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Wang J, Zheng C, Xiao W. Zebrafish as a Model for Studying Virus-Host Interactions. Methods Mol Biol 2025; 2940:261-272. [PMID: 40515918 DOI: 10.1007/978-1-0716-4615-1_23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/16/2025]
Abstract
During the long evolutionary process, viruses and hosts have engaged in a game of immune escape and viral clearance, resulting in an exquisite model of virus-host interaction. An appropriate animal model can help unravel the mysteries of this process. Owing to their susceptibility to multiple viruses, small size, high fecundity, low cost, ease of visualization, and efficient gene editing, zebrafish (Danio rerio) have recently emerged as a powerful vertebrate model system for studying virus-host interactions. In this chapter, we describe in detail the experimental protocols for analyzing virus-host interactions in a zebrafish model in which spring viremia of carp virus (SVCV) is used as an example. These protocols include the generation of knockout zebrafish via CRISPR/Cas9 technology, methods for viral infection of zebrafish larvae or adults, analysis of in vivo viral resistance of host genes, analysis of host and viral gene expression in zebrafish larvae via qRT-PCR and Western blotting, and detection of host protein binding to viral proteins via immunoprecipitation. These experimental protocols provide an effective reference for studying virus-host interactions in a zebrafish model.
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Affiliation(s)
- Jing Wang
- Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture, Institute of Hydrobiology, Chinese Academy of Sciences, Hubei Hongshan Laboratory, Wuhan, China
- University of Chinese Academy of Sciences, Beijing, China
| | - Chunfu Zheng
- University of Chinese Academy of Sciences, Beijing, China
| | - Wuhan Xiao
- Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture, Institute of Hydrobiology, Chinese Academy of Sciences, Hubei Hongshan Laboratory, Wuhan, China.
- University of Chinese Academy of Sciences, Beijing, China.
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2
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Wu XM, Zheng SY, Chang MX. Zebrafish as a Model for Investigating Antiviral Innate Immunity. Methods Mol Biol 2025; 2854:221-236. [PMID: 39192133 DOI: 10.1007/978-1-0716-4108-8_22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/29/2024]
Abstract
Zebrafish is a widely used model organism in genetics, developmental biology, pathology, and immunology research. Due to their fast reproduction, large numbers, transparent early embryos, and high genetic conservation with the human genome, zebrafish have been used as a model for studying human and fish viral diseases. In particular, the ability to easily perform forward and reverse genetics and lacking a functional adaptive immune response during the early period of development establish the zebrafish as a favored option to assess the functional implication of specific genes in the antiviral innate immune response and the pathogenesis of viral diseases. In this chapter, we detail protocols for the antiviral innate immunity analysis using the zebrafish model, including the generation of gene-overexpression zebrafish, generation of gene-knockout zebrafish by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, methods of viral infection in zebrafish larvae, analyzing the expression of antiviral genes in zebrafish larvae using qRT-PCR, Western blotting and transcriptome sequencing, and in vivo antiviral assays. These experimental protocols provide effective references for studying the antiviral immune response in the zebrafish model.
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Affiliation(s)
- Xiao Man Wu
- Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture (CAS), Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China
| | - Si Yao Zheng
- Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture (CAS), Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China
- College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China
| | - Ming Xian Chang
- Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture (CAS), Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China.
- College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China.
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3
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Fajar S, Dwi SP, Nur IS, Wahyu AP, Sukamto S M, Winda AR, Nastiti W, Andri F, Firzan N. Zebrafish as a model organism for virus disease research: Current status and future directions. Heliyon 2024; 10:e33865. [PMID: 39071624 PMCID: PMC11282986 DOI: 10.1016/j.heliyon.2024.e33865] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2023] [Revised: 06/22/2024] [Accepted: 06/28/2024] [Indexed: 07/30/2024] Open
Abstract
Zebrafish (Danio rerio) have emerged as valuable models for investigating viral infections, providing insights into viral pathogenesis, host responses, and potential therapeutic interventions. This review offers a comprehensive synthesis of research on viral infections using zebrafish models, focusing on the molecular mechanisms of viral action and host-virus interactions. Zebrafish models have been instrumental in elucidating the replication dynamics, tissue tropism, and immune evasion strategies of various viruses, including Chikungunya virus, Dengue virus, Herpes Simplex Virus type 1, and Influenza A virus. Additionally, studies utilizing zebrafish have evaluated the efficacy of antiviral compounds and natural agents against emerging viruses such as SARS-CoV-2, Zika virus, and Dengue virus. The optical transparency and genetic tractability of zebrafish embryos enable real-time visualization of viral infections, facilitating the study of viral spread and immune responses. Despite challenges such as temperature compatibility and differences in host receptors, zebrafish models offer unique advantages, including cost-effectiveness, high-throughput screening capabilities, and conservation of key immune pathways. Importantly, zebrafish models complement existing animal models, providing a platform for rapid evaluation of potential therapeutics and a deeper understanding of viral pathogenesis. This review underscores the significance of zebrafish research in advancing our understanding of viral diseases and highlights future research directions to combat infectious diseases effectively.
