The genomic landscape of swine influenza A viruses in Southeast Asia.

Swine are a primary source for the emergence of pandemic influenza A viruses. The intensification of swine production, along with global trade, has amplified the transmission and zoonotic risk of swine influenza A virus (swIAV). Effective surveillance is essential to uncover emerging virus strains; however gaps remain in our understanding of the swIAV genomic landscape in Southeast Asia. More than 4,000 nasal swabs were collected from pigs in Cambodia, yielding 72 IAV-positive samples by RT-qPCR and 45 genomic sequences. We unmasked the cocirculation of multiple lineages of genetically diverse swIAV of pandemic concern. Genomic analyses revealed a novel European avian-like H1N2 swIAV reassortant variant with North American triple reassortant internal genes, that emerged approximately seven years before its first detection in pigs in 2021. Using phylogeographic reconstruction, we identified south central China as the dominant source of swine viruses disseminated to other regions in China and Southeast Asia. We also identified nine distinct swIAV lineages in Cambodia, which diverged from their closest ancestors between two and 15 B.P., indicating significant undetected diversity in the region, including reverse zoonoses of human H1N1/2009 pandemic and H3N2 viruses. A similar period of cryptic circulation of swIAVs occurred in the decades before the H1N1/2009 pandemic. The hidden diversity of swIAV observed here further emphasizes the complex underlying evolutionary processes present in this region, reinforcing the importance of genomic surveillance at the human-swine interface for early warning of disease emergence to avoid future pandemics.

Impact of a family-centred clinical care programme on short-term outcomes of very low-birth weight infants.

AIM: We evaluated the effects of a family-centred clinical care pathway and case management programme on short-term clinical outcome in a cohort of very low-birth weight (VLBW) infants. METHODS: The programme, named NeoPAss, was developed at the Department of Neonatology Children's hospital Passau in 2013. Short-term outcomes of infants were compared to matched controls from the Bavarian neonatology surveillance database before (n = 111; 2008-2012) and after implementation (n = 170; 2014-2017). RESULTS: After implementation the rate of late-onset sepsis was significantly lower (2.5% vs. 10.7%, p = 0.005) and the length of stay was significantly shorter (VLBW 28 to 31 weeks' gestational age (GA) 47.5 vs. 53.1 days, p = 0.047; <28 weeks' GA 79.4 vs. 91.9 days, p = 0.007) in the intervention group compared to controls. Infants were discharged with significantly lower weight (mean 2351 vs. 2539 g, p = 0.013). There was no statistically significant difference in the rate of intraventricular haemorrhage (3.7% vs. 8.2%), necrotizing enterocolitis (0.6% vs. 1.9%) and bronchopulmonary dysplasia (0% vs. 6.9%). CONCLUSION: Our data confirm that of other studies demonstrating a beneficial effect of family-centred care programmes and provides evidence that structured parental involvement is not associated with increased risk of infection in a VLBW cohort.

ISU FLUture: a veterinary diagnostic laboratory web-based platform to monitor the temporal genetic patterns of Influenza A virus in swine.

BACKGROUND: Influenza A Virus (IAV) causes respiratory disease in swine and is a zoonotic pathogen. Uncontrolled IAV in swine herds not only affects animal health, it also impacts production through increased costs associated with treatment and prevention efforts. The Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) diagnoses influenza respiratory disease in swine and provides epidemiological analyses on samples submitted by veterinarians. DESCRIPTION: To assess the incidence of IAV in swine and inform stakeholders, the ISU FLUture website was developed as an interactive visualization tool that allows the exploration of the ISU VDL swine IAV aggregate data in the clinical diagnostic database. The information associated with diagnostic cases has varying levels of completeness and is anonymous, but minimally contains: sample collection date, specimen type, and IAV subtype. Many IAV positive samples are sequenced, and in these cases, the hemagglutinin (HA) sequence and genetic classification are completed. These data are collected and presented on ISU FLUture in near real-time, and more than 6,000 IAV positive diagnostic cases and their epidemiological and evolutionary information since 2003 are presented to date. The database and web interface provides rapid and unique insight into the trends of IAV derived from both large- and small-scale swine farms across the United States of America. CONCLUSION: ISU FLUture provides a suite of web-based tools to allow stakeholders to search for trends and correlations in IAV case metadata in swine from the ISU VDL. Since the database infrastructure is updated in near real-time and is integrated within a high-volume veterinary diagnostic laboratory, earlier detection is now possible for emerging IAV in swine that subsequently cause vaccination and control challenges. The access to real-time swine IAV data provides a link with the national USDA swine IAV surveillance system and allows veterinarians to make objective decisions regarding the management and control of IAV in swine. The website is publicly accessible at http://influenza.cvm.iastate.edu .

