EVALUATION OF EMERGENCY VACCINATION TO STOP THE SPREAD OF PRRS VIRUS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1002307
• Project Start Date: Jan 15, 2014
• Project End Date: Sep 30, 2014
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801

Performing Department
Veterinary Research & Extension

Non Technical Summary
The most recent estimate of the economic impact of a porcine reproductive and respiratory syndrome (PRRS) to the pork industry is $650 million dollar per year. Although not representative of the whole industry, the available data indicates that for the last 4 years the annual cumulative incidence of new PRRS infections in sow farms during the PRRS season is approximately 40-50%. The current intervention strategies that have been tried to control PRRS outbreaks are not adequate to deal with this costly disease. In this project we will examine the ability of a novel live virus vaccine, which has a unique ability to stimulate the innate immune system, to provide rapid protection against a virulent PRRS virus challenge. Given the high incidence of new PRRS outbreaks, the development of effective intervention strategies to control a PRRS virus outbreak and minimize its impact is a high priority and could have significant impact in efforts to control this disease.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3510	1101	50%
311	3510	1090	50%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1090 - Immunology; 1101 - Virology;

Keywords

prrs virus

vaccination

immunity

innate immunity

Goals / Objectives
Project Objective: To determine the efficacy of the G16X vaccine to provide rapid protective immunity from a heterologous PRRS virus challenge and decrease its spread.

Project Methods
To test the ability of the G16X virus to protect rapid protective immunity we will use thirty 6-week-old SPF pigs of similar body weight (±3 lb.) (free of all major swine pathogens including PRRS virus, influenza virus mycoplasma and circovirus) which will be randomly allocated into 5 groups and housed in isolation cubicles (3 pigs per cubicle) at two suites in the Bio-containment Facility at the University of Illinois. After a seven-day acclimation period, animals in groups 3, 4 and 5, will be vaccinated with the G16X virus either 7, 4 or 1 day prior to challenge, respectively. The G16X vaccine will be given intramuscularly in the rump area at a dose of 2 x 104 TCID50 dose in a 2 ml volume. All of the animals in groups 2, 3, 4 and 5 will be challenged with an intranasal aerosol of the virulent field virus isolate NADC20 (2 x 105 TCID50 in a 2 ml volume) using a MAD 300 device (Wolfe Tory Medical, USA). Animals in group 2 will be injected with 2 ml of spent culture supernatant diluted with PBS to the same amount as the vaccine. The unvaccinated and challenged animals assigned to group 2 will serve to establish the severity of clinical syndrome resulting from the infection by the virulent NADC-20 PRRS virus, while the animals allocated to group 1 will be used to provide the normal parameters of growth and health. All of the methods and procedures are well established in our laboratory and have been performed successfully numerous times (Calzada-Nova, et al., 2012). To monitor the spread of the virus, 2 sentinel pigs will be moved one day after challenge to each of the pens housing virus-challenged pigs. The level of viremia will be monitored at 4, 7, 10 and 14 days after challenge. The animals will be monitored for clinical signs (morbidity) for a total of 14 days. Clinical monitoring will include weight gain, rectal temperature and clinical symptoms as previously described (Calzada-Nova et al., 2012). The IFN-alpha levels in the serum will be measured by ELISA from serum samples collected at 1, 4, 10 and 14 days after virulent virus challenge.

