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United States Department of Agriculture

Agricultural Research Service

Research Project: Intervention Strategies to Control Viral Diseases of Swine
2012 Annual Report

1a.Objectives (from AD-416):
Objective 1: Identify mechanisms of porcine respiratory and reproductive syndrome virus (PRRSV) pathogenesis and develop vaccination strategies to enhance immunity and prevent the infection of swine herds. Subobjective 1.1: Identify PRRSV determinants of viral pathogenesis, immune evasion, and transmission. Subobjective 1.2: Use reverse genetic technology to design live attenuated vaccine strains that provide broad protection against PRRSV infection and allow differentiation of infected from vaccinated animals. Objective 2: Identify mechanisms of swine influenza virus (SIV) pathogenesis and develop vaccination strategies that provide broad cross-protection against subtypes of SIV that are emerging worldwide. Subobjective 2.1: Improve SIV vaccination and control programs through development of a repository of characterized emerging SIV subtypes and genotypes. Subobjective 2.2: Determine the role of the triple reassortant internal gene cassette (TRIG) or other genetic elements in the adaptation of novel reassortant influenza viruses to pigs. Subobjective 2.3: Discover novel influenza vaccine platforms that elicit cross-protection against emerging SIV subtypes. Objective 3: Identify viral factors contributing to porcine circovirus associated disease (PCVAD) complex and develop novel vaccination strategies to enhance the protection of swine herds. Subobjective 3.1: Identify unique endemic or novel swine viruses that are associated with PCVAD. Subobjective 3.2: Determine the role of these viruses, including PCV2, in inducing PCVAD. Subobjective 3.3: Discover novel vaccine platforms designed to improve control and prevention of PCVAD. Objective 4: Develop methods to modulate innate and adaptive immune responses to swine viral pathogens. Subobjective 4.1: Identify innate defense mechanisms associated with disease resistance to viral pathogens. Subobjective 4.2: Discover novel biotherapeutics or other intervention strategies to ameliorate the effects of diseases caused by priority viral diseases of swine.

1b.Approach (from AD-416):
The pathogenesis of disease caused by swine viral pathogens will be investigated in swine disease models to investigate methods of intervention. Animal experiments conducted involve one of four general designs:.
1)disease pathogenesis and transmission studies,.
2)vaccine efficacy studies,.
3)neonatal studies, and.
4)a gnotobiotic model in sterile-filtered pig isolators to study the effects of a single pathogen on the pig. Knowledge obtained will be applied to break the cycle of transmission of these swine pathogens through development of better vaccines or other novel intervention strategies. A major research approach will be the use of reverse engineering and infectious clones to identify virulence components of each virus under study through mutational studies. Development of vaccines that provide better cross-protective immunity than what is currently available with today’s vaccines will be approached through vaccine vector platform development, attenuated strains for vaccines and other novel technologies. A key approach in the study of disease pathogenesis is to better understand the host response to viral infection to various viruses. This research on comparative host transcriptomics will provide insights on viral pathogenesis and possible virulence factors that will enable rational design of more effective vaccines and target possible novel intervention strategies.

3.Progress Report:
In support of Objective 1, pathogenesis studies of Asian HP-PRRSV were compared with U.S. isolates and found 100% mortality in 4-week-old pigs and <50% mortality in 10-week-old pigs. To understand the severe disease we investigated gene expression profiles in lymphnodes after infection. We constructed chimeric viruses made up of nonstructural protein 2 (nsp2) from viruses with a range of virulence and differing substantially in nucleotide sequence to examine the role of nsp2 in PRRSV virulence. We constructed infectious clones of moderately (U.S.) and highly pathogenic (Asian) PRRSV clones with a deletion in nsp2 to assess virus attenuation for vaccine studies. We developed a cloning strategy to engineer small immunogenic tags into deleted regions of nsp2 using a licensed vaccine strain.

