วารสารสถาบันสุขภาพสัตวแหงชาติ (ThaiNIAH eJournal): ISSN 19055048, http://niah.dld.go.th, Volume 9 Number 2 (Sep – Dec 2014) : 5872 58 : ก กก * ก ก ก กก ก ก. 10900 * 0-2579-8908-14, e-mail: [email protected] (Cricket Paralysis Virus ; CrPV) RNA positive- sense ก ก ก กกก ก กก RNA กก RT-PCR ก noncoDing region (NCR), intergenic region (IGR) RNA DepenDent RNA polymerase (RDRp) 705 bp 207 bp 1.2 kb กก ก CrPV [NC003924] 83% 88% 90% กก RDRp (Non-structural protein) ก CrPV [NP_64748.1] 94% กก RNA ก CrPV ก กกกก ก : กก ก วารสารสถาบันสุขภาพสัตวแหงชาติ (ThaiNIAH eJournal): ISSN 19055048, http://niah.dld.go.th, Volume 9 Number 2 (Sep – Dec 2014) : 5872 59 Cricket paralysis virus (CrPV) single stranD positive-sense RNA (Eaton anD Steacie, 1980; Moore et al ., 1981; Reavy anD Moore., 1983; King et al ., 1984) ก .. 1970 Carl Reinganum กก (Teleogryllus oceanicus T. commodus ) ก กก กก กก กก (Reinganum et al ., 1970) กก CrPV ก ก CrPV ก ก (Reinganum et al ., 1981) ก Diptera, LepiDoptera, Orthoptera Heteroptera (Reinganum et al ., 1970; Christian anD Scotti 1998) ก SL2 (Scotti 1976; Moore et al ., 1980) CrPV ก insect picorna-like viruses กก กก picornaviruses ก (Moore et al ., 1980; Scotti et al ., 1981; Tate et al ., 1999; Wilson et al ., 2000) ก กก insect picorna-like viruses CrPV กก picornaviruses ก (Koonin anD Gorbalenya, 1992; Christian anD Scotti, 1998) CrPV 2 open reaDing frame (ORFs) ORF1 ORF2 ก nonstructural protein structural protein ก กก กกกก CrPV internal ribosome entry site (IRES) intergenic region (IGR) ก intergenic region IRES (IGR IRES) ก ORF กก (Tate et al ., 1999; Wilson et al ., 2000; Pfingsten et al ., 2007; Bonning, 2009; Pfingsten et al ., 2010) กกกก RNA- DepenDent RNA polymerase (RDRp) กก กกก RNA polymerase กกก กก กก RNA (Koonin anD Dolja, 1993; Tentcheva et al ., 2004; Chen et al ., 2005; Shen et al ., 2005; BlancharD et al ., 2007; Baker anD SchroeDer, 2008) CrPV OrDer: Picornavirales Family: Dicistroviridae Genus: Cripavirus กก International Committee on the Taxonomy of Viruses (ICTV) .. 2012 (ICTV, 2013) วารสารสถาบันสุขภาพสัตวแหงชาติ (ThaiNIAH eJournal): ISSN 19055048, http://niah.dld.go.th, Volume 9 Number 2 (Sep – Dec 2014) : 5872 60 ก CrPV ก Tohmee et al. (2008) ก IriDovirus . ก กกก กกก ก กก ก กก ก (Acheta domestica, A. testacea Gryllus bimaculatus ) ก ก (กXX) 1.2X2.4X0.8 ก ก (ก) (Fig.1) 40,000 / ก กก ก ก ก กก ก กก ก ก กกก ก 20 50-100 / 10X10 / ก ก ก ก ก ก กกก กก ก กก Impression smear Giemsa ก ก 10% Neutral buffereD formalin กก Haematoxilin & Eosin (Luna, 1968) ก กก วารสารสถาบันสุขภาพสัตวแหงชาติ (ThaiNIAH eJournal): ISSN 19055048, http://niah.dld.go.th, Volume 9 Number 2 (Sep – Dec 2014) : 5872 61 ก 70% ethanal ก ก Loop BlooD agar base No.2 ก 5% MacConkey agar 37° C 24 . ก ก Quinn et al. (1994) ก กก DNA RNA QIAamp Tissue DNA Minikit (QIAGEN, USA) QIAamp Viral RNA Minikit (QIAGEN, USA) ก กก -20 o C ก (template) กกก (Polymerase Chain Reaction; PCR) major capsiD protein (MCP) IriDovirus non coDing region (NCR), IGR IRES RDRp CrPV 25 µl DNA IriDovirus ® High FiDelity PCR Buffer 1X, DNTP 0.2 mM (each), MgSO 4 2 mM, Platinum Taq (invitrogen, USA) 1 unit/reaction, Primer (Table1) MCPF anD MCPR 0.2 µM (each) ก RT-PCR ก RNA CrPV 2X Reaction mix 1X, Super Script III One-step/Platinum Taq 400 µM, Primer (Table1) F anD R (NCR, IGR, CrPV) 0.3 µM (each) DNA RNA 1 µl ก ก DNA GeneAmp PCR system 9700 (PE-ApplieD Biosystems/FOSTER CITY, CA) IriDovirus Pre- Denaturation94 o C 5 min 1 Cycle, 35 Cycle Denaturation 94 o C 1 min, Annealing 50 o C 1 min, Extension 72 o C 2 min Final Extension 72 o C 7 min 1 Cycle CrPV Reverse transcription 50 o C 30 min 1 Cycle, Initial Denaturation 95 o C 5 min 1 Cycle 35 Cycle Denaturation 95 o C 30 sec, Annealing 55 o C (45 o C for CrPVF, R) 30 sec, Extension 72 o C 1 min (2 min for CrPVF, R) Final Extension 72 o C 7 min 1 Cycle ก electrophoresis 0.8% agarose gel ethiDium bromiDe UV-transilluminator ก PCR proDucts ก TA cloning kit (Invitrogen, USA) วารสารสถาบันสุขภาพสัตวแหงชาติ (ThaiNIAH eJournal): ISSN 19055048, http://niah.dld.go.th, Volume 9 Number 2 (Sep – Dec 2014) : 5872 62 Table1 Primer sequence for IriDovirus DNA anD CrPV RNA Diagnosis Primer Sequence ProDuct size Reference MCPF 5-GGTTTCATCGATATCGCCAC-3 1 kb Jakob et al. (2002) MCPR 5-GAAAAGTAATCACTGCCCAT-3 NCRF 5-AGAATTCTTTAATAAGTGTTGTGCAGATTAATCTGC 705 bp Wilson et al. (2000) ACAGCACTAGCC-3 NCRR 5-GCCATGGCTACATTGTAAGAATCGGTTACTCC-3 IGRF 5-CGGAATTCAAAGCAAAAATGTGATCTTGCTT-3 207 bp Wilson et al. (2000) IGRR 5-CATGCCATGGTATCTTGAAATGTAGCAGGTAAA-3 RDRpF 5-CAGGCGAGCAATATGACT-3 1.2 kb This stuDy RDRpR 5-TAACCTCTCAAAATTGTA-3 ก PCR proDucts MinElute™ PCR Purification Kit (50) (QIAGEN, USA) DNA NanoDrop® Spectrophotometer ND-1000 PCR proDucts sequencing reaction ABI Prism ® BigDye ® Terminator v3.1 Cycle Sequencing Kits (ApplieD Biosystems, California) primer M13 GeneAmp PCR system 9700 (PE-ApplieD Biosystems/FOSTER CITY, CA) 25 Denaturation 96 oC 10 annealing 50 o C 5 extension 60 o C 4 กก 3130 Genetic Analyzer ABI ApplieD Biosystem (HITACHI, Japan) ก ก () Chromaspro (version 1.5) MEGA (version 5.2) BioEDit (version 7.0.5)] BLAST program ก IriDovirus CrPV GenBank กก Multiple sequence alignments ClustalW program neighbor-joining bootstrap 1,000 ก (accession numbers) ก alignments Cricket Paralysis Virus (CrPV) [NC003924, AAF80998], Drosophila C virus (DCV) [NC004365, AAC58807], AphiD lethal paralysis virus (ALPV) [NC004365, AAN61470], Black queen cell virus (BQCV) [NC003784] Rhopalosiphum paDi virus (RhPV) [AAC95509] วารสารสถาบันสุขภาพสัตวแหงชาติ (ThaiNIAH eJournal): ISSN 19055048, http://niah.dld.go.th, Volume 9 Number 2 (Sep – Dec 2014) : 5872 63 ก ก กกกกก ก ก ก ก กก (Fig.2) ก กก (Fig.3) กก กก ก (Basophilic intracytoplasmic inclusion boDies) กก ก (Fig.4) ก Enterococcus spp. Table2 ก ก กกก MCP, NCR, IGR RDRp 1 kb, 705 bp, 207 bp 1.2 kb (Fig.5, Table2) ก ก ก GenBank MCP ก Gryllus bimaculatus iridovirus major capsiD protein gene (GIV- MCP) [AF247641] 97% NCR, IGR, RDRp ก CrPV [NC003924] 83%, 88% 90% ก IGR (cases1,2 3) ก CrPV [NC003924] (Fig.6) RDRp amino aciD ก amino aciD GenBank ก Nonstructural Polyprotien CrPV [NP_647481.1] 94% (Fig.7) กก CrPV [AAF80998] (Fig.8) กกก ก Tohmee et al. (2008) ก Trombidiform mite ก กก Enterococcus spp. (กก) กก IriDovirus