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Affiliation(s)
- Sofyantoro Fajar
- Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
| | - Sendi Priyono Dwi
- Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
| | | | | | - Mamada Sukamto S
- Department of Pharmacy, Faculty of Pharmacy, Hasanuddin University, Makassar, 90245, Indonesia
| | | | - Wijayanti Nastiti
- Faculty of Biology, Universitas Gadjah Mada, Yogyakarta, 55281, Indonesia
| | - Frediansyah Andri
- Research Center for Food Technology and Processing (PRTPP), National Research and Innovation Agency (BRIN), Yogyakarta 55861, Indonesia
| | - Nainu Firzan
- Department of Pharmacy, Faculty of Pharmacy, Hasanuddin University, Makassar, 90245, Indonesia
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Bastos TSB, de Paula AGP, Dos Santos Luz RB, Garnique AMB, Belo MAA, Eto SF, Fernandes DC, Ferraris FK, de Pontes LG, França TT, Barcellos LJG, Veras FP, Bermejo P, Guidelli G, Maneira C, da Silveira Bezerra de Mello F, Teixeira G, Pereira GAG, Fernandes BHV, Sanches PRS, Braz HLB, Jorge RJB, Malafaia G, Cilli EM, Olivier DDS, do Amaral MS, Medeiros RJ, Condino-Neto A, Carvalho LR, Machado-Santelli GM, Charlie-Silva I, Galindo-Villegas J, Braga TT. A novel insight on SARS-CoV-2 S-derived fragments in the control of the host immunity. Sci Rep 2023; 13:8060. [PMID: 37198208 PMCID: PMC10191404 DOI: 10.1038/s41598-023-29588-8] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2022] [Accepted: 02/07/2023] [Indexed: 05/19/2023] Open
Abstract
Despite all efforts to combat the pandemic of COVID-19, we are still living with high numbers of infected persons, an overburdened health care system, and the lack of an effective and definitive treatment. Understanding the pathophysiology of the disease is crucial for the development of new technologies and therapies for the best clinical management of patients. Since the manipulation of the whole virus requires a structure with an adequate level of biosafety, the development of alternative technologies, such as the synthesis of peptides from viral proteins, is a possible solution to circumvent this problem. In addition, the use and validation of animal models is of extreme importance to screen new drugs and to compress the organism's response to the disease. Peptides derived from recombinant S protein from SARS-CoV-2 were synthesized and validated by in silico, in vitro and in vivo methodologies. Macrophages and neutrophils were challenged with the peptides and the production of inflammatory mediators and activation profile were evaluated. These peptides were also inoculated into the swim bladder of transgenic zebrafish larvae at 6 days post fertilization (dpf) to mimic the inflammatory process triggered by the virus, which was evaluated by confocal microscopy. In addition, toxicity and oxidative stress assays were also developed. In silico and molecular dynamics assays revealed that the peptides bind to the ACE2 receptor stably and interact with receptors and adhesion molecules, such as MHC and TCR, from humans and zebrafish. Macrophages stimulated with one of the peptides showed increased production of NO, TNF-α and CXCL2. Inoculation of the peptides in zebrafish larvae triggered an inflammatory process marked by macrophage recruitment and increased mortality, as well as histopathological changes, similarly to what is observed in individuals with COVID-19. The use of peptides is a valuable alternative for the study of host immune response in the context of COVID-19. The use of zebrafish as an animal model also proved to be appropriate and effective in evaluating the inflammatory process, comparable to humans.
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Affiliation(s)
| | | | | | - Anali M B Garnique
- Department of Cell Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
| | | | - Silas Fernandes Eto
- Center of Excellence in New Target Discovery (CENTD) Special Laboratory, Butantan Institute, São Paulo, Brazil
- Center of Innovation and Development, Laboratory of Development and Innovation, Butantan Institute, São Paulo, Brazil
| | | | - Fausto Klabund Ferraris
- Department of Pharmacology and Toxicology, Oswaldo Cruz Foundation, FIOCRUZ, Rio de Janeiro, Brazil
| | - Leticia Gomes de Pontes
- Laboratory of Human Immunology, Department Immunology, Institute Biomedical Sciences, University São Paulo, São Paulo, Brazil
| | - Tábata Takahashi França
- Laboratory of Human Immunology, Department Immunology, Institute Biomedical Sciences, University São Paulo, São Paulo, Brazil
| | - Leonardo José Gil Barcellos
- Laboratory of Fish Physiology, Graduate Program of Bioexperimentation, University of Passo Fundo, Santa Maria, Brazil
- Graduate Program of Pharmacology, Federal University of Santa Maria, Santa Maria, Brazil
| | - Flavio P Veras
- Center of Research in Inflammatory Diseases, Ribeirão Preto Medical School, University of Sao Paulo, Ribeirão Preto, São Paulo, Brazil
- Department of Pharmacology, Ribeirao Preto Medical School, University of São Paulo, Ribeirao Preto, São Paulo, Brazil
| | - Pamela Bermejo
- Laboratório de Genômica e bioEnergia (LGE), Institute of Biology - Unicamp, Campinas, Brazil
| | - Giovanna Guidelli
- Laboratório de Genômica e bioEnergia (LGE), Institute of Biology - Unicamp, Campinas, Brazil
| | - Carla Maneira
- Laboratório de Genômica e bioEnergia (LGE), Institute of Biology - Unicamp, Campinas, Brazil
| | | | - Gleidson Teixeira
- Laboratório de Genômica e bioEnergia (LGE), Institute of Biology - Unicamp, Campinas, Brazil
| | | | - Bianca H Ventura Fernandes
- Laboratório de Controle Genético e Sanitário, Diretoria Técnica de Apoio ao Ensino e Pesquisa, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
| | - Paulo R S Sanches
- Instituto de Química, Universidade Estadual Paulista, Araraquara, SP, Brazil
| | - Helyson Lucas Bezerra Braz
- Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Fortaleza, CE, Brazil
| | - Roberta Jeane Bezerra Jorge
- Department of Physiology and Pharmacology, School of Medicine, Federal University of Ceará, Fortaleza, CE, Brazil
| | - Guilherme Malafaia
- Biological Research Laboratory, Goiano Federal Institute, Urutai Campus, Urutaí, GO, Brazil
| | - Eduardo M Cilli
- Instituto de Química, Universidade Estadual Paulista, Araraquara, SP, Brazil
| | | | - Marcos Serrou do Amaral
- Institute of Physics, Federal University of Mato Grosso do Sul, Campo Grande, MS, 79070-900, Brazil
| | - Renata J Medeiros
- Laboratory of Physiology, INCQS/Fiocruz Zebrafish Facility, Department of Pharmacology and Toxicology, National Institute for Quality Control in Health, Rio de Janeiro, Brazil
| | - Antonio Condino-Neto
- Laboratory of Human Immunology, Department Immunology, Institute Biomedical Sciences, University São Paulo, São Paulo, Brazil
| | - Luciani R Carvalho
- Laboratório de Controle Genético e Sanitário, Diretoria Técnica de Apoio ao Ensino e Pesquisa, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil
| | - Glaucia M Machado-Santelli
- Laboratory of Cellular and Molecular Biology, Department of Cell and Developmental Biology, Institute of Biomedical Science, University of Sao Paulo, University of São Paulo, São Paulo, Brazil
| | - Ives Charlie-Silva
- Department of Pharmacology, University of São Paulo-ICB/USP, São Paulo, Brazil.
| | - Jorge Galindo-Villegas
- Department of Genomics, Faculty of Biosciences and Aquaculture, Nord University, Bodø, Norway.
| | - Tárcio Teodoro Braga
- Department of Pathology, Federal University of Parana, Curitiba, Brazil.
