Biological and molecular studies of a cypovirus from the black fly Simulium ubiquitum (Diptera: Simuliidae)

Biological and molecular studies of a cypovirus from the black fly Simulium ubiquitum (Diptera: Simuliidae)

Journal of Invertebrate Pathology 95 (2007) 26–32 www.elsevier.com/locate/yjipa Biological and molecular studies of a cypovirus from the black Xy Sim...

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Journal of Invertebrate Pathology 95 (2007) 26–32 www.elsevier.com/locate/yjipa

Biological and molecular studies of a cypovirus from the black Xy Simulium ubiquitum (Diptera: Simuliidae) Terry B. Green a, Susan White a, Shujing Rao b, Peter P.C. Mertens b, Peter H. Adler c, James J. Becnel a,¤ b

a ARS, CMAVE, 1600-1700 S.W. 23rd Drive, Gainesville, FL 32608, USA Pirbright Laboratory, Institute for Animal Health, Ash Road Pirbright, Woking, Surrey, GU24 0NF, UK c Division of Entomology, Clemson University, 114 Long Hall, Clemson, SC 29634-0315, USA

Received 20 July 2006; accepted 24 October 2006 Available online 16 January 2007

Abstract A cypovirus from the black Xy Simulium ubiquitum (SuCPV) was isolated and examined using biological and molecular techniques. SuCPV produces small (typically 0.25 m), polyhedral shaped inclusion bodies (polyhedra), in which the virus particles become multiply embedded. SuCPV is the third cypovirus isolated from Diptera, but the Wrst from Simuliidae that has been characterized using molecular analyses. SuCPV has a genome composed of 10 segments of dsRNA, with an electrophoretic migration pattern that is diVerent from those of recent UsCPV-17 and CrCPV-17 isolates from the mosquitoes Uranotaenia sapphirina and Culex restuans, respectively. The SuCPV electropherotype appears to show signiWcant diVerences from those of the previously characterized lepidopteran cypoviruses. Sequence analysis of SuCPV segment 10 shows that it is unrelated to either of the two CPV isolates from Diptera or to the CPV species for which Seg-10 has been previously characterized from Lepidoptera. A comparison of the terminal regions of SuCPV genome segments to those of CPV-1, 2, 4, 5 14, 15, 16, 17, 18, and 19 also revealed only low levels of conservation. We therefore, propose that SuCPV is classiWed within a new Cypovirus species, which we have tentatively identiWed as Cypovirus-20. We have therefore referred to this virus isolate as S. ubiquitum CPV-20 (SuCPV-20). © 2006 Elsevier Inc. All rights reserved. Keywords: Simulium ubiquitum Cypovirus; Black Xy; Transmission; Reoviridae; Morphology; Electropherotype; Diptera

1. Introduction Cypoviruses have been isolated mainly from insects in the orders Lepidoptera, Diptera, and Hymenoptera. The cypovirus genome usually consists of 10 double-stranded RNA (dsRNA) segments (Mertens et al., 2004; Hukuhara and Bonami, 1991; Payne and Rivers, 1976), packaged as exactly one copy of each segment, within each singleshelled, icosahedral and turreted virus particle. The cypoviruses replicate within the cytoplasm of infected insect cells and typically produce inclusion bodies (polyhedra) that are composed primarily of a single viral protein (polyhedrin),

*

Corresponding author. Fax: +1 352 374 5966. E-mail address: [email protected] (J.J. Becnel).

