Pandemic Potential Research Articles

Thapsigargin: An Unlikely Drug in the Battle Against Viral Pandemics

Antiviral resistance is an increasingly categorized phenomenon, and with the high pandemic potential of emergent pathogens such as coronaviruses, the need for novel therapeutic agents is dire. Thapsigargin, a potent endoplasmic reticulum stress inducer found in the Mediterranean region, has shown significant promise in combatting replication of several viruses including coronaviruses by harnessing the host's innate immune system, even being more efficacious than some antivirals currently used. There is a major gap in knowledge regarding the exact mechanism of action that permits Thapsigargin to be an incredible endoplasmic reticulum stress inducer and an potent antiviral. We hypothesize that interferon 1 production and SERCA-inhibition caused by thapsigargin mediates the antiviral effects observed. This review outlines what is currently known about this novel compound, the gap in research, and details methodologies including gene expression analyses, protein quantification, and mammalian cell culturing, to address the gap and answer the hypothesis.

Characterization of H5N1 avian influenza virus isolated from bird in Russia with the E627K mutation in the PB2 protein

Currently A(H5Nx) avian influenza viruses are globally widespread and continue to evolve. Since their emergence in 2020 novel highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b reassortant viruses have become predominant in the world and caused multiple infections in mammals. It was shown that some of A(H5N1) viruses mostly isolated from mammals contain an E627K mutation in the PB2 protein which can lead to adaptation of influenza viruses to mammalian cells. In 2023 in Russia we have isolated two highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses from birds one of which contained an E627K mutation in the PB2 protein. This virus had increased virulence in mice. Limited airborne transmission of the virus with the PB2-E627K mutation was observed between ferrets, in which infectious virus was detected in the nasal washings of the three of the twelve recipient ferrets, and clinical symptoms of the disease were observed in one case. Both viruses showed dominant binding to avian-type sialoside receptors, which was most likely the reason for the limited transmissibility. Thus, this study indicates a possible limited increase in the pandemic potential of A(H5N1) 2.3.4.4b viruses and highlights the importance of continuous avian influenza surveillance for pandemic preparedness and response.

Gender differences in correlation of biochemical parameters with the severity of COVID pneumonia and the need for oxygen/mechanical support

Background:COVID-19 pandemic had caused a global medical, economic and social problems. High infection rate, heterogenous presentation, lack of previous data and no standardized treatment had led to a need for further analysis, in order to be prepared for any potential new pandemics. We analyzed any possible correlation between gender, laboratory findings and severity of disease with need for oxygen or mechanical ventilation support. Methods: 100 patients with confirmed SARS-CoV-2 virus infection enrolled. Baseline characteristics that included age, sex, smoking history, BMI, oxygen therapy or mechanical ventilation support need were recorded. Type and severity of radiological finding determined by chest CT scan. Results: Majority of our patients were over 61 years old (58.6%), male (57.6%), and had severe radiological finding (bilateral pneumonia 29.3%, ARDS 35.4%), with only 20.2% had not required any oxygen supplementation. Regarding gender and laboratory findings, men have shown statistically significant higher values of CRP, lymphocytes, LDH and ferritin (96.4 vs 87.1, p=0.014; 1.17 vs 0.84, p=0.048; 674.8 vs 609.1, p=0.031; 1263 vs 578.4, p=0.001, respectfully). Severe radiological finding had shown positive correlation with the need for HFNC and/or (N)IMV (p=0.021 and p=0.032. respectfully), as well as with higher values of WBC, LDH and ferritin (p=0.042, p=0.035 and p=0.017. respectfully). Conclusions: There is a difference between presentation of the disease, as well as analyzed laboratory markers between sexes. The difference is most likely multifactorial and should require further research in order to discover other risk and prognostic factors.

