Dedicated to All my Teachers DECLARATION I declare that the thesis entitled "Genetic Characterization of Nucleopolyhedrovirus Isolated from Hyposidra talaca Walker (Lepidoptera: Geometridae), a Tea Pest in Terai Region of Darjeeling Foothills, India." has been prepared by me under the guidance of Professor Min Bahadur, Department of Zoology, University of North Bengal. No part of this thesis has formed the basis for the award of any degree or fellowship previously Bappya Chosh 19/D2012 Bappaditya Ghosh Department of Zoology University of North Bengal Raja Rammohunpur, Siliguri District: Darjeeling, West Bengal-734013, India Date: UNIVERSITY OF NORTH BENGAL AccREDITED BY NAAC wITH GRADE B** DEPARTMENT OF ZOOLDGY P.O. North Bengal University Raja Rammohunpur, Dist. Darjeeling West Bengal, India, PIN 734013 DST-FIST & UGC SAP Sponsored CERTIFICATE We certify that Mr. Bappaditya Ghosh has prepared the thesis entitled Genetic Characterization of Nucleopolyhedrovirus Isolated from Hyposidra talaca Walker (Lepidoptera: Geometridae), a Tea Pest in Terai Region of Darjeeling Foothills, India." for the award of Ph.D. degree of the University of North Bengal, under our guidance. He has carried out the work at the Department of Zoology, University of North Bengal. No part of this thesis has formed the basis for the award of any degree or fellowship previously. Supervisor Prof. Min Bahadur Department of Zoology University of North Bengal Date: 19 Sepimbov, a022 Co-supervisor Prof. Ananda Mukhopadhyay Department of Zoology University of North Bengal Date: 9 Sept. Bo ) Phone: (0353) 2776353, Fax (0363) 2699001, E-mail: zoology@nbu.ac.in, Vist us at: www.nbu.ac.l Ourigingal y rn Document Information Analyzed document Bappaditya Ghosh_Zoology pdf (D143186448) Submitted 2022-08-23 13.36:00 Submitted by University of North Bengal Submitter email nbuplg@ntbu ac. in Simitarity 0% Analysis address nbuplg.nbu@analysis.urkund.com Sources included in the report URL https.//www.researchgate.net/publication/267101495Genomic Sequencing_and_Analysis._of_Sucrajujuba_Nucleopolyhedrovirus gp 1 Fetched 2021-05-01 22:30 32 Entire Document 5 Chapter 1: Introduction, Review of Literature, Materials and Methods 6 Section 2: Introduction Tea cultivation in India Tea. Camellia sinensis (L) OKuntze, is the most common and widely consumed refreshing dink througnout the world. It is a resolutely and skilfully managed monoculture crop cultivated on large as well as small-scale between lattudes 41°N and 16'S with an annual precipitation of 1000-5000 mm and temperature of 8-35 0C (Hazarika et al, 2008) As per 64th annual report (2017-18) of Tea Board India", India s the World's second largest producer of tea sharing 23% (1325 05 Million Kg) of global tea production, while China is the first producing 45%. However, India exported 14% of world tea which comes after Kenya (23%). China (20%) and Srilanka (16%). India is also one of the largest tea consumers in the world accounting 19% of global tea consumption. Almost 76% of produced tea in India is consumed within the Country itself During 2017-18, 256.57 Million Kg of tea was exported from India with Cost, Insurance, and Freight (CIF) value of Rs. 5064.88 Crs, white 20 59 Million Kg with CIF value of Rs 288 56 Crs was imported into lIndia (Anorymous, 2018). Tea is the chief foliar crop in northern part of West Bengal. Assam, Sikkim, Tripura, Nilgiri of Tamit Nadu and aso grown in small scale in Himachal Pradesh, Kerala, Karnataka and Orissa (Fig 1). The Assan and Darjeeling tea are very popular in India and exclusively cultivated in large number of tea plantations in the Terai-Dooars region of West Bengal and Assam. In India tea is cultivated over an area of 6.36.55707 hectare, of which 337/690 35 hectare (53%) is situated in Assam and 1.48,12174 hectare (237%) in West Bengal including both big and small growers The economy of both of these states largely depends on the production of tea. In the northern part of West Berngal concerning the Himalayan foothils and plains, the Terai plantation is on the Western flank of the mighty Teesta river and the Dooars plantation stretches on the eastern terrain of the river continuing upto the state of Assam Looper pests in the Tea plantations of Terai-Dooars region of India Tea plantations of the Terai and Dooars region of West Bengal and Assam are experiencing severe attacks of different leaf eating lepidopteran pests, among which, Biston (Buzura) 7 suppressaria Guen (Lepidoptera. Geometridae) was reported as a major