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

Outputs
Target Audience: The target audience consists of scientists involved in investigating the mechanisms of virus virulence and vaccine development. Additionally, swine veterinarians and pork producers would be interested in having a better understanding of the methods to stimulate protective immunity against porcine reproductive and respiratory syndrome (PRRS) virus. Changes/Problems: The initial goal of the project was the to determine the efficacy of the G16X vaccine to provide rapid protective immunity from a heterologous PRRS virus challenge and decrease its spread. During the interim period between the submission of the proposal for funding and the decision for it to be funded, it became clear that it was very unlikely that the proposed experimental approach as described was going to yield the intended outcome. This was because we came to the realization that for our proposed approach to work, it required that the vaccine virus being used to perform the emergency vaccination should have to be able to stimulate a strong IFN-alpha response in alveolar macrophages. The G16X vaccine is able to stimulate effective protective immunity provided that a period of three to fourweeks before virulent virus challenge is allowed. Since the G16X virus strain is only able to stimulate a weak IFN-alpha response in alveolar macrophages, administration of the G16X vaccine virus within days before or after virus challenge was unlikely to provide protective immunity in an emergency situation as we had proposed. Our way of thinking changed because previously our studies were focused on understanding the mechanisms by which PRRS virus inhibits an IFN-alpha response in macrophages. However, our more recent studies led us to the realization that it was the lack of stimulation of IFN-alpha rather an active mechanism of type I IFN signaling suppression that was most likely responsible for the immune evasion strategy utilized by PRRS virus to defeat the immune system of its host. For this reason it was decided to change the focus of our research so that we could examine the mechanism by which PRRS virus evades the immune response of its host cell by not stimulating IFN-alpha production. Since we had a very unique set of tools to address this issue, namely the 1198 PRRS virus strain with the ability to stimulate a strong IFN-alpha response in porcine alveolar macrophages as well as the NSP2 revertant 1198B (which has lost the ability to stimulate IFN-alpha) we decided that the best use of the funding provided to us was to examine this issue. The outcome of the project was the discovery of a unique mechanism of immune evasion that had not been previously contemplated. A manuscript describing the results of this project is being prepared. We anticipate that our findings will lead to strategies that enable the stimulation of protective immunity against PRRS virus in emergency as well as non-emergency situations. What opportunities for training and professional development has the project provided? The graduate student and other personnel involved in this project acquired knowledge and experience on methods to measureand assess the innate immune response of porcine macrohages against a virus. How have the results been disseminated to communities of interest? The data is being prepared as a manuscript for publication in a referred journal. 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 most recent estimate of the economic impact of porcine reproductive and respiratory syndrome (PRRS) to the pork industry is $650 million dollar per year. Although not representative of the whole industry, the available data indicates that for the last 4 years the annual cumulative incidence of new PRRS infections in sow farms during the PRRS season is approximately 40-50%. None of the intervention strategies that have been tried to control PRRS outbreaks are adequate to deal with this costly disease. A likely reason for this situation is that PRRS virus posses unique properties that allow it to evade the immune system. A major feature exhibited by PRRS virus is the lack of stimulation of interferon (IFN)-alpha production. Thus, the aim of this project was to determine the mechanism behind the failure of PRRS virus to stimulate the production of IFN-alpha of porcine alveolar macrophages. We examined the ability of several wild-type PRRS virus isolates to stimulate porcine alveolar macrophages to secrete IFN-alpha. The host cell used in this study consisted of the porcine alveolar macrophage cell line ZMAC. The ZMAC cells are readily infected by PRRS virus and are fully functional with regard to the type I IFN signaling pathway, as evidenced by the fact that they produce large amounts of IFN-alpha in response to their stimulation with synthetic double stranded RNA, namely polyinosine-polycytidylic acid (polyI:C). PolyI:C is a well known toll-like receptor 3 (TLR3) agonist that stimulates IFN-alpha production in myeloid and non-myeloid cells. While all of the wild-type PRRS virus tested failed to stimulate IFN-alpha production, a laboratory strain mutant termed 1198 exhibited the ability to stimulate copious amounts of IFN-alpha. Using a comparative approach, our experiments were focused on understanding the molecular basis of the IFN-alpha-stimulatory phenotype of the 1198 strain and the lack of stimulation by the wild-type PRRS virus strains as well as two other laboratory strains that are nearly isogenic to 1198. In these studies the 1198 strain proved to further distinguish itself from all other PRRS virus strains in that, unlike all other strains tested, it stimulated the phosphorylation of IRF3 and induced type I IFN gene transcription. Extensive analyses revealed wild-type PRRS virus exhibits a novel mechanism of immune evasion that is missing in the 1198 strain. Using the PRRS virus strains that are nearly isogenic to the 1198 strain, we were able to determine that the mechanism of immune evasion was attributable to a unique mutation in the non-structural protein 2 (NSP2). The role of the NSP2 mutation in the IFN-alpha stimulatory phenotype was confirmed by a revertant mutant of the 1198 strain, mutant 1198B, which had lost a unique amino acid change in NSP2. A manuscript describing this novel mechanism of immune evasion is being prepared.

Publications