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Research Project: Intervention Strategies to Control Endemic and New and Emerging Viral Diseases of Swine

Location: Virus and Prion Research

2020 Annual Report

1. Identify pathogenic mechanisms of swine Nidovirales, including identifying the pathogenic mechanisms of Porcine Respiratory and Reproductive Syndrome Virus (PRRSV), and the pathogenic mechanisms of Porcine Epidemic Diarrhea Virus (PEDV). 2. Discover and assess vaccines that can reduce or prevent economic losses from swine viral diseases, including identifying mechanisms to modulate innate and adaptive immune responses to swine viral pathogens and investigating technologies to override vaccine interference from passively acquired immunity. 3. Determine evolutionary antigenic and pathogenic properties of economically significant swine viral pathogen, including identifying and monitoring genetic and antigenic evolution in Nidovirales and emerging viral pathogens. 4. Identify mechanisms of pathogenesis, transmission, and immunity for emerging viral diseases of swine, starting with evaluating the onset and duration of Seneca A virus immunity in swine.

This research project will focus on swine diseases caused by viruses that are top concerns for United States pork producers: porcine reproductive and respiratory syndrome, porcine coronaviruses, and new and emerging diseases such as Seneca A virus. These pathogens will be examined in the laboratory as well as in swine disease models to investigate mechanisms of pathogenesis, transmission, immunity, evolution and methods of intervention. Animal experiments to be conducted involve one of three general designs: 1) disease pathogenesis and transmission studies, 2) vaccine efficacy studies, 3) sow/neonatal studies. Knowledge obtained will be applied to break the cycle of transmission of these swine pathogens through development of better vaccines or other novel intervention strategies. A major research approach will be the use of reverse engineering and infectious clones to identify virulence components of each virus under study through mutational studies. Development of vaccines that provide better cross-protective immunity than what is currently available with today’s vaccines will be approached through vaccine vector platform development, attenuated strains for vaccines and other novel technologies. A key approach in the study of disease pathogenesis is to better understand the host response to viral infection to various viruses. This research on comparative host transcriptomics will provide insights on viral pathogenesis and possible virulence factors that will enable rational design of more effective vaccines and target possible novel intervention strategies.

