This article was downloaded by: 10.3.98.104 On: 25 Sep 2021 Access details: subscription number Publisher: CRC Press Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place, London SW1P 1WG, UK
Molecular Detection of Animal Viral Pathogens
Dongyou Liu
Taura Syndrome Virus
Publication details https://www.routledgehandbooks.com/doi/10.1201/b19719-ch3 Parin Chaivisuthangkura, Akapon Vaniksampanna, Phongthana Pasookhush, Siwaporn Longyant, Paisarn Sithigorngul Published online on: 09 Aug 2016
How to cite :- Parin Chaivisuthangkura, Akapon Vaniksampanna, Phongthana Pasookhush, Siwaporn Longyant, Paisarn Sithigorngul. 09 Aug 2016, Taura Syndrome Virus from: Molecular Detection of Animal Viral Pathogens CRC Press Accessed on: 25 Sep 2021 https://www.routledgehandbooks.com/doi/10.1201/b19719-ch3
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The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The publisher shall not be liable for an loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material. Downloaded By: 10.3.98.104 At: 12:21 25 Sep 2021; For: 9781498700375, chapter3, 10.1201/b19719-ch3 and controlof TS [15]. be themostsuccessfulhusbandry practicefortheprevention shrimp stocksofP. vannamei andP. stylirostris hasproven to has beenapplied. The development ofspeci cpathogen-free spawned eggs/nauplii usingpolymerasechainreaction(PCR) infection duringshrimpfarming, screening ofbroodstockand treatments available. Therefore, toreduce theriskof TSV tally veri ed. Currently, therearenovaccines orchemotherapy [5,6,8,14], but vertical transmission hasnotbeenexperimen- by cannibalismorcontaminatedwater hasbeendocumented and lessthan5g[6,8,13]. The horizontaltransmission of TSV with TS aretypicallysmalljuveniles weighingbetween0.05g lations ofpostlarval, juvenile, andsubadultshrimp.Shrimp mortalities rangingfrom40%to>90%ofculturedpopu - by the virus [6–12]. The diseaseoften results incumulative P. duorarum References...... 3.3 3.2 such as and blueshrimpP. stylirostris. However, othershrimpspecies [5]. The principal hostsof TSV arewhiteshrimpP. vannamei studies usingspeci cpathogen-freeP. vannameiasthehost drome virus(TSV),was identi edin1994throughinfectivity and Indonesia[2–4]. The viraletiologicagent, Taura syn- tries, includingtheUnitedStates, Taiwan, China, Thailand, Taura River inEcuador[1]. TS laterspreadtomany coun- in Taura syndrome(TS)ofpeneidshrimpwas describedin1992 3.1 3.1 CONTENTS Penaeus vannameiharvested fromshrimp farms nearthe
Conclusions 3.2.2 Sample Preparation...... 3.2.1 Methods .. lnclFaue n ahgnss...... 3.1.4 Pathogenesis and Features Clinical 3.1.3 Biology, Genome Organization, Epidemiology...... and 3.1.2 3.1.1 Introduction 3 INTRODUCTION P. setiferus, , 3.2.2.2 Quantitative RT-PCR...... Quantitative 3.2.2.2 3.2.2.1 Detection Procedures Detection 3.1.4.3 3.1.4.2 3.1.4.1 Diagnosis Classi cation P. indicus,andMetapenaeusensiscanbeinfected ...... Siwaporn Longyant, Paisarn and Sithigorngul Parin Chaivisuthangkura, Akapon Vaniksampanna, Phongthana Pasookhush, Virus Syndrome Taura ...... P. monodon, Gel-Based RT-PCR Gel-Based Immunological Techniques Techniques Molecular Conventional Techniques ...... P. chinensis, ...... P. aztecus, ing the 3 the ing ssRNA genome is 10,205 nucleotides exclud length, in - anonenveloped shape. with TSV The is small, icosahedral 3.1.2 approximately 1.338 gmL include of TSV abuoyant virions Characteristics density of 3.1.1 Aparavirus on Taxonomy novelCommittee the into of Viruses genus [17]. Presently, classi ed TSV by International been has the TSVthat is closely virus cricketparalysis-like the to related revealed comparison sequence and genomic characterization Picornaviridae family of amember as the 3 the (ssRNA) RNA stranded genome is polyadenylated that at ofa size 31–32 nm apositive and diameter, in single- sense Picornavirales represent 92% of the TSV genome, and the remaining 8%represent 92% TSV of the genome, remaining the and 55 kDa), (40 VP2 (24 kDa), kDa). VP3 ORF2 and and ORF1 major capsid VP1 (CP2, proteins, including three proteins, of sequences structural the (RdRp).merase contains ORF2 poly RNA-dependent RNA an of and helicase, a protease, nucleotides [17]. acidof sequence consists ORF1 amino The (ORFs)frames region intergenic by (IGR) an separated of 207 ′ end [16]. classi ed was initially virus Therefore, the C g and ′ -poly(A) tail, and contains two large open reading -poly(A) two reading open large contains and tail, lassifi eno in a new family Dicistroviridae anew in family e Me pide [18]. C o ation M rganization iology −1 −1 in CsCl, icosahedral morphology, icosahedral CsCl, in , B iology , [5,16]. However, [5,16]. However, in the order order the in 20 23 23 22 22 22 19 19 19 19 21 21 17 17 17 17 - Downloaded By: 10.3.98.104 At: 12:21 25 Sep 2021; For: 9781498700375, chapter3, 10.1201/b19719-ch3 infection in lymphoid organs was observed in shrimp with with lymphoid in shrimp infection was in organs observed the and acuteinfections, with was most prevalent shrimp in epithelium cuticular the in Infection mesoderm. and ectoderm [27]. variant America Colombia)and anew and subcluster Central Mexico the in in two to new (Venezuelaleading subclusters South America in group diversityrevealed Americas the the of in TSV CP2 previous the Colombia from differs and results isolates. The anew forms CO 10 cluster the that strain indicated sequence acid phylogenetic The lyzed. amino CP2 on the analysis based CO 10 called - was ana farms Colombian from lated shrimp [12,25,26].group 2010, In anewly of TSV iso emerged strain Venezuelan the group, and Belize group, the Asian Southeast group, the Americas the are variants These protein. structural VP1 of sequence the the to haveants according identi ed been TSVamong isolates. Currently, atleast four genotypic vari genetic relationship the establish to used region been has (CP1) (CP3) 0.8%) VP3 and (both [24]. CP2 Therefore, the acid (3.5%) sequence its amino in variation greater VP2 than step[23]. initial the cupied during - unoc Psite Asite, 40S remained the the whereas in structure by placementwas established the tRNA–mRNA-like of the mRNA IRES-driven of Speci cally, the ORF mechanism. the byunprecedented an translation initiated TSVthat IRES indicated codon. Moreover, structure anticodon/mRNA the tRNA of the that to (A)aminoacyl similar site amanner in atthe center decoding ribosomal occupiedthe of TSV IRES [22]. analysis PKI revealed that structure Acryomicroscopy of for IGR-IRES function is essential the I(PKI), pseudoknot one of pseudoknots, which, called three IGR-IRESs contain IGR-IRES [21]. distinct TSV astructurally that had The in vitro and IRES, predicted the in stem-loop structure internal TSVsuggested an that had search [20].ylated tRNAs structure Acomputational-based - elongation aminoac eukaryotic and factors 1A 2, units, and sub ribosomal only puri ed system containing reconstituted a synthesis in protein direct and ribosomes recruit ments can [20]. polyprotein TSV ofcodon the structural GCU-alanine of positionedcodon the two upstream codons alanyl-tRNA consists of an untranslated region (UTR). In the 3 region the (UTR). In consists untranslated of an 18 tRNA non-AUG startcodonanddidnotinvolve initiator methionyl- a from IGR-IRES started shown from TSV that translation AUG an [19]. not did require initiation IRES the It been has (Met-tRNAi). Therefore, tRNA methionine initiator in triplet anticodon the and codon initiation the between interactions absenceof base-pair the was in shown occur to cistroviruses - dis in of translation initiation IGR-IRES-mediated tions, the - initia translation [17].(IRES) IRES-mediated other Unlike site entry ribosome byinternal an was translated TSV ORF2 suggested that viruses cricketparalysis-like ofmembers the [17]. Sequencehomology IGR of TSV the other between and putative polyadenylation (AAUAAA) signal was identi ed Infection of TSV occurs in tissues derived from the the tissues derived from of in TSVInfection occurs VP1 (equivalent capsid to 2; protein CP2) demonstrates absence factors,of TSV initiation the IGR-IRES eleIn Met . Translation from the TSV IGR-IRES initiated with with . Translation TSV the IGR-IRES initiated from Ala even though there was an in-frame AUG even in-frame was an there though translation analysis showed translation ′ -UTR, no -UTR, - - - - ( [31]. Trichocorixa reticulata water boatman the addition, In a vector of for TSV environments transmission aquatic to may as serve seabird wild tissues. Therefore, this shrimp consumption of viral-infected h after for upparticles to 6 ( feces of seagulls [10,28–30]. organs of enteric infection TSV the in Histological infections. analysis revealedchronic no signs of could be genetically resistant animals. Therefore, one of animals. genetically resistant