Progress 06/15/18 to 06/14/23

Outputs
Target Audience:This project aimed to understand the basis for the modulation of host immunity by the PRRS virus and to investigate the mechanism for enhanced clinical disease during coinfection with a secondary pathogen on swine farms. The target audience includes investigators in virology, immunology, and vaccinology. University educators, pork producers, swine practitioners, and those in the veterinary pharmaceutical industry will also benefit from our research. New information obtained from this project has been delivered to the target audience via oral and poster presentations and abstracts at various scientific conferences. Scientific discoveries and systematic reviews derived from this project have been published as research articles in microbiology, immunology, and veterinary journals. Changes/Problems:This project has been completed and this is the final report. No changes or problems are associated with this report. What opportunities for training and professional development has the project provided?Three graduate students have participated in this project and received research training. H. Ke completed the program in 2019 and went to Harvard University as a post-doctoral fellow for further training, and J. Kim graduated in 2020 and returned to her home country. C.M. Su graduated in July 2023 and went to Boston University as a post-doctoral fellow. All of them contributed to this project actively and productively. The students also received opportunities to present their research data at various scientific conferences. They gained experience in public presentations and in-person interactions with other students and scientists. These students learned how to prepare manuscripts and address reviewer comments. By the time of their graduation, the students fully developed professional competency as young scientists. The NIFA funding was a critical component for the professional development of graduate students. How have the results been disseminated to communities of interest?We have disseminated new scientific findings to researchers, educators, and stakeholders, mainly through journal publications, conference presentations, and research seminars. During the NIFA funding period, we have published 19 journal articles, 1 book chapter, and 3 thesis/dissertations. We have also presented 32 conference papers at various national and international scientific meetings, including the Conference of Research Workers in Animal Disease, PRRS Symposium, American Society for Virology meetings, International Nidovirus Symposium, and International Immunology Congress, plus 23 seminars at different institutions. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? PRRSV emerged 3 decades ago and still remains one of the most economically significant diseases in the swine industry worldwide, including the US. A limited number of vaccines are available, but their protective efficacies are less satisfactory, and the disease continues to cause economic losses to the US pork industry. Our study aims to develop a better vaccine candidate to control PRRS. When virus infects, the host produces interferons (type I IFNs) quickly and eliminates the infecting virus, and thus IFNs are considered the most potent antiviral cytokines. We and others, however, have shown that PRRS virus suppresses the IFN production during infection of pigs and weakens the host immune response. As a result, PRRS virus-infected pigs cannot effectively eliminate the virus, so that the virus can persist within infected pigs for an extended period. An additional host response to PRRS virus-infection is production of proinflammatory cytokines. An inflammatory response is critical for a host to resolve infection, but it can also benefit PRRS virus to replicate. Production of proinflammatory cytokines recruits fresh macrophages to infection sites, and PRRSV can infect newly recruited fresh macrophages for further replication. Clinically, increased production of proinflammatory cytokines will lead to more severe disease, especially when pigs are coinfected with a bacterial pathogen, which is common in pig farms. Co-infection contributes to the development of porcine respiratory disease complex (PRDC), a clinically severe disease. It is evident that PRRS virus has evolved to counteract such critical host responses; viral suppression of IFNs and over-production of inflammatory cytokines. The current project aims to remove these functions from PRRS virus and to generate a new virus that may serve as a better vaccine candidate. The newly generated PRRS virus will increase the host immune responses and reduce clinical severity during co-infection with a secondary pathogen. Aim 1 is complete: We discovered that PRRS virus suppressed the host cell gene expression by inhibiting mRNA translation. The PRRS virus protein nsp1-beta bound to the Nup62 host protein and disrupted the nuclear gate, which blocked the host mRNAs export and translation. As a result, PRRS virus can replicate better because viral mRNAs translation occurs normally but host cell mRNA translation is inhibited. By mutational studies, we identified the leucine at position 126 of nsp1-beta was critical for this function, and L216 was mutated to alanine. The leucine to alanine mutation at 126 (L126A) restored the IFN production. Aim 2 is complete: We have previously developed a reverse genetics system for PRRS virus, which allows to engineer the viral genome. We also developed an extra-long inverse PCR technology to introduce specific mutations or deletions to the PRRS virus genome. The substitution of leucine to alanine at position 126 of the nsp1-beta protein was accomplished using this technology, and a mutant PRRS virus was generated. The viral mutant virus showed the biological phenotype of type I IFN suppression-negative. Aim 3 is complete: Nuclear factor (NF)-kB is the critical cellular factor for the production of proinflammatory cytokines. During PRRS virus infection, NF-kB is activated and triggers proinflammatory cytokine productions. We discovered that the viral nucleocapsid (N) protein was the NF-kB activator. We also determined the underlying mechanism for NF-kB activation by N protein. The viral N protein was found to bind to the cellular protein PIAS1. PIAS1 is a repressor for NF-kB, and we showed that the binding of viral N to PIAS1 conferred NF-kB activation. Subsequently, we mapped the PIAS1 binding motif to 41-49 of N. Two lysine residues at positions 43 and 44 and another lysine at 49 were deleted from N, and this mutation did not activate NF-kB. Phenotypically, this mutant virus produced lower levels of proinflammatory cytokines compared to wild-type PRRSV in cells. Aim 4 is complete: Based on the results from Aims 1, 2, and 3 and by using the reverse genetics and the extra-long PCR technology, a double-mutant PRRS virus was constructed. This double-mutant virus (L126 mutation in nsp1-beta and lysine mutations in N) was viable and rescued. The titer of the double-mutant virus was 2 logs lower than that of the wild-type virus, and this could be attributed to the higher level of IFNs production, which would be antiviral. The phenotype of the double-mutant PRRS virus was IFN suppression-negative and NF-kB activation-negative as expected. Using porcine alveolar macrophage cells (PAM-Cl3), which was established as an immortalized cell line by a NIFA-funded investigator at the Utah State University, we confirmed the decreased production proinflammatory cytokines and increased production of IFNs by the double-mutant PRRS virus. Aim 5 is complete:In this objective of the project, we examined the phenotype of double-mutant PRRSV during co-infection with a secondary pathogen. Respiratory pathogens frequently found in swine farms include influenza virus, porcine circovirus, PRRS virus, Mycoplasma, Actinobacillus pleuropneumonia, Pasteurella multocida, Streptococcus suis, and others. To test our hypothesis, we chose Streptococcus suis (S. suis) as a secondary pathogen. We initially planned this study to begin in years 3-4, but the animal experiment was delayed by 1 year to years 4-5 (2022-2023), due to the COVID-19 pandemic and related restrictions to various facilities. During the delated period, we continued to explore additional roles of the nsp1-beta protein of PRRS virus. Interestingly, we found that the viral nsp1-beta protein downregulated PML expression. PML is a promyelocytic leukemia protein localized in the nuclear bodies and participates in various cellular functions, including immune response and antiviral function. We found that nsp1-beta protein specifically bound to PML and promoted PRRSV replication. This discovery paves a novel way to investigating viral strategies to evade host innate immunity. For PRDC and co-infection studies, we first established an in vitro model in PAMs.In PAMs, the double-mutant virus did not suppress IFN expression but decreased the NF-kB-dependent inflammatory cytokine productions compared to those for wild-type PRRSV. Co-infection of PAMs with the mutant PRRSV and S. suis also decreased the production of NF-kB-directed inflammatory cytokines. To further examine the cytokine profiles and the disease severity by the double-mutant virus in natural host animals, 6 groups of pigs, 7 animals per group, were used for co-infection with the mutant PRRSV and S. suis. The double-mutant PRRSV was clinically attenuated in pigs, and the expressions of proinflammatory cytokines and chemokines were significantly reduced after bacterial co-infection. Compared to the wild-type PRRSV and S. suis co-infection control, pigs coinfected with the double-mutant PRRSV exhibited milder clinical signs, lower titers and shorter duration of viremia, and lower expression of proinflammatory cytokines. In conclusion, our study demonstrates that genetic modification of the type I IFN suppression and NF-kB activation functions of PRRSV may allow us to design a novel vaccine candidate to alleviate the clinical severity of PRRS and PRDC during bacterial co-infection.

