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As part of the routine MICU respiratory therapy protocol, mechanical ventilation parameters are recorded every 4 hours. All patients are managed according to a mechanical ventilation protocol that incorporates the use of nonconventional modes when a lung protective strategy on conventional modes failed to provide adequate oxygenation. The following criteria were used to define the analyzed parameters: 1) mode of ventilation: the mode of ventilation that was used for the longest time for a given day; 2) PaO 2 /FiO 2 : worst daily ratios were recorded; 3) plateau pressure (Pplat): for patients on volume control ventilation the airway pressure was measured after a 5-second inspiratory hold without concomitant active inspiratory efforts, and for patients on pressure control ventilation (PCV) the highest total system pressure (PEEP + inspiratory pressure) was recorded; 4) positive end expiratory pressure (PEEP): the value corresponding to the highest PEEP for the day was recorded; 5) tidal volume (Vt): the largest daily volume was recorded. Respiratory data were captured on the first day of intubation (day 1) and then on subsequent days 3, 7, and 14 of mechanical ventilation. There were no differences in ventilator protocols or management between the two groups.

An itemized bill of individual charges for each patient was obtained from the hospital billing office and was organized by billing code into the following categories: room/board, pharmacy, supplies, laboratory, radiology, surgical (including procedures performed under general anesthesia), blood products, respiratory services, dialysis, and miscellaneous (which included some professional fees, nonsurgical procedures and phlebotomy, and diagnostics not included in the other categories, such as electroencephalograms, electrocardiograms, echocardiograms, cardiac catheterizations, and vascular studies). The values represent the hospital charges for the aforementioned services rather than the actual reimbursement, which may be subject to more variability. The single-center nature of the study removes interfacility differences in clinical and billing practices.

Continuous data that were normally distributed are presented as the mean ± SD and were analyzed by the Student's t test. The chi-square and Fisher exact tests were used to evaluate differences in proportions between patient groups. In instances where the data were not normally distributed, the groups were compared with the Wilcoxon rank-sum test. Differences were considered statistically significant if the p value was <0.05.

Fifty-one patients were identified in the acute lung injury screening database between September 2009 and March 2010. Twenty-two met criteria for ALI and did not have confirmed or suspected H1N1 infection and were thus included in the noninfluenza group (ALI/ARDS secondary to noninfluenza etiologies). Thirty-six patients in the H1N1 patient log had confirmed influenza A testing. Of those, 23 had ALI requiring mechanical ventilation (MV) during their MICU stay and were included in our analysis.

Demographics, presenting symptoms, past medical history, and acuity on admission are shown in Table 1 . Patients in the influenza group tended to be younger with a higher BMI. Patients in the influenza group presented more often with lower respiratory infection (100 vs. 73%, p = 0.135) and had increased requirement for mechanical ventilation on admission to the ICU (96 vs. 68%, p = 0.022). On the other hand, the noninfluenza group had a higher propensity to present with shock requiring vasopressors (45 vs. 22%, respectively, p = 0.07). The primary cause of ALI in the H1N1 group was pneumonia (n = 23), whereas in the noninfluenza group the etiologies were more varied, including pneumonia (n = 9), sepsis (n = 5), aspiration of gastric contents (n = 2), transfusion reaction (n = 1), and other (n = 5). Whereas seven patients (30%) in the H1N1 group were considered healthy, only one patient (5%) in the noninfluenza group had no comorbid medical conditions on admission to the ICU (Table 1) . This difference is reflected in the lower mean APACHE III score on admission to the ICU in the H1N1 group (66 ± 20 vs. 89 ± 32, p = 0.015), despite similar SOFA scores (8.3 ± 3.4 and 9.2 ± 4.1, p = 0.44).

There were no statistically significant differences between the two groups for initial laboratory data, including white blood cell count, platelets, serum creatinine, bilirubin, and creatinine kinase. The number of patients who developed acute renal failure that required dialysis throughout their ICU stay was the same (n = 8) in both groups. SOFA scores on days 1, 3, 7, and 14 of mechanical ventilation indicate that patients in the noninfluenza group had more severe organ failure during their ICU stay (p = 0.017; Table 2 ). Table 3 shows oxygenation index and mechanical ventilation related parameters on days 1, 3, 7, and 14. There was a nonsignificant trend toward worsening hypoxia in the H1N1 group, despite significantly higher PEEP and Pplat on days 1, 3, and 14. Tidal volumes were comparable throughout. Plateau pressures in the H1N1 group were high due to the relative decrease in pulmonary compliance in H1N1-related lung injury. Four patients in both groups were ventilated with airway pressure release ventilation (APRV). More patients in the influenza group required rescue therapies on day 1 of mechanical ventilation (4 vs. 0, respectively, p = 0.108); however, similar numbers of patients in both groups required rescue therapies over the duration of MV (7 and 5 patients, respectively). Rescue therapies in the H1N1 group included inhaled NO (n = 4), ECMO (n = 2), prone ventilation (n = 3), and high-frequency ventilation (n = 1), and in the noninfluenza group only inhaled NO (n = 3) and prone ventilation (n = 2).

Mechanical ventilation days were comparable between groups (22 ± 17 vs. 19 ± 15 days for groups I and II, respectively, p = 0.53) as were 28-day ventilator-free days (5 ± 7.6 and 4.6 ± 9, p = 0.88). Four patients in the H1N1 group and seven in the noninfluenza group underwent a tracheostomy procedure. Hospital and ICU LOS were comparable (median ± IQR: 16 ± 22 vs. 24.5 ± 26.5 and 12 ± 15 vs. 17 ± 25.5 days for the influenza group and II, respectively, Wilcoxon p = 0.17 and 0.45). Mortality was significantly higher for patients in the noninfluenza group (77 vs. 39%, p = 0.016). Interestingly, a Kaplan-Meier curve of ICU mortality (Figure 1) indicates that patients in the H1N1 group were more likely to be discharged alive from the ICU when the length of stay was greater than 25 days, despite a trend toward higher mortality within the first 2 weeks.

Even though all charges were higher in the noninfluenza group, only the difference in blood products utilized in the ICU was significant (4 ± 6 vs. 21 ± 25 thousands of U.S. dollars, Wilcoxon p < 0.001; Table 4 ). Differences in ICU charges in pharmacy (p = 0.23), supplies (p = 0.09), radiology (p = 0.08), and miscellaneous (p = 0.09) were large but not significant due to considerable variation. The proportion of charges in each of the major categories was similar between the groups (Figure 2 ). The average total ICU cost per patient (253 ± 193 vs. 350 ± 270 thousands of U.S. dollars, Wilcoxon p = 0.19) and the average ICU cost per patient per day (13 ± 4 vs. 15 ± 6 thousands of U.S. dollars, Wilcoxon p = 0.06) tended to be higher in the noninfluenza group.

The fall of 2009 heralded the influx of patients suffering from severe hypoxic respiratory complications secondary to the pandemic H1N1 influenza to ICUs across the country. Due to the severity of pulmonary disease that many of these patients experienced, perception among treating clinicians was that these patients would have

a All values expressed as mean ± SD. Using mixed models, the overall p value comparing the influenza and noninfluenza groups is 0.017. The trend over time was not significant (p = 0.1). worse outcomes and consume more resources, as measured by hospital charges, than patients who developed ALI from other etiologies. We demonstrated that, contrary to what was perceived, pandemic influenza A ALI/ ARDS was associated with a lower acuity and, consequently, lower hospital mortality that ALI/ARDS from other etiologies, and had a similar ICU and hospital LOS. ICU and total hospital charges reflected a trend toward higher overall charges for room and board, blood products, pharmacy, and overall charge per patient in the noninfluenza group.

In accordance with other descriptive reports of pandemic influenza [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] , patients who tested positive for H1N1 infection, tended to be young (no patients >64 years old), obese (15 had BMI >30 kg/m 2 ), and in relatively good health (30% with no comorbid medical conditions). There were no pregnant patients in either group. Compared with other studies of pandemic influenza patients who required mechanical ventilation, SOFA scores (mean 8.3) were similar, although APACHE II (25 ± 9) scores were higher [5] [6] [7] [8] 14, 16, 17, 23] . The degree of respiratory compromise in our patients was more severe than other reports judging by the higher PEEP requirements and longer duration of mechanical ventilation, which was roughly double that reported in other studies [4] [5] [6] 8, 11, 13, 14, 16, 23] . Plateau pressures in these studies were not consistently reported. However, despite significantly longer ventilation duration and prolonged ICU and hospital stays, the mortality in our cohort was not higher than that seen in other studies, which ranged from 22-41% in patients who required mechanical ventilation [4] [5] [6] 8, 11, 13, 14, 16, 23] .

Looking at the different patient characteristics between groups, it may be tempting to postulate that the higher rate of patients with pulmonary ARDS in the H1N1 group, in contrast to prevalent nonpulmonary ARDS in the noninfluenza group, would correlate with a higher PEEP response among the latter [24] . Our findings suggest the contrary. Patients in the H1N1 group had higher mean plateau pressure, likely indicative of lower compliance. The similarity of PaO 2 /FiO 2 ratios in the two groups may be a reflection of higher PEEP values used in the H1N1 group for lung recruitment, rather than being indicative of comparable degrees of lung injury. Although assessing recruitability from this retrospective analysis is difficult and may be inaccurate, the higher PEEP used and the implication of lower compliance observed are predictors of potentially recruitable lung [24] . These observations support the recent call for a reevaluation of the ALI and ARDS criteria to account for this heterogeneity in the patient population [25] .

A number of important differences between the two cohorts emerged as well. As expected, the noninfluenza group was older, had more comorbid medical conditions, and less often presented to the ICU with respiratory failure. The degree of ventilator support was significantly higher in the H1N1 group on days 1, 3, and 14, and there was a trend to more severe hypoxemia during that time as well. Nevertheless, the use of use of APRV and rescue therapies was comparable in both groups. Despite more severe respiratory compromise, H1N1 patients did not have longer time on the ventilator, longer ICU or hospital stays, or higher mortality. Although SOFA scores were similar, the noninfluenza group had significantly higher APACHE III scores, likely secondary to points assigned to comorbid medical conditions. The high acuity of illness, as well as the presence of severe comorbidities, such as solid and hematologic oncologic conditions (7 patients), chronic renal insufficiency (6 patients), and cirrhosis of the liver (4 patients), likely contributed to the poor outcomes in the noninfluenza group. Conversely, despite more severe respiratory compromise, patients in the H1N1 group were more likely to recover due to their younger age and better overall health histories.

The 77% mortality in the noninfluenza group was much higher than typically reported in clinical trials, with one notable exception [26] . However, reports from tertiary care centers involving patient cohorts with similar underlying comorbid conditions have reported equally high mortality rates [27] . Our observation brings up an interesting point, namely the difference between the reported mortality in clinical trials and the observed mortality in a similar clinical condition affecting patients that would have been excluded from such trials due to coexisting comorbidities. A Kaplan-Meier plot of ICU mortality (Figure 1) indicates that although patients in the H1N1 group were less likely to survive the first 14 days of ICU care, those that did survive past day 25 were more likely to be discharged alive from the hospital. Patients in the noninfluenza group were unlikely to survive if their ICU length of stay exceeded 3 weeks. ARDS is among the most expensive conditions encountered in the ICU [28] . In 1984, Bellamy and Oye described the charges of patients with ARDS, with the most expensive being room and board (30%), clinical laboratory (24%), pharmacy (14%), and inhalation therapy and ventilation (8%) [27] . Twenty-five years later, our study indicates that the aforementioned categories continue to represent the most expensive charges incurred by ARDS patients in the ICU.