0022-2011/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2006.10.006

within which the virus particles can become either singly or (more usually) multiply embedded. Cypoviruses usually infect the midgut cells, particularly of the insect larval stages and can produce chronic rather than fatal disease. Sixteen diVerent virus species (Cypovirus-1 to Cypovirus-16) have already been formally recognized within the genus Cypovirus, family Reoviridae (Mertens et al., 2004). These diVerent species can be distinguished by diVerences in the migration patterns of their dsRNAs during electrophoresis, variations in RNA sequences (both in the coding regions and conserved terminal regions) and by antigenic variation in the viral proteins (Mertens et al., 2004, 1999; Payne and Rivers, 1976). Further Cypovirus species (CPV-17, 18, and 19) have been proposed, based on isolates from mosquitoes (CPV-17—Green et al., 2006; Shapiro et al., 2005) and from the winter moth Operophtera brumata (CPV-18 and 19—

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Graham et al., 2006) that have been characterized and compared to the existing species by electropherotype analysis and sequencing of the viral genome. Historically, hundreds of CPV isolates have been identiWed from diVerent species of Lepidoptera, based on the similarities in their virion, inclusion bodies and infection of midgut epithelial cells. However, most of these viruses have remained uncharacterized and are consequently unclassiWed by the International Committee for the Taxonomy of Viruses (ICTV), due (in large part) to an inability to recover enough of each virus for molecular studies (such as electropherotyping, serological analysis, or sequencing). We report the isolation of a CPV from the black Xy Simulium ubiquitum and present molecular data to support its classiWcation as a member of a new Cypovirus species that we have tentatively identiWed as Cypovirus-20. The new virus isolate can therefore be named as ‘Simulium ubiquitum CPV-20’ (SuCPV-20). 2. Materials and methods 2.1. Field collection and gross pathology Simulium larvae were collected twice in Hatchet Creek at Hwy 24 in Alachua County, Florida (N29.73066 W82.24906), in April and May 2005. The larvae were held in creek water and returned to the laboratory for examination. Individual larvae were removed from the plant material and examined against a dark background, using a dissecting microscope. Diseased larvae were segregated by pathogen types, and samples of diseased and healthy larvae were placed in 70% ethanol for identiWcation. 2.2. Ultrastructural studies (electron microscopy) Dissected midguts of infected black Xy larvae were processed for electron microscopy as described by Shapiro et al. (2004). BrieXy, dissected midguts were Wxed in 2.5% gluteraldehyde for 2 h, postWxed in 2% osmium tetroxide for 1 h, dehydrated in an ethanol series and embedded in epon-araldite. Thin sections were stained in uranyl acetate and lead citrate and examined and photographed at 75 kV. 2.3. PuriWcation of virus Midguts of SuCPV-infected larvae were dissected and homogenized in deionized water (25 from the Wrst collection and 50 from the second collection). The suspension was placed on a continuous 0.1 mM NaOH : HS-40 Ludox® gradient and centrifuged at 16,000g for 30 min. The resulting band was removed and suspended in 0.1mM NaOH and spun again at 16,000g for 20 min and this washing was repeated three times. The Wnal pellet was suspended in 0.1mM NaOH.

