SUMMARY NLR genes mediate host immunity to various pathogenic stimuli. However, in vivo evidence for NLR involvement in viral sensing has not been widely investigated and remains controversial. As an ultimate test of the physiologic role of NLRP3 during RNA viral infection, this work explores the in vivo role of NLRP3 inflammasome components during influenza virus infection. Mice lacking Nlrp3, ASC, or Caspase-1, but not Nlrc4, exhibit dramatically increased mortality but reduced immune response following influenza virus exposure. Utilizing analogs of dsRNA (poly(I:C)) and ssRNA (ssRNA40), we demonstrate that NLRP3-mediated response can be activated by RNA species. Mechanistically, NLRP3 inflammasome activation by influenza virus is dependent upon lysosomal maturation and reactive oxygen species. Inhibition of ROS induction eliminated IL-1β production in animals during influenza infection. Together, these data place the NLRP3 inflammasome as an essential component in host defense against influenza infection through the sensing of viral RNA.

Gene therapy for cystic fibrosis (CF) lung disease requires efficient gene transfer to airway epithelial cells after intralumenal delivery. Most gene transfer vectors so far tested have not provided the efficiency required. Although human respiratory syncytial virus (RSV), a common respiratory virus, is known to infect the respiratory epithelium, the mechanism of infection and the epithelial cell type targeted by RSV have not been determined. We have utilized human primary airway epithelial cell cultures that generate a well-differentiated pseudostratified mucociliary epithelium to investigate whether RSV infects airway epithelium via the lumenal (apical) surface. A recombinant RSV expressing green fluorescent protein (rgRSV) infected epithelial cell cultures with high gene transfer efficiency when applied to the apical surface but not after basolateral inoculation. Analyses of the cell types infected by RSV revealed that lumenal columnar cells, specifically ciliated epithelial cells, were targeted by RSV and that cultures became susceptible to infection as they differentiated into a ciliated phenotype. In addition to infection of ciliated cells via the apical membrane, RSV was shed exclusively from the apical surface and spread to neighboring ciliated cells by the motion of the cilial beat. Gross histological examination of cultures infected with RSV revealed no evidence of obvious cytopathology, suggesting that RSV infection in the absence of an immune response can be tolerated for >3 months. Therefore, rgRSV efficiently transduced the airway epithelium via the lumenal surface and specifically targeted ciliated airway epithelial cells. Since rgRSV appears to breach the lumenal barriers encountered by other gene transfer vectors in the airway, this virus may be a good candidate for the development of a gene transfer vector for CF lung disease.Human respiratory syncytial virus (RSV) is the most important viral agent causing serious pediatric respiratory disease worldwide (5). RSV infection causes common-cold-like symptoms that progress to lower respiratory tract disease in 25 to 40% of infected infants and results in hospitalization for 0.1 to 1.0% of those infected. Almost everyone has been infected by RSV by 2 years of age. The immunity induced by RSV infection typically is incomplete, and reinfection is common, although subsequent infections are partially restricted and the disease severity is reduced (5).There is a relative lack of direct, detailed information on the characteristics of RSV infection, spread, and pathology in the upper and lower respiratory tract of humans. RSV infects the superficial layer of the respiratory epithelium and has the capacity to spread throughout the conducting airways. However, the details of RSV infection remain unclear, including whether there is a cell specificity to infection, the extent of virus infection and its relationship to disease manifestation and severity, the extent and cause of tissue damage, and whether the formation of syncytia that are so prominent in nonpola...

