Ali, Abdul Manaf

Ideris, Aini

Omar, Abdul Rahman

Sipat, Abdullah

Tan, Wen Siang

Yusoff, Khatijah

Universiti Putra Malaysia Institutional Repository

Agricultural SCiencesMolecular Characterization of Newcastle Disease VirusKhatiJah Yueoff, Alnllderifi, AI7c1ulManaf All, AI7c1ullahSipat. Wen Slang Tan, Al7c:1ulRahman OmarDepartment of Biochemistryand Microbiology,FacultyScience and Environmental StudiesUniversiti Putra Malaysia43400 UPM, Serdang, SelangorMalaysiaTelephoneNumber of Corresponding Author: 03-89466692E-mail cllCorrespondingAuthor:khatijah@ftas.upm.edu.myKey 'Words: Newcastle disease virus, molecular biology, diagnostics, vaccines, biotechnologyIntroductionNOV is a major pathogen in poultry. Control of the virus is mainly through vaccination. Any effort to develop an effectivevaccine against the disease would require a detailed understanding of their molecular biology and mechanism of infection Theglycoprotein genes, haemagglutinin-neuraminidase (HN) and fusion (F) are immunogenic and are involved in viralpathogenesis. In addition, the large (L) polymerase protein as well as the phosphoprotein (P) and nuceloprotein (P) have beenshown to be involved in viral transcription. Their molecular characterization .and interactions would benefit in thedevelopment of a subunit vaccine against the virus. This project has been extended from phase I of RM7. We havesuccessfully cloned the HN and F genes into baculovirus vector and transfected into insect tissue cultures. There wasexpression of the recombinant proteins which are potential candidates for subunit vaccine development. This project wasundertaken to continue such studies. Specific regions of the NOV strain AF2240 genome were amplified and sequencedbefore being studied in detail. In addition, various peptides from biopanning experiments against the virus will be used todevelop a diagnostic assay for NOV and its potential as anti-viral peptide was evaluated.Materials and MethodsViruses in the study included several NOV field isolates and reference strains, including the strain AF2240. The viruses weregrown, purified and their genomic RNAs were extracted. Primers were constructed to amplify specific regions in the genomeby RT-PCR. The amplified products were analysed by restriction enzyme analysis and sequencing. The NP, P, M, F, HN andparts of the L genes of NOV strain AF2240 were cloned into Escherichia coli and sequenced. Diagnostic kits for NOVidentification were developed using (i) RT nested PCR-ELISA assay and (ii) a recombinant phage which was isolated frombiopanning experiments. The HN and F genes were subcloned into the Baculovirus expression system. All of the expressedgene products were then studied in detail. Specific peptides sequences which bind to NOV were determined throughbiopanning with a phage display library. Various chimaeras and mutants are currently being constructed and their biologicalfunctions are determined.Results and DiscussionDiagnosis of NDV. Various NDV isolates could be distinguished by sequence analysis of the cleavage site of the F proteingene. A nested RT-PCR ELISA diagnostic kit was developed for the determination and identification of NOV. This kit ismore sensitive and specific then the current serological tests. We have filed a patent in Malaysia. The NDV kit has beenincluded as a finalist in the FEER-HP "Young Inventors Awards" and won a Silver Medal in the Expo Science & Technology2001 organised by the Ministry of Science, Technology and the Environment of Malaysia and a consolation prize in the UPMInventors and Innovations Award 2000 competition.Sequence determinations of the various genes of NDV and their expressions. The sequences of all the genes of velogenic-viscerotropic NOV strain AF2240, except the L gene, have been completely determined and each given EMBUGenBankdatabase accession numbers. The HN, F, NP, P gene sequences have been published and the remaining gene sequences are inthe process of being