Progress Report
In support of Objective 1, prior studies of small non-coding RNAs (sncRNAs) have been used to identify the possible gene expression changes that occur during pathogen challenges that cause porcine illness. The expression of well-studied sncRNAs, such as microRNAs (miRNAs), have been shown to both bolster and depress the immune system of pigs infected with highly pathogenic porcine reproductive and respiratory virus (HP-PRRSV). Unfortunately, there is little known about another class of sncRNA referred to as tRNA fragments (tRFs). It is possible that these tRFs, like miRNAs, have modulating effects on the immune response in virally infected livestock. ARS scientists in Ames, Iowa, initiated a study to identify the presence of the tRFs in HP-PRRSV infected pigs. Discovery and mapping of possible porcine tRFs from domestic pig (Sus scrofa domesticus)11.1 reference genome were conducted using the whole blood of both healthy and HP-PRRSV infected pigs confirming the presence of circulating tRFs during healthy and infected states and paves a way for the identification of these rarely studied sncRNAs and how their expression changes during HP-PRRSV infection. In support of Objective 1, the impact of PRRSV and Influenza B coinfection on antiviral response in the porcine lung during infection was investigated by ARS scientists in Ames, Iowa. Activation of the antiviral response leads to host protection through expression of interferon-stimulated genes that aid to clear viruses. Control, porcine reproductive and respiratory syndrome virus (PRRSV), influenza B virus (FluB), and FluB/PRRSV coinfected swine lung tissue was assessed at different times after infection. Gene expression analysis was performed. The study found thousands of statistically significant differentially expressed genes (DEGs) for the different treatment groups over the course of the experiment. At early times after infection, the FluB and FluB/PRRSV coinfection groups showed greater expression of antiviral genes than the PRRSV group. All groups showed a reduction in upregulation for antiviral genes at later times post-infection. The downregulated genes for these comparisons indicated changes in pathways affecting lung development, wound healing, and cellular integrity. For all three treatment groups, the neutrophil degranulation pathway activity, associated with inflammation, increased, which may be related to lung lesions with these respiratory infections. The FluB infection increased expression for interferon and antiviral genes that lead to viral mRNA degradation and assembly and transcription inhibition. In comparison, the level of expression of antiviral genes in the PRRSV group decreased across time, which is a possible explanation as to why PRRSV infections persist while FluB infections are cleared. In support of Objective 1, investigators have shown coronaviruses (CoV) are capable of suppressing the host immune response by generating specific proteins capable of inhibiting interferons, called interferon antagonists. Using an infectious clone of CoV porcine epidemic diarrhea virus (icPEDV), collaborators at Loyola University generated viruses with inactive versions of interferon antagonist viral proteins. To evaluate clinical disease and virus shedding, ARS scientists in Ames, Iowa, infected piglets with either wild-type icPEDV or icPEDV-mut4 (virus with four mutations in the interferon antagonist viral proteins). icPEDV-mut4 infection of piglets did not induce diarrhea, although virus replication was detected in gut epithelial cells, and low levels of virus shedding was detected. Importantly, icPEDV-mut4 infection elicited IgG and neutralizing antibody responses to PEDV, and the mutations in the virus were stable throughout the infection. Inactivating the CoV interferon antagonists is an approach for generating candidate vaccines to limit the replication and disease caused by enteric CoVs. In support of Objective 1, recombinant porcine delta coronavirus virus (PDCoV) was produced in collaboration with scientists at Loyola University. The recombinant PDCoV was inoculated in parallel with wild-type PDCoV into neonatal piglets by ARS scientists in Ames, Iowa. The recombinant virus produced clinical signs that were indistinguishable from the native virus. The recombinant will now be utilized to prepare a vaccine that has been modified to inhibit interferon antagonists that are specific regions of the virus capable of suppressing the host immune response. In support of Objective 2, the replication-competent expression of IFNs using a PRRSV infectious clone has been characterized for the viral replication kinetics, IFN expression and stability, and ARS scientists in Ames, Iowa, have conducted proof-of-concept studies in pigs. Compared with a commercial vaccine, it was shown that some vaccine candidates were more effective in protecting pigs from a field-isolate challenge. In support of Objective 2, ARS scientists in Ames, Iowa, completed a Senecavirus A (SVA) vaccine study. SVA causes a vesicular disease in swine that is recognized as blister-like skin lesions located around the hoof and on the snout. Beginning in the summer of 2015 in the United States, there was a dramatic increase of swine vesicular disease cases from which SVV was isolated. These lesions are indistinguishable from vesicular disease caused by foot-and-mouth disease virus (FMDV), which can rapidly spread among livestock causing an economically devastating disease that can affect the food supply. Although the United States is free of FMDV, there is constant vigilance for this disease, so every time a lesion is observed a foreign animal disease investigation must be initiated, which can become an economic burden to government agencies and the swine industry. The study used sows and demonstrate efficacy of an inactivated whole virus mixed with an adjuvant to function as a vaccine against heterologous challenge. Protection was provided to piglets by passive maternal immunity in the colostrum from vaccinated sows. In support of Objective 4, ARS scientists in collaboration with Iowa State University scientists conducted an animal study demonstrating transmission of atypical porcine pestivirus (APPV) from persistently infected gilts to naïve contacts. The APPV persistently infected gilts were bred to observe whether they gave birth to piglets with congenital tremors, a neonatal pig disease recently shown to be caused by APPV. The piglets were normal at birth and did not exhibit congenital tremors. In support of Objective 4, ARS scientists in Ames, Iowa, in a collaboration with Iowa State University scientists conducted a study to validate the use of a whole-virus pseudorabies virus (PRV) assay to test oral fluids for PRV antibodies. Oral fluids and serum were collected from individual animals from three treatment groups: challenged with wild-type PRV, vaccinated with a commercial PRV vaccine and vaccinated and then challenged with wild-type PRV. These samples were utilized for assay validation that demonstrated that oral fluids could be used to test for PRV antibody.

1. Developed multiple porcine epidemic diarrhea (PEDV) virus strains with inactive interferon inhibition proteins. PEDV is a coronavirus (CoV), that appeared suddenly in the U.S. in 2013 and led to dramatic losses to the swine herd and economic losses to producers. No efficacious modified-live vaccine is available and wild-type CoVs are capable of suppressing the host immune response against the virus. Using an infectious clone of porcine epidemic diarrhea virus (icPEDV), Loyola University researchers, working in collaboration with ARS researchers in Ames, Iowa, generated viruses with inactive versions of three interferon antagonist regions individually, or combined in one virus designated icPEDV-mut4. Cells were infected with these mutant viruses and showed higher levels of interferon mRNA as compared to icPEDV infection, consistent with inactivation of interferon antagonists. One icPEDV mutant elicited the most robust interferon responses, which severely limited virus replication. These viral mutants will be inoculated into swine to test for their ability to protect against wild-type PEDV in neonatal pigs.

