neighboring cells, microvillar larvae were not infected. None of the other mosquitoes membranes, or the basal membrane. exposed to UrsaNPV developed patent infections. At the initial stages of occlusion, virions were either UrsaCPV was also transmitted to both species of Ur. occluded singly in small globular or pleomorphic oc- lowii and Ur. saphhirina (9.2 7.5%, n ¼ 2 and clusion bodies (OBs) or were directly incorporated into 13.8 6.1%, n ¼ 6) respectively. Of the other mosqui- existing OBs (Fig. 14). Smaller OBs coalesced into toes tested, Ae. aegypti larvae were infected with Ur- roughly tetrahedral structures that fused producing fi- saCPV (7.8 2.4%, n ¼ 8) and Cx. quinquefasciatus nal dumbbell, or bow-tie shaped OBs (Figs. 4, 12, and were also infected with UrsaCPV (54.9 12.5%, n ¼ 2). 14). Mature OBs were large, measuring up to 10–15 lm in length and 2–3 lm in diameter (Fig. 12). The OBs 3.4. Ultrastructural studies lacked the polyhedron envelope and the crystalline protein lattice characteristic of most NPVs. Each OB Electron microscopy of posterior midgut tissues re- contained numerous rod-shaped virions. Each virion vealed UrsaNPV infection in the nuclei of the epithelial consisted of one nucleocapsid, an intermediate layer, cells (Figs. 4, 5) with all the nuclei approximately in the and an outer envelope (Fig. 12 inset). Approximate same stage of virogenesis. We did not observe infection lengths of virions and nucleocapsids were 200 and in regenerative cells. Most of the epithelial cell nuclei 130 nm, approximate diameters were 60 and 33 nm, were heavily infected and had developing or mature respectively. occlusion bodies. Virogenic stroma observed in some of Ultrastructural studies of the posterior midgut of the nuclei appeared as a loose granular material, lo- UrsaNPV infected Ur. lowii larvae (laboratory trans- cated centrally and surrounded by virions at different missions) showed viral infection in epithelial cell (Figs. stages of envelopment and occlusion. Also present were 15 and 16). This was a dual UrsaNPV/UrsaCPV infec- occlusion bodies of various size and maturation (Fig. tion. Nuclear infection in Ur. lowii looked similar to 5). Empty capsids or nucleocapsids were located within what we found in the original viral host, Ur. sapphirina. the virogenic stroma with little or no evidence for nucleocapsid envelopment (Fig. 6). Occasionally, large parallel arrays of nucleocapsids and empty capsids 4. Discussion were observed in virogenic stroma (Fig. 7). Nucleo- capsids, up to 400–500 nm in length, were adjacent to UrsaNPV infection is lethal to Uranotaenia mosqui- shorter nucleocapsids, approximately 200 nm in length. toes and kills the larvae within 3–5 days pi. The devel- Shorter nucleocapsids were uniform in size. Singly en- opment and ultrastructural morphology of UrsaNPV veloped and non-enveloped nucleocapsids were scat- from Ur. sapphirina were in general similar to those re- tered throughout the nucleus and some non-enveloped ported from other mosquito hosts (Federici, 1980; nucleocapsids were associated with the envelope pre- Federici and Lowe, 1972; Moser et al., 2001; Stiles et al., cursors. These envelope precursors were spherical 1983). Virus development was tissue specific and was membranous structures (vesicles) found in the nuclei. limited to the nuclei of the epithelial cells of gastric caeca Envelopment was initiated when either the ends or and posterior portion of the midgut. No infection was sides of nucleocapsids attached to the membrane of the observed in regenerative cells or the cardia. Virions were vesicle (Figs. 8 and 9). Upon contact, the membrane rod-shaped, contained one nucleocapsid and were sim- surrounded the nucleocapsids until they were com- ilar in size to other mosquito NPVs. Formation of the pletely enveloped and some nucleocapsids moved to a parallel arrays of nucleocapsids of various lengths was position within the vesicles (Fig. 9). similar to this process observed in Oc. triseriatus (Fed- A number of virions were found budding singly erici and Lowe, 1972), Oc. sollicitans (Federici, 1980), through the nuclear membranes and some were found in and Cx. nigripalpus (Moser et al., 2001). Presence of the cytoplasm. Virions budding through the nuclear short and long nucleocapsids, where the combined membranes acquired these membranes and budded off length of two short ones was approximately that of the as small vesicles containing virions (Figs. 10 and 11). long nucleocapsid was also similar to what was observed These vesicles were observed only near the nuclei. Viri- in Oc. triseriatus (Federici and Lowe, 1972), Oc. sollic- 100 A.M. Shapiro et al. / Journal of Invertebrate Pathology 86 (2004) 96–103

