Biological and Molecular Studies of a Cypovirus from the Black Fly

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,¤

a ARS, CMAVE, 1600-1700 S.W. 23rd Drive, Gainesville, FL 32608, USA b 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 within which the virus particles can become either singly or (more usually) multiply embedded. Cypoviruses usually Cypoviruses have been isolated mainly from insects in infect the midgut cells, particularly of the insect larval the orders Lepidoptera, Diptera, and Hymenoptera. The stages and can produce chronic rather than fatal disease. cypovirus genome usually consists of 10 double-stranded Sixteen diVerent virus species (Cypovirus-1 to Cypovirus-16) RNA (dsRNA) segments (Mertens et al., 2004; Hukuhara have already been formally recognized within the genus and Bonami, 1991; Payne and Rivers, 1976), packaged as Cypovirus, family Reoviridae (Mertens et al., 2004). These exactly one copy of each segment, within each single- diVerent species can be distinguished by diVerences in the shelled, icosahedral and turreted virus particle. The cypovi- migration patterns of their dsRNAs during electrophoresis, ruses replicate within the cytoplasm of infected insect cells variations in RNA sequences (both in the coding regions and typically produce inclusion bodies (polyhedra) that are and conserved terminal regions) and by antigenic variation composed primarily of a single viral protein (polyhedrin), 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 * Corresponding author. Fax: +1 352 374 5966. (CPV-17—Green et al., 2006; Shapiro et al., 2005) and from E-mail address: [email protected] (J.J. Becnel). the winter moth Operophtera brumata (CPV-18 and 19—

Graham et al., 2006) that have been characterized and com- were exposed to 5 LE (larval equivalent) of S. ubiquitum pared to the existing species by electropherotype analysis infected with CPV in 100 ml of 0 or 10 mM MgCl2 at room and sequencing of the viral genome. temperature and then transferred to 15 °C for two days. Historically, hundreds of CPV isolates have been identi- Larvae were examined two and Wve days post inoculum Wed from diVerent species of Lepidoptera, based on the simi- (p.i.) for signs of disease. larities in their virion, inclusion bodies and infection of midgut epithelial cells. However, most of these viruses have 2.5. Analysis of SuCPV by electropherotype remained uncharacterized and are consequently unclassiWed by the International Committee for the Taxonomy of Viruses Genomic dsRNAs of SuCPV was extracted from puri- (ICTV), due (in large part) to an inability to recover enough Wed polyhedra, using a QIAampViral Mini Kit from of each virus for molecular studies (such as electropherotyp- Qiagen (Green et al., 2006; Shapiro et al., 2005; Rao et al., ing, serological analysis, or sequencing). We report the isola- 2003; Hagiwara et al., 2002). SuCPV RNA was analyzed on tion of a CPV from the black Xy Simulium ubiquitum and a 1% agarose gel. Ethidium bromide was integrated into the present molecular data to support its classiWcation as a mem- gel at a Wnal concentration of 0.5 mg/ml. Genomic RNA ber of a new Cypovirus species that we have tentatively iden- from UsCPV-17 and CrCPV-17 was also analyzed in the tiWed as Cypovirus-20. The new virus isolate can therefore be same gel to compare the electrophoretic proWle of cypo- named as ‘Simulium ubiquitum CPV-20’ (SuCPV-20). viruses isolated from Diptera. Seg-10 sequences from UsCPV-17 (AY876384) and CrCPV-17 (DQ212785) 2. Materials and methods genomes are accessible in GenBank.