2. Developed a modified-live attenuated porcine epidemic diarrhea virus (PEDV) vaccine. Currently, available killed or subunit vaccines have proven insufficient in controlling PEDV, which is a problematic disease that results in neonatal piglet death. Using an infectious clone (icPEDV-mut4) that had been specifically altered to inactivate domains responsible for inhibition of interferon, ARS researchers in Ames, Iowa, evaluated clinical disease and virus shedding when piglets were infected with either wild-type icPEDV or icPEDV-mut4. icPEDV-mut4 infection of piglets did not induce diarrhea, although virus replication was detected in gut epithelial cells and low levels of virus shedding. Importantly, icPEDV-mut4 infection elicited IgG and neutralizing antibody responses to PEDV, and the mutations in the virus were stable throughout the infection. Inactivating these three CoV interferon antagonists is an approach for generating candidate vaccines to limit the replication and disease caused by enteric CoVs. This will be of interest to veterinarians and swine vaccine manufacturers.

3. Characterized the miRNAs and tRNA genes that affect host homeostasis in young pigs. A non-coding RNA (ncRNA) is an RNA molecule that is not translated into a protein. Noncoding RNAs belong to several groups and have regulatory roles in cellular processes. Abundant and functionally important types of non-coding RNAs include transfer RNAs (tRNAs) and small RNAs such as microRNAs (miRNAs). For decades, researchers have been exploring the many complications to swine health caused by porcine reproductive and respiratory virus (PRRSV) in order to find ways to reduce losses in commercial pig populations. ARS researchers in Ames, Iowa examined the expression profile of miRNA and tRNA expressed in whole blood between healthy and highly pathogenic PRRSV-infected pigs, in order to determine if ncRNA is involved in involved in viral entry, proliferation, and pro-inflammatory signaling networks that underlie the ability of HP-PRRSV to hinder host homeostasis. Overall, the results will serve to bring researchers closer to elucidating how gene function in the pig can become dysregulated due to PRRSV through changes in miRNA and tRNA expression.

4. Characterization of gene expression profiles of following swine infection with key viral pathogens. Porcine reproductive and respiratory syndrome virus (PRRSV) is a major respiratory pathogen of swine that has become extremely costly to the swine industry worldwide, often causing losses in production and animal life due to ease of spread. However, the intracellular changes that occur in pigs following viral respiratory infections are still scantily understood for PRRSV, as well as other viral respiratory infections. ARS researchers in Ames, Iowa, endeavored to acquire a better understanding of PRRS disease by comparing gene expression changes that occur in tracheobronchial lymph nodes of pigs infected with either PRRSV, porcine circovirus type 2 (PCV2), or swine influenza A virus (IAV-S) infections. The study identified and compared gene expression changes in the tracheobronchial lymph nodes of pigs following infection by PRRSV, PCV2, IAV, or sham inoculation. Total RNA was pooled for analysis by a process called Digital Gene Expression Tag Profiling (DGETP). The results showed that PRRSV, IAV-S and PCV-2 viral infections followed a clinical course typical of experimental infection of young pigs. Gene expression results uncovered genes related to shared and unique host immune responses to PRRSV, PCV2 and IAV-S. By testing and observing the host response to other respiratory viruses, our study has elucidated similarities and differences that can assist in the development of vaccines and therapeutics that shorten or prevent a chronic PRRSV infection.

5. Developed a method to catalog gene function by applying bioinformatics techniques to older, yet to be analyzed genetic sequence data. This methodology aids in determining which genes are expressed and how they might interact with other genes. Compiling this information provides insight into how genes with similar function could be grouped together, or how they might interact to form a response pathway to a specific stimulus. One way of gathering this information is to run tests that allow for similar genes to be grouped together by a common theme, such as disease resistance, and then compare, contrast, and characterize these genes and regulatory pathways response to major infections to verify the grouping. ARS researchers in Ames, Iowa, constructed a pipeline through the use of open-sourced freely available software and genomic databases (termed the (w)HOL(e)ISTIC Gene Ontology enrichment) to provide a method to group similar genes together to examine the different processes affecting the results of an experiment. This will benefit researchers investigating host gene expression responses to disease allowing for a better understanding of disease resistance.

