Fatal Case of Exercise Collapse Associated with Sickle Cell Trait with Novel Underlying LAMA2 Mutation.

ABSTRACT: Sickle cell trait is typically thought to be an asymptomatic carrier state, but it is rarely associated with exertional rhabdomyolysis in cases termed Exercise Collapse Associated with Sickle Cell Trait (ECAST). In a subset of these cases, underlying disease contributes to the development and/or severity of the ensuing medical complications. We describe the first ever case of ECAST reported in a previously asymptomatic, multiply deployed, highly physically active service member with an underlying heterozygous LAMA2 mutation. Moreover, the mutation identified via whole exome sequencing is a novel, likely pathogenic variant that has yet to be described in the literature.

Myeloid/Lymphoid Neoplasm with FGFR1 Rearrangement Presenting with Polycythemia Vera and T-cell Acute Lymphoblastic Leukemia.

Myeloid/lymphoid neoplasm with fibroblast growth factor 1 rearrangements (MLN-FGFR1) represents a rare group of hematologic neoplasms, with approximately 100 cases reported to date. A 69-year-old woman with a history of polycythemia and leukocytosis, with negative molecular testing for JAK2, CALR, and MPL, presented with diffuse adenopathy. A lymph node (LN) biopsy revealed effacement by T-lymphoblasts, consistent with T-cell acute lymphoblastic lymphoma (T-ALL). A staging bone marrow (BM) biopsy demonstrated trilineage hyperplasia, which, taken together with the patient's elevated hemoglobin and low serum erythropoietin level, fulfilled diagnostic criteria for polycythemia vera. Karyotype and fluorescence in situ hybridization on both the BM and LN demonstrated a FGFR1 rearrangement due to t(8;13), consistent with MLN-FGFR1. Whole genome sequencing on the LN additionally identified a pathogenic frameshift mutation of ASXL1 NC_000020.11:g32434646dup NM_015338.6(ASXL1):c.1934dup p.(Gly646Trpfs) predicted to result in loss of protein function, a finding also observed in 8.1% of BM reads. Both the BM and LN harbored missense variants in HDAC4 NM_001378414.1(HDAC4):c.[2763G>A]; [2763=] p.(Met921Ile) and CHEK2 NM_007194.4(CHEK2):c.[538C>T];[538=] p.(Arg180Cys), with an unknown significance. Despite initial response to Mini-CVD + venetoclax, the patient subsequently experienced rapid clinical deterioration and death. We report the second case of MLN-FGFR1 with an ASXL1 mutation and the first case with HDAC4 and CHEK2 variants.

Sequencing SARS-CoV-2 from antigen tests.

Genomic surveillance empowers agile responses to SARS-CoV-2 by enabling scientists and public health analysts to issue recommendations aimed at slowing transmission, prioritizing contact tracing, and building a robust genomic sequencing surveillance strategy. Since the start of the pandemic, real time RT-PCR diagnostic testing from upper respiratory specimens, such as nasopharyngeal (NP) swabs, has been the standard. Moreover, respiratory samples in viral transport media are the ideal specimen for SARS-CoV-2 whole-genome sequencing (WGS). In early 2021, many clinicians transitioned to antigen-based SARS-CoV-2 detection tests, which use anterior nasal swabs for SARS-CoV-2 antigen detection. Despite this shift in testing methods, the need for whole-genome sequence surveillance remains. Thus, we developed a workflow for whole-genome sequencing with antigen test-derived swabs as an input rather than nasopharyngeal swabs. In this study, we use excess clinical specimens processed using the BinaxNOW™ COVID-19 Ag Card. We demonstrate that whole-genome sequencing from antigen tests is feasible and yields similar results from RT-PCR-based assays utilizing a swab in viral transport media.

Whole-genome Sequencing of SARS-CoV-2: Using Phylogeny and Structural Modeling to Contextualize Local Viral Evolution.

