2012 Annual Report
1a.Objectives (from AD-416):
Foot-and-mouth disease virus (FMDV) causes a highly contagious, viral disease of domestic and wild cloven-hoofed animals effecting cattle, swine, sheep, goats and deer, which is characterized by fever, lameness and vesicular lesions and results in a high morbidity but low mortality in adult animals. FMD is the number one foreign animal disease threat to the United States livestock industry. Disease outbreaks result in the inhibition of trade in susceptible animals and their products. This research project will provide an understanding of virus-host interactions and disease pathogenesis that will aid in the development of alternative vaccine candidates and biotherapeutics.
The objectives of this research project are:
1. Develop an understanding the mechanisms of FMDV host-pathogen interactions at the molecular and in vivo levels will aid in the development of improved disease control measures.
2. Develop new and improve current novel vaccines and biotherapeutic countermeasures that will enable rapid and effective prevention of FMDV infection in natural hosts and thereby contribute to global eradication of FMD.
3. Determine the epidemiology of FMDV in enzootic regions by biological characterization of variant FMDV strains and identifying the epidemiological importance of persistence on transmission and maintenance.
1b.Approach (from AD-416):
1. To gain an understanding of the mechanisms of FMDV host-pathogen interaction, we will determine the molecular mechanisms of FMDV pathogenesis and viral-host interactions responsible for virulence, transmission and host-range specificity. Parental wild-type and attenuated viruses will be compared both in cell culture and in susceptible animals to identify virulence determinants; comparative pathogenesis studies will be conducted with multiple serotype and attenuated viruses to determine FMDV tissue distribution in susceptible hosts. The mechanisms mediating long term immunity induced by FMDV infection will be studied as well as the mechanisms of FMDV persistence and development of the carrier state.
2. The development and continued improvement of biotherapeutic countermeasures will be achieved through the discovery of biotherapeutic platforms to improve early protection against infection. Improved vaccine platforms will be identified by examining a series of vaccine approaches including inactivated vaccines derived from genetically engineered attenuated viruses with DIVA markers, live-attenuated vaccines, and adenovirus vectored FMDV. Studies will focus on the identification of the mechanisms to broaden the immune response of vaccines and to increase the duration of immunity conferred.
3. The characterization of FMDV epidemiological factors will be achieved through the collection of field samples and corresponding epidemiological information supplied by collaborating international partners from countries in which FMDV is currently circulating. Transmission, carrier state and epidemiological information will be compiled to gain a better understanding of FMD transmission and viral ecology in enzootic regions.
A 2nd generation replication-defective adenovirus, Ad5-FMD, vectored vaccine containing the coding regions for viral capsid, 3C and 2B proteins induced an enhanced cell mediated immune response and increased protection in cattle compared to a 1st generation vector. We initiated swine potency studies combining Ad5-A24 with various adjuvants.
We began development with a CRADA partner of a 2nd generation inactivated FMDV leader (L) deleted marker vaccine and companion DIVA diagnostic tests. The 3D test was transferred to APHIS for validation and a 3ABC-cELISA companion test was improved. Prototype DIVA ELISA tests and reagents were transferred to APHIS and industry. We demonstrated that a live-attenuated FMDV vaccine candidate with mutations in the L coding region is a virulent but can protect swine from challenge with virulent FMDV as early as 2 days post vaccination. We are introducing DIVA markers into this virus.
We examined approaches to improve FMDV biotherapeutics. Bovine interferon (IFN) lambda induced systemic antiviral responses and up-regulation of IFN stimulated genes. Porcine IFN lambda was cloned and an Ad5 vector was constructed. We demonstrated the effectiveness of Ad5-pIFNalpha alone or in combination with an adjuvant to rapidly protect swine against FMDV. We identified and cloned a number of bovine type I IFN genes and tested their ability to induce antiviral activity. 1 IFNalpha and 1 IFNbeta inducing high antiviral activity in vitro were cloned in an Ad5 vector to test in cattle. By microarray analysis we found that tissue tropism of FMDV is associated with differential expression of genes involved in cell attachment. Analysis of FMDV-specific T cell responses showed that 6 BoLA class I and 8 BoLA class II encoded proteins are expressed by 90 percent of the U. Vermont research herd that are used in FMDV-specific T cell studies. These genes have been expressed by U. Copenhagen collaborators for identification of FMDV T cell epitopes.
We showed that expression of the cellular proteins Sam68 and Beclin 1 are required for efficient FMDV replication.
In pathogenesis studies cattle were inoculated with virulent or a mutant FMDV. At the tissue level, both viruses initiated infection at several sites throughout the respiratory tract with similar predilection for epithelial cells of the lungs and pharynx; but the virulent virus achieved higher viral loads. Virulent FMDV entered the circulation, distributed throughout the body and replicated to high titers in selected lesion sites. In studies to characterize host-pathogen interactions during the FMD carrier state, issue-specific analyses implicated nasopharyngeal epithelium sites, the same sites colonized in initial infection, as the sites of virus persistence and various cytokines were downregulated in these tissues. Viruses from these animals had changes in the genome.
In international collaborations in FMD endemic countries we collected viral strains and obtained data on vaccine matching of the strains associated to outbreaks. We are also continuing studies to determine viral ecology and epidemiology including the role of Asian buffalo on FMDV transmission.