The overall similarity of charges in room and board and respiratory therapy between the two groups is likely indicative of the comparative durations of hospitalization and mechanical ventilation. Interestingly, despite higher ventilatory requirements and more severe hypoxemia in the H1N1 group, respiratory charges were similar between the two groups, suggesting that the high cost of maintaining a patient on mechanical ventilation is independent of the degree of ventilator support necessary. Thus, respiratory charges are more likely a reflection of duration of mechanical ventilation rather than the degree of ventilator support necessary. Absolute ICU charges for room and board, blood products, pharmacy, radiology, average daily charge, and overall charge per patient were larger in the noninfluenza group. ICU charges for blood products in the noninfluenza group were greater by a factor of four, and pharmacy charges double that of the H1N1 group. This finding is likely a reflection of the higher prevalence of underlying comorbid medical conditions in the noninfluenza group, such as malignancy and cirrhosis, which require expensive medications and predispose to anemia. Moreover, the high mortality in this cohort likely precluded even higher hospital charges. Nevertheless, the H1N1 cohort amassed charges of similar magnitude to the most ill and expensive patients in the ICU, indicating the abundant health care resources consumed by severe pandemic influenza infection.

There are a number of limitations to our study. As a retrospective chart review rather than a prospective investigation, the information was culled from sources that were at times incomplete. Second, the study contained a relatively small number of patients, and measures taken to ensure internal validity of each group, such as limiting the influenza group to confirmed H1N1 infection and the noninfluenza group to the duration of the influenza season, further limited its size. Additionally, whereas our study provides descriptive information relevant to the patient population of our institution and tertiary referral centers with similar acuity, other ICUs may be exposed to a different cohort of patients. On the other hand, as a single-center study, potential differences in clinical and billing practices could be minimized. Although a comprehensive charge profile of each patient was generated, trends in the timing of charges could not be obtained. Finally, the hospital charge data were mined from an extensive database divided by charge coding, and therefore, some charges may have been mislabeled or inappropriately categorized.

Our study provides interesting observations about the clinical course, outcomes, and cost of the H1N1 influenza pandemic. Although patients with severe pulmonary complications of pandemic influenza infection have poor oxygenation and require significant ventilatory support and rescue therapies, their younger age and tendency to have fewer comorbid medical conditions contribute to their improved prognosis compared with patients with ALI from other causes. Both groups of patients consume enormous amounts of hospital resources, and physicians and policy makers must be aware of this when future pandemics arise. Identification of a Conserved B-cell Epitope on Reticuloendotheliosis Virus Envelope Protein by Screening a Phage-displayed Random Peptide Library BACKGROUND: The gp90 protein of avian reticuloendotheliosis-associated virus (REV-A) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported. METHODS AND RESULTS: This study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb) A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched (213)SVQYHPL(219) of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that (213)SVQYHPL(219) was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group. CONCLUSIONS AND SIGNIFICANCE: We identified (213)SVQYHPL(219) as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group. Reticuloendotheliosis viruses (REVs) are a group of viruses in the family Retroviridae, specifically gammaretroviruses in the same genus as mammalian C-type retroviruses [1] . The REV group includes defective REV-T [2, 3] , non-defective REV-A [4, 5] , chick syncytial virus (CSV) [6] , duck infectious anemia virus [7] , and spleen necrosis virus (SNV) [8] . Except for the defective REV-T, all isolated REV strains belong to a single serotype [5] and their genetic sequences show little variation [9] .

REV genome consists of three structural genes (gag, pol and env) flanked by long-terminal repeats (LTRs) [10] . The major mature env gene products of REVs are the surface glycoprotein (gp90) and the transmembrane protein (gp20) [11, 12] . The gp90 protein containing both continuous and discontinuous epitopes functions as the immunodominant protein [13] and is responsible for eliciting REV antibodies. Previous studies indicated that the Cterminal epitope of gp90 was exposed on the outer surface of the REV-A-infected cell [12] . However, the epitope identified in REV gp90 protein has not been finely mapped, and the core sequence of the epitope needs to be determined.

Detailed analysis of epitopes is important for the understanding of immunological events, and the development of epitopebased marker vaccines and diagnostic tools for various diseases [14, 15] . In this study, we prepared a neutralizing monoclonal antibody (mAb) against gp90 protein from the REV-A strain HLJ07I, and used it to screen a phage-displayed random 12mer peptide library for the linear B-cell epitope. This study describes the first identification of the precise location of the epitope on gp90 protein. The information provided in this study will facilitate the development of specific serological diagnosis of REV infection, and will contribute to the rational design of vaccines by further understanding of the antigenic structure of gp90.

Care of laboratory animals and animal experimentation were performed in accordance with animal ethics guidelines and approved protocols. All animal studies were approved by the Animal Ethics Committee of Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (SYXK (H) 2006-032).

REV-A Strain HLJ07I (GenBank accession No. GQ375848) was isolated from Heilongjiang Province in China in 2007. Chicken embryo fibroblasts (CEFs) were prepared as primary cultures from 10-day-old chicken embryos as previously described [16] and were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum plus antibiotics. Viruses were grown in CEFs and incubated at 37uC with 5% CO 2 for 5 days. The suspension was frozen and thawed three times to disrupt cells and release virus, and then clarified by two centrifugation steps (2000 g for 15 min, and 10,000 g for 60 min). Virus present in the upper phase was precipitated with 10% (w/v) polyethylene glycol 6000 (PEG 6000) for 4 hours at 4uC. Precipitates were collected by centrifugation at 9,000 g for 30 minutes and resuspended in TNE buffer (50 mM tris-HC1, pH 7.5; 0.1 M NaC1, 10 mM EDTA). Finally, they were centrifuged through a 30% (w/v) sucrose cushion for 90 minutes at 200,000 g and resuspended in TNE buffer. The purified virus was analyzed in SDS-PAGE.

Six-week-old female BALB/c mice were subcutaneously immunized with 100 mg of the purified recombinant gp90 protein emulsified with an equal volume of Freund's complete adjuvant (Sigma, St. Louis, MO, USA). Two boosters of the Freund's incomplete adjuvant (Sigma, St. Louis, MO, USA) emulsified antigen were given at two week interval. Two weeks after the third immunization, the mice were intraperitoneally boosted with 100 mg antigen alone. Three days later, the spleen cells from immunized mice were fused with myeloma cells SP2/0 (SP2/0-Agl4; ATCC CRL 1581) [17] , using 50% (wt/vol) polyethylene glycol and 10% dimethyl sulfoxide (DMSO) (vol/vol) (Sigma, St Louis, MO, USA). Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA). The hybridomas producing mAbs were cloned three times by limiting dilution of the cells. Antibody subtype identification was performed using SBA Clonotyping TM System/HRP Kit (Southern Biotech, Birmingham, AL, USA).

Plates were coated with 100 mL/well of purified REV gp90 antigen diluted in carbonate-bicarbonate buffer (pH 9.6) for incubation overnight at 4uC. Following 4 washes with 200 mL/ well of PBS/0.05% Tween-20, the plates were blocked with 200 mL/well of blocking buffer (PBS containing 5% skim milk) for 1 h at 37uC. The supernatant of hybridoma culture (100 mL/well) was added in duplicate and the plates were incubated for 1 h at 37uC. After washing three times with PBS, 100 mL of horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG, 1:5,000 dilution,Sigma, St Louis, MO, USA) was added to each well and incubated for 1 h at 37uC. Plates were washed three times with PBS and incubated with 100 mL/well of o-phenylenediamine dihydrochloride (OPD, Sigma, St Louis, MO, USA) containing 0.3% H 2 O 2 for 5 minutes at room temperature in the dark. The reaction was stopped with 50 mL/well of 2 M H 2 SO 4 and the absorbance measured at 492 nm.

About 70-80% confluent CEF cells in 96-well plates were infected with REV-A HLJ07I at a MOI of 0.2. At 5 days postinfection, the infected cells were fixed with icy cold ethanol absolute for 15 min at 4uC, and air dried. The fixed cells were incubated with mAb A9E8, REV-A-positive chicken serum, antiporcine IFN-c mAb (Sigma, St Louis, MO, USA), or REV-Anegative chicken serum for 1 h at 37uC. After washing three times with PBS, 50 mL/well of FITC-conjugated goat anti-mouse IgG or FITC-conjugated rabbit anti-chicken IgG (Sigma, St Louis, MO, USA) at 1:100 dilutions were added and incubated for 1 h at 37uC. The cells were rinsed three times with PBS and once with deionized water, and mounted in 50 mL of 90% glycerol in PBS, and then observed under the Nikon Eclipse Ti-E microscope equipped with NIS-Elements AR software.

The micro-neutralization assay was modified from a previously described procedure [18] . The ascitic fluid was heat inactivated for 30 min at 56uC, and two fold serial dilutions were incubated with 2610 3 tissue culture infective doses 50% (TCID 50 /mL ) of REV-A in a 96-well micro-plate. Four uninfected control wells were included on each plate as control wells. After 2 h incubation at 4uC, 100 mL of CEF cells at 1.5610 5 cells/mL was added to each well. The plates were incubated for 5 days at 37uC and 5% CO 2 . The monolayers were washed with PBS and fixed in icy cold ethanol for 15 minutes. The presence of viral gp90 protein was detected by ELISA with the mAb A9E8. The absorbance was measured at 492 nm with an ELISA microplate reader. The average A492 was determined for quadruplicate wells of virusinfected and uninfected control wells, and a neutralizing endpoint was determined by using a 50% specific signal calculation. The endpoint titer was expressed as the reciprocal of the highest dilution of ascitic fluid with A492 value less than X, where 6= [(average A492 of infected wells) 2 (average A492 of control wells)]/2+ (average A492 of control wells).

The Ph.D.-12 TM Phage Display Peptide Library Kit was purchased from New England BioLabs Inc. The dodecapeptide library consisted of 2.7610 9 electroporated sequences (1.5610 13 pfu/mL). The mAb was purified from the ascites uid of mice inoculated with the hybridma cells secreting A9E8 by affinity chromatography using rProtein G Agorose (Invitrogen, Carlsbad, CA,USA) according to the manufacturer's instructions. The concentration of the purified protein was determined using the Bradford Protein Assay Kit (Beyotime, Shanghai, China). Three successive rounds of biopanning were carried out according to the manufacturer's instruction manual. Briey, one well of a 96well microtiter plate was coated with 10 mg/mL of mAb A9E8 in coating buffer (0.1 M NaHCO 3 , pH 8.6) overnight at 4uC, followed by blocking with blocking buffer (0.1 M NaHCO 3 , pH 8.6, 0.02% NaN3, and 5 mg/ml BSA) for 2 h at 4uC. The phage library (1.5610 11 phages/100 mL) was added to the blocked wells and the plate incubated for 1 h at room temperature. The unbound phages were removed by successive washings with TBS buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing gradually increased concentrations (0.1%, 0.3%, and 0.5%) of Tween-20, and the bound phages were eluted by 0.2 M glycine-HCl containing 1 mg/mL BSA (pH 2.2) and immediately neutralized with 1 M Tris-HCl (pH 9.1). The eluted phages were amplified by infecting E. coli (ER2738), and were titered on LB/IPTG/Xgal plates for the subsequent rounds of selection. The output to input ratio was calculated as follows: (titer of the amplified eluent phages/titer of the input phages (1.5610 11 ))6100%.

After three rounds of biopanning, eight individual phage clones were selected for target binding in ELISA as described in the manufacturer's instructions. Briey, 96-well plates were coated with 100 ng of purified mAb A9E8, or anti-porcine IFN-c mAb (Sigma, St Louis, MO, USA) as negative controls overnight at 4uC. The coated wells were blocked for 2 h at room temperature and then the phages (10 10 pfu/100 mL/well) diluted in blocking solution were added. The plates were incubated for 1 h at room temperature followed by washing ten times with TBST. Bound phages were subjected to reaction with horseradish peroxidase (HRP)-conjugated sheep anti-M13 antibody (Pharmacia, Piscataway, NY, USA), followed by color development with substrate solution containing o-phenylenediamine (OPD).

