Timothy R. Peters

AP-3 Directs the Intracellular Trafficking of HIV-1 Gag and Plays a Key Role in Particle Assembly

Gag proteins direct the process of retroviral particle assembly and form the major protein constituents of the viral core. The matrix region of the HIV-1 Gag polyprotein plays a critical role in the transport of Gag to the plasma membrane assembly site. Recent evidence indicates that Gag trafficking to late endosomal compartments, including multivesicular bodies, occurs prior to viral particle budding from the plasma membrane. Here we demonstrate that the matrix region of HIV-1 Gag interacts directly with the delta subunit of the AP-3 complex, and that this interaction plays an important functional role in particle assembly. Disruption of this interaction eliminated Gag trafficking to multivesicular bodies and diminished HIV particle formation. These studies illuminate an early step in retroviral particle assembly and provide evidence that the trafficking of Gag to late endosomes is part of a productive particle assembly pathway.

Exposure to Influenza Virus Aerosols During Routine Patient Care

BACKGROUND Defining dispersal of influenza virus via aerosol is essential for the development of prevention measures. METHODS During the 2010-2011 influenza season, subjects with influenza-like illness were enrolled in an emergency department and throughout a tertiary care hospital, nasopharyngeal swab specimens were obtained, and symptom severity, treatment, and medical history were recorded. Quantitative impaction air samples were taken not ≤0.305 m (1 foot), 0.914 m (3 feet), and 1.829 m (6 feet) from the patients head during routine care. Influenza virus was detected by rapid test and polymerase chain reaction. RESULTS Sixty-one of 94 subjects (65%) tested positive for influenza virus. Twenty-six patients (43%) released influenza virus into room air, with 5 (19%) emitting up to 32 times more virus than others. Emitters surpassed the airborne 50% human infectious dose of influenza virus at all sample locations. Healthcare professionals (HCPs) were exposed to mainly small influenza virus particles (diameter, <4.7 µm), with concentrations decreasing with increasing distance from the patients head (P < .05). Influenza virus release was associated with high viral loads in nasopharyngeal samples (shedding), coughing, and sneezing (P < .05). Patients who reported severe illness and major interference with daily life also emitted more influenza virus (P < .05). CONCLUSIONS HCPs within 1.829 m of patients with influenza could be exposed to infectious doses of influenza virus, primarily in small-particle aerosols. This finding questions the current paradigm of localized droplet transmission during non-aerosol-generating procedures.

Reovirus σNS and μNS Proteins Form Cytoplasmic Inclusion Structures in the Absence of Viral Infection

ABSTRACT Reovirus replication occurs in the cytoplasm of infected cells and culminates in the formation of crystalline arrays of progeny virions within viral inclusions. Two viral nonstructural proteins, σNS and μNS, and structural protein σ3 form protein-RNA complexes early in reovirus infection. To better understand the minimal requirements of viral inclusion formation, we expressed σNS, μNS, and σ3 alone and in combination in the absence of viral infection. In contrast to its concentration in inclusion structures during reovirus replication, σNS expressed in cells in the absence of infection is distributed diffusely throughout the cytoplasm and does not form structures that resemble viral inclusions. Expressed σNS is functional as it complements the defect in temperature-sensitive, σNS-mutant virus tsE320. In both transfected and infected cells, μNS is found in punctate cytoplasmic structures and σ3 is distributed diffusely in the cytoplasm and the nucleus. The subcellular localization of μNS and σ3 is not altered when the proteins are expressed together or with σNS. However, when expressed with μNS, σNS colocalizes with μNS to punctate structures similar in morphology to inclusion structures observed early in viral replication. During reovirus infection, both σNS and μNS are detectable 4 h after adsorption and colocalize to punctate structures throughout the viral life cycle. In concordance with these results, σNS interacts with μNS in a yeast two-hybrid assay and by coimmunoprecipitation analysis. These data suggest that σNS and μNS are the minimal viral components required to form inclusions, which then recruit other reovirus proteins and RNA to initiate viral genome replication.