In support of Objective 2, we assessed susceptibility of pigs to selected influenza virus H9N2 subtype isolates. Although pigs were infected with H9N2 viruses only mild disease was observed but it supports the “mixing vessel” hypothesis demonstrating a need for ongoing swine influenza virus (SIV) surveillance. Human and swine H3N2 with either a 2009 pandemic matrix gene or a North American swine-lineage matrix gene were compared in pigs for virulence and transmission properties. Naturally occurring H1N1pdm09 and endemic SIV reassortant viruses were identified in U.S. swine, fully sequenced, and compared to human H3N2v viruses identified between July 2011-January 2012. A vaccine study was conducted to evaluate protection by a fully licensed commercial vaccine against a newly emerged H3N2 reassortant SIV. Selected human and swine isolates of H1N1 and H1N2 were used to immunize pigs to generate reference antisera for use in antigenic mapping of delta cluster U.S. swine H1 influenza viruses; this mapping will aid in understanding vaccine and diagnostic failures, and aid in vaccine strain selection. A subset of H1 viruses was subjected to whole genome sequencing and genetic analysis to complement the antigenic analysis. We compared monovalent and bivalent whole virus inactivated vaccines and duration between vaccination and challenge to investigate vaccine associated enhanced respiratory disease (VAERD) using inactivated influenza vaccines that contain a mismatched strain to the challenge virus. The role of the HA, NA, and NP in VAERD was investigated using reverse genetic engineered viruses.

In support of Objective 3 we used a two-dimensional chloroquine gel system to identify DNA replicative intermediates of porcine circovirus similar to geminiviruses. We also developed a method to use pyrosequencing to identify and clone novel single-stranded DNA swine viruses and to examine the virus flora present in healthy as well as diseased swine. Several novel swine viruses were identified and partial genomic clones obtained.

In support of Objective 4 we assessed the efficacy of a cytokine to enhance innate immunity to help combat secondary bacterial infections caused by HP-PRRSV infection. Although innate immunity was enhanced in treated pigs, the cytokine did not alter the infection but it produced unexpected findings providing insight into HP-PRRSV virulence.

1. Designed and developed novel PRRSV cDNA clones for vaccine testing. Porcine reproductive and respiratory syndrome virus (PRRSV) causes dramatic losses to the swine industry worldwide. The virus persists in its host for weeks to months thus showing it has mechanisms to evade host immune response. Currently available vaccines would be of limited usefulness in the event of a PRRSV eradication program and next generation vaccines with Differentiating Infected from Vaccinated Animals (DIVA) traits are needed to facilitate an eradication effort. ARS scientists at the National Animal Disease Center in Ames, Iowa designed and developed six unique PRRSV infectious cDNA clones which will enable the development of next generation vaccines with DIVA characteristics urgently needed by swine practitioners and producers.

2. Host gene responses in pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV), highly pathogenic PRRSV (HP-PRRSV), swine influenza virus (SIV), porcine circovirus type 2 (PCV2), or pseudorabies (PRV). PRRSV causes highly significant losses to the swine industry worldwide. The PRRSV negatively affects the pig's immune system, which explains in part why PRRS is difficult to control. To understand how the pig's immune response is being thwarted by PRRSV, ARS scientists at the National Animal Disease Center in Ames, Iowa completed a series of studies comparing how the immune system reacts to 4 different viral pathogens to find which genes get turned on and which ones get turned off (called the transcriptome) 2 weeks after infection. PRRSV reduces the diversity of host genes expressed following infection compared with SIV and PCV2. This "silencing" effect on the transcriptome has not previously been reported, and may allow the virus to persist in the pig longer than SIV or PCV2 and give PRRSV a greater chance to mutate and stay ahead of neutralizing antibody responses that eventually develop (albeit more slowly than SIV or PCV2). This is the first study of its kind and provides novel gene expression information for scientists to evaluate in their search for more effective vaccines.

3. Host gene responses studied in pigs infected with highly pathogenic strains of Asian porcine reproductive and respiratory syndrome virus (HP-PRRSV). The first direct comparison study between HP-PRRSV and contemporary U.S. PRRSV strains for their effects on the tracheobronchial lymph node transcriptome was completed. In 2006 a syndrome with very high morbidity and mortality was recognized in growing pigs in China that was called porcine high fever disease and was later shown to be caused by HP-PRRSV strains. In collaboration with an international team of scientists, ARS scientists at the National Animal Disease Center in Ames, Iowa demonstrated that these HP-PRRSV strains cause severe disease with extensive secondary bacterial infections and high mortality in the absence of foreign animal disease co-infections. Major changes in gene activation occurred in response to infection with both PRRSV strains, however the degree of up or down-regulation of genes following infection was much greater with the HP-PRRSV strain. These findings will aid scientists investigating virulence factors of PRRSV that can be manipulated to produce safer and more efficacious vaccines.

