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United States Department of Agriculture

Agricultural Research Service

Research Project: Intervention Strategies to Control Viral Diseases of Swine
2013 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, Asian highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) was compared in swine to several U.S. strains. A germ-free study was done to evaluate HP-PRRSV in the absence of bacteria. Severe disease was induced, indicating a primary role for the virus. We are rescuing viruses with chimeric nonstructural protein 2 (nsp2) to examine its role in virulence. We are also rescuing virus of a moderate strain and HP-PRRSV with a nsp2 deletion to assess virus attenuation. In support of Objective 2, we assessed susceptibility of pigs to avian H7N9 influenza A virus from the recent Chinese epidemic. Although the virus did replicate in pigs, only minimal disease was observed and pig-to-pig transmission seems unlikely. In support of Objective 2, human trivalent inactivated influenza vaccine was used in pigs to evaluate protection from heterologous H1N2 infection or homologous H1N1pdm09 virus. The vaccine prevented disease from homologous challenge and did not cause vaccine-associated enhanced respiratory disease (VAERD) with heterologous challenge. In contrast, a hemagglutinin (HA) subunit vaccine caused enhanced macroscopic lesions and higher virus titers, suggesting immunity against the HA protein alone is sufficient for inducing VAERD. In support of Objective 2, two influenza virus genomes are being cloned to evaluate the triple reassortant internal gene cassette or H1N1pdm09 backbone in adapting the human influenza surface proteins HA and neuraminidase (NA) for swine. In support of Objective 3, we produced vectors containing porcine circovirus 2 (PCV2) constructs with or without the immunodominant sequence that induces antibodies cross reactive with nuclear antigens. We will next evaluate whether either construct will induce protective immunity. In support of Objective 3, we developed a method to clone novel swine and avian viruses. Single-stranded DNA viruses were identified and genomic clones were obtained. In support of Objective 3, we determined that two replication initiation proteins are essential for PCV to produce progeny viruses. In support of Objective 4, we identified and compared gene expression changes in lymph nodes of germ-free pigs following single viral infections with PRRSV, HP-PRRSV, swine influenza virus, PCV2, or pseudorabies. Interferon-induced genes were differentially expressed, and 11 distinct genes were discovered that could be used for vaccine development. In support of Objective 4, we developed standardized transcriptome profiling tools and developed whole transcriptome target sequencing (WTTS). WTTS will increase the ability to sequence genes/transcripts with low levels of expression. In support of Objective 4, we established RNA-seq procedures for genome-wide profiling of signature genes in porcine innate immune cells. Our data revealed family-wide differential expression and will aid in discovery of antiviral regulation genes. In support of Objective 4, we established a globinRNA removal protocol from whole blood for transcriptomics to provide major insights into host response mechanisms during viral infection. This provides increased accuracy and reproducibility.


4.Accomplishments
1. Examined new and potentially dangerous strains of PRRSV. Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant reproductive losses in the sow herd and respiratory disease in growing pigs. It is the top disease problem for pig producers in the United States and many other parts of the world. In 2006, a new strain of PRRSV emerged in Chinese swine herds that were suffering dramatic losses, and this strain eventually spread to other South-eastern Asian countries including Vietnam. These viruses have become known as "highly-pathogenic PRRSV" (HP-PRRSV), which means they cause more severe disease than PRRSV strains we have seen before. These Asian HP-PRRSV strains, foreign to this country, are a continued threat to our nation's swine and agricultural economy. ARS scientists at the National Animal Disease Center in Ames, Iowa compared the genetic make-up of two different lineages of HP-PRRSV, one from China and one from Vietnam, and their ability to cause disease in US swine. The Chinese strain produced more severe clinical disease than the strain from Vietnam, but the infection of both result resulted in disease that was more severe than that seen with strains of PRRSV that are currently circulating in the US. The genetic differences between the two HP-PRRSV strains suggest that we may pinpoint crucial components to target that weaken the virus and its ability to cause disease in pigs. These findings reveal that the HP-PRRSV strains are evolving, but are highly virulent in U.S. swine. The core knowledge acquired as a result of these studies will be used to develop a vaccine against HP-PRRSV in order to prevent its spread.

