Sex presents evolutionary costs and benefits, leading to the expectation that the amount of genetic exchange should vary in conditions with contrasting cost-benefit equations. Like eukaryotes, viruses also engage in sex, but the rate of genetic exchange is often assumed to be a relatively invariant property of a particular virus. However, the rates of genetic exchange can vary within one type of virus according to geography, as highlighted by phylogeographic studies of cystoviruses. Here we merge environmental microbiology with experimental evolution to examine sex in a diverse set of cystoviruses, consisting of the bacteriophage ϕ6 and its relatives. To quantify reassortment we manipulated – by experimental evolution – electrophoretic mobility of intact virus particles for use as a phenotypic marker to estimate genetic exchange.We generated descendants of ϕ6 that exhibited fast and slow mobility during gel electrophoresis. We identified mutations associated with slow and fast phenotypes using whole genome sequencing and used crosses to establish the production of hybrids of intermediate mobility. We documented natural variation in electrophoretic mobility among environmental isolates of cystoviruses and used crosses against a common fast mobility ϕ6 strain to monitor the production of hybrids with intermediate mobility, thus estimating the amount of genetic exchange. Cystoviruses from different geographic locations have very different reassortment rates when measured against ϕ6, with viruses isolated from California showing higher reassortment rates than those from the Northeastern US.The results confirm that cystoviruses from different geographic locations have remarkably different reassortment rates –despite similar genome structure and replication mechanisms– and that these differences are in large part due to sexual reproduction. This suggests that particular viruses may indeed exhibit diverse sexual behavior, but wide geographic sampling, across varying environmental conditions may be necessary to characterize the full repertoire. Variation in reassortment rates can assist in the delineation of viral populations and is likely to provide insight into important viral evolutionary dynamics including the rate of coinfection, virulence, and host range shifts. Electrophoretic mobility may be an indicator of important determinants of fitness and the techniques herein can be applied to the study of other viruses. Sex – defined as the exchange of genetic material between individuals during reproduction – has evolutionary benefits and costs. Asexual populations maintain co-adapted gene complexes, but sexSex can have profound effects on viral evolutionary dynamics including host range emergence, escape from immunity, increased virulence, among others. However, systematic information on the rates of genetic exchange among different viruses is scarce. In partwithin a particular virus group[Reported variation in reassortment among viruses is likely influenced by the dramatic differences in genome structure, expression and replication. A less us group. Geograpus group. Thus, wCystoviridae, ϕ6, was isolated from Pseudomonas-infected bean straw by Vidaver[Pseudomonas syringae pv phaseolicola[Here we investigate the rates of reassortment in a diverse set of cystoviruses, most of which were isolated from the environment, primarily from natural settings. Cystoviruses are lytic, lipid enveloped, dsRNA viruses with tripartite genomes of ~13 kbp composed of a small, medium, and large segment. The first member of the  Vidaver and rema Vidaver and natu Vidaver,14. The seolicola, which hseolicola, the hosTwo recent studies concluded that reassortment rates in naturally isolated cystoviruses differed by geographic region. Phylogenetic analyses of partial genome sequences of cystoviruses isolated from plants in California and the We used ϕ6, a common lab strain, as a reference point to assay other cystoviruses, to estimate their propensity to hybridize (reassort). We employed experimental evolution to generate variability in electrophoretic mobility for use as a novel phenotypic marker for examining hybridization rates. Electrophoresis has been used previously to investigate intact virus particles’ properties, includiHere we: 1) present the results of experimental evolution to select for slow and fast electrophoretic mobility in bacteriophage ϕ6; 2) sequence the genomes of selected strains to reveal sequence changes associated with mobility phenotypes; 3) demonstrate the utility of mobility differences to detect hybridization; 4) apply the technique to determine whether and at what rate a collection of environmental isolates of cystoviruses can hybridize with ϕ6; and 5) examine differences in the hybridization rate according to geography.Pseudomonas syringae pv phaseolicola (Pp) . Growth, plating, dilution, and incubation were conducted at 25°C in LC media[Bacteriophage strains used in experiments are listed in Table LC media at a pH LC media. CrossesLC media, but at 7-1011 particles/mL) with 5 μL of blue/orange loading dye at 70 V at 4°C in a 0.8% agarose/TAE gel. We rinsed the gel with distilled water and sampled (poked) two times with an 8-channel pipette outfitted with tips. The sampling points were offset in order to create 16 sampling points along the length of the 9.5 cm gel tray. After sampling, we immediately inserted the pipette tips into a microtiter plate with LC media and serially diluted. We plated serial dilutions from the microtiter plate on a lawn of Pp to obtain a virus concentration for each sampling point. We used sampling points as reference points to assign Rf values , relative to the migration of xylene cyanol, in order to control for differential migration during separate gel runs. We tabulated and graphed the concentration of phage at each sampling point  in order to obtain a distribution of the abundance of phage particles throughout the length of the gel lane.We sought to use electrophoretic mobility of whole, intact virus particles as a phenotypic marker. We took the following steps to measure electrophoretic mobility of strains: We ran approximately 20 μL of lysate  on an agarose gel, as described above, until xylene cyanol reached a pre-determined point on the gel (corresponding to sampling point 6). We determined mobility as described above. In order to select for fast and slow moving phage particles, we excised a section of the gel corresponding to either tail of the mobility distribution . We placed this gel section in LC media, serially diluted, and plated on a To identify the genetic changes in the strains subjected to experimental evolution of electrophoretic mobility, we sequenced whole genomes of ϕ6-Fast1, ϕ6-Slow1, ϕ6-Fast2, ϕ6-Slow2, as well as the ancestor, ϕ6. We extracted genomic material from high titer lysates representing a population sample of each strain, using a QiaAMP Viral RNA extraction kit . We reverse transcribed extractions by RT-PCR with Superscript polymerase and random hexamer primers . We purified PCR amplicons of the genome using gel extraction  and sequenced using standard Sanger sequencing methods. Sequences are available in Genbank under the following accession numbers: KF615858 - KF615869.In order to confirm that ϕ6 strains selected for slow and fast electrophoretic mobility would be useful in studies of reassortment, we crossed slow and fast phage lysates in a single burst experiment at a mulThe gel assay uses a phenotypic trait to estimate genetic reassortment. We took additional steps to ensure that intermediate phenotypes corresponded to hybrid genotypes, and thus, that the phenotypic measurements would reasonably estimate genetic exchange. Single burst lysates are composed of the assembled viruses resulting from the coinfection of parental strains. It is possible that during coinfection, strains may exchange proteins with other strain(s) that do not correspond to their genotypes. This is known as phenotypic mixing. When viral lysate has been prepared after a single burst, these assembled viruses have not been given the chance to express their own genotypes, because they have not infected a bacterial cell. Thus, it is in principle possible that some viral proteins do not reflect the genotype of an individual virion. Since single burst lysates were run directly on the gel (in absence of bacteria), it is possible to have a mismatch between phenotypic and genotypic mixing (reassortment). To exclude this possibility, we plated single burst lysates resulting from a cross of ϕ6-Fast1 and ϕ6-Slow1 as single plaques, harvested into a new lysate, and then subjected to the same gel assay. The only difference between these lysates, is that the latter has allowed individual virions to reproduce clonally in cells to allow expression of their genotypes. We compared the results of these gel runs using two sample statistical tests. Additionally, we isolated 10 clones from the single burst lysate and determined the mobility of each of those, to determine whether intermediate phenotypes were recovered from clones.The single burst lysate resulting from a cross contains viral progeny with some combination of the six parental segments. When run on a gel, progeny phage with both parental and hybrid segment combinations are represented in the distribution generated by sampling the gel. We crossed all cystoviruses isolated from the environment against a standard: the population lysate of a ϕ6 strain, ϕ6-Fast1, with manipulated electrophoretic mobility from selection experiments described above. Using a known standard allowed quantification of differences in hybridization rate across environmental strains. We used self-crossing of parental phages  in a single burst experiment with the same MOI (10), as a control. Additionally, we used ϕH2 in a tesPp lawn. This modification was repeatable and increased throughput in the measurement of electrophoretic mobility. We generated mobility distributions using data from three independent gel runs of the same single burst lysate of each cross.We quantified electrophoretic mobility as described above, except that we diluted by serially poking the pipette tips directly on a plate with a mixtools[mixtools use an expectation maximization algorithm (EM) to find maximum likelihood estimates of parameters of incomplete observations. The EM algorithm has a stochastic element, so we run models with varying inputs to verify results. In particular, we tested an unsupervised classification (i.e. no prior information of means or variances of components) with three components to verify if the assigned components coincided with the parental controls and an intermediate hybrid. We used the package mclust 3[We used finite Gaussian mixture models to determine the proportion of parental and hybrid progeny represented in the single burst lysate. We used the package mixtools in the Rmixtools to estim mclust 3 – which t-test), and c) likelihood ratio bootstrapping (mixtools) and Bayesian Information Criterion (mclust 3) of mixture models with different numbers of components determined a two component mixture was more likely.Crosses between strains may sometimes fail to yield hybrid progeny. Since hybrids may be present at low frequencies and may not be detected by our method, we adopted conservative criteria