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BACKGROUND: Co-morbidity with respiratory viruses including influenza A, cause varying degree of morbidity especially in TB patients compared to general population. This study estimates the risk factors associated with influenza A (H1N1)pdm09 in TB patients with ILI. METHODS: A cohort of tuberculosis (TB) patients who were admitted to and enrolled in a TB Directly Observed Therapy Program (DOTs) in tertiary care hospitals of Lahore (Mayo Hospital and Infectious Disease Hospital) were followed for 12 weeks. At the start of study period, to record influenza-like illness (ILI), a symptom card was provided to all the participants. Every participant was contacted once a week, in person. When the symptoms were reported by the participant, a throat swab was taken for the detection of influenza A (H1N1)pdm09. A nested case control study was conducted and TB patients with ILI diagnosed with influenza A (H1N1)pdm09 by conventional RT-PCR were selected as cases, while those who tested negative by conventional RT-PCR were enrolled as controls. All cases and controls in the study were interviewed face-to-face in the local language. Epidemiological data about potential risk factors were collected on a predesigned questionnaire. Logistic analysis was conducted to identify associated risk factors in TB patients with ILI. RESULTS: From the main cohort of TB patients (n = 152) who were followed during the study period, 59 (39%) developed ILI symptoms; of them, 39 tested positive for influenza A (H1N1)pdm09, while 20 were detected negative for influenza A (H1N1)pdm09. In univariable analysis, four factors were identified as risk factors (p < 0.05). The final multivariable model identified one risk factor (sharing of towels, P = 0.008)) and one protective factor (wearing a face mask, p = < 0.001)) for influenza A (H1N1)pdm09 infection. CONCLUSION: The current study identified the risk factors of influenza A (H1N1)pdm09 infection among TB patients with ILI.
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Subtipo H1N1 del Virus de la Influenza A , Gripe Humana , Tuberculosis , Humanos , Gripe Humana/epidemiología , Gripe Humana/virología , Masculino , Factores de Riesgo , Pakistán/epidemiología , Femenino , Adulto , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Estudios de Casos y Controles , Persona de Mediana Edad , Tuberculosis/epidemiología , Tuberculosis/complicaciones , Adulto Joven , Adolescente , AncianoRESUMEN
The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2.
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Transporte Activo de Núcleo Celular , COVID-19 , Proteínas de Transporte Nucleocitoplasmático , ARN Mensajero , Proteínas de Unión al ARN , SARS-CoV-2 , Proteínas no Estructurales Virales , Humanos , SARS-CoV-2/metabolismo , SARS-CoV-2/patogenicidad , SARS-CoV-2/genética , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Animales , COVID-19/virología , COVID-19/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Replicación Viral , Núcleo Celular/metabolismo , Células Vero , Virulencia , Chlorocebus aethiops , Células HEK293RESUMEN
Hybrid immunity (vaccination + natural infection) to SARS-CoV-2 provides superior protection to re-infection. We performed immune profiling studies during breakthrough infections in mRNA-vaccinated hamsters to evaluate hybrid immunity induction. The mRNA vaccine, BNT162b2, was dosed to induce binding antibody titers against ancestral spike, but inefficient serum virus neutralization of ancestral SARS-CoV-2 or variants of concern (VoCs). Vaccination reduced morbidity and controlled lung virus titers for ancestral virus and Alpha but allowed breakthrough infections in Beta, Delta and Mu-challenged hamsters. Vaccination primed for T cell responses that were boosted by infection. Infection back-boosted neutralizing antibody responses against ancestral virus and VoCs. Hybrid immunity resulted in more cross-reactive sera, reflected by smaller antigenic cartography distances. Transcriptomics post-infection reflects both vaccination status and disease course and suggests a role for interstitial macrophages in vaccine-mediated protection. Therefore, protection by vaccination, even in the absence of high titers of neutralizing antibodies in the serum, correlates with recall of broadly reactive B- and T-cell responses.
