Risk Assessment in Agricultural Biotechnology: Proceedings of the International Conference August 7988 This conference was sponsored by the following: * The College of Agricultural and Environmental Sciences University of California, Davis * The University of California Biotechnology Research and Education Program * The University of California Toxic Substances Research and Teaching Program * National Association of State Universities and Land Grant Colleges * United States Department of AgricultureORDERING INFORMATION For Information about ordering this publication, write to Publications Division of Agriculture and Natural Resources University of California 6701 San Pablo Avenue Oakland, California 94608-1239 or telephone (415) 642-2431 Publication number 1928 © 1990 by the Regents of the University of California All rights reserved No part of this publication may be reproduced, storad in a retcieval system, or transmitted, in any form or by any means, electronic, ‘mechanical, photocopying, recording, or otherwise, without the - ‘written pertnission of the publisher and the author. 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Nor does the University of California disceiminate on the basis of ancestry, sexual orientation, Imarltal status, citizenship, medical condition (as defined in section 12926 of the Califomia Government Code) or because individuals are special and 122 others, saw the OTA’s report on field esting in draft form. I found the draft heavily biased soward the conclusion that biotechnology is inher- sntly risky. The published version, evidently a prod- tact of much searing criticism, is greatly improved, better balanced and provides a convenient and use- ful summary of the issues. The OTA’s most recent report covers funding, a subject dear to all our hearts. The summary is useful but OTA is unwilling to take a strong position in its list of options for Congress. Clear statements of the consequences of various actions, especially of inade- ‘quate support and, therefore, lack of action, are too often missing. A second agency, the U.S. General Accounting Of fice, was asked by the House Committee on Energy and Commerce to “review the federal risk manage- ment of genetically engineered organisms intended for agriculture and health uses in the environment” (General Accounting Office, 1988). The authors of this report took a different tack from OTA. The report, was evidently not sent out for wide review before it was published. Instead, GAO sent a final draft to USDA, EPA and HHS (Department of Health and Human Services) for comments. These comments and GAO's replies are presented in an appendix Which reveals major differences of opinion among federal agencies, The USDA conducted a series of regional meetings this year on “Biotechnology and the Public” designed to bring agricultural scientists from industry, univer- sities, and state and federal government together with media representatives and interested members of the public (legislators, teachers, business people, &tc.) to discuss the issues. I took part in the Northeast meeting held in New Brunswick, New Jersey on April 18-20. In my view, it did not meet its intended objec- es since it was largely an exercise in preaching to the converted. There were relatively few media rep- ‘esentatives and general public present, and there Was little that was new for those who did attend. Awide range of other publications, too numerous to list here, have also appeared. Prominent among these is the 1987 report by the U.S. National Acad- emy of Science (National Academy of Science, 1987) which identified key issues. This meeting in Davis is the latest in a series of such events, which will proba- bly continue for as long as the risk associated with release of genetically engineered organisms remains a major concern, What has happened in the United States has been paralleled by similar developments in Western Eu- rope and other parts of the world. International meetings on the subject of risk assessment include those sponsored by OECD (Organization for Eco- nomicand Commercial Development) and COGENE (Committee on Genetic Experimentation of the International Council of Scientific Unions). Of great interest is the combined special issue of the two Elsevier journals, Trends in Ecology and Evolution with Trends in Biotechnology, devoted to the planned re- lease of genetically engineered organisms which appeared in April 1988 (Hodgson and Sugden, 1988). Much time, effort, and many words have thus been devoted to the questions before us at this meeting. Although an interested party, I will try to discount my own prejudices as I review them. Process or Product? In the early debates on the risk of working under containment in the laboratory, it became clear that those in favor of strong regulation believed that the process of recombinant DNA technology was inher- ently hazardous. DNA splicing was new in the 1970s. ‘There appeared to be a possibility that novel geno- types hazardous to man or environment might be re- covered by recombining the genomes of organisms that had been isolated for millions of years. Might “oncogenes be recovered in isolating and cloning ver- tebrate (especially mammalian) DNA in Escherichia coli, which could be passed on and expressed in labo- ratory workers who accidentally consumed the re- combinant bacteria? As the subject advanced and knowledge grew, it became clear that the new techniques of molecular biology were far more precise in bringing about ge-netic changes than either tandom mutation or con- ventional crossbreeding and selection, The foreign DNA to be introduced via transformation could be defined as a nucleotide sequence and its properties in the new host organism confirmed. However, as 1 shall mention later, we still have too little control over the site of integration in the organism receiving the introduced DNA sequences. Everyone has agreed that it would be possible deliberately to design re- combinant products that are undesirable or poten- tially dangerous and that these should be controlled. However, the debate continues on whether or not the safety of recombinant products can be predicted with sufficient accuracy so that they can be released with minimal safeguards in the confidence that they will not be hazardous to man or the envizonment. ‘Those advocating only regulation of the product accuse those who wish to regulate both the product and the process of irrational fear. The latter accuse the former of scientific arrogance. The argument is, largely philosophical. It is clearly best resolved by a compromise that recognizes different degrees of hypothetical risk among engineered products based ‘on the genetic relatedness and properties of the parent materials. These, in turn, can be subject to corresponding levels of stringency in regulation. As several commentators have pointed out (Tudge, 1988) this strategy carries with it the obligation to review the products of any deliberate exercise in genetic recombination—whether by the new techniques of molecular biology or by conventional breeding— before release to determine whether or not they represent a risk. Familiar Risks Modern man is much more concemed with the nutritional value of his food than ever before, There isa growing demand for better balanced diets, greater choice, and improved food value, which crop and animal breeders must address. A recent book by Tudge (1988) sets out the challenge to plant breeders. ‘The question of risk assessment does not often arise in practice of conventional plant and animal breed- ing. It is clear that it should have arisen in certain cases; for example, in determining whether a new cultivar of a food crop is safe to eat. A late blight- resistant potato named Lenape was derived by hy- bridization in the United States with Solanum demis- sum, a wild relative of S. tuberosum. Lenape had to be withdrawn after it had been released by the breeder because the alkaloid content of its tubers was too high. Alkaloid-sensitive people became ill after eat- ing them. Asa result, tests for tuber alkaloid content are now a routine part of the testing of new potato cultivars. They are also an intrinsic part of trials for “value in cultivation and use” (VCU) in the European Plant Variety Rights scheme. The Lenape experience ‘was, in part, responsible for the expectation in the United States that all new cultivars of food crops should be generally recognized as safe (GRAS) before release and if there is any reason to believe that they are not safe, they must be tested and shown to besafe. ‘An important feature of the cereal VCU trials in Britain is the requirement that they be carried out at three widely separated locations over two successive years. Varieties that perform well in official VCU trials are selected for third-year trials on a larger scale. Satisfactory data from these trials can lead to recom- mended status for outstanding entries. The Recom- mended List is prepared annually in November each year by the National Institute of Agricultural Botany (NIAB) in Cambridge for the benefit of its members. The lists for cereals and certain other crops function © like the journal Consumer Reports and provide farm- ers with a guide to the “best buys” among the major agronomic crops in Britain. Varieties on the NIAB Recommended Lists are much in demand by facmers: and can be very profitable for their owners. Varieties that reach the VCU trials have already been subject : to rigorous testing by the breeders. Nevertheless, some varieties have been rejected due to weaknesses revealed following exposure to different seasonal and regional environments, thus proving the effec-, tiveness of the testing scheme, The most dramatic, example of failure to assess adequately plant-breed- ing- related risks was the major impact on the United States hybrid maize crop in 1970 of the extreme sus, ceptibility of hybrids based on Texas male sterile oy toplasm to a novel race of the southern com leaf blight pathogen. In its 1974 report on genetic vul nerability, a National Academy of Sciences commit; tee attributed the catastrophe to a failure to assess We; consequences of using this cytoplasm for hybrid se i production on so large a scale (Levin, 1988). |While most examples, except for Lenape potato, cepresent hazard to the pocketbook rather than to human health or the environment, they demon- strate the need for general concern over any release of a new and unknown genotype. Does the new material have a drawback or defect that introduces «isk? This question will be especially important in the ‘rst years of testing genetically engineered crops if only because of the present lack of precision in determining the sites of chromosomal integration in sansformed plants and animals. Will important functions be compromised because of unintended genetic effects due to site of integration (position 2ffects)? Already we know that the first corn plants cegenerated after transformation were sterile. Had hey been normal in the greenhouse and in the first year of field trials, would they be so in all seasons and n all locations? Clearly, testing will be required to provide answers to these and other questions. iotechnology will present other risks. Currently, here is considerable interest in inserting into crop slants the deita-endotoxin genes of Bacillus thuring- ensis (Bt) that confer resistance to lepidopteran and ‘oleopteran larvae. This will almost certainly result, n development of resistance in target insects, unless neasures such as integrated control or sparing use of hese forms are taken to reduce the intense selection, or resistance that will otherwise result from wide- pread use, Because the Bt spray is so short-lived in jature, it seems to have had little effect on the ievelopment of resistant forms. Compromising the 4se of Bt as a safe insecticide would be unfortunate. ‘he Bt example illustrates one of the risks of imple- nenting agricultural biotechnology. I agree