to conclude that the single-burst lysate was a two-component mixture, i.e. a hybridization rate of zero. We determined that a lysate from a cross was a two component mixture if: a) the proportion of a component in a mixture was lower than 0.01 or the means of mixture components differed by less than 0.1, b) the component distribution means were not significantly different  at a high MOI (10). The distribution of the single burst lysate created from the cross of ϕ6-Slow and ϕ6-Fast falls in between parentals with intermediate mean mobility  at the expense of hybrid and ϕ6-Fast1 phenotypes , but with a lysate derived from single plaques of the single burst lysate. These samples were chosen to capture the different hybridization patterns we observed, including samples that yielded no hybrids. The hybridization rate estimate obtained from these samples was not different from the single burst lysates and captured the range of hybridization dynamics (Table To explicitly test the influence of geography on reassortment rates, we compared hybridization estimates from samples of known geographic origin and found that California strains had higher hybridization rates than those isolated in the Northeasten United States (Wilcoxon rank sum test: W = 28, HL∆ = 0.166, HL∆ 95% CI = 0.043-0.344, p = 0.021, Figure Cystoviridae, or may be applied to other viruses.Electrophoretic mobility was useful as a phenotypic marker for estimating hybridization, as shown previously for ϕX174. Here weGenome sequencing of fast and slow-moving ϕ6 strains identified genetic changes that occurred during the experimental evolution regime. These sequence changes may affect phenotypic traits that underlie electrophoretic mobility. While a variety of factors can cause differences in electrophoretic mobility on an agarose gel, charge, mass, and shape are likely candidates for component traits underlying mobility. The genetic mutations observed suggest potential phenotypes. First, all strains exhibited some genetic change in the same medium segment untranslated region Figure . This reWe note that this study documents natural variability in the electrophoretic mobility of phages isolated from the environment. While electrophoretic mobility was used purely as a convenient phenotypic marker for this study, the traits underlying electrophoretic mobility in viruses are very likely of evolutionary significance. These traits can be major determinants of fitness for microbes and viruses. A variety of microbes change charge to avoid host immune defenses,29. SizePhylogenetic analyses have suggested differences in reassortment among cystoviruses in different locations,14, findEvidence from laboratory experiments with ϕ6 suggest that the rates of reassortment are influenced by the coinfection regime in which the virus evolved. If thisInsights into reassortment and rates of coinfection have important implications for virus population structure in nature, which remains largely unexplored-33. UncoIn sum, electrophoretic mobility is a useful, selectable phenotypic marker for studies of virus sex and could itself be an indicator of important determinants of virus fitness. The results confirm that cystoviruses from different geographic locations have remarkably different reassortment rates –despite similar genome structure and replication mechanisms– and that these differences are in large part due to sexual reproduction. This suggests that particular viruses may indeed exhibit diverse sexual behavior, but wide geographic sampling, across varying environmental conditions may be necessary to characterize the full repertoire, as the scale at which viral populations occur may be vastly different than for macro-organisms. Variation in reassortment rates suggests varied cost-benefit equations for sex, having implications for local evolutionary dynamics and a suite of virus characteristics including rates of coinfection, virulence and host range shifts.The data set supporting the results of this article is available in the Dryad repository, DOI: 10.5061/dryad.6qh25, and genome sequences have been deposited in GenBank under the following accession numbers: KF615858 - KF615869.The authors declare no competing interests.SLDM and LC conceived and designed the study. OT and PET assisted in study design and analysis. SLDM and OT conducted experimental evolution of electrophoretic mobility. DG, KB, and PET conducted genome sequencing and analysis. SLDM conducted electrophoresis mobility assays, statistical analyses, and drafted the manuscript. SLDM, OT, DG, KB, PET, and LC participated in manuscript preparation and approved the final version. All authors read and approved the final manuscript.


Objective. Chemotherapy-associated peripheral neuropathy (CAPN) is a painful side-effect of chemotherapy. This study assesses healthcare and workloss costs of CAPN patients with breast, ovarian, head/neck, or non-small cell lung cancer (NSCLC) from a third-party payor/employer perspective. Research Design and Methods. Patients with qualifying tumors, and claims for chemotherapy and services indicative of peripheral neuropathy (PN) within 9-months of chemotherapy (cases) were identified in a administrative claims database. Cases were matched 1 : 1 to controls with no PN-related claims based on demographics, diabetes history and propensity for having a diagnosis of PN during the study period (based on resource use and comorbidities in a 3-month baseline period). Average all-cause healthcare costs, resource use and workloss burden were determined. Results. Average healthcare costs were $17,344 higher for CAPN cases than their non-CAPN controls, with outpatient costs being the highest component . On average, each CAPN case had 12 more outpatient visits than controls, and spent more days in the hospital. Workloss burden was higher for cases but not statistically different from controls. Conclusion. This study establishes that breast, ovarian, head/neck, or NSCLC patients with CAPN have significant excess healthcare costs and resource use. Chemotherapy-associated peripheral neuropathy (CAPN) is a neurological side effect of chemotherapy characterized by loss of sensation in the hands and feet, burning or tingling in limbs, and, in some cases, loss of hearing and blurred vision. The neuropathic symptoms are progressive and tend to increase as chemotherapy treatment proceeds. In addition, comorbid conditions may exacerbate the severity of CAPN . For exaThe chemotherapy drugs most commonly associated with CAPN are taxanes (paclitaxel and docetaxel), vinca alkaloids (vincristine and vinorelbine), and platinums . The incidence of CAPN varies by drug and dose and can range across products from 4–92% , 2, 5, 6Few data exist regarding the health outcomes of CAPN patients, CAPN's effects on chemotherapy treatment, and associated costs. However, Berger suggests neuropathies in general can lead to adverse outcomes and higher costs . In partCalhoun conducted a pilot study on the medical and workloss costs associated with chemotherapy-induced toxicities in women with ovarian cancer . Using sTo the authors' knowledge, no study has quantified the comprehensive health outcomes, medical costs, and workloss burden of CAPN patients with breast, ovarian, head/neck, or nonsmall cell lung cancer (NSCLC). The purpose of the current study is to assess health outcomes as well as the healthcare  and workloss cost burden of CAPN patients (cases) in these 4 tumor types from a third-party payor/employer perspective. The first objective is to compare the healthcare costs of CAPN cases with those of matched controls who have the same cancer but no CAPN. The second objective is to compare workloss costs in patients with and without CAPN. The third objective is to compare the healthcare costs of CAPN cases and non-CAPN controls who have comorbid diabetes. By examining these 4 tumor types, this study captures the use of the chemotherapeutic agents most commonly associated with CAPN.Data were obtained from a database of privately insured administrative claims records (Ingenix Employer Database) that included approximately 8 million beneficiaries from 40 large US-based companies (1999–2006). The companies have operations nationwide in a broad array of industries and job classifications. The database contains de-identified beneficiary information including demographics , enrollment, and medical and pharmacy claims. Utilization measures include date of service, diagnoses, procedures, and actual payments to providers. Pharmaceutical drug claims include National Drug Code (NDC), fill date, days of supply, quantity, and actual payments. In addition, disability claims and employee wage information were available for employees in 23 companies.Three analytic samples consisting of CAPN cases and matched non-CAPN controls were used for this study. The main sample, consisting of cases and controls without diabetes, was used to evaluate the healthcare costs and resource use associated with CAPN patients. A sample of cases and controls with diabetes was used in the secondary analysis to evaluate the costs and resource use associated with diabetic CAPN patients. Finally, a subsample of employed cases and controls was used to assess the workloss burden associated with CAPN patients. Patients under age 65 were eligible for inclusion in the main sample if they had at least 1 claim with a diagnosis for 1 or more of the following cancers from 1999–2005: NSCLC, breast, ovarian, or head and neck. The cancer types were identified using ICD-9-CM codes see . To idenA sample of patients with 1 of the 4 tumor types, CAPN, and diabetes were selected for the secondary analysis. The patient selection criteria were the same as in the Main CAPN sample with the exception of the diabetes criteria. Specifically, diabetic CAPN patients were identified as those patients with a diagnosis of ICD-9-CM code 250.x anytime from at least 3 months up to 1 year prior to the index date or during the 12 months following the index date.A subsample of patients from the Main CAPN and Diabetic CAPN samples who were employees with disability data were selected for the workloss cost analysis. Note that this sample will not contain all employed persons from the main and diabetic CAPN samples but rather will only include the employees of the subset of companies with disability data available.The study period encompassed the 12 months following the index date. Since the perspective adopted was that of the payor and the payor incurs no cost for patients who withdraw from coverage, no requirements were placed on the length of continuous eligibility following the index event. The baseline period for assessing patient history extended 3 months prior to the index date except for ascertaining the presence of diabetes. In all cases, the patients were required to have a minimum of 3 months eligibility prior to the index date to qualify for the study.Cases in the main CAPN sample and the diabetes CAPN sample were separately matched to controls selected from among the set of NSCLC, breast, ovarian, and head/neck cancer patients receiving chemotherapy who did not have a diagnosis for CAPN-related symptoms at any time in the claims history and were under age 65. Controls were matched to cases based on age, gender, employment status (employee versus nonemployee), cancer type, index date of chemotherapy, length of followup (controls were required to have postindex eligibility of at least as long as their matched case), and the estimated likelihood of developing CAPN. Each case was matched 1 : 1 to a control using an optimal matching algorithm . LikelihThe time period over which controls contributed to the outcomes measures was truncated at the end of the observation period for their matching case .Healthcare costs were stratified into 3 mutually exclusive groups: chemotherapy costs, drug costs, and medical costs. Costs were computed as the paid (reimbursed) amount by the insurer to the health-care provider and were annualized to 2006 U.S. Dollars using the Consumer Price Index for medical care. Chemotherapy costs included medical claims with a chemotherapy procedure code and pharmacy claims for oral chemotherapy agents, identified by NDCs. Drug costs included all pharmaceutical claims other than chemotherapy claims. Medical costs included claims for inpatient, emergency department (ED), and outpatient/other care. Costs included all claims associated with any service provided to the case/control during the study period regardless of diagnosis, procedure, or drug. The subset of pharmaceutical and medical costs that could be directly attributed to CAPN was estimated as follows: CAPN-related drug costs included those for drugs potentially used for CAPN  and CAPN-related medical costs included those from claims with a primary or secondary diagnosis of a CAPN-related symptom see .Healthcare resource use consisted of hospitalizations, emergency department (ED), and outpatient/other services (reported by type of visit). The resource use components were defined using provider specialty codes and/or place of service codes on the claims. All claims during the study period were included in assessing resource use, regardless of the underlying reason for a visit. In addition, the proportion of patients using CAPN-related drugs, and specific chemotherapy agents were measured.Workloss days and costs consisted of disability and medically related absenteeism. Workloss costs during the 12-month study period included actual employer payments for disability days plus imputed costs for medically related absenteeism. Medically related absenteeism costs were imputed by multiplying the number of days with medical services resource use by the employee's wage: each hospitalization day accounted for a full day of workloss, and outpatient visits accounted for half a day of workloss. As with healthcare costs, total workloss costs were not limited to only those related to a particular condition.Patient characteristics included demographics, employment status (employee versus nonemployee), cancer type, cancer stage (metastatic versus not metastatic), and comorbidities identified using claims during baseline period and the 12-month study period. Metastatic cancer was defined using claims with diagnoses for metastases (ICD-9-CM codes 196.0-199.1). The CCI was calculated from the claims data and individual physical comorbidities included in the index which were also identified . t-tests. Excess costs of CAPN cases compared with controls  were compared between diabetic and nondiabetic patients using a 2-sample t-test. All analyses were conducted using SAS version 9.1 . P-values less than or equal to 0.05 were considered statistically significant.Baseline characteristics  were summarized as proportions of the sample with the characteristic. Continuous measures  were summarized by mean and standard deviation. Comparisons of matched pairs of categorical variables used McNemar tests. Comparisons of the differences in continuous measures between cases and controls used paired The main study sample for the healthcare cost and resource use analysis contained 454 cases and controls without diabetes see . The diaP = 0.0412) and uncomplicated hypertension , whereas the control group had a higher rate of complicated hypertension . Resource use was generally the same between cases and controls; however, there were more cases with neurology specialist visits than controls . The number of neurology visits and neurology costs were not significantly different.Tables P < 0.0001). There were no differences in the classes of chemotherapy agents used by cases and controls. The most common chemotherapeutic agents used by cases and controls were taxanes  and platinums . Significantly more cases used a CAPN-related drug than controls . Cases had substantially higher rates and amounts of use of both inpatient and outpatient visits during the study period. More cases were hospitalized at least once compared with controls . CAPN cases had significantly higher rates and amounts of use for all outpatient components except for primary care visits and lab/pathology. More cases saw a neurologist than did controls .CAPN cases had significantly higher rates and counts of comorbidities and resource use during the study period compared with matched non-CAPN controls (Tables P < 0.0001). Mean excess annual per patient healthcare costs for cases versus controls were $36,660 for head and neck cancer, $18,790 for nonsmall cell lung cancer, $16,940 for breast cancer, and $5,140 for ovarian and all were statistically significant (data not shown). Cases had significantly higher component costs compared with controls. Outpatient costs were the highest component for both cases and controls. However, the excess costs of outpatient and inpatient components were similar with cases having excess annual outpatient costs of $8,092 per patient (P < 0.0001) and excess annual inpatient costs of $7,552 per patient (P < 0.0001). Annual oncology-related costs  were $22,453 for cases compared with $19,362 for controls, with the majority of costs being chemotherapy costs . CAPN-related drug and medical costs accounted for approximately 2% of total healthcare costs.There were no statistically significant differences in workloss measures between the subset of 78 employees with CAPN and their matched non-CAPN controls . More caAverage annual workloss costs were approximately 25% higher for cases than controls  with a $2,255 annual per patient difference . Cases hP = 0.0374), complicated hypertension , and a higher CCI than controls . Resource use was generally well balanced; however, cases did have more primary care visits than controls . There were no statistically significant differences in baseline costs.Tables P = 0.0236). More cases were hospitalized compared with controls  though the days per capita were not statistically different . Six times as many cases had a neurology specialist visit during the study period compared with controls . Cases also had 18.4 more outpatient visits during the study period on average than the controls (P < 0.0001). CAPN-related drugs were used by 93% of cases compared with 65% of controls (P = 0.0003).Cases had significantly higher rates and counts of resource use during the study period compared with controls . The diabetic case's annual per capita excess costs were higher than the control's costs , however, this difference was not statistically significant.For cases, annual per capita healthcare costs were $76,555 versus $54,816 per control, with an excess annual per patient cost of $21,739  experience a large burden in terms of out-of-pocket costs  and time spent on medical care.CAPN can lead to increased costs as a result of services specifically aimed at mitigating the PN  and secondary effects, such as switches in chemotherapy regimens or exacerbated cancer. This study separately estimated the costs for CAPN-related services and found they accounted for less than 2% of healthcare costs during the 12-month study period. While a significantly higher proportion of CAPN patients did use the pharmacologic treatments used to manage neuropathic symptoms, these drugs are often generic or lower cost relative to cancer treatments and overall healthcare costs. CAPN-related medical costs were low suggesting that the excess utilization demonstrated by cases was not directly attributed to CAPN. However, the CAPN-related costs as measured here may understate the true burden related specifically to CAPN. First, because there are no specific diagnoses for CAPN, PN-related diagnoses may be underreported on claims. In addition, the secondary effects of CAPN mentioned above are not included in the CAPN-related subset of costs presented here but rather are included in the chemotherapy, drug, and medical costs.Overall, this study finds that patients with CAPN experience significantly increased costs and resource use. It is important not to conclude that all of the excess costs are caused by CAPN. Since the cases and controls were matched during the baseline period, the results suggest that CAPN is a serious condition that should be carefully monitored in clinical practice. Improvements to the diagnostic tools for CAPN severity and investigation of therapies that treat CAPN without negatively impacting the cancer treatment could benefit both patients and payors.Calhoun's pilot study of chemotherapy-induced toxicity found that the direct CAPN costs for women with ovarian cancer are $688 for episodes up to 9 months, compared with our findings that CAPN-related costs are on average $1,320 over a period up to 12 months [Healthcare costs and resource use were calculated for diabetic CAPN patients as a secondary analysis as literature suggests that diabetes may exacerbate the risk and severity of CAPN. With the prevalence of diabetes increasing, it is important to assess how diabetic CAPN patients may differ from controls and nondiabetic CAPN patients. The results demonstrated that diabetes may, indeed, be associated with increased cost in patients with CAPN, as excess costs of diabetic CAPN patients were 25% higher than for nondiabetic CAPN patients. However, the comparison between diabetic and nondiabetic CAPN patients was not statistically significant, perhaps due to the small sample of diabetic CAPN patients. In addition, although diabetic cases were balanced with their controls in terms of costs and resource use, a higher proportion of cases had metastatic cancer and the CCI of cases was significantly higher than controls. Both these factors could impact the excess cost findings.This study is limited by the lack of clinical measures, which is common to research that uses claims data. While propensity score matching was used to adjust for many baseline group differences, the possibility exists of other confounding factors not available in this database. Moreover, because CAPN does not have a specific diagnosis code and is not consistently recorded in claims data, there are challenges in identifying the condition. Physicians may not report CAPN unless the impairment is severe enough to affect a patient's activities of daily living or warrant alterations to cancer treatment . Thus, tCAPN patients are associated with significantly higher healthcare costs and resource use in patients with breast, ovarian, head/neck, or NSCLC. The excess healthcare cost of CAPN is underestimated when only the cost for medical or pharmacy claims directly for CAPN is considered. Improvements in clinical assessments and treatments for CAPN would be useful for patients and payors.