2. Discovered that a PRRSV protein is associated with the virus particle. Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant reproductive losses in the sow herd and respiratory disease in growing pigs. It is the top disease problem for pig producers in the United States and many other parts of the world, causing hundreds of millions of dollars in losses. Current vaccines against PRRSV are not very protective and thus need to be improved. Viruses are made up of genetic material that encodes proteins. Some of these proteins make up the virus structure itself while other proteins are produced after the virus infects a cell to help it replicate. ARS scientists at the National Animal Disease Center in Ames, Iowa discovered that a protein originally not thought to be a structural protein of PRRSV, called nonstructural protein 2 (nsp2), is actually present as part of the virus particle itself. This is important because proteins that are part of the virus particle itself interact with cells when infecting the pig and are potential targets for the immune system, thus making them good candidates for vaccines that could lessen the impact of this virus on the swine industry.

3. Designed novel PRRSV vaccine candidates for evaluation as a next generation DIVA (Differentiating Infected from Vaccinated Animals) vaccine. Porcine reproductive and respiratory syndrome virus (PRRSV) causes highly significant losses to the swine industry worldwide. The industry would like to be able to eradicate this virus from swine herds in the United States. One tool that must be in place for this to occur is an efficacious DIVA vaccine. The purpose of this type of vaccine in an eradication program is to be able to differentiate an animal that has been vaccinated from an animal that is actively infected with the virus with a blood test so that infected animals can be eliminated from the herd before they spread the virus to other animals. ARS scientists at the National Animal Disease Center in Ames, Iowa designed and developed six unique PRRSV vaccines with DIVA characteristics. These vaccine candidates were tested in swine to confirm that they were stable and that they can function as a backbone for next generation vaccines. These types of vaccines will be important to reduce disease associated with PRRSV and eliminate the virus from the swine population in the United States decreasing losses this virus causes to the swine industry each year.

4. The molecular immune response to influenza virus was investigated in the germ-free pig model. The immune system helps protect animals from disease. It can be divided into two compartments, the humoral and cellular immune system that work together against infectious agents. Humoral immunity involves the production of antibodies by B cells, a type of cell that circulates in the blood. Cellular immunity involves T cells, another type of blood cell that can "attack" infectious agents using several mechanisms. Viruses and bacteria are constantly mutating trying to improve their chances to infect animals. Vaccines can be used to enhance the immune system to minimize or prevent infections. To better understand how a vaccine might be improved, it is necessary to understand more about the immune response. ARS scientists at the National Animal Disease Center in Ames, Iowa investigated the development of the humoral immune response against swine influenza virus using germ-free pigs as the animal model. This response involves the development of a specific class of antibodies suggesting if this response could be enhanced, then the overall immune response against influenza virus might be enhanced. The knowledge gained from this germ-free pig study will be applied in developing future vaccines in order to stimulate a specific class of antibodies to better fight swine influenza.

5. Genotype patterns of contemporary reassorted H3N2 virus in U.S. swine were determined. Although it is not uncommon for influenza A viruses found circulating in swine to occasionally infect humans, there have been >300 swine-origin influenza A viruses (called H3N2v) detected in humans since July 2011 in the United States. Most of the human cases had direct exposure to pigs in various settings, most notably in agricultural fairs. ARS scientists at the National Animal Disease Center in Ames, Iowa performed the largest sequence analysis at a whole-genome scale to date of influenza viruses collected from U.S. swine during 2009-2012, including 119 swine H3N2 viruses. The H3N2 swine viruses comprised two main groups: approximately half had all eight genes found in viruses isolated from North American swine from 1998-2009, and the remaining viruses had mixed (reassorted) with the 2009 pandemic H1N1 into at least ten distinct patterns. Importantly, the reassortant H3N2 swine with the same genetic pattern as the H3N2v human cases were the most frequently detected genotype, indicating a possible genetic fitness for this gene combination in swine and a potential for spillover to humans. Although the swine viruses had a diverse genetic make up, all human H3N2v viruses studied were genetically similar, suggesting the requirements for virus transmission from swine to humans is more stringent than what is observed for transmission among swine. Understanding these requirements may allow prediction of which animal influenza viruses may be of greater risks to humans, aiding in pandemic preparedness.