- Graduate Program in Biosciences and Biotechnology, Instituto Carlos Chagas, Fiocruz-Parana, Brazil.
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Maleski ALA, Rosa JGS, Bernardo JTG, Astray RM, Walker CIB, Lopes-Ferreira M, Lima C. Recapitulation of Retinal Damage in Zebrafish Larvae Infected with Zika Virus. Cells 2022; 11:1457. [PMID: 35563763 PMCID: PMC9100881 DOI: 10.3390/cells11091457] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2022] [Revised: 03/24/2022] [Accepted: 04/07/2022] [Indexed: 02/06/2023] Open
Abstract
Zebrafish are increasingly being utilized as a model to investigate infectious diseases and to advance the understanding of pathogen-host interactions. Here, we take advantage of the zebrafish to recapitulate congenital ZIKV infection and, for the first time, demonstrate that it can be used to model infection and reinfection and monitor anti-viral and inflammatory immune responses, as well as brain growth and eye abnormalities during embryonic development. By injecting a Brazilian strain of ZIKV into the yolk sac of one-cell stage embryos, we confirmed that, after 72 h, ZIKV successfully infected larvae, and the physical condition of the virus-infected hosts included gross morphological changes in surviving embryos (84%), with a reduction in larval head size and retinal damage characterized by increased thickness of the lens and inner nuclear layer. Changes in locomotor activity and the inability to perceive visual stimuli are a result of changes in retinal morphology caused by ZIKV. Furthermore, we demonstrated the ability of ZIKV to replicate in zebrafish larvae and infect new healthy larvae, impairing their visual and neurological functions. These data reinforce the deleterious activity of ZIKV in the brain and visual structures and establish the zebrafish as a model to study the molecular mechanisms involved in the pathology of the virus.
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Affiliation(s)
- Adolfo Luis Almeida Maleski
- Immunoregulation Unit of the Laboratory of Applied Toxinology (CeTICs/FAPESP), Butantan Institute, São Paulo 05503-900, Brazil; (A.L.A.M.); (J.G.S.R.); (J.T.G.B.); (M.L.-F.)
- Laboratory of Neuropharmacological Studies (LABEN), Post-Graduation Program of Pharmaceutical Science, Federal University of Sergipe, São Paulo 05503-009, Brazil;
| | - Joao Gabriel Santos Rosa
- Immunoregulation Unit of the Laboratory of Applied Toxinology (CeTICs/FAPESP), Butantan Institute, São Paulo 05503-900, Brazil; (A.L.A.M.); (J.G.S.R.); (J.T.G.B.); (M.L.-F.)
| | - Jefferson Thiago Gonçalves Bernardo
- Immunoregulation Unit of the Laboratory of Applied Toxinology (CeTICs/FAPESP), Butantan Institute, São Paulo 05503-900, Brazil; (A.L.A.M.); (J.G.S.R.); (J.T.G.B.); (M.L.-F.)
| | | | - Cristiani Isabel Banderó Walker
- Laboratory of Neuropharmacological Studies (LABEN), Post-Graduation Program of Pharmaceutical Science, Federal University of Sergipe, São Paulo 05503-009, Brazil;
| | - Monica Lopes-Ferreira
- Immunoregulation Unit of the Laboratory of Applied Toxinology (CeTICs/FAPESP), Butantan Institute, São Paulo 05503-900, Brazil; (A.L.A.M.); (J.G.S.R.); (J.T.G.B.); (M.L.-F.)
| | - Carla Lima
- Immunoregulation Unit of the Laboratory of Applied Toxinology (CeTICs/FAPESP), Butantan Institute, São Paulo 05503-900, Brazil; (A.L.A.M.); (J.G.S.R.); (J.T.G.B.); (M.L.-F.)
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6
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Sullivan C, Soos BL, Millard PJ, Kim CH, King BL. Modeling Virus-Induced Inflammation in Zebrafish: A Balance Between Infection Control and Excessive Inflammation. Front Immunol 2021; 12:636623. [PMID: 34025644 PMCID: PMC8138431 DOI: 10.3389/fimmu.2021.636623] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2020] [Accepted: 04/21/2021] [Indexed: 12/16/2022] Open
Abstract
The inflammatory response to viral infection in humans is a dynamic process with complex cell interactions that are governed by the immune system and influenced by both host and viral factors. Due to this complexity, the relative contributions of the virus and host factors are best studied in vivo using animal models. In this review, we describe how the zebrafish (Danio rerio) has been used as a powerful model to study host-virus interactions and inflammation by combining robust forward and reverse genetic tools with in vivo imaging of transparent embryos and larvae. The innate immune system has an essential role in the initial inflammatory response to viral infection. Focused studies of the innate immune response to viral infection are possible using the zebrafish model as there is a 4-6 week timeframe during development where they have a functional innate immune system dominated by neutrophils and macrophages. During this timeframe, zebrafish lack a functional adaptive immune system, so it is possible to study the innate immune response in isolation. Sequencing of the zebrafish genome has revealed significant genetic conservation with the human genome, and multiple studies have revealed both functional conservation of genes, including those critical to host cell infection and host cell inflammatory response. In addition to studying several fish viruses, zebrafish infection models have been developed for several human viruses, including influenza A, noroviruses, chikungunya, Zika, dengue, herpes simplex virus type 1, Sindbis, and hepatitis C virus. The development of these diverse viral infection models, coupled with the inherent strengths of the zebrafish model, particularly as it relates to our understanding of macrophage and neutrophil biology, offers opportunities for far more intensive studies aimed at understanding conserved host responses to viral infection. In this context, we review aspects relating to the evolution of innate immunity, including the evolution of viral pattern recognition receptors, interferons and interferon receptors, and non-coding RNAs.