In September 2010, an unusually high number of wild birds (partridges and pheasants) died in Cádiz in southwestern Spain. Reverse transcription PCR and virus isolation detected flavivirus infections. Complete nucleotide sequence analysis identified Bagaza virus, a flavivirus with a known distribution that includes sub-Saharan Africa and India, as the causative agent. An essential feature of certain emerging pathogens is their ability to expand their geographic ranges. Several arboviral diseases, particularly those caused by flaviviruses, have been found in new areas beyond their usual ranges. The best example of this phenomenon was introduction of West Nile virus into the Americas in 1999  in Spain that was caused by a flavivirus, Bagaza virus (BAGV). This virus was first isolated in Bagaza, Central African Republic, in 1966, from a pool of mixed-species female Alectoris rufa) died on several hunting properties in southwestern Cádiz, the southernmost province in Andalusia, Spain. Clinical signs included weakness, prostration, lack of motor coordination, weight loss, and white diarrhea. Some common pheasants (Phasianus colchicus) were also affected. These findings coincided in time with the first cases of West Nile virus (WNV) infection in horses detected in Spain  specific for lineage 1 and lineage 2 WNV , indicatwww.ncbi.nlm.nih.gov). Our isolates had >90% homology with 2 BAGV strains in GenBank .cDNA obtained after real-time RT-PCR (252-bp fragment) or heminested RT-PCR (214-bp fragment) amplification of samples was subjected to nucleotide sequencing. Resulting nucleotide sequences were compared with those in GenBank by using BLAST analysis . Two full-length genome sequences obtained from heart  and brain , were identical.To assess phylogenetic relationships between these new BAGVs and other flaviviruses, the complete BAGV sequence obtained in this study (accession no. HQ644143) or a partial envelope (E) protein–coding gene subregion were aligned with other complete flaviviral genomes in GenBank by using the ClustalW algorithm in MEGA5 (Flavivirus species (Bagaza virus). The tree based on the E region grouped Israel turkey meningoencephalitis virus and BAGV within the same cluster and showed that both viruses are closely related to Ntaya virus (Sequence of BAGV from Spain was closely related to the 2 unique full-length BAGV sequences available in GenBank and showed greater similarity with the strain from Africa (94.1% nt identity) than with the strain from India (92.8% nt identity). Identity between the isolates from Africa and India was 95.0%, indicating that they are more related to each other than to BAGV from Spain. A total of 742 nt and 637 nt differences (70 nt and 47 aa differences) were observed between BAGV from Spain and the isolates from India and Africa, respectively. Genetic relatedness between all 3 viruses was high (>92%), which indicates that they belong to the same ya virus .BAGV has been detected and isolated in Cádiz, Spain. It appeared to seriously affect partridges and, to a lesser extent, pheasants, and caused an unusually high number of deaths in these birds. No signs of infection and no deaths were observed for other bird species. However, whether other bird species are susceptible to disease caused by BAGV should be determined because this virus is similar to Israel turkey meningoencephalitis virus, a relevant pathogen for turkeys. Also, other vertebrates could be at risk for infection with this virus. Thus, experimental studies on the pathogenicity of this virus in specific vertebrates should be conducted.,Transcontinental spread of flaviviruses has been often associated with bird migrations (


Pteropus giganteus, n = 127) of 288 total serum samples, obtained from bats in 9 different taxa, neutralized lyssaviruses Aravan and Khujand. The infection occurs in bats in Bangladesh, but virus prevalence appears low.Lyssavirus surveillance in bats was performed in Bangladesh during 2003 and 2004. No virus isolates were obtained. Three serum samples (all from  Lyssavirus genus, family Rhabdoviridae. In the Americas, bats maintain the circulation of different lineages of rabies virus   and later placed at –80°C. Carcasses of representative specimens were placed in formalin for archival purposes.Bat brains collected in 2003 N = 212,  were tesThe presence of virus-neutralizing antibodies was determined by an adaptation of the rapid fluorescent focus inhibition test (RFFIT), as described . Four diNo evidence of lyssavirus antigen was detected in any bat brain by DFAT, and no neurotropic viruses were isolated by mouse inoculation. If a limited number of deaths occurred during the initial MIT (1 or 2 mice of 5 infected), those effects were not reproduced during the subpassage by filtration, which suggests that bacterial contamination of the field samples caused the death of mice during the initial MIT.Pteropus giganteus)   capturedLyssavirus genus (We have presented serologic evidence of lyssavirus infection in bats from Bangladesh. Antigenic cross-reactivity has been reported among Aravan, Khujand, and other members of the P. giganteus in Bangladesh. Thus, it is not surprising that all brain samples collected were negative for detection of lyssavirus antigen. Because different lyssaviruses and bat species are found in Asia, and therefore different virus-host interactions would be expected in the region, extrapolating antibody-positive/virus-positive ratios, as have been estimated from American (,Based upon this preliminary survey, a low prevalence of lyssavirus infection appears in Further surveillance for Asian bat lyssaviruses should be conducted. Public health authorities need to be aware of the potential for bats to transmit lyssaviruses, and public education of this potential should be enhanced. Frugivorous bats forage in fruit plantations in many regions of the Old World tropics, including Bangladesh. Indigenous people capture and may consume these animals. Direct contact between humans and bats frequently occurs during these interactions. Absence of current information on human rabies after bat exposure may be a result of inadequate education, incomplete surveillance, and lack of characterization of viruses from rabies cases (


LRR-RLP gene family in the model tree poplar (Populus trichocarpa). In total, 82 LRR-RLP genes have been identified in P. trichocarpa genome, among which 66 are organized in clusters of up to seven members. In these clusters, LRR-RLP genes are interspersed by orphan, poplar-specific genes encoding small proteins of unknown function (SPUFs). In particular, the nine largest clusters of LRR-RLP genes (47 LRR-RLPs) include 71 SPUF genes that account for 59% of the non-LRR-RLP gene content within these clusters. Forty-four LRR-RLP and 55 SPUF genes are expressed in poplar leaves, mostly at low levels, except for members of some clusters that show higher and sometimes coordinated expression levels. Notably, wounding of poplar leaves strongly induced the expression of a defense SPUF gene named Rust-Induced Secreted protein (RISP) that has been previously reported as a marker of poplar defense responses. Interestingly, we show that the RISP-associated LRR-RLP gene is highly expressed in poplar leaves and slightly induced by wounding. Both gene promoters share a highly conserved region of ~300 nucleotides. This led us to hypothesize that the corresponding pair of proteins could be involved in poplar immunity, possibly as a ligand/receptor couple. In conclusion, we speculate that some poplar SPUFs, such as RISP, represent candidate endogenous peptide ligands of the associated LRR-RLPs and we discuss how to investigate further this hypothesis.In plants, cell-surface receptors control immunity and development through the recognition of extracellular ligands. Leucine-rich repeat receptor-like proteins (LRR-RLPs) constitute a large multigene family of cell-surface receptors. Although this family has been intensively studied, a limited number of ligands has been identified so far, mostly because methods used for their identification and characterization are complex and fastidious. In this study, we combined genome and transcriptome analyses to describe the  Plants possess cell-surface receptors able to recognize extracellular molecules for subsequent intracellular signaling  family is constituted of small peptides (~23 amino acids) encoded by larger pro-AtPep precursor proteins of ~100 amino acids , which are often cleaved from larger precursor proteins of unknown function (~100–200 amino acids) and released into the apoplast where they function as messengers . Moreover, transcriptomic data highlighted a coordinated expression for a pair of physically associated LRR-RLP and SPUF genes in response to wounding, suggesting that the gene products might be functionally linked.Here, we report the genome-wide analysis of the LRR-RLP genes were identified in the poplar genome  are depicted in Figure A total of 82 me Table . This vas Figure . Sequencd Figure . LRR-RLPLRR-RLP-associated genes  in the poplar genome sequence, and found a total of 375 LRR-RLP-associated genes (Table LRR-RLP-associated genes) encode small proteins (less than 200 amino acids) of unknown function that we abbreviated SPUF  and chromosome 12 . Both the SPUF and the LRR-RLP from chromosome 12 are the closest paralogs of SPUFs and LRR-RLPs from the cluster 15    Figure . POPTR_0) Figure . Among t) Figure . Interess Figure . Similar levels <000 , which compiles expression data from rust-infected leaves, mature leaves, leaf disks, seeds, stems and suspension cells from poplar. Expression data were available for 47 LRR-RLP and 36 SPUF genes. A large majority (42 LRR-RLPs and 34 SPUFs) was expressed at low levels and did not exhibit any particular expression profile or obvious regulation in the analyzed conditions .To extend our survey of s Figure . NeverthLRR-RLP and SPUF genes that likely participate to defense mechanisms, we compared transcript expression profiles between excised poplar leaf discs and entire leaves  by using whole-genome poplar oligoarrays. As expected, the expression of genes classically associated with wounding such as wound-response proteins, chitinases, or protease inhibitors were induced in leaf discs   that were slightly (<2 fold) induced by wounding  have an inducible expression in response to stresses like wounding or pathogen attack  Figure . This ob) Figure . Similarg Figure . Interesg Figure . Second,RISP and LRR-RLP9 genes have been analyzed to identify putative common regulatory elements explaining the observed co-expression. Whereas we did not detect particular signature or conservation in the 0 to −700 nucleotide region, we identified a stretch of ~300 bp with almost 90% nucleotide identity in the −700 to −1000 region  seem to be present at different positions in the poplar genome , unresolved scaffold 580, a poorly assembled part of poplar genome, we sought to investigate further the physical association between RISP and LRR-RLP580 by using a combination of PCR amplification and sequencing approaches  and a reverse primer that cannot discriminate between the regions downstream the LRR-RLP9 and LRR-RLP580 genes (primer 4), both sequences being too similar , we amplified, from the 8 kb PCR product as a template, two fragments of ~5 and 3 kb, respectively, . RISP and RISP-like proteins show 63% identity and both carry a conserved predicted signal peptide Figure . In the e Figure . Conside Figures , 6A. Usir Figure . By usin, Figure . Sequencs Figure . AltogetLRR-RLP genes are arranged into large clusters which are interspaced by genes coding SPUFs. Based on the observed co-regulation upon wounding of RISP and its physically associated LRR-RLP and on the presence of similar regulatory elements in their promoter regions, we hypothesize that both gene products may be functionally linked and by