Describes the development of a reverse transcription-polymerase chain reaction and an enzyme immunoassay for the detection of an Australian Norwalk-like virus group 2b virus (Camberwell) and related viruses and their application to clinical samples.Thesis (M.Sc.) -- University of Adelaide, Dept. of Microbiology and Immunology, 2000

Wati, Satiya

Adelaide Research & Scholarship

r-l,ã.ooRapid Detection of Norwalk-Like Viruses (NLVx)Søtþa WatiB. App. Sc (Hons'¡Infectious Diseases LaboratoriesInstitute of Medical and Veterinary ScienceAdelaideSouth AustraliaDepartment of Microbiology and ImmunologyUniversity of AdelaideAdelaideSouth Australiarl. t * *!* {< tl. {. !ß rß rß t {. ¡Ë {.A thesß submitted to the Universìty of Adeløide infuffilment of the reqairementsforthe degree of Master of ScienceJuly, 1999ConlentsAbstractAcknowledgementsAbbreviations ...... .........xiiCHAPTER 1. fntuoduction1.1 Review of gastroenteritis causing agents .1.1.1 Bacterial and protozoal gastroenteritis . . . .1^l.2 Viral gastroenteritisI.l.2.l Rotavirus (Reoviridae).....I .l .2.2 Adenoviru s (Adenoviridae)II2J5LI.2.3 Astrovirus (Astroviridae).51.1.2.4 Sapporo-like viruses (SLVs) and Norwalk-like viruses gflvs) (Caliciviridae). . . . . ....61.2 History - Discovery of NorwalkJike viruses 71.41.4.r1.4.21.4.31.3 Significance of NLVs in relation to bacterial and other viral gastroenteritis ...........8Classification of NLV ................9Genomic organisation of NLVs ...Comparison of NLV and SLV genomic organisation ..Antigenic diversity of NLVs .10t21.5 Ultra structure of NLV ........13vu1.6 Physicochemical properties of NLVs ................141.8 Clinical Presentation of NLV infection ...............161.7 Pathogenesis ......... .....151.9 Immunity .........,171.10.1 Transmission ......LID.2 Incidence of NLV in Australia ........Maintenance of SF21 cellsInfection of SF21 cells with recombinant baculovirus ...... ...Harvest of infected SF21 cells ......Quantitation of virus particles .....Electron Microscopy (EM)Density of CAM VLPsFractionation of antisera with ammonium sulphateI9.2tl.l 1.1 Electron microscopy . 22l.Il.2 Immunoassays using human convalescent sera and antigen purified from faece s . . .... . .231.1 1.3 New approaches to rapid laboratory diagnosis of NLV infection ......231.11.3.1 Recombinant NLV enzyme immunoassay 24I.IT.3.2 NLV RT-PCR . . . 25l.l2 Project Aims..25CHAPTER 2. General Materials and Methods2.1 Commonly used reagents ......... ..............261.11 Diagnosis ..........222.2.12.2.22.2.32.2.42.2.52.2.62.2.72.2.832.3232Detection of CAM VLP proteins .....ltJJ2.2.8.1 Separation of proteins in I0%o SDS PAGE gels . .2.2.8.2 Western blot and detection of CAM VLP proteins .2.2.9 Hirt extraction of plasmid DNA from recombinant baculovirus ... ..2.2.10 RNA extraction using the Boom method ... .. .2.2.11 Chomczynski and Sacchi (CS) method ........2.2.11.1 RNA extraction using the chomczynski and Sacchi (cs) method .....2.2.11.2 RNA extraction using the modified Chomczynski and Sacchi (CS) method .2.2.12 DNaseI treatment of extracted RNA2.2.13 RNA electrophoresis on Northern gel ...2.2.14 Poliovirus culture ..2.2.15 Plaque assay for determining poliovirus plaque forming units þfu) ..2.2.16 Negative controls used in EIA and all RT-PCR2.2.17 Processing of faecal specimens to be tested by EIA or RT-pCR2.2.18 Titanru one tube RT-PCR system ...2.2.19 RT-PCR of poliovirus using Corbett Thermocycler...,2.2.20 RT-PCR of rotavirus using Corbett Thermocycler ... .2.2.21RT-PCR of NLV using Corbett Thermocycler .........2.2.22 Detection of amplicons ..2.2.22.1 Electrophoresis of PCR products2.2.22.2 Digoxigenin (DIG) ELISA .. . ..2.2.23 Sensitivity of RT-PCR amplification ....2.2.23.1Competent Escherichia coli (E.coli) strain DH5a2.2.23 .2 Plasmid vectors2.2.23.3 Cloning of the 205bp NLV oDNA products into plasmid DNA ..2.2.23.4 DNA ligation2.2.23.5 Transformation of plasmid into bacteria2.2.23.6 Selection of colonies .....2.2.23.7 confirmation of 205bp NLV cDNA in recombinant plasmid DNA .,.........33.........33...... ...34.........34.........35,........353636....37....37. . ..38. . ..38, ....38....40.....40....40.....412.2.23.8 