The Role of the Environment in the Evolution of Tolerance and Resistance to a Pathogen.

Defense against parasites can be divided into resistance, which limits parasite burden, and tolerance, which reduces pathogenesis at a given parasite burden. Distinguishing between the two and understanding which defense is favored by evolution in different ecological settings are important, as they lead to fundamentally different evolutionary trajectories of host-parasite interactions. We let the mosquito Aedes aegypti evolve under different food levels and with either no parasite, a constant parasite, or a coevolving parasite (the microsporidian Vavraia culicis). We then tested tolerance and resistance of the evolved lines on a population level at the two food levels. Exposure to parasites during evolution increased resistance and tolerance, but there were no differences between the lines evolved with coevolving or constant parasites. Mosquitoes that had evolved with food restriction had higher resistance than those evolved with high food but similar tolerance. The mosquitoes that had restricted food when being tested had lower tolerance than those with normal food, but there was no difference in resistance. Our results emphasize the complexity and dependence on environmental conditions of the evolution and expression of resistance and tolerance and help to evaluate some of the predictions about the evolution of host defense against parasites.

The pervasiveness and plasticity of circadian oscillations: the coupled circadian-oscillators framework.

MOTIVATION: Circadian oscillations have been observed in animals, plants, fungi and cyanobacteria and play a fundamental role in coordinating the homeostasis and behavior of biological systems. Genetically encoded molecular clocks found in nearly every cell, based on negative transcription/translation feedback loops and involving only a dozen genes, play a central role in maintaining these oscillations. However, high-throughput gene expression experiments reveal that in a typical tissue, a much larger fraction ([Formula: see text]) of all transcripts oscillate with the day-night cycle and the oscillating species vary with tissue type suggesting that perhaps a much larger fraction of all transcripts, and perhaps also other molecular species, may bear the potential for circadian oscillations. RESULTS: To better quantify the pervasiveness and plasticity of circadian oscillations, we conduct the first large-scale analysis aggregating the results of 18 circadian transcriptomic studies and 10 circadian metabolomic studies conducted in mice using different tissues and under different conditions. We find that over half of protein coding genes in the cell can produce transcripts that are circadian in at least one set of conditions and similarly for measured metabolites. Genetic or environmental perturbations can disrupt existing oscillations by changing their amplitudes and phases, suppressing them or giving rise to novel circadian oscillations. The oscillating species and their oscillations provide a characteristic signature of the physiological state of the corresponding cell/tissue. Molecular networks comprise many oscillator loops that have been sculpted by evolution over two trillion day-night cycles to have intrinsic circadian frequency. These oscillating loops are coupled by shared nodes in a large network of coupled circadian oscillators where the clock genes form a major hub. Cells can program and re-program their circadian repertoire through epigenetic and other mechanisms. AVAILABILITY AND IMPLEMENTATION: High-resolution and tissue/condition specific circadian data and networks available at http://circadiomics.igb.uci.edu. CONTACT: pfbaldi@ics.uci.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Inter-species prediction of protein phosphorylation in the sbv IMPROVER species translation challenge.

MOTIVATION: Animal models are widely used in biomedical research for reasons ranging from practical to ethical. An important issue is whether rodent models are predictive of human biology. This has been addressed recently in the framework of a series of challenges designed by the systems biology verification for Industrial Methodology for Process Verification in Research (sbv IMPROVER) initiative. In particular, one of the sub-challenges was devoted to the prediction of protein phosphorylation responses in human bronchial epithelial cells, exposed to a number of different chemical stimuli, given the responses in rat bronchial epithelial cells. Participating teams were asked to make inter-species predictions on the basis of available training examples, comprising transcriptomics and phosphoproteomics data. RESULTS: Here, the two best performing teams present their data-driven approaches and computational methods. In addition, post hoc analyses of the datasets and challenge results were performed by the participants and challenge organizers. The challenge outcome indicates that successful prediction of protein phosphorylation status in human based on rat phosphorylation levels is feasible. However, within the limitations of the computational tools used, the inclusion of gene expression data does not improve the prediction quality. The post hoc analysis of time-specific measurements sheds light on the signaling pathways in both species. AVAILABILITY AND IMPLEMENTATION: A detailed description of the dataset, challenge design and outcome is available at www.sbvimprover.com. The code used by team IGB is provided under http://github.com/uci-igb/improver2013. Implementations of the algorithms applied by team AMG are available at http://bhanot.biomaps.rutgers.edu/wiki/AMG-sc2-code.zip. CONTACT: meikelbiehl@gmail.com.