Publications

• Type: Journal Articles Status: Published Year Published: 2023 Citation: Su, C-M, Du, Y., Rowland, R.R., Wang, Q. & Yoo, D. (2023) Reprogramming viral immune evasion for a rational design of next-generation vaccines. Frontiers in Immunology. 14:117200. https://doi.org/10.3389/fimmu.2023.1172000
• Type: Book Chapters Status: Published Year Published: 2022 Citation: Wang, L. & Yoo, D. (2022). Animal Coronaviruses. In: Animal Coronaviruses. Edited by: L. Wang, pp 3-19. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2091-5_1
• Type: Journal Articles Status: Published Year Published: 2022 Citation: Wu, X., Hu, Y., Sui, C., Pan, L., Yoo, D., Miller, L., Lee, C., Cong, X., Li, J., Du, Y. & Qi, J. (2022). Multiple Site SUMOylation of FMDV 3C Protease and Its Negative Role in Viral Replication. Journal of Virology. 96:e0061222. https://doi.org/10.1128/jvi.00612-22
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Yoo, D., Su, C-M, Han, M., Everett, R. D. 2023. Nonstructural protein 1 of arteriviruses downregulates promyeocytic leukemia protein expression and promotes viral replication. XVIth International Nidovirus Symposium, Montreux, Switzerland, May 14-18.
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Su, C-M., Zuckermann, F., Husmann, R., Roady, P., Kim, J., Lee, Y-M., Yoo, D. 2023. Immune outcome of IFN suppression-negative and NF-?B activation-negative PRRSV infection in vitro and in vivo. 103rd Conference of Research Workers in Animal Diseases. Chicago, IL. Jan 20-24. (This paper was awarded as the 2nd prize winner selected by The American College of Veterinary Microbiologists. Oral Presentation)
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Yoo, D., Su, C-M, Han, M. 2023. Nonstructural protein 1-beta of PRRSV suppresses PML protein expression and promotes viral replication. 103rd Conference of Research Workers in Animal Diseases. Chicago, IL. Jan 20-24.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Yoo, D., Su, C-M., Kim, J., Zuckermann, F.A. Husmann, R. Roady, P., Kim, Y.M. Lee, Redirecting viral immune evasion to a novel vaccine design: IFN suppression-negative and NF-kB activation-negative PRRSV in pigs during coinfection with Streptococcus suis. NC-1202 PD Workshop. 103rd Conference of Research Workers in Animal Diseases. Chicago, IL. Jan 20-24.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Kim, J., Davies, C., Gowen, B., Yoo, D., Lee, Y.M. 2023. A panel of pig macrophage-derived cell clones that differs in ability to support various steps of PRRSV replication. 103rd Conference of Research Workers in Animal Diseases. Chicago, IL. Jan 20-24.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Su, C-M, Kim, J., Zuckermann, F. A, Husmann, R., Roady, P., Kim, J., Lee, Y.M, D. Yoo. 2022. IFN suppression-negative and NF-?B activation-negative PRRSV in pigs during coinfection with Streptococcus suis. 2022 North American PRRS Symposium-International Conference of Swine Viral Disease. Chicago, IL, Dec 2-4.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Yoo, D. Su, C-M. 2022. Porcine reproductive and respiratory syndrome virus induces degradation of the promyelocytic leukemia protein and promotes viral replication. 2022 North American PRRS Symposium-International Conference of Swine Viral Disease. Chicago, IL, Dec 2-4.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Su, C-M, Yoo, D. 2022. Generation of the IFN suppression-negative and NF-?B activation-negative porcine reproductive and respiratory syndrome virus and the immune outcome with mutant virus infection. 41st American Society for Virology Annual Meeting. University of Wisconsin, Madison, WI. July 16-20.
• Type: Theses/Dissertations Status: Published Year Published: 2023 Citation: Chia-Ming Su (2023) Innate immunomodulatory strategies and pathogenic basis for nidoviruses. PhD Dissertation deposited on July 14, 2023. University of Illinois at Urbana-Champaign, Urbana IL.

Progress 06/15/21 to 06/14/22

Outputs
Target Audience:This project aims to understand the basis for PRRSV modulation of host immunity and to investigate the mechanism for enhanced clinical disease during coinfection with a secondary pathogen on swine farms. The target audience includes researchers in virology, immunology, and vaccinology. University educators, pork producers, swine practitioners, and those in the veterinary pharmaceutical industry will also benefit from our research. New information obtained from this project has been delivered to the target audience through oral presentations, poster presentations, and abstracts at scientific conferences. Manuscripts have been prepared and published in various journals Changes/Problems:We initially planned to conduct an infection study in pigs in 2020-2021.However, the animal experiment has been delayed unexpectedly for two reasons: 1) The COVID-19 pandemic has delayed the scheduled pig experiment due to limited access to laboratories and animal facilities. 2) The Co-PI (Dr. R. Rowland), who was supposed to conduct the pig experiment at the Kansas State University, relocated to the University of Illinois at Urbana-Champaign (UIUC) in 2020. His relocation to Illinois delayed establishing his lab settings, especially for animal experiments described in the NIFA project. To resolve the delay, I submitted a request for the no-cost extension and the modification of the experimental protocol, including the change of Co-PI to Dr. F. Zuckermann and the change of location for the pig experiment from Kansas State University to the University of Illinois. Dr. Zuckermann is a renowned swine immunologist, and the request has been approved by the NIFA office. The IACUC protocol has also been approved by the University of Illinois, and the animal holding facility has been secured to begin the experiment on July 7th, 2022. Thus, I should be able to complete the entire project by the end of the extended granting period, which is June 2023. Despite the delay due to the pandemic and the Co-PI relocation, we published two peer-reviewed articles in scientific journals and four presentations at four different conferences during the reporting period (07/2021-06/2022). What opportunities for training and professional development has the project provided?One student (CMS) participates in this project. CMS is a 3rd year PhD student and is actively working on this project. I run weekly lab meetings, and the students in my lab learn their presentation skills and receive suggestions and comments from me. The student has attended scientific conferences and gained experience in public presentations. As COVID-19 restrictions have become lifted, the student has been allowed to travel and experience in-person interactions. The student has also received training in writing manuscripts and addressing reviewer comments. How have the results been disseminated to communities of interest?The current project is the continuation of the previous project, and we have been productive despite the COVID-19 pandemic. During the current reporting period, we have published two peer-reviewed articles in Scientific Reports and the Journal of Virology. We have also presented four papers at four different scientific meetings. The meetings include the 102nd Conference of Research Workers in Animal Disease inChicago, the 2021 North American PRRS Symposium, Veterinary Research Day, and the 19th Annual Great Plains Infectious Disease Conference in Columbus, MO. What do you plan to do during the next reporting period to accomplish the goals?The nationwide success in the COVID-19 vaccination program has allowed the State of Illinois to move to Phase Five (complete restoration to normal activities), and accordingly, the University of Illinois has returned to normal in all academic activities, including research. In the next reporting period, we will conduct the pig infection study and complete Aim 5. Specifically, we plan to study clinical outcome, production of type I interferons, and production of NF-kB-mediated proinflammatory cytokines in pigs during coinfection with the mutant PRRSV generated from Aim 4 and the bacterial pathogen Streptococcus suis. We have developed a series of PRRSV mutants and selected a particular mutant that shows a unique phenotype of increased production of type I IFN and reduced proinflammatory cytokines in cells. Such phenotype will be examined in pigs in vivo during coinfection of the mutant PRRSV and a bacterial pathogen. Streptococcus suis is a common pathogen in swine farms, and we plan to examine the clinical outcome during co-infection with the mutant PRRSV and Streptococcus suis. We have prepared both pathogens for the infection study. We have received approval from NIFA to change the Co-PI from Dr. Rowland to Dr. Zuckermann and the location of the animal study from Kansas to Illinois. The IACUC protocol has also been approved by the University of Illinois Animal Committee, and the infection study will begin on July 7th, 2022. The sample analyses can be completed in the following several months without any problems. A manuscript will be prepared based on the data from infection studies and from Aims 2 and 3. I anticipated completing the entire project by the end of the extended granting period, which is June 2023.