The coxsackievirus and adenovirus receptor (CAR) mediates viral attachment and infection, but its physiologic functions have not been described. In nonpolarized cells, CAR localized to homotypic intercellular contacts, mediated homotypic cell aggregation, and recruited the tight junction protein ZO-1 to sites of cell-cell contact. In polarized epithelial cells, CAR and ZO-1 colocalized to tight junctions and could be coprecipitated from cell lysates. CAR expression led to reduced passage of macromolecules and ions across cell monolayers, and soluble CAR inhibited the formation of functional tight junctions. Virus entry into polarized epithelium required disruption of tight junctions. These results indicate that CAR is a component of the tight junction and of the functional barrier to paracellular solute movement. Sequestration of CAR in tight junctions may limit virus infection across epithelial surfaces.G roup B coxsackieviruses and a number of adenovirus serotypes initiate infection by binding to the coxsackievirus and adenovirus receptor (CAR) (1-3). CAR is a 46-kDa integral membrane protein with a typical transmembrane region, a long cytoplasmic domain, and an extracellular region composed of two Ig-like domains (1, 2). Both adenovirus (4) and coxsackievirus (5) interact with the N-terminal domain. Homologs of human CAR have been characterized in mice (2, 6), rats, pigs, dogs (7), and zebrafish (8). The murine and human proteins are very similar (91% amino acid identity within the extracellular domain, 77% within the transmembrane domain, and 95% identity within the cytoplasmic domain). Variant isoforms of CAR, which differ only at the C terminus and which most likely result from alternative splicing, have been identified in mice (6), humans, and rats (7). Despite evidence of its evolutionary conservation in mammals and nonmammalian vertebrates, the function of CAR is not known.Tight junctions are continuous circumferential intercellular contacts at the apical poles of lateral cell membranes, appearing in electron micrographs as a series of discrete contacts between the plasma membranes of adjacent cells (9). Tight junctions form a barrier that regulates the paracellular transit of water, solutes, and immune cells across an epithelium (10), and are essential for establishing cell polarity by separating the apical and basolateral domains of polarized epithelial cells (11). ZO-1, the first tight junction protein identified (12, 13), is an intracellular peripheral membrane scaffolding protein important for tight junction structure and assembly. In the present study, we found that in polarized epithelial cells CAR is expressed at the tight junction, where it associates with ZO-1 and functions in the barrier to the movement of macromolecules and ions. Materials and MethodsCell Culture. T-84, CALU-3, and 16HBE14o-cells were grown in 10% CO 2 in a 1:1 mixture of DMEM and Ham's F-12 medium with 10% FCS. To establish polarized monolayers, 5 ϫ 10 5 cells per well were plated on 12-mm diameter polyester filters with a ...

Outbreaks from zoonotic sources represent a threat to both human disease as well as the global economy. Despite a wealth of metagenomics studies, methods to leverage these datasets to identify future threats are underdeveloped. In this study, we describe an approach that combines existing metagenomics data with reverse genetics to engineer reagents to evaluate emergence and pathogenic potential of circulating zoonotic viruses. Focusing on the severe acute respiratory syndrome (SARS)-like viruses, the results indicate that the WIV1-coronavirus (CoV) cluster has the ability to directly infect and may undergo limited transmission in human populations. However, in vivo attenuation suggests additional adaptation is required for epidemic disease. Importantly, available SARS monoclonal antibodies offered success in limiting viral infection absent from available vaccine approaches. Together, the data highlight the utility of a platform to identify and prioritize prepandemic strains harbored in animal reservoirs and document the threat posed by WIV1-CoV for emergence in human populations.A lthough previously associated with upper respiratory infections, the emergence of severe acute respiratory coronavirus (SARS-CoV) in [2002][2003], and more recently, Middle East respiratory syndrome (MERS)-CoV underscores the threat of cross-species transmission leading to virulent pandemic viral infections (1, 2). Whereas prevailing research suggests that SARSCoV emerged from viruses in the Chinese horseshoe bat, identifying a progenitor strain that used human angiotensin converting enzyme 2 (ACE2) had proven elusive (3, 4). However, recent metagenomics studies isolated several SARS-like virus sequences that share ≥90% genome-wide homology and represented the closest sequences to the epidemic strains (5, 6). Importantly, researchers also isolated replication competent virus; WIV1-CoV, part of the Rs3306 cluster, could use ACE2 orthologs and mediated low-level replication in human cells (5). Overall, the evidence indicates that SARS-CoV likely emerged from Chinese horseshoe bats and that similar viruses are still harbored in these populations.The identification of WIV1-CoV and its capacity to use ACE2 orthologs offers a warning for possible reemergence and provides an opportunity to prepare for a future CoV outbreak. To achieve this goal, a new platform is required to translate metagenomics findings; the approach must generate critical diagnostic reagents, define emergence potential of novel strains, and determine efficacy of current therapeutics. Building on this premise, we developed a framework to examine circulating CoVs using reverse genetic systems to construct full-length and chimeric viruses. The results indicate that viruses using WIV1-CoV spike are poised to emerge in human populations due to efficient replication in primary human airway epithelial cell cultures. However, additional adaptation, potentially independent of the spike protein receptor-binding domain, is required for pathogenesis and epidemic disease. Importantly,...