The TCF C-clamp DNA binding domain expands the Wnt transcriptome via alternative target recognition.

LEF/TCFs direct the final step in Wnt/ß-catenin signalling by recruiting ß-catenin to genes for activation of transcription. Ancient, non-vertebrate TCFs contain two DNA binding domains, a High Mobility Group box for recognition of the Wnt Response Element (WRE; 5'-CTTTGWWS-3') and the C-clamp domain for recognition of the GC-rich Helper motif (5'-RCCGCC-3'). Two vertebrate TCFs (TCF-1/TCF7 and TCF-4/TCF7L2) use the C-clamp as an alternatively spliced domain to regulate cell-cycle progression, but how the C-clamp influences TCF binding and activity genome-wide is not known. Here, we used a doxycycline inducible system with ChIP-seq to assess how the C-clamp influences human TCF1 binding genome-wide. Metabolic pulse-labeling of nascent RNA with 4'Thiouridine was used with RNA-seq to connect binding to the Wnt transcriptome. We find that the C-clamp enables targeting to a greater number of gene loci for stronger occupancy and transcription regulation. The C-clamp uses Helper sites concurrently with WREs for gene targeting, but it also targets TCF1 to sites that do not have readily identifiable canonical WREs. The coupled ChIP-seq/4'Thiouridine-seq analysis identified new Wnt target genes, including additional regulators of cell proliferation. Thus, C-clamp containing isoforms of TCFs are potent transcriptional regulators with an expanded transcriptome directed by C-clamp-Helper site interactions.

Circadian clock regulates the host response to Salmonella.

Organisms adapt to day-night cycles through highly specialized circadian machinery, whose molecular components anticipate and drive changes in organism behavior and metabolism. Although many effectors of the immune system are known to follow daily oscillations, the role of the circadian clock in the immune response to acute infections is not understood. Here we show that the circadian clock modulates the inflammatory response during acute infection with the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). Mice infected with S. Typhimurium were colonized to higher levels and developed a higher proinflammatory response during the early rest period for mice, compared with other times of the day. We also demonstrate that a functional clock is required for optimal S. Typhimurium colonization and maximal induction of several proinflammatory genes. These findings point to a clock-regulated mechanism of activation of the immune response against an enteric pathogen and may suggest potential therapeutic strategies for chronopharmacologic interventions.

GRHL3/GET1 and trithorax group members collaborate to activate the epidermal progenitor differentiation program.

The antagonistic actions of Polycomb and Trithorax are responsible for proper cell fate determination in mammalian tissues. In the epidermis, a self-renewing epithelium, previous work has shown that release from Polycomb repression only partially explains differentiation gene activation. We now show that Trithorax is also a key regulator of epidermal differentiation, not only through activation of genes repressed by Polycomb in progenitor cells, but also through activation of genes independent of regulation by Polycomb. The differentiation associated transcription factor GRHL3/GET1 recruits the ubiquitously expressed Trithorax complex to a subset of differentiation genes.

Quality assessment of glucose measurement with regard to epidemiology and clinical management of diabetes mellitus in Germany.

