Tropism for ciliated cells is the dominant driver of influenza viral burst size in the human airway.

Influenza viruses pose a significant burden on global human health. Influenza has a broad cellular tropism in the airway, but how infection of different epithelial cell types impacts replication kinetics and burden in the airways is not fully understood. Using primary human airway cultures, which recapitulate the diverse epithelial cell landscape of the human airways, we investigated the impact of cell type composition on virus tropism and replication kinetics. Cultures were highly diverse across multiple donors and 30 independent differentiation conditions and supported a range of influenza replication. Although many cell types were susceptible to influenza, ciliated and secretory cells were predominantly infected. Despite the strong tropism preference for secretory and ciliated cells, which consistently make up 75% or more of infected cells, only ciliated cells were associated with increased virus production. Surprisingly, infected secretory cells were associated with overall reduced virus output. The disparate response and contribution to influenza virus production could be due to different pro- and antiviral interferon-stimulated gene signatures between ciliated and secretory populations, which were interrogated with single-cell RNA sequencing. These data highlight the heterogeneous outcomes of influenza virus infections in the complex cellular environment of the human airway and the disparate impacts of infected cell identity on multiround burst size, even among preferentially infected cell types.

NK Cell and Monocyte Dysfunction in Multisystem Inflammatory Syndrome in Children.

Multisystem inflammatory syndrome in children (MIS-C) is a severe complication of SARS-CoV-2 infection characterized by multiorgan involvement and inflammation. Testing of cellular function ex vivo to understand the aberrant immune response in MIS-C is limited. Despite strong Ab production in MIS-C, SARS-CoV-2 nucleic acid testing can remain positive for 4-6 wk postinfection. Therefore, we hypothesized that dysfunctional cell-mediated Ab responses downstream of Ab production may be responsible for delayed clearance of viral products in MIS-C. In MIS-C, monocytes were hyperfunctional for phagocytosis and cytokine production, whereas NK cells were hypofunctional for both killing and cytokine production. The decreased NK cell cytotoxicity correlated with an NK exhaustion marker signature and systemic IL-6 levels. Potentially providing a therapeutic option, cellular engagers of CD16 and SARS-CoV-2 proteins were found to rescue NK cell function in vitro. Taken together, our results reveal dysregulation in Ab-mediated cellular responses of myeloid and NK cells that likely contribute to the immune pathology of this disease.

Single cell resolution of SARS-CoV-2 tropism, antiviral responses, and susceptibility to therapies in primary human airway epithelium.

The human airway epithelium is the initial site of SARS-CoV-2 infection. We used flow cytometry and single cell RNA-sequencing to understand how the heterogeneity of this diverse cell population contributes to elements of viral tropism and pathogenesis, antiviral immunity, and treatment response to remdesivir. We found that, while a variety of epithelial cell types are susceptible to infection, ciliated cells are the predominant cell target of SARS-CoV-2. The host protease TMPRSS2 was required for infection of these cells. Importantly, remdesivir treatment effectively inhibited viral replication across cell types, and blunted hyperinflammatory responses. Induction of interferon responses within infected cells was rare and there was significant heterogeneity in the antiviral gene signatures, varying with the burden of infection in each cell. We also found that heavily infected secretory cells expressed abundant IL-6, a potential mediator of COVID-19 pathogenesis.

Summer Outbreak of Severe RSV-B Disease, Minnesota, 2017 Associated with Emergence of a Genetically Distinct Viral Lineage.

BACKGROUND: Respiratory syncytial virus (RSV) typically causes winter outbreaks in temperate climates. During summer 2017, the Minnesota Department of Health received a report of increased cases of severe RSV-B infection. METHODS: We compared characteristics of summer 2017 cases with those of 2014-2018 summers. To understand the genetic relatedness among viruses, we performed high-throughput sequencing of RSV from patients with a spectrum of illness from sites in Minnesota and Wisconsin. RESULTS: From May to September 2017, 58 RSV cases (43 RSV-B) were reported compared to 20-29 cases (3-7 RSV-B) during these months in other years. Median age and frequency of comorbidities were similar, but 55% (24/43) were admitted to the ICU in 2017 compared to 12% in preceding 3 years (odds ratio, 4.84, P < .01). Sequencing was performed on 137 specimens from March 2016 to March 2018. Outbreak cases formed a unique clade sharing a single conserved nonsynonymous change in the SH gene. We observed increased cases during the following winter season, when the new lineage was the predominant strain. CONCLUSIONS: We identified an outbreak of severe RSV-B disease associated with a new genetic lineage among urban Minnesota children during a time of expected low RSV circulation.

