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ABADRL plays a key role in handling threats to the US food system from outside pathogens. Its value is recognized by various outside organizations. Two such organizations have written reports about the necessity of ABADRL in this role. Those reports are listed below.
The RAND Report
7/14/2003
PANEL REPORT
EXPERT PANEL REVIEW OF THE
ARTHROPOD-BORNE ANIMAL DISEASE RESEARCH LABORATORY (ABADRL)
Laramie, Wyoming
8-10 April, 2003
EXECUTIVE SUMMARY
On 7-10 April, 2003, this review panel met in Laramie, Wyoming to assess ABADRL’s research relevance, quality, capacity and impacts. ABADRL has a unique role in the federal government, as the only national laboratory with the mandate to conduct research on arthropod-borne virus diseases of significance to large domestic animals. The panel concluded that ABADRL research has never been more relevant than it is today to the nation’s livestock industry and USDA needs. The core of the laboratory’s research program continues to be bluetongue (BT) and the related epizootic hemorrhagic disease (EHD) from an integrated host-virus-vector perspective. This work alone would justify the existence of the laboratory, since BT remains one of the most significant livestock diseases and is of considerable economic consequence, not only as a cause of direct loss, but also indirectly as a non-tariff trade barrier to international commerce in animal germplasm. Work has begun on vesicular stomatitis (VS), also a significant cause of disease during sporadic outbreaks in the US, and of concern because of its clinical similarity to foot and mouth disease. Most recently, the laboratory has added West Nile (WN) to its research portfolio as an emerging disease in the US. This broadens the scope of the laboratory’s mission, taking on the responsibility to respond to arthropod-borne viral diseases that are introduced into the U.S. accidentally or deliberately that involve domestic animals – a unique dimension within its unique mission.
The quality of ABADRL research within the integrated host-vector-virus triad has been uneven in recent years. The field investigations, vector-pathogen studies and development of diagnostics are nationally and internationally recognized. Their seminal work on Culicoides taxonomy and its relationship to BT virus transmission and geographic distribution has brought significant benefit to the US cattle industry through the recognition of BT virus-free areas where virus vector-incompetent Culicoides occur and where livestock can be exported without problems. The difference in BT virus susceptibility opens the door to pioneering work on mechanisms of vector susceptibility to BT virus in the immediate future. VS virus research is relatively new. Observations of the relationship of VS virus and grasshoppers as non-biting insect hosts would revolutionize concepts of maintenance and spread of this virus. The confirmation of these observations in the field is urgently needed. WN research is just beginning, but offers the laboratory the opportunity to apply its integrated host-virus-vector approach to an understanding of how this virus is maintained and spread in nature, particularly in the intermountain region where it has been less well studied. Studies relevant to all of these viruses are also being done. Development of vaccines and diagnostic reagents using robust yeast systems is exciting. The review panel was enthusiastic about the development of PCR and SERS diagnostic tests. Good progress is being made on vector genomic studies.
The Panel’s greatest concern centered on the capacity of the laboratory to adequately meet its mission, especially considering the inevitable expectation that ABADRL would deal with emerging diseases. The availability of a broader and deeper range of scientific expertise and adequate BSL-3 laboratory and animal space are very serious constraints that require urgent attention by the agency. Existing physical facilities are old, inadequate and must be replaced as soon as possible. Patching up these facilities has not been satisfactory, is expensive and the resulting down-time has caused inordinate delays in conducting research projects, especially those requiring large animal experimentation. These laboratory and animal holding facilities were not designed to meet contemporary Ag BSL-3 requirements and continued attempts to retrofit them to meet this containment level will never be fully satisfactory. Although the current scientific and support staff are highly competent and dedicated, they face two problems: (1) too many projects, and (2) limited availability of key areas of expertise that would lead to collaboration between ABADRL and University of Wyoming scientists. The ABADRL scientists have taken on an inordinate number of projects. Until the panel requested it, neither the laboratory staff collectively, nor the individual scientists had prioritized the research projects. Involvement in an overwhelming number of projects may account for the relatively modest publication record in recent years. The panel strongly recommends that fewer projects be undertaken and that publication efforts be increased. Scientific leadership in virus-vector-host research requires more expertise than the current scientific staff can supply. Future innovative research will require expertise in veterinary immunology (to understand host-virus and host-vector relationships), molecular biology (to bridge genomic information and functional genomics to better understand vector-virus relationships), population entomology (to understand vectorial capacity on a landscape scale) and veterinary epidemiology (to characterize factors that influence disease ecology, assess risk, and develop predictive models). This expertise can be incorporated into research by hiring four additional scientists within the laboratory and/or by collaborative research with specialists in other institutions (particularly universities) or organizations. Support of the laboratory to position it to better meet important national needs will require efforts by the agency, the laboratory’s stakeholders and the scientific community dedicated to animal health.
Background
Several years have passed since the last ABADRL review. Since then, there have been significant changes in personnel and new arthropod-borne disease problems have arisen. These circumstances, and the recent arrival of a new Research Leader, make this a propitious time for assessment of past accomplishments, current activities and future directions.
The ABADRL has undergone an interesting evolutionary process. It was originally established as a pathology laboratory of the USDA Meat Inspection Service in Denver, Colorado, in 1955. Two years later it became the diagnostic laboratory for bluetongue in Lakewood, Colorado. In 1985 the scope and mission of the laboratory was expanded, renamed ABADRL and relocated to the campus of the University of Wyoming (UW) in Laramie.
The current vision statement of the laboratory is, “Safe, economical and competitive production of healthy U.S. livestock and livestock products for domestic and world markets.” The four-fold mission statement includes:
- Definition of host-vector-pathogen interactions,
- Development of new diagnostic tools
- Development of effective disease control and management strategies
- Transfer of information and technology to the livestock industry, and action and regulatory agencies.
In April, 2003, a review panel was convened to assess ABADRL’s research relevance, quality, capacity and impacts. The panel membership and their affiliations are listed in Appendix A.
