Dr. Ying Fang - Emerging swine disease diagnostics and characterization: connecting basic research to real-world applications
•Download as PPTX, PDF•
2 likes•199 views
Emerging swine disease diagnostics and characterization: connecting basic research to real-world applications - Dr. Ying Fang, Kansas State University, from the 2017 North American PRRS/National Swine Improvement Federation Joint Meeting, December 1‐3, 2017, Chicago, Illinois, USA. More presentations at http://www.swinecast.com/2017-north-american-prrs-nsif-joint-meeting
Recommended
More Related Content
What's hot
What's hot (20)
Similar to Dr. Ying Fang - Emerging swine disease diagnostics and characterization: connecting basic research to real-world applications
Similar to Dr. Ying Fang - Emerging swine disease diagnostics and characterization: connecting basic research to real-world applications (20)
More from John Blue
More from John Blue (20)
Recently uploaded
Recently uploaded (20)
Dr. Ying Fang - Emerging swine disease diagnostics and characterization: connecting basic research to real-world applications
- 1. Ying Fang Kansas State University yfang@vet.k-state.edu
- 2. An integrated effort for combating emerging diseases Vaccine Challenge Animal Model Vaccine Candidate Genetics of Host Response Genes Confer Resistance DIVA Rowland and Fang, unpublished data Vaccine Design Isolation/Construction Infection/Disease Animal Model Molecular Diagnostic Tests Epidemiology/EcologyDiagnostic Reagent Production Antibody Detection Assays A Novel Viral Sequence Immunological Detection Molecular Detection In vitro Characterization
- 3. Molecular tools for pathogen discovery Courtesy of Crystal Jaing, Lawrance Livermore National Laboratory
- 4. Adaptation of Lawrence Livermore Microbial Detection Array at K-State Gardner, Jaing C, et al., 2010, BMC Genomics 11:668; Jaing C, 2017, unpublished Jaing Rowland Multiple probes target conserved and unique genomic regions of a microbial strain
- 5. Straightforward assay procedures and automated analysis algorithm with web interface Unknown Sample Isolate DNA/RNA Amplification if needed Label with fluorescent dye Hybridize sample on array Detect signal on fluorescent scanner Data analysis Courtesy of Crystal Jaing, Lowrance Livermore National Laboratory
- 6. Application of LLMDA for analysis of swine samples I: inoculum S: serum O: oral fluid T: tonsil
- 7. New diagnostic reagents and assays for emerging pathogens Molecular Diagnostic Tests Diagnostic Reagent Production Antibody Detection Assays A Novel Viral Sequence Immunological Detection Molecular Detection
- 8. Atypical porcine pestivirus (APPV) • mAbs against E2, Erns Porcine circovirus (PCV3, PCV2b/2d) • mAbs against N Porcine epidemic diarrhea virus (PEDV): • mAbs against S, S2, N, nsp8 Porcine parainfluenza virus-1 (PPIV-1): • mAbs against F Seneca Valley virus (SVV): • mAbs against VP1 and VP2 Monoclonal antibodies for emerging swine pathogens Anti-PEDV S2 Distal jejunum Anti-APPV Erns Lymph node
- 9. SHIC grants for q(RT)-PCR test development at K-State Bai J. et al., Detection and differentiation of PCV3 from PCV2a, PCV2b and PCV2d strains. Abstract #188 Li Y. et al., Development of a TaqMan quantitative RT-PCR test for porcine parainfluenza virus 1. Abstract #46 Liu X. et al., Multiplex real-time RT-PCR assay for simultaneous detection and differentiation of swine influenza C, D, and B viruses. Abstract #42 Peddireddi L. et al., Development of sensitive and reliable diagnostic assay to detect atypical porcine pestivirus. Abstract #54
- 10. SHIC grants for antibody detection assay at K-State Bai J., et al. Development and evaluation of antibody detection assay for PCV3 virus. Liu X., et al. Development of antibody detection assays for swine influenza B, C, D viruses. Marthaler D., et al. Development and validation of a rapid ELISA against porcine teschovirus in serum and oral fluid. Marthaler D., et al. Development and validation of ELISA against porcine sapelovirus in serum and oral fluid.