2.1. Field collection and gross pathology 2.6. cDNA synthesis, ampliWcation by PCR, cloning and sequencing Simulium larvae were collected twice in Hatchet Creek at Hwy 24 in Alachua County, Florida (N29.73066 The conditions for cDNA synthesis, ampliWcation by W82.24906), in April and May 2005. The larvae were held PCR, cloning, and sequencing were similar to the proto- in creek water and returned to the laboratory for examina- cols published by Shapiro et al. (2005) and Green et al. tion. Individual larvae were removed from the plant mate- (2006). BrieXy, RNA was isolated from puriWed virions, rial and examined against a dark background, using a using a QIAampViral Mini Kit (Qiagen). An anchor dissecting microscope. Diseased larvae were segregated by primer was ligated to the dsRNAs, using T4 RNA ligase pathogen types, and samples of diseased and healthy larvae (New England Biolab). cDNA synthesis was then per- were placed in 70% ethanol for identiWcation. formed with a primer part that is complementary to the anchor using AMV reverse transcriptase (Promega). PCR 2.2. Ultrastructural studies (electron microscopy) was completed using the Advantage 2 PCR Kit (Clon- tech) with primer 5-15-1. The PCR conditions were 95 °C Dissected midguts of infected black Xy larvae were pro- for 15 s of denaturing and 68 °C for 3 min of annealing/ cessed for electron microscopy as described by Shapiro extension (24 cycles). et al. (2004). BrieXy, dissected midguts were Wxed in 2.5% The PCR products of SuCPV were separated on a 1% gluteraldehyde for 2 h, postWxed in 2% osmium tetroxide agarose gel. The expected size of Seg-10 was »850 bp. The for 1 h, dehydrated in an ethanol series and embedded in Seg-10 DNA band was then excised, puriWed, A-tailed by epon-araldite. Thin sections were stained in uranyl acetate Taq enzyme (10 min at 72 °C with 200 nM dATP in and lead citrate and examined and photographed at 75 kV. 1£ Taq buVer), and cloned into pGEM-T Easy vector. The cloned insert from Seg-10 was ampliWed by PCR, 2.3. PuriWcation of virus using SP6 and T7 primer pairs, and was conWrmed by the lengths of the PCR product and by restriction mapping. Midguts of SuCPV-infected larvae were dissected and Three clones from Seg-10 were completely sequenced homogenized in deionized water (25 from the Wrst collection using SP6 and T7 primers, with a dye terminator cycle and 50 from the second collection). The suspension was placed sequencing ready reaction kit on a Beckman CEQ8000 on a continuous 0.1mM NaOH : HS-40 Ludox® gradient and system. The sequences obtained were then used to design centrifuged at 16,000g for 30min. The resulting band was primers for direct sequencing of the RT-PCR products removed and suspended in 0.1mM NaOH and spun again at from Seg-10, which conWrmed the original sequence data 16,000g for 20min and this washing was repeated three times. from the cloned cDNAs. The Wnal pellet was suspended in 0.1mM NaOH. 2.7. Homology analysis 2.4. Horizontal transmission (Cation Assays) Homology searches of the nucleotide and predicted Four-day old Culex quinquefasciatus (Say), Anopheles amino acid sequence of SuCPV were performed using quadrimaculatus (Say) and Anopheles albimanus (Wied.) BLAST (NCBI). 28 T.B. Green et al. / Journal of Invertebrate Pathology 95 (2007) 26–32

3. Results ous Ludox gradient. The estimated density was 1.200 § 0.001 (n D 2). 3.1. Field collection and gross pathology 3.3. Ultrastructural studies Hatchet Creek drains a woodland swamp area in North- ern Alachua County, Florida, USA. At the collection site, Inclusion bodies of this virus were localized in the cyto- however, the creek crosses under the highway and is not plasm of epithelial cells in the gastric caeca and posterior shaded by tree canopy, allowing grasses and other aquatic midgut of larval black Xies (Fig. 1a and b). Non-occluded vegetation to grow. The average conductivity of the stream and occluded viral particles were distributed throughout was 65 § 5 S/cm, with total dissolved solids of 43 § 4 ppm the cytoplasm, but not observed within nuclei (Fig. 2a). The and temperature of 18.9 § 0.8 °C (n D 2). There were three normal cytoplasmic organelles and ribosomes were gener- species in the collections: Simulium jonesi Stone & Snoddy, ally not present in the regions where virus replication was Simulium slossonae Dyar & Shannon, and S. ubiquitum active but could be found in the surrounding regions Adler, Currie & Wood (formerly an unnamed species in the (Fig. 2c). When viewed by negative staining and electron Simulium tuberosum complex). However, only S. slossonae microscopy, the non-occluded virions were spherical and and S. ubiquitum were infected with the CPV. Table 1 gives approximately 50 nm in diameter. Each virion consisted of 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.

