Yellow Head Virus: Transmission and Genome Analyses
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The University of Southern Mississippi The Aquila Digital Community Dissertations Fall 12-2008 Yellow Head Virus: Transmission and Genome Analyses Hongwei Ma University of Southern Mississippi Follow this and additional works at: https://aquila.usm.edu/dissertations Part of the Aquaculture and Fisheries Commons, Biology Commons, and the Marine Biology Commons Recommended Citation Ma, Hongwei, "Yellow Head Virus: Transmission and Genome Analyses" (2008). Dissertations. 1149. https://aquila.usm.edu/dissertations/1149 This Dissertation is brought to you for free and open access by The Aquila Digital Community. It has been accepted for inclusion in Dissertations by an authorized administrator of The Aquila Digital Community. For more information, please contact [email protected]. The University of Southern Mississippi YELLOW HEAD VIRUS: TRANSMISSION AND GENOME ANALYSES by Hongwei Ma Abstract of a Dissertation Submitted to the Graduate Studies Office of The University of Southern Mississippi in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy December 2008 COPYRIGHT BY HONGWEI MA 2008 The University of Southern Mississippi YELLOW HEAD VIRUS: TRANSMISSION AND GENOME ANALYSES by Hongwei Ma A Dissertation Submitted to the Graduate Studies Office of The University of Southern Mississippi in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Approved: December 2008 ABSTRACT YELLOW HEAD VIRUS: TRANSMISSION AND GENOME ANALYSES by I Iongwei Ma December 2008 Yellow head virus (YHV) is an important pathogen to shrimp aquaculture. Among 13 species of naturally YHV-negative crustaceans in the Mississippi coastal area, the daggerblade grass shrimp, Palaemonetes pugio, and the blue crab, Callinectes sapidus, were tested for potential reservoir and carrier hosts of YHV using PCR and real time PCR. The YHV replicated in the daggerblade grass shrimp, and was still detectable on 36 d post-inoculation, causing 8% mortality after injection. However, YHV did not replicate in the blue crab. These data suggest that the daggerblade grass shrimp could act as a reservoir host for YHV. Storage conditions of hemolymph samples containing YHV may result in a decline of virus or exhibit false-negative results in viral detection. The YHV-positive hemolymph was stored at either 4 or 25 °C for a 6-d period, the viral load number at 4 °C was not significantly different from that stored at 25 °C. The only difference was between the samples stored for 6-d at either 4 or 25 °C and those stored at -80 °C, suggesting that shrimp hemolymph can be stored at either 4 or 25 °C for 3-5 d without a significant reduction in measured YHV RNA levels. The whole genome of 3 isolates in Penaeus monodon obtained from Thailand in 1992, 1995, and 1999 was sequenced. The only indel event in the coding region was located in the 5' end of ORFla, containing a segment of 12 nt. Other indels occurred in the non-coding region. The 3' untranslated region (UTR) forms a putative pseudoknot, ii with an octonucleotide motif being the counterpart in the 3' UTR of Coronavims. The phytogeny of Nidovirales was reconstructed based on 48 nidovirus proteome sequences, demonstrating a consistency with the contemporary phytogeny. Recombination in RNA viruses plays a major role in virus diversity and evolution. Seven recombination events were detected among the 4 YHV genomes with high statistical support. The divergence times for the most recent common ancestor of the YHV lineage were dated back to 1970-1980s based on 3 recombination-free data sets. These values were consistent with shrimp culture practice in Asia. in ACKNOWLEDGEMENTS I would like to express my gratitude to the faculty, staff, and graduate students at the USM Guif Coast Research Laboratory lor their kindness and support. I especially owe much gratitude to my mentor, Dr. Robin M. Overstreet. for providing me with the opportunity and for providing his guidance and dedication to this research. I would also like to thank members of my committee, Drs. Marius Brouwer, Richard Heard, and Jeffrey Lotz, for their time and expert advice. I want to thank Dr. Lotz's laboratory members, Mrs. Verlee Breland and Mr. John Ogle, for most willingly providing me with many of the SPF Litopenaeus vannamei that allowed this study to be a success, and Mrs. Marie Mullen for histology help. Thanks are also extended to Dr. Brouwer's laboratory members, including Mrs. Thea Brouwer, Mrs. Rachel Ryan, and Dr. Alexander Pozhitkov, for their valuable technique support and permission to use their facilities. Our Parasitology laboratory members contributed much of their wisdom, experience, time, and love to my dissertation. Mrs. Jean A. Jovonovich not only provided her molecular technique but also helped purchasing reagents as well as reviewing most of my manuscripts. Mr. Walter Grater taught me molecular technique. Mr. Joshua Cook, Mr. Jody Peterson, Ronnie Palmer, and Drs. S. Ashton Bullard and Stephen Curran helped me collect crustaceans. Mrs. Tershara Matthews and Kimberly Lamey contributed much assistance in the wet laboratory and with H&E staining. 