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United States Department of Agriculture

Agricultural Research Service

Research Project: SWINE VIRAL DISEASES PATHOGENESIS AND IMMUNOLOGY
2011 Annual Report


1a.Objectives (from AD-416)
Obj. 1: Identify mechanisms of PRRS virus (PRRSV) pathogenesis to develop vaccination strategies to enhance or improve immunity against PRRSV. Sub-objective 1.2: Use reverse genetic technology to identify determinants of viral pathogenesis, immune evasion, and transmission. Sub-objective 1.3: Use reverse genetic technology to design live attenuated vaccine strains that provide broad protection against PRRSV infection and allow the differentiation of infected from vaccinated animals. Obj. 2: Identify mechanisms of SI virus (SIV) pathogenesis and develop vaccination strategies to enhance or provide broad cross-protection for circulating subtypes of SIV. Sub-objective 2.4: Characterize humoral and cell mediated immune responses elicited by influenza vaccines administered in the face of maternal antibodies. Sub-objective 2.5: Characterize heterologous protection provided by humoral and cell mediated immune responses elicited by influenza vaccines. Obj. 3: Identify the host-pathogen interactions and environmental factors that lead to PCVAD and discover effective measures to prevent, control, and eliminate this emerging disease from U.S. swine herds. Obj. 4: Develop methods of modulation of innate and adaptive immune responses to swine viral pathogens with an emphasis on modulating the effects of innate immunity on pathogenesis of viral diseases.


1b.Approach (from AD-416)
For improved PRRSV control, identify strategies for improved immunoprophylaxis by testing vaccine strategies with recombinant adenoviruses expressing selected PRRS viral gene constructs to increase safety and efficacy of PRRS vaccines. For improved SIV control, identify mechanisms of SIV pathogenesis and develop vaccination strategies to enhance cross-protection for circulating subtypes of SIV. Investigate the role of avian polymerase genes in adaptation of novel reassortant SIVs to pigs. Study specific regions within identified genes that confer growth advantages. Maintain a contemporary repository for emerging SIV subtypes and genotypes and combine with novel vaccine approaches for improved SIV vaccines. Develop vaccine strategies that have broader subtype coverage through use of better cross-reacting isolates, novel combinations of adjuvants and/or cytokines, and different routes of vaccination. Specific aims: A) Genetic, antigenic and pathogenic characterization of novel isolates; B) Evaluation of new inactivated vaccines against current isolates; and C) Evaluation of genetically engineered, modified-live vaccines against current isolates. For improved control of PCV type 2, conduct research to identify mechanisms of PCV2 pathogenesis in PMWS and perform genetic analysis of the replication and virulence mechanisms of PCV2 to develop vaccination strategies against porcine circoviruses. Goal is to develop recombinant virus vaccines against PMWS by attenuation of the viral replication and virulence mechanisms; and develop and evaluate multiplex diagnostic assays to detect pathogens involved in PCVAD, determine the role of endemic and novel swine viruses in inducing PCVAD, and evaluate genetic and biological determinants that lead to PCVAD. Development of methods for modulation of innate and adaptive immune responses to swine viral pathogens will focus on modulating the effects of innate immunity on pathogenesis of viral diseases. Evaluate whether early serum IFN-gamma response is caused by the interaction of PRRSV structural proteins with components of the hosts' immune system. Ameliorate clinical disease through prophylactic or metaphylactic administration of granulocyte-colony stimulating factor in an attempt to reduce the severity or duration of viral pneumonia associated with PRRSV and SIV. Investigate the B cell response to these swine viruses with a focus on immunoglobulin class switch recombination and diversification of the VDJ repertoire. Changes in B cells correlate with the appearance of neutralizing antibody; understanding the virulence mechanisms contributing to the delayed development of neutralizing antibody against PRRSV may provide essential insights into improved control of PRRSV shedding in vaccinated and infected pigs.


