ANIMAL CORONAVIRUSES.

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Preface Contents Contributors Part I: Reviews Chapter 1: Animal Coronaviruses 1 Introduction 2 Bovine Coronavirus (BCoV) and Bovine-Like Coronavirus 3 Canine Coronavirus (CCoV) 4 Cetacean Coronavirus 5 Equine Coronavirus (ECoV) 6 Feline Coronavirus (FCoV) 7 Infectious Bronchitis Virus (IBV) 8 Porcine Coronavirus 9 Turkey Coronavirus (TCoV) 10 SARS-CoV-2 in Animals 10.1 Experimental Infection in Animals 10.1.1 High Susceptibility Group 10.1.2 Low Susceptibility Group 10.1.3 Insusceptibility Group 10.2 Natural Infection in Animals 10.2.1 Animals in Order Carnivora, Suborder Caniformia 10.2.2 Animals in Order Carnivora, Suborder Feliformia 10.2.3 Animals in the Order Artiodactyla 10.2.4 Animals in Order Primate, Suborder Haplorhini 11 Conclusion References Chapter 2: Feline Coronavirus: Insights into the Pathogenesis and Diagnosis 1 Overview 1.1 Introduction and Historical Aspects 1.2 Taxonomy 1.3 Morphology 1.4 Structural Protein, Non-structural Proteins and Accessory Proteins 1.5 Viral Entry and Replication 1.6 Type I and Type II Feline Coronaviruses 2 Pathogenesis 2.1 Feline Enteric Coronavirus 2.2 Origin of Feline Infectious Peritonitis Virus 2.2.1 Internal Mutation Theory 2.2.2 Quasispecies Theory 2.2.3 Immune Response Related to Viral-Host Interaction 2.3 Clinical and Pathological Features of FIP 2.3.1 Prevalence 2.3.2 Transmission, Shedding, and Persistence 2.3.3 Clinical Features 3 Diagnosis of FCoVs 3.1 Detection of Antibodies 3.2 Analysis of Effusion Samples 3.3 Molecular Detection of Viral RNA 3.4 Mutation Analysis 3.5 Histological Examination 3.6 Immunostaining 4 Treatment 5 Conclusions References Chapter 3: Animal Models for Studying COVID-19 Pathology and Infection Abbreviations 1 Introduction 2 COVID-19 in Humans 3 Ideal Animal Model Characteristics for COVID-19 4 Animal Models for COVID-19 4.1 Nonhuman Primates (NHPs) 4.2 Hamster 4.3 hACE2 Transgenic Mice 4.4 Ferrets 4.5 Cats 5 Other Perspective Species 6 Conclusions References Part II: Virus Isolation Chapter 4: Isolation of Porcine Epidemic Diarrhea Virus from Clinical Samples 1 Introduction 2 Materials 2.1 Solutions and Reagents 2.2 Equipment/Tools 3 Methods 3.1 Preparation of Vero Cell Plates/Flasks 3.2 Clinical Sample Preparation 3.3 Virus Inoculation 3.4 Observe CPE and Harvest Samples 4 Notes References Chapter 5: Isolation and Characterization of Porcine Epidemic Diarrhea Virus Using Suspension Vero Cells 1 Introduction 2 Materials 2.1 Sample Processing 2.2 Cell Culture Preparation 2.3 Inoculation of Cell Culture 3 Methods 3.1 Processing of Small Intestine Tissues and Fecal Samples 3.2 Preparation of Cell Culture 3.3 Inoculation of Cell Culture and Assessment of Virus Isolation 3.4 Electron Microscopy 3.5 IFA 3.6 Viral Replication Kinetics 3.7 Sequence Analysis of the Clinical PEDV Isolate 4 Notes References Chapter 6: Isolation and Propagation of Porcine Deltacoronavirus in Cell Culture and Embryonated Chicken Eggs 1 Introduction 2 Materials 2.1 Sample Collection and Processing 2.2 Cell Culture, Virus Propagation, and Titration 2.3 Plaque Assay for Purification of PDCoV 2.4 Inoculation of Embryonated Chicken Eggs 3 Methods 3.1 Sample Collection and Preparation 3.2 Preparation of Cell Cultures 3.2.1 Preparation of LLC-PK Cells 3.2.2 Preparation of ST Cells 3.3 Isolation of PDCoV from LLC-PK or ST Cells 3.4 Propagation (Serial Passage) of PDCoV in LLC-PK or ST Cells 3.5 Isolation and Propagation of PDCoV