could be survivors the that speculated researchers and technicians including staff various and survived, 20%–30% of shrimp Colombia. and of time, Ecuador At industries that shrimp [36]. Taiwan, Americas, Thailand the and of those to TSV isolates from isolates were similar 96%–99% nucleotide of sequences VP1, partial two TSVon the Korean of stocks P. Korea to imported via duced vannamei it and was TSV suggested that was intro Korea occurred, of P. mortality vannamei mass 2004, In Taiwan and China [35]. from were of cially imported shrimp over time, stock that brood 150,000 During mid-2002. in of P. legal vannamei importation after 2003 in con rmed was elsewhereTS outbreak and rst the [2]. Thailand, In Ecuador spawners from and postlarvae by contaminated Taiwan to TSV that was transferred was assumed 1998. It low as to abruptly 10% as volume of early the in produced 1998 late in 1999, production early to dropped shrimp and Taiwan in Asia occurred in TS outbreak rst 1990s. The the Asia during in species shrimp aprimary which became live of P. infected Asia to due importation to vannamei America Mexico and Belize, [34]. TSV may have South from spread Salvador, El Nicaragua, Guatemala, Brazil, Honduras, Peru, including America, Central South and in countries other in farms losses [6]. on shrimp impact million an caused TS also Texas Central and of Southern US$10 to TSled in outbreaks of Hawaii, Texas, [34]. Florida the and that It was estimated progressivelyspread states including UnitedStates, the the to up US$100 to caused [6]. of damage worth TS has million forpose ariskaspotentialcarriers [33]. TSV transmission palaemonidshrimpspeciescan and crabs suggest these that at15 results These d. but no occurred reaction feeding after lanchesteri M. of case at15 the In d. postchallenge but no occurred reaction a strong RT-PCR at5 d P. was reaction in observed stylirostris TSV-infectingwith P. vannamei (RT-PCR) at10, 15, 50 days and (d), respectively, feeding after gave reaction chain polymerase strong reverse transcription showed results The U. that vocans Sesarma mederi species,crab Sesarma lanchesteri Macrobrachium and styliferus Palaemon species, including ponds shrimp two palaemonid shrimp found near ve with native common crustaceans Thailand [6,32]. gut assay the (ISH) in ization active TS, was shown TSV be to positive using Corixidae In TSV vector studies, it has been demonstrated that the the that TSV demonstrated In vector it been studies, has During the mid-1990s, the TSV devastation caused During the to 1992 in [1] Ecuador in appeared TS rst may have and A study on potential carriers of TSV was in conducted carriers A study on potential ), an aquatic insect collected from ponds with with ponds from collected insect ), aquatic an Molecular Detection of Animal Viral Pathogens Viral of Animal Detection Molecular , alightRT-PCR was reaction at2d observed Larus atricilla Larus , Scylla serrata carcasses. Also after feeding, feeding, after Also carcasses. ) can contain infectious TSV infectious contain ) can , S. serrata S. shrimp cultured in in cultured shrimp , and Uca, and vocans , and S. mederi , and in situ , and three three , and hybrid . Based . Based - - . ,
Downloaded By: 10.3.98.104 At: 12:21 25 Sep 2021; For: 9781498700375, chapter3, 10.1201/b19719-ch3 Taura Syndrome Virus Taura Syndrome their succeeding molt. Shrimp that survive the next survive the molting that molt. Shrimp succeeding their during black these ofwith lesions atsome mortality risk are Shrimp cuticle occurs. the necrosis acutephase where the lesions or under in melanized variably sized domly distributed - ran demonstrate typically shrimp The infection. the from [6,42,43].molting during acute phase the in occurs often population. Death pond may last for farm several affected days an in a shrimp However, 24 h. survive less than typically in acutephase the and despitegut, empty stop having feeding lethargic, an are shrimps visibleand tissue. epithelial of Infected necrosis the fan, tail the in especially sion chromatophores cuticular of red coloration red to expan duesuch the to asoft shell, as pink symptoms demonstrate acutephase the in shrimp Moribund water. surface pond edge and the approaching shrimp bund massive sudden is often and mori with onset of mortality phases. chronic including and phases acute, transition, distinct three P.TSV in infection vannamei 