INTRODUCTION: The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has created a global pandemic resulting in over 1 million deaths worldwide. In the Department of Defense (DoD), over 129,000 personnel (civilians, dependents, and active duty) have been infected with the virus to date. Rapid estimations of transmission and mutational patterns of virus outbreaks can be accomplished using whole-genome viral sequencing. Deriving interpretable and actionable results from pathogen sequence data is accomplished by the construction of phylogenetic trees (from local and global virus sequences) and by the creation of protein maps, to visualize and predict the effects of structural protein amino acid mutations. MATERIALS AND METHODS: We developed a sequencing and bioinformatics workflow for molecular epidemiological SARS-CoV-2 surveillance using excess clinical specimens collected under an institutional review board exempt protocol at Joint Base San Antonio, Lackland AFB. This workflow includes viral RNA isolation, viral load quantification, tiling-based next-generation sequencing, sequencing and bioinformatics analysis, and data visualization via phylogenetic trees and protein mapping. RESULTS: Sequencing of 37 clinical specimens collected at JBSA/Lackland revealed that by June 2020, SAR-CoV-2 strains carrying the 614G mutation were the predominant cause of local coronavirus disease 2019 infections. We identified 109 nucleotide changes in the coding region of the SARS-CoV-2 genome (which lead to 63 unique, non-synonymous amino acid mutations), one mutation in the 5'-untranslated region (UTR), and two mutations in the 3'UTR. Furthermore, we identified and mapped six additional spike protein amino acid changes-information which could potentially aid vaccine design. CONCLUSION: The workflow presented here is designed to enable DoD public health officials to track viral evolution and conduct near real-time evaluation of future outbreaks. The generation of molecular epidemiological sequence data is critical for the development of disease intervention strategies-most notably, vaccine design. Overall, we present a streamlined sequencing and bioinformatics methodology aimed at improving long-term readiness efforts in the DoD.

Associations between Physiological Biomarkers and Psychosocial Measures of Pregnancy-Specific Anxiety and Depression with Support Intervention.

Stress and anxiety significantly impact the hypothalamic-pituitary axis, and in pregnancy, the subsequent maternal-fetal response can lead to poor outcomes. The objective of this study was to assess the association between psychosocial measures of pregnancy-specific anxiety and physiologic inflammatory responses. Specifically, to determine the effectiveness of the Mentors Offering Maternal Support (M-O-M-STM) program to reduce psychosocial anxiety and associated inflammatory response. In conjunction with measures of pregnancy-specific anxiety and depression, serum biomarkers (IL-2, IL-6, IL-10, IL1-B, TNF-α, CRH, CRP, and cortisol) were analyzed for each trimester throughout pregnancy. Results demonstrated that women receiving the M-O-M-STM intervention had longitudinally sustained lower TNF-α/IL-10 ratios than the control group, and it was significantly associated with psychosocial measures of anxiety, specifically for fears of labor and spouse/partner relationships. Additionally, the anxiety of spouse/partner relationships was significantly associated with IL-6/IL-10 ratios. The findings highlight the important counter-regulatory relationship between anti- and pro-inflammatory cytokines and provide insight into the distinct physiologic responses to pregnancy-specific anxiety with early prenatal intervention.