Research Relevance
Pathogens transmitted by arthropod vectors continue to cause inestimable morbidity and mortality in animals and humans throughout the world. Indeed, in the US, two arthropod-borne diseases, vesicular stomatitis and bluetongue, cause significant adverse economic impacts on U.S. producer groups due to direct effects on livestock health and further severe economic losses due to the trade barriers attributable to these diseases. The Office of International Epizootics (OIE) classifies both bluetongue and vesicular stomatitis as highest priority “List A” diseases. West Nile is a contemporary example of the unexpected appearance and rapid spread of a foreign arthropod-borne disease that affects humans, domestic animals and wildlife. The ABADRL is the only national laboratory with the mandate to provide research on these types of emerging diseases of agricultural concern.
ABADRL provides the ability to address arthropod-borne animal diseases in all aspects of their epidemiology (i.e., virus, vector, and vertebrate host) and this approach is highly encouraged. To ensure success, each component of the disease triad must be investigated, and it is difficult to predict a priori which research will lead to the most efficacious control and surveillance strategies. Clearly, however, emphasis should be directed to those aspects of the problems that can be addressed best by ABADRL (e.g., large animal disease pathogenesis, vaccinology, and diagnosis, vector-virus-large animal host interactions, and long-term field studies). In addition, this synergy should make research components greater than the sum of the parts.
In summary, ABADRL research is relevant to the livestock industry and agency needs. The ABADRL is the only USDA laboratory devoted to arbovirus diseases. Its mission is clearly central to program areas: 103 – Animal Health and 104 – Veterinary, Medical, and Urban Entomology.
1. Is research on bluetongue and epizootic hemorrhagic disease of deer (BT/EHD) relevant? This orbivirus program is the cornerstone of ABADRL research. It is the longest functioning program and it has made significant contributions to the nation and the world. This program is uniquely poised to provide cutting-edge leadership in livestock diseases in several areas.
- Is the direction of the research program appropriate to the agency mission and national programs? This program is directly aligned with the agency mission and national programs. Indeed, BT remains one of the most significant livestock diseases. The OIE estimates worldwide losses due to bluetongue alone at $3,000,000,000 per year, in large part because it is a non-tariff barrier to international trade. The ABADRL is the only USDA laboratory capable of addressing this disease in all of its component aspects. Similarly, EHD remains an ongoing threat to the cattle industry.
- What is the potential value of the research to intended users? One of the most impressive advances in livestock arbovirus disease research and in addressing trade barrier/regionalization issues has been the ABADRL program to apply population genetics to Culicoides variipennis. This program resulted in recognition of the C. variipennis complex, the realization that C. sonorensis was the critical vector, and in the relaxation of trade barriers from areas where C. variipennis predominated (e.g., northeastern U.S.). Similarly the program has developed effective and widely used diagnostic techniques for BT virus infection, has provided potentially important information concerning environmental/ecological determinants of C. sonorensis, and insights into the molecular and anatomic determinants of productive vector infection.
- Suggestions for improving the relevance of the research:
· The seminal work on C. variipennis complex taxonomy needs to be expanded.
· It was disappointing in the project prioritization by the laboratory scientists to see the focus devolve into diagnosis. This is clearly important but ABADRL needs to exploit its uniqueness to address its portfolio of diseases. The overwhelming emphasis on diagnostics in the list of laboratory priorities, although important, should not preclude a balanced research agenda with significant attention to other areas in the virus-vector-host triad.
- Is research on vesicular stomatitis relevant? Vesicular stomatitis (VS) virus has caused recurring epizootics for many decades. VS epizootics in the western U. S. in 1995 and 1997 resulted in estimated losses of $50,000,000. Because the signs and symptoms of VS cannot be distinguished from foot and mouth disease, rapid differentiation of these two diseases is of concern to emergency foreign animal disease control and eradication programs.
- Is the direction of the research program appropriate to the agency mission and national programs? Yes, this is essentially the only research group that has the continuity to pursue VS investigations. As usual, VS has disappeared from the national agenda in the absence of a recent outbreak, but past sporadic epizootics suggest that it will likely reappear.
- What is the potential value of the research to intended users? VS remains a very important disease because it is designated an OIE List A pathogen. The need to discriminate between VS and FMD in the field is important in terms of biodefense needs. Intended users will benefit from scientifically sound research that will provide an understanding of virus maintenance, persistence, and transmission mechanisms in nature. More insights into these mechanisms may well provide invaluable new approaches for the surveillance and control of VS.
- Suggestions for improving the relevancy of the research. The investigators are beginning to address a number of aspects, including new diagnostics, vaccines, and field/epidemiological insights. In this regard, there is substantial enthusiasm by the review panel for the studies to develop PCR and SERS diagnostics. Similarly, the group is uniquely poised to investigate new approaches for immunization against VS virus (as well as other pathogens) based upon understanding of the vector-host interface. Such investigations into broad spectrum approaches to controlling vector-borne pathogens offers great potential, but the ultimate utility of such investigations will require the work to be done in the actual vertebrate host, not small animal models. Thus, given the emerging bovine genome, this group is uniquely poised to lead the field in such efforts.
