Enhancing Biosecurity Oversight in Malaysia with Dual Use Case Studies

1. Enhancing Biosecurity Oversight in Malaysia with Dual Use Case Studies

2. This case study exercise was developed by Gryphon Scientific and the Science and Technology Research Institute For Defence (STRIDE) for the workshop titled “Workshop on Enhancing Biosecurity Oversight in Malaysia with Dual Use Case Studies” This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License License: https://creativecommons.org/licenses/by-nc-sa/4.0/

3. Case Study Exercise Song Y et al. (2017) Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes.

4. Learning Goals and Objectives Learning goals: • Appreciate the role of risk analysis – including risk identification, risk assessment, risk management, and risk communication – in the conduct of responsible research • Develop skills for risk analysis of life sciences research • Learn to communicate about risk analysis with colleagues and other stakeholders • Learn to apply risk analysis and mitigation concepts to your own work Objectives: • Identify and assess risks in the context of life sciences research • Determine strategies to minimize risk, while maintaining the utility and quality of scientific work

5. Discussion • In groups, discuss the case study article: • Research objective • Background information • Methodology • Results and conclusions • If applicable, any risks and mitigation practices included in the research article • We will reconvene to review your findings

6. Research Objective • The researchers recoded and synthesized the poliovirus genome • Genome recoding: DNA or RNA sequences are substituted with different sequences that encode the same proteins • The researchers conducted the study to: • Determine how tolerant poliovirus is to large-scale genome recoding • Evaluate a proposed mechanism of +ssRNA virus assembly, which suggests that dozens of specific sequences spread throughout the genome determine packaging specificity • This mechanism was proposed in studies by other research groups

7. Background on Polio Polio • Polio is a disease caused by infection with poliovirus • Symptoms can range from flu-like illness to neurological issues including paralysis • Most infected people do not develop symptoms • Paralysis affects 1 in 200 people infected with poliovirus and can cause permanent disability and death • Poliovirus is highly infectious and transmissible between humans • Because of global efforts, polio has been eradicated in many countries • Since 1988, when the Global Polio Eradication Initiative was launched, cases have fallen 99% • Malaysia’s last reported case was in 1996 • However, polio is not eradicated worldwide, and resurgence of infections has been observed in several countries Poliovirus • Poliovirus is a small, nonenveloped virus in the family Picornaviridae • It has a small (~7500 base pairs), single-stranded RNA genome • In a 2002 study by Cello et al., poliovirus became the first infectious agent to be synthesized chemically, a landmark achievement in gene synthesis World Health Organization. Poliomyeltis. http://www.who.int/en/news-room/fact-sheets/detail/. Last Updated March 14 2018. Accessed July 26, 2018. World Health Organization. Polio Reported Cases. http://apps.who.int/immunization_monitoring/globalsummary/timeseries/tsincidencepolio.html. Last Updated July 15, 2018. Accessed July 26, 2018. Cello J, Paul AV, Wimmer E. (2002) Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. Science. 297 (5583): 1016-1018.

8. Background on Genome Recoding • Codons are three-base DNA sequences encoding an amino acid, or protein building block • Many codons are synonymous, meaning that changes can be made to the DNA without changing the amino acid sequence • So, some amino acids may be encoded by 2 or more codons Codons A and B are synonymous, but C is not amino acid 1 codon A amino acid 1 codon B amino acid 2 codon C • Different organisms have different codon bias: not all synonymous codons are translated with equal efficiency by the organism (or, in the case of viruses, the host organism) • Generally, more efficiently translated codons will be overrepresented Organism α Overrepresented: Underrepresented: Organism α Overrepresented: Underrepresented: codon B codon A codon A codon B • Different organisms also have different codon pair bias: not all synonymous sets of two codons are translated with equal efficiency • Codon pair bias sometimes can run counter to codon bias codon C codon C In genome recoding, the genome sequence of a virus (or organism) is rewritten and synonymous codons or codon pairs are substituted such that the same proteins are encoded by different underlying sequences that may have different translation efficiency. These changes can result in changes to the functionality of the virus or organism.

