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Highly pathogenic avian influenza viruses (HPAIV) H5N1 clade 2.3.4.4b continue to have unprecedented global impacts on wild birds and mammals, with especially significant mortality observed in colonial surface-nesting seabirds and in some marine mammal species. In July of 2023 H5N1 HPAIV 2.3.4.4b was detected in Caspian terns nesting on Rat Island, Washington USA. An estimated 1,800-1,900 adult terns populated the breeding colony, based on aerial photographs taken at the start of the outbreak. On a near-weekly basis throughout July and August, we counted and removed carcasses, euthanized moribund birds, and collected swab and tissue samples for diagnostic testing and next-generation sequencing. We directly counted 1,101 dead Caspian tern adults and 520 dead chicks, indicating a minimum 56% loss of the adult colony population and potential impacts to reproductive success. Combining the observed mortality on Rat Island with HPAI-related Caspian tern deaths recorded elsewhere in Washington and Oregon, we estimate that 10-14% of the Pacific Flyway population was lost in the summer of 2023. Comparatively few adult Glaucous-winged gulls (hybrids) nesting on Rat Island died (~3% of the local population), although gull chick mortality was high. Sixteen harbor seals in the immediate or nearby area stranded during the outbreak, and H5N1 HPAIV was detected in brain and/or lung tissue of five seals. These cases are the first known detections of HPAIV in a marine mammal on the Pacific coast of North America. Phylogenetic analyses support the occurrence of at least three independent avian-mammalian virus spillover events (tern or gull to harbor seal). Whole genome sequencing indicated that H5N1 HPAIV may have been introduced to Washington from Caspian terns in Oregon. Ongoing monitoring and surveillance for H5N1 HPAIV in the marine environment is necessary to understand the epidemiology of this virus, assess conservation impacts to susceptible species, and provide support for data-driven management and response actions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Haman, Pearson, Brown, Frisbie, Penhallegon, Falghoush, Wolking, Torrevillas, Taylor, Snekvik, Tanedo, Keren, Ashley, Clark, Lambourn, Eckstrand, Edmonds, Rovani-Rhoades, Oltean, Wilkinson, Fauquier, Black and Waltzek.)

Rapid growth in aquaculture has resulted in high-density production systems in ecologically and geographically novel conditions in which the emergence of diseases is inevitable. Well-characterized methods for detection and surveillance of infectious diseases are vital for rapid identification, response, and recovery to protect economic and food security. We implemented a proof-of-concept approach for virus detection using a known high-consequence fish pathogen, infectious salmon anemia virus (ISAV), as the archetypal pathogen. In fish infected with ISAV, we integrated histopathology, virus isolation, whole-genome sequencing (WGS), electron microscopy (EM), in situ hybridization (ISH), and reverse transcription real-time PCR (RT-rtPCR). Fresh-frozen and formalin-fixed tissues were collected from virus-infected, control, and sham-infected Atlantic salmon ( Salmo salar ). Microscopic differences were not evident between uninfected and infected fish. Viral cytopathic effect was observed in cell cultures inoculated with fresh-frozen tissue homogenates from 3 of 3 ISAV-infected and 0 of 4 uninfected or sham-infected fish. The ISAV genome was detected by shotgun metagenomics in RNA extracted from the medium from 3 of 3 inoculated cell cultures, 3 of 3 infected fish, and 0 of 4 uninfected or sham-infected fish, yielding sufficient coverage for de novo assembly. An ISH probe against ISAV revealed ISAV genome in multiple organs, with abundance in renal hematopoietic tissue. Virus was detected by RT-rtPCR in gill, heart, kidney, liver, and spleen. EM and metagenomic WGS from tissues were challenging and unsuccessful. Our proof-of-concept methodology has promise for detection and characterization of unknown aquatic pathogens and also highlights some associated methodology challenges that require additional investigation., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to research, authorship, and/or publication of this article.

We performed a diagnostic disease investigation on a cohort of coho salmon ( Oncorhynchus kisutch ) fingerlings in Alaska exhibiting anorexia, gaping mouths, anemia, and increased mortality. Histologic examination revealed mild-to-severe myocardial degeneration and lymphohistiocytic and neutrophilic myocarditis, moderate splenic histiocytosis, and mild renal histiocytosis. Piscine orthoreoviruses 1 and 3 were not detected by molecular methods, and no other viruses could be cultured on 3 common diagnostic fish cell lines. De novo assembly produced a viral genome of 10 linear segments with >80% homology to piscine orthoreovirus 2 (PRV2) encoding all 11 PRV2 proteins. An in situ hybridization probe using RNAscope was developed against 697 viral nucleotides identified by sequencing, which revealed viral genome in heart, spleen, gill, kidney, liver, blood, and the lamina propria of the intestines. Our findings are supportive of a novel piscine orthoreovirus most closely related to PRV2 associated with morbidity and mortality of coho salmon in the northeastern Pacific., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to research, authorship, and/or publication of this article.

