DETECTION AND IDENTIFICATION OF POTYVIRUSES AND GEMINIVIRUSES IN VIETNAM by Cuong Viet Ha Tropical Crops and Biocommodities Domain Institute of Health and Biomedical Innovation A thesis submitted for the degree of Doctor of Philosophy to the Queensland University of Technology 2007 ABSTRACT Prior to the commencement of this project, few plant viruses had been identified from Vietnam despite virus-like symptoms being commonly observed on many crops and weeds. In limited surveys in the late 1990’s, preliminary evidence was obtained indicating that potyviruses and geminiviruses were causing significant diseases. As a result, this study was aimed at developing generic PCR-based methods for the rapid detection of viruses belonging to viruses in the families Potyviridae and Geminiviridae in plant samples collected from Vietnam, and to characterise the viruses at the molecular level. Novel degenerate PCR primers were developed for the identification of begomoviruses. Using these primers, 17 begomoviruses species infecting seven crop and nine weed species in Vietnam were identified and characterised. Sequence analyses showed that ten of the viruses (six monopartite and four bipartite) were new species. Of the seven previously characterized begomoviruses, five were identified in Vietnam for the first time. Additionally, eight DNA-ß and three nanovirus-like DNA- 1 molecules were also found associated with the monopartite viruses. Five of the DNA-β molecules were putatively novel. Two novel bipartite begomoviruses, named Corchorus yellow vein virus (CoYVV) and Corchorus golden mosaic virus (CoGMV), were isolated from jute plants. Analysis of these viruses showed that they were more similar to New World begomoviruses than to viruses from the Old World. This was based on the absence of an AV2 open reading frame, the presence of an N-terminal PWRLMAGT motif in ii the coat protein and phylogenetic analysis of the DNA A and DNA B nucleotide and deduced amino acid sequences. This is the first known occurrence of Old World viruses bearing features of New World viruses, and their presence in Vietnam suggests the presence of a “New World” virus in the Old World prior to Gondwana separation. Other interesting features relating to begomoviruses identified in Vietnam were; (i) the detection of several recombination events, particularly between the newly identified Tomato yellow leaf curl Vietnam virus (TYLCVNV), and the previously characterised, Tomato leaf curl Vietnam virus (ToLCVV), (ii) the identification of new natural hosts of Sida leaf curl virus (SiLCV), Papaya leaf curl China virus (PaLCuCNV) and Alternanthera yellow vein virus (AlYVV), (iii) the first report of variation in the geminivirus stem-loop nonanucleotide sequence (CoGMV sequence was TATTATTAC rather than TAATATTAC) and (iv) the first report of different stem sequences in the stem-loop structure of two genomic components from a bipartite begomovirus, Kudzu mosaic virus (KuMV). The sequence and phylogenetic analyses of the begomoviruses and begomovirus- associated DNAs identified in this study suggested that South East Asia, and Vietnam in particular, may be a centre of begomovirus diversity. Two pairs of degenerate primers, designed in the CI gene (CIFor/CIRev) and HC-Pro gene (HPFo/HPRev), were developed for the detection of viruses in the genus Potyvirus. Using these primers, three novel potyviruses from Vietnam were detected, namely Telosma mosaic virus (TelMV) infecting telosma (Telosma cordata), Peace lily mosaic virus (PeLMV) infecting peace lily (Spathiphyllum patinii) and Wild tomato mosaic virus (WTMV) infecting wild tomato (Solanum torvum). The fragments amplified by the two sets of primers enabled additional PCR and complete iii genomic sequencing of these three viruses and a Banana bract mosaic virus (BBrMV) isolate from the Philippines. All four viruses shared genomic features typical of potyviruses. Sequence comparisons and phylogenetic analyses indicated that WTMV was most closely related to Chilli veinal mottle virus (ChiVMV) and Pepper veinal mottle virus (PVMV) while PeLMV, TelMV were related to different extents with members of the BCMV subgroup. The incidence of potyviruses infecting plants in Vietnam was investigated using the potyvirus-specific primers. Fifty two virus isolates from 13 distinct potyvirus species infecting a broad range of crops were identified in Vietnam by PCR and sequence analysis of the 3’ region of the genome. The viruses were Bean common mosaic virus (BCMV), Potato virus Y (PVY), Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Chilli veinal mottle virus (ChiVMV), Zucchini yellow mosaic virus (ZYMV), Leek yellow stripe virus (LYMV), Shallot yellow stripe virus (SYSV), Onion yellow dwarf virus (OYDV), Turnip mosaic