Jamestown Canyon virus (JCV) is a bunyavirus and arbovirus that causes viral neuroinvasive disease in North America. JCV neuropathogenesis is understudied, and no pro-viral host factors for cellular infection have been identified. Here, we assessed the role of low-density lipoprotein receptor-related protein 1 (LRP1; also known as CD91), which has been identified as a host entry factor for other bunyaviruses, in mediating JCV infection with a focus on neurons. Both neuronal and non-neuronal immortalized cell lines deficient for murine Lrp1 displayed reduced binding, internalization, and infection with JCV. Furthermore, a soluble extracellular domain of human LRP1 can bind directly to JCV, and the same region of LRP1 can neutralize JCV infection. Primary neurons, where Lrp1 was highly expressed, were permissive for JCV infection. Treatment of primary neurons with the murine Lrp1 ligand receptor-associated protein (RAP) resulted in reduced infectivity with JCV. Finally, treatment of Lrp1 knockout cells with RAP further reduced JCV infection, suggesting that other low-density lipoprotein receptors may mediate JCV entry in the absence of Lrp1. Together, these results support LRP1 as an important cellular factor for efficient neuronal infection by JCV. Emerging support for the use of LRP1 for viral entry by multiple bunyaviruses also makes LRP1 a promising target for antiviral development.IMPORTANCEJamestown Canyon virus (JCV), an emerging mosquito-transmitted virus in North American white-tailed deer, causes several cases of severe neurologic disease in humans each year. Our results on the use of low-density lipoprotein receptor (LDLR)-related protein 1 by JCV for efficient cellular infection of neurons underscore the significance of the LDLR family of receptors in viral infection. Recent studies also highlight the emerging use of the LDLR family of receptors for virus entry by the bunyavirus and alphavirus family members. Defining cellular factors that mediate infection by mosquito-transmitted viruses is critically important to the prototype pathogen approach for combating infectious diseases and countermeasure development.

BackgroundAllergic airway disease (AAD) is a growing concern in industrialized nations and can be influenced by fungal exposures. Basidiomycota yeast species such as Cryptococcus neoformans are known to exacerbate allergic airway disease; however, recent indoor assessments have identified other Basidiomycota yeasts, including Vishniacozyma victoriae (syn. Cryptococcus victoriae), to be prevalent and potentially associated with asthma. Until now, the murine pulmonary immune response to repeated V. victoriae exposure was previously unexplored. ObjectiveThis study aimed to compare the immunological impact of repeated pulmonary exposure to Cryptococcus yeasts. MethodsMice were repeatedly exposed to an immunogenic dose of C. neoformans or V. victoriae via oropharyngeal aspiration. Bronchoalveolar lavage fluid (BALF) and lungs were collected to examine airway remodeling, inflammation, mucous production, cellular influx, and cytokine responses at 1 day and 21 days post final exposure. The responses to C. neoformans and V. victoriae were analyzed and compared. ResultsFollowing repeated exposure, both C. neoformans and V. victoriae cells were still detectable in the lungs 21 days post final exposure. Repeated C. neoformans exposure initiated myeloid and lymphoid cellular infiltration into the lung that worsened over time, as well as an IL-4 and IL-5 response compared to PBS-exposed controls. In contrast, repeated V. victoriae exposure induced a strong CD4(+) T cell-driven lymphoid response that started to resolve by 21 days post final exposure. DiscussionC. neoformans remained in the lungs and exacerbated the pulmonary immune responses as expected following repeated exposure. The persistence of V. victoriae in the lung and strong lymphoid response following repeated exposure were unexpected given its lack of reported involvement in AAD. Given the abundance in indoor environments and industrial utilization of V. victoriae, these results highlight the importance to investigate the impact of frequently detected fungal organisms on the pulmonary response following inhalational exposure. Moreover, it is important to continue to address the knowledge gap involving Basidiomycota yeasts and their impact on AAD.