1 am grateful to Drs. D. V. Lightner and K. F. J. Tang of the University of Arizona for kindly providing 3 viral isolates and details of real time PCR method and to Dr. N. Sittidilokratna of BIOTEC, Bangkok, Thailand, and Dr. P. J. Walker of CSIRO iv Livestock Industries, Australian Animal Health Laboratory, Geeiong, Victoria, Australia, for sharing the sequence of the Chachoengsao 1998 YHV strain. I would like to thank Dr. Leigh Owens of the James Cook University for providing the gill-associated virus sample and Dr. Charles Laird of the University of Washington for reviewing the last chapter. The research was supported by U.S. Department of Agriculture, CSREES Award No. 2006-38808-03589; U.S. Department of Commerce, NOAA, Award No. NA08NOS4730322 and Subaward No. NA17FU2841; and the National Science Foundation, Award No. 0529684. I am especially grateful to my wife Lirong Liu, my son Zihao Ma, and my relatives and friends for their continuous support, patience, and encouragement. v TABLE OF CONTENTS ABSTRACT ii ACKNOWLEDGEMENTS iv LIST OF ILLUSTRATIONS. viii LIST OF TABLES x LIST OF ABBREVIATIONS xi CHAPTER I. INTRODUCTION 1 Yellow Flead Disease and Yellow Head Virus Shrimp Viral Disease Shrimp Susceptibility to YFIV and Antiviral Research Study Objectives II. DAGGERBLADE GRASS SHRIMP (PALAEMONETES PUGIO): A RESERVOIR HOST FOR YELLOW HEAD VIRUS (YHV) 29 Abstract Introduction Materials and Methods Results Discussion III. STABLE YELLOW HEAD VIRUS (YHV) RNA DETECTION BY QRT- PCR DURING SIX-DAY STORAGE 48 Abstract Introduction Materials and Methods Results Discussion IV. GENOME ANALYSIS OF THREE ISOLATES OF YELLOW HEAD VIRUS (YHV) AND PHYLOGENY OF NIDOVIRALES 59 Abstract Introduction Materials and Methods Results Discussion VI V. EVOLUTION OF YELLOW HEAD VIRUS THROUGH RECOMBINATION 82 Abstract Introduction Materials and Methods Results Discussion REFERENCES 104 vn LIST OF ILLUSTRATIONS The genome organization of Nidovirales 7 Schematic organization of Okavirus 12 RNA secondary structure models for the ribosomal frameshift (RFS) sites in Nidovirales 14 qRT-PCR quantification of YHV RNA levels in the grass shrimp Palaemonetes pugio at different time periods after injection 38 Semi-nested RT-PCR amplification of RNA from grass shrimp 39 Semi-nested RT-PCR amplification of YHV in hemolymph of blue crab administered by feeding YHV-positive Litopenaeus vannamei tissue 39 The qRT-PCR quantification of YHV RNA levels in hemolymph of blue crab at different times 40 The qRT-PCR quantification of RNA levels in hemolymph of blue crab and white shrimp at different times 41 Conventional RT-PCR amplification of RNA from aliquoted hemolymph from Litopenaeus vannamei stored at different temperatures over 6 days 52 Standard curve generated from qRT-PCR and a 10-fold dilution series 53 Quantitative analysis of log scale in Litopenaeus vannamei hemolymph at 4 and 25 °C for 6 d storage 54 Schematic genomic organization of Okavirus 67 The indel event in the coding region of ORFla and comparison in the intergenic regions 68 RNA secondary structure models for the ribosomal frameshift (RFS) sites and 3' untranslated region (UTR) of Roniviridae 70 Alignments and topology of the putative Okavirus hydrophobic transmembrane (TM) domains in pp 1 a 73 Predicted topology of Okavirus ORF3 and alignment of the transmembrane (TM) domains of okaviruses 74 Unrooted NJ tree by analysis of 48 amino acid sequences of Nidovirales using CVTree program 79 viii 18. Cumulative substitution rates of 4 coding genes 89 19. Recombinant regions detected within yellow head virus (YHV) sequences based on the RDP3 program 91 20. Detection of recombination on ORF1 a (A), ORF1 b (B), and ORF3 (C) of Okavirus 93 21. Phylogenetic analyses and divergence dates of Okavirus lineages based on alignments of 3 data sets 95 Supplement 1. RDP3 genome analysis of Okavirus (RDP3 project file) 100 2 A. Tracer statistics of data set from 5' end of ORF 1 a for Okavirus 101 2B. Tracer statistics of data set from 3' end of ORFla for Okavirus 102 2C. Tracer statistics of data set from 3' end of ORF3 for Okavirus 103 IX LIST OF TABLES Comparison of the characteristics of the Rcnivirkiae with ihose of other families in Nidovirales 9 Penaeid viruses 21 Experimental infection of yellow head virus in decapod and stomatopod crustaceans from coastal Mississippi determined by RT-PCR 37 Comparison of YHV log viral copy/jig hemolymph