virus (TuMV), Dasheen mosaic virus (DsMV), Sweet potato feathery mottle virus (SPFMV) and a novel potyvirus infecting chilli, which was tentatively named Chilli ringspot virus (ChiRSV). With the exception of BCMV and PVY, this is first report of these viruses in Vietnam. Further, rabbit bell (Crotalaria anagyroides) and typhonia (Typhonium trilobatum) were identified as new natural hosts of the Peanut stunt virus (PStV) strain of BCMV and of DsMV, respectively. Sequence and phylogenetic analyses, based on the nucleotide sequence of the entire CP-coding region of all 52 virus isolates, revealed considerable variability in BCMV, SCMV, PVY, ZYMV and DsMV. The phylogenetic analyses also suggested the possible presence of ancestral groups of BCMV, SCMV and ZYMV in Vietnam. iv Keywords: ssDNA viruses, Geminiviridae, begomovirus, ssDNA satellites, begomovirus-associated DNA β, begomovirus-associated DNA 1, ssRNA viruses, Potyviridae, potyvirus, degenerate primer, nanovirus, Vietnam. v PUBLICATIONS Publications related to this PhD thesis 1. Ha, C., Coombs, S., Revill, P., Harding, R., Vu, M., and Dale, J. (2006) Corchorus yellow vein virus, a New World geminivirus from the Old World. Journal of General Virology 87: 997-1003. 2. C. Ha, S. Coombs, P. Revill, R. Harding, M. Vu and J. Dale. (2007). Molecular characterization of begomoviruses and DNA satellites from Vietnam – additional evidence that New World geminiviruses were present in the Old World prior to continental separation. Accepted for publication in Journal of General Virology. 3. C Ha, S. Coombs, P. Revill, R. Harding, M. Vu and J. Dale. (2007). Design and application of two novel degenerate primer pairs for the detection and complete genomic characterization of potyviruses. Accepted for publication in Archives of Virology. 4. C. Ha, P. Revill, R. Harding, M. Vu and J. Dale. (2007). Identification and sequence analysis of potyviruses infecting crops in Vietnam. Accepted for publication in Archives of Virology. Papers unrelated to this PhD thesis 5. Revill, P.A., Ha, C.V., Porchun, S.C., Vu, M.T., and Dale, J.L. (2003) The complete nucleotide sequence of two distinct geminiviruses infecting cucurbits in Vietnam. Archives of Virology 148: 1523-1541. 6. Revill, P.A., Ha, C.V., Lines, R.E., Bell, K.E., Vu, M.T., and Dale, J.L. (2004) PCR and ELISA-based virus surveys of banana, papaya and cucurbit crops in Vietnam. Asia Pacific Journal of Molecular Biology and Biotechnology 12: 27 - 32. 7. Bell, K.E., Dale, J.L., Ha, C.V., Vu, M.T., and Revill, P.A. (2002) Characterisation of Rep-encoding components associated with banana bunchy top nanovirus in Vietnam. Archives of Virology 147: 695-707. vi 8. Bateson, M.F., Lines, R.E., Revill, P., Chaleeprom, W., Ha, C.V., Gibbs, A.J., and Dale, J.L. (2002) On the evolution and molecular epidemiology of the potyvirus Papaya ringspot virus. Journal of General Virology 83: 2575-2585. vii TABLE OF CONTENTS ABSTRACT.................................................................................................................ii PUBLICATIONS…………………………................................................................vi TABLE OF CONTENTS..........................................................................................viii LIST OF ABBREVIATIONS....................................................................................xii STATEMENT OF ORIGINAL AUTHORSHIP.......................................................xiv ACKNOWLEDGEMENTS.......................................................................................xv CHAPTER 1: AIMS AND OBJECTIVES………………………………………..1 CHAPTER 2: LITERATURE REVIEW……………………………………… 5 2.1. THE FAMILY GEMINIVIRIDAE ……………………………………..…… 6 2.1.1. INTRODUCTION ...........................................................................................6 2.1.2. TAXONOMY...................................................................................................6 2.1.3. GENOME ORGANIZATION.........................................................................11 2.1.3.1. Genome organization of begomoviruses.......................................................11 2.1.3.2. Genome organization of mastreviruses.........................................................15 2.1.3.3. Genome organization of curtoviruses and topocuviruses..............................16 2.1.4. FUNCTIONS OF GENES................................................................................16 2.1.4.1. Replication-associated protein (Rep).............................................................16 2.1.4.2. Coat protein (CP)...........................................................................................18 2.1.4.3. Genes on DNA-B of bipartite begomovirus..................................................21 2.1.4.4.