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1.  Hantavirus Fever without Pulmonary Syndrome in Panama 
In Panama, hantavirus pulmonary syndrome (HPS) is caused by Choclo virus, a species phylogenetically related to Andes and Maporal viruses. Up to 60% of the population has been positive for specific serum antibody in community-based surveys, but mortality is very uncommon. In four western Panama clinics, we tested individuals presenting with a severe febrile prodrome for acute hantavirus (HV) infection by immunoglobulin M enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction as well as clinically similar infections, such as dengue and leptospirosis. From 2006 to 2009, at least 21% of 117 patients diagnosed with HV infection had HV Fever (HF) with no evidence of pulmonary edema (no respiratory distress or radiographic lung infiltrates), and 44% of patients had very mild HPS (radiographic pulmonary edema but no respiratory insufficiency). HV infection caused by Choclo virus in Panama presents often as HF, which contrasts with HV in the Americas but is consistent with the high seroprevalence in endemic regions.
PMCID: PMC3771286  PMID: 23836565
2.  Antibody against Arenaviruses in Humans, Southwestern United States 
Emerging Infectious Diseases  2014;20(9):1592-1593.
PMCID: PMC4178406  PMID: 25153356
arenaviruses; viruses; antibodies; humans; zoonoses; vector-borne infections; Arizona; New Mexico; southwestern United States
3.  Delta Inulin Polysaccharide Adjuvant Enhances The Ability Of Split-virion H5N1 Vaccine To Protect Against Lethal Challenge In Ferrets 
Vaccine  2011;29(37):6242-6251.
The reduced immunogenicity of the H5 hemagglutinin (HA), compared to seasonal HA serotypes, has stimulated searches for effective adjuvants to improve H5 vaccine efficacy. This study examined the immunogenicity and protective efficacy in ferrets immunized with a split-virion H5N1 vaccine combined with Advax™, a novel delta inulin-based polysaccharide adjuvant technology that has previously demonstrated ability to augment humoral and cellular immunity to co-administered antigens.
Ferrets were vaccinated twice 21 days apart with 7.5 µg or 22.5 µg of a split-virion preparation of A/Vietnam/1203/2004 with or without adjuvant. An additional group received just one immunization with 22.5 µg HA plus adjuvant. Serum antibodies were measured by hemagglutination inhibition and microneutralization assays. Vaccinated animals were challenged intranasally 21 days after the last immunization with 106 EID50 of the homologous strain. Morbidity was assessed by observed behavior, weight loss, temperature, cytopenias, histopathology, and viral load.
No serum neutralization antibody was detected after two immunizations with unadjuvanted vaccine. Two immunizations with high or low dose adjuvanted vaccine stimulated high neutralizing antibody titers. Survival was 100% in all groups receiving adjuvanted-vaccine including the single dose group, compared to 67% survival with unadjuvanted vaccine, and 0% survival in saline or adjuvant-alone controls. Minimal morbidity was seen in all animals receiving adjuvanted vaccine, and was limited to rhinorrhea and mild thrombocytopenia, without fever, weight loss, or reduced activity. H5N1 virus was cleared from the nasal wash by day 4 post-challenge only in animals receiving adjuvanted vaccine which also prevented viral invasion of the brain in most animals.
In this initial study, Advax™ adjuvant formulations improved the protective efficacy of a split-virion H5N1vaccine as measured by significantly enhanced immunogenicity, survival, and reduced morbidity.
PMCID: PMC3156374  PMID: 21736913
4.  Elevated VEGF Levels in Pulmonary Edema Fluid and PBMCs from Patients with Acute Hantavirus Pulmonary Syndrome 
Advances in Virology  2012;2012:674360.