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were exposed to 5 LE (larval equivalent) of S. ubiquitum infected with CPV in 100 ml of 0 or 10 mM MgCl2 at room temperature and then transferred to 15 °C for two days. Larvae were examined two and Wve days post inoculum (p.i.) for signs of disease. 2.5. Analysis of SuCPV by electropherotype Genomic dsRNAs of SuCPV was extracted from puriWed polyhedra, using a QIAampViral Mini Kit from Qiagen (Green et al., 2006; Shapiro et al., 2005; Rao et al., 2003; Hagiwara et al., 2002). SuCPV RNA was analyzed on a 1% agarose gel. Ethidium bromide was integrated into the gel at a Wnal concentration of 0.5 mg/ml. Genomic RNA from UsCPV-17 and CrCPV-17 was also analyzed in the same gel to compare the electrophoretic proWle of cypoviruses isolated from Diptera. Seg-10 sequences from UsCPV-17 (AY876384) and CrCPV-17 (DQ212785) genomes are accessible in GenBank. 2.6. cDNA synthesis, ampliWcation by PCR, cloning and sequencing The conditions for cDNA synthesis, ampliWcation by PCR, cloning, and sequencing were similar to the protocols published by Shapiro et al. (2005) and Green et al. (2006). BrieXy, RNA was isolated from puriWed virions, using a QIAampViral Mini Kit (Qiagen). An anchor primer was ligated to the dsRNAs, using T4 RNA ligase (New England Biolab). cDNA synthesis was then performed with a primer part that is complementary to the anchor using AMV reverse transcriptase (Promega). PCR was completed using the Advantage 2 PCR Kit (Clontech) with primer 5-15-1. The PCR conditions were 95 °C for 15 s of denaturing and 68 °C for 3 min of annealing/ extension (24 cycles). The PCR products of SuCPV were separated on a 1% agarose gel. The expected size of Seg-10 was »850 bp. The Seg-10 DNA band was then excised, puriWed, A-tailed by Taq enzyme (10 min at 72 °C with 200 nM dATP in 1£ Taq buVer), and cloned into pGEM-T Easy vector. The cloned insert from Seg-10 was ampliWed by PCR, using SP6 and T7 primer pairs, and was conWrmed by the lengths of the PCR product and by restriction mapping. Three clones from Seg-10 were completely sequenced using SP6 and T7 primers, with a dye terminator cycle sequencing ready reaction kit on a Beckman CEQ8000 system. The sequences obtained were then used to design primers for direct sequencing of the RT-PCR products from Seg-10, which conWrmed the original sequence data from the cloned cDNAs. 2.7. Homology analysis

2.4. Horizontal transmission (Cation Assays) Four-day old Culex quinquefasciatus (Say), Anopheles quadrimaculatus (Say) and Anopheles albimanus (Wied.)

Homology searches of the nucleotide and predicted amino acid sequence of SuCPV were performed using BLAST (NCBI).

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ous Ludox gradient. 1.200 § 0.001 (n D 2).

3. Results

The

estimated

density

was

3.1. Field collection and gross pathology 3.3. Ultrastructural studies Hatchet Creek drains a woodland swamp area in Northern Alachua County, Florida, USA. At the collection site, however, the creek crosses under the highway and is not shaded by tree canopy, allowing grasses and other aquatic vegetation to grow. The average conductivity of the stream was 65 § 5 S/cm, with total dissolved solids of 43 § 4 ppm and temperature of 18.9 § 0.8 °C (n D 2). There were three species in the collections: Simulium jonesi Stone & Snoddy, Simulium slossonae Dyar & Shannon, and S. ubiquitum Adler, Currie & Wood (formerly an unnamed species in the Simulium tuberosum complex). However, only S. slossonae and S. ubiquitum were infected with the CPV. Table 1 gives the proportion of the community and the estimated infection rate of each species. S. ubiquitum accounted for half of the larvae collected, and the infection rate was 10.4% and 14.6% in each collection. S. slossonae accounted for 12.4% of the community but only 1.3% was infected with CPV. The CPV-infected larvae were readily identiWed when examined against a black background. The posterior portion of the midgut and the gastric caeca appeared whitish with irregularly shaped white granules in these areas (arrows in Fig. 1a and b). When examined by phase microscopy, the granules were observed to be the swollen cytoplasm of midgut cells Wlled with small angular microscopic granules with the nuclei conspicuously compressed.

Inclusion bodies of this virus were localized in the cytoplasm of epithelial cells in the gastric caeca and posterior midgut of larval black Xies (Fig. 1a and b). Non-occluded and occluded viral particles were distributed throughout the cytoplasm, but not observed within nuclei (Fig. 2a). The normal cytoplasmic organelles and ribosomes were generally not present in the regions where virus replication was active but could be found in the surrounding regions (Fig. 2c). When viewed by negative staining and electron microscopy, the non-occluded virions were spherical and approximately 50 nm in diameter. Each virion consisted of

3.2. Virus puriWcation The CPV from the dissected midguts was centrifuged at 16,000g for 30 min and formed a single band in a continu-