with -evin (1988) that these do not differ from the risks we wre already familiar with in introducing new forms 'y plant and animal introduction or by conven- ional breeding. We already know how to protect, vurselves against these risks by tests and trials de- igned for each species. We should all be able to agree hat each new case should be evaluated as a product ‘efore planned release, without regard to the process y which it was made, The tests and criteria used hould be tailored specifically to each case. The point t which a test or trial should be stopped and meas- {88s taken to prevent further introduction should be ‘early defined. As we gain experience from different cases, we should be able to modify and simplify procedures, The Risks of Not Implementing Biotechnology Even in 1988, a year when US. agricultural productivity is being severely tested by drought, it is difficult for people outside agriculture to compre- hend the importance of both safeguarding and im- proving production. They question why we should constantly strive to do better when we read of over- production, subsidies, surplus, waste, overeating, and obesity; not tospeak of pollution from fertilizers, pesticides, feed lots, and soil erosion. We know full well that all of this is supported by squandering irreplaceable fossil fuel resources. ‘The solutions to these defects in agriculture depend ‘on improving our understanding of, and hence our ability to manipulate, the systems and interactions in ways that are beneficial. Iwill not review here the compelling evidence for the importance of sustain- ing agricultural productivity to feed and support ourselves and the developing nations. The Consulta- tive Group on Intemational Agricultural Research has recognized the role that biotechnology will play in helping the International Agricultural Research Centers serve the needs of their client national sys- tems. Thus CIMMYT (Centro Intemacional de Mejo- ramiento de Maiz y Trigo) and IRRI (International Rice Research Institute) are now establishing labora- tories which use DNA probes to test the application ofrestriction fragment length polymorphisms (RFLPs) in breeding programs for maize and rice. The CIP (Centro Internacional de la Papa) has active research in the use of tissue culture for potato breeding and multiplication. The JLRAD (Intemational Labora- tory for Research on Animal Diseases), in Kenya, has for some years had an active research program using molecular biology as a tool in investigating animal virus diseases. These and other activities will test the application of new methods and will educate scien- tists in the benefits of technology transfer. The devel- ‘oping world has much to gain from improvements in efficiency in plant and animal improvement. And there are other longer-term challenges where Dio- technology is expected to be extremely useful.10 ‘The greenhouse effect is currently a source of con- cern to atmospheric physicists and others whoworry about the consequences of a slow but inexorable increase in global temperatures, as a result of increas- ing CO, concentrations that trap the radiant energy of the sun. Measures to combat this problem must first address the problem of reducing the sources of CO, emissions, Agriculture and forestry have an im- portant role in reducing CO, directly through pho- tosynthesis. Current work on the molecular biology of this process is laying the foundations for increas- ing its efficiency. However, the prospect of introduc- ing the C4 photosynthetic pathway into C3 crop species is still remote, Nevertheless, the use of bio- technology to accelerate the development of better adapted crop plants and trees to be used on land areas that aze either unsuitable (too hot, cold, dry, wet, saline, ot toxic for other reasons) or are defor- ested will have shorter-term benefits. The resultant increase in vegetation cover will reduce, if only slightly, the CO, content of the atmosphere. At the~ same time this vegetation should benefit from the effect that higher concentrations of CO, have on increasing photosynthesis, I have referred already to the consumption of non- renewable fossil fuel resources. In many countries in. the developing world there is an urgent need for fuel, crops for cooking and heating. There is now a grow- ing interest in developing renewable resources. The suggestion has been made by several people that needed research and development programs should be financed by a tax on fossil fuels. Only by such means will we be able to arrest the massive destruc- tion of trees and forests, which in turn leads to soil erosion and further deterioration of the environ- ment and the atmosphere. Leguminous crops that, fix their own nitrogen have attracted much atten- tion and are attractive as fuel crops. I need not stress, the importance of exploiting these phenomena to meet such pressing global problems. ‘We have learned recently that the widespread use of freon, halon, and other fluorohydrocarbons is re- sponsible for partial destruction of the ozone layer in, the stratosphere, which absorbs much of the UV radiation in sunlight. Plant life is remarkably well adapted to resist high exposure to UV irradiation without harmful effects. However, if crop plants are exposed throughout their lifespans to higher UV intensities, can we be certain that this will not be damaging? While I am not advocating breeding programs for UV resistance as a high priority, ! am reminded of the successful breeding for resistance to atmospheric pollutants that threatened the Con- necticut shade tobacco industry in the 1960s. Again we may note the need for applicable technology. We are increasingly concerned about the effects of agriculture and industry in polluting the environ- ment. Many people advocate alternative approaches to agriculture that reduce the farmers’ dependence on pesticides and chemical fertilizers, The effects of these agents in contaminating ground water and wells may soon lead to legislation that will force agriculture to reduce their use. For this reason, Dio- logical nitrogen fixation and engineered resistance to pests and diseases are of high priority to the many research groups with short- to mid-term goals for agricultural biotechnology. Biotechnology has much to offer to environmental science. Ideas include organisms designed to degrade particular compounds, probes to detect metabolic activities in microbial populations, and better meth- ods for engineering treatment and restoration pro- grams, These have special appeal, but we cannot a= ford to invent and release cures that are worse than. the wrongs we try to right. Although we hope not to find ourselves forced to breed trees that are resistant to acid rain and other industrial pollutants because of failure to tackle these problems at their source, biotechnology will offer information and methods. From these brief comments it should be clear that the nature of the challenges facing those who would improve our food and fiber crops, and at the same time safeguard the quality of our living space on earth, is greater than at any time before, We must move forward because the human costs of not doing soare so great. However, we must do so carefully with the confidence that appropriate safeguards will gi us, In this way we can minimize the two kinds of risks. Acknowledgments | IhankGraham Jenkins, Lauraand Tors Meagher, andJudyS00% for comments and helpful suggestions.Literature Cited jeneral Accounting Office. 1988, Biotechnology: Managing the Uisks of Field Testing Genetically Engineered Organisms, Report to hhe Chairman, Subcommittee on Oversight and investigations, Committee on Energy and Commerce, House of Representatives. SAO/RCED-88.27. Gaithersburg, MD. Yodgson, J.,and A.M, Sugden, eds. 1988. Planned Release of Ge- saically Engineered Organisms. Trends in Biotechology/Trends in Zeology and Evolution—Special Publication. Elsevier, Cambridge. Levin, S.A. 1988. Safety standards for the environmental release of senctically engineered organisms, pp. 47-49.1nJ. Hodgson and AM. .aden (eds.), Planned Release of Genetically Engineered Organisms. "Isevier, Cambridge. National Academy of Sciences, Committeeon the Introduction of Genetically Engineered Organisms into the Environment, 987. Introduction of Recombinant DNA. Engineered Organisms into hie Environment: Key Issues. Washington, D.C. dffice of Technology Assessment: 1987. New Developmentsin Motechnology—Background Paper: Public Perceptions of Biotech- tology. OTA-BP-BA.45, US. Government Printing Office, Wash- ngton, D.C. Office of Technology Assessment. 1988. New Developmentsin Hlotechnology—Field- Testing Engineered Organisms: Genetic and ‘eological{ssues. OTA-BA-350. U.S. Government Printing Office, Washington, D.C. ffice of Technology Assessment. 1988, New Developments in Biotechnology-—Special Report. U.S. Investment in Biotechnology. JTA-BA-360, US. Government Printing Office, Washington, dc. Tudge,. 1988, Food Cops forthe Future: Te Development of Plant Resouces Blackwell, Oxtord, 225 pp nN12 Section Il 4 Hypovirulence of Cryphonectria (Endothia) parasi Mother Nature's Example for Fungal Biotechnolog: Neal K. Van (Formerly) Department of Biology, Utah State Unis Logan, Utah : Department of Plant Pathology, Texas A&M Univ College Station, Texas 77843 Abstract Hypovirulence of Cryphonectria (Endothia) parasitica is characterized by a reduction in virulence and sporulation by this filamentous fungus. Transmissible hypovitulence has proved to be effective in the control ‘of the serious disease of chestnut caused by this pathogen. Double.stranded (ds)RNAs, which likely are the genomes of mycovituses, are responsible for transmissible hypovirulence. The dsRNAs affect the fungus by Tegulating expression of specific gene products, some of which prestumably are important for virulence expression and sporulation. Transmissible hypovitulence providles a model for the control of a plant disease, 435 well as a model for how cytoplasmic agents can move in populations of filamentous Fungi. The Nature of Fungi “Der gut Herr Gott said ‘let there be rot’” (Updike, 1985). This is the first line of John Updike’s poem about the important role of rot and decay in nature. ‘The poem is also a tribute to the chief rotters, the fungi, the simple multicellular microbes that ate so adaptable and competitive as heterotrophs that they are probably found wherever there is life, to utilize the reduced carbon of other living organisms. Fungi have been studied for a variety of reasons. Plant pathologists have been particularly interested in them because the fungi are the most numerous and serious causal agents of plant disease. On the positive side, fungi are useful sources of valuable products of metabolism, and they are model organ- isms for the study of genetics, As a result, fungi are prime candidates for genetic engineering, particu- larly for the production of commercially valuable products by fermentation. With proper contain- ‘ment stich uses pose little threat to the environment, and will not be considered in this paper. Fungi, however, will likely play important roles in agricul. tural biotechnology, in the protection of managed “The teat ofthis chapter was prepared ftom a transcrip ofthe author’ conference presentation and natural plant communities, and as “rotte environmentally dangerous man-made carbon ucts. These uses of fungi could require the rele genetically engineered fungal strains into the ronment, and so we must know the potential fi vironmental damage that could result in the t these recombinant organisms, Fungi can exct nuclear, mitochondrial, and cytoplasmic gen« tween different strains under laboratory condi (Fincham et al., 1979). What has been learned : this genetic exchange and the implications 0 genetic exchange for the use of these organist biotechnology, are addressed in this paper. Fungi as Organisms for Genetic Recombination Fungi have a major advantage over most e yotic systems as tools