Background: During the last decade, Germany has seen an increased prevalence and a redistribution from undetected to diagnosed diabetes mellitus. Due to this substantial epidemiological development, the number of people with documented type 2 diabetes was 8.7 million in 2022. An estimated two million undiagnosed subjects are to be added. Beyond that, the life expectancy of diabetic subjects is increasing due to more responsive health systems in terms of care. Possible reasons include improved screening of at-risk individuals, the introduction of HbA1c for diagnosis in 2010, and the higher use of risk scores. Additionally, quality aspects of the laboratory methodology should be taken into consideration. Methods: Epidemiology and clinical management of diabetes in Germany are presented in the light of publications retrieved by a selective search of the PubMed database. Additionally, the data from German external quality assessment (EQA) surveys for the measurands glucose in plasma and HbA1c in whole blood, reviewed from 2010 until 2022, were evaluated. Above this, data concerning the analytical performance of near-patient glucometer devices, according to the ISO norm 15197:2013, were analyzed. Results: Two laboratory aspects are in good accordance with the observation of an increase in the diabetes mellitus prevalence when retrospectively reviewing the period 2010 to 2022: First, the analytical performance according to the ISO norm 15197:2013 of the glucometer devices widely used by patients with diabetes for the glucose self-testing, has improved during this period. Secondly, concerning the EQA program of INSTAND, the number of participating laboratories raised significantly in Germany. The spreads of variations of the specified results for plasma glucose remained unchanged between 2010 and 2022, whereas for HbA1c a significant decrease of the result scattering could be observed. Conclusion: These retrospectively established findings testify to an excellent analytical quality of laboratory diagnostics for glucose and HbA1c throughout Germany which may be involved in a better diagnosis and therapy of previously undetected diabetes mellitus.

Detection and Characterization of Influenza A Virus Endemic Circulation in Suckling and Nursery Pigs Originating from Vaccinated Farms in the Same Production System.

Inactivated influenza A virus (IAV) vaccines help reduce clinical disease in suckling piglets, although endemic infections still exist. The objective of this study was to evaluate the detection of IAV in suckling and nursery piglets from IAV-vaccinated sows from farms with endemic IAV infections. Eight nasal swab collections were obtained from 135 two-week-old suckling piglets from four farms every other week from March to September 2013. Oral fluid samples were collected from the same group of nursery piglets. IAV RNA was detected in 1.64% and 31.01% of individual nasal swabs and oral fluids, respectively. H1N2 was detected most often, with sporadic detection of H1N1 and H3N2. Whole-genome sequences of IAV isolated from suckling piglets revealed an H1 hemagglutinin (HA) from the 1B.2.2.2 clade and N2 neuraminidase (NA) from the 2002A clade. The internal gene constellation of the endemic H1N2 was TTTTPT with a pandemic lineage matrix. The HA gene had 97.59% and 97.52% nucleotide and amino acid identities, respectively, to the H1 1B.2.2.2 used in the farm-specific vaccine. A similar H1 1B.2.2.2 was detected in the downstream nursery. These data demonstrate the low frequency of IAV detection in suckling piglets and downstream nurseries from farms with endemic infections in spite of using farm-specific IAV vaccines in sows.

Monitoring emerging pathogens using negative nucleic acid test results from endemic pathogens in pig populations: Application to porcine enteric coronaviruses.

This study evaluated the use of endemic enteric coronaviruses polymerase chain reaction (PCR)-negative testing results as an alternative approach to detect the emergence of animal health threats with similar clinical diseases presentation. This retrospective study, conducted in the United States, used PCR-negative testing results from porcine samples tested at six veterinary diagnostic laboratories. As a proof of concept, the database was first searched for transmissible gastroenteritis virus (TGEV) negative submissions between January 1st, 2010, through April 29th, 2013, when the first porcine epidemic diarrhea virus (PEDV) case was diagnosed. Secondly, TGEV- and PEDV-negative submissions were used to detect the porcine delta coronavirus (PDCoV) emergence in 2014. Lastly, encountered best detection algorithms were implemented to prospectively monitor the 2023 enteric coronavirus-negative submissions. Time series (weekly TGEV-negative counts) and Seasonal Autoregressive-Integrated Moving-Average (SARIMA) were used to control for outliers, trends, and seasonality. The SARIMA's fitted and residuals were then subjected to anomaly detection algorithms (EARS, EWMA, CUSUM, Farrington) to identify alarms, defined as weeks of higher TGEV-negativity than what was predicted by models preceding the PEDV emergence. The best-performing detection algorithms had the lowest false alarms (number of alarms detected during the baseline) and highest time to detect (number of weeks between the first alarm and PEDV emergence). The best-performing detection algorithms were CUSUM, EWMA, and Farrington flexible using SARIMA fitted values, having a lower false alarm rate and identified alarms 4 to 17 weeks before PEDV and PDCoV emergences. No alarms were identified in the 2023 enteric negative testing results. The negative-based monitoring system functioned in the case of PEDV propagating epidemic and in the presence of a concurrent propagating epidemic with the PDCoV emergence. It demonstrated its applicability as an additional tool for diagnostic data monitoring of emergent pathogens having similar clinical disease as the monitored endemic pathogens.