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Affiliation(s)
- Con Sullivan
- College of Arts and Sciences, University of Maine at Augusta, Bangor, ME, United States
| | - Brandy-Lee Soos
- Department of Molecular and Biomedical Sciences, University of Maine, Orono, ME, United States
| | - Paul J. Millard
- Department of Environmental and Sustainable Engineering, University at Albany, Albany, NY, United States
| | - Carol H. Kim
- Department of Biomedical Sciences, University at Albany, Albany, NY, United States
- Department of Biological Sciences, University at Albany, Albany, NY, United States
| | - Benjamin L. King
- Department of Molecular and Biomedical Sciences, University of Maine, Orono, ME, United States
- Graduate School of Biomedical Science and Engineering, University of Maine, Orono, ME, United States
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7
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Bove G, Mehnert AK, Dao Thi VL. iPSCs for modeling hepatotropic pathogen infections. IPSCS FOR STUDYING INFECTIOUS DISEASES 2021:149-213. [DOI: 10.1016/b978-0-12-823808-0.00013-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2025]
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Bailone RL, Fukushima HCS, Ventura Fernandes BH, De Aguiar LK, Corrêa T, Janke H, Grejo Setti P, Roça RDO, Borra RC. Zebrafish as an alternative animal model in human and animal vaccination research. Lab Anim Res 2020; 36:13. [PMID: 32382525 PMCID: PMC7203993 DOI: 10.1186/s42826-020-00042-4] [Citation(s) in RCA: 67] [Impact Index Per Article: 13.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2020] [Accepted: 03/19/2020] [Indexed: 02/07/2023] Open
Abstract
Much of medical research relies on animal models to deepen knowledge of the causes of animal and human diseases, as well as to enable the development of innovative therapies. Despite rodents being the most widely used research model worldwide, in recent decades, the use of the zebrafish (Danio rerio) model has exponentially been adopted among the scientific community. This is because such a small tropical freshwater teleost fish has crucial genetic, anatomical and physiological homology with mammals. Therefore, zebrafish constitutes an excellent experimental model for behavioral, genetic and toxicological studies which unravels the mechanism of various human diseases. Furthermore, it serves well to test new therapeutic agents, such as the safety of new vaccines. The aim of this review was to provide a systematic literature review on the most recent studies carried out on the topic. It presents numerous advantages of this type of animal model in tests of efficacy and safety of both animal and human vaccines, thus highlighting gains in time and cost reduction of research and analyzes.
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Affiliation(s)
- Ricardo Lacava Bailone
- Ministry of Agriculture, Livestock and Supply, Federal Inspection Service, São Carlos, SP Brazil
- São Paulo State University, Botucatu, SP Brazil
| | - Hirla Costa Silva Fukushima
- Health and Biological Sciences Center, Federal University, Federal University of São Carlos, São Carlos, SP Brazil
| | | | - Luís Kluwe De Aguiar
- Department of Food Technology and Innovation, Harper Adams University, Newport, UK
| | - Tatiana Corrêa
- Department of Genetic and Evolution, Federal University of São Carlos, São Carlos, SP Brazil
| | - Helena Janke
- Department of Genetic and Evolution, Federal University of São Carlos, São Carlos, SP Brazil
| | - Princia Grejo Setti
- Department of Genetic and Evolution, Federal University of São Carlos, São Carlos, SP Brazil
| | | | - Ricardo Carneiro Borra
- Department of Genetic and Evolution, Federal University of São Carlos, São Carlos, SP Brazil
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9
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Chua SCJH, Tan HQ, Engelberg D, Lim LHK. Alternative Experimental Models for Studying Influenza Proteins, Host-Virus Interactions and Anti-Influenza Drugs. Pharmaceuticals (Basel) 2019; 12:E147. [PMID: 31575020 PMCID: PMC6958409 DOI: 10.3390/ph12040147] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2019] [Revised: 09/11/2019] [Accepted: 09/12/2019] [Indexed: 12/14/2022] Open
Abstract
Ninety years after the discovery of the virus causing the influenza disease, this malady remains one of the biggest public health threats to mankind. Currently available drugs and vaccines only partially reduce deaths and hospitalizations. Some of the reasons for this disturbing situation stem from the sophistication of the viral machinery, but another reason is the lack of a complete understanding of the molecular and physiological basis of viral infections and host-pathogen interactions. Even the functions of the influenza proteins, their mechanisms of action and interaction with host proteins have not been fully revealed. These questions have traditionally been studied in mammalian animal models, mainly ferrets and mice (as well as pigs and non-human primates) and in cell lines. Although obviously relevant as models to humans, these experimental systems are very complex and are not conveniently accessible to various genetic, molecular and biochemical approaches. The fact that influenza remains an unsolved problem, in combination with the limitations of the conventional experimental models, motivated increasing attempts to use the power of other models, such as low eukaryotes, including invertebrate, and primary cell cultures. In this review, we summarized the efforts to study influenza in yeast, Drosophila, zebrafish and primary human tissue cultures and the major contributions these studies have made toward a better understanding of the disease. We feel that these models are still under-utilized and we highlight the unique potential each model has for better comprehending virus-host interactions and viral protein function.
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Affiliation(s)
- Sonja C J H Chua
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore.
- NUS Immunology Program, Life Sciences Institute, National University of Singapore, Singapore 117456, Singapore.
- CREATE-NUS-HUJ Molecular Mechanisms of Inflammatory Diseases Programme, National University of Singapore, Singapore 138602, Singapore.
| | - Hui Qing Tan
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore.
- NUS Immunology Program, Life Sciences Institute, National University of Singapore, Singapore 117456, Singapore.
| | - David Engelberg
- CREATE-NUS-HUJ Molecular Mechanisms of Inflammatory Diseases Programme, National University of Singapore, Singapore 138602, Singapore.
- Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545, Singapore.
- Department of Biological Chemistry, The Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel.
| | - Lina H K Lim
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117593, Singapore.
- NUS Immunology Program, Life Sciences Institute, National University of Singapore, Singapore 117456, Singapore.
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10
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Wrighton PJ, Oderberg IM, Goessling W. There Is Something Fishy About Liver Cancer: Zebrafish Models of Hepatocellular Carcinoma. Cell Mol Gastroenterol Hepatol 2019; 8:347-363. [PMID: 31108233 PMCID: PMC6713889 DOI: 10.1016/j.jcmgh.2019.05.002] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/11/2019] [Revised: 05/03/2019] [Accepted: 05/03/2019] [Indexed: 12/16/2022]
Abstract
The incidence of hepatocellular carcinoma (HCC) and the mortality resulting from HCC are both increasing. Most patients with HCC are diagnosed at advanced stages when curative treatments are impossible. Current drug therapy extends mean overall survival by only a short period of time. Genetic mutations associated with HCC vary widely. Therefore, transgenic and mutant animal models are needed to investigate the molecular effects of specific mutations, classify them as drivers or passengers, and develop targeted treatments. Cirrhosis, however, is the premalignant state common to 90% of HCC patients. Currently, no specific therapies are available to halt or reverse the progression of cirrhosis to HCC. Understanding the genetic drivers of HCC as well as the biochemical, mechanical, hormonal, and metabolic changes associated with cirrhosis could lead to novel treatments and cancer prevention strategies. Although additional therapies recently received Food and Drug Administration approval, significant clinical breakthroughs have not emerged since the introduction of the multikinase inhibitor sorafenib, necessitating alternate research strategies. Zebrafish (Danio rerio) are effective for disease modeling because of their high degree of gene and organ architecture conservation with human beings, ease of transgenesis and mutagenesis, high fecundity, and low housing cost. Here, we review zebrafish models of HCC and identify areas on which to focus future research efforts to maximize the advantages of the zebrafish model system.
