In This Issue:
Cassava Biotechnology: a Response to Questions Raised
1996 Risk Assessment Grants Announced
If You Have Something to Patent, Heads up
Update on Virus Recombination in Transgenic Crops
Risk Assessment of Transgenic Plants: Going Beyond the Crossroads
Transposons for Insect Transformation
Genetically Engineered Mosquitoes Resistant to Dengue Virus Transmission
Agbiotech Investors Report
Biotech Supply Companies Serve Web Customers
Three reports of progress in cassava biotechnology in the June '96 issue of Nature Biotechnology were accompanied by an editorial and Research News analysis which questioned the ultimate utility of the effort. The commentary prompted a detailed response from Ann Marie Thro, Coordinator of the Cassava Biotechnology Network. A slightly shortened version of her reply appears as a Letter to the Editor in the current issue of Nature Biotechnology (August 1996, p. 929). The exchange, although centered on a single crop, is representative of the larger debate over appropriate use of the technology, particularly with respect to projects intended to benefit developing countries. In the interest of furthering the discussion, the following excerpts from Dr. Thro's response are reprinted here with permission.
"... (T)he author of the editorial "Transforming the root of the problem" has accurately identified a "root" problem for cassava research: establishing and funding the linkages for "grabbing hold of innovations and transforming them in to useful products" ... and getting them to the users. The difficulties cited by the author apply not only to cassava biotechnology innovations, but to all efforts to enhance cassava's value for food security and economic development. But cassava is NOT in danger of becoming "the wrong kind of icon for biotechnology". On the contrary. Through efforts such as the Cassava Biotechnology Network (CBN), founded in 1988, the donor community of the developed world is collaborating with national agricultural research programs of cassava-growing countries, international agricultural research centers and others, working hard with small-scale farmers and processors ... to make sure that cassava research -- including biotechnology -- is properly targeted and delivered.
"... CBN's members include over 300 cassava biotechnologists engaged in strategic research or biotechnology applications. Two-thirds of these work in 25 cassava-growing countries, with the remainder in two international research centers and 13 economically-advanced countries. Our members also include another 300 researchers who use or will use cassava biotech innovations in applied research on plant breeding, plant health, variety multiplication, post harvest processing, and product development and marketing; with collaborators in socio-economics and technology transfer programs. Non-governmental organizations and farmer/processor cooperatives are among other CBN members.
"... It was clear from the onset of cassava biotech research that a network would be needed to bring a critical mass of researchers to the task. The author of the Research News analysis article, "Milestones in crop biotechnology ... cassava ... maize" perhaps conjectures that the successful laboratories reporting from the UK, Switzerland, and the USA/France achieved their breakthroughs in isolation. In fact, cassava transformation research began when the international centers CIAT (Centro Internacional de Agricultural Tropical) and IITA (International Institute for Tropical Agriculture) approached these very labs and several others for help in developing biotechnology tools for cassava. CBN was born out of the need to maintain the connections between these founding members and link them to applied cassava research. For the research reported in Nature Biotechnology June '96, all three labs were working with CIAT and with other CBN members (including China, Uganda, Zimbabwe...), and their results would not have been achievable alone.
"... In vitro micropropagation biotechnologies for conservation and exchange of cassava genetic diversity have already been transferred to national programs in about 20 cassava-growing countries, providing a base of skills and infrastructure for the new genetic transformation methods. The "Milestones" author's statements, "It remains to be seen if, how, when, and at what cost the technology will be transferred to the countries where it will be of practical benefit" and "there is very little, if any, effort toward the biotechnological improvement of crops in the developing nations" are highly valid concerns but underestimate the degree of attention in fact being paid to these issues in cassava R&D.
"... The editorial on "the root of the problem" is correct, however, when it implies that the cassava R&D cycle is under funded (hopelessly so, concludes the editorial) in view of cassava's importance to so many in the world, including some of the poorest farming communities and nations. Funding for cassava research, a small fraction of that spent on other major world crops, comes from contributions from national governments, bilateral and multilateral donors, and private foundations. CBN fully agrees with the "Milestones" author in calling for more international funding for biotech research on crops important to developing countries, in developing countries. It is important to remember that this call must be made not only to the international donors but also to the voting public of the donor countries. The public in economically advanced countries are almost entirely unaware of issues in cassava, food security, and agroeconomic development.