tea pest for several decades from Assam and Terai-Dooars region of North Bengal (Das, 1965) Recent studies reveal that a few other species of geometrid pests are attacking tea plantations among which the pest species. Hyposidra talaca Walker (Lepidoptera Geometridae), early caterpilar of which are commonly known as Black Inch Looper Fig 2), has taken over as the major defoliating pest in tea plantations (Das et al, 2010a). Loopers of this species that prinmarily feed on a number of forest plants and weeds in India, Malayasia and Thiland (Das and Mukhopadhyay, 2009, Mathew et al, 2005, Winotai et al., 2005) have turned to tea as an active defolating insect pest in plantations of Assam and Darjeeling Terai-Dooars of North Bengal (Basu Majumdar, 2004, Das et al, 2010). A substantial loss in tea production due to this defoliating lepidopteran pest has been reported from these regions (Gurusubramanian et al, 2008; Hazarika et al. 2008), that severely has affected the economy of the country (Roy and Muraleedharan, 2014) Management of Tea pest population To manage pest infestations, there are two main methods of pest control chemical and biological The looper pests have so far been controlled by regular application of synthetic insecticides especially (Fig 3), organophosphates and pyrethroids, but, the pests are gradually becoming less susceptible, and have developed some extent of resistance to the pesticides (Das et al, 2010b, Das and Mukhopadhyay. 2008) often resulting in their control failures (Sannigrahi and Talukdar. 2003). Moreover, the applcation of these synthetic chemical insecticides is one of the major source of pollution of sol and water (Saravanan et al. 2009, Singh et al. 2017) and reported to be hazardous to the non-target organisms including human (Azmi et al. 2006, Mobed et al. 1992, Saravanan et al, 2009, Velmurugan et al. 2006) Because of these harmful effects, application of a number of these insecticides has been banned by the EPA of the USA and even by the Tea Research Organizations of India. The registration of many other insecticides have been reviewed (Chattopadhyay et al., 2008) and recommended under the Plant Protection Codes (PPC). Therefore, concerning the fact of development of pesticide resistance in insect pests and hazardous effects of the chemical pesticides, the organic tea has become more acceptable especialy for export and health conscious tea consumers than the chemically managed conventional tea. In view of this, the 6oppdAby Chosh A Signature of the Candidate Signature of the Supervisor Dr. Min Bahedur Professor Departmem of Zoology University of North Benga! Siliguri-734013 West Bengal Acknowledgement I take this opportunity to express my deep sense of gratitude and indebtedness to those who helped me in carrying out this investigation. My parents have been a constant source of inspiration throughout my doctoral study, without their support and encouragement it was quite impossible to finish such hard work. I am immensely indebted and sincerely grateful to my honourable supervisor Prof. Min Bahadur, Genetics and Molecular Biology Laboratory, Department of Zoology, University of North Bengal and honourable co-supervisor Prof. Ananda Mukhopadhyay who introduced me to the exciting field of research and extended the fullest possible help and unconditional encouragement. Their invaluable suggestions, guidance and supervision throughout my work help me to solve problems and to take correct decisions during hard times of my research work. I am thankful to Prof. Soumen Bhattacharjee, Head, Department of Zoology, University of North Bengal for ensuring the necessary facilities in the Department, particularly the instrumentation facility. I express my thanks to my respected teachers Prof. Joydeb Pal, Prof. Sudip Barat and Prof. Tapas Kumar Chaudhury, Prof. Dhiraj Saha, Dr Tilak Saha (Associate Professor), Dr Sourav Mukherjee (Assistant Professor), Dr Ritwik Mondal (Assistant Professor), Dr Arpan Kumar Maiti (Assistant Professor), Dr Subhra Prkash Hui for their valuable guidance during my work. I extend my thanks to Prof. Sudip Barat and Prof. Dhiraj Saha, previous Head, Department of Zoology, University of North Bengal, for providing all the necessary facilities from the Department. I would like to sincerely acknowledge UGC for providing financial support as a fellowship under the UGC-BSR scheme, Bahadur Shah Zafar Marg, New Delhi, India (Ref. No. 4-1/2006(BSR)/7- 134/2007 (BSR) dated 25.02.2013). I am indebted to Prof. Soumen Bhattacharjee