A recombinant porcine reproductive and respiratory syndrome virus type 2 field strain derived from two PRRSV-2-modified live virus vaccines.

A porcine reproductive and respiratory syndrome virus (PRRSV) type 2 (PRRSV-2) isolate was obtained from lung samples collected from a 4.5-month-old pig at a wean-to-finish site in Indiana, USA, although no gross or microscopic lesions suggestive of PRRSV infection were observed in the lung tissue. Phylogenetic and molecular evolutionary analyses based on the obtained virus sequences indicated that PRRSV USA/IN105404/2021 was a natural recombinant isolate from Ingelvac PRRS® MLV and Prevacent® PRRS, which are PRRSV-2-modified live virus vaccines commercially available in the United States. This study is the first to report the detection of a PRRSV-2 recombinant strain consisting entirely of two modified live virus vaccine strains under field conditions. Based on clinical data and the absence of lung lesions, this PRRSV-2 recombinant strain was not virulent in swine, although its pathogenicity needs to be confirmed by clinical trials.

Use of the ISU FLUture multisequence identity tool for rapid interpretation of swine influenza A virus sequences in the United States.

Rapid and reliable identification of the hemagglutinin (HA) and neuraminidase (NA) genetic clades of an influenza A virus (IAV) sequence from swine can inform control measures and multivalent vaccine composition. Current approaches to genetically characterize HA or NA sequences are based on nucleotide similarity or phylogenetic analyses. Public databases exist to acquire IAV genetic sequences for comparison, but personnel at the diagnostic or production level have difficulty in adequately updating and maintaining relevant sequence datasets for IAV in swine. Further, phylogenetic analyses are time intensive, and inference drawn from these methods is impacted by input sequence data and associated metadata. We describe here the use of the IAV multisequence identity tool as an integrated public webpage located on the Iowa State University Veterinary Diagnostic Laboratory (ISU-VDL) FLUture website: https://influenza.cvm.iastate.edu/. The multisequence identity tool uses sequence data derived from IAV-positive cases sequenced at the ISU-VDL, employs a BLAST algorithm that identifies sequences that are genetically similar to submitted query sequences, and presents a tabulation and visualization of the most genetically similar IAV sequence and associated metadata from the FLUture database. Our tool removes bioinformatic barriers and allows clients, veterinarians, and researchers to rapidly classify and identify IAV sequences similar to their own sequences to augment interpretation of results.

Implementing a user-friendly format to analyze PRRSV next-generation sequencing results and associating breeding herd production performance with number of PRRSV strains and recombination events.

The open reading frames (ORF)5 represents approximately 4% of the porcine reproductive and respiratory syndrome virus (PRRSV)-2 genome (whole-PRRSV) and is often determined by the Sanger technique, which rarely detects >1 PRRSV strain if present in the sample. Next-generation sequencing (NGS) may provide a more appropriate method of detecting multiple PRRSV strains in one sample. This work assessed the effect of PRRSV genetic variability and recombination events, using NGS, on the time-to-low prevalence (TTLP) and total losses in breeding herds (n 20) that detected a PRRSV outbreak and adopted measures to eliminate PRRSV. Serum, lung or live virus inoculation material collected within 3-weeks of outbreak, and subsequently, processing fluids (PFs) were tested for PRRSV by RT-qPCR and NGS. Recovered whole-PRRSV or partial sequences were used to characterize within and between herd PRRSV genetic variability. Whole-PRRSV was recovered in five out of six (83.3%) lung, 16 out of 22 (72.73%) serum and in five out of 95 (5.26%) PF. Whole-PRRSV recovered from serum or lung were used as farm referent strains in 16 out of 20 (80%) farms. In four farms, only partial genome sequences were recovered and used as farm referent strains. At least two wild-type PRRSV strains (wt-PRRSV) were circulating simultaneously in 18 out of 20 (90%) and at least one vaccine-like strain co-circulating in eight out of 20 (40%) farms. PRRSV recombination events were detected in 12 farms (59%), been 10 out of 12 between wt-PRRSV and two out of 12 between wt-PRRSV and vaccine-like strains. Farms having ≥3 strains had a 12-week increase TTLP versus herds ≤2 strains detected. Farms with ≤2 strains (n 10) had 1837 and farms with no recombination events detected (n 8) had 1827 fewer piglet losses per 1000 sows versus farms with ≥3 PRRSV strains (n 8) or detected recombination (n 10), respectively. NGS outcomes and novel visualization methods provided more thorough insight into PRRSV dynamics, genetic variability, detection of multiple strains co-circulating in breeding herds and helped establish practical guidelines for using PRRSV NGS outputs.