Hantavirus pulmonary syndrome is characterized by vascular permeability, hypoxia, and acute pulmonary edema. Vascular endothelial growth factor (VEGF) is induced by hypoxia, potently induces vascular permeability, and is associated with high-altitude-induced pulmonary edema. Hantaviruses alter the normal regulation of β3 integrins that restrict VEGF-directed permeability and hantavirus infected endothelial cells are hyperresponsive to the permeabilizing effects of VEGF. However, the role of VEGF in acute pulmonary edema observed in HPS patients remains unclear. Here we retrospectively evaluate VEGF levels in pulmonary edema fluid (PEF), plasma, sera, and PBMCs from 31 HPS patients. VEGF was elevated in HPS patients PEF compared to controls with the highest levels observed in PEF samples from a fatal HPS case. VEGF levels were highest in PBMC samples during the first five days of hospitalization and diminished during recovery. Significantly increased PEF and PBMC VEGF levels are consistent with acute pulmonary edema observed in HPS patients and HPS disease severity. We observed substantially lower VEGF levels in a severe HPS disease survivor after extracorporeal membrane oxygenation. These findings suggest the importance of patients' VEGF levels during HPS, support the involvement of VEGF responses in HPS pathogenesis, and suggest targeting VEGF responses as a potential therapeutic approach.
PMCID: PMC3432326  PMID: 22956954
5.  Exhaled Aerosol Transmission of Pandemic and Seasonal H1N1 Influenza Viruses in the Ferret 
PLoS ONE  2012;7(4):e33118.
Person-to-person transmission of influenza viruses occurs by contact (direct and fomites) and non-contact (droplet and small particle aerosol) routes, but the quantitative dynamics and relative contributions of these routes are incompletely understood. The transmissibility of influenza strains estimated from secondary attack rates in closed human populations is confounded by large variations in population susceptibilities. An experimental method to phenotype strains for transmissibility in an animal model could provide relative efficiencies of transmission. We developed an experimental method to detect exhaled viral aerosol transmission between unanesthetized infected and susceptible ferrets, measured aerosol particle size and number, and quantified the viral genomic RNA in the exhaled aerosol. During brief 3-hour exposures to exhaled viral aerosols in airflow-controlled chambers, three strains of pandemic 2009 H1N1 strains were frequently transmitted to susceptible ferrets. In contrast one seasonal H1N1 strain was not transmitted in spite of higher levels of viral RNA in the exhaled aerosol. Among three pandemic strains, the two strains causing weight loss and illness in the intranasally infected ‘donor’ ferrets were transmitted less efficiently from the donor than the strain causing no detectable illness, suggesting that the mucosal inflammatory response may attenuate viable exhaled virus. Although exhaled viral RNA remained constant, transmission efficiency diminished from day 1 to day 5 after donor infection. Thus, aerosol transmission between ferrets may be dependent on at least four characteristics of virus-host relationships including the level of exhaled virus, infectious particle size, mucosal inflammation, and viral replication efficiency in susceptible mucosa.
PMCID: PMC3317934  PMID: 22509254
6.  Primary pneumonic plague in the African Green monkey as a model for treatment efficacy evaluation 
Primary pneumonic plague is rare among humans but treatment efficacy may be tested in appropriate animal models under the FDA ‘Animal Rule’.
Ten African Green monkeys (AGM) inhaled 44 to 255 LD50 doses of aerosolized Y. pestis strain CO92. Continuous telemetry, arterial blood gases, chest radiography, blood culture, and clinical pathology monitored disease progression.
Onset of fever, >39°C detected by continuous telemetry, 52 to 80 h post-exposure Was the first sign of systemic disease and provides a distinct signal for treatment initiation. Secondary endpoints of disease severity include tachypnea, measured by telemetry, bacteremia, extent of pneumonia imaged by chest x-ray, and serum lactate dehydrogenase enzyme levels.
Inhaled Y. pestis in the AGM results in a rapidly progressive and uniformly fatal disease with fever and multifocal pneumonia, serving as a rigorous test model for antibiotic efficacy studies.
PMCID: PMC2991483  PMID: 20722770
plague; pneumonia; African Green monkey; telemetry
7.  Incidence Rate for Hantavirus Infections without Pulmonary Syndrome, Panama 
Emerging Infectious Diseases  2011;17(10):1936-1939.
During 2001–2007, to determine incidence of all hantavirus infections, including those without pulmonary syndrome, in western Panama, we conducted 11 communitywide surveys. Among 1,129 persons, antibody prevalence was 16.5%–60.4%. Repeat surveys of 476 found that patients who seroconverted outnumbered patients with hantavirus pulmonary syndrome by 14 to 1.
PMCID: PMC3310655  PMID: 22000376
hantavirus; Bunyaviridae; epidemiology; hantavirus pulmonary syndrome; viruses; Panama; dispatch
8.  Transmission of Aerosolized Seasonal H1N1 Influenza A to Ferrets 
PLoS ONE  2011;6(9):e24448.