for his technical suggestions during my research work. The contributions of my grandfather, Dr. Radhagobinda Ghosh, and my uncle Mr. Gour Chandra Ghosh, in my life, are undeniable. Without them I would never come to this stage. I am grateful to my wife Mrs Dipa Mandal for her continuous encouragement, co-operation and moral support. i I am grateful to Dr Mahua Rudra and Mr Goutam Debnath for their initial guidance and unconditional cooperation as my elder sister and elder brother, respectively. I am grateful to all my family members, my brothers, my sisters, and my in-laws for their continuous support. Special thanks will go to one of my best friends, Mr Tanmoy Dutta, my sister, Ms Farha Yasmin, and my brothers, Mr Sudipta Kumar Roy, and Mr Debabrata Mandal for their continuous support and encouragement. I am also grateful to Mr Binoy Kishore Rai for his initial guidance during my research work. I am also thankful to Dr Susmita Dutta, Mr Akash Chowdhury, Mrs Swati Singh, Mrs Trisita Mazumdar, Mr Saugata Ghosh, Mr Debojit Dutta, Ms Arpita Ray for their untiring help and support throughout my work. I would like to thank Mrs Jayashree Saren, Ms Ananya Das, Mr Ronit Dey, Mr Prasanta Saha, Mr Tanmoy Dutta, Dr Subhashis Paul, Mr Sourav Sarkar, Mr Debojit Dutta and Mr Saugata Ghosh, for cooperating me during sample collection. I am thankful to Dr Sangita Khewa Subba and Dr Kumar Basnet for providing some samples. I extend my thanks to Dr Soma Das, Dr Ritesh Biswa, Dr Sangita Khewa Subba, Dr Anjali Prasad, Dr Kumar Basnet, Mr Mrinal Ray, Ms Jayashree Saren, Dr Priyankar Dey, Dr Somit Dutta, Dr Subhrajyoti Roy, Dr Pokhraj Guha, Dr Avishek Das, Dr Rudra Prasad Roy, Ms Ruksa Nur, Dr Dawa Bhutia, Mr Sanjib Sarkar, Dr Tanmay Mukhopadhyay, Dr Sutanuka Chattaraj, Dr Uttara Dey Bhowmick, Dr Subhashis Paul, Mr Tanmoy Dutta, Ms Naznin Islam, Mr Debabrata Modak, Mr Sourav Sarkar, Mr Sona Sutradhar Dr Minu Bharti, Dr Priyanka Rai, Mr Abhishek Subba, Mr Manas Pratim Modak, Mr Sagar Sarkar, Ms Sashwati Ghosh for extending their helping hands. Thanks are also due to the non-teaching staff of the Dept. of Zoology for their assistance in departmental facilities. I am also thankful to all administerial staff for their help during my Ph.D. work. In the end, I thank all those who helped me or were associated with my work directly or indirectly. ii Preface The application of synthetic chemical pesticides to control different agricultural pests is a global concern because of their adverse effects on the environment and non-target organisms including humans. Moreover, due to the irrational use of chemical pesticides, insect pests may gradually develop resistance to synthetic chemical pesticides leading to control failure. Hence, the development of a pest management strategy that is non-polluting, eco-friendly, target- specific, and sustainable will be highly acceptable. In this regard, microorganisms as biopesticides/ bioinsecticides may be an alternative to synthetic chemical pesticides. Among several microorganisms, baculoviruses, particularly nucleopolyhedroviruses (NPVs) are well known as bioinsecticides in the integrated pest management (IPM) strategies because of their host specificity and proved to be safe to the environment, humans, other plants, and natural enemies of pests. However, as the mode of action of NPVs is slower than chemical pesticides, genetic modifications to enhance the killing efficiency of NPVs are highly required for better pest control and management. Therefore, the studies to explore the genome of pest- specific NPVs will help design an effective biopesticide. The present study has been contemplated to characterize the genome of NPVs isolated from Hyposidra talaca, a major lepidopteran tea pest in the Terai-Dooars region of the northern part of West Bengal. As the tea plantations of the Terai-Dooars regions are facing immense defoliation by the Hyposidra talaca larvae resulting in huge economic loss, several synthetic chemical pesticides are regularly applied in the tea plantations of the Terai-Dooars regions to manage the pest problem. Therefore, the development of Hyposidra talaca nucleopolyhedrovirus (HytaNPV) into a potential biopesticide will be beneficial to minimize the use of chemical pesticides. In this context, the characterization of the genome and evolutionary study of HytaNPV will be very helpful in designing a virus-based pesticide. This thesis has been divided into several chapters. A brief introduction, review of literature, objectives, and