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- Arzt, Jonathan
- Rieder, Aida - Elizabeth
- Zhu, James
- Borca, Manuel
- 2023
- 2022
- Publications
- Animal Health (103)

Research Project: Intervention Strategies to Respond, Control, and Eradicate Foot-and-Mouth Disease Virus (FMDV) **Location:
                            Foreign Animal Disease Research** 2022 Annual Report **Objectives**
                                

OBJECTIVE 1.  Develop vaccines engineered for the control and eradication of Foot-and-Mouth Disease Virus (FMDV).  
Sub-objective 1.A Determine the mechanisms of protective immunity to FMDV conferred by multiple Foot-and-Mouth Disease (FMD) vaccine platforms. 
Sub-objective 1.B Determine the mechanisms mediating duration of immunity to FMDV.
Sub-objective 1.C Develop vaccine platforms that will provide cross-protective immunity against different FMDV subtypes.
Sub-objective 1.D Develop vaccines that reduce the incidence and accelerate clearance of persistent FMDV infections in cattle. 
Sub-objective 1.E Develop vaccines that can be rapidly manufactured to respond to new FMDV outbreaks.

Objective 2.  Develop biotherapeutics that can rapidly control the spread of FMD.  
Sub-objective 2.A Develop biotherapeutic platforms that induce rapid onset of immunity as a companion to an effective FMD vaccination. 
Sub-objective 2.B Develop biotherapeutics that alone or in vaccine formulations can reduce or abrogate FMDV persistence. 

Objective 3.  Elucidate the host-pathogen interactions of FMDV.   
Sub-objective 3.A Identify viral determinants of FMD that control virulence in susceptible hosts.  
Sub-objective 3.B Determine the virus/host interactions associated with FMDV persistence.  

Objective 4.  Characterize the ecology of FMDV in endemic regions.  
Sub-objective 4.A Determine drivers of FMDV transmission and maintenance in endemic regions. 
Sub-objective 4.B Determine factors that drive viral evolution and the mechanisms that lead to the emergence and spread of new FMDV strains.
Sub-objective 4.C Support the epidemiological analysis of data collected from countries with FMDV epidemics. **Approach**
                                

1.	The development of intervention strategies to control and eradicate Foot-and-Mouth Disease Virus (FMDV) will be achieved through research on novel Foot-and-Mouth Disease (FMD) vaccine platforms including of marker modified live-attenuated FMDV vaccine candidates such as FMDV-LL3B3D, second–generation Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) vaccines, and cross-protective vaccines against multiple subtypes. 

2.  Combinations of vaccine and biotherapeutics / and or adjuvants will be investigated as a way to induce mucosal immunity necessary not only to prevent disease but also to decrease persistent infection. These vaccine/adjuvant formulations will be tested using alternate routes such as transdermal and by direct mucosal delivery.

3. The host-pathogen interactions of FMDV will be determined through: the identification of viral determinants of FMDV that control virulence in susceptible hosts, determining virus/host interactions associated with the FMDV life cycle, and determining the mechanisms of protective immunity to FMDV. The molecular basis for FMDV-host interactions that impact virulence and their specific contributions to virulence will be determined. In addition, the interactions of the virus with specific tissues at the primary infection sites will be studied by characterizing infected tissues at the cellular and subcellular level as well as utilizing transcriptomic analyses with micro arrays and next generation RNAsequencing. Bioinformatic analyses will be extensively applied in order to understand species specific factors mediating the establishment and maintenance of persistent infections. The withinhost FMDV genomic evolution will be characterized through an examination of sitespecific mutational pressure, genomic variation and potential adaption to the host.
The immune mechanisms affecting protective immunity against FMDV will be determined through the analysis of CD4 helper and CD8 cytotoxic T cell responses to FMDV vaccination and B-cell responses to FMDV in peripheral blood and lymphoid tissue.

