Journal of Invertebrate Pathology 109 (2012) 11–19

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Journal of Invertebrate Pathology

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The complete genome sequence of a single-stranded RNA from the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) ⇑ Omaththage P. Perera a, , Gordon L. Snodgrass a, Kerry C. Allen a, Ryan E. Jackson a, James J. Becnel b, Patricia F. O’Leary c, Randall G. Luttrell a a Southern Management Research Unit, USDA-ARS, Stoneville, MS 38776, USA b Mosquito and Research Unit, USDA-ARS Center for Medical and Veterinary Entomology, Gainesville, FL 32606, USA c Cotton Incorporated, 6399 Weston Parkway, Cary, NC 27513, USA article info abstract

Article history: The complete genome sequence of a single-stranded RNA virus infecting the tarnished plant bug, Lygus Received 12 April 2011 lineolaris (Palisot de Beauvois), was identified by sequencing cDNA prepared from collected from Accepted 19 August 2011 the Mississippi Delta. The 9655 nucleotide positive-sense single-stranded RNA genome of the L. lineolaris Available online 10 September 2011 single-stranded RNA virus (LyLV-1) contained a single open reading frame of 8958 nucleotides encoding a 2986 amino acid genome polypeptide. The open reading frame was flanked by untranslated regions of Keywords: 603 and 69 nucleotides at the 50- and 30- ends of the genome, respectively. Database searches and homology Lygus based modeling was used to identify four proteins (VP1-VP4), helicase/AAA-ATPase, cysteine prote- Single-stranded RNA virus ase (C3P), protease 2A, and the RNA-directed RNA polymerase (RdRp). In addition, a region with weak sim- Pathogen Picorna-like ilarity to the eukaryotic structural maintenance of chromosome (SMC) domain was identified near the Tarnished plant bug amino-terminal of the polyprotein and adjacent to the VP1 domain. The amino acid sequence of LyLV-1 Iflaviridae was approximately 44.4% similar to that of sacbrood virus (SBV) of the honey . The genomic organization Iflavirus of both showed remarkable similarity with the exception of highly divergent amino acid regions Transovarial transmission flanking fairly conserved structural and non-structural polypeptide regions. High similarity to the SBV gen- ome and similarities in the genome organization and amino acid sequence with the viruses of the family Iflaviridae suggested that LyLV-1 was a novel member of this family. Virus particles were 39 nm in diameter and appeared to transmit vertically via eggs. Although this virus may only cause covert infections under normal conditions, the potential for using this virus in biological control of L. lineolaris is discussed. Published by Elsevier Inc.

1. Introduction may lead to significant economic losses include infectious flacherie virus (IFV) and sacbrood virus (SBV) outbreaks in the silk worm A large number of insect viruses with small RNA genomes and moth, L., and the honey bee, Apis mellifera L., respec- morphological resemblance to vertebrate have been tively (Chen et al., 2006b; Isawa et al., 1998; Liu et al., 2010). characterized since the first discovery of sacbrood viruses of honey Monocistronic positive-sense single-strand RNA viruses that in- (Bailey et al., 1964, 1963; Reinganum et al., 1970; van Oers, fect insects are generally termed ‘‘picorna-like’’ viruses and the 2010; White, 1917). While some of these viruses cause only latent International Committee on Taxonomy of Viruses (ICTV) has classi- infections without much adverse effect on the host under most fied them in the genus Iflavirus of the family Iflaviridae in the order conditions (Baker and Schroeder, 2008; Valles et al., 2004; Yue (van Oers, 2010). The IFV isolated from infected larvae et al., 2007), some may cause debilitating lethal infections in the of the silkworm moth (Isawa et al., 1998) is the type species of the host depending on the mode of transmission or other environmen- genus Iflavirus. In addition to IFV, this genus contains the deformed tal factors that affect the health of the host (Bacandritsos et al., wing virus (DWV) (Lanzi et al., 2006) and the SBV of the honey bee 2010; Mockel et al., 2011). Examples of lethal infections that (Ghosh et al., 1999), the Varrora destructor virus-1 (VDV-1) (Ongus et al., 2004), the Perina nuda virus (PnV) (Wang et al., 1999), and the Ectropis obliqua virus (EoV) (Wang et al., 2004). Genome se- quences of a large number of Iflavirus isolates are available in the public databases (i.e. http://www.picornavirales.org/iflaviridae/ Abbreviations: cDNA, complementary DNA; PCR, polymerase chain reaction. ⇑ Corresponding author. Address: 141 Experimental Station Road, Stoneville, MS iflavirus_seq.htm). In this report, we describe a novel single-stranded 38776, USA. Fax: +1 662 686 5421. RNA virus resembling the genome structure of Iflaviridae from the E-mail address: [email protected] (O.P. Perera). tarnished plant bug, Lygus lineolaris (Palisot de Beauvois).

