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In late 2019, SARS-CoV-2 spilled over from an animal host into humans, where it efficiently spread, resulting in the COVID-19 pandemic. Through both natural and experimental infections, we learned that many animal species are susceptible to SARS-CoV-2. Importantly, animals in close proximity to humans, including companion, farmed, and those at zoos and aquariums, became infected, and many studies demonstrated transmission to/from humans in these settings. In this study, we first review the literature of SARS-CoV-2 infections in tigers and lions and compare species, sex, age, virus and antibody detection assay, and types, frequency, and length of clinical signs, demonstrating broad heterogeneity among infections. We then describe a SARS-CoV-2 outbreak in lions, tigers, and hyenas at Denver Zoo in late 2021. Animals were tested for viral RNA (vRNA) for 4 months. Lions had significantly more vRNA in nasal swabs than both tigers and hyenas, and many individual lions experienced viral recrudescence after weeks of undetectable vRNA. Infectious virus was correlated with high levels of vRNA and was more likely to be detected earlier during infection. Four months post-infection, all tested animals generated robust neutralizing antibody titers. Animals were infected with Delta lineage AY.20 identical to a variant circulating at less than 1% in Colorado humans at that time, suggesting a single spillover event from an infected human spread within and between species housed at the zoo. Better understanding of epidemiology and susceptibility of SARS-CoV-2 infections in animals is critical to limit the current and future spread and protect animal and human health.IMPORTANCESurveillance and experimental testing have shown many animal species, including companion, wildlife, and conservatory, are susceptible to SARS-CoV-2. Early in the COVID-19 pandemic, big cats at zoological institutions were among the first documented cases of naturally infected animals; however, challenges in the ability to collect longitudinal samples in zoo animals have limited our understanding of SARS-CoV-2 kinetics and clearance in these settings. We measured SARS-CoV-2 infections over 4 months in lions, tigers, and hyenas at Denver Zoo and detected viral RNA, infectious virus, neutralizing antibodies, and recrudescence after initial clearance. We found lions had longer and higher levels of virus compared to the other species. All animals were infected by a rare viral lineage circulating in the human population, suggesting a single spillover followed by interspecies transmission. These data are important in better understanding natural SARS-CoV-2 spillover, spread, and infection kinetics within multiple species of zoo animals.

This study evaluates the efficacy of Bifidobacterium longum subsp. longum BL21 in mitigating symptoms of polycystic ovarian syndrome (PCOS) in DHT-induced PCOS model mice. It focuses on BL21's role in modulating metabolic dysregulation, inflammation, and neuroprotection via the gut-brain-ovary axis. Employing an 8-week treatment regimen, this research assessed the effects of BL21 on prenatal androgen-induced PCOS in ICR mice. Evaluations included body weight, glucose tolerance tests, serum analyses of BDNF, inflammatory markers, sex hormone levels, and 16S rRNA gene sequencing for gut microbiota diversity and composition. Twenty-four ICR mice with induced PCOS served as subjects to examine the probiotic's impact. Mice were administered a daily oral dose of 1 × 10⁹ CFU of BL21 continuously for a total of 8 weeks. BL21 significantly enhanced sex hormone levels (P < 0.05), particularly those of follicle-stimulating hormone (FSH) and estradiol (E2), indicating improved ovarian function and offering a novel PCOS treatment approach. The intervention notably curbed weight gain and improved glucose tolerance in PCOS mice (P < 0.05). BL21 reduced inflammatory markers such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and lipopolysaccharides (LPS), while increasing IL-10, BDNF, FSH, and E2 levels (P < 0.05 for all). It also enriched gut microbiota diversity, enhancing populations of Bifidobacterium and Lactobacillus. Correlation analyses underscored the positive shifts in microbiota linked to beneficial hormonal and inflammatory profiles. BL21 shows promise in alleviating PCOS symptoms through metabolic regulation, inflammation reduction, and neuroprotection, validating its potential in integrated therapeutic strategies.IMPORTANCEPolycystic ovarian syndrome (PCOS) is a prevalent endocrine disorder affecting women of reproductive age, characterized by metabolic irregularities, hormonal imbalances, and chronic inflammation. Existing treatments are often inadequate, addressing symptoms without targeting the underlying etiological factors. The investigation of Bifidobacterium longum subsp. longum BL21 as a probiotic intervention offers a novel approach by potentially regulating the gut-brain-ovary axis. This could lead to innovative therapeutic strategies that not only manage but also potentially reverse the multifaceted symptoms of PCOS, enhancing quality of life and reproductive health.