4.The characterization of the ecology of FMDV in endemic regions, including determining drivers of FMDV transmission and maintenance in endemic regions, characterizing factors driving FMDV emergence and spread, and the characterization of the role of the Asian buffalo in the transmission and maintenance of FMDV in the context of tolerance to infection will be analyzed. Efforts will focus on the characterization of the ecology of FMDV in endemic regions in Asia and Africa, including determining the factors driving viral transmission and maintenance.
Factors driving FMDV emergence and spread of novel FMDV strains in endemic settings will be characterized. The role of Asian buffalo in maintenance and transmission of FMDV in endemic settings will be assessed. **Progress Report**
                                

In October 2021, the Foot-and-Mouth Disease (FMD) research team at Foreign Animal Disease Research Unit (FADRU) kicked off this new project which continues the research performed under 8064-32000-061-000D, Intervention Strategies to Support the Global Control and Eradication of Foot-and-Mouth Disease Virus (FMDV). We achieved many of the milestones for the year despite the challenges of the ongoing COVID pandemic, restricted access to the laboratory and ever-increasing staff duties associated with the upcoming facility closure and mission transfer to National Bio and Agro-Defense Facility (NBAF), currently projected for 2024. NBAF transition tasks that detracted from scientific staff ability to conduct research included ongoing refinement of biorepository inventories, large scale destruction of biorepository samples not going to NBAF, refinement of equipment procurement for the new laboratory, safety and freedom testing of samples and continuation of the large-scale scanning of tens of thousands of pages of laboratory records for transition to the NBAF. 
Scientific progress was accrued in all objectives. In Objective 1: Develop vaccines engineered for the control and eradication of FMDV, Subobjective 1.A, Determine the mechanisms of protective immunity to FMDV conferred by multiple FMD vaccine platforms, progress included the development of multiple antibody panels for flow cytometry that allow for the characterization of several cell populations including CD4 and CD8 memory and effector T cells, gamma-delta T cells and NK cells. New protocols for ex vivo stimulation of cells from vaccinated and infected animals have been standardized and are now in use for the analysis of the immune response in different vaccination and biotherapeutic treatment studies in cattle and pigs. We have also validated methods for the quantitative assessment of the humoral immune response, including immunoglobulin isotyping. Recent outputs achieved from these newly standardized methods include correlation between protection by mosaic FMD vaccine and lack of correlation between cellular response and protection during superinfection studies.  
Under Sub-objective 1B Determine the mechanisms mediating duration of immunity to FMDV, novel recombinant FMDV vaccine candidates of serotypes O and Asia have been shown to be more stable at low PH and able to maintain their integrity at slightly higher temperatures. A comparative analysis of their biophysical properties was performed using Thermal and pH stability, Thermofluor assay, and time-response effect of live viruses stored for longer periods of time at different temperatures. Among these candidates, selected type O mutants, formulated as inactivated antigen with selected adjuvants will be assessed for their protective immunity and challenged at different times post vaccination. In addition to this approach, work has continued on the development of modified live vaccine (MLV) candidates, including sequence deoptimization of non-structural protein regions in combination with mutations to generate DIVA capabilities, demonstrated attenuated viral properties in vitro, and importantly, displayed reduced pathogenic characteristics compared to parental virus in cattle. Preliminary analysis of the induced immune response indicates their potential to be developed as vaccine candidates, although further animal studies need to be performed to evaluate efficacy. 