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Affiliation(s)
- Paul J Wrighton
- Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts
| | - Isaac M Oderberg
- Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts
| | - Wolfram Goessling
- Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts; Harvard Stem Cell Institute, Cambridge, Massachusetts; Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts; Broad Institute, Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts; Division of Health Sciences and Technology, Harvard and Massachusetts Institute of Technology, Boston, Massachusetts; Division of Gastroenterology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts.
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11
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Cazorla-Vázquez S, Steingruber M, Marschall M, Engel FB. Human cytomegaloviral multifunctional protein kinase pUL97 impairs zebrafish embryonic development and increases mortality. Sci Rep 2019; 9:7219. [PMID: 31076608 PMCID: PMC6510723 DOI: 10.1038/s41598-019-43649-x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2018] [Accepted: 04/26/2019] [Indexed: 12/27/2022] Open
Abstract
Cytomegalovirus is a worldwide-distributed human pathogen, which is the leading cause of congenital virus infection, affecting 0.5 to 2% of live births. To date, it is largely unclear which molecular mechanisms underlie the symptomatic outcomes. This is mainly due to species specificity and limited homology among cytomegalovirus genomes. As it is not possible to infect model organisms with human cytomegalovirus, the aim of this study was to develop a heterologous system allowing in the future the elucidation of the pathological role of individual viral proteins. As a model organism the zebrafish has been chosen due to its ease of manipulation and characterization as well as its large offspring. As cytomegalovirus model protein, pUL97 was characterized because it is multiply involved in virus-host interaction. Here, we show in zebrafish embryos, that (i) pUL97 can be expressed in zebrafish, (ii) increasing pUL97 expression levels quantitatively correlate with both minor and major pathological defects, (iii) pUL97 expression impairs cell cycle progression and induces cell death, (iv) active pUL97, but not an inactive mutant, induces excess mortality, and (v) co-administration of a pUL97 inhibitor reduces embryonic pathology. Collectively, these data indicate the suitability of zebrafish to elucidate the pathological role of human cytomegaloviral proteins.
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Affiliation(s)
- Salvador Cazorla-Vázquez
- Experimental Renal and Cardiovascular Research, Department of Nephropathology, Institute of Pathology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Schwabachanlage 12, 91052, Erlangen, Germany
| | - Mirjam Steingruber
- Institute for Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Schlossgarten 4, 91054, Erlangen, Germany
| | - Manfred Marschall
- Institute for Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Schlossgarten 4, 91054, Erlangen, Germany
| | - Felix B Engel
- Experimental Renal and Cardiovascular Research, Department of Nephropathology, Institute of Pathology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Schwabachanlage 12, 91052, Erlangen, Germany.
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12
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Burm R, Collignon L, Mesalam AA, Meuleman P. Animal Models to Study Hepatitis C Virus Infection. Front Immunol 2018; 9:1032. [PMID: 29867998 PMCID: PMC5960670 DOI: 10.3389/fimmu.2018.01032] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2018] [Accepted: 04/25/2018] [Indexed: 12/18/2022] Open
Abstract
With more than 71 million chronically infected people, the hepatitis C virus (HCV) is a major global health concern. Although new direct acting antivirals have significantly improved the rate of HCV cure, high therapy cost, potential emergence of drug-resistant viral variants, and unavailability of a protective vaccine represent challenges for complete HCV eradication. Relevant animal models are required, and additional development remains necessary, to effectively study HCV biology, virus–host interactions and for the evaluation of new antiviral approaches and prophylactic vaccines. The chimpanzee, the only non-human primate susceptible to experimental HCV infection, has been used extensively to study HCV infection, particularly to analyze the innate and adaptive immune response upon infection. However, financial, practical, and especially ethical constraints have urged the exploration of alternative small animal models. These include different types of transgenic mice, immunodeficient mice of which the liver is engrafted with human hepatocytes (humanized mice) and, more recently, immunocompetent rodents that are susceptible to infection with viruses that are closely related to HCV. In this review, we provide an overview of the currently available animal models that have proven valuable for the study of HCV, and discuss their main benefits and weaknesses.
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Affiliation(s)
- Rani Burm
- Laboratory of Liver Infectious Diseases, Department of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine and Health Sciences, Ghent University, Gent, Belgium
| | - Laura Collignon
- Laboratory of Liver Infectious Diseases, Department of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine and Health Sciences, Ghent University, Gent, Belgium
| | - Ahmed Atef Mesalam
- Laboratory of Liver Infectious Diseases, Department of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine and Health Sciences, Ghent University, Gent, Belgium.,Therapeutic Chemistry Department, National Research Centre (NRC), Cairo, Egypt
| | - Philip Meuleman
- Laboratory of Liver Infectious Diseases, Department of Clinical Chemistry, Microbiology and Immunology, Faculty of Medicine and Health Sciences, Ghent University, Gent, Belgium
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13
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Wang J, Zhou B, Ge R, Song TS, Yu J, Xie J. Degradation characterization and pathway analysis of chlortetracycline and oxytetracycline in a microbial fuel cell. RSC Adv 2018; 8:28613-28624. [PMID: 35542450 PMCID: PMC9084353 DOI: 10.1039/c8ra04904a] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2018] [Accepted: 07/31/2018] [Indexed: 11/21/2022] Open
Abstract
The wide presence of antibiotics in the environment has raised concerns about their potential impact on ecological and human health.