Impacts
What was accomplished under these goals? PRRSV is still one of the most economically significant diseases in the swine industry worldwide, including in the US. Vaccines are available, but their protective efficacies are less satisfactory, and the disease continues to cause economic losses to the US pork industry. Our study aims to develop a better vaccine candidate to control PRRS. We and others have shown that PRRSV inhibits the IFN production during infection of pigs and weakens the host immune response. As a result, PRRSV-infected pigs cannot effectively eliminate the virus, and the virus can persist within infected animals for an extended period. Another host response to PRRSV infection is the production of proinflammatory cytokines. The inflammatory response is critical for hosts to resolve infections but can benefit PRRSV. Production of proinflammatory cytokines recruits fresh macrophages to infection sites, and PRRSV can infect newly recruited macrophages for effective replications. Clinically, increased production of proinflammatory cytokines will lead to more severe disease, especially when pigs are coinfected with a bacterial pathogen common in swine farms. The current project aims to remove those functions (IFN suppression and enhanced production of proinflammatory cytokines) from PRRSV and generate a new virus that may be used as a novel vaccine; The newly generated PRRSV is anticipated to increase the host immune responses and reduce clinical severity during coinfection with a secondary pathogen. We have made good research progress during the current reporting period of 06/2021-06/2022. Aim 1 is complete. We found that PRRSV infection suppressed the host mRNA translation. This suppression was mediated by blocking the host mRNA transport from the nucleus to the cytoplasm by the PRRSV nsp1-beta protein. We found that the nsp1-beta-protein of PRRSV is bound to the Nup62 cellular protein, which disintegrates the pore structure and blocks the host mRNAs nuclear export. As a result, PRRSV could grow better since PRRSV mRNAs are exclusively translated without competition with cellular mRNA. By mutational studies, we identified that leucine at position 126 was critical for this function, and this residue was mutated to alanine. The leucine to alanine mutation at 126 also restored the IFN production and antiviral proteins. This mutation has been incorporated into the mutant PRRSV. Aim 2 is complete. The reverse genetics system that we developed for PRRSV allows us to engineer the viral genome. We also developed an extra-long inverse PCR technology to introduce specific mutations or deletions to the viral genome. The substitution of leucine to alanine at position 126 of the nsp1-beta protein was accomplished, and a mutant virus was generated. The viral mutant virus showed the biological phenotype of type I IFN suppression-negative. Aim 3 is complete. During PRRSV infection, NF-kB is activated, which triggers the production of proinflammatory cytokines. We found that the viral nucleocapsid (N) protein was the NF-kB activator. We also determined the underlying mechanism for the NF-kB activation. The N protein interacted with the protein inhibitor of activated STAT1 (PIAS1). PIAS1 is a repressor for NF-kB, and we showed that the binding of PRRSV N to PIAS1 released NF-kB for activation. We identified the PIAS1 binding motif in PRRSV N as amino acid positions 41-49. Two lysine residues at positions 43 and 44 and another lysine at 49 were deleted from N, and the deletion mutant did not activate NF-kB. Phenotypically, this double mutant produced lower levels of proinflammatory cytokines compared to wild-type PRRSV. Aim 4 is complete. By using the reverse genetics and the extra-long PCR technology, a double-mutant virus was generated based on the results from Aims 1, 2, and 3. This double-mutant (L126 mutation in nsp1-beta and lysine mutant in N) was viable. The titer of the double-mutant virus was 2 logs lower than that of the wild-type virus, but was still infectious. Its phenotype was IFN-suppression-negative and NF-kB-activation-negative as anticipated. Porcine alveolar macrophage cells were developed as an immortalized cell line by an investigator at the Utah State University, and we obtained these cells (PAM Cl3). Using PAM Cl3 cells, proinflammatory cytokine productions were determined by infecting with the double mutant PRRSV, and the results were validated in the primary target cells for PRRSV. Aim 5:This aim is to examine the double mutant PRRSV in pigs for clinical attenuation during co-infection with a secondary pathogen.We initially planned this study to begin in years 3-4 (2020-2021).However, the animal experiment was delayed as explained in the "Changes and Problems" section. We requested a no-cost extension along with the protocol modifications. NIFA has approved this request, and thus, the animal infection experiment will be conducted in 2022. In the meantime, we continued to explore additional roles of the nsp1-beta protein of PRRSV. Interestingly, we found that the viral nsp1-beta protein downregulated PML expression. PML is a promyelocytic leukemia protein localized in the PML nuclear bodies and participates in various cellular functions, including immune response, apoptosis, cell growth, and autophagy. We found that nsp1-beta protein specifically bound to PML and showed by gene silencing technology that PRRSV replicated better by inhibiting the PML expression. The PML degradation was a post-translational event. It was not due to the proteolytic activity of the nsp1-beta nor ubiquitination of PML. We are currently investigating the possible involvement of autophagy. The PML degradation is an important viral evasion strategy for PRRSV to combat the host's innate immunity for better viral replication regardless of the mechanistic basis.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Sui, C., Jiang, D., Wu, X., Liu, S., Li, F., Pan, L., Cong, X., Li, J., Yoo, D., Rock, D., Miller, L., Lee, C., Qi, J., & Du, Y. 2021. Inhibition of Antiviral Innate Immunity by Foot-and-Mouth Disease Virus Lpro through Interaction with N-terminal Domain of Swine RNase L. Journal of Virology 95:e0036121.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Su, C-M, & Yoo, D. 2021. Mediation of type 1 IFN signaling by PRRSV nsp5-related protein. 2021 North American PRRS Symposium. Chicago IL. Dec 3-4.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Su, C-M., Wang, L., & Yoo, D. 2021. NF-kB activation and proinflammatory cytokine productions by ORF7a protein of SARS-CoV-2. 102nd Conference of Research Workers in Animal Diseases. Chicago, IL. Dec. 4-7.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Su, C-M, & Yoo, D. 2021. Activation of NF-kB and related proinflammatory cytokine induction by SARS-CoV-2 proteins. 19th Annual Great Plains Infectious Disease Conference. University of Missouri, Columbus, MO, Nov. 5-6.
