Tying the knot: Unraveling the intricacies of the coronavirus frameshift pseudoknot.

Understanding and targeting functional RNA structures towards treatment of coronavirus infection can help us to prepare for novel variants of SARS-CoV-2 (the virus causing COVID-19), and any other coronaviruses that could emerge via human-to-human transmission or potential zoonotic (inter-species) events. Leveraging the fact that all coronaviruses use a mechanism known as -1 programmed ribosomal frameshifting (-1 PRF) to replicate, we apply algorithms to predict the most energetically favourable secondary structures (each nucleotide involved in at most one pairing) that may be involved in regulating the -1 PRF event in coronaviruses, especially SARS-CoV-2. We compute previously unknown most stable structure predictions for the frameshift site of coronaviruses via hierarchical folding, a biologically motivated framework where initial non-crossing structure folds first, followed by subsequent, possibly crossing (pseudoknotted), structures. Using mutual information from 181 coronavirus sequences, in conjunction with the algorithm KnotAli, we compute secondary structure predictions for the frameshift site of different coronaviruses. We then utilize the Shapify algorithm to obtain most stable SARS-CoV-2 secondary structure predictions guided by frameshift sequence-specific and genome-wide experimental data. We build on our previous secondary structure investigation of the singular SARS-CoV-2 68 nt frameshift element sequence, by using Shapify to obtain predictions for 132 extended sequences and including covariation information. Previous investigations have not applied hierarchical folding to extended length SARS-CoV-2 frameshift sequences. By doing so, we simulate the effects of ribosome interaction with the frameshift site, providing insight to biological function. We contribute in-depth discussion to contextualize secondary structure dual-graph motifs for SARS-CoV-2, highlighting the energetic stability of the previously identified 3_8 motif alongside the known dominant 3_3 and 3_6 (native-type) -1 PRF structures. Using a combination of thermodynamic methods and sequence covariation, our novel predictions suggest function of the attenuator hairpin via previously unknown pseudoknotted base pairing. While certain initial RNA folding is consistent, other pseudoknotted base pairs form which indicate potential conformational switching between the two structures.

Shapify: Paths to SARS-CoV-2 frameshifting pseudoknot.

Multiple coronaviruses including MERS-CoV causing Middle East Respiratory Syndrome, SARS-CoV causing SARS, and SARS-CoV-2 causing COVID-19, use a mechanism known as -1 programmed ribosomal frameshifting (-1 PRF) to replicate. SARS-CoV-2 possesses a unique RNA pseudoknotted structure that stimulates -1 PRF. Targeting -1 PRF in SARS-CoV-2 to impair viral replication can improve patients' prognoses. Crucial to developing these therapies is understanding the structure of the SARS-CoV-2 -1 PRF pseudoknot. Our goal is to expand knowledge of -1 PRF structural conformations. Following a structural alignment approach, we identify similarities in -1 PRF pseudoknots of SARS-CoV-2, SARS-CoV, and MERS-CoV. We provide in-depth analysis of the SARS-CoV-2 and MERS-CoV -1 PRF pseudoknots, including reference and noteworthy mutated sequences. To better understand the impact of mutations, we provide insight on -1 PRF pseudoknot sequence mutations and their effect on resulting structures. We introduce Shapify, a novel algorithm that given an RNA sequence incorporates structural reactivity (SHAPE) data and partial structure information to output an RNA secondary structure prediction within a biologically sound hierarchical folding approach. Shapify enhances our understanding of SARS-CoV-2 -1 PRF pseudoknot conformations by providing energetically favourable predictions that are relevant to structure-function and may correlate with -1 PRF efficiency. Applied to the SARS-CoV-2 -1 PRF pseudoknot, Shapify unveils previously unknown paths from initial stems to pseudoknotted structures. By contextualizing our work with available experimental data, our structure predictions motivate future RNA structure-function research and can aid 3-D modeling of pseudoknots.

Effects of message delivery on cross-cultural biosecurity compliance: Insights from experimental simulations.

