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Background According to the National Electronic Injury Surveillance System, there are an estimated 9,400 riding lawn mower injuries in the United States in pediatric patients per year. Previous case series report a 7-12.5% infection rate after injury. We report our experience of lawn mower injury-related infections over a 4-year period. Methods Cases of lawn mower injury at our center were identified by the ICD-10 code W28.XXXA for contact with a powered lawn mower between 2017-2020. Patient demographics, injury background, microbial results, and antimicrobial use were collected via manual chart review. Results During the 4-year period, 25 patients presented to UPMC Children’s Hospital of Pittsburgh who had a diagnosis of lawn mower injury. The median age of the patients was 6.5 years old. Most cases sustained foot injury (68%, 17/25), followed by leg (24%, 6/25), and hand or arm (24%, 6/25). Perioperative antibiotics depended on the extent of the damage, intent to amputate or salvage a limb, and extent of obvious contamination. These included 24-48 hours of piperacillin-tazobactam for 44% (11/25) of patients, ampicillin/sulbactam for 32% (8/25) of patients, and cefazolin for 24% (6/25) patients. Four of the 25 patients (16%) had infection after the initial injury as defined as growth of organisms from a wound culture collected more than 48 hours after the initial injury and debridement. Antimicrobials for the treatment of infection were further tailored to the culture results. From the four patients with infection after initial debridement or amputation, pathogens included: Serratia marcescens, Staphylococcus aureus, Myroides odartus, Stenotrophomonas, Prevotella, Aeromonas, Coagulase negative Staphylococcus, Enterobacter cloacae, Sphingomonas, Bacillus species, Pseudomonas aeruginosa, Candida albicans, Rhizopus, and Trichosporon. Conclusion Infections after lawn mower injures are infrequent with early, aggressive surgical debridement. However, infections following lawn mower injuries are frequently polymicrobial and involve a wide variety of pathogens. Obtaining repeat cultures during subsequent surgical debridements can be useful to track persistent growth and identify new and uncommon pathogens. Disclosures All Authors: No reported disclosures.

Background Seasonal influenza causes substantial morbidity and mortality in older adults. High-dose inactivated influenza vaccine (HD-IIV), with increased antigen content compared to standard-dose influenza vaccines (SD-IIV), is licensed for use in people aged ≥65 years. We sought to evaluate the effectiveness of HD-IIV and SD-IIV for prevention of influenza-associated hospitalizations. Methods Hospitalized patients with acute respiratory illness were enrolled in an observational vaccine effectiveness study at 8 hospitals in the United States Hospitalized Adult Influenza Vaccine Effectiveness Network during the 2015–2016 and 2016–2017 influenza seasons. Enrolled patients were tested for influenza, and receipt of influenza vaccine by type was recorded. Effectiveness of SD-IIV and HD-IIV was estimated using a test-negative design (comparing odds of influenza among vaccinated and unvaccinated patients). Relative effectiveness of SD-IIV and HD-IIV was estimated using logistic regression. Results Among 1487 enrolled patients aged ≥65 years, 1107 (74%) were vaccinated; 622 (56%) received HD-IIV, and 485 (44%) received SD-IIV. Overall, 277 (19%) tested positive for influenza, including 98 (16%) who received HD-IIV, 87 (18%) who received SD-IIV, and 92 (24%) who were unvaccinated. After adjusting for confounding variables, effectiveness of SD-IIV was 6% (95% confidence interval [CI] −42%, 38%) and that of HD-IIV was 32% (95% CI −3%, 54%), for a relative effectiveness of HD-IIV versus SD-IIV of 27% (95% CI −1%, 48%). Conclusions During 2 US influenza seasons, vaccine effectiveness was low to moderate for prevention of influenza hospitalization among adults aged ≥65 years. High-dose vaccine offered greater effectiveness. None of these findings were statistically significant.