The positive phage clones identified by phage ELISA were sequenced with the 296 gIII sequencing primer 59-TGA GCG GAT AAC AAT TTC AC-39 as described in the manufacturer's instructions.

A series of complementary oligonucleotides (Table 1) coding for wild-type and truncated motif SVQYHPL were synthesized, annealed, and cloned into the BamHI/XhoI sites of the prokaryotic expression vector pGEX-6p-1 (Pharmacia, Piscataway, NY, USA), producing a group of recombinant plasmids. All the resulting recombinant plasmids were validated by restriction analysis and DNA sequencing. Expression plasmids were transformed into BL21 (DE3) competent cells, followed by the addition of 1 mM isopropyl-D-thioga-lactopyranoside (IPTG; GE Healthcare, USA) for induction.

Approximately equivalent amount of each GST fusion protein was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% SDS-PAGE). The gel was either stained with commassie blue staining solution or electrophoretically transferred to nitrocellulose membrane. After being blocked with 5% skim milk in PBS overnight at 4uC, the membrane was incubated with mAb A9E8 (diluted 1:2,000 in PBS) or REV-A-positive chicken serum (diluted 1:100 in PBS) at 37uC for 1 h. After being washed three times with PBST, the membrane was probed with a 1:5,000 dilution of HRP-conjugated goat anti-mouse IgG

To investigate the conservation of the epitope among REV viruses, sequence alignment of the epitope and the corresponding regions on gp90 proteins of 32 REV-A strains, one REV-T strain, four SNV strains and one CSV strain was performed using the DNASTAR Lasergene program (Windows version; DNASTAR Inc., Madison, WI, USA).

Purified gp90 protein was used to immunize BALB/c mice. After cell fusion and screening, several hybridoma cell lines were generated, which produced gp90-reactive mAbs. One monoclonal antibody produced by the line designated as A9E8 was selected for strong reactivity with recombinant gp90 protein in Western blot ( Figure 1A ) and in an indirect ELISA (data not shown). It also showed strong reactivity with purified whole virus in Western blot ( Figure 1B) and could be used to detect REV-A antigen by an indirect immunofluorescence assay (IFA; Figure 1C ). The mAb A9E8 was compose of an IgG2b heavy chain paired with a k-type light chain, as determined using the SBA Clonotyping TM System/ HRP Kit. The titers of antibody in hybridoma cell culture supernatants and in ascites were measured by indirect ELISA and determined to be 1:3,200 and 1:128,000, respectively.

The neutralizing activities of the mAb A9E8 were then determined by a micro-neutralization assay on CEF cells using REV-A HLJ07I. The mAb A9E8 neutralized the virus with a neutralization titer (NT 50 ) of 100.

To determine the epitope recognized by mAb A9E8, biopanning of a phage displayed 12-mer random peptide library was performed using the affinity purified mAb A9E8. After three rounds of biopanning, an enrichment of phages bound to the mAb A9E8 was obtained. The output to input ratios of the three rounds of biopanning were 0.00008%,0.038% and 0.79%.

Eight phage clones were selected for reactivity with the mAb A9E8 after three rounds of biopanning and enrichment of the phages binding to the mAb A9E8. These selected clones were further evaluated by Phage ELISA for reactivity with the mAb A9E8 and a negative control mAb (anti-porcine IFN-c). As shown in Figure 2 , all the selected eight phage clones (A1-A8) showed specific reactivity with A9E8 (OD492 nm .1.10), but not with anti-porcine IFN-c mAb (OD492 nm ,0.15). The eight phage clones were sequenced, and were shown to display a consensus sequence SVQYHPL, which was identical to the motif 213 SVQYHPL 219 at the C-terminus of the gp90 protein of REV-A strain HLJ07I (Table 2) .

To verify whether the identified motif represented an epitope recognized by the mAb A9E8, a DNA fragment coding for the motif SVQYHPL was expressed as a GST fusion protein (GST-H7wt) in E. coli. Western blot analysis showed that the fusion protein was recognized by the mAb A9E8 ( Figure 3A ) and REV-A infected chicken antiserum ( Figure 3B ), indicating that the motif represented a linear B-cell epitope.

To define the epitope precisely, four mutants with deletions at C-and N-termini of the motif SVQYHPL (Table 1) were constructed to express the GST fusions GST-H7DS, GST-H7DL, GST-H7DSV, and GST-H7DPL representing -VQYHPL, SVQYHP-, -QYHPL and SVQYH-(deletions were shown as dashes) in E. coli, respectively. We found that only the full-length SVQYHPL polypeptide (GST-H7wt) was recognized by the mAb A9E8 ( Figure 3A ). Removal of one or more amino acids at either the amino or carboxyl terminus of the peptide abolished antibody binding, indicating that the peptide SVQYHPL represented the minimal requirement for the reactivity of the epitope with A9E8. Minimal Unit of the Epitope with the Maximal Binding Activity to mAb A9E8

To investigate minimal unit of the epitope with the maximal binding activity to mAb A9E8, a series of GST-fusion proteins were expressed with extended amino acid residues at both N and C termini of the motif SVQYHPL (Table 1) . These GST-fusion proteins were subjected to SDS-PAGE and testing for reactivity with mAb A9E8 in Western blot. Fusion proteins GST-R1 (SVQYHPLA), GST-R2 (SVQYHPLAL) and GST-R3 (SVQYH-PLALP) reacted strongly with mAb A9E8 in Western blot ( Figure 4) . The GST-R2 and GST-R3 showed similar binding activity to the GST-R1, indicating that alanine alone significantly increased binding activity of the core epitope to mAb A9E8. In contrast, GST-fusion proteins with extended amino acid residues at the N terminus of the motif SVQYHPL showed no increased binding activity compared with GST-H7wt in Western blot (data not shown). Taken together, these results showed that SVQYH-PLA was the minimal unit of the epitope with the maximal binding activity to mAb A9E8.

To investigate the conservation of the SVQYHPL epitope, we aligned the epitope identified in this study with REVs gp90 coding regions available in GenBank. The alignment results showed that all amino acids in the motif were identical among all REV strains ( Figure 5 ), indicating that the motif represented a conserved epitope on the gp90 protein of REVs. Figure 2 . Detection of the selected phages for antibody binding by Phage ELISA. Eight phage clones selected after three rounds of biopanning were added to the microplate wells (10 10 pfu/100 mL/well) coated with the mAb A9E8 or anti-porcine IFN-c mAb (negative control) (100 ng/well ), and incubated for 1 h at room temperature. Bound phages were subjected to reaction with horseradish peroxidase (HRP)-conjugated anti-M13 antibody, followed by color development with substrate solution containing o-phenylenediamine (OPD). Three independent assays were performed for each selected phage. doi:10.1371/journal.pone.0049842.g002 Table 2 . Sequence comparison of random peptide inserts displayed on the positive phages.

Amino acid sequence of the insert a

Conservative amino acid motifs are bold and underlined. doi:10.1371/journal.pone.0049842.t002

The gp90 protein of REV is an important antigenic protein and is associated with virus neutralization, which is the major candidate antigen for vaccine development and disease serological diagnosis [12, 13] . Studies showed that recombinant gp90 protein expressed in Pichia pastoris induced a protective immune response against REV in chickens [19] . Precise mapping of epitopes in gp90 is important for understanding antibody-mediated protection and developing epitope-based marker vaccines and diagnostic tools. Cui et al. [20] reported the generation and partial characterization of a panel of 11 mAbs against the nondefective REV Strain T, and showed that the epitope was on the viral envelope glycoprotein. However, they only identified the relative regions in REV envelope glycoprotein recognized by the mAbs, and did not map the fine locations of the epitopes. To our knowledge, there has been no report on linear epitope mapping of the gp90 of REV.

Mapping epitopes using monoclonal antibodies has become a powerful tool to study protein structure and has been used to diagnose diseases and design marker vaccines [21, 22, 23] . In this study, we described the generation and epitope mapping of a gp90 protein specific mAb, and demonstrated that the epitope was conserved among the REV group. Precise analysis of REV-A gp90 protein epitope will provide the fundamental information for development of epitope-based vaccines and diagnostic tools for REV-A and/or other REV group infection.

Phage display is an in vitro selection technique in which a peptide or protein is genetically fused to a coat protein of bacteriophage and the fused peptide or protein is displayed on the exterior surface of the phage virion. The phage displayed random peptide library is a powerful and high throughput tool for rapid mapping of epitopes [24] .

In this study, we generated a gp90-specific mAb A9E8 using recombinant gp90 protein expressed in E. coli. The mAb A9E8 showed strong reactivity against purified whole virus in Western blot and could be used to detect REV-A antigen by an indirect immunofluorescence assay. The linear epitope recognized by the mAb A9E8 was defined as SVQYHPL by screening a random phage display peptide library. This peptide sequence was identical to 213 SVQYHPL 219 of the gp90 protein of REV-A. N-or Cterminal deletions of amino acids of this epitope demonstrated that 213 SVQYHPL 219 is the minimal requirement for recognition by A9E8. Fusion proteins GST-R1 with extended amino acid residues at the C terminus of the motif SVQYHPL showed increased binding activity compared with that of GST-H7wt in Western blot, indicating that alanine alone significantly increased binding activity of the core epitope to mAb A9E8. Thus, the peptide SVQYHPLA was determined to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8.

The peptide was also recognized by REV-A-positive chicken serum, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding reactivity. Sequence alignments of REV-A strains, REV-T strain and five other REV strains demonstrated that the motif was highly conserved among REV viruses, indicating that it is a broad group-specific epitope. Since A9E8 was identified as a neutralizing mAb, the epitope identified with A9E8 in this study was a neutralizing epitope.

Many neutralizing epitopes have been mapped in the variable regions of the proteins of viruses, including infectious bursal disease virus [25] , infectious bronchitis virus [26] , hepatitis C virus [27] , and HIV [28] . Some neutralizing epitopes, however, are highly conserved across most of the viruses in the same group [29, 30] . A novel epitope was mapped within the highly conserved flavivirus fusion loop peptide 98 DRXW 101 by phage-display biopanning and structure modeling using mAb 2A10G6 that had broad cross-reactivity with dengue virus (DENV) 1-4, yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) viruses. This mAb potently neutralized DENV 1-4, YFV, and WNV and conferred protection against lethal challenge with DENV 1-4 and WNV in murine model. Further functional studies revealed that 2A10G6 blocked infection at a step after viral attachment. These results show that the broad cross-reactivity epitope recognized by neutralizing mAb 2A10G6 is highly conserved among DENV 1-4, YFV and WNV [29] . An epitope recognized by mAb 51 belonging to isotype IgM was mapped to 215 KQEKD 219 of the VP1 capsid protein of Enterovirus 71 (EV71), which possessed neutralizing activity in vitro and provided 100% in vivo passive protection against lethal challenge with EV71 strain HFM 41. BLAST analyses of the neutralizing epitope revealed that it was highly conserved among all EV71 strains, but not coxsachievirus 16 [30] . In this study, the epitope recognized by neutralizing mAb A9E8 was mapped to a highly conserved region of the gp90 protein among REVs, which would be useful for development of REV marker vaccines and diagnostic techniques.