- Is research on West Nile and other emerging arthropod-borne virus diseases relevant? In the new global economy, GATT and NAFTA trade agreements provide exciting new opportunities for U.S. agriculture, but also bring new threats for frequent introduction of diseases. Importation of vectors and pathogens would seem to be inevitable with increased movement of animals and products, and with increased trade in general. Indeed, many arthropod-borne diseases of major concern to the livestock industry in the U.S. have recently demonstrated their ability to move into new areas with devastating consequences. Arthropod-borne viruses of great concern to the U.S. such as Rift Valley fever (RVF), Japanese encephalitis, and West Nile (WN) viruses have demonstrated their ability to become established in new areas or reappear in previously endemic regions of the world, thereby causing significant morbidity and mortality. Furthermore, a number of arboviruses such as RVF, are potential bioterroism agents; introduction into the US would have enormous economic as well as public health consequences. The introduction of WN virus into the United States in 1999 demonstrates U.S. vulnerability to emerging diseases and highlights the critical deficits in facilities, infrastructure, organization and human resources needed to address such introductions quickly. Trained veterinarians and large animal BSL-3 facilities to address the many research needs associated with such an introduction were and are in woefully short supply. Such emergences and the subsequent anthrax bioterrorist events of 2001 also emphasized the limits to our knowledge and capacity to predict and respond quickly and effectively. Certainly, emergence and resurgence of arthropod- and rodent-borne diseases will continue and increase in the U.S. and elsewhere in the world. The seriousness of this threat is underscored in two recent reports from the National Academies -- “Counteracting Agricultural Bioterrorism” by the National Research Council (Committee on Biological Threats to Agricultural Plants and Animals), and “Microbial Threats to Health – Emergence, Detection, and Response” by the Institute of Medicine (Committee on Microbial Threats to Health in the 21st Century), which describe the threats of emerging diseases and bioterrorism acts to animal and human health in the U.S. Both of these reports address the critical threats of emerging diseases to the nation, and state the need to have the trained personnel and containment facilities to address emerging diseases in a timely and efficacious manner. A broader scope of the ABADRL to address these threats will require a redefinition of the animal species with which the laboratory deals. This review panel certainly agrees with the reports of the national academies, and strongly supports USDA-ABADRL efforts to meet these urgent national needs.
The USDA – ABADRL is clearly of national and international importance in this regard. The ABADRL has the unique responsibility to carry out research on arthropod-borne diseases of large animals. Strictly speaking, WN falls outside the food/fiber large animal species, and focuses instead on horses and wildlife. The agency leadership and laboratory scientists have arrived at a clear mutual understanding of the breadth of their mandate for WN. The laboratory needs to extend this understanding to other viruses that pose threats upon introduction. This is the only federal research laboratory with the mandate to meet this national need.
- Is the direction of the WNV research program appropriate to the agency mission and national programs? The panel believes that the laboratory should develop broad-based platforms and field capabilities that can be used for a wide range of agents. Most of the proposed projects are appropriate and involve development of new diagnostics and vaccines. However, some projects are clearly less appropriate or urgent than others:
· The project to develop antiviral prophylactic treatment for WNV infection in horses is difficult to justify in terms of practical application in the field.
The ABADRL agrees with the Expert Panel’s opinion and will give this project a low priority.
· Similarly, the project to assess recombination potential between WN virus and SLE virus are not of great priority. This project could yield interesting results, but considering the number of projects of greater significance and investigator’s time constraints, this should not be a priority project.
The outlined experiment to assess potential recombination between WNV and SLE was modified to accommodate the concerns of the panel. Instead of a two-stage initial experiment (cell culture then insects) the scientific staff has cut back to a single insect experiment comprised of superinfection of Culex spp. insects, separation of progeny by plaque assay, and microarray analysis to assess recombination frequency. A significant investment of time and expense had already been expended at the time of the review by both the ABADRL scientific staff and external collaborators, and this experiment was also added to the NP104 Action Plan between the time of the review and the receipt of the Panel’s report. In addition, this experiment was only “moved up” on the priority list in response to the breakdown of our containment facilities, since it could be done in the temporarily leased BSL-3 space on the main campus, as well as in collaboration with insect facilities at the Arthropod-borne and Infectious Diseases Laboratory, Colorado State University. We hope that cutting back on the future investment of time and expense in this experiment without eliminating it is an acceptable balance between the conflicting imperatives of responsibility to collaborators and respect for the recommendations of the panel.
- What is the potential value of the research to intended users? Development of new rapid diagnostics and vaccines, provision of new information on potential vectors of the pathogen, and assessment of existing strategies for prevention of infection are all very worthwhile and would be of great value for users.
- Suggestions for improving the relevancy of the research:
· The list of projects did not include straightforward risk assessment of the threat of WN to equine animals (horses, donkeys and mules) and perhaps other livestock animals, such as emu and ostrich, which the ABADRL team is uniquely positioned to do. Indeed, WN could serve as the model for ABADRL being able to address the risk to domestic large animals of introduced or emerging viral diseases. As noted above, this laboratory and its personnel could provide unparalleled biodefense capacity in the event of bioterrorism events.
· Development of WN virus vaccines and diagnostic reagents using robust yeast systems offers a potentially significant alternative to conventional vaccines for each of the pathogens of interest and many other livestock diseases.
· Diagnostics remains a strong suit of the group, and of course they are uniquely positioned to develop these tests in large animals and in livestock vectors.
· Field investigations and control strategies are a strong part of the research portfolio at ABADRL, and provide a truly unique capacity to conduct translation studies of new products and knowledge concerning livestock arbovirus diseases that are unparalleled elsewhere. These field studies should be continued.
· Communication with stakeholder groups to inform them of research results and for laboratory scientists to understand emerging needs that they have is essential and should be actively continued as a mechanism to maintain relevance. This could be accomplished through establishment of focus groups.
· The scope of the ABADRL mission should be expanded to include rapid response to national disease emergencies. This will require the development of interdisciplinary teams to carry out research required to respond quickly to emergencies. These rapid response teams must integrate field and laboratory investigations at molecular, whole organism and population levels. Again, balance between the host-vector-virus elements will be essential. Recent and proposed work with WN virus infections is an important step in developing this capacity and should be continued.
· Investigators should resist the temptation to take on initiatives that fall outside the scope of the national programs.
Research Quality
The success of ABADRL will ultimately depend upon the quality of its personnel at all levels and the support that they receive. The laboratory has a competent, well-trained staff working in an important scientific research niche. However, it has been difficult to maintain research strength in all three elements of the host-virus-vector triad. Work with animals has lagged, in large part due to turn-over of veterinary medical officers (VMOs), which has been detrimental to carrying out critical animal experiments. The laboratory now has three VMOs and the expectation is that these experiments will begin as soon as facilities permit.