Antimicrobial resistance (AMR) in pathogens important to aquatic animal health is of increasing concern but vastly understudied. Antimicrobial therapy is used to both treat and prevent bacterial disease in fish and is critical for a viable aquaculture industry and for maintenance of wild fish populations. Unfortunately, phenotypic antimicrobial susceptibility testing is technically difficult for bacteria recovered from aquatic animal hosts resulting in challenges in resistance monitoring using traditional methods. Whole-genome sequencing provides an appealing methodology for investigation of putative resistance. As part of the ongoing efforts of the FDA CVM Vet-LIRN to monitor AMR, source laboratories cultured and preliminarily identified pathogenic bacteria isolated from various fish species collected in 2019 from across the United States. Sixty-one bacterial isolates were evaluated using whole-genome sequencing. We present here the assembled draft genomes, AMR genes, predicted resistance phenotypes, and virulence factors of the 61 isolates and discuss concurrence of the identifications made by source laboratories using matrix-assisted laser desorption/time-of-flight mass spectrometry., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to research, authorship, and/or publication of this article. The views expressed in this manuscript are those of the authors and may not reflect the official policy of the Department of Health and Human Services, the U.S. Food and Drug Administration, or the U.S. Government.

Asymptomatic malarial parasitemia represents the largest reservoir of infection and transmission, and the impact of coinfection with HIV-1 on this reservoir remains incompletely described. Accordingly, we sought to determine the prevalence of asymptomatic malarial parasitemia in Kombewa, Western Kenya, a region that is endemic for both malaria and HIV-1. A total of 1,762 dried blood spots were collected from asymptomatic adults in a cross-sectional study. The presence of parasitemia was first determined by a sensitive Plasmodium genus-specific 18S assay, followed by less sensitive species-specific DNA-based quantitative polymerase chain reaction (PCR) assays. The prevalence of asymptomatic malarial parasitemia by 18S genus-specific PCR assay was 64.4% (1,134/1,762). Of the 1,134 malaria positive samples, Plasmodium falciparum was the most prevalent species (57.4%), followed by Plasmodium malariae (3.8%) and Plasmodium ovale (2.6%) as single or mixed infections. As expected, the majority of infections were below the detection limit of microscopy and rapid diagnostic tests. HIV-1 prevalence was 10.6%, and we observed a significant association with malarial parasitemia by χ2 analysis (P = 0.0475). Seventy-one percent of HIV-1 infected volunteers were positive for Plasmodium 18S (132/186), with only 29% negative (54/186). In HIV-1-negative volunteers, the proportion was lower; 64% were found to be positive for 18S (998/1,569) and 36% were negative (571/1,569). Overall, the prevalence of asymptomatic malarial parasitemia in Western Kenya is high, and knowledge of these associations with HIV-1 infection are critically important for malaria elimination and eradication efforts focused on this important reservoir population.

Malaria parasites require pantothenate from both human and mosquito hosts to synthesize coenzyme A (CoA). Specifically, mosquito-stage parasites cannot synthesize pantothenate de novo or take up preformed CoA from the mosquito host, making it essential for the parasite to obtain pantothenate from mosquito stores. This makes pantothenate utilization an attractive target for controlling sexual stage malaria parasites in the mosquito. CoA is synthesized from pantothenate in a multi-step pathway initiated by the enzyme pantothenate kinase (PanK). In this work, we manipulated A. stephensi PanK activity and assessed the impact of mosquito PanK activity on the development of two malaria parasite species with distinct genetics and life cycles: the human parasite Plasmodium falciparum and the mouse parasite Plasmodium yoelii yoelii 17XNL. We identified two putative A. stephensi PanK isoforms encoded by a single gene and expressed in the mosquito midgut. Using both RNAi and small molecules with reported activity against human PanK, we confirmed that A. stephensi PanK manipulation was associated with corresponding changes in midgut CoA levels. Based on these findings, we used two small molecule modulators of human PanK activity (PZ-2891, compound 7) at reported and ten-fold EC 50 doses to examine the effects of manipulating A. stephensi PanK on malaria parasite infection success. Our data showed that oral provisioning of 1.3 nM and 13 nM PZ-2891 increased midgut CoA levels and significantly decreased infection success for both Plasmodium species. In contrast, oral provisioning of 62 nM and 620 nM compound 7 decreased CoA levels and significantly increased infection success for both Plasmodium species. This work establishes the A. stephensi CoA biosynthesis pathway as a potential target for broadly blocking malaria parasite development in anopheline hosts. We envision this strategy, with small molecule PanK modulators delivered to mosquitoes via attractive bait stations, working in concert with deployment of parasite-directed novel pantothenamide drugs to block parasite infection in the human host. In mosquitoes, depletion of pantothenate through manipulation to increase CoA biosynthesis is expected to negatively impact Plasmodium survival by starving the parasite of this essential nutrient. This has the potential to kill both wild type parasites and pantothenamide-resistant parasites that could develop under pantothenamide drug pressure if these compounds are used as future therapeutics for human malaria.