for the study of mole biology: the ease by which they can be geneti manipulated. The value of combining genetics molecular biology in the study of celitilar proc has been elegantly and repeatediy demonstrate researchers using the common brewer's yeast charomyces cerevisiae (Strathern et al,, 1981). Th amentous fungi Neurospora crassa and Asper, nenidulans are also useful model systems, particularly for studying the cellular and developmental biology ‘ofmulticellular organisms (Bennett and Lasure, 1985). ‘The tools developed to study these organisms are ‘useful in the exploitation of fungi for biotechnology. oes Genetic transformation, the single most important tool for recombinant genetic studies, has been suc- cessful with most filamentous fungal systems stud- ied to date. Spheroplasts of filamentous fungi have pen transformed with vectors based on either © complementation of mutants at loci such as TrpC, ‘ArgB, and AmdS or direct selection based on genes for resistance to antibiotics such as hygromycin B or benomyl (Hynes, 1986). The genes and promoters can be heterologous, although, as expected, the source of the promoter can affect the success or effi- ciency of transformation (Turgeon et al. 1987). Transformation of filamentous fungi usually results in the integration of the transformed DNA into the chromosomal DNA (Hynes, 1986). Autonomously replicating vectors based upon mitochondrial DNA d (mtDNA) plasmids are being developed. However, ‘od: rearrangements of these plasmids are common and thus currently limit their usefulness (Kinsey, 1985). ai A high-copy number, cytoplasmically replicating en. vector for filamentous fungi, is needed. Be Transformation has been used in filamentous fungi to isolate genes by complementation as well as for gene amplification, gene disruption, and heteroto- gous gene expression (Hynes, 1986). The basic tools for molecular genetic manipulation of filamentous fungi thus are available. The use of these tools by bio- technologists for construction of strains with unique and desirable properties is just beginning. The pri- mary limitation now for the construction of recom- binant fungal strains for beneficial uses is the imagi- nation of researchers. tar ilar ally nd ses age Nature of the Fungal Genome Meiosis arose early in the evolution of eukaryo- tes and consequently this is the mechanism em- by ployed by most eukaryotes for genetic recombina- ac- tion. As a result of meiosis, the alternation between fil- diploid and haploid nuclei governs the life cycles of Jus Most multicellular organisms. The variety of ways that have evolved to maintain a separation of hap- 13 Joid nuclei and to assure their eventual union illus- trates the complexity that can arise from a very simple process. The fungi probably have evolved examples of all mechanisms of alternation of genera- tions that are found in other organisms, as well as some which are unique to fungi (Burnett, 1975). Itis thus difficult to generalize about the ploidy levels and life cycles of fungi. The ploidy levels of many fungi are still not known. The most commonly encountered fungi are Ascomycetes; if the Deutero- mycetes are included with the Ascomycetes, they comprise 70 percent of the fungal species (Burnett, 1986). Most species of these groups have haploid nuclei during most of their life cycles. Although many fungi are haploid, the number of chromosomes per cell can be quite variable. In Ba- sidiomycetes, after plasmogamy, the number of nuclei per cell is carefully maintained at two. In the As- comycetes, the number varies (Burnett, 1975). How ‘genome expression is regulated when more than one nucleus is present within a cell is not known, Likewise, the number of copies of the mtDNA chro- mosome within each cell is poorly known. The size of mtDNA chromosomes within fungi vary widely, from 18.9 kb in Torulopsis glabrata to 176.3 kb in Agaricus bitorquis (Scazzocchio, 1986). Although ‘mtDNA genome size differsamong fungi, thegenome content is highly conserved (Gray, 1982). Plasmids are found within mitochondria of many fungi. One type of plasmid is defective mtDNA, that is, part of the mtDNA which is autonomously replicating along. with the normal mtDNA chromosome. Defective mtDNA may be suppressive relative to normal mtDNA. Mitochondria may also contain plasmids of unknown function that are not homologous to the mtDNA chromosome (Lambowitz et al., 1986), Cyto- plasmic plasmids are essentially unknown in the fungi. Many fungi contain “viruses” in their cytoplasm. The fungal viruses that have been studied do not fit the definition of a virus since they are not infective. ‘These viruses always remain in the host cytoplasm and are transmitted only by cytoplasmic fusion or by spores, These modes are more typical of plasmids than of viruses (Buck, 1986). One group of fungal viruses, the killer viruses, may benefit the resident14 host. Some do not have recognizable phenotypes, and, as expected, some fungal viruses damage their hosts, such as, the mushroom virus diseases (Van Zaayen, 1979), d-factors of Ophiostroma ulmi (Brasier, 1986), and hypovitulence of Cryphonectria parasitica (Van Alfen, 1986). The extensive studies by Wickner and his associates (Wickner et al., 1986) of the genetics of maintenance of killer viruses suggest a ighly evolved relationship between the viruses and the yeast ceils. There is much that is not known about the nature of the fungal viruses, but in many respects they are as much plasmid as virus. Potential for Gene Movement in Fungi Eukaryotes have evolved the ability to recom- bine chromosomes through cytoplasmic and nu- clear fusion, In most eukaryotes this movement of nuclei from one individual into another is limited to specific cells, The dikaryon of the Basidiomycetes is the result of all cells of the thallus having received nuclei from two different individuals, In the As- comycetes, only specialized cells receive nuclei that will be involved in sexual recombination, but so- matic tissue can contain nuclei contributed by different individuals after anastomosis of the vegeta- tive hyphae. The formation of such heterokaryons theoretically allows nuclei to move from one strain, into another (Burnett, 1975). Anastomosis between fungal hyphae is common between clonal strains, but in many fungi such anastomosis between geneti- cally different strains is regulated by vegetative com- patibility (vc) genes (Fincham et al., 1979). In fungi where ve genes control anastomosis, both strains must contain the same alleles of the ve genes for stable anastomosis to occur. Experiments indicate that heterokaryons form only when ve genes allow anastomosis to proceed. Even when anastomosis is not prevented, heterokaryons usually form in the laboratory only under forced conditions, i.e,, by complementation of mutants. Although heterokary- ons are known to form in nature, their role and importance are still debated (Cooke and Rayner, 1984; Lane, 1981), The potential for interstrain movement of nuclei by heterokaryon formation must be considered by biotechnologists using recombi- nant DNA methods. Surprisingly, although there is clear evidence nuclei migrate across cytoplasmic bridges fo, during anastomosis (Burnett, 1975), there is evidence that mitochondria move. In a stuc mitochondrial inheritance of the basidiom Coprinus cinereus, it was found that when dikar are formed, a separate contact celi can be ident where two strains anastomose. Mitochondria both anastomosing strains apparently enter contact cell. The mtDNAs do not move beyonce contact cell, whereas the nuclei do (Economou ¢ 1987). Cytoplasmic double-stranded (dsRNA, the gen of most fungal viruses, moves readily across cyto micbridges formed by vegetatively compatiblestr ‘This movement between compatible strains verts virulent strains of the chestnut blight pa gen, C. parasitica, into hypovirulent strains (A nostakis, 1984). Infection of the population o: virulent fungus by the dsRNA viruses is now b used to control chestnut blight in Europe (Gr and Berthelay-Sauret, 1978). In this fungus theds can sometimes move between strains of diffe compatibility types. The incompatibility reac Limits, but does not prevent the spread of the ds (Anagnostakis and Waggoner, 1981). In cont vegetative incompatibility in the fungus O. prevents movement of its viruses between strait different ve types (Brasier, 1988). Movement of genetic elements between som cells of fungi clearly occurs, but itis regulated. Te ‘genes appear to have evolved to prevent moven Of genetic elements between different strains, dsRNA viruses probably were the major selec pressure for evolution of ve genes. The ve gi clearly act as a defense against viruses in the ft (Caten, 1972). Vegetative incompatibility also vents formation of heterokatyons. Heterokary provide many of the same advantages to the ce diploids (complementation of lethal recessive g¢ and increased genetic diversity within a cell) would seemingly be favored in selection, The di vantages associated with transmissible cytoplas elements apparently outweighed the selective vantages conferred by heterokaryon formation. hypothesis is based on what little is known about movement of genetic elements in fungi under nial conditions. The ready movement of cytoplasmic ‘ements and nuclei, but not mitochondria, between ‘vegetatively compatible strains suggests that there is jnuch to discover about the regulation of gene movement between somatic cells of fungi. Hypovirulence of C. parasitica: a Paradigm The assessment of risks involved in release of Lg recombinant fungi must be based on the types of *E recombinant genes to be introduced and on the * normal genomes in which the recombinant genes © will reside. Recombinant fungi are not likely to be re- 5 leased into the environment in the near future. The ~ first field testing of recombinant fungi will probably involve the degradation of man-made chemicals and ‘oe lignin, or their use as agents for the biocontrol of ig. Weeds or other pests. Recombinant DNA methods he, Will probably be utilized to enhance existing proper- ties of fungi. Truly novel uses of fungi based on ite fecombinant DNA technology have not yet been xq identified and tested. Transmissible hypovirulence ni_ of C. parasitica, the causal agent of chestnut blight, is on. @ Naturally occurring example of how recombinant ta DNA methods might be used to control fungal dis) st, €88¢S Or mitigate damage caused by fungi. It isalso an. mm example of how specific genes can move through a ‘o) Population of fungi, Chestnut blight is a very seri- ‘ous canker disease of both natural and cultivated populations of chestnut trees in North America and tic Europe (fig. 1). The chestnut trees have almost been vc eliminated from their natural range in North Amer- ni ica (Roane etal, 1986). in Europe the disease was also the €Pidemic until less virulent populations of the fun- ye SUS appeared. These populations contained dsRNA ies Vituses, which reduce both virulence expression, as measured by canker expansion, and sporulation. Hypovirulent forms of the fungus disseminated by researchers and farmers appear to be effectively controlling chestnut blight in Europe (Grente and Berthelay-Sauret, 1978). In North America, hypovir- ulent strains of the fungus, which contain dsRNA, @. Rave been found in widely scattered parts of the continent. These strains were identified because the ig. cankers were noninvasive. These North American Populations of hypovirulent forms of the fungus appear to be controlling chestnut blight only in areas of Michigan (Fulbright et al., 1983). ng tis 15 Figute 1. American chestnut tree artificially inoculated with virulent (upper three inoculations) and hypovirulent (lower inoculation) strains of C. parasitica. A plug oF agar containing the fungus was placed in a wound made with a cork borcr. The virulentstrainsof the funguscause a sunken canker. The hypovir- ulent strain invaded only a small ring of host tissue around the wound, The