Characterization and immunological studies of fish nervous necrosis virus

CHARACTERIZATION AND IMMUNOLOGICAL STUDIES OF FISH NERVOUS NECROSIS VIRUS ASHOK HEGDE NATIONAL UNIVERSITY OF SINGAPORE 2003 CHARACTERIZATION AND IMMUNOLOGICAL STUDIES OF FISH NERVOUS NECROSIS VIRUS BY ASHOK HEGDE (MFSc) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2003 ACKNOWLEDGEMENTS First of all, I would like to express my deep sense of gratitude and appreciation to my PhD supervisor, Associate Professorial Fellow Sin Yoke Min and co-supervisor Professor LamToong Jin for their kind guidance, consistent encouragement and critical comments throughout the course of my PhD programme. I sincerely thank my Ph.D. committee members, Associate Professor Wong Sek Man and Dr. Chen Changlin for their advice and timely help during the course of my study. I admire necessary help provided by Dr. Qin Qiwei, in my research work. My lab mates, Jeffry Seng Eng Kuan, Lam Siew Hong, Huang Weidong, Lim Sze Yun, Jeanne, Christine Fock and Christina Liew have been very friendly and helpful during my research work. Lab technicians, Mrs. Wei Fong and Mr. Loh Mun Seng have been very helpful in providing necessary help. I extend my heartfelt thanks to all of them. I appreciate the help of Madam Loy Gek Luan in taking excellent electron micrograph pictures. Mrs. Ngoh and Dr. Chang Siew Fong of Agricultural and Veterinary Authority of Singapore have been very helpful in providing virus and cells whenever necessary. I remain grateful to them. I acknowledge the research collaboration with Singapore Fish Breeding and Immunization Centre, Teo Way Yong (Pte) Ltd, Singapore. My friends, Srinivasa Rao, Narayana Murthy, Sudha P.M, Sasi Nayar, Rahul Sen, Eeshwarappa Sridhara, Satish, Venugopal, Somashekhar Gouri, Bhinu, Adaikalam, Dr. Adrian Elangovan, Dr. Sugumar, Dr. Byrappa Venkatesh, Dr. Satish R.L and Dr. Konda Reddy have been very helpful during the years of my stay here. Affection of my parents, brother, sisters, uncles and aunts has been the pillar of inspiration for my success. I greatly acknowledge their moral support. I would like to thank all my past and present I teachers for constantly inspiring me to study. Last, but not the least, I acknowledge the Research Scholarship provided by the National University of Singapore to my PhD degree. II TABLE OF CONTENTS TITLE ACKNOWLEDGEMENTS TABLE OF CONTENTS I III LIST OF TABLES X LIST OF FIGURES XI LIST OF ABBREVIATIONS XIII SUMMARY GENERAL INTRODUCTION I. REVIEW OF LITERATURE I.1 Fish viral diseases I.2 Nodaviridae I.2.1 Alphanodavirus I.2.1.1 Virion properties and molecular biology 14 1.2.1.2 Host range 17 I.2.2 Betanodavirus 18 1.2.2.1 Background 18 I.2.2.2 Members of the family 19 I.2.2.3 Host range 20 I.2.2.4 Clinical signs 20 I.2.2.5 Experimental infection experiments 24 I.2.2.6 Histopathology 26 I.2.2.7 Ultrastructural studies 28 III I.2.2.8 Diagnosis 28 I.2.2.8.1 Molecular diagnostics 28 I.2.2.8.2 Immununological detection 29 I.2.2.8.3 Histopathology 30 I.2.2.8.4 Electron microscopy 30 I.2.2.8.5 Cell culture 31 I.2.2.9 Viral structure and composition 32 I.2.2.10 Antigenic similarity 33 I.2.2.11 Structural studies 34 I.2.2.12 Epidemiology 34 I.2.2.13 Control 36 CHAPTER ONE CHARACTERIZATION AND PATHOGENICITY STUDIES OF A NERVOUS NECROSIS VIRUS ISOLATED FROM GROUPER, EPINEPHELUS TAUVINA IN SINGAPORE 38 Abstract 38 1.1 Introduction 39 1.2 Materials and Methods 40 1.2.1 Cell culture 40 1.2.2 Viral isolate 40 1.2.3 Tissue culture infective dose 50 (TCID 50 ) 41 1.2.4 Purification of virus 41 IV 1.2.5 Electron microscopy 42 1.2.6 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) 42 1.2.7 N-terminal sequence analysis 43 1.2.8 Polymerase