In Sub-objective 1.C Develop vaccine platforms that will provide cross-protective immunity against different FMDV subtypes, there were important advances. Specifically, mosaic capsids for serotypes O and Asia were designed, cloned and expressed using full length FMDLL3B3D backbones. These mosaic vaccine candidates demonstrated desirable in vitro growth characteristics and induced cross-protective immunity when formulated as inactivated vaccine for protection against heterologous challenge in cattle. The antigenic profile of the mosaic capsids was assessed using immunological methods including ImmunoDOT, Western Blot and peptide arrays. Novel tools to examine Th2/Th1 immune responses were developed simultaneously and successfully applied in these studies. In Sub-objective 1.D Develop vaccines that reduce the incidence and accelerate clearance of persistent FMDV infections in cattle, sample processing and data analysis was completed for one animal experiment in which cattle were vaccinated with a deoptimized (LAV-precursor) FMDV. Additionally, preliminary evaluations were performed on the potential impact on the FMDV carrier state associated with use of the LL3B3D-mosaic vaccines. Description of the output of both of these endeavors is limited by CRADAs and/or patent applications. Sub-objective 1.E Develop vaccines that can be rapidly manufactured to respond to new FMDV outbreaks. There were also achievements in the advanced development of this vaccine platform. Specifically, in collaboration with our industry partner, under a CRADA partnership, progress continued toward the production of FMD-LL3B3D inactivated vaccines and for the experimental small-scale production of pre-Master Seeds in production suspension BHK cells. A number of relevant vaccine strains for several serotypes have been synthesized by the commercial partner and premaster seed virus stocks have been successfully produced at Plum Island Animal Disease Center (PIADC). A production facility on the U.S. mainland was built by our CRADA partner, where master stocks of the main serotypes is starting this year.
In Objective 2, Develop biotherapeutics that can rapidly control the spread of FMD, two novel type I IFN subtypes (one bovine and one porcine) were designed, cloned, and expressed in Ad5 vectors for delivery. Both molecules demonstrated strong antiviral activity in cell culture and will be further evaluated in FMD-susceptible natural host species. Novel chemically modified IFN molecules were identified and preliminarily successfully evaluated in cell culture to limit FMDV growth. In collaboration with the Canadian Food Inspection Agency, we demonstrated that porcine interferon alpha delivered by and adenovirus 5 vector is an effective biotherapeutic to control and limit the spread of Ebola virus in pigs.
Within Objective 3, Elucidate the host-pathogen interactions of FMDV, 3A. Identify viral determinants of FMDV that control virulence in susceptible hosts, substantial progress was made in identifying novel mutations in Lpro outside the active site that are important for virulence. Virus bearing novel mutations in Lpro were significantly attenuated in cells and in pigs. Mechanistic studies demonstrated that mutant viruses were specifically impaired in their ability to neutralize the expression of interferon in the host. In addition, novel conserved secondary RNA structures were identified as potential virulence elements. Additionally, substantial progress was made towards subobjective 3.B “Determine the virus/host interactions associated with FMDV persistence.” Specifically, several studies were executed to investigate the effect of coinfection with distinct viral strains in cattle. The various outputs of this work included the discovery of partial protection against superinfection with heterologous strains and the finding that during dual infection of cattle with 2 distinct strains, recombination of the viral genomes may occur during the first 3 days of infection. 
There was also progress on viral ecology studies (Objective 4: Characterize the ecology of FMDV in endemic regions) including milestones regarding studies executed in various countries in Asia and Africa, addressing various aspects of subclinical and clinical infections. To address Sub-objective 4.A Determine drivers of FMDV transmission and maintenance in endemic regions, field work from Vietnam culminated in the finding that FMDV surveillance could be adequately achieved through sampling of asymptomatic buffalo and cattle at slaughterhouses. Subobjective 4.B Determine factors that drive viral evolution and the mechanisms that lead to the emergence and spread of new FMDV strains, was advanced through the large-scale acquisition of sequences of newly described FMDV strains from Pakistan and Vietnam which were subsequently used in longitudinal viral evolution studies. Additionally, spatiotemporal metadata corresponding to field FMDVs in Uganda were extensively examined. This information along with next generation sequencing data for serotype O and A isolates in Uganda were analyzed and are being prepared for release to the public domain. Novel regression methods were developed that may provide tools for targeted FMDV control strategies in endemic areas. **Accomplishments**
                                

