Location: Virus and Prion Research
2017 Annual Report
Objectives
1. Identify pathogenic mechanisms of swine Nidovirales, including identifying the pathogenic mechanisms of Porcine Respiratory and Reproductive Syndrome Virus (PRRSV), and the pathogenic mechanisms of Porcine Epidemic Diarrhea Virus (PEDV).
2. Discover and assess vaccines that can reduce or prevent economic losses from swine viral diseases, including identifying mechanisms to modulate innate and adaptive immune responses to swine viral pathogens and investigating technologies to override vaccine interference from passively acquired immunity.
3. Determine evolutionary antigenic and pathogenic properties of economically significant swine viral pathogen, including identifying and monitoring genetic and antigenic evolution in Nidovirales and emerging viral pathogens.
4. Identify mechanisms of pathogenesis, transmission, and immunity for emerging viral diseases of swine, starting with evaluating the onset and duration of Seneca A virus immunity in swine.
Approach
This research project will focus on swine diseases caused by viruses that are top concerns for United States pork producers: porcine reproductive and respiratory syndrome, porcine coronaviruses, and new and emerging diseases such as Seneca A virus. These pathogens will be examined in the laboratory as well as in swine disease models to investigate mechanisms of pathogenesis, transmission, immunity, evolution and methods of intervention. Animal experiments to be conducted involve one of three general designs: 1) disease pathogenesis and transmission studies, 2) vaccine efficacy studies, 3) sow/neonatal studies. 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.
Progress Report
In support of Objective 1, to identify the pathogenic mechanisms of porcine respiratory and reproductive syndrome virus (PRRSV) on the activation of the immune response, RNA transcript analyses on infected and control monocyte-derived cells were completed. Such research uncovered networks of predicted protein-protein interactions and biological processes related to both low virulence and highly pathogenic PRRSV infection. The analysis revealed the ability of PRRSV to affect cell activation. Genes showing variability in expression were related to cellular structure and inflammatory immune responses. These results supply novel insight into the interplay of PRRSV pathogenicity and immune system evasion.
In support of Objective 1, expression analysis of the type and quantity of small non-coding RNAs between healthy and PRRSV-infected pigs showed the largest change in gene expression occurs 3 days after infection and affects all small non-coding types. Numerically, from 3 to 8 days post-infection, the transfer RNA fragments demonstrated a lower reduction than the micro RNAs, and were more stable than microRNA or other small non-coding RNA types. This information helps in understanding how gene functions in the pig can become dysregulated by PRRSV and how the pig’s immune system responds to the virus.
In support of Objective 1, to define regions of PRRSV that influence virus pathogenesis, full-genome clones of PRRSV were produced that possessed changes in nonstructural protein 2 (nsp2). Nsp2 possesses a domain involved in immune evasion, it exists in several forms and varies dramatically between different strains of PRRSV. In collaboration with scientists from the University of Georgia, we exchanged the nsp2 segments between viruses of differing virulence and introduced mutations to assess any potential role these segments may have in determining virulence. The viruses grow in tissue culture and the next experiments will involve administering the mutant viruses in vivo to assess the impact of changes in nsp2 in pigs and their potential for use in preventative measures.
In support of Objective 1, ARS researchers at Ames, Iowa working with scientists from the University of Georgia, studied the enzymatic activity of a domain in nonstructural protein 3 of porcine epidemic diarrhea virus (PEDV) and porcine delta coronavirus (PDCoV). The research found striking differences in this domain between the two viruses, and will be used to further investigate viral virulence traits.
In support of Objective 1, to develop tools to investigate coronavirus pathogenesis, a complete copy of the genome of PDCoV was cloned into a replicating plasmid. In addition, along with researchers from Loyola University and the University of Georgia, a recombinant PEDV was produced and mutated in a specific region of nonstructural protein 3 to reduce the ability of the protease to inhibit host responses.
In support of Objective 2, a modified attenuated vaccine of PRRSV was used to prepare novel candidate vaccine constructs. One region of the attenuated vaccine was amplified and will be used to join to another section of the genome of more contemporary viruses found on U.S. swine farms.
In support of Objective 2, animal studies were conducted to investigate field observations that traditional use of live-virus inoculation in breeding age gilts to induce PRRSV protection is now failing because of some inherent change in contemporary field isolates.
In support of Objective 2, conducted genome-wide RNA profiling of signature genes in activated porcine monocytic innate immune cells to identify mechanisms that modulate innate and adaptive immune responses to swine viral pathogens. From this research, the diverse antiviral properties that interferon and interferon-stimulated gene families have on swine viral pathogens were determined. The data revealed different expression levels of inflammatory cytokines, chemokines, receptors, interferon-regulatory factors and interferon-stimulated gene families in PRRSV-infected macrophages setting the stage for development of novel therapies and vaccine strategies.