Widespread Lichtheimia Infection in a Patient with Extensive Burns: Opportunities for Novel Antifungal Agents.

The Mucorales fungi-formerly classified as the zygomycetes-are environmentally ubiquitous fungi, but generally rare causes of clinical infections. In the immunocompromised host, however, they can cause invasive, rapidly spreading infections that confer a high risk of morbidity and mortality, often despite surgical and antifungal therapy. Patients with extensive burn injuries are particularly susceptible to skin and soft-tissue infections with these organisms. Here, we present a case of Lichtheimia infection in a patient with extensive full-thickness burns that required significant and repeated surgical debridement successfully treated with isavuconazole and adjunctive topical amphotericin B washes. We also review the available literature on contemporary antifungal treatment for Lichtheimia species and related Mucorales fungi.

Detection of Influenza C Viruses Among Outpatients and Patients Hospitalized for Severe Acute Respiratory Infection, Minnesota, 2013-2016.

Background: Existing literature suggests that influenza C typically causes mild respiratory tract disease. However, clinical and epidemiological data are limited. Methods: Four outpatient clinics and 3 hospitals submitted clinical data and respiratory specimens through a surveillance network for acute respiratory infection (ARI) from May 2013 through December 2016. Specimens were tested using multitarget nucleic acid amplification for 19-22 respiratory pathogens, including influenza C. Results: Influenza C virus was detected among 59 of 10 202 (0.58%) hospitalized severe ARI cases and 11 of 2282 (0.48%) outpatients. Most detections occurred from December to March, 73% during the 2014-2015 season. Influenza C detections occurred among patients of all ages, with rates being similar between inpatients and outpatients. The highest rate of detection occurred among children aged 6-24 months (1.2%). Among hospitalized cases, 7 required intensive care. Medical comorbidities were reported in 58% of hospitalized cases and all who required intensive care. At least 1 other respiratory pathogen was detected in 40 (66%) cases, most commonly rhinovirus/enterovirus (25%) and respiratory syncytial virus (20%). The hemagglutinin-esterase-fusion gene was sequenced in 37 specimens, and both C/Kanagawa and C/Sao Paulo lineages were detected in inpatients and outpatients. Conclusions: We found seasonal circulation of influenza C with year-to-year variability. Detection was most frequent among young children but occurred in all ages. Some cases that were positive for influenza C, particularly those with comorbid conditions, had severe disease, suggesting a need for further study of the role of influenza C virus in the pathogenesis of respiratory disease.

Case Report: Locally Acquired Leptospirosis in a Minnesota Boy and His Dog.

Leptospirosis affects numerous animal species, including domestic dogs, but documented transmission to humans is rare. Here, we describe epidemiologically linked cases in a 12-year-old Minnesota boy and his pet dog. While human leptospirosis is often thought of as a disease of tropical locations, this case report describes a rare documented example of local transmission in the northern United States, a region historically not perceived to be at high risk of Leptospira species transmission to humans. This case highlights an unusual presentation, with facial nerve palsy, underappreciated epidemiological risks, and diagnostic challenges of this reemerging infection.

Antibody-mediated cellular responses are dysregulated in Multisystem Inflammatory Syndrome in Children (MIS-C).

Multisystem Inflammatory Syndrome in Children (MIS-C) is a severe complication of SARS-CoV-2 infection characterized by multi-organ involvement and inflammation. Testing of cellular function ex vivo to understand the aberrant immune response in MIS-C is limited. Despite strong antibody production in MIS-C, SARS-CoV-2 nucleic acid testing can remain positive for 4-6 weeks after infection. Therefore, we hypothesized that dysfunctional cell-mediated antibody responses downstream of antibody production may be responsible for delayed clearance of viral products in MIS-C. In MIS-C, monocytes were hyperfunctional for phagocytosis and cytokine production, while natural killer (NK) cells were hypofunctional for both killing and cytokine production. The decreased NK cell cytotoxicity correlated with an NK exhaustion marker signature and systemic IL-6 levels. Potentially providing a therapeutic option, cellular engagers of CD16 and SARS-CoV-2 proteins were found to rescue NK cell function in vitro. Together, our results reveal dysregulation in antibody-mediated cellular responses unique to MIS-C that likely contribute to the immune pathology of this disease.