- 11. New Pathogen characterization Vaccine Challenge Animal Model Vaccine Design Isolation/Construction Infection/Disease Animal Model Diagnostic Reagent Production A Novel Viral Sequence Immunological Detection Molecular Detection In vitro Characterization
- 12. Characterization of newly emerging viral pathogens of swine
- 13. Application of reverse genetics in new pathogen characterization PRRSV Reverse Genetics
- 14. New Pathogen Characterization Application of Reverse Genetic Technology Fang & Snijder, 2010, Virus Res.
- 15. Construction of full-length cDNA infectius clone of SVV Fang & Snijder, 2010, Virus Res.pACYC177 Vector Chen et al., 2016, Virology, 497:111-124.
- 16. In vitro recovery and characterization of recombinant virus derived from SVV full-length cDNA clone pKS15-01-Clone Fang & Snijder, 2010, Virus Res. Chen et al., 2016, Virology, 497:111-124. Recombinant viruses possess similar in vitro growth properties with that of parental virus.0 12 24 36 48 0 3 6 9 Cloned virus EGFP virus Parental virus Hours post infection VirusTiter(logTCID50/ml) EGFP virusCloned virusParental virus
- 17. In vivo growth properties of the parental and recombinant SVV in pigs In vivo growth properties of parental and recombinant SVV in pigs
- 18. Clinical observations in nursery pigs SVV infectious clone as an useful tool to study the pathogenesis of vesicular disease dorsal snout lesion coronary band lesion Rectal temperature Clinical score Chen et al., 2016, Virology, 497:111-124. Vesicular lesions were NOT observed in recombinant virus-infected pigs; parental virus-infected pigs
- 19. New Pathogen Characterization Elucidating molecular mechanisms for the emergence of new pathogens Fang & Snijder, 2010, Virus Res.
- 20. Recombination Frequency in Enteroviruses Recombination frequencies: Intratypic >> Intertypic >> Interspecies Lauber C, Gorbalenya AE. J Virol. 2012 Simon-Loriere E, Holmes EC. Nat Rev Microbiol. 2011. Enterovirus Picornaviridae
- 21. NGS detected EVG-ToV recombinant in a diarrhea case from swine herd at North Carolina Enterovirus (EV) G-Torovirus recombinant ToV papain-like protease (PLP) 3Cpro cleavage site 3Cpro cleavage site Torovirus PLP (ToV-PLP) Shang, et al. 2017, J. Virol. 91(14) Ben Hause
- 22. Phylogeny of recombinant ToV-PLP to other viral PLPs ToV-PLP vs others AA identity Toroviral PLPs 54%-68% Coronaviral PLPs 16%-18% Picornaviral PLPs 12%-24% Shang, et al. 2017, J. Virol. 91(14)
- 23. EVG-ToV from TX Recombinant EVG-ToV travels around world EVG-ToV from Belgium
- 24. Structural modeling and comparison of ToV-PLP with other viral and host PLPs
- 25. ToV-PLP exhibits in vitro cleavage activities on K63-linked Poly-Ub chain and proISG15 K63 Ub3-7 ISG15 precursor (proISG15) Shang, et al. 2017, J. Virol. 91(14)
- 26. ToV-PLP functions as an innate immune antagonist Shang, et al. 2017, J. Virol. 91(14)
- 27. pCMV-EVG PLP-KO Construction of ToV-PLP knockout recombinant virus pCMV-EVG Shang, et al. 2017, J. Virol. 91(14)
- 28. Rescue of ToV-PLP knockout mutant in infected cells Shang, et al. 2017, J. Virol. 91(14)
- 29. In vitro growth kinetics of ToV-PLP knockout mutant Shang, et al. 2017, J. Virol. 91(14)
- 30. Deubiquitination and deISGylation activities of ToV-PLP in the context of viral infection Deubiquitination DeISGylation
- 31. ToV-PLP functions as an innate immune antagonist in the context of viral infection Shang, et al. 2017, J. Virol. 91(14) EVG gains function through the recombinant event that enhances its ability to evade host immunity Broad implications for other picornaviral and/or nidoviral species
- 32. An integrated effort for combating emerging diseases Vaccine Challenge Animal Model Vaccine Candidate Genetics of Host Response Genes Confer Resistance DIVA Rowland and Fang, unpublished data Vaccine Design Isolation/Construction Infection/Disease Animal Model Molecular Diagnostic Tests Epidemiology/EcologyAntigen, Antibody Production Antibody Detection Assays A Novel Viral Sequence Immunological Detection Molecular Detection In vitro Characterization
- 33. Summary Timely identify viral infection is a key approach to prevent the spreading of new pathogens to larger animal population; Adapt current/innovative technologies for new pathogen discovery and development of diagnostic assays; Understand viral pathogenesis and host immunity provide a basis to develop efficient approaches for emerging disease control; Understand the linkage between basic and applied science; Apply basic knowledge and research tools to field application; Open interaction, communication and collaboration among researchers, diagnosticians, and practitioners;