Figs. 4–12. Transmission electron micrographs of UrsaNPV infection in Uranotaenia sapphirina. (4) Midgut epithelial cell with infected nucleus showing mature dumbbell-shaped occlusion body. (5) Midgut epithelial cell with infected nucleus showing virogenic stroma located centrally in the nucleus. (6) Virogenic stroma with empty capsids and nucleocapsids and no evidence for nucleocapsid envelopment. (7) Parallel array of nucleo- capsids in virogenic stroma showing short and long nucleocapsids where short nucleocapsids are uniform in length and the combined length of two short ones is approximately that of the long nucleocapsid (inset). (8) Centrally located in the nucleus membranous envelope precursors surrounded by nucleocapsids. (9) Membranous envelope precursors surrounded by nucleocapsids showing one enveloped virion that moved to a position within the vesicle. Inset shows several nucleocapsids getting surrounded by the membrane while moving towards the center of the vesicle and one enveloped virion already inside. (10) Budding of a virion through the nuclear envelope, shown with white arrow. Note the presence of the viral envelope and acquisition of the nuclear membranes. N-nucleus. (11) Vesicles containing virions in the cytoplasm near the nucleus, shown with white arrows. (12) Mature dumbbell or bow-tie shaped occlusion body with numerous virions. Inset shows a UrsaNPV virion. itans (Federici, 1980), in Oc. epactius (Stiles et al., 1983) ids in Ur. sapphirina might be formed by cleavage of the and in Cx. nigripalpus (Moser et al., 2001). As suggested long ones, and may be a characteristic feature of mos- for Oc. sollicitans by Federici (1980), short nucleocaps- quito NPVs. The enveloping process was similar to the A.M. Shapiro et al. / Journal of Invertebrate Pathology 86 (2004) 96–103 101

Figs. 13–16. Transmission electron micrographs of UrsaNPV infection in Uranotaenia sapphirina. (13) Non-enveloped nucleocapsids (arrows) located in the part of cytoplasm that faces the basal membrane (BM). Inset shows a non-enveloped nucleocapsid (arrow) in the cytoplasm next to the Golgi bodies (G) and endoplasmic reticulum (ER). (14) Infected nucleus showing a singly occluded virion that is in the process of coalescing with a large occlusion body (white arrow) and non-occluded virions attaching to the same occlusion body (black arrow). (15 and 16) Transmission electron micrographs of a dual UrsaNPV/UrsaCPV infection in Ur. lowii. (15) An epithelial midgut cell at the early stage of UrsaNPV virogenesis in the nucleus (N) (virions are shown with a black arrow) and cytoplasm filled with occluded UrsaCPV virions (white arrow) and UrsaCPV stroma (S). (16) An epithelial midgut cell at the later stage of UrsaNPV virogenesis with forming occlusion bodies in the nucleus (N) (black arrow) and scattered occluded virions of UrsaCPV (white arrow) in the cytoplasm. one in Oc. sollicitans described by Federici (1980) and in appeared as well defined spherical membranous vesicles Oc. epactius (Stiles et al., 1983) with a few differences. while in Oc. sollicitans and Oc. epactius they were ir- For example, the envelope precursors in Ur. sapphirina regular ribbons or pleomorphic vesicles. 102 A.M. Shapiro et al. / Journal of Invertebrate Pathology 86 (2004) 96–103

Virions of Ur. sapphirina had two phenotypes: bud- Thus far, mosquito NPVs fall into two groups based ded virions (BVs), budding through the nuclear mem- on OB formation; those where small globular occlusion branes, and occlusion derived virions (ODVs). Although bodies are formed that do not coalesce (CuniNPV we use the common term ‘‘budded virus’’, the meaning is (Moser et al., 2001), WysmNPV (Hall and Fish, 1974)) slightly different from what it stands for in lepidopteran and those that coalesce into larger OBs of various size hosts, where BVs refer to those virions that cross the and shape (UrsaNPV, OcsoNPV (Federici, 1980)). plasma membrane. Budding of the virions through the Transmission of UrsaNPV was enhanced by magne- nuclear membranes observed in Ur. sapphirina was sium, similar to what was observed for CuniNPV, al- generally similar to this process previously described in though the level of enhancement was much lower for Cx. nigripalpus (Moser et al., 2001). Small differences UrsaNPV than for CuniNPV. We have no data on could be due to the fact that in Cx. nigripalpus this calcium effects on UrsaNPV transmission. We also have process was