1. Is the BT/EHD research of high quality?
a. How good is the current BT/EHD research program compared with
other research programs in the field? The group is unique, thus it is difficult to compare and contrast the program with others. The field aspects of the program have been exceptionally productive. Frankly, overall research productivity has been modest in the past few years for a number of reasons.
b. What is the quality of the BT/EHD published research? A number of recent publications have been in good journals, but the number of publications in the most recent years has been modest.
c. Is the BT/EHD research being performed at the forefront of scientific and technological knowledge? Some aspects of the research are indeed in the forefront in the area:
· The field studies, vector-pathogen studies and diagnostics development are certainly nationally and internationally recognized.
· The field research defining spatial distributions of vectors is very interesting and will provide the foundation for exceptional intralaboratory interactions from a population genomics standpoint.
d. Suggestions for improving the quality of BT/EHD research. The group is uniquely poised to assume a leadership role in the post genomic era. The group would be at the absolute forefront of the field by addressing these general areas:
· Exploitation of the emerging bovine genome in arbovirus disease and vaccine studies,
· Exploitation of genomic and proteomic studies of C. sonorensis to develop novel vaccine and control strategies.
· Exploitation of existing knowledge to develop a population genomics approach to control BT.
Although there was not enough time to review all individual projects in detail, the panel was concerned that the group is not sufficiently focused. There are many more projects proposed than can be adequately pursued. For example:
· The genomics work on C. sonorensis is of interest to many different areas. Current emphasis is on the midgut response to virus infection. This very interesting basic research essentially constitutes an entirely new research program. We question if resources are adequate to generate significant, usable data in this area. The panel suggests that work in this area should proceed with caution to limit adverse impacts on projects addressing more immediate laboratory priorities.
· The group has shown that proteolytic processing of the BT virion generates secreted virus particles (SVPs), thereby exposing VP7 which is the ligand for a cellular receptor. What is the midgut receptor? What are the vector enzymes that proteolytically process the virion? This information can be exploited for use in development of vaccine strategies, population genetic markers, and in field epidemiology programs.
· Similarly protein (s) in saliva that perturb the immune response to BT/EHD virus infections in vertebrates would also provide a foundation for the strategies listed above and merits additional research. Recent studies at ABADRL have confirmed that Culicoides saliva facilitates VS virus replication in vitro. Does this effect also apply to BT and EHD viruses? If so, analyses of Culicoides saliva would have obvious broad application to understanding vector competence of this insect. However, the strategy for these analyses should be focused (in contrast to addressing the question, “What are the components of Culicoides saliva?”), given the incredible diversity and bioactivities of insect salivary gland proteins that have been described to date. Using the potentiation of viral replication in vitro as an assay, active salivary proteins could be identified using blocking antisera. The same polyclonal antisera could be used to identify salivary proteins by Western analysis. Eventual identification of these proteins using standard biochemical techniques would be supported by the availability of a salivary gland cDNA library. Recombinant salivary gland proteins could then be utilized to confirm bioactivity and also support efforts to determine whether salivary proteins can act as adjuvants for increased vaccine efficacy.
· The field studies must be continued, but modern technologies should be brought to bear on seemingly intractable issues. For example, important ongoing studies have delineated BT risk based on vector distribution. These studies should be completed and extended/focused to include the mapping of genetic markers associated with susceptibility. Expansion of GIS to include remote sensing may provide a valuable addition to this research. Fine scale mapping may delineate large geographical areas that actually are BT virus free with a low risk of transmission under a variety of climate regimes. GIS mapping and remotely sensed data will likely be useful in the development of models to forecast years of high epizootic risk.
· The overwintering mechanism of BT and EHD viruses is still not known, although ABADRL scientists have reported important preliminary findings. These results require verification by partial sequencing and virus isolation after blind passage in Culicoides and other insect cell lines. These studies must be completed as soon as possible and published because of their potential importance to BT epidemiology and to our general understanding of how viruses overwinter in their insect hosts. If these results are confirmed, then additional studies are essential. For example, the identification of RNA segments in overwintering larvae suggests that BT virus can be transmitted vertically. Laboratory experiments should be done to verify that vertical transmission can occur in C. sonorensis is capable of vertical passage. If an overwintering mechanism can be discerned, then models based on the correlation of outbreaks with persistence in the environment may lead to outbreak prediction based upon larval surveillance.
· Development of new diagnostic tools for BT and EHD viruses:
o General screening assays are needed that will detect ALL serotypes of BT and EHD viruses, including exotic strains. Focusing on selected serotypes alone may not detect introductions of new serotypes. However, serotype-specific assays are needed to identify serotypes cycling currently and to track these specific virus signatures spatially and over time. Real-time PCR, serotype-specific antigen (direct) ELISA, and antibody (indirect) ELISA are proposed by laboratory scientists. The development of all three technologies, while laudable, will be very time consuming. The development of real-time PCR capability for monitoring virus strains currently in the U.S. and for surveillance for introduced strains will require significantly less time than will development of multiple serotype-specific ELISAs.
o Multiplexing will also allow for simultaneous sensitive detection of multiple virus types in individual samples. The BT/EHD team leaders are encouraged, therefore, to concentrate on real-time PCR strategies.
o In the absence of detectable virus, indirect ELISAs can be utilized to monitor exposure of livestock to BT and EHD viruses. As such, the development of indirect ELISAs will meet a critical need.
o Collaboration with Veterinary Diagnostics, Inc. is very commendable and should be expanded, if possible, to pursue commercial development of the indirect ELISA.
