Relative Contribution of Diagnostic Testing to the Diagnosis of Respiratory Syncytial Virus in Hospitalized Adults in the United States.

BACKGROUND: Respiratory syncytial virus (RSV) is a leading cause of acute respiratory illness (ARI) in older adults. Optimizing diagnosis could improve understanding of RSV burden. METHODS: We enrolled adults ≥50 years of age hospitalized with ARI and adults of any age hospitalized with congestive heart failure or chronic obstructive pulmonary disease exacerbations at two hospitals during two respiratory seasons (2018-2020). We collected nasopharyngeal (NP) and oropharyngeal (OP) swabs (n=1558), acute and convalescent sera (n=568), and expectorated sputum (n=153) from participants, and recorded standard-of-care (SOC) NP results (n=805). We measured RSV antibodies by two immunoassays and performed BioFire testing on respiratory specimens. RESULTS: Of 1,558 eligible participants, 92 (5.9%) tested positive for RSV by any diagnostic method. Combined NP/OP PCR yielded 58 positives, while separate NP and OP testing identified 11 additional positives (18.9% increase). Compared to Study NP/OP PCR alone, the addition of paired serology increased RSV detection by 42.9% (28 vs 40) among those with both specimen types, while the addition of SOC swab RT-PCR results increased RSV detection by 25.9% (47 vs 59). CONCLUSIONS: The addition of paired serology testing, SOC swab results, and separate testing of NP and OP swabs improved RSV diagnostic yield in hospitalized adults.

Influenza Vaccine Effectiveness Pre-pandemic Among Adults Hospitalized With Congestive Heart Failure or Chronic Obstructive Pulmonary Disease and Older Adults.

BACKGROUND: Data are limited on influenza vaccine effectiveness (VE) in the prevention of influenza-related hospitalizations in older adults and those with underlying high-risk comorbidities. METHODS: We conducted a prospective, test-negative, case-control study at 2 US hospitals from October 2018-March 2020 among adults aged ≥50 years hospitalized with acute respiratory illnesses (ARIs) and adults ≥18 years admitted with congestive heart failure (CHF) or chronic obstructive pulmonary disease (COPD) exacerbations. Adults were eligible if they resided in 1 of 8 counties in metropolitan Atlanta, Georgia. Nasopharyngeal and oropharyngeal swabs were tested using BioFire FilmArray (bioMérieux, Inc.) respiratory panel, and standard-of-care molecular results were included when available. Influenza vaccination history was determined from the Georgia vaccine registry and medical records. We used multivariable logistic regression to control for potential confounders and to determine 95% confidence intervals (CIs). RESULTS: Among 3090 eligible adults, 1562 (50.6%) were enrolled. Of the 1515 with influenza vaccination history available, 701 (46.2%) had received vaccination during that season. Influenza was identified in 37 (5.3%) vaccinated versus 78 (9.6%) unvaccinated participants. After adjustment for age, race/ethnicity, immunosuppression, month, and season, pooled VE for any influenza-related hospitalization in the eligible study population was 63.1% (95% CI, 43.8-75.8%). Adjusted VE against influenza-related hospitalization for ARI in adults ≥50 years was 55.9% (29.9-72.3%) and adjusted VE against influenza-related CHF/COPD exacerbation in adults ≥18 years was 80.3% (36.3-93.9%). CONCLUSIONS: Influenza vaccination was effective in preventing influenza-related hospitalizations in adults aged ≥50 years and those with CHF/COPD exacerbations during the 2018-2020 seasons.

Functional Antibody Responses to Severe Acute Respiratory Syndrome Coronavirus 2 Variants in Children With Coronavirus Disease 2019, Multisystem Inflammatory Syndrome in Children, and After Two Doses of BNT162b2 Vaccination.