In summary, a highly conserved neutralizing linear B-cell epitope on the gp90 protein of REV-A was identified in this study. The identified conserved epitope may have potential for development of REV specific diagnostic assays and epitope-based marker vaccines. Identification of an Epitope on REV gp90 Protein PLOS ONE | www.plosone.org Lower Respiratory Tract Infection in a Renal Transplant Recipient: Do not Forget Metapneumovirus Human metapneumovirus (hMPV) is emerging as a cause of a severe respiratory tract infection in immunocompromised patients. hMPV pneumonia has only been seldom reported in nonpulmonary solid organ transplanted patients, such as renal transplant recipients. We report here a case of a 39-year-old patient presenting with fever, cough, and interstitial opacities on CT scan diagnosed as a nonsevere hMPV pneumonia 11 years after a renal transplantation. Infection resolved spontaneously. Differential diagnosis with Pneumocystis pneumonia was discussed. We review the medical literature and discuss clinical presentation and detection methods that can be proposed in solid organ transplant recipients. Respiratory viruses such as respiratory syncytial virus (RSV), influenza, parainfluenza viruses (PIV), and adenovirus are commonly associated with mild to severe symptoms, depending on the immune status. Human Metapneumovirus (hMPV) was the sixth most frequent viral infection in patients hospitalized for respiratory illness [1] . hMPV is a nonsegmented, enveloped, negative single-stranded RNA virus [2] responsible for lower respiratory tract infections (LRI), especially in extreme ages [3, 4] . It has a seasonal distribution and occurs mainly in winter and spring, with an incubation period usually between 4 and 6 days [5] . hMPV is now widely recognized as an opportunistic infection in immunocompromised hosts such as hematopoietic stem cell transplant (HSCT) and pulmonary transplant recipients, leading to a significant respiratory morbidity [6] . Although its detection is not yet routinely performed, hMPV appears to account for 9% of acute pneumonia in patients with haematologic malignancies (including HSCT), in a similar proportion to RSV [7] . This rate is close to that reported in lung transplant recipients, ranging from 6% to 12% of LRI [6, 8] .

In contrast, it has been seldom reported in other SOT settings such as renal transplantation [9] .

A 39-year-old patient with an 11-year history of kidney transplantation for severe amyloidosis was referred to the Centre d'Infectiologie Necker Pasteur for acute fever for 2 days. After 8 years of transplantation, he was treated for graft rejection by corticosteroids. Clinical course was uneventful, except for recurrent prostatitis. His current immunosuppressive regimen consisted of mycophenolate mofetil except for sore throat and rhinorrhea. Biological analyses showed an elevated C-reactive protein blood level (157 mg/L, normal <5 mg/L) but normal blood leukocytes and neutrophil counts. Blood lymphocyte count was low (0.94 G/L), with CD4+ T cells accounting for 39.5% of total lymphocytes (0.372 G/L). HIV serology was negative. Blood and urine cultures were sterile and initial chest radiograph was normal. Nonproductive cough without dyspnea or chest pain appeared on day 3 of hospitalization. Oxygen saturation in ambient air was 92%. Chest auscultation was normal. As the cough increased, a thoracic computed tomography (CT) scan was performed on day 6 and revealed bilateral ground glass infiltrates mainly located in subpleural and peripheral areas, associated with bilateral pleural effusion ( Figure 1 ). No mediastinal adenopathy was seen. Because of clinical and radiological presentation suggesting Pneumocystis jirovecii pneumonia, trimethoprim-sulfamethoxazole was initiated the same day. Nasopharyngeal aspirates were screened by direct immunofluorescence with specific monoclonal antibodies to RSV, influenza virus A and B, PIV, adenovirus, and hMPV (Argène, Verniolle, France) on day 7. Immunofluorescence was strongly positive for hMPV and negative for other viruses. Blood cultures and S. pneumoniae and L. pneumophila urinary antigen detections were negative. As Pneumocystis pneumonia was initially suspected, bronchoalveolar lavage (BAL) fluid analysis performed on day 11 demonstrated 450.10 3 cells/mL (macrophages 72%, neutrophils 17%, and lymphocytes 11%). Microbiological studies did not reveal any bacterial or fungal microorganisms. Gomori-Grocott staining for Pneumocystis jiroveci detection, indirect immunofluorescence, and polymerase chain reaction (PCR) for P. jiroveci were negative. hMPV was also detected in BAL fluid by direct immunofluorescence. As all microbiological investigations were negative except for hMPV, antibiotics were discontinued; respiratory symptoms spontaneously improved within 6 days. Thus, decreased immunosuppression or other medications such as ribavirin or intravenous immunoglobulin were not considered. The patient was discharged on day 14.

This is the second hMPV pneumonia in a kidneytransplanted recipient described in the literature. The first reported case was a severe LRI requiring transient intensive care unit stay [9] . It occurred three years after kidney transplantation, while receiving immunosuppressive regimen consisting of ciclosporine (125 mg b.i.d), azathioprine (75 mg/d), and prednisone (10 mg/d). Compared to this case, our patient had mild symptoms, mainly cough and upper respiratory symptoms. He was also less immunosuppressed without corticosteroids regimen.

In solid organ transplanted patients, hMPV is responsible for LRI and may lead to hospitalization and significant respiratory illness in up to 63% of cases [6, 8] . As initial clinical symptoms are nonspecific, thoracic CT scan can be more helpful than chest X-ray, which is less sensitive. Consolidation, nodular infiltrates, and pleural effusions may be seen. Subpleural and basal areas are usually observed, and bilateral locations are seen in 50% of cases, as in our case [10] . Whereas crazy paving, network of a smooth linear pattern superimposed on an area of ground-glass opacity, is unusual, bronchiectasis is common, up to 68% in the series by Wong et al. [10] .

Of note, lymphopenia, as noticed in our patient, is the most common feature reported in HSCT patients with hMPV, accounting for 73% of patients in one series [7] . This illustrates that although innate immune responses are stimulated upon hMPV exposure, adaptive immunity also appears important to control hMPV. As for other paramyxoviruses, the matrix proteins are involved in the induction of proinflammatory and Th1 responses by dendritic cells and macrophages (i.e., production of interleukin-2 and interferon-γ) [11] . Inflammation may cause diffuse alveolar damage and hyaline membrane formation as shown by histopathology investigations [12] .

Apart from other respiratory viral infections occurring in SOT recipients, differential diagnoses of hMPV-associated LRI include severe bacterial and fungal pneumonitis, particularly Pneumocystis pneumonia. Ribavirin, previously shown active in a mouse model of infection [13] , has been suggested as a potential antiviral therapy in HSCT and lung transplant recipients with hMPV-associated LRI [14, 15] . In our case and in the other case of the literature [9] , ribavirin was not used because the diagnosis was made retrospectively after the patient's spontaneous clinical improvement.

In conclusion, hMPV has to be considered as a potential cause of LRI in kidney transplant recipients and may mimic Pneumocystis pneumonia. A prompt recognition would have avoided antibiotic use and further diagnostic studies such as bronchoscopy. Its early detection using immunofluorescence and/or RT-PCR must be proposed routinely in transplantation settings. In addition, early recognition could improve the implementation of appropriate infection control practices to prevent viral spread of this potential lifethreatening infection in immunocompromised patients. What was the primary mode of smallpox transmission? Implications for biodefense The mode of infection transmission has profound implications for effective containment by public health interventions. The mode of smallpox transmission was never conclusively established. Although, “respiratory droplet” transmission was generally regarded as the primary mode of transmission, the relative importance of large ballistic droplets and fine particle aerosols that remain suspended in air for more than a few seconds was never resolved. This review examines evidence from the history of variolation, data on mucosal infection collected in the last decades of smallpox transmission, aerosol measurements, animal models, reports of smallpox lung among healthcare workers, and the epidemiology of smallpox regarding the potential importance of fine particle aerosol mediated transmission. I introduce briefly the term anisotropic infection to describe the behavior of Variola major in which route of infection appears to have altered the severity of disease. Controversy exists regarding the best method of protecting the public against the potential release of smallpox as a biological weapon (Bicknell, 2002; Fauci, 2002; Halloran et al., 2002; Kaplan et al., 2002; Mack, 2003) . Infectious disease modeling plays an important role in this dialog, and the biology of the transmission pathway, the focus of this review, is critical to producing appropriate predictive models and understanding which controls will work best under varying conditions (Ferguson et al., 2003) .

The rapidity with which smallpox would spread in a developed nation is not known and is a major source of uncertainty in models used for public health planning (Ferguson et al., 2003) . The basic reproductive number (R 0 ), which describes the tendency of a disease to spread, has been estimated for smallpox from historical data and outbreaks in developing countries (Gani and Leach, 2001; Eichner and Dietz, 2003) . Because R 0 is a function of the contact rate between individuals, it can be affected by changes in the environment (Anderson and May, 1991) . A potentially important difference between contemporary environments and those used to estimate R 0 is that today many buildings, including hospitals, mechanically recirculate air. If smallpox was almost entirely transmitted by mucosal contact with large droplets (aerodynamic diameters >10 µm), which can only occur following "face-toface" exposure over distances of a few feet, then change in the built environment would not change the contact rate between individuals. If, however, smallpox was frequently transmitted from person-to-person by airborne droplet nuclei [fine particles with aerodynamic diameters of ≤2.5 µm capable of remaining suspended in air for hours and of depositing in the lower lung (Hinds, 1999) ] then mechanically recirculated air systems would increase the contact rate, R 0 , the risk of epidemic spread, and the difficulty of hospital infection control. Unfortunately, leading authorities disagree regarding the relative importance of fine and large particle routes of transmission; some state that smallpox was transmitted primarily via airborne droplet nuclei, (Henderson et al., 1999) while others emphasize "face-to-face" contact and state that, airborne transmission was rare (Centers for Disease Control, 2002; Mack, 2003) . This paper reviews the evidence for each of these modes of transmission.

Prior to Jenner, variolation, (Fenner et al., 1988) inoculation of variola into the skin or nasal mucosa, was used to reduce the risk of smallpox. Jenner himself was variolated as a child. Skin inoculation with a small amount of fresh pustule fluid, likely to have contained large numbers of infectious virions, produced a local lesion with satellite pustules, but generalized rash was reported to be less severe and mortality rates were usually 10-fold lower than with naturally acquired disease (Fenner et al., 1988) . In China, variolation was frequently performed by inoculation of the nasal mucosa. Some accounts describe blowing carefully aged scabs compounded with plant material into the nose (MacGowan, 1884). Other reports suggests that nasal insufflation was considered relatively ineffective and that nasal insertion of cotton pledgets impregnated with powdered scabs or smeared with vesicle contents was preferred (Wong and WU, 1936; Miller, 1957) . Descriptions of the latter method do not include ageing infectious material before use.

Because natural infection was thought to occur via large droplets deposited on the upper respiratory mucosal, the success of nasal inoculation in producing low mortality rates has been hard to understand. A theory suggested by Henderson to the author of a smallpox history, (Hopkins, 1983, p. 114 ) "is that virus inhaled naturally was in sufficiently small particles to be deposited deep within the lung, whereas particles inoculated by nasal insufflation may have been much larger and were likely to implant in the nose or throat where [only] a local lesion might be produced." The relative importance of age and health of inoculated subjects, infectious dose, and route of exposure are not known.

However, it appears that inoculation via the skin or nasal mucosa tended to produce modified disease. If true, this would indicate that natural transmission did not occur via direct skin or mucosal contact. Figure 1 shows graphically a how these different routes of exposure may have produced altered patterns of viral replication within the host and resulted in different risks of extensive viremia and severe disease.

If natural smallpox was initiated through the upper respiratory mucosa, then an early asymptomatic mucosal infection would be expected. To investigate this, Sarkar and colleagues performed pharyngeal swab surveys of household contacts (Sarkar et al., 1973a 4-8 days following onset of rash in the index cases. They found that contacts with positive throat cultures often did not develop smallpox. In one survey, (Sarkar et al., 1973a) 10% (Westwood et al., 1966) of 328 contacts had positive swabs, but only 12% (Kaplan et al., 2002) of those with positive swabs developed smallpox. Among 59 unvaccinated contacts 27% (Miller, 1957) were culture positive, but only one developed smallpox. All subjects were vaccinated at the time of examination. However, vaccination four or more days after exposure is usually considered to be too late to prevent disease. The observation that disease did not develop in 94% of persons with mucosal infection suggests that, even in unvaccinated contacts, mucosal infection may not have been sufficient to initiate disease.