In vitro transcription2.2.23.9 Quality and quantity of RNA2.2.24 Purification of DNA for sequencing ..4t4l41.42.42.43.4344.44452.2.25 Sequencing analysis ......lll2.2.26 Restriction enzyme digestions .2.2.27 Phenol extraction and ethanol precipitation of nucleic acids.2.2.28 List of suppliers..47..47483.1 Introduction..........CHAPTER 3. Development of a NLV (camberwell) Enryme rmmunoassay3.1.1 Aims3.1.2 Baculovirus expression system . .. . .3.1.3 Baculovirus expression of NLV virus-like particles (VLPs)Materials and Methods .......... ..........51RecombinantBaculoviruscontainingORF2ofCAMandantiserafordetection............5lVerification of CAM ORF2 oDNA in clone obtained from Monash University . . . . . . . . ....51Expression of CAM VLPs 52Time course experiments to determine optimal day of harvest of CAM VLPs anddistribution of these VLPs (cell or supernatant)..........53..........53. ..... .....53Production of Recombinant CAM VLPs in Cell FactoriesPurification of CAM VLPs.....3.2.103.2.t|3.2.12Quantitation of purified CAM VLPs. .....54Production of anti-CAM serum in rabbits and guinea pig .. .......54Immunoreactivity of purif,red CAM VLPs to CAM VLP antisera produced in rabbits andguineapigs.. ....55Dilution of purified recombinant Camberwell capsid protein in I\Yo faecal suspension..55EIA to detect CAM antigen ........55Determination of EIA cutoff value...... .... . ...5649.50.s0.503.23.2.13.2.23.2.33.2.43.2.53.2.63.2.73.2.83.2.93.3 Results... ...............573 .3 . 1 Verification of CAM ORF2 sequence in recombinant Baculovirus. . . . . .573.3.2 Expression of recombinant CAM VLPs 573.3.3 Purification of CAM VLPs . . . . .IY573.3.4 Density of CAM VLPs in Caesium chloride3.3.5 Electron microscopy4.L2 Problems associated with NLV RT-PCR4.1.3 Aims...4.1.4 TitanrM One Tube RT-PCR System (Boehringer Mannheim)3.3.6 Immunoreactivity of purified CAM VLPs to CAM VLP antisera produced in rabbits andguinea pigs... .. . .. 593.3.7 Optimization of Cam VLP antisera for CAM EIA ........593.3.8 Sensitivity of EIA 593.3.9 Specificity of CAM EIA..60CHAPTER 4. Development of RT-PCR for detection of NLV RNA4.1.1 Reverse transcription polymerase chain reaction (RT-PCR)3.4 Discussion .........604.1 Introduction..... .......634.L5 RNA extraction methods .....4.2 Materials and Methods .......... ..........674.2.1 Comparison of in-house RNA extraction methods from 4549 tissue culture cells andfaecal suspensions .......684.2.2 Development of RT-PCR for poliovirus, rotavirus and NLV .....674.2.3 Sensitivity of RT-PCR in terms of poliovirus plaque forming unit (pfu)4.2.4 Sensitivity of RT-PCR as judged by end point titration of rotavirus . . 684.2.5 Testing of routine specimens for rotaviral RNA by RT-PCR .....694.2.6 Sensitivity of amplif,rcation of NLV RNA and oDNA ......694.2.6.1 Determination of level of sensitivity of Titan RT-PCR in terms of copy numbers . . ..694.2.6.2 DeterminationoflevelofsensitivityofTitanPCRwithouttheRTstep ........704 .2.6 .3 Determination of level of sensitivity of PCR using Taq polymerase . . . . ..7 04.34.3.14.3.24.3.34.3.44.3.54.3.64.3.74.3.84.3.94.3.t04.3.trResultsExtraction of total RNA from cells and faecal specimensDNaseI treatment of RNA used for amplification ........,RT-PCR of poliovirus RNASensitivity of PCR for DNA alone ....76.76Sensitivity of RT-PCR in terms of poliovirus plaque forming units 73RT-PCR of rotavirus RNA 73Routine Specimens tested for rotaviral RNA by RT-PCR 74Sensitivity of RT-PCR as judged by endpoint titration of rotavirus faecal samples . . . . . . .7 4Sensitivity of RT-PCR as judged by endpoint titration of rotavirus in cell culture fluid ..75Extraction of NLV RNA and optimizationof NLV RT-PCR .... 