- 34. Kansas State University: • Gary Anderson • Jianfa Bai • Alfonso Clavijo • Ben Hause • Richard Hesse • Jamie Henningson • Xuming Liu • Douglas Marthaler • Saurav Misra • Megan Niederwerder • Lalitha Peddireddi • Raymond Rowland Funding Sources • Swine Health Information Center • US Department of Agriculture • K-State CVM, DMP and KSVDL • Midwest Veterinary Services Midwest Veterinary Services: • Kelly Lechtenberg • Robin Schroeder Fairmont Veterinary Clinic: • Chase Stahl Iowa State University: • Phillip Gauger • Pablo Pineyro South Dakota State University: • Travis Clement Smithfield Hog Production: • Emily Byers Carthage Veterinary Service: • John Prickett Lawrence Livermore National lab: • Crystal Jaing • James Tissen Acknowledgement Fang laboratory at K-State: • Yanhua Li • Pengcheng Shang • Rui Guo • Fangfeng Yuan • Russell Ransburgh • Longchao Zhu • Zhenhai Chen • Xinyu Yan • Sailesh Menon • Tao Wang • Yongning Zhang • Hewei Zhang • Kash Kruep • Alaina Littlejohn • William Patterson • Tori Matta
Editor's Notes
- 1
- As we all known, emerging swine infectious diseases have increased significantly during the past decade. The timely control and prevention of new disease outbreak require integration of a wide range of expertise and application of modern technologies. In fact, the development of new pathogen detection methods, pathogen characterization, and subsequently development of diagnostic assays, vaccines, and other control measure are all initiated from the discovery of a novel viral sequence.
- Molecular tools, including next generation sequencing and pathogen microarray, have played a key role in discovery of emerging pathogens. Kevin Lahmers is going to present the next generation sequencing technology shortly.
- I would like to give you a brief overview of the pathogen array technology that we currently adapted from Lawrence Livermore National Laboratory. Dr. Bob Rowland initially established the collaboration with Lawrence Livermore group. This is Crystal Jaing from Lawrence Livermore, who initially developed this pathogen array. The most recent version of the array can detect over 10K species of microbes simultaneously, including 4219 viruses and 5367 bacteria. The microarray targets both conserved and unique genomic regions of sequenced microbial strains. Multiple microbe targets provide confirmation and can serve as an internal validation mechanism. Such design provides an efficient method for microbial detection and discovery. It creates the opportunity to simultaneously query hundreds of thousands to several million sequence-specific DNA signatures, all in parallel.
- The microarray procedure is straight forward. Ease of use by diagnostic personnel is provided by an automated data analysis algorithm, Composite Likelihood Maximization Method (CLiMax), which is integrated with a web interface that enables LLMDA data analysis within 30 minutes. The LLMDA allows for the detection of any bacteria or virus on a tested sample within 24 hours. Automated analysis algorithm with web interface to speed up time to answer
- This is the initial study from Dr. Rowland and Jaing’s lab to investigate the utility of the Lawrence Livermore Microbial Detection Array to evaluate known and unknown microbes in serum, oral fluid, and tonsil from pigs experimentally coinfected with Porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine circovirus-2 (PCV-2). The oral fluid sample appears the most informative. I would like to point out here is that porcine parainfluenza virus was detected in this study, which was the first report of the PPIV appearance in the US swine herd.
- Once a novel viral sequence is discovered, new diagnostic reagents and assays are urgently needed for pathogen detection and characterization.
- This is a panel of monoclonal antibodies that we generated in recent years; and they have been distributed to different diagnostic and research labs.
- With the support from Swine Health Information Center, molecular detection assays have been developed for these emerging pathogens. If you are interested in knowing the details on any of these PCR tests, you can discuss with the PI of each project during poster presentation.
- And we just received the SHIC grants to develop antibody detection assays for this panel of emerging swine pathogens. We welcome collaboration on these projects.