extension we suggest that some SPUFs could represent candidate endogenous ligands of their genome associated LRR-RLPs. Below, we did not discuss the genomic organization of LRR-RLP in poplar as this is not different from other plant species, but we rather discuss how to validate LRR-RLP/SPUF interactions and whether such genomic analysis can be extended to other species.In this study, by analysing the LRR-RLP gene family in poplar, we observed that Prf gene clusters with several Pto resistance genes and it has been shown that the proteins physically interact . However, the density of SPUF genes in A. thaliana LRR-RLP clusters is lower compared with the average number of SPUFs in the predicted proteome (6 vs. 13%)  and known defense-related secreted proteins   Table , which wLRR-RLP genes have been identified in the P. trichocarpa “Nisqually-1” genome sequence, version 2.2, hosted on the Phytozome portal (http://www.phytozome.net/poplar) by amino acid sequence homology searches using tomato Cf-9 protein sequence as a query, then complemented with recursive searches with poplar LRR-RLP sequences representative from each clade (A–D). We considered only the LRR-RLP genes presenting the Cf-9 canonical domains and more than 15 LRRs , by using default parameters  . All protein-coding genes present in the 50 kb vicinity of poplar and A. thaliana LRR-RLP genes were inspected manually on the Phytozome portal. This limit was determined to maximize the genomic area investigated and to have a single LRR-RLP in the genome portion analyzed. All genes coding for a small protein of unknown function, defined as proteins with a size below 200 amino acids and with no annotation and no pfam available, were retained and designated as SPUF genes. Only 12 needed a manual annotation for start and stop codons  and SecretomeP 2.0 (http://www.cbs.dtu.dk/services/SecretomeP/) servers and amino acid sequence alignments were executed on the Multalin website . Poplar and A. thaliana SPUF paralogs have been searched in respective predicted proteomes by amino acid homology searches, whereas homologs in other plants as well as conserved domains have been searched in the non-redundant protein database at NCBI .on Table , Text S1ns Table . Signal P. trichocarpa × P. deltoides) leaf disks  collected at six different time-points from 18 to 48 h after inoculation with M. larici-populina (GSE39727 at NCBI GEO) were compared with strictly identical experiments performed on entire leaves . Poplar cultivation and inoculation procedures have been carried out as previously reported  and in the downstream conserved sequence of both LRR-RLP9 and LRR-RLP580 (primer 4: 5′GAGATGCTAATGGGATGAGGTTT3′). The LongRange PCR Kit (Qiagen) was used to amplify an 8 kb fragment using 25 ng of P. trichocarpa genomic DNA as a template. Two additional primers (primer 2: 5′CATGCAAGTGGTTATTGCTCA3′ and primer 3: 5′TGAGCAATAACCACTTGCATG3′) were designed in the conserved coding sequence of both LRR-RLPs and two fragments (using the 1–2 and the 3–4 primer pairs) were amplified from 1 ng of the purified 8 kb PCR fragment using the LongRange PCR Kit. The amplified fragments were purified with the QIAquick PCR purification kit (Qiagen) and directly sequenced according to GenomeLab Dye terminator cycle sequencing with Quick Start kit  on a Genetic Analysis System CEQ-8000 (Beckman Coulter). The sequence coding the extracellular domain of the chimera between LRR-RLP 580 and LRR-RLP 9 has been amplified from P. trichocarpa cDNAs using primer5: 5′ggggggCATATGTTGTCTTCAAATTTCTCCTCT3′; and primer6: 5′ggggggGGATCCTTATGCTTTCCATCCAAATCCATC3′ and cloned into the NdeI and BamHI restriction sites (underlined in the primers) of a pET28a vector before sequencing.Genomic DNA was isolated using the DNeasy Plant Mini Kit  from 100 mg of in silico analysis, transcriptome analyses, PCR cloning of the LRR-RLP extracellular part and drafted the manuscript. Stéphane Hacquard performed the PCR amplification/sequencing of the genomic fragment containing RISP and the LRR-RLP genes. Sébastien Duplessis performed the promoter analysis and contributed the unpublished RNA-Seq data. All authors contributed to the conceptual design of the experiments, participated in the writing of the manuscript, have read and approved the final manuscript.Benjamin Petre carried out the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cryptococcus gattii in a patient from New Mexico. The patient had no history of recent travel to known C. gattii-endemic areas. Multilocus sequence typing revealed that the isolate belonged to the major molecular type VGIII. Virulence studies in a mouse pulmonary model of infection demonstrated that the strain was less virulent than other C. gattii strains. This represents the first documented case of C. gattii likely acquired in New Mexico.We report a case of fatal disseminated infection with  Cryptococcus gattii was first isolated from a pediatric patient in the Congo Eucalyptus trees in Australia, where rural aboriginals have frequently been reported with infection C. gattii infections were only rarely reported from temperate climates of North America C. gattii infections has been reported from British Columbia (BC), and more recently from the U.S. Pacific Northwest (Washington and Oregon) C. gattii was previously believed to be a tropical fungus, these outbreaks demonstrate a larger ecological niche. Investigations into the source of the outbreak in the temperate climate of Vancouver Island and the Pacific Northwest (PNW) provide evidence that decaying wood from tree hollows, soil, fresh and saltwater may all be sources of C. gattiiC. neoformans and C. gattii are the only two major species of the genus Cryptococcus considered to be pathogenic in humans. Prior to the AIDS epidemic, cryptococcal infections were rare. Since the molecular characterization of C. neoformans and C. gattii and classification into separate species, ecological and clinical differences have been reported. C. gattii tends to be seen clinically in immunocompetent patients, although the spectrum of infection can involve immunocompromised patients as well, whereas C. neoformans is primarily an opportunistic pathogen. Both C. neoformans and C. gattii have a propensity to cause pulmonary and central nervous system (CNS) disease. In patients from Papua New Guinea and Australia, C. gattii is more likely than C. neoformans to cause cryptococcomas in the lung and brain, which may be mistaken for malignancies (as in this case) or abscesses C. neoformans, patients with C. gattii cerebral involvement were found to have more severe neurological sequelae such as hydrocephalus and focal CNS findings, including ataxia, hearing loss, altered mentation, papilledema, and higher intracranial pressures often requiring surgical procedures for management. Infections due to C. gattii follow a prolonged clinical course and are slower to respond to antifungal therapy; however mortality does not appear to be higher than C. neoformans infections The onset of cryptococcal infection is often subacute and nonspecific, with headache being the most common presenting symptom C. gattii infection in New Mexico, from a patient without a recent history of travel to a known C. gattii-endemic area. Because the patient was immunocompetent and New Mexico is not currently known to be a C. gattii-endemic region, the infection went unrecognized until autopsy.We report the first documented case of 9/L, normal range 4–10.6), and hyponatremia . A chest radiograph showed a focal infiltrate or mass estimated to be 4×8 cm in the lingula as well as additional perihilar infiltrates, particularly in the right upper lobe. A computed tomography of the head without contrast revealed no evidence of focal lesions, and an opacified left maxillary sinus suggestive of sinusitis. The patient was begun on empiric ceftriaxone and azithromycin for possible community-acquired pneumonia, and admitted for further evaluation of headache and confusion.The patient was a 56-year-old Hispanic man who was brought to the emergency room (ER) in late April 2010 for evaluation of a 2.5-month history of intermittent, progressively worsening headache. The patient was noted to have mild confusion. He reported no fever, chills, dyspnea, nausea, vomiting or diarrhea. He had returned home in February 2010 after release from a two-month incarceration in a state prison in Santa Rosa, NM. Upon entering his home, he found that his dogs had expired, and his house was extensively contaminated with dog feces. Several days after cleaning his house, he reported experiencing a headache which worsened over the next 2.5 months. In the ER, the patient was afebrile and his physical and neurological exam was unremarkable. Initial serum laboratory tests indicated mild leukocytosis  and tachycardic (P 129). He responded to naloxone and labetalol. On exam, pupils were noted to be initially unequal  but were reported equal after administration of naloxone. After becoming more alert, the patient stated that he had developed progressive, severe weakness over the past 3 weeks resulting in difficulty lifting his extremities. He was essentially bed-bound as a result. Furthermore, he reported intermittent, severe headache with associated neck pain. The patient also noted “ringing in his ears” for the preceding four days, with difficulty hearing anything else.A portable chest radiograph revealed no change in the lingular mass and new scattered areas of patchy infiltrate in both lung fields. A head CT without contrast was unchanged from the prior CT. Empiric intravenous vancomycin and piperacillin-tazobactam were given after the collection of blood cultures for treatment of possible healthcare-associated pneumonia. A repeat brain MRI the next day was also unchanged. Empiric azithromycin was added on day two of the patient's hospitalization. The patient was found unresponsive on hospital day two and expired shortly thereafter.A post-mortem examination was performed. Sections through the cerebral hemispheres demonstrated multiple, small vesicular lesions predominantly in the bilateral basal ganglia and also scattered throughout the bilateral white matter tracts . No lesiCryptococcus species  on June 23, 2010.Blood cultures collected on admission to the hospital as well as postmortem bacterial and fungal cultures of the lungs and heart blood grew C. gattii emergence in the Pacific Northwest U.S. and British Columbia (PNW), MLST analysis, using the seven loci of the consensus MLST typing scheme was performed C. gattii isolates from the U.S. and neighboring Mexico were analyzed and a neighbor-joining dendrogram was constructed based on the seven concatenated MLST sequences  than mice infected with R265 or H99 .Our findings represent the first documented case of C. gattii infection. However, due to the initial clinical impression of lung malignancy, the patient's immunocompetent status and lack of a travel history did not heighten clinical awareness of a cryptococcal infection in time to initiate antifungal therapy. Because the clinical presentation was most suggestive of a lung mass or malignancy, and the actual diagnosis is infrequently seen in this area, further testing for invasive fungal infection was not performed. The patient had widely disseminated infection with extensive inflammation, and likely succumbed to overwhelming systemic infection and sepsis, rather than elevated intracranial pressure with brainstem herniation, based on post-mortem findings. To date, C. gattii infections in the United States have been reported in Washington, Oregon, California, and North Carolina, in which all patients had some geographical exposure to endemic regions. C. gattii CNS disease who had no history of recent overseas travel C. gattii infections, suggest that there might be a broader distribution of the pathogen than is currently recognized.In general, cryptococcal