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Su, C-M, & Yoo, D. 2021.Cellular response to IFN suppression-negative and NF-?B activation-negative porcine reproductive and respiratory syndrome virus infection. UIUC College of Veterinary Medicine Annual Research Day, Urbana IL, April 27.
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Su, C-M, Wang, L., & Yoo, D. 2021. Activation of NF-?B and Induction of Proinflammatory Cytokine Expressions mediated by ORF7a Protein of SARS-CoV-2. Scientific Reports 11:13464.

Progress 06/15/20 to 06/14/21

Outputs
Target Audience:This project aims to understand the basis for viral modulation of host immunity and investigate the mechanism for enhanced clinical disease during coinfection with other pathogens on swine farms. The target audience includes researchers in the area of virology, immunology, and vaccinology, and university educators. Pork producers, swine practitioners, and those in the veterinary pharmaceutical industry will also benefit from our research. New information obtained from this project has been delivered to the target audience via oral presentations, poster presentations, and abstracts at professional conferences. Manuscripts have been prepared and published in various scientific journals Changes/Problems:The COVID-19 pandemic and the further spread in the US have been a significant obstacle in making desirable progress of the project. The State of Illinois issued a 'stay-at-home' order in Spring 2020, and the University suspended most research activities on campus. The lockdown and limited activities continued until recently, in 2021. The University continued to implement actions to maximize university members' health and safety, including faculty and graduate students. The COVID-19 lockdown of the University has caused a significant delay in the research progress. We initially planned to conduct an animal study in 2020-2021.However, the animal experiment has been delayed unexpectedly for two reasons: 1) As stated above, the COVID-19 pandemic has delayed the scheduled pig experiment due to limited access to laboratories and animal facilities. 2) The collaborator, who intended to conduct the pig experiment at Kansas, has relocated to the University of Illinois at Urbana-Champaign (UIUC) in 2020. Since his relocation to Illinois, the collaborator has been busy establishing a functional research laboratory. Nonetheless, we are currently working hard to catch up on the delayed progress. I still expect a significant backlog for the use of the UIUC animal facility, primarily due to the COVID-19-related delay for all animal researchers of the University. In the meantime, we have been focusing on laboratory experiments. During the pandemic, we noticed some pathogenic similarities between PRRSV-infected pigs and COVID-19 human patients. Through laboratory experiments, we found that the accessory proteins of SARS-CoV-2 were also NF-kB activators as with the PRRSV nucleocapsid protein (A manuscript has been prepared, reviewed, and is currently under revision for final acceptance). This finding expands our understanding of immune evasion and modulation of cytokine response of the host as a common strategy for PRRSV and coronaviruses. Despite the delay due to the pandemic, we have published a total of six peer-reviewed articles in scientific journals and given three presentations remotely at two conferences.A recent nationwide success in the vaccination program will likely be possible for the State of Illinois to move into Phase Five (complete restoration to normal activities) in June 2021. We will try our best to catch up on the progress. The future progress is mainly dependent on the smooth transition to the new normal. What opportunities for training and professional development has the project provided?During the reporting period, two students (JK and CMS) participated in this project. One student (JK) completed the program in 2020 and received an MSc degree. Another student (CMS) is a 2nd-year PhD student and is actively working on this project. I run weekly lab meetings, and these students in my lab learned their data presentation skills and received suggestions and comments for the following experiments. Both students have participated in scientific conferences and gained experience in public presentations. Unfortunately, because of the COVID-19 pandemic, travels were not allowed during the reporting period, and the conference presentations were done remotely by Zoom, limiting their interactions with other researchers. The students have also received training in writing manuscripts and thesis, and addressing reviewer comments. Such activities have stimulated student's aspirations and research motivations. How have the results been disseminated to communities of interest?The current project is the continuation of the previous project, and we have been productive despite the COVID-19 pandemic. We have published six peer-reviewed articles in various scientific journals during the reporting period, including Virus Research, Vaccines, Viruses, and Journal of Veterinary Science. We have also attended virtual scientific conferences and presented three papers. The meetings include the 101st Conference of Research Workers in Animal Disease inChicago, and Veterinary Research Day of the University of Illinois at Urbana-Champaign. One graduate student completed the program during the reporting period, and a thesis was prepared and reviewed by the graduate committee. What do you plan to do during the next reporting period to accomplish the goals??We plan to study the cellular outcome of NF-kB-mediated proinflammatory cytokines during coinfection with PRRSV and a secondary viral or bacterial pathogen in cells and pigs. We have generated a series of PRRSV mutants. Those mutants will be examined for their biological phenotypes in cells, including IFN suppression and cytokine production in cells when coinfected with either Streptococcus suis (as a common bacterial pathogen) or swine influenza virus (as a common viral pathogen). We will then prepare the mutant viruses in large amounts to conduct animal experiments in pigs for clinical outcomes, proinflammatory cytokines, and interferon productions during coinfection with PRRSV and a secondary pathogen. At this time, the satisfactory progress will be dependent on the smooth transition from current COVID-19 restrictions to the new normal.

Impacts
What was accomplished under these goals? PRRS remains one of the most economically significant diseases in the swine industry worldwide, including in the US. Vaccines are available, but their protective efficacies are less satisfactory, and the disease continues to cause economic losses to the US pork industry. In response to viral infection, host cells produce a large number of cytokines and chemokines. Among them, type I interferons (IFNs) are major antiviral cytokines produced soon after infection to eliminate invading viruses. We and others have shown that PRRSV inhibits the IFN production during infection of pigs and disarms the host immune response. As a result, PRRSV-infected pigs cannot effectively eliminate the virus, and the virus can persist within infected animals for an extended period. Another response of the host to PRRSV infection is the production of proinflammatory cytokines. The inflammatory response is helpful for hosts to resolve the infection condition but can be beneficial for PRRSV. The production of proinflammatory cytokines recruits fresh macrophages to infection sites, and PRRSV can infect newly recruited macrophages for continuous and efficient replications. Increased production of proinflammatory cytokines will lead to a more severe clinical outcome, especially when pigs are coinfected with other bacterial pathogens commonly found in swine farms. The current project aims to remove the above two viral functions from PRRSV; 1) IFN suppression and 2) increased production of proinflammatory cytokines. The newly generated PRRS virus will increase the host immune responses during infection and reduce