published. The heat stability of the HN protein was studied. The NP and P gene sequences have beenfiled for patents in Malaysia and US. The NP protein was expressed in E. coli as ring and herringbone-like structures. Thesestructures were shown to be able to carry extra peptide fragments at the C-terminal end and can act as antigenic carriers. Thishas been filed for patent.Cloning and expression of HN and F genes of NDV. The recombinant HN protein has been shown to be immunogenic. TheHN genes of V4(UPM), V4(QUE) and AF2240 have also been cloned into Baculovirus and the expressed recombinantproteins were studied for the heat stability. In addition, the HN and F genes have also been cloned into Pichia pas/oris andeukaryotic expression vectors for the development of alternative recombinant vaccines. Some positive results have beenobtained for these recombinant proteins expressed as DNA vaccines. The expression of these proteins in E. coli are beingstudied in detail.Biopanning of NDV proteins through use 0/ a phage display library. Various kinds of tests have been developed to distinguishthe different strains of NOV. Unfortunately, these tests are often laboratory specific, expensive or tedious and they were notable to distinguish between the vaccine strains (mesogenic and lentogenic strains) and the field isolates (velogenic strains)which are the etiologic agents for the disease. We have developed a novel peptide (Malaysian Patent Pending PI 20013687)203UPM Research Report 1997-2000, Vol II,Section 2-Extended Abstractsthat can distinguish between vaccinated chickens and those that were infected with the field isolates ofNDV. It was found thatthis form of NDV typing was not previously reported, and furthermore it is the first invention that can distinguish thevelogenic from the mesogenic strains. This invention is therefore useful as a routine diagnostic test to locate the source of anepidemic. In addition, this peptide is able to inhibit the replication of the virus and may be used as an antiviral drug. Ionaddition, two anti-NDV peptides have been constructed and shown to inhibit NDV replication.NDV proteins interactionsWork on the protein-protein interactions of the recombinant proteins are being carried out todetermine the mechanism(s) of virus-cell interactions. Chimaeras comprising various NDV protein segments with the NPprotein have been constructed and their immunogenicity tested. These results are published and a patent has been filed inMalaysia and the US.ConclusionsDiagnostic test for NDV has been developed. The complete sequences of the NP, P, M, F and HN genes of NDV strainAF2240 were determined and given EMBUGenBank database accession numbers. The F and HN genes of NDV strainAF2240 were cloned and expressed in the Baculovirus, E. coli, yeast and other expression systems. Anti-NDV pep tides havebeen developed. The receptor and protein-protein interactions of the NDV proteins are being studied in detail. The NP proteincan be expressed as a ring structure in E. coli and may be suitable as a carrier in future drug delivery system.Benefits from the studyDevelopment of diagnostic kits and subunit vaccines for NDV; patents for the PCR-ELISA kit and NP and P gene sequences;and training of molecular biologists.Patent(s), if applicableNucleotide sequences of the nucleocapsid (NP) and phosphoprotein (P) genes of a Malaysian velogenie Newcastle diseasevirus strain AF2240 and the production of the NP and P proteins in Escherichia coli (Malaysian Patent PendingPI 20004837).US Patent being filed (App. No. 09/970,851)Detection of Newcastle disease virus (Malaysian Patent Pending: PI 20005526)Peptides that inhibit the propagation of Newcastle disease virus (Malaysian Patent PendingPI 20013687)Nucleocapsid protein of Newcastle disease virus as a carrier for immunogens (Malaysian Patent Pending: P120021709)Stage of Commercialization, if applicable:NilProject Publications in Refereed JournalsOng, H.KA, Ali, A.M., Omar, A.R., Tan, W.S. and