Sarkar and colleagues also showed that the oropharyngeal excretion of virus was greatest during the first days after the rash erupted and generally resolved at most 2 weeks following onset of rash (Sarkar et al., 1973b) . Rao et al. found that oropharyngeal excretion was greatest in the most severe, hemorrhagic cases and corresponded with the period of infectiousness (Rao et al., 1968) . In contrast to oropharyngeal excretion, scabs contained large quantities of virus regardless of disease severity and were shed for another week or more after throat cultures were negative. Scabs alone, however, were not associated with further cases (Rao et al., 1968; Mitra et al., 1974) .

The apparent lack of infectiousness of scab associated virus has been attributed to encapsulation with inspissated pus (Fenner et al., 1988 ). Henderson's theory about the importance of small particles may provide a straightforward mechanism for why encapsulated virus, simply by entrapment in large particles, had low infectious potential. Sarkar et al. (1973a) were concerned that asymptomatic contacts could have been infectious because their throat swab viral titers were similar to those of milder smallpox cases. A paradox arose from these data because there was never evidence of infection arising from asymptomatic household contacts. Yet, oropharyngeal secretions were thought to be the primary source of infectious virus particles. An explanation may be that oropharyngeal excretion of virus was merely temporally correlated with excretion of virus from elsewhere in the respiratory tract and not the actual source of fine particles virus aerosols.

The large spray of particles from sneezing visualized by high speed photography consists of particles down to about 10 µm in diameter (Papineni and Rosenthal, 1997) . Smaller particles may also be dislodged from the upper airways by the turbulence of sneezing, coughing, and talking, but will mostly be larger than 2.5 µm in diameter. Recent studies, however, show that the healthy lung generates abundant fine particles (100-1000/l with size <0.3 µm diameter) during normal breathing (Fairchild and Stampfer, 1987 ) that do not arise from the oropharynx; condensates of these particles are the subject of recent reviews (Mutlu et al., 2001; Hunt, 2002) . Such particles could carry variola virus (0.2-0.3 µm diameter), would remain airborne in indoor air for many hours, and would be deposited primarily in the lower airways after inhalation.

There is some evidence that variola was present in the lung and potentially available for aerosolization. Animals infected by inhalation produced high concentrations of variola in the lung (Hahon and Wilson, 1960) . Fenner et al. (1988) regarded bronchitis and pneumonitis as a part of the normal smallpox syndrome, especially in the more severe cases which were also the most infectious, (Rao et al., 1968) although specific lesions were less frequent in the lower trachea and bronchi. Systematic evaluations of viral excretion in the lower respiratory tract of non-fatal cases were not reported. Thus, if some degree of pneumonitis with pulmonary excretion of virus and exhalation of fine particle variola aerosols was a feature of clinical smallpox but was not a feature asymptomatic household contact with positive throat cultures, then the paradox would be resolved.

Air sampling for viruses is a difficult undertaking and the literature on the subject remains sparse in comparison with that for bacteria and fungi (Sattar and Ijaz, 2002) . Only three attempts to detect airborne variola were published. The earliest attempt used highly inefficient methods and was negative (Meiklejohn et al., 1961) . In a subsequent study, Downie and colleagues used short duration, low volume air sampling with liquid impingers and obtained 5 positive samples out of 47 attempts to sample exhaled breath of patients (Downie et al., 1965) . Assuming that each positive sample represented a single infectious particle, the concentration of airborne infectious particles was 0.85/m 3 ; higher concentrations were observed close to shaken bed sheets. Concentrations were likely to have been underestimated because of several frequently encountered problems with air sampling for viruses including failure of impingers to retain particles less than 1 µm in diameter that represent the majority of particles in exhaled breath, culture of only a portion of the impinger fluid, uncertain suitability of sampling fluid for virus survival, and loss of infectivity due to sampling trauma (Spendlove and Fannin, 1982) .

In the 1970s, Thomas Figure 3 .1] appears to have frequently been less extensive after dermal inoculation and nasal insufflation compared with naturally acquired infection. This may have been due to less extensive lymphatic replication of virus and limited viremia by dermal and nasal routes as compared with infection via lower respiratory tract deposition. The size of the arrows represents the historically reported proportions of cases following each pathway. The size of the X on each image represents the reported mortality rate from each pathway. For natural infection, the ordinary-type rash and flat and hemorrhagic rashes are shown.

efficiency for submicrometer particles) for long duration large air volume viral sampling (Thomas, 1970a) . He showed that 23% of naturally airborne rabbit pox particles were ≤2.5 µm and 71% were between 2.5 and 10 µm (Thomas, 1970b) . Both Thomas and Westwood et al. (1966) in a room supplied with 10 ACH containing 7-9 infected rabbits. Westwood et al. probably obtained higher concentrations because they used an electrostatic precipitator allowing higher efficiency collection of submicrometer particles compared with Thomas's slit sampler. Thomas also studied convalescent cases of variola minor (Thomas, 1974) . One patient with relatively active lesions produced an average concentration of approximately 1 PFU/m 3 . Unfortunately the samples were collected late in the disease when the patient was probably minimally infectious, based on comparison with epidemiological data (Rao et al., 1968; Eichner and Dietz, 2003) . The airborne virus observed appears to have been due to resuspension and is unlikely to be representative of the airborne concentration of respirable variola earlier in the course of the infection. The method used would also not have been able to collect submicrometer viral aerosol particles.

Overall, the air sampling studies suggest that animals and people infected with poxviruses generated respirable aerosols, but that air concentrations may have been low, or airborne virus was present in submicrometer particles that could not be collected the instruments available. Because detection of virus aerosols is subject to potentially large losses in sampling equipment, especially when sampling dilute natural aerosols over extended periods, and because plaque assays may not accurately represent the infectivity of virus deposited in human airways at 100% relative humidity, (Spendlove and Fannin, 1982; Ijaz, 1987, 2002) the available data can be considered a lower limit on concentration of infectious natural poxvirus aerosols.

Experimental aerosol data suggested that poxvirus, which survived the trauma of artificial aerosolization, remained infectious for significant periods of time. Aerosols of vaccinia demonstrated a half-life of about 6 h at 22 • C and relative humidity ≤50% with reduced stability at higher relative humidity and temperature (Harper, 1961) . Variola appeared to have a similar half-life and not to be affected by relative humidity at 26.67 • C (Mayhew and Hahon, 1970) . Other experiments demonstrated that airborne vaccinia is highly sensitive to inactivation by germicidal ultraviolet light (Edward et al., 1943; Jensen, 1964) . Westwood et al. (1966) demonstrated that inhalation of a single PFU of a submicrometer vaccinia aerosol was sufficient to infect rabbits. Airborne rabbit pox was similarly infectious. They demonstrated rabbit-to-rabbit airborne transmission of rabbit pox in each of seven trials by placing uninfected rabbits in separate cages in the same room with infected animals. They also infected rhesus monkeys using submicrometer aerosols of variola.

In one of the earliest extensive animal models of smallpox, Brinckerhoff and Tyzzer (1906) reported the effect of inoculating cynomologus monkeys with variola at different sites. Inoculation of mucus membranes of the lip, palate, and nose produced local lesions, but generalized rash occurred in only 10% of animals. Inoculation through the skin produced a local lesion and a generalized eruption in 70-80% of animals. Animals inoculated by scratching the tracheal mucosa through a rigid bronchoscope all developed a generalized rash, and one developed a variolous bronchitis and pneumonia. Laryngeal instillation of dry pustule contents produced infections while instillation of powdered crusts did not. Inhalation exposures to an atomizer spray of vesicle contents infected only one of five monkeys; however, the particle size distribution and type of atomizer were not reported.

Hahon and Wilson demonstrated that infection of Macaca irus with high dose [5 × 10 5 PFU] fine particle (<5 µm) variola aerosols produced a disease that simulated human smallpox (Hahon and Wilson, 1960; Hahon, 1961) . The initial site of virus replication was the lung, with subsequent appearance of virus in the nasopharynx and nares. Peak concentrations of virus per gram of tissue were higher in the lung than in the upper respiratory tract; the peak in lung tissue occurred during the incubation period and lung levels declined during the secondary viremia and exanthem. Whether the time course and viral concentrations in lung in this animal model produced by inhalation of high dose aerosols mimicked that in humans with natural infection is doubtful. However, it may be relevant to the first generation of cases exposed to concentrated aerosols in a biological attack. In a relatively recent experiment, (Kalter et al., 1979) a female chimpanzee became infected with variola while housed in the same room, but without direct contact, with two infected chimpanzees. She developed a generalized rash and was reported to have had more severe constitutional symptoms than the other chimpanzees infected by dermal inoculation or direct contact. The authors concluded that she was infected via aerosol.

The animal data show that artificial respirable aerosols were effective means of producing poxvirus infections, that the infectious dose by the airborne route could be very low, and that animal-to-animal airborne transmission of rabbitpox and variola was observed. They also suggest that inoculation of mucus membranes was less effective at producing a generalized rash than was exposure of the lower respiratory tract.

Two reports, one from the 1940s and one from the 1960s showed that, during epidemics, staff in smallpox hospitals who had been repeatedly vaccinated sometimes developed malaise, fever, and pneumonitis without evidence of infection with smallpox or other viruses, and without evidence of allergic reaction to other agents (Howat and Arnott, 1944; Morris Evans and Foreman, 1963) . In one outbreak, after investigation of other possible causes, the authors attributed the phenomena to an allergic reaction to inhaled variola. The pulmonary focus of the reaction suggests that there were significant concentrations of respirable variola in the vicinity of smallpox patients. Concentrations of respirable variola high enough to elicit allergic reactions, if true, raise a significant concern for the likelihood of airborne transmission.

Fomites, particularly exposure of laundry workers to contaminated bedding, were implicated in a few reported outbreaks (Cramb, 1951) . However, during the eradication campaign careful epidemiologic investigation rarely implicated fomites as a source of infection (Fenner et al., 1988) . Laundry was contaminated by scabs containing large amounts of virus, and with respiratory secretions containing virus in smaller particles (Downie et al., 1965) . Very large particles with diameters greater than 50-100 µm are easily reaerosolized. Thus, the rarity of clear evidence of transmission due to fomites would be surprising, if exposure of upper respiratory mucosa to virus in large particles were an efficient means of initiating infection. However, the probability of reaerosolizing particles ≤10 µm from surfaces is extremely low because surface forces tend to bind particles more avidly the smaller the particle (Hinds, 1999) . Thus, the rarity of smallpox transmission via fomites suggests that mucosal exposure was not the primary means of transmission and is consistent with a preference for infection via the lower respiratory tract. The rarity of transmission on crowded buses and trains could be evidence that airborne transmission was not important. However, Fenner et al. (1988) state that transmission on public transport was rare because patients seldom traveled after becoming ill. They showed that transmission did occur on public transport by reporting a case of confluent smallpox who traveled early in her illness and infected five persons on a bus. If most patients who traveled were convalescent so that they no longer had virus in respiratory secretions and only shed virus in large particles from scabs, which were rarely associated with transmission of infection, (Rao et al., 1968 ) then lack of transmission on buses and trains was consistent with a preference for airborne transmission. Mack (1972) emphasized that 85% of cases had clear-cut exposures to known cases. However, the remaining 15% had no obvious exposure suggesting that a small number of more distant or casual contacts transmitted infection as would be expected if smallpox were transmitted by dilute virus aerosols. For example, in the 1947 New York outbreak one secondary case was seven floors away in the hospital (Weinstein, 1947) . Dispersal of smallpox downwind of hospitals was the only obvious explanation for a small number of cases in a British outbreak (Bradley, 1963; Westwood, 1963) . Unexplained introductions of smallpox into Pakistani towns was greatest in towns with facilities for treatment of smallpox, (Thomas et al., 1972) which may suggest that relatively casual contact, or down wind dispersal were capable of occasionally spreading infection.