disease appears to be rare in New Mexico. Between 2003 and 2007, we estimate there were at least one to two confirmed cases of cryptococcal disease in the Albuquerque area, which is home to the four largest hospitals in New Mexico. Starting in 2008, the number of confirmed cases increased with three cases in 2008, eight cases in 2009 and at least five cases to date in 2010. The autopsy report of this patient led to the diagnosis of disseminated C. gattii isolate indicates it does not represent the migration of the PNW outbreak strains southward to New Mexico. Because of the lack of a clonal relationship to isolates from Mexico, it also does not likely represent the recent migration of C. gattii isolates northward from Mexico. Rather, this isolate likely represents the emergence of a clonal genotype that, based on geography of the common isolates has probably persisted in the U.S. for quite some time. A recent literature review C. gattii are most common in South America but they are also found in North America, Central America, Australasia and Southeast Asia. It was also recently reported that the majority of C. gattii isolates from HIV+ patients in California are VGIII The non-VGII genotype of this C. gattii causes granulomatous pulmonary and disseminated disease in mouse inhalation models of infection, irrespective of the mouse strain used. In terms of genotypic variation in virulence, VGIIa isolates from the U.S. and from the Vancouver outbreak are more virulent in mice than VGIIb isolates from either region C. gattii cases reported to the CDC between 2004 and 2010, 50% were molecular type VGIIa, 10% were VGIIb, and only 3% were VGIII C. gattii infections are immunologically similar in mice and infected humans with respect to cytokine profiles C. gattii replicates intracellularly within macrophages. It has been demonstrated that intracellular replication rate within murine macrophages in vitro is correlated with in vivo virulence in mice in vitro and are more virulent in vivo than VGIIb isolates. Furthermore, intracellular replication rate in murine macrophages correlates very highly with the intracellular replication rate in human macrophages, suggesting there might be a correlation between genotype and human virulence and disease C. gattii R4569 and other isolates within murine and human macrophages will be needed to support this hypothesis. The major clinical point from these in vivo studies is that despite strain R4569 being less virulent in this mouse infection model is that it is still highly capable of causing severe morbidity and mortality, particularly in the absence of timely diagnosis and therapy.C. gattii infection in the U.S. due to VGIII isolates that may be unrecognized outside of HIV+ patients. However, the underlying level of infection due to C. gattii is currently unknown because many of the cases of cryptococcosis in the U.S. are diagnosed using an antigen test and most cases outside of the Pacific Northwest are assumed to be caused by C. neoformans. The heterogeneity of the VGIII isolates in this study, as compared to the almost exclusively clonal nature of the current VGII PNW emergence isolates, would be consistent with a long-term presence in the U.S. and a possibly unrecognized, long term, low level, endemicity. It is also interesting to note that among HIV+ patients in California in the 1990's almost all of the infections due to C. gattii were caused by isolates of the VGIII genotype Cryptococcus clinical isolates due to the implications for public health and for understanding the epidemiological distribution of this fungal pathogen. In addition, there is epidemiological evidence that there are substantive differences in the clinical presentation of C. gattii and the course of disease compared to C. neoformansBased on the few previous reports C. gattii increase both in the United States and worldwide, epidemiologic surveillance will play an important role in identifying new ecological niches or reservoirs for this emerging fungal pathogen. Since not all clinical and reference laboratories are equipped to differentiate between C. gattii and C. neoformans, a heightened clinical awareness of the disease will be required to prompt further identification and genotyping to track the distribution of C. gattii.As infections due to The authorized representative of the patient in this manuscript has given written informed consent (as outlined in the PLoS consent form) to publication of their case details. These animal experiments were approved by The University of Texas at San Antonio Institutional Animal Care and Use Committee (IACUC), approved protocol number MU021-11/1A2, and mice were handled according to IACUC guidelines. An IRB waiver was provided by the New Mexico Veterans Healthcare System Office of Research.URA5 sequencing as previously described Fungal identification was performed at the Fungus Testing Laboratory  using canavanine-glycine-bromthymol blue (CGB) agar and URA5, IGS, PLB1, SOD1, GPD1, LAC1, and CAP59 gene fragments were amplified as described by Meyer and colleagues et alC. gattii MLST database at mlst.mycologylab.org. The compiled sequences were compared to C. gattii sequences collected during the U.S. PNW surveillance (SRL) or during a previous surveillance in Mexico . Phylogenetic relationships, the neighbor-joining tree and bootstrap values were calculated using the Mega 4.1 software package C. gattii MLST database at mlst.mycologylab.org.The isolate was subtyped using The ISHAM consensus multilocus sequence typing (MLST) scheme d) mice, four to six weeks of age , were used throughout these studies. Mice were housed at The University of Texas at San Antonio Small Animal Laboratory vivarium and handled according to guidelines approved by the Institutional Animal Care and Use Committee.Female BALB/c (H-25 colony forming units (CFU) of C. neoformans strain H99 or C. gatti strains R265 (VGIIa), R272 (VGIIb), or R4569 (isolate from this case) in 50 µl of sterile PBS introduced directly into the nares. The strains R265 and R272 were provided by Joseph Heitman . The inocula used for nasal inhalation were verified by quantitative culture on yeast peptone dextrose (YPD) agar. The mice were fed ad libitum and monitored by inspection twice daily. Mice were either euthanized when moribund or at day 50 post-inoculation. For histopathology (three mice per group), lungs were transcardially perfused with PBS then inflated with 10% formalin via an intratracheal catheter. Lungs were tied off, dissected, and stored in formalin until paraffin embedding, sectioned, and stained by hematoxylin and eosin . For colony-forming unit (CFU) determination in surviving mice (four to five mice per group), lung tissues were excised using aseptic technique, homogenized in 1 ml of sterile PBS, and cultured by 1∶10 dilutions on YPD agar supplemented with chloramphenicol . CFUs were enumerated following incubation at 30°C for 48 h.Pulmonary cryptococcal infections were initiated by nasal inhalation as previously described


To determine prevalence of antibodies against Schmallenberg virus in adult cows and proportion of infection transmitted to fetuses, we tested serum samples from 519 cow/calf pairs in Belgium in spring 2012. Of cattle within 250 km of location where the virus emerged, ≈91% tested positive for IgG targeting nucleoprotein. Risk for fetal infection was ≈28%. Significance level was set at p<0.05.During February 13–April 22, 2012, serum samples were obtained at random from blood drawn by field veterinarians from 519 cow/calf pairs at 209 farms located in southeastern  of 471 , with a progressive decrease of apparent seroprevalence among calves born to seropositive cows: 28.4% , 21.0% , and 19.2% . Thus, the apparent rate of the virus crossing the placenta seems to decrease over time. Because the biology of the new virus would not have changed in a few weeks, this decreased rate of transmission could be attributed to the gradual increase in the relative proportion of cows that were infected before the development of the placenta enables placental transfer of the virus, i.e., before the 30th day of gestation (In conclusion, this study confirms that the emerging virus was absent from the area examined in spring 2011 and provides evidence that 1 year later almost all adult cattle had seroconverted. Furthermore, the results suggest that the risk for infection of the fetus in an immunologically naive herd is ≈28% and that in utero infections can occur without sequelae visible at birth if the infection occurs when the fetus’s immune system is mature enough to control virus spread. In the case of Akabane virus, the cow’s natural immunity prevents subsequent infections of the fetus (


In vitro HPAIV H5N1 infection of AM compared to that of macrophages cultured from monocytes resulted in a lower percentage of infected cells (up to 25% vs up to 84%), lower virus production and lower TNF-alpha induction. In vitro infection of AM with H3N2 or H1N1 virus resulted in even lower percentages of infected cells (up to 7%) than with HPAIV H5N1, while virus production and TNF-alpha induction were comparable. In conclusion, this study reveals that macrophages cultured from monocytes are not a good model to study the interaction between AM and these influenza virus strains. Furthermore, the interaction between HPAIV H5N1 and AM could contribute to the pathogenicity of this virus in humans, due to the relative high percentage of infected cells rather than virus production or an excessive TNF-alpha induction.Highly pathogenic avian influenza virus (HPAIV) of the subtype H5N1 causes severe, often fatal pneumonia in humans. The pathogenesis of HPAIV H5N1 infection is not completely understood, although the alveolar macrophage (AM) is thought to play an important role. HPAIV H5N1 infection of macrophages cultured from monocytes leads to high percentages of infection accompanied by virus production and an excessive pro-inflammatory immune response. However, macrophages cultured from monocytes are different from AM, both in phenotype and in response to seasonal influenza virus infection. Consequently, it remains unclear whether the results of studies with macrophages cultured from monocytes are valid for AM. Therefore we infected AM and for comparison macrophages cultured from monocytes with seasonal H3N2 virus, HPAIV H5N1 or pandemic H1N1 virus, and determined the percentage of cells infected, virus production and induction of TNF-alpha, a pro-inflammatory cytokine.  