0022-2011/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.jip.2011.08.004 12 O.P. Perera et al. / Journal of Invertebrate Pathology 109 (2012) 11–19

2. Materials and methods Full length cDNA was also synthesized from the pooled mRNA using the BD SMART RACE cDNA synthesis kit (BD Clontech, Moun- 2.1. RNA sample preparation tain View, CA) with modified 50- and 30-RACE adapters as described in the Evrogen Trimmer Direct cDNA normalization kit (Evrogen, Total RNA for initial nucleotide sequence analysis by Illumina Moscow, Russia). Briefly, mRNA was annealed with Evrogen’s Genome Analyzer II was obtained from L. lineolaris adults and CDS 3 M 30-adapter primer (50-TAAGCAGTGGTATCAACGCAGGCAG- nymphs collected from Washington County, Mississippi in August CAGTACTGCTGTTTTTTTTTTTTTTTTTTTVN-30) by heating at 70 °C 2008. All RNA extractions were performed using the TriZol reagent for 3 min followed by 2 min incubation at 42 °C. The first strand (Invitrogen, Carlsbad, CA). Insects were homogenized in 400 llof cDNA synthesis was carried out using the reagents provided with Trizol reagent and 80 ll of chloroform was added to the homoge- the BD SMART cDNA synthesis kit with the exception of the BD nate after a 10 min incubation. The tubes were agitated to mix SMART T7 oligonucleotide which was replaced by modified the TriZol and chloroform and held at room temperature for Evrogen 50-RACE adapter (50-AAGCAGTGGTATCAACGCAGAGTCAG 10 min. Tubes were then centrifuged for 10 min at 12,000 g to CAGGAATCTGCTGAGGCCATTACGGCCGGG-30). The first-strand separate aqueous and organic phases and the aqueous phase cDNA was subjected to 10 rounds of PCR amplification with 50- containing total RNA was carefully aspirated and transferred to RACE and 30-RACE primers (50-AAGCAGTGGTATCAACGCAGAGT - nuclease-free tubes. Total RNA was precipitated by adding 240 ll 30 and 50-AAGCAGTGGTATCAACGCAGGCAGC-30, respectively) in a of 100% isopropanol followed by 15 min centrifugation at 100 ll volume with a 20 s denaturing step at 95 °C and a 5 min 12,000 g at 4 °C. Total RNA from 50 insects was pooled and mes- annealing/extension step at 68 °C. senger RNA (mRNA) was purified using the Poly A + tract mRNA purification system following the manufacturer’s instructions (Pro- 2.3. Cloning and identification of the genome mega, Madison, WI). Briefly, 1 llof10lM biotin labeled oligo (dT) primer was added to 10 lg of pooled total RNA in a 20 ll volume. Segments of the genome of the L. lineolaris single-stranded RNA The RNA and oligo (dT) mixture was incubated at 65 °C for 5 min, virus (LyLV-1) were identified in an assembly of approximately 18 chilled on ice for 1 min, and spun briefly to collect the liquid on the million Illumina sequence reads obtained from cDNA produced bottom of the tube. The mixture was incubated at room tempera- from a pool of approximately 100 L. lineolaris (nymphs and adults). ture for 10 min. Streptavidin coated magnetic beads were washed Assembly of sequence reads was performed using DNASTAR NGen 3 times in 500 ll of 0.5 SSC (75 mM sodium chloride, 7.5 mM so- 2.3 software (DNASTAR, Madison, WI). Assembled sequence con- dium citrate, pH 7.0) and re-suspended in 100 ll of 0.5 SSC. The tigs were exported as a FASTA format file for bioinformatics analy- RNA and oligo (dT) mixture was added to the washed magnetic sis using BLAST2GO software (Götz et al., 2008). BLAST function of beads and incubated at room temperature for 10 min to facilitate the BLAST2GO software identified 4 sequence segments with high binding of biotin labeled oligo (dT) to streptavidin coated paramag- similarity to the SBV of the honey bee (Ghosh et al., 1999). These netic beads. Paramagnetic beads and bound mRNA were captured sequence segments were aligned with the SBV genome (Ghosh using a magnetic stand and the beads were washed 3 times with et al., 1999; Accession: AF092924) for orderly arrangement of the 0.1 SSC buffer (15 mM sodium chloride, 1.5 mM sodium citrate, segments along the SBV genome. Primers were developed to PCR pH 7.0). Messenger RNA was eluted by adding 10 ll of water and amplify additional nucleotide sequences from the cDNA toward the mRNA was quantified using a spectrophotometer. assembling the full length genome sequence of the LyLV-1. Primers designed to the sequence segments were used to amplify genome sections from cDNA to bridge missing sections. Finally, full length 2.2. cDNA synthesis and sequencing cDNA prepared using rapid amplification of cDNA ends (RACE) was used to amplify the genome in 2 overlapping segments. Final gen- Double-stranded complementary DNA (cDNA) was synthesized ome assembly was used as a reference to overlay original sequence using the SuperScript III double strand cDNA synthesis kit (Invitro- reads to manually curate the genome. Primer sequences used to gen, Carlsbad, CA). Purified mRNA (200 ng) was annealed with 1 ll obtain the genome sequence are given in Table 1 and the relative of 10 lM oligo (dT) primer supplied with the kit in a 10 ll volume locations of primers are shown in Fig. 1A. by heating at 65 °C for 5 min and chilling on ice for 1 min. First strand cDNA synthesis was carried out in a 20 ll volume that con- 2.4. Verification of virus replication in L. lineolaris tained 4 llof5 first strand synthesis buffer (250 mM Tris–HCl, pH 8.3, 375 mM KCL, 15 mM MgCl2), 1 ll of 0.1 M dithiothreitol Total RNA was extracted from whole insects as well as dissected (DTT), 2 ll of 10 mM dNTP mix, and 2 units of SuperScript III re- fat body tissue and a small amount of this RNA was used to synthe- verse transcriptase. The reaction was incubated at 42 °C for 2 h size cDNA with an oligo d(T)20 primer. The cDNA was PCR ampli- and heated to 75 °C for 10 min to deactivate the enzyme. To syn- fied with primer 1111F and 1112R to verify the presence of thesize the second strand of the cDNA, the reaction was chilled LyLV-1. All PCR amplifications were carried out in 25 ll reactions on ice and then 91 ll of water, 30 llof5 second strand buffer containing 1X PCR buffer (20 mM Tris_HCl pH 8.4, 50 mM KCl),