Rift Valley fever (RVF) is an arboviral disease of humans and livestock responsible for severe economic and human health impacts. In humans, RVF spans a variety of clinical manifestations, ranging from an acute flu-like illness to severe forms of disease, including late-onset encephalitis. The large variations in human RVF disease are inadequately represented by current murine models, which overwhelmingly die of early-onset hepatitis. Existing mouse models of RVF encephalitis are either immunosuppressed, display an inconsistent phenotype, or develop encephalitis only when challenged via intranasal or aerosol exposure. In this study, the genetically defined recombinant inbred mouse resource known as the Collaborative Cross (CC) was used to identify mice with additional RVF disease phenotypes when challenged via a peripheral foot-pad route to mimic mosquito-bite exposure. Wild-type Rift Valley fever virus (RVFV) challenge of 20 CC strains revealed three distinct disease phenotypes: early-onset hepatitis, mixed phenotype, and late-onset encephalitis. Strain CC057/Unc, with the most divergent phenotype, which died of late-onset encephalitis at a median of 11 days post-infection, is the first mouse strain to develop consistent encephalitis following peripheral challenge. CC057/Unc mice were directly compared to C57BL/6 mice, which uniformly succumb to hepatitis within 2–4 days of infection. Encephalitic disease in CC057/Unc mice was characterized by high viral RNA loads in brain tissue, accompanied by clearance of viral RNA from the periphery, low ALT levels, lymphopenia, and neutrophilia. In contrast, C57BL/6 mice succumbed from hepatitis at 3 days post-infection with high viral RNA loads in the liver, viremia, high ALT levels, lymphopenia, and thrombocytopenia. The identification of a strain of CC mice as an RVFV encephalitis model will allow for future investigation into the pathogenesis and treatment of RVF encephalitic disease and indicates that genetic background makes a major contribution to RVF disease variation.

BackgroundThree SARS-CoV-2 vaccines, two based on mRNA, BNT162b2 and mRNA-1273, and one based on an adenovirus platform, Ad26.COV2.S, received emergency use authorization by the U.S. Food and Drug Administration in 2020/2021. These vaccines displayed clinical efficacy in initial studies against confirmed COVID-19 of 95.0%, 94.1%, and 66.9%, respectively.MethodsIndividuals receiving one of these vaccines were invited to participate in a prospective longitudinal comparative study of immune responses elicited by the three vaccines. In this observational cohort study, humoral responses were evaluated using a SARS-CoV-2 receptor-binding domain (RBD) ELISA and a SARS-CoV-2 virus neutralization assay at mean of 21-31 days and 45-63 days following each initial vaccination. IFN-γ ELISPOT assays were conducted with peripheral blood mononuclear cells obtained at a median of 45-63 days after each initial vaccination.ResultsThe two mRNA-based platforms elicited similar RBD ELISA responses and neutralizing antibody responses. The adenovirus-based vaccine elicited significantly lower RBD ELISA and SARS-CoV-2 virus neutralization activity. The mRNA-1273 vaccine elicited significantly higher spike glycoprotein-specific T cell responses than either the BNT162b2 or the Ad26.COV2.S vaccines.ConclusionsBoth mRNA based vaccines elicited higher magnitude humoral responses than Ad26.COV2.S and mRNA1273 elicited the highest magnitude of T cell response. Neutralizing antibody titers correlated with reported estimates of vaccine efficacy.Summary of key pointsWe compared antigen specific humoral and T cell responses following vaccination with BNT162b2, mRNA-1273, or Ad26.COV2.S. Both mRNA based vaccines elicited higher magnitude humoral responses than Ad26.COV2.S and mRNA1273 elicited the highest magnitude of T cell response.

In the United States, annual vaccination against seasonal influenza is recommended for all persons aged ≥6 months (https://www.cdc.gov/flu/protect/whoshouldvax.htm). Effectiveness of seasonal influenza vaccine varies by season. During each influenza season since 2004-05, CDC has estimated the effectiveness of seasonal influenza vaccine to prevent laboratory-confirmed influenza associated with medically attended acute respiratory illness (ARI). This interim report uses data from 3,254 children and adults enrolled in the U.S. Influenza Vaccine Effectiveness Network (U.S. Flu VE Network) during November 23, 2018-February 2, 2019. During this period, overall adjusted vaccine effectiveness against all influenza virus infection associated with medically attended ARI was 47% (95% confidence interval [CI] = 34%-57%). For children aged 6 months-17 years, overall vaccine effectiveness was 61% (44%-73%). Seventy-four percent of influenza A infections for which subtype information was available were caused by A(H1N1)pdm09 viruses. Vaccine effectiveness was estimated to be 46% (30%-58%) against illness caused by influenza A(H1N1)pdm09 viruses. CDC recommends that health care providers continue to administer influenza vaccine because influenza activity is ongoing and the vaccine can still prevent illness, hospitalization, and death associated with currently circulating influenza viruses, or other influenza viruses that might circulate later in the season. During the 2017-18 influenza season, in which influenza A(H3N2) predominated, vaccination was estimated to prevent 7.1 million illnesses, 3.7 million medical visits, 109,000 hospitalizations, and 8,000 deaths (1). Vaccination can also reduce the severity of influenza-associated illness (2). Persons aged ≥6 months who have not yet been vaccinated this season should be vaccinated.