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-

Fig. 2. Transmission electron micrographs of SuCPV-20 infections in the Table 1 midgut epithelial cells of the posterior midgut of a Weld-collected Simu- Community and infection rate of each Simulium species from Hatchet lium ubiquitum larva. (a) A midgut epithelial cell with SuCPV-20 inclusion Creek, Alachua County, Florida bodies dispersed in the cytoplasm. (b) An inclusion body of SuCPV-20 in Simulium species Proportion of community Infection rate 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 S. jonesi 36.9 § 6.5% 0.0% cytoplasmic organelles. Adjacent regions appear to have the normal com- S. slossonae 12.4 § 2.1% 1.3 § 0.03% plement of organelles. (d) Mature inclusion bodies that are polyhedral in S. ubiquitum 50.7 § 4.4% 12.5 § 2.1% 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. T.B. Green et al. / Journal of Invertebrate Pathology 95 (2007) 26–32 29 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 symp- toms 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 W V Fig. 3. Electrophoretic Separation of Diptera CPVs on a 1% agarose gel. pro le of the SuCPV dsRNAs was clearly di erent from KB Marker (lane 1), SuCPV-20 (lane 2), UsCPV-17 (lane 3), and CrCPV- those of the other CPVs previously isolated from Diptera 17 (lane 4). The published segment-10 sizes of UsCPV-17 and CrCPV-17 (UsCPV-17 and CrCPV-17) demonstrating that it belongs are 892 bp and 890 bp, respectively. to a distinct electropherotype (Fig. 3, lanes 3 and 4, respectively) (Shapiro et al., 2005; Green et al., 2006). The size of 20 were also diVerent from those previously published for the SuCPV RNAs (estimated basepairs (bp)) from Seg-1 to isolates of CPV-1, 2, 4, 5, 14, 15, 16, 18, and 19 (Mertens Seg-10 were 3700, 3650, 3600, 3100, 2200, 1750, 1400, 1400, et al., 2004—www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/ 1250, and 850, respectively, as determined by comparison CPV-RNA-Termin.htm). with 1 kb marker and published sequence sizes for the genome segments of CrCPV-17 and UsCPV-17. 3.8. Homology with other CPV polyhedrins

3.6. Sequence determination and analysis of segment 10 Alignment of the Seg-10, using (bl2seq) (Tatusova and Madden, 1999) from SuCPV to CrCPV-17 and UsCPV-17 Sequence analysis of SuCPV Seg-10 demonstrated that it nucleotide sequences, showed no sequence identity (data is 836 bp in length (Fig. 4) (GenBank Accession No. not shown). Moreover, a BLAST nucleotide search of DQ834386) and contains a single large open reading frame SuCPV-20 illustrated no signiWcant homology to previ- (ORF) from nucleotides 38–766. The ORF encodes a ously characterized cypoviruses. However, a BLAST pro- predicted protein of 243 amino acids with an estimated tein search of the SuCPV Seg-10 predicted open reading molecular mass of »27 kDa. frame revealed low but signiWcant homology to the polyhedrin proteins of isolates belonging to the Cypovirus-4, 3.7. Comparison of RNA termini and homology to other Cypovirus-16 and Cypvirus-17 species. The isolates from CPV polyhedrins the Cypovirus-4 species, which include AaCPV-4 (from Antheraea assamensis), AmCPV-4 (from Antheraea The terminal sequence of SuCPV-20 Seg-10 was mylitta), and ApCPV-4 (from Antheraea proylei), have a 5Ј-AGAAAACƒACCAUGGC-3Ј (Table 2). Although the 34% sequence identity to SuCPV (Qanungo et al., 2000); 5Ј terminal region of SuCPV RNAs contain four of the Wve the isolate of Choristoneura fumiferana cypovirus-16 conserved bases also found in CPV-17 (UsCPV-17 and (CfCPV-16) had a 25% identity (Echeverry et al., 1997); CrCPV-17, which have a conserved sequence of while members of the Cypovirus-17 species, UsCPV-17 5Ј-AGAACƒUACACU-3Ј), the 3Ј terminal sequences are and CrCPV-17, have 20% identity (Green et al., 2006; very diVerent (Table 2). The terminal sequences of SuCPV- Shapiro et al., 2005). 30 T.B. Green et al. / Journal of Invertebrate Pathology 95 (2007) 26–32