6. Determined conditions that contribute to influenza vaccine-associated enhanced respiratory disease. Influenza A virus causes a respiratory disease in swine which is similar to that in humans. Inactivated vaccines work well when pigs are exposed to influenza viruses represented 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 vaccinated pigs with a poorly matched inactivated influenza A vaccine followed by infection with the pandemic human influenza A virus 2009 H1N1, and demonstrated that not only was the vaccine ineffective at reducing disease, more severe clinical disease and lung lesions occurred in vaccinated pigs compared to non-vaccinated pigs. Vaccinated pigs rapidly demonstrated greater lung damage compared to non-vaccinated pigs, and the pneumonia was more severe. Elevated immune factors that are associated with inflammation and disease were detected in the lungs of vaccinated pigs. These results demonstrate the need for active surveillance and monitoring the quality of match between vaccine strains and strains infecting swine herds to prevent vaccine mismatch in the swine population. In addition, development of improved vaccines that stimulate immune responses that are effective at protecting against many strains influenza virus will be important to prevent infection and clinical disease and reduce the burden of this economically important disease.

7. Determined that antibodies induced by PCV2 cross react with pig cells. Porcine circovirus type 2 (PCV2) has been implicated as the cause of a collection of disease syndromes in swine labeled porcine circovirus associated disease (PCVAD). Preliminary data had originally suggested that one of two proteins of the virus induces antibodies in swine that cross react with the nucleus of pig cells known as antinuclear antibodies. Antibodies against pig cells can induce autoimmune-like disease syndromes where the immune system that normally attacks invading pathogens instead attacks the animals own cells. ARS scientists at the National Animal Disease Center in Ames, Iowa determined that it was a protein that was a part of the capsid, the outer protein cover of the virus, which induced these antinuclear antibodies. These findings will help us develop more efficacious vaccines against PCV2 that do not have side effects and help eliminate the disease syndromes in swine and economic losses to the swine industry associated with this virus.

8. Novel viruses discovered in pig feces. Infectious swine diseases can be caused by a variety of viruses, many of which are unknown. New sequencing and analyses methods have been established for the discovery of novel viruses. This technology enables detection of viruses (without prior knowledge) that might not otherwise be detected by routine diagnostic methods that will lead to significant advances in our capacity for pathogen discovery. ARS scientists at the National Animal Disease Center in Ames, Iowa in collaboration with scientists at the University of California at San Francisco and Purdue University established procedures using pig fecal samples that allow rapid discovery and identification of existing or newly emerging viruses. Using these methods, novel viruses were identified that are related to viruses detected in chimpanzees and cattle, and these viruses may be classified as a new group. To date, there is no association between this group of viruses and disease, suggesting they may be part of a growing list of viruses that infect animals with no known consequences. The ability to elucidate the complete pig virome, which is all the viruses that are found in the pig, allows better understanding of the infectious disease processes in swine.

9. Discovery of a novel picornavirus from a turkey with gastro-intestinal disease. A new disease appeared in turkeys in the United States causing diarrhea and weakness with no known cause. Developments in technology have enabled the discovery of viruses without the need to isolate the virus. ARS scientists at the National Animal Disease Center in Ames, Iowa in collaboration with scientists at the Blood Systems Research Institute in San Francisco and Purdue University used this technique on fecal samples collected from these sick turkey poults. A novel virus was identified and named turkey avisivirus. It is closely related to a turkey virus recently discovered in Hungary using similar techniques. The Hungarian turkey virus also was isolated from sick birds. The discovery of this virus will allow future work to determine the prevalence of these new viruses and whether they are the cause of the disease, hopefully allowing us to eliminate or control an emerging viral threat to the turkey industry.


Review Publications
Cheung, A.K. 2012. Replicative intermediates of porcine circovirus in animal tissue cultured cells or in bacteria undergoing copy-release replication. Virology. 434(1):38-42.

Nelson, M.I., Gramer, M.R., Vincent, A.L., Holmes, E.C. 2012. Global transmission of influenza viruses from humans to swine. Journal of General Virology. 93(10):2195-2203.

Lager, K.M., Ng, T.F., Bayles, D.O., Alt, D.P., Delwart, E.L., Cheung, A.K. 2012. Diversity of viruses detected by deep sequencing in pigs from a common background. Journal of Veterinary Diagnostic Investigation. 24(6):1177-1179.