3.Progress Report
We compared the pathogenicity of a Chinese porcine reproductive and respiratory syndrome virus (PRRSV) isolate rescued from a cDNA clone, a Vietnamese PRRSV isolate derived in our laboratory from a cDNA clone, and the North American PRRSV prototype - VR2332 using U.S. swine under ABSL3 containment. The Chinese virus induced a fatal disease for all inoculated pigs. In contrast, the North American prototype produced a mild disease and under conditions of the study, the Vietnamese isolated produced a severe disease with low mortality. We compared nucleotide sequences of the two Asian strains and found differences throughout the PRRSV genome that are being investigated for clues into the highly pathogenic nature of these viruses. The Asian strains result in an overgrowth of commensal bacteria, contributing to severe clinical disease. We conducted a vaccine efficacy study with a commercial PRRSV vaccine and found partial, statistically significant protection against the Chinese highly pathogenic PRRSV isolate (HP-PRRSV).

We identified a region of nsp2 of PRRSV as a potential virulence trait. To test this region as a potential vaccine, we selected regions of this viral gene in various lengths and engineered them into replication defective adenovirus vectors. The vector constructs were confirmed for correct sequence, grown up and purified for vaccination of swine.

Studies were conducted to define the kinetics of lesion development in vaccine enhancement of respiratory disease (VERD) using an inactivated influenza vaccine that contains a mismatched strain to the challenge virus. Studies compared the inactivated vaccine that induces VERD with an attenuated modified live virus vaccine containing the same parental virus strain. A separate vaccine study was conducted to evaluate a replication deficient adenovirus vectored vaccine containing the hemagglutinin (HA) and/or nucleoprotein (NP) genes in comparison with the inactivated vaccine. Immune assays have been developed and utilized in the vaccine studies to identify differences in responses to vaccines that may allow for predicting the outcome and understanding the mechanism underlying VERD. Human and swine isolates of H1N1 and H1N2 were utilized to immunize pigs to generate immune sera for use in antigenic mapping of the delta cluster of U.S. swine H1 influenza viruses. Antigenic mapping will aid in understanding vaccine and diagnostic failures and aid in vaccine strain selection. A subset of the H1 viruses was subjected to whole genome sequencing and genetic analysis to complement the antigenic analysis.

We are adapting high through-put pyrosequencing technology to examine the complete virus profile of pigs with or without porcine circovirus associated disease (PCVAD). The information obtained will facilitate determining virus candidates or interactions that lead to PCVAD.


4.Accomplishments
1. Demonstrated highly pathogenic strains of Asian porcine reproductive and respiratory syndrome virus (HP-PRRSV) cause severe disease in healthy U.S. pigs. In 2006, reports of the sudden appearance of a novel porcine high fever disease syndrome in Asia were attributed to highly pathogenic strains of PRRSV. The emergence and discovery of these highly virulent strains of PRRSV in China compelled ARS scientists at the National Animal Disease Center, Ames, IA, to investigate the clinical repercussions of this virus if the strain were to appear in the U.S. We demonstrated that in the absence of foreign animal disease co-infections and other basic differences between Asian swine and high health U.S. swine, these Asian strains, cause severe disease and high mortality. Asian HP-PRRSV strains appear to severely impact the pig's immune system resulting in overgrowth of commensal bacteria, contributing to the high morbidity and mortality of this disease. An internationally approved vaccine was tested for its efficacy; although it provided some protection it was not ideal for protection against the highly pathogenic strain; therefore a new vaccine must be developed in order to fully protect U.S. swine. These results can be used by scientists to investigate virulence traits of PRRSV and to develop better vaccines for controlling disease losses caused by highly pathogenic strains of PRRSV.