· Characterization of vector and BT/EHD virus transmission:
o Experiments are needed to determine the median infectious virus dose required to infect C. sonorensis, to determine the viremia profiles in cattle and other animals sharing vector habitats, and to determine the role of cattle infection in virus amplification in nature. Experimental studies should focus on using recent low passage isolates. If findings are negative, then field examination of study sites are needed to suggest alternative competent hosts.
o Culicoides genes that respond to BT/EHD virus infection: Significant published work by Campbell and Wilson indicate that a putative RNA editase (CsRED1), putative receptors involved in cell differentiation and putative translation machinery components, are induced in the midgut at 1d after feeding on EHD virus and in the insect carcass >1d after feeding. The most striking increase was noted in CsRED1, the putative RNA editase homolog. As would be expected during viral infection, the virus appears to subvert host cell machinery for propagation of viral progeny. The scientific team proposes that microarray analyses based on virus-inducible genes may be useful to predict virus prevalence in insect populations. With the possible exception of CsRED1, which is dramatically induced by the virus, it is possible that conclusive links between changes in gene expression, which may be subtle and associated with both normal and abnormal physiologies, and virus infection will not be established. As such, the panel suggests that the scientific team proceed with caution, perhaps focusing on the possibility that CsRED1 and as yet unidentified putative antiviral gene products may be more useful markers of viral infection. The production of EST and tissue-specific cDNA libraries should focus on salivary glands and midgut tissue to support ongoing efforts with BT and EHD viruses to identify midgut receptors for these viruses and salivary gland proteins that may enhance viral infection or prove useful as adjuvants for vaccine development.
o The panel suggests that additional work on fat body and ovary libraries is of secondary interest or premature at this time.
o Midgut microflora of Culicoides: A wealth of literature has demonstrated the practical application of paratransgenesis for the triatominae, which possess natural life history behaviors that are consistent with environmental introduction, infection, and transfer to progeny of transgenic endosymbiotic gut microbes. In light of these findings, the scientific team proposes extensive analyses of the microbial fauna in larval aquatic habitats and midgut flora of Culicoides, with the aim of identifying gut endosymbionts suitable for paratransgenesis. The panel agrees that gut microflora have significant impacts on insect health and survival and perhaps also influence vector competence based on published literature, but the long term applicability of the research is difficult to discern and the application of paratransgenesis to this vector is not likely to be feasible. To help focus and refine this work, the panel urges the scientific team to pursue communication with Dr. Edward Walker, an expert in this area, at Michigan State University.
o Larval habitats of Culicoides: Characterization of the larval habitats of Culicoides has significant practical implications for predicting and controlling BT/EHD virus transmission and should be carried to completion. Habitat characteristics can be used to predict Culicoides species composition and, hence, disease risk, can be used to support claims of “disease-free” status and will facilitate focused insect control for disease transmission intervention.
· Development of effective BT/EHD virus vaccines: Two major strategies were presented to develop a vaccine based on cell-mediated immunity against the range of virus serotypes. The first strategy is based on immunization of animals, presumably mice, with a viral expression library (ELI), analyses of protection following viral challenge, and iterative rounds of immunization with sub-pools of the library. A second strategy is based on transformation of dendritic cells with the ELI and reaction of these cells with immune cells from virus-inoculated hosts. With the availability of agriculturally relevant hosts and unique facilities for containment, the review panel urges the scientific team to focus on these hosts whenever possible. For example, because the proposed studies are time-demanding, the review panel suggests that the more conservative in vitro strategy may be best for preliminary work, utilizing dendritic cells prepared from large animal hosts. In addition, harvesting of immune cells from virus-inoculated large animals can be coordinated with numerous ongoing challenge studies in ABADRL. Further, the scientific team should consider the possibility of including potential transmission-blocking components (e.g., VP2 and VP7, the attachment proteins for mammalian and insect cells, respectively) into the vaccine strategy.
· Characterization of BT/EHD viruses and the host response to virus infection. This research encompasses disparate studies of the effects of EHD virus infection on pregnant cattle, analyses of endothelial cell responses to EHD virus infection, transformation and transgenesis of Culicoides cells and whole insects, and characterization of receptors and replication strategies for viruses in insect and mammalian cells.
o Clarification of the pathology of EHD virus infection in pregnant cattle is of fundamental importance and should be carried to completion. These observations are necessary to confirm or refute anecdotal evidence of this phenomenon and, as such, to justify control and surveillance efforts.
o Justification for the analyses of endothelial cell responses to EHDV is presumably based upon the hypothesis that more permissive viral replication in endothelial cells contributes to greater clinical pathology. Although these experiments would contribute to our understanding of the species-specific virulence of EHDV, it is unclear how these observations would lead to enhanced screening and control efforts. The review panel lauds the collaborative nature of these studies, but suggests that ABADRL should play a supporting, rather than lead, role.
o Transformation and transgenesis strategies can benefit from significant momentum of similar strategies with mosquitoes, but the investment of effort to bring the concept of transgenesis to fruition is not insignificant. The panel urges the scientific team to consider valuable short-term goals that can be achieved along the way instead of focusing solely on long-term potential outcomes. Indeed, the technology and data that are the foundations of transgenesis can lead to the development and utilization of much-needed population genetic markers for analyses of purportedly resistant and susceptible populations of Culicoides and in vitro cell transformation tools to discern gene function (e.g., BT virus receptor and CsRED1).
o Characterization of receptors and replication strategies of EHD and BT viruses in insect and mammalian cells has several important implications and should be carried to completion. First, identification of viral receptors and anti-receptor immunity that can block viral infection could lead to inclusion of transmission-blocking strategies in vaccine development. Second, the discovery that divergent viruses can utilize a common receptor on mammalian cells (Bergelson et al. 1997 Science 275:1320-3) suggests that divergent viruses may exploit a similar strategy for invasion of more primitive insect cells. The findings of Bergelson et al. suggest that ABADRL scientists should be cognizant of the possibility that multiple viruses may utilize a common receptor, thereby significantly expanding the practical application of transmission-blocking strategies in the insect host. Third, understanding of replication strategies may facilitate rational drug design to block viral infection, but this goal would require significant collaborative efforts to complement expertise at ABADRL and would likely not be attainable in the short-term.