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COVID-19 , SARS-CoV-2 , Animales , Cricetinae , Humanos , Vacuna BNT162 , Infección Irruptiva , COVID-19/prevención & control , Mesocricetus , Anticuerpos Neutralizantes , Complicaciones Posoperatorias , ARN Mensajero/genética , Inmunidad , Anticuerpos Antivirales , VacunaciónRESUMEN
Strongyloides hyperinfection syndrome is a rare manifestation caused by the Strongyloides stercoralis parasite and has mortality rates close to 90% if left untreated. Corticosteroids are commonly implicated as a trigger for hyperinfection syndrome in patients with Strongyloides autoinfection, and it has been suggested that even a single dose of corticosteroids can trigger hyperinfection syndrome. Here, we report a case of hyperinfection syndrome eight days after administering a single 8 mg dose of dexamethasone for fetal lung development before a late preterm, emergency cesarean section (C-section) delivery secondary to placental abruption. Prior to the C-section, the patient had been exhibiting signs of autoinfection syndrome, cough, and abdominal pain, for several months. Following corticosteroid administration, she had sequelae of Strongyloides hyperinfection syndrome, including gram-negative bacteremia, undulating fevers, protein wasting enteropathy, and hypersensitivity pneumonitis. Sputum cultures were positive for Strongyloides, and after treatment with ivermectin and albendazole, the patient fully recovered. Strongyloides hyperinfection syndrome is a documented consequence of short courses of corticosteroids. Still, this case is unique because the patient only received a single dose of corticosteroids before developing hyperinfection syndrome. Clinicians must recognize patients at risk for Strongyloides hyperinfection syndrome and understand the risks of administering corticosteroids to patients harboring the parasite.
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SARS-CoV-2 variants of concern (VOCs) emerged during the COVID-19 pandemic. Here, we used unbiased systems approaches to study the host-selective forces driving VOC evolution. We discovered that VOCs evolved convergent strategies to remodel the host by modulating viral RNA and protein levels, altering viral and host protein phosphorylation, and rewiring virus-host protein-protein interactions. Integrative computational analyses revealed that although Alpha, Beta, Gamma, and Delta ultimately converged to suppress interferon-stimulated genes (ISGs), Omicron BA.1 did not. ISG suppression correlated with the expression of viral innate immune antagonist proteins, including Orf6, N, and Orf9b, which we mapped to specific mutations. Later Omicron subvariants BA.4 and BA.5 more potently suppressed innate immunity than early subvariant BA.1, which correlated with Orf6 levels, although muted in BA.4 by a mutation that disrupts the Orf6-nuclear pore interaction. Our findings suggest that SARS-CoV-2 convergent evolution overcame human adaptive and innate immune barriers, laying the groundwork to tackle future pandemics.
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COVID-19 , SARS-CoV-2 , Humanos , COVID-19/virología , Inmunidad Innata/genética , Pandemias , SARS-CoV-2/genéticaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes several proteins that inhibit host interferon responses. Among these, ORF6 antagonizes interferon signaling by disrupting nucleocytoplasmic trafficking through interactions with the nuclear pore complex components Nup98-Rae1. However, the roles and contributions of ORF6 during physiological infection remain unexplored. We assessed the role of ORF6 during infection using recombinant viruses carrying a deletion or loss-of-function (LoF) mutation in ORF6. ORF6 plays key roles in interferon antagonism and viral pathogenesis by interfering with nuclear import and specifically the translocation of IRF and STAT transcription factors. Additionally, ORF6 inhibits cellular mRNA export, resulting in the remodeling of the host cell proteome, and regulates viral protein expression. Interestingly, the ORF6:D61L mutation that emerged in the Omicron BA.2 and BA.4 variants exhibits reduced interactions with Nup98-Rae1 and consequently impairs immune evasion. Our findings highlight the role of ORF6 in antagonizing innate immunity and emphasize the importance of studying the immune evasion strategies of SARS-CoV-2.
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COVID-19 , SARS-CoV-2 , Proteínas Virales , Humanos , COVID-19/virología , Inmunidad Innata , Interferones/genética , Interferones/metabolismo , SARS-CoV-2/genética , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Hybrid immunity to SARS-CoV-2 provides superior protection to re-infection. We performed immune profiling studies during breakthrough infections in mRNA-vaccinated hamsters to evaluate hybrid immunity induction. mRNA vaccine, BNT162b2, was dosed to induce binding antibody titers against ancestral spike, but inefficient serum virus neutralization of ancestral SARS-CoV-2 or variants of concern (VoCs). Vaccination reduced morbidity and controlled lung virus titers for ancestral virus and Alpha but allowed breakthrough infections in Beta, Delta and Mu-challenged hamsters. Vaccination primed T cell responses that were boosted by infection. Infection back-boosted neutralizing antibody responses against ancestral virus and VoCs. Hybrid immunity resulted in more cross-reactive sera. Transcriptomics post-infection reflects both vaccination status and disease course and suggests a role for interstitial macrophages in vaccine-mediated protection. Therefore, protection by vaccination, even in the absence of high titers of neutralizing antibodies in the serum, correlates with recall of broadly reactive B- and T-cell responses.