BACKGROUND: Although neutralizing antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) correlate with protection against coronavirus disease 2019 (COVID-19), little is known about the neutralizing and antibody-dependent cell-mediated cytotoxicity (ADCC) responses to COVID-19, multisystem inflammatory syndrome in children (MIS-C), and COVID-19 vaccination in children. METHODS: We enrolled children 0-21 years of age with a history of COVID-19 (n = 13), MIS-C (n = 13), or 2 doses of BNT162b2 vaccination (n = 14) into a phlebotomy protocol. We measured pseudovirus neutralizing and functional ADCC antibodies to SARS-CoV-2 variants, including Omicron (B.1.1.529). RESULTS: The primary BNT162b2 vaccination series elicited higher neutralizing and ADCC responses with greater breadth to SARS-CoV-2 variants than COVID-19 or MIS-C, although these were diminished against Omicron. CONCLUSIONS: Serologic responses were significantly reduced against variants, particularly Omicron.

An RSV Live-Attenuated Vaccine Candidate Lacking G Protein Mucin Domains Is Attenuated, Immunogenic, and Effective in Preventing RSV in BALB/c Mice.

BACKGROUND: Respiratory syncytial virus (RSV) is a leading viral respiratory pathogen in infants. The objective of this study was to generate RSV live-attenuated vaccine (LAV) candidates by removing the G-protein mucin domains to attenuate viral replication while retaining immunogenicity through deshielding of surface epitopes. METHODS: Two LAV candidates were generated from recombinant RSV A2-line19F by deletion of the G-protein mucin domains (A2-line19F-G155) or deletion of the G-protein mucin and transmembrane domains (A2-line19F-G155S). Vaccine attenuation was measured in BALB/c mouse lungs by fluorescent focus unit (FFU) assays and real-time polymerase chain reaction (RT-PCR). Immunogenicity was determined by measuring serum binding and neutralizing antibodies in mice following prime/boost on days 28 and 59. Efficacy was determined by measuring RSV lung viral loads on day 4 postchallenge. RESULTS: Both LAVs were undetectable in mouse lungs by FFU assay and elicited similar neutralizing antibody titers compared to A2-line19F on days 28 and 59. Following RSV challenge, vaccinated mice showed no detectable RSV in the lungs by FFU assay and a significant reduction in RSV RNA in the lungs by RT-PCR of 560-fold for A2-line19F-G155 and 604-fold for A2-line19F-G155S compared to RSV-challenged, unvaccinated mice. CONCLUSIONS: Removal of the G-protein mucin domains produced RSV LAV candidates that were highly attenuated with retained immunogenicity.

Characterization of ß-d-N4-Hydroxycytidine as a Novel Inhibitor of Chikungunya Virus.

Chikungunya virus (CHIKV) represents a reemerging global threat to human health. Recent outbreaks across Asia, Europe, Africa, and the Caribbean have prompted renewed scientific interest in this mosquito-borne alphavirus. There are currently no vaccines against CHIKV, and treatment has been limited to nonspecific antiviral agents, with suboptimal outcomes. Herein, we have identified ß-d-N4-hydroxycytidine (NHC) as a novel inhibitor of CHIKV. NHC behaves as a pyrimidine ribonucleoside and selectively inhibits CHIKV replication in cell culture.

Long-term Health Outcomes After Hospital Discharge Among Children Hospitalized for MIS-C or COVID-19, September 29, 2021, to June 21, 2022.