2. Is the VS research of high quality? The sporadic recurrence of VS outbreaks in the Mountain West offers the opportunity to make significant contributions to the understanding of disease reoccurrence and virus transmission in this region, as a necessary first step toward control and prevention.
a. How good is the current VS research program compared with other research programs in the field? No other group in the intermountain region is working to characterize VS virus maintenance and transmission. Other groups are working in subtropical or tropical ecosystems that are very different from field sites being studied by ABADRL. The work in coastal Georgia has emphasized the importance of identifying insect vectors. Work on VS virus maintenance in enzootic foci in Costa Rica and Colombia are taking similar approaches to field work proposed by ABADRL scientists while studies on VS pathogenesis at Plum Island are restricted to the laboratory.
b. What is the quality of the published VS research? The three peer-reviewed publications since 1995 are in good journals. As additional work on VS is carried out, the expectation is that additional published papers will follow.
c. Is the VS research being performed at the forefront of scientific and technological knowledge?
· Development of new diagnostic tools for VS encompasses the development of real-time PCR, a SERS-based diagnostic, and analysis of the persistence of VS virus RNA in convalescent animals. Together, these efforts are likely to yield significant tools for detection and surveillance for VS virus. Real-time PCR can significantly improve response time for outbreak control, while the understanding of RNA persistence will have significant implications on how the real-time assay(s) are designed. In addition, understanding the persistence of VS virus RNA is critical to understanding the epidemiology of VS and may reveal that this virus can be exploited as a vaccine vector. Finally, the development of SERS technology is the result of a productive collaboration with the UW Chemistry Department. The panel encourages the continuation of collaborative efforts such as these.
· Non-biting insect vectors. Although R. Hanson and K. Johnson suggested a potential role for non-biting insects in the dissemination and maintenance of VS virus many years ago, there has been no evidence to support this speculation until the recent work of ABADRL scientists. Significant ABADRL analyses support the possibility that grasshoppers could play an important role in VS virus transmission and outbreak patterns. Publication of these results and continuation of these studies to determine whether this phenomenon is “field reality” versus “lab artifact” are absolutely critical. If grasshoppers collected in VS-endemic regions outside of the U.S. confirm that grasshoppers are indeed VS virus vectors, this work will open up a completely new area of research for arthropod-borne viruses – that is, virus transmission by non-bloodfeeding arthropods. The ecology and behavior of these grassland insects could explain many mysteries of VS outbreaks and position ABADRL scientists to be at the forefront of these new discoveries. The significance of this work cannot be overstated and the ABADRL/UW multi-disciplinary teamwork that led to these observations should be a model for other studies.
d. Suggestions for improving the quality of VS research.
· New approaches for VS diagnosis (SERUS) and preparation of vaccines and diagnostics (yeast expression systems) are very exciting and should be pursued vigorously.
· It is critical to demonstrate the transmission potential of C. sonorensis for VS viruses before embarking on laborious and extensive midgut receptor studies. It is important to know if C. sonorensis can transmit VS Indiana and VS New Jersey by bite, and if so, what the extrinsic incubation period and relative vectorial efficiency are. This is a high priority study for VS research. If life-long infection of midges results, demonstration of transovarial infection (or not) would be useful to know as well. There is still the old dilemma of how hematophagous insects become infected in the absence of a demonstrable viremia. Feeding experiments with C. sonorensis on VS-infected (presumably non viremic) hosts or co-feeding (infected and non-infected C. sonorensis feeding simultaneously on susceptible hosts) might help to address the viremia dilemma, or at least discard these possibilities. ABADRL is uniquely positioned to do these kinds of experiments in the hosts most affected in nature in the Rocky Mountain region -- cattle and horses. But given that the lab scientists are already over-extended, the priority assigned to these viremia-related experiments will need to be carefully weighed.
· Because of the predominant role of sand flies in transmission of VS virus elsewhere in the world, it is important to determine if they are an enzootic vector of VS virus in the SW US in areas where the disease has occurred periodically. Current efforts, however, have been limited by the difficulties of collecting sand flies and by a lack of facilities and expertise for sand fly rearing. The panel strongly recommends communicating, and possibly collaborating with Dr. Edgar Rowton of the Department of Entomology, Walter Reed Army Institute of Research, a recognized expert in sand fly biology in the laboratory and in the field, to address this relevant question.
· Studies should continue on convalescent VS virus infections in cattle, because this could be important in virus persistence in areas of disease occurrence and in virus dispersal through movement of animals during commercial activities. Factors stimulating relapse and subsequent transmission to insects also should be examined.
· Based on laboratory studies with the Cocal strain of VS virus done many years ago, it would be valuable to know if insect feeding lesions promote virus availability to other insects. If so, which insects? Can these studies be done in the field to examine natural associations? Observations that virus is available to other insects may be important to understanding spread of VS virus, but how can this information be used?
3. Is the West Nile research of high quality? This activity is too new to evaluate. There are opportunities to determine proactively the epizootiology and spread of WN virus in the intermountain region focusing on the host-vector-virus triad. Collaboration and coordination with other relevant agencies (CDC, USGS/BRD, USDA/APHIS, and responsible state agencies) will be essential to avoid duplication of effort and to focus on the unique capabilities of the laboratory, such as exploring the possible role of non-mosquito biting insects in WN virus transmission. The laboratory should avoid taking on routine service testing of mosquito collections and sera from domestic and wild animals and focus on relevant researchable questions and development of diagnostic tools.
Research Capacity
To meet its mission to address arboviruses affecting livestock in the US, the ABADRL staff is comprised of veterinarians, medical entomologists, and virologists whose research focuses on important indigenous arbovirus diseases of livestock. The ABADRL large animal BSL-3 containment facilities are unique in the USDA, and indeed in the nation, in providing the ability to address critical emerging and indigenous livestock arbovirus disease research needs. The biocontainment facilities of USDA – ABADRL comprise an important component of the national laboratory capacity available to address bioterrorism or emerging disease events in the U.S. as they occur. The facilities and staff of ABADRL should also be critical to the national response to the emergence of such large animal zoonotic diseases such as new variant Creutzfeld Jacob, RVF, Nipah, and others. Thus, the USDA-ABADRL program, personnel, and facilities are central to the national biodefense effort, and it is critical that the capacity of the laboratory in terms of personnel and facilities be adequate to meet this challenge. Capacity is necessary in order to:
· Identify pathogens. General diagnostics are needed to rapidly identify new agents of livestock disease. The ability to modify current diagnostic platforms for new agents is imperative.