Influenza virus is a major cause of morbidity and mortality worldwide, yet little quantitative understanding of transmission is available to guide evidence-based public health practice. Recent studies of influenza non-contact transmission between ferrets and guinea pigs have provided insights into the relative transmission efficiencies of pandemic and seasonal strains, but the infecting dose and subsequent contagion has not been quantified for most strains. In order to measure the aerosol infectious dose for 50% (aID50) of seronegative ferrets, seasonal influenza virus was nebulized into an exposure chamber with controlled airflow limiting inhalation to airborne particles less than 5 µm diameter. Airborne virus was collected by liquid impinger and Teflon filters during nebulization of varying doses of aerosolized virus. Since culturable virus was accurately captured on filters only up to 20 minutes, airborne viral RNA collected during 1-hour exposures was quantified by two assays, a high-throughput RT-PCR/mass spectrometry assay detecting 6 genome segments (Ibis T5000™ Biosensor system) and a standard real time RT-qPCR assay. Using the more sensitive T5000 assay, the aID50 for A/New Caledonia/20/99 (H1N1) was approximately 4 infectious virus particles under the exposure conditions used. Although seroconversion and sustained levels of viral RNA in upper airway secretions suggested established mucosal infection, viral cultures were almost always negative. Thus after inhalation, this seasonal H1N1 virus may replicate less efficiently than H3N2 virus after mucosal deposition and exhibit less contagion after aerosol exposure.
PMCID: PMC3176225  PMID: 21949718
9.  Confirmation of Choclo Virus as the Cause of Hantavirus Cardiopulmonary Syndrome and high serum antibody prevalence in Panama 
Journal of medical virology  2010;82(9):1586-1593.
Choclo virus (CHOV) was described in sigmodontine rodents, Oligoryzomys fulvescens, and humans during an outbreak of hantavirus cardiopulmonary syndrome (HCPS) in 1999 to 2000 in western Panama. Although HCPS is rare, hantavirus-specific serum antibody prevalence among the general population is high suggesting that CHOV may cause many mild or asymptomatic infections. The goals of this study were to confirm the role of CHOV in HCPS and in the frequently detected serum antibody and to established the phylogenetic relationship with other New World hantaviruses. CHOV was cultured to facilitate the sequencing of the small (S) and medium (M) segments and to perform CHOV-specific serum neutralization antibody assays.
Sequences of the S and M segments found a close relationship to other Oligoryzomys-borne hantaviruses in the Americas, highly conserved terminal nucleotides, and no evidence for recombination events. The maximum likelihood and maximum parsimony analyses of complete M segment nucleotide sequences indicate a close relationship to Maporal and Laguna Negra viruses, found at the base of the South American clade. In a focus neutralization assay acute and convalescent sera from 6 Panamanian HCPS patients neutralized CHOV in dilutions from 1:200 to 1:6400. In a sample of antibody-positive adults without a history of HCPS, 9 of 10 sera neutralized CHOV in dilutions ranging from 1:100 to 1:6400. Although cross-neutralization with other sympatric hantaviruses not yet associated with human disease is possible, CHOV appears to be the causal agent for most of the mild or asymptomatic hantavirus infections, as well as HCPS, in Panama.
PMCID: PMC2927102  PMID: 20648614
hantavirus; phylogeny; neutralizing antibody; Bunyaviridae
10.  Higher Level of Replication Efficiency of 2009 (H1N1) Pandemic Influenza Virus than Those of Seasonal and Avian Strains: Kinetics from Epithelial Cell Culture and Computational Modeling▿  
Journal of Virology  2010;85(2):1125-1135.