4. Vaccine-associated enhanced respiratory disease (VAERD) characterized in pigs. Influenza A virus causes a respiratory disease in pigs similar to that in humans. Inactivated influenza virus vaccine use in swine has increased over the past 10 years in an effort to prevent disease and transmission of the virus. Inactivated vaccines work well when pigs are exposed to influenza viruses used in the vaccine, however vaccine efficacy is reduced when pigs are infected with new strains. ARS scientists at the National Animal Disease Center in Ames, Iowa found that pigs administered an inactivated swine influenza A vaccine followed by infection with the pandemic human influenza A virus (2009) demonstrated more severe disease compared to non-vaccinated pigs infected with the same virus. Pigs with VAERD demonstrated greater percentages of affected lungs compared to controls, the microscopic damage was more severe with distinct lesions, and had elevated immune factors associated with inflammation and disease in the lungs. Active surveillance and monitoring of the quality of match between vaccine strains and strains infecting swine herds is needed to prevent vaccine mismatch and VAERD in commercial swine. Future vaccines that stimulate improved immune responses for differing influenza viruses will be important to prevent infection and clinical disease in commercial swine production, as well as potential virus transmission to humans.

5. Viral metagenomics method discovers novel viruses in pig intestines. Deep sequencing and metagenomics analyses methods were developed to characterize the virome of healthy and diseased pigs. Traditional diagnostic methods used to detect pathogens are dependent on knowing possible pathogens because diagnostic tests are specific for known pathogens. Use of genetic sequence-independent viral metagenomics methods avoids many potential limitations of traditional methods and can detect uncharacterized viruses or multiviral infections. ARS scientists at the National Animal Disease Center in Ames, Iowa in collaboration with scientists at the University of California at San Francisco, established procedures with fecal samples from a group of pigs, that allows rapid discovery and identification of existing or newly emerging viruses. A surprising number and spectrum of viruses were detected that covered 7 different virus families, and there were more RNA viruses than DNA viruses detected. This technology enables detection of viruses that might not otherwise be detected by routine diagnostic methods and such capacity will lead to significant advances in pathogen discovery, full genome sequencing, transcriptomics, metagenomics, and microbial ecology.

6. Novel A(H3N2) influenza viruses characterized in swine. Influenza (flu) A viruses occasionally spread between pigs and humans, and causes a respiratory disease in swine similar to that in humans. The best known example of this is the 2009 pandemic virus (pH1N1) that was generated from 2 different swine viruses and gained the ability to infect people, and then was spread by people to pigs around the world. Between July and December 2011, a new virus was detected in 12 humans in the United States. This virus contained genetic material from swine flu viruses as well as the pH1N1 and has been called A(H3N2)v by the World Health Organization. ARS scientists at the National Animal Disease Center in Ames, Iowa compared the properties of H3N2 viruses isolated from U.S. human and swine that inherited genes from typical swine flu viruses and the 2009 pH1N1 virus with different combinations to determine the ability of these viruses to spread in the swine population. Our results suggest that although certain combinations of gene segments may be required for the viruses to survive in pigs, all 3 viruses tested were able to infect and spread among pigs. The A(H3N2)v from a human did not have any increased ability to infect, spread, or cause disease in pigs. This new information helps us to understand how these viruses are changing and to detect when viruses are shared between pigs and people.

7. Genetic evolution of novel reassortant swine influenza A viruses. Influenza A virus causes a respiratory disease in swine similar to that in humans. In collaboration with NIH scientists, ARS scientists at the National Animal Disease Center in Ames, Iowa investigated genetic evolution of novel reassortant swine influenza A viruses detected in the United States and Canada between 2009-2011 with a focus on H3N2 viruses. Analyses included A(H3N2)v viruses that infected humans in the United States since July 2011. The A(H3N2)v is distinct from contemporary H3N2 circulating in humans and the human vaccine, and hence represents a potential pandemic threat. Monitoring and reporting evolutionary dynamics of gene segments in swine at a detailed level is required to understand how these novel H3N2 viruses emerged in swine and to assess the potential epidemic and/or pandemic threat posed to humans.

8. Validation of a pseudorabies diagnostic test. A critical need for the current pseudorabies virus (PRV) surveillance program is the rapid detection of PRV and a diagnostic assay for use in feral and domestic swine to detect an acute pseudorabies virus (PRV) infection was evaluated. Pseudorabies is a viral disease in swine that is endemic in most parts of the world but was eradicated from the United States commercial swine population in 2004; however, pseudorabies remains at significant levels in the large and increasing feral pig population and represents a vulnerability to our PRV control and eradication programs. For this reason, ARS scientists at the National Animal Disease Center in Ames, Iowa in collaboration with Animal Plant and Health Inspection Service scientists at the National Veterinary Services Laboratory in Ames, Iowa conducted a series of studies to develop and validate a real-time polymerase chain reaction (PCR) assay for its capability to detect and differentiate field and vaccine strains of PRV in tissues of experimentally infected domestic swine. Diagnostic performance of the real-time PCR assay indicates that it is a sensitive, rapid and accurate assay that can provide reliable results in early stages of an infection before antibodies develop. These findings will benefit diagnostic laboratories and regulatory agencies tasked with testing and eradicating PRV from various populations of swine around the world.