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Affiliation(s)
- Ji Wang
- Institute of Botany
- Jiangsu Province and Chinese Academy of Sciences
- Nanjing 210014
- PR China
- State Key Laboratory of Materials-Oriented Chemical Engineering
| | - Boyi Zhou
- State Key Laboratory of Materials-Oriented Chemical Engineering
- Nanjing Tech University
- Nanjing 211816
- PR China
- College of Life Science and Pharmaceutical Engineering
| | | | - Tian-shun Song
- State Key Laboratory of Materials-Oriented Chemical Engineering
- Nanjing Tech University
- Nanjing 211816
- PR China
- College of Life Science and Pharmaceutical Engineering
| | - Jinping Yu
- Institute of Botany
- Jiangsu Province and Chinese Academy of Sciences
- Nanjing 210014
- PR China
| | - Jingjing Xie
- State Key Laboratory of Materials-Oriented Chemical Engineering
- Nanjing Tech University
- Nanjing 211816
- PR China
- College of Life Science and Pharmaceutical Engineering
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14
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Kassar TC, Magalhães T, S JVJ, Carvalho AGO, Silva ANMRDA, Queiroz SRA, Bertani GR, Gil LHVG. Construction and characterization of a recombinant yellow fever virus stably expressing Gaussia luciferase. AN ACAD BRAS CIENC 2017; 89:2119-2130. [PMID: 28746549 DOI: 10.1590/0001-3765201720160196] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2016] [Accepted: 06/20/2016] [Indexed: 11/22/2022] Open
Abstract
Yellow fever is an arthropod-borne viral disease that still poses high public health concerns, despite the availability of an effective vaccine. The development of recombinant viruses is of utmost importance for several types of studies, such as those aimed to dissect virus-host interactions and to search for novel antiviral strategies. Moreover, recombinant viruses expressing reporter genes may greatly facilitate these studies. Here, we report the construction of a recombinant yellow fever virus (YFV) expressing Gaussia luciferase (GLuc) (YFV-GLuc). We show, through RT-PCR, sequencing and measurement of GLuc activity, that stability of the heterologous gene was maintained after six passages. Furthermore, a direct association between GLuc expression and viral replication was observed (r2=0.9967), indicating that measurement of GLuc activity may be used to assess viral replication in different applications. In addition, we evaluated the use of the recombinant virus in an antiviral assay with recombinant human alfa-2b interferon. A 60% inhibition of GLuc expression was observed in cells infected with YFV-GLuc and incubated with IFN alfa-2b. Previously tested on YFV inhibition by plaque assays indicated a similar fold-decrease in viral replication. These results are valuable as they show the stability of YFV-GLuc and one of several possible applications of this construct.
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Affiliation(s)
- Telissa C Kassar
- Departamento de Virologia e Terapia Experimental, Centro de Pesquisas Aggeu Magalhães/CPqAM, Fundação Oswaldo Cruz/FIOCRUZ, Av. Professor Moraes Rego, s/n, Cidade Universitária, 50740-465 Recife, PE, Brazil
| | - Tereza Magalhães
- Departamento de Virologia e Terapia Experimental, Centro de Pesquisas Aggeu Magalhães/CPqAM, Fundação Oswaldo Cruz/FIOCRUZ, Av. Professor Moraes Rego, s/n, Cidade Universitária, 50740-465 Recife, PE, Brazil
| | - José V J S
- Departamento de Virologia e Terapia Experimental, Centro de Pesquisas Aggeu Magalhães/CPqAM, Fundação Oswaldo Cruz/FIOCRUZ, Av. Professor Moraes Rego, s/n, Cidade Universitária, 50740-465 Recife, PE, Brazil
| | - Amanda G O Carvalho
- Departamento de Virologia e Terapia Experimental, Centro de Pesquisas Aggeu Magalhães/CPqAM, Fundação Oswaldo Cruz/FIOCRUZ, Av. Professor Moraes Rego, s/n, Cidade Universitária, 50740-465 Recife, PE, Brazil
| | - Andréa N M R DA Silva
- Departamento de Virologia e Terapia Experimental, Centro de Pesquisas Aggeu Magalhães/CPqAM, Fundação Oswaldo Cruz/FIOCRUZ, Av. Professor Moraes Rego, s/n, Cidade Universitária, 50740-465 Recife, PE, Brazil
| | - Sabrina R A Queiroz
- Departamento de Virologia e Terapia Experimental, Centro de Pesquisas Aggeu Magalhães/CPqAM, Fundação Oswaldo Cruz/FIOCRUZ, Av. Professor Moraes Rego, s/n, Cidade Universitária, 50740-465 Recife, PE, Brazil
| | - Giovani R Bertani
- Departamento de Bioquímica, Universidade Federal de Pernambuco/UFPE, Av. Professor Moraes Rego, s/n, Cidade Universitária, 50670-420 Recife, PE, Brazil
| | - Laura H V G Gil
- Departamento de Virologia e Terapia Experimental, Centro de Pesquisas Aggeu Magalhães/CPqAM, Fundação Oswaldo Cruz/FIOCRUZ, Av. Professor Moraes Rego, s/n, Cidade Universitária, 50740-465 Recife, PE, Brazil
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15
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Luo K, Li Y, Xia L, Hu W, Gao W, Guo L, Tian G, Qi Z, Yuan H, Xu Q. Analysis of the expression patterns of the novel large multigene TRIM gene family (finTRIM) in zebrafish. FISH & SHELLFISH IMMUNOLOGY 2017; 66:224-230. [PMID: 28461211 DOI: 10.1016/j.fsi.2017.04.024] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/01/2017] [Revised: 04/24/2017] [Accepted: 04/27/2017] [Indexed: 06/07/2023]
Abstract
Tripartite motif (TRIM) proteins are receiving increased research interest because of their roles in a wide range of cellular biological processes in innate immunity. In zebrafish (Danio rerio), the functions of the finTRIM (ftr) family are unclear. In the present study, we investigated the expression pattern of ftr12, ftr51, ftr67, ftr82, ftr83, and ftr84 in zebrafish for the first time. The results showed that ftr12, ftr67, and ftr84 are maternally expressed in the oocyte and highly expressed at the early stage (0-4 hpf) of embryo (P < 0.05), suggesting their involvement in the embryonic innate defense system. The ftr82 gene was highly expressed at 8 hpf (P < 0.05), which implied that the embryos could synthesize their own immunity-related mRNAs. However, ftr51 and ftr83 were highest at 8 hpf (2.33 and 51.53 relative to β-actin respectively) and might mediate embryonic development. The expression levels of ftr12, ftr51, and ftr67 were highest in the gill, intestines, and liver, respectively. Ftr82, ftr83, and ftr84 were predominantly expressed in the kidney, suggesting that these finTRIMs might play roles in both immunity and non-immunity-related tissue compartments. Zebrafish embryonic fibroblast (ZF4) cells were infected with Grass carp reovirus (GCRV) and Spring viremia of carp virus (SVCV). During GCRV infection, the expression of ftr12 was significantly upregulated from 12 h to 24 h; and ftr51 and ftr67 increased from 3 h to 12 h. The expressions of ftr82, ftr83, and ftr84 were only upregulated at 12 h, 12 h, and 24 h, respectively. All of these genes were significantly downregulated at 48 h (P < 0.05). Challenge with SVCV upregulated the expressions of ftr12 and ftr51 at 12 h and 48 h (P < 0.05), respectively, and ftr67 reached its highest expression level at 3 h. ftr82 showed only a slight upregulation at 6 h and 48 h, and ftr83 and ftr84 were consecutively increased, reaching their highest levels at 12 h (P < 0.05). Meanwhile, ftr67 and ftr83 were significantly downregulated at 48 h (P < 0.05). Our research demonstrated that ftr12, ftr51, ftr67, ftr82, ftr83, and ftr84 probably have important roles in innate immune responses and in non-immunity-related tissues.