Genetic and Antigenic Characterization of an Expanding H3 Influenza A Virus Clade in U.S. Swine Visualized by Nextstrain.

Defining factors that influence spatial and temporal patterns of influenza A virus (IAV) is essential to inform vaccine strain selection and strategies to reduce the spread of potentially zoonotic swine-origin IAV. The relative frequency of detection of the H3 phylogenetic clade 1990.4.a (colloquially known as C-IVA) in U.S. swine declined to 7% in 2017 but increased to 32% in 2019. We conducted phylogenetic and phenotypic analyses to determine putative mechanisms associated with increased detection. We created an implementation of Nextstrain to visualize the emergence, spatial spread, and genetic evolution of H3 IAV in swine, identifying two C-IVA clades that emerged in 2017 and cocirculated in multiple U.S. states. Phylodynamic analysis of the hemagglutinin (HA) gene documented low relative genetic diversity from 2017 to 2019, suggesting clonal expansion. The major H3 C-IVA clade contained an N156H amino acid substitution, but hemagglutination inhibition (HI) assays demonstrated no significant antigenic drift. The minor HA clade was paired with the neuraminidase (NA) clade N2-2002B prior to 2016 but acquired and maintained an N2-2002A in 2016, resulting in a loss of antigenic cross-reactivity between N2-2002B- and -2002A-containing H3N2 strains. The major C-IVA clade viruses acquired a nucleoprotein (NP) of the H1N1pdm09 lineage through reassortment in the replacement of the North American swine-lineage NP. Instead of genetic or antigenic diversity within the C-IVA HA, our data suggest that population immunity to H3 2010.1 along with the antigenic diversity of the NA and the acquisition of the H1N1pdm09 NP gene likely explain the reemergence and transmission of C-IVA H3N2 in swine. IMPORTANCE Genetically distinct clades of influenza A virus (IAV) in swine undermine efforts to control the disease. Swine producers commonly use vaccines, and vaccine strains are selected by identifying the most common hemagglutinin (HA) gene from viruses detected in a farm or a region. In 2019, we identified an increase in the detection frequency of an H3 phylogenetic clade, C-IVA, which was previously circulating at much lower levels in U.S. swine. Our study identified genetic and antigenic factors contributing to its resurgence by linking comprehensive phylodynamic analyses with empirical wet-lab experiments and visualized these evolutionary analyses in a Nextstrain implementation. The contemporary C-IVA HA genes did not demonstrate an increase in genetic diversity or significant antigenic changes. N2 genes did demonstrate antigenic diversity, and the expanding C-IVA clade acquired a nucleoprotein (NP) gene segment via reassortment. Virus phenotype and vaccination targeting prior dominant HA clades likely contributed to the clade's success.

Characterization of a 2016-2017 Human Seasonal H3 Influenza A Virus Spillover Now Endemic to U.S. Swine.