- The availability of diagnostic reagents and assays allowed us to characterize these emerging pathogens; and we further applied basic research tools to facilitate in depth characterization of these viral pathogens.
- During the past three year, with a collaborative effort among researchers, diagnosticians, and field practitioners, we have been characterized a panel of emerging swine pathogens, including atypical porcine pestivirus, porcine parainfluenza virus, porcine circovirus, Seneca Valley virus (SVV), and recombinant enterovirus/torovirus.
- particularly, the application of reverse genetics system accelerated the structure-function analysis of viral RNA and protein sequences. This system also facilitates studies into host immune responses and viral immune evasion and pathogenesis. Recently, we adapted our PRRSV reverse genetics system for study the pathogenic mechanisms of Seneca Valley virus.
- Currently, reverse genetics system has been developed for SVV and EVG for disease characterization, which are based on PRRSV reverse genetics system as template (an old template for new application)
- To construct the full length cDNA infectious clone, we used a newly emerging SVV strain KS15-01. Five separate fragments ABCDE flanked with unique restriction enzyme sites were amplified from viral genome and assembled together into pACYC177 vector that we used for PRRSV infectious clone construction previously. To explore the potential use as a viral backbone for expressing marker genes, we also constructed an infectious clone expressing EGFP, in which EGFP was fused with teschovirus 2A element and then inserted between 2A and 2B site in SVV genome.
- Viable recombinant viruses were recovered from cell culture. Cloned virus and EGFP-tagged virus showed similar in vitro growth characteristics with that of parental virus.
- Subsequently, we measured virus titer to determine the level of viral replication and shedding in pigs. At 3 and 7 dpi, Similar levels of SVV titer were detected in all the samples from parental and cloned virus group pigs. At 14 dpi, all infected pigs almost cleared out of the viruses from blood circulation, nasal and fecal samples; however, it is worth to note here, a certain level of viral RNA still detected in the oral fluid samples. The oral fluid contains gingival crevicular fluid, which is an oral mucosal transudate derived from the oropharyngeal mucosa into the gingival crevices of the mouth. Since oropharyngeal tonsil is identified as the site for primary some picornavirus infection and viral (RNA/antigen) persistence, we are wondering whether oral fluid could be used as a diagnostic sample for detecting the persistence of SVV.
- So, these results that I showed so far suggest that cloned virus and parental virus have the similar in vitro and in vivo growth ability. However, when we compared the clinical diseases among the different groups of pigs, we found that the parental virus-infected pigs showed severe clinical signs, and vesicular lesions were observed on the pig snout and coronary bands, but vesicular lesions were not observed in recombinant virus-infected pigs. This provides us an important tool to further study the pathogenic mechanisms of vesicular disease. On the other hand, the cloned virus with decreased virulence could be a potential backbone for vaccine development in the future.
- Using the reverse genetics, we have been also exploring the molecular mechanisms underlying the emergence of new pathogens. I am going to show you an example in this area of study.
- Family Picornaviridae is well-known for its high genetic diversity. What we are interested in this study is the recombination of enterovirus. Recombination is known as an important driving force which shapes genetic diversity of enterovirus. Most of recombination events are intratypic. Less happening between different types or species.
- In 2015, fecal samples from neonatal pigs with diarrhea symptoms were submitted to Kansas State Veterinary Diagnostic Laboratory for diagnostic testing. Ben Hause initially performed NGS metagenomic sequencing and discovered a novel Enterovirus G containing approximate 600nt foreign gene insertion at 2C/3A junction region. Subsequently, we isolated the virus and confirmed this insertion by Sanger sequencing. BLAST search results showed that the inserted gene is most homologous to the torovirus PLP gene. Interestingly, the recombinant PLP gene in enterovirus genome is flanked by two possible 3C cleavage sites.
- The gene insertion shows 54% to 68% amino acid identity with torovirus PLPs. Phylogenetic analysis further confirmed that this foreign gene forms a well-supported clade with torovirus PLPs. So, we designated this foreign gene as torovirus-derived PLP.
- Almost at the same time, two independent studies also reported the identification of recombinant EVG-ToV chimera from swine herd at TX and Belgium. Phylogenetic analysis showed these novel EVG-ToV recombinants form a well-support clade, and interestingly, they are most closely related to a group of EVGs detected in 2012 from pigs in the ThanhBinh and CaoLanh areas of Vietnam, but these vietnam viruses do not contain the ToV gene insertion.