BACKGROUND: The long-term effects of children hospitalized with multisystem inflammatory syndrome in children (MIS-C) or acute COVID-19 are not well known. Our objective was to determine long-term outcomes. METHODS: Children hospitalized with MIS-C or COVID-19 at 3 US hospitals from March 2020, through February 2021 were followed to assess health through 2 years post-hospitalization using medical records and patient surveys. RESULTS: Medical record abstraction was performed for 183 patients hospitalized with MIS-C, 53 of whom participated in surveys, and 97 patients hospitalized with COVID-19, 35 of whom participated in surveys. Patients with MIS-C were younger (median, 9 vs. 14 years of age for COVID-19 patients; P = 0.004), more frequently male (62% vs. 39%; P < 0.001) and had more cardiac (14% vs. 2%; P = 0.001) and neurologic sequelae (8% vs. 1%; P = 0.023). Children with COVID-19 more often had other comorbidities (59% vs. 19%; P < 0.001). Full mental recovery at the time of survey 2 (median, 16 months post-hospitalization for patients with MIS-C and 20 months for patients with COVID-19) was 85% and 88%, respectively; full physical recovery was 87% and 81%, respectively; and nearly all had resumption of normal activities. Patients with MIS-C reported more frequent headache at 1 month (45% vs. 20%; P = 0.037). Patients with COVID-19 were more likely to report cough at 1 month (37% vs. 17%; P = 0.045). Fatigue persisted >1 year in 15%-20% of patients in both groups. CONCLUSIONS: Approximately 20% of children with MIS-C and COVID-19 continued to have symptoms including fatigue and headache >1 year after hospital discharge. The duration of these findings emphasizes the importance of providers following patients until sequelae have resolved.

Plasma Cell-free RNA Signatures of Inflammatory Syndromes in Children.

Inflammatory syndromes, including those caused by infection, are a major cause of hospital admissions among children and are often misdiagnosed because of a lack of advanced molecular diagnostic tools. In this study, we explored the utility of circulating cell-free RNA (cfRNA) in plasma as an analyte for the differential diagnosis and characterization of pediatric inflammatory syndromes. We profiled cfRNA in 370 plasma samples from pediatric patients with a range of inflammatory conditions, including Kawasaki disease (KD), Multisystem Inflammatory Syndrome in Children (MIS-C), viral infections and bacterial infections. We developed machine learning models based on these cfRNA profiles, which effectively differentiated KD from MIS-C - two conditions presenting with overlapping symptoms - with high performance (Test Area Under the Curve (AUC) = 0.97). We further extended this methodology into a multiclass machine learning framework that achieved 81% accuracy in distinguishing among KD, MIS-C, viral, and bacterial infections. We further demonstrated that cfRNA profiles can be used to quantify injury to specific tissues and organs, including the liver, heart, endothelium, nervous system, and the upper respiratory tract. Overall, this study identified cfRNA as a versatile analyte for the differential diagnosis and characterization of a wide range of pediatric inflammatory syndromes.

Clinical Features and Outcomes of Pediatric and Adult Patients Hospitalized for Coronavirus Disease 2019: A Comparison Across Age Strata.

Background: Coronavirus disease 2019 (COVID-19) continues to cause hospitalizations and severe disease in children and adults. Methods: This study compared the risk factors, symptoms, and outcomes of children and adults hospitalized for COVID-19 from March 2020 to May 2023 across age strata at 5 US sites participating in the Predicting Viral-Associated Inflammatory Disease Severity in Children with Laboratory Diagnostics and Artificial Intelligence consortium. Eligible patients had an upper respiratory swab that tested positive for severe acute respiratory syndrome coronavirus 2 by nucleic acid amplification. Adjusted odds ratios (aOR) of clinical outcomes were determined for children versus adults, for pediatric age strata compared to adolescents (12-17 years), and for adult age strata compared to young adults (22-49 years). Results: Of 9101 patients in the Predicting Viral-Associated Inflammatory Disease Severity in Children with Laboratory Diagnostics and Artificial Intelligence cohort, 1560 were hospitalized for COVID-19 as the primary reason. Compared to adults (22-105 years, n = 675), children (0-21 years, n = 885) were less commonly vaccinated (14.3% vs 34.5%), more commonly infected with the Omicron variant (49.5% vs 26.1%) and had fewer comorbidities (P < .001 for most comparisons), except for lung disease (P = .24). After adjusting for confounding variables, children had significantly lower odds of receiving supplemental oxygen (aOR, 0.57; 95% confidence interval, .35-.92) and death (aOR, 0.011; 95% confidence interval, <.01-.58) compa--red to adults. Among pediatric age strata, adolescents 12-17 years had the highest odds of receiving supplemental oxygen, high-flow oxygen, and ICU admission. Among adults, those 50-64 years had the highest odds of mechanical ventilation and ICU admission. Conclusions: Clinical outcomes of COVID-19 differed across pediatric and adult age strata. Adolescents experienced the most severe disease among children, whereas adults 50-64 years experienced the most severe disease among adults.