Detection of group A Streptococcus in tonsils from pediatric patients reveals high rate of asymptomatic streptococcal carriage

BackgroundGroup A Streptococcus (GAS) causes acute tonsillopharyngitis in children, and approximately 20% of this population are chronic carriers of GAS. Antibacterial therapy has previously been shown to be insufficient at clearing GAS carriage. Bacterial biofilms are a surface-attached bacterial community that is encased in a matrix of extracellular polymeric substances. Biofilms have been shown to provide a protective niche against the immune response and antibiotic treatments, and are often associated with recurrent or chronic bacterial infections. The objective of this study was to test the hypothesis that GAS is present within tonsil tissue at the time of tonsillectomy.MethodsBlinded immunofluorescent and histological methods were employed to evaluate palatine tonsils from children undergoing routine tonsillectomy for adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis.ResultsImmunofluorescence analysis using anti-GAS antibody was positive in 11/30 (37%) children who had tonsillectomy for adenotonsillar hypertrophy and in 10/30 (33%) children who had tonsillectomy for recurrent GAS pharyngitis. Fluorescent microscopy with anti-GAS and anti-cytokeratin 8 & 18 antibodies revealed GAS was localized to the tonsillar reticulated crypts. Scanning electron microscopy identified 3-dimensional communities of cocci similar in size and morphology to GAS. The characteristics of these communities are similar to GAS biofilms from in vivo animal models.ConclusionOur study revealed the presence of GAS within the tonsillar reticulated crypts of approximately one-third of children who underwent tonsillectomy for either adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis at the Wake Forest School of Medicine.Trial RegistrationThe tissue collected was normally discarded tissue and no patient identifiers were collected. Thus, no subjects were formally enrolled.

Effectiveness of live attenuated influenza vaccine and inactivated influenza vaccine in children 2–17 years of age in 2013–2014 in the United States

BACKGROUND A postmarketing observational study was initiated to evaluate quadrivalent live attenuated influenza vaccine (LAIV) effectiveness in children aged 2-17 years in the United States. METHODS Children and adolescents aged 2-17 years seeking outpatient care for febrile acute respiratory illness <5 days duration were enrolled at 4 geographically diverse sites during the 2013-2014 influenza season. Nasal swabs were tested for influenza using reverse transcription polymerase chain reaction. Vaccination status was documented from medical records or immunization registries. Children who received ≥1 dose of influenza vaccine ≥14 days before study visit were considered vaccinated. Vaccine effectiveness (VE) was estimated as 100×(1-adjusted odds ratio), where the odds of interest are the odds of vaccine exposure among influenza cases and test-negative controls. RESULTS In total, 1033 children and adolescents were included in the analysis. Influenza was detected in 14% (145/1033) of all children, with 74% (108/145) of the influenza cases due to A/H1N1pdm09 strains, 21% (31) to influenza B, and 4% (6) to influenza H3N2. LAIV did not show significant effectiveness against A/H1N1pdm09 (VE 13% [95% CI: -55 to 51]) but was effective against B/Yamagata strains (82% [95% CI: 12-96]). Inactivated influenza vaccine was effective against A/H1N1pdm09 (74% [95% CI: 50-86]) and B/Yamagata (70% [95% CI: 18-89]). CONCLUSIONS LAIV provided significant protection against B/Yamagata influenza but not against A/H1N1pdm09 in children aged 2-17 years in 2013-2014, resulting in a proposed change of the 2015-2016 formulation with a new and more heat-stable A/H1N1pdm09 LAIV strain.