Impact of the 100 days mission for vaccines on COVID-19: a mathematical modelling study

The COVID-19 pandemic has underscored the beneficial impact of vaccines. It also highlighted the need for future investments to expedite an equitable vaccine distribution. The 100 Days Mission aims to develop and make available a new vaccine against a future pathogen with pandemic potential within 100 days of that pathogen threat being recognised. We assessed the value of this mission by estimating the impact that it could have had on the COVID-19 pandemic. Using a previously published model of SARS-CoV-2 transmission dynamics fitted to excess mortality during the COVID-19 pandemic, we projected scenarios for three different investment strategies: rapid development and manufacture of a vaccine, increasing manufacturing capacity to eliminate supply constraints, and strengthening health systems to enable faster vaccine roll-outs and global equity. Each scenario was compared against the observed COVID-19 pandemic to estimate the public health and health-economic impacts of each scenario. If countries implemented non-pharmaceutical interventions (NPIs) as they did historically, the 100 Days Mission could have averted an estimated 8·33 million deaths (95% credible interval [CrI] 7·70-8·68) globally, mostly in lower-middle income countries. This corresponds to a monetary saving of US$14·35 trillion (95% CrI 12·96-17·87) based on the value of statistical life-years saved. Investment in manufacturing and health systems further increases deaths averted to 11·01 million (95% CrI 10·60-11·49). Under an alternative scenario whereby NPIs are lifted earlier on the basis of vaccine coverage, the 100 Days Mission alone could have reduced restrictions by 12 600 days (95% CrI 12 300-13 100) globally while still averting 5·76 million deaths (95% CrI 4·91-6·81). Our findings show the value of the 100 Days Mission and how these can be amplified through improvements in manufacturing and health systems equity. However, these investments must be enhanced by prioritising a more equitable global vaccine distribution. Schmidt Science Fellowship in partnership with the Rhodes Trust, WHO, UK Medical Research Council, Coalition for Epidemic Preparedness Innovations.

Expression of Toll-like Receptor Genes and Antiviral Cytokines in Macrophage-like Cells in Response to Indole-3-carboxylic Acid Derivative

Ongoing outbreaks and often rapid spread of infections caused by coronaviruses, influenza, Nipah, Dengue, Marburg, monkeypox, and other viruses are a concern for health authorities in most countries. Therefore, the search for and study of new antiviral compounds are in great demand today. Since almost all viruses with pandemic potential have immunotoxic properties of various origins, particular attention is paid to the search and development of immunomodulatory drugs. We have synthesised a new compound related to indole-3-carboxylic acid derivatives (hereinafter referred to as the XXV) that has antiviral and interferon-inducing activity. The purpose of this work is to study the effect of the XXV on the stimulation of the expression of toll-like receptor genes, interferons, and immunoregulatory cytokines in a macrophage-like cell model. In this study, real-time PCR methods were used to obtain data on the transcriptional activity of genes in macrophage-like cells. Stimulation of the genes of toll-like receptors TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9 was detected. A high-fold increase in stimulation (from 6.5 to 16,000) of the expression of the TLR3 and TLR4 genes was detected after 4 h of exposure to the XXV. Increased activity of interferon (IFNA1, IFNA2, IFNB1, IFNK, and IFNλ1) genes with simultaneous stimulation of the expression of interferon receptor (IFNAR1 and IFNAR2) genes and signalling molecule (JAK1 and ISG15) genes was detected. Increased fold stimulation of the expression of the cytokine genes IL6, TNFA, IL12A, and IL12B was also observed. Thus, it is shown that the XXV is an activator of TLR genes of innate immunity, which trigger signalling mechanisms of pathogen “recognition” and lead to stimulation of the expression of genes of proinflammatory cytokines and interferons.

Acquisition of a multibasic cleavage site does not increase MERS-CoV entry into Calu-3 human lung cells.