clinical severity in pigs during coinfection with a secondary pathogen. We have made relatively good research progress during the reporting period between 6/15/2020-6/14/2021 despite the COVID-19 pandemic. Aim 1: We found that PRRSV infection suppressed the host mRNA translation. This suppression was mediated by blocking the host mRNA transport from the nucleus to the cytoplasm by the PRRSV nsp1-beta protein. Pores are present on the nuclear membrane of the cellto allow host mRNA transport and translation, and we found that the PRRSV nsp1-beta-protein bound to the cellular protein constituting the pore structure. As a result, the pore structure was disintegrated, host mRNAs transport was inhibited, and mRNA translation could not occur. In turn, PRRSV could grow efficiently since PRRSV mRNAs are produced in the cytoplasm and thus do not require the pore structure for translation. We mapped the interactive domain with the nuclear pore to amino acid positions 126-135 in the nsp1-beta protein. By mutational studies, we found the residues at 126 and 135 were critical for this function. Mutant nsp1-beta were constructed to change these amino acids individually, and as predicted, the mutants lost the suppression function for type I IFNs and a variety of antiviral proteins. However, these mutant viruses replicated at a lower efficiency than the wild-type virus. We examined other residues and found the amino acid at position 129 also had a similar suppressive function to 126 and 135. Subsequently, we constructed another mutant that contained a similar role but grew better than the previous two mutants. Aim 2: Previously, we developed a reverse genetics system for PRRSV, and this system allows us to engineer the viral genome. We also developed an extra-long inverse PCR technology to introduce specific mutations or deletion in the viral genome. Subsequently, a single amino acid at position 129 of the nsp1-beta protein was deleted from the virus. We successfully deleted the amino acid at position 129 and rescued the mutant virus. This mutant was genetically stable. The mutant virus showed the biological phenotype of type I IFN suppression-negative. Aim 3: During PRRSV-infection, NF-kB is activated, which is then triggers proinflammatory cytokine expressions. By examining all PRRSV gene products individually in cells, we found that the nucleocapsid (N) protein was the NF-kB activator. We also found that the N protein specifically interacted with the protein inhibitor of activated STAT1 (PIAS1). PIAS1 is known as a repressor for a subunit of NF-kB in the nucleus and suppresses NF-kB activation. We found that the interaction of PRRSV N protein with PIAS1 released NF-kB and so NF-kB became activated. This series of interactions triggered the expression of proinflammatory cytokines. We mapped the PIAS1 interactive domain in the PRRSV N protein and found that the active domain lay in amino acid positions 41-47 in the N protein. Using the full-length infectious clone and the extra-long inverse PCR technique, three amino acids were deleted from this domain, and the deletion mutant PRRSV was successfully generated. This NLS-deletion mutant PRRSV was infectious and produced lower levels of proinflammatory cytokines compared to those of wild-type PRRSV. Aim 4: Next, we attempted to introduce double-deletion mutations to the PRRSV genome; a single amino acid deletion at position 129 in the nsp1-beta-protein (as described in Aim 2) and three-amino acid deletion in the N protein (as described in Aim 3). Subsequently, we were able to generate a double-deletion PRRSV mutant. The titer of the double-deletion mutant PRRSV was 2 logs lower than that of wild-type PRRSV. Currently, this mutant virus is being characterized for its abilities in cells for type I IFN induction and NF-kB activation. Once the genotype at the genome level and the phenotypes for interferons and cytokines are characterized, we will prepare large-scale production of the mutant virus for clinical studies in natural host animals. During the pandemic, we noticed some pathogenic similarities between PRRSV-infected pigs and COVID-19 human patients. Through laboratory experiments, we found that the accessory proteins of SARS-CoV-2 were also NF-kB activators as with the PRRSV nucleocapsid protein (A manuscript has been prepared, reviewed, and is currently under revision for final acceptance). This finding expands our understanding of immune evasion and modulation of cytokine response of the host as a common strategy for PRRSV and coronaviruses. Aim 5:This aim is to examine the mutant PRRSV for clinical attenuation in pigs during co-infection with a secondary pathogen. We initially planned this study to start sometime during year 3.However, the animal experiment has been delayed unexpectedly for two reasons: 1) The COVID-19 pandemic has delayed the scheduled pig experiment due to limited access to laboratories and animal facilities. 2) The collaborator, who intended to conduct the pig experiment, has relocated to the University of Illinois at Urbana-Champaign in 2020. Since his relocation to Illinois, the collaborator has been busy establishing a functional research laboratory. Nonetheless, we are currently working hard to catch up on the delayed progress. I expect a significant backlog for the use of the animal facility primarily due to the COVID-19-related delay for all animal researchers of the University. In the meantime, we would like to focus on the laboratory experiments.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: An, T.Q., Li, J.N., Su, C-M., & Yoo, D. 2020. Molecular and cellular mechanisms for PRRSV pathogenesis and host response to infection. Virus Research. 286:197980 (Invited Review Article). https://doi.org/10.1016/j.virusres.2020.197980
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Zhang, G., Li, B., Yoo, D., Qin, T., Zhang, X., Jia, Y., & Cui, S. 2021. Animal coronaviruses and SARS-CoV-2. Transboundary Emerging Disease. 68:10971110. https://onlinelibrary.wiley.com/doi/10.1111/tbed.13791 (E-published in 2020)
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Yoo, H.S. & Yoo, D. 2020. COVID-19 and veterinarians for one health, zoonotic- and reverse-zoonotic transmissions. Journal of Veterinary Science. 21:e51. https://doi.org/10.4142/jvs.2020.21.e51
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Liang, W., Zhou, D., Geng, C., Yang, K., Duan, Z., Guo, R., Liu, W., Yuan, F., Liu, Z., Gao, T., Zhao, L., Yoo, D., & Tian, Y. 2020. Isolation and evolutionary analyses of porcine epidemic diarrhea virus in Asia. PeerJ. e10114. https://peerj.com/articles/10114/
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Hicks, J.A. Yoo, D. & Liu, H.C. 2020. Transcriptional immune signatures of alveolar macrophages and the impact of the NLRP3 inflammasome on porcine reproductive and respiratory syndrome virus (PRRSV) replication. Viruses 12, 1299; https://www.mdpi.com/1999-4915/12/11/1299
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Su, C-M, Rowland, R.R.R., & Yoo, D. 2021. Recent advances in PRRS virus receptors and the targeting of receptor-ligand for control. Vaccines 9:354-368 (Invited Review Article). https://doi.org/10.3390/vaccines9040354
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Kim, J., Su, C-M. & Yoo, D. 2020. Immunological responses are dampened during co-infection of NF-kB activation-negative PRRSV and Streptococcus suis. 101st Conference of Research Workers in Animal Diseases. Chicago, IL. Dec 3-7.
• Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Su, C-M. & Yoo, D. 2020. Inhibition of type 1 IFN signaling by PRRSV nsp5 through blocking STAT2 nuclear translocation. 101st Conference of Research Workers in Animal Diseases. Chicago, IL. Dec 3-7.
• Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Su, C-M., Wang, L. & Yoo, D. 2021. Activation of NF-kB and induction of proinflammatory cytokine expressions mediated by ORF7a protein of SARS-CoV-2. CVM Research Day. University of Illinois at Urbana-Champaign. Urbana, IL. April 23 (This paper was awarded Honorable Mention).