The pathogenicity and transmission of influenza A viruses are likely determined in part by replication efficiency in human cells, which is the net effect of complex virus-host interactions. H5N1 avian, H1N1 seasonal, and H1N1 2009 pandemic influenza virus strains were compared by infecting human differentiated bronchial epithelial cells in air-liquid interface cultures at relatively low virus particle/cell ratios. Differential equation and computational models were used to characterize the in vitro kinetic behaviors of the three strains. The models were calibrated by fitting experimental data in order to estimate difficult-to-measure parameters. Both models found marked differences in the relative values of p, the virion production rate per cell, and R0, an index of the spread of infection through the monolayer, with the values for the strains in the following rank order (from greatest to least): pandemic strain, followed by seasonal strain, followed by avian strain, as expected. In the differential equation model, which treats virus and cell populations as well mixed, R0 and p varied proportionately for all 3 strains, consistent with a primary role for productivity. In the spatially explicit computational model, R0 and p also varied proportionately except that R0 derived for the pandemic strain was reduced, consistent with constrained viral spread imposed by multiple host defenses, including mucus and paracrine antiviral effects. This synergistic experimental-computational strategy provides relevant parameters for identifying and phenotyping potential pandemic strains.
PMCID: PMC3019989  PMID: 21068247
11.  Enhanced Immunogenicity, Mortality Protection, and Reduced Viral Brain Invasion by Alum Adjuvant with an H5N1 Split-Virion Vaccine in the Ferret 
PLoS ONE  2011;6(6):e20641.
Pre-pandemic development of an inactivated, split-virion avian influenza vaccine is challenged by the lack of pre-existing immunity and the reduced immunogenicity of some H5 hemagglutinins compared to that of seasonal influenza vaccines. Identification of an acceptable effective adjuvant is needed to improve immunogenicity of a split-virion avian influenza vaccine.
Methods and Findings
Ferrets (N = 118) were vaccinated twice with a split-virion vaccine preparation of A/Vietnam/1203/2004 or saline either 21 days apart (unadjuvanted: 1.9 µg, 7.5 µg, 30 µg, or saline), or 28 days apart (unadjuvanted: 22.5 µg, or alum-adjuvanted: 22.5 or 7.5 µg). Vaccinated animals were challenged intranasally 21 or 28 days later with 106 EID50 of the homologous strain. Immunogenicity was measured by hemagglutination inhibition and neutralization assays. Morbidity was assessed by observed behavior, weight loss, temperature, cytopenias, histopathology, and viral load.
No serum antibodies were detected after vaccination with unadjuvanted vaccine, whereas alum-adjuvanted vaccination induced a robust antibody response. Survival after unadjuvanted dose regimens of 30 µg, 7.5 µg and 1.9 µg (21-day intervals) was 64%, 43%, and 43%, respectively, yet survivors experienced weight loss, fever and thrombocytopenia. Survival after unadjuvanted dose regimen of 22.5 µg (28-day intervals) was 0%, suggesting important differences in intervals in this model. In contrast to unadjuvanted survivors, either dose of alum-adjuvanted vaccine resulted in 93% survival with minimal morbidity and without fever or weight loss. The rarity of brain inflammation in alum-adjuvanted survivors, compared to high levels in unadjuvanted vaccine survivors, suggested that improved protection associated with the alum adjuvant was due to markedly reduced early viral invasion of the ferret brain.
Alum adjuvant significantly improves efficacy of an H5N1 split-virion vaccine in the ferret model as measured by immunogenicity, mortality, morbidity, and brain invasion.
PMCID: PMC3110201  PMID: 21687736
12.  Levofloxacin Cures Experimental Pneumonic Plague in African Green Monkeys 
Yersinia pestis, the agent of plague, is considered a potential bioweapon due to rapid lethality when delivered as an aerosol. Levofloxacin was tested for primary pneumonic plague treatment in a nonhuman primate model mimicking human disease.
Methods and Results
Twenty-four African Green monkeys (AGMs, Chlorocebus aethiops) were challenged via head-only aerosol inhalation with 3–145 (mean = 65) 50% lethal (LD50) doses of Y. pestis strain CO92. Telemetered body temperature >39°C initiated intravenous infusions to seven 5% dextrose controls or 17 levofloxacin treated animals. Levofloxacin was administered as a “humanized” dose regimen of alternating 8 mg/kg and 2 mg/kg 30-min infusions every 24-h, continuing until animal death or 20 total infusions, followed by 14 days of observation. Fever appeared at 53–165 h and radiographs found multilobar pneumonia in all exposed animals. All control animals died of severe pneumonic plague within five days of aerosol exposure. All 16 animals infused with levofloxacin for 10 days survived. Levofloxacin treatment abolished bacteremia within 24 h in animals with confirmed pre-infusion bacteremia, and reduced tachypnea and leukocytosis but not fever during the first 2 days of infusions.
