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Characterization of the Matrix Proteins of the Fish Rhabdovirus, Infectious Hematopoietic Necrosis Virus

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Characterization of the Matrix Proteins of the Fish Rhabdovirus, Infectious Hematopoietic Necrosis Virus AN ABSTRACT OF THE THESIS OF Patricia A. Ormonde for the degree of Master of Science presented on April 14. 1995. Title: Characterization of the Matrix Proteins of the Fish Rhabdovinis, Infectious Hematopoietic Necrosis Virus. Redacted for Privacy Abstract approved: Jo-Ann C. ong Infectious hematopoietic necrosis virus (1HNV) is an important fish pathogen enzootic in salmon and trout populations of the Pacific Northwestern United States. Occasional epizootics in fish hatcheries can result in devastating losses of fish stocks. The complete nucleotide sequence of IHNV has not yet been determined. This knowledge is the first step towards understanding the roles viral proteins play in IHNV infection, and is necessary for determining the relatedness of IHNV to other rhabdoviruses. The glycoprotein, nucleocapsid and non-virion genes of IHNV have been described previously; however, at the initiation of this study, very little was known about the matrix protein genes. Rhabdoviral matrix proteins have been found to be important in viral transcription and virion assembly. This thesis describes the preliminary characterization of the M1 and M2 matrix proteins of IHNV. In addition, the trout humoral immune response to M1 and M2 proteins expressed from plasmid DNA injected into the fish was investigated. This work may prove useful in designing future vaccines against IHN. The sequences of M1 phosphoprotein and M2 matrix protein genes of IHNV were determined from both genomic and mRNA clones. Analysis of the sequences indicated that the predicted open reading frame of M1 gene encoded a 230 amino acid protein with a estimated molecular weight of 25.6 kDa. Further analysis revealed a second open reading frame encoding a 42 amino acid protein with a calculated molecular weight of 4.8 kDa. This second protein is highly basic and arginine rich, reminiscent of the small proteins encoded within the phosphoprotein gene of vesicular stomatitis virus (VSV) and rabies virus (RV). The putative M2 gene open reading frame encoded a 195 amino acid protein with a calculated weight of 22 kDa. In addition, the intergenic regions of the fish rhabdoviruses were compared. From the alingments of the intergenic sequence, the probable consensus transcriptional initiation signal for IHNV mRNA was determined. The amino acid sequences of the IHNV matrix proteins share sequence homology with that of the hirame rhabdovirus (HRV), viral hemorrhagic septicemia virus (VHSV) and no other rhabdoviruses for which there are available sequence data. Since previous studies indicated that the M1 phosphoprotein and M2 matrix proteins could act as immunological adjuvants, plasmids were constructed for the expression of M1 or M2. The genetic immunization was carried out by direct injection of the plasmid DNA into rainbow trout. The specificity of the antibody response to the matrix proteins was determined by western blotting and serum antibody levels were monitored by enzyme-linked immunosorbant assays. CHARACTERIZATION OF THE MATRIX PROTEINS OF THE FISH RHABDOVIRUS, INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS by Patricia A. Ormonde A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Completed April 14, 1995 Commencement June, 1995 Master of Science thesis of Patricia A. Ormonde presented on April 14, 1995 APPROVED: Redacted for Privacy Maj ofessor, representing Microbiology Redacted for Privacy Chair of Department of Microbiology Redacted for Privacy Dean of Gr I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Redacted for Privacy Patricia A. Ormonde, Author TABLE OF CONTENTS Chapter Page 1. Introduction 1 2. Literature Review 3 Infectious Hematopoietic Necrosis Virus 3 Rhabdovirus Matrix Proteins 9 3. Sequence Analysis of the Matrix Protein Genes of the Fish Rhabdovirus, Infectious Hematopoietic Necrosis Virus. 13 Abstract. 14 Introduction 14 Materials and Methods 16 Results 18 Discussion 31 References 42 4. Humoral Immune Response in Rainbow trout (Oncorhynchus mykiss) Following Injection with Plasmids Encoding the Ml and M2 Protein Genes of Infectious Hematopoietic Necrosis Virus 45 Abstract. 46 Introduction 46 Materials and Methods 48 Results and Discussion 54 References 61 5. Summary 63 BIBLIOGRAPHY 65 LIST OF FIGURES Figure Page 3.1 Nucleotide and deduced amino acid sequence of the IHNV (RBI) M1 phosphoprotein 21 3.2 Alignment of deduced amino acid sequences of IHNV (RB1), HRV and VHSV (07-71) phosphoproteins 23 3.3 Nucleotide and deduced amino acid sequence of the IHNV (RB1) matrix protein 27 3.4 Alignment of deduced amino acid sequences of IHNV (RB1), HRV and VHSV (07-71) matrix proteins 29 3.5 Predicted secondary structure of IHNV M1 (RB1) 33 3.6 Predicted secondary structure of IHNV M2 (RB1) 35 3.7 The phylogenetic tree for the phosphoproteins of the rhabdoviruses 38 3.8 The phylogenetic tree for the matrix proteins of the rhabdoviruses 40 4.1 Auto radiograph of