In support of Objective 2, expression analysis of the type and quantity of small non-coding RNAs was completed comparing healthy and PRRSV-infected pigs to elucidate when the largest change in gene expression occurs, and if all categories of small non-coding RNAs are affected. Transfer RNA fragments were less reduced in number than the microRNAs and appear to be more stable across time points than microRNA or other non-coding RNAs. This information helps in understanding how gene function in the pig can become dysregulated by PRRSV, in conjunction with how the pig's immune system responds to the virus.
In support of Objective 3, PRRSV open reading frame 5 nucleotide sequences of field strains detected in the United States by scientists at Iowa State University were used by ARS researchers at Ames, Iowa to compare them to those sequences from China deposited at the National Center for Biotechnology Information. The data obtained indicate that the United States and China are experiencing different rates of PRRSV evolution.
In support of Objective 4, the entire genome of 17 PRRSV isolates obtained from scientists at Iowa State University were sequenced and it was discovered that the isolates, originally thought to be similar based on a small region of the genome, were very dissimilar at the whole genome level. The isolate genomes were analyzed for evidence of viral recombination and several instances of viral recombination were detected in most of the 17 isolates, showing that viral recombination occurs at a high frequency in infected swine herds. Four genomically distinct isolates were chosen for swine infection experiments that resulted in a spectrum of diseases, two of which were much more pathogenic than the others, and one which produced very mild disease.
Accomplishments
1. Described recombination within a set of diverse porcine reproductive and respiratory syndrome virus (PRRSV) field isolates. ARS researchers in Ames, Iowa processed 17 isolates that had emerged in the United States in 2015 for next generation sequencing and assembled them into complete viral genomes. Results revealed that the viruses were very dissimilar in all parts of their genomes. Further evolutionary analyses, comparing the isolates to unique prototype index genomes, revealed several common areas where the viruses had recombined. The data indicates the remarkable ability of PRRSV to undergo high frequency recombination in the field. Three viral isolates were used to challenge swine. One isolate was shown to produce enhanced clinical disease. The viral strain will be used in our formulation of new vaccine candidates.
2. Investigated the ecology and protective immune response of Senecavirus A (SVA). SVA is a swine virus that has recently emerged as a problem in U.S. swine. ARS researchers at Ames, Iowa demonstrated that 1) wild-type SVA infection can induce a protective immune response with a duration for at least 4-5 months, 2) SVA transmission can occur for at least 2 weeks post-infection to age-matched sows, and 3) environmental contamination may be a likely source of SVA detected in sows moving from farm to eventual slaughter. This information will help in developing response strategies at slaughter houses, which can help in developing control programs on the farm.
3. Demonstrated the utility of modifications to nonstructural protein 2 (nsp2) porcine reproductive and respiratory syndrome virus (PRRSV). ARS researchers at Ames, Iowa investigated the stability of mutant viruses. Next generation sequencing showed that three inserted small tags were all stable (except for one mutant) over 10 cell passages. The rate of viral replication and the plaque size of all mutants were not affected. However, insertion of any tag near the beginning of the protein could be detected in several viral RNAs, whereas tag insertion near the end of the protein was only detected in genome length viral RNA. In addition, infected cell immunofluorescence examination suggests that the two different nsp2 insertions resulted in proteins localizing to discrete areas around the cell nucleus. The mutant viruses will be used to investigate the role of nsp2 in pathogenesis, and this knowledge will enhance our ability to produce better vaccines.
4. Characterized proteases of porcine epidemic diarrhea virus (PEDV) and porcine delta coronavirus (PDCoV). ARS researchers in Ames, Iowa worked with investigators at University of Georgia to investigate the preferred substrates of proteases located in nonstructural protease 3. These domains cleave the substrates off of necessary immune molecules that results in a poor immune response. PEDV was found to prefer a substrate called ubiquitin whereas PDCoV preferred a protein called immune stimulating gene product 15. These findings may aid in future vaccine design.
5. Produced cDNA copies of porcine epidemic diarrhea virus (PEDV) and porcine delta coronavirus (PDCoV). ARS researchers in Ames, Iowa developed strategies to clone PEDV and PDCoV into a replicating vector for mammalian cells. After gene synthesis, three individual overlapping segments of PDCoV and the vector were assembled into one clone. Collaborators from Loyola University in Chicago developed a method to enzymatically connect fragments of cDNA to synthetically produce genomic length PEDV RNA which can be used to generate infectious PEDV. This live virus can be used to study the effect of targeted viral mutations that may contribute to the production of novel vaccines.