geographical differences in the ability of trans- missible hypovirulence to control chestnut blight has prompted considerable speculation concerning differences in the nature of the disease between the ‘two continents, The most widely accepted hypothe- sis for the difference in spread of the dsRNA viruses between the two populations is that there are fewer ve types of the fungus in Europe than in North America (Anagnostakis, 1984). The greater diversity ‘of ve types in North America is thought to slow the spread of the viruses through the fungal population (fig. 2). The greater number of ve types in North America appears to be the result of the sexual repro- duction of the fungus in North America, but not in Europe (E. Gobbi, personal communication, Plant Defense Institute, University of Udine, Udine, Italy). The sexually reproducing population in North America continually produces new ve types by the reassortment of the ve loci.16 Paired inoculations demonstrate that the dsRNA does not move as readily between strains of different ve types.as it does between strains of the same vctype (Anagnostakis and Waggoner, 1981). It is important tonote, however, that the dsRNA can move between many strains of different vc types. This suggests that the vegetative incompatibility of C. parasitica is ini- tiated after cytoplasmic contact isestablished. Strains have been identified which are easily converted to hypovirulence by the dsRNA from incompatible strains (Kuhlman et al., 1984). These strains may be better able to maintain dsRNA than other strains or they may have a slower incompatibility response. The difference in ability of strains to exclude the dsRNA during an incompatibility response indicates that the degree of incompatibility is variable. It is not known what regulates the extent of the incompati- bility response. Vegetative incompatibility in the Dutch elm disease fungus, O. ulmi, is a barrier to the COTe O@: Nuclei Z 2 Mitochondria 1 ds RNA in Vesicles, | | | | | Figure 2. Diagrammatle representation of how dslINA in fungal vesicles is spread by hyphal anastomosis ofa virulent (lower) and ypovirulent (upper) straim of the fungus. Theze is no evidence of regular transfer of mitechondrta and nuclei with the dsRNA. during anastomosis, Vegetative incompatibility reduces the success of hyphal anastomos! transfer of dsRNA. The dsRNA-infected stra pear to be a factor in the selection of some } tions of the fungus because of the low virul dsRNA strains and because the dsRNA is not transmitted between strains of these popu rasier, 1988), In C. parasitica, this selection populations of dsRNA-containing strains di appear to be occurring because the dsRNA ma readily cross vc barriers. The dsRNA moves t the population of the fungus as a disease, which the fungus has only vegetative incom ity as a defense. The disease is maintained population because of the survival of infecte viduals. If the dsRNA rapidly killed infecte infected cells would have little opportunity t tomose with other strains before death. Th dsRNA will probably spread through enough population of the fungus in North America t chestnut blight. ‘The spread of the dsRNA responsible for hy ulence is an example of how a cytoplasmic ge: spread in a population of fungi, The vc genes c may have evolved to limit such movement. Ir such as O. ulmi, the genes apparently are prev spread of the dsRNA (Brasier, 1988). It is not whether the inability of the vc genes to previ spread of dsRNA in C. parasitica is a faunct fungal genes or viral genes. Hypovirulence parasitica is effective because it is able to sprt tween different ve types. Before recombinan methods can be used to emulate hypoviruler must understand how the virus moves with fungus. ‘One of the interesting aspects of hypovirulenc parasiticais that transmissible hypovirulence is by a group of viruses whose genomes differ ¢ that their RNAs do not hybridize with each (L'Hostis et al., 1984). The colony phenotypes hypovirulent strains are determined by which virus is present, suggesting that there is a spec in the response of the fungus to the virus (El 1985). In spite of the diversity of RNA sequen indicated by molecular hybridization analysi the various colony phenotypes found whe viruses ate present, the virus-infected fungal are nearly uniformly hypovirulent, and all reduced sporulation, A better understanding5 aif the viruses cause hypovirulence and reduce sporula~ pul tion may indicate how hypovirulence can be mim- ce @ icked in other fungi. adi tion We are studying the relationship between the viruses ing and hypovirulence by examining how the viruses + nd‘ affect specific fungal mRNAs and proteins, which “noi were detected by differential hybridizations and two sugé dimensional (2-D) protein gels, respectively. The aing strains used in these studies were isogenic, except tibilg that one of the paired strains contained a virus. The 1 the isogenic strains were constructed by transferring the indi viral dsRNA, but not the nuclear or mtDNA, into celly. strain EP1SS by hyphal anastomosis. The lack of inas. transfer of nuclei and mitochondria was confirmed , the by using strains with nuclear auxotrophic markers f thy and unique restriction fragment length polymor- {fea phisms of mtDNA in the donor and recipient strains, of the fungus, The strain resulting from transfer of the dsRNA (UEP1) is thus isogenic with EP153 except, ‘vie: for the presence of the virus in UEP1. The 2-D protein may gels showed that presence of the virus caused a ung down-regulation of about nine major polypeptides ung (fig. 3). Most of these polypeptides were not detect- ating able in virus-containing strains (Powell and Van cows Alfen, 1987a). We also learned that the same poly- t the Peptides were down-regulated by two different viral nd strains, one from North America and one from Eu- of, Tope. These viruses do not show sequence similarity i be by molecular hybridization, The fact that these two DNA viruses have similar effects on the fungus simplifies 2, we Our efforts to understand the mechanism of viral the tegulation of fungal virulence and sporulation. The number of major polypeptides that are down- of C tegulated by the viruses suggests that the viruses do ‘used NOt directly affect the genes encoding these polypep- nugh tides, It s more likely that a regulatory gene(s) or a ‘ther Sene encoding a product important in a number of fthe Cellular processes is affected. Support for this hy- RNA Pothesis comes from analysis of a mutant of C. icity Parasitica that has a phenotype indistinguishable ton, fom a European dsRNA-containing hypovirulent 's, as Stain. Electrophoresis gels of this non-dsRNA-con- and taining mutant show that many of the same poly- the Peptides down-regulated by the viruses are also down- ‘ains Tegulated in this mutant (Van Alfen, unpublished). how his mutant (pig) has been mapped to a singie locus how in the nucleus. Itis probably a mutant of a gene that wv Figure 3. Regulation of protein expression by dsRNA in the fungus C. parasitica, Two-dimensional gels of total proteins of virulent strain EPISS/2. and the isogenelc hypovirulent strain UEPI. Gels A represent high molecular weight proteins and gels Brepresent low molecular weight proteins. The protein labeled ¢ is present in both high and low molecular weight gels. regulates cellular processes; the type of regulation is not yet known, ‘One of the polypeptides regulated by the viruses was in the cell culture fluid in which the fungus grew. We have isolated and purified this protein. Purity of the protein is indicated by a single homogeneous pro- tein detected by SDS-polyacrylamide gel electro- phoresis and high performance liquid chromatogra- phy. The purified protein was used to prepare a poly- clonal antibody from rabbits. Western blotting showed that this antibody was specific for the pro- tein, Using this antibody, conjugated to acid phosphatase, on sectioned hyphal tissue, we found that the protein accumulated on the surface of hyphae. Amino acid analysis indicates that the pro- tein has a molecular weight of about 22,000 (22 k protein). We have sequenced part of the protein; the amino terminal end is very rich in glycine, much of it in a repeating sequence of -gly-ser-. The glycine- rich nature of the protein is similar to a recently dis- covered type of plant ceil wall protein (Condit andMeagher, 1986). We find that the protein has lectin- like properties. It agglutinates specific types of red blood cells and this agglutination is inhibited by cell wall preparations of the fungus. We also found that the protein is tissue specific, and is found only on sporulation-competent hyphae. The protein is pro- duced in large amounts by virulent, sporulating cultures, but only trace amounts are found in virus- infected or pig mutant strains. Down-regulation of this protein, and perhaps others, may reduce sporu- lation by virus-infected and pig mutant strains of the fungus (C.E. Carpenter, R,I. Mueller, P. Kazmierczak, and N.K. Van Alfen, submitted for publication). Differential hybridization procedures detected two very abundant fungal polyadenylated RNAs [poly(A) RNAs], which are down-regulated when the viruses are present (Powell and Van Alfen, 1987b). These poly(A) RNAs have been mapped toa 4.2 kb sequence of the nuclear DNA. Sequencing and Northern blot data suggest that neither of the sequences encodes the 22 k protein described above. These two poly(A) RNAs are the most abundantly produced poly(A) RNAS of the fungal cell. Virus infection completely stops the accumulation of these poly(A) RNAS. We don’t yet know whether the viruses regulate the amount of these poly(A) RNAs by transcriptional regulation or mRNA turnover, In any case, the regu- lation is specific to these and probably a limited number of other poly(A) RNAs. We have no evidence ‘that these viruses affect transcription or mRNA turn- ‘over in a general way. Preliminary findings concerning how the viruses affect the fungus have enabled us to make a few conclusions. First, it is clear that the viruses have specific effects. Hypovirulence and the reduction of sporulation are not a result of general and nonspe- cific effects of the viruses on fungal cellular pro- cesses. Second, the viruses appear to regulate mRNA accumulation. Third, different viruses affect thesame specific genes or gene products of the fungus. Hypovirulence of C. parasitica is a useful model for biotechnologists. The spread of the dsRNA through the populations of the fungus is an example of the movement of genetic elements in nature. We have some insights into the factors that affect the spread of the dsRNA, but much remains to be learned, Study of this system should help clarify the constraint fungal anastomosis, the primary method of 5 movement between fungi. In attempting to un stand how dsKNA moves, we are comparing movement of dsRNA to that of specific nuclear mtDNA types. This fungus is particularly well su for these studies because the mtDNA is large polymorphic. Hypovirulence might be a useful model for control of fungal diseases of plants using a specific, biological agent. What we have lear about hypovirulence of C. parasitica indicates this system will help in the construction of cyto, mically transmissible genetic elements, which cause hypovirulencein other fungi. The most ser limitation to achieving this goal is the lack of | copy number cytoplasmic vectors for transfor tion of filamentous fungi. The availability of : vectors will make it possible to disrupt specific j functions important to pathogenesis in other f using antisense RNA. Our studies of C. paras suggest that transmissible genetic elements can cessfully control specific diseases or problems car by filamentous fungi, if the fungus is not an obli parasite and if the fungus is not severely debilité Conclusion Many filamentous fungi have the abilit anastomose with closely related strains. This an: mosis of vegetative hyphae is generally not relate the sexual cycle, but