Location: Virus and Prion Research2010 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, one approach will 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, one approach will identify mechanisms of SIV pathogenesis and develop vaccination strategies to enhance cross-protection for circulating subtypes of SIV. We will investigate the role of avian polymerase genes in adaptation of novel reassortant SIVs to pigs. We will study specific regions within identified genes that confer growth advantages. Another approach will maintain a contemporary repository for emerging SIV subtypes and genotypes and combine with novel vaccine approaches for improved SIV vaccines. Vaccine strategies will be developed that have broader subtype coverage through by use of better cross-reacting isolates, novel combinations of adjuvants and/or cytokines and different routes of vaccination. Specific aims are: 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, we will conduct research to identify mechanisms of PCV type 2 (PCV2) pathogenesis in PMWS and perform genetic analysis of the replication and virulence mechanisms of PCV2 to develop vaccination strategies against porcine circoviruses. The goal is to develop recombinant virus vaccines against PMWS by attenuation of the viral replication and virulence mechanisms. In addition we will 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 finally evaluate genetic and biological determinants that lead to PCVAD. Our approach to develop 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. We will evaluate whether the early serum IFN-gamma response is caused by the interaction of PRRSV structural proteins with components of the hosts' immune system. Another approach will be to 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. Another approach will be to investigate the B cell response to these swine viruses with a focus on immunoglobulin class switch recombination and diversification of the VDJ repertoire. These 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 the improved control of PRRSV shedding in vaccinated and infected pigs.
3. Progress Report
The project plan involves 4 objectives: 1) Identify mechanisms of PRRS virus (PRRSV) pathogenesis to develop vaccination strategies to enhance or improve immunity against PRRSV. 2) Identify mechanisms of SI virus (SIV) pathogenesis and develop vaccination strategies to enhance or provide broad cross-protection for circulating subtypes of SIV. 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. 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. Reverse engineered mutations in selected strains of PRRSV were used to identify virulence factors contributing to disease. Selected regions of the PRRSV genome identified as possible virulence genes were cloned into a replication defective human adenovirus vector for the purpose of vaccinating pigs in future disease trials. We have been working to develop an infectious clone of a Vietnamese PRRSV isolate obtained during a porcine high fever syndrome outbreak. Viruses derived from strain VR-2332 with selected nsp2 deletion mutants and tagged with c-myc and/or influenza HA were propagated and studied using pulse-chase, immunoprecipitation and/or western blot analyses. We discovered the nsp2 protein is expressed in at least six isomers in infected MARC-145 cells and these isomers are present on purified virions. A pig study was conducted to assess in vivo growth of nsp2 deletion mutants and their effect on immune responses; the animal study was recently completed and laboratory analyses remain underway. For objective 2, we compared the pathogenesis and transmissibility of 2 isolates of the 2009 A/H1N1 influenza pandemic virus in pigs and have found differences in the transmissibility of selected pandemic virus isolates in pigs. In addition, we continue to make progress in understanding the pathogenesis of enhanced pneumonia associated with inactivated influenza vaccines that contain a mismatched strain from the challenge virus. For objective 3, we constructed 8 recombinant adenoviruses containing various constructs of the capsid and replicase genes of PCV. These vectors will be tested as potential vaccine candidates against PCV2. For objective 4 of our project plan, we assessed the capacity of a re-engineered cytokine (granulocyte colony stimulatory factor) to enhance innate immunity in pigs. The predicted unique ability of this modified cytokine to induce sustained elevated neutrophil levels was confirmed and additional disease prevention efficacy studies are now underway. In support of objectives 1-3, we conducted a series of animal studies investigating changes in the transcriptome during the host immune response, as well as host cytokine production as a consequence of infection by each of the following viruses: PRRSV, SIV, PCV2 and pseudorabies.
1. Demonstrated efficacy of commercially-available swine influenza virus vaccines versus a homologous vaccine (i.e., from the same strain as the challenge virus) against the 2009 A/H1N1 pandemic virus. The 2009 pandemic A/H1N1 influenza virus has gene segments of swine origin but some gene segments are quite genetically distinct from previously circulating North American swine influenza strains; although its hemagglutinin gene is related to North American H1 SIV, it was unknown if current vaccines for U.S. swine would cross-protect against the pandemic A/H1N1. ARS scientists in Ames, Iowa evaluated the efficacy of inactivated vaccines prepared with North American swine influenza viruses and an experimental 2009 A/H1N1 vaccine to protect pigs from 2009 pandemic A/H1N1. All vaccines provided partial protection ranging from reduction of pneumonia lesions to significant reduction in virus replication. Commercially-available vaccines provided partial protection; however, none were able to prevent all nasal shedding or clinical disease. An experimental homologous 2009 A/H1N1 vaccine provided optimal protection with no shedding or clinical disease. Based on cross-protection demonstrated with vaccines evaluated, the U.S. swine herd likely has significant immunity to the 2009 A/H1N1 from prior vaccination or natural exposure. However, the experimental vaccine worked best to protect swine by limiting shedding and potential transmission of 2009 A/H1N1 from pigs to people. The pig-passaged virus from this experimental vaccine was transferred to the Center for Veterinary Biologics (CVB) and made available as a master seed for commercial manufacture of swine influenza vaccine for pigs. In December 2009, a conditional license for a vaccine with this virus was granted by USDA-APHIS-CVB to a commercial company and in September 2010, full licensure was granted to the company from the USDA for this vaccine to help protect pigs from the 2009 pandemic H1N1 strain of influenza.