Human-to-human transmission of the highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) is currently inefficient. However, there is concern that the virus might mutate and thereby increase its transmissibility and thus pandemic potential. The pandemic SARS-CoV-2 depends on a highly cleavable furin motif at the S1/S2 site of the viral spike (S) protein for efficient lung cell entry, transmission, and pathogenicity. Here, by employing pseudotyped particles, we investigated whether augmented cleavage at the S1/S2 site also increases MERS-CoV entry into Calu-3 human lung cells. We report that polymorphism T746K at the S1/S2 cleavage site or optimization of the furin motif increases S protein cleavage but not lung cell entry. These findings suggest that, unlike what has been reported for SARS-CoV-2, a highly cleavable S1/S2 site might not augment MERS-CoV infectivity for human lung cells.IMPORTANCEThe highly cleavable furin motif in the spike protein is required for robust lung cell entry, transmission, and pathogenicity of SARS-CoV-2. In contrast, it is unknown whether optimization of the furin motif in the spike protein of the pre-pandemic MERS-CoV increases lung cell entry and allows for robust human-human transmission. The present study indicates that this might not be the case. Thus, neither a naturally occurring polymorphism that increased MERS-CoV spike protein cleavage nor artificial optimization of the cleavage site allowed for increased spike-protein-driven entry into Calu-3 human lung cells.

Human-forest proximity and the risk infectious diseases in a changing world

Abstract Background Emerging Infectious Diseases (EIDs) pose a significant threat to global human health; they can lead to epidemics and pandemics with severe repercussions on human life, economic performance, and societal well-being. Despite the recognized negative impact of the EIDs, current research lacks a comprehensive global framework to assess the significance and interaction of various environmental and anthropogenic factors driving emergence of these diseases. Methods In this study, we examine both environmental and anthropogenic factors and their relation to the emergence of EIDs, focusing on historical trends from 1975 to 2020. We focus on 10 diseases with pandemic and epidemic potential. Spatial data on outbreaks in humans and animals from 1975 to 2020 were retrieved from the GIDEON database. Using Bayesian statistical models, we pinpoint regions at increased risk, and we focus on a new indicator based on the human-forest proximity. Conclusions Our results show that human-forest proximity together with biodiversity loss, and high population density increase the likelihood of diseases being transmitted from animals to humans, influencing where outbreaks occur among human populations. Lastly, we introduce a country vulnerability index that combines our risk assessment with the IHRC3 indicator from the WHO, which gauges a country’s capability to handle zoonotic diseases. These combined indices help us to assess each country’s ability to respond to an outbreak. The study also addresses data limitations and proposes directions for future research, emphasizing the importance of our findings in shaping public health policies and decision-making. Key messages • Anthropogenic drivers shape the distribution of outbreaks in human populations. • Human-forest proximity exacerbates the likelihood of Emerging Infectious Disease.

An international comparison of Long-term Care Facility outcomes during the COVID-19 pandemic

Abstract Background The COVID-19 pandemic has had huge repercussions for public health internationally and different countries had different outcomes based on preconceived policy interventions. Lessons influencing future policy have been learned, notably how best to manage vulnerable populations, particularly those living in long-term care facilities (LTCFs). In Ireland LTCF residents accounted for 56% of deaths during the first wave of the pandemic and it is imperative to establish the causes of this. International research has highlighted how factors including county incidence rate, facility size, occupancy, LTCF ownership and compliance with health inspection regulations influenced LTCF COVID-19 outcomes. The aim of this research is to explore the impact of these factors in an Irish context and compare to best international standards. Methods The main outcome considered was deaths per 100 beds. Data on crude mortality were available for 580 Irish LTCFs up to 28 May 2020. Data regarding occupancy, ownership, facility size and compliance with regulations were obtained for these LTCFs from a novel dataset of 1130 available LTCF regulatory inspection reports between 2018- 2022. Regression analysis was carried out to show how LTCF mortality was predicted by examined variables. Results The most significant predictor of deaths from COVID-19 in Irish LTCFs resulting from this research was LTCF occupancy (Odds ratio:1.029, significance <0.001). Regional community incidence, in concordance with international literature was also an important factor. Other stated predictors in this model were either non-significant or only marginally significant. These results are analysed in conversation with international research of the same variables. Conclusions To protect LTCF residents for potential future pandemics, attention should be paid to monitoring and suppressing local incidence rates and moves should be implemented towards designing smaller facilities with individual rooms for vulnerable residents. Key messages • Irish long-term care pandemic mortality was predicted by high occupancy of facilities and high regional COVID-19 incidence. • Comparing International COVID-19 outcomes in long-term care facilities can provide guidelines for the future.