Background: Effective biosecurity communication of transmission risks and associated protective behaviors can reduce the impacts of infectious diseases in US animal agriculture. Yet, more than 1/5 of animal production workers speak a language other than English at home, and more than 40 percent are less than fluent in English. Communicating with these workers often involves translating into their primary languages. However, communication strategies targeting different cultural groups are not well-understood. Aims: To identify cross-linguistic risk communication strategies to facilitate compliance, we hypothesized that uncertainty avoidance cultures associated with the languages might affect biosecurity compliance contingent upon two additional covariates: (1) the risk of acquiring an infection and (2) the delivery method of the infection risk. Methods: We designed an experimental game simulating a line of separation (LOS) biosecurity tactic in a swine production facility, where participants were tasked with completing tasks inside and outside of the facility. Data were collected using games in the two most spoken languages in the US: English (EN) and Spanish (SP). Participants made binary decisions about whether to use the LOS biosecurity tactic based on the risk information provided. Mixed-effect logistic models were used to test the effects of covariates on using the LOS tactic by different language groups. Results: We found that biosecurity compliance rates of participants who took the experiments in the language associated with high and low uncertainty cultures showed no significant differences. However, there are substantial differences in how risk information is perceived between the two language groups under different infection risks. Specifically, and counterintuitively, SP participants were more risk-averse in gain scenarios but more risk-taking in loss scenarios. These differences are most pronounced in numeric risk messaging, indicating that numbers may not be the best way to communicate risk information regarding biosecurity cross-culturally. Conclusions: When confronted with situational biosecurity decisions, risk perception and preferences vary by language group. Effective biosecurity communication needs to account for these differences and not assume that direct translation of risk messages will result in comparable compliance.

Comparing behavioral risk assessment strategies for quantifying biosecurity compliance to mitigate animal disease spread.

Understanding the impact of human behavior on the spread of disease is critical in mitigating outbreak severity. We designed an experimental game that emulated worker decision-making in a swine facility during an outbreak. In order to combat contamination, the simulation features a line-of-separation biosecurity protocol. Participants are provided disease severity information and can choose whether or not to comply with a shower protocol. Each simulated decision carried the potential for either an economic cost or an opportunity cost, both of which affected their potential real-world earnings. Participants must weigh the risk infection vs. an opportunity cost associated with compliance. Participants then completed a multiple price list (MPL) risk assessment survey. The survey uses a context-free, paired-lottery approach in which one of two options may be selected, with varying probabilities of a high and low risk payouts. We compared game response data to MPL risk assessment. Game risk was calculated using the normalized frequency of biosecurity compliance. Three predominant strategies were identified: risk averse participants who had the highest rate of compliance; risk tolerant participants who had the lowest compliance rate; and opportunists who adapted their strategy depending on disease risk. These findings were compared to the proportion of risk averse choices observed within the MPL and were classified into 3 categories: risk averse, risk tolerant and neutral. We found weak positive correlation between risk measured in our experimental game compared to the MPL. However, risk averse classified participants in the MPL tended to comply with the biosecurity protocol more often than those classified as risk tolerant. We also found that the behavioral risk clusters and categorization via the MPL were significantly, yet weakly associated. Overall, behavioral distributions were skewed toward more risk averse choices in both the MPL and game. However, the MPL risk assessment wasn't a strong predictor for observed game behavior. This may indicate that MPL risk aversion metrics might not be sufficient to capture these simulated, situational risk aversion behaviors. Experimental games have a large potential for expanding upon traditional survey instruments by immersing participants in a complex decision mechanism, and capturing dynamic and evolving behavioral signals.

Connecting livestock disease dynamics to human learning and biosecurity decisions.