Aspergillus versicolor is ubiquitous in the environment and is particularly abundant in damp indoor spaces. Exposure to Aspergillus species, as well as other environmental fungi, has been linked to respiratory health outcomes, including asthma, allergy, and even local or disseminated infection. However, the pulmonary immunological mechanisms associated with repeated exposure to A. versicolor have remained relatively uncharacterized. Here, A. versicolor was cultured and desiccated on rice then placed in an acoustical generator system to achieve aerosolization. Mice were challenged with titrated doses of aerosolized conidia to examine deposition, lymphoproliferative properties, and immunotoxicological response to repeated inhalation exposures. The necessary dose to induce lymphoproliferation was identified, but not infection-like pathology. Further, it was determined that the dose was able to initiate localized immune responses. The data presented in this study demonstrate an optimized and reproducible method for delivering A. versicolor conidia to rodents via nose-only inhalation. Additionally, the feasibility of a long-term repeated exposure study was established. This experimental protocol can be used in future studies to investigate the physiological effects of repeated pulmonary exposure to fungal conidia utilizing a practical and relevant mode of delivery. In total, these data constitute an important foundation for subsequent research in the field.

Fungi are found ubiquitously in the environment and chronic repeated exposures have been associated with pulmonary and cognitive effects in humans. Previous studies have shown that two prominent indoor fungal contaminants, Stachybotrys chartarum and Aspergillus versicolor, are associated with allergic airway disease, airway remodeling, and Th2-mediated immunological effects. To understand the mechanisms of disease development, genetically modified murine models are an invaluable tool. To examine the immunotoxicological response to each fungal species, a nose-only acoustical generation system was used to deliver dry aerosolized spores to C57BL/6J (WT) and IL-13 and RAG-2 knockout mice, twice weekly for 4 or 13 weeks. Twenty-four-hours after final exposure, the immune response was examined via flow cytometry, antibody quantification, and histology. Wild-type mice had a strong inflammatory immune response to repeated fungal exposure by 13 weeks, which was significantly diminished in knockout mice. The response to S. chartarum resulted in increased T cells and eosinophils, whereas A. versicolor exposure in WT mice was characterized by an increase of B cells in the airways and total serum IgE antibodies. Mice lacking IL-13 or RAG-2 demonstrated a significant reduction of each, indicating that the cytokine IL-13 is necessary for the adaptive immune response to fungal exposure, as are mature B and T cells, as indicated in RAG-2-/- mice. This study identifies the mechanistic role of IL-13 during fungal exposure. Understanding the interplay of the immune system and indoor fungal contaminants is vital for identifying potential biomarkers and benchmarks for assessment of disease development.

Rift Valley fever virus (RVFV) is an emerging arbovirus found in Africa. While RVFV is pantropic and infects many cells and tissues, viral replication and necrosis within the liver play a critical role in mediating severe disease. The low-density lipoprotein receptor–related protein 1 (Lrp1) is a recently identified host factor for cellular entry and infection by RVFV. The biological significance of Lrp1, including its role in hepatic disease in vivo, however, remains to be determined. Because Lrp1 has a high expression level in hepatocytes, we developed a mouse model in which Lrp1 is specifically deleted in hepatocytes to test how the absence of liver Lrp1 expression affects RVF pathogenesis. Mice lacking Lrp1 expression in hepatocytes showed minimal RVFV replication in the liver, longer time to death, and altered clinical signs toward neurological disease. In contrast, RVFV infection levels in other tissues showed no difference between the two genotypes. Therefore, Lrp1 is essential for RVF hepatic disease in mice.

Pathogenic and non-pathogenic Cryptococcus yeast species are detected in indoor environments, and epidemiological data suggests varying potential impacts of exposure on allergic airway disease. However, the impact of non-pathogenic Cryptococcus species has yet to be examined. Mice were exposed via oropharyngeal aspiration to either a single or repeated dose (six exposures every other day) of 104 Cryptococcus neoformans cells or 106 Vishniacozyma victoriae (syn. Cryptococcus victoriae) cells. Circulating immunoglobulins, infiltrating immune cells, and lung histopathology were analyzed. Repeated exposure to Cryptococcus neoformans and Vishniacozyma victoriae resulted in increased neutrophils and monocytes, and decreased macrophages in the bronchoalveolar lavage fluid (BALF). Repeated Cryptococcus neoformans exposure increased T-cells and CD4+-T cells in the BALF, whereas exposure to Vishniacozyma victoriae increased only lung monocytes. Repeated exposure to each species did not significantly impact serum IgE or IgG levels, although Cryptococcus neoformans exposure initiated a trend towards increased IgG. Lung inflammation and airway epithelium changes were characteristic of both repeated exposures. Following repeated exposure, GMS-stained yeast of each species were observed within alveolar macrophages and associated with the foci of lung inflammation. Although no yeast were observed in the lungs following a single dose of Cryptococcus neoformans, Vishniacozyma victoriae cells were frequently seen within macrophages near terminal bronchioles. Pathogenic Cryptococcus neoformans induced a stronger immune response compared to Vishniacozyma victoriae, evidenced by an increase in T-cell responses following Cryptococcus neoformans exposure. Future studies will aim to understand the impact these species have in a model of allergic airway disease.