Seroprevalence studies are important for understanding the dynamics of local virus transmission and evaluating community immunity. To assess the seroprevalence for SARS-CoV-2 in Allegheny County, an urban/suburban county in Western PA, 393 human blood samples collected in Fall 2020 and February 2021 were examined for spike protein receptor-binding domain (RBD) and nucleocapsid protein (N) antibodies. All RBD-positive samples were evaluated for virus-specific neutralization activity. Our results showed a seroprevalence of 5.5% by RBD ELISA, 4.5% by N ELISA, and 2.5% for both in Fall 2020, which increased to 24.7% by RBD ELISA, 14.9% by N ELISA, and 12.9% for both in February 2021. Neutralization titer was significantly correlated with RBD titer but not with N titer. Using these two assays, we were able to distinguish infected from vaccinated individuals. In the February cohort, higher median income and white race were associated with serological findings consistent with vaccination. This study demonstrates a 4.5-fold increase in SARS-CoV-2 seroprevalence from Fall 2020 to February 2021 in Allegheny County, PA, due to increased incidence of both natural disease and vaccination. Future seroprevalence studies will need to include the effect of vaccination on assay results and incorporate non-vaccine antigens in serological assessments.

Annual seasonal influenza epidemics of variable severity result in significant morbidity and mortality in the United States (U.S.) and worldwide. In temperate climate countries, including the U.S., influenza activity peaks during the winter months. Annual influenza vaccination is recommended for all persons in the U.S. aged 6 months and older, and among those at increased risk for influenza-related complications in other parts of the world (e.g. young children, elderly). Observational studies have reported effectiveness of influenza vaccination to reduce the risks of severe disease requiring hospitalization, intensive care unit admission, and death. A diagnosis of influenza should be considered in critically ill patients admitted with complications such as exacerbation of underlying chronic comorbidities, community-acquired pneumonia, and respiratory failure during influenza season. Molecular tests are recommended for influenza testing of respiratory specimens in hospitalized patients. Antigen detection assays are not recommended in critically ill patients because of lower sensitivity; negative results of these tests should not be used to make clinical decisions, and respiratory specimens should be tested for influenza by molecular assays. Because critically ill patients with lower respiratory tract disease may have cleared influenza virus in the upper respiratory tract, but have prolonged influenza viral replication in the lower respiratory tract, an endotracheal aspirate (preferentially) or bronchoalveolar lavage fluid specimen (if collected for other diagnostic purposes) should be tested by molecular assay for detection of influenza viruses. Observational studies have reported that antiviral treatment of critically ill adult influenza patients with a neuraminidase inhibitor is associated with survival benefit. Since earlier initiation of antiviral treatment is associated with the greatest clinical benefit, standard-dose oseltamivir (75 mg twice daily in adults) for enteric administration is recommended as soon as possible as it is well absorbed in critically ill patients. Based upon observational data that suggest harms, adjunctive corticosteroid treatment is currently not recommended for children or adults hospitalized with influenza, including critically ill patients, unless clinically indicated for another reason, such as treatment of asthma or COPD exacerbation, or septic shock. A number of pharmaceutical agents are in development for treatment of severe influenza.