2. Assessed ability of U.S. veterinary diagnostic laboratories to detect highly pathogenic strains of Asian porcine reproductive and respiratory syndrome virus (HP-PRRSV). In 2006, reports of the sudden appearance of a novel porcine high fever disease syndrome in Asia were attributed to highly pathogenic strains of PRRSV. The emergence and discovery of these highly virulent strains of PRRSV in China compelled ARS scientists at the National Animal Disease Center, Ames, IA, to investigate the ability of our veterinary diagnostic laboratory network to detect these recently emerged strains of PRRSV in case they should enter the U.S. swine herd. We completed a blind panel study using a subgenomic clone of HP-PRRSV transcribed into RNA and distributed to three major U.S. diagnostic laboratories that collaborated on this project. These 3 diagnostic laboratories were chosen on the basis they receive the vast majority of swine diagnostic lab submissions in the U.S. All laboratories readily detected HP-PRRSV and all three laboratories were capable of generating the correct nucleotide sequence for open reading frame 5 of HP-PRRSV. This study confirms the ability of U.S. veterinary diagnostic laboratories to detect and correctly identify these recently emerged strains of HP-PRRSV should they enter the U.S. swine herd.

3. Demonstrated the time course of lung lesion development and pro-inflammatory cytokine response in pigs with vaccine-associated enhanced respiratory disease (VERD). Influenza A virus causes a respiratory disease in swine similar to that in humans and is considered one of the three most important swine respiratory pathogens. Killed 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. In fact, vaccinating with a killed influenza virus can increase the severity of disease when pigs are exposed to a new strain of influenza virus compared to pigs that are not vaccinated. ARS scientists at the National Animal Disease Center, Ames, IA, have now shown that VERD in pigs vaccinated with a delta-cluster virus (first introduced into pigs from humans around 2003) is evident as little as 24 hours after challenge with the pandemic human influenza A virus (2009). Pigs that were vaccinated with a mismatched virus strain exhibited greater percentages of pneumonia compared to non-vaccinated pigs, and the microscopic character of the pneumonia was more severe with distinct types of lung damage. Elevated immune factors associated with inflammation and disease were detected in the lungs at all time points tested. Active surveillance and monitoring of the quality of match between vaccine strains and strains infecting swine herds is necessary to prevent vaccine mismatch in the swine population. Future vaccines that stimulate improved immune responses across differing influenza viruses will be important to prevent infection and clinical disease and reduce the burden of this economically important disease. Since influenza viruses from swine may infect people, controlling influenza in the swine population has important implications to human health as well.

4. DNA vaccine elicits protective immune responses against pandemic and classic swine influenza viruses in pigs. Swine influenza is a highly contagious viral infection in pigs that significantly impacts the pork industry due to weight loss and secondary infections. There is also the potential of a significant public health threat, highlighted by the 2009 H1N1 pandemic virus. ARS scientists at the National Animal Disease Center, Ames, IA, in collaboration with scientists from the Vaccine Research Center at the National Institutes of Health, demonstrated a DNA vaccine encoding an influenza hemagglutinin protein gene could induce a protective immune response when given intramuscularly with a needle and syringe, or with a commercial needleless injection device that uses pressure to create a fine stream of liquid that penetrates the skin, delivering doses of vaccine to the desired depth. When compared to the production of traditional swine influenza vaccines, the DNA vaccine technology could reduce the time required to produce a new vaccine in response to the emergence of novel influenza viruses. Likewise, when compared to the intramuscular injection of traditional swine influenza vaccines, the use of a needleless device with a DNA vaccine could reduce the time and effort required to vaccinate pigs.

5. Demonstrated the evolution and movement of H1N1 and H1N2 influenza viruses in the U.S. pig population. Today's modern swine production practices involve the daily movement of large numbers of pigs between different geographic locations in the United States. This movement of pigs affects the distribution of swine pathogens. Genetic sequence data from swine influenza viruses was analyzed for understanding the way these viruses are evolving and moving in the swine population in the USA. The study considered the role of the long-distance transport of pigs in the United States in the spread of influenza viruses of the A/H1N1 and A/H1N2 subtypes that were transmitted from humans to swine in 2003. Using the most intensive nationwide sampling of swine influenza viruses to date (n = 1,416 H1 hemagglutinin sequences), it was found that large-scale movements of swine are important in the spread of influenza viruses from the South to the grain-rich Midwestern U.S., where swine are grown to maturity. This study reveals the effects of multi-site, animal husbandry production practices in the U.S. swine industry on the spread and evolutionary dynamics of an economically important pathogen in the swine industry that also presents a public health threat. Further understanding of the role of long-distance pig transport in the ecology and evolution of swine influenza viruses in the U.S. will guide animal health officials to prepare targeted surveillance and mitigation strategies in the future.