Review Publications
Butler, J.E., Sun, X., Wertz, N., Lager, K.M., Chaloner, L., Urban Jr., J., Francis, D.L., Nara, P.L., Tobin, G.J. 2011. Antibody repertoire development in fetal and neonatal piglets XXI. Usage of most VH genes remains constant during fetal and postnatal development. Molecular Immunology. 49(3):483-494.

Cheung, A.K. 2012. Porcine circovirus: transcription and DNA replication. Virus Research. 164(1-2):46-53.

Ciacci Zanella, J.R., Vincent, A.L., Zanella, E.L., Lorusso, A., Loving, C.L., Brockmeier, S.L., Gauger, P.C., Janke, B.H., Gramer, M.R. 2012. Comparison of human-like H1 (delta-cluster) influenza A viruses in the swine host. Influenza Research and Treatment. Available:

de Groot, R.J., Cowley, J.A, Enjuanes, L., Faaberg, K.S., Perlman, S., Rottier, P.J.M., Snijder, E.J., Ziebuhr, J. and Gorbalenya, A.E. 2011. Order Nidovirales. In: King, A.M.Q., Adams, M.J., Carstens, E.B., Lefkowitz, E.J., editors. Virus Taxonomy, Ninth Report of the International Committee on Taxonomy of Viruses. London: Elsevier Inc. p. 785-795.

Faaberg, K.S., Balasuriya, U.B., Brinton, M.A., Gorbalenya, A.E., Leung, F.C-C., Nauwynck, H., Snijder, E.J., Stadejek, T., Yang, H. and Yoo, D. 2011. Equine arteritis virus. In: King, A.M.Q., Adams, M.J., Carstens, E.B. Carstens, Lefkowitz, E.J., editors. Virus Taxonomy, Ninth Report of the International Committee on Taxonomy of Viruses. London: Elsevier Inc. p. 796-805.

Faaberg, K.S. 2007. Arterivirus structural proteins and assembly. In: Perlman, S., Gallaher , T., and Snjder, E., editors. The Nidoviruses. Washington, DC: ASM Press. p. 211-234.

Gauger, P.C., Faaberg, K.S., Guo, B., Kappes, M.A., Opriessnig, T. 2012. Genetic and phenotypic characterization of a 2006 United States porcine reproductive and respiratory virus isolate associated with high morbidity and mortality in the field. Virus Research. 163(1):98-107.

Gauger, P.C., Lager, K.M., Vincent, A.L., Opriessnig, T., Cheung, A.K., Butler, J.E., Kehrli, Jr., M.E. 2011. Leukogram abnormalities in gnotobiotic pigs infected with porcine circovirus type 2. Veterinary Microbiology. 154(1-2):185-190.

Gauger, P.C., Lager, K.M., Vincent, A.L., Opriessnig, T., Kehrli, Jr., M.E., Cheung, A.K. 2011. Postweaning multisystemic wasting syndrome produced in gnotobiotic pigs following exposure to various amounts of porcine circovirus type 2a or type 2b. Veterinary Microbiology. 153(3-4):229-239.

Gorres, J.P., Lager, K.M., Kong, W.P., Royals, M., Todd, J.P., Vincent, A.L., Wei, C.J., Loving, C.L., Zanella, E.L., Janke, B., Kehrli, Jr., M.E., Nabel, G.J., Rao, S.S. 2011. DNA vaccination elicits protective immune responses against pandemic and classic swine influenza viruses in pigs. Clinical and Vaccine Immunology. 18(11):1987-1995.

Guo, B., Vorwald, A.C., Alt, D.P., Lager, K.M., Bayles, D.O., Faaberg, K.S. 2011. Large scale parallel pyrosequencing technology: PRRSV strain VR-2332 nsp2 deletion mutant stability in swine. Virus Research. 161(2):162-169.