1. Improved capacity for vaccine production in the United States for foot-and-mouth disease (FMD). FMD continues to be a major threat to U.S. agriculture. Currently, FMD vaccine production involves the use of live (virulent) FMD virus, which is not allowed in the U.S., and therefore requires vaccines to be purchased from overseas manufacturers. ARS researchers have developed a new FMD vaccine platform, based on a genetically modified virus that is safer than the parent virus. Importantly, the use of this modified virus has now been approved for research and development in the U.S. mainland, at a new multi-million-dollar facility built by the industry partner, specifically for this purpose. This new product bridges the long-standing need for a rationally designed, vaccine platform for safe production of efficacious FMD vaccines in the U.S with the capability to differentiate naturally infected animals from vaccinated animals (DIVA). Full-scale production of this product will ultimately allow for much more rapid responses to potential outbreaks of FMD. This government-industrial partnership was selected as the 2022 Excellence in Technology Transfer Award from the Federal laboratory Consortium for Technology transfer (FLC). 2. Discovery of unique, recombinant foot-and-mouth disease viruses through superinfection of asymptomatic carriers. ARS researchers in Orient Point, New York, have a long track record of discoveries regarding foot-and-mouth disease virus (FMDV) infection in cattle. Cumulative research efforts through the last decade have demonstrated that the virus is capable of persisting without causing disease in a small subset of cells within the nasopharynx (throat) of cattle that have recovered from the disease; this “carrier state” of FMDV is widely believed to be a dead end, specifically, that is non-transmissible to other animals. In a recent body of work ARS scientists demonstrated that when persistently infected carriers of FMDV were exposed to a different strain of the virus, the two viruses exchanged genetic material (recombined) to give rise to new, recombinant viruses containing distinct parts of each of the parental virus strains. This new discovery demonstrates a process whereby new strains of FMDV may evolve and emerge in the field, and contributes towards improvements in preparedness for a potential outbreak of FMD in the U.S. homeland. The work was published in the journal Pathogens in 2022 (https://www.mdpi.com/2076-0817/11/6/644). 3. Antiviral approach for blocking Ebola virus infection in pigs. ARS researchers in Orient Point, New York, have a long history of developing novel antiviral products to protect livestock from exotic viral diseases, particularly foot-and-mouth disease (FMD). One highly successful strategy has been the use of interferons (IFN), to rapidly block virus replication in infected animals. Over the last decade several high-profile outbreaks of Ebola virus (EBOV) in Africa and the discovery that EBOV can infect pigs led to the concern that pigs could potentially transmit the virus to humans. In order to seek a system to rapidly control EBOV infection in pigs, ARS scientists collaborated with counterparts at the Canadian Food Inspection Agency to test if a synthetically produced genetic virus that delivers the IFNa gene would protect pigs from EBOV infection, similar to how it had previously been shown to protect against FMD virus. The collaboration was highly productive and demonstrated that pre-treatment of pigs with IFNa is highly effective at protecting pigs from EBOV infection and virus shedding which ultimately would be likely to prevent transmission to humans. These findings highlight the versatility of the ARS-developed platform against multiple highly important viral diseases. This work was published in Pathogens in 2022, doi: (https://www.mdpi.com/2076-0817/11/4/449). **Review Publications**
                            *Holinka-Patterson, L.G., Fish, I., Bertram, M.R., Hartwig, E.J., Smoliga, G.R., Stenfeldt, C., Rodriguez, L.L., Arzt, J. 2022. Genome of bovine viral diarrhea virus (BVDV) contaminating a continuous LFBK-aVß6 cell line. Microbiology Resource Announcements. https://doi.org/10.1128/mra.01167-21.*
                                *Palinski, R., Brito, B., Jaya, F., Sangula, A., Gakuya, F., Bertram, M.R., Pauszek, S.J., Hartwig, E.J., Smoliga, G.R., Obanda, V., Omondi, G., Vanderwaal, K., Arzt, J. 2022. Viral population diversity during co-infection of foot-and-mouth disease virus serotypes SAT1 and SAT2 in African buffalo in Kenya. Viruses. https://doi.org/10.3390/v14050897.*
                                *Bertram, M.R., Stenfeldt, C., Holinka-Patterson, L.G., Fish, I., Farooq, U., Ahmedc, Z., Hartwig, E.J., Smoliga, G.R., Rodriguez, L.L., Arzt, J., Naeem, K., Meek, H.C., Pauszek, S.J. 2022. Foot-and-mouth disease virus Asia-1 lineage Sindh-08 from outbreaks in Pakistan 2011-2012. Microbiology Resource Announcements. https://journals.asm.org/doi/10.1128/mra.00312-22.*
                                *Fish, I., Bertram, M.R., Stenfeldt, C., Smoliga, G.R., Hartwig, E.J., Holinka-Patterson, L.G., De Los Santos, T.B., Spinard III, E.J., Medina, G.N., Arzt, J., Azzinaro, P.A. 2022. Foot-and-Mouth Disease virus interserotypic recombination in superinfected carrier cattle. Pathogens.  https://doi.org/10.3390/pathogens11060644.*
                                *Stenfeldt, C., Bertram, M.R., Holinka-Patterson, L.G., Fish, I., Farooq, U., Ahmed, Z., Hartwig, E.J., Smoliga, G.R., Rodriguez, L.L., Arzt, J., Naeem, K., Meek, H.C., Pauszek, S.J. 2022. Foot-and-mouth disease virus serotype Asia-1 obtained from subclinically infected Asian buffalo (Bubalus bubalis) in Pakistan. Microbiology Resource Announcements. https://doi.org/10.1128/mra.00312-22.*


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