Levofloxacin cures established pneumonic plague when treatment is initiated after the onset of fever in the lethal aerosol-challenged AGM nonhuman primate model, and can be considered for treatment of other forms of plague. Levofloxacin may also be considered for primary presumptive-use, multi-agent antibiotic in bioterrorism events prior to identification of the pathogen.
Author Summary
Yersinia pestis is the causative agent of bubonic plague as well as a rare severe form known as primary pneumonic plague resulting from the inhalation of contaminated aerosols. The relative ease of aerosol preparation and high virulence makes Y. pestis a dangerous bioweapon. The current study describes the treatment of established pneumonic plague with the widely available, broad-spectrum fluoroquinolone antibiotic levofloxacin in a nonhuman primate model. African green monkeys inhaled a target dose of 100 lethal doses for 50% of animals (LD50) and were monitored for fever and vital signs by telemetry. Fever was the first sign of illness, correlating with bacteremia but preceding radiographic pneumonia, and initiated intravenous levofloxacin treatment in doses designed to mimic antibiotic levels achieved in humans. All animals treated with saline died and all animals completing 10 days of treatment survived, with resolution of high fever within 24–48 hours. We conclude that levofloxacin may be an appropriate broad-spectrum antibiotic for presumptive therapy in an aerosolized bioweapons attack and should be studied for treatment of bubonic plague.
PMCID: PMC3035670  PMID: 21347450
13.  Milestones in Progression of Primary Pneumonic Plague in Cynomolgus Macaques▿  
Infection and Immunity  2010;78(7):2946-2955.
Vaccines against primary pneumonic plague, a potential bioweapon, must be tested for efficacy in well-characterized nonhuman primate models. Telemetered cynomolgus macaques (Macaca fascicularis) were challenged by the aerosol route with doses equivalent to approximately 100 50% effective doses of Yersinia pestis strain CO92 and necropsied at 24-h intervals postexposure (p.e.). Data for telemetered heart rates, respiratory rates, and increases in the temperature greater than the diurnal baseline values identified the onset of the systemic response at 55 to 60 h p.e. in all animals observed for at least 70 h p.e. Bacteremia was detected at 72 h p.e. by a Yersinia 16S rRNA-specific quantitative reverse transcription-PCR and was detected later by the culture method at the time of moribund necropsy. By 72 h p.e. multilobar pneumonia with diffuse septal inflammation consistent with early bacteremia was established, and all lung tissues had a high bacterial burden. The levels of cytokines or chemokines in serum were not significantly elevated at any time, and only the interleukin-1β, CCL2, and CCL3 levels were elevated in lung tissue. Inhalational plague in the cynomolgus macaque inoculated by the aerosol route produces most clinical features of the human disease, and in addition the disease progression mimics the disease progression from the anti-inflammatory phase to the proinflammatory phase described for the murine model. Defined milestones of disease progression, particularly the onset of fever, tachypnea, and bacteremia, should be useful for evaluating the efficacy of candidate vaccines.
PMCID: PMC2897392  PMID: 20385751
14.  Convalescent Pulmonary Dysfunction Following Hantavirus Pulmonary Syndrome in Panama and the United States 
Lung  2010;188(5):387-391.
The objective of this study was to document persistent pulmonary symptoms and pulmonary function abnormalities in adults surviving hantavirus pulmonary syndrome (HPS). Acute infection by most hantaviruses result in mortality rates of 25–35%, while in Panama the mortality rate of 10% is contrasted by an unusually high incidence. In all types of HPS, the viral prodrome, cardiopulmonary phase due to massive pulmonary capillary leak syndrome, and spontaneous diuresis are followed by a convalescent phase with exertional dyspnea for 3–4 weeks, but the frequency of persistent symptoms is not known. In this observational study of a convenience sample, 14 survivors of HPS caused by Choclo virus infection in Panama and 9 survivors of HPS caused by Sin Nombre virus infection in New Mexico completed a questionnaire and pulmonary function tests up to 8 years after infection. In both groups, exertional dyspnea persisted for 1–2 years after acute infection in 43% (Panama) and 77% (New Mexico) of survivors surveyed. Reduction in midexpiratory flows (FEF25–75%), increased residual volume (RV), and reduced diffusion capacity (DLCO/VA) also were common in both populations; but the severity of reduced expiratory flow did not correlate with exertional dyspnea. Symptoms referable to previous hantavirus infection had resolved within 3 years of acute infection in most but not all patients in the Panama group. Temporary exertional dyspnea and reduced expiratory flow are common in early convalescence after HPS but resolves in almost all patients.