- To obtain insights into potential functions of the ToV PLP within the context of EVG, we explored the structural homology between this PLP and other viral and host proteases. Model prediction pinpointed structural similarities between the recombinant torovirus-derived PLP and the leader protease of the Foot and Mouth Disease Virus, although the torovirus-derived PLP and FMDV-Lpro only have 24% amino acid identity at the sequence level. Like FMDV leader protease, recombinant torovirus PLP adopts a minimal core papain-like fold with a characteristic arrangement of its Cys-His-Asp catalytic triad. Furthermore, torovirus-derived PLP also exhibits varied structural homology to coronavirus PLPs and host ubiquitin proteases. These proteases have been previously reported to possess deubiquitinating activity. The major distinction is that the minimal core domain of the ToV-PLP lack the primarily β-stranded fingers domains that bridge the N-terminal thumb subdomain and C-terminal palm.
- To further verify that the ToV-PLP domain could directly target the conjugation of polyubiquitin chain and ISG15. We carried out in vitro ubiquitin deconjugation assays. Purified ToV-PLP was co-incubated with K-48, K-63 and M1-linked polyubiquitin chains and ISG15 precursor. Results proved the robust cleavage activities of ToV-PLP on K63 and K48-link polyUb chain and ISG15 precursor. Here, I only show K63 result as a example. The cleavage activity on linear M1-linked polyubuiqitin is less efficient as K48 and K63.
- Since ubiquitination affects innate immune gene signaling pathways, we investigated whether ToV-PLP also influences innate immune gene expression in a host cell context. We first carried out luciferase reporter assays using an luciferase reporter plasmid that expresses the firefly luciferase under the control of the IFN-β promoter. As expected, expression of ToV-PLP significantly inhibited the luciferase gene expression, while the innate immunity suppression effect was decreased by catalytic triad mutations. Consistently, quantitative RT-PCR of IFNB1 showed a similar result.
- To explore the potential contribution of torovirus PLP recombination to growth and fitness of enterovirus, we generated a full-length cDNA infectious clone. Using this infectious clone, the PLP gene knockout mutant was generated.
- The rescue of PLP knockout virus was confirmed by IFA and western blot using specific monoclonal antibodies. As you can see, the western blot result shows that the ToV-PLP was deleted from the enterovirus polyprotein.
- In vitro characterization showed that the cloned virus showed similar growth kinetics with parental virus. While the PLP-knockout mutant has impaired growth property in comparison with cloned virus.
- To determine whether knock out of ToV-PLP affects innate immune suppression ability of the virus, we initially analyzed DUB and de-ISGylation activities of wild-type and mutant ToV-PLP knockout viruses. Compared to uninfected cells, the amount of Ub-conjugated proteins was decreased by about 95% in both parental and cloned virus. In contrast, Ub-conjugated proteins were elevated 8.7-fold in PLP-KO infected cells. Consistently, the ISG15-conjugated proteins in cells infected with vPLP-KO was elevated 47-fold compared to cells infected with parental and cloned virus.
- Finally, we investigated the effect of PLP knockout on innate immune gene expression in infected cells. Swine testicle cells were infected with parental, cloned and PLP-KO viruses, respectively. Results consistently showed parental and cloned virus largely suppressed the expression of type I interferons, type III interferons, IFN transcription factor IRF7, and IFN stimulated gene ISG15 to a similar level. In contrast, vPLP-KO exhibited a reduced ability to antagonize the expression of innate immune genes. So, these data indicate that ToV-PLP functions as a innate immune antagonist. EVG gains function through this cross-order recombination event, which enhances its ability to evade host immunity. This study provides an example in how recombination drives virus evolution, and has broad implications for other picornaviruses and nidoviruses.
- So, in summary, the studies that I just presented represent our onging collaborative effort to apply current knowledge and technologies for emerging swine disease control and prevention.
- We have been adapting current/innovative technologies for new pathogen discovery and development of diagnostic assays in order to timely identify the emerging viral infection; We have also applied our basic knowledge and research tools for understanding viral biology and host immunity, which provides a basis to develop efficient approaches for emerging disease control. Finally, which is most importantly, in order to achieve our goals, it is essential to open interaction, cooperation among researchers, practitioners and diagnosticians.