can result in the exchang genetic elements. Viruses of fungi spread by hy anastomosisand consequently the fungus hasevo mechanisms to limit anastomosis. The import: of hyphal anastomosis in genetic element exch: in nature is not known, but under laboratory co tions, heterokaryons are easily formed in this ‘The little evidence availablé concerning mt! suggests that it does not move from strain to stra this manner, Hypovirulence of C. parasitica demonstrates readily a cytoplasmically transmissible genetic ment can spread throughout the population fungus, even across genetic barriers. It is alsoam_ of how cytoplasmic elements regulate specific tree eee 1 xt ed at 1% ill us ae a ne agi ica ac ate 2. to to te nal re ace age di ay, NE i ale: it de its Acknowledgments ‘The editorial assistance of Kurt Gutknecht and Dane Hansen is, appreciated, The work reported here was supported by the USDA Competitive Research Grants Office grant number 87-PSTY-9 (0243 and the Utah State Agricultural Experiment Station. References Anagnostakis, $.L,, 1984, The mycelial biology of Endothia para- sitica. ll. Vegetative incompatibility. tn The Ecology and Physiology of the Fargal Mycelium, (D.H. Jennings and A.D.M, Rayner, eds.) pp. 499.507, Cambridge University Press, Cambridge, U.K. Anagnostakis, S.L. and P.W. Waggoner, 1981, Hypovirulence, vegetative incompatibility, and the growth of cankers of chestnut blight. Phytopathology 71:1198-1202, Bennett, J.W.and L.L. Lasure, 1985, Gene Manipulationsin Fun, ‘Academic Press, Orlando, FL. Brasier, CM, 1986, Ted: factor in Ceratocystis ulmi -its biolos- cal characteristics and implications for Dutch el asease. In Fungal Virology (KW. Buck, ed.) pp. 177-220, CRC Press, Roca Raton, Brasier, C.M., 1988, Rapid changes in genetic structure of epidemic populations of Ophiostrome ulmi, Nature 332:538.541. Buck, K.W., 1986, Fungal virology—an overview. In Fungal Vitol- oy (KW. Buck, ed.) pp.1-84, CRC Press, Boca Raton, FL. Burnett, J41., 1975, Mycogenetics, John Wiley and Sons, London, Burnett, J.H., 1986, Aspectsof the macro- and microsevolution of the fungi. in Bvolutionary Biology of the Fungi (A.D.M. Rayner, CM. Brasier, and D. Moore, eds.) pp. 1-18, Cambridge University Press, Cambridge, U.K. Caten, C.E., 1972, Vegetative incompatibility and cytoplasmic infec tion in fungi}. Gen. Microbiol, 72:221.229. Condit, C.M. and RB. Meagher, 1986, A gene encoding a novel 43.8. The practical useful- ness of the correlation between increased stability (higher Tm) and necrogenicity in tomato is that temperature-gradient gel analysis will detect the ap- pearance of such variants during serial propagation Passages of CMV and sat-RNA. For example, dsRNA ‘of $52, after serial passage in tobacco, was tested necrogenic in tomato. This has been confirmed by temperature-gradient gel electrophoresis, where a mixture of four bands with the major band in the higher Tm-range characteristic for necrogenicdsRNA was found (Tien et al., 1987). The phenomenon of necrogenic sat-RNA of CMV. emergence upon serial passage in tobacco is now well known and can probably be ascribed to the existence of variant populations of CMV sat-RNA where in certain host plants specific variants of greater “stabil- ity” are selected and preferentially replicated (Gar- cia-Luque et al., 1984). Gel electrophoretic methods that can detect the emergence of such variants have been described (Kaper, et al., 1976), but these analyze the single-stranded form of sat-RNA. Because the single-stranded form lacks the well-defined molecu- lar shape of the double-stranded molecule, this method does not have similar resolution power; moreover, unlike the double-stranded molecule, single-stranded molecule of sat-RNA does not yet allow the theoretical development of a rationale for its behavior in gel electrophoresis. In addition to examining the uniformity of sat-RNA in the control agents, temperature-gradient gel electrophoresis could be applied to analyze the emergence of sat- RNA variants when the agents are used in the field (Tien et al., 1987). Preparation and Application of sat-RNA Control Agents In consideration of the stability of the agents, the isolates selected from local lesions on C. quinoa were propagated in tomato, Infected dry-tissues used as inoculum for the agents should be from prepara- tions of no more than four passages, according to our experience. One tube in the stock is used before each32 producing season to multiply the agents. The origi- nal stock of $52 still maintains its property of being non-necrogenic in tomato (Tien et al., 1987). Multiplication of the agents can be on seedlings of tomato, since tomato is a kind of host that can keep the proper balance between genomic RNA and sat- RNA; but large-scale multiplication becomes diffi- cult because of the small amount of leaf-tissue of to- mato. The tobacco variety ‘sansum NN’ is another multiplying host, however no more than one pas- sage can be used and still keep the sat-RNA proper- ties. A large amount of tobacco leaf-tissues with Infective agents can be obtained when the plant seedings are grown in insect-proof greenhouses at 20° to 25°C. The leaves were harvested 7 to 10 days after inoculation (Tien et al., 1987). The sat-RNA control agents were extracted as fol- lows: Add 0.5M citrate buffer (pH 6.5) containing 0.1%-mercaptocthanol to the leaves (1.5/1, V/W), homogenize for 2 to 3 minutes and centrifuge or filter to remove the pellets. Add chloroform (10%, V/ ‘V) to the supernatant or filtrate, shake for 20 minutes and centrifuge at 5,000 rpm for 20 minutes. Collect the supernatant, add PEG6000 (7g PEG/100 ml), store at 4°C for 3 hours, then centrifuge at 8,000 rpm for 20 minutes. Suspend the pellets in 0,00SM boric acid buffer (pH9.0, 1 ml buffer/10g leaves), centri- fuge the solution at 8,000 rpm for 30 minutes, and use the supernatant for protective inoculation (Tien et al,, 1987). After several dilutions, the extracts of the agents were inoculated in C. quinoa and tomato for testing infec- tivity by local lesion number and examining non- necrogenicity. Only those that were symptomless, and therefore non-nectogenic, were used as protec- tive inocula, Fresh leaves were stored at 4°C without loss of infec- tivity for 7 days, and partially purified extracts at 4°C for 1 to 2 months (Tien et al., 1987). To apply the agents to the field, the extractants were diluted several times. After adding 0.5g 400 to 600 mesh carborundum to 100 mi diluted solution, the solution was sprayed on seedlings by a spray-gun under a pressure of 4 to 5 Kgfem?. A toot dip inoculation also can be used for roc plant species such as tomato. Before young see were planted in fields, the roots were washed water to remove soil, then immersed in the ¢ extracts for 30 to 60 minutes, This method suitable for the plant that does not have ro abundant as pepper roots (Tien et al., 1985). Effects of sat-RNA Control Agen on CMV-Caused Diseases and Production of. Le a and Ton in Fields During a period of 5 years (1981-1985), a of tests using $51 and $52 on pepper to contro! diseases in the field around a number of loc throughout China showed that diseases we duced and production of the fruits was incr Data obtained from three of the localities, Bc Handan, and Yantai, showed that $51 and S: creased incidence of the diseases, and in part gave an effective decrease of disease index by ‘Table 1. Application of $52 on controlling CMV Induce tomato diseases and field production of fru in 1987 Lowility Arca Treatment Disease Frultyild 1 (ou) Index Kg/mmu Increase % Beijing 2100 $52. mos 41727 349 Control 47.3 2093.9 Shanghai 2703 $52 = wo a6 Control 2487.0 Shenyang 1105352 16a wea S17 Contiol 47.8 1484S Lanzhou 1400 $52. — sees Bs: Conmol = 29280, Talyuan 1254 852 302 48000 200 Conta! $3.3 4000.0, Handan 2450. $52 192 3570 28.0 Control $8.9. 2855.0 ‘Tort 11082 (0667 hectare) nese unit for aves (rmrig:to 82.8% and an evident increase of fruit yields by 1 10.8% to $5.6% (Tien et al., 1987), Application of $52 ton tomato showed a similar result. A 3-year field test itesneat Taiyuan (1983-1985) gave a decrease of disease néindex by 31.2% to 39.7% and an increase of fruit S tyields by 31.7% to 44.9% (Guo et al, 1986). “Since 1986, these control agents have been put into application in large areas and the plants tested have increased from pepper to tomato, tobacco, cucum- ‘ber, and other crops. The results obtained from these tttextensive tests reconfirmed the antiviral and yield- promoting effect of these control agents. Table 1 miindicates under field conditions the antiviral and M yield-promoting effects of SS2 agent on CMV severely itvaffected tomato on a Large scale. i sq In some large cities (e.g., Beijing, Shanghai, and Sh- igenyang) and medium-sized cities (e.g., Lanzhou and Taiyuan), the proportion of total tomato and pepper iigcultivating areas using $52 is about 3%. In some ‘gsmall cities (e.g., Handan and Anda), the proportion- ate use of $52 agent in tomato is as high as 60% and in pepper greater than 80%. The use of sat-RNA as a biological control agent on a _large scale will save manpower and increase eco- slenomic value by allowing for the use of electro-driven mechanical inoculation machines. The ratio of eco- ‘wsnomic value between control-agent-used fields and “Snon-used fields is then increased even further. 3% Sat-RNA Biological Control Agents Increase Plant Resistance to Fungus Diseases 11 In the process of using sat-RNA as biological control agents against CMV on tomato and pepper, itwas observed that the agents also caused resistance 20 some fungi. For instance, tomato plants treated with $52 had strong resistance to Phytophthora ,silféstans and Cladosporum fulrum (Tien et al., 1985) ont When sat-RNA was applied to cucumber, increased aoresistance to CMV was not observed because CMV “esistance is already high in Chinese cucumber varie- geit®S. However, it was found that the treated plants ‘equited resistance to P. cubensis, which can cause “revere disease on cucumbers (Table 2). Cucumber 33 ‘Table2. Control of cucumber downy mildew by sat-RNA biologl- cal control agent $52 in Anda, Heilongjiang province, 1987 Treatment ‘Saeed ——Uateated—‘Tisated x30 contol Unueated Diseased anb(®) 26.6 s20 83.4 Disease Inox 86 us a2 Fruit Yield (kg/m) 137400 woso0 42 Net income per mu (f) 9613.0 sano na Tested area = 400 au plants treated with $52 showed resistance to P. cuben- ‘sis during early stages of growth, when nontreated fields begin to show symptoms and require 1 or 2 fungicide sprays. In order to demonstrate the protective effect of 352 biological control agent against CMV on tobacco plants, we treated tobacco plants with 52. The treated plants were not only resistant to CMV, but also to Alternaria altemata, In the Yianbian Korean ‘Autonomous Area, jilim province, when the tobacco variety G-140 was treated with S52, protection against CMV and A. alternata was observed; yield and quality of tobacco leaves was also improved (Cui et al., 1989; Table 3). In 1988, as many as 20,000 mu of field in this area used S52 contro! agent to prevent disease caused by CMV and A. alternata, Table 3, Effect of satellite RNA control agent on CMV and A. altemnata- caused diseases and yields of tobacco ow Aalterata Yield ‘Treat: Diseased Disease Diseased Disease Ke/om Superior ment Plants% Index Plants % Index Quay Leaves ssi ne ut 52 30810309 sz 2G 8733025 ae Contol 494 457 1000421113834 Sat-RNA Biological Control Agent Stimulates Early Maturation of Fruits Viral infections can cause changes in develop- ment of infected plants; some infections will acceler- ate plant development and cause early aging. For instance, even though N14, a mild strain of TMV, which is used as vaccine, does not create symptoms on tobacco and pepper plants, it can accelerate plant growth and make treated plants flower and mature earlier (Tien et al., 1985). The CMV $82 that is attenuated by sat-RNA can also cause early maturation in tomato and pepper. For instance, in Handan city a 3-year experiment indi- cated that $82 can make pepper flower 3 to 7 days. earlier than nontreated plants and early yield is increased by 23.3 to 34.3%. The increase in early fruit production can greatly promote economic income (Table 4), Transgenic Plants by Expression of Sat-RNA for the Control of Virus Diseases The feasibility of using sat-RNA as a biological control agent for viral diseases (Tien and Chang, 1983) raises the theoretical possibility of construct- ing virus-resistant sat-cDNA transgenic plants. Baulcombe et al., (1986) first introduced the 1.3 or 2.3 units of complementary DNA of CMV sat-RNA I- 17N into tobacco plants and achieved its expression. ‘They also regenerated CMV-resistant transgenic to- bacco plants. Table 4. Effects of satellite RNA contol agent $52 on flowering date and eatly stage fruit yleld of pepper (Handan) Treatment Flowering Yield of earlystage Total yieia Year Date Days ahead Kg/m Increase Kgim Increase of control % % 1982 Sz IM 7079 343 52660 165 Contr! 