3.1.3 pathway, (4) (5) antioxidation, and [41]. protease (2) transducers, signal (3) and coagulation, proteins, proPO adhesive regulators, cues such immune as ognition through activity. response includedimmune (1) These pathogen- rec revealed expressed genes several involved differentially the in of gene expression susceptible and resistant shrimp between comparison RNA-seq. The usingconducted high-throughput of TSV-resistant susceptible and P. vannamei regions [40]. farming shrimp many in ger amajor considered be to threat industry, TSV and is no lon farming bene t shrimp to the of availability Therefore, TSV-resistant the is of great shrimp worldwide. hatcheries shrimp stock commercial to brood have TSV-resistant programs distributed breeding shrimp eral exposure [39]. after 100% survival sev decade, past Over the generation per 15 exhibited several generations, and families P. selecting OI been had vannamei [38]. control families unselected with selection The compared 18.4% one generation of TSV to after survival in increase to TSV. resistance for and growth an demonstrated line One on aselection based index weighted program ing equally - OI Hawaii, (Waimanalo, UnitedStates)the abreed operated (OI). Institute From 1995 by Oceanic the operated 1998, to improve to P. in TSV program resistance breeding vannamei aselective initiated Program Farming Shrimp U.S. Marine ous levels [37]. previ to rates survival simple scheme ofcess increased this suc generations,- the two three to selection Within process. for next the parents generation asimple as in them mass used and ponds infected from to selectsurvivors the gram Colombia a pro in producers initiated major shrimp the In the transition phase, some affected shrimp may recover shrimp some phase, affected transition the In outbreak of acutephase disease the During hemolymph study in hemocytes and transcriptomic rst The the UnitedStates, the in TSV to response outbreaks In C lini C al f eatures
and exhibits gross pathology exhibits gross in p for for TSV resistance over athogenesis in in was recently P. vannamei ------, , This problem avoided be can This bypH (~6.0–7.0) using a neutral false-negative to genomic leading RNA [28]. results probe 4.0) of Davidson’s xative acid causes hydrolysis of TSV acidic pH (~3.5– highly The solution 24–48 h. for more than Davidson’s in preserved acid–formaldehyde–alcohol acetic tissueswere shrimp the negative if may results obtained be histopathology of diagnosis TSV However, infection. false- gets [29,45,46]. for routine used the been ISH has method The identi cationlowing successful of TSV-speci c the gene tar assays molecular early consistThe of ISH RT-PCR and fol 3.1.4.2 some gave cells positive [10,43,44]. for signals virus the (MAbs), monoclonal with antibodies immunohistochemistry vessel. assayeda central When ISH via TSV with or probe vacuolated lack oferative and cells, nodules of staining, pale spheroids, prolif has which usually are phase chronic the in display lymphoidand signs no of gross The infection. organ lesions cuticle on the melanized mostly sized and disappear [43,44]. H&E with staining normal appear corrosive on the observed cuticle.be lymphoid The may organ black spots.Invasion Vibrio of the irregular the to rise giving conspicuous melanized, become are and necrosis site atthe of accumulation hemocytes and in ltration areas, severity. and abundance affected the both lesions in In decline cuticular acute-phase typical which the in phase transition [6,42,43]. remnants basophilic pale inclusions the show to (H&E) staining, eosin eosinophilic and hematoxylin In appearance. in or “buckshot” “peppered” as described often are and diameter in 20 µm 1to from range inclusion spherical present as that and bodies abundant are cells necrotic of the remnants Cytoplasmic areas. infected the throughout nuclei scattered are karyorrhectic and Pyknotic lymphoid and observed. is occasionally organ muscle, heart, underlining the connective in tissue.subcuticular Infection epithelium and cuticular the of in necrosis areas multifocal as Histopathology present acuteTSV-infected changes in shrimp 3.1.4.1 3.1.4 [6,42,43]. tubules lymphoid for vessel organ normal is typical central that the tive vacuolated alack of nodulesand cells, of staining), pale of lymphoid The organ shrimp. uninfected than stress salinity show butbehave will slightly less normally low to tolerance and may They apersistent infection. appear as life out their [6,42,43]. bacteria opportunistic from epithelium of orsystematic to cuticular infection destruction of aconsequence widespread as either osmotic to failure due occur may also cuticle. death phase, the transition the In from lesions the and disappear look normal typically process P. vannamei In shrimp with chronic infection, the irregularly shaped shaped irregularly the infection, chronic with shrimp In may acuteinfection undergo the survive the that Shrimp - TSV through may carry phase, shrimp chronic the In d