Possible Donor-Derived Infection in a Pediatric Liver Transplant Patient With Granulomatous Hepatitis.

Pediatric liver transplant recipients are a high-risk group for the development of adenovirus hepatitis and other manifestations of disseminated adenoviral disease. The risk is greatest during periods of increased immunosuppression, including immediately post-transplantation and following treatment for rejection. Manifestations of adenovirus hepatitis are heterogeneous with a wide spectrum of clinical severity, ranging from mild, focal disease to fulminant liver failure. Here we report a case of liver transplantation-associated adenovirus hepatitis presenting with fever and multifocal liver lesions. The diagnosis was not clinically suspected due to atypical imaging findings and pathology. Non-targeted metagenomic sequencing of plasma cell-free DNA facilitated and expedited the diagnosis. Confirmatory conventional testing was obtained, allowing for appropriate initiation of targeted treatment in this patient.

Use of Cidofovir for Safe Transplantation in a Toddler with Acute Liver Failure and Adenovirus Viremia.

Background: Since October 2021, there have been more than 500 cases of severe hepatitis of unknown origin in children reported worldwide, including 180 cases in the U.S. The most frequently detected potential pathogen to date has been adenovirus, typically serotype 41. Adenovirus is known to cause a self-limited infection in the immunocompetent host. However, in immunosuppressed individuals, severe or disseminated infections may occur. Method: We present the case of a two-year-old female who presented with cholestatic hepatitis and acute liver failure (ALF). Work up for etiologies of ALF was significant for adenovirus viremia, but liver biopsy was consistently negative for the virus. The risk for severe adenoviral infection in the setting of anticipated immunosuppression prompted us to initiate cidofovir to decrease viral load prior to undergoing liver transplantation. Result: Our patient received a successful liver transplant, cleared the viremia after 5 doses of cidofovir, and continues to maintain allograft function without signs of infection at the time of this report, 5 months posttransplant. Conclusion: Recent reports of pediatric hepatitis cases may be associated with adenoviral infection although the exact relationship is unclear. There is the possibility of the ongoing SARS-CoV-2 environment, or other immunologic modifying factors. All patients presenting with hepatitis or acute liver failure should be screened for adenovirus and reported to state health departments. Cidofovir may be used to decrease viral load prior to liver transplantation, to decrease risk of severe adenoviral infection.

Innate immune signaling induces high levels of TC-specific deaminase activity in primary monocyte-derived cells through expression of APOBEC3A isoforms.

In HIV-1-infected individuals, G-to-A hypermutation is found in HIV-1 DNA isolated from peripheral blood mononuclear cells (PBMCs). These mutations are thought to result from editing by one or more host enzymes in the APOBEC3 (A3) family of cytidine deaminases, which act on CC (APOBEC3G) and TC (other A3 proteins) dinucleotide motifs in DNA (edited cytidine underlined). Although many A3 proteins display high levels of deaminase activity in model systems, only low levels of A3 deaminase activity have been found in primary cells examined to date. In contrast, here we report high levels of deaminase activity at TC motifs when whole PBMCs or isolated primary monocyte-derived cells were treated with interferon-alpha (IFNalpha) or IFNalpha-inducing toll-like receptor ligands. Induction of TC-specific deaminase activity required new transcription and translation and correlated with the appearance of two APOBEC3A (A3A) isoforms. Knockdown of A3A in monocytes with siRNA abolished TC-specific deaminase activity, confirming that A3A isoforms are responsible for all TC-specific deaminase activity observed. Both A3A isoforms appear to be enzymatically active; moreover, our mutational studies raise the possibility that the smaller isoform results from internal translational initiation. In contrast to the high levels of TC-specific activity observed in IFNalpha-treated monocytes, CC-specific activity remained low in PBMCs, suggesting that A3G deaminase activity is relatively inhibited, unlike that of A3A. Together, these findings suggest that deaminase activity of A3A isoforms in monocytes and macrophages may play an important role in host defense against viruses.

