MECHANISMS OF CELLULAR RESISTANCE TO VIRAL INFECTION

• Sponsoring Institution: State Agricultural Experiment Station
• Project Status: TERMINATED
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 1003993
• Project No.: MIN-63-031
• Project Start Date: Aug 8, 2014
• Project End Date: Jun 30, 2016

Project Director
Murtaugh, MI, P.

Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108

Performing Department
Veterinary Biomedical Sciences

Non Technical Summary
Porcine reproductive and respiratory syndrome virus (PRRSV) causes a severe, economically devastating disease in MN and US swine herds. Vaccination strategies often fail to prevent disease and biosecurity with air filtration is enormously expensive and fails occasionally. Improved methods are difficult to develop because a basic understanding of the host-pathogen interaction remains unknown. Discovery and characterization of viral restriction factors, proteins that suppress (restrict) viral growth in permissive cells, has provided novel insights and paths to preventive possibilities, in various examples, especially HIV infection of humans. We discovered age-related restriction of PRRSV growth in porcine lung macrophages, suggesting an age-dependent expression of a viral restriction factor. We hypothesize that porcine macrophage expression of an anti-PRRSV restriction factor is a cellular mechanisms of resistance to viral infection, and that age-dependent expression accounts for the observed reduction of PRRSV growth in adult pigs. We propose to identify all of the proteins produced in infected and uninfected macrophages from young and old pigs and compare to find anti-viral restriction factors in old pigs. The complete set of proteins will be identified by sequencing total cellular RNA and bioinformatic analysis. Future aims will focus on functional tests of restriction factor activities identified here. Unbiased, reproducible screening by comprehensive analysis of gene expression is a novel discovery approach to screen for viral restriction factors in food animal species. The results will be highly significant since restriction factors frequently are broadly active on other viruses, and provide new insights into host-pathogen interactions. The proposed studies are directly relevant to US animal agriculture and will provide the key investigator, with preliminary data for development of a nationally competitive, independent research program.

Animal Health Component

100%

Research Effort Categories

Basic

90%

Applied

10%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3510	1090	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1090 - Immunology;

Keywords

swine

prrs

immunology

macrophage

pcr

sequencing

protein

virus

viral disease

Goals / Objectives
The major goal is to gain a better fundamental understanding of the interaction of Porcine reproductive and respiratory syndrome virus (PRRSV) and its host, the pig, so that better tools can be developed for control and prevention of PRRSV infection and disease. The objective of the proposed research is to discover and characterize viral restriction factors, i.e. proteins that suppress (restrict) viral growth in permissive cells, which appear to be expressed in an age-dependent manner in adult swine. The objective will be carried out through experimental infection studies using lung macrophages from young and old pigs.

Project Methods
Lung macrophage cells, which are the standard type of natural host cell used to study PRRSV growth, will be isolated from young and old pigs and incubated in the laboratory with and without PRRSV.The results of the incubation will be evaluated by PCR to determine the extent of viral infection, and cellular RNA will be isolated for high throughput sequencing to identify all of the genes that are expressed in the presence and absence of viral infection in young and old macrophages. A difference analysis will be carried out to screen for restriction factors that prevent or reduce viral growth in aged macrophages.Evaluation of progress and success will be accomplished through milestones marking completion of macrophage collection from young and old pigs, completion of cell culture experiments, completion of viral growth determinations, completion of high throughput sequencing, completion of data analysis, and completion of report preparation.

