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Molecular and Biological Characterization of a Cypovirus from the Mosquito Culex Restuans

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Molecular and Biological Characterization of a Cypovirus from the Mosquito Culex Restuans Journal of Invertebrate Pathology 91 (2006) 27–34 www.elsevier.com/locate/yjipa Molecular and biological characterization of a Cypovirus from the mosquito Culex restuans Terry B. Green a,¤, Alexandra Shapiro a, Susan White a, Shujing Rao b, Peter P.C. Mertens b, Gerry Carner 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 Clemson University, 114 Long Hall, Clemson, SC 29634, USA Received 4 August 2005; accepted 11 October 2005 Abstract A cypovirus from the mosquito Culex restuans (named CrCPV) was isolated and its biology, morphology, and molecular characteris- tics were investigated. CrCPV is characterized by small (0.1–1.0 m), irregularly shaped inclusion bodies that are multiply embedded. Lab- oratory studies demonstrated that divalent cations inXuenced transmission of CrCPV to Culex quinquefasciatus larvae; magnesium enhanced CrCPV transmission by »30% while calcium inhibited transmission. CrCPV is the second cypovirus from a mosquito that has been conWrmed by using molecular analysis. CrCPV has a genome composed of 10 dsRNA segments with an electropherotype similar to the recently discovered UsCPV-17 from the mosquito Uranotaenia sapphirina, but distinct from the lepidopteran cypoviruses BmCPV-1 (Bombyx mori) and TnCPV-15 (Trichoplusia ni). Nucleotide and deduced amino acid sequence analysis of CrCPV segment 10 (polyhe- drin) suggests that CrCPV is closely related (83% nucleotide sequence identity and 87% amino acid sequence identity) to the newly char- acterized UsCPV-17 but is unrelated to the 16 remaining CPV species from lepidopteran hosts. A comparison of the terminal segment regions of CrCPV and UsCPV-17, an additional method for diVerentiating various Cypovirus species, revealed a high level of conserva- tion. Therefore, we propose that CrCPV is a member of the Cypovirus-17 group and designate this species as CrCPV-17. 2005 Elsevier Inc. All rights reserved. Keywords: Culex restuans Cypovirus; Transmission; Divalent cations; Mosquito; Morphology; Genomic; Electropherotype 1. Introduction inclusion bodies by the insect, the alkaline pH and protein- ases of the intestinal track break down the polyhedrin and Occluded RNA viruses of insects (formerly cytoplasmic release the virions, which enter and replicate in the cyto- polyhedrosis viruses) have been classiWed in the family Reo- plasm of midgut epithelial cells (Mertens et al., 1999, 2004b; viridae within the genus Cypovirus (CPVs). CPV virions are Zhang et al., 1999). Most CPV infections produce chronic embedded (occluded) by a polyhedrin protein to form the disease with low mortality although some are pathogenic resulting inclusion body (Xia et al., 1991). Zhang et al. (Mertens et al., 1999, 2004b). (1999) reported that CPVs have a single-shelled capsid CPV genomes usually consist of 10 double-stranded RNA instead of a multiple-shelled organization as found in other (dsRNA) segments (Seg-1 to Seg-10) (Fujii-Kawata et al., Reoviridae members. These icosahedral, cubic, or some- 1970). An eleventh segment has been shown in cypoviruses times irregular particles infect midgut cells of a wide range from TnCPV-15 (Trichoplusia ni), AmCPV-4 (Antheraea of insects (Payne and Rivers, 1976). After ingestion of the mylitta) and BmCPV-1 (Bombyx mori). (Arella et al., 1988; Qanungo et al., 2002; Rao et al., 2003). The RNA segments are composed of plus-strand mRNA and its complementary * Corresponding author. Fax: +1 352 374 5966. minus-strand in an end to end based-pair conWguration with E-mail address: [email protected] (T.B. Green). the exception of a protruding 5Ј cap on the plus-strand 0022-2011/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2005.10.007 28 T.B. Green et al. / Journal of Invertebrate Pathology 91 (2006) 27–34 (Furuichi and Miura, 1975). Based on the migration pattern through a 400 mesh nylon screen. The Wltrate was layered of the genome segments on a 1% agarose or 3–5% polyacryl- on top of an HS-40 Ludox continuous gradient and centri- amide gel, CPVs are classiWed into 17 distinct electrophore- fuged at 16,000g for 30 min. The resulting band containing types (Mertens et al., 1989, 1999, 2004b; Payne and Mertens, puriWed virus formed at an estimated density of 1983; Payne and Rivers, 1976; Shapiro et al., 2005). 