6. Improved computer modeling to predict susceptibility of different species to infection with severe acute respiratory coronavirus 2 (SARS-CoV2). As a group of obligate pathogens, viruses need to enter a cell to replicate. Viral entry is a process that begins with attachment between a virus protein and a cell receptor(s), which allows for the virus to be internalized into the cell. Once inside the cell, the virus initiates replication starting the race between host immunity and a productive infection. For coronaviruses, the spike protein protruding from the viral surface is responsible for cell receptor binding and mediating viral entry. Several groups have reported that SARS-CoV2 uses the same angiotensin-converting enzyme 2 (ACE2) as the primary receptor for cell attachment, similar to SARS-CoV discovered in 2003. SARS-CoV-2 has been shown to have higher receptor affinity to human ACE2, which may contribute to the apparent faster spread of human SARS-CoV2 infection. For cross-species animal tropism, the potential infectivity of SARS-CoV2 in both wild and domestic animals, and potential for zoonotic transmission is a big public health concern that involves two aspects: (1) screening to identify the animal species that serve as a virus reservoir originally passing SARS-CoV2 to humans; and (2) the existing risk of infected people to pass the virus to animals, particularly the domestic species, thus potentially forming into an amplifying zoonotic cycle to worsen SARS-CoV2 evolution and prevalence. ARS researchers in Ames, Iowa, evaluated cross-species ACE2 genetic (and especially epigenetic) diversity in the regulation of ACE2 expression and functionality to determine the cell tropism and susceptibility of different animal species to SARS-CoV2. The analysis was used to predict the potential of livestock transmission of SARS-CoV2, and it also revealed that domestic animals including dogs, pigs, cattle and goats may evolve ACE2-related immunogenetic diversity to restrict SARS-CoV2 infections.

7. Developed and improved diagnostic assay for detection of pseudorabies virus (PRV) antibodies. PRV infection can produce severe disease in pigs and significant economic loss to producers. PRV has been eradicated from the US domestic swine herd, but the virus can be found in some feral swine populations making ongoing PRV control a challenge. Improving PRV control involves rapid testing of large groups of swine, which can be done by testing oral fluid samples for antibodies against the virus. ARS researchers in Ames, Iowa, conducted a PRV challenge study to produce oral fluids that were tested using a commercially available PRV antibody test. Results indicated good diagnostic performance and excellent repeatability; therefore, oral fluids could be a useful tool for PRV surveillance and detection. This will benefit veterinarians and pork producers as they improve rapid response plans for PRV control.

Review Publications
Miller, L.C., Fleming, D.S., Lager, K.M. 2020. Comparison of the transcriptome response within the swine tracheobronchial lymphnode following infection with PRRSV, PCV-2 or IAV-S. Pathogens. 9(2):99.
Fleming, D.S., Miller, L.C. 2019. Differentially expressed miRNAs and tRNA genes effect host homeostasis during highly pathogenic porcine reproductive and respiratory syndrome virus infections in young pigs. Frontiers in Genetics. 10:691.
Cheng, T., Buckley, A.C., Van Geelen, A., Lager, K.M., Henao-Diaz, A., Poonsuk, K., Pineyro, P., Ji, J., Wang, C., Main, R., Zimmerman, J., Gimenez-Lirola, L. 2020. Detection of pseudorabies virus antibody in swine oral fluid using a serum whole-virus indirect ELISA. Journal of Veterinary Diagnostic Investigation.
Lager, K.M., Buckley, A.C. 2019. Porcine anti-viral immunity: how important is it? Frontiers in Immunology. 10:2258.
Fleming, D.S., Herring, A.D., Miller, L.C., Gill, C.A. 2019. Transcriptomic analysis among sub-clinically ill cattle following bovine respiratory disease vaccine protocol and challenge with bovine viral diarrhea virus. BioMed Central (BMC) Immunology.
Fleming, D.S., Miller, L.C. 2020. (w)HOL(e)ISTIC gene ontology and pathway analysis of data using open source web tools. Genomics Data.