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Nuclear export of influenza A virus (IAV) mRNAs occurs through the nuclear pore complex (NPC). Using the Auxin-Induced Degron (AID) system to rapidly degrade proteins, we show that among the nucleoporins localized at the nucleoplasmic side of the NPC, TPR is the key nucleoporin required for nuclear export of influenza virus mRNAs. TPR recruits the TRanscription and EXport complex (TREX)-2 to the NPC for exporting a subset of cellular mRNAs. By degrading components of the TREX-2 complex (GANP, Germinal-center Associated Nuclear Protein; PCID2, PCI domain containing 2), we show that influenza mRNAs require the TREX-2 complex for nuclear export and replication. Furthermore, we found that cellular mRNAs whose export is dependent on GANP have a small number of exons, a high mean exon length, long 3' UTR, and low GC content. Some of these features are shared by influenza virus mRNAs. Additionally, we identified a 45 nucleotide RNA signal from influenza virus HA mRNA that is sufficient to mediate GANP-dependent mRNA export. Thus, we report a role for the TREX-2 complex in nuclear export of influenza mRNAs and identified RNA determinants associated with the TREX-2-dependent mRNA export.
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Transporte Activo de Núcleo Celular , Gripe Humana , Orthomyxoviridae , Transporte de ARN , Humanos , Transporte Activo de Núcleo Celular/genética , Núcleo Celular/metabolismo , Gripe Humana/metabolismo , Poro Nuclear/genética , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Orthomyxoviridae/genética , Transporte de ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Influenza B virus (IBV) strains are one of the components of seasonal influenza vaccines in both trivalent and quadrivalent formulations. The vast majority of these vaccines are produced in embryonated chickens' eggs. While optimized backbones for vaccine production in eggs exist and are in use for influenza A viruses, no such backbones exist for IBVs, resulting in unpredictable production yields. To generate an optimal vaccine seed virus backbone, we have compiled a panel of 71 IBV strains from 1940 to present day, representing the known temporal and genetic variability of IBV circulating in humans. This panel contains strains from the B/Victoria/2/87-like lineage, B/Yamagata/16/88-like lineage and the ancestral lineage that preceded their split to provide a diverse set that would help to identify a suitable backbone which can be used in combination with hemagglutinin (HA) and neuraminidase (NA) glycoproteins from any IBV strain to be incorporated into the seasonal vaccine. We have characterized and ranked the growth profiles of the 71 IBV strains and the best performing strains were used for co-infection of eggs, followed by serial passaging to select for high-growth reassortant viruses. After serial passaging, we selected 10 clonal isolates based on their growth profiles assessed by hemagglutination and plaque-forming units. We then generated reverse genetics systems for the three clones that performed best in growth curves. The selected backbones were then used to generate different reassortant viruses with HA/NA combinations from high and low titer yielding wild type IBV. When the growth profiles of the recombinant reassortant viruses were tested, the low titer yielding HA/NA viruses with the selected backbones yielded higher titers similar to those from high titer yielding HA/NA combinations. The use of these IBV backbones with improved replication in eggs might increase yields for the influenza B virus components of seasonal influenza virus vaccines.
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Common variable immunodeficiency (CVID) is a primary immunodeficiency disorder that causes decreased immunity and increased susceptibility to infections. It affects B lymphocyte differentiation, resulting in predominantly bacterial and less frequently viral, fungal, and protozoal infections. The respiratory and gastrointestinal tracts where antibody defences are essential are usually affected. Individuals with CVID are also predisposed to developing lymphoid and gastrointestinal malignancies. We present two cases with rare infectious and oncological complications of CVID, including a patient with Mycobacterium avium complex-intracellular infection and ovarian cancer, and another patient with group B Streptococcus empyema of the lung with acute myeloid leukaemia. The main objective of this study is to highlight how CVID-induced hypogammaglobulinaemia can lead to rare infections and malignancies. The management of these complications can vary according to severity, but an awareness of their existence is crucial to diagnose them promptly in an already immunocompromised CVID patient.