Variants in SARS-CoV-2 associated with mild or severe outcome.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic is a global public health emergency causing a disparate burden of death and disability around the world. The viral genetic variants associated with outcome severity are still being discovered. METHODS: We downloaded 155 958 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from GISAID. Of these genomes, 3637 samples included useable metadata on patient outcomes. Using this subset, we evaluated whether SARS-CoV-2 viral genomic variants improved prediction of reported severity beyond age and region. First, we established whether including genomic variants as model features meaningfully increased the predictive power of our model. Next, we evaluated specific variants in order to determine the magnitude of association with severity and the frequency of these variants among SARS-CoV-2 genomes. RESULTS: Logistic regression models that included viral genomic variants outperformed other models (area under the curve = 0.91 as compared with 0.68 for age and gender alone; P < 0.001). We found 84 variants with odds ratios greater than 2 for outcome severity (17 and 67 for higher and lower severity, respectively). The median frequency of associated variants was 0.15% (interquartile range 0.09-0.45%). Altogether 85% of genomes had at least one variant associated with patient outcome. CONCLUSION: Numerous SARS-CoV-2 variants have 2-fold or greater association with odds of mild or severe outcome and collectively, these variants are common. In addition to comprehensive mitigation efforts, public health measures should be prioritized to control the more severe manifestations of COVID-19 and the transmission chains linked to these severe cases.Lay summary: This study explores which, if any, SARS-CoV-2 viral genomic variants are associated with mild or severe COVID-19 patient outcomes. Our results suggest that there are common genomic variants in SARS-CoV-2 that are more often associated with negative patient outcomes, which may impact downstream public health measures.

Successful Expectant Management of Nonocclusive Thrombosis in Simultaneous Pancreas-Kidney Transplantation.

BACKGROUND: Simultaneous pancreas-kidney (SPK) transplantation can be complicated by thrombosis in the early post-transplant period. METHODS: We performed a single-center retrospective study examining risk factors, management, and outcomes of modern era SPK transplants. We reviewed 235 recipients over 10 years (January 1, 2008, to September 1, 2017). We used multivariate analysis to examine donor, recipient, and operative risk factors for thrombosis. RESULTS: Forty-one patients (17%) had a thrombosis diagnosed on postoperative imaging, but 61% of these patients (n = 25/41) did not lose their graft secondary to the thrombosis. Nine patients (22%) were managed with watchful waiting and serial imaging, 12 (29%) were managed with therapeutic anticoagulation, and 4 (10%) required laparotomy and graft thrombectomy. Sixteen of 235 pancreas grafts (6.8%) required pancreatectomy, and 10 of these cases occurred in the first half of the study, before 2012. The risk of thrombosis leading to graft loss increased 11.2-fold in recipients with a body mass index (calculated as weight in kilograms divided by height in meters squared) > 25 compared with others (odds ratio, 11.2; 95% CI, 1.1-116.7; P = .043). CONCLUSIONS: The majority of SPK transplants (61%) complicated by thrombosis of the pancreatic graft were salvaged by use of imaging, anticoagulation, and in select cases, laparotomy and graft thrombectomy.

Adverse Childhood Experience, Genes, and PTSD Risk in Soldiers: A Methylation Study.