Background Rift Valley fever virus (RVFV) is a zoonotic arbovirus endemic to large areas of Africa and the Arabian peninsula that causes a significant annual burden of disease in ruminant animals, and is associated with periodic outbreaks in human populations. RVFV causes a wide spectrum of disease in infected patients ranging from a self-limiting febrile illness to hepatitis, encephalitis, hemorrhagic fever, and death. Despite the significant economic and public health impact of RVFV, there are no currently available vaccines or therapeutics available for RVFV. Methods Previous work has established that vaccination with an attenuated strain of RVFV, RVFV-ΔNSs, confers protection against subsequent lethal challenge in a murine model of infection. To characterize mechanistic immune correlates of protection, we vaccinated C57BL/6 mice with RVFV-ΔNSs then depleted vaccinated mice of various memory lymphocyte subsets, including CD4+, CD8+, CD4+/CD8+ T-cells before challenging mice with a lethal dose of WT RVFV. To assess for the role of antibodies in providing protection, we depleted mice of their B cells prior to vaccination with RVFV-ΔNSs, confirmed the lack of RVFV specific antibodies, and then challenged them with lethal WT RVFV. To determine the minimal immunologic factors required for protection, we vaccinated a series of donor mice, transferred either immunoglobulins or splenocytes to naïve recipient mice, then challenged them with WT RVFV. Results Vaccination with RVFV-ΔNSs protected mice from subsequent challenge, and this protection was not abrogated by depletion of CD4+, CD8+, or combined CD4+ and CD8+ lymphocytes, suggesting that RVFV-specific T-cells are dispensable for protection in the presence of virus specific antibodies. Vaccinated mice depleted of CD20+ B-cells were also protected against WT challenge, suggesting that B-cells/antibodies are also dispensable for protection in the presence of an intact T-cell response. Passive transfer of antibodies from vaccinated mice to recipient mice protected them against subsequent WT challenge, demonstrating that antibodies are sufficient for protection. Adoptive transfer of total splenocytes protected mice against subsequent WT challenge, indicating that lymphocytes are also sufficient to mediate protection. Conclusions In this study we defined the mechanistic immune correlates of protection following vaccination with attenuated RVFV-ΔNSs against WT RVFV in a murine model. Here, we have shown that both humoral and cellular immunity are sufficient to mediate protection in recipient animals, and can compensate for each other when one is missing. These results further our understanding of the protective immunologic response to infection by RVFV, and will help inform further vaccine development efforts against this important emerging pathogen.

Purpose of review Influenza causes a range of illnesses in children, from uncomplicated self-limited illness to severe disease and death. This review provides an update on the severity and burden of influenza in US children over recent seasons. Recent findings The 2017-2018 influenza season was widespread and severe across all ages, including children. Disease severity is influenced by influenza virologic characteristics and host factors, as well as public health interventions such as influenza vaccination and antiviral treatment. In recent influenza A (H3N2)-predominant seasons (2016-2017 and 2017-2018), influenza vaccination effectiveness was higher in younger children compared with older children and adolescents, although the reasons for this are unclear. Interestingly, even in seasons when influenza A (H3N2) viruses predominate, influenza A (H1N1)pdm09 and B viruses can play a large role in severe pediatric disease. Although children less than 5 years of age and those with underlying medical conditions are at increased risk for severe disease, influenza-associated hospitalizations and deaths occur every season in healthy children. Summary Influenza causes a substantial burden of outpatient visits, hospitalizations, and deaths among children. Ongoing research is important to better characterize factors that contribute to influenza severity, and to identify strategies to improve the impact of influenza vaccination and treatment.

OBJECTIVES: National guidelines recommend influenza testing for children hospitalized with influenza-like illness (ILI) during influenza season and treatment of those with confirmed influenza. Using quality improvement methods, we sought to increase influenza testing and treatment of children admitted to our hospital medicine service with ILI from 65% to 90% during the 2014–2015 influenza season. METHODS: We targeted several key drivers using multiple plan-do-study-act cycles. Interventions included awareness modules, biweekly flyers, and failure tracking. ILI admissions (fever plus respiratory symptoms) were reviewed weekly once surveillance data revealed elevated influenza activity. Appropriate testing and treatment of ILI was defined as influenza testing and/or treatment within 24 hours of admission unless a known cause other than influenza was present. We used statistical process control charts to track progress using established quality improvement methods. Appropriate testing and treatment was also assessed in the 2016–2017 influenza season by using similar methods, although no new interventions were introduced. RESULTS: For the 2014–2015 season, appropriate testing and treatment increased from a baseline mean of 65% to 91% within 3 months. For the 2016–2017 season, appropriate testing and treatment remained at a mean of 80% throughout the influenza season. CONCLUSIONS: Appropriate influenza testing and treatment increased to 90% in children with ILI during the 2014–2015 season. Improvements were sustained in a subsequent influenza season. Our initiative improved recognition of influenza and likely increased treatment opportunities. Future work should be focused on wider implementation and further reducing variation.