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Agammaglobulinemia , Inmunodeficiencia Variable Común , Infección por Mycobacterium avium-intracellulare , Neoplasias Ováricas , Humanos , Femenino , Inmunodeficiencia Variable Común/complicaciones , Inmunodeficiencia Variable Común/diagnóstico , Carcinoma Epitelial de Ovario , Complejo Mycobacterium aviumRESUMEN
Semen traits play the vital role in determining the fertility of a broiler breeder flock; however, it can be influenced by several factors. This experiment was carried out to assess some of these factors affecting the semen. A total of 89 male birds and 960 hens of 20-week-old broiler breeder (2215 g ± 7.5%) were divided into two main groups; one was kept in cages (AIC) and another group was kept on deep litter floor (AIF), while both these groups were subjected to AI. The male birds of aforementioned groups (44 males and 480 females) were further divided into 4 sub-groups (11 males and 120 females) to execute different semen collection frequencies i.e., 2, 3, 4, and 5th days' interval. The impact of time duration between semen collection and insemination on sperm kinematics was monitored. The quantitative and qualitative analysis of semen including sperm concentration and sperm kinematics of the collected semen was conducted through a computer-assisted sperm analyzer (CASA) and ONGO machine (working on the CASA principle). Resultantly, the data revealed that the studied parameters of semen were deteriorated with the progression of age of male birds, while the group of males with standard body weight produced the best semen quantitatively and qualitatively followed by overweight particularly during the post peak phase (46-65 = 20 weeks). Although the 3rd day, semen collection frequency was found better for quality, the higher quantity of semen was achieved when males were being collected at the intervals of 4th and 5th day respectively regardless of housing systems. Significant decline in sperm kinematics was recorded with the progression of semen holding duration at the temperature of poultry farm. Furthermore, the highest contamination of E. coli, Salmonella, and Mycoplasma gallisepticum was recorded in the reproductive tract of hens and semen of the AIF group as compared to AIC. Thus, conclusion can be settled that the semen properties are significantly affected by age, body weight, and semen collection intervals in both housing systems, while sperm kinematics is being disrupted with the progression of holding duration. Although housing systems could affect the semen insignificantly, yet lesser contamination was recorded in semen and in the reproductive tract of hens of AIC.
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Pollos , Semen , Animales , Masculino , Femenino , Escherichia coli , Vivienda , Espermatozoides , Fertilidad , Peso CorporalRESUMEN
We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection both in vitro , and in the Syrian golden hamster model in vivo . In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants. ONE SENTENCE SUMMARY: SARS-CoV-2 ORF6 subverts bidirectional nucleo-cytoplasmic trafficking to inhibit host gene expression and contribute to viral pathogenesis.
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Due to differences in human and murine angiotensin converting enzyme 2 (ACE-2) receptor, initially available SARS-CoV-2 isolates could not infect mice. Here we show that serial passaging of USA-WA1/2020 strain in mouse lungs results in "mouse-adapted" SARS-CoV-2 (MA-SARS-CoV-2) with mutations in S, M, and N genes, and a twelve-nucleotide insertion in the S gene. MA-SARS-CoV-2 infection causes mild disease, with more pronounced morbidity depending on genetic background and in aged and obese mice. Two mutations in the S gene associated with mouse adaptation (N501Y, H655Y) are present in SARS-CoV-2 variants of concern (VoCs). N501Y in the receptor binding domain of viruses of the B.1.1.7, B.1.351, P.1 and B.1.1.529 lineages (Alpha, Beta, Gamma and Omicron variants) is associated with high transmissibility and allows VoCs to infect wild type mice. We further show that S protein mutations of MA-SARS-CoV-2 do not affect neutralization efficiency by human convalescent and post vaccination sera.