Alveolar macrophages (AM), which reside in the alveolar lumen, usually dampen down the host immune response to incoming pathogens. However, they are thought to increase inflammation during highly pathogenic avian influenza virus (HPAIV) H5N1 infections, which cause severe and often fatal disease in humans. This is based on experiments with human macrophages cultured from monocytes rather than with human AM. Here we show that human AM, collected via broncho-alveolar lavage from healthy volunteers, can become infected with HPAIV H5N1. However, this results in neither induction of the pro-inflammatory cytokine TNF-alpha nor virus production. Therefore, AM are most likely not responsible for the excessive cytokine response or high viral load during human HPAIV H5N1 infections as assumed previously. These data significantly changes our insight into the pathogenesis of HPAIV H5N1 pneumonia in humans, indicating that other cells than AM must be responsible for the excessive pro-inflammatory cytokine profile observed during HPAIV H5N1 infections. Seasonal, pandemic and zoonotic influenza A virus infections cause substantial morbidity and mortality in humans. Seasonal influenza virus infections in humans are usually mild, causing pneumonia in a minority of infected individuals. Pandemic influenza virus infections vary in their disease outcome. The 1918 Spanish flu caused pneumonia more often than the recent pandemic H1N1 (pH1N1) virus. Zoonotic influenza virus infections in humans vary from self-limiting conjunctivitis to severe, often fatal pneumonia. The currently ongoing outbreak of highly pathogenic avian influenza virus (HPAIV) of the subtype H5N1 in poultry is sporadically transmitted to humans, in which it causes severe pneumonia with a case-fatality rate of about 60% ex vivo lung cultures, influenza virus antigen was detected in type II pneumocytes and alveolar macrophages The differences in pathogenesis of different influenza virus infections in humans, and thereby their disease outcome are only partly understood. Early events after infection of the human alveoli—the site of pneumonia—contribute to protection against or the development of pneumonia. Previously we have shown that different influenza viruses attach to different cell types in the human alveoli. Seasonal influenza viruses and pH1N1 virus attach predominantly to type I pneumocytes Lung macrophages can be distinguished in alveolar, pleural, interstitial and intravascular macrophages, but the AM is the most important cell of the innate immune system in the human alveoli The AM has a protective role in the lung to prevent loss of function. Therefore, immune responses need to be balanced to protect the lungs from invading pathogens without excessive inflammation in vitro infections of macrophages cultured from monocytes in the presence of human serum (MM). In MM, HPAIV H5N1 infects up to 100% of the cells, which results in a productive infection. Infection of MM with HPAIV H5N1 also results in an excessive immune response, marked by the high induction of the pro-inflammatory cytokine TNF-alpha, which is not observed after infection with seasonal H1N1 or H3N2 virus The role of the AM in the pathogenesis of HPAIV H5N1-induced pneumonia has been studied by ex vivo cultured lung biopsies. Also we examined whether monocytes cultured in the presence of GM-CSF (GM-MM)—which is abundantly present in the alveolar lining fluid—instead of human serum would develop a phenotype more similar to that of the AM To address this question, we compared the effect of seasonal H3N2 virus, pH1N1 virus or HPAIV H5N1 infection both on AM from broncho-alveolar lavages (BAL) of healthy volunteers, and on MM. In addition, we determined the percentage of AM infected in experimentally infected ex vivo lung cultures, although percentages varied largely. In the double-staining, influenza virus antigen was present in the nucleus, indicative for virus replication, whereas HAM65, a macrophage marker, was present in the cytoplasm  . This inThis is the first time that the role of AM during HPAIV H5N1 virus infection has been studied. This study shows that AM are more susceptible to HPAIV H5N1 infection than to seasonal H3N2 or pH1N1 virus infection but, that this infection results neither in virus production nor in an excessive immune response.Macrophages cultured from monocytes do not respond the same way as AM to two out of three influenza viruses included in this study: there are significant differences between MM and AM in the percentages of cells infected, cytokine response and virus production after infection with HPAIV H5N1 and seasonal H3N2 virus. Therefore, MM are not suitable to study the interaction between AM and these influenza virus strains.ex vivo lung cultures and in AM collected from BAL. This fits with the results of our virus attachment studies, in which HPAIV H5N1 attached more abundantly to AM than H3N2 virus HPAIV H5N1 infected more AM than seasonal H3N2 or pH1N1 viruses, in both the HPAIV H5N1 infection in AM did not result in significantly higher induction of TNF-alpha mRNA compared to seasonal H3N2 or pH1N1 virus. This is in contrast with the significantly higher TNF-alpha mRNA levels in MM after HPAIV H5N1 virus infection compared to seasonal H3N2 and pH1N1 virus infection observed in our and previous studies TNF-alpha, and the cascade it induces, is known to attract neutrophils and other leukocytes in the lung and is thought to play an important role in the pathogenesis of acute respiratory distress syndrome and multiple organ dysfunction syndrome The lack of virus production in AM after infection with influenza viruses included in this study corresponds with a previous study in which no virus release in AM was detected after infection with a seasonal H1N1 or H3N2 virus During influenza virus pneumonia, there is an influx of a variety of leukocytes, including monocytes Interestingly, macrophages that were cultured in the presence of GM-CSF, resembled AM in shape, but differed in their response to influenza virus infection. In fact, the percentages of cells infected of GM-MM resembled that of the MM more closely. Although GM-CSF is abundantly present in the human alveolus and is thought to be responsible for the AM phenotype Overall, the pH1N1 virus used in this study did not infect high percentages of either AM or MM. Even compared to seasonal H3N2 virus, pH1N1 virus infected lower percentages of cells. Whether this is a unique feature of pH1N1 virus or a common feature of pandemic influenza viruses remains to be determined. Overall, this observation fits with the relatively mild disease caused by pH1N1 virus infection In conclusion, we have shown that the MM are unsuitable to study the interaction between AM and these influenza virus strains, which might be due to their difference in phenotype; alternatively activated versus classically activated. In contrast to MM, AM do not induce excessive TNF-alpha after HPAIV H5N1 infection. However, AM are more abundantly infected by HPAIV H5N1 than by seasonal H3N2 virus or pH1N1 virus. We speculate that this relatively high percentage of AM infection by HPAIV H5N1 may contribute to the unusual high pathogenicity of HPAIV H5N1 for the human lung.The collection of AM via a broncho-alveolar lavage was approved by the Dutch Medical Ethical Committee . All study participants provided written informed consent for the collection of samples and subsequent analysis.Viruses used for these experiments were seasonal H3N2 virus (A/Netherlands/213/03), a pandemic H1N1 virus (A/NL/602/09) and a HPAIV H5N1 (A /Vietnam/1194/04). All virus stocks were prepared on Madin-Darby Canine Kidney cells (MDCK).ex vivo infections. Biopsies were made with a 3-mm-biopsy punch and cultured overnight in F12K  supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine and 5% FCS. After washing, to remove FCS, lung biopsies were infected with 107 tissue culture infectious dose (TCID)50/ml of seasonal H3N2 virus, pH1N1 virus or HPAIV H5N1 at room temperature on a rocker for 1 hour. Lung biopsies were washed and cultured in F12K supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine at 37°C in 95% O2 and 5% CO2. After 24 hours, biopsies were collected in 10% neutral-buffered formalin, embedded in paraffin, and sectioned at 3 µm.Surgically removed lung tissues, without any histological evidence for respiratory disease, were used for All lung biopsy sections were stained for pancytokeratin to determine the condition of the alveolar epithelium. Lung biopsy sections of which the epithelium in more than 50% of the alveoli was desquamated were excluded. Lung biopsy sections infected with seasonal H3N2 virus (n = 5) pH1N1 virus (n = 3), HPAIV H5N1 (n = 6), or uninfected lung biopsy sections were stained for influenza A virus nucleoprotein as described earlier 1 >85% predicted, Tiffeneau-index >0.7), and a normal thorax X-ray underwent a BAL. The BAL was performed according to standard clinical procedures. A total of 3 times 50 ml warm, sterile 0.9% NaCl was instilled and aspirated sequentially. The middle pulmonary lobe was selected for sampling in all cases.Six non-smoking volunteers (age >18 years) free of any respiratory symptoms, with a normal lung function  supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine. After 2 hours, non-adherent cells were removed. To confirm that cells isolated were macrophages, cells were stained for CD68  using the same protocol as for the influenza nucleoprotein staining described below.6 cells/ml in teflon flasks  in RPMI-1640  supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine and 5% human AB serum. After 7 days, monocyte-derived macrophages (MM) from each donor were harvested from the teflon flask and seeded independently in flat-bottom culture plates. After 2 hours, non-adherent cells were removed. Cells were stained for CD68 as described below.From each donor that underwent the BAL, peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood using a Lymphoprep gradient (density 1.007±0.001 g/ml (Axis-shield PoC AS)). Subsequently, monocytes were purified using a Percoll gradient ). Monocytes from each donor were cultured in suspension at a concentration of 1*10g. The top layer, mainly consisting of thrombocytes, was discarded. After this extra procedure, PBMC and monocytes were subsequently isolated and purified as described above. For comparison, from each blood bank donor, half of the monocytes were cultured in teflon flasks as described above, and the other half was cultured in RPMI supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 5% FCS and GM-CSF . GM-CSF cultured MM (GM-MM) were harvested after 7 days. Cells were stained for CD68 as described below.Blood was diluted in PBS and centrifuged for 10 minutes at 220 AM (n = 6), MM  and GM-MM (n = 4) were seeded in a concentration of 50 000 cells per wells. MM obtained from peripheral blood of the AM donors were compared directly to AM from the same donor in below infection experiments. Cells were infected with a multiplicity of infection (MOI) of 2 with seasonal H3N2 virus, pH1N1 virus or HPAIV H5N1 for 1 hour. After 8 hours post infection (hpi), cells were fixed in the plate with 80% acetone and stored at −20°C. Plates were thawed, and washed with PBS before incubation with the primary antibody, a mouse-anti-influenza A nucleoprotein (Hb65) followed by peroxidase-labeled goat-anti-mouse IgG2a. Peroxidase was revealed using AEC resulting in a red precipitate. Influenza virus positive and -negative cells were counted in five 40x high power fields.AM (n = 6), MM (n = 6) and GM-MM (n = 4) were seeded in a concentration of 100 000 cells per well and infected with a MOI of 0.1 with seasonal H3N2 virus, pH1N1 virus or HPAIV H5N1 for 1 hour. Supernatants were sampled at 4, 8 and 24 hpi. Virus titrations on these supernatants were performed by end-point titration in MDCK cells as described previously 5′-TCCACTGGCGTCTTCAC, reverse primer 5′-GGCAGAGATGATGACCCTTTT for GAPDH; forward primer 5′-GGCAAAGATCCAAGATAAGGAA, reverse primer 5′-GGACCAAGTGCAGAGTGGAC for UBC; forward primer 5′-GAGCCCAAGGGCTACCAT, reverse primer 5′-GGGTTATGCTGGTTGTACAGG for TGF-beta; forward primer 5′-CAGCCTCTTCTCCTTCCTGAT, reverse primer 5′-GCCAGAGGGCTGATTAGAGA for TNF-alpha. Probes from the universal probe library from Roche were used; probe 45 for GAPDH; probe 11 for UBC; probe 29 for TNF-alpha and TGF-beta.AM (n = 6 per treatment) and MM (n = 6 per treatment) were seeded in a concentration of 100 000 cells per well and infected with an MOI of 2 with seasonal H3N2 virus, pH1N1 virus or HPAIV H5N1 for 1 hour, or exposed to 100 ng/ml LPS for 1 hour. At 8 and 24 hpi, cells were harvested in lysis buffer, and mRNA was isolated using an mRNA capture kit  according to manufacturer's instructions. Subsequently, cDNA was made using Superscript III reverse transcriptase (Invitrogen). Taqman analysis was used to determine target gene RNA expression as described previously Statistical analyses were performed using SPSS 15 software. The Wilcoxon test was used for analysis of paired samples, and the Mann-Whitney test for unpaired samples.TNF-alpha – NM_000594.2 (NCBI); TGF-beta – NM_000660.3 (NCBI); GAPDH – NM_002046.3 (NCBI) and UBC – NM_021009.4 (NCBI).