INTRODUCTION: Epigenetics can serve as a marker of susceptibility to many known psychiatric diseases. DNA methylation patterns of multiple genes have been studied in both civilian populations and military personnel with post-traumatic stress disorder (PTSD). Many of these genes serve various functions that span the hypothalamic-pituitary-adrenal axis, immune system, and central nervous system (CNS) growth factors and neurotransmission. It is thought that the methylation levels of such genes may be able to identify individuals who are at higher risk of developing PTSD. Our study seeks to establish whether previously reported PTSD genes possess a particular methylation pattern that is predictive of PTSD in active duty military members with combat exposure. MATERIALS AND METHODS: This is an institutional review board (IRB)-approved, cross-sectional, case control, gene-environment interaction study. About 170 active military members with and without PTSD were recruited. Patients with a history of structural brain damage, traumatic brain injury (TBI) resulting in loss of consciousness, predeployment diagnosis of PTSD or anxiety disorder, and predeployment prescription of an antidepressant or psychoactive medication were excluded. Validated measures of childhood trauma and adversity (adverse childhood experience [ACE] score), PTSD symptoms (PTSD check-list military version [PCL-M]), and combat exposure scales (CES) were measured via validated questionnaires for all subjects. After extracting DNA from peripheral blood provided by the 170 subjects, we determined methylation percentages, via pyrosequencing assays, for nine target areas within the following seven genes: BDNF, NR3C1, MAN2C1, TLR8, SLC6A4, IL-18, and SKA2. These genes are commonly reported in the literature as being highly correlated with PTSD and early-life traumatic experiences.Methylation levels were measured as a percentage at specific sites within the previously mentioned genes. Data were examined with SPSS v 22.0 Statistics and JMP v13.1 software using a general linear model for methylation × trauma (CES scores) split by diagnosis of PTSD or not, methylation versus childhood trauma (ACE scores), and methylation versus PTSD severity (PCL-M score). Two-way ANOVA was performed to control for antidepressant use. A two-tailed Student t-test was performed for PTSD analyses and was correlated with PTSD diagnosis, demographic information as well as ACE score, PCL-M score, and CES scores. RESULTS: Differentially methylated sites that were highly associated with PTSD diagnosis were found in three of seven candidate genes: BDNF, NR3C1, and MAN2C1. When compared to controls, patients with PTSD diagnosis had significantly lower levels of methylation, even after controlling for antidepressant use. PCL-M, ACE, and CES scores were significantly associated with PTSD diagnosis. CONCLUSION: Our study suggests that methylation of key genes involved in synaptic plasticity and the hypothalamic-pituitary-adrenal axis is associated with lower levels of methylation in military PTSD subjects exposed to combat when compared to their non-PTSD counterparts. Strengths of this study include controlling for antidepressant use and excluding TBI patients. Similar studies in an active duty population of this size are scarce. What is not clear is whether methylation changes are driving PTSD symptomology or whether they are merely a marker of disease. Future areas of research include prospective studies that measure methylation pre- and postcombat exposure in the same individual.

Temporal shifts in the collective dermatologic microbiome of military trainees.

BACKGROUND:  New military members undergo a highly-regimented 7-week training course during which trainees live and work within the same group of approximately 50 subjects for nearly 24 hours a day. This creates an optimal environment for assessing the impact of communal living on the collective skin microbiome. PURPOSE: The objective of this pilot study was to investigate dynamic changes of the skin microbiome in basic military trainees (BMT), in light of the unique environmental influences faced by this population. PATIENTS AND METHODS:  We evaluated collective changes in the skin microbiome of normal healthy adult basic trainees in response to communal living and universal Group A Strep prophylaxis with penicillin over the course of their initial 7-week training course. Samples from 10 flights of trainees were collected by swabbing upon arrival at Lackland AFB for their training (week 0) which is prior to prophylaxis with penicillin, at the 4 week point, and at the conclusion of their 7-week course of basic military training. Three separate high-throughput sequencing platforms and three bioinformatic pipeline analysis tools were utilized to assess the data. RESULTS: At all three time points we found that the top three bacterial genus identified were Propionibacterium, Staphylococcus, and Corynebacterium. We detected a community membership difference between the initial week 0 samples and the week 4 and 7 samples. A strong inverse correlation between Propionibacterium and Staphylococcus was noted with Propionibacterium being high at week 0 and much lower at weeks 4 and 7; conversely, Staphylococcus was low at week 0 and higher at weeks 4 and 7, this relationship was noted in both the individual and collective specimens. CONCLUSION: The collective dermatologic microbiome in the military trainee population examined exhibited a relative increase in Staphylococcus and Corynebacterium abundance coupled with a relative decrease in Propionibacterium abundance in this observational pilot study. Additional studies are needed to further assess the causal impact of communal living and widespread penicillin chemoprophylaxis.

Intramuscular cobinamide versus saline for treatment of severe hydrogen sulfide toxicity in swine.