Genetic ancestry effects on the response to viral infection are pervasive but cell type specific.

Humans differ in their susceptibility to infectious disease, partly owing to variation in the immune response after infection. We used single-cell RNA sequencing to quantify variation in the response to influenza infection in peripheral blood mononuclear cells from European- and African-ancestry males. Genetic ancestry effects are common but highly cell type specific. Higher levels of European ancestry are associated with increased type I interferon pathway activity in early infection, which predicts reduced viral titers at later time points. Substantial population-associated variation is explained by cis-expression quantitative trait loci that are differentiated by genetic ancestry. Furthermore, genetic ancestry­associated genes are enriched among genes correlated with COVID-19 disease severity, suggesting that the early immune response contributes to ancestry-associated differences for multiple viral infection outcomes.

12 Tips for Engaging Medical Students in Health Communications.

This article was migrated. The article was marked as recommended. The proliferation of misinformation during the COVID-19 pandemic provides a clear example of the harms that can occur when medical professionals do not engage with the public regarding health topics. To address this need for accessible, accurate medical information, we taught medical students a COVID-19-specific curriculum tailored to sharing this information with the lay public via social media. Through active learning, students developed their understanding of disease-specific pathophysiology, prevention techniques, treatments, and public health interventions while practicing new skills in public communication as health professionals. After two cohorts completed the course, students' high-quality medical information about COVID-19 reached >100,000 viewers. To further broaden the impact, we shared the course curriculum through the Association of American Medical College (AAMC) iCollaborative. This curriculum provides a model for future engagement of medical students in health communication with lay audiences.

Feasibility of SARS-CoV-2 Antibody Testing in Remote Outpatient Trials.

Background: During the coronavirus disease 2019 (COVID-19) pandemic, clinical trials necessitated rapid testing to be performed remotely. Dried blood spot (DBS) techniques have enabled remote HIV virologic testing globally, and more recently, antibody testing as well. We evaluated DBS testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody testing in outpatients to assess seropositivity. Methods: In 2020, we conducted 3 internet-based randomized clinical trials and offered serologic testing via self-collected DBS as a voluntary substudy. COVID-19 diagnosis was based on the Centers for Disease Control and Prevention case definition with epidemiological link to cases. A minority reported polymerase chain reaction (PCR) testing at an outside facility. We tested for anti-SARS-CoV-2 immunoglobulin via antibody detection by agglutination-PCR (ADAP) and compared the results with enzyme-linked immunosorbent assay (ELISA). Results: Of 2727 participants in the primary studies, 60% (1648/2727) consented for serology testing; 56% (931/1648) returned a usable DBS sample. Of those who were asymptomatic, 5% (33/707) had positive ADAP serology. Of participants with a positive PCR, 67% (36/54) had positive SARS-CoV-2 antibodies. None of those who were PCR-positive and asymptomatic were seropositive (0/7). Of 77 specimens tested for concordance via ELISA, 83% (64/77) were concordant. The challenges of completing a remote testing program during a pandemic included sourcing and assembling collection kits, delivery and return of the kits, and troubleshooting testing. Self-collection was successful for >95% of participants. Delays in US mail with possible sample degradation and timing of DBS collection complicated the analysis. Conclusions: We found remote antibody testing during a global pandemic feasible although challenging. We identified an association between symptomatic COVID-19 and positive antibody results at a similar prevalence as other outpatient cohorts.

Geographic Variability and Pathogen-Specific Considerations in the Diagnosis and Management of Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a rare but serious primary immunodeficiency with varying prevalence and rates of X-linked and autosomal recessive disease worldwide. Functional defects in the phagocyte nicotinamide adenine dinucleotide phosphate oxidase complex predispose patients to a relatively narrow spectrum of bacterial and fungal infections that are sometimes fastidious and often difficult to identify. When evaluating and treating patients with CGD, it is important to consider their native country of birth, climate, and living situation, which may predispose them to types of infections that are atypical to your routine practice. In addition to recurrent and often severe infections, patients with CGD and X-linked female carriers are also susceptible to developing many non-infectious complications including tissue granuloma formation and autoimmunity. The DHR-123 oxidation assay is the gold standard for making the diagnosis and it along with genetic testing can help predict the severity and prognosis in patients with CGD. Disease management focuses on prophylaxis with antibacterial, antifungal, and immunomodulatory medications, prompt identification and treatment of acute infections, and prevention of secondary granulomatous complications. While hematopoietic stem-cell transplantation is the only widely available curative treatment for patients with CGD, recent advances in gene therapy may provide a safer, more direct alternative.