6. Identified a genetic marker associated with antinuclear antibody development in pigs exposed to porcine circovirus type 2 antigen. In late 2005, a postweaning, high mortality syndrome spread rapidly through swine dense areas of the United States. Porcine circovirus type 2 (PCV2) was soon implicated as the causative agent of a collection of disease syndromes labeled porcine circovirus associated disease or PCVAD. Previously we discovered evidence for development of antinuclear antibodies (ANA) that recognize nuclear antigens or double stranded DNA in sera of germ-free pigs infected with PCV2. We also have detected ANA in conventionally reared pigs following exposure to PCV2 virus or vaccination and that the level of ANA correlates to disease severity. Suspecting a possible genetic factor involvement with ANA development, ARS scientists at the National Animal Disease Center, Ames, IA, collaborated with ARS scientists at US-Meat Animal Research Center, Clay Center, NE, and have obtained preliminary results identifying a single nucleotide polymorphism in two loci that appear to be linked to the development of ANA in pigs exposed to PCV2 antigens. One locus was on chromosome 7 in the region of the swine major histocompatibility complex that has known involvement with ANA development in humans. These data may help to explain some of the variability reported by veterinarians in the response to vaccination and why some but not all pigs had severe forms of PCVAD.

7. Completed a study on the genetic relationship and prevalence of six porcine bocaviruses among diseased pigs in swine herds from ten Chinese provinces. In late 2005, a postweaning, high mortality syndrome spread rapidly through swine dense areas of the United States. Porcine circovirus type 2 (PCV2) was soon implicated as the etiological agent of a collection of disease syndromes labeled porcine circovirus associated disease or PCVAD. ARS scientists at the National Animal Disease Center, Ames, IA, previously discovered porcine parvovirus type 4 (now named porcine bocavirus.
2)in U.S. pigs with PCVAD; however, it was unknown what if any relationship porcine bocavirus 2 had to PCVAD in pigs. Traditionally it was believed that clinical disease following PCV2 infection is dependent on an immunostimulatory co-factor such as a viral co-infection as had been demonstrated with other parvoviruses. To further our understanding of the epidemiology of porcine bocavirus 2 and PCVAD we established a collaboration to determine how prevalent this virus was in a large swine producing country. We found four porcine bocavirus species were highly prevalent in Chinese swine herds. Porcine bocavirus co-infections with two, three, four or five PoBoVs were detected and were quite common. Furthermore, many mixed-infections with viruses from other families (porcine reproductive and respiratory syndrome virus, classic swine fever virus and porcine circovirus type.
2)were detected. However, no clear relationship between the presence of these porcine bocaviruses among clinically sick or healthy pigs was established. These data help scientists rule out certain viruses as causes of disease to better focus swine health research management strategies in the future.

8. Validated a real-time PCR assay for Pseudorabies virus surveillance purposes. Pseudorabies virus (PRV) is of significant economic importance for the swine industry worldwide. PRV is the cause of Aujeszky's disease, also known as Pseudorabies. The main symptoms are related to respiratory and nervous systems, which are the preferred site for PRV. Also, PRV can cause a high mortality in neonatal piglets, abortion in sows, and loss of body condition in the growing pigs. The feral swine population is known to be infected with PRV strains, and is considered to be a threat for the commercial swine industry. A critical need for the current PRV surveillance program in the United States is the rapid detection of PRV. Real-time polymerase chain reaction (real-time PCR) is a valuable diagnostic technique that can rapidly identify infectious agents in clinical specimens. A real-time PCR assay was designed based on the gB and gE genes to identify PRV in diagnostic samples. Using virus isolation as the previous best standard test, the assay performed well in a variety of diagnostic matrices. Diagnostic performance of the real-time PCR assay developed as a testing method indicates that it is a rapid, accurate assay that is adaptable to a variety of PCR platforms currently in use by diagnostic laboratories around the world and can provide reliable results on an array of clinical samples.