Molecular Techniques Molecular Conventional Techniques iagnosis in this phase usually has spheroids has (prolifera usually - phase this in spp. may 19 - - - Downloaded By: 10.3.98.104 At: 12:21 25 Sep 2021; For: 9781498700375, chapter3, 10.1201/b19719-ch3 FrigItlieeTcnlg oprto, Taiwan). Technology IntelliGene Corporation, (Farming RT-PCR Prevention TSV and Detection System IQ2000 kit ve nested less sensitive of times commercial that than wascoupled approximately dot-blotting with procedure employed of for detection TSV speci c [52–55]. NASBA loop- including (NASBA) nucleic ampli cation acid–based and [51]. sensitivity ~50 from size ~70–100 to and its speci city bp enhanced amplicon the increasing that indicated results the tion, and RT-PCR [50]. earlier assay described TSV, by single lower afforded the which that times is 20 than However,virus. copies assay of asensitivity has the of 2000 oligonucleotide TaqMan for and speci c probes each primers of detection WSSV,the of sets TSV IHHNV, and using three multiplex developed was also method real-time PCR The for RT-PCRprobe by [15]. OIE recommended assay been has [49]. shrimp TaqMan infected acutely This chronically and both of detect to 100 used copies could be limit and detection isolate RT-PCR ofor an YHV. real-time a has method This shrimp healthy from was RNA with observed ampli cation No Colombia, Mexico, Thailand. and Indonesia, Belize, TSV with Hawaii,ampli cation isolates Texas, from strated region ORF1 TSV of the the from genomic sequence, demon RT-PCR, a TaqMantime and designed probe using primers RT-PCRreal-time assays were developed [49–51]. real- The P.in vannamei WSSV with coinfection detect to TSV used and could be and plex RT-PCR for of detection TSV WSSV and was developed [47]. addition single to RT-PCR(IMNV) In multi methods, myonecrosis or infectious yellow-head virus (YHV), virus (WSSV), virus white spot syndrome (IHHNV), virus necrosis hematopoietic and hypodermal infectious to cross-reactivity Venezuela and Asia, Southeast Americas, displayed and no fourisolates phylogenetic from including the Belize, lineages 7171F/7511R TSV detect successfully to were demonstrated primers 9992F/9195R The by OIE. recommended primers genome, more sensitive RT-PCR 100 the times than using the improved of the RT-PCRlimit assay was copies 20 TSV of the 7511R,and a341 detection The which amplify bp fragment. [47] al. using newly 7171F designated designedprimers, et purposes. diagnostic and lance (OIE) Epizooties des [15] International Of ce the for surveil [43] TSV known genetic variants all by is recommended and TSV genome [17,46]. RT-PCR This assay allows of detection 9195R (CP1) primer and VP2 the in is located gene of the 3 the near is located 9992F genome. Primer a231 (bp) TSV amplify of sequence pair base the primers TSV cDNA library. Using RT-PCR one-step the the approach, 9195R and 9992F called on a cloneof based primers from Davidson’s in (24–48 h) xation solution [28]. proper RNA-friendly xative xative called or through 20 The alternative nucleic alternative The techniques, acid ampli cation RT-PCR SYBRThe Green was evaluated for TSV- detec addition gel-based to In conventional RT-PCR, quantitative improvedAn RT-PCR assay was developed by Navarro [46] of case RT-PCR, the designedapair In et al. Nunan mediated isothermal ampli cation (LAMP), were also (LAMP), ampli cation isothermal mediated [48]. ′ end IGR, of the - - - including VP1, VP2 and VP3, were also developed [60–62]. wereincluding developed [60–62]. VP3, VP1, also and VP2 capsid p structural recombinant against MAbs or Venezuela Belize, Mexico,from Nicaragua, [25,26,59]. TSV isolates Hawaii, it and notUnited States, does recognize TSV with react Taiwan, isolates from can MAb the this while However, blot western via immunohistochemistry. dot