"Molecular Biological investigations on viral diseases affecting farmed penaeid shrimps in Kerala" THESIS SUBMITTED TO THE COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN MOLECULAR BIOLOGY (Under the faculty of Marine Sciences) By Toms C. Joseph Reg. No 2826 January 20 I 0 MICROBIOLOGY. FERMENTATION AND BIOTECHNOLOGY DIVISION CENTRAL INSTITUTE OF FISHERIES TECHNOLOGY (Indian Council of Agricultural Research) COCHIN-682 029 Declaration I hereby declare that this thesis is a record of bonafide research carried out by me under the supervision and guidance of Dr. P. K Surendran, my supervising guide and Dr. Nirma\'-l Thampuran, my Co-guide and it has not previously formed the basis for award of any degree, diploma, associateship, fellowship or other similar title or recognition to me, from this or any other University or Society pd. Place: Cochin Date: 1I/01}to Toms C. Joseph 2666845.266846.2666847.2666848.2668145 www.cift.res.in Telephone 1 2666763.2666764.2666765.2666766 Fax: 0091·484 ·2668212 2668576. 2668577, 2668578. 2668579. 2668580 E·mail: enk_ciftaris@sanchametin [email protected] 4; ;~fi (01 q IH 4 f€)l M' eJ) PI ctn <H '&.'I Ft CENTRAL INSTITUTE OF FISHERIES TECHNOLOGY ~~~~ "(C'"A 3!R'!'f (Indian Council of Agricultural Research) :mm, m., - r~f4jlI5'i ~ m. ~ 682 029 Wiltingdon Island. Matsyapuri P. 0 .. Cochin -682 029 CERTIFICATE This is to certify that this thesis entitled "Molecular Biological investigations on viral diseases affecting farmed penaeid shrimps in Kerala" embodies the result of original work conducted by Sri. Toms C. Joseph, under our supervision from November 2004 to December 2009. We further certify that no part of this thesis has previously formed the basis for the award to the candidate, of any degree, diploma, associateship, fellowship or other similar titles of this or any other University or Society. He has passed the PhD qualifying examination of the Cochin University of Science and Technology, held in March 2006. Place: Cochin Date: \\ -0\ -11>\1:> _ ),l~A Or. Nirmala Thampuran Dr. P K Surendran Co-guide Supervising Guide Principal Scientist (Retd.) and Principal Scientist (Retd.) and Fonner Head of Division Former Head of Division Microbiology, Fennentation and Microbiology, Fermentation and Biotechnology Division Biotechnology Division Central Institute of Fisheries Technology Central Institute of Fisheries Technology Dedicated to llly beloved parents All praise and glory to Almighty God for the wisdom and perseverance that he has bestowed upon me throughout my life. I would like to express my deep and sincere gratitude to my supervising guide, Dr. P. K Surendran, Principal Scientist (Retired) and former Head of Microbiology, Fermentation and Biotechnology Division of Central Institute of Fisheries Technology for his support, guidance, advice throughout the research project, as well as his pain-staking effort in proof reading the drafts. Indeed, without his guidance, I would not be able to put the thesis together. I would like to thank my Co-guide, Dr. Nirmala Thampuran, Principal Scientist (Retired) and former Head of Microbiology, Fermentation and Biotechnology Division of Central Institute of Fisheries Technology for her encouragement and insightful comments throughout my study period. My thanks are due to Dr. K. V Lalitha, Head of Microbiology, Fermentation and Biotechnology Division of Central Institute of Fisheries Technology for her valuable suggestions and constant encouragement which gave an impetus for completing the thesis work. I am sincerely thankful to Dr. B Meenakumari, Director, CIFT and Dr. K Devadasan, former Director of CIFT for providing all the facilities required for carrying out the research work. I am indebted to my colleagues at MFB Division Sri. V. N Nambiar, Sri. Rakesh Kumar and Dr. Sanjoy Das for their support and suggestions throughout the study. I also express my heartfelt thanks to Sri. N. M Vasu, Smt. Rekha, Sri. Raman Nampoothiri, Smt. Mythri, Sri. Sukumaran Nair, Sri. J Samarajan (late), Smt. Leena, Smt. Vijayakumari, Sri. Bijoy, Smt. Ammini and Sri. Deepakwin for their love and support through out my study. My heartfelt thanks to Ms. Roswin and Mr. Anburajan for all the help and support rendered during the study. I would like to thank my parents who through my childhood and study career had always allowed me to follow my heart and inquisitive mind in any direction that took me. My parents inculcated in me the values of life. My special gratitude is due to my brother for his loving support. I owe my loving thanks to my wife Lovely, my daughter Liza and sons J oseph and Zachariah. Without their encouragement and understanding it would have been impossible for me to finish this work. I also thank all the staff of C1FT for their care, help and affection. Toms C. Joseph CONTENTS No. Title Page No. t. Introduction 1 2. Review of literature 7 2.1 Shrimp aquaculture 7 2.2 Diseases affecting shrimp 8 2.2.1 Viral