Helicobacter pylori is a major causative agent in several upper gastroduodenal tract diseases, including gastric cancer. The development of methods to genetically manipulate H. pylori by natural transformation has allowed a greater understanding of its biology and role in these diseases. Nevertheless, the transformation methods used for H. pylori are time-consuming, requiring growth of these fastidious and slow-growing bacteria from -80°C stocks. The aim of the study was to develop a more rapid and convenient method for generating H. pylori mutants. We describe here a method in which competent H. pylori bacteria can be stored at -80°C and used in transformations on the day of resuscitation, similar to methods routinely used for Escherichia coli. This means that transformation can be performed at will and that transformants can be obtained within days, rather than weeks. Furthermore, we show that bacteria remain competent for at least six months storage at -80°C and that the method is applicable to strains with varying levels of natural competence. Transformation efficiencies of the bacteria varied between 10¹ and 10⁶ transformants/total colony-forming units/µg donor DNA, depending on the strain. We suggest that this improved method will facilitate studies on H. pylori and, moreover, may be applicable to other naturally transformable pathogens with fastidious growth requirements and requiring ultra-low temperature refrigeration for long-term preservation.IMPORTANCEGenetic manipulation is an important tool in the study of pathogenic bacteria and their interactions with the host. Many pathogenic bacteria are naturally transformable; however, transformation experiments can be impeded by the slow-growing and fastidious nature of some species. One such bacterium is Helicobacter pylori, which requires resuscitation from -80°C and multiple subcultures prior to transformation. The method described in the current study uses a simple modification of a conventional method of natural transformation. Using this method, competent H. pylori bacteria can be stored for long periods (at least six months) and resuscitated as needed for use in experiments. The method circumvents the need for multiple and lengthy subcultures prior to transformation, nor does it involve costly materials, complicated procedures, or sophisticated equipment. Thus, we describe a simple, inexpensive, and time-efficient method that may have broader applications for use with other fastidious bacteria.

Chronic wasting disease (CWD) is a highly contagious prion disease occurring in free-ranging and farmed cervids. CWD continues to spread uncontrolled across North America, and cases continue to be detected almost every year in the Republic of Korea. CWD-infected animals contaminate the soil by releasing infectious prions through their excreta, and shed prions accumulate and remain infectious in the soil for years. Given that the upper soil levels can become contaminated with prions and serve as infectivity reservoirs facilitating horizontal transmission of CWD, the ability to detect prions in the soil is needed for monitoring and managing CWD spread. Using the protein misfolding cyclic amplification (PMCA) technique, we investigated whether prions could be amplified and detected in farm soil experimentally exposed to CWD-infected brain homogenate as well as in the soil of CWD-affected farms. From each soil sample, we performed 10 serial extractions and used these 10 extracts as PMCA templates. Here, we show that prion seeding activity was detected in extracts from farm soil following 4 years of incubation with CWD-infected brain homogenate. More importantly, 13 of 38 soil samples collected from six CWD-affected farms displayed prion seeding activity, with at least one soil sample in each farm being PMCA positive. Mouse bioassays confirmed the presence of prion infectivity in the soil extracts in which PMCA seeding activity was detected. This is the first report describing the successful detection of prions in soil collected from CWD-affected farms, suggesting that PMCA conducted on serial soil extracts is a sensitive means for prion detection in CWD-contaminated soil.IMPORTANCEChronic wasting disease (CWD) is a highly contagious prion disease affecting free-ranging and farmed cervids. CWD continues to spread uncontrollably across North America, and multiple cases are detected annually in the Republic of Korea. Prions shed from CWD-infected animals remain infectious in the soil for years, serving as infectivity reservoirs that facilitate horizontal transmission of the disease. Therefore, the ability to detect CWD prions in soil is crucial for monitoring and managing the spread of the disease. In this study, we have demonstrated for the first time that prions in the soil of CWD-affected farms can be reliably detected using a combination of serial soil extraction and a prion amplification technique. Our data, in which at least one soil sample tested positive for CWD in each of the six CWD-affected farms analyzed, suggest that the approach employed in this study is a sensitive method for prion detection in CWD-contaminated soil.