9. Effects of Pseudorabies virus infection on the Tracheobronchial lymph node transcriptome. Pseudorabies virus (PRV) is of significant economic importance for the swine industry worldwide. PRV is the cause of Aujeszky's disease, also known as Pseudorabies. The main symptoms are related to respiratory and nervous systems, which are the preferred site for PRV. Also, PRV can cause a high mortality in neonatal piglets, abortion in sows, and loss of body condition in the growing pigs. In an effort to understand comparative differences in how several viruses affect the pig's immune system versus other endemic swine viruses ARS scientists at the National Animal Disease Center, Ames, IA, collaborated with ARS scientists at US-Meat Animal Research Center, Clay Center, NE, to investigate how a feral isolate of PRV alters gene expression in the draining lymph node of the pig's lung to better understand the physiopathology of infection and the immune response at a cellular level. The experimental results have been integrated with previous studies to develop a robust model of swine respiratory virus infection. These results are now being used by ARS scientists to characterize regulatory pathways controlling the pig's immune response to virus challenge, develop information on cellular response networks and to identify genes to target for improving disease resistance.


Review Publications
Cheung, A.K., Greenlee, J.J. 2011. Identification of an amino acid domain encoded by the capsid gene of porcine circovirus type 2 that modulates intracellular viral protein distribution during replication. Virus Research. 155(1):358-362.

Gauger, P.C., Vincent, A.L., Loving, C.L., Lager, K.M., Janke, B.H., Kehrli, Jr., M.E., Roth, J.A. 2011. Enhanced pneumonia and disease in pigs vaccinated with an inactivated human-like (delta-cluster) H1N2 vaccine and challenged with pandemic 2009 H1N1 influenza virus. Vaccine. 29(15):2712-2719.

Huang, L.V., Zhai, S.L, Cheung, A.K., Zhang, H.B., Long, J.X., Yuan, S.S. 2010. Detection of a novel porcine parvovirus, PPV4, in Chinese swine herds. Virology Journal. 7(1):333.

Kitikoon, P., Sreta, D., Nuntawan Na Ayudhya, S., Wongphatcharachai, M., Lapkuntod, J., Prakairungnamthip, D., Bunpapong, N., Suradhat, S., Thanawongnuwech, R., Amonsin, A. 2011. Brief report: molecular characterization of a novel reassorted pandemic H1N1 2009 in Thai pigs. Virus Genes. 43(1):1-5.

Larson, L.J., Henningson, J., Sharp, P., Thiel, B., Deshpande, M.S., Davis, T., Jayappa, H., Wasmoen, T., Lakshmanan, N., Schultz, R.D. 2011. Efficacy of canine influenza virus (H3N8) vaccine to decrease severity of clinical disease after cochallenge with canine influenza virus and Streptococcus equi subsp. Zooepidemicus. Clinical and Vaccine Immunology. 18(4):559-564.

Lorusso, A., Vincent, A.L., Harland, M.L., Alt, D., Bayles, D.O., Swenson, S.L., Gramer, M.R., Russel, C.A., Smith, D.J., Lager, K.M., Lewis, N.S. 2011. Genetic and antigenic characterization of H1 influenza viruses from United States swine from 2008. Journal of General Virology. 92(Pt 4):919-930.

Ma, W., Lager, K.M., Lekcharoensuk, P., Ulery, E.S., Janke, B.H., Solorzano, A., Webby, R.J., Garcia-Sastre, A., Richt, J.A. 2010. Viral reassortment and transmission after coinfection of pigs with classical H1N1 and triple reassortant H3N2 swine influenza viruses. Journal of General Virology. 91(Pt 9):2314-2321.