Many viruses invade mucosal surfaces to establish infection in the host. Some viruses are restricted to mucosal surfaces, whereas others disseminate to sites of secondary replication. Studies of strain-specific differences in reovirus mucosal infection and systemic dissemination have enhanced an understanding of viral determinants and molecular mechanisms that regulate viral pathogenesis. After peroral inoculation, reovirus strain type 1 Lang replicates to high titers in the intestine and spreads systemically, whereas strain type 3 Dearing (T3D) does not. These differences segregate with the viral S1 gene segment, which encodes attachment protein σ1 and nonstructural protein σ1s. In this study, we define genetic determinants that regulate reovirus-induced pathology following intranasal inoculation and respiratory infection. We report that two laboratory isolates of T3D, T3D C and T3D F , differ in the capacity to replicate in the respiratory tract and spread systemically; the T3D C isolate replicates to higher titers in the lungs and disseminates, while T3D F does not. Two nucleotide polymorphisms in the S1 gene influence these differences, and both S1 gene products are involved. T3D C amino acid polymorphisms in the tail and head domains of σ1 protein influence the sensitivity of virions to protease-mediated loss of infectivity. The T3D C polymorphism at nucleotide 77, which leads to coding changes in both S1 gene products, promotes systemic dissemination from the respiratory tract. A σ1s-null virus produces lower titers in the lung after intranasal inoculation and disseminates less efficiently to sites of secondary replication. These findings provide new insights into mechanisms underlying reovirus replication in the respiratory tract and systemic spread from the lung.

Following attachment and internalization, mammalian reoviruses undergo intracellular proteolytic disassembly followed by viral penetration into the cytoplasm. The initiating event in reovirus disassembly is the cathepsin-mediated proteolytic degradation of viral outer capsid protein σ3. A single tyrosine-to-histidine mutation at amino acid 354 (Y354H) of strain type 3 Dearing (T3D) σ3 enhances reovirus disassembly and confers resistance to protease inhibitors such as E64. The σ3 amino acid sequence of strain type 3 Abney (T3A) differs from that of T3D at eight positions including Y354H. However, T3A displays disassembly kinetics and protease sensitivity comparable with T3D. We hypothesize that one or more additional σ3 polymorphisms suppress the Y354H phenotype and restore T3D disassembly characteristics. To test this hypothesis, we engineered a panel of reovirus variants with T3A σ3 polymorphisms introduced individually into T3D-σ3Y354H. We evaluated E64 resistance and in vitro cathepsin L susceptibility of these viruses and found that one containing a glycine-to-glutamate substitution at position 198 (G198E) displayed disassembly kinetics and E64 sensitivity similar to those properties of T3A and T3D. Additionally, viruses containing changes at positions 233 and 347 (S233L and I347T) developed de novo compensatory mutations at position 198, strengthening the conclusion that residue 198 is a key determinant of σ3 proteolytic susceptibility. Variants with Y354H in σ3 lost infectivity more rapidly than T3A or T3D following heat treatment, an effect abrogated by G198E. These results identify a regulatory network of residues that control σ3 cleavage and capsid stability, thus providing insight into the regulation of nonenveloped virus disassembly.