materials and methods were provided in Chapter 1. Chapter 2 comprises the results of the survey and sampling, restriction endonuclease fragment analyses and partial restriction map of HytaNPV genome, characterization of selected genes and phylogenetic analyses and Chapter 3 contains the detailed discussion of the findings of the present study, conclusion and summary. Chapter 4 comprises the list of research articles, and the papers presented at different conferences under the Appendix, along with the Bibliography and Index. iii Abbreviations %idN : percentage of identical sites for nucleotide %idA : percentage of identical sites for amino acid acc. no. : accession number (NCBI) °N : degree North °S : degree South µg : microgram µl : micro liter 0 C : degree centigrade AcNPV : Autographa californica NPV AdhoNPV : Adoxophyes honmai NPV AdorGV : Adoxophyes orana GV AdorNPV : Adoxophyes orana NPV AgipNPV : Agrotis ipsilon NPV AgseGV : Agrotis segetum GV AgseNPV : Agrotis segetum NPV alk-exo : alkaline exonuclease AM : anti meridian AngeNPV : Anticarsia gemmatalis NPV AnpeNPV : Antheraea pernyi NPV ApciNPV : Apocheima cinerarium NPV BLAST : Basic local alignment search tool BLASTN : nucleotide BLAST BLASTP : protein BLAST BmNPV : Bombyx mori NPV BomaNPV : Bombyx mandarina NPV bp : base pair BSA : bovine serum albumin Bt : Bacillus thuringiensis BusuNPV : Buzura suppressaria NPV BVs : budded virus CapoNPV : Catopsilia pomona NPV cds : coding sequence ix CfGV : Choristoneura fumiferana GV CfNPV : Choristoneura fumiferana NPV ChchNPV : Chrysodeixis chalcites NPV ChmuNPV : Choristoneura murinana NPV ChocNPV : Choristoneura occidentalis NPV ChroNPV : Choristoneura rosaceana NPV CIF : Cost, Insurance, and Freight ClanGV-H : Clostera anachoreta GV ClanGV-HB : Clostera anastomosis GV ClbiNPV : Clanis bilineata NPV cN : number of conserved sites for nucleotides CnmeGV : Cnaphalocrocis medinalis GV CoveNPV : Condylorrhiza vestigialis NPV CrluGV : Cryptophlebia leucotreta GV Crs : Crores CuniNPV : Culex nigripalpus NPV CypoGV : Cydia pomonella GV DapuNPV : Dasychira pudibunda NPV DekiNPV : Dendrolimus kikuchii Matsumura NPV DisaGV : Diatraea saccharalis GV dN : number of variable sites for nucleotides dA : number of variable sites for amino acids DNA : deoxyribonucleic acid dNTPs : deoxyribonucleotide tri-phosphates EcobNPV : Ectropis Obliqua NPV EDTA : Ethylenediaminetetraacetic acid egt : ecdysteroid glucosyltransferase EPA : Environmental Protection Agency EpapGV : Epinotia aporema GV EppoNPV : Epiphyas postvittana NPV ErelGV : Erinnyis ello GV et al : and others EuprNPV : Euproctis (=Arna) pseudoconspersa NPV g : gravity (for centrifugation) x GM : genetically modified gm : gram GTR : General time reversible (model) GV(s) : granulovirus(es) HearGV : Helicoverpa armigera GV HearNPV : Helicoverpa armigera NPV HycuNPV : Hyphantria cunea NPV HytaNPV Hyposidra talaca nucleopolyhedrovirus HytaNPV-ID1 : Hyposidra talaca nucleopolyhedrovirus Dooars isolate HytaNPV-ITK1 : Hyposidra talaca nucleopolyhedrovirus Terai isolate HytaNPV-R : Hyposidra talaca NPV as reference HzNPV : Helicoverpa zea NPV in silico in or on a computer: done or produced by using computer software or simulation in vitro : outside the living body and in an artificial environment IPM : integrated pest management kb : kilo base kDa : kilo dalton Kg : kilogram LafiNPV : Lambdina fiscellaria NPV LC50 : median lethal concentration LD50 : median lethal dose LdNPV : Lymantria dispar NPV Lef : late expression factor LeseNPV : Leucania separata NPV LG : Le Gascuel LoobNPV : Lonomia obliqua NPV LT50 : median lethal time LyxyNPV : Lymantria xylina NPV MabrNPV : Mamestra brassicae NPV MacoNPV-A : Mamestra configurata NPV-A MacoNPV-B : Mamestra configurata NPV-B MaviNPV : Maruca vitrata NPV MEGA : Molecular Evolutionary Genetics Analysis xi : : MgCl2 : magnesium chloride ml : milliliter mm : millimetre mM : millimolar MNPV : multiple-nucleopolyhedrovirus MolaGV : Mocis latipes GV Na2CO3 : Sodium carbonate NaCl : Sodium chloride NCBI : National Centre for Biotechnology Information nd : non-synonymous mutation NeabNPV : Neodiprion abietis NPV NeleNPV : Neodiprion lecontei NPV NeseNPV : Neodiprion sertifer NPV ng : nanogram NPV(s) : nucleopolyhedrovirus(es) OB : Occlusion body ODVs : Occlusion-derived virus OpNPV : Orgyia pseudotsugata NPV ORF : Open reading frame OrleNPV : Orgyia leucostigma NPV PCR : Polymerase Chain Reaction PeluNPV : Perigonia lusca NPV PespNPV : Peridroma sp NPV pH : potential of Hydrogen PhcyNPV : Philosamia