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Background Children can present with overlapping symptoms of acute respiratory illness (ARI) and acute gastroenteritis (AGE). In these cases, it is unclear if the etiologic agent is a respiratory pathogen, gastrointestinal pathogen, or both. Methods We analyzed data collected in Nashville, TN (12/01/2016–2/28/2020) as part of the New Vaccine Surveillance Network, a prospective ARI/AGE surveillance study. Children (< 18 years old) who presented to the emergency department or were admitted with fever and/or respiratory symptoms for < 14 days were enrolled as ARI subjects and had mid-turbinate nasal ± throat swabs collected, while children with ≥1 episode of vomiting and/or ≥3 episodes of diarrhea in 24 hours were enrolled as AGE subjects and had stool collected. Children who met both sets of criteria were dually enrolled. Respiratory specimens were tested for common respiratory viruses by molecular testing and stool specimens were tested for common gastrointestinal (GI) pathogens by Luminex GI Pathogen Panel. We compared detection groups using Pearson’s χ2 test. C. difficile detection in children < 2 years old was considered asymptomatic carriage (n=32). Results We identified 501 dual enrollees, among whom 279 (55.7%) had both a respiratory and stool specimen tested. Overall, 127 (45.5%) had only a respiratory virus detected, 33 (11.8%) had only a GI pathogen detected, 39 (14.0%) had both, and 77 (27.6%) had no detection (Table 1). Vomiting and diarrhea were frequently reported ( >50%) in all dual enrollees whether or not a pathogen was detected (Figure 1). Cough was detected in high frequency in all groups with pathogen detection. Children with respiratory-only or dual detection had a higher frequency of wheezing and shortness of breath than those with GI-only or no detection. The distribution of pathogens did not significantly differ between single and co-detected cases (Figure 2). Table 1Demographic characteristics of N=279* children presenting with symptoms of acute respiratory illness and acute gastroenteritis in Nashville, TN, stratified by detection status.Figure 1Distribution of signs and symptoms in N=279 children presenting with symptoms of acute respiratory illness and acute gastroenteritis in Nashville, TN, stratified by detection status. p values represent omnibus comparisons of all four groups.Figure 2Distribution of pathogens detected in N=279 children presenting with symptoms of acute respiratory illness and acute gastroenteritis in Nashville, TN, stratified by detection status. Conclusion Children presenting with overlapping symptoms of ARI and AGE were more likely to have an ARI-associated virus. Lower respiratory symptoms (namely, wheezing and shortness of breath) were more specific for ARI-associated viral detection compared with other signs and symptoms. Disclosures Natasha B. Halasa, MD, Quidel: Grant/Research Support|Quidel: equipment donation|Sanofi: Grant/Research Support|Sanofi: HAI testing and vaccine donation.

Background Detections of influenza A subtype specific antibody responses are often complicated by the presence of cross-reactive antibodies. We developed two novel multiplex platforms for antibody detection. The multiplexed magnetic fluorescence microsphere immunoassay (MAGPIX) is a high throughput laboratory-based assay. Chembio Dual Path Platform (DPP) is a portable and rapid test that could be used in the field. Methods Twelve recombinant globular head domain hemagglutinin (GH HA1) antigens from A(H1N1)pdm09 (pH1N1), A(H2N2), A(H3N2), A(H5N1), A(H7N9), A(H9N2), A(H13N9), B/Victoria lineage, B/Yamagata lineage viruses, and protein A control were used. Human sera from U.S. residents either vaccinated (with H5N1 or pH1N1) or infected with pH1N1 influenza viruses, and sera from live bird market workers in Bangladesh (BDPW) were evaluated. GH HA1 antigens and serum adsorption using full ectodomain recombinant hemagglutinins from A(H1N1) and A(H3N2) were introduced into the platforms to reduce cross-reactivity. Results Serum adsorption reduced cross-reactivity to novel subtype HAs. Compared to traditional hemagglutination inhibition or microneutralization assays, when serum adsorption and the highest fold rise in signals were used to determine positivity, the correct subtype-specific responses were identified in 86% to 100% of U.S. residents exposed to influenza antigens through vaccination or infection (N=49). For detection of H5N1 specific antibodies in sera collected from BDPW, H5 sensitivity was 100% (6/6) for MAGPIX, 83% (5/6) for DPP; H5 specificity was 100% (15/15) and cross-reactivity against other subtype was 0% (0/6) for both platforms. Conclusion MAGPIX and DPP platforms can be utilized for high-throughput and in-field detection of novel influenza virus infections. This article is protected by copyright. All rights reserved.

Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb, and reverses HIV-1 latency., A leading pharmacological strategy toward HIV cure requires “shock” or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription.

OBJECTIVE To evaluate rabies virus (RABV) characterization data obtained from animal specimens submitted to the US public health rabies surveillance system and propose a standardized approach to sample selection for RABV characterization that could enhance early detection of important rabies epizootic events in the United States. SAMPLE United States public health rabies surveillance system data collected from January 1, 2010, through December 31, 2015. PROCEDURES Data were reviewed to identify RABV-positive specimens for which virus characterization would likely provide information regarding any of 4 overarching events (discovery of novel variants, translocation of RABV variants, host-shift events, and any unusual rabies-related event) that could substantially alter animal rabies epizootiology in the United States. These specimens were designated as specimens of epizootiological importance (SEIs). Estimates of the additional number of specimens that public health laboratories could expect to process each year if all SEIs underwent RABV characterization were calculated. RESULTS During the 6-year period, the mean annual number of SEIs was 855 (95% CI, 739 to 971); the mean number of SEIs that underwent virus characterization was 270 (95% CI, 187 to 353). Virus characterization of all SEIs would be expected to increase the public health laboratories’ test load by approximately 585 (95% CI, 543 to 625) specimens/y. CONCLUSIONS AND CLINICAL RELEVANCE Prioritization of RABV characterization of SEIs may improve early detection of rabies events associated with RABV host shifts, variant translocations, and importation. Characterization of SEIs may help refine wildlife rabies management practices. Each public health laboratory should evaluate testing of SEIs to ensure diagnostic laboratory capacity is not overstretched.

Monkeypox virus (MPXV) is a member of the genus Orthopoxvirus, endemic in Central and West Africa. This viral zoonosis was introduced into the United States in 2003 via African rodents imported for the pet trade and caused 37 human cases, all linked to exposure to MPXV-infected black-tailed prairie dogs (Cynomys ludovicianus). Prairie dogs have since become a useful model of MPXV disease, utilized for testing of potential medical countermeasures. In this study, we used recombinant MPXV containing the firefly luciferase gene (luc) and in vivo imaging technology to characterize MPXV pathogenesis in the black-tailed prairie dog in real time. West African (WA) MPXV could be visualized using in vivo imaging in the nose, lymph nodes, intestines, heart, lung, kidneys, and liver as early as day 6 post infection (p.i.). By day 9 p.i., lesions became visible on the skin and in some cases in the spleen. After day 9 p.i., luminescent signal representing MPXV replication either increased, indicating a progression to what would be a fatal infection, or decreased as infection was resolved. Use of recombinant luc+ MPXV allowed for a greater understanding of how MPXV disseminates throughout the body in prairie dogs during the course of infection. This technology will be used to reduce the number of animals required in future pathogenesis studies as well as aid in determining the effectiveness of potential medical countermeasures.

Many broadly neutralizing antibodies (bnAbs) bind to conserved areas of the hemagglutinin (HA) stalk region and can inhibit the low pH induced HA conformational changes necessary for viral membrane fusion activity. We developed and evaluated a high-throughput virus-free and cell-free ELISA based low pH induced HA Conformational Change Inhibition Antibody Detection Assay (HCCIA) and a complementary proteinase susceptibility assay. Human serum samples (n = 150) were tested by HCCIA using H3 recombinant HA. Optical density (OD) ratios of mAb HC31 at pH 4.8 to pH 7.0 ranged from 0.87 to 0.09. Our results demonstrated that low pH induced HA conformational change inhibition antibodies (CCI) neutralized multiple H3 strains after removal of head-binding antibodies. The results suggest that HCCIA can be utilized to detect and characterize CCI in sera, that are potentially broadly neutralizing, and serves as a useful tool for evaluating universal vaccine candidates targeting the HA stalk.