Background Seasonal influenza causes substantial morbidity and mortality, and older adults are disproportionately affected. Newer vaccines have been developed for use in people 65 years and older, including a trivalent inactivated vaccine with a 4-fold higher dose of antigen (IIV-HD). In recent years, the use of IIV-HD has increased sufficiently to evaluate its effectiveness compared with standard-dose inactivated influenza vaccines (IIV-SD). Methods Hospitalized patients with acute respiratory illness were enrolled in an observational vaccine effectiveness study at 8 hospitals in 4 states participating in the United States Hospitalized Adult Influenza Vaccine Effectiveness Network during the 2015–2016 and 2016–2017 influenza seasons. Predominant influenza A virus subtypes were H1N1 and H3N2, respectively, during these seasons. All enrolled patients were tested for influenza virus with polymerase chain reaction. Receipt and type of influenza vaccine was determined from electronic records and chart review. Odds of laboratory-confirmed influenza were compared among vaccinated and unvaccinated patients. Relative odds of laboratory-confirmed influenza were determined for patients who received IIV-HD or IIV-SD, and adjusted for potential confounding variables via logistic regression. Results Among 1,744 enrolled patients aged ≥ 65 years, 1,105 (63%) were vaccinated; among those vaccinated, 621 (56%) received IIV-HD and 484 (44%) received IIV-SD. Overall, 315 (18%) tested positive for influenza, including 97 (6%) who received IIV-HD, 86 (5%) who received IIV-SD, and 132 (8%) who were unvaccinated. Controlling for age, race, sex, enrollment site, date of illness, index of comorbidity, and influenza season, the adjusted odds of influenza among patients vaccinated with IIV-HD vs. IIV-SD were 0.72 (P = 0.06, 95% CI: 0.52 to 1.01). Conclusion Comparison of high-dose vs. standard-dose vaccine effectiveness during 2 recent influenza seasons (1 H1N1 and 1 H3N2-predominant) suggested relative benefit (nonsignificant) of high-dose influenza vaccine in protecting against influenza-associated hospitalization among persons aged 65 years and older; additional years of data are needed to confirm this finding. Disclosures H. K. Talbot, sanofi pasteur: Investigator, Research grant. Gilead: Investigator, Research grant. MedImmune: Investigator, Research grant. Vaxinnate: Safety Board, none. Seqirus: Safety Board, none.

Positive-strand RNA viruses induce modifications of cytoplasmic membranes to form replication complexes. For coronaviruses, replicase nonstructural protein 4 (nsp4) has been proposed to function in the formation and organization of replication complexes. Murine hepatitis virus (MHV) nsp4 is glycosylated at residues Asn176 (N176) and N237 during plasmid expression of nsp4 in cells. To test if MHV nsp4 residues N176 and N237 are glycosylated during virus replication and to determine the effects of N176 and N237 on nsp4 function and MHV replication, alanine substitutions of nsp4 N176, N237, or both were engineered into the MHV-A59 genome. The N176A, N237A, and N176A/N237A mutant viruses were viable, and N176 and N237 were glycosylated during infection of wild-type (wt) and mutant viruses. The nsp4 glycosylation mutants exhibited impaired virus growth and RNA synthesis, with the N237A and N176A/N237A mutant viruses demonstrating more profound defects in virus growth and RNA synthesis. Electron microscopic analysis of ultrastructure from infected cells demonstrated that the nsp4 mutants had aberrant morphology of virus-induced double-membrane vesicles (DMVs) compared to those infected with wt virus. The degree of altered DMV morphology directly correlated with the extent of impairment in viral RNA synthesis and virus growth of the nsp4 mutant viruses. The results indicate that nsp4 plays a critical role in the organization and stability of DMVs. The results also support the conclusion that the structure of DMVs is essential for efficient RNA synthesis and optimal replication of coronaviruses.