B cells and humoral immune responses play an important role in the pathogenesis and diagnosis of multiple sclerosis (MS). A characteristic finding in patients with MS is a polyspecific intrathecal B cell response against neurotropic viruses, specifically against measles virus, rubella virus, and varicella zoster virus, also known as an MRZ reaction (MRZR). Here, we correlated from the routine clinical diagnostics individual IgG antibody indices (AIs) of MRZR with magnetic resonance imaging (MRI) findings in patients with first MS diagnosis.U test) and the sum of lesions on cMRI and sMRI (p = 0.0413). Varicella zoster virus AI also showed a correlation with T2 lesion load on cMRI but did not reach statistical significance (p = 0.2893).MRZR was determined in 68 patients with a clinically isolated syndrome (CIS) or early relapsing-remitting MS (RRMS). Absolute AI values for measles virus, rubella virus, and varicella zoster virus were correlated with T2 lesion load and gadolinium enhancing lesions on cerebral MRI (cMRI) and cMRI combined with spinal MRI (sMRI). Measles virus AI correlated significantly with T2 lesion load on cMRI (p = 0.0312, Mann-Whitney The results confirm MRZR as part of the polyspecific immune reaction in MS with possible prognostic impact on MRI and clinical parameters.Furthermore, the data indicate that intrathecal measles virus IgG production correlates with disease activity on cMRI and sMRI in patients with early MS. Great effort has done into defining prognostic markers in multiple sclerosis (MS) over recent years. To date, a combination of clinical, cerebrospinal fluid (CSF) and MRI variables are used to predict disease course. Research has shown high T2 lesion load on cMRI at diagnosis is associated with a more active disease course CSF is a promising source of biochemical markers in MS, as its compounds are relevant not only in the diagnosis of MS but may also reflect disease activity. Detection of oligoclonal bands in CSF and at least two positive AI against measles virus, rubella virus and varicella zoster virus of ≥1.5 (MRZR), are important markers in the diagnosis of MS. In 80–100% of patients with MS, a polyspecific intrathecal B cell response in the form of a positive MRZR is detectable in CSF and is accepted as being highly specific for this disease In the present study, we correlated from the routine clinical diagnostics absolute AIs for measles virus, rubella virus, and varicella zoster virus (VZV) with magnetic resonance imaging (MRI) parameters of disease activity in patients with a first diagnosis of clinically isolated syndrome (CIS) or early RRMS.61patients with relapsing-remitting MS according to the revised McDonald criteria from 2010 Antibodies against measles virus and VZV were measured in serum and CSF samples using commercial enzyme-linked immunosorbent assays  according to the manufacturer's instructions. Antibodies against rubella virus were also measured by ELISA . A standard curve consisting of serial dilutions of a standard human plasma  was included on each ELISA plate. The same batch of SHP was used for all experiments. Total albumin and IgG concentration in serum (IgG (serum)) and CSF were measured nephelometrically .spec CSF/IgGspec serum)/ = Qspec/QIgG if QIgG<QLim, or AI = Qspec/QLim if QIgG>QLim. QLim was calculated as described previously . AI values ≥1.5 were considered as indicative of an intrathecal virus-specific antibody production.AI values were calculated either with the formula: AI =  axial slices with and without application of gadolinium as well as T2-weighted SE axial slices. Hyperintense lesion on T2-weighted MRI lesions >3 mmStatistical analysis: Statistical significance of differences in absolute AI values between patients with high and low lesion loads in T2-weighted images and patients with and without Gd-enhancing lesions were analysed by Mann-Whitney U test using GraphPad Prism 5 software. P values <0.05 were considered significant.The demographic data of all patients included in this study are shown in But rubella virus AI also showed a correlation with Gd-enhancing lesions on combined cerebral and spinal MRI but did not reach statistical significance (p = 0.0611). Furthermore, none of the other AIs correlated with the presence of Gd-enhancing lesions see .We also performed a Spearman Rank Correlation for single AIs and the number of T2 or Gd-enhancing lesions, which did not yield any statistically significant results see .We investigated the relationship between the levels of the individual IgG AIs that form the MRZ reaction and quantitative MRI outcomes in patients with MS. Higher anti-measles AI levels were associated with a higher T2 lesion load in cMRI and sum of T2 lesions in cerebral and spinal MRI. There was no link between individual MRZ-IgG AIs and Gd-enhancing lesions as a marker of acute inflammation on cMRI.MRZ reaction in CSF is positive in chronic CNS autoimmune diseases (e.g. neurolupus or CNS-vasculitis), but has been proposed as a specific marker for autoimmunity in MS On the other hand MRI T2 lesion load has an important clinical impact in diagnosis of MS and therapeutic decisions. The number of lesions in CIS correlates with the risk of conversion to clinically definite MS and is used to monitor disease activity In conclusion, our data suggest that measles AIs correlate with MRI markers in early MS and increased measles AI levels are a marker of increased disease activity. The study underlines the relevance of humoral immunity in MS pathogenesis and supports MRZR as an important tool not only in MS diagnosis but also to estimate disease activity. Additional longitudinal studies are needed to investigate the relationship between MRZ AIs and MRI parameters on one hand and clinical aspects like neurocognitive function and disability progression on the other.Figure S1Measle virus IgG AI and T2 lesion load in MRI. Correlation between number of T2 lesions in cerebral MRI (A), including 67 patients, and sum of lesions in cerebral and spinal MRI (sMRI) (B) from 43 patients with IgG AI for measles virus.(TIF)Click here for additional data file.Figure S2Correlation between symptom duration and total intrathecal IgG synthesis. Correlation of time between first symptoms (FS) and lumbal puncture (LP) and total intrathecal IgG synthesis from 65 patients.(TIF)Click here for additional data file.Figure S3Total intrathecal IgG and T2 and Gd-enhancing lesion load in MRI. Comparison of mean total intrathecal IgG for patients with <6 lesions and ≥6 lesions in cMRI (A) and both cMRI and sMRT (B). Comparison of mean total intrathecal IgG for patients with Gd-enhancing and no Gd-enhancing lesions in cMRI (C) and both cMRI and sMRI (D).(TIF)Click here for additional data file.


To the Editor: West Nile virus (WNV) infections were reported in horses in Morocco in 1996, 2003 , and 31% were male. Cohort B consisted of 200 persons living in the region of Rabat , where the WNV outbreaks among horses were described in 1996 (5 cells/mL) of a Vero cell suspension with 5% fetal calf serum. This mixture was incubated for 5–6 days at 37°C until cytopathic effects were observed in a negative control well containing a 50% tissue culture infectious dose of virus. Serum samples were screened in duplicate at dilutions of 1:6, 1:18, and 1:54. Samples that neutralized the virus, characterized by absence of cytopathic effects, at 1 of the dilutions tested were retested in 4 replicates to confirm the result. We then titrated the samples by testing 6 serial dilutions ranging from 1:6 to 1:1,458. Titers were calculated by using the Spearman-Kärber method  had WNV neutralizing bodies . Titers determined by the micro virus-neutralization test ranged from 18 to 2,630 .The prevThe low prevalence of WNV neutralizing antibodies  in persons from Meknes (cohort A) suggests a low level of WNV circulation in the area. This finding is likely related to the ecosystem of this region , which is unfavorable for survival of the vectors. Infections are more likely the result of travel to areas where WNV is endemic.In persons from Rabat (cohort B), the medium prevalence reported  confirms human infection with epidemic WNV strains found in horses during WNV epidemics of 1996 and 2010. However, this prevalence is lower than that for persons in Kenitra (cohort C) . High titers were obtained in cohort C, which is located in an extremely humid area that includes several wetlands and rice fields flooded through regulated channels from the Sebou River. In addition, this region includes 2 natural bird reserves along a principal migratory Europe–sub-Saharan route. These conditions are favorable for WNV circulation and are likely related to the high prevalence of WNV infection registered in this region.We found evidence for local circulation of WNV in Morocco. The Moroccan WNV strains most often cause mild and self-limiting illnesses. These illnesses are difficult to distinguish from many other febrile illnesses, making it less likely that viral testing would be performed for WNV. Our results show that WNV is an emerging disease in Morocco, and a national response plan should be implemented by public health authorities.