1)VIt 445.2 46085 1983 $82 AV #7073 233 S4050 15.2 Control 28/V 5736 6065 1984 S52 S38 Stas may aar3s tad Control 18/VL 415.0 3089.0 In our laboratory, Zhong et al., (1987) synt! and cloned the complementary DNA of CN RNA-1, and by sequence analysis the cDNA clo shown to contain the full length sequence RNA-1. Then the CDNA monomer was inserti expression vector ROK II, comprising T-DNA sequence and the promoter of cauliflower virus 38s RNA and the terminal sequence nopaline synthase gene of Agrobacterium T-[ Wa Shixuan et al., (1988). The resultant plas referred to as pRI. Under the help of plasm 2013, the pRI was introduced into A, fume containing Ti-plasmid by triparental matin; discs of tobacco G-140, which is widely u China, were infected by A. tumefaciens. Tran plants were regenerated from callus in kana medium and were then inoculated with a sa free CMV isolate. The transformed plants dev. vein clearing the same as control plants 7 day inoculation. The RNA was isolated from plant and assayed for the presence of satellite dsR polyacrylamide gel electrophoresis, Sat-RN Present in large amounts in all transformed except one. No sat-RNA was detected in non formed plants. In non-transformed plants, or transformed plant that did not express sat more severe symptoms developed, and 3 week inoculation new leaves showed severe mosaic toms and the plants became stunted. In contr the transgenic plants that expressed sat-RNA, clearing symptoms attenuated and did not severe mosaic or stunting. The plants grew aln well as non-infected ones. Finally, half-leaf t infectivity on C. quinoa, which is a local lesio: of CMV, were used to determine the titre of bi cally active virus in the transgenic plants, Nir days after inoculation, 42 local lesions develo, 20 half leaves when inoculated with extracts CMV-infected transgenic plants expressing sat In contrast, 446 local lesions developed whe inoculum was from transgenic plants expressi sat-RNA. Our experiment shows that most « transgenic plants incorporate satellite CDNA ar produce large amounts of sat-RNA after CMV tion. This sat-RNA can interfere with the replic of the CMV and attenuate symptom developm transgenic plants.The most conspicuous difference between ours and stBaulcombe's transgenic tobacco plants is that the ‘Wigat-cDNA we use is a monomer. Experiments proved sithat the insertion of sat-cDNA monomer into to- ipacco genome generated sat-RNA with biological ac- tétivity. Moreover, as the transcript of CDNA monomer sidoes not need processing, biologically active sat- tRNA may be expected to be generated more rapidly \lin monomer inserted plants than in dimer inserted ¢plants. In measuring the resistance of sat-cDNA pimonomer-transgenic tobacco to CMY, it was found lethat the transgenic plants inhibited CMV replication Leboth in inoculated leaves and in later systemically in- | fected leaves. The dimer transgenic tobacco was re- ansistant to CMV only in systemically infected leaves so{Harrison, 1987). The delay in resistance is probably liidue to the time needed for processing of sat-RNA piimer into biologically active monomer. ie ssin the spring of 1988, we planted 85 descendant « plants from transgenic tobacco in the field and used w28 non-transgenic plants of same variety as controls, afhese transgenic descendants grew normally in the afield and possessed comparatively strong resistance ts0 CMV infection (Table 5). The influence on tobacco Nyvality and production is still under measurement. tthe application of transgenic plants is undoubtedly madvantageous in viral disease resistance, variant se- tection and mutation of sat-RNA, and release of sat- eRNA from the transgenic plants. But when the sat- GNA was introduced into plant by the Ti-plasmid, it stlid not create resistance to fungus diseases and early saturation of fruits, which presumably are caused roy helper virus. Table 6 lists the advantages and dis- ostlvantages of sat-RNA biological control agent and gat-cDNA transgenic plants in field application. di o _ poacco to CMV in the field N 12DIES. Resistance of progeny of CMV satelite cDNA transgenic f v ‘No.of plants in cpbsceo isease grade Diseased Disease fe O12 3-4 Plans% Index tic — wanenic 38:22 21 4 0 ss m4 neransgenic ontiel 0.227 82 100 na 35 Table 6. Comparison of using satellte RNAanditscDNA transgenic plants in agriculture Satelite RNA biological (Characteristics Satelite DNA, control agent tuansgenie plants Resistance to “The resistance ischange- ‘The resistance is helper and able dueto the time of stronger and related viruses Inoculation and soon more stable Resistance to Yer No Fungus disease Application “To be used every growing Resistant variety frequency season arlymatuation Yes No of alts Selection of Temay occur even when The possibility is SaURNA varlants using eDNA transcript for expected to be uring application inoculum reduced Mutation of satRNA The mutation frequency ‘of RNA is higher than “The mutation frequeney DNA is DNA ower than RNA Release of at-RNA Releases posible before Release s ‘wild Helper vis possible nly tnfection ster wid helper ins nection Discussion ‘The mechanism of sat-RNA for the control of cMv It has been asked whether the protection pro- vided by the sat-RNA control agent is due to conven- tional cross-protection on the part of the attenuated CMV, or whether the presence of the sat-RNA itself, by outcompeting viral RNAs, prevented any signifi cant natural infections by CMV (Kaper et al., 1983). Comparative studies on cross-protection among CMV strains and its satellite protection effects were done by Wu Gusui et al. (1988). Tobacco and pepper seedlings were preinoculated by CMV with or with- out sat-RNA and then challenged by virulent CMV at different times, Protection effects were detected by a disease survey, protein A sandwich ELISA for the challenge CMY strain and dot-blot for sat-RNA. The36 disease index developed after challenge inoculation indicated that plants preinoculated with CMV with- out sat-RNA showed slight cross-protection in the first 10 to 15 days, but more severe symptoms were obtained from then on. Nevertheless, the plants preinoculated with CMV containing sat-RNA showed more effective protection against the challenges, especially after 15 days, The results demonstrate that the protection provided by CMV with sat-RNA is different from conventional cross-protection. How- ever, in plants the protection power of the former is strengthened by the latter, Interference with CMV replication and system ex- pression in sat-cDNA transgenic plants gives further evidence for the direct effect of sat-RNA on CMV infection. It is reasonable to assume that the interfer- ence with virus production by sat-RNA can be ex- plained by competition between sat-RNA and CMV genomic RNAs for replication. Another fact is that large amounts of double-stranded sat-RNA exist in plants treated with control agent (Yang et al., 1986) and in sat-cDNA transgenic plants after CMV infec- tion (Wu Gusui et al., 1989). The apparent link between double-stranded sat-RNA reduced viral synthesis and disease attenuation can be rationalized by a disease regulatory mechanism based on the putative replication competitive between sat-RNA and CMV (Kaper and Tousignant, 1984). On the other hand, accumulation of double-stranded sat- RNA might induce the production of some anti-viral factors, analogous to interferon and animal viruses. Safety of using sat-RNA In agriculture Considering concern for public safety, it is ap- propriate and beneficial to summarize the results and knowledge we have obtained during 7 years of, experiments using sat-RNA control agent. 1. The sat-RNA and CMV variants we use for con- structing sat-RNA control agent are already exist- ing in nature and will not cause necrosis on tomato plants or aggravate symptoms on other plants, 2. Wehave made a survey of viral diseases on plants, especially on CMV-infected crops in areas where sat-RNA control agents have been used for 7 years, So ar there are no new types of viral or more severe symptoms. 3. Through electrophoresis we analyzed sat-1 areas where sat-RNA control agents havi applied and found no obvious increasin dency in the amount of sat-RNA, It is pr due to several factors: (i) sat-RNA control contains a large quantity of sat-RNA con it, and after inoculation CMV is gradually difficult to detect. Single-stranded sat-RN seems to be disappearing, but double-st: sat-RNA can be occasionally detected; (i) In infected by sat-RNA containing CMY, th concentration is low. Thetransmission effi of CMV S51 by Myzus persicae was much and the incubation period was longer the of CMV alone. To reach the same transn efficiency as CMV without sat-RNA, ten ti many aphids were needed. Acknowledgments ‘The author wishes to express sincere thanks to Drs. J.M D.C. Baulcombe, D. Riesner, RB. Franski, and J, Rar providing us experimental materials iterature Cited Baulcombe, D.C. , G.R. Saunders, M.W. Bevan, M.A and B. D. Harrison. 1986. Expression of biologically ac satellite-RNA fiom the nuclear genome of transformed plants 321: 446-449, Cui, Y.H., MH. He, andZ.8. Han, 1989. Protection effect satelite RNA biological control agent on productive tobac. eld. Acta Virologica Sinica (In Press). Francki, RB. 1985. Plant virus satellites. Ann. Rev. Mi 39:151-174, Guo, LY., X.H. Zhang, B.G. Qin, and P. Tien, 1986. 4 RNA as a biological control agent CMV-SS2 and control 0} virus disease. Acta Phytopatha Sinica 17:256. Harrison, B.D., M.A. Mayo and D.C. Baulcombe. 198 resistance in transgenic plants that express cucumber mos satellite RNA. Nature 328:799-802. Kaper, J.W.andM.E. Tousignant. 1984. Viral satellites: rucleie acids capable of modulating disease expression. En 8(4),Kaper, J.M., ME. Tousignant, and I. Lot. 1976. A low molecu- neigh RNA associated with adivided genome plant viru: Defective “pretelite RNA? Biochemical and Biophysicsl Research Commu- ‘peation 72:1237-1243, {urant, A.F. and M.A. Mayo. 1982. Satellites of plant viruses. in. Rev. Phytopathol. 20:49-70, ee 5 tectrophoresis-Thermodynamic analysis of nucleic acids and proteins ‘Lin puafed form and in cellular extracts, Blophiysical Chemistry 1078:238-246, aly {Tian, W.HL, S.X. Cao, Q. Sun, $.P. Chang, XH. Chang, and P. “Tien, 1984, Satelite RNA asa biological conto agent of cucumber BWipmatic virus. Biological properties of CMV SS1-contained satel it}ite RNA. Acta Phytopathological Sinica 15:145-149, Fy yeTien, P. 1985. Biological control of plant virus diseases. Chinese J. tpBlological Control 12):41-45.. “Tren, p.,G. Steger, V. Rosenbaum, J. Kaper, and D. Riesner. °3'1987. Double-stranded cucumovirus associated RNA 5: Experimental analysis of necogenie and nor-necagenic variants by temperature sraient se electrophoresis. Nucleic Acids Research Vol. $069- 5083. Tien, P. BLY. Qin, LY. Kang, and X.H1.2hang. 1985. Mild strains acne of pant vires, Academic Press Tien, P, and X.H1. Chang. 