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COVID-19 , Evasión Inmune , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Anciano , Animales , COVID-19/virología , Humanos , Sueros Inmunes , Ratones , Mutación , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Concerns that infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), may cause new-onset diabetes persist in an evolving research landscape, and precise risk assessment is hampered by, at times, conflicting evidence. Here, leveraging comprehensive single-cell analyses of in vitro SARS-CoV-2-infected human pancreatic islets, we demonstrate that productive infection is strictly dependent on the SARS-CoV-2 entry receptor ACE2 and targets practically all pancreatic cell types. Importantly, the infection remains highly circumscribed and largely non-cytopathic and, despite a high viral burden in infected subsets, promotes only modest cellular perturbations and inflammatory responses. Similar experimental outcomes are also observed after islet infection with endemic coronaviruses. Thus, the limits of pancreatic SARS-CoV-2 infection, even under in vitro conditions of enhanced virus exposure, challenge the proposition that in vivo targeting of ß cells by SARS-CoV-2 precipitates new-onset diabetes. Whether restricted pancreatic damage and immunological alterations accrued by COVID-19 increase cumulative diabetes risk, however, remains to be evaluated.
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COVID-19 , Diabetes Mellitus , Células Secretoras de Insulina , Humanos , Páncreas , SARS-CoV-2RESUMEN
SARS-CoV-2 lineages have diverged into highly prevalent variants termed "variants of concern" (VOCs). Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to understand their impact on transmissibility and virus pathogenicity and fitness. We demonstrate that the substitution S:655Y, represented in the gamma and omicron VOCs, enhances viral replication and spike protein cleavage. The S:655Y substitution was transmitted more efficiently than its ancestor S:655H in the hamster infection model and was able to outcompete S:655H in the hamster model and in a human primary airway system. Finally, we analyzed a set of emerging SARS-CoV-2 variants to investigate how different sets of mutations may impact spike processing. All VOCs tested exhibited increased spike cleavage and fusogenic capacity. Taken together, our study demonstrates that the spike mutations present in VOCs that become epidemiologically prevalent in humans are linked to an increase in spike processing and virus transmission.
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COVID-19 , SARS-CoV-2 , Humanos , Mutación , SARS-CoV-2/genética , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
RATIONALE: In the United States, non-tuberculous mycobacterium (NTM) infections are considered an important cause of morbidity and mortality, especially in people with progressive lung disease. The state of Florida has an extremely high incidence and prevalence of NTM disease which is likely a rapidly emerging infection in the state due to environmental and demographic factors. OBJECTIVES: Adjemian et al. [1] To determine the burden of NTM disease of patients admitted to a large Central Florida academic center, Falkinham [2] to identify the most common risk factors associated with developing NTM disease in this area, and Sfeir et al. [4] to categorize antimicrobial susceptibilities and genetic resistance markers. METHODS: We conducted a retrospective case review from January 1, 2011 to December 31, 2017 in a large university-associated metropolitan hospital in west-central Florida. NTM infections were identified using TheraDoc® during the study period with the inclusion criteria of any inpatient admission, culture confirmed NTM at any site, and age ≥ 12 years. Demographic variables (including residential zip code) and comorbidity data (including solid organ transplant status, HIV status and subsequent testing results, intrinsic pulmonary disease, and cancer diagnosis of any site) were collected for each patient. Microbiologic data collected included NTM species/subspecies, anatomic location of specimen collection, antimicrobial susceptibility including minimum inhibitory concentration (MIC). All collected data were analyzed within Stata/IC14.2. Geospatial relationships between zip codes, diagnosis type, and co-morbidities were computed using Arc GIS Pro. RESULTS: Our results demonstrated that a substantial number of our inpatient cases with NTM were of the M. abscessus group, and with M. avium complex and M. fortuitum also representing the pathogen in numerous cases. Novel findings included compilation of the first hospital wide comprehensive NTM resistance plot to our knowledge. Our results did show a concordance with previous data with expected predominance of NTM inpatient cases in Caucasian males with pre-existing pulmonary disease, though additional work could be done with isolates within the transplant and immunosuppressed populations. CONCLUSIONS: Our data set demonstrates the most common species/subspecies of NTM infections and their associated conditions seen at our central Florida hospital, and includes an antimicrobial sensitivity analysis in toto. This could be insight into the possible prevalence of NTM in the area, and provides the foundation for future studies on both the acquisition and prevention for NTM infections in central Florida.