"... In the short and medium term, cassava R&D will remain humanitarian aid and a public investment in economic development. On this basis, and in the short time span of 25 years (compare a century of maize research or 50+plus years of soybean research, with investments dwarfing that in cassava), cassava research has already had impact: successful biocontrol of insect pests in Africa and South America; improved drought tolerance and increased yields of high quality cassava in Southeast Asia and Brazil; new products from cassava in Nigeria, Colombia, and Ecuador; new methods of safe international exchange of genetic diversity in vegetative form ... [etc.]
The letter concludes "... By working together to make best use of scarce resources, the cassava R&D community will continue to deliver useful technologies to cassava users. Cassava research does and will have "significance beyond the merely technical"."
CBN maintains a website at http://www.ciat.cgiar.org/cassava/cbn/cbn.html
Award recommendations for this year's USDA Biotechnology Risk Assessment Research Grants Program have been announced. The following proposals have been recommended for funding:
Recombination of Viral and Transgene RNAs in Susceptible and Resistant Plants. Jozef Bujarski, Northern Illinois University. $145,000 2 yr.
Risk Assessment of Transgenic Entomopathogenic Nematodes for Biological Control. Randy Gaugler, Rutgers University. $145,000 2 yr.
Risk Assessment of Plant Viruses Genetically Engineered for Vaccine Production. Rose Gergerich, University of Arkansas. $114,169 2 yr.
Inadvertent Movement of Recombinant Baculoviruses by Non-target Endoparasitoids. Kevin Heinz, Texas A&M University. $196,866 3 yr.
Field Test of a Transgenic Arthropod. Marjorie Hoy, Univ. of Florida. $50,220 1 yr.
Risk Assessment of DNA Transfer Between Recombinant and Natural Baculoviruses. Susumu Maeda, University of California. $185,578 3 yr.
Fitness of Recombined Tombaviral Genomes. Ulrich Melcher, Oklahoma State University. $203,256 3 yr.
Reproductive Manipulation of Fishes: Ecologically Safe Assessment. William Shelton, Univ. of Oklahoma. $20,013 1 yr.
The Spread and Ecological Fitness of Fitness-related Transgenes in Wild Sunflowers. Allison Snow, Ohio State University. $210,000 3 yr.
Assessment of the Risk of Bacterial Spread in the Phyllosphere. Christen Upper, University of Wisconsin. $205,000 3 yr.
For information, contact USDA/NRI Program Director Dr. Edward Kaliekau, 202-401-1931.
Can a non-inventor's use of an invention in a private laboratory result in a loss of patent rights in the invention? A recent decision of the United States Court of Appeals for the Federal Circuit shows that indeed this can happen.
Consider the following scenario. You devise an expression vector for testing plant promoters that contains a novel marker gene downstream of a multiple cloning site. Knowing that your colleague is starting a project on root-specific promoters, you send her a sample of your expression vector. Your colleague finds the vector to be very suitable for her study, and she uses it in her laboratory to screen promoter fragments. Eventually, you decide to obtain a patent for the expression vector, and you file the application over a year from the day that your colleague began to use it. Unfortunately, your colleague's use of the vector probably created a "public use" statutory bar that destroyed your patent rights (1). This is one lesson of Baxter International, in which the Federal Circuit decided that a non-inventor's use of an invention in his laboratory at the National Institutes of Health created a public use bar that invalidated a patent for the invention (2).
Baxter International began with a patent infringement suit that Baxter brought against COBE Laboratories. Inc. As a defense to infringement, COBE Labs asserted that Baxter's patent was invalid because the claimed invention had been in public use for more than one year before the filing date of the patent application. Baxter responded that its patent was valid because the inventor was entitled to an "experimental use exception" to the public use bar. According to Baxter, the non-inventor's use of the invention was not publicly known or accessible, and ethical constraints would have precluded those who saw the invention in the non-inventor's NIH laboratory from revealing their knowledge of it.