Yusoff, K. 1999. N-linked glycosylatedHN protein of NOV strain AF2240 expressed inBaculovirus-infectedSt9 cells. Journal of Biochemistry,Molecular Biologyand Biophysics3: 147-15I.Ong, H.K.A., Ali, A,M., Omar, A.R. and Yusoff, K. 1999. Cloning and the steady-state expression of the HN gene from Newcastle diseasevirus strain AF2240 in St9 insect cells. Cytotechnology32: 243-25 I.Kho, C.L, Mohd. Azmi, M.L., Arshad, S.S. and Yusoff, K. 2000. Performance of an RT nested PCR ELISA for detection of Newcastledisease virus. Journal of VirologicalMethods 86: 71-83.Salih, 0., Omar, A.R. Ali, A.M. and Yusoff, K. 2000. Nucleotide sequence analysis of the F protein gene of a Malaysian velogenic NOVstrain AF2240. Journal of Biochemistry,Molecular Biologyand Biophysics4: 5 I-57.Tang, Y.K., Ong, H.KA, Tan W.S. and Yusoff, K. 2000. Heat stability assay on the haemagglutinin (HA) and neuraminidase (NA) activitiesof Newcastle disease virus (NOV) strains AF2240, V4UPM and V4QUE. MalaysianJournal of Biochemistryand MolecularBiology5: 79.(Abstract only)Salih, 0., Omar, A.R. Ali, A.M. and Yusoff, K. 2001. Analysis ofa recombinant Baculovirus expressing the fusion glycoproteingene of theMalaysian velogenic-viscerotropic Newcastle disease virus strain AF2240. Journal of Biochemistry Molecular Biology and Biophysics 5:67-74.Kho, C.L., Tan, W.S. and Yusoff, K. 2001. Sequence analysis of the nucleoprotein ofa Malaysian heat resistant NOV straineomparisonwithother members of Paramyxoviridae. Journal of BiochemistryMolecular Biologyand Biophysics5463471.Yusoff, K. and Tan, W.S. 200 I. NOV:macromoleculaes and opportunities Avian Pathology30: 439455.Kho, C.L., Tan, W.S. and Yusoff, K. 2001. Production of the nucleocapsid protein of Newcastle disease virus in Escherichia coli and itsassembly into ring- and nu~leocapsid-likeparticles. Journal of Microbiology39: 293-299.Kho,CL, Tan, W.S. and Yusoff, K. 2002. Cloning and expression of the phosphoproteingene of Newcastledisease virus in Escherichia coli.Journal of Molecular Biology,Biochemistryand Biophysics 61 17-121.204Agricultural SciencesRamanujarn, P., Tan, W.S., Nathan, S. and YusolT, K. 2002. Novel peptides that inhibit the replication of Newcastle disease virus. Archivesof Virology 147: 981-993.Rabu, A., Tan, W.S., Kho, C.L. Omar, A.R. and YusofT, K. 2002. Potential of the nucleocapsid protein of Newcastle disease virus as a carrierfor antigenic polypeptides. Acta Virologica 46: 211-217.Kho, C.L., Tan, W.S., Tey, RT. and YusolT, K. 2003. Identification of nucleocapsid protein self-assembly domain of Newcastle disease virus.Journal of General Virology 842163-2168.Loke, C.F., Raha, A.R., Omar, A.R. and YusolT, K. 2003. Protection against Newcastle disease virus by DNA vaccines. Vaccine (submitted)Kho, C.L., Tan, W.S., Tey, B.T. and YusolT, K. 2003. NOV nucleocapsid: self assembly and length determination domains. Archives ofVirology (revised).Loke, C.F., Raha, A.R., Omar, A.R. and YusolT, K. 2003. Enhanced expression of F and HN Proteins of NOV in a mammalian expressionsystem by alteration of their Kozak Sequences. Plasmid (submitted)Loke, C.F., Raha, A.R., Omar, A.R. and YusofT, K. 2003. Immunogenicity of chickens immunized with plasmids encoding the F glycoproteinsof Newcastle disease virus. Immunopathology (submitted)Ramanujarn, P., Tan, W.S., Nathan, S. and YusofT, K. 2003. NOV-phage interaction: pathotyping and affinity constants. Biotechniques(submitted)Project Publications in Conference ProceedingsOng, H.K.A., Ali, A.M., Omar, A.R., Ideris, A., Poh, Y.M. and YusofT, K. 1999. Electron microscopy and immunological studies of therecombinant baculovirus expressing the HN gene of NOV strain At2240. In: Proceedings of the 11111National Biotechnology Seminar: 107-109.Chang, L.Y., Raha, A.R., Sipat, A.B. and YusofT, K. 1999. Cloning ofa thermostable xylanase gene from Bacillus coagulans ST-6 and an Mgene from Newcastle disease virus into Lactococcal expression vector