(100 mM Tris–HCl, pH 6.9, 450 mM KCl, 10 mM EDTA, 1 mM ß- 2 mM MgCl2, 400 lM dNTP, 200 lM each forward and reverse Nicotinamide Adenine Dinucleotide (b-NAD), 50 mM (NH4)2SO4, primers, and 0.5 units of recombinant Taq DNA polymerase (Invit- 3 ll of 10 mM dNTP, 1 llofE. coli DNA ligase (10 u/ll), 1 llof rogen, Carlsbad, CA), unless noted otherwise. A thermal cycler pro- E. coli RNase H (2 u/ll), and 4 llofE. coli DNA polymerase I gram (PTC200; BioRad) with 95 °C initial denaturation for 1 min, (10 u/ll) were added. The reaction was mixed, centrifuged briefly, followed by 35 cycles of 15 s denaturation at 95 °C, 15 s annealing and incubated at 16 °C for 2 h to complete second strand cDNA at 56 °C, and 2 min extension at 72 °C was used to amplify the DNA synthesis. Resulting double-stranded cDNA was purified using made from viral RNA. Two micrograms of total RNA from whole Invitrogen low-elution cDNA purification columns. This double- body or fat body tissues of insects positive for LyLV-1 were stranded cDNA was submitted to the National Center for Genomics digested with 100 units of DNase I for 30 min at room temperature Resources, Santa Fe, New Mexico for sequencing with the Illumina followed by DNase I inactivation at 70 °C for 10 min. The RNA sam- Genome Analyzer II platform. Nucleotide sequence reads (36 ples were purified using Invitrogen RNA purification columns. Two nucleotides long) were obtained using standard protocols devel- primers were designed to bind the negative-sense strand of the oped for the Illumina Genome Analyzer II platform. LyLV-1 genomic RNA sequence from 130–148 (primer 1721F) O.P. Perera et al. / Journal of Invertebrate Pathology 109 (2012) 11–19 13

Table 1 Primers used for cloning, sequencing, negative strand synthesis, and amplification of the Lygus lineolaris single-stranded virus. Lower case letters in primers 1721–1728 indicate artificial anchor sequences added to the 50-end of each primer. Forward and reverse primers are designated by ‘‘F’’ and ‘‘R’’ at the end of the primer number and nucleotide positions of the primers indicate the 50–30 position of each primer in the LyLV-1 genomic RNA sequence.

Primer # Primer sequence Nucleotide Remarks (orientation) positions 1024F ATTCAACACGGAGTGGTTCG Forward primer for L. lineolaris b-tubilin 1023R TTATGGAGGAATGCGAGTCG Reverse primer for L. lineolaris b-tubilin 1028F GCAACGACAACTGTCATCCAGTC Forward primer for L. lineolaris actin 1031R TTACCAATAGCAATAATTTGACCG Reverse primer for L. lineolaris actin 1111F TTGGTTGGAGTTTTCCTGGCTCTAG 4441–4465 Forward (sense) primer for LyLV-1 detection 1112R CTGGTGTAATACTGGAGAGACGCGA 4842–4866 Reverse (anti-sense) primer for LyLV-1 detection 1721F ctacgactgacattagggagaagagGGCAACTTTAGGTTCCGGG 130–148 Negative strand cDNA synthesis from 130 nt with artificial anchor sequence (in lower case) 1722F gatcacgagaagacacagtagTTGGTTGGAGTTTTCCTGGCTCT 4441–4463 Negative strand cDNA synthesis from 4441 nt. Artificial anchor sequence (in lower case) 1723F ctacgactgacattagggagaagagGGCA For 1721F Artificial anchor primer to 1721F 1724F gatcacgagaagacacagtagTTGGT For 1722F Artificial anchor primer to 1722F 1725R acgactgacattagggagaagagCGATCAAGAAAAACATCCACCG 6290–6269 For second strand cDNA synthesis from 6290 nt with artificial anchor sequence (in lower case) 1726R cgatcacgagaagacacagtagCCTTCAACCGGCGTAACGTCAT 2708–2687 For second strand cDNA synthesis from 2708 nt with artificial anchor sequence (in lower case) 1727R acgactgacattagggagaagagCGAT For 1725R Artificial anchor primer to 1725R 1728R gatcacgagaagacacagtagCCTT For 1726R Artificial anchor primer to 1728R