2. Demonstrated an Asian isolate of the porcine reproductive and respiratory syndrome virus (PRRSV) rescued from an infectious cDNA clone is capable of causing a severe form of PRRS in swine under experimental conditions. PRRSV causes respiratory and reproductive disease and is the number one swine disease problem in the United States, causing an estimated $570 million annual loss. In 2006, PRRSV isolates in Asia were linked to a syndrome termed porcine high fever disease. Isolates similar to these strains have not been seen in the United States. In an effort to examine the pathogenicity of these Asian strains, ARS scientists at the National Animal Disease Center in Ames, Iowa obtained from a collaborator a cDNA clone prepared from a Chinese PRRSV strain isolated from a porcine high fever outbreak. Virus was successfully rescued from the cDNA (i.e., grown in tissue culture in the lab) and examined for its in vitro growth characteristics, as well as, for its ability to infect and cause disease in pigs. The rescued virus was found to replicate well under laboratory conditions. In addition, ARS scientists at the National Animal Disease Center produced a small subgenomic segment to serve as a positive control for diagnostic testing by veterinary diagnostic laboratories that is currently being evaluated. In addition, RNA extracted from a Vietnamese PRRSV isolate from an outbreak of porcine high fever disease was obtained from the National Veterinary Services Laboratories-APHIS was used to amplify approximately 10.4 out of 15.3 kilobases of genomic material; our goal is to develop an infectious clone from the RNA from the Vietnamese isolate for comparison with the Chinese isolate in pigs. These results can be used by scientists to investigate virulence traits of the PRRSV and to evaluate vaccines to better control losses from this disease.
3. Genetic and antigenic characterization of H1 influenza viruses from United States swine prior to the emergence of the 2009 pandemic H1N1. Swine play an important role in the evolution of influenza A viruses. Prior to introduction of the 2009 pandemic H1N1 virus from humans into pigs, four phylogenetic clusters of the hemagglutinin (HA) gene from H1 influenza viruses co-circulated in U.S. swine. Viruses from the classical H1N1 swine lineage evolved to form alpha-, beta-, and gamma-clusters whereas viruses with HA genes from human seasonal H1 viruses emerged in 2003 to form a delta-cluster. Limited sequence information was available regarding the six genes that make up the triple reassortant internal gene cassette in contemporary H1 influenza viruses of swine. Information regarding antigenic relatedness of H1 viruses was lacking. ARS scientists at the National Animal Disease Center in Ames, Iowa, and their collaborators characterized twelve H1 isolates from 2008 by sequencing and genetic analysis of all eight gene segments, and by studying immune cross-reactivity (antigenic relatedness). Genetic analysis found each of the four previously described genetic clusters of H1 influenza viruses of swine represented among the 2008 swine influenza isolates. Genetic diversity was demonstrated in all gene segments, but most notably in the HA gene. Genetic evolution of the neuraminidase (NA) gene was comparable to that of the HA gene. Gene segments from the 2009 A/H1N1 pandemic virus formed genetic clusters (groupings) separate from North American swine lineage viruses, suggesting progenitors of the pandemic virus were not present in U.S. pigs immediately prior to 2009. Immune cross-reactivity studies demonstrated that viruses in different genetic clusters are also antigenically divergent or seen differently by the immune system. Results can be used by scientists to make informed decisions for vaccine strain selection.
4. Demonstrated the presence of antinuclear antibodies (ANA) in gnotobiotic pigs infected with porcine circovirus. Porcine circovirus type 2 (PCV2) causes post-weaning-multisystemic-wasting-syndrome (PMWS), a swine disease first observed in Canada in 1991. It is characterized by general wasting, respiratory disease, jaundice and pale mucous membranes in young pigs resulting in production losses and variable mortality. In experimental studies, an unusual feature of disease is a delayed onset of clinical disease (~3 weeks) following infection with virus, but a then sudden decline in health of diseased piglets. ARS scientists at the National Animal Disease Center discovered the development of ANA that recognize nuclear antigens or double stranded DNA in sera of germ-free pigs infected with PCV2. Finding ANA and a PCV2 capsid peptide that induced antibodies cross-reactive with porcine nuclear antigens suggests an autoimmune component to the pathogenesis of PCV2-induced disease in germ-free pigs. The presence and role of ANA in conventionally-reared pigs infected with PCV2 remains to be determined but when detected, ANA are typically associated with autoimmune disease. These results can be used by other scientists investigating virulence factors of PCV2 and how it causes disease.