Like the pathogenic yeast Cryptococcus neoformans, Vishniacozyma victoriae (syn. Cryptococcus victoriae), a non-pathogenic yeast, has been associated with allergic airway disease. Although V. victoriae is frequently detected in indoor environments, the impact of repeated exposure is currently unknown. In this study, mice were exposed via oropharyngeal aspiration to V. victoriae or C. neoformans every other day for a total of six exposures, and responses were analyzed 1 day and 21 days post final exposure. As expected, repeated exposure to C. neoformans resulted in an increase of myeloid and lymphoid cells in the bronchoalveolar lavage fluid (BALF), granuloma formation with intralesional yeast, and peribronchiolar and perivascular inflammation 1 day post exposure. This response was exacerbated 21 days post exposure as the yeast cells replicated. Repeated exposure to V. victoriae resulted in an increase in eosinophils, Ly6Chi/int monocytes, and CD103+ dendritic cells in the BALF, and mild perivascular and peribronchiolar inflammation 1 day post exposure. Surprisingly, V. victoriae cells were present in the lungs of exposed mice 21 days post exposure, and the response shifted toward a nodular lymphocytic response with an increase of CD4+ T cells in the BALF. Lastly, C. neoformans exposure was predicted to activate antigen presentation, B cell development and Th2 proteomic pathways, whereas V. victoriae was predicted to activate VDR/RXR activation, Granzyme A signaling, and IGF-1 signaling pathways 21 days post exposure. The results of this study suggest that exposure to environmentally ubiquitous V. victoriae may exacerbate allergic airway disease, although not to the extent of exposure to pathogenic C. neoformans. Interagency Agreement between NIEHS & NIOSH: AES 12007001-1-0-6

Indoor environments contain a broad diversity of non-pathogenic Basidiomycota yeasts, but their role in exacerbating adverse health effects has remained unclear. To understand the role of Vishniacozyma victoriae exposure and its impact on human health. A qPCR assay was developed to detect and quantify an abundant indoor yeast species, Vishniacozyma victoriae (syn. Cryptococcus victoriae), from homes participating in the New York City Neighborhood Asthma and Allergy Study (NAAS). We evaluated the associations between V. victoriae, housing characteristics, and asthma relevant health endpoints. V. victoriae was quantified in 236 of the 256 bedroom floor dust samples ranging from less than 300–45,918 cell equivalents/mg of dust. Higher concentrations of V. victoriae were significantly associated with carpeted bedroom floors (P = 0.044), mean specific humidity (P = 0.004), winter (P < 0.0001) and spring (P = 0.001) seasons, and the presence of dog (P = 0.010) and dog allergen Can f 1 (P = 0.027). V. victoriae concentrations were lower in homes of children with asthma vs. without asthma (P = 0.027), an association observed only among the non-seroatopic children.

Aspergillus versicolor is an environmentally ubiquitous fungal species that is particularly abundant in environments with water infiltration. Respiration of fungal products, including conidia, hyphal fragments, mycotoxins, and other secondary metabolites may cause adverse pulmonary health effects including asthma and allergy. In the present study, pulmonary immune responses in B6C3F1 mice were examined following 1, 2, 4, 8, and 13 weeks of repeated respiratory challenge to 3×105A. versicolor conidia. Increased alveolar macrophages, inflammatory monocytes, neutrophils, and eosinophils, which peaked between 4 and 8 weeks, were detected in the lungs of mice exposed to viable conidia. Type 2 innate lymphoid cells (ILC2s) peaked between 8 and 13 weeks compared to total T cells which increased between 2 and 8 weeks and decreased at 13 weeks. Protein concentrations of the Th2 cytokines IL4, IL5, and IL13 were highest in bronchoaveolar lavage fluid of exposed mice following 4 weeks and exhibited a time-dependent reduction at 8 and 13 weeks. Inhalation of conidia for 13 weeks led to goblet cell hyperplasia, vascular wall thickening, and perivascular inflammation. Finally, mixed perivascular inflammation and smooth muscle hypertrophy were sustained at 4 weeks post-exposure. These data indicate that exposure to A. versicolor elicits a Th2-biased immune response that is driven by innate cell populations and is associated with the induction of remodeling of the pulmonary vasculature.