Using Embryonated Chicken Eggs 3.6 Titration of Infectious PDCoV by TCID50 Assay 3.7 Titration and Purification of PDCoV in ST Cells by Plaque Assay (See Note 17) 4 Notes References Chapter 7: Isolation and Propagation of Infectious Bronchitis Virus (Avian Coronavirus) in Chicken Embryonated Eggs 1 Introduction 2 Materials 2.1 Samples (See Note 1) 2.2 Media, Reagents, and Biologics 2.3 Other Materials 2.4 Equipment 3 Methods 3.1 Preparation or Organ Macerates (See Note 4) 3.2 Preparation of Swabs Samples 3.3 Egg Preparation 3.4 Egg Inoculation Through Chorioallantoic Sac Route 3.5 Screening by Rapid Plate Hemagglutination Assay 4 Notes References Part III: Diagnostic Approaches by Detection of Viral Nucleic Acid or Protein Chapter 8: Real-Time Reverse Transcription-Polymerase Chain Reaction for Simultaneous Detection of Bovine Coronavirus and Othe... 1 Introduction 2 Materials 2.1 Sample Handling 2.2 RNA Extraction 2.3 Real-Time PCR Setup 3 Methods 3.1 Sample Handling 3.2 RNA Extraction 3.3 Real-Time PCR Setup 4 Notes References Chapter 9: TaqMan Real-Time RT-PCR Detection of Porcine Epidemic Diarrhea Virus 1 Introduction 2 Materials 2.1 Samples Preparation 2.2 RNA Extraction 2.3 Two-Step Real-Time RT-PCR 2.3.1 Materials for the RT Step (Standard First-Strand Synthesis of cDNA) 2.3.2 Materials for Real-Time PCR 3 Methods 3.1 Fecal Samples Preparation 3.2 RNA Extraction 3.3 TaqMan Real-Time RT-PCR Assay 3.3.1 Standard First-Strand Synthesis of cDNA 3.3.2 TaqMan Real-Time PCR Assay 3.4 Standard Curve 4 Notes References Chapter 10: Real-Time RT-PCR for Detection of Feline Coronavirus 1 Introduction 2 Materials 2.1 Sample Handling 2.2 Nucleic Acid Extraction 2.3 FCoV One-Step RT-PCR 3 Methods 3.1 Sample Handling 3.2 RNA Extraction 3.3 Prepare Real-Time RT-PCR 3.4 Set Up Real-Time PCR on Applied Biosystems (ABI) 7500 Real-Time PCR System 3.5 Result Interpretation 4 Notes References Chapter 11: SARS-CoV-2 Real-Time RT-PCR Assay in Animals 1 Introduction 2 Materials 2.1 Sample Handling 2.2 Nucleic Acid Extraction 2.3 SARS-CoV-2 One-Step RT-PCR 3 Methods 3.1 Sample Handling 3.2 RNA Extraction 3.2.1 RNA Extraction Using BioSprint 96 One-for-all Vet Kit 3.2.2 RNA Extraction Using MagMAX Pathogen RNA/DNA Kit (See Note 2) 3.3 Prepare Real-Time RT-PCR 3.4 Set up Real-Time RT-PCR on Applied Biosystems (ABI) 7500 Real-Time PCR System 3.5 Result Interpretation 4 Notes References Chapter 12: Real-Time PCR and Antigen-Capture ELISA for the Diagnosis of Porcine Deltacoronavirus Infection 1 Introduction 2 Materials 2.1 qPCR 2.1.1 Sample Collection and Processing 2.1.2 RNA Extraction 2.1.3 Reverse Transcription 2.1.4 TaqMan Real-Time PCR Assay 2.1.5 SYBR Green I Real-Time PCR 2.2 DAS-ELISA for Detection of PDCoV 2.2.1 Prepare the DAS-ELISA Kit for PDCoV 2.2.2 DAS-ELISA for Detection of Samples 3 Methods 3.1 qPCR for Detection of PDCoV 3.1.1 Sample Collection and Processing 3.1.2 Viral RNA Extraction 3.1.3 Reverse Transcription to cDNA 3.1.4 TaqMan Real-Time PCR Assay (See Note 11) 3.1.5 SYBR Green I Real-Time PCR 3.2 DAS-ELISA for Detection of PDCoV 3.2.1 Prepare the DAS-ELISA Kit for PDCoV Detection 3.2.2 DAS-ELISA for Detection of Samples 4 Notes References Part IV: Serology Chapter 13: Identification of a Neutralizing Epitope-Containing Segment in Turkey Coronavirus Spike Protein for Diagnostic Ser... 1 Introduction 2 Materials 2.1 Construction of TCoV S Fragments in Expression Vectors 2.1.1 Virus Preparation 2.1.2 RNA Extraction and Viral cDNA Preparation by Reverse