1983. Control of two seed-horne virus diseases in China by the use of protective inoculation, VNM 2020 5/ 09. iaBlen,P. Kil Zhang, HS. Qh, RY. Qin, and GS. Wa, 1987, tSaeite RNA forthe contol of plan diseases caused by cxcumber ‘Smt vas. tn. App Bol. $:143-152 Tien, P,, XH. Zhang, BS. Qiu, B.Y. Qin, X.C. Yang, LY. Kang, 2yiP GS, Wa. 1985. A new method to control plant virus diseases “Musing satelite RNA as a Biological control agent of plant diseases ‘caused by cucumber mosaic virus. Kexue Tongbao (Science Bulle- tin) 1985:69-70, bWu, 6.5, LY. Kang, and P. Tien. 1989. The effect of satellite RNA on croseprotection among cucumber mosaic vis strains. Ann. App. pflol 1189-496, Wu, $.X., 8H. Zhao, CX. Zhang GJ. Wang, X. C. Wang, X. Wang, and P. Tien. 1988. Transgeni tobacco plants resistant 10 cer mos vis (CMY by expressing its satelite RNA, Kexse Vongbao (Science Bulletin) 6:480. Wu, S.X,, S.H. Zhao, GJ. Wang, C.X. Zhong, X.C. Yang, X. Wang, ond P. Tien, 1989. Construction of cucumber mosaic viras ‘Shesistant tobacco plants by expressing its satellite RNA. Scientia Sinica (In Press). 37 Yang, X., B.G. Qin, X.X. Liang, and P. Tien. 1986. Satellite RNA 4 a biolosical control agent of diseases caused by cucumber mosaic virus, I. Effectof satellite RNA on the content of double stranded vial BNAlnCMV-infectedtissues, Acta Microbiologica Sinica 26(2):120- 128. Zhong, C,, X.H. Chang, and P. Tien. 1987. Field experiments of control of pepper mosaic diseases using vaccines $S1 and N14 in yantai, Acta. Virologica Sinica 1987:251-253.38 7 Poxvirus-Derived Recombinant Veterinary Vaccine Enzo Paole ‘Wadsworth Center for Laboratories and Research, New York State Department of Hi Empire State F Albany, New York 1; *Virogenetics, Inc., P.O. Box 2108, Empire State F Albany, New York 1: Abstract ‘The technology for developing vaccinia virus as a vector for the expression of specific foreign Immunogens has been extended to other members of the poxvirus family. Specifically, members of the avipox genus have been engineered to express foreign genes. Fowlpox virus, the prototype specles of the genus, has becn engineered to express genes from pathogens of interest to the poultry industry. Chickens and turkeys immunized with 2 fowlpox virus recombinant expressing the hemagglutinin from a highly virulent avian influenza virus were completely protected against a lethal challenge dose. ‘Surprisingly, when a fowlpox virus recombinant expressing the rabies glycoprotein was Inoculated into non. avian species, protective immunity was demonstrated against a live rabies virus challenge. In non-avian species fowlpox is not capable of productively replicating, yet expression of the foreign gene Is apparently sufficient to induce protective immunity. In this report [ will present some recent work, particularly from my laboratory, which involves the use of viral vectors for applications as vaccine ve- hicles. What I will talk about are specific applications to the veterinary field. The viruses that I will be referting to are in the poxvirus family. Figure 1 is an electronphotomicrograph of vaccinia virus which is the prototype species of the orthopox virus genus. What should I tell you about vaccinia as a virus? It is one of the most complicated of the viruses that we know. It is a double-stranded DNA virus that repli- cates, forall practical purposes, within the cytoplasm of the infected cell. It has a very complex genome en- coding roughly 250 proteins. Of these proteins, ap- proximately half are expressed early, that is to say prior to DNA replication. The other half are ex- pressed late, after DNA replication begins. I will tell you something about the use of this virus as a vaccine, Recall, if you will, that vaccinia has been used since 1796, when it was introduced by Jenner as “The tent of thls chapter was prepared from a transcript ofthe author conference presentation | | Figure 1. Electronmicrograph of negatively stained pact! vaccinia virus. Photo generously provided by W. Sam’ ‘Wadsworth Center for Laboratorlesand Research, New Yor Department of Health. agi2 vaccine against smallpox. The success of the virus “as a vaccine was attested to in 1980 when the World Health Organization declared that smallpox, as a Jnuman infectious disease, had been eradicated from the globe. This is a truly wonderful and unique yaccomplishment in terms of human infectious dis- “eases. 2 % Advantages of Vaccinia Virus as a Vector for Vaccine Production ‘There are properties of the virus that approach ‘the ideal in terms of a live vaccine: (1) at least under ‘conditions traditionally used, the vaccine is very inexpensive to prepare, costing a few cents per dose, (2) the virus is very stable and can be stored at 37°F foras long as a month without any significant loss in, infectivity, (3) the administration of the virus is very simple, being applied as a dermal abrasion with a bifurcated needle. Vaccinia, therefore, requires nei- ther expensive equipment nor very highly trained ‘medical personnel. These properties made us iden- tify vaccinia, a few years ago, as a vehicle to explore in terms of constructing engineered, live vaccines using recombinant DNA technology. “Protocol for Insertion of Foreign Genes into Vaccinia Virus Recombinants Figure 2 schematically shows how we go about making a recombinant poxvirus. What we do is isolate a foreign gene of interest and insert it at a locus in a cloned segment of DNA derived from vaccinia virus. The insertion locus of course must not, Sisrupt the flow of essential genetic information, pothertse we would not derive viable progeny. More Sophisticated engineering at the termini of the for- eign gene is done to facilitate expression. The chim- eric construct is amplified with a convenient cloning vehicle (like the plasmid PBR322) to have sufficient Quantities of the material for the next step, which is to transfect it into a tissue culture cell. The tissue Culture cell is simultaneously infected with a wild- type vaccinia virus, which goes through its standard replication cycle of uncoating, early gene expression, ~DNAreplication, and late gene expression and matu- “tation, What happensiis that within the cytoplasm of jitvelntected cella replicating JNA molecule localizes, itself in close proximity with the input chimeric - RECOMBINANT OHA Figure2, General protocol for generating recombinant poxvituses, A cloned foreign gene Inserted into a non-essential locus of a cloned vaccinia DNA fragment Is Introduced into tissue culture cells additionally Infected with virus. In vivo recombination Inserts the forelgn DNA into an intact viral genome that is packaged Into Infectious recombinant virus. gene, What can happen, and does happen at a frequency of approximately 0.1 percent, is that there ishomologous recombination between the sequences flanking the foreign gene and the homologous se- quences on the replicating vaccinia DNA molecule. This recombination event inserts the foreign genetic element into a novel recombinant DNA molecule that can itself be replicated and packaged into ma ture progeny virus. The recombinant virus can be identified and isolated by a variety of techniques, One of the advantages of vaccinia virus is that it has40 a very broad host range. Thus vaccinia virus can be useful for the inoculation of humans as well asa wide variety of animal species. A Polyvalent Vaccine Vehicle Another potential advantage of vaccinia virus is that there is a great plasticity in the viral genome, which is to say that the virus can be infectious while retaining a very large quantity of foreign DNA. One can consider the construction of polyvalent vac- cines, which, by a single inoculation, could immu- nize the recipient against a number of different infectious diseases. We estimate currently that there is easily room for about three dozen different genes. Figure 3 showsan example in which we have inserted three foreign genetic elements into a single vaccinia virus recombinant. The three foreign genes that we inserted were the genes encoding the herpes simplex virus glycoprotein D (HSV-gD), the gene encoding the hepatitis B virus surface antigen (HBsAG), and the cDNA copy of the RNA segment encoding the influenza virus hemagglutinin (Inf-HA). They are localized roughly in the particular configuration shown in Figure 3. Each is driven by a specific vaccinia virus promoter. The Southern blot (Fig. 3) demonstrates that the genes have indeed been in- seried into the virus at the locations predicted, What we then did was to take this vaccinia virus recombinant, expressing three foreign genes, and inoculate it into two rabbits. We demonstrated that there was indeed a serological response elicited in these rabbits against all three foreign antigens: hepa- titis B virus surface antigen, the influenza virus hemagglutinin, and the herpes simplex virus glycoprotein D, These responses were analyzed sepa- rately by various serological tests, and the data are shown in Table 1. The results demonstrate the immunological response to multiple foreign anti- gens expressed by a single vaccinia virus recombi- nant, aswell as to the many proteins expressed by the virus itself, This data encourages the use of members of the poxvirus family as vectors for eliciting polyva- lent immunity against infectious diseases. An Avian Poxvirus Vector Let me switch subjects for a while. As | men- tioned, one of the advantages of vaccinia virus is that | = fF | . css sso— E Figure 3. Thetop of the figure shows the structure ofthe w virus recombinant harboring the HSV-gD, HBsAg, and coding sequences, The leftmost 30 Kb of the vaccinia S ‘genome encompassing the terminal HinilllF, M,K, anit F fragments are shown. The restriction sites identifled (Gambil) and H (Hindlll). The X denotes the deletion of a mately 0.6 kb of vaccinia sequences described in the constz ‘of pMP62-15 (Perkuset a, 1986). Thebottom of the figure Souther blot hybridization analysisof the vaccinia virus binant vP168. DNA was extracted from purified vaccini: digested with restriction endonucleases, andblottedtoGenc Plus after electrophocetie separation of the DNA fragmec ‘agarose gel. vP168 DNA wascligested with Hindlll lane2). Bam dane 4 and lanes 6 to8) and probed with amixtur 110 5) or single (lanes 6 0 8) 32P-labeled nick-translated HSV-gD, and inf-HA sequences, Purified DNA fragmentscx ingthe coding sequences or HBsAg (lane 1), HSV-gD (lane Inf-HA (ane 8) were run as starcards Sizesof the fragmen obtained with Hindif-cleaved lambda DNA as marke above flgure is reproduced from Perkuset al, 1985, with~ gates Immunoogieal response to vaccination with arecomis- ant vaccinia expressing multiple foreign genes Gnoculation (RlAunits) ~— InfHA HSV vaccinia : 7 re i Ce er 2 tao Sane 3 aan Sam 1 om ay Bsco_ tan : wedi akon at 26 (nude 3 © eo : oma ; in i am § we ‘Rabbits were inoculated with 5 x 10" plaque-forming unitsin 0.5 ‘nleitherintravenously (rabbit 257) or intradermally (rabbit 288) ‘with avaccinia recombinant expressing the HBsAg, HSV-gD, and. InGHA genes, and the Immunological response was followed. ‘Sera were tested for antibodies to HBsAg with the commercially available AUSAB radioimmunoassay (RIA) kit; titersare expressed, In RIA units per milliliter ofserum asdefined by the manufacturer (Abbott). Antibodies to InfHA were measured by hemagglutinin, Inhibition tests performed with chicken erythrocytes and four ‘hemagglutinin units. The reciprocal of serum dilution is ineli- fated. Plaque-reduction assays monitoring reduction of HSV of vaccinia virus Infectivity were performed on CV-1 cells. The —teciprocal of serum dilution giving greater than $0 percent oe luton inplaquenumbersshown,Datarefrom Pckusel, 2eit has a wide host range, both among animal species eug8 Well as different tissue culture systems. However, tothere are other members of the poxvirus family that cottave a much more restricted host range, For ex- “ample, swinepox is able to replicate only in swine; ssmembers of the avipox group, such as fowlpox virus, ‘wivhich is the prototype species, can productively ian®€Plicate only in avian species (Matthews, 1982). sh waiWhat we elected to do initially was to look at the \.sivipox virus group, fowlpox in particular, so that we “ould, if you will, kill two birds with one stone, A mowlDox-virus-derived vector could be used specifi ™allyin the poultry industry. Furthermore, we wanted Ounderstand the basis of host restriction, especially 1 terms of productive replication of fowlpox virus. 