Miller, L.C., Fleming, D., Arbogast, A., Bayles, D.O., Guo, B., Lager, K.M., Henningson, J.N., Schlink, S.N., Yang, H.-C., Faaberg, K.S., Kehrli, Jr., M.E. 2012. Analysis of the swine tracheobronchial lymph node transcriptomic response to infection with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus. BioMed Central (BMC) Veterinary Research. 8(1):208. Available: http://www.biomedcentral.com/1746-6148/8/208.

Guo, B., Lager, K.M., Henningson, J.N., Miller, L.C., Schlink, S.N., Kappes, M.A., Kehrli, Jr., M.E., Brockmeier, S.L., Nicholson, T.L., Yang, H., Faaberg, K.S. 2013. Experimental infection of United States swine with a Chinese highly pathogenic strain of porcine reproductive and respiratory syndrome virus. Virology. 435(2):372-384.

Jiang, Z., Zhou, X., Michal, J.J., Wu, X.-L., Zhang, L., Zhang, M., Ding, B., Liu, B., Manoranjan, V.S., Neill, J.D., Harhay, G.P., Kehrli, Jr., M.E., Miller, L.C. 2013. Reactomes of porcine alveolar macrophages infected with porcine reproductive and respiratory syndrome virus. PLoS One. 8(3):e59229.

Nelson, M.I., Detmer, S.E., Wentworth, D.E., Tan, Y., Schwartzbard, A., Halpin, R.A., Stockwell, T.B., Lin, X., Vincent, A.L., Gramer, M.R., Holmes, E.C. 2012. Genomic reassortment of influenza A virus in North American swine, 1998-2011. Journal of General Virology. 93(Pt 12):2584-2589.

Butler, J.E., Sun, X., Wertz, N., Vincent, A.L., Zanella, E.L., Lager, K.M. 2013. Antibody repertoire development in fetal and neonatal piglets. XVI. Influenza stimulates adaptive immunity, class switch and diversification of the IgG repertoire encoded by downstream C-gamma genes. Immunology. 138(2):134-144.

Sun, X., Wertz, N., Lager, K.M., Butler, J.E. 2012. Antibody repertoire development in fetal and neonatal piglets. XV. Porcine circovirus type 2 infection differentially affects serum IgG levels and antibodies to ORF2 in piglets free from other environmental factors. Vaccine. 31(1):141-148.

Kitikoon, P., Nelson, M.I., Killian, M.L., Anderson, T.K., Koster, L., Culhane, M.R., Vincent, A.L. 2013. Genotype patterns of contemporary reassorted H3N2 virus in US swine. Journal of General Virology. 94(Pt 6):1236-1241.

Li, X., Galliher-Beckley, A., Nietfeld, J.C., Faaberg, K.S., Shi, J. 2013. MontanideTM Gel01 ST adjuvant enhances PRRS modified live vaccine efficacy by regulating porcine humoral and cellular immune responses. World Journal of Vaccines. 3(1):1-9.

Chitko McKown, C.G., Chapes, S.K., Miller, L.C., Riggs, P.K., Ortega, M.T., Green, B.T., McKown, R.D. 2013. Development and characterization of two porcine monocyte-derived macrophage cell lines. Results in Immunology. 3:26-31.

Ng, T.F.F., Cheung, A.K., Wong, W., Lager, K.M., Kondov, N.O., Cha, Y., Murphy, D.A., Pogranichniy, R.M., Delwart, E. 2013. Divergent picornavirus from a turkey with gastro-intestinal disease. Genome Announcements. 1(3):e00134-13.

Epperson, S., Jhung, M., Richards, S., Quinlisk, P., Ball, L., Moll, M., Boulton, R., Haddy, L., Biggerstaff, M., Brammer, L., Trock, S., Burns, E., Gomez, T., Wong, K.K., Katz, J., Lindstrom, S., Klimov, A., Bresee, J.S., Jernigan, D.B., Cox, N., Finelli, L., Influenza A(H3N2)v Virus Investigation Team (Vincent, A.L.). 2013. Human infections with influenza A(H3N2) variant virus in the United States, 2011-2012. Clinical Infectious Diseases. 57(S1):S4-S11.

Last Modified: 12/26/2014
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