Table 1 Community and infection rate of each Simulium species from Hatchet Creek, Alachua County, Florida Simulium species S. jonesi S. slossonae S. ubiquitum

Proportion of community 36.9 § 6.5% 12.4 § 2.1% 50.7 § 4.4%

Infection rate 0.0% 1.3 § 0.03% 12.5 § 2.1%

Fig. 2. Transmission electron micrographs of SuCPV-20 infections in the midgut epithelial cells of the posterior midgut of a Weld-collected Simulium ubiquitum larva. (a) A midgut epithelial cell with SuCPV-20 inclusion bodies dispersed in the cytoplasm. (b) An inclusion body of SuCPV-20 in the process of growing by the accretion of virions. (c) Mature inclusion bodies developing in a region of the cytoplasm that is devoid of normal cytoplasmic organelles. Adjacent regions appear to have the normal complement of organelles. (d) Mature inclusion bodies that are polyhedral in shape with angular sides.

Fig. 1. (a) Intact SuCPV-20 infected Simulium ubiquitum larva. (b) Dissected midgut of S. ubiquitum infected with SuCPV-20. Arrows indicate infected cells in the posterior midgut and gastric caeca.

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a poorly deWned capsid (icosahedral in shape, approximately 30 nm in diameter) with an electron-dense core of approximately 25 nm in diameter (Fig. 2b). It appeared likely that individual or small groups of virions were initially occluded by the inclusion body protein and grew by accretion as additional virions became associated with the original core (Fig. 2b and c). The symmetry of mature inclusion bodies was icosahedral with angular sides and contained upwards of 50 virions in a cross section. Mature inclusion bodies were typically 0.25 m in diameter with a small portion attaining a maximum diameter of approximately 1.0 m. 3.4. Horizontal transmission The mosquitoes C. quinquefasciatus, A. quadrimaculatus, and A. albimanus exposed to the SuCPV showed no symptoms of disease after two and Wve day examinations. The addition of magnesium ions, which has been shown to increase the level of infection in CrCPV and UsCPV, had no eVect on the transmission of the virus (Shapiro et al., 2005; Green et al., 2006). 3.5. Electrophoretic separation of dsRNA from SuCPV The SuCPV genome separated into 10 distinct segments on a 1% agarose gel (Fig. 3, lane 2). The electrophoretic proWle of the SuCPV dsRNAs was clearly diVerent from those of the other CPVs previously isolated from Diptera (UsCPV-17 and CrCPV-17) demonstrating that it belongs to a distinct electropherotype (Fig. 3, lanes 3 and 4, respectively) (Shapiro et al., 2005; Green et al., 2006). The size of the SuCPV RNAs (estimated basepairs (bp)) from Seg-1 to Seg-10 were 3700, 3650, 3600, 3100, 2200, 1750, 1400, 1400, 1250, and 850, respectively, as determined by comparison with 1 kb marker and published sequence sizes for the genome segments of CrCPV-17 and UsCPV-17. 3.6. Sequence determination and analysis of segment 10 Sequence analysis of SuCPV Seg-10 demonstrated that it is 836 bp in length (Fig. 4) (GenBank Accession No. DQ834386) and contains a single large open reading frame (ORF) from nucleotides 38–766. The ORF encodes a predicted protein of 243 amino acids with an estimated molecular mass of »27 kDa. 3.7. Comparison of RNA termini and homology to other CPV polyhedrins The terminal sequence of SuCPV-20 Seg-10 was 5⬘-AGAAAACƒACCAUGGC-3⬘ (Table 2). Although the 5⬘ terminal region of SuCPV RNAs contain four of the Wve conserved bases also found in CPV-17 (UsCPV-17 and CrCPV-17, which have a conserved sequence of 5⬘-AGAACƒUACACU-3⬘), the 3⬘ terminal sequences are very diVerent (Table 2). The terminal sequences of SuCPV-

Fig. 3. Electrophoretic Separation of Diptera CPVs on a 1% agarose gel. KB Marker (lane 1), SuCPV-20 (lane 2), UsCPV-17 (lane 3), and CrCPV17 (lane 4). The published segment-10 sizes of UsCPV-17 and CrCPV-17 are 892 bp and 890 bp, respectively.

