Comparing Nasopharyngeal and Midturbinate Nasal Swab Testing for the Identification of Severe Acute Respiratory Syndrome Coronavirus 2.

Testing of paired midturbinate (MT) nasal and nasopharyngeal (NP) swabs, collected by trained personnel from 40 patients with coronavirus disease 2019 (COVID-19), showed that more NP (76/95 [80%]) than MT swabs tested positive (61/95 [64%]) (P = .02). Among samples collected a week after study enrollment, fewer MT than NP samples were positive (45% vs 76%; P = .001).

Tumor Necrosis Factor Alpha-Induced Recruitment of Inflammatory Mononuclear Cells Leads to Inflammation and Altered Brain Development in Murine Cytomegalovirus-Infected Newborn Mice.

Congenital human cytomegalovirus (HCMV) infection is a significant cause of abnormal neurodevelopment and long-term neurological sequelae in infants and children. Resident cell populations of the developing brain have been suggested to be more susceptible to virus-induced cytopathology, a pathway thought to contribute to the clinical outcomes following intrauterine HCMV infection. However, recent findings in a newborn mouse model of the infection in the developing brain have indicated that elevated levels of proinflammatory mediators leading to mononuclear cell activation and recruitment could underlie the abnormal neurodevelopment. In this study, we demonstrate that treatment with tumor necrosis factor alpha (TNF-α)-neutralizing antibodies decreased the frequency of CD45+ Ly6Chi CD11b+ CCR2+ activated myeloid mononuclear cells (MMCs) and the levels of proinflammatory cytokines in the blood and the brains of murine CMV-infected mice. This treatment also normalized neurodevelopment in infected mice without significantly impacting the level of virus replication. These results indicate that TNF-α is a major component of the inflammatory response associated with altered neurodevelopment that follows murine CMV infection of the developing brain and that a subset of peripheral blood myeloid mononuclear cells represent a key effector cell population in this model of virus-induced inflammatory disease of the developing brain.IMPORTANCE Congenital human cytomegalovirus (HCMV) infection is the most common viral infection of the developing human fetus and can result in neurodevelopmental sequelae. Mechanisms of disease leading to neurodevelopmental deficits in infected infants remain undefined, but postulated pathways include loss of neuronal progenitor cells, damage to the developing vascular system of the brain, and altered cellular positioning. Direct virus-mediated cytopathic effects cannot explain the phenotypes of brain damage in most infected infants. Using a mouse model that recapitulates characteristics of the brain infection described in human infants, we have shown that TNF-α plays a key role in brain inflammation, including recruitment of inflammatory mononuclear cells. Neutralization of TNF-α normalized neurodevelopmental abnormalities in infected mice, providing evidence that virus-induced inflammation is a major component of disease in the developing brain. These results suggest that interventions limiting inflammation associated with the infection could potentially improve the neurologic outcome of infants infected in utero with HCMV.

Altered cleavage of human factor VIII at the B-domain and acidic region 3 interface enhances expression after gene therapy in hemophilia A mice.

BACKGROUND: Variants of human factor VIII (hFVIII) have been developed to further understand the structure and function of hFVIII and improve gene-based therapeutics. We have previously characterized several hFVIII variants of the furin cleavage site (1645-1648) with improved secretion. We have also identified a second cleavage site in the acidic region 3 (a3) (1657-1658) that becomes the primary hFVIII intracellular cleavage position in the absence of the furin site. We tested a hypothesis that modification of this site may confer additional functional advantages to hFVIII. OBJECTIVES: The aim of this study was to conduct the biochemical and functional characterization of hFVIII variants of the furin cleavage site, the a3 cleavage site, or in combination, both in vitro and in vivo after AAV mediated gene therapy. METHODS: Recombinant hFVIII variants of the furin cleavage site (hFVIII-Δ3), the a3 cleavage site (hFVIII-S1657P/D1658E [SP/DE]), or in combination (hFVIII-Δ3-SP/DE) were purified and characterized in vitro and in vivo. RESULTS: Recombinant hFVIII-Δ3, hFVIII-SP/DE, and hFVIII-Δ3-SP/DE variants all had comparable specific activity to B-domain deleted (BDD) hFVIII. Hemophilia A mice tolerant to hFVIII did not develop immune responses to hFVIII after protein challenge with these variants or after adeno-associated virus (AAV) delivery. Following AAV delivery, hFVIII-Δ3-SP/DE resulted in expression levels that were 2- to 5-fold higher than those with hFVIII-BDD in hemophilia A mice. CONCLUSION: The novel hFVIII-Δ3-SP/DE variant of the furin and a3 cleavage sites significantly improved secretion compared with hFVIII-BDD. This key feature of the Δ3-SP/DE variant provides a unique strategy that can be combined with other approaches to further improve factor VIII expression to achieve superior efficacy in AAV-based gene therapy for hemophilia A.