Conserved sites on the influenza H1 and H3 hemagglutinin recognized by human antibodies.

Monoclonal antibodies (mAbs) targeting the influenza hemagglutinin (HA) have the potential to be used as prophylactics or templates for next-generation vaccines that provide broad protection. Here, we isolated broad, subtype-neutralizing mAbs from human B cells targeting the H1 or H3 HA head as well as a unique mAb targeting the stem. The H1 mAbs target the previously defined lateral patch epitope on H1 HAs and recognize HAs from 1933 to 2021 in addition to a swine H1N1 virus with pandemic potential. Using directed evolution, we improved the neutralization potency of these H1 mAbs towards a contemporary H1 strain. Using deep mutational scanning of four antigenically distinct H1N1 viruses, we identified potential viral escape pathways. For the H3 mAbs we used cryo-EM to define the targeted epitopes: one mAb recognizes the side of the H3 head, accommodating the N133 glycan and a pocket underneath the receptor binding site. The other H3 mAb recognizes an epitope in the HA stem that overlaps with previously characterized mAbs, but with distinct antibody variable genes and mode of recognition. Collectively, these mAbs identify common sites recognized by broad, subtype-specific mAbs that may be elicited by next-generation vaccines.

Development of learning objectives to support undergraduate virology curriculum guidelines.

It has become increasingly important for microbiology educators to help students learn critical concepts of the discipline. This is particularly true in virology, where current challenges include increasing rates of vaccine hesitancy, misinformation about the COVID-19 pandemic, and controversy surrounding research on pathogens with pandemic potential. Having students learn virology can attract more people to the field and increase the number of people who can engage in meaningful discourse about issues relating to the discipline. However, the limited number of virologists who teach undergraduates, combined with the fact that many institutions lack stand-alone virology courses, results in virology often being taught as a limited number of lectures within an undergraduate microbiology course (if it is covered at all), which may or may not be taught by an individual trained as a virologist. To provide a framework to teach virology to undergraduate students, a team of virology educators, with support from the American Society for Virology (ASV), developed curriculum guidelines for use in a stand-alone undergraduate virology course or a virology section within another course (D. B. Kushner et al., J Virol 96:e01305-22, 2022, https://doi.org/10.1128/jvi.01305-22). These guidelines are available at the ASV website (https://asv.org/curriculum-guidelines/). To assist educators in implementing these guidelines, we created examples of measurable learning objectives. This perspective provides details about the virology curriculum guidelines and learning objectives and accompanies the perspective by Boury et al. in this issue of the Journal of Microbiology & Biology Education (25:e00126-24, 2024, https://doi.org/10.1128/jmbe.00126-24) about the recent revision of the microbiology curriculum guidelines overseen by the American Society for Microbiology.

Impact of visitation restrictions in neonatal intensive care units during the COVID-19 pandemic on parents in northern Hokkaido, Japan.

Medical institutions restricted visitation to neonatal intensive care units (NICUs) during the coronavirus disease (COVID-19) pandemic. Therefore, this study aimed to investigate the impact of COVID-19 NICU visitor restrictions on parents. We conducted a questionnaire of 378 parents of infants who were hospitalised for more than 1 week at two NICUs in our area and discharged between 1 April 2020 and 31 March 2022. While the visiting rules for NICUs during this period varied depending on the phase of the COVID-19 epidemic, generally, only parents were allowed to visit the NICUs for a few hours daily. A total of 157 parents (A University Hospital (AMUH), n = 79; AK General Hospital (AKGH), n = 78) responded to the survey (41.3% response rate), with 40% stating that their emotions towards their infants were affected by restrictions due to the COVID-19 pandemic. Parents' feelings towards their infants were influenced by mode of delivery, parity, duration of hospitalisation and opportunities to visit the NICU. More than 70% of respondents felt stressed due to COVID-19 restrictions, and anxiety and emotional stress related to visitor restrictions were significantly affected by the number and duration of visits and allowability of family member visits. Parents felt that the restrictions had a negative impact on breastfeeding, overall infant care and infant-family bonding. As parents felt an impact on the bond with their infant during visitation restrictions, it is essential for facilities to implement measures to protect infant-family bonding during potential future pandemics.