Fig. 1. (A) Schematic of relative positions of the primers used for cDNA synthesis from positive and negative strand genomic RNA and amplification of the Lygus lineolaris picorna-like virus genome (not drawn to a scale). GeneRacer RNA oligonucleotide, LyLV-1 genomic RNA, poly (A+) tail, and the GeneRacer 30- anchor sequences are represented by the open box, solid line, AAAA, and the hatched line, respectively. Open arrow heads (?) represent the primers used for amplifying the LyLV-1 genome, solid arrow heads with dotted lines ( ) represent the primers used for synthesizing negative strand cDNA, and small solid arrow heads with dashed lines ( ) represent the primers used for amplifying cDNA synthesized from negative strand genomic RNA. (B) Schematic of peptide domains identified in the polyprotein of the Lygus lineolaris single-stranded RNA virus. Protein domains identified by SMART and CDD database searches are shown on the solid line representing the polyprotein. Capsid protein domains identified by SWISS-MODEL structure database are shown in color below the polyprotein sequence. VP1-VP4: Capsid proteins 1–4, 2A: Protease 2A, TM: transmembrane domain, RdRp: RNA-directed RNA polymerase. All domains are drawn proportional to the polyprotein sequence. and 4441–4165 (primer 1722F). Artificial anchor sequences were reactions and incubated at room temperature for 60 min to frag- added to the 50-ends of these primers (Table 1 and Fig. 1A). Each ment and digest residual RNA. The reactions were purified using primer was used in a separate cDNA synthesis reaction using Invitrogen low elution cDNA purification columns. The primers approximately 0.5 lg of DNase I treated total RNA. Primers with 1726R and 1725R were added to the purified cDNA with primers sense strand sequence of b-tubulin mRNA (1024F) and anti-sense 1721F and 1722F, respectively and primers 1023R, and 1028F were strand sequence of actin mRNA (1031R) were used as internal neg- added to both sets of cDNA. These reaction mixes were subjected ative and positive reverse transcription (RT) controls, respectively. to 10 cycles of PCR amplification in 50 ll reactions using the same Negative control (no RT) RNA was produced by setting up separate reaction and cycling conditions listed above except for an RT reactions as described above with primers 1721F and 1722F, increased extension time of 4 min at 72 °C. At the end of PCR but without SuperScript III reverse transcriptase. After incubation amplification, the reactions were purified using QIAquick PCR puri- at 46 °C for 2 h, the SuperScript III reverse transcriptase was inac- fication columns (Qiagen, Valencia, CA) to remove residual re- tivated by heating at 70 °C for 10 min. The cDNA was precipitated agents and primers. Nucleic acids bound to the columns were by adding two volumes of 100% ethanol and centrifuging at eluted with 30 ll of 10 mM Tris-HCl, pH 8.0 and 2 ll of this eluate 16,000 g for 30 min at 4 °C. Nucleic acid pellets were resuspended was used in PCR amplifications with primers designed to the arti- in distilled water and incubated with 20 units of exonuclease I ficial anchor sequences of LyLV-1 primers used for first strand (New England BioLabs, Ipswich, MA) in 1X buffer (67 mM Gly- cDNA synthesis and PCR amplification. Primer pairs 1723F/1728R cine-KOH, 6.7 mM MgCl2, 10 mM 2-Mercaptoethanol, pH 9.5) for and 1724F/1727R were used to amplify cDNA synthesized and 60 min at 37 °C to remove residual unincorporated primers. Exo- amplified with primer pair 1721F/1726R and 1722F/1725R, respec- nuclease I was inactivated by heating for 20 min at 80 °C. Esche- tively (Table 1 and Fig. 1A). Internal controls b-tubulin and actin richia coli RNase H and RNase A (5 units each) was added to the were amplified from cDNA synthesized using primer pairs 1024F/ 14 O.P. Perera et al. / Journal of Invertebrate Pathology 109 (2012) 11–19

1023R and 1028F/1031R, respectively. Amplicons were gel purified transferred to a sterile 1.5 ml centrifuge tube and total RNA was and cloned into pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA) and extracted using the TriZol reagent. This RNA was used to synthe- recombinant clones were submitted to the USDA-ARS Genomic and size cDNA using the primer 1725R for PCR amplification with prim- Bioinformatics Research Unit, Stoneville, MS for sequencing with ers 1111F and 1112R. cDNA synthesized using the mRNA obtained ABI 3730xl instrument using Sanger chemistry. from the eggs of an uninfected colony and adult insects was used as negative controls and the cDNA from a infected adult was used 2.5. Purification and electron microscopy of the virus particles as positive control for the PCR amplification.

