Location: Virus and Prion Research
2018 Annual Report
Objectives
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.
Approach
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, to define regions of porcine reproductive and respiratory syndrome virus (PRRSV) that influence virus pathogenesis, full-genome clones of PRRSV were produced that possessed changes in nonstructural protein 2 (nsp2). Nsp2 possesses a domain involved in immune evasion, it exists in several forms and varies dramatically between different strains of PRRSV. In collaboration with scientists from the University of Georgia, we exchanged the nsp2 segments between viruses of differing virulence and introduced mutations to assess any potential role these segments may have in determining virulence. The additional recombinant viruses were analyzed for phenotypic properties as well as deriving the complete viral genome sequences at MARC-145 cell passages 3 and 10. We are presently studying the effect of the mutant viruses on infected MARC-145 cell host RNAs involved in the cytokine pathway as well as in porcine alveolar macrophages. The next experiments will involve administering the mutant viruses in vivo to assess the impact of changes in nsp2 in pigs and their potential for use in preventative measures.
In support of Objective 1, a recombinant porcine epidemic diarrhea virus (PEDV) was produced in collaboration with researchers from Loyola University, and subsequently mutated in a specific region of nonstructural protein 3 and another region in nonstructural protein 15 to reduce the ability of the protease to inhibit host interferon (IFN) responses. The parental and mutant viruses were administered to newborn pigs in a pilot study investigating the attenuation of the mutant viruses to serve as a potential vaccine, with one mutant virus showing some reduction in viral shedding.
In support of Objective 2, groups of genes that are predicted to share the same regulatory elements that are knocked down in a coordinated fashion early in viral infection were identified. PRRSV appears to reduce the diversity of host genes expressed following infection compared with swine influenza virus and porcine circovirus type 2. This silencing effect on the host transcriptome has not previously been reported. This is the first comparative evaluation of gene expression in pigs experimentally infected with respiratory viruses and provides novel gene expression information for scientists to evaluate in their search for more effective vaccines. Evolutionary characterization of the porcine interferon-induced genes and genomic organization found 11 distinct genes that have potential antiviral functions in the pig.
In support of Objective 2, a modified attenuated PRRSV vaccine was used to prepare novel candidate vaccine constructs by conjoining sections of vaccine virus with sections of contemporary PRRSV found on U.S. swine farms. The conjoined viruses or chimeras produced viable virus with MARC-145 cell culture growth characteristics equal to the parent modified attenuated vaccine. Similarly, a chimeric vaccine construct was made using a section of an HP-PRRSV isolate, a PRRSV strain not yet detected in U.S. swine, to explore the potential to quickly respond to foreign PRRSV intrusions by changing the current vaccine to better match these foreign viruses. The next experiments will involve administering the mutant viruses in a pig model to examine their abilities to protect against contemporary strains of PRRSV.
In support of Objective 3, a United States Swine Pathogen Database was implemented and is housed on SCINet, an intranet infrastructure utilizing an Internet2 backbone, which is specifically designed to move large data sets quickly and is a project of the USDA Agricultural Research Service. The United States Swine Pathogen Database, presently not available to the public, contains genetic sequences from a number of pathogens along with the date and place of detection as well as other information useful in molecular evolution. Presently, the database contains all relevant information from GenBank®, the NIH genetic sequence database, and deposited sequences by collaborators from the South Dakota Animal Disease Research and Diagnostic Laboratory and the Kansas State Veterinary Diagnostic Laboratory. A sequence annotation tool was developed to process all sequences quickly. The database will eventually harbor sequences from two additional laboratories, Iowa State University Veterinary Diagnostic Laboratory, and University of Minnesota Veterinary Diagnostic Laboratory and they will be available to researchers around the globe by password accession.
In support of Objective 4, a pig study was completed that demonstrated Seneca Valley virus (SVV) isolates collected before 2015 could induce vesicular-like lesions similar to contemporary SVV isolates and that antiserum against the "old" and "new" isolates could cross-neutralize each other. Prior to 2015 in the United States, SVV was sporadically isolated from swine and sometimes associated with a vesicular-like disease. Although attempts were made, the vesicular disease was not reproduced with the "old" isolates. Beginning in late 2015 multiple cases of a vesicular–like disease occurred from which SVV could be isolated. The disease was reproduced by ARS scientists in Ames, Iowa using a 2015 SVV isolate provided by Iowa State University collaborators. To address the question is there a difference in the old vs. new SVV isolates, pigs were inoculated with 1 of 3 old or with 1 of 3 new SVV isolates. Each isolate induced lesions in respective pigs and induced antibodies that would cross-react among all of the 6 SVV isolates. The results suggest the explanation for the sudden increase in SVV vesicular-like cases in 2015 and subsequent years is not simply explained by the emergence of a more pathogenic lineage.
In support of Objective 4, a pig study was completed demonstrating that inactivated whole virus SVV could be used as a vaccine which demonstrates the potential for vaccines to be used in SVV control programs.