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 ruses are the most common viruses in black Xy larvae Comparison of cypovirus conserved terminal regions (Weiser and Undeen, 1981), they have remained unclassi- Virus stain Terminal region sequences Wed due to the lack of molecular and genomic data. This CPV-1 5Ј-AGUAAƒGUUAGCC-3Ј study has provided these data for a viral isolate from the CPV-2 5Ј-AGUUUUAƒUAGGUC-3Ј black Xy S. ubiquitum, verifying that SuCPV is a new mem- CPV-4a 5Ј-AAUCGACƒUCGUAUG-3Ј ber of the cypoviruses. Ј Ј CPV-5 5 -AGUUƒUUGC-3 The cypoviruses are currently classiWed into 16 distinct CPV-14 5Ј-AGAAƒAGCU-3Ј CPV-15 5Ј-AUUAAAAƒGC-3Ј species (recognized by ICTV), (with three further provi- CPV-16 5Ј-AGUUUUUƒUUUGUGC-3Ј sional species: CPV-17 to CPV-19), which can be distin- CPV-17 5Ј-AGAACƒUACACU-3Ј guished by genomic RNA electrophoretype, by sequence CPV-18 5Ј-AGUAAAƒGUUAGCU-3Ј comparisons and/or by serological assays. Most of these Ј Ј CPV-19 5 -AACAAAƒUUUGC-3 species are from Lepidoptera, although the isolates of CPV- SuCPV-20 5Ј-AGAAAACƒ.ACCAUGGC-3Ј 17 are from Diptera (Green et al., 2006; Shapiro et al., 2005; a Genome segment 9 only of ApCPV-4, AaCPV-4, and AmCPV-4. Mertens et al., 2004, 1999; Payne and Rivers, 1976; Mertens et al., 1989; Payne and Mertens, 1983). Initially, it was con- sidered likely that the sequence of Seg-10 from SuCPV 4. Discussion would show signiWcantly higher similarities to the other Dipteran CPV isolates of CPV-17, from mosquitoes There are approximately 1800 black Xy species through- (Uranotaenia sapphirina UsCPV - 17 (Shapiro et al., 2005) out the world, representing less than 2% of all Diptera and Culex restuans CrCPV-17 (Green et al., 2006; Shapiro (Crosskey, 2002; Adler et al., 2004). S. ubiquitum is a newly et al., 2005)). However, electrophoretic and sequence analy- recognized species within the S. tuberosum group (Adler sis, which are two of the established parameters used to et al., 2004). The S. tuberosum group contains 10 named identify and distinguish individual Cypovirus species species in the Nearctic Region (United States of America, (Mertens et al., 2004) demonstrated that this is not the case. Canada, and Northern Mexico) and more than 30 in the The 10 dsRNAs from SuCPV-20 have a migration pattern Palearctic Region (Adler et al., 2004). Although cypovi- that is clearly diVerent to that of CPV-17. Unfortunately no T.B. Green et al. / Journal of Invertebrate Pathology 95 (2007) 26–32 31 sequence data are currently published for many of the other from North American black Xies can be found in Adler cypoviruses isolates originally identiWed as distinct types et al. (2004). At least 10 species of black Xies have been (species) by electropherotyping (Payne and Rivers, 1976) reported to harbor cypoviruses, which represent the most including types 3, 6–13. However, despite the use of diVer- common viruses of simuliids in North America, but the ent electrophoresis conditions to analyse the genomic dsR- biology of these disease agents remains virtually unknown. NAs of many of the lepidopteran cypoviruses in earlier This study is an important Wrst step in clarifying the taxo- publications (CPV types 1–16) (Payne and Rivers, 1976; nomic status of cypoviruses in black Xies and establishing Payne et al., 1977; Fouillaud and Morel, 1994; Belloncik their relationship to cypoviruses from other insects. et al., 1996; Echeverry et al., 1997), the diVerences between Sequencing the entire genome of SuCPV-20, along with their migration patterns and those of the SuCPV isolate that of CrCPV-17 and UsCPV-17 from mosquitoes, is pres- described here, suggest that this new isolate belongs to a ently underway and will provide crucial information that distinct electropherotype. should facilitate studies to investigate viral-host relation- A BLAST search using the SuCPV-20 nucleotide ships at the molecular level. It is anticipated that these data sequence, revealed no signiWcant similarity to any of the will also provide new reagents and techniques (e.g. RT- previously characterized cypoviruses (including types 1, 2, PCR primers, probes, expressed antigens and antibodies) to 4, 5, 14–19). However, a BLAST search using the SuCPV- investigate the Weld dynamics of cypovirus infections and to 20 polyhedrin sequence found low but signiWcant levels of explore their possible use to control black Xies. sequence identity with the polyhedrins of CPV-4 (from silk- worms), CPV-16 (from budworms), and CPV-17 (from Acknowledgments mosquitoes). Although the Wrst four terminal bases at the 5Ј end of SuCPV-20 are similar to those of the mosquito CPV- The authors acknowledge the support of Sasha Shapiro 17s, they do not contain conserved 3Ј terminal sequences and Heather Furlong (USDA/ARS Gainesville) and fund- that have been identiWed in any of the other cypoviruses, ing support by EU contract: ReoID (contract number (although sequence data are not available for CPV-3, 6, 7, QLK2-CT-2000-00143). We also express our gratitude to 8, 9, 10, 11, 12, and 13—see http://www.iah.bbsrc.ac.uk/ Yoshifumi Hashimoto (USDA/ARS, Gainesville) and dsRNA_virus_proteins/CPV-RNA-Termin.htm). The elec- Lawrence A. Lacey (USDA/ARS/Yakima) for their critical trophoretic and sequence comparison data collectively sup- review of this work. port the classiWcation of SuCPV within a new Cypovirus species (CPV-20.) References There are few reports on the biological and morphological features of black Xy cypoviruses. 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