20 were also diVerent from those previously published for isolates of CPV-1, 2, 4, 5, 14, 15, 16, 18, and 19 (Mertens et al., 2004—www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ CPV-RNA-Termin.htm). 3.8. Homology with other CPV polyhedrins Alignment of the Seg-10, using (bl2seq) (Tatusova and Madden, 1999) from SuCPV to CrCPV-17 and UsCPV-17 nucleotide sequences, showed no sequence identity (data not shown). Moreover, a BLAST nucleotide search of SuCPV-20 illustrated no signiWcant homology to previously characterized cypoviruses. However, a BLAST protein search of the SuCPV Seg-10 predicted open reading frame revealed low but signiWcant homology to the polyhedrin proteins of isolates belonging to the Cypovirus-4, Cypovirus-16 and Cypvirus-17 species. The isolates from the Cypovirus-4 species, which include AaCPV-4 (from Antheraea assamensis), AmCPV-4 (from Antheraea mylitta), and ApCPV-4 (from Antheraea proylei), have a 34% sequence identity to SuCPV (Qanungo et al., 2000); the isolate of Choristoneura fumiferana cypovirus-16 (CfCPV-16) had a 25% identity (Echeverry et al., 1997); while members of the Cypovirus-17 species, UsCPV-17 and CrCPV-17, have 20% identity (Green et al., 2006; Shapiro et al., 2005).

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Fig. 4. Nucleotide and Predicted Protein Sequence of Seg-10 of SuCPV-20 (243 amino acids). The initiation and termination codons for SuCPV-20 are underlined. The translated amino acid is listed below the nucleotide sequence. Table 2 Comparison of cypovirus conserved terminal regions Virus stain

Terminal region sequences

CPV-1 CPV-2 CPV-4a CPV-5 CPV-14 CPV-15 CPV-16 CPV-17 CPV-18 CPV-19 SuCPV-20

5⬘-AGUAAƒGUUAGCC-3⬘ 5⬘-AGUUUUAƒUAGGUC-3⬘ 5⬘-AAUCGACƒUCGUAUG-3⬘ 5⬘-AGUUƒUUGC-3⬘ 5⬘-AGAAƒAGCU-3⬘ 5⬘-AUUAAAAƒGC-3⬘ 5⬘-AGUUUUUƒUUUGUGC-3⬘ 5⬘-AGAACƒUACACU-3⬘ 5⬘-AGUAAAƒGUUAGCU-3⬘ 5⬘-AACAAAƒUUUGC-3⬘ 5⬘-AGAAAACƒ.ACCAUGGC-3⬘

a

Genome segment 9 only of ApCPV-4, AaCPV-4, and AmCPV-4.

4. Discussion There are approximately 1800 black Xy species throughout the world, representing less than 2% of all Diptera (Crosskey, 2002; Adler et al., 2004). S. ubiquitum is a newly recognized species within the S. tuberosum group (Adler et al., 2004). The S. tuberosum group contains 10 named species in the Nearctic Region (United States of America, Canada, and Northern Mexico) and more than 30 in the Palearctic Region (Adler et al., 2004). Although cypovi-