Approximately 500 insects from a LyLV-1 infected colony were 3. Results used to purify virus particles for electron microscopy using the method described in Valles and Hashimoto (2009). Insects were 3.1. Genome composition and organization homogenized in 25 ml of NT buffer (100 mM Tris–HCl, 10 mM NaCl, pH 7.4). The homogenate was filtered using a fine mesh ny- The 9655 nucleotide (nt) genome of the LyLV-1 was deposited lon cloth to remove most debris and then clarified by extraction in the GenBank database under the accession number JF720348. with an equal volume of chloroform. Discontinuous gradients of The nucleotide composition of the genome is 2556, 1927, 2529, 1.2 g/ml and 1.5 g/ml CsCl were prepared in 13.5 ml QuickSeal and 2642 A, C, G, and U, respectively. The G + C content of the tubes (Beckman Coulter, Brea CA) and the clarified insect homoge- LyLV-1 genome is fairly high at 46.2% when compared to other Ifl- nate was applied on top of the 1.2 g/ml CsCl solution. Discontinu- aviruses whose G + C content range from 33.6% in Brevicoryne ous gradients were centrifuged for 2 h at 150,000 g in a Beckman brassicae virus (BBV) to 44.5% in Ectropis obliqua Picorna-like virus ultra centrifuge using Type 50 Ti rotor. Material accumulated at (EoV), and is similar to the 46.3% G + C content of the poliovirus the interface of 1.2 and 1.5 g/ml CsCl layers was aspirated using (Racaniello and Baltimore, 1981). The genome has a 603 nt 50- a 5 ml syringe fitted with a 21 gauge needle. The density of this untranslated region (UTR) followed by a single open reading frame fraction was adjusted with saturated CsCl to 1.38 g/ml and centri- spanning 8958 nt from 604 to 9561 nt, and a 69 nt 30-UTR prior to a fuged for overnight at 200,000 g in a SW 41 Ti rotor. A gray color poly (A) of varying length. The translation initiation codon (AUG) band of virus particles observed at approximately 1.32 g/ml was starting at the nucleotide position 604 is located in the context recovered and used for negative staining. of the consensus invertebrate initiation sequence (ANNAUGG; A10ll drop of CsCl gradient purified viral suspension was ap- N = any nucleotide). Therefore, it is most likely the translation ini- plied to a formvar coated grid for approximately 5 min. The excess tiation site for the polyprotein. The polyprotein of 2986 amino was removed from the side with a sliver of filter paper. A 10 ll acids has a calculated molecular mass of 334.4 kDa, an isoelectric drop of 2% (w/v) aqueous phosphotungstic acid (PTA) adjusted to point of 6.34, and 16.2 charge at pH 7. pH 7.5 with 1 N NaOH was applied to the grid for 2 min, then the excess was removed and the grid was allowed to air dry prior to 3.2. Similarity searches viewing. The stained specimens were viewed and photographed with a Hitachi H-600 transmission electron microscope at 75 kv. Basic Local Alignment and Search Tool (BLAST) (Altschul et al., 1997) searches of databases using the deduced amino acid se- 2.6. Detection of virus in L. lineolaris eggs quence of the polyprotein indicated that LyLV-1 is highly similar to SBV strains of the honey bee (family: Iflaviridae; NP_049374.1, Parafilm gel packs prepared with 4% carrageenan (Sigma– AAL79021.1, and ADN38255.1) with bit scores and expected Aldrich, St. Louis, MO) was placed in a cage containing infected probabilities ranging from 513–580 and 2 10140 2 10162, adult L. lineolaris for 2 days to collect eggs. Gel packs were removed respectively. Nucleotide sequences of LyLV-1 and the SBV of honey from the cage and washed with running tap water for 15 min fol- bee (NP_049374.1) shared 49.5% nucleotide identities while the lowed by a 10 min wash with distilled water. The gel pack was amino acid sequences showed 30.4% identities and 44.4% consen- then immersed in 500 ml of 1% bleach solution for 2 min to remove sus positions. In addition to SBV strains, several other members any external contaminants and washed with three changes of of Iflaviridae, including full genome peptide sequences of the 500 ml of distilled water. Parafilm layer containing eggs was re- (DWV; ADL66818.1, score:253, E-value: 4E- moved by cutting along the edges of the gel pack and eggs were re- 64), Kakugo virus (kV; YP_015696.1, score: 251, E-value: 9E-64), leased into a sterilized glass beaker containing autoclaved distilled Varroa destructor virus (VDV; YP_145791.1, score: 246, E-value:E- water. Eggs were collected by filtering with a sterile cheese cloth 63), and Brevicoryne brassicae picorna-like virus (BrBV; and dipped in a 1% bleach solution for 1 min followed by 3 washes YP_001285409.1, score: 243, E-value: 4E-61) showed high similar- with distilled water. This process not only removed any external ity to the respective LyLV-1 amino acid sequence domains. contaminants, but also de-chorionated the eggs. The eggs were Alignment of genomic polypeptide sequences of LyLV-1, SBV, and

Table 2 Major polypeptide domains identified in the genomic polypeptide of the Lygus lineolaris picorna-like virus and corresponding polypeptide regions of the sacbrood virus (SBV; Accession # NP_049374.1) of the honey bee genomic polypeptide.

Domain Name AA Position Best match (AA position) Description VP2 81–342 SBV (78–293) Capsid protein 2 VP4 346–425 SBV (346–423) Capsid protein 4 VP3 426–713 SBV (427–711) Capsid protein 3 VP1 725–986 SBV (756–873) Capsid protein 1 2A 1038–1210 SBV (928–1004) Protease 2A domain TM 1275–1297 Transmembrane domain Helicase/AAA-ATPase 1443–1584 SBV (1370–1512) RNA helicase/AAA-ATPase C3P 2237–2438 SBV (2104–2306) Peptidase C3 RdRP 2481–2983 SBV (2341–2843) RNA dependent RNA polymerase SMC 708–866 Structural maintenance of chromosomes O.P. Perera et al. / Journal of Invertebrate Pathology 109 (2012) 11–19 15

