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Influenza pandemics are associated with severe morbidity, mortality, and social and economic disruption. Every summer in the United States, youths attending agricultural fairs are exposed to genetically diverse influenza A viruses (IAVs) circulating in exhibition swine, resulting in over 450 lab-confirmed zoonotic infections since 2010. Exhibition swine represent a small, defined population (∼1.5% of the U.S. herd), presenting a realistic opportunity to mitigate a pandemic threat by reducing IAV transmission in the animals themselves. Through intensive surveillance and genetic sequencing of IAVs in exhibition swine in six U.S. states in 2018 ( n  = 212), we characterized how a heterogeneous circuit of swine shows, comprising fairs with different sizes and geographic coverage, facilitates IAV transmission among exhibition swine and into humans. Specifically, we identified the role of an early-season national show in the propagation and spatial dissemination of a specific virus (H1δ-2) that becomes dominant among exhibition swine and is associated with the majority of zoonotic infections in 2018. These findings suggest that a highly targeted mitigation strategy, such as postponing swine shows for 1 to 2 weeks following the early-season national show, could potentially reduce IAV transmission in exhibition swine and spillover into humans, and this merits further study. IMPORTANCE The varying influenza A virus (IAV) exposure and infection status of individual swine facilitates introduction, transmission, and dissemination of diverse IAVs. Since agricultural fairs bring people into intimate contact with swine, they provide a unique interface for zoonotic transmission of IAV. Understanding the dynamics of IAV transmission through exhibition swine is critical to mitigating the high incidence of variant IAV cases reported in association with agricultural fairs. We used genomic sequences from our exhibition swine surveillance to characterize the hemagglutinin and full genotypic diversity of IAV at early-season shows and the subsequent dissemination through later-season agricultural fairs. We were able to identify a critical time point with important implications for downstream IAV and zoonotic transmission. With improved understanding of evolutionary origins of zoonotic IAV, we can inform public health mitigation strategies to ultimately reduce zoonotic IAV transmission and risk of pandemic IAV emergence., (Copyright © 2020 American Society for Microbiology.)

While working overnight at a swine exhibition, we identified an influenza A virus (IAV) outbreak in swine, Nanopore sequenced 13 IAV genomes from samples we collected, and predicted in real time that these viruses posed a novel risk to humans due to genetic mismatches between the viruses and current prepandemic candidate vaccine viruses (CVVs). We developed and used a portable IAV sequencing and analysis platform called Mia (Mobile Influenza Analysis) to complete and characterize full-length consensus genomes approximately 18 h after unpacking the mobile lab. Exhibition swine are a known source for zoonotic transmission of IAV to humans and pose a potential pandemic risk. Genomic analyses of IAV in swine are critical to understanding this risk, the types of viruses circulating in swine, and whether current vaccines developed for use in humans would be predicted to provide immune protection. Nanopore sequencing technology has enabled genome sequencing in the field at the source of viral outbreaks or at the bedside or pen-side of infected humans and animals. The acquired data, however, have not yet demonstrated real-time, actionable public health responses. The Mia system rapidly identified three genetically distinct swine IAV lineages from three subtypes, A(H1N1), A(H3N2), and A(H1N2). Analysis of the hemagglutinin (HA) sequences of the A(H1N2) viruses identified >30 amino acid differences between the HA1 of these viruses and the most closely related CVV. As an exercise in pandemic preparedness, all sequences were emailed to CDC collaborators who initiated the development of a synthetically derived CVV. IMPORTANCE Swine are influenza virus reservoirs that have caused outbreaks and pandemics. Genomic characterization of these viruses enables pandemic risk assessment and vaccine comparisons, though this typically occurs after a novel swine virus jumps into humans. The greatest risk occurs where large groups of swine and humans comingle. At a large swine exhibition, we used Nanopore sequencing and on-site analytics to interpret 13 swine influenza virus genomes and identified an influenza virus cluster that was genetically highly varied to currently available vaccines. As part of the National Strategy for Pandemic Preparedness exercises, the sequences were emailed to colleagues at the CDC who initiated the development of a synthetically derived vaccine designed to match the viruses at the exhibition. Subsequently, this virus caused 14 infections in humans and was the dominant U.S. variant virus in 2018.

Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-negative bacterium that utilizes phosphite as the sole electron source for homoacetogenic CO2 reduction or sulfate reduction. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidation via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence analysis of two positive clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidation. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD-ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidation ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidation activity was measured in cell extracts of D. balticum transconjugants, suggesting that all genes required for phosphite oxidation were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence analysis and enzyme assays.

Enrichment was performed to isolate organisms that could utilize reduced phosphorus compounds as their sole phosphorus sources. One isolate that grew well with either hypophosphite or phosphite was identified by 16S rRNA gene analysis as a strain of Alcaligenes faecalis. The genes required for oxidation of hypophosphite and phosphite by this organism were identified by using transposon mutagenesis and include homologs of the ptxD and htxA genes of Pseudomonas stutzeri WM88, which encode an NAD-dependent phosphite dehydrogenase (PtxD) and 2-oxoglutarate-dependent hypophosphite dioxygenase (HtxA). This organism also has the htxB, htxC, and htxD genes that comprise an ABC-type transporter, presumably for hypophosphite and phosphite transport. The role of these genes in reduced phosphorus metabolism was confirmed by analyzing the growth of mutants in which these genes were deleted. Sequencing data showed that htxA, htxB, htxC, and htxD are virtually identical to their homologs in P. stutzeri at the DNA level, indicating that horizontal gene transfer occurred. However, A. faecalis ptxD is very different from its P. stutzeri homolog and represents a new ptxD lineage. Therefore, this gene has ancient evolutionary roots in bacteria. These data suggest that there is strong evolutionary selection for the ability of microorganisms to oxidize hypophosphite and phosphite.

A9145 is a basic, water-soluble, antifungal antibiotic which is produced in a complex organic medium by Streptomyces griseolus. The metabolite has a molecular weight of 510, and contains adenine as well as sugar hydroxyl and amino groups. Although glucose, fructose, glucose polymers, and some long-chain fatty acid methyl esters supported biosynthesis, oils were superior, with cottonseed oil being preferred. Several ions and salts, especially Co(2+), PO(4) (3-), and CaCO(3), were stimulatory. Adenine, nucleosides, and some amino acids increased the accumulation of A9145 in shaken-flask fermentors. Enrichment of the culture medium with tyrosine afforded maximal enhancement of antibiotic production in both flask and tank fermentors. Control of the dissolved O(2) level was also critical, the optimal concentration being 3 x 10(-2) to 4.5 x 10(-2) mumole of O(2)/ml. Optimization of various fermentation parameters increased antibiotic titers approximately 135-fold in shaken flask fermentors and 225-fold in stirred vessels.