The evolution of oxygenic photosynthesis in the Cyanobacteria was one of the most transformative events in Earth history, eventually leading to the oxygenation of Earth's atmosphere. However, it is difficult to understand how the earliest Cyanobacteria functioned or evolved on early Earth in part because we do not understand their ecology, including the environments in which they lived. Here, we use a cutting-edge bioinformatics tool to survey nearly 500,000 metagenomes for relatives of the taxa that likely bookended the evolution of oxygenic photosynthesis to identify the modern environments in which these organisms live. Ancestral state reconstruction suggests that the common ancestors of these organisms lived in terrestrial (soil and/or freshwater) environments. This restricted distribution may have increased the lag between the evolution of oxygenic photosynthesis and the oxygenation of Earth's atmosphere.IMPORTANCECyanobacteria generate oxygen as part of their metabolism and are responsible for the rise of oxygen in Earth's atmosphere over two billion years ago. However, we do not know how long this process may have taken. To help constrain how long this process would have taken, it is necessary to understand where the earliest Cyanobacteria may have lived. Here, we use a cutting-edge bioinformatics tool called branch water to examine the environments where modern Cyanobacteria and their relatives live to constrain those inhabited by the earliest Cyanobacteria. We find that these species likely lived in non-marine environments. This indicates that the rise of oxygen may have taken longer than previously believed.

Coronaviruses (CoV) emerge suddenly from animal reservoirs to cause novel diseases in new hosts. Discovered in 2012, the Middle East respiratory syndrome coronavirus (MERS-CoV) is endemic in camels in the Middle East and is continually causing local outbreaks and epidemics. While all three newly emerging human CoVs from the past 20 years (SARS-CoV, SARS-CoV-2, and MERS-CoV) cause respiratory disease, each CoV has unique host interactions that drive differential pathogeneses. To better understand the virus and host interactions driving lethal MERS-CoV infection, we performed a longitudinal multi-omics analysis of sublethal and lethal MERS-CoV infection in mice. Significant differences were observed in body weight loss, virus titers, and acute lung injury among lethal and sub-lethal virus doses. Virus-induced apoptosis of type I and II alveolar epithelial cells suggests that loss or dysregulation of these key cell populations was a major driver of severe disease. Omics analysis suggested differential pathogenesis was multi-factorial with clear differences among innate and adaptive immune pathways as well as those that regulate lung epithelial homeostasis. Infection of mice lacking functional T and B cells showed that adaptive immunity was important in controlling viral replication but also increased pathogenesis. In summary, we provide a high-resolution host response atlas for MERS-CoV infection and disease severity. Multi-omics studies of viral pathogenesis offer a unique opportunity to not only better understand the molecular mechanisms of disease but also to identify genes and pathways that can be exploited for therapeutic intervention all of which is important for our future pandemic preparedness.IMPORTANCEEmerging coronaviruses like SARS-CoV, SARS-CoV-2, and MERS-CoV cause a range of disease outcomes in humans from an asymptomatic, moderate, and severe respiratory disease that can progress to death but the factors causing these disparate outcomes remain unclear. Understanding host responses to mild and life-threatening infections provides insight into virus-host networks within and across organ systems that contribute to disease outcomes. We used multi-omics approaches to comprehensively define the host response to moderate and severe MERS-CoV infection. Severe respiratory disease was associated with dysregulation of the immune response. Key lung epithelial cell populations that are essential for lung function get infected and die. Mice lacking key immune cell populations experienced greater virus replication but decreased disease severity implicating the immune system in both protective and pathogenic roles in response to MERS-CoV. These data could be utilized to design new therapeutic strategies targeting specific pathways that contribute to severe disease.

Lori Huberman works in the field of fungal genetics, with an emphasis on investigating the genetic mechanisms fungi use to sense and respond to the nutrients and toxins in their environment. In this mSphere of Influence article, she reflects on how "Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons" by K. M. Wetmore, M. N. Price, R. J. Waters, J. S. Lamson, et al. (mBio 6:e00306-15, 2015, https://doi.org/10.1128/mBio.00306-15) made an impact on her by establishing technologies that open realistic possibilities for developing high-throughput screening methods to correlate phenotype to genotype in diverse fungal species.