Some well-known hospital-associated outbreaks make it clear that airborne transmission at a distance of more than a few feet did occur occasionally (Wehrle et al., 1970) . But, these examples were rare. However, because highly infectious disseminators are rare in other airborne infectious diseases, (Riley, 1980; Olsen et al., 2003) the rarity of superspreaders in smallpox is not an indication that transmission by less infectious cases was necessarily by a different route.

To examine whether the available data on variola aerosols is consistent with Mack's observation regarding known contacts, we can apply a standard Poisson probability model of airborne infection to estimate how long a susceptible person would need to be in a patient's room to have a reasonably high probability of contracting disease (Riley et al., 1978; Rudnick and Milton, 2003) . If, we assume that inhalation and lower respiratory deposition of one PFU of variola was sufficient to cause infection, as for rabbits exposed to vaccinia and rabbit pox, (Westwood et al., 1966) and if a patient's room contained between 0.5 and 5 PFU/m 3 in particles with a 25% lower respiratory deposition fraction (consistent with the literature discussed above), a susceptible individual breathing at 8 l/min would have needed to spend between 1.7 and 16.7 h in the patient's room to have a 63% probability of becoming infected. Outside of the patient's room, aerosol concentrations would have been much lower. If most patients stayed at home in small buildings or in hospitals without mechanically recirculated air, the risk of infection would have been significantly lower outside of patients' rooms, consistent Mack's (1972) observation that 85% of cases arose from identifiable contacts. Thus, a predominance of identifiable face-to-face contacts among cases is not strong evidence against transmission by fine particle aerosols.

The weight of evidence suggests that fine particle aerosols were the most frequent and effective mode of smallpox transmission because this would explain the relatively low mortality after variolation, the rarity of transmission by fomites, resolve the paradox of mucosal infection, and be consistent with "smallpox handler's lung" and with animal and virus aerosol experimental data. Certainly other modes of transmission occurred; fullblown disease could result from inoculation through the skin, the nasal mucosa, or the conjunctiva. Thus, smallpox cannot be classified as an "obligate" airborne infectious disease, such as tuberculosis (Riley et al., 1995) (sometimes referred to as a "true" airborne infection), because it was capable of initiating disease via infection of tissues outside of the lower respiratory tract. However, smallpox also cannot be classified as an isotropic infection (formerly termed "opportunistically" airborne infectious disease) because it appeared not to have been transmitted with equal effectiveness and virulence by all routes, whether aerosol, large droplet, or direct contact and skin inoculation. Smallpox appears to have been most effectively and virulently transmitted by fine particle aerosols and therefore should be classified as an anisotropic infection; an infection where route of transmission influences either virulence and or probability of infection, formerly called a "preferentially" airborne infectious disease.

Current recommendations for control of secondary smallpox infections emphasize transmission "by expelled droplets to close contacts (those within 6-7 feet)" (Centers for Disease Control, 2002 Control, , 2003 . Recommendations include vigilant maintenance of standard, droplet, and airborne precautions. However, emphasis on spread via large droplets may reduce the vigilance with which more difficult airborne precautions are maintained. High concentrations of variola in the lung during the incubation and prodromal periods in monkeys after simulated use of variola as a bioweapon (Hahon, 1961 ) may indicate that first generation cases after an attack with a concentrated aerosol may be more infectious than expected based on historical data. Moreover, because airborne precautions are not routine for all hospitalized patients, and because first generation cases will probably not be initially suspected to have smallpox, it is likely that they will not be placed on airborne precautions until well into their infectious period. Therefore, the extent of transmission to a second generation in the contemporary hospital environment may be greater than expected based on historical estimates.

These considerations suggest that models of a potential smallpox attack should incorporate an aerobiological perspective to predict how the infection might propagate in the modern environment. It is particularly important to examine smallpox transmission in hospitals because hospitals have previously been identified as the major site of transmission in developed countries and ill patients will inevitably gravitate to hospitals, at least early in the outbreak before alternatives exist (Mack, 1972 (Mack, , 2003 . Additional attention to prevention of airborne transmission in hospitals from unrecognized cases may not only be an important aspect of public health preparedness for smallpox, but may also benefit society by reduced morbidity and disruption from SARS and other emergent airborne infections. Chitinase Dependent Control of Protozoan Cyst Burden in the Brain Chronic infections represent a continuous battle between the host's immune system and pathogen replication. Many protozoan parasites have evolved a cyst lifecycle stage that provides it with increased protection from environmental degradation as well as endogenous host mechanisms of attack. In the case of Toxoplasma gondii, these cysts are predominantly found in the immune protected brain making clearance of the parasite more difficult and resulting in a lifelong infection. Currently, little is known about the nature of the immune response stimulated by the presence of these cysts or how they are able to propagate. Here we establish a novel chitinase-dependent mechanism of cyst control in the infected brain. Despite a dominant Th1 immune response during Toxoplasma infection there exists a population of alternatively activated macrophages (AAMØ) in the infected CNS. These cells are capable of cyst lysis via the production of AMCase as revealed by live imaging, and this chitinase is necessary for protective immunity within the CNS. These data demonstrate chitinase activity in the brain in response to a protozoan pathogen and provide a novel mechanism to facilitate cyst clearance during chronic infections. The brain has unique structures in place to limit access of immune cells and molecules. Although this can provide protection against an overambitious inflammatory response it may also lead to the high prevalence of latent and chronic infections that can persist at this site. Removal of such pathogens has its own particular problems in an organ dense with sensitive neurons and stringent gateways for immune cell infiltration. Toxoplasma gondii is a common intracellular protozoan parasite that forms a chronic infection in the brain for the lifetime of the host. The infection is controlled, in part, through the effector mechanisms of macrophages that result in the conversion of fast replicating tachyzoites to the slow replicating, cyst forming bradyzoites [1] [2] [3] . Cysts can form in all tissues but exist predominantly in the brain for the lifetime of the host requiring a continuous immune response to prevent cyst reactivation and Toxoplasmic encephalitis, a common cause of AIDS related fatalities [4, 5] . The infection-induced immune response in the brain consists of activated CNS resident cells including astrocytes and microglia, infiltrating CD4+ and CD8+ T cells, peripheral macrophages and substantial tissue remodeling [6] [7] [8] . Such immune activity in the brain is often associated with a pathological outcome yet despite the high prevalence of infection Toxoplasma is seemingly controlled without adverse neurological damage. The mechanisms that are involved in the trafficking and control of such a potentially pathological immune response within the CNS are only beginning to be understood [6, [8] [9] [10] [11] . The cyst and cyst-forming bradyzoites are poorly immunogenic [12, 13] and although we have known for some time that T cells are required to prevent cyst reactivation [4, 5, 14] , very little is understood about the biology of this structure in the brain. Although anti-Toxoplasma drugs are available that efficiently control the tachyzoite, there are as yet no therapies available that can effectively remove the cyst form of the parasite. Thus, the continuous presence of Toxoplasma cysts in the brain presents a critical and constant danger for the immune compromised patient.

It is widely believed that cysts remain intracellular within neurons possibly minimizing their contact with host defense systems [15] . However it has been known for some time that cyst burden reaches a peak, declines and becomes stable over time pointing to some form of effector mechanism that can target this stage of the parasite [16] . Studies have implicated CD8+ T cell production of perforin in cyst clearance with perforin deficient mice exhibiting higher cyst burden and susceptibility at the chronic stage of infection [17, 18] . Nevertheless, histological analysis from these studies as well as recent live imaging of cell interactions in the CNS [19] demonstrates monocyte accumulation and contact with cysts.

In recent years, our understanding of macrophages has expanded and we now appreciate these cells' remarkable plasticity. Thus, although whole populations of macrophages can become polarized to classical or alternative phenotypes associated with protection against protozoan and helminth pathogens respectively, the ability to respond and adapt to local stimuli in the environment is paramount [20] [21] [22] [23] [24] . The role of classically activated macrophages in the control of T. gondii infection is well documented. These cells are a source of IL-12, reactive oxygen and nitrogen species, and GTPases that enable the direct killing of the parasite [6, [25] [26] [27] [28] [29] [30] . However, here we describe a population of CXCR3+ macrophages in the brain following T. gondii infection. These cells express the scavenger receptors MMR and stabilin-1 and produce arginase in response to the presence of Toxoplasma cysts. In addition to these traditional signs of alternative activation, these studies demonstrate that macrophages respond to chitin present in the cyst wall and produce the true mammalian chitinase, AMCase. Finally we show that this chitinase activity destroys cysts and is essential for the control of cyst burden within the chronically infected brain.

Recent studies have identified a substantial increase in tissue remodeling in the brain during chronic T. gondii infection [8] . Additionally, there is a continuous need for the clearance of debris from ruptured cysts and dead cells in the brain [31] . To investigate if AAMØ, known for their role in tissue remodeling and homeostatic clearance, are present during such an event in the CNS, macrophage populations from the infected brain were phenotypically analyzed for the expression of known markers of alternative activation. One of the key molecules that has been associated with a tissue remodeling macrophage phenotype in the CNS is the expression of CXCR3 on microglia [32, 33] . CXCR3 is required for protective immune responses to T. gondii primarily due to its role in Th1 cell recruitment and most recently for T cell search strategies in the brain [34] [35] [36] [37] . Indeed CXCR3 and its ligands are significantly upregulated in the brain at a timepoint associated with significant T cell influx into the CNS following infection ( Figure S1A , B) with ,35% of T cells expressing CXCR3 ( Figure 1A ). However, in addition to this well characterized role on T cells, there is a small but distinct population of macrophages that express high levels of CXCR3 (,10% of total macrophages)( Figure 1B ). There is also constitutive, although lower, expression of CXCR3 by CNS resident microglia, which remains unchanged following infection ( Figure S1C ).

To confirm that expression of CXCR3 is associated with alternative activation of macrophages the expression of the scavenger receptor 'macrophage mannose receptor' (MMR; also known as Mrc1 and CD206), a key indicator of the AAMØ phenotype [38] was analyzed. Here we show that MMR expression is limited to macrophages and microglia that also express CXCR3 (Figures 1C and S1D ). In contrast these cells did not express IL-10 ruling out an anti-inflammatory phenotype ( Figure S1E ) [39] . Depletion using blocking antibodies to CXCR3 or its ligand, CXCL10 led to a significant decrease in T cell recruitment and a reciprocal increase in parasite burden ( Figure S2 ). However, in addition, the proportion of macrophages in the brain was significantly reduced ( Figure 1D ) despite no defect in macrophage-attracting chemokines ( Figures 1E, F) , confirming a role for CXCR3 in the maintenance of this cell population.

To quantify MMR expression by macrophages and microglia in the infected brain, qRT-PCR was performed on magnetically isolated CD11b+ cells from the brains of naive and infected animals. Our results show an approximate 3-fold increase in MMR expression in macrophage populations from infected mice over naïve ( Figure 1G ). Confirmation of this population in the brain was revealed by immunohistochemical analysis. MMR+ macrophages were observed as small and discrete populations of IBA-1+ or tomato lectin+ cells confirming the source of MMR on macrophages or microglia ( Figures 1H, I) . A further functional marker of alternative activation is the scavenger receptor stabilin-1 [40] . Stabilin-1 is involved in the clearance of cell corpses as well as the uptake of extracellular matrix components [41, 42] . Expression of MMR co-localized with stabilin-1 and microglia/ macrophage markers, confirming that these cells display an alternatively activated phenotype ( Figures 1H, I) . These cell populations were frequently found in close proximity with intact and degrading T. gondii cysts in the CNS ( Figure 1J and S3 ).