· Characterize pathogenesis. Pathogenesis models are needed to assess rapidly the impact of new agents on the livestock industry. Molecular epidemiology, sequencing and large animal models are needed.
· Understand pathogen epidemiology. Collaboration is needed to provide epidemiological insights in vector-host-pathogen systems to identify needed laboratory investigation.
· Control and prevent disease. Rapid systems for vaccine development are needed.
Because needed research capacity for work on BT/EHD, VS and WN overlap significantly, this general assessment covers all three disease areas. The panel’s general conclusion is that the ABADRL current research capacity is not adequate to accomplish its mission. The panel is concerned that currently available facilities, funding, and staff numbers compromise the ability of ABADRL scientists to accomplish the goals of the respective projects. There is also concern about the focus of the group; clearly the overwhelming number of projects that the laboratory staff envisions exceeds the capacity of limited personnel and resources to address all of them well.
1. Facilities, land, laboratories and equipment.
Assessment :
· One cross-cutting problem is clearly the lack of functional BSL-3 facilities. This situation negatively impacts each of the programs and indeed almost 20 separate projects are awaiting the availability of the facilities. The laboratory leases some facilities from the University. The responsibility for maintenance between ABADRL and the University is not clearly defined and should be clarified. In addition, leases have been reduced to one year because of the University’s demolition and construction plans. These plans add an element of uncertainty about facilities availability that complicates realistic research planning by ABADRL. Timely, joint University-ABADRL contingency planning is necessary to assure continuity of ABADRL’s research activities as construction goes forward. Statements given to the panel by University administrators clearly indicates a high degree of support for ABADRL that should facilitate continuous dialogue and joint planning.
· Despite pressing needs, it is difficult for the panel to recommend more than the minimal necessary repairs to the Round Building facility to return it to service. Its future as an Ag BSL-3 facility is clearly limited and institution of major renovations is contraindicated. This facility could provide functional BSL-2 space to support a new on-site BSL-3 building. In addition, it is also debatable whether fixing the large animal facility is worthwhile. There is no doubt that there is a compelling need for new facilities, but whether or not they should be built near existing facilities in Laramie or elsewhere in the region is debatable. In the years that USDA-ABADRL has been located at the University Wyoming; few interactive and collaborative projects have emerged. Unless more UW faculty are hired who have common interests and share goals with ABADRL scientists, growth in collaborations appears unlikely.
· Increased vector studies will require additional insectary space. This situation will become more acute when the University’s scheduled demolition of the current insectary building occurs.
· The laboratories appear to be well equipped.
Suggestions:
- The BSL-3 laboratories and animal holding facility must be returned to service and adequately maintained as soon as possible.
- Large animal experiments addressing host-virus and host-vector relationships are behind schedule. Although some animal experiments require BSL-3 containment others do not. Those experiments not requiring BSL-3 containment should be initiated as soon as possible.
- Current lease arrangements with the University should be renegotiated to define responsibility for maintenance and extended to multi-year contracts.
- New BSL-2 and -3 laboratories, including insectary space and animal holding facilities, should be built as soon as possible. The stakeholders should assist in obtaining funding and University endorsement of the project should be obtained.
- The new tissue culture laboratory will bring additional clean (virus-free) space to the laboratory. Apparently, budgetary constraints resulted in construction of two large rooms, rather than a series of small rooms. This may result in cross-contamination of cell lines, requiring considerable caution during routine operations.
2. Personnel, including support staff.
Assessment:
· Another cross-cutting problem is the excessive number of projects and activities of the research scientists. Some 49 projects ongoing or proposed were mentioned during the introduction to the program. This is clearly overwhelming to the scientific staff and facilities, and fundamentally bespeaks of an emphasis on activity rather than productivity as indicated by completed projects leading to publications. Even after the prioritization requested by the review committee, there was still an inordinate number of projects. Not even filling currently open positions would allow the lab to address this list of projects in an efficient manner. The preferred alternative would be to fill open positions and to hire additional scientists in specialties not covered adequately at present. Alternatively, projects should be prioritized and efforts focused on the highest priority ones, eliminating or postponing lower priority ones.
· The unit needs to focus and to develop cross-cutting approaches that will help them expeditiously address the diseases in their portfolio. Similarly, in the BT/EHD and other disease programs there seems to be little exploitation of the individual strengths and training of the respective scientists to most expeditiously address research needs. For example, veterinarians are proposing in vitro molecular virology studies, non-veterinarians are proposing pathogenesis studies in bovines, one VMO is heavily involved in facilities maintenance. Productivity of high quality research leading to publications and presentations requires that the scientists work in areas of their greatest strengths.
· In discussion of the laboratory’s priority needs, it was clear that additional expertise is required for the laboratory to achieve a leadership role in the animal disease field and move into exciting new scientific areas to better accomplish its mission.
Suggestions
- Continuously prioritize projects, establish realistic time-lines and evaluate progress at regular intervals. Low priority projects should be eliminated or postponed.
- Scientists should be involved in projects that correspond with their individual strengths and expertise.
- In order to strategically position the laboratory to carry out research in new, fast-moving areas, and more effectively accomplish its mission, new expertise will be required in:
o Veterinary immunology to understand virus-host and vector-host interactions. Immunology is a very fast-moving field. However, many small laboratory animal models cannot be extrapolated arbitrarily to large animals. A bovine immunologist is needed to provide in-house expertise to other members of the ABADRL teams working on host-virus and host-vector relationships.
o Molecular biology to bridge genomic information and functional genomics,
o Population entomology to understand vectorial capacity, and
o Veterinary epidemiology to characterize factors that influence arboviral disease ecology and risk assessment, and to develop predictive models.