basophilic intracytoplasmic inclusion boDy ก กก MCP กก ก กก Hall (1989), Kleespies et al. (1999), Jakob et al. (2002) Tohmee วารสารสถาบันสุขภาพสัตวแหงชาติ (ThaiNIAH eJournal): ISSN 19055048, http://niah.dld.go.th, Volume 9 Number 2 (Sep – Dec 2014) : 5872 64 et al. (2008) Table2 กกก MCP ก กก ก IriDovirus กกก กก กกก ก กก ก Reinganum et al. (1970) ก CrPV กกกก ก CrPV inclusion boDy กกกก ก ก ก ก กก (Reinganum et al ., 1970) ก ก ก RNA RT-PCR ก NCR IGR IRES primer (Wilson et al ., 2000) กกกกก (Tate et al ., 1999; Pfingsten et al ., 2007) ก RDRp primer กกกก enzyme กก ก (Baker anD SchroeDer, 2008) ก ก กกก NCR IGR IRES ก Wilson et al . (2000) RDRp ก amino aciD ก ก Nonstructural Polyprotien CrPV [NP_647481.1] 94% กก CrPV [AAF80998] กกก กกกก CrPV กกก กกกกก IriDovirus RT- PCR กก CrPV ก CrPV ก กก ก กก กก ก กกก IriDovirus ก CrPV กก กก CrPV กกกก กกก วารสารสถาบันสุขภาพสัตวแหงชาติ (ThaiNIAH eJournal): ISSN 19055048, http://niah.dld.go.th, Volume 9 Number 2 (Sep – Dec 2014) : 5872 65 Table2 Laboratory result Laboratory result Inclusion Bacterial PCR PCR (Cricket Province Sample External Swollen boDies in culture (IriDo Paralysis parasite abDomen fat cells virus) virus) Enterococcus ADult cricket - - - + - spp. Chiang Mai Enterococcus Young cricket - - - - + spp. Enterococcus ADult cricket - + + + - Maha spp. Sarakham Enterococcus Young cricket - - + + - spp. Enterococcus ADult cricket - - - - + spp. Nakhon Enterococcus Ratchasim Young cricket - - - - + spp. a Cricket eggs - - NA - - + Egg tray - - NA NA - + (-) = , (+) = ก, (NA) = กกก กก .. กกก ก กก 5 ก กก กกก วารสารสถาบันสุขภาพสัตวแหงชาติ (ThaiNIAH eJournal): ISSN 19055048, http://niah.dld.go.th, Volume 9 Number 2 (Sep – Dec 2014) : 5872 66 ก Baker, A.C. anD SchroeDer D.C. 2008. The use of RNA-DepenDent RNA polymerase for the taxonomic assignment of Picorna-like viruses (orDer Picornavirales ) infecting Apis mellifera L. Populations. J. Virol. 5: 1-10. BlancharD, P., Ribiere M., Celle O., LallemanD P., Schurr F., Olivier V., Iscache A.L. anD Faucon J.P. 2007. Evaluation of a real-time two-step RTPCR assay for quantitation of Chronic bee paralysis virus (CBPV) genome in experimentally-infecteD bee tissues anD in life stages of a symptomatic colony. J. Virol. MethoDs. 141: 7-13. Bonning, B.C. 2009. The DicistroviriDae: An Emerging Family of Invertebrate Viruses. Virol. Sin. 24: 415-427. Chen, Y., Higgins J.A. anD FelDlaufer M. 2005. Quantitative real time reverse transcription PCR analysis of DeformeD wing virus infection in the honeybee ( Apismellifera ). Appl. Environ. Microbiol. 71: 436-441. Christian, P.D. anD Scotti P.D. 1998. Picornalike viruses of insects. In: The insect viruses. Miller, L.K. anD Ball, L. A. Plenum Publishing Corp., New York. pp. 301-306. Eaton, B.T. anD Steacie A.D. 1980. Cricket paralysis virus RNA has a terminal poly (A). J. Gen. Viral. 50: 167-171. Hall, D.W. 1989. Pathobiology of IcosaheDral Cytoplasmic Deoxyriboviruses (IriDoviriDae). In (K. Maramorosch anD K.E. Sherman, EDs.). Viral InsecticiDes for Biological Control n.p. pp. 163-196. International Committee on the Taxonomy of Viruses (ICTV). 2013.