cynthia ricini NPV Pif : per os infectivity factor PiraGV : Pieris rapae GV PlxyGV : Plutella xylostella GV PlxyNPV : Plutella xylostella NPV PM : post meridian POBs : polyhedral occlusion bodies PPC : Plant Protection Codes PsinNPV : Chrysodeixis (=Pseudoplusia) includens NPV PsunGV : Pseudaletia unipuncta GV xii REN : Restriction endonuclease RFLP : Restriction fragment length polymorphism RoNPV : Rachiplusia ou NPV s/v : transition over transversion sd : synonymous mutations SDS : sodium dodecyl sulphate SeNPV : Spodoptera exigua NPV SfGV : Spodoptera frugiperda GV SfNPV : Spodoptera frugiperda NPV SNPV : single nucleopolyhedrovirus SpliNPV-A : Spodoptera litura NPV SpliNPV-B : Spodoptera littoralis NPV SujuNPV : Sucra jujuba NPV TAE : Tris-acetate EDTA ThorNPV : Thysanoplusia orichalcea NPV TnGV : Trichoplusia ni GV TnNPV : Trichoplusia ni NPV UrprNPV : Urbanus proteus NPV USA : United States of America UV : ultra violet VLF : very late factor w/v : weight per volume λ DNA : lambda DNA xiii Single letter code for nucleotide A adenine nucleotide G guanine nucleotide C cytosine nucleotide T thymine nucleotide S guanine nucleotide/cytosine nucleotide W adenine nucleotide/ thymine nucleotide Y pyrimidine nucleotide R purine nucleotide H adenine nucleotide/ cytosine nucleotide / thymine nucleotide M adenine nucleotide/ cytosine nucleotide (with nitrogen base having amino group) Single letter code for amino acid G Glycine P Proline A Alanine V Valine L Leucine I Isoleucine M Methionine C Cysteine F Phenylalanine Y Tyrosine W Tryptophan H Histidine K Lysine R Arginine Q Glutamine N Asparagine E Glutamic Acid D Aspartic Acid S Serine T Threonine xiv List of Figures Figure 1-1: Tea growing regions in India (Map not to scale). ................................................. 10 Figure 1-2: Caterpillar of Hyposidra talaca. ........................................................................... 11 Figure 1-3: Application of Chemical pesticides in tea plantations. ......................................... 12 Figure 1-4: Transmission electron micrographs of occlusion bodies (MNPV, SNPV and GV), and other forms of baculoviruses, BV (budded virions), ODV (occlusion-derived virions) and nucleocapsids (NC). The picture was taken from King et al. (2012). ................. 16 Figure 1-5: Morphology of MNPV, SNPV, Budded virus, Occlusion derived virus and Occlusion body. The Picture was taken from King et al. (2012). ........................................... 17 Figure 1-6: NPV infected dead caterpillar of H. talaca. ......................................................... 18 Figure 1-7: Infection cycle of nucleopolyhedrovirus through insect larva. ............................ 19 Figure 1-8: Map showing the sampling sites of Terai and the Dooars region in the present study. ....................................................................................................................................... 42 Figure 1-9: Culture of H. talaca larvae in the laboratory. (a) culture of healthy larvae (3 rd -4 th instar), (b) culture of infected larvae (3 rd – 5 th instar). ............................................................ 44 Figure 2-1: (a) The caterpillar of Hyposidra talaca, a leaf-eating tea pest, (b) NPV-infected dead H. talaca in tea plantation, (c) Non-infected H. talaca caterpillars collected from the tea plantation. ................................................................................................................................ 58 Figure 2-2: Map showing the sampling sites of Terai and the Dooars region in the present study. ....................................................................................................................................... 58 Figure 2-3: The figure showing the mean number of collected non-infected (green line) and NPV-infected (red line) H. talaca larvae collected from four different tea plantations in the Terai regions of Darjeeling foothills, during 2013-15. ............................................................ 59 Figure 2-4: (a) The pellet of polyhedra OBs isolated from NPV infected dead cadavers of H. talaca after centrifugation (b) HytaNPV polyhedra OBs suspended in distilled water. ......... 61 Figure 2-5: The polyhedra OBs under the compound microscope (400X). ............................ 61 Figure 2-6: NPV-infected dead H. talaca larvae obtained in the laboratory after oral inoculation with OBs (a and b). ............................................................................................... 