Biothermodynamic analysis of the Dengue virus: Empirical formulas, biosynthesis reactions and thermodynamic properties of antigen-receptor binding and biosynthesis

After the experience with the COVID-19 pandemic, WHO has issued a warning about the possible causes of future pandemics. One such causative agent is the Dengue virus. Until now, we have had information mostly on biological properties of the Dengue virus and very little information about its chemical and thermodynamic properties. To be better prepared for a potential Dengue pandemic, the goal of this paper is to chemically and thermodynamically characterize the Dengue virus, as well as to describe the biophysical basis of the virus-host interactions of the Dengue virus. To that goal, the empirical formula was determined, as well as biosynthesis reactions and thermodynamic properties of antigen-receptor binding and thermodynamic properties of biosynthesis and multiplication of the Dengue virus. A model was developed of virus-host interactions between the Dengue virus and its host tissues, based on nonequilibrium thermodynamics.

An mRNA vaccine candidate encoding H5HA clade 2.3.4.4b protects mice from clade 2.3.2.1a virus infection

Highly pathogenic avian influenza (HPAI) H5 viruses from different clades have been circulating globally, threatening wild/domestic birds and mammals. Given frequent spillovers and high mortality among mammals, coupled with our inability to predict which clade of H5 virus has pandemic potential, cross-clade protective HPAI H5 vaccines are urgently needed. Here, we demonstrate the applicability of a lipid nanoparticle-based mRNA vaccine modality to induce cross-protective immunity against lethal HPAI virus infection.

How a paramyxovirus fusion/entry complex adapts to escape a neutralizing antibody

Paramyxoviruses including measles, Nipah, and parainfluenza viruses are public health threats with pandemic potential. Human parainfluenza virus type 3 (HPIV3) is a leading cause of illness in pediatric, older, and immunocompromised populations. There are no approved vaccines or therapeutics for HPIV3. Neutralizing monoclonal antibodies (mAbs) that target viral fusion are a potential strategy for mitigating paramyxovirus infection, however their utility may be curtailed by viral evolution that leads to resistance. Paramyxoviruses enter cells by fusing with the cell membrane in a process mediated by a complex consisting of a receptor binding protein (HN) and a fusion protein (F). Existing atomic resolution structures fail to reveal physiologically relevant interactions during viral entry. We present cryo-ET structures of pre-fusion HN-F complexes in situ on surfaces of virions that evolved resistance to an anti-HPIV3 F neutralizing mAb. Single mutations in F abolish mAb binding and neutralization. In these complexes, the HN protein that normally restrains F triggering has shifted to uncap the F apex. These complexes are more readily triggered to fuse. These structures shed light on the adaptability of the pre-fusion HN-F complex and mechanisms of paramyxoviral resistance to mAbs, and help define potential barriers to resistance for the design of mAbs.

Immunogenicity and Protectivity of Sputnik V Vaccine in hACE2-Transgenic Mice against Homologous and Heterologous SARS-CoV-2 Lineages Including Far-Distanced Omicron BA.5

Background: The SARS-CoV-2 virus continuously acquires mutations, leading to the emergence of new variants. Notably, the effectiveness of global vaccination efforts has significantly declined with the rise and spread of the B.1.1.529 (Omicron) variant. Methods: The study used virological, immunological and histological research methods, as well as methods of working with laboratory animals. In this study, we evaluated the Gam-COVID-Vac (Sputnik V), an adenoviral vaccine developed by the N.F. Gamaleya National Research Center for Epidemiology and Microbiology, and conducted experiments on hemizygous K18-ACE2-transgenic F1 mice. The variants studied included B.1.1.1, B.1.1.7, B.1.351, B.1.1.28/P.1, B.1.617.2, and B.1.1.529 BA.5. Results: Our findings demonstrate that the Sputnik V vaccine elicits a robust humoral and cellular immune response, effectively protecting vaccinated animals from challenges posed by various SARS-CoV-2 variants. However, we observed a notable reduction in vaccine efficacy against the B.1.1.529 (Omicron BA.5) variant. Conclusions: Our results indicate that ongoing monitoring of emerging mutations is crucial to assess vaccine efficacy against new SARS-CoV-2 variants to identify those with pandemic potential. If protective efficacy declines, it will be imperative to develop new vaccines tailored to current variants of the virus.

Simulation of Epidemic Dynamics Using a Multi-Agent Model: Analysis of Social Distancing Strategies and Their Impacts on Public Health and Economy

Infectious disease epidemics have played a crucial role in shaping public health responses, particularly in global health crises. This study emerges as part of the efforts to prepare effective responses to potential future pandemics, leveraging lessons learned during the COVID-19 crisis. The research uses an adapted compartmental epidemiological model and a synthetic multi-agent community to investigate how social variables influence epidemic forecasts in socioeconomically vulnerable regions. Focusing on the simulation of epidemic dynamics in the socio-economically disadvantaged neighbourhood of Ilha Joana Bezerra in Recife, this study examines the impacts of social distancing strategies and other control measures, such as face masks and moderate social isolation. Through the adapted SEPAI3R3O model, which includes compartments for pre-symptomatic and asymptomatic states, this study provides a detailed analysis of disease dynamics in contexts characterised by high social vulnerability. The results underscore the importance of public health policies adapted to socio-economic factors, emphasising the need for continuous preparedness to manage future epidemic threats in vulnerable communities effectively.

A protocol for a living mapping review of global research funding for infectious diseases with a pandemic potential - Pandemic PACT.

The COVID CIRCLE initiative Research Project Tracker by UKCDR and GloPID-R and associated living mapping review (LMR) showed the importance of sharing and analysing data on research at the point of funding to improve coordination during a pandemic. This approach can also help with research preparedness for outbreaks and hence our new programme the Pandemic Preparedness: Analytical Capacity and Funding Tracking Programme (Pandemic PACT) has been established. The LMR described in this protocol builds on the previous UKCDR and GloPID-R COVID-19 Research Project database with addition of the priority diseases from the WHO Blueprint list plus initial additions of pandemic influenza, mpox and plague. We capture data on new funding commitments directly from funders and map these against a core ontology (aligned to existing research roadmaps). We will analyse regularly collated new research funding commitments to provide an open, accessible, near-real-time overview of the funding landscape for a wide range of infectious disease and pandemic preparedness research and assess gaps. The periodicity of updates will be increased in the event of a major outbreak. We anticipate that this LMR and the associated online tool will be a useful resource for funders, policy makers and researchers. In the future, our work will inform a more coordinated approach to research funding by providing evidence and data, including identification of gaps in funding allocation with a particular focus on low- and middle-income countries.