· The addition of scientists to meet current research needs and future opportunities will require additional support staff for laboratory work and facilities maintenance.
· Additional expertise should be tapped by increasing strategic partnerships through active collaboration with scientists at University of Wyoming and elsewhere. The University partnerships should be broadened to expand graduate and postdoctoral training involving laboratory facilities and personnel, and by University faculty hires in areas where synergy with laboratory scientists can occur.
3. Funds (hard and soft).
Assessment:
- Apparently ARS budgeting is based on a standard figure per scientist-year. It was not clear to the review panel whether the additional expense that large animal work and operating at Ag BSL-3 level containment require are factored into annual budget requests. If these costs are not factored in, they should be in order to realistically provide adequate resources that will assure timely project completion.
- With few exceptions, laboratory scientists did not report that they are actively seeking extramural competitive funds to supplement ARS core funds.
Suggestions:
- ARS budgeting should recognize and provide for the actual expenses involved in Ag BSL-3 operations and large animal research.
- Laboratory scientists at all levels should be aggressively seeking extramural funding for high priority projects through competitive grant programs. In those instances where federal employees are precluded from applying as PIs, they should form strategic alliances with university colleagues with corresponding interests who are eligible to apply.
Contribution to National and International Leadership In Relevant Fields
1.Benchmark the Unit’s research programs in relationship to national and international leadership.
· The population genetics and field investigations of BT are exceptional and should be expanded to include a population genomics approach. This approach could provide a foundation for risk assessment for BT that is urgently needed and that will serve as a model for other diseases.
· The vector-BT work is unique and has also provided a basis for novel control and vaccine approaches.
· The unit is positioned to advance development of BT diagnostics and therapeutics with post ELISA and RT-PCR diagnostics. These developments are highly encouraged as is the use of the yeast system for new vaccines and diagnostics.
· The novel vaccine approaches under development could position the laboratory in a leadership position and every effort should be made to enhance this line of research. The role of insect saliva as a possible adjuvant for yeast-produced vaccines is cutting-edge and a potentially valuable approach to development of a variety of vaccines.
· The VS program is more recent than the BT/EHD effort. Obviously the lab needs to become the source for VS information that would serve as the basis for formulation of animal health policy in the western states and indeed in the nation and the world.
· ABADRL scientists are just beginning their studies on WNV. Some of the projects that capitalize on the unique capacity of the laboratory, such as development of yeast antigens, have great potential to make ABADRL scientists the leaders in the field.
2.Suggestions for improving national and international leadership.
· The scientists and research staff must be more proactive in publishing their research findings. This is essential to maintain scientific standing of ABADRL within the research community and is important to them as individual scientists. A strong publication record will increase the competitiveness of ABADRL scientists for internal USDA/ARS support and for extramural funds, hone the quality and focus of their research through exposure to the peer review process and stimulate continuous reassessment of research goals.
· Participation in regional, national, international scientific and stakeholder meetings is critical to the mission of the ABADRL and to the establishment of leadership in the field. Meeting participation (oral presentations and posters), therefore, should be facilitated for Category I Scientists and Research Associates as well as for Category III Scientists and Graduate Students. Because published data are typically 1-2 years old, annual meeting attendance is critical to learn of new and relevant data and research directions, to network with scientists in the field, and to develop new and productive research collaborations, all of which are critical to attaining scientific leadership. Without cutting-edge scientific interaction, it is likely that ABADRL scientists, research staff and their graduate students will gradually lose scientific competitiveness and, hence, they will not be properly positioned to serve at the forefront of critical national needs in livestock health and biosecurity. The panel urges that the ARS administration facilitate and support meeting participation.
· ABADRL scientists and research staff should be encouraged to take advantage of leadership opportunities such as offering special training courses, organizing specialized meetings or workshops in their fields, serving on committees or as officers of professional societies, and serving on editorial boards of scientific journals.
· The ABADRL should not try to address WN research needs in the same depth as BT and VS. Scientists and research staff should focus upon a reasonable number of high priority questions that are appropriate to the unique capabilities of the staff and facilities. For example, ABADRL is poised to address questions related to risk of infection for selected large animals and of transmission by alternate arthropod vector species, diagnostics, vaccines, and control. By contributing in these areas the laboratory will become an integral part of the national and international leadership in WN research.
Acknowledgements
The panel thanks the ABADRL staff for their hospitality and the excellent organization of the review. The information provided by the Laboratory staff and Research Leader has been extremely useful in understanding current research activities and future plans. We appreciate the candor of the communication. The participation and contributions by the Area Administrator and National Program Leaders were also valuable.
APPENDIX A
REVIEW PANELISTS
Dr. Harvey Artsob
Chief, Zoonotic Diseases and Special Pathogens
National Microbiology Laboratory
Health Canada
Winnipeg, Manitoba, Canada
Dr. Barry J. Beaty
Arthropod Infectious Disease Laboratory
Dept. of Microbiology, Immunology and Pathology
Colorado State University
Ft. Collins, CO
Dr. Bob Bohlender
Chairman, Research Committee
National Cattleman’s Beef Association
North Platte, NE
Dr. Jim Logan
State Veterinarian
Wyoming Livestock Board
Cheyenne, WY
Dr. Shirley Luckhart
Dept. of Biochemistry
Virginia Tech University
Blacksburg, VA
Dr. Brian McCluskey
USDA-APHIS-CAHM
Ft. Collins, CO
Dr. William K. Reisen
Arbovirus Field Station
School of Veterinary Medicine
University of California, Davis
Bakersfield, CA
Dr. Thomas M. Yuill (Panel Chair)
Institute for Environmental Studies and
Department of Pathobiological Studies
University of Wisconsin-Madison
Madison, WI
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