Role of cognitive parameters in dengue hemorrhagic fever and dengue shock syndrome.

Dengue is becoming recognized as one of the most important vector-borne human diseases. It is predominant in tropical and subtropical zones but its geographical distribution is progressively expanding, making it an escalating global health problem of today. Dengue presents with spectrum of clinical manifestations, ranging from asymptomatic, undifferentiated mild fever, dengue fever (DF), to dengue hemorrhagic fever (DHF) with or without shock (DSS), a life-threatening illness characterized by plasma leakage due to increased vascular permeability. Currently, there are no antiviral modalities or vaccines available to treat and prevent dengue. Supportive care with close monitoring is the standard clinical practice. The mechanisms leading to DHF/DSS remains poorly understood. Multiple factors have been attributed to the pathological mechanism, but only a couple of these hypotheses are popular in scientific circles. The current discussion focuses on underappreciated factors, temperature, natural IgM, and endotoxin, which may be critical components playing roles in dengue pathogenesis.

Mortality from septic shock in a dengue infected patient: a case report.

Dengue infection can be associated with secondary infections which may be challenging to recognize due to the overlap with the symptoms of dengue infection. We report here the case of a 48 year old Chinese female with dengue fever with a fatal secondary bacterial infection due to Enterococcus faecium.

SARS-CoV-2 Antibody Profiles in Maternal Serum and Breast Milk Following mRNA COVID-19 Vaccination: A Longitudinal Prospective Observational Cohort Study.

COVID-19 vaccination during pregnancy protects infants against symptomatic COVID-19. Vaccination of lactating mothers may offer additional protection, but our understanding of immune responses in breast milk is limited. We, therefore, performed a single-center prospective cohort study of lactating mothers who received a COVID-19 mRNA primary vaccine series to evaluate the durability, breadth, and neutralizing capacity of the antibody responses in breast milk. Spike IgG- and IgA-binding antibodies of ancestral SARS-CoV-2 in serum and breast milk were quantified over 9 months using Meso Scale Discovery (MSD) V-PLEX assays, and ancestral titers were compared to four variants of concern (Alpha, Beta, Delta, Gamma) at a single time point. Neutralizing antibodies against ancestral SARS-CoV-2 and Omicron BA.4/5 were compared before and after vaccination using a pseudovirus-neutralization assay. Eleven lactating mothers received either Pfizer BNT162b2 (7/11) or Moderna mRNA-1273 (4/11) vaccine primary series. IgG and IgA titers increased in serum and breast milk following each dose, peaking 1-4 weeks after series completion. Titers remained significantly elevated for 7-9 months, except for in breast milk IgA which returned to baseline within 1 month. Furthermore, binding antibodies against all included variants were detected in breast milk collected 1-3 weeks after series completion. However, while vaccination induced a strong neutralizing response against ancestral SARS-CoV-2 in serum and more modest response in breast milk, it did not induce neutralizing antibodies against Omicron BA.4/5 in either specimen type. This study demonstrates that maternal COVID-19 mRNA vaccination may enhance immune protection for infants through breast milk via increased IgG- and IgA-binding-and-neutralizing antibodies; although, variant-specific boosters may be required to optimize immune protection.

Serologic responses to COVID-19 vaccination in children with history of multisystem inflammatory syndrome (MIS-C).