An important feature of AAMØ is the cell's ability to produce arginase-1, which acts on its substrate, L-arginine to produce Lornithine, a precursor to collagen [43] . L-arginine is also the substrate for NO synthase and the two enzymes compete for substrate availability and are regulated by Th1 and Th2 type cytokines [44, 45] . Previous studies have demonstrated that direct infection of macrophages by T. gondii tachyzoites can induce arginase expression via STAT-6 dependent and independent pathways [46] [47] [48] . Furthermore these studies imply that such an induction is a survival strategy enlisted by the parasite to inhibit killing via NO. To assess whether or not macrophages and microglia in the infected brain produce arginase, CD11b+ BMNCs from infected mice were isolated and analyzed for arginase-1 expression by qRT-PCR. Our results show almost a 2fold increase in arginase-1 expression in cells from infected brains over naïve ( Figure 1K ). Thus, during chronic T. gondii infection there is a population of AAMØ in the CNS characterized by expression of CXCR3, MMR, stabilin-1 and the production of arginase-1.

Alternatively activated macrophages secrete an active chitinase in the CNS in response to chitin in the cyst wall During chronic infection there are several forms of the parasite that could be the source of the infection-associated stimulus for

Described here is a novel mechanism of protozoan cyst clearance in the CNS during chronic infection. These data show the presence of a population of alternatively activated macrophages in the brain that secrete the active chitinase, AMCase, in response to chitin in the cyst wall. Using both chemical and genetic inhibition in vitro, it is revealed that this enzyme is required for efficient degradation and destruction of the cyst. The necessity for AMCase is demonstrated in vivo, as the absence of the enzyme resulted in a significant increase in cyst burden and decrease in survival during chronic infection. Together, these data identify an important mechanism of parasite control and cyst clearance in the CNS. Currently, no therapies exist that lead to the total clearance of this parasite from the brain. Therefore, developing an understanding of the natural mechanisms of cyst clearance has the potential to lead to new and effective therapies for this and other chronic infections. alternative activation of macrophages in the CNS. Since latent cysts are the most prevalent form of infection in the brain, an attractive candidate for the source of this stimulus is the presence of chitin in the cyst wall [49, 50] as it has been shown that the presence of chitin induces the recruitment of macrophages that have an alternatively activated phenotype [51, 52] . To determine if sources of T. gondii can induce alternative activation, tachyzoites, bradyzoites, and cysts were added to bone marrow derived macrophage (BMDM) cultures and the production of urea, a downstream product of arginase activity, was measured [53] . In addition, soluble antigen derived from freeze-thawed tachyzoites (sTAg) and whole cysts (cystAg) was assessed for their ability to induce urea (Figure 2A) . Our results show a baseline production of urea in unstimulated (media) macrophage cultures, possibly due to the presence of M-CSF [54] . This significantly increased (p,0.001) during AAMØ polarization with IL-4. Despite the known ability of tachyzoites to induce arginase production [46] , tachyzoite infection of macrophages did not lead to significant production of urea ( Figure 2A ). This can be attributed to the use of a type II strain which is a weak inducer of arginase-1 [47, 48] . Importantly, addition of cysts or cyst antigen but not ''naked'' bradyzoites, did lead to a significant increase in urea production although not as great as induction of alternative activation by IL-4 [38, 55] . This points to components of the cyst wall as the stimulus for AAMØ. Taken together, these data demonstrate that macrophages can be alternatively activated by the presence of T. gondii cysts, but not free replicating parasites.

Chitinase activity has been demonstrated in certain populations of AAMØ in both mice and humans [52, 56, 57] . Chitinolytic activity by macrophages has also been implicated in host defense against chitin-containing fungal pathogens [56, 58] . To test whether or not chitinase activity is induced by Toxoplasma infection, a chitinase assay was performed on whole brain lysates from naïve and infected animals. Three chitin substrates labeled with 4-methylumbelliferone (4MU) were used to assess the type of chitinase activity present. Upon hydrolysis, 4MU is released and can be measured fluorometrically to determine chitolytic activity. Our data reveal that chitinase activity is significantly increased in the brains of infected mice as compared to the naïve group in only one of the three substrates ( Figure 2B ). This substrate, 4MU Nacetyl-b-D-glucosaminide, is suitable for detection of exochitinase activity where the enzyme degrades the non-reducing end of the chitin [59] . Several studies have linked chitinases and chitinase-like proteins to inflammation [58] [59] [60] . This family of 18 glycosyl hydrolases is typically induced during Th2 type immune responses and plays a role in tissue remodeling, fibrosis, and the modulation of both the innate and adaptive immune response [60] . Acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT1) are unique members of this family in that they possess an enzymatically active domain that hydrolyzes the b 1-4 linkages that exist in chitin [56, 58] . Analysis using qRT-PCR demonstrated a significant upregulation of AMCase but not CHIT1. In addition, the chitinase-like protein, Ym-1 (Chi313) was also upregulated following infection ( Figure S4 ). This molecule is known to inhibit IL-12 production and induce alternative activation in macrophages [25, 61, 62] . In contrast to Ym-1, that has been associated with AMCase production by macrophages in the lung and airway [63] , Ym-2 and BRP-39 were not upregulated in infected brains ( Figure 2C , S4). Since chitin has been shown to activate and recruit AAMØ, it is possible that the cyst wall may serve as the stimulus for chitinase activity in this population of cells. To test this further, BMDM were co-cultured with different forms of the parasite. The addition of tachyzoites, bradyzoites or sTAg did not lead to chitinase production ( Figure 2D ). In contrast, live cysts and cyst antigen led to a significant increase in chitinase activity that was abolished following chitinase treatment of cysts ( Figure 2D , S4). Furthermore, treatment with IL-4 to induce alternative activation in macrophages did not lead to increased chitinase activity. Indeed measurement of IL-4, IL-4Ra and the IL-4dependent RELM-a [64] [65] [66] in the brains of chronically infected mice showed no significant increase over naïve mice ( Figure S4 ). These data suggest that the presence of chitin in the cyst wall induces a phenotype of macrophage characterized by the production of the enzymatically active chitinase, AMCase and is distinct from IL-4 induced activation.

Previous work has shown macrophages in close association with rupturing cysts [17, 31] and the presence of an active chitinase could point to a role for these cells in the breakdown of cysts within the brain. Recognition of chitin by macrophages is size dependent and likely contact dependent [49, 58, 67] . To test this, we cocultured cysts separated from macrophages using 5 mm transwell inserts and assayed for urea and chitinase activity as previously described ( Fig. 2A, 2D ). Our results show no increase in either urea production or chitinase activity from macrophages that have been separated from cysts, confirming that the observed alternative activation is dependent on contact with cysts or cyst antigen.

Immunohistochemical analysis of the location of AMCase secreting macrophages in the infected brain shows them in close proximity with tissues cysts (Figures 3A-C) . As a proportion of macrophages and microglia in the brain, alternatively activated cells are in the minority and it was not possible to find such cells in the naïve brain. However, cysts are easily identifiable with a highly spherical distinct morphology, can stain non-specifically and specifically with antibodies and individual bradyzoites within the cyst are visible by DAPI staining. Examination of chitinase localization in the infected brain revealed distinct cytoplasmic staining of several cells, nearly all of which were within 75 mm of a cyst (Fig. 3A) . Although there were cells that were AMCase positive yet did not stain positively for macrophage/microglial markers, there were clearly several macrophages in close association with cysts that displayed chitinase activity polarized to the cyst wall ( Figure S5 , arrows). AmCase activity was also observed in macrophages surrounding cysts that seemed to be in the process of lysing or cysts that had been lysed (Figures 3B,C and Video S1). The examples provided show the destruction of the spherical cyst (Video S1) and escaping parasites visualized using anti-Toxoplasma antibodies. Directly at the point of rupture there are AMCase expressing macrophages (Figures 3B). Taken together, these data suggest that the induction of chitinase activity in macrophages occurs in close proximity with the cyst wall and that this distinct population of macrophages is responsible for attacking the long-term chronic cyst stage of Toxoplasma via chitinase activity.

The prevailing view is that cysts in the brain remain intracellular within neurons and that CD8+ T cell production of perforin is responsible for cyst clearance in the brain although the exact mechanism of cyst destruction has yet to be described [17, 18] . In order to determine whether or not macrophage chitinase activity could be responsible for the direct lysis of cysts, BMDM were co-cultured with cysts; with and without the chitinase inhibitor allosamidin. Cultures were observed for 14 hours capturing images every 10 minutes. Cysts observed in the absence of macrophages remained intact for the entire time course suggesting no parasite intrinsic mechanism of cyst destruction ( Figure 4A ; Video S2). In contrast, the addition of 20 mg/ml trichoderma chitinase to cyst cultures led to rapid rupture of the cysts within an average of 4 hrs, releasing bradyzoites into the media ( Figure 4B ; Video S3). Strikingly, cysts that were cultured with macrophages came under vigorous attack. This involved efficient and rapid migration of macrophages toward the cyst creating clusters of macrophages that could be seen pulling at the cyst wall ( Figure 4C ; Video S4). In these cultures most of the cysts were destroyed during the observation period with the average survival time of 9.5 hours ( Figure 4E ). In contrast cysts cultured in the presence of macrophages and the chitinase inhibitor allosamidin survived significantly longer than in untreated cultures. Although there appeared to be no defect in the recruitment and activity of macrophages to cysts with similar clustering and 'pulling' of the cyst wall, the majority of cysts survived the entire 14 hour period when treated with either 100 mM or 10 mM allosamidin ( Figure 4D ; Video S5). Decreasing concentrations of allosamidin led to a dose dependent decrease in cyst survival time ( Figure 4E ). These results demonstrate that macrophages can induce cyst lysis in a chitinase dependent manner.

Although both AMCase and CHIT1 are upregulated in certain bacterial and nematode infections [68, 69] only AMCase was significantly increased in the brain following Toxoplasma infection ( Figure 2C ). To confirm that AMCase is responsible for the observed chitinase activity, we performed a chitinase assay similar to that in Figure 1D using BMDM from WT and AMCase null mice ( Figure 5A ). Our results reveal a severe defect in chitinase production by AMCase null macrophages. Indeed, these cells showed a significantly lower baseline level of chitinase and were unresponsive to the addition of cysts. To test whether the ability to destroy cysts is dependent on this enzyme, BMDM from WT and AMCase2/2 mice were fluorescently labeled and cultured with Me49-RFP expressing cysts and cyst lysis time imaged as before. Using fluorescently labeled parasites enhanced the ability to see escaping parasites from lysing cysts. Results, as before, demonstrated that WT non-polarized macrophages were able to lyse cysts in ,10 hours ( Figures 5B and 5D ; Videos S6 and S10). In contrast, cysts cultured with AMCase2/2 macrophages had a significantly increased survival time over WT macrophages consistent with AMCase being the source of chitinase activity required to lyse cysts ( Figures 5C and 5D ; Videos S7 and S11). To determine the requirement for macrophage polarization in their ability to lyse cysts, macrophages were treated with cytokines to polarize them to classical or alternative phenotypes prior to cyst addition. In line with the lack of chitinase induction, IL-4 priming had no effect on cyst survival time, suggesting that cytokineinduced alternative activation does not enhance the ability to destroy cysts ( Figures 5D; Video S9) . In contrast, macrophages that were classically polarized with LPS and IFN-c showed a defect in chitinase activity and cyst destruction (Figures 5D and 5E ; Video S9). Suggesting that polarization of macrophages is required but that chitin is the most likely source of alternative activation and not IL-4. Taken together, these data demonstrate that macrophages lyse cysts in an AMCase-dependent manner in vitro.