INTRODUCTION: Hydrogen sulfide (H2S) is found in petroleum, natural gas, and decaying organic matter. Terrorist groups have attempted to use it in enclosed spaces as a chemical weapon. Mass casualty scenarios have occurred from industrial accidents and release from oil field sites. There is no FDA approved antidote for sulfide poisoning. We have previously reported that intravenous cobinamide is effective for sulfide poisoning. A rapid-acting antidote that is easy to administer intramuscularly (IM) would be ideal for use in a prehospital setting. In this study, we assessed survival in sulfide-poisoned swine treated with IM cobinamide. METHODS: Eleven swine (45-55 kg) were anesthetized, intubated, and instrumented with continuous femoral and pulmonary artery pressure monitoring. After stabilization, anesthesia was adjusted such that animals ventilated spontaneously with a FiO2 of 0.21. Sodium hydrosulfide (NaHS, 8 mg/mL) was infused intravenously at 0.9 mg/kg.min until apnea or severe hypotension. Animals were randomly assigned to receive cobinamide (4 mg/kg), or no treatment at the apnea/hypotension trigger. The NaHS infusion rate was sustained for 1.5 min post trigger, decreased to 0.2 mg/kg.min for 10 min, and then discontinued. RESULTS: The amount of NaHS required to produce apnea or hypotension was not statistically different in both groups (cobinamide: 9.0 mg/kg ±6.1; saline: 5.9 mg/kg ±5.5; mean difference: -3.1, 95% CI: -11.3, 5.0). All of the cobinamide treated animals survived (5/5), none of the control (0/6) animals survived (p < .01). Mean time to return to spontaneous ventilation in the cobinamide treated animals was 3.2 (±1.1) min. Time to return to baseline systolic blood pressure (±5%) in cobinamide-treated animals was 5 min. CONCLUSION: Intramuscular cobinamide was effective in improving survival in this large swine model of severe hydrogen sulfide toxicity.

Long-term clinical benefit and cost-effectiveness of an 8-week multimodal knee osteoarthritis management program incorporating intra-articular sodium hyaluronate (Hyalgan®) injections.

BACKGROUND: Given the poor long-term effectiveness of focused nonsurgical knee osteoarthritis (OA) treatments, alternative therapies are needed for patients who have unsuccessfully exhausted nonsurgical options. METHODS: A telephone interview was conducted in patients who participated in a single 8-week multimodal knee OA treatment program (mean follow-up: 3.7 years, range: 2.7-4.9 years). The program consisted of five intra-articular knee injections of sodium hyaluronate (Hyalgan®), with each injection given 1 week apart, structured physical therapy, knee bracing, and patient education. Clinical outcomes included knee pain severity, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) subscores, current medication use, and history of total knee arthroplasty. Base-case, subgroup, and sensitivity analyses were conducted to determine the incremental cost-effectiveness ratio (ICER) of the treatment program with comparisons made to historical literature controls undergoing usual care. RESULTS: A total of 218 patients (54%) provided long-term follow-up data. Knee pain severity decreased 60% and WOMAC subscores decreased 33%-42% compared to baseline (all p<0.001). Total knee arthroplasty was performed in 22.8% (81/356) of knees during followup. The treatment program was highly cost-effective compared to usual care with a base-case ICER of $6,000 per quality-adjusted life year (QALY). Results of subgroup analyses, one-way deterministic sensitivity analyses, and second-order probabilistic sensitivity analyses resulted in ICERs ranging from $3,996 to $10,493 per QALY. The percentage of simulations with an ICER below willingness-to-pay limits was 97.2%, 98.9%, and 99.4% for the $50,000, $100,000, and $150,000 per QALY thresholds, respectively. CONCLUSION: Participation in a single 8-week knee OA treatment program, which included one cycle of five intra-articular knee injections of sodium hyaluronate given at weekly intervals, is highly cost-effective and provides clinically meaningful reductions in patient symptoms that are maintained over 3.7 years mean follow-up.

Rotavirus NSP4: Cell type-dependent transport kinetics to the exofacial plasma membrane and release from intact infected cells.