Progress 08/08/14 to 06/30/16

Outputs
Target Audience:The target audience is scientists, animal health experts including swine veterinarians, production animal specialists including swine producers, and consumers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided training and professional development for undergraduate students at the University of Minnesota Morris andColorado State University, a Brazilian exchange undergraduate student at the University of Wisconsin, River Falls, a high school student from Wayzata High School, Wayzata, Minnesota, and a professional development sabbatical experience by a Professor of Chemistry at St. Thomas University, St. Paul MN. How have the results been disseminated to communities of interest?Publications in the scientific literature, reports and presentations at stakeholder meetings and events, and in communications with relevant commodity groups and end users. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The impact of the project accomplishments has been to revolutionize our understanding of mechanisms of anti-PRRSV immunity in swine and to help explain age-dependent differences in resistance and susceptibility to viral disease. The findings help to explain some of the variation in vaccine efficacy when used in the field by showing that PRRSV grows better in young animals than in older adults. Because the strength of an immune response is related to the amount of virus that is produced, the reduced growth of PRRSV in older animals results in some of the animals being less well protected than other animals. The results also show that there is a strong anti-viral response in infected pigs, which is contrary to numerous publications using artificial systems that do not accurately reproduce the true environment that PRRSV encounters in the pig, its natural host. The information revealed here will help guide future vaccine development. The impacts were arrived at by working with the same cells that the virus infects in the pig, and by obtaining the pigs from commercial sources. The significance of these steps is that the findings are directly relevant to PRRSV problems in the field. There is no need for a leap of faith that is required when studies use non-natural cells or artificial systems in which PRRSV genes are manipulated in ways that are not based on using PRRSV isolates that are obtained in the field. Major activities included establishment of a large bank of alveolar macrophage preparations from individual pigs of various ages from neonates to aged adults, experimental expression analysis of surface markers CD163 and CD169 on macrophages from young and old pigs, growth characteristics of PRRSV on same said cells, and gene expression profiles of infected and uninfected alveolar macrophages from young and old pigs. Results showed that there was a huge difference in rate of viral infection with young pig macrophages being relatively highly susceptible compared to adult pig macrophages even though there was no difference at all in cell surface expression of CD163 and CD169. Since these markers are involved in PRRSV infection, it meant that whatever difference existed in infection differences must have been due to the intracellular milieu. Gene expression profiling showed that old macrophages were distinctly different from young macrophages and that there was a large and robust type 1 interferon response to infection in young and old macrophages. The significance of this observation is that it contradicts the common perception of immune evasion by PRRSV. It simply is not true, and it means that the overall effect of virus infection on pig health needs to consider that immune response capabilities are intact.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Robinson SR, Li J, Nelson EA, Murtaugh MP. 2015. Broadly neutralizing antibodies against the rapidly evolving porcine reproductive and respiratory syndrome virus. Virus Res. 203:56-65.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Loving C, Osorio FA, Murtaugh MP, Zuckermann FA. 2015. Innate and adaptive immunity against porcine reproductive and respiratory syndrome virus. Vet. Immunol. Immunopath. 167:1-14.
• Type: Theses/Dissertations Status: Published Year Published: 2015 Citation: Robinson SR. 2015. Mechanisms of immune protection against porcine reproductive and respiratory syndrome virus (PRRSV). PhD dissertation, UMN.
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Rahe M, Murtaugh MP. 2016. Mechanisms of humoral immunity to porcine reproductive and respiratory syndrome virus. Inter. J. Immunol. Immunother.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Swine producers, swine veterinarians, and swine health and disease researchers, nationally and internationally. Changes/Problems:Major problems in the previous reporting period were overcome. What opportunities for training and professional development has the project provided?PhD graduate students Sally Robinson, DVM, and Michael Rahe, DVM, and Xiong Wang received training and professional development opportunities in viral immunology, moleular virology, and infectious disease resistance; Qinye Song, PhD, visiting scientist from Hebei Agricultural University, China, gained experience in swine immunology and virology; and researchassociate Cheryl Dvorak, PhD, in project administration, personnel supervision and mentoring, scientific laboratory management, and program administration. How have the results been disseminated to communities of interest?Participation in the Allen D. Leman Swine Conference is a central avenue of dissemination as it provides direct interaction with the key communities of interest. Additional methods include consultation with swine veterinarians and producers on disease problems in the field, presentation at national and international meetings to scientists and veterinarians, and publication in esteemed scientific journals. What do you plan to do during the next reporting period to accomplish the goals?Key experiments were repeated with mycoplasma-free PRRSV and data are being analyzed. Manuscript and thesispreparation is in progress.