Understanding the serological responses to COVID-19 vaccination in children with history of MIS-C could inform vaccination recommendations. We prospectively enrolled seven children hospitalized with MIS-C and measured SARS-CoV-2 binding IgG antibodies to spike protein variants longitudinally pre- and post-Pfizer-BioNTech BNT162b2 primary series COVID-19 vaccination. We found that SARS-CoV-2 variant cross-reactive IgG antibodies variably waned following acute MIS-C, but were significantly boosted with vaccination and maintained for up to 3 months. We then compared post-vaccination binding, pseudovirus neutralizing, and functional antibody-dependent cell-mediated cytotoxicity (ADCC) titers to the reference strain (Wuhan-hu-1) and Omicron variant (B.1.1.529) among previously healthy children (n = 16) and children with history of MIS-C (n = 7) or COVID-19 (n = 8). Despite the breadth of binding antibodies elicited by vaccination in all three groups, pseudovirus neutralizing and ADCC titers were significantly reduced to the Omicron variant.

Treatments and Severe Outcomes for Patients Diagnosed With MIS-C at Four Children's Hospitals in the United States, March 16, 2020-March 10, 2021.

BACKGROUND: Clinical management of multisystem inflammatory syndrome in children (MIS-C) has varied over time and by medical institution. METHODS: Data on patients with MIS-C were collected from 4 children's hospitals between March 16, 2020 and March 10, 2021. Relationships between MIS-C treatments and patient demographics, clinical characteristics, and outcomes were described. Propensity score matching was utilized to assess the relative risk of outcomes dependent on early treatment with intravenous immunoglobulin (IVIG) or low-dose steroids, controlling for potential confounding variables. RESULTS: Of 233 patients diagnosed with MIS-C, the most commonly administered treatments were steroids (88.4%), aspirin (81.1%), IVIG (77.7%) and anticoagulants (71.2%). Compared with those patients without respiratory features, patients with respiratory features were less likely to receive IVIG and steroids on the same day (combination treatment) (44.1%). Controlling for confounding variables, patients receiving IVIG within 1 day of hospitalization were less likely to have hospital length of stay ≥8 days (RR = 0.53, 95% CI: 0.31-0.88). Patients receiving low-dose steroids within 1 day of hospitalization were less likely to develop ventricular dysfunction (RR = 0.45, 95% CI: 0.26-0.77), have increasingly elevated troponin levels (RR = 0.55, 95% CI: 0.40-0.75) or have hospital length of stay ≥8 days (RR = 0.46, 95% CI: 0.29-0.74). CONCLUSION: Treatments for MIS-C differed by hospital, patient characteristics and illness severity. When IVIG and low-dose steroids were administered in combination or low-dose steroids were administered alone within 1 day of hospitalization, the risk of subsequent severe outcomes was decreased.

Dengue virus-induced hemorrhage in a nonhuman primate model.

Lack of a dengue hemorrhagic animal model recapitulating human dengue virus infection has been a significant impediment in advancing our understanding of the early events involved in the pathogenesis of dengue disease. In efforts to address this issue, a group of rhesus macaques were intravenously infected with dengue virus serotype 2 (strain 16 681) at 1 x 10(7) PFU/animal. A classic dengue hemorrhage developed 3 to 5 days after infection in 6 of 6 animals. Blood chemistry appeared to be normal with exception of creatine phosphokinase, which peaked at 7 days after infection. A modest thrombocytopenia and noticeable neutropenia concomitant with slight decrease of hemoglobin and hematocrit were registered. In addition, the concentration of D-dimer was elevated significantly. Viremia peaked at 3 to 5 days after infection followed by an inverse relationship between T and B lymphocytes and a bimodal pattern for platelet-monocytes and platelet-neutrophil aggregates. Dengue virus containing platelets engulfed by monocytes was noted at 8 or 9 days after infection. Thus, rhesus macaques inoculated intravenously with a high dose of dengue virus produced dengue hemorrhage, which may provide a unique platform to define the early events in dengue virus infection and help identify which blood components contribute to the pathogenesis of dengue disease.

Role of microparticles in dengue virus infection and its impact on medical intervention strategies.