IFV are given in Supplementary material S.1. Other database se- represents the super family 3 helicases, forms a hexameric ring and quences with high similarity to the LyLV-1 genomic polypeptide participates in viral replication (Hickman and Dyda, 2005). included RNA-directed RNA polymerase (RdRp) domains of picor- Helicase/ATPase of the RNA viruses is also thought to form a hex- na-like viruses of the brown bat, Eptesicus fuscus (EFV; americ ring (Papageorgiou et al., 2010). The SWISS-MODEL ADR79389), and the tsetse fly, Glosina morsitans morsitans (GMMV; matched the helicase domain of LyLV-1 polyprotein with a ADD18747). Similarity searches with the Simple Modular Architec- structure of the D2 hexamerization domain (PDB: 1nsf) of n-ethyl- ture Research Tool (SMART: http://smart.embl-heidelberg.de) and maleimide sensitive factor (nsf) from Chinese hamster, Cricetulus Conserved Domain Database (CDD: http://www.ncbi.nlm.nih.gov/ griseus (Neuwald, 1999; Yu et al., 1998) indicating the presence Structure/cdd/cdd/shtml) with the LyLV-1 polyprotein identified of a putative hexamerization domain in the helicase of the LyLV-1. all structural and non-structural domains commonly found in in- sect picorna viruses except the capsid protein 4 (Table 2). The pep- 3.3.3. Proteases tide domains identified in the genomic polypeptide of the LyLV-1 A protease domain similar to a conserved 3C cysteine protease by homology based modeling tools available at the SWISS-MODEL domain (pfam00548, 170 amino acids) was identified in the LyLV-1 bioinformatics server (Arnold et al., 2006; Guex and Peitsch, 1997; polyprotein from amino acid position 2229 to 2421 (score: 53.8; Kiefer et al., 2009; Schwede et al., 2003) are shown in the Fig. 1B. E-value: 4.7E-07). This region also showed somewhat lower simi- Complete SWISS-MODEL outputs, including secondary structure larity to the Tungro spherical virus-type peptidase (pfam12381) of the genomic polyprotein (Zdobnov and Apweiler, 2001) and ter- present in a number of positive single-stranded plant viruses. tiary structure predicted for the capsid proteins, helicase, 3C prote- Homology searches identified structural models of cysteine prote- ase, and the RdRp are given in Supplementary material S.2. ase 2A of human coxavirus (PDB:1z8rA) and human Alignments of peptide domain core sequences of capsid proteins, (PDB: 2hrvB). LyLV-1 amino acid residues 1025–1187 matched helicase, peptidase, and RNA directed RNA polymerase (RdRp) from the PDB crystal structure1z8rA (13–149 amino acids of 150 amino the conserved domain databases (Marchler-Bauer et al., 2009, acid peptide) with a certainty of 99.7% (score:155.6, E-value: 5.4E- 2007) with corresponding LyLV-1 domains are given in Supple- 17) and the PDB crystal structure 2hrvB (26–135 amino acids of mentary material S.3. 139 amino acid peptide) with a certainty of 99.6% (score:139.5, E-value:4.2E-15). The amino acid identities between putative 3.3. Peptide domains LyLV-1 protease 2A domain and the polypeptides 1z8rA and 2hrvB were 16% and 10%, respectively. 3.3.1. Capsid proteins Crystal structures of the capsid proteins of the genus Iflavirus 3.3.4. RNA-directed RNA polymerase (RdRp) and peptide phylogeny are not available yet and therefore, database searches were analysis performed to identify putative capsid protein domains. Two pic- Conserved amino acid blocks of the the RdRp domain located at orna like-virus capsid domains similar to the 178 amino acid the carboxy-terminal from residues 2652–2925 are the most con- conserved domain cd00205 were identified between the amino served region between the polyproteins of LyLV-1 and other posi- acids 177–388 and 445–684 of the LyLV-1 polyprotein. SMART tive single-stranded RNA viruses (Koonin, 1991). The RdRP analysis also identified a region similar to the capsid protein of domains of several viruses from the super family Picornavirales the cricket paralysis virus (pfam:CRPV_capsid) in the LyLV-1 aligned using MEGA v5.03 software (Tamura et al., 2007) and the polyprotein from amino acid position 901–1102. This region tree generated with the neighbor-joining method (Saitou and was also identified in the polyproteins of the SBV (NP_ Nei, 1987) implemented in MEGA software are shown in Figs. 2 049374.1) and DWV (NP_853560.2) in amino acid positions and 3, respectively. Amino acid sequence alignment showed fairly 819–988 and 919–1148, respectively. conserved blocks of sequences typical of RdRp domains of picorna- Based on homology modeling (SWISS-MODEL), LyLV-1 polypro- like viruses. With 10,000 bootstrap replicates and SINV-1 anchored tein amino acid regions 178–388, 445–684, and 719–1006 were to the root, the RdRp sequence of LyLV-1 grouped 51% of the time identified as capsid proteins 2, 3, and 1, respectively. These regions with SBV which in turn grouped 94% of the bootstrap replicates overlapped the capsid peptide domains identified using the CDD with EFV (Fig. 3). This high similarity between RdRp domains of and SMART analyses. Analyses performed using BLAST, SMART, LyLV-1 and SBV as well as 44% overall sequence similarity to SBV and SWISS-MODEL tools matched the LyLV-1 amino acid region is the highest observed between LyLV-1 and any of the Iflaviruses from 708–866 as a part of structural maintenance of chromosome described to date. (SMC) proteins (Accession # TIGR02168). The two sequences were 20% identical and 31% similar and overlapped with the amino ter- 3.3.5. Transmembrane regions minal of the predicted capsid protein 1 (VP1) of LyLV-1. SMART analysis identified two putative transmembrane re- gions from residues 1235–1254 (TLFRHLIGVSLTSLSEYGLI) and 3.3.2. RNA helicase and AAA-ATPase domains 1275–1297 (GIGPALVGVFLALAGTIVGQILS) of the LyLV-1 polypro- Overlapping RNA helicase and ATPase domains were identified tein. Further analysis of this region of LyLV-1 and SBV using the in the polyprotein from residue 1484–1538 (E-value: 5.51013) and topology prediction software (mobyle.pasteur.fr) identified corre- 1445–1558 (E-value 0), respectively. This helicase/AAA-NTPase do- sponding transmembrane domains in the polyprotein of both main was previously identified in the viruses of Iflaviridae and Pic- viruses. The analyses also confirmed the second transmembrane ornaviridae. The helicases in small DNA and RNA viruses have been domain of both viruses with certainty while the first domain classified under the super family 3 of five RNA helicase super fam- was classified as a putative transmembrane region. No other ilies (Gorbalenya et al., 1990; Iyer et al., 2004). Crystal structures of structural or non-structural peptides were identified in this re- several of these super family 3 helicases indicated structural gion, and therefore, the function of the transmembrane region resemblance to P-loop NTPases (AAA + ATPases) which are in- remains to be identified. volved in energy-dependent unfolding of macromolecules (Iyer et al., 2004; Li et al., 2003; Lupas and Martin, 2002; Tucker 3.4. Verification of replication in insects and Sallai, 2007). Although core helicase/ATPase domains are fairly conserved, the amino-terminal sequence is poorly conserved, and Nucleotide sequences of the clones selected from the cDNA syn- therefore, often difficult to identify. Simian virus 40 helicase, which thesized with primer pairs 1721F/1726R and 1722F/1725R verified 16 O.P. Perera et al. / Journal of Invertebrate Pathology 109 (2012) 11–19