The contribution of non-pathogenic Cryptococcus species to respiratory morbidity has recently been reported. A quantitative polymerase chain reaction (qPCR) assay was developed to quantify Vishniacozyma victoriae (syn. Cryptococcus victoriae) in dust samples derived from the NYC Neighborhood Asthma and Allergy Study (NAAS).

Carpet and rugs currently represent about half of the United States flooring market and offer many benefits as a flooring type. How carpets influence our exposure to both microorganisms and chemicals in indoor environments has important health implications but is not well understood. The goal of this manuscript is to consolidate what is known about how carpet impacts indoor chemistry and microbiology, as well as to identify the important research gaps that remain. After describing the current use of carpet indoors, questions focus on five specific areas: 1) indoor chemistry, 2) indoor microbiology, 3) resuspension and exposure, 4) current practices and future needs, and 5) sustainability. Overall, it is clear that carpet can influence our exposures to particles and volatile compounds in the indoor environment by acting as a direct source, as a reservoir of environmental contaminants, and as a surface supporting chemical and biological transformations. However, the health implications of these processes are not well known, nor how cleaning practices could be optimized to minimize potential negative impacts. Current standards and recommendations focus largely on carpets as a primary source of chemicals and on limiting moisture that would support microbial growth. Future research should consider enhancing knowledge related to the impact of carpet in the indoor environment and how we might improve the design and maintenance of this common material to reduce our exposure to harmful contaminants while retaining the benefits to consumers.

More than 180 individual phages infecting hosts in the phylum Actinobacteria have been sequenced and grouped into Cluster A because of their similar overall nucleotide sequences and genome architectures. These Cluster A phages are either temperate or derivatives of temperate parents, and most have an integration cassette near the centre of the genome containing an integrase gene and attP. However, about 20% of the phages lack an integration cassette, which is replaced by a 1.4 kbp segment with predicted partitioning functions, including plasmid-like parA and parB genes. Phage RedRock forms stable lysogens in Mycobacterium smegmatis in which the prophage replicates at 2.4 copies/chromosome and the partitioning system confers prophage maintenance. The parAB genes are expressed upon RedRock infection of M. smegmatis, but are downregulated once lysogeny is established by binding of RedRock ParB to parS-L, one of two centromere-like sites flanking the parAB genes. The RedRock parS-L and parS-R sites are composed of eight directly repeated copies of an 8 bp motif that is recognized by ParB. The actinobacteriophage parABS cassettes span considerable sequence diversity and specificity, providing a suite of tools for use in mycobacterial genetics.

Allergic airway diseases such as asthma continue to increase in incidence in industrialized nations like the United States. These diseases are complex inflammatory processes involving numerous cells and mediators and are strongly influenced by fungal exposures. Recent developments in fungal detection methods have highlighted the contribution of Basidiomycota yeast species in indoor environments such as Vishniacozyma (syn. Cryptococcus) victoriae. However, despite the high levels of this yeast detected in indoor environments, very little is known about it or its role in respiratory morbidity. V. victoriae is phylogenetically similar to pathogenic Cryptococcus neoformans but lacks a capsule and is not known to be pathogenic. Epidemiological-based studies suggested varied impacts of Cryptococcus yeast species and allergic airway disease. For these reasons, this dissertation addresses the knowledge gap regarding the pulmonary inflammatory response to V. victoriae. First, V. victoriae was quantified in indoor environmental samples, and associations between this yeast and housing, environmental, and health data were examined. Then, the pulmonary immune response to repeated exposures to either V. victoriae or C. neoformans in mice was analyzed and compared. Lastly, the impact of repeated exposures to each yeast was examined in the context of an allergic airway disease model and proteomic analyses were conducted to start to identify potential mechanisms impacted because of V. victoriae exposure. Together, these findings show that exposure to Cryptococcus yeast species induces significant pulmonary inflammation and that the persistence of V. victoriae and C. neoformans following repeated exposure elicits unique pulmonary inflammatory responses. These results establish a crucial need for further exploration into yeast exposures, specifically Basidiomycota yeasts, and lay the groundwork for future investigations and policy decisions regarding exposure to yeast.