Transocular Entry of Seasonal Influenza–Attenuated Virus Aerosols and the Efficacy of N95 Respirators, Surgical Masks, and Eye Protection in Humans

BACKGROUND The efficacy of barrier precautions to prevent influenza transmission is unknown. METHODS Twenty-eight participants were exposed to monodispersed live attenuated influenza vaccine (LAIV) particles (4.9 μm) in 6 groups: group 1, no precautions; group 2, ocular exposure only; group 3, surgical mask without eye protection; group 4, surgical mask with eye protection; group 5, fit-tested N95 respirator without eye protection; and group 6, fit-tested N95 respirator with eye protection. Influenza was detected by reverse-transcription polymerase chain reaction (RT-PCR) and culture in nasal washes. Exact 95% confidence intervals (CIs) were calculated. RESULTS Influenza was detected in 4 of 4 participants in group 1 (95% CI, 0-.60), 3 of 4 in group 2 (95% CI, .006-.806]), 5 of 5 in group 3 (95% CI, 0-.522), 5 of 5 in group 4, (95% CI, 0-.522), 3 of 5 in group 5 (95% CI, .053-.853), and 1 of 5 in group 6 (95% CI, .05-.72). RT-PCR revealed significant differences between group 1 and all other groups except group 3. CONCLUSIONS Transocular transmission of LAIV occured in most participants suggesting the necessity of eye protection. An N95 respirator provided the best guard further enhanced by eye protection.

One third of middle ear effusions from children undergoing tympanostomy tube placement had multiple bacterial pathogens

BackgroundBecause previous studies have indicated that otitis media may be a polymicrobial disease, we prospectively analyzed middle ear effusions of children undergoing tympanostomy tube placement with multiplex polymerase chain reaction for four otopathogens.MethodsMiddle ear effusions from 207 children undergoing routine tympanostomy tube placement were collected and were classified by the surgeon as acute otitis media (AOM) for purulent effusions and as otitis media with effusion (OME) for non-purulent effusions. DNA was isolated from these samples and analyzed with multiplex polymerase chain reaction for Haemophilus influenzae, Streptococcus pneumoniae, Alloiococcus otitidis, and Moraxella catarrhalis.Results119 (57%) of 207 patients were PCR positive for at least one of these four organisms. 36 (30%) of the positive samples indicated the presence of more than one bacterial species. Patient samples were further separated into 2 groups based on clinical presentation at the time of surgery. Samples were categorized as acute otitis media (AOM) if pus was observed behind the tympanic membrane. If no pus was present, samples were categorized as otitis media with effusion (OME). Bacteria were identified in most of the children with AOM (87%) and half the children with OME (51%, p < 0.001). A single bacterial organism was detected in middle ear effusions from children with AOM more often than those with OME (74% versus 33%, p < 0.001). Haemophilus influenzae was the predominant single organism and caused 58% of all AOM in this study. Alloiococcus otitidis and Moraxella catarrhalis were more frequently identified in middle ear effusions than Streptococcus pneumoniae.ConclusionsHaemophilus influenzae, Streptococcus pneumoniae, Alloiococcus otitidis, and Moraxella catarrhalis were identified in the middle ear effusions of some patients with otitis media. Overall, we found AOM is predominantly a single organism infection and most commonly from Haemophilus influenzae. In contrast, OME infections had a more equal distribution of single organisms, polymicrobial entities, and non-bacterial agents.

Multicenter Surveillance of Streptococcus pneumoniae Isolates From Middle Ear and Mastoid Cultures in the 13-Valent Pneumococcal Conjugate Vaccine Era

BACKGROUND Streptococcus pneumoniae is a common cause of otitis media (OM) in children; mastoiditis remains an important complication of OM. Limited data are available on the impact of the 13-valent pneumococcal conjugate vaccine (PCV13) on pneumococcal otitis. METHODS Investigators from 8 childrens hospitals in the United States prospectively collected pneumococcal isolates from middle ear or mastoid cultures from children from 2011 to 2013. Serotype and antibiotic susceptibilities were determined and PCV13 doses for children documented. RESULTS Over the 3-year period, the proportion of isolates included in PCV13 (plus a related serotype) decreased significantly (P = .0006) among the middle ear/mastoid isolates (2011, 50% [74/149]; 2012, 40.5% [47/116]; 2013, 29% [34/118]). The number of serotype 19A isolates in 2013 (n = 12, 10.2% of total) decreased 76% compared with the number of 19A isolates in 2011 (n = 50, 33.6% of total). Of the children from whom serotype 19A was isolated (n = 93), 55% had previously received <3 doses of PCV13. The most common non-PCV13 serotypes for the combined years were 35B (n = 37), 21 (n = 20), 23B (n = 20), 15B (n = 18), 11 (n = 17), 23A (n = 14), 15A (n = 14), and 15C (n = 14). The proportion of isolates with a penicillin minimal inhibitory concentration >2 µg/mL decreased significantly over the 3 years (2011, 22% [35/154]; 2012, 20% [24/118]; 2013, 10% [12/120]; P < .02). CONCLUSIONS The number of pneumococcal isolates and the percentage of isolates with high-level penicillin resistance from cultures taken from children with OM or mastoiditis for clinical indications have decreased following PCV13 use, largely related to decreases in serotype 19A isolates.