The court, however, reminded Baxter that a public use includes any use of the claimed invention by a non-inventor who is under no limitation, restriction or obligation of secrecy to the inventor. Moreover, the experimental use exception is limited to the inventor, to people working for the inventor, or to people who are under the direction and control of the inventor. In this case, those who observed the invention in the non-inventor's laboratory were under no duty to treat their knowledge as confidential. Accordingly, the court decided that Baxter's patent was invalid due to the public use bar.
In her dissent, Judge Newman declared that Baxter International establishes a new rule of law, that an unpublished use of an invention in a private laboratory is public use. Yet the outcome in Baxter International is consistent with another Federal Circuit case, in which the court decided that the use of an invention by Monsanto researchers, who had no connection with the inventor, created a public use bar (3). Here, the court emphasized that it was irrelevant that visitors to the laboratory who may have seen the invention in use by Monsanto workers, had no idea of the true nature of the invention. The important point was that the Monsanto scientists' knowledge and use alone provided a sufficient basis to invoke the bar.
Baxter International does provide some guidance for determining whether a use is an experimental use. The court's evidentiary factors include the length of the test period, whether the inventor received payment for the testing, an agreement by the user to maintain the use confidential, and the existence of test records.
The most significant factor for determining experimental use, however, is the extent of control that the inventor maintains over the testing. This point was highlighted by another recent opinion of the Federal Circuit, which stated that the "factor of control is critically important, because, if the inventor has no control over the alleged experiments, he is not experimenting" (4). The court further explained that an inventor is not experimenting if the inventor fails to inquire about the testing or fails to receive reports concerning test results. By the way, the inventor in this case, like the hypothetical inventor of the expression vector, simply gave copies of his invention to friends without any provision for follow-up involvement.
References
1. "A person shall be entitled to a patent unless . . . the invention was . . . in public use or on sale in this country, more than one year prior to the date of the application for patent in the United States . . ." 35 U.S.C. Section 102(b).
2. Baxter International, Inc. and Baxter Healthcare Corp. v. COBE
Laboratories, Inc. and COBE BCT, Inc., No. 95-1407 (Fed. Cir. July 10,
1996).
3. National Research Development v. Varian Associates, 30 USPQ2d 1537 (Fed.
Cir. 1994).
4. Lough v. Brunswick Corp., 39 USPQ2d 1100, 1105 (Fed. Cir. 1996).
Phillip B.C. Jones
Foley & Lardner, Washington, D.C.
pbcj@ari.net
Concerns over the potential for genetically engineered virus resistant plants to cause environmental problems have focused on whether novel viruses could be produced by RNA recombination. The process, which involves the exchange of template RNAs during virus replication, results in a product that combines two originally distinct RNA templates. Such exchanges, occurring in the cells of an infected host plant, are not limited to viral RNAs but include messenger RNA transcripts of host genes as well. Consequently, while expression of a viral transgene, in many cases the capsid protein gene, confers virus resistance to a transgenic plant, the viral sequences in that mRNA are available for recombination with the RNA of a challenging virus. Data addressing this issue were presented in four talks at the 8th Symposium on Environmental Releases of Biotechnology Products: Risk Assessment Methods and Research Progress held in Ottawa, Ontario June 23-26, 1996.
Zhongguo Xiong, University of Arizona, described recombination in plants transformed with a gene of red clover necrotic mosaic virus. The virus has a bipartite genome, which means it is comprised of two separate RNAs both of which are required for normal infection. The transgenic plants contained sequences from RNA 2 encoding the virus movement protein, but not flanking sequences necessary for viral RNA replication. In Xiong's experiments, these transgenic plants were challenged with RNA 1 alone. Recombination between the movement protein transcript and the inoculated viral RNA restored viral replication signals to the transcript and converted it to its complete viral form thus reestablishing the bipartite virus. Suitable markers confirmed these recombination events.
James Schoelz, University of Missouri, reported that recombination involving a transgene also occurs in cauliflower mosaic virus (CaMV), a DNA virus which replicates through an RNA intermediate. Transgenic Nicotiana bigelovii plants expressing gene VI from a CaMV isolate that infects this host were challenged under conditions of either strong or moderate selection pressure. Strong selection pressure was simulated by inoculating the transgenic plants with a CaMV strain that does not naturally infect N. bigelovii . Viral recombination with the transgene converted the challenging virus to a N. bigelovii systemic pathogen and viable recombinants were recovered at a high frequency. Moderate selection pressure was simulated by inoculating with a CaMV strain that could infect N. bigelovii. In this case, recombinants were isolated, but at a lower frequency. In competition assays, recombinant viruses from both experiments out-competed wild type virus.
Based on these and other recombination studies, it is apparent that viral transgenes are available for recombination with challenging viruses. Do virus resistant transgenic plants provide an enhanced opportunity for RNA recombination? To determine this, it is necessary first to assess how often recombination occurs in natural mixed infections, that is, in nontransgenic plants simultaneously infected with two (or more) viruses.
In our laboratory, mixed infections of two related bromoviruses, brome mosaic virus and cowpea chlorotic mottle virus, were established in nontransgenic N. benthamiana plants. No recombination events were detected within the capsid genes of viruses recovered from the multiply-infected plants. Healthy plants were also inoculated with functional chimeric versions of the same viruses, in which the capsid protein genes had been exchanged. When mixed infections were established with these modified viruses, recombination was detected readily within the recovered viral RNAs. Thus modification of the virus genomes either destabilized viral RNA such that recombination was encouraged or provided selection pressure for a more efficient virus. The rate of recombination in these mixed infections was similar to that seen in transgenic plants although it was more difficult than anticipated to establish the mixed infections.
In the field, virus resistant transgenic plants are still susceptible to infection by viruses other than the targeted pathogen. In cases where the plant is not a host for the secondary virus and infection and replication are limited to a few cells, there will be strong selection pressure for the ability to establish a systemic infection. It is not clear if the transgene, derived from a virus capable of systemically infecting that host, can provide a challenging virus with sequences that will enable it to become a systemic pathogen. But it is clear that the opportunity for recombination exists.
Therefore, it seems prudent to design transgenes with a minimal propensity for recombination with a challenging virus. Suggestions to this end include limiting the transgene to the smallest segment that affords adequate virus resistance and avoiding sequences that encourage RNA recombination. Such sequences include viral RNA replication binding sequences and sequences identified as recombination hot spots.
Richard Allison
Michigan State University
22923mgr@msu.edu
Pat Traynor, Editor
RISK ASSESSMENT OF TRANSGENIC PLANTS: GOING BEYOND THE CROSSROADS
Biotechnology has recently provided agriculture with new plant varieties
that promise to increase yields, decrease chemical usage, and provide food
of higher quality. Many of these features, such as altered fruit ripening
and modified oil profiles, are ecologically innocuous and an economic boon.
However, many plants are being engineered for fitness-related traits such
as insect and disease resistance -- traits that would help them survive
outside of cultivation and affect ecology. So it is not surprising that many
researchers are investigating the ecological risks of transgenic plants.
The Eighth Symposium on Environmental Releases of Biotechnology Products was held this June in Ottawa Canada, where an estimated 250 participants heard data from research on the risks of biotechnology. Aptly, the theme of the conference was "Risk Assessment at the Crossroads." More than in any other such conference, researchers presented hard data on the ramifications of transgenic plant release on agriculture and nature. For example, Allison Snow demonstrated that the out crossing barrier from cultivated to wild sunflower is weak, and that gene transfer to wild sunflower will very likely occur. Jack Brown showed that transgenic canola is able to pass transgenes on to several wild relatives, and work from my lab has shown that insecticidal transgenic canola is more fit than non-transgenic canola in the presence of ubiquitous herbivorous insects. The cases of sunflower and canola are examples of what many may consider problem crops because wild relatives grow in close proximity to crop sites. Thus we are beginning to see that there are certain imminent risks involved with releasing certain crop/transgene combinations.
The question we are currently facing is -- What will happen now that we are at the crossroads of risk assessment? Tomorrow the crossroads will be in the rearview mirror, and we will have decided on a path for each crop/transgene combination. There is limited data demonstrating certain risks in small organismal and population experiments. One lesson from the past is that risks identified in small realistic experiments translate into larger risks in large-scale releases which may not be easily identifiable from the outset. For example, chestnut blight killed a few trees when it was first introduced, but soon thereafter, it drastically altered the ecology of the Appalachian hardwood forest. The large-scale suite of ecological effects from blight introduction could not easily have been predicted a priori. Yet no one will contest that it would have been better to avoid chestnut blight altogether rather than try somehow to "manage" it later.
At the crossroads, one road is called "avoidance," and the other is called "management and mitigation." The road of avoidance is fraught with short-term economic potholes for companies should certain biotechnology products not be released because the short- or long-term risks outweigh the benefits. Such decisions are not easy to make, since the costs may be in the form of hard-to-measure, long-term ecological effects not only in this country but other countries as well. Furthermore the benefits are generally economic and will likely be geographically localized. Therefore, unless regulatory agencies critically analyze risk assessment data, and take to heart recommendations from unbiased scientists, the short-term economic incentives for releasing some products will likely win by default. The road to management and mitigation will have been taken.
This road is not a smooth one for many of its travelers, however lots of vendors make lots of money along the way. Unlike choosing, perhaps temporarily, the road of "avoidance", the decision to opt for management and mitigation is irreversible. There is no way to recall a deregulated and widely-dispersed transgenic plant, if that plant or its relatives can survive outside of cultivation. On this road we must devise creative methods to contain transgenes, and/or mitigate the ecological damages of escape. If all long-term ecological effects were knowable from short-term research, the task would be less daunting. However, ecology teaches that all levels of biology are interwoven and not immediately manifested. There are various levels of ecological effects: from whole organisms, to populations, to communities, to ecosystems, and beyond. Risk assessment data obtained thus far for any transgenic plant has not progressed beyond the population level. The reasons for this are varied. For example if organism- or population-level effects are not present, then higher-level ecological effects will likely not be present either. Another reason why community and ecosystem-level data on transgenic plants does not exist is that experiments at these levels are usually long-term ones. Transgenic plants have not existed long enough to enable researchers to perform the studies.
As alluded to at the beginning, I am not suggesting that the only road we should ever take is the avoidance trail. There are many data indicating that genetic engineering per se does not translate into ecological risk. In fact, transgenic plants are emerging as powerful tools in ecological research and potential solutions to long-standing environmental problems such as metal contamination in soil. However, it may be of great collective benefit to avoid the commercialization and large-scale release of any crop engineered with fitness-altering genes and capable of interbreeding with wild relatives. For example, most people want to avoid creating insect-resistant transgenic weeds. This should be quite simple since it is relatively quick and easy to investigate organismal- and population-level effects of suspected high-risk transgenic plants. If effects at these levels are discovered, it is almost certain that unknown effects at higher ecological levels will exist, too, and it would makes sense, then, to test for community and ecosystem effects. High risks would imply the avoidance of commercialization and release. The alternative is to merely speculate on ecological effects (or ignore hard data), release the plants, and attempt to implement management strategies when a problem appears.
I would like to invite discussion from the scientific, regulatory, environmental and industrial communities about our upcoming decisions concerning which road we will take for certain crop/transgene combinations. My opinion is certainly not entirely unbiased. It comes from one who performs molecular biology and ecological research in a mid-sized university. I present scientific data that is unbiased, but my opinions are colored by my desire to be liked, published and funded! It is impossible for those who discuss these issues to be divorced from their biases, but it would be nice to know what those biases are. I am encouraged by where biotechnology has come from, and with most of the directions it is heading. But if we do not carefully assess where we want it to take us in the future, we will certainly not arrive where we had hoped.
C. Neal Stewart, Jr.
University of North Carolina - Greensboro
nstewart@goodall.uncg.edu
Transposons, also called "jumping genes", are DNA sequences which are capable of moving themselves in and out of genomes. Ever since the P element was utilized to genetically transform Drosophila melanogaster in the early 1980's, transposons have been a major focus of insect transgenesis research. However, the P element was found not to work in insects other than D. melanogaster and its drosophilid relatives. Researchers had tried for many years to get the P element to work in medfly, but gave up and turned to another element called minos, which was eventually used to transform the medfly. Several other transposable elements, such as hobo and mariner, are being investigated as transformation vectors for other economically important insects.
Transposable elements of the class to which P, mariner, and hobo belong work by using a cut-and-paste mechanism. The transposon encodes a gene for a transposase, an enzyme that finds its particular target site on the chromosome and makes a cut into which the transposable element (DNA) inserts itself. When the element later "jumps" to a new site, the same transposase acts to excise the DNA. In order for a transposon to be useful as a transformation vector, molecular biologists use a dual system in which one plasmid carries the transposable element with a reporter gene or some other gene of interest in place of the transposase coding region, and a helper plasmid carries the functional transposase gene. The helper plasmid mediates insertion of the transposable element into the chromosome, but is not capable of transposition or integration itself. Once inserted, the modified element is unable to excise itself.
A Symposium on "Evolutions and Revolutions in Insect Genetic Transformation" was held at the Pacific Branch Entomological Society of America meeting this past June in Big Sky, Montana. At the meeting, scientists from around the country discussed the current usefulness of various transposons in transforming insects. The element hobo, which has been used to stably transform Drosophila, is being studied in tephritid and drosophilid fruit flies, including the medfly, at the USDA-ARS lab in Gainesville, FL, and at the University of Hawaii. Element excision was found to occur in a D. melanogaster strain which did not have functional hobo only when the helper plasmid was injected. However, with the exception of D. saltans, medfly dark pupae, and Bactrocera dorsalis, hobo was found to be mobile in all other tephritid and drosophilid species tested which did not have functional hobo. This indicates that there might be hobo-like elements present in many fly species which are capable of "cross-mobilization". Although hobo has been shown to function in a number of different insects, its utility may be limited if integration of a foreign gene is not stable due to cross-mobilization.
Another transposable element found in Lepidoptera is being investigated as a potential gene vector at Notre Dame University and at the USDA-ARS lab in Gainesville, FL. Reports by Malcolm Fraser and his research group at Notre Dame have detailed the mobility characteristics of IFP2, now called piggyBac (1,2). This transposable element has been found repeatedly as an insertion within a baculovirus genome.
At the recent ESA Symposium, Dr. Fraser addressed the potential for piggyBac to serve as a transformation vector for lepidopteran insects. Plasmid DNA with the piggyBac element carrying an insecticide resistance marker was coinjected with a helper plasmid into preblastoderm Trichoplusia ni (cabbage looper) eggs. Only one female survived the insecticide selection and was crossed with a wild type male. To determine if piggyBac had integrated into the chromosome of the female, genomic DNA of offspring was analyzed by polymerase chain reaction. Insertion events were found in a portion of the offspring, and further analysis suggested that three to seven copies of piggyBac had integrated into the genome of the transformed parent. Similar results have also been obtained for the Indian meal moth, Plodia interpunctella.
Though the potential for piggyBac to be used routinely in insect genetic transformation seems great, there are a couple of problems which may hamper its applicability. There was a small background level of the piggyBac element showing up in the offspring of the putative transgenic female. The background may arise from the presence of endogenous piggyBac element itself or a piggyBac-like element which may be capable of cross-mobilization. In the latter case, the usefulness of piggyBac for stable transformation is clearly limited. It remains to be seen whether using this element in a lepidopteran species not closely related to cabbage looper will eliminate the background signal. Plans are underway to use the piggyBac element in codling moth and pink bollworm, two species which are relatively distant lepidopteran cousins of the cabbage looper. Another problem with piggyBac is that because it inserts at multiple sites, it may cause gene mutations that are deleterious or lethal. Also, multiple copies of a foreign gene may lead to detrimental effects on the gene's expression or unknown effects on the functions of other genes.
Typically, scientists rely on "brute force" to genetically transform insects, that is, the microinjection of DNA into thousands of preblastoderm eggs followed by a chance event whereby the foreign DNA sneaks into the genome during DNA replication when chromosome breakage may occur. The search continues to find suitable transposable element vectors, because they will dramatically minimize the brute force and luck needed for stable insect transformation.
References
1. Fraser, M., et al. 1995. Virology 211: 397-407.
2. Fraser, M., et al. 1996. Insect Molecular Biology 5 (2):
141-151.
Holly J. Ferguson, Research Entomologist
USDA-ARS Yakima Agricultural Research Laboratory
fergie@yarl.gov
Mosquito-transmitted diseases, such as malaria and dengue fever, affect hundreds of millions of people yearly. It has been estimated that dengue virus, which is transmitted by Aedes mosquitoes, infects 100 million people. In an effort to limit the spread of mosquito-transmitted diseases, two strategies that do not involve insecticide spraying are being evaluated. A novel biological control method is in development which aims to eradicate mosquito populations by genetically engineering bacteria to produce mosquitocidal toxins (July 96 issue of ISB/NBIAP News Report). An alternative approach to eradication involves the development of virus-resistant mosquitoes, which are incapable of propagating the virus and subsequently spreading disease.
In the May 10 issue of Science, scientists from the Arthropod-Borne and Infectious Diseases Laboratory in Colorado describe genetically engineered mosquitoes resistant to dengue type 2 virus. The natural lifecycle of this virus involves the transfer of the virus to a female mosquito following the taking of a blood meal from an infected human. The virus enters the insect's midgut, replicates and spreads to other organs including the salivary glands. The cycle is completed by transmission of the virus to an uninfected human upon taking of the next bloodmeal.
Dengue virus contains a sense strand RNA genome that is translated into a single polyprotein, which is subsequently cleaved into individual viral proteins. The strategy that has been utilized to block viral replication involves the synthesis of antisense RNA. In theory antisense RNA, which has a complementary sequence to the target RNA, forms a RNA:RNA duplex that inhibits translation of the target RNA. A recombinant Sindbis virus has been constructed which expresses an antisense RNA targeted to the premembrane coding region of dengue viral RNA. The premembrane protein was chosen because it is an essential component of dengue virus assembly and because the premembrane coding region is located at the start of the viral RNA. Thus inhibition of translation of the polyprotein is predicted to affect the production of all dengue viral proteins.
Only mosquitoes which were simultanenously inoculated with the recombinant antisense Sindbis virus and wild type dengue virus were unable to support replication of dengue type 2 virus in their salivary glands. Interference was specific since there was no inhibition of replication with dengue type 3 or type 4 viruses. However, the present work only shows that transient expression of the antisense construct is effective at blocking viral replication. Clearly a long term goal is to develop transgenic mosquitoes that constitutively express the antisense RNA and become "immune" to viral infection. These genetically engineered mosquitoes would then no longer serve as competent disease vectors.
One limitation to the present approach is the fact that Sindbis virus, while innocuous to mosquitoes, is a human pathogen although its pathogenicity in humans is not well characterized. Thus Sindbis represents only a model system and other viral vectors that are more benign to humans need to be developed. Hopefully in the near future, a mosquito bite will cease to be a potentially deadly event and will only be a nuisance.
Eric A. Wong
Department of Animal and Poultry Sciences
Virginia Tech
ewong@vt.edu
The stock market correction that some analysts had been predicting appears to have come to fruition in July. Technology stocks of all kinds lost value, including biotechnology stocks. In fact, biotech stocks have been hinting of a correction since mid-June, before the rest of the market fell.
Reasons given for the slide include the fall of high technology stocks in general, primarily computer-related technology companies, because many of the same investors pursue these two areas. Also, certain FDA panel reviews of new drug applications received more restricted approvals than had been hoped for by investors. Finally, certain biotechnology company stocks were likely overvalued, and warranted correction (1). This is relevant to agbiotech companies given the influence of biopharmaceutical stocks on the overall performance of the biotechnology sector.