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Affiliation(s)
- Kai Luo
- Engineering Research Centre of Ecology and Agricultural Use of Wetland, Ministry of Education, Jingzhou 434020, China
| | - Youshen Li
- Engineering Research Centre of Ecology and Agricultural Use of Wetland, Ministry of Education, Jingzhou 434020, China
| | - Lihai Xia
- Engineering Research Centre of Ecology and Agricultural Use of Wetland, Ministry of Education, Jingzhou 434020, China
| | - Wei Hu
- Engineering Research Centre of Ecology and Agricultural Use of Wetland, Ministry of Education, Jingzhou 434020, China; School of Animal Science, Yangtze University, Jingzhou 434020, China
| | - Weihua Gao
- Engineering Research Centre of Ecology and Agricultural Use of Wetland, Ministry of Education, Jingzhou 434020, China; School of Animal Science, Yangtze University, Jingzhou 434020, China
| | - Liwei Guo
- Engineering Research Centre of Ecology and Agricultural Use of Wetland, Ministry of Education, Jingzhou 434020, China
| | - Guangming Tian
- Engineering Research Centre of Ecology and Agricultural Use of Wetland, Ministry of Education, Jingzhou 434020, China
| | - Zhitao Qi
- Engineering Research Centre of Ecology and Agricultural Use of Wetland, Ministry of Education, Jingzhou 434020, China
| | - Hanwen Yuan
- College of Marine and Biotechnology, Guangxi University for Nationalities, Nanning, Guangxi 530006, China; Guangxi Key Laboratory of Marine Natural Products and Combinatorial Biosynthesis Chemistry, Nanning, Guangxi 530006, China.
| | - Qiaoqing Xu
- Engineering Research Centre of Ecology and Agricultural Use of Wetland, Ministry of Education, Jingzhou 434020, China; School of Animal Science, Yangtze University, Jingzhou 434020, China.
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16
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Sullivan C, Lage CR, Yoder JA, Postlethwait JH, Kim CH. Evolutionary divergence of the vertebrate TNFAIP8 gene family: Applying the spotted gar orthology bridge to understand ohnolog loss in teleosts. PLoS One 2017; 12:e0179517. [PMID: 28658311 PMCID: PMC5489176 DOI: 10.1371/journal.pone.0179517] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2017] [Accepted: 05/30/2017] [Indexed: 02/06/2023] Open
Abstract
Comparative functional genomic studies require the proper identification of gene orthologs to properly exploit animal biomedical research models. To identify gene orthologs, comprehensive, conserved gene synteny analyses are necessary to unwind gene histories that are convoluted by two rounds of early vertebrate genome duplication, and in the case of the teleosts, a third round, the teleost genome duplication (TGD). Recently, the genome of the spotted gar, a holostean outgroup to the teleosts that did not undergo this third genome duplication, was sequenced and applied as an orthology bridge to facilitate the identification of teleost orthologs to human genes and to enhance the power of teleosts as biomedical models. In this study, we apply the spotted gar orthology bridge to help describe the gene history of the vertebrate TNFAIP8 family. Members of the TNFAIP8 gene family have been linked to regulation of immune function and homeostasis and the development of multiple cancer types. Through a conserved gene synteny analysis, we identified zebrafish orthologs to human TNFAIP8L1 and TNFAIP8L3 genes and two co-orthologs to human TNFAIP8L2, but failed to identify an ortholog to human TNFAIP8. Through the application of the orthology bridge, we determined that teleost orthologs to human TNFAIP8 genes were likely lost in a genome inversion event after their divergence from their common ancestor with spotted gar. These findings demonstrate the value of this enhanced approach to gene history analysis and support the development of teleost models to study complex questions related to an array of biomedical issues, including immunity and cancer.
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Affiliation(s)
- Con Sullivan
- Department of Molecular and Biomedical Sciences, University of Maine, Orono, Maine, United States of America
- Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, Maine, United States of America
| | - Christopher R. Lage
- Program in Biology, University of Maine - Augusta, Augusta, Maine, United States of America
| | - Jeffrey A. Yoder
- Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, North Carolina, United States of America
| | - John H. Postlethwait
- Institute of Neuroscience, University of Oregon, Eugene, Oregon, United States of America
| | - Carol H. Kim
- Department of Molecular and Biomedical Sciences, University of Maine, Orono, Maine, United States of America
- Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, Maine, United States of America
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17
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Modelling viral infections using zebrafish: Innate immune response and antiviral research. Antiviral Res 2017; 139:59-68. [DOI: 10.1016/j.antiviral.2016.12.013] [Citation(s) in RCA: 55] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2016] [Accepted: 12/21/2016] [Indexed: 12/20/2022]
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18
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Abstract
Zebrafish (Danio rerio) has become an increasingly important model for in vivo and in vitro studies on host-pathogen interaction, offering scientists with optical accessibility and genetic tractability, and a vertebrate-type immunity that can be separated into innate and adaptive ones. Although it is shown in previous studies that few species of viruses can naturally infect zebrafish, the spring viraemia of carp virus (SVCV), a rhabdovirus that causes contagious acute hemorrhagic viraemia in a variety of cyprinid fishes, can infect zebrafish by both injection and static immersion methods in laboratory conditions. In addition, SVCV can infect zebrafish fibroblast cell line (ZF4 cells), together with the Epithelioma papulosum cyprini (EPC) cell line (EPC cells), a common cell line used widely in fish disease research. The infection and propagation of SVCV in zebrafish and especially in these cell lines can be employed conveniently in laboratory for functional assays of zebrafish genes. The zebrafish, ZF4 and EPC cell, and SVCV can serve as a simple and efficient model system in understanding host-virus interactions. In the present chapter, we provide detailed protocols for the host-virus interaction analysis based on zebrafish embryos, ZF4/EPC cells, and SVCV, including infection methods of zebrafish embryos and cell lines, analyses of immune responses by quantitative PCR (qPCR) and RNA sequencing (RNA-Seq), antiviral assays based on ZF4 and EPC cells, and the analysis of host-virus interaction using luciferase assays. These protocols should provide efficient and typical means to address host-virus interactions in a more general biological sense.