Accomplishments
1. Characterized the pathogenesis of Seneca Valley virus in nursery-aged pigs. Foot-and-mouth disease virus (FMDV) causes a vesicular disease in livestock that is recognized as a blister-like skin disease located around the hoof, on the nose, and sometimes in the mouth of cattle and swine. Because FMDV can rapidly spread among livestock it can cause 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. In swine, sporadic cases of vesicular disease are identified each year that are not caused by FMDV, but are attributed to Seneca Valley virus (SVV) suggesting this virus may be the cause of the disease, and of the FMDV false alarm. 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. This change in the "normal" SVV ecology raised questions. One question was about the potential effect stress might have on causing the upsurge in SVV cases. ARS researchers at Ames, Iowa completed a study to better characterize how SVV causes disease in swine, as well as look at the effect stress has on this disease since cases often appeared during times of stress such as animal movement. Using a pig "stress model" that used an immunosuppressive drug, "stress" did not enhance the incidence of disease when compared to the SVV only infected group. This finding indicates changes in the virus and/or current swine production led to hundreds of SVV cases being reported since 2015.
2. Identified small non-coding RNAs of the pig's tissues that may regulate gene silencing in PRRSV-infected animals. The commercial pig industry has long suffered economic losses due to infections from porcine reproductive and respiratory syndrome virus (PRRSV). The virus is easily spread among herds and in its low pathogenicity form leads to respiratory illness, reduced birthweights, stillborn piglets, sterility, and in its highly pathogenicity form causes death among older pigs and breeding age animals. It has been established that reduced susceptibility to PRRSV has a genetic component that may take the form of small non-coding RNA (sncRNA) molecules that function as regulators of host and viral gene expression. In order to identify differences in sncRNA expression between healthy and highly pathogenic PRRSV (HP-PRRSV) challenged pigs, a transcriptomic analysis of porcine whole blood from control and infected pigs was examined for changes in expression profiles associated with the virus by ARS researchers in Ames, Iowa. The results revealed multiple classes of sncRNA were both present and differentially expressed during HP-PRRSV infection. Some of the sncRNA identified were previously only seen during other viral respiratory infections and cancer. By assessment of the expression changes in sncRNA during infections, researchers can move closer to a discernment of how PRRSV disrupts host homeostasis and possibly uncover additional channels of detecting or destroying the virus.
3. Compared the host transcriptome changes following infection with three respiratory viruses of swine. The intracellular changes that occur in pigs following viral respiratory infections are still scantily understood for porcine reproductive and respiratory syndrome virus (PRRSV), as well as other viral respiratory infections. The aim of this study was to acquire a better understanding of PRRS disease by comparing gene expression changes that occur in tracheobronchial lymph nodes (TBLN) of pigs infected with either PRRSV, porcine circovirus type 2 (PCV2), or swine influenza A virus (IAV-S) infections. ARS researchers in Ames, Iowa identified and compared gene expression changes in the TBLN of pigs following infection by PRRSV, PCV2, IAV, or sham inoculation. The results showed that PRRSV, IAV-S and PCV-2 viral infections followed a clinical course in the pigs typical of experimental infection of young pigs with these viruses. Gene expression results echoed this course, as well as uncovered genes related to host immune responses to the 3 viruses. By testing and observing the host response to other respiratory viruses, the study has elucidated similarities and differences that can assist in development of vaccines and therapeutics that shorten or prevent a chronic PRRSV infection.
Review Publications
Spear, A., Wang, F.X., Kappes, M.A., Das, P.B., Faaberg, K.S. 2018. Progress toward an enhanced vaccine: eight marked attenuated viruses to porcine reproductive and respiratory disease virus. Virology. 516:30-37. https://doi.org/10.1016/j.virol.2017.12.029.
Miller, L.C., Fleming, D.S., Li, X., Bayles, D.O., Blecha, F., Sang, Y. 2017. Comparative analysis of signature genes in PRRSV-infected porcine monocyte-derived cells to different stimuli. PLoS One. 12(7):e0181256. https://doi.org/10.1371/journal.pone.0181256.
Fleming, D.S., Miller, L.C. 2018. Identification of small non-coding RNA classes expressed in swine whole blood during HP-PRRSV infection. Virology. 517:56-61. https://doi.org/10.1016/j.virol.2018.01.027.
van Geelen, A.G.M., Anderson, T.K., Lager, K.M., Das, P.B., Otis, N.J., Montiel, N.A., Miller, L.C., Kulshreshtha, V., Buckley, A.C., Brockmeier, S.L., Zhang, J., Gauger, P.C., Harmon, K.M., Faaberg, K.S. 2018. Porcine reproductive and respiratory disease virus: evolution and recombination yields distinct ORF5 RFLP 1-7-4 viruses with individual pathogenicity. Virology. 513:168-179.