BACKGROUND: Rotavirus NSP4 localizes to multiple intracellular sites and is multifunctional, contributing to RV morphogenesis, replication and pathogenesis. One function of NSP4 is the induction of early secretory diarrhea by binding surface receptors to initiate signaling events. The aims of this study were to determine the transport kinetics of NSP4 to the exofacial plasma membrane (PM), the subsequent release from intact infected cells, and rebinding to naïve and/or neighboring cells in two cell types. METHODS: Transport kinetics was evaluated using surface-specific biotinylation/streptavidin pull-downs and exofacial exposure of NSP4 was confirmed by antibody binding to intact cells, and fluorescent resonant energy transfer. Transfected cells similarly were monitored to discern NSP4 movement in the absence of infection or other viral proteins. Endoglycosidase H digestions, preparation of CY3- or CY5- labeled F(ab)2 fragments, confocal imaging, and determination of preferential polarized transport employed standard laboratory techniques. Mock-infected, mock-biotinylated and non-specific antibodies served as controls. RESULTS: Only full-length (FL), endoglycosidase-sensitive NSP4 was detected on the exofacial surface of two cell types, whereas the corresponding cell lysates showed multiple glycosylated forms. The C-terminus of FL NSP4 was detected on exofacial-membrane surfaces at different times in different cell types prior to its release into culture media. Transport to the PM was rapid and distinct yet FL NSP4 was secreted from both cell types at a time similar to the release of virus. NSP4-containing, clarified media from both cells bound surface molecules of naïve cells, and imaging showed secreted NSP4 from one or more infected cells bound neighboring cell membranes in culture. Preferential sorting to apical or basolateral membranes also was distinct in different polarized cells. CONCLUSIONS: The intracellular transport of NSP4 to the PM, translocation across the PM, exposure of the C-terminus on the cell surface and subsequent secretion occurs via an unusual, complex and likely cell-dependent process. The exofacial exposure of the C-terminus poses several questions and suggests an atypical mechanism by which NSP4 traverses the PM and interacts with membrane lipids. Mechanistic details of the unconventional trafficking of NSP4, interactions with host-cell specific molecules and subsequent release require additional study.

Department of Defense influenza and other respiratory disease surveillance during the 2009 pandemic.

The Armed Forces Health Surveillance Center's Division of Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) supports and oversees surveillance for emerging infectious diseases, including respiratory diseases, of importance to the U.S. Department of Defense (DoD). AFHSC-GEIS accomplishes this mission by providing funding and oversight to a global network of partners for respiratory disease surveillance. This report details the system's surveillance activities during 2009, with a focus on efforts in responding to the novel H1N1 Influenza A (A/H1N1) pandemic and contributions to global public health. Active surveillance networks established by AFHSC-GEIS partners resulted in the initial detection of novel A/H1N1 influenza in the U.S. and several other countries, and viruses isolated from these activities were used as seed strains for the 2009 pandemic influenza vaccine. Partners also provided diagnostic laboratory training and capacity building to host nations to assist with the novel A/H1N1 pandemic global response, adapted a Food and Drug Administration-approved assay for use on a ruggedized polymerase chain reaction platform for diagnosing novel A/H1N1 in remote settings, and provided estimates of seasonal vaccine effectiveness against novel A/H1N1 illness. Regular reporting of the system's worldwide surveillance findings to the global public health community enabled leaders to make informed decisions on disease mitigation measures and controls for the 2009 A/H1N1 influenza pandemic. AFHSC-GEIS's support of a global network contributes to DoD's force health protection, while supporting global public health.

Diagnosis of 2009 pandemic influenza A (pH1N1) and seasonal influenza using rapid influenza antigen tests, San Antonio, Texas, April-June 2009.

Clinicians frequently use influenza rapid antigen tests for diagnostic testing. We tested nasal wash samples from 1 April to 7 June 2009 from 1538 patients using the QuickVue Influenza A+B (Quidel) rapid influenza antigen test and compared the results with real-time reverse transcription polymerase chain reaction (rRT-PCR) assay (gold standard). The prevalence of 2009 pandemic influenza A (pH1N1) was 1.98%, seasonal influenza type A .87%, and seasonal influenza type B 2.07%. The sensitivity and specificity of the rapid test for pH1N1 was 20% (95% CI, 8-39) and 99% (95% CI, 98-99), for seasonal influenza type A 15% (95% CI, 2-45) and 99% (95% CI, 98-99), and for influenza type B was 31% (95% CI, 9-61) and 99% (95% CI, 98-99.7). Rapid influenza antigen tests were of limited use at a time when the prevalence of pH1N1 and seasonal influenza in the United States was low. Clinicians should instead rely on clinical impression and laboratory diagnosis by rRT-PCR.