Dengue virus (DV) is one of the most important vector-borne diseases in the world. It causes a disease that manifests as a spectrum of clinical symptoms, including dengue hemorrhagic fever. DV is proficient at diverting the immune system to facilitate transmission through its vector host, Aedes spp. mosquito. Similar to other vector-borne parasites, dengue may also require a second structural form, a virus of alternative morphology (VAM), to complete its life cycle. DV can replicate to high copy numbers in patient plasma, but no classical viral particles can be detected by ultra-structural microscopy analysis. A VAM appearing as a microparticle has been recapitulated with in vitro cell lines Meg01 and K562, close relatives to the cells harboring dengue virus in vivo. VAMs are likely to contribute to the high viremia levels observed in dengue patients. This review discusses the possible existence of a VAM in the DV life cycle.

Serologic and Cytokine Signatures in Children With Multisystem Inflammatory Syndrome and Coronavirus Disease 2019.

BACKGROUND: The serologic and cytokine responses of children hospitalized with multisystem inflammatory syndrome (MIS-C) vs coronavirus disease 2019 (COVID-19) are poorly understood. METHODS: We performed a prospective, multicenter, cross-sectional study of hospitalized children who met the Centers for Disease Control and Prevention case definition for MIS-C (n = 118), acute COVID-19 (n = 88), or contemporaneous healthy controls (n = 24). We measured severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (RBD) immunoglobulin G (IgG) titers and cytokine concentrations in patients and performed multivariable analysis to determine cytokine signatures associated with MIS-C. We also measured nucleocapsid IgG and convalescent RBD IgG in subsets of patients. RESULTS: Children with MIS-C had significantly higher SARS-CoV-2 RBD IgG than children with acute COVID-19 (median, 2783 vs 146; P < .001), and titers correlated with nucleocapsid IgG. For patients with MIS-C, RBD IgG titers declined in convalescence (median, 2783 vs 1135; P = .010) in contrast to patients with COVID-19 (median, 146 vs 4795; P < .001). MIS-C was characterized by transient acute proinflammatory hypercytokinemia, including elevated levels of interleukin (IL) 6, IL-10, IL-17A, and interferon gamma (IFN-γ). Elevation of at least 3 of these cytokines was associated with significantly increased prevalence of prolonged hospitalization ≥8 days (prevalence ratio, 3.29 [95% CI, 1.17-9.23]). CONCLUSIONS: MIS-C was associated with high titers of SARS-CoV-2 RBD IgG antibodies and acute hypercytokinemia with IL-6, IL-10, IL-17A, and IFN-γ.

Effectiveness of liquid soap and hand sanitizer against Norwalk virus on contaminated hands.

Disinfection is an essential measure for interrupting human norovirus (HuNoV) transmission, but it is difficult to evaluate the efficacy of disinfectants due to the absence of a practicable cell culture system for these viruses. The purpose of this study was to screen sodium hypochlorite and ethanol for efficacy against Norwalk virus (NV) and expand the studies to evaluate the efficacy of antibacterial liquid soap and alcohol-based hand sanitizer for the inactivation of NV on human finger pads. Samples were tested by real-time reverse transcription-quantitative PCR (RT-qPCR) both with and without a prior RNase treatment. In suspension assay, sodium hypochlorite concentrations of >or=160 ppm effectively eliminated RT-qPCR detection signal, while ethanol, regardless of concentration, was relatively ineffective, giving at most a 0.5 log(10) reduction in genomic copies of NV cDNA. Using the American Society for Testing and Materials (ASTM) standard finger pad method and a modification thereof (with rubbing), we observed the greatest reduction in genomic copies of NV cDNA with the antibacterial liquid soap treatment (0.67 to 1.20 log(10) reduction) and water rinse only (0.58 to 1.58 log(10) reduction). The alcohol-based hand sanitizer was relatively ineffective, reducing the genomic copies of NV cDNA by only 0.14 to 0.34 log(10) compared to baseline. Although the concentrations of genomic copies of NV cDNA were consistently lower on finger pad eluates pretreated with RNase compared to those without prior RNase treatment, these differences were not statistically significant. Despite the promise of alcohol-based sanitizers for the control of pathogen transmission, they may be relatively ineffective against the HuNoV, reinforcing the need to develop and evaluate new products against this important group of viruses.