diseases 9 2.2.2 Bacterial diseases 11 2.2.3 Fungal diseases 12 2.2.4 Parasitic diseases 12 2.3.1 White spot syndrome virus 13 2.3.1.1 Spread of WSSV infection 13 2.3.1.2 Morphology of WSSV 14 2.3.1.3 Genome and classification 14 2.3.1.4 Genetic, anti genic and virulence variability in WSSV strains 16 2.3.1.5 Structural proteins 18 2.3.1.6 Clinical signs and pathology 20 2.3.1.7 Pathogenesis 21 2.3.1.8 Control of WSSV 24 2.3.1.9 Host range 25 2.3.1.10 Incidence of WSSV infection 27 2.3 .1.11 Variation of structural proteins among isolates 29 2.3.1.12 Natural epidemics 32 2.3.1.13 Transmission of virus 33 2.3.1.14 Diagnostic methods for WSSV 35 2.3.1.14.1 Field methods 35 2.3.1.14.2 Serological methods 36 2.3.1.14.3 Polymerase chain reaction 37 2.3.1.14.4 Real time PCR 38 2.3.1.14.5 Other diagnostic methods 40 2.3.2 Infectious hypodermal haematopoietic necrosis virus 41 2.3.2.1 IHHN symptoms in P. stylirostris 41 2.3.2.2 IHHN disease in P. vannamei 42 2.3.2.3 Sequence variation 43 2.3.2.4 Genome 44 2.3.2.5 Diagnostic methods 45 2.3.2.5.1 Histological method 45 2.3.2.5.2 Polymerase chain reaction 45 2.3.3 Monodon Baculo virus 46 2.3.3.1 Pathogenicity and virulence 46 2.3.3.2 Incidence of infection 47 2.3.3.3 Diagnosis of infection 48 2.3.4 Hepatopancreatic parvo virus 49 2.3.4.1 Occurrence 49 2.3.4.2 Sequence variation among HPV isolates 49 2.3.4.3 Host range 50 2.3.4.4 Mode of transmission 52 2.3.4.5 Symptoms of infection 52 2.3.4.6 Detection methods 52 2.3.5 Multiple infections 53 2.3.6 Yellow Head Virus 55 2.3.6.1 Virus morphology and structure 56 2.3.6.2 Morphogenesis 56 2.3.6.3 Infection state 57 2.3.6.4 Transmission 57 2.3.6.5 Host range 57 2.3.6.6 Detection and diagnosis 58 2.3.6.7 Histopathology 58 2.3.6.8 Molecular diagnostic techniques 59 2.3.7 Taura Syndrome virus 59 2.3.8 Whitish muscle disease (WMD) 61 2.3.8.1 Pathogen and mode of infection 61 2.3.8.2 Transmission 62 2.3.8.3 Signs and symptoms 62 2.3 .8.4 Diagnostic methods 63 2.3.8.5 Reverse-transcriptase polymerase chain reaction 63 3. Materials and methods 64 3.1 Materials 64 3.l.1 Animals 64 3.1.2 Molecular Biology Reagents 67 3.1.3 Agarose gel electrophoresis reagents 67 3.1.4 Equipments 68 3.2 Methods 68 3.2.1 Extraction of viral DNA 68 3.2.2 Detection of WSSV in farmed and wild decapods by nested PCR 69 3.2.3 PCR Amplification of structural genes 71 3.2.4 Cloning of PCR products in pTZ57R1T vector 73 3.2.5 Polymerase Chain Reaction for detection of multiple viral infections 74 3.2.6 Real-time SYBR Green PCR primer 78 3.2.7 Plasmid standard for quantification by SYBR Green PCR 78 3.2.8 SYBR Green PCR for quantification of WSSV 79 3.4.9 Melting curve analysis of the peR product 79 3.4.10 Analysis of data 79 4 Results and Discussion 80 4.1 Nested peR method to determine incidence of white spot syndrome virus 80 in farmed and wild crustaceans 4.1.1 Nested peR for detection of WSSV 80 4.1.2 Detection of WSSV in shrimps and decapods from farm environment 81 4.1.3 Detection ofWSSV in shrimps from Vembanad estuary 84 4.1.4 Detection of WSSV in wild captured decapods from sea landings 85 4.2 Insilico analysis to compare variations in structural protein sequences of 89 an Indian isolate of WSSV with sequences in databank 4.2.1 Comparison ofVP28 gene sequence of the Indian isolate ofWSSV 90 obtained in the study with that available from the GenBank 4.2.2 Comparison ofVP26 gene sequence of the Indian isolate ofWSSV 92 obtained in the study with that available from the GenBank 4.2.3 Comparison of VP 19 gene sequence of the Indian isolate of WSSV 93 obtained in the study with that available from the GenBank 4.2.4 Comparison ofVP281 gene sequence of the Indian isolate ofWSSV 94 obtained in the study with that available from the GenBank 4.2.5 Comparison ofVP466 gene sequence of the Indian isolate ofWSSV 95 obtained in the study with that available from the GenBank 4.3 PCR amplification to determine the prevalence of multiple viral infections 99 in penaeid postlarvae 4.3.1 Prevalence of WSSV in postlarvae from hatcheries 100 4.3.2 Prevalence of MBV in postlarvae from hatcheries 101 4.3.3 Prevalence of HPV in postlarvae from hatcheries 102 4.3.4 Prevalence of IHHNV in postlarvae from hatcheries 103 4.3.5 Prevalence of mUltiple viral infections in postlarvae from hatcheries 104 4.4 A Real-time SYBR Green PCR Assay for quantification of WSSV 107 infection in postlarvae of P. monodon 4.4.1 Standard curve and limit of detection 107 4.4.2 WSSV quantification in postJarvae from four hatcheries 109 5. Summary and conclusions 113 5.1 Detection of WSSV In shrimps and other decapods from farm 112 environment 5.2 Detection of WSSV in shrimps from Vembanad estuary 113 5.3 Detection of WSSV in wild captured decapods from sea landings 113 5.4 Insilico analysis to compare variations in structural protein sequences of 115 an Indian isolate ofWSSV with sequences in databank 5.5 Comparison of VP28 gene sequence of the Indian isolate of WSSV 115