In 2017, the Iowa State University Veterinary Diagnostic Laboratory detected a reverse-zoonotic transmission of a human seasonal H3 influenza A virus into swine (IAV-S) in Oklahoma. Pairwise comparison between the recently characterized human seasonal H3 IAV-S (H3.2010.2) hemagglutinin (HA) sequences detected in swine and the most similar 2016-2017 human seasonal H3 revealed 99.9% nucleotide identity. To elucidate the origin of H3.2010.2 IAV-S, 45 HA and 27 neuraminidase (NA) sequences from 2017 to 2020 as well as 11 whole-genome sequences (WGS) were genetically characterized. Time to most recent common human ancestor was estimated between August and September 2016. The N2 NA was of human origin in all but one strain from diagnostic submissions with NA sequences, and the internal gene segments from WGS consisted of matrix genes originating from the 2009 pandemic H1N1 and another 5 internal genes of triple reassortant swine origin (TTTTPT). Pigs experimentally infected with H3.2010.2 demonstrated efficient nasal shedding and replication in the lungs, mild pneumonia, and minimal microscopic lung lesions and transmitted the virus to indirect contact swine. Antigenically, H3.2010.2 viruses were closer to a human seasonal vaccine strain, A/Hong Kong/4801/2014, than to the H3.2010.1 human seasonal H3 viruses detected in swine in 2012. This was the second sustained transmission of a human seasonal IAV into swine from the 2010 decade after H3.2010.1. Monitoring the spillover and detection of novel IAV from humans to swine may help vaccine antigen selection and could impact pandemic preparedness. IMPORTANCE H3.2010.2 is a new phylogenetic clade of H3N2 circulating in swine that became established after the spillover of a human seasonal H3N2 from the 2016-2017 influenza season. The novel H3.2010.2 transmitted and adapted to the swine host and demonstrated reassortment with internal genes from strains endemic to pigs, but it maintained human-like HA and NA. It is genetically and antigenically distinct from the H3.2010.1 H3N2 introduced earlier in the 2010 decade. Human seasonal IAV spillovers into swine become established in the population through adaptation and sustained transmission and contribute to the genetic and antigenic diversity of IAV circulating in swine. Continued IAV surveillance is necessary to detect emergence of novel strains in swine and assist with vaccine antigen selection to improve the ability to prevent respiratory disease in swine as well as the risk of zoonotic transmission.

High-throughput prediction of protein antigenicity using protein microarray data.

MOTIVATION: Discovery of novel protective antigens is fundamental to the development of vaccines for existing and emerging pathogens. Most computational methods for predicting protein antigenicity rely directly on homology with previously characterized protective antigens; however, homology-based methods will fail to discover truly novel protective antigens. Thus, there is a significant need for homology-free methods capable of screening entire proteomes for the antigens most likely to generate a protective humoral immune response. RESULTS: Here we begin by curating two types of positive data: (i) antigens that elicit a strong antibody response in protected individuals but not in unprotected individuals, using human immunoglobulin reactivity data obtained from protein microarray analyses; and (ii) known protective antigens from the literature. The resulting datasets are used to train a sequence-based prediction model, ANTIGENpro, to predict the likelihood that a protein is a protective antigen. ANTIGENpro correctly classifies 82% of the known protective antigens when trained using only the protein microarray datasets. The accuracy on the combined dataset is estimated at 76% by cross-validation experiments. Finally, ANTIGENpro performs well when evaluated on an external pathogen proteome for which protein microarray data were obtained after the initial development of ANTIGENpro. AVAILABILITY: ANTIGENpro is integrated in the SCRATCH suite of predictors available at http://scratch.proteomics.ics.uci.edu. CONTACT: pfbaldi@ics.uci.edu

Spatial and temporal coevolution of N2 neuraminidase and H1 and H3 hemagglutinin genes of influenza A virus in US swine.

The neuraminidase (NA) and hemagglutinin (HA) are essential surface glycoproteins of influenza A virus (IAV). In this study, the evolution of subtype N2 NA paired with H1 and H3 subtype HA in swine was evaluated to understand if the genetic diversity of HA and NA were linked. Using time-scaled Bayesian phylodynamic analyses, the relationships of paired swine N2 with H1 or H3 from 2009 to 2018 were evaluated. These data demonstrated increased relative genetic diversity within the major N2 clades circulating in swine in the USA (N2.1998 between 2014 and 2017 and N2.2002 between 2010 and 2016). Preferential pairing was observed among specific NA and HA genetic clades. Gene reassortment between cocirculating influenza A strains resulted in novel pairings that persisted. The changes in genetic diversity in the NA gene were quantified using Bayesian phylodynamic analyses, and increases in diversity were observed subsequent to novel NA-HA reassortment events. The rate of evolution among NA-N2 clades and HA-H1 and HA-H3 clades were similar. Bayesian phylodynamic analyses demonstrated strong spatial patterns in N2 genetic diversity, but frequent interstate movement of rare N2 clades provided opportunity for reassortment and emergence of new N2-HA pairings. The frequent regional movement of pigs and their influenza viruses is an explanation for the documented patterns of reassortment and subsequent changes in gene diversity. The reassortment and evolution of NA and linked HA evolution may result in antigenic drift of both major surface glycoproteins, reducing vaccine efficacy, with subsequent impact on animal health.