and 1A1, designated MAbs VP1, to speci c TSV detect to was used antigen TSV[58]. the as using puri ed produced of One the mouse polyclonalChicken and were MAbs and TSV antisera 3.1.4.3 including WSSV, [57]. YHV, IHHNV and TSV-speci c viruses showed and noshrimp to cross-reaction of RT-PCR. that than HRCA higher 100 The was times test sensitivityTSV The detection. was approximately 10 copies, (HRCA) was test developed assay astrip with combined for [56]. risk contamination reduced with kit, sitive RT-PCR gel to agarose RT-LAMP IQ2000-nested and sen - RT-LAMPis equally this coupledthat turbidimeter with showed results The tubes. reaction the bidity without opening tur the measure to used was designedand table turbidimeter turbidity, in resulting amplicons, a por LAMP of ampli ed phosphate (Mg phosphate pyro magnesium produced ampli cation Given LAMP the samples could between occur. of contamination risk tion, the - detec for must opened be further tube reaction which the WSSV, with reactivity [54]. IHHNV, or YHV RT-PCR TSV to no speci city with cross- high exhibited and of nested that to comparable limit assay adetection had This 10 min. available are results within the and amplicons LAMP appropriate the into is immersed LFD the probe. Without equipment, relying on special (FITC)-labeled a uorescein with hybridized been had that ucts - prod [54]. biotin-labeled LAMP onstrated detected strip This ow was amplicons dem dipstick for (LFD) of detection LAMP false-positive [55]. reactions eliminate to DBH capacity the products, the has LAMP to speci c probe of hybridization utilization the to Due products. aberrant the production of templates couldthe to lead target of the trations However, samples low or very sometRNA cases, in concen- parvovirus. hepatopancreatic and monodon baculovirus, including WSSV, viruses, shrimp IHHNV, IMNV, GAV, was no cross-reaction There with other method. IQ2000 RT-PCR nested sensitivity same the as the exhibited and cons RT-LAMP (DBH)hybridization detect to was used ampli WSSV and [53].including YHV dot-blot colorimetric The pathogens was nucleic with observed shrimp other acidsfrom RT-PCR nested No the kit. sitivity ampli cation than IQ2000 RT-PCR the sitivity by but OIE less than sen recommended (RT-LAMP)ampli cation 10 more sen demonstrated times [52].YHV, IMNV and (GAV), virus including gill-associated viruses RNA shrimp No nontarget with false-positive was reaction obtained Recently, circle ampli cation rolling ahyperbranched As the early RT-LAMP procedures included RT-LAMP early a step in the As procedures developmentFurther of RT-LAMP lateral with combined RT gel–based loop-mediated agarose The isothermal
Immunological Techniques Molecular Detection of Animal Viral Pathogens Viral of Animal Detection Molecular 2 P 2 O 7 ) by-products correlated with the amount amount the ) by-products with correlated buffer containing isothiocyanate isothiocyanate roteins, roteins, ------Downloaded By: 10.3.98.104 At: 12:21 25 Sep 2021; For: 9781498700375, chapter3, 10.1201/b19719-ch3 Taura Syndrome Virus Taura Syndrome ethanol-preserved tissues. ethanol-preserved available are for and TSV frozen, isolation RNA fresh, from for RT-PCR assays. extraction kits Various RNA commercial used be can hemolymph, pleopods, or whole shrimp small Tissue samples such purposes. as diagnostic and monitoring for by OIE TSV the RT-PCR recommended are methods 3.2.1 3.2 regions. geographic ous of for detection may most useful the TSV be vari isolates from VP3 and VP1, to combination of the MAbs-speci c that VP2, 3.1). lightTSV (Figure and infection chronic detect It is likely to used could be MAbs these Through immunohistochemistry, muscle; tissue;skeletal M, hemolymph; V, H, connective vessel.S, subcuticular central epithelium; E, of TSV infection. however, immunoreactivity strong H&E staining; in it demonstrated normal histologically (3) demonstrated Note lesion areas. the in (*) scattered are appearance “buckshot” as referred remnants cytoplasmic eosinophilic and nuclei (arrow) karyorrhectic and of pyknotic staining basophilic Intensely staining. brown as appeared Positiveeosin. immunoreactivity with counterstained of TSV and VP2 to speci c antibody monoclonal with stained (b) were immunohistochemically and tissues (H&E) (3) (1) and in (2) Two lymphoid organ. epithelium, eosin heart, and cuticular hematoxylin (a)with sections: consecutive were stained tissues 3.1FIGURE