Transcriptase 2.1.3 Construction of TCoV S Fragments in Expression Vector 2.2 Selection of TCoV S Fragments Containing Antigenic Epitopes by Immunofluorescent Antibody Assay 2.2.1 Cell Preparation 2.2.2 Immunofluorescent Antibody Assay 2.3 Expression and Purification of TCoV S Fragments Containing Antigenic Epitopes in E. coli 2.3.1 Protein Expression 2.3.2 Protein Purification 2.4 Expression and Purification of TCoV S Fragments Containing Antigenic Epitopes in E. coli 2.5 Identification of a Neutralizing Epitope-Containing Segment by VN Assay for Antibodies to TCoV S Fragments 3 Methods 3.1 Construction of TCoV S Fragments in Expression Vectors 3.2 Selection of TCoV S Fragments Containing Antigenic Epitopes by Immunofluorescent Antibody Assay 3.3 Expression and Purification of TCoV S Fragments Containing Antigenic Epitopes in E. coli 3.4 Generation of Polyclonal Antibodies to TCoV S Fragments 3.5 Identification of a Neutralizing Epitope-Containing Segment by Virus Neutralization (VN) Assay for Antibodies to TCoV S Fr... 4 Notes References Chapter 14: Antibody-Capture Enzyme-Linked Immunosorbent Assay for Detecting Protective Antibodies to Turkey Coronavirus Based... 1 Introduction 2 Materials 2.1 Construction of TCoV S Fragments in the Expression Vector 2.1.1 TCoV Virus Preparation 2.1.2 RNA Extraction and Viral cDNA Preparation by Reverse Transcriptase 2.1.3 Construction of TCoV S Fragments in Expression Vector 2.2 Expression and Purification of 4F/4R or Mod4F/Epi4R TCoV S Segments 2.2.1 Protein Expression 2.2.2 Protein Purification 2.3 SDS-PAGE and Western Blotting 2.4 ELISA with 4F/4R or Mod4F/Epi4R Segment as Coating Antigen 3 Methods 3.1 Construction of TCoV S Fragments in the Expression Vector 3.2 Expression and Purification of 4F/4R or Mod4F/Epi4R TCoV S Segments 3.3 SDS-PAGE and Western Blotting 3.4 ELISA with 4F/4R or Mod4F/Epi4R Segment as Coating Antigen 4 Notes References Chapter 15: ELISA, Virus Neutralization, and Immunofluorescence Assays for Detection of Antibodies Against Porcine Deltacorona... 1 Introduction 2 Materials 2.1 Blocking ELISA for PDCoV Antibody Detection 2.2 Indirect ELISA for PDCoV Antibody Detection 2.3 Virus Neutralization for Detection of PDCoV Antibody 2.4 Immunofluorescence Antibody Assay for PDCoV 3 Methods 3.1 Blocking ELISA for PDCoV Antibody Detection 3.2 Indirect ELISA for PDCoV Antibody Detection 3.3 Virus Neutralization for Detection of PDCoV Antibody 3.4 Immunofluorescence Antibody Assay for PDCoV 4 Notes References Chapter 16: Luminescent Immunoprecipitation System (LIPS) Development for the Specific Detection of PDCoV and SARS-CoV-2 Antib... 1 Introduction 2 Materials 2.1 Reagents 2.2 Equipment and Supplies 3 Methods 3.1 Construction of Expression Plasmids 3.2 Transfection of HEK293T Cells 3.3 Western Blot to Confirm N-Luc-Antigen Expression 3.4 Confirm N-Luc-Antigen Expression Using Luciferase Assay 3.5 Sample Testing 4 Notes References Chapter 17: Construction of a Mouse-Feline Chimeric Neutralizing Antibody Against SARS-CoV-2 1 Introduction 2 Materials 2.1 Immunization 2.2 Cell Fusion 2.3 Indirect Fluorescent Assay 2.4 Hybridoma Cloning 2.5 Isotyping of mAb 2.6 Construction of Chimeric Mouse-Feline Antibody 2.7 Antibody Purification 2.8 Lowry Assay 2.9 Production of Pseudotyped Virus Expressing SARS-CoV-2 S Protein 2.10 Pseudotyped Virus-Based Neutralization Assay 3 Methods 3.1 Immunization 3.2 Cell Fusion 3.2.1 Myeloma Preparation 3.2.2 Mouse Splenocyte Preparation 3.2.3 Fusion of Myeloma Cells