The acceleration of animal disease spread worldwide due to increased animal, feed, and human movement has driven a growing body of epidemiological research as well as a deeper interest in human behavioral studies aimed at understanding their interconnectedness. Biosecurity measures can reduce the risk of infection, but human risk tolerance can hinder biosecurity investments and compliance. Humans may learn from hardship and become more risk averse, but sometimes they instead become more risk tolerant because they forget negative experiences happened in the past or because they come to believe they are immune. We represent the complexity of the hog production system with disease threats, human decision making, and human risk attitude using an agent-based model. Our objective is to explore the role of risk tolerant behaviors and the consequences of delayed biosecurity investments. We set up experiment with Monte Carlo simulations of scenarios designed with different risk tolerance amongst the swine producers and we derive distributions and trends of biosecurity and porcine epidemic diarrhea virus (PEDv) incidence emerging in the system. The output data allowed us to examine interactions between modes of risk tolerance and timings of biosecurity response discussing consequences for disease protection in the production system. The results show that hasty and delayed biosecurity responses or slow shifts toward a biosecure culture do not guarantee control of contamination when the disease has already spread in the system. In an effort to support effective disease prevention, our model results can inform policy making to move toward more resilient and healthy production systems. The modeled dynamics of risk attitude have also the potential to improve communication strategies for nudging and establishing risk averse behaviors thereby equipping the production system in case of foreign disease incursions.

In Silico Analysis to Explore Lineage-Independent and -Dependent Transcriptional Programs Associated with the Process of Endothelial and Neural Differentiation of Human Induced Pluripotent Stem Cells.

Despite a major interest in understanding how the endothelial cell phenotype is established, the underlying molecular basis of this process is not yet fully understood. We have previously reported the generation of induced pluripotent stem cells (iPS) from human umbilical vein endothelial cells and differentiation of the resulting HiPS back to endothelial cells (Ec-Diff), as well as neural (Nn-Diff) cell lineage that contained both neurons and astrocytes. Furthermore, the identities of these cell lineages were established by gene array analysis. Here, we explored the same arrays to gain insight into the gene alteration processes that accompany the establishment of endothelial vs. non-endothelial neural cell phenotypes. We compared the expression of genes that code for transcription factors and epigenetic regulators when HiPS is differentiated into these endothelial and non-endothelial lineages. Our in silico analyses have identified cohorts of genes that are similarly up- or downregulated in both lineages, as well as those that exhibit lineage-specific alterations. Based on these results, we propose that genes that are similarly altered in both lineages participate in priming the stem cell for differentiation in a lineage-independent manner, whereas those that are differentially altered in endothelial compared to neural cells participate in a lineage-specific differentiation process. Specific GATA family members and their cofactors and epigenetic regulators (DNMT3B, PRDM14, HELLS) with a major role in regulating DNA methylation were among participants in priming HiPS for lineage-independent differentiation. In addition, we identified distinct cohorts of transcription factors and epigenetic regulators whose alterations correlated specifically with the establishment of endothelial vs. non-endothelial neural lineages.

Message Delivery Strategy Influences Willingness to Comply With Biosecurity.

As the Covid-19 pandemic continues worldwide, it has become increasingly clear that effective communication of disease transmission risks associated with protective behaviors is essential, and that communication tactics are not ubiquitously and homogenously understood. Analogous to Covid-19, communicable diseases in the hog industry result in millions of animal deaths and in the United States costs hundreds of millions of dollars annually. Protective behaviors such as preventative biosecurity practices are implemented to reduce these costs. Yet even with the knowledge of the importance of biosecurity, these practices are not employed consistently. The efficacy of biosecurity practices relies on consistent implementation and is influenced by a variety of behavioral factors under the umbrella of human decision-making. Using an experimental game, we collected data to quantify how different messages that described the likelihood of a disease incursion would influence willingness to follow biosecurity practices. Here we show that graphical messages combined with linguistic phrases demarking infection risk levels are more effective for ensuring compliance with biosecurity practices, as contrasted with either simple linguistic phrases or graphical messages with numeric demarcation of risk levels. All three of these delivery methods appear to be more effective than using a simple numeric value to describe probability of infection. Situationally, we saw greater than a 3-fold increase in compliance by shifting message strategy without changing the infection risk, highlighting the importance of situational awareness and context when designing messages.