Novel Influenza A (H1N1) outbreak at the U.S. Air Force Academy: epidemiology and viral shedding duration.

BACKGROUND: The U.S. Air Force Academy is an undergraduate institution that educates and trains cadets for military service. Following the arrival of 1376 basic cadet trainees in June 2009, surveillance revealed an increase in cadets presenting with respiratory illness. Specimens from ill cadets tested positive for novel influenza A (H1N1 [nH1N1])-specific ribonucleic acid (RNA) by real-time reverse transcriptase-polymerase chain reaction. PURPOSE: The outbreak epidemiology, control measures, and nH1N1 shedding duration are described. METHODS: Case patients were identified through retrospective and prospective surveillance. Symptoms, signs, and illness duration were documented. Nasal-wash specimens were tested for nH1N1-specific RNA. Serial samples from a subset of 53 patients were assessed for presence of viable virus by viral culture. RESULTS: A total of 134 confirmed and 33 suspected cases of nH1N1 infection were identified with onset date June 25-July 24, 2009. Median age of case patients was 18 years (range, 17-24 years). Fever, cough, and sore throat were the most commonly reported symptoms. The incidence rate among basic cadet trainees during the outbreak period was 11%. Twenty-nine percent (31/106) of samples from patients with temperature <100 degrees F and 19% (11/58) of samples from patients reporting no symptoms for > or = 24 hours contained viable nH1N1 virus. Of 29 samples obtained 7 days from illness onset, seven (24%) contained viable nH1N1 virus. CONCLUSIONS: In the nH1N1 outbreak under study, the number of cases peaked 48 hours after a social event and rapidly declined thereafter. Almost one quarter of samples obtained 7 days from illness onset contained viable nH1N1 virus. These data may be useful for future investigations and in scenario planning.

Full-length, glycosylated NSP4 is localized to plasma membrane caveolae by a novel raft isolation technique.

Rotavirus NSP4, initially characterized as an endoplasmic reticulum intracellular receptor, is a multifunctional viral enterotoxin that induces diarrhea in murine pups. There have been recent reports of the secretion of a cleaved NSP4 fragment (residues 112 to 175) and of the association of NSP4 with LC3-positive autophagosomes, raft membranes, and microtubules. To determine if NSP4 traffics to a specific subset of rafts at the plasma membrane, we isolated caveolae from plasma membrane-enriched material that yielded caveola membranes free of endoplasmic reticulum and nonraft plasma membrane markers. Analyses of the newly isolated caveolae from rotavirus-infected MDCK cells revealed full-length, high-mannose glycosylated NSP4. The lack of Golgi network-specific processing of the caveolar NSP4 glycans supports studies showing that NSP4 bypasses the Golgi apparatus. Confocal imaging showed the colocalization of NSP4 with caveolin-1 early and late in infection, elucidating the temporal and spatial NSP4-caveolin-1 association during infection. These data were extended with fluorescent resonance energy transfer analyses that confirmed the NSP4 and caveolin-1 interaction in that the specific fluorescently tagged antibodies were within 10 nm of each other during infection. Cells transfected with NSP4 showed patterns of staining and colocalization with caveolin-1 similar to those of infected cells. This study presents an endoplasmic reticulum contaminant-free caveola isolation protocol; describes the presence of full-length, endoglycosidase H-sensitive NSP4 in plasma membrane caveolae; provides confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins; and provides a final plasma membrane destination for Golgi network-bypassing NSP4 transport.