with Mouse Splenocytes 3.3 Hybridoma Screening 3.3.1 Monitor Cells 3.3.2 Indirect Fluorescent Assay 3.4 Hybridoma Cloning 3.5 Isotyping of mAbs 3.6 Construct Chimeric Antibody 3.6.1 Isolation of Cellular RNA (Modified Procedure from SV Total RNA Isolation System) 3.6.2 Amplification of Variable Regions of Heavy Chain and Light Chain 3.6.3 Cloning and Identification of Positive Clones 3.6.4 Sequence Analysis of Variable Regions 3.6.5 Creation of Chimeric Mouse-Feline Constructs 3.6.6 Chimeric mAb Expression and Identification 3.6.7 Concentration and Purification of Chimeric mAb 3.6.8 Measure Antibody Concentration by Lowry Assay 3.7 Neutralization Assay 3.7.1 Production of Pseudotyped Virus Expressing SARS-CoV-2 S Protein 3.7.2 Pseudotyped Virus-Based Neutralization Assay 3.7.3 Luciferase Assay 4 Notes References Chapter 18: Microneutralization Test of Avian Infectious Bronchitis Virus Using Vero Cells 1 Introduction 2 Materials 2.1 Virus Titration Assay 2.2 Serum Virus Neutralization Assay 3 Methods 3.1 Virus Titration 3.1.1 IBV Stock Preparation 3.1.2 Vero Cell Preparation 3.1.3 Virus Titration 3.2 Serum Neutralization Assay 3.2.1 Vero Cell Preparation 3.2.2 Neutralization Reaction 3.2.3 Virus Back Titration 3.2.4 Calculation of Virus Neutralization Titer 4 Notes References Part V: Disease-Targeted Animal Species as Animal Models Chapter 19: Pig Bioassay to Assess Infectivity of Porcine Epidemic Diarrhea Virus Detected in Various Sample Matrices 1 Introduction 2 Materials 2.1 Ethics Approval 2.2 Source of Piglets 2.3 Screening Piglets 2.4 Feeding Piglets 2.5 Inoculation of Piglets 2.6 Rectal Swab Collection 2.7 Necropsy 2.8 Processing Samples for Testing 2.9 Nucleic Acid Extraction 2.10 Quantitative PEDV Real-Time RT-PCR 2.11 Histopathological Examination and Immunohistochemistry Staining 3 Methods 3.1 Screening Piglets 3.2 Preparation of Inoculum 3.3 Pig Study Design (Fig. 1) 3.4 Sample Processing and PEDV PCR Testing 3.5 Examinations of Histopathological Lesions and Immunohistochemistry Staining 3.6 Analysis of Results and Interpretation 4 Notes References Chapter 20: Diagnostic Investigation and Confirmation of Emerging Turkey Coronaviral Enteritis 1 Introduction 2 Materials 2.1 Detection of TCoV in the Intestine Samples from Clinically Suspected Turkeys 2.1.1 Immunofluorescence Antibody Assay 2.1.2 Virus Isolation and Purification 2.1.3 Quantitative Reverse Transcription Polymerase Chain Reaction 2.2 TCoV Sequencing 2.3 Infectious Challenge 2.4 TCoV Antibody Detection by TCoV Spike Protein ELISA 3 Methods 3.1 Detection of TCoV in the Intestine Samples from Clinically Suspected Turkeys 3.1.1 Immunofluorescence Antibody (IFA) Assay 3.1.2 Virus Isolation and Purification 3.1.3 Quantitative Reverse Transcription Polymerase Chain Reaction 3.1.4 Virus Titration by IFA 3.2 TCoV Sequencing 3.3 Infectious Challenge 3.4 TCoV Antibody Detection by TCoV S Protein ELISA 3.5 Cross-neutralization 4 Notes References Part VI: Next-Generation Sequencing and Bioinformatics Chapter 21: Whole Genome Sequencing of Porcine Epidemic Diarrhea Virus and Porcine Deltacoronavirus 1 Introduction 2 Materials 2.1 Equipment 2.2 Disposable Materials and Reagents 3 Methods 3.1 RNA Extraction 3.1.1 Preprocess the Intestine Tissue and Fecal Homogenate Samples 3.1.2 Preprocess Cell Culture Samples 3.1.3 Prepare the Lysis, Wash, and Elution for KingFisher 96 3.1.4 Load Samples and Run the KingFisher 96 Automatic RNA Extraction Program 3.1.5 Collect Extracted RNA 3.2 DNase Digestion