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Fc-dependent effector functions are an important determinant of the in vivo potency of therapeutic antibodies. Effector function is determined by the combination of FcRs bound by the antibody and the cell expressing the relevant FcRs, leading to antibody-dependent cellular cytotoxicity (ADCC). A number of ADCC assays have been developed; however, they suffer from limitations in terms of throughput, reproducibility, and in vivo relevance. Existing assays measure NK cell-mediated ADCC activity; however, studies suggest that macrophages mediate the effector function of many antibodies in vivo. Here, we report the development of a macrophage-based ADCC assay that relies on luciferase expression in target cells as a measure of live cell number. In the presence of primary mouse macrophages and specific antibodies, loss of luciferase signal serves as a surrogate for ADCC-dependent killing. We show that the assay functions for a variety of mouse and human isotypes with a model antigen/antibody complex in agreement with the known effector function of the isotypes. We also use this assay to measure the activity of a number of influenza-specific antibodies and show that the assay correlates well with the known in vivo effector functions of these antibodies.
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In addition to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), humans are also susceptible to six other coronaviruses, for which consecutive exposures to antigenically related and divergent seasonal coronaviruses are frequent. Despite the prevalence of COVID-19 pandemic and ongoing research, the nature of the antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unclear. Here we longitudinally profile the early humoral immune response against SARS-CoV-2 in hospitalized coronavirus disease 2019 (COVID-19) patients and quantify levels of pre-existing immunity to OC43, HKU1 and 229E seasonal coronaviruses, and find a strong back-boosting effect to conserved but not variable regions of OC43 and HKU1 betacoronaviruses spike protein. However, such antibody memory boost to human coronaviruses negatively correlates with the induction of IgG and IgM against SARS-CoV-2 spike and nucleocapsid protein. Our findings thus provide evidence of immunological imprinting by previous seasonal coronavirus infections that can potentially modulate the antibody profile to SARS-CoV-2 infection.
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Anticuerpos Antivirales/sangre , COVID-19/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Anciano , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos , COVID-19/sangre , COVID-19/transmisión , COVID-19/virología , Reacciones Cruzadas , Femenino , Humanos , Masculino , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/patogenicidadRESUMEN
The pulmonary effects of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus that causes coronavirus disease (COVID-19), are well documented; however, more evidence is needed to understand its effect on multiple organ systems. We present the case of a 69-year-old male with dyspnea for two weeks and bilateral conjunctivitis who tested positive for SARS-CoV-2. He was found to be hypoxic, requiring supplemental oxygen. On hospital day two, he complained of worsening left eye pain with the development of a left lower eyelid ulcer. He underwent a CT of facial bones, which showed findings consistent with pre-septal cellulitis and abscess. Samples from bilateral conjunctival secretions and left lower eyelid ulcer tested positive for herpes simplex virus-1 (HSV-1), and negative for SARS-CoV-2. He received supportive care, antibiotics, and famciclovir with almost complete resolution of his ocular complaints. This case illustrates an atypical COVID-19 presentation and raises concern as to how this virus modulates the immune system, allowing for concurrent viral infections.
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The current COVID-19 (coronavirus disease 19) pandemic, caused by SARS-CoV-2, disproportionally affects the elderly and people with comorbidities like obesity and associated type 2 diabetes mellitus. Small animal models are crucial for the successful development and validation of antiviral vaccines, therapies and to study the role that comorbidities have on the outcome of viral infections. The initially available SARS-CoV-2 isolates require adaptation in order to use the mouse angiotensin converting enzyme 2 (mACE-2) entry receptor and to productively infect the cells of the murine respiratory tract. We have "mouse-adapted" SARS-CoV-2 by serial passaging a clinical virus isolate in the lungs of mice. We then used low doses of this virus in mouse models for advanced age, diabetes and obesity. Similar to SARS-CoV-2 infection in humans, the outcome of infection with mouse-adapted SARS-CoV-2 resulted in enhanced morbidity in aged and diabetic obese mice. Mutations associated with mouse adaptation occurred in the S, M, N and ORF8 genes. Interestingly, one mutation in the receptor binding domain of the S protein results in the change of an asparagine to tyrosine residue at position 501 (N501Y). This mutation is also present in the newly emerging SARS-CoV-2 variant viruses reported in the U.K. (20B/501Y.V1, B1.1.7 lineage) that is epidemiologically associated with high human to human transmission. We show that human convalescent and post vaccination sera can neutralize the newly emerging N501Y virus variant with similar efficiency as that of the reference USA-WA1/2020 virus, suggesting that current SARS-CoV-2 vaccines will protect against the 20B/501Y.V1 strain.