chain reaction (PCR) 43 1.2.9 Polyclonal antisera against the purified virus and coat protein 44 1.2.10 Western blotting 45 1.2.11 In vivo infection experiment 45 1.2.12 Histopathology of infected larvae 46 1.2.13 Isolation of ETNNV from artificially-infected sea bass larvae 46 1.3 Results 46 1.3.1 Susceptibility of cell line to virus infection 46 1.3.2 Electron microscopy 47 1.3.3 SDS-PAGE, N-terminal sequence analysis 47 1.3.4 Polymerase chain amplification of virus coat protein gene 47 1.3.5 Antibodies and Western blotting 52 1.3.6 Experimental infection 52 1.4 Discussion 56 V CHAPTER TWO DETERMINATION OF GENOME SEQUENCE OF EPINEPHELUS TAUVINA NERVOUS NECROSIS VIRUS AND EXPRESSION OF RECOMBINANT COAT PROTEIN 61 Abstract 61 2.1 Introduction 63 2.2 Materials and Methods 64 2.2.1 RNA extraction and RT-PCR 64 2.2.2 Cloning and sequencing 66 2.2.3 Sequence alignment and phylogenetic analysis 66 2.2.4 Expression and purification of recombinant coat protein in E. coli 2.2.5 67 Polyclonal antisera against the purified recombinant coat protein 68 2.2.6 Western blot detection of recombinant protein 69 2.2.7 Design of specific primers for detection of NNV 69 2.2.8 Detection of ETNNV 70 2.2.9 Sensitivity of PCR 70 VI 2.3 Results 71 2.3.1 Nucleotide and deduced amino acid sequence of ETNNV 71 2.3.2 Comparison with other fish nodaviruses 76 2.3.2.1 Nucleotide sequence 76 2.3.2.1 Deduced amino acid sequence 76 2.3.3 SDS-PAGE analysis and western blotting 77 2.3.4 Detection of ETNNV using conserved primers 77 2.4 Discussion 87 CHAPTER THREE NODAVIRUS INFECTION IN FRESHWATER ORNAMENTAL FISH, GUPPY, POICELIA RETICULATA - COMPARATIVE CHARACTERIZATION AND PATHOGENICITY STUDIES 91 Abstract 91 3.1 Introduction 92 3.2 Materials and Methods 93 3.2.1 Isolation and propagation of virus 93 3.2.2 Determination of TCID 50 94 3.2.3 Viral purification 94 3.2.4 SDS-PAGE and Western Blotting 95 3.2.5 RNA isolation, RT-PCR, cloning and sequencing 95 3.2.6 Sequence analysis 96 VII 3.2.7 Experimental infection of guppy fry 96 3.3 Results 97 3.3.1 Cytopathic effects 97 3.3.2 SDS-PAGE and Western Blotting 97 3.3.3 RT-PCR, sequencing and analysis of coat protein gene 102 3.3.4 Experimental infection 106 3.4 Discussion 106 CHAPTER FOUR EVALUATION OF RECOMBINANT COAT PROTEIN OF NERVOUS NECROSIS VIRUS AS A VACCINE IN FISH 112 Abstract 112 4.1 Introduction 113 4.2 Materials and Methods 114 4.2.1 Formalin inactivation of ETNNV 114 4.2.2 Preparation of recombinant coat protein for immunization 115 4.2.2.1 Preparation of crude bacterial lysate 115 4.2.2.2 Preparation of pure recombinant protein 115 4.2.3 Immunization of gouramy 116 4.2.4 Immunization of guppy 117 4.2.5 In vitro neutralization of ETNNV and GNNV with rabbit antibody 118 VIII Arthur, J.R., Subhasinghe, P. 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Fish Pathol. 35, 95-96. 162 [...]... affecting fish 10 I.2 Important clinical features of viral nervous necrosis virus (VNN) of larval and juvenile fish I.3 21 Geographical and species distribution of clinical nervous necrosis virus (VNN) 22 2.1A Primers used for amplification of RNA1 65 2.1B Primers used for amplification of RNA2 65 3.1 Comparison of nucleotide and amino acid sequences in the open reading frame of different fish nervous necrosis. .. significant fish virus diseases comprise of six RNA virus and five DNA virus diseases The RNA viruses include, Orthomyxoviridae-Infectious salmon anemia virus (ISAV); Rhabdoviridae-Infectious hematopoietic necrosis virus (IHNV), Spring viramia of carp virus (SVCV), Viral haemorrhagic septicaemia virus (VHSV), Nodaviridae -Nervous necrosis virus (NNV), 8 and Birnaviridae-Infectious pancreatic necrosis virus. .. and pathogenicity