Background: Speed congenics is an important tool for creating congenic mice to investigate gene functions, but current SNP genotyping methods for speed congenics are expensive. These methods usually rely on chip or array technologies, and a different assay must be developed for each backcross strain combination. "Next generation" high throughput DNA sequencing technologies have the potential to decrease cost and increase flexibility and power of speed congenics, but thus far have not been utilized for this purpose., Results: We took advantage of the power of high throughput sequencing technologies to develop a cost-effective, high-density SNP genotyping assay that can be used across many combinations of backcross strains. The assay surveys 1640 genome-wide SNPs known to be polymorphic across > 100 mouse strains, with an expected average of 549 ± 136 SD diagnostic SNPs between each pair of strains. We demonstrated that the assay has a high density of diagnostic SNPs for backcrossing the BALB/c strain into the C57BL/6J strain (807-819 SNPs), and a sufficient density of diagnostic SNPs for backcrossing the closely related substrains C57BL/6N and C57BL/6J (123-139 SNPs). Furthermore, the assay can easily be modified to include additional diagnostic SNPs for backcrossing other closely related substrains. We also developed a bioinformatic pipeline for SNP genotyping and calculating the percentage of alleles that match the backcross recipient strain for each sample; this information can be used to guide the selection of individuals for the next backcross, and to assess whether individuals have become congenic. We demonstrated the effectiveness of the assay and bioinformatic pipeline with a backcross experiment of BALB/c-IL4/IL13 into C57BL/6J; after six generations of backcrosses, offspring were up to 99.8% congenic., Conclusions: The SNP genotyping assay and bioinformatic pipeline developed here present a valuable tool for increasing the power and decreasing the cost of many studies that depend on speed congenics. The assay is highly flexible and can be used for combinations of strains that are commonly used for speed congenics. The assay could also be used for other techniques including QTL mapping, standard F2 crosses, ancestry analysis, and forensics.

Antifolate resistance is significant in Kenya and presumed to result from extensive use and cross-resistance between antifolate antimalarials and antibiotics, including cotrimoxazole/Bactrim used for HIV-1 chemotherapy. However, little is known about antifolate-resistant malaria in the context of newly diagnosed HIV-1 co-infection prior to administration of HIV-1 chemotherapy. Blood samples from a cross-sectional study of asymptomatic adult Kenyans enrolled during voluntary HIV testing were analyzed by PCR for Plasmodium spp. More than 95% of volunteers with identifiable parasite species (132 HIV-1 co-infected) were infected with Plasmodium falciparum alone or P. falciparum with Plasmodium ovale and/or Plasmodium malariae . Deep sequencing was used to screen for mutations in P. falciparum dihydrofolate reductase (dhfr) (N51I, C59R, S108N, I164L) and dihydropteroate synthase (dhps) (S436H, A437G, K540E, A581G) from 1133 volunteers. Individual mutations in DHPS but not DHFR correlated with HIV-1 status. DHFR haplotype diversity was significantly different among volunteers by gender and HIV-1 status. DHPS haplotype diversity by HIV-1 status was significantly different between volunteers paired by age and gender, indicating that patterns of resistance were independent of these variables. Molecular simulations for a novel DHPS mutation (I504T) suggested that the mutated protein has increased affinity for the endogenous ligand DHPPP and decreased affinity for drug binding. A sub-group of monoclonal infections revealed that age and parasitemia were not correlated and enabled identification of a rare septuple-mutant haplotype (IRNL-HGEA). In our study, adult Kenyans newly diagnosed with HIV-1 infection were predominantly infected with moderately resistant P. falciparum , with patterns of infecting parasite genotypes significantly associated with HIV-1 status. Together with the discovery of DHPS I504T, these data indicate that antifolate resistance continues to evolve in Kenya. Further, they highlight the need to understand the effects of associated mutations on both fitness and resistance of P. falciparum in the context of HIV-1 co-infection to better inform treatment for asymptomatic malaria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Torrevillas, Garrison, McKeeken, Patel, Van Leuven, Dizon, Rivas, Hathaway, Bailey, Waitumbi, Kifude, Oyieko, Stewart and Luckhart.)