in vitro translation products from pCR3-M1, pCR3-M2 55 4.2 Immunoblot analysis of the in vitro translation products 57 LIST OF TABLES Table Page 2.1 Classification scheme of the Rhabdoviridae family 6 3.1 Deoxynucleotide primers used in sequencing and PCR reactions 17 3.2 Nucleotide sequence alignments of the intergenic regions of various fish rhabdoviruses 19 3.3 Percentage similarity and identity of amino acid sequences between IHNV Ml and other rhabdovirus phosphoproteins 25 3.4 Percentage similarity and identity of amino acid sequences between IHNV M2 and other rhabdovirus matrix proteins 31 CHARACTERIZATION OF THE MATRIX PROTEINS OF THE FISH RHABDOVIRUS, INFECTOUS HEMATOPOIETIC NECROSIS VIRUS CHAPTER 1 INTRODUCTION Infectious hematopoietic necrosis virus (IHNV) is an important fish pathogen enzootic in salmon and trout populations of the Pacific Northwestern United States. Occasional epizootics in fish hatcheries can result in devastating losses of fish stocks. This thesis describes the characterization of the matrix proteins of IHNV. Sequence analysis of the matrix proteins of IHNV The complete nucleotide sequence of IHNV has not yet been determined. This knowledge is the first step towards understanding the roles viral proteins play in IHNV infection, and is necessary for determining the relatedness of IHNV to other rhabdoviruses. The glycoprotein, nucleocapsid and non-virion genes of IHNV have been described previously; however, at the initiation of this study, very little was known about the matrix protein genes. Rhabdoviral matrix proteins have been found to be important in viral transcription and virion assembly. The third chapter of this thesis describes the preliminary characterization of the Ml and M2 matrix proteins of IHNV. The sequences of Ml phosphoprotein and M2 matrix protein genes of IHNV were determined from both genomic and mRNA clones. Analysis of the sequences indicated that the predicted open reading frame of Ml gene encoded a 230 amino acid protein with a estimated molecular weight of 25.610a. Further analysis revealed a second open reading frame encoding a 42 amino acid protein with a calculated molecular weight of 4.8 kDa. This second protein is highly basic and arginine rich, reminiscent of the small proteins encoded within the phosphoprotein gene of vesicular 2 stomatitis virus (VSV) and rabies virus (RV). The putative M2 gene open reading frame encoded a 195 amino acid protein with a calculated weight of 22 kDa. In addition, the intergenic regions of the fish rhabdoviruses were compared. From the alignments of the intergenic sequence, the probable consensus transcriptional initiation signal for IHNV mRNA was determined. The amino acid sequences of the IHNV matrix proteins share sequence homology with that of the hirame rhabdovirus (HRV), viral hemorrhagic septicemia virus (VHSV) and no other rhabdoviruses for which there are available sequence data. Humoral response in rainbow trout following DNA injection Since previous studies indicated that the Ml phosphoprotein and M2 matrix proteins could act as immunological adjuvants, plasmids were constructed for the expression of M1 or M2. The fourth chapter of this thesis describes the genetic immunization by direct injection of the plasmid DNA into rainbow trout. The specificity of the antibody response to the matrix proteins was determined by immunoblotting and serum antibody levels were monitored by enzyme-linked immunosorbant assays. This work may prove useful in designing future vaccines against IHN. 3 CHAPTER 2 LITERATURE REVIEW Infectious Hematopoietic Necrosis Virus The Rhabdoviridae family is a large, diverse group of single-stranded RNA viruses. Over 150 rhabdoviruses have been described, approximately half of them in animals and the other half in plants (Peters, 1991). Infectious hematopoietic necrosis virus (IHNV), the causative agent of infectious hematopoietic necrosis diseasein salmon and trout, is one of many rhabdoviruses that infect fish. Distribution and host range of IHNV Infectious hematopoietic necrosis virus infects trout and salmon in the Pacific Northwest of the United States and elsewhere. IHNV causes serious disease outbreaks in hatchery-reared trout and salmon and has been spread to other parts of the world by the movement of infected eggs and fry (Hill, 1992). The virus has been isolated from rainbow trout (Oncorhynchus mykiss) in France (Laurencin 1987), Taiwan (Chen et al. 1985), Italy (Bova et al. 1987) and more recently in Belgium (Hill 1992) and Korea (Park et al. 1993). In Japan, IHNV has become a serious pathogen with yearly outbreaks in hatcheries (Fukuda et al. 1992). IHNV is known to infect chinook salmon (0. tshawytscha), sockeye salmon (0. nerka), rainbow trout (0. mykiss), cutthroat trout (0. clarki), Atlantic salmon (Salmo salar) and kokanee salmon or land-locked sockeye salmon (Pi lcher and Fryer 1980; Wolf 1988). The disease is most severe in hatchery- reared fry where losses can sometimes approach 100%. Near yearling and yearling fish are also susceptible, but in older fish the histopathologic changes may be less severe and the mortality rates lower(Wolf 1988). Coho salmon (0.
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