Fig. 2. Alignment of the conserved amino acid motifs of the RNA-directed RNA polymerase domains (RdRp) of Lygus lineolaris virus 1 (LyLV-1) with those of Brevicoryne brassicae virus (BrBV-1; YP_001285409.1), deformed wing virus (DWV; Accession # ADL66818.1), Eptesicus fuscus picorna-like virus (EFV; Accession # ADR79389), Ectropis obliqua picorna-like virus (EoV; Accession # AAQ64627.1), infectious flacherie virus (IFV; Accession # BAA25371.1), Perina nuda virus (PnV; Accession # AAL06289.1), sacbrood virus (SBV; Accession # NP_049374.1), Solenopsis invicta virus 1 (SINV-1; Accession # NC_006559), and Varroa destructor virus (VDV; Accession # YP_145791.1). Amino acids conserved among all RdRp domains are shown in white text against black background and those that are conserved or similar in the majority of RdRp domains are shown in white text in dark gray background. Conserved blocks of amino acids are in black text in light gray background.

the presence of synthetic anchor nucleotide sequences at the ends primers that could have contributed to amplification artifacts. L. of the PCR products. Primers 1721F and 1722F were designed to lineolaris actin sense primer 1028F would not prime the mRNA bind the negative sense strand at the nucleotide positions 130– present in the reactions. Lack of any amplicons in corresponding 148 and 4441–4463, respectively, of the LyLV-1 genomic RNA. RT reactions with this primer served as an internal negative Amplification of products from cDNA synthesized using these neg- control. Amplification of products from cDNA synthesized with ative strand-specific primers indicated the presence of full length b-tubulin anti-sense primers 1023R served as an internal positive negative-strand RNA in the insects. Digestion of RT reactions with control. In addition, lack of PCR amplifications in ‘‘no RT’’ controls exonuclease I and purification of cDNA with columns that elimi- (cDNA synthesis reactions without reverse transcriptase) with nated small nucleic acid fragments prevented carryover of excess primers for LyLV-1, b-tubulin, or actin served as a control for O.P. Perera et al. / Journal of Invertebrate Pathology 109 (2012) 11–19 17

Fig. 3. Bootstrap consensus tree generated by neighbor joining method implemented in MEGA 5.0 software using the eight conserved motifs of the RNA-directed RNA polymerase domains (RdRp) of Brevicoryne brassicae virus (BrBV-1; YP_001285409.1), deformed wing virus (DWV; Accession # ADL66818.1), Eptesicus fuscus picorna-like virus (EFV; Accession # ADR79389), Ectropis obliqua picorna-like virus (EoV; Accession # AAQ64627.1), infectious flacherie virus (IFV; Accession # BAA25371.1), Perina nuda virus (PnV; Accession # AAL06289.1), sacbrood virus (SBV; Accession # NP_049374.1), Solenopsis invicta virus 1 (SINV-1; Accession # NC_006559), and Varroa destructor virus (VDV; Accession # YP_145791.1). Numbers on each node represents the percentage of 10,000 bootstrap replicates contributed to each branch. contamination with exogenous or endogenous DNA. Amplification structure, a prominent system of protrusions could clearly be seen of products from fat body cDNA synthesized using primers specific on the outer surface of LyLV-1. In addition, empty viruses of the to negative strand genomic RNA, especially with the primer 1721F LyLV-1 showed a relatively thicker capsid protein layer than those designed to the 50-UTR, indicated the presence of full length nega- seen in the electron micrographs of DWV, IFV, and SBV (Bailey tive strand genomic RNA in the LyLV-1 in infected L. lineolaris. et al., 1964; Lanzi et al., 2006; Xie et al., 2009).