s METHODS aM
ple P. vannamei TSV-infected P. naturally from of tissues immunohistochemistry and Histopathology p 3 2 1 reparation (a) M M M V S V V E E H - tion of 500 µL Wash Buffer I, centrifuged at 8000 ×g Wash at8000 tion µL of 500 Buffer centrifuged I, at 15°C–25°C addi is washed the via column The for 15 min. incubated and collection tube the with combined column spin solution the to incubation buffer, prepared is added the and of of DNase DNase solution Iworking µL 90 with is mixed For isolation, each ow-through 10 the is discarded. then µL (13,000 speed ×g atmaximal centrifuged and volume µL) (maximal 700 collection tube the with combined column spin the to is applied lute Next, mixture ethanol. the of abso µL 200 with is mixed supernatant The centrifuge. amicro in speed atmaximum for 2 min lysate is centrifuged homogenizer, using the homogenized arotor–stator and and disrupted tissuesare The tube. 1.5 microcentrifuge free mL of mg) anuclease- tissue (max. amount in 20–25 appropriate of Lysis/Binding µL Germany), the to Buffer is added 400 (b) In case of the High Pure RNA Tissue RNA (Roche, Kit Penzberg, Pure High of case the In 50 µm with asymptomatic infection infection asymptomatic with : Lymphoid organ ) for 30 s, and for 15 s, 21 - - - Downloaded By: 10.3.98.104 At: 12:21 25 Sep 2021; For: 9781498700375, chapter3, 10.1201/b19719-ch3 et al. [46]. by et al. Nunan described on that is based detection text for TSV succeeding the in outlined method The product. RT-PCR, the in used of forare ampli cation a231 speci c bp Two 9195R, and oligonucleotide 9992F designated primers, 3.2.2.1 3.2.2 for used be RT-PCRsthen for at−80°C analysis. or stored later ×g at8000 centrifuged and column spin ofthe to 100 elution µL directly buffer is added and tube onto is placed anewcolumn 1.5 microcentrifuge mL Finally, spin ow-through the the is discarded. then and 2 min, at13,000 ×g Wash µL tion ofcentrifuged 300 and Buffer II is washed by addi column the Wash the with Then, Buffer II. stepis repeated washing the and ow-throughthe is discarded, 22 Total volume RNA sample rTth DNA polymerase2.5U EZbuffer5× Manganese acetate(2.5 mM) 9195R (0.46 µM) 9992F (0.46 µM) dNTPs (300 µMeach) Diethyl pyrocarbonate (DEPC)-treatedwater Component Temperature The GeneAmp GC Sequence 9195R 231bp 9992F Product Primers 3.2.2.1.1 controls shouldbeperformedalongwiththeno-templatecontrol. described inthesucceedingtext. The TSV positive andnegative Forster City, CA,USA)isusedfortheampli cationreactions 2. 4. 3. 1.
spin down all reagents before opening the tubes.) reagents the before downspin opening all specimen). per are ( listed umes RT-PCR the follows as Prepare mix reaction (vol 60°C for60°C 7 min. extension for extension at60°C 45 nal s, at and at94°Cditions: for denaturation 45 s, annealing/ for cycles 40 ampli ed are following using the con completion the of RT, After samples the for 2 min. followed for by at60°C 30 min, 94°C proceed then cycler. athermal in sample RT tubes the Place may 14,000 ×g at amicrocentrifuge in sample tubes Spin the RT-PCRAdd tube. the PCR a0.2 to mL mix reaction d
Gel-Based RT-PCRGel-Based ete Procedure C ® tion EZrTthRNA PCRkit(AppliedBiosystems, for 30temperature. satroom 5′-TCA ATGGCT AGA 5′-AAGTAG ACA p TGG TCC-3′ GCC GCGCTT-3′ ro C edures for 1 min. The eluted RNA can eluted can RNA The for 1 min. Note 0 63°C 69°C 50% 55% : Vortex and Volume (µL) 50 10 1 10 Variable Variable Variable Variable Variable - - for for - Total volume DEPC-treated water RNA sample(5–50ng) TSV1075R (0.3 µM) TSV1004F (0.3 µM) probe TSV-P1(0.1 µM)TaqMan MultiScribe andRNaseInhibitorMix 40× Master mixwithouturacil-N-glycosylase(UNG) 2× Component
AF277675). TSV1004F primers The (5 region ORF1 TSV of the to the genomespeci c (GenBank [49]. al. TaqMan and designedprimers The et are probe by text Tang follows described succeeding procedure the TSV genome [49]. qRT-PCR the The in outlined method approximately the 100 in sequence copies target of the sensitive, sensitivity speci c. The of and qRT-PCR is of detection TSVThe using quantitative RT-PCR is rapid, 3.2.2.2
AAA CGA CATAAA T-3 3.2.2.2.1 run. included each in TSV-positive control should no-template be the control and 3 on the cent dyes 5 (FAM) on the 5-carboxyl uorescein ACA ATA GAG TTC CAT C-3 TaqManment. The TSV-P1 probe (5 AAC ACA TGG CAC TGT-3 N,N,N 2. 4. 6. 5. 3. 1.