• Type: Theses/Dissertations Status: Awaiting Publication Year Published: 2020 Citation: Kim, J. 2020. Cytokine modulations by type I interferon suppression-negative and NF-kB activation-negative porcine reproductive and respiratory syndrome virus. MSc Thesis, University of Illinois at Urbana-Champaign (The public viewing of this thesis is delayed until 2022 due to the confidentiality)

Progress 06/15/19 to 06/14/20

Outputs
Target Audience:The current project aims to understand the basis for viral modulation of host immunity and to investigate the mechanism for enhanced clinical disease during coinfection with a bacterial pathogen on farm. The primary target audiences of the project include researchers in virology, immunology, and vaccinology. University educators, pork producers, swine practitioners, and those in the veterinary pharmaceutical industry will also gain benefits from our research. New information obtained from the project has been delivered to target audiences via oral presentations, poster presentations, and abstracts at variousmeetings and professional conferences. Manuscripts have been published in scientific journals. Changes/Problems:In the begining of March2020,the COVID-19 outbreaks and further spreadbecame widen in the country.The State of Illinois issued a 'stay-at-home' orderin the mid-March, and all the research activities at the University were suspended. The lockdown has been continuing until the time the current annualreport is submitted (May 29, 2020). According to the state progress moving into Phase Three (of the Five Phases) of Gov. J. B. Pritzker's Restore Illinois plan, it is possible that some employees will start to return to on-campus work begining June 1. Nevertheless, the university will continue to implement actions to maximize the health and safety of the university members. The University lockdown due to the COVID-19 pandemic has caused a significant delay for research progress. We will try ourbest tocatch up the progress, but the future progress is largely dependent on the smooth transition to the new normal. What opportunities for training and professional development has the project provided?During the reporting period, two PhD students (JK and CMS) have participated in this project. One student (JK) is a 2nd-year student, and the other student (CMS) is a 1st-year student in the program. I run weekly-lab meetings, and the students in my lab practice their presentation skills for data and receive suggestions for subsequent experiments. The students have participated in various scientific conferences at the national and international levels and gained experience in conference presentations and interaction with other researchers. The students have also received training in writing manuscripts and addressing reviewer comments. Such training has stimulated the student's aspiration and research motivation. How have the results been disseminated to communities of interest?The current project is continuation of the previous project, and we have been productive. During the reporting period, we have published 3 peer-reviewed papers in various journals including the Journal of Virology. We have also attended scientific conferences and symposiums and presented 6 papers. These conferences include the North American PRRS Symposium in Chicago, Asian Pig Veterinary Society (IPVS) Congress in Seoul, Korea, and International Immunology Congress in Beijing, China. These conferences are represented by researchers, university educators, veterinarians, and commodity representatives. We have exchanged new scientific findings and up-to-date information on swine infectious diseases. While I was on my sabbatical leave for 1 semester in Korea and China, I was invited to give seminars at various institutions, and used these opportunities to disseminate our research findings. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, I plan to study the cellular ourcome of NF-kB-mediated proinflammatory cytokines during coinfection with bacterial pathogens. We havegenerated a series ofPRRSV mutants, and those mutants will be examined for their biological phenotypes, including IFN suppression and cytokine productions in cells when coinfected with Streptococcus suis which is a common bacterial pathogen found in swine farms. I plan tocomplete charaterization of the mutant viruses and prepare the mutants inlarge quantities, so that we can conduct animal experiments in the future.

Impacts
What was accomplished under these goals? PRRS remains one of the most significant diseases in pigs worldwide. Vaccines are available but they are less satisfactory, and the disease continues to cause economic losses to the pork industry in the US. In response to viral infection, host cells produce a variety of cytokines and chemokines. Type I interferons (IFNs) are produced immediately after infection, and IFNs are the principle cytokines responsible for eliminating invading viruses. We and others have shown that PRRSV inhibits IFN production and disarms the host immune system. As a result, PRRSV-infected pigs are unable to effectively eliminate the virus, and the virus can persist for up to 6 months within infected animals. Another response of host to PRRSV is production of proinflammatory cytokines. The production of proinflammatory cytokines recruits fresh macrophages to infection sites, and PRRSV can infect freshly recruited macrophages for continuous and efficient replication. Increased production of proinflammatory cytokines leads to a more severe clinical outcome when pigs are coinfected with other bacterial pathogens which is commonplace on farms. The current project aims to disable these two functions of PRRSV; IFN suppression function and increased production of proinflammatory cytokines. Newly generated PRRSV will increase the host immune responses during infection and reduce a clinical severity in pigs during coinfection with a secondary pathogen. During the reporting period between 6/15/2019-6/14/2020, we have made a good research progress. Specific Aim 1: Previously, we showed that host mRNA translation was inhibited in PRRSV-infected cells. This inhibition was mediated by PRRSV nsp1 protein which blocks mRNA transport from the nucleus to the cytoplasm. PRRSV nsp1 protein disintegrated the nuclear pore complex on the nuclear membrane during infection. We discovered that nsp1 bound specifically to the cellular protein nucleoporin 62 (Nup62). The interactive domain in Nup62 was mapped to the 328-522 residues. For nsp1, the interaction domain was mapped in the SAP domain of the nsp1β subunit. Further studies demonstrated that mutation in the SAP domain did not allow the interaction between nsp1β and Nup62 and consequently did not inhibit the host mRNA translation. A series of virus mutants was generated by reverse genetics. In SAP-mutant virus-infected cells, expressions of type I interferons (IFNs), IFN-stimulated genes (ISGs), IFN-induced proteins with tetratricopeptide repeats 1 and 2 (IFITs 1 and 2), and IFN regulatory factor 3 (IRF3) were decreased. The mutant PRRSV replicated at the lower efficiency that that of wild-type PRRSV but its replication was rescued to the level of wild-type PRRSV by nsp1β complementation. These results demonstrate a new strategy of PRRSV for host immune evasion and enhanced replication during infection. These results were presented at scientific conferences and published in journals. Specific Aim 2: Genetic manipulation of the PRRSV genome is technically difficult, but the reverse genetic system, that we developed in our laboratory, has made it possible. The infectious clone for PRRSV contains the full-length genomic cDNA copy of PRRSV in the plasmid backbone and thus is approximately 30 kb in length. We developed an extra-long inverse PCR technique, which allowed us to introduce specific mutations or deletions in any region of the viral genome. Using this reverse genetic system, a single amino acid at position 135 of the nsp1β protein was deleted from the virus. The deletion mutant virus was successfully generated and was genetically stable. This mutant virus was characterized for its replication and phenotypes for IFN suppression-negative and NF-kB activation-negative. Specific Aim 3: During PRRSV-infection, NF-kB is activated, which then stimulates the expression of proinflammatory cytokines. We cloned all 22 viral genes, and each gene product was examined individually for their ability for NF-kB activation. We found that nucleocapsid (N) protein was the NF-kB activator. The N protein was interactive with the cellular protein PIAS1 (protein inhibitor of activated STAT1) during infection, indicating that PAIS1 is a binding partner of N in the cell. PIAS1 functions as a repressor for p65 (a subunit of NF-kB) in the nucleus and suppresses p65 activation. We found that the binding of N to PIAS1 released p65 from NF-kB complex so that NF-kB becomes activated and triggers expression of proinflammatory cytokines. We mapped the binding region of N to PIAS1, and this region overlapped the nuclear localization signal (NLS) of N. The mutation of NLS abolished the NF-kB activity of N, demonstrating the positive correlation between N-PIAS1 binding and NF-kB activation. This study unveiled the role of N for NF-kB activation and thus proinflammatory cytokine expression. The NLS-knocked-out N protein was NF-kB activation-negative and was unable to express proinflammatory cytokines. Using the full-length infectious clone and extra-long inverse PCR techniques, NLS-deletion mutant PRRSV was successfully generated. This NLS-deletion mutant PRRSV was infectious and produced lower levels of proinflammatory cytokines than those of wild-type PRRSV. Specific Aim 4: We attempted to introduce double deletions to the PRRSV; a single amino acid deletion at position 135 in the nsp1β protein (as described in Specific Aim 2) and an NLS deletion in the N protein (as described in Specific Aim 3). We were able to generate a double-deletion mutant PRRSV. This virus was examined for IFN induction and NF-kB activation in cell culture. As anticipated, the viral phenotype was IFN-suppression-negative and NF-kB activation-negative. The titer of the double-deletion PRRSV mutant was 2 logs lower than that of wild-type PRRSV. Specific Aim 5: Examination of the mutant PRRSV for clinical attenuation in pigs during co-infection. This study will be conducted in year 4.