62 Figure 2-7: Number of H. talaca larvae orally infected by OBs (blue bar) in the laboratory and the number of larvae that died due to NPV infection (red bar). ....................................... 62 Figure 2-8: Agarose gel electrophoresis of DNA extracted from OBs isolated from Hyposidra talaca. .................................................................................................................... 63 1 Figure 2-9: Electrophoregrams of restriction digestion of Terai isolate, HytaNPV-ITK1. A. BamHI, B. BglI, C. EcoRI, D. HindIII, E. KpnI, F. PstI, and G. XhoI .................................... 68 Figure 2-10: Electrophoregrams of restriction digestion of Dooars isolate, HytaNPV-ID1. A. BamHI, B. BglI, C. EcoRI, D. HindIII, E. KpnI, F. PstI, and G. XhoI. ................................... 69 Figure 2-11: Restriction map of HytaNPV-R genome for BamHI, HindIII and XhoI ............. 73 Figure 2-12: Restriction map of HytaNPV-R genome for EcoRI and PstI.............................. 76 Figure 2-13: Restriction map of HytaNPV-R genome for BglI and KpnI ............................... 77 Figure 2-14: Position and details of the six genes used in the present study (purple arrow) and the binding sites of the primers used to amplify respective genes of the HytaNPV genome. HytaNPV-R 139.089 kb (MH261376.1; Nguyen et al., 2018) was used as a reference template. Details of the genes were highlighted in purple boxes and details of the primers have been represented without any highlighted box. Coloured circles were used to distinguish the primers used for different genes. ..................................................................... 84 Figure 2-15: Electrophoregrams showing PCR products of HytaNPV DNA. HytaNPV-ITK1 and HytaNPV-ID1 represent the Terai and Dooars isolate, respectively and F stands for ‘fragment’ (see Table 2-13). .................................................................................................... 86 Figure 2-16: Electrophoregram of sequencing of lef-8 gene. .................................................. 87 Figure 2-17: Electrophoregram of sequencing of pif-2 gene. .................................................. 88 Figure 2-18: Primer binding, amplicon and sequence details of polyhedrin gene using HytaNPV-R as reference (the amplified portion was shown in purple colour on the template DNA). ....................................................................................................................................... 90 Figure 2-19: Nucleotide and translated amino acid sequence alignment of the polyhedrin gene of HytaNPV-ITK1 and HytaNPV-ID1 with HytaNPV-R as a reference. Similar sequences were highlighted in grey background. .................................................................... 92 Figure 2-20: Restriction sites present in the polyhedrin of different HytaNPV isolates. ........ 93 Figure 2-21: Primer binding, amplicon and sequence details of the lef-8 gene using HytaNPV-R as reference. ......................................................................................................... 94 Figure 2-22: Nucleotide and translated amino acid sequence alignment of the lef-8 gene of HytaNPV-ITK1 and HytaNPV-ID1 with HytaNPV-R as a reference. Similar sequences were highlighted in grey background. .............................................................................................. 96 Figure 2-23: Restriction sites present in the lef-8 of different HytaNPV isolates. .................. 99 Figure 2-24: Primer binding, amplicon and sequence details of the lef-9 gene using HytaNPV-R as a template. ..................................................................................................... 100 2 Figure 2-25: Nucleotide and translated amino acid sequence alignment of lef-9 gene of HytaNPV-ITK1 and HytaNPV-ID1 with HytaNPV-R as reference. Similar sequences were highlighted in grey background. ............................................................................................ 102 Figure 2-26: Restriction sites present in the lef-9 of different HytaNPV isolates. ................ 104 Figure 2-27: Primer binding, amplicon and sequence details of the pif-1 gene using HytaNPV-R as a template...................................................................................................... 105 Figure 2-28: Nucleotide and translated amino acid sequence alignment of pif-1 gene of HytaNPV-ITK1 and HytaNPV-ID1 with HytaNPV-R as reference. Similar sequences were highlighted in grey background. ............................................................................................ 