ruses are the most common viruses in black Xy larvae (Weiser and Undeen, 1981), they have remained unclassiWed due to the lack of molecular and genomic data. This study has provided these data for a viral isolate from the black Xy S. ubiquitum, verifying that SuCPV is a new member of the cypoviruses. The cypoviruses are currently classiWed into 16 distinct species (recognized by ICTV), (with three further provisional species: CPV-17 to CPV-19), which can be distinguished by genomic RNA electrophoretype, by sequence comparisons and/or by serological assays. Most of these species are from Lepidoptera, although the isolates of CPV17 are from Diptera (Green et al., 2006; Shapiro et al., 2005; Mertens et al., 2004, 1999; Payne and Rivers, 1976; Mertens et al., 1989; Payne and Mertens, 1983). Initially, it was considered likely that the sequence of Seg-10 from SuCPV would show signiWcantly higher similarities to the other Dipteran CPV isolates of CPV-17, from mosquitoes (Uranotaenia sapphirina UsCPV - 17 (Shapiro et al., 2005) and Culex restuans CrCPV-17 (Green et al., 2006; Shapiro et al., 2005)). However, electrophoretic and sequence analysis, which are two of the established parameters used to identify and distinguish individual Cypovirus species (Mertens et al., 2004) demonstrated that this is not the case. The 10 dsRNAs from SuCPV-20 have a migration pattern that is clearly diVerent to that of CPV-17. Unfortunately no

T.B. Green et al. / Journal of Invertebrate Pathology 95 (2007) 26–32

sequence data are currently published for many of the other cypoviruses isolates originally identiWed as distinct types (species) by electropherotyping (Payne and Rivers, 1976) including types 3, 6–13. However, despite the use of diVerent electrophoresis conditions to analyse the genomic dsRNAs of many of the lepidopteran cypoviruses in earlier publications (CPV types 1–16) (Payne and Rivers, 1976; Payne et al., 1977; Fouillaud and Morel, 1994; Belloncik et al., 1996; Echeverry et al., 1997), the diVerences between their migration patterns and those of the SuCPV isolate described here, suggest that this new isolate belongs to a distinct electropherotype. A BLAST search using the SuCPV-20 nucleotide sequence, revealed no signiWcant similarity to any of the previously characterized cypoviruses (including types 1, 2, 4, 5, 14–19). However, a BLAST search using the SuCPV20 polyhedrin sequence found low but signiWcant levels of sequence identity with the polyhedrins of CPV-4 (from silkworms), CPV-16 (from budworms), and CPV-17 (from mosquitoes). Although the Wrst four terminal bases at the 5⬘ end of SuCPV-20 are similar to those of the mosquito CPV17s, they do not contain conserved 3⬘ terminal sequences that have been identiWed in any of the other cypoviruses, (although sequence data are not available for CPV-3, 6, 7, 8, 9, 10, 11, 12, and 13—see http://www.iah.bbsrc.ac.uk/ dsRNA_virus_proteins/CPV-RNA-Termin.htm). The electrophoretic and sequence comparison data collectively support the classiWcation of SuCPV within a new Cypovirus species (CPV-20.) There are few reports on the biological and morphological features of black Xy cypoviruses. The Wrst report was from Stegopterna mutata (Malloch) larvae collected in Newfoundland, Canada (Bailey et al., 1975; Bailey, 1977), with an additional report from Simulium donovani Vargas (as Simulium aureum) from Guatemala, Central America (Takaoka, 1980). Each of these isolates was restricted to midgut cells of larval black Xies, with incidence of infection ranging from 1 to 5% for St. mutata and 0.5–14% for S. donovani. Infection rates for SuCPV-20 in S. ubiquitum were in a comparable range to these previous reports, with an average infection level of 12.5%. Bailey (1977) was able to experimentally transmit the cypovirus from St. mutata to black Xy larvae in the laboratory but the results were sporadic. Although the present study did not attempt to infect black Xy larvae with SuCPV-20 due to diYculties in maintaining black Xy larvae under laboratory conditions, eVorts to infect mosquito larvae were unsuccessful. Ultrastructural features of the inclusion bodies for black Xy cypoviruses have been reported only for St. mutata (Bailey et al., 1975). This isolate is similar to SuCPV-20 in that the inclusion bodies are polyhedral in shape and contain multiple spherical virions that range in size from approximately 0.5 to 1.0 m. Inclusion body formation was restricted to the cytoplasm of midgut epithelial cells mainly in the gastric caeca and the posterior midgut. Simuliidae are hosts for a variety of natural enemies, and a complete review of the pathogens and parasites isolated