As obligate intracellular parasites, viruses must coopt basic cellular processes to enter host cells and deliver their genomes to the appropriate intracellular site for replication (45). Viral entry steps include attachment of the virus to the cell surface, penetration of the virus into the cell interior, disassembly of the viral capsid, and activation of the viral genetic program. These events are essential for the virus to transition from the extracellular environment to the cellular compartment in which viral transcription and replication occur. Entry steps also play key roles in viral pathogenesis, as these events often determine cell tropism within the infected host. Mammalian orthoreoviruses (reoviruses) are important models for studies of virus cell entry and the pathogenesis of viral disease. Reoviruses form nonenveloped, double-shelled particles that contain a segmented, double-stranded RNA genome (70). Virtually all mammals, including humans, serve as hosts for reovirus infection (84). However, reovirus causes disease primarily in the very young (44, 77, 79). Newborn mice infected with reovirus sustain injury to a variety of organs, including the brain, heart, and liver (5, 56, 84). Mechanisms of reovirus-induced disease, including cellular determinants of viral spread and tropism, are only partially understood. Reovirus entry into cells is initiated by the attachment of virions to cell surface receptors via the σ1 protein (41, 85) and internalization into cells by receptor-mediated endocytosis (6, 7, 24, 76). In cellular endosomes, virions undergo stepwise disassembly, forming discrete intermediates, the first of which is the infectious subvirion particle (ISVP) (7, 14, 74, 76). ISVPs are generated by proteolytic removal of the σ3 protein and cleavage of the μ1 protein to form particle-associated fragments δ and ϕ. Following formation of ISVPs, σ1 is shed and the μ1 cleavage fragments undergo conformational rearrangement, yielding the ISVP* (11, 12). ISVP*s penetrate endosomes to deliver transcriptionally active viral cores into the cytoplasm (54, 55). Endocytic proteases cathepsins B and L catalyze reovirus virion-to-ISVP disassembly in murine fibroblasts, although cathepsin L is the major mediator of this process (23). These proteases are expressed in most organs, including the intestine, brain, heart, and liver (78). In P388D cells, a macrophage-like cell line, cathepsin S, mediates the uncoating of some reovirus strains (28). Cathepsin S expression is largely restricted to cells and tissues of the immune system (16), which may be important during enteric infection, as reovirus replication in the intestine occurs in mononuclear cells of Peyer's patches (25, 51). Cathepsin S is known to be expressed in mononuclear cells, including alveolar macrophages in the lung (72, 73) and microglial cells in the brain (61). Cathepsins B, L, and S are responsible for unique, tissue-specific activities (65). Cathepsin B modulates pathological trypsinogen activation (30) and apoptosis induced by tumor necrosis factor alpha (29). Cathepsin L is required for hair follicle cycling and epidermal homeostasis (68). By virtue of its activity at neutral pH (9, 39), cathepsin S is thought to participate in remodeling of the extracellular matrix (72, 89). Functions of cathepsins B, L, and S intersect in the regulation of adaptive immunity. Cathepsin L cleaves the invariant chain in cortical thymic epithelial cells (52) and is hypothesized to mediate efficient endosomal protein fragmentation to ensure diverse peptide generation in the thymus (33, 43). Through these functions, cathepsin L serves to facilitate positive selection of CD4+ T cells (15, 35). Cathepsin S cleaves the invariant chain in peripheral antigen-presenting cells, leading to CD4+ T-cell activation (53). Both cathepsin L (32) and cathepsin S (67) participate in NK1.1+ T-cell selection in the thymus through proteolytic processing in thymocytes and antigen-presenting cells, respectively. As a result, cathepsin L-deficient (Ctsl−/−) and cathepsin S-deficient (Ctss−/−) mice have impairments in both CD4+ and NK1.1+ T-cell activities. Cathepsin S also processes antigen in endosomes for cross-presentation via the major histocompatibility complex class I pathway (71). Like cathepsins L (34) and S (63), cathepsin B processes endocytosed antigen for display by major histocompatibility complex class II molecules (46, 48). However, cathepsin B-deficient mice (Ctsb−/−) do not display overt immunodeficiency (65). Underscoring the importance of endosomal cathepsin proteases in host functions, viruses have usurped these enzymes to allow entry into the cytoplasm. In addition to reovirus, cathepsins catalyze proteolytic events required for membrane fusion of several important pathogens. Ebola virus requires both cathepsin B and cathepsin L for efficient cell entry (13), while severe acute respiratory syndrome coronavirus requires cathepsin L but also can utilize cathepsins B and S (36, 75). Hendra (58) and Nipah (57) viruses utilize cathepsin L for fusion protein processing, most likely at the stage of virion assembly (47). Despite the importance of cathepsins in viral growth, nothing is known about the function of these proteases in the pathogenesis of viral disease. To determine the role of cathepsin proteases in viral virulence, we studied reovirus disease by using mice lacking a single cathepsin. Mice deficient for cathepsin B, L, or S were monitored for survival, disease symptoms, and viral replication following reovirus infection. We found that following peroral inoculation of reovirus, cathepsin deficiency leads to decreased viral replication in sites of secondary replication. However, Ctsl−/− and Ctss−/− mice succumb to doses of virus nonlethal to wild-type (wt) and Ctsb−/− animals. Although viremia is not affected by cathepsin deficiency, we observed alterations in disease pathogenesis in the hearts, livers, and brains of cathepsin-deficient animals. Furthermore, treatment of wt mice with an inhibitor of cathepsin L reduces disease severity. These studies demonstrate that cathepsin activity plays a key role in viral pathogenesis and identify a new target for antiviral drug development.