75Determination of the sensitivity of Titan RT-PCR4.4 I)iscussionCHAPTER 5. Testing for NLV faecal samples by CAM EIA and RT-PCR775.15.1.15.25.2.r5.2.25.2.3Aims 81Materials and Methods .......... ..........81Gastroenteritis outbreak specimens in Adelaide (August to Decemb er, 1996) . . . . ..g 1Further optimization of NLV RT-PCR to increase sensitivityComparison of commercial RNA extraction methods to increase sensitivity ofRT-PCR82825.2.4 Concentration of NLV by Polyetþlene glycol MV/ 8000 precipitation (PEG ppt)and Immuno precipitation (Immuno ppt)5.2.4.I Concentration of NLV in 41 specimens (Adelaide NLV outbreaþ which werenegative by RT-PCR5.2.5 Controls for RT-PCR and Nested PCR .. .5.2.6 Nested PCR5.2.6.1 Nested PCR of NLV using Corbett thermocycler8383848484,845.2.6.2 Sensitivity of nested PCR .vl5.2.6.3 Further amplifications by nested PCR of specimens which were negative for NLVRNA by RT-PCR5.2.7 NLV nucleotide sequences of specimens from the 1996 Adelaide gastroenteritisoutbreak 865.3 Results ..............875.3.1 Detection of NLV by CAM EIA in stored specimens from 1996 Adelaidegastroenteritis outbreak 875.3.2 Detection of NLV RNA by RT-PCR in the outbreak specimens 875.3.3 Attempts to increase sensitivity of RT-PCR . . . . . ... .. gg5.3.3.1 Comparison of two commercial RNA extraction methods to the Boom method toincrease sensitivity of RT-PCR 885.3.3.2 Comparison of two different concentration methods to increase sensitivity of NLVRT-PCR - precipitation by specific antibodies (Immuno ppt) or with polyethyleneglycol (PEG ppÐ ....... 885.3.3.3 Repeat of RT-PCR on PEG ppt and Qiagen extracted RNA of the 41 first roundPCRNLV negative specimens ........895.3.3.4 Nested PCR . 905.3.4 Summary of CAM EIA, RT-PCR and nested PCR results on the 65 Adelaide85outbreak specimens5.3.5 Inhibition of RT-PCR Enzymes5.3.6 Sequencing5.3.6.1 Sequencingof 205bpRT-PCRand lOgbpnestedPCRproduct.... .........925.3.6.2 Sequencing of 1443bp nucleotides from the ORFl region . . . 935.3.7 Testing of routine specimens sent to the IMVS for gastroenteritis investigation byCAM EIA and NLV RT-PCR ..5.4 Discussion 95CHAPTER 6. General discussion and future work....... ....102929I9294vlrAbstractNLVs (Norwalk-like viruses) are members of the caliciviridae family and are common causes ofepidemic gastroenteritis characterized by short (24-48 hours) incubation and duration. They arecurrently under diagnosed because of limited availability of convenient diagnostic tests e.g. cellculture systems are not available for this group of viruses. Recent advances including the cloningand sequencing of several strains of this virus has enabled the detection of NLVs in faecalsamples. This study describes the development of an RT-PCR and aî eîzlme immunoassay forthe detection of an Australian NLV group 2b virus (Camberwell) and related viruses and theirapplication to clinical samples.A recombinant baculovirus containing the complete Camberwell capsid protein was provided byAssoc. Prof. Peter Wright (Monash University). Virus-like particles (VLPs) produced by thisconstruct were purified and used to produce hyperimmune rabbit and guinea pig antisera for usein an antigen capture enzyme immunoassay (EIA) with a sensitivity of 0.1ng of purified VLPsNo cross-reaction was observed when a range of gastroenteritis causing bacteria and viruses wereused to test the CAM EIA. When applied to a clinical outbreak 31165 (48%) specimens werefound to be EIA positive. In contrast 1000 consecutive routine specimens submitted to ourlaboratory were EIA negative.A one tube RT-PCR was also developed for NLV Group 2 (Camberwell-like) viruses. Due to thenon-availability of known NLV positive specimens in preliminary experiments, stock cultures ofpoliovirus and rotavirus positive (by EIA) faecal samples were used for initial optimization of theRT-PCR. The sensitivity of RT-PCR for the three viruses was determined to be 16 pfu forvlllpoliovirus, ca 2.5 x 102 viral particles/ml for rotavirus and 2 x l}a copies of in vitro transcribedNLV RNA. However, when the outbreak faeces were tested only 24 of the 65 (37%) outbreakspecimens were positive on first round NLV RT-PCR.Further optimization including an improved faecal RNA extraction method (Qiagen RNeasyPlant RNA kit) and concentration of NLVs by Immuno and PEG precipitation methods allimproved the sensitivity of the first round RT-PCR but failed to find any additional positiveoutbreak specimens. However, a nested PCR was found to increase the sensitivity of NLV RT-PCR by 100 fold in vitro and found an additional 6 