PMCID: PMC2939327  PMID: 20524006
Hantavirus; Pneumonia; Pulmonary function test
15.  Choclo Virus Infection in the Syrian Golden Hamster 
Andes virus and Choclo virus are agents of hantavirus pulmonary syndrome. Andes virus in hamsters almost always causes a disease that is pathologically indistinguishable from fatal hantavirus pulmonary syndrome. The purpose of this study was to assess the pathogenicity of Choclo virus in hamsters. None of 18 hamsters infected with Choclo virus exhibited any symptom of disease. No evidence of inflammation or edema was found in the lungs of the 10 animals killed on days 7, 9, 11, 13, and 16 post-inoculation or in the lungs of the 8 animals killed on day 28 post-inoculation; however, hantavirus antigen was present in large numbers of endothelial cells in the microvasculature of the lungs of the animals killed on days 7, 9, 11, and 13 post-inoculation. These results suggest that infection in the microvasculature of lung tissue alone does not result in the life-threatening pulmonary edema in hamsters infected with Andes virus.
PMCID: PMC2689364  PMID: 18385367
16.  Immunization with Recombinant V10 Protects Cynomolgus Macaques from Lethal Pneumonic Plague▿  
Infection and Immunity  2008;76(12):5588-5597.
Vaccine and therapeutic strategies that prevent infections with Yersinia pestis have been sought for over a century. Immunization with live attenuated (nonpigmented) strains and immunization with subunit vaccines containing recombinant low-calcium-response V antigen (rLcrV) and recombinant F1 (rF1) antigens are considered effective in animal models. Current antiplague subunit vaccines in development for utilization in humans contain both antigens, either as equal concentrations of the two components (rF1 plus rLcrV) or as a fusion protein (rF1-rLcrV). Here, we show that immunization with either purified rLcrV (a protein at the tip of type III needles) or a variant of this protein, recombinant V10 (rV10) (lacking amino acid residues 271 to 300), alone or in combination with rF1, prevented pneumonic lesions and disease pathogenesis. In addition, passive immunization studies showed that specific antibodies of macaques immunized with rLcrV, rV10, or rF1, either alone or in combination, conferred protection against bubonic plague challenge in mice. Finally, we found that when we compared the reactivities of anti-rLcrV and anti-rV10 immune sera from cynomolgus macaques, BALB/c mice, and brown Norway rats with LcrV-derived peptides, rV10, but not rLcrV immune sera, lacked antibodies recognizing linear LcrV oligopeptides.
PMCID: PMC2583586  PMID: 18794281
17.  Rodent-associated Bartonella Febrile Illness, Southwestern United States 
Emerging Infectious Diseases  2006;12(7):1081-1086.
Patients showed seroconversion to rodent-associated Bartonella antigens, but not to Bartonella pathogenic for humans.
Serum specimens from 114 patients hospitalized with a febrile illness were tested with an indirect immunofluorescence assay (IFA) using Bartonella antigens prepared from 6 species of sigmodontine rodents and 3 known human Bartonella pathogens: B. henselae, B. quintana, and B. elizabethae. Acute- and convalescent-phase serum samples from 5 of these patients showed seroconversion with an IFA titer >512 to rodent-associated Bartonella antigens. The highest titer was against antigen derived from the white-throated woodrat (Neotoma albigula), although this rodent is not necessarily implicated as the source of infection. Three of the 5 who seroconverted showed no cross-reaction to the 3 Bartonella human pathogens. Common clinical characteristics were fever, chills, myalgias, leukopenia, thrombocytopenia, and transaminasemia. Although antibodies to Bartonella are cross-reactive, high-titer seroconversions to rodent-associated Bartonella antigens in adults with common clinical characteristics should stimulate the search for additional Bartonella human pathogens.
PMCID: PMC3291043  PMID: 16836824
Bartonella; liver function tests; hepatic injury; leukopenia; thrombocytopenia; fever; zoonosis; research

Results 1-17 (17)