studies of NNV, and to test the efficacy of recombinant coat protein of NNV as vaccine against two different isolates of fish nervous necrosis virus obtained from marine and freshwater fish in this region There are four major objectives in the present study, viz: 1) To characterize and study the pathogenicity of nervous necrosis virus (ETNNV) isolated from the marine fish, grouper, Epinephelus... diseases of fish are a major threat to cultured fish especially during their early age Fish diseases caused by viruses have been of intensive investigation since 1960s However, developments in study of many of the fish virus diseases have been significantly hampered by lack of cell culture systems, ultrastructural and molecular studies to understand the biology of viral diseases Prevention and control of fish. .. alphanodaviruses that primarily infect insects and the betanodaviruses that infect fish (Ball et al., 2000) I.2.1 Alphanodavirus The genus Alphanodavirus consists of eight virus species, of which seven are accepted by the International Committee on Taxonomy of Viruses (ICTV) and one is tentatively classified as a member 9 Table- I 1 List of viruses affecting fish Viruses References Single stranded (-) RNA viruses... could be tested and monitored (Ellis, 2001) Nervous necrosis virus has been one of the major pathogens of marine fish over the last ten years (Munday and Nakai, 1997; Munday et al., 2002) Devastating losses, often reaching 100% cumulative mortality of the affected population within a short span of 4 - 7 days have been experienced in fish hatcheries due to fish nervous necrosis virus (NNV) As of now, NNV... The DNA viruses include, Iridoviridae-Epizootic haematopoietic necrosis virus (EHNV), Red sea bream iridovirus (RSIV), White sturgeon iridovirus (WSIV); Herpesviridae-Oncorhynchus masou virus (OMV), Salmonid herpesvirus 2 (SaHV-2), and Channel catfish herpesvirus (CCHV) As per the recent review by Essbauer and Ahne (2001), viruses belonging to 11 families of RNA viruses and 4 families of DNA viruses... goal for fish disease specialists around the world because of their economic importance Several methods of control and prevention of fish diseases have been in use, viz drug therapy, immunization, test and slaughter, quarantine and restriction of movement of fish stock, destruction and reduction of link in the disease transmission cycle and limitation of release of toxic substances The drug and antibiotic... 2000) The ICTV accepted fish nodavirus species are: Barfin flounder nervous necrosis virus (BFNNV), Dicentrarchus labrax nervous necrosis virus (DlEV), Japanese flounder nervous necrosis virus (JFNNV), Lates calcarifer encephalitis virus (LcEV), Redspotted grouper nervous necrosis virus (RGNNV), Striped jack nervous necrosis virus (SJNNV) and Tiger puffer 19 ... ETNNV on fish larvae 55 1.7 Histopathology of artificially- infected fish 57 2.1A cDNA nucleotide sequences and deduced amino acid sequences of RNA1 and RNA2 of ETNNV 2.1B 72 cDNA nucleotide sequence and deduced amino acid sequence of RNA2 75 2.2A Comparison of nucleotide and amino acid sequences 78 2.2B Multiple sequence alignment of the ORF of ETNNV, SJNNV, DLEV, and, T2 region (variable region) of AH95Ori, . CHARACTERIZATION AND IMMUNOLOGICAL STUDIES OF FISH NERVOUS NECROSIS VIRUS ASHOK HEGDE NATIONAL UNIVERSITY OF SINGAPORE 2003 CHARACTERIZATION AND IMMUNOLOGICAL. clinical features of viral nervous necrosis virus (VNN) of larval and juvenile fish 21 I.3 Geographical and species distribution of clinical nervous necrosis virus (VNN) 22 2.1A Primers. STUDIES OF FISH NERVOUS NECROSIS VIRUS BY ASHOK HEGDE (MFSc) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF