In December 1992, APHIS held a meeting to discuss the use of exotic viruses under contained conditions.
UNITED STATES DEPARTMENT OF AGRICULTURE
EDITED BY THOMAS PIRONE, JAMES WHITE, AND SALLY VAN WERT
The first topic dealt with the concept and definition of exotic viruses (outside the current definition used by APHIS). An important point is that although a virus might not be considered exotic because its nomenclatural equivalent exists in the U.S., its strains or isolates may differ considerably in host range or other significant biological properties. Requests for permits should detail how the viruses proposed for importation differ from strains existing in the U.S.
The subsequent topics were discussed in roughly the order on the handout (see attached). Although the meeting focus was on use of exotic geminiviruses, many of the issues discussed and recommendations proposed would be applicable to other exotic viruses.
Should the use of exotic geminiviruses be allowed? There was general agreement that use of exotic viruses should be allowed, provided appropriate conditions for containment can be designed. There was no support for allowing the use of exotic viruses as challenge inoculum in field situations. This included the inoculation of plants by novel methods (e.g. particle gun or Agro-infection) that circumvent the use of insect vectors.
Factors to be considered in permitting use of exotic viruses included the presence or absence of susceptible hosts (of viruses, vectors or both) both in the field and in greenhouse cultivation, the importance and intensity of commercial cultivation of susceptible crops in the area, and the ability of the vector to survive under adverse climatic conditions. Since these and other potentially relevant factors are interactive, it was agreed that the requirements for confinement would need to be determined on a case-by-case basis.
What should be done about the reconstruction of infectious viruses from nonregulated entities? The scientists agreed that a logical approach would be to add to the existing regulations the requirement that if partial sequences, and/or noninfectious extracts are to be used to produce infectious viruses or if the sequences are to be used to produce transgenic plants, APHIS permission must be obtained. The oversight of these types of experiments currently appears to reside with local Institutional Biosafety Committees (IBC), but several participants expressed the view that most IBCs were not set up to deal with plant issues and that there are no official guidelines to aid them in knowing how to deal with such issues. Such guidelines are very much needed.
Description of appropriate containment conditions. Requests by scientists for use of exotic viruses under contained conditions can be divided into two categories. Requests to work with the exotic virus and the insect vector, or requests to use the virus alone. The major difference between these two categories is the former requires containment of the viruliferous insect while the latter requires that insect vectors or potential vectors be excluded from the facility (Note: to work with an exotic insect, a scientist must obtain permission to import the insect from APHIS via PPQ Form 526).
It was generally agreed that plants infected with exotic virus be maintained under specifically defined containment conditions, and that these should include restriction to isolated, limited-access facilities. The use of exotic viruses with their insect vectors under contained conditions should only be allowed under the most stringent containment conditions. These conditions should be virtually identical to those used at Fort Detrick or be restricted in other appropriate manner that would preclude survival of viruliferous insects. One example of the latter would be use of viruliferous insects only during months when the vector could not survive outside because of cold temperature and the lack of host plants. Also, consideration must be given to the nature of the insect vectors. Aphids and leafhoppers can be contained, whiteflies are difficult to contain, thrips are even more difficult, and mites are virtually impossible to keep contained.
Three subsets exist for requests which do not include use of the insect vector: (1) the insect vector is present in the State, (2) the insect vector is not present in the State but is present in the U.S., and (3) the insect vector is not present in the U.S. but related insect species that may be vectors are present.
When the insect vector is present in the State (subset 1), the scientists did not support permitting the growing of virus-infected plants in ordinary, single-paned, screened glasshouses. The growing of virus-infected plants in a glasshouse could be permitted if exclusion of the insect vector could be assured by physical means (facilities equivalent to Fort Detrick) or by growing the plants during periods where the insect vectors are not present. To permit the use of exotic virus in laboratories and growth chambers in a State where the insect vector is present the primary objective would be to reduce the probability that the insect vector could enter and leave the facility and then transmit the virus to a susceptible plant. To meet this objective the following questions would need to be asked during the facility inspection: is the facility secure; are the windows sealed shut; are the vents and air conditioning ducts screened to exclude insect vectors; are the in-coming plants insect- free; are growth chambers locked; are virus-infected plants disposed of by means which preclude exposure to the insect vector; and have procedures been established to detect and eliminate insect vectors if detected in the facility?
For subsets 2 and 3, factors needing consideration for permitting the use of exotic viruses included the availability of adequate data to ascertain whether or not vectors or potential vectors are in nearby laboratories or the immediate geographical area; whether or not - susceptible hosts (for viruses, vectors or both) are present both in field and in greenhouse cultivation; the importance and intensity of commercial cultivation of susceptible crops in the area; and the ability of the vector to survive under adverse climatic conditions. Since these and other potentially relevant factors are interactive, it was agreed that the requirements for confinement would need to be determined an a case-by-case basis. These requirements could vary from a screened greenhouse to isolated, locked growth chambers under negative pressure with HEPA filtration and strict limited access with intensive monitoring for insects and potential escapes. Particular attention should be paid to the proximity of geminivirus-infected plants to ornamentals such as poinsettia, hibiscus, and other ornamentals which are commonly infested with geminivirus whitefly vector Bemesia tabaci. Some participants suggested that only plants grown from seed be permitted in greenhouses containing geminivirus- infected plants. Special efforts are needed to determine if another researcher in the facility has permission to work with an exotic insect vector that could transmit the exotic virus.
It would be the researcher's responsibility to provide the data about the biology of the insect vector, its presence or absence in their State or research facility, and other related information. The responsibility for ensuring that confinement conditions are followed, instructing greenhouse managers, and ensuring proper disposal of infected plant material, appears to rest with the researcher, the institution, and its IBC, with the attendant shortcoming discussed above. Currently, APHIS inspects facilities that work with exotic viruses yearly to verify that containment conditions are being followed.
Flow and dissemination of information. It was suggested that BBEP ask researchers whose protocols for confinement and related approaches have been approved and tested to briefly document their success, need for modification, and so forth, so that the information could be disseminated. The suggestion was made that such information could be published in an appropriate format in a relevant journal. BBEP could also develop a list of resource persons willing to provide input when novel situations arise. Examples might include advice as to whether properties of an exotic strain are sufficiently different to pose a potential problem and the likely efficacy of proposed procedures for vector exclusion.
Regulation of release of transgenic plants. Numerous coat protein transgenics have been field tested without apparent mishap, and requests for large-scale tests and commercialization are pending. The question of potential problems was addressed and the possibility was raised of recombination involving unrelated viruses infecting the transgenic plant and the plant-expressed viral sequences. There is evidence for some viruses that recombinants do occur. Thus questions arise as to which viruses/virus sequences can recombine, how often recombination occurs, and how likely is it to result in an adverse product, e.g. a virus with increased virulence or expanded host range.
It was agreed that studies examining these questions should be conducted, and several approaches were suggested; these ranged from using the systems most likely to produce a recombinant and attempting to select for one with adverse properties, to large-scale sampling in and around an ongoing field test to look for such events. It was agreed that funding is needed for evaluation of the recombination phenomenon. In the view of the chairman such studies should be initiated but it is questionable whether sufficient relevant information for a realistic assessment is likely to be forthcoming soon.
Finally, since it is becoming increasingly evident that particular regions of viral genomes may have multiple, often uncharacterized, functions, it was agreed that the use of sequences of exotic viruses for the production of virus-resistant transgenics should only be considered if there is evidence that sequences from indigenous viruses cannot be used to achieve the desired resistance.
The Animal and Plant Health Inspection Service under the Federal Plant Pest Act is charged with ensuring that exotic pests are not introduced into the U.S. One of the foundations of APHIS regulations under the Federal Plant Pest Act is the importation and interstate movement of exotic pathogens is based on concept of living infectious organisms. Thus, once the exotic organism is no longer infectious, it is no longer is no longer regulated. Recent advances in biotechnology allow scientists to import materials, that are not currently deemed organism by APHIS, but can be manipulated to regenerate viruses or viroids that are not widely prevalent in the U.S.
The purpose of this meeting is to:
(1) Discuss if the use of exotic viruses in laboratories should be allowed.
(2) Discuss possible containment protocols for the use of plants infected with exotic viruses in laboratory setting.
(3) Give guidance on how APHIS can obtain from the scientific community in a timely fashion, information on exotic viruses and their vectors.
The focus will be on the use of nonmechanically transmitted viruses (geminiviruses) laboratory settings.
Permit requirements for the importation and interstate movement of noninfectious nucleic acids containing partial or complete viral sequences.
Currently, APHIS importation or interstate movement permits are not required for the following: certain microorganisms harboring plasmids containing partial genomic sequences of exotic viruses; noninfectious extracts containing exotic virus genomic and subgenomic nucleic acids; or plasmids containing the complete or partial sequence of an exotic virus. However, using recombinant DNA methods, scientists have been able to reconstruct or produce infectious exotic virus from this material, which is does not require an APHIS permit.
Permit requirement for the importation and interstate movement Of nonmechanically transmissible exotic viruses.
APHIS will not consider issuing a permit that will result in research utilizing infection of plants with an exotic virus by an exotic vector unless the facility meets stringent containment conditions (e.g Ft. Detrick). Since the majority of plant viruses are readily transmitted by mechanical inoculation, this has not been a major impediment to most types of experimentation with exotic viruses. However, certain important virus groups, such as luteoviruses and some geminiviruses, are only efficiently transmitted by specific insect vectors. Fortunately, recent advances in technology, have increased the researcher's ability to infect plants with these viruses in the absence of their vectors. Therefore, APHIS expects to see an increase in permit requests for the importation and interstate movement of these types of viruses.
GENERAL ISSUES DEALING WITH WORKING WITH EXOTIC VIRUSES
Should researchers be allowed to work with nucleic acids of exotic viruses where the infectious virus is not present?
Should researchers be allowed to maintain plants-infected with exotic viruses outside of specifically defined containment conditions? Should use of plants infected with exotic viruses be allowed when the insect vector is not present in the United States?
Should use of plants infected with exotic viruses be allowed when the insect vector is widely prevalent in the United States? 'When the vector is located only in certain geographical sections of U.S.? When related insect species (biotypes), that are not known to be vectors, are present?
Should the use of plants infected with exotic viruses be allowed when the insect vector is present as a research organism in the building that the virus-infected plants are? Related insect species?
DEFINING CONTAINMENT PARAMETERS
The next series of questions address possible containment, monitoring protocols that would allow the use exotic-virus infected plants under certain conditions. The questions should be answered with respect to whether (1) vector in not present in US, (2) vector is not present in local area, (3) where taxonomically related insect species are present in the locality.
Can infected plants be grown in a greenhouse? Growth chambers?
Should the facilities be locked at all times? Should the facilities have restricted access?
Would placing restrictions on the growing of virus-infected plants to time periods where insect vector could not survive outdoors be sufficient containment? Where there are no susceptible plants in the region? Where the insect can not survive overwinter?
Are there any physical containment procedures (e.g. use of yellow sticky boards, filters, trap plants) that would assist in ensuring that insects could be contained? That potential insect vectors entering a facility could be killed or trapped?
Assuming appropriate containment conditions can be designed to work with these plants, who should have responsibility in ensuring that the containment conditions are being followed? Researcher? Institutional Biosafety Committee? State Regulatory Official?
How can the research community assist BBEP in responding to rapidly evolving research techniques and meet our 60-day turn around time for issuance of movement permit? In providing resource people? Informing the researcher community? Informing the State regulatory officials?
Participants in APHIS-BBEP Meeting use of Exotic Viruses under Contained Conditions
Dr. Tom Pirone, Chair
Dr. James Duffus
Dr. Osmatt Azzam
Dr. Sandy Lazarowitz
Dr. Judith Brown
Dr. Gus de Zoeten
Dr. Conrad Istock
Dr. Tom German
Dr. Sue Tolin
Dr. Jane Rissler
Dr. Cliff Gabriel
Dr. Jim White
Mrs. E. Dianne Hatmaker
Dr. Sally van Wert
Dr. Larry McDaniel
Dr. Vernon Damsteegt
Last Modified: October 10, 2007