9. Methodology Recoded (modified) genome design: • Using a computer program, designed several modified variants of poliovirus: • Max variants with substitution of synonymous codon pairs that are overrepresented • Min variants with substitution of synonymous codon pairs that are underrepresented • SD (“scrambling design”) variants with substitution of synonymous codons by random shuffling • For each type of variant, several designs were created, in which the modification was applied to different segments of the genome Viral synthesis: • Chemically synthesized DNA fragments were ordered from DNA synthesis providers, and then assembled into complementary DNA (cDNA) constructs using a methodology similar to the Cello 2002 methodology • cDNA was transcribed into the RNA genome, in vitro, using RNA polymerase • RNA genome was transfected into a human cell line (HeLa R19) and incubated until cytopathic effects were observed, which indicated production of poliovirus

10. Methodology (2) Growth properties: Growth phenotype was determined based on the number of passages through a human cell line (HeLa R19) until full cytopathic effects were observed (if at all); viral titer and plaque size after transfection also were measured Specific infectivity: Specific infectivity, defined as virus particles per plaque-forming unit (PFU), were determined by infecting two different human cell lines (HeLa R19 and A549) with wild-type virus and two variants of modified virus Other assays: Variants of modified viruses were characterized by DNA sequencing, protein measurement, and temperature sensitivity; a software program was used to model RNA structures

11. Now, apply the risk analysis framework

12. Risk Analysis Framework Scientific Activities Risk Analysis Risk Identification Plan Project Risk Assessment Continuously identify, assess, manage risks Risk Management Conduct Research Publish or Present Findings Risk Communication Although risk analysis can be distilled into four steps – risk identification, risk assessment, risk management, and risk communication – the process is continuous and linked with the scientific process

13. Risk Identification Song Y et al. (2017) Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

14. Risk Assessment Song Y et al. (2017) Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

15. Risk Management Song Y et al. (2017) Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

16. Results and Conclusions Results Growth: • Wild-type viruses displayed full cytopathic effects (CPE) upon transfection, after 0 passages • Min variants all grew less well than the wild-type virus. They either failed to display CPE or displayed CPE after 3 passages • SD variants grew less well than the wild-type viruses or comparably well to the wild-type virus, displaying CPE at 0-3 passages, with poorer growth in variants with modifications across more genome domains • Max variants all grew comparably well to the wild-type virus, displaying CPE at 0 passages and mostly large plaques, even with more genome domains (2-3 of 3 domains) modified • Plaque size may be a marker of virulence Specific infectivity: • A Min variant, with modification in 1 of 3 genome domains, displayed specific infectivity < 1% of the specific infectivity of the wild-type virus • A Max variant, with modifications throughout 3 of 3 genome domains (1297 nucleotides changed in the ~7500 nucleotide genome), displayed specific infectivity similar to the specific infectivity of the wild-type virus, 84% and 101% in two different human cell lines.

17. Results and Conclusions (2) Conclusions • Poliovirus is fairly tolerant to genome recoding. The virus can retain much of its growth and infectivity capability after substitution with synonymous codons and codon pairs, as long as the substitution scheme is designed carefully to maintain high levels of overrepresented/preferred codons • According to the authors, this study suggests that a proposed mechanism of +ssRNA virus assembly, in which dozens of specific sequences spread throughout the genome would determine packaging specificity, is likely incorrect • Heavily modified variants that have lost the RNA sequence and RNA secondary structures implicated in this model still display efficient replication and packaging

18. Risk Communication Song Y et al. (2017) Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes

19. Reflection Think about how to apply the risk analysis framework to your own research, using a past, current, or planned project You may use the questions below to guide your thinking:

20. Materials were adapted from: • Workshop on Enhancing Biosecurity Oversight in Malaysia with Dual Use Case Studies by Gryphon Scientific and STRIDE, licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License • License: https://creativecommons.org/licenses/by-nc-sa/4.0/ • This license allows sharing and adaptation of the materials. • International Engagement: Secure Science, Technology, and Research - BMENA Case Studies by American Association for the Advancement of Science’s Center for Science, Technology and Security Policy (AAAS CSTSP), licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 United States License • International Engagement: Secure Science, Technology, and Research - BMENA Case Studies: https://www.aaas.org/report/BMENA-risk-analysis-training • License: https://creativecommons.org/licenses/by-nc-sa/3.0/us/ • This license allows sharing and adaptation of the materials. References • Cello J, Paul AV, Wimmer E. (2002) Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. Science. 297 (5583): 1016-1018. • Song Y et al. (2017) Limits of variation, specific infectivity, and genome packaging of massively recoded poliovirus genomes. Proc Natl Acad Sci U S A. 114 (41): E8731-E8740. • World Health Organization. Poliomyeltis. http://www.who.int/en/news-room/fact-sheets/detail/. Last Updated March 14 2018. Accessed July 26, 2018. • World Health Organization. Polio Reported Cases. http://apps.who.int/immunization_monitoring/globalsummary/timeseries/tsincidencepolio.html. Last Updated July 15, 2018. Accessed July 26, 2018.