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Yoo D., Kim H., Lee JY, & Yoo HS. 2020. African swine fever: Etiology, epidemiological status in Korea and perspectives on control. Journal of Veterinary Science. 21:e38. doi.org/10.4142/jvs.2020.21.e38
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ke, H., Lee, S., Kim, J., Liu, H.C., & Yoo, D. 2019. Interaction of PIAS1 with PRRS virus nucleocapsid protein mediates NF-kB activation and triggers proinflammatory mediators during viral infection. Scientific Reports 9, 11042.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, L., Lanka, S., Cassout, D., Mateus-Pinilla, Li, G., Wilson, W.C., Yoo, D., Shelton, P., & Fredrickson, R.L. 2019. Inter-serotype reassortment among epizootic hemorrhagic disease viruses in the United States. Transboundary Emergring Diseases. 66:1809-1820. doi:10.1111/tbed.13257.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Hou, Y., Ke, H., Kim, J., Yoo, D., Su, Y., Boley, P., Saif, L. J., & Wang, Q 2019. Engineering an effective and safe live attenuated PEDV vaccine candidate via inactivation of the viral 2-O methyltransferase and the endocytosis signal of the spike protein. J. Virol. 93: e00406-19. doi:10.1128/JVI.00406-19.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kim J. & Yoo D. 2019. Expression attenuation of proinflammatory cytokines by NF-?B activation and type I IFN suppression-negative PRRSV. North American PRRS Symposium. Chicago, IL USA. Nov 2-3.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Yoo, D., Ke, H., Lee, S., Kim, J., & Liu, H.C. 2019. Arterivirus nucleocapsid protein binds to protein inhibitor of activator STAT1 and triggers NF-kB activation to produce proinflammatory mediators during viral infection. 17th International Immunology Congress, Beijing, China. Oct 19-23.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Yoo, D., Han, M., Kim, J., Gustin, K.E., & Ke, H. 2019. Porcine reproductive and respiratory syndrome virus nsp1-beta protein interacts with nucleoporin 62 to promote viral replication and evasion from antiviral immunity. Asian Pig Veterinary Society Congress. Seoul, Korea, Aug 25-28
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Yoo, D., Hou, Y., Ke, H., Kim, Su, Y., Bopley P., Chepngeno, J., Vlasova, A., Saif, L., & Wang Q. 2019. Double-inactivation of nsp16 methyl transferase activity and S protein endocytosis signal increases innate immune response and confers complete protection from PEDV infection. Asian Pig Veterinary Society Congress. Seoul, Korea, Aug 25-28
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Viral immune evasion and a novel approach to vaccine developments. 2019. 3rd International Symposium on Immunology and Biotechnology, Seoul National University, Nov 2
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Porcine arterivirus and a novel design for vaccine candidates. 2019. International Joint Symposium of Biosafety & Korea Zoonosis Research Institutes and Jeonbuk National University. Iksan, Chollapuk-do. Korea, Dec 4
• Type: Websites Status: Published Year Published: 2019 Citation: Ke, H., Han, M., Kim, J., Kurt E. Gustin, & Yoo, D. 2019. Porcine reproductive and respiratory syndrome virus nsp1-beta interaction with the nucleoporin 62 facilitates virus replication in vitro. J. Virol. 93: e00469-19. This article was chosen as a cover image article of Vol 93, Issue 14, July 15, 2019, and published at the website. https://jvi.asm.org/content/93/14.cover-expansion

Progress 06/15/18 to 06/14/19

Outputs
Target Audience:The current project aims to understand the basis for viral modulation of host immunity and to investigate the mechanism for enhanced clinical disease during coinfection with a bacterial pathogen on farm. The primary target audiences of the project include researchers in virology, immunology, and vaccinology. University educators, pork producers, swine practitioners, and those in the veterinary phamaceutical industry will also gain benefits from our reserach. New information obtained from the project will be delivered to target audiencesvia oral presentations, poster presentations, and abstracts at various meetings and professional conferences. Manuscripts will be prepared and published in scientificjournals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the reporting period,two PhD levelgraduate students and one undergraduate were involved in thisproject. For PhD students, one was a 1st year student and another was in the 5th year of the program. The entry level students worked together with the senior graduate student and received hands-on training in the laboratory. Through the weekly lab meetings, the students learned how to present their research data and received comments for further experiments. The students participated in various scientificconferences at the national and international levels, and gained experience in communicating with other researchers. Students learned how to write and prepare manuscripts, and how to address reviewer comments and suggestions. The acceptance of a manuscriptprovides a major motivation to graduate students towards their research. During the reporting period, one graduate studentcompleted the program and received a PhD degree in May 2019. This student was able to publish four papers as the 1st-authorand three papers as a coauthor in 5 years on swine infectious diseases. After graduationin May 2019, this student moved to Harvard University to further his research as a post-doctoral fellow. How have the results been disseminated to communities of interest?The current research is based on the findings from the previous project, and thus, we have been very productive from the first year of the project. We have published a total of 4 articles in virology journals including the Journal of Virology, Virology, Virus Research, and the Journal of Virological Methods. These are the mainstream virology journals. In addition, we have attended various scientific conferences and presented a total of 9papers. The conferences include the99th Conference of Research Workers in Animal Diseases (CRWAD) in Chicago, 2018 International Pig Veterinary Society (IPVS)Congress/International PRRS Symposium in Chongqing China, and the 61st American Association of Veterinary Laboratory Diagnosticians (AAVLD)/122nd U.S. Animal Health Association (USAHA) Annual Meeting in Kansas City, KS.These are the meetings that researchers, educators, veterinarians, and commodity representatives gather together to exchange new information and scientific findings. A graduate student completed the PhD program during the reporting period, and received a PhDdegree. A thesis has been deposited to the library of the University of Illinois. The thesis research isavailableonline. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? PRRSstill remains one of the most economically significant swine diseases in the U.S. Commercial vaccines are available, but they are less satisfactory, and the disease continues to damage the U.S. pork industry. Many studies have demonstrated that PRRSV carries the ability to suppress the host innate immunity. As a result, virus-infected pigs are unable to effectively eliminate the invading virus, and PRRSV persists in infected animals. In response to virus infection, host cells produce a variety of cytokines and chemokines. One arm of the host responses is the type I interferons (IFNs) that are principle antiviral proteins. PRRSV is known to suppress IFN-signaling pathways to disarm the host immune system. Another arm of the host response is the production of proinflammatory cytokines. The production of proinflammatory cytokines recruits fresh macrophages to infection sites and in turn, PRRSV can infect fresh target cells for its replication. Once proinflammatory cytokines are elevated, it leads to a more severe clinical outcome when secondary infection occurs by other bacterial pathogens on farms. The current project aims to remove the two functions from PRRSV; IFN-mediated antiviral suppression function and NF-kB mediated proinflammatory cytokine production function. The newly generated PRRSV will increase the host immune responses during PRRS infection while reducing the clinical severity in pigs during coinfection with a secondary pathogen. During the reporting period, we have made good research progress. Specific Aim 1: We have shown that PRRSV blocks host mRNA nuclear export to the cytoplasm and have identified viral nsp1 is the protein disintegrating the nuclear pore complex in virus-infected cells. Subsequently, we have discovered that the cellular protein nucleoporin 62 (Nup62) binds to nsp1. The binding region in Nup62 is mapped to the C-terminal 328-522 residues. For the nsp1 protein, the binding region is mapped to the SAP domain within nsp1β. Mutational studies show that mutation in the SAP domain of nsp1β does not allow the binding of nsp1β to Nup62 and does not inhibit the host mRNA nuclear export. A mutant PRRSV has been generated by reverse genetics and this virus replicates at a slower rate with a lower titer than wild-type virus. In cells infected with PRRSV, the production of type I interferons, IFN-stimulated genes, IFN-induced proteins with tetratricopeptide repeats 1 and 2, and IFN regulatory factor 3 was decreased. As a consequence, the growth of the mutant PRRSV was rescued to the level of wild-type PRRSV. These findings are attributed to the Nup62 disintegration by PRRSV, which results in the increased viral protein production and decreased host protein production including antiviral proteins in the cytoplasm. Our study reveals a new strategy of PRRSV for immune evasion and enhanced replication during infection. Specific Aim 2: Our infectious cDNA clones for PRRSV are approximately 30 kb in size including the plasmid backbone, and it is technically difficult to introduce specific mutations. We have optimized the extra-long inverse PCR technology which allows us to introduce specific mutations in any region of the full-length cDNA clone. Using this technology, we have deleted a triplet codon for the active site amino acid from the nsp1β gene in the viral genome. This virus has successfully been rescued from the infectious clone and passaged three times in cell culture. We have made a stock virus as 'passage 4' virus, and this virus will be characterized for its phenotype for IFN suppression-negative, reversion-resistant infectious PRRSV. Specific Aim 3: PRRSV activates NF-kB during infection and upregulates proinflammatory cytokine productions. We have cloned all 22 viral protein-coding sequences and examined for NF-kB activation. We have found PRRSV N is the NF-kB activator. Subsequently, protein inhibitor of activated STAT1 (PIAS1) has been identified as the cellular protein binding to N. PIAS1 is known to bind to p65 in the nucleus and blocks its DNA binding, and thus functions as a repressor of NF-kB. Binding of N to PIAS1 releases p65 for NF-kB activation. We have mapped the binding region of N to PIAS1. Surprisingly, this region overlaps the nuclear localization signal (NLS) of N, and the mutation of NLS abolishes NF-kB activation, demonstrating the positive correlation between N-binding to PIAS1 and NF-kB activation. Our study reveals the role of N in the nucleus for NF-kB activation and proinflammatory cytokine production. The NLS-knockout N protein is NF-kB activation-negative. Using the full-length PRRSV infectious cDNA clones and the extra-long inverse PCR technique, NLS-deletion mutant cDNA clone has been constructed, and subsequently, progeny viruses have been successfully rescued from mutated clones by reverse genetics. This NLS-deletion mutant PRRSV is replicating and infectious. We have made a virus stock as 'passage 4', and this virus will be characterized for its phenotype for NF-kB activation and proinflammatory cytokines in cells. Specific Aim 4 will be conducted in the coming year. A double-mutant PRRSV will be constructed using NLS-deleted virus as a backbone. SAP mutation will be introduced in the NLS-deletion virus to knockout IFN suppression function by nsp1β. Thus, this virus will be negative for IFN suppression and NF-kB activation. Specific Aim 5: Examine clinical attenuation of the double-mutant PRRSV in pigs during co-infection. This study will be conducted in year 4.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Ke, H., Han, M., Zhang, Q., Rowland, R.R., Kerrigan, M. and Yoo, D. 2018. Type I interferon suppression-negative and host mRNA nuclear retention-negative mutation in nsp1-beta confers attenuation of porcine reproductive and respiratory syndrome virus in pigs. Virology 517: 177-187.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Hicks, J.A., Yoo, D. and Liu, H.C. 2018. Interaction of porcine reproductive and respiratory syndrome virus major envelope proteins GP5 and M with the cellular protein snapin. Virus Res. 249: 85-92.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Wang, L., Bowman, A., Lanka, S., Yoo, D., Zhang, Y., Li, G., Fredrickson, R. and Eggett, T.E. 2019. Development of a triplex real-time RT-PCR assay for detection and differentiation of three genotypes of porcine hemagglutinating encephalomyelitis virus. J. Virol. Methods 269: 13-17.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ke, H., Han, M., Kim, J., Kurt E. Gustin and Yoo, D. 2019. Porcine reproductive and respiratory syndrome virus nsp1-beta interaction with the nucleoporin 62 (Nup62) facilitates virus replication in vitro. J. Virol. 83: (On-line published on May 1). This article was chosen as a cover image article, Volume 93, Issue 14, July 15, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ke, H., Han, M., Zhang, Q., Rowland, R.R., Kerrigan M. and Yoo, D. 2018. A novel approach to PRRS vaccine development: IFN suppression and host mRNA nuclear retention-negative mutant virus confers clinical attenuation. 25th International Pig Veterinary Society Congress/International PRRS Symposium. Chongqing, China. June 10-15.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ke, H., Lee, S. and Yoo, D. 2018. PRRSV nucleocapsid protein binding to PIAS1 activates NF-kB for production of proinflammatory mediators. 25th International Pig Veterinary Society Congress/International PRRS Symposium. Chongqing, China. June 10-15.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zhang Q., Ke, H., Blikslager, A., Fujita, T. and Yoo, D. 2018. Restriction of type III interferon antiviral cytokines by PEDV and the role of nsp1. 25th International Pig Veterinary Society Congress/International PRRS Symposium. Chongqing, China. June 10-15.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zhang, Q., Ke, H. and Yoo, D. 2018. Regulation of type III interferons by PEDV is mediated through suppression of IRF1 and peroxisome biogenesis. 25th International Pig Veterinary Society Congress/International PRRS Symposium. Chongqing, China. June 10-15.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Ke, H. and Yoo, D. 2018. Interaction of PRRSV nsp1-beta and the cellular protein nucleoporin 62 inhibits host mRNA nuclear export and host cell gene expression. 25th International Pig Veterinary Society Congress/International PRRS Symposium. Chongqing, China. June 10-15.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Yoo, D. and Ke, H. 2018. PRRSV nsp1 binding to Nup62 and disintegration of nuclear pore complex downregulates host cell gene expression. 99th Conference of Research Workers in Animal Diseases. Chicago, IL. Dec 2-4.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Yoo, D., Ke, H., Han, M., Zhang, Q., Rowland, R.R. and Sheahan, M. 2018. Immune evasion of PRRS virus and a novel strategy for vaccine design. 99th Conference of Research Workers in Animal Diseases. Chicago, IL. Dec 2-4.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Ke, H. and Yoo, D. 2019. Nuclear localization signal of porcine reproductive and respiratory syndrome virus nucleocapsid (N) protein is essential for NF-kB activation. CVM Research Day. University of Illinois at Urbana-Champaign. Urbana, IL. April 26.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kim, J. and Yoo, D. 2019. Generation of type I interferon suppression-negative and NF-kB activation-negative PRRSV and characterization of their phenotypes in cells. CVM Research Day. University of Illinois at Urbana-Champaign. Urbana, IL. April 26.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wang, L., Eggett, T.E., Li, G., Zhang, Y., Lanka, S., Fredrickson, R.L, Yoo, D. and Bowman, A.S. 2018. Development of a triplex real-time RT-PCR assay for detection and differentiation of three genotypes of porcine hemagglutinating encephalomyelitis virus. 61st Am. Ass. Vet. Lab. Diagn/122nd USAHA Annual Meeting, Kansas City, KS. Oct 18-22.
• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Hicks, J.A., Yoo, D. and Liu, H.C. 2018. Identification of potential regulators of the porcine immunomodulatory microRNA, miR-146a. 25th International Pig Veterinary Society Congress/International PRRS Symposium. Chongqing, China. June 10-15.
• Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Hanzhong Ke. 2019. Innate immune modulation and pathogenic basis for porcine reproductive and respiratory syndrome virus. PhD Thesis, University of Illinois at Urbana-Champaign, Urbana, IL.