41 A fowlpox virus vector would allow us to address some basic issues and also raise other questions which I'll get to in a bit. ‘There are essentially two preconditions necessary for the construction of a viral vector. One is the identi- fication of sites within the genome at which inser- tion of foreign genetic elements will not disrupt the flow of essential genetic information. The other precondition is that one knows what regulatory elements are going to be required to allow expression. of the foreign gene. In terms of identification of nonessential sites, we approached that question with fowlpox virus empirically, as we have for other studies with vaccinia virus (Perkusetal., 1985; Perkus etal., 1986; Pat etal., 1982), To fulfill the second requitement, we asked the question of whether fowlpox virus could recognize vaccinia virus regula- tory elements. We had in the laboratory a number of plasmids with various reporter genes driven by a number of vaccinia virus promoters from different ‘temporal classes. To save time and effort, we carried outa transient expression assay to determine whether fowlpox virus would recognize vaccinia virus regula- tory elements in a “trans” situation. The particular reporter gene used was the hepatitis B virus surface antigen, That gene was fused to either strictly tempo- rally regulated early or late promoters or to an early- late constitutive promoter. Then we transfected these plasmids into tissue culture cells, primary chicken embryo fibroblasts, infected with either fowlpox or vaccinia virus. We tested whether the hepatitis B virus surface antigen was expressed by serological screcning. As shown by this kind of transient expres. sion (Table 2), fowlpox virus indeed is able to recog- nize vaccinia virus regulatory elements. Vaccinia promoters work rather well in fowlpox-regulated systems, Thus we went ahead and utilized vaccinia virus promoters for constructing fowlpox- virus- based recombinant viruses. One of the first fowlpox virus recombinant vectors that we put together was a vector for specific use in poultry. We took a CDNA copy of the hemagglutinin ‘gene, which was derived froma highly virulent avian influenza virus (Kawaoka et al., 1987), inserted it into fowlpox and assayed for the expression of the avian influenza virus hemagglutinin. As shown in Figure 4, thereisindeed expression, as detected by immunopre-42, ‘Table 2. Recognition of vaccinia virus promoters in cells infected with fowlpox virus Promoter Class infecting Virus ‘P/N Ratio Eaaly Fowlpox 308 Vaccinia 336 Late Fooripox sus Vaccinia “7 Earlyfate Fowipox 05 vaccinia 422 None Fow pox. Vaccinia Earlyflate None 13 Baty, late or eailyate temporally regulated vaccinia promoters fused to “HHepettisB virus sequences encoding the surfaceantigen were transfected into primary chick embryo fibroblasts infected with either forslpox oF ‘vaccinia virss. Expression of the Hepatitis B virus surface antigen was rionitored using the AUSRIA kit (Abbott Laboratories) and noted as P/N ratios a described in the manufacturers protocel. Figure 4. Immunoprecipi- tation of radiolabeled avian influenza virus hemagglutinin synthe- sized in chick embryo fibroblasts infected with a recombinant fowlpox vitus. Lanes acdrepresent molecular weight mark- fers, uninfected cell ex: tracts, cell extracts in- fected with parental or recombinant fowlpox virus respectively. Data from Taylor et al., 19882. 25-7 \ cipitation from metabolically labeled infectec of a precursor polypeptide and the two exp cleavage products, with appropriate mole weights of 63, 44, and 23 kd. In this particular we wanted to demonstrate not only that the he glutinin gene was expressed by fowlpox virv also that it was processed and transported cor: Figure 5 shows that we can detect the hemaggh ‘on the surface membrane of cells infected v. recombinant fow|pox virus by immunofluoresc No immunofluorescence appeared in the cel fected by the wildtype virus. These data st proper synthesis, processing and transport « avian influenza virus hemagglutinin by a for virus recombinant. Chickens vaccinated with this recombinant for virus and challenged with live avian influenza were protected (Tables 3 and 4). Birds were nated at either 2 days or 4 weeks of age with ei fowlpox virus recombinant expressing the influenza virus hemagglutinin or a classicall pared killed vaccine preparation given as i Figure 5. Expression of the avian influenza virus hemagg ‘moleculeon the surfaccof primary chickembryo fibrobla figure shows identical fields of infected cells under phase (@and o) ot fluorescence (b and a) microscopy. Figs. a 31 chick embryo cells infected with wildtype parental fowl. whereas Figs. ¢ and d are infected with the fowlpo recombinant, Data reproduced from Taylor et al,, 198 permission.sGfable 3. Protection of chickens vaccinated with a fowlpox virus gecombinant expressing the HS hemagglutinin ene “Ageof Protection Virus detected Niirust Vaccine chickens sick/dead/total trachea cloaca gyre Tecombinant Pde OT —«OD OO AGEN) fowlpox Seeks 0/015 os os ee re infreund's Seeks 10/5 os os © Fowlpox Zaye O96 parent Seeks 45/5 oss tt None 2dsye 10910? Le 2dayss — 2/1/2 22 22 Seeks 27215 oss pichiciens were challenged with approximately 10°1,, of the highly Ingtbogeatc A/Turkeyeland/1375/83 (I5N8) influenea vius by ade iqnstering 0.1 ml to the nares of each bid. Two-day-o bind were alleged 6 weeks after vaccination abd S.weekold bids were chal feaged at 5 weeks postvaccination. “Sick” birds showed swelling and. “igyanonis of the face and comb and hemorchage ofthe legs such bits PAbequenly could net stand (Pe binds were sampled 3 days after infection by tracheal and cloacal Shabbing Virus was detected by Inoculation of embyonated exgs ZNon-vaccinated bids wore housed and mized wth the recombinant ~Yaccinate group of 10 birds to test for spread ofthe recombinant vin Fin one goup, 4 ofthe birds died the day before sampling, ise mociied from Tayior et al (19883). Emulsion. The vaccinated birds and appropriate Fontrols (Table 3), were challenged with 10° LD,, of the highly pathogenic homologous avian influenza ‘rus, the avian influenza virus from which we had ‘#erived the cDNA copy of the hemagglutinin gene. What is demonstrated in Table 3 is that upon chal- lenge with the homologous virus none of the birds jecame sick and none died from the challenge. No irus- spread, either to the trachea or cloaca, could be letected (Table 3). Chickens inoculated with wild- ype fowlpox virus or not vaccinated, as well as ~ontact controls, were sensitive to challenge demon- strating that there had been no spread of the recom- spinant fowlpox visus to this group of chickens. [We challenged another group of birds vaccinated in *} similar protocol with a heterologous avian influ- vihza virus. The heterologous virus differed in the wiemagglutinin gene by approximately fifteen per- “ent in the amino acid composition. What is demon- 4B ‘Table 4. Protection of chickens vaccinated with a fowlpox virus, recombinantexpressing the avian influenza virushemagg)utinin challenged with a heterologous influenza virus, A/Chick/Penn/ 1370/83 Challenge ‘Age of Protection Virus detected vim! Vaccine chickent slck/dead/tota] Trachea Cloaca Ciena Recombinant 2days _ o/0/10 sno o/10 (HSN2) towlpox — S weeks 0/06 sis 6 Inactivated days 0/08 28 08 imfreund’s S weeks 010/5 3S OS Fowlpox — 2days_— 10/1/10, ano 10/10 parent Sweoks 5/05 ss 98 None 2 days 99 9 2 days 22 2a 5 weeks 555 ‘Chickens were challenged with approximately 10° LD, af the highly pathogenic A/Chick/Penn/¥370(83 (15N2) vis by administering 0.1 amlto the nares of ech bid. Twosday-old birds were challenged 6 weeks ater vacinationand Sveek-old birds werechallenged $ weeks afer Vac ‘ination, "Sick" binds showed sweling and cyanosis the face andcomb and hemorthage ofthe logs Such sick binds Frequently could not stand Ss were sampled for virus spcad 3 days post-challenge by tacheal and cloacal swabs Non-vaccinatedcontotswerehoused withthe bles yaclnated with the recombinant fowipox vss to test for tansmision Table modified from Taylor et al, 1988, strated in Table 4 is that with the same vaccine regimen the chickens vaccinated with the recombi- nant fowlpox virus did not show any symptoms of illness. All survived the challenge. However, spread of influenza virus in the trachea or cloaca was de- tected. The contact control birds also succumbed to the challenge, which again indicated that the recom- binant fowipox did not spread. The efficacy of a fowlpox-virus-based vaccine was similar to an inactivated preparation given in complete Freund's adjuvant (Tables 3 and 4). Table § shows the serology demonstrable in these birds. In the birds that were vaccinated and subse- quently challenged with the homologous influenza virus, we could detect hemagglutinin-inhibiting and virus-neutralizing antibodies. Under these condi- tions there was no sickness, no death, and no detect- able spread of the virus after challenge. However, when we screened the sera for reactivity against the“4 Table 5, Serological response induced by inoculation with a recombinant fowlpox virus or an Inactivated influenza virus va Ai tes? ‘Neutilization of infec Chabenge Age of ‘Tyfetand ‘cuPenn Vaccine ‘chickens Post Post? Post Post Post Post.2 Recombinant aay = ise a0 S 70 2,506 Fowlpos S weeks 100 480 «10 20 160 10,00 Inactivated 2days 30 7 30 50 65 3,000 sm Freund's S weeks 350 600 380 200 240 2,506 Fowlpox 2aays <0 16xy? «10 20 <10 30 parent 5 weeks <10 42802) <10 o <0 10,000 None 2days <0 soa) 10 » <10 30 5 weeks «10 2,0003) <0 o 10 % CkPenn Recombinant 2days 15 600 «10 90 10 % sN2) Fowlpox 5 woeks 892,500 10 300 «10 2,506 Inactivated 2days o 300 Py 70 10 49 in Freund's S week 300 500 100 200 150 1,506 Fowlpox days <10 6) 10 10 <10 « parent S weeks <10 80 <10 140 =10 « None 2days <10 6) 10 160 <10 2% "The Soak old bieds were bled before vaccination and testedin Hl and neutvalization tests. None contained detectablean srenot shown, The 2-day-old chickens wore bled at 6 weeks post.vaccination (Post1) and the S week old birds Were blad at S weeks postvace! (@ost-); both groups were bled 2 weeks after challenge (Post.2). The igurvsare the man antibody titers from the same groups of chlckens de fn Tables 3 and 4, The numbers in parenthesis ar birds that survived challenge. femagglutination lnhibition titer. Data derived from Taylor etal, 1968 ‘Table 6, Protection of turkeys against challenge with avian influenza virus mediated by a fowlpox based recombinant Hlantibody Neutraizingantibor Protection Virus detection (yitre) yi) Vaccine ‘Age of binds sickidead/total trachea cloaca Post1— Post2 Post:l Post Recomb, 2aays Ts ws aE <0 10 a 732 Fowlpox weeks 26 216 16 =10 640 10s 4.16 Contact 2days 2212 2 22 <10 ead a ead Controls ‘weeks 212 22 22 <10 ead 4 0S >4 0 005 0 1 3 1 o4 05 4 0 4 0 2 4 1 > 2 04 0 4 0 2 5 454 2 54 0 2 0 4 6 Mo 4 m4 0 2 0 2 ‘Table 12. Protection of cats and dogs Immuntzed with a binant fowlpox virus expressing the rabies glycoprotein alive rabies virus challenge Titerat 94 days Day of deat Animal Vaccination’ —_postinoculatian postchallen Guns a 1 Survived 701s RAP 13 Survived 8271 None o 8 110 None o 2 Tal None 0 B T42 None ° 2 og 426 RAP 12 Survived 427 REP 19 Survived 55. None ° 1S 8240 None ° 6 fm aliquot of 2x po ofa Fowipox virus ecorbinant was Inactivated at °C overnight in the presence of 0.1% Beta propiolactone. Antibody to rabies glveoprtein, Sanuibody to fowipon vis antigens.

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