Metwally, S., Mohamed, F., Faaberg, K., Burrage, T., Prarat, M., Moran, K., Bracht, A., Mayr, G., Berninger, M., Koster, L., To, T.L., Nguyen, V.L., Reising, M., Landgraf, J., Cox, L., Lubroth, J., Carrillo, C. 2010. Pathogenicity and molecular characterization of emerging porcine reproductive and respiratory syndrome virus in Vietnam in 2007. Transboundary and Emerging Diseases. 57(5):315-329.

Miller, L.C., Neill, J.D., Harhay, G.P., Lager, K.M., Laegreid, W.W., Kehrli, Jr., M.E. 2010. In-depth global analysis of transcript abundance levels in porcine alveolar macrophages following infection with porcine reproductive and respiratory syndrome virus. Advances in Virology. 2010:Article 864181. Available: http://downloads.hindawi.com/journals/av/2010/864181.pdf.

Nelson, M.I., Lemey, P., Tan, Y., Vincent, A., Lam, T.T-Y., Detmer, S., Viboud, C., Suchard, M.A., Rambaut, A., Holmes, E.C., Gramer, M. 2011. Spatial dynamics of human-origin H1 influenza A virus in North American swine. PLoS Pathogens. 7(6):e1002077.

Shi, M., Lam, T.T., Hon, C.C., Hui, R.K., Faaberg, K.S., Wennblom, T., Murtaugh, M.P., Stadejek, T., Leung, F.C. 2010. Molecular epidemiology of PRRSV: a phylogenetic perspective. Virus Research. 154(1-2):7-17.

Trible, B.R., Kerrigan, M., Crossland, N., Potter, M., Faaberg, K., Hesse, R., Rowland, R.R. 2011. Antibody recognition of porcine circovirus type 2 capsid protein epitopes after vaccination, infection, and disease. Clinical and Vaccine Immunology. 8(5):749-757.

Wisedchanwet, T., Wongphatcharachai, M., Boonyapisitsopa, S., Bunpapong, N., Kitikoon, P., Amonsin, A. 2011. Genetic characterization of avian influenza subtype H4N6 and H4N9 from live bird market, Thailand. Virology Journal. 8:131.

Chen, L.M., Rivailler, P., Hossain, J.M., Carney, P., Balish, A., Perry, I., Davis, C.T., Garten, R., Shu, B., Xu, X., Klimov, A., Paulson, J.C., Cox, N.J., Swenson, S., Stevens, J., Vincent, A., Gramer, M., Donis, R.O. 2011. Receptor specificity of subtype H1 influenza A viruses isolated from swine and humans in the United States. Virology. 412(2):401-410.

Cheung, A.K., Long, J.X., Huang, L., Yuan, S.S. 2011. The RNA profile of porcine parvovirus 4, a boca-like virus, is unique among the parvoviruses. Archives of Virology. 56(11):2071-2078.

Zhang, H.B., Huang, L., Lin, T., Sun, C.Q., Deng, Y., Wei, Z.Z., Cheung, A.K., Long, J.X., Yuan, S.S. 2011. Porcine bocaviruses: genetic analysis and prevalence in Chinese swine population. Epidemiology and Infection. 139(10):1581-1586.

Nfon, C.K., Berhane, Y., Hisanaga, T., Zhang, S., Handel, K., Kehler, H., Labrecque, O., Lewis, N.S., Vincent, A.L., Copps, J., Alexandersen, S., Pasick, J. 2011. Characterization of H1N1 swine influenza viruses circulating in Canadian pigs in 2009. Journal of Virology. 85(17):8667-8679.

Opriessnig, T., Shen, H.G, Pal, N., Ramamoorthy, S., Huang, Y.W., Lager, K.M., Beach, N.M., Halbur, P.G., Meng, X.J. 2011. A live-attenuated chimeric porcine circovirus type 2 (PCV2) vaccine is transmitted to contact pigs but is not upregulated by concurrent infection with porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV) and is efficacious in a PCV2b-PRRSV-PPV challenge model. Clinical and Vaccine Immunology. 18(8):1261-1268.

Last Modified: 10/24/2014
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