Effects of Social Cues on Biosecurity Compliance in Livestock Facilities: Evidence From Experimental Simulations.

Disease outbreaks in U.S. animal livestock industries have economic impacts measured in hundreds of millions of dollars per year. Biosecurity, or procedures intended to protect animals against disease, is known to be effective at reducing infection risk at facilities. Yet, to the detriment of animal health, humans do not always follow biosecurity protocols. Human behavioral factors have been shown to influence willingness to follow biosecurity protocols. Here we show how social cues may affect cooperation with a biosecurity practice. Participants were immersed in a simulated swine production facility through a graphical user interface and prompted to make a decision that addressed their willingness to comply with a biosecurity practice. We tested the effect of varying three experimental variables: (1) the risk of acquiring an infection, (2) the delivery method of the infection risk information (numerical vs. graphical), and (3) the behavior of an automated coworker in the facility. We provide evidence that participants changed their behavior when they observed a simulated worker making a choice to follow or not follow a biosecurity protocol, even though the simulated worker had no economic effect on the participants' payouts. These results advance the understanding of human behavioral effects on biosecurity protocol decisions, demonstrating that social cues need to be considered by livestock facility managers when developing policies to make agricultural systems more disease resilient.

Using experimental gaming simulations to elicit risk mitigation behavioral strategies for agricultural disease management.

Failing to mitigate propagation of disease spread can result in dire economic consequences for agricultural networks. Pathogens like Porcine Epidemic Diarrhea virus, can quickly spread among producers. Biosecurity is designed to prevent infection transmission. When considering biosecurity investments, management must balance the cost of protection versus the consequences of contracting an infection. Thus, an examination of the decision making processes associated with investment in biosecurity is important for enhancing system wide biosecurity. Data gathered from experimental gaming simulations can provide insights into behavioral strategies and inform the development of decision support systems. We created an online digital experiment to simulate outbreak scenarios among swine production supply chains, where participants were tasked with making biosecurity investment decisions. In Experiment One, we quantified the risk associated with each participant's decisions and delineated three dominant categories of risk attitudes: risk averse, risk tolerant, and opportunistic. Each risk class exhibited unique approaches in reaction to risk and disease information. We also tested how information uncertainty affects risk aversion, by varying the amount of visibility of the infection as well as the amount of biosecurity implemented across the system. We found evidence that more visibility in the number of infected sites increases risk averse behaviors, while more visibility in the amount of neighboring biosecurity increased risk taking behaviors. In Experiment Two, we were surprised to find no evidence for differences in behavior of livestock specialists compared to Amazon Mechanical Turk participants. Our findings provide support for using experimental gaming simulations to study how risk communication affects behavior, which can provide insights towards more effective messaging strategies.

Emulating Agricultural Disease Management: Comparing Risk Preferences Between Industry Professionals and Online Participants Using Experimental Gaming Simulations and Paired Lottery Choice Surveys.