107 Figure 2-29: Restriction sites present in the pif-1 of different HytaNPV isolates ................. 109 Figure 2-30: Primer binding, amplicons and sequence details of the pif-2 gene using HytaNPV-R as a template...................................................................................................... 110 Figure 2-31: Nucleotide and translated amino acid sequence alignment of the pif-2 gene of HytaNPV-ITK1 and HytaNPV-ID1 with HytaNPV-R as a reference. Similar sequences were highlighted in grey background. ............................................................................................ 112 Figure 2-32: Restriction sites present in the pif-2 of different HytaNPV isolates. ................ 113 Figure 2-33: Primer binding, amplicon and sequence details of the pif-3 gene using HytaNPV-R as a template...................................................................................................... 114 Figure 2-34: Nucleotide and translated amino acid sequence alignment of the pif-3 gene of HytaNPV-ITK1 and HytaNPV-ID1 with HytaNPV-R as a reference. Similar sequences were highlighted in grey background. ............................................................................................ 116 Figure 2-35: Restriction sites present in the pif-3 of different HytaNPV isolates. ................ 117 Figure 2-36: Partial restriction map of HytaNPV-ITK1 (Terai isolate) using HytaNPV-R (Nguyen et al., 2018) as a template. ...................................................................................... 122 Figure 2-37: Partial restriction map of HytaNPV-ID1 (Dooars isolate) using HytaNPV-R (Nguyen et al., 2018) as a template. ...................................................................................... 123 Figure 2-38A & B: Maximum likelihood tree based on concatenated sequence alignment of six genes together using nucleotide substitution. Bootstrap values were shown at each node. The Bar/scale represents the number of substitutions per site. ............................................. 126 Figure 2-39A & B: Maximum likelihood tree based on concatenated sequence alignment of six genes together using amino acid substitution. Bootstrap values were shown at each node. The Bar/scale represents the number of substitutions per site. ............................................. 128 Figure 2-40: Maximum likelihood tree based on concatenated sequence alignment of six genes together using nucleotide (a) and amino acid (b) substitution. Bootstrap values were shown at each node. The Bar/scale represents the number of substitutions per site. ............ 136 3 List of Tables Table 1-1: List of virus families and the corresponding genera pathogenic to insects (summarized from King et al. (2012). ..................................................................................... 14 Table 1-2: Genome size of different host-specific baculovirus. .............................................. 25 Table 1-3: Core genes found in Baculoviruses [Adapted form Rohrmann, (2011)]. .............. 28 Table 1-4: List of the primers used to amplify different genes and respective annealing temperature and duration of extension in each cycle of the PCR program. ............................ 47 Table 1-5: List of baculovirus sequences used for phylogenetic analysis in the present study.50 Table 2-1: HytaNPV isolates of the present study and the reference ...................................... 55 Table 2-2: Numbers and size of restriction fragments of HytaNPV DNA. ............................. 66 Table 2-3: In vitro restriction endonuclease fragment profile (BamHI, BglI, EcoRI, HindIII) of HytaNPV-ITK1 (Terai) and HytaNPV-ID1 (Dooars). Fragment size was mentioned in kb.70 Table 2-4: In vitro restriction endonuclease fragment profile (KpnI, PstI, XhoI) of HytaNPV- ITK1 (Terai) and HytaNPV-ID1 (Dooars). Fragment size was mentioned in kb. .................. 71 Table 2-5: In silico BamHI and HindIII fragment profiles of the complete genome of HytaNPV-R mentioning the positions of the restriction fragments along with its flanked restriction sites ......................................................................................................................... 73 Table 2-6: In silico XhoI fragment profile of complete genome of HytaNPV-R mentioning the positions in the genome of the restriction fragments along with its flanked restriction sites. ......................................................................................................................................... 