The larval environment of holometabolous insects determines many adult life history traits including, but not limited to, rate and success of development and adult lifespan and fecundity. The ancient stress signaling hormone abscisic acid (ABA), released by plants inundated with water and by leaf and root fragments in water, is likely ubiquitous in the mosquito larval environment and is well known for its wide ranging effects on invertebrate biology. Accordingly, ABA is a relevant stimulus and signal for mosquito development. In our studies, the addition of ABA at biologically relevant levels to larval rearing containers accelerated the time to pupation and increased death of A. stephensi pupae. We could not attribute these effects, however, to ABA-dependent changes in JH biosynthesis-associated gene expression, 20E titers or transcript patterns of insulin-like peptide genes. Adult females derived from ABA-treated larvae had reduced total protein content and significantly reduced post blood meal transcript expression of vitellogenin , effects that were consistent with variably reduced egg clutch sizes and oviposition success from the first through the third gonotrophic cycles. Adult female A. stephensi derived from ABA-treated larvae also exhibited reduced lifespans relative to controls. Collectively, these effects of ABA on A. stephensi life history traits are robust, durable and predictive of multiple impacts of an important malaria vector spreading to new malaria endemic regions., (Copyright © 2020 Taylor, Olds, Haney, Torrevillas and Luckhart.)

The World Health Organization estimates that more than half of the world's population is at risk for vector-borne diseases, including arboviruses. Because many arboviruses are mosquito borne, investigation of the insect immune response will help identify targets to reduce the spread of arboviruses. Here, we use a genetic screening approach to identify an insulin-like receptor as a component of the immune response to arboviral infection. We determine that vertebrate insulin reduces West Nile virus (WNV) replication in Drosophila melanogaster as well as WNV, Zika, and dengue virus titers in mosquito cells. Mechanistically, we show that insulin signaling activates the JAK/STAT, but not RNAi, pathway via ERK to control infection in Drosophila cells and Culex mosquitoes through an integrated immune response. Finally, we validate that insulin priming of adult female Culex mosquitoes through a blood meal reduces WNV infection, demonstrating an essential role for insulin signaling in insect antiviral responses to human pathogens., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Abscisic acid (ABA) is an ancient stress hormone and is detectable in a wide variety of organisms where it regulates innate immunity and inflammation. Previously, we showed that oral supplementation with ABA decreased parasitemia in a mouse model of malaria, decreased liver and spleen pathology and reduced parasite transmission to mosquitoes. Here, we report that higher circulating ABA levels were associated with a reduced risk of symptomatic malaria in a cohort of Plasmodium falciparum-infected Ugandan children. To understand possible mechanisms of ABA protection in malaria, we returned to our mouse model to show that ABA effects on Plasmodium yoelii 17XNL infection were accompanied by minimal effects on complete blood count and blood chemistry analytes, suggesting a benefit to host health. In addition, orally delivered ABA induced patterns of gene expression in mouse liver and spleen that suggested enhancement of host anti-parasite defenses. To test these inferences, we utilized passive immunization and knockout mice to demonstrate that ABA supplementation increases circulating levels of protective, parasite-specific IgG and requires caspase-1 to reduce parasitemia. Collectively, ABA induces host responses that ameliorate infection and disease in an animal model and suggest that further studies of ABA in the context of human malaria are warranted.

Background: Abscisic acid (ABA) is naturally present in mammalian blood and circulating levels can be increased by oral supplementation. We showed previously that oral ABA supplementation in a mouse model of Plasmodium yoelii 17XNL infection reduced parasitemia and gametocytemia, spleen and liver pathology, and parasite transmission to the mosquito Anopheles stephensi fed on these mice. Treatment of cultured Plasmodium falciparum with ABA at levels detected in our model had no effects on asexual growth or gametocyte formation in vitro. However, ABA treatment of cultured P. falciparum immediately prior to mosquito feeding significantly reduced oocyst development in A. stephensi via ABA-dependent synthesis of nitric oxide (NO) in the mosquito midgut., Results: Here we describe the mechanisms of effects of ABA on mosquito physiology, which are dependent on phosphorylation of TGF-β-activated kinase 1 (TAK1) and associated with changes in homeostatic gene expression and activity of kinases that are central to metabolic regulation in the midgut epithelium. Collectively, the timing of these effects suggests a transient physiological shift that enhances NF-κB-dependent innate immunity without significantly altering mosquito lifespan or fecundity., Conclusions: ABA is a highly conserved regulator of immune and metabolic homeostasis within the malaria vector A. stephensi with potential as a transmission-blocking supplemental treatment.