1.157 § 0.002 (mean § SE), n D 6. The viral band was recov- With the exception of a single isolate from mosquitoes, ered, washed in 0.1 mM NaOH three times, and held in Uranotaenia sapphirina (UsCPV-17) (Shapiro et al., 2005), 0.1 mM NaOH at 8 °C. only lepidopteran cypoviruses have been characterized (Mertens et al., 2004b). However, there are many additional 2.4. Cation assays cypoviruses (>230) from other lepidopteran, dipteran, and hymenopteran hosts that have been reported but not classi- Groups of 100 mosquito larvae (third star) were exposed Wed (Andreadis, 1981; Mertens et al., 2004b). One of these is to puriWed virus (»10 larval equivalents (LE) of CrCPV) in an unclassiWed CPV from the mosquito Culex restuans 100 ml deionized water in the presence of 0 or 10 mM (Andreadis, 1986). Here, we report on the re-isolation of MgCl2, and/or 10 mM CaCl2, and 0.04% alfalfa/pig chow this CPV from Cx. restuans (named CrCPV) and present (2:1) for food. Controls included exposure of larvae to only molecular evidence that CrCPV is most closely related to 10 mM MgCl2, 10 mM CaCl2 or virus alone. The bioassays the recently isolated UsCPV-17 and propose that it be des- were repeated for a total of three trials. The larvae were ignated as CrCPV-17. examined 3 days after exposure under a dissecting micro- scope against a dark background and checked for visible 2. Materials and methods signs of infection. 2.1. Field collections and gross pathology of mosquitoes 2.5. Ultrastructural studies (electron microscopy) Mosquito larvae were collected from various permanent Dissected midguts of fourth instar larvae infected with water habitats in the environs of Alachua County, Florida, CrCPV were processed for electron microscopy as USA. Cx. restuans (the target host of this study) is a sea- described in Shapiro et al. (2004). BrieXy, dissected midguts sonal species in this region and is most prevalent during the were Wxed in 2.5% glutaraldehyde for 2 h, postWxed in 2% winter months of January through March. Larvae were col- osmium tetroxide for 1 h, dehydrated in an ethanol series, lected from the Weld sites following previously described and embedded in epon–araldite. Thin sections were stained protocols (Becnel et al., 2001) and returned to the labora- in uranyl acetate and lead citrate, examined, and photo- tory for processing. Larvae were separated by species, and graphed at 75 kV. placed on a clear petri dish with a minimal amount of water for examination. Infected midgut cells were detected using a 2.6. Electropherotype analysis of CrCPV dissecting scope by viewing larvae from the ventral side on a dark background. The total number of larvae, the propor- A QIAampViral Mini Kit from Qiagen was used to tion of each species present, and the percent infection were extract genomic dsRNAs of CrCPV from puriWed polyhe- recorded from each collection. dra (Hagiwara et al., 2002; Rao et al., 2003). After precipi- tating, washing, drying, and resuspending the RNA in 2.2. Virus production RNase free water, »100 ng was analyzed on a 1% agarose gel. Ethidium bromide was integrated into the gel at a Wnal Culex quinquefasciatus was used as the host in all labora- concentration of 0.5 mg/ml. Genomic RNA from BmCPV- tory bioassays because Cx. restuans has not been colonized. 1, TnCPV-15, and UsCPV-17 was added to the gel to com- Groups of 3000 early fourth instar Cx. quinquefasciatus lar- pare the electrophoretic proWle of diVerent cypoviruses. The vae (5 days old) were held in 500 ml water without food at sequences of BmCPV-1, TnCPV-15, and Seg-10 of UsCPV- 24 °C overnight, then exposed to 100 homogenized CrCPV 17 (Accession No. AY876384) genomes are accessible in infected cadavers in 10 mM MgCl2 with food (0.2% alfalfa/ GenBank. pig chow 2:1) and held at 27 °C for an additional 24 h. The To compare structural protein proWles of CrCPV with following day, the larvae and exposure media were trans- that of UsCPV, puriWed virions were denatured by boiling W V ferred to trays to a nal volume of 3 L (MgCl2 concentra- in protein sample bu er and analyzed by electrophoresis tion of 1.7 mM). Larvae were held for an additional 3 days using a standard 12% SDS–polyacrylamide gel (PAGE) and examined under a dissecting microscope for patent (Laemmli, 1970). infections. 2.7. cDNA synthesis, ampliWcation by PCR, cloning, and 2.3. PuriWcation of CrCPV sequencing Groups of between 100 and 500 infected larvae were The conditions for cDNA synthesis, ampliWcation by homogenized in 5–10 ml of deionized water and strained PCR, cloning, and sequencing are similar to the protocols T.B. Green et al. / Journal of Invertebrate Pathology 91 (2006) 27–34 29 published by Shapiro et al. (2005). BrieXy, RNA was iso- (7.2§6.4%), and Culex nigripalpus in one collection (1 of 5 lated using a QIAampViral Mini Kit (Qiagen) after puriW- larvae for 20% infection rate). Culex territans were present in cation of the CrCPV. The dsRNAs were then ligated to the three of the collections, but none were infected. anchor primer using T4 RNA ligase (New England Biolab). AMV reverse transcriptase (Promega) was used to synthe- 3.2. Cation assays size cDNA. PCR was completed (Advantage 2 PCR Kit (Clontech)) using the primer 5-15-1 that was complemen- Laboratory transmitted infections in Cx.
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