74 Table 2-7: In silico EcoRI and PstI fragment profiles of the complete genome of HytaNPV-R mentioning the position in the genome of the restriction fragments along with its flanked restriction sites. ........................................................................................................................ 75 Table 2-8: In silico KpnI and BglI fragment profile of complete genome of HytaNPV-R mentioning the position in the genome of the restriction fragments along with its flanked restriction sites. ........................................................................................................................ 77 Table 2-9: Comparisons of the number and size of restriction fragments between in silico digestion of HytaNPV-R complete genome and in vitro digestions of HytaNPV-ITK1 and HytaNPV-ID1 .......................................................................................................................... 79 Table 2-10: Comparisons of in silico and in vitro restriction endonuclease fragment profiles (BamHI, BglI, EcoRI & HindIII) of HytaNPV isolates (HytaNPV-ITK1, HytaNPV-ID1 and HytaNPV-R) ............................................................................................................................ 80 5 Table 2-11: Comparisons of in silico and in vitro restriction endonuclease fragment profiles (KpnI, PstI, XhoI) of HytaNPV isolates (HytaNPV-ITK1, HytaNPV-ID1 and HytaNPV-R).81 Table 2-12: Position and size of the six genes (used for analyses in the present study) in the HytaNPV-R genome (Nguyen et al., 2018). ............................................................................ 83 Table 2-13: Details of sequences, binding sites (nucleotide position) of primers used to amplify specific genes of HytaNPV and length of the respective PCR products. Binding sites of the primers were shown using HytaNPV-R (MH261376.1; Nguyen et al., 2018) as a reference ................................................................................................................................... 85 Table 2-14: Details of gene sequences submitted to NCBI GenBank database. The location of each sequence has been shown using HytaNPV-R (MH261376.1; Nguyen et al, 2018) as reference. .................................................................................................................................. 89 Table 2-15: Blast results of polyhedrin sequence. ................................................................... 91 Table 2-16: Blast results lef-8 sequence................................................................................... 95 Table 2-17: Blast results lef-9 sequence ................................................................................ 101 Table 2-18: Blast results pif-1 sequence ................................................................................ 106 Table 2-19: Blast results pif-2 sequence ................................................................................ 111 Table 2-20: Blast results pif-3 sequence. ............................................................................... 115 Table 2-21: Pairwise and overall comparisons of the gene sequences of HytaNPV isolates using HytaNPV (MH261376.1) as reference. cN = number of conserved sites for nucleotides, dN = number of variable sites for nucleotides, %idN = % of identical sites for nucleotide, nd = non-synonymous substitution, sd = synonymous substitution, dA = number of variable sites for amino acids, %idA = % of identical sites for amino acids. ............................................... 119 Table 2-22: Details of the non-synonymous nucleotide substitutions and respective amino acid substitutions using HytaNPV-R as reference. ................................................................ 120 Table 2-23: list of NPVs considered for detailed phylogenetic analysis. .............................. 131 Table 2-24: Pairwise distance matrix representing p-distance based on nucleotide substitutions (lower half) and transition/transversion ratio (upper half) of a concatenated sequence alignment of six genes together. ............................................................................. 133 Table 2-25: Pairwise distance matrix representing p-distance based on amino acid substitution of a concatenated sequence alignment of six genes together. ............................ 134 6