Potential Impact of Acceleration of the Pertussis Vaccine Primary Series for Infants

OBJECTIVE. This study estimates the potential impact, on rates of pertussis infections, hospitalizations, and deaths among infants in the United States, of administering the first dose of diphtheria and tetanus toxoids and acellular pertussis vaccine at 6 weeks rather than 2 months of age. METHODS. We used existing data to estimate current US rates of pertussis infections, hospitalizations, and deaths according to age and infant population in 2004. We then estimated the potential impact of accelerating the administration of the first dose of diphtheria and tetanus toxoids and acellular pertussis vaccine from 2 months to 6 weeks of age, an alternative schedule consistent with current vaccination guidelines. We used Poisson distribution analysis to determine 95% confidence intervals for projected rates of pertussis disease. RESULTS. Acceleration of administration of the first dose of diphtheria and tetanus toxoids and acellular pertussis vaccine from 2 months to 6 weeks of age is expected to prevent 1236 cases of pertussis, 898 hospitalizations, and 7 deaths attributable to pertussis per year in the United States. These decreases represent 9% reduction in cases, 9% reduction in hospitalizations, and 6% reduction in deaths attributable to pertussis among infants <3 months of age. Acceleration of the second and third doses by 2 weeks is expected to prevent an additional 923 cases, 520 hospitalizations, and 2 deaths attributable to pertussis each year. CONCLUSION. Acceleration of administration of diphtheria and tetanus toxoids and acellular pertussis vaccine from 2 months to 6 weeks should reduce the burden of pertussis among young infants.

Human metapneumovirus nucleoprotein and phosphoprotein interact and provide the minimal requirements for inclusion body formation

Human metapneumovirus (HMPV) is a recently discovered paramyxovirus of the subfamily Pneumovirinae, which also includes avian pneumovirus and human respiratory syncytial virus (HRSV). HMPV is an important cause of respiratory disease worldwide. To understand early events in HMPV replication, cDNAs encoding the HMPV nucleoprotein (N), phosphoprotein (P), matrix protein (M), M2-1 protein and M2-2 protein were cloned from cells infected with the genotype A1 HMPV wild-type strain TN/96-12. HMPV N and P were shown to interact using a variety of techniques: yeast two-hybrid assays, co-immunoprecipitation and fluorescence resonance energy transfer (FRET). Confocal microscopy studies showed that, when expressed individually, fluorescently tagged HMPV N and P exhibited a diffuse expression pattern in the host-cell cytoplasm of uninfected cells but were recruited to cytoplasmic viral inclusion bodies in HMPV-infected cells. Furthermore, when HMPV N and P were expressed together, they also formed cytoplasmic inclusion-like complexes, even in the absence of viral infection. FRET microscopy revealed that HMPV N and P interacted directly within cytoplasmic inclusion-like complexes. Moreover, it was shown by yeast two-hybrid analysis that the N-terminal 28 aa are required for the recruitment to and formation of cytoplasmic inclusions, but are dispensable for binding to HMPV P. This work showed that HMPV N and P proteins provide the minimal viral requirements for HMPV inclusion body formation, which may be a distinguishing characteristic of members of the subfamily Pneumovirinae.