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from North American black Xies can be found in Adler et al. (2004). At least 10 species of black Xies have been reported to harbor cypoviruses, which represent the most common viruses of simuliids in North America, but the biology of these disease agents remains virtually unknown. This study is an important Wrst step in clarifying the taxonomic status of cypoviruses in black Xies and establishing their relationship to cypoviruses from other insects. Sequencing the entire genome of SuCPV-20, along with that of CrCPV-17 and UsCPV-17 from mosquitoes, is presently underway and will provide crucial information that should facilitate studies to investigate viral-host relationships at the molecular level. It is anticipated that these data will also provide new reagents and techniques (e.g. RTPCR primers, probes, expressed antigens and antibodies) to investigate the Weld dynamics of cypovirus infections and to explore their possible use to control black Xies. Acknowledgments The authors acknowledge the support of Sasha Shapiro and Heather Furlong (USDA/ARS Gainesville) and funding support by EU contract: ReoID (contract number QLK2-CT-2000-00143). We also express our gratitude to Yoshifumi Hashimoto (USDA/ARS, Gainesville) and Lawrence A. Lacey (USDA/ARS/Yakima) for their critical review of this work. References Adler, P.H., Currie, D.C., Wood, D.M., 2004. The Black Flies (Simuliidae) of North America. Cornell University Press, Ithaca, NY, 941 pp. Bailey, C.H., 1977. Field and laboratory observations on a cytoplasmic polyhedrosis virus of blackXies (Diptera: Simuliidae). J. Invertebr. Pathol. 29, 69–73. Bailey, C.H., Shapiro, M., Granados, R.R., 1975. A cytoplasmic polyhedrosis virus from the larval blackXies Cnephia mutata and Prosimulium mixtum (Diptera: Simuliidae). J. Invertebr. Pathol. 25, 273–274. Belloncik, S., Liu, J., Su, D., Arella, M., 1996. IdentiWcation and characterization of a new cypovirus type 1, isolated from Heliothis armigera. J. Invertebr. Pathol. 67, 41–47. Crosskey, R.W., 2002. Second Update to the Taxonomic and Geographical Inventory of World BlackXies (Diptera: Simuliidae). The Natural History Museum, London. Echeverry, F., Bergeron, J., Kaupp, W., Guertin, C., Arell, M., 1997. Sequence analysis and expression of the polyhedrin gene of Christoneura fumiferana cytoplasmic polyhedrosis virus (CfCPV). Gene 198, 399–406. Fouillaud, M., Morel, G., 1994. Characterization of cytoplasmic and nuclear polyhedrosis viruses recovered from the nest of Polistes hebraeus F. (Hymenoptera; Vespidae). J. Invertebr. Pathol. 64, 89–95. Graham, R.I., Rao, S., Possee, R.D., Sait, S.M., Mertens, P.P., Hails, R.S., 2006. Detection and characterisation of three novel species of reovirus (Reoviridae), isolated from geographically separate populations of the winter moth Operophtera brumata (Lepidoptera: Geometridae) on Orkney. J. Invertebr. Pathol. 91, 79–87. Green, T.B., Shapiro, A., White, S., Rao, S., Mertens, P.P.C., Carner, G., Becnel, J.J., 2006. Molecular and biological characterization of a Cypovirus from the mosquito Culex restuans. J. Invertebr. Pathol. 91, 27–34. Hagiwara, K., Rao, S., Scott, S.W., Carner, G.R., 2002. Nucleotide sequences of segments 1, 3 and 4 of the genome of Bombyx mori cypovirus 1 encoding putative capsid proteins VP1, VP3 and VP4, respectively. J. Gen. Virol. 83, 1477–1482.

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