of RNA and RNA Cleanup 3.2.1 Prepare DNase I Stock Solution (See Note 2) 3.2.2 DNase I Treatment 3.2.3 RNA Cleanup 3.3 Synthesis of Double-Stranded cDNA 3.3.1 Preparation of Primer Sets 3.3.2 Synthesis of the First Strand 3.3.3 Synthesis of the Second Strand 3.4 Bead Cleanup 3.5 Illumina Nextera XT DNA Library Preparation and Sequencing on the Illumina MiSeq 3.6 Bioinformatics Analysis and Viral Genome Assembly 4 Notes References Chapter 22: Whole Genome Sequencing of Avian Infectious Bronchitis Virus by iSeq100 Platform and MinION 1 Introduction 2 Materials 2.1 Sample 2.2 RNA Extraction 2.3 Real-Time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) Reaction 2.4 Sequencing Using iSeq 100 Platform 2.4.1 SISPA Method (Sequence-Independent, Single-Primer Amplification) 2.4.2 Illumina Nextera DNA Flex Library Preparation 2.5 Sequencing Using MinION 2.5.1 Reagents and Supplies 2.5.2 Direct RNA Sequencing 2.5.3 Direct cDNA Sequencing 2.5.4 cDNA-PCR Sequencing 2.6 Next-Generation Sequencing on iSeq 100 2.7 Next-Generation Sequencing on MinION 2.8 Sequence Assembly and Analysis 2.8.1 iSeq Reads (See Note 1) 2.8.2 MinION Reads (See Note 1) 3 Methods 3.1 Viral RNA Extraction 3.2 Real-Time RT-PCR Reaction 3.3 Sequencing on iSeq 100 3.3.1 Purify PCR Products Using Agencourt AMPure XP Beads 3.3.2 Library Preparation Using DNA Flex Kit 3.4 Sequencing on MinION (See Note 6) 3.4.1 Hardware and Software Used for MinION Sequencing 3.4.2 Check Flow Cell 3.4.3 Sequencing IBV by Direct RNA Sequencing (See Note 7) 3.4.4 Sequencing IBV by Direct cDNA Sequencing Reverse Transcription and Strand Switching RNA Degradation and Second-Strand Synthesis End-Prep Adapter Ligation AMPure XP Bead Binding to Purify Adapted and Tethered DNA Library 3.4.5 Sequencing IBV by cDNA-PCR Reverse Transcription and Strand Switching Selecting for Full-Length Transcripts by PCR Adapter Addition 3.4.6 Priming and Loading the SpotON Flow Cell 3.4.7 Pooling of Libraries 3.5 Sequence Assembly and Analysis (See Note 1) 3.5.1 Sequence Assembly and Analysis of iSeq 100 Reads 3.5.2 Sequence Assembly and Analysis of MinION Reads 4 Notes References Chapter 23: Phylogenetic and Recombination Analysis of Animal Coronaviruses 1 Introduction 2 Materials 2.1 Database 2.2 Sequence Format Converter 2.2.1 Software 2.2.2 Web Server 2.3 Multiple Sequence Alignment (MSA) 2.3.1 Software 2.3.2 Web Server 2.4 Model Test 2.4.1 Software 2.4.2 Web Server 2.5 Phylogeny Inference 2.5.1 Software 2.5.2 Web Server 2.6 Tree Visualization and Annotation 2.7 Recombination Analysis 3 Methods 3.1 Building the Sequence Dataset 3.2 Multiple Sequence Alignment 3.3 Alignment Optimization 3.4 Model Test 3.4.1 Model Test by MEGA X 3.4.2 Model Test by jModelTest 3.4.3 Model Test by SMS (Smart Model Selection) 3.5 Sequence Format Conversion 3.6 Phylogeny Inference and Statistical Support of Tree Topology 3.6.1 Distance-Based Phylogeny Neighbor-Joining Phylogeny by MEGA Neighbor-Joining Phylogeny by Seaview 3.6.2 Character-Based Phylogeny Maximum Parsimony Phylogeny by MEGA Maximum Parsimony Phylogeny by Seaview Maximum Likelihood Phylogeny by MEGA Maximum Likelihood Phylogeny by Seaview Maximum Likelihood Phylogeny by IQ-TREE 3.6.3 Bayesian Method Bayesian Inference by MrBayes Bayesian Inference by MrBayes Server 3.7 Phylogenetic Tree Labeling and Re-rooting the Outgroup 3.7.1 Display Statistical Values of Phylogenetic Topology 3.7.2 Highlight a Virus 3.7.3 Re-root a Phylogeny 3.8 Recombination Analysis 4 Notes References Index