6. Analyzed gene expression changes during pseudorabies virus (PRV) infection. Pseudorabies virus causes severe disease in swine and is an economically important disease or disease threat in most swine producing countries. As the pig responds to a PRV infection, changes in metabolism reflect changes in the expression of specific genes. Gene expression describes the regulation of the pig's metabolic processes, and gene expression profiling is the process of determining which genes are active in a specific cell or group of cells. Variation in gene expression profiles can act as an important indicator of disease or predisposition to disease. ARS researchers at Ames, Iowa characterized core gene expression changes during PRV infection, giving insight into how the virus affects the host and how the host is trying to combat the infection, and leading to a greater understanding of how to build better vaccines which may help in the control of pseudorabies.
7. Identified signature gene response of immune cells caused by porcine reproductive and respiratory syndrome virus (PRRSV). Monocytic cells are one of the cell types that are intricately involved in the animal's response to disease. Following infection, the monocytic cell becomes activated which can occur by direct contact with an infectious agent, or indirectly through stimulation of the cell by specific proteins produced by other cells in the body. Activated monocytic cells then become polarized (meaning the cell has developed a certain response against a virus or bacteria). Here, ARS researchers at Ames, Iowa studied the direct involvement of polarization of monocytes during infection. Understanding the complex nature of the protective immune response may be critical to improving vaccines.
8. Annotation of interferon (IFN) gene families in swine and across 155 animal genomes. Innate immune interferons (IFNs), particularly type I IFNs, are primary mediators regulating antiviral immunity. These antiviral cytokines have evolved remarkable molecular and functional diversity to confront ever-evolving viral threats. ARS researchers at Ames, Iowa showed that pigs have the largest and an expanding type I IFN family, consisting of nearly 60 functional genes that encode seven IFN subtypes including multigene subtypes of one class of IFN (IFN-alpha). Whereas subtypes such as IFN-alpha and IFN-beta have been widely studied, the unconventional IFN-omega subtype has barely been investigated. Cross-species comparison revealed that porcine IFN-omega has evolved several novel features such as emerging forms that have much higher antiviral potency than IFN-alpha, high antiviral activity in cells of humans and other mammalian species, and potential action through unusual pathways. This study revealed the antiviral potency of porcine IFN-omega.
9. Described the interaction of type I interferons (IFNs) with a signaling pathway that underlies porcine reproductive and respiratory syndrome virus (PRRSV) infection. Targeting on macrophages, ARS researchers at Ames, Iowa elaborated the direct involvement of the mTOR (mechanistic target of rapamycin) signaling pathway during PRRSV infection. Comprehensive understanding of the immunological impact may become increasingly important to understand host-virus interactions of existing and emerging pathogens, with application to the development of novel therapies and vaccine strategies.
Review Publications
Crawford, K., Kulshreshtha, V., Lager, K.M., Miller, L.C., Faaberg, K.S. 2016. Status of vaccines for porcine epidemic diarrhea virus in the United States and Canada. Virus Research. 226:108-116.
Fleming, D.S., Miller, L.C. 2016. Leading edge analysis of transcriptomic changes during pseudorabies virus infection. Genomics Data. 10:104-106.
Gerber, P.F., Xiao, C.T., Lager, K., Crawford, K., Kulshreshtha, V., Cao, D., Meng, X.J., Opriessnig, T. 2016. Increased frequency of porcine epidemic diarrhea virus shedding and lesions in suckling pigs compared to nursery pigs and protective immunity in nursery pigs after homologous re-challenge. Veterinary Research. 47(1):118.
Liu, Q., Miller, L.C., Blecha, F., Sang, Y. 2017. Reduction of infection by inhibiting mTOR pathway is associated with reversed repression of type I interferon by porcine reproductive and respiratory syndrome virus. Journal of General Virology. 98(6):1316-1328. doi:10.1099/jgv.0.000802.
Niederwerder, M.C., Nietfeld, J.C., Bai, J., Peddireddi, L., Breazeale, B., Anderson, J., Kerrigan, M.A., An, B., Oberst, R.D., Crawford, K., Lager, K.M., Madson, D.M., Rowland, R.R., Anderson, G.A., Hesse, R.A. 2016. Tissue localization, shedding, virus carriage, antibody response, and aerosol transmission of porcine epidemic diarrhea virus following inoculation of 4-week-old feeder pigs. Journal of Veterinary Diagnostic Investigation. 28(6):671-678.