Killian, M.L., Swenson, S.L., Vincent, A.L., Landgraf, J.G., Shu, B., Lindstrom, S., Xu, X., Klimov, A., Zhang, Y., Bowman, A.S. 2013. Simultaneous infection of pigs and people with triple-reassortant swine influenza virus H1N1 at a U.S. county fair. Zoonoses and Public Health. 60(3):196-201.

Kitikoon, P., Vincent, A.L., Gauger, P.C., Schlink, S.N., Bayles, D.O., Gramer, M.R., Darnell, D., Webby, R.J., Lager, K.M., Swenson, S.L., Klimov, A. 2012. Pathogenicity and transmission in pigs of the novel A(H3N2)v influenza virus isolated from humans and characterization of swine H3N2 viruses isolated in 2010-2011. Journal of Virology. 86(12):6804-6814.

Ma, W., Lager, K.M., Li, X., Janke, B.H., Mosier, D.A., Painter, L.E., Ulery, E.S., Ma, J., Lekcharoensuk, P., Webby, R.J., Richt, J.A. 2011. Pathogenicity of swine influenza viruses possessing an avian or swine-origin PB2 polymerase gene evaluated in mouse and pig models. Virology. 410(1):1-6.

Opriessnig, T., Gauger, P.C., Faaberg, K.S., Shen, H., Beach, N.M., Meng, X., Wang, C., Halbur, P.G. 2012. Effect of porcine circovirus type 2a or 2b on infection kinetics and pathogenicity of two genetically divergent strains of porcine reproductive and respiratory syndrome virus in the conventional pig model. Veterinary Microbiology. 158(1-2):69-81.

Schaefer, R., Zanella, J.R.C., Brentano, L., Vincent, A.L., Ritterbusch, G.A., Silveira, S., Caron, L., Mores, N. 2011. Isolation and characterization of pandemic H1N1 influenza viruses in pigs in Brazil. Pesquisa Veterinaria Brasileira. 31(9):761-767.

Sinha, A., Schalk, S., Lager, K.M., Wang, C., Opriessnig, T. 2012. Singular PCV2a or PCV2b infection results in apoptosis of hepatocytes in clinically affected gnotobiotic pigs. Research in Veterinary Science. 92(1):151-156.

Thontiravong, A., Tantilertcharoen, R., Tuanudom, R., Sreta, D., Thanawongnuwech, R., Amonsin, A., Oraveerakul, K., Kitikoon, P. 2011. Single-step multiplex reverse transcription-polymerase chain reaction assay for detection and differentiation of the 2009 (H1N1) influenza A virus pandemic in Thai swine populations. Journal of Veterinary Diagnostic Investigation. 23(5):1017-1021.

Zanella, E.L., Miller, L.C., Lager, K.M., Bigelow, T.T. 2012. Evaluation of a real-time polymerase chain reaction assay for pseudorabies virus surveillance purposes. Journal of Veterinary Diagnostic Investigation. 24(4):739-745.

Lu, Z., Zhang, J., Huang, C.M., Go, Y.Y., Faaberg, K.S., Rowland, R.R., Timoney, P.J., Balasuriya, U.B. 2012. Chimeric viruses containing the N-terminal ectodomains of GP5 and M proteins of porcine reproductive and respiratory syndrome virus do not change the cellular tropism of equine arteritis virus. Virology. 432(1):99-109.

Nan, Y., Wang, R., Shen, M., Faaberg, K.S., Samal, S.K., Zhang, Y.J. 2012. Induction of type I interferons by a novel porcine reproductive and respiratory syndrome virus isolate. Virology. 432(2):261-270.

Nelson, M.I., Vincent, A.L., Kitikoon, P., Holmes, E.C., Gramer, M.R. 2012. The evolution of novel reassortant A/H3N2 influenza viruses in North American swine and humans, 2009-2011. Journal of Virology. 86(16):8872-8878.

Sun, X., Wertz, N., Lager, K., Sinkora, M., Stepanova, K., Tobin, Butler, J.E. 2012. Antibody repertoire development in fetal and neonatal piglets. XXII. Lambda rearrangement precedes kappa rearrangement during B-cell lymphogenesis in swine. Immunology. 137(2):149-159.

Sun, X.Z., Wertz, N., Lager, K.M., Tobin, G., Butler, J.E. 2012. Antibody repertoire development in fetal and neonatal piglets. XXIII: fetal piglets infected with a vaccine strain of PRRS virus display the same immune dysregulation seen in isolator piglets. Vaccine. 30(24):3646-3652.

Last Modified: 4/18/2014
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