The consequences of chitinase dependent cyst lysis in the CNS could potentially benefit either the host or the parasite. If the escaping bradyzoites were quickly killed by macrophages or associated immune cells, we would expect this mechanism to benefit the host and result in a lower parasite burden. Conversely, if bradyzoites are able to propagate and infect new cells, this could be a mechanism that promotes the persistence of the parasite in the brain. To investigate the role of AMCase in the brain in vivo, we infected WT and AMCase deficient mice and analyzed the immune response and parasite burden in the absence of chitinase activity. To determine if AMCase is required during the acute stage of infection, tissue samples from lungs were taken at day 7 and analyzed for parasite burden by qPCR. No significant differences in lung parasite load were found and serum cytokine concentrations were equivalent throughout acute infection ( Figure 6A-F) . Thus a lack of AMCase does not lead to deficient immune responses early on during infection in the periphery. At 5 weeks post infection, when systemic inflammation has been controlled and parasites are located solely in the brain predominantly as cysts containing bradyzoites [67, 70] parasite burden was evaluated. In the absence of AMCase, there was a significant increase (p = 0.0014) of approximately 2-fold in the total number but not the size of cysts in the brain (Figures 6G and 6H) . Differences in cyst burden were not observed at 3 weeks post infection ( Figure S6 ), a period representing the transition between acute and chronic infection, further suggesting that the increase in cyst burden is occurring due to events within the CNS during chronic infection. In addition, total parasite burden in the CNS as measured by qPCR was significantly greater by more than 2-fold (p = 0.0055) ( Figure 6I ) correlating with the appearance of more cysts histologically ( Figure 6J ). In addition, parasite burden was evaluated using RT-qPCR with stage-specific primers identifying tachyzoite (SAG1), bradyzoite (SAG4), and cyst (MAG1) specific transcripts [71] (Figure S6 ). Our results show similar increases in parasite burden for all three transcripts, suggesting that cyst lysis is also an important mechanism to control the cell invasive forms of the parasite. Flow cytometric analysis revealed no differences in infiltrating CD4+, CD8+ T cells, or macrophage populations ( Figure 6K) . Therefore, the increase in parasite burden is not due to a defect in infiltrating effector immune cells. Furthermore, AMCase2/2 mice failed to survive and succumbed to infection beginning at six weeks (p = 0.0177) ( Figure 6L ). Although some acute mortality was noted over several experiments significance was only achieved when chronic mortality was included. These results demonstrate that AMCase activity is required for the protective immune response to T. gondii during chronic infection in the brain and that AMCase mediated cyst lysis in the CNS is a beneficial mechanism for the host to control parasite burden at non-lethal levels.

Chronic infections represent a continuous battle between the host's immune system and pathogen replication. Many protozoan parasites and fungal pathogens have evolved a cyst lifecycle stage that provides it with increased protection from environmental degradation as well as endogenous host mechanisms of attack [72] [73] [74] . In the case of Toxoplasma, these cysts are predominantly found in the immune protected brain making clearance of the parasite more difficult and resulting in a lifelong infection. Here we describe three novel findings 1) despite a dominant Th1 immune response during Toxoplasma infection there exists a population of macrophages in the infected brain which display a distinct alternatively activated phenotype; 2) these cells are responsible for chitinase dependent lysis of Toxoplasma cysts and 3) this chitinase activity is through the production of AMCase which is required for protective immune responses.

Multiple studies have demonstrated the role of CXCR3 and its ligands in the migration of activated T cells during Th1 immune responses including to sites of infection [75, 76] . It is also known that the chemokines CXCL9 and CXCL10 are induced by the presence of the proinflammatory cytokine, IFN-c [34, 36, 37, 77] . More recently, however, the function of this family of chemokines has expanded to include neural-glial signaling following brain lesion where injured neurons upregulate CXCL10 and recruit CXCR3 expressing microglia to phagocytose denervated dendrites [33] . Consistent with this, another recent study has implicated CXCR3 in the function of perivascular macrophages and their ability to remodel the vasculature during stress [78] . We noted upregulation of CXCR3, CXCL9 and CXCL10 in the brain during chronic Toxoplasma infection. Furthermore, CXCR3 was preferentially expressed on macrophages expressing the scavenger receptors MMR and stabilin-1, suggesting an alternatively activated phenotype for these cells. Previous studies have established important functions of AAMØ in the context of helminth infection and wound healing [43, 79, 80] however not during an infection that generates such a polarized Th1 immune response such as Toxoplasma. T. gondii is known to exploit the arginine metabolic pathway and induce arginase-1 expression in macrophages thereby suppressing nitric oxide production but this does not lead to the alternative activation of these cells [46] [47] [48] . Instead our data point to a role for the cyst being the source of alternative activation of macrophages and the subsequent ability of these cells to lyse cysts via destruction of the chitin in the cyst wall. Thus, we observed a contact dependent significant increase in arginase activity following treatment with cysts and cystAg suggesting that this induction is not a result of infection by the replicating parasite, but rather by the presence of chitin in the cyst wall. The weak induction of arginase activity observed also points to a limited role for arginase-1 in the chitin-induced phenotype.

Chitin is found in the exoskeletons of insects, fungal cell walls, sheaths of parasitic nematodes, and is a component of the T. gondii cyst wall [49, 50] . The presence of this exogenous molecule can induce the recruitment of AAMØ, basophils, neutrophils, and eosinophils [52, [81] [82] [83] [84] . Active chitinases such as AMCase and chitotriosidase are secreted by macrophages in response to chitincontaining pathogens and has been shown to inhibit hyphal growth of chitinous fungi such as Candida and Aspergillus [56, 58] . Despite the link between chitin and the recruitment of type 2 inflammation in the lung [52] , a recent study has demonstrated no role for AMCase in the generation of allergic airway pathology [85] . In this study we demonstrate for the first time, macrophage chitinase activity in response to a protozoan pathogen. Chitin recognition is thought to be a size dependent process and involve a combination of TLR2 and scavenger receptors such as MMR and dectin-1 [49, 58] . Here we have demonstrated the presence of such scavenger receptors in association with cysts and it will be of interest in future studies to determine the role of these molecules in cyst containment during Toxoplasma infection. Independent of the receptors involved it is likely that this is a contact-dependent process and indeed, cysts were unable to induce urea production or chitinase activity in macrophages when separated by transwell membranes. Furthermore, analysis of the location of AMCase producing cells in the brain finds them reliably close to cysts and often in direct contact with reactivating or rupturing cysts. These images show that despite the presence of many DAPI positive cells surrounding the cyst structure, the escape of parasites through the cyst wall occurs juxtaposed to the macrophage or AMCase activity. Our data suggest that the presence of cyst antigens induces alternative activation of macrophages and that these antigens are required for macrophages to produce chitinase even in the presence of IL-4. Thus alternative activation of macrophages is not sufficient for AMCase production and chitinase activity. The significant increase of the non-enzymatically active chitinase-like molecule YM-1 in infected brains is consistent with previous reports of AMCase and YM-1 being co-expressed specifically in macrophages and not epithelial cells [63] .

Live imaging in vitro demonstrated AMCase dependent degradation of cysts as shown using both a chitinase inhibitor and AMCase2/2 macrophages. Although there is no evidence of an active chitinase produced by T. gondii (ToxoDB), the similar cyst survival times observed for AMCase-null macrophages and chitinase-inhibited macrophages exclude the possibility that bradyzoites are the source of enzymatic activity and are breaking out of the cyst. The chitin dependent induction of chitinase activity implies that macrophages have access to the chitin in the cyst wall Figure 6 . AMCase2/2 mice have a higher parasite burden in the brain and succumb to infection during the chronic stage. (A-F) C57Bl/6 (WT) and AMCase2/2 mice were infected with the Me49 strain of T. gondii. Serum was isolated from whole blood samples at days 3, 7 and 14 post infection and analyzed for (A) IFN-c, (B) IL-6, (C) MCP-1, (D) TNF-a, (E) IL-12p70 (F) At day 7 DNA was isolated from the lungs and analyzed for parasite burden using qPCR. Results are displayed as parasites per mg tissue. (G-K) C57Bl/6 (WT) and AMCase2/2 mice were infected with the Me49 strain of T. gondii and sacrificed at 5 weeks following infection. Brains were harvested and analyzed for cyst burden, cellular composition and histology. (G) Cyst counts were obtained from homogenized whole brain samples. (H) Cyst area, 20 cysts from each mouse were photographed microscopically and cyst area was determined using ImageJ software. (I) DNA from brains of WT and AMCase2/2 was isolated and analyzed for parasite burden using qPCR. (J) Whole brains were fixed, frozen and stained for H&E to examine cyst burden and pathology. (K) BMNCs were isolated and analyzed for expression of CD4+ T cells, CD8+ T cells, macrophages (CD45 hi /CD11b+) and microglia (CD45 hi /CD11b+) by flow cytometry. Significance was determined using log rank test with p = 0.0177. Data are representative of at least 2 individual experiments with a minimum of n = 4 and are represented as mean 6 SEM, ns = not significant, * p,0.05, ** p,0.01. (L) Survival data from C57Bl/6 (squares, n = 7) and AMCase2/2 (triangles, n = 7). Data are representative of 4 individual experiments with C57Bl/6 (n.40) and AMCase2/2 (n = 40) and significance tested using Logrank (Mantel-Cox) and Gehan-Breslow Wilcoxan test * p,0.05. doi:10.1371/journal.ppat.1002990.g006 prior to chitinase-mediated cyst destruction. The prevailing view from ultrastructure studies is that cysts remain intracellular within neurons [15, 86, 87] (A. Koshy and J. Boothroyd personal communication) yet analysis of cyst burden over time shows a reduction in cyst numbers implying some form of effector mechanism in place [16] . Several studies have demonstrated perforin dependent control of cyst burden during chronic infection [17, 18] . We suggest that instead of a direct effect of perforin on cysts, it is most likely that perforin production by CD8+ T cells may initiate this process by lysing the cyst infected cell, thus exposing the cyst wall to chitinase activity from macrophages. This model would explain the many observations of macrophages in close association with rupturing cysts [8, 17, 19] . Of note, we found that BALB/c macrophages that are more easily alternatively activated had enhanced cyst lysis activity when compared to C57Bl/6 ( Figure S7 ). This may be one explanation for the increased resistance to toxoplasmic encephalitis in BALB/c mice [30, 88] .

Although AMCase activity is not required for protective immunity during acute infection it is required for protection during the chronic stage of infection. Our observation of a higher cyst count and parasite burden as well as decreased survival in AMCase-null mice points to a specific and important role for chitinase mediated cyst lysis in the brain. Thus, within the brain, cyst containment seems as important as the killing of free parasites in the control of pathology. In addition, continuous chitinmediated attack by macrophages and the release of parasites from latent cysts will provide a constant source of antigenic stimulation for the immune response. This latter discovery may provide an explanation for the continuous recruitment of T cells into the brain.

It has been apparent for some time that cyst numbers in the brain can be controlled endogenously yet identification of the exact effector mechanisms has not been so apparent. In these studies we demonstrate the presence of a distinct population of macrophages in the brain during chronic Toxoplasma infection, which express CXCR3, MMR, stabilin-1 and arginase-1. Furthermore these macrophages have chitinase activity, are localized to cysts and are observed in association with cyst degradation. The mechanism of cyst lysis is dependent on AMCase and this enzyme is required for survival during chronic infection to reduce parasite burden. The continuous presence of Toxoplasmic cysts in the brain and other tissues presents a constant threat of reactivation to the immune compromised patient. Mechanisms that enhance cyst removal or prevent their reactivation during Toxoplasma or other protozoan infections would provide a novel line of anti-parasitic therapies.

The experiments in this study were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Institutional Animal Care and Use Committee at University of California, Riverside. All efforts were made to minimize animal suffering during the course of these studies.