described for the earlier stages of virogen- no evidence on the influence of a cypoviral infection on esis, when occlusion of virions had not begun, while Ur. NPV transmission. A dual NPV/CPV infection was sapphirina material available for EM in this study was in previously described in a different mosquito, Oc. sollic- the later stages of development. Virions of Ur. sapphi- itans (Clark and Fukuda, 1971). In that study, the lab- rina budded singly through the nuclear membranes. oratory tests showed that the presence of CPV in the They were enveloped in contrast to what was observed larvae may have predisposed them to NPV infections. in Cx. nigripalpus (Moser et al., 2001) where the budded In conclusion, developmental and structural features virions (budding singly and in groups) were naked nu- of UrsaNPV were generally similar to other mosquito cleocapsids with no viral envelope. After budding NPVs, with major differences in occlusion bodies shape through the nuclear membranes, the virions of Ur. and size. This study provides information on the natural sapphirina were surrounded by these membranes and history of UrsaNPV and conditions for its transmission, budded off as vesicles containing virions. The nuclear thus broadening our understanding of the biology membranes were probably dissolved soon after budding mosquito NPVs and adding to the knowledge of NPVs since these vesicles were observed only in the proximity in general. of the nuclei and the majority of the virions found in the cytoplasm lacked these acquired membranes. Interest- ingly, some of the virions observed in the cytoplasm Acknowledgments were naked nucleocapsids lacking the viral envelope. They were found primarily in the part of the cytoplasm The authors thank Dr. James Maruniak (University that faced the basal membrane. We did not observe vi- of Florida) for his helpful suggestions and comments on rions crossing plasma membranes toward neighboring an earlier draft of the manuscript. We also appreciate cells, microvillar membranes, or the basal membrane. the technical support of Greg Allen and Heather Fur- Similar results were found in Cx. nigripalpus (Moser long (USDA/ARS Gainesville). et al., 2001). There is no evidence yet of lateral trans- mission by BVs within the midgut of mosquitoes, while in lepidopteran hosts this is a well documented event (Adams and McClintock, 1991). Thus, the mechanism References by which the virus spreads from cell to cell in the mos- quito midgut is not known. Adams, J.R., McClintock, J.T., 1991. Baculoviridae. Nuclear polyhe- ODVs were occluded only after envelopment was drosis viruses of . In: Adams, J.R., Bonami, J.R. (Eds.), complete. They were occluded either singly by deposi- Atlas of Invertebrate Viruses. CRC Press Inc, Boca Raton, pp. 89– tion of protein around individual virions or the virions 204. Becnel, J.J., White, S.E., Moser, B.A., Fukuda, T., Rotstein, M.J., attached to existing OBs. Formation of the OBs by at- Undeen, A.H., Cockburn, A., 2001. Epizootiology and transmis- tachment of the virions to existing pleomorphic occlu- sion of newly discovered baculovirus from the mosquito Culex sions and the coalescence of the OBs was similar to the nigripalpus and C. quinquefasciatus. J. Gen. Virol. 82, 275–282. process of occlusion body formation in Oc. sollicitans Clark, T.B., Chapman, H.C., Fukuda, T., 1969. Nuclear-polyhedrosis (Federici, 1980). This was different from that observed virus infections in Louisiana mosquitoes. J. Invertebr. Pathol. 14, 284–286. in Cx. nigripalpus where OBs seemed to form by con- Clark, T.B., Fukuda, T., 1971. Field and laboratory observations of densation of protein around groups of virions without two viral deseases in Aedes sollicitans (Walker) in southeastern coalescence into larger OBs (Moser et al., 2001). The Louisiana. Mosq. News 31 (2), 193–199. absence of a polyhedron envelope and crystalline pro- Federici, B.A., 1980. Mosquito baculovirus: sequence of morphogen- tein structure in UrsaNPV are similar to other mosquito esis and ultrastructure of the virion. Virology 100, 1–9. Federici, B.A., 1985. Viral pathogens of mosquito larvae. Bull. Am. NPVs. The most distinguishing features of this virus Mosq. Control Assoc. 6, 62–74. were its large size and characteristic dumbbell or bow-tie Federici, B.A., 1997. Baculovirus pathogenesis. In: Miller, L.K. (Ed.), shape exhibited by mature OBs. The Baculoviruses. Plenum Press, New York, pp. 33–59. A.M. Shapiro et al. / Journal of Invertebrate Pathology 86 (2004) 96–103 103

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