EIA positive specimens to be nested PCRpositive. None of the EIA negative specimens became positive. Sequencing of all RT-PCR andnested PCR products showed that they were all identical and showed 960/o homology to theCamberwell strain.This study suggests that NLV antigen capture EIA and RT-PCR assays may be of similarsensitivity and can provide complementary information during the investigation of outbreaks. Thedevelopment of EIAs for representative strains of other NLV (and Sapporo Like Viruses)genogroups may therefore permit the production of clinically useful diagnostic EIAs. Furtherdevelopment of the RT-PCR assays will require the use of consensus primers for each of theNLV groups 1,2 and SLV viruses.1XDeclørationThis thesis contalns no material which has been accepted for the award of any other degree ordiploma and to the best of my knowledge and belief, the thesis contains no material previouslypublished or written by another person, except where due reference is made in the text. Inaccordance with the University of Adelaide regulations, I give my consent to this thesis beingmade available for photocopying and loan if accepted for the award of the degree.Satiya WatixAcknowledegmentsMany thanks to Dr Tuck Weng Kok and Dr Geoff Higgins for giving me the opportunity to studyin the Viral Detection Laboratory and for their supervision.I would also like to acknowledge the collaboration and helpful discussions with Assoc. Prof.Peter 'Wright (Monash University, Melbourne) and his staff as well as the provision of the CAMclones, antisera, positive Camberwell specimens and Australian NLV isolate sequences.Thank you to all the team of the Virus Detection Laboratory for their kindness, help andavailability. Special thanks to Adriana Sharley for the graphic work, Sonia Schepetiuk for adviceand discussions, Lyn Payne and Sharon Blake for proof-reading and all the past and present staffof VDL for the entertainment during the long working hours.Special gratitude to fellow peers, John Karlis, Budimen Bela and Adam Davis for the technicaladvice and continual assistance during the course of my degree. Thanks also to Helen Hockingand Julie Calvert for assistance in obtaining reagents and also encouragement and friendshipduring my studies.Thank you to Pete Grant for the photographic work and Rodney Ratcliff for help with alignmentofsequences.Finally I would like to thank my family for all their support and in particular my sister Sanyogitafor the generous donation of a computer.xlcae.g.i.e.OCbpCAMcDNAdATPDDWdCTPdGTPDIGDNADNaseIdNTPDTTdTTPEIAEDTAEMFBSg (units)IEMkbKDALBI'rgplpMmgAbbreviafionsapproximatelyfor examplethat isgreater thandegrees Celsiusbase pairCamberwellcomplementary DNA2' -deoxyadenosine 5' -triphosphatedouble distilled water2' -deoxy cytidine 5' -triphosphate2' -deoxyguanosine 5' -triphosphatedigoxigenindeoxyribonucleic aciddeoxyribonuclease-l2' -deoxynucleoside 5' -triphosphatedithiothreitol2' -deoxythymidine 5' -triphosphateenzyme immunoassayetþlenediaminetetraacetic acidelectron microscopyfetal bovine serumgravity forceImmune electron microscopykilobase pairkilodaltonLuria-Bertani mediummicrogrammicrolitremicromole per litre (micromolar)milligramxllmin(s)mlmMMOPSMV/ngnmNLVNVODORFPAGEPBSPCRPEGp.i.ppmpptRIARNARNase ArNTPrRNArpmRTSDSSLVSSCTCAUTPVLPsX-galminute(s)millilitremillimole per litre (millimolar)3 - fN-morpholinolpropanesulfonic acidmolecular weightnanogramnanometreNorwalk-like virusNorwalk virusoptical densityopen reading framepolyacrylamide gel electrophoresisplasmid Bluescript (SK)polymerase chain reactionpolyethylene glycolpost infectionparts per millionprecipitationradioimmunoassayribonucleic acidribonuclease Aribonucleoside 5' -tripho sphateribosomal RNArevolutions per minutereverse transcriptasesodium dodecyl sulfateSapporo-like virusstandard saline citratetrichloroacetic aciduridine 5'-triphosphateviruslike particles5 -bromo-4-chloro-3 -indolyl- B-D- galactosidexlu

Rapid detection of Norwalk-like viruses (NLVs)

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Rapid detection of Norwalk-like viruses (NLVs)

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