pMG236e. In: Proceedings of the 11111National BiotechnologySeminar: 289- 290.Ramanujam, P., Tan, W.S., Nathan, S. and YusofT, K. 1999. A heptapeptide containing four hydroxyl groups interacts with Newcastle diseasevirus. In91l1 Scientific Meeting of Malaysian Society for Molecular Biology and Biotechnology. pp. 20.Wong, S.K., Tan, W.S., Tan, C.S. and Yusoff, K. 1999. Cloning of the envelope glycoprotein genes (HN and F) of Newcastle disease virusstrain AF2240 into Pichia pastoris. In: 9111Scientific Meeting of Malaysian Society for Molecular Biology and Biotechnology. pp. 45.Ramanujarn, P., Tan, W.S., Nathan, S. and YusofT, K. 1999. Selection of peptide ligands that interact with Newcastle disease virus. In: 3"'.UNESCO National Workshop on Promotion of Microbiology in Malaysia. pp. 38.Tan, W.S., Aziz, H.A., Tamanujam, P. and Yusoff, K. 1999. Determination of the binding affinity of a fusion phage that interacts withNewcastle disease virus. In: 22'" Microbiology Symposium, pp 102.Wong S.K., Tan W.S., Tan, C.S. and YusolT, K. 2000. In vitro transcription and translation of truncated hemagglutinin-neuraminidase (HN)gene from a local Newcastle disease virus (NOV) isolate. In: 10111Scientific Meeting of Malaysian Society for Molecular Biology andBiotechnology: 42.Kho, C.L., Tarn, W.S. and YusolT, K. 2000. Cloning and expression ofNP protein of Newcastle disease virus strain AF2240 in Escherichiacoli. In: 10111Scientific Meeting of Malaysian Society for Molecular Biology and Biotechnology: 42.Loke, C.F., Raha, A.R., Omar, A.R. and Yusoff, K. 2000. Cloning and expression of the fusion (F) and haemagglutinin-neurminidase (HN)genes from Newcastle disease virus (NOV) strain AF2240 in a mammalian expression system. In: 10111Scientific Meeting of MalaysianSociety for Molecular Biology and Biotechnology: 43.Rabu, A., Kho, C.L., Tan, W.S. and YusofT, K. 2000. Fusion of the immunogenic peptides of haemagglutinin-neuraminidase (HN)glycoprotein to the nucleocapsid (NP) protein of NOV. In: 10<1>Scientific Meeting of Malaysian Society for Molecular Biology andBiotechnology: 43.Ramanujarn, P., Tan, W.S., Nathan, S. and YusofT, K. 2000. Biophysical properties of'pbages that interact with Newcastle disease virus. In:10111Scientific Meeting of Malaysian Society for Molecular Biology and Biotechnology: 44.Tang, Y.K., Ong, H.K.A., Tan, W.S. and YusolT, K. 2000. Heat stability assay on the haemagglutinin (HA) and neuraminidase (NA) activitiesof Newcastle disease virus (NOV) strains AF2240, V4UPM and V4QUE. In: Proceedings of Annual Conference of the Malaysian Societyfor Biochemistry and Molecular Biology 25: 34 .205UPM Research Report 1997-2000, Vol II, Section 2-Extended AbstractsRamanujam, P., Tan, W.S., Nathan, S. and YusofT, K. 2000. Inhibition of Newcastle disease virus propagation with synthetic peptides. In:Proceedings of the 12'" National Biotechnology Seminar: 23-24.Rabu, A., Kho, C.L, Tan, W.S. and YusolT, K. 2000. Construction of chimeric proteins of Newcastle disease virus (NDV). In: Proceedingsof the 12'" National Biotechnology Seminar: 52.Wong, S.K., Tan, W.S., Tan, C.S. and YusofT, K. 2000. Salt-induced expression of the extracellular domains of the envelope proteins ofNewcastle disease virus in E. coli. In: Proceedings of the 12th National Biotechnology Seminar53-54.Kho, c.L., Tan, W.S. and YusofT, K. 2000. Cloning and expression of the phosphoprotein (P) of Newcastle disease virus strain AF2240 inEscherichia coli system. In: Proceedings of the 3011.Symposium of the Malaysian Society for Microbiology79-80.Chiew Ling Kho, Wen Siang Tan and Khatijah YusofT 2001. Newcastle disease virus nucleocapsid protein expressed in Escherichia coliassembles into ring-like and herringbone-like structures. In: II th Scientific Meeting of Malaysian Society for Molecular Biology andBiotechnology: 34.Ramanujam, P., Tan, W.S., Nathan, S. & YusolT, K. 2001. Identification of the binding site of a synthetic peptide that interacts withNewcastle