Virological Characteristics of Hospitalized Children With SARS-CoV-2 Infection.

BACKGROUND AND OBJECTIVES: In children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, virological characteristics and correlation with disease severity have not been extensively studied. The primary objective in this study is to determine the correlation between SARS-CoV-2 viral load (VL) in infected children with age, disease severity, and underlying comorbidities. METHODS: Children <21 years, screened for SARS-CoV-2 at the time of hospitalization, who tested positive by polymerase chain reaction were included in this study. VL at different sites was determined and compared between groups. RESULTS: Of the 102 children included in this study, 44% of the cohort had asymptomatic infection, and children with >1 comorbidity were the most at risk for severe disease. VL in children with symptomatic infection was significantly higher than in children with asymptomatic infection (3.0 × 105 vs 7.2 × 103 copies per mL; P = .001). VL in the respiratory tract was significantly higher in children <1 year, compared with older children (3.3 × 107 vs 1.3 × 104 copies per mL respectively; P < .0001), despite most infants presenting with milder illness. Besides the respiratory tract, SARS-CoV-2 RNA was also detectable in samples from the gastrointestinal tract (saliva and rectum) and blood. In 13 children for whom data on duration of polymerase chain reaction positivity was available, 12 of 13 tested positive 2 weeks after initial diagnosis, and 6 of 13 continued to test positive 4 weeks after initial diagnosis. CONCLUSIONS: In hospitalized children with SARS-CoV-2, those with >1 comorbid condition experienced severe disease. SARS-CoV-2 VL in the respiratory tract is significantly higher in children with symptomatic disease and children <1 year of age.

Virus-induced cochlear inflammation in newborn mice alters auditory function.

Although human cytomegalovirus (HCMV) is a known cause of sensorineural hearing loss in infants with congenital HCMV (cCMV) infections, mechanisms that contribute to sensorineural hearing loss (SNHL) in infants with cCMV infection are not well defined. Using a murine model of CMV infection during auditory development, we have shown that peripheral infection of newborn mice with murine CMV (MCMV) results in focal infection of the cochlea and virus-induced cochlear inflammation. Approximately 50%-60% of infected mice exhibited increased auditory brainstem response (ABR) thresholds across a range of sound frequencies. Histological analyses of the cochlea in MCMV-infected mice with elevated ABR thresholds revealed preservation of hair cell (HC) number and morphology in the organ of Corti. In contrast, the number of spiral ganglion neurons (SGN), synapses, and neurites connecting the cochlear HC and SGN nerve terminals were decreased. Decreasing cochlear inflammation by corticosteroid treatment of MCMV-infected mice resulted in preservation of SGN and improved auditory function. These findings show that virus-induced cochlear inflammation during early auditory development, rather than direct virus-mediated damage, could contribute to histopathology in the cochlea and altered auditory function without significant loss of HCs in the sensory epithelium.

Ganciclovir prophylaxis improves late murine cytomegalovirus-induced renal allograft damage.