Impacts
What was accomplished under these goals? Large-scale isolation of macrophages was accomplished from neonatal, weaned, and adult swine from peripheral lung lavages. Macrophage cultures were established for each age of the donor swine, and infection conditions for type 1 and type 2 PRRSV were optimized. Infection experiments were carried out and infection levels, CD163 and CD169 expression levels, and virus production levels were determined. RNA was collected and submitted for RNAseq analysis. Pathogen screening was performed and mycoplasma contamination of PRRSV stocks was detected. Mycoplasma-free stocks were re-established from type 1 and type 2 PRRSV. RNA Seq experiments were performed and data are being analyzed.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Li, J. and Murtaugh, M.P. 2015. Functional analysis of porcine reproductive and respiratory syndrome virus N-glycans in infection of permissive cells. Virology. 477:82-88.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Li, J., S. Tao, R. Orlando, and M.P. Murtaugh. 2015. N-glycosylation profiling of porcine reproductive and respiratory syndrome virus envelope glycoprotein 5. Virology 478:86-98.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Robinson, S.R., J. Li, E.A. Nelson, and M.P. Murtaugh. 2015. Broadly neutralizing antibodies against the rapidly evolving porcine reproductive and respiratory syndrome virus. Virus Res. 203:56-65.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Wang, X., D. Marthaler, A. Rovira, S. Rossow, and M.P. Murtaugh. 2015. Emergence of a virulent porcine reproductive and respiratory syndrome virus in vaccinated herds in the United States. Virus Res. 210:34-41.

Progress 08/08/14 to 09/30/14

Outputs
Target Audience: Swine producers, swine veterinarians, and swine health and disease researchers, nationally and internationally. Changes/Problems: It was discovered that PRRSV stocks used in the infection assays were contaminated with laboratory varieties of mycoplasma. We are determining if key experiments need to be repeated, which will delay slightly completion of the study. What opportunities for training and professional development has the project provided? PhD graduate students Sally Robinson, DVM, and Michael Rahe, DVM, and Xiong Wang received training and professional development opportunities in viral immunology, moleular virology, and infectious disease resistance; Qinye Song, PhD, visiting scientist from Hebei Agricultural University, China, gained experience in swine immunology and virology; and research associate Cheryl Dvorak, PhD, in project administration, personnel supervision and mentoring, scientific laboratory management, and program administration. How have the results been disseminated to communities of interest? Participation in the Allen D. Leman Swine Conference is a central avenue of dissemination as it provides direct interaction with the key communities of interest. Additional methods include consultation with swine veterinarians and producers on disease problems in the field. What do you plan to do during the next reporting period to accomplish the goals? Data analysis of the RNAseq experiment is underway. Key experiments may be repeated with mycoplasma-free PRRSV. Manuscript preparation is in progress.

Impacts
What was accomplished under these goals? Large-scale isolation of macrophages was accomplished from neonatal, weaned, and adult swine from peripheral lung lavages. Macrophage cultures were established for each age of the donor swine, and infection conditions for type 1 and type 2 PRRSV were optimized. Infection experiments were carried out and infection levels, CD163 and CD169 expression levels, and virus production levels were determined. RNA was collected and submitted for RNAseq analysis. Pathogen screening was performed and mycoplasma contamination of PRRSV stocks was detected. Mycoplasma-free stocks were re-established from type 1 and type 2 PRRSV.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2015 Citation: Robinson SR, Li J, Nelson EA, Murtaugh MP. 2015. Broadly neutralizing antibodies against the rapidly evolving Porcine reproductive and respiratory syndrome virus. Virus Res.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Murtaugh M, Rahe M, Robinson S, Taylor J, Jenkins M. 2014. Identification of PRRSV cross-neutralizing memory B cells. J. Immunol. 192:208.14.