Mitigating the spread of disease is crucial for the well-being of agricultural production systems. Implementing biosecurity disease prevention measures can be expensive, so producers must balance the costs of biosecurity investments with the expected benefits of reducing the risk of infections. To investigate the risk associated with this decision making process, we developed an online experimental game that simulates biosecurity investment allocation of a pork production facility during an outbreak. Participants are presented with several scenarios that vary the visibility of the disease status and biosecurity protection implemented at neighboring facilities. Certain rounds allowed participants to spend resources to reduce uncertainty and reveal neighboring biosecurity and/or disease status. We then test how this uncertainty affects the decisions to spend simulation dollars to increase biosecurity and reduce risk. We recruited 50 attendees from the 2018 World Pork Expo to participate in our simulation. We compared their performance to an opportunity sample of 50 online participants from the survey crowdsourcing tool, Amazon Mechanical Turk (MTurk). With respect to biosecurity investment, we did not find a significant difference between the risk behaviors of industry professionals and those of MTurk participants for each set of experimental scenarios. Notably, we found that our sample of industry professionals opted to pay to reveal disease and biosecurity information more often than MTurk participants. However, the biosecurity investment decisions were not significantly different during rounds in which additional information could be purchased. To further validate these findings, we compared the risk associated with each group's responses using a well-established risk assessment survey implementing paired lottery choices. Interestingly, we did not find a correlation in risk quantified with simulated biosecurity investment in comparison to the paired lottery choice survey. This may be evidence that general economic risk preferences may not always translate into simulated behavioral risk, perhaps due to the contextual immersion provided by experimental gaming simulations. Online recruitment tools can provide cost effective research quality data that can be rapidly assembled in comparison to industry professionals, who may be more challenging to sample at scale. Using a convenience sample of industry professionals for validation can also provide additional insights into the decision making process. These findings lend support to using online experimental simulations for interpreting risk associated with a complex decision mechanism.

Risk Attitudes Affect Livestock Biosecurity Decisions With Ramifications for Disease Control in a Simulated Production System.

Hog producers' operational decisions can be informed by an awareness of risks associated with emergent and endemic diseases. Outbreaks of porcine epidemic diarrhea virus (PEDv) have been re-occurring every year since the first onset in 2013 with substantial losses across the hog production supply chain. Interestingly, a decreasing trend in PEDv incidence is visible. We assert that changes in human behaviors may underlie this trend. Disease prevention using biosecurity practices is used to minimize risk of infection but its efficacy is conditional on human behavior and risk attitude. Standard epidemiological models bring important insights into disease dynamics but have limited predictive ability. Since research shows that human behavior plays a driving role in the disease spread process, the explicit inclusion of human behavior into models adds an important dimension to understanding disease spread. Here we analyze PEDv incidence emerging from an agent-based model (ABM) that uses both epidemiological dynamics and algorithms that incorporate heterogeneous human decisions. We investigate the effects of shifting fractions of hog producers between risk tolerant and risk averse positions. These shifts affect the dynamics describing willingness to increase biosecurity as a response to disease threats and, indirectly, change infection probabilities and the resultant intensity and impact of the disease outbreak. Our ABM generates empirically verifiable patterns of PEDv transmission. Scenario results show that relatively small shifts (10% of the producer agents) toward a risk averse position can lead to a significant decrease in total incidence. For significantly steeper decreases in disease incidence, the model's hog producer population needed at least 37.5% of risk averse. Our study provides insight into the link between risk attitude, decisions related to biosecurity, and consequent spread of disease within a livestock production system. We suggest that it is possible to create positive, lasting changes in animal health by nudging the population of livestock producers toward more risk averse behaviors. We make a case for integrating social and epidemiological aspects in disease spread models to test intervention strategies intended to improve biosecurity and animal health at the system scale.

Willingness to Comply With Biosecurity in Livestock Facilities: Evidence From Experimental Simulations.

Disease in U.S. animal livestock industries annually costs over a billion dollars. Adoption and compliance with biosecurity practices is necessary to successfully reduce the risk of disease introduction or spread. Yet, a variety of human behaviors, such as the urge to minimize time costs, may induce non-compliance with biosecurity practices. Utilizing a "serious gaming" approach, we examine how information about infection risk impacts compliance with biosecurity practices. We sought to understand how simulated environments affected compliance behavior with treatments that varied using three factors: (1) the risk of acquiring an infection, (2) the delivery method of the infection risk message (numerical, linguistic and graphical), and (3) the certainty of the infection risk information. Here we show that compliance is influenced by message delivery methodology, with numeric, linguistic, and graphical messages showing increasing efficacy, respectively. Moreover, increased situational uncertainty and increased risk were correlated with increases in compliance behavior. These results provide insight toward developing messages that are more effective and provide tools that will allow managers of livestock facilities and policy makers to nudge behavior toward more disease resilient systems via greater compliance with biosecurity practices.