BACKGROUND: Human cytomegalovirus (CMV) infection is associated with inferior survival in renal transplant patients, and ganciclovir (GCV) prophylaxis is associated with improved survival. In a murine CMV (MCMV) renal transplantation model, ganciclovir prophylaxis improved innate infiltrates and allograft damage during the period of prophylaxis. In this study, late effects were examined after the discontinuation of prophylaxis. METHODS: MCMV D+/R- and D-/R- allogeneic transplants were performed with cyclosporine immunosuppression. One D+/R- cohort received ganciclovir prophylaxis for 14 days after transplantation followed by 28 days without ganciclovir. At 42 days after transplantation, grafts were analyzed for histologic tissue damage and immune infiltrates. Another D+/R- cohort was treated with anti-NK1.1 antibodies for 14 days after transplantation and compared with animals without natural killer (NK) cell depletion. RESULTS: At day 42, MCMV-infected transplants had higher damage scores (15.6±0.6) compared with uninfected transplants (8.3±0.9; P<0.01), which improved in ganciclovir-treated allografts (9.5±1.4). MCMV-infected grafts contained greater frequencies of NK cell and myeloid infiltrates compared with uninfected grafts (P<0.05), which decreased in the ganciclovir-treated grafts. NK cell depletion improved allograft histology of MCMV-infected grafts. CONCLUSIONS: MCMV infection exacerbates late renal allograft damage and is associated with NK and myeloid cell infiltrates. Ganciclovir prophylaxis reduces allograft injury and NK cell and myeloid infiltrates even after the cessation of prophylaxis. NK cell depletion in MCMV-infected transplants also improves histology. These results suggest that ganciclovir prophylaxis may have a long-term beneficial effect on CMV-infected renal allografts and suggest a potential role for NK cells in the pathogenesis of CMV-associated allograft injury.

Dysregulated TLR3-dependent signaling and innate immune activation in superoxide-deficient macrophages from nonobese diabetic mice.

In type 1 diabetes (T1D), reactive oxygen species (ROS) and proinflammatory cytokines produced by macrophages and other innate immune cells destroy pancreatic ß cells while promoting autoreactive T cell maturation. Superoxide-deficient nonobese diabetic mice (NOD.Ncf1(m1J)) are resistant to spontaneous diabetes, revealing the integral role of ROS signaling in T1D. Here, we evaluate the innate immune activation state of bone marrow-derived macrophages (BM-Mϕ) from NOD and NOD.Ncf1(m1J) mice after poly(I:C)-induced Toll-like receptor 3 (TLR3) signaling. We show that ROS synthesis is required for efficient activation of the NF-κB signaling pathway and concomitant expression of TLR3 and the cognate adaptor molecule, TRIF. Poly(I:C)-stimulated NOD.Ncf1(m1J) BM-Mϕ exhibited a 2- and 10-fold decrease in TNF-α and IFN-ß proinflammatory cytokine synthesis, respectively, in contrast to NOD BM-Mϕ. Optimal expression of IFN-α/ß is not solely dependent on superoxide synthesis, but requires p47(phox) to function in a NOX-independent manner to mediate type I interferon synthesis. Interestingly, MHC-II I-A(g7) expression necessary for CD4 T cell activation is increased 2-fold relative to NOD, implicating a role for superoxide in I-A(g7) downregulation. These findings suggest that defective innate immune-pattern-recognition receptor activation and subsequent decrease in TNF-α and IFN-ß proinflammatory cytokine synthesis necessary for autoreactive T cell maturation may contribute to the T1D protection observed in NOD.Ncf1(m1J) mice.

A potential role for the nucleolus in L1 retrotransposition.

Determining the subcellular localization of the L1 ORF2 protein (ORF2p) has been impossible to date because of technical limitations in detecting either endogenous or overexpressed forms of the protein. Here we report visualization of the full-length ORF2p in cultured human cells following expression in a modified vaccinia virus/T7 RNA polymerase (MVA/T7RP) system. The MVA/T7RP system was used to ascertain subcellular localization of L1 ORF1p and ORF2p both as fusions with green fluorescent protein and by immunocytochemistry. Full-length ORF2p was predominantly cytoplasmic, while carboxy-terminal-deleted ORF2p localized additionally to the nucleolus. We mapped a functional nucleolar localization signal in ORF2p. ORF1p appeared in the cytoplasm with a speckled pattern and colocalized with ORF2p in nucleoli in a subset of cells. These findings help explain the presence of chimeras between L1s and small RNA gene sequences recently discovered in the human genome.