Single cell resolution of SARS-CoV-2 tropism, antiviral responses, and susceptibility to therapies in primary human airway epithelium.

The human airway epithelium is the initial site of SARS-CoV-2 infection. We used flow cytometry and single cell RNA-sequencing to understand how the heterogeneity of this diverse cell population contributes to elements of viral tropism and pathogenesis, antiviral immunity, and treatment response to remdesivir. We found that, while a variety of epithelial cell types are susceptible to infection, ciliated cells are the predominant cell target of SARS-CoV-2. The host protease TMPRSS2 was required for infection of these cells. Importantly, remdesivir treatment effectively inhibited viral replication across cell types, and blunted hyperinflammatory responses. Induction of interferon responses within infected cells was rare and there was significant heterogeneity in the antiviral gene signatures, varying with the burden of infection in each cell. We also found that heavily infected secretory cells expressed abundant IL-6, a potential mediator of COVID-19 pathogenesis.

T cells contain an RNase-insensitive inhibitor of APOBEC3G deaminase activity.

The deoxycytidine deaminase APOBEC3G (A3G) is expressed in human T cells and inhibits HIV-1 replication. When transfected into A3G-deficient epithelial cell lines, A3G induces catastrophic hypermutation by deaminating the HIV-1 genome. Interestingly, studies suggest that endogenous A3G in T cells induces less hypermutation than would be expected. However, to date, the specific deaminase activity of endogenous A3G in human CD4+ T cells has not been examined directly. Here, we compared deaminase activity of endogenous and exogenous A3G in various human cell lines using a standard assay and a novel, quantitative, high-throughput assay. Exogenous A3G in epithelial cell lysates displayed deaminase activity only following RNase treatment, as expected given that A3G is known to form an enzymatically inactive RNA-containing complex. Surprisingly, comparable amounts of endogenous A3G from T cell lines or from resting or activated primary CD4+ T cells exhibited minimal deaminase activity, despite RNase treatment. Specific deaminase activity of endogenous A3G in H9, CEM, and other T cell lines was up to 36-fold lower than specific activity of exogenous A3G in epithelial-derived cell lines. Furthermore, RNase-treated T cell lysates conferred a dose-dependent inhibition to epithelial cell lysates expressing enzymatically active A3G. These studies suggest that T cells, unlike epithelial-derived cell lines, express an unidentified RNase-resistant factor that inhibits A3G deaminase activity. This factor could be responsible for reduced levels of hypermutation in T cells, and its identification and blockade could offer a means for increasing antiretroviral intrinsic immunity of T cells.

Assembly of immature HIV-1 capsids using a cell-free system.

For many years it has been known that viral capsid proteins are capable of self-assembly, but increasing evidence over the past decade indicates that in cells HIV-1 capsid assembly occurs via a complex but transient series of steps requiring multiple viral-host interactions. To better understand the biochemistry of HIV assembly, our group established a cell-free system that faithfully reconstitutes HIV-1 Gag synthesis and post-translational events of capsid assembly using cellular extracts, albeit more slowly and less efficiently. This system allowed initial identification of interactions that occur very transiently in cells but can be tracked in the cell-free system. Analysis of the cell-free system revealed that Gag progresses sequentially through a step-wise, energy-dependent series of assembly intermediates containing cellular proteins. One of these cellular proteins, the ATPase ABCE1, has been shown to play a critical role in the assembly process. The existence of this energy-dependent assembly pathway was subsequently confirmed in cellular systems, further validating the cell-free HIV-1 capsid assembly system as an excellent tool for identifying mechanisms underlying HIV-1 capsid formation. Here we describe how to assemble immature HIV-1 capsids in a cell-free system and separate assembly intermediates by velocity sedimentation.