B-152 Analysis of carbonyl compounds in exhaled breath for detection of COVID-19

Abstract Background COVID-19 pandemic has caused enormous loss of lives and economic disruption. The global crisis has resulted in more than 700 million infections and 7 million deaths worldwide. Rapid screening and detection of highly infectious respiratory diseases is a key for preventing and curbing future epidemic and pandemic. The objective of this work is to develop a novel breath analysis technique for detection of COVID-19 using chemoselective capture of carbonyl compounds in exhaled breath for analysis by gas chromatography-mass spectrometry (GC-MS). Respiratory viral infection causes oxidative stress and inflammation in its host body which leads to production of higher levels of some carbonyl compounds through oxidation of lipids and could be detected in exhaled breath. This breath analysis technique can be rapidly adapted for screening infectious respiratory diseases for future epidemic and potential pandemic. Methods A total of 237 subjects were enrolled in this study. Of these, 142 were COVID-19 positive and 95 were negative. One liter exhaled breath in Tedlar bags from both COVID-19 positive and negative subjects confirmed by reverse transcriptase polymerase chain reaction between April 2022 and September 2023. The collected one liter breath samples were completely evacuated through a cartridge packed with silica particles in 2-3 minutes. Silica particles were coated with a reactive aminooxy agent for capture of carbonyl compounds in the breath samples through oximation reactions. The captured carbonyl compounds were eluted by methanol and the eluted solutions were analyzed by GC-MS for calculation of each compound concentration in the exhaled breath samples. Biostatistics was used to identify significant carbonyl compounds (defined as p value < 0.05) as markers of CPOVID-19 infection. A machine learning algorithm was implemented with the captured carbonyl compounds as input features for differentiation of COVID-19 positive from negative subjects. Results A total of more than 30 carbonyl compounds were detected in exhaled breath samples of all subjects. 10 features including 3 individual carbonyl compounds and 7 compound ratios show significant differences (p value <0.05) between COVID-19 positive and negative groups. The developed machine learning and artificial intelligence (AI) algorithms successfully separated COVOD-19 positive from negative with an accuracy of 95.4%, sensitivity of 96.5%, specificity of 93.7%, and AUROC=0.974. Conclusions The analysis of carbonyl compounds in exhaled breath by GC-MS has great potential for non-invasive detection of COVID-19. The method could be rapidly adapted for detection of other respiratory infectious diseases with a small sample size and data set for training of the machine learning and AI algorithms.

Discriminating North American Swine Influenza Viruses with a Portable, One-Step, Triplex Real-Time RT-PCR Assay, and Portable Sequencing.

Swine harbors a genetically diverse population of swine influenza A viruses (IAV-S), with demonstrated potential to transmit to the human population, causing outbreaks and pandemics. Here, we describe the development of a one-step, triplex real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay that detects and distinguishes the majority of the antigenically distinct influenza A virus hemagglutinin (HA) clades currently circulating in North American swine, including the IAV-S H1 1A.1 (α), 1A.2 (β), 1A.3 (γ), 1B.2.2 (δ1) and 1B.2.1 (δ2) clades, and the IAV-S H3 2010.1 clade. We performed an in-field test at an exhibition swine show using in-field viral concentration and RNA extraction methodologies and a portable real-time PCR instrument, and rapidly identified three distinct IAV-S clades circulating within the N.A. swine population. Portable sequencing is used to further confirm the results of the in-field test of the swine triplex assay. The IAV-S triplex rRT-PCR assay can be easily transported and used in-field to characterize circulating IAV-S clades in North America, allowing for surveillance and early detection of North American IAV-S with human outbreak and pandemic potential.

Utilizing machine learning and hemagglutinin sequences to identify likely hosts of influenza H3Nx viruses

Influenza is a disease that represents both a public health and agricultural risk with pandemic potential. Among the subtypes of influenza A virus, H3 influenza virus can infect many avian and mammalian species and is therefore a virus of interest to human and veterinary public health. The primary goal of this study was to train and validate classifiers for the identification of the most likely host species using the hemagglutinin gene segment of H3 viruses. A five-step process was implemented, which included training four machine learning classifiers, testing the classifiers on the validation dataset, and further exploration of the best-performing model on three additional datasets. The gradient boosting machine classifier showed the highest host-classification accuracy with a 98.0 % (95 % CI [97.01, 98.73]) correct classification rate on an independent validation dataset. The classifications were further analyzed using the predicted probability score which highlighted sequences of particular interest. These sequences were both correctly and incorrectly classified sequences that showed considerable predicted probability for multiple hosts. This showed the potential of using these classifiers for rapid sequence classification and highlighting sequences of interest. Additionally, the classifiers were tested on a separate swine dataset composed of H3N2 sequences from 1998 to 2003 from the United States of America, and a separate canine dataset composed of canine H3N2 sequences of avian origin. These two datasets were utilized to look at the applications of predicted probability and host convergence over time. Lastly, the classifiers were used on an independent dataset of environmental sequences to explore the host identification of environmental sequences. The results of these classifiers show the potential for machine learning to be used as a host identification technique for viruses of unknown origin on a species-specific level.