One of the factors that could have sent the sector into an even more dramatic tailspin was recent quarterly earnings announcements of some of the industry's top tier companies. Fortunately, the sector's lead company, Amgen, showed stronger than expected second quarter earnings, outpacing analyst expectations by 6 cents per share (2). Second quarter earnings of $178 million represented a 30 percent increase over last year's second quarter earnings.
As is the case with biopharmaceutical stocks, agbiotech firms posted mixed quarterly results. Dekalb Genetics had stronger than expected earnings for their third quarter, posting a gain of 51 cents per share and well above analyst expectations of approximately 29 cents (3). Dekalb has seen its stock price steadily rise over the last year from around $13 a share to over $30 a share, and was most recently buoyed by the awarding of a patent for using a "gene gun" for insertion of genetic material into corn to confer insect or herbicide resistance. The longer term impact of the patent is as yet unclear, as rival Pioneer Hi-Bred has announced its intention to challenge the Dekalb patent.
Another mainstay of the agbiotech sector, Mycogen, announced less impressive quarterly results, posting earnings per share of 11 cents, down from 36 cents for the same period in 1995. Revenues were up from 1995, increasing to $80.4 million, strengthened by its United AgriSeeds unit which was acquired earlier in the year. Planting delays were partly to blame for other sales decreases that impacted quarterly earnings (4). One securities firm downgraded its recommendation for Mycogen based on the disappointing quarterly earnings (5).
Despite Wall Street's recent dissatisfaction with biotech stocks, some analysts believe that select biotechnology sector stocks will begin to recover in the fall, although much of this optimism is based on strong fundamentals in the biopharmaceutical sector. Its unclear how agbiotech stocks will perform and what the impact will be of recent events in the agbiotech sector, including the questions being cast over the effectiveness of Monsanto's genetically engineered bollworm resistant cotton (6).
References from Wall Street Journal Interactive Edition (wsj.com), Dow Jones News Service:
1) Eisinger, J., Biotechs Hurt In Tech Stock Flight; Recovery Seen. July 15,
1996
2) Rundle, R.L., Amgen Net Income Rises 30% Surpassing Analysts' Forecasts.
July 19, 1996.
3) Earnings Surprise Summary. July 12, 1996.
4) Mycogen Corp. 3Q Net 11c A Shr Vs 36c. July 10, 1996.
5) NatWest Cuts Mycogen To Hold From Accumulate. July 11, 1996.
6) Buggy Cotton Crops Cast Doubt on Engineered Seeds. July 23, 1996.
William O. Bullock
Institute for Biotechnology Information
Research Triangle Park, NC
http://www.biotechinfo.com
Research in biotechnology is more resource-intensive than any other branch of science, as there is a continuous need for sophisticated laboratory reagents including enzymes, oligonucleotides, radiolabeling compounds, as well as equipment such as better thermal cyclers and faster centrifuges. New products are released into the market practically every day but researchers often have only limited time to scan product brochures and catalogues. Increasingly, biotechnology vendors are placing technical and pricing information for their products on the Internet. Scientists can use efficient search engines to learn about these products 'on demand'. The discerning equipment or reagent buyer can now tap into the Cyberspace to comparison shop for molecular biology research products and services, place orders, obtain technical information such as experimental protocols, and also communicate with technical service specialists.
While there are hundreds of biotechnology supply companies represented on the World Wide Web, fortunately there are a few web directories that link these companies in an alphabetical manner and offer a brief description.
Following are among such directories:
Biological Data Transport: http://www.data-transport.com/
BioSupply Net: http://www.biosupplynet.com/bsn/
BioTechnet: http://www.biotechniques.com/
All three sites feature announcements of new products while Biological Data Transport has links to auctions of used lab equipment.
C. S. Prakash
Center for Plant Biotechnology Research
Tuskegee University
prakash@acd.tusk.edu
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