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Affiliation(s)
- Peng Fei Zou
- College of Fisheries, Jimei University, 43 Yindou Road, Xiamen, Fujian Province, 361021, China
| | - Pin Nie
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei Province, 430072, China.
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19
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Sullivan C, Matty MA, Jurczyszak D, Gabor KA, Millard PJ, Tobin DM, Kim CH. Infectious disease models in zebrafish. Methods Cell Biol 2016; 138:101-136. [PMID: 28129840 DOI: 10.1016/bs.mcb.2016.10.005] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
In recent years, the zebrafish (Danio rerio) has developed as an important alternative to mammalian models for the study of hostpathogen interactions. Because they lack a functional adaptive immune response during the first 4-6weeks of development, zebrafish rely upon innate immune responses to protect against injuries and infections. During this early period of development, it is possible to isolate and study mechanisms of infection and inflammation arising from the innate immune response without the complications presented by the adaptive immune response. Zebrafish possess several inherent characteristics that make them an attractive option to study hostpathogen interactions, including extensive sequence and functional conservation with the human genome, optical clarity in larvae that facilitates the high-resolution visualization of host cell-microbe interactions, a fully sequenced and annotated genome, robust forward and reverse genetic tools and techniques (e.g., CRISPR-Cas9 and TALENs), and amenability to chemical studies and screens. Here, we describe methods for studying hostpathogen interactions both through systemic infections and through localized infections that allow analysis of host cell response, migration patterns, and behavior. Each of the methods described can be modified for use in downstream applications that include ecotoxicant studies and chemical screens.
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Affiliation(s)
- C Sullivan
- University of Maine, Orono, ME, United States
| | - M A Matty
- Duke University School of Medicine, Durham, NC, United States
| | | | - K A Gabor
- National Institute of Environmental Health Sciences, Durham, NC, United States
| | - P J Millard
- University of Maine, Orono, ME, United States
| | - D M Tobin
- Duke University School of Medicine, Durham, NC, United States
| | - C H Kim
- University of Maine, Orono, ME, United States
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20
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Han R, Zhao Q, Zong S, Miao S, Song W, Wang L. A novel TRIM family member, Trim69, regulates zebrafish development through p53-mediated apoptosis. Mol Reprod Dev 2016; 83:442-54. [PMID: 27031046 DOI: 10.1002/mrd.22643] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2015] [Accepted: 03/16/2016] [Indexed: 01/02/2023]
Abstract
Trim69 contains the hallmark domains of a tripartite motif (TRIM) protein, including a Ring-finger domain, B-box domain, and coiled-coil domain. Trim69 is structurally and evolutionarily conserved in zebrafish, mouse, rat, human, and chimpanzee. The role of this protein is unclear, however, so we investigated its function in zebrafish development. Trim69 is extensively expressed in zebrafish adults and developing embryos-particularly in the testis, brain, ovary, and heart-and its expression decreases in a time- and stage-dependent manner. Loss of trim69 in zebrafish induces apoptosis and activates apoptosis-related processes; indeed, the tp53 pathway was up-regulated in response to the knockdown. Expression of human trim69 rescued the apoptotic phenotype, while overexpression of trim69 does not increase cellular apoptosis. Taken together, our results suggest that trim69 participates in tp53-mediated apoptosis during zebrafish development. Mol. Reprod. Dev. 83: 442-454, 2016. © 2016 Wiley Periodicals, Inc.
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Affiliation(s)
- Ruiqin Han
- National State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
| | - Qing Zhao
- National State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
| | - Shudong Zong
- National Research Institute for Family Planning, WHO Collaboration Center of Human Reproduction, Beijing, China
| | - Shiying Miao
- National State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
| | - Wei Song
- National State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
| | - Linfang Wang
- National State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China
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21
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Lu JW, Ho YJ, Yang YJ, Liao HA, Ciou SC, Lin LI, Ou DL. Zebrafish as a disease model for studying human hepatocellular carcinoma. World J Gastroenterol 2015; 21:12042-12058. [PMID: 26576090 PMCID: PMC4641123 DOI: 10.3748/wjg.v21.i42.12042] [Citation(s) in RCA: 42] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/16/2015] [Revised: 05/28/2015] [Accepted: 08/31/2015] [Indexed: 02/07/2023] Open
Abstract
Liver cancer is one of the world’s most common cancers and the second leading cause of cancer deaths. Hepatocellular carcinoma (HCC), a primary hepatic cancer, accounts for 90%-95% of liver cancer cases. The pathogenesis of HCC consists of a stepwise process of liver damage that extends over decades, due to hepatitis, fatty liver, fibrosis, and cirrhosis before developing fully into HCC. Multiple risk factors are highly correlated with HCC, including infection with the hepatitis B or C viruses, alcohol abuse, aflatoxin exposure, and metabolic diseases. Over the last decade, genetic alterations, which include the regulation of multiple oncogenes or tumor suppressor genes and the activation of tumorigenesis-related pathways, have also been identified as important factors in HCC. Recently, zebrafish have become an important living vertebrate model organism, especially for translational medical research. In studies focusing on the biology of cancer, carcinogen induced tumors in zebrafish were found to have many similarities to human tumors. Several zebrafish models have therefore been developed to provide insight into the pathogenesis of liver cancer and the related drug discovery and toxicology, and to enable the evaluation of novel small-molecule inhibitors. This review will focus on illustrative examples involving the application of zebrafish models to the study of human liver disease and HCC, through transgenesis, genome editing technology, xenografts, drug discovery, and drug-induced toxic liver injury.
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