Quantification of Norwalk virus inocula: Comparison of endpoint titration and real-time reverse transcription-PCR methods.

Human noroviruses (NoV) are the leading cause of epidemic acute gastroenteritis. In order to fully characterize features such as persistence and infectious dose, precise quantification of virus concentration is necessary. The purpose of this study was to compare two methods [endpoint titration RT-PCR and quantitative RT-PCR (RT-qPCR)] with respect to quantification of Norwalk virus (NV) in inocula made from purified stock suspensions of human fecal specimens. A full-length NV RNA transcript was developed to facilitate quantification using RT-qPCR and provided log linear detection in the range of 49-4.9 x 10(4) genome equivalent copies (GEC) per reaction. Endpoint titration RT-PCR was used to estimate PCR detection units, and RT-qPCR was used to estimate genome copies in two NV inocula (8fIIa and 8fIIb) used in previous human challenge studies. Overall, RT-qPCR was 1.1-1.6 log(10) more sensitive (lower detection limit) than endpoint titration RT-PCR when the same RNA release method, PCR primers and thermocycle program were used. These findings have important implications for many experimental interpretations, not the least of which is estimating the median infectious dose in human challenge studies.

Jak Inhibitors Modulate Production of Replication-Competent Zika Virus in Human Hofbauer, Trophoblasts, and Neuroblastoma cells.

Zika Virus (ZIKV) is a flavivirus that has been implicated in causing brain deformations, birth defects, and microcephaly in fetuses, and associated with Guillain-Barre syndrome. Mechanisms responsible for transmission of ZIKV across the placenta to the fetus are incompletely understood. Herein, we define key events modulating infection in clinically relevant cells, including primary placental macrophages (human Hofbauer cells; HC), trophoblasts, and neuroblastoma cells. Consistent with previous findings, HC and trophoblasts are permissive to ZIKV infection. Decrease of interferon signaling by Jak ½ inhibition (using ruxolitinib) significantly increased ZIKV replication in HC, trophoblasts, and neuroblasts. Enhanced ZIKV production in ruxolitinib-treated HC was associated with increased expression of HLA-DR and DC-SIGN. Nucleoside analogs blocked ruxolitinib-mediated production of extracellular virus. Although low-level ZIKV infection occurred in untreated HC and trophoblasts, replicating virions were incapable of infecting naive Vero cells. These deficient virions from untreated HC have "thin-coats" suggesting an immature structure. Blocking Jak ½ signaling (with ruxolitinib) restored replication competence as virions produced under these conditions confer cytopathic effects to naive Vero cells. These data demonstrate that Jak-STAT signaling directly impacts the ability of primary placental cells to produce replication-competent virus and is a key determinant in the production of mature virions in clinically relevant cells, including HC and trophoblasts. Design of targeted agents to prevent ZIKV replication in the placenta should consider Jak ½ signaling, the impact of its block on ZIKV infection, and subsequent transmission to the fetus.

Characterization of dengue virus 2 growth in megakaryocyte-erythrocyte progenitor cells.

Megakaryocyte-erythrocyte progenitor (MEP) cells are potential in vivo targets of dengue virus (DENV); the virus has been found associated with megakaryocytes ex vivo and platelets during DENV-induced thrombocytopenia. We report here that DENV serotype 2 (DENV2) propagates well in human nondifferentiated MEP cell lines (Meg01 and K562). In comparison to virus propagated in Vero cells, viruses from MEP cell lines had similar structure and buoyant density. However, differences in MEP-DENV2 stability and composition were suggested by distinct protein patterns in western blot analysis. Also, antibody neutralization of envelope domain I/II on MEP-DENV2 was reduced relative to that on Vero-DENV2. Infectious DENV2 was produced at comparable kinetics and magnitude in MEP and Vero cells. However, fewer virion structures appeared in electron micrographs of MEP cells. We propose that DENV2 infects and produces virus efficiently in megakaryocytes and that megakaryocyte impairment might contribute to dengue disease pathogenesis.