48°C for 30 min, initial denaturation at 95°C denaturation for initial 48°C for 30 min, qRT-PCR the Perform cycling consisting of RT at suitable). also are brands and 7500 7300, and (newerABI PRISM 7000, models SystemSequence Detection (Applied Biosystems); 5700 GeneAmp the in tubes reaction the Place agarose gel, stained with ethidium bromide (0.5 bromide gmL ethidium with gel, stained agarose using 2.0% products a10 PCR ofAnalyze the µL tubes. of wall the inner condensation atthe droplets the down pull to tubes reaction the spin tion, pulse RT-PCR of the Following- termination reac the the sample under an ultraviolet an sample under lightsource. the acid). visualize Then, ethylenediaminetetraacetic electrophoresis 0.5×and in acid, TBE (Tris, boric recommended.) before is use mix down reaction the spinning and Biosystems, no. 4309169). part ( (AppliedTaqMan RT-PCR One-Step mixture master qRT-PCR the follows as Prepare mix reaction using earlier. described method tissue using the shrimp from RNA Extract ′ ,N′
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end (Applied Biosystems, no. Cat. 450025). The Quantitative RT-PCR Quantitative -tetramethyl-6-carboxyrhodamine (TAMRA) (TAMRA) -tetramethyl-6-carboxyrhodamine Procedure Molecular Detection of Animal Viral Pathogens Viral of Animal Detection Molecular ′ ) and TSV1075R) and (5 ′ ) generatea72 bp DNA frag ′ ) is labeled uores with - ′ -CAG CAC TGA CGC CGC CAC TGA -CAG Note ′ ′ -TTG ACC GGC -GGG AGC TTA-GGG : Vortexing Volume (µL) ′ 25 Variable Variable Variable Variable Variable 0.63 12.50 end and and end –1 ), ), -
Downloaded By: 10.3.98.104 At: 12:21 25 Sep 2021; For: 9781498700375, chapter3, 10.1201/b19719-ch3 Taura Syndrome Virus Taura Syndrome REFERENCES Dicistroviridae is novel classi ed the to genus Aparavirus TSV comparison, sequence and on genomic characterization Based over and shrimp. 40% between of 90% infected talities mor in results usually TS disease countries, many to spread P. vannamei white shrimp pathogen cultured TSV in is amajor viral 3.3
TSV is no longer considered amajor threat. regions, farming industry. shrimp many aconsequence, in As farming worldwide,eries shrimp bene t the to great giving have TSV-resistant- hatch distributed stock shrimp to brood programs breeding shrimp several past the the years, During vannamei P. improve to in TSV programs available, resistance breeding RT-PCRnested available. is also kit sensitivities.TSV different with detection commercial The weremonoclonal developed techniques antibody-based for However,nostic purposes. several or assays such LAMP as TSVof diag the genomic and for sequence TSV surveillance region ORF1 TaqMan and the designedfrom probes primers using qRT-PCR and TSV known genetic variants, all detect can 9195R, and 9992F RT-PCR called ofwhich primers a pair with VP1. of sequence the the Venezuela/Aruba, and to according Belize/Nicaragua, Asia, have including Mexico, identi ed, ones in been Southeast IGR. Currently, atleast within four genotypiclocated lineages IRES by an is translated TSV suggested that ORF2 been has IGR. by It an genome two of ORFs separated TSV contains 2. 1. As no vaccine or chemotherapy treatment are presently are or chemotherapy noAs vaccine treatment of use the recommends OIE For detection, molecular 6. 5.
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