disease virus. In: II th Scientific Meeting of Malaysian Society for Molecular Biology and Biotechnology: 38.Tang, Y.K., Ong, H.K. A., Tan, W.S. and YusofT, K. 2001. Cloning and expression of the haemagglutinin-neuraminidase (HN) gene ofavirulent Newcastle disease virus strains V4QUE and V4UPM in baculovirus expression system. In: II th Scientific Meeting of MalaysianSociety for Molecular Biology and Biotechnology: 43.Wong, S.K., Tan, W.S., Tan, C.S., Omar, A.R. and YusofT, K. 2001. Immunogenic properties of the recombinant HN and F proteins ofNewcastle disease virus produced in Escherichia coli. In: II th Scientific Meeting of Malaysian Society for Molecular Biology andBiotechnology: 43.Rabu, A., Tan, W.S., Kho, C.L., Omar, A.R. and Yusolf, K. 200 I. Immunogenic properties of the nucleocapsid (NP) fusion proteins ofNewcastle disease virus. In: Proceedings of the 31dNational Biotechnology Seminar: 4748.Kho, C.L., Tan, W.S. and YusofT, K.2001. Mutagenesis of the nucleocapsid protein of Newcastle disease virus. In: Proceedings of the 3rdNational Biotechnology Seminar: 51-53.Ramanujam, P., Tan, W.S., Nathan, S. and YusofT, K. 2001. Detection of Newcastle disease virus pathotypes with a recombinant phage. In:Proceedings of the 3rdNationai Biotechnology Seminar: 70-71.Tan, S.W., Aini, I., Omar, A.R., Yusolf, K. and Tan, W.S. 2001. Phylogenetic analysis of recently isolated Newcastle disease virus (NDV).In: Proceedings of the 3 rdNational Biotechnology Seminar: 79-8 I.Wong, S.K., Tan, W.S., Tan, C.S., Omar, A.R. and Yusolf, K. 2001. Cloning of the genes encoding envelope glycoproteins of Newcastledisease virus (NDV) strain AF2240 into yeast expression vector and bacterial secretion vector. Proceedings of the 3n1 NationalBiotechnology Seminar: 83-85.Graduate ResearchName ofGraduateResearch Topic FieldExpertiseof DegreeAwardedGraduation Year14 Kho ChiewLingNucleocapsid (NP) and Phospho-(P)proteins of Newcastle disease virus:Identification of regions on NP thatform particles and interact with PMolecularBiologyPhD 2003140meimaSalihSequencing, cloning and expression ofthe Newcastle disease virus fusionprotein gene of strain AF2240MolecularBiologyPhD 199915 Alan OngHan KiatCloning and expression of thehaemagglutinin-neuraminidase (HN)gene from Newcastyle disease virus(NDV) strain AF2240 in Baculovirus(AcNPV)MolecularBiologyPhD 199915 WongingSing Cloning and expression of the genesencoding the envelope proteins ofNewcastle disease virusMolecularBiologyPhD 2003206Agricultural Sciences15 PriadarishiniRamanujamPeptide ligands that interact withNewcastle disease virus: selection,characterization and applicationsMolecularBiologyPhD 200315 LokeFungChui Towards the development of DNA Molecularvaccine against Newcastle disease BiologyPhD 2002virusSequence determination of the large LKusumanin protein gene of Newcastle diseasevirus strain AF2240______ g~tLa=s~~~~~--~~--~~----~----~~~------~~-------------Tang Yik Thermostability of the recombinant Molecular MSKiong haemagglutinin-neuraminidase Biologyglycoprotein of Newcastle disease15 Eni MolecularBiologyMS 2003213 2003virus214. Amir Rabu Nucleocapsid protein of Newcastledisease virus as an antigenic carrierMolecularBiologyMS 2002215. Chang Li Yen Cloning and expression of thexylanase gene from Bacilluscoagu/ans and the M gene ofNewcastle disease virus InLactococcus lactisMolecularBiologyMS 2001216. Kho Chiew Development of an RT Nested PCR- MolecularLing ELISA diagnostic test for the Biologydetection of NDVMS 1999217 Siti Fatimah Sequence determination of the matrixPutery bt gene in NDV strain AF2240JemainMolecularBiologyMS 1998IRPA Project number: 01-02-04-0107UPM Research Cluster: BAB(An update of the abstract published in UPM Research Report 1997-2000.)207

Molecular characterization of Newcastle disease virus

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Molecular characterization of Newcastle disease virus

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