3.6. Detection of LyLV-1 in eggs 3.5. Purification and electron microscopy of the virus particles Virus genomic RNA was detected in the eggs obtained from the Isopycnic separation of virus particles by CsCl gradient centrifu- infected colony by PCR. No PCR amplification was observed in the gation resulted in a virus particle band approximately at 1.35 g/ml. cDNA prepared from the eggs of the uninfected colony as well as Electron microscopy of negatively stained virus particles (Fig. 4) re- the uninfected adults. Extensive washing and bleaching of gel vealed intact (white arrow) and empty (black arrow) viruses in the packs and eggs was employed to remove any surface contami- preparation. Diameters of intact and empty virions were 38.7 nants, including virus particles. The detection of viral genomic (±0.05) and 40.2 (±0.9) nm (N = 10), respectively, indicating that RNA in eggs collected from a LyLV-1 infected colony indicates a the LyLV-1 is considerably larger than other Iflaviruses such as possible vertical transmission of LyLV-1. DWV, IFV, and SBV which are approximately 30 nm in diameter. Three-dimensional structure of IFV determined by Cryo-electron microscopy indicated a slightly protruding ridge and plateau sys- 4. Discussion tem on a relatively smooth surface (Xie et al., 2009). Although the resolution of the images of the negatively stained virus was Fragments of a novel positive-sense single-stranded RNA virus not sufficient to make an accurate determination of the surface of L. lineolaris identified in a transcriptome sequencing project were used to obtain the complete genome by sequencing DNA fragments generated through RT-PCR. The positive-sense single- stranded RNA genome of LyLV-1 had 9655 nucleotides and was polyadenylated at the 30-end. The RNA genome contained a single open reading frame that coded for a putative genomic polyprotein of 2986 amino acids. Various proteomic analysis tools identified putative polypeptide regions that formed four viral capsid proteins (VP1-VP4), a helicase/AAA-ATPase, two protease domains (2A and C3), and a RNA-directed RNA polymerase (RdRp). The amino acid sequence of LyLV-1 polyprotein showed the highest overall simi- larity to that of SBV. In a phylogenetic analysis using the conserved RdRp domains of the viruses of Iflaviridae, , and Pic- ornaviridae, LyLV-1 formed a branch with SBV and IFV which indi- cated that LyLV-1was most likely a member of Iflaviridae. However, significant differences in morphology and size observed between LyLV-1and other Iflaviruses warrant further investigations prior to placing LvLy-1 in Iflaviridae. Successful synthesis of cDNA from the negative-sense strand (with primers designed to 50-UTR and the coding region of the LyLV-1 genomic RNA and PCR amplifica- Fig. 4. Electron micrograph of negatively stained virus particles purified by CsCl tion of specific fragments using artificial primer binding regions gradient centrifugation. White arrow (inset) indicates an intact virus particle with a solid core and the black arrow indicates an empty virus particle without genomic incorporated to these primers) indicated the presence of nega- DNA. Measured diameters of intact and empty virus particles are 39 and 40 nm, tive-strand genomic RNA and replication of LyLV-1 in L. lineolaris. respectively. Negative strand detection by RT-PCR can lead to false positives 18 O.P. Perera et al. / Journal of Invertebrate Pathology 109 (2012) 11–19 due to a variety of reasons such as self-, random-, or false-priming viruses and other insect pathogens have not been studied in the (Moison et al., in press; Craggs et al., 2001; Sangar and Carroll, tarnished plant bug. Our discovery of the LyLV-1 virus makes this 1998). Our design of reverse transcription (sense) and antisense research possible. PCR primers containing artificial nucleotide sequences at the 50- end and the use of primers binding to these artificial anchor se- 5. Conclusions quences for PCR amplification eliminated the possibility of false positives due to priming issues. Digestion of unincorporated prim- We have identified and characterized a novel positive sense sin- ers, and purification of cDNA using columns selective for larger nu- gle-stranded RNA virus (LyLV-1) from the tarnished plant bug, L. cleic acids eliminated complications due to primer carryover. In lineolaris. The genomic polyprotein sequence of this virus, coded addition, verification amplification products for correct priming by a single open reading frame, was most similar to that of sac- and the presence of artificial anchor sequences at both ends of brood virus of the honey bee and possessed characteristics typical the amplicons by nucleotide sequencing provided additional of the genus Iflavirus. Putative capsid protein, helicase, protease, molecular evidence for negative strand detection and the replica- and RNA-directed RNA polymerase domains were identified using tion of LyLV-1 in L. lineolaris. Finally, purification of large quantities homology modeling and database searches. The detection of nega- of virus particles through gradient centrifugation of homogenates tive-strand RNA in the fat body tissues of infected insects and iso- of infected insects provided the ultimate proof for LyLV-1 replica- lation of virus particles by gradient centrifugation indicated active tion in L. lineolaris. The presence of negative strand LyLV-1 genomic replication of LyLV-1 after infection. The detection of viral genomic RNA in the fat body tissue also demonstrated that this virus repli- RNA from surface sterilized eggs indicated a transovarial pathway cated in internal body tissues other than the gut. of LyLV-1 transmission. Transovarial transmission of a virus was previously docu- mented for Rhopalosiphum padi virus (RhPV) and the Kashmir bee virus (KBV) (Chen et al., 2006a; D’Arcy et al., 1981). Detection of Disclosure statement LyLV-1 in surface sterilized eggs indicated a transovarial route of viral transmission. Although we detected the virus in ovaries dis- No conflicts of interest exists between authors or any company, sected from LyLV-1 infected females, it was not possible to elimi- organization mentioned in the manuscript. nate contamination of the sample with virus particles present in other tissues during dissection of ovarian tissues. Role of the funding source Picorna-like viruses are pathogenic to many insects, including the honey bee (SBV, , etc.), (pea Partial funding for this study was provided by the Cotton Incor- virus), (Nasonia vitripennis virus), crickets (cricket porated grant # 08-471. Funding source had no influence in study paralysis virus), and fire (de Miranda et al., 2010; Hashimoto design, in the collection, analysis, and interpretation of data, in the and Valles, 2008; Valles and Hashimoto, 2009; Valles et al., 2004, writing of the report or in the decision to submit the paper for 2007; van den Heuvel et al., 1997). These pathogens may have publication. the potential to be biological control agents (Lacey et al., 2001), and some of these viruses such as CrPV and SINV-3, have been con- Acknowledgments sidered for controlling pests (Manousis and Moore, 1987; Valles et al., 2010). The utility of these viruses may lie in the fact that they We thank Calvin Pierce III for technical support, Charles Lanford establish in hosts by overcoming and weakening host immune de- and Arnell Patterson for insect collections and Drs. Kent Shelby and fenses. A host already infected with one species of pathogen may Steven Valles for critically reading a previous version of the manu- mount a weaker immune defense against a different pathogen, script. Sanger dideoxy sequencing of clones was performed at the facilitating rapid establishment of the pathogen and causing death USDA-ARS Genomics and Bioinformatics Research Unit, Stoneville, of the host. Additive or synergistic effects have been reported for MS. This work is partly funded by the Cotton Incorporated grant some combinations of pathogens. For example, the parasitic nem- 08-471 to the corresponding author. Mention of trade names or atode Heterorhabditis indica and the pathogenic fungus Metarhizi- commercial products in this publication is solely for the purpose um anisopliae showed additive effects against larvae of the pecan of providing specific information and does not imply recommenda- weevil, Curculio caryae, when applied together (Shapiro-Ilan tion or endorsement by the US Department of Agriculture. et al., 2004). In the same study, a non-pathogenic fungus and a bac- terium showed additive effects when combined with a moderately Appendix A. Supplementary material pathogenic fungus. 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