5. Demonstrated changes in cytokine levels in draining lymph nodes of pig lungs infected with pseudorabies virus. Pseudorabies virus (PRV) is a pathogen that is capable of producing fatal encephalitis in newborn pigs, respiratory disorders in growing pigs and reproductive failure in pregnant sows. PRV has been eradicated from commercial pigs in the U.S. but remains present in feral swine populations. Good biosecurity practices for commercial pig producers protect U.S. swine populations but it is important to be able to rapidly detect PRV should there be a breach of biosecurity. As part of studies designed to validate diagnostic tests for their ability to detect feral swine isolates, ARS scientists at the National Animal Disease Center in Ames, Iowa and their collaborators evaluated the pig's immune response following infection with PRV. Several cytokines (i.e., hormones of the immune system) including interferon (IFN)-alpha, interleukin (IL)-1beta, IL-12, and IFN-gamma were increased in draining lymph nodes of lungs of pigs infected with PRV as early as 1 day post infection (dpi) whereas IL-18 was decreased from 3 to 6 dpi. These findings demonstrate that a feral swine isolate of PRV can deviate the pig's early immune response such that it allows the virus to more efficiently establish an infection and cause disease. These results can be used by other scientists investigating how viruses evade host protection mechanisms so that more effective controls might be developed against viral diseases of swine.
6. Demonstrated enhanced pneumonia and disease in pigs vaccinated with an inactivated human-like swine H1N2 vaccine and challenged with the pandemic 2009 A/H1N1 influenza virus. Swine influenza A viruses (SIV) endemic in North America include H1N1, H1N2, and H3N2 derived from swine, avian and human influenza viruses with a triple reassortant internal gene constellation. Four H1 phylogenetic clusters designated as alpha, beta, gamma and delta co-circulate in U.S. swine. Although the 2009 pandemic H1N1 (pH1N1) is genetically related to the gamma cluster HA of North American viruses, substantial antigenic drift has occurred and considerable antigenic distance exists between pH1N1 and human-like delta-cluster SIV. Control of SIV has relied on the use of commercially available or autogenous inactivated influenza vaccines in the U.S. swine population. ARS scientists at the National Animal Disease Center in Ames, Iowa and their collaborators demonstrated an enhancement of disease and pathologic changes in the lungs of pigs vaccinated with a virus with the H1 HA derived from human seasonal influenza virus and challenged with 2009 A/H1N1 pandemic virus. These data suggest that the mismatched inactivated vaccine-induced immune response contributes to enhanced disease, consistent with previous findings by our group. This phenomenon is of potential concern for the U.S. swine population due to the concurrent circulation of genetically diverse H1 SIV among swine vaccinated with inactivated virus vaccines that are potentially mismatched to the circulating strains. These results emphasize the need for proper swine influenza vaccine strain selection for vaccination and can be used by scientists devising improved influenza vaccines for swine.
7. Demonstrated comparative virulence of 4 PRRSV isolates in swine. Porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory and reproductive disease and is the number one swine disease problem in the United States, causing an estimated $570 million annual loss. Using a high-through-put, massively parallel, DNA sequencing-by-synthesis approach ARS scientists at the National Animal Disease Center determined the full genome sequences of 4 field isolates of PRRSV and then conducted studies in pigs with three of these PRRSV isolates along with a different strain of known virulence to evaluate PRRSV replication and associated disease. One of the isolates studied replicated with faster kinetics than all others. From this work ARS scientists have selected one PRRSV strain for infectious clone development for future studies investigating mechanisms of pathogenesis and vaccinology.
8. Demonstrated stability of nsp2 deletion mutants following one or two passages through swine. Porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory and reproductive disease and is the number one swine disease problem in the United States, causing an estimated $570 million annual loss. ARS scientists at the National Animal Disease Center have been investigating the role of a particular region of the virus genome (nsp2) in the pathogenesis of PRRS in pigs and have identified regions of PRRSV nsp2 that may contain a virulence factor. A high-through-put, DNA sequencing approach was used to examine the stability of PRRSV mutants passed one or two times in pigs. The engineered mutations in each of 15 viruses were stable in the viruses recovered from the infected pigs demonstrating the application of this technology to verify the presence of particular mutations in the virus and their association with disease measures. These results can be used by scientists devising improved PRRSV vaccines for swine.
Cheung, A.K., Wu, G., Wang, D., Bayles, D.O., Lager, K.M., Vincent, A.L. 2010. Identification and Molecular Cloning of a Novel Porcine Parvovirus. Archives of Virology. 155(5):801-806.