Improved Foot-and-Mouth Disease (FMD) vaccine potency in swine. New Ad5-FMD vaccine candidates require relatively high doses to be effective. ARS researchers at Greenport, New York, have combined a second generation replication-defective Ad5 vector containing not only the immunologically relevant FMDV capsid proteins and 3C proteinase required for capsid protein processing, but also contains the nonstructural protein 2B. With this new vaccine applied with various adjuvants to multiple sites under the skin (subcutaneously) we were able to reduce as much as 1000-fold the vaccine dose required to induce protection against FMDV compared to our first generation vaccine. This dose reduction represents an important improvement and a potential cost reduction for the production of Ad5 vaccines.
New means to induce rapid protection against Foot-and-Mouth Disease. Foot-and-mouth disease virus (FMDV) grows and spreads very rapidly. To control the disease, protective measures must be started quickly. ARS researchers at Greenport, New York, have identified several approaches. 1- The use of a stimulant (poly ICLC) of the immune system, which can fully protect swine against Foot and Mouth Disease (FMD) as early as one day after vaccination. 2-Bovine IFN lambda (bIFNlambda), to protect cattle against FMD. These and other approaches have significant potential as rapid acting countermeasures for the control of FMD outbreaks.
Elucidated novel aspects of Foot-and-Mouth Disease Virus (FMDV) infection in one of its natural hosts (cattle). There is a critical need to identify events associated in FMDV infection of cattle; specifically, in order to create better vaccines, one must first characterize how the virus attacks the host and how the host responds. ARS researchers at Greenport, New York have now identified subtle differences in the way cattle respond during the early phases of infection with FMD viruses. By conducting controlled aerosol exposure using a nebulizer to simulate the natural route, of infection we have identified that in the early phases of infection, cattle have a robust immune response consisting of largely interferons. These chemical messengers activate a highly complex network of gene expression alterations known as the “antiviral state”. This information is being used in the development of vaccines and biotherapeutics by modulating the immune response to artificially create a similar “antiviral state” by delivering the same interferons exogenously.
Combination of biotherapeutics and Foot-and-Mouth Disease (FMD) virus vaccines are being developed to induce rapid and long-lasting protection in cattle. There is a need to develop biotherapeutics to induce immediate protection in order to limit or inhibit disease spread prior to induction of vaccine induced immunity. In preliminary studies, ARS researchers at PIADC, Greenport, New York have demonstrated that a combination of a replication-defective human adenovirus (Ad5) vector containing bovine IFN lambda (Ad5-boIFNlambda) and an Ad5-FMD vaccine can induce rapid protection in bovines. This combination has been shown to induce systemic antiviral activity and a localized induction of interferon stimulated genes in epithelial tissues that are known targets for FMDV growth. This combination provides a broader innate immune response to infection and provides rapid and long-lasting protection to animals against FMD in an emergency outbreak situation.
Biswal, J.K., Sanyal, A., Rodriguez, L.L., Subramaniam, S., Arzt, J., Sharma, G.K., Hammond, J.M., Parida, S., Mohapatra, J.K., Mathapati, B.S., Dash, B.B., Ranjan, R., Rout, M., Venketaramanan, R., Misri, J., Krishna, L., Prasad, G., Pathak, K.M., Pattnaik, B. 2012. Foot-and-mouth disease: global status and Indian perspective. Indian Journal of Animal Sciences. 82(2):109-131.
Montiel, N., Smoliga, G.R., Arzt, J. 2012. Early detection and visualization of human adenovirus serotype 5-viral vectors carrying foot-and-mouth disease virus or luciferase transgenes in cell lines and bovine tissues. Vaccine. 30(9):1690-1701.
Diaz San Segundo, F.C., Weiss, M., Perez-Martin, E., Dias, C.C., Grubman, M.J., De Los Santos, T.B. 2012. Inoculation of swine with foot-and-mouth disease SAP-mutant virus induces early protection against disease. Journal of Virology. 86(3):1316-1327.
Pandya, M., Pacheco Tobin, J., Bishop, E.A., Kenney, M.A., Milward, F., Doel, T., Golde, W.T. 2012. An alternate delivery system improves vaccine performance against foot-and-mouth disease virus (FMDV). Vaccine. 30(20):3106-3111.
Moraes, M.P., Diaz San Segundo, F.C., Dias, C.C., Pena, L., Grubman, M.J. 2011. Increased efficacy of an adenovirus-vectored foot-and-mouth disease capsid subunit vaccine expressing nonstructural protein 2B is associated with a specific T cell response. Vaccine. 29(51):9431-9440.
Perez-Martin, E., Weiss, M., Diaz San Segundo, F.C., Pacheco Tobin, J., Arzt, J., Grubman, M.J., De Los Santos, T.B. 2012. Bovine type III interferon significantly delays and reduces the severity of foot-and-mouth disease in cattle. Journal of Virology. 86(8):4477-4487.
Lawrence, P.J., Schafer, E.A., Rieder, A.E. 2012. The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells. Virology. 425:40-52.
Borca, M.V., Pacheco Tobin, J., Holinka-Patterson, L.G., Hartwig, E.J., Carrillo, C., Garriga, D., Kramer, E., Rodriguez, L.L., Piccone, M.E. 2012. Role of the Arg56 of the structural protein VP3 of foot-and-mouth disease virus (FMDV) 01 campos in virus virulence. Virology. (422):37-45.