Human enteric virus infections range from gastroenteritis to life threatening diseases such as myocarditis and aseptic meningitis. Rotavirus is one of the most common enteric agents and mortality associated with infection can be very significant in developing countries. Most enteric viruses produce diseases that are not distinct from other pathogens, and current diagnostics is limited in breadth and sensitivity required to advance virus detection schemes for disease intervention strategies. A spectroscopic assay based on surface enhanced Raman scattering (SERS) has been developed for rapid and sensitive detection of rotavirus. The SERS method relies on the fabrication of silver nanorod array substrates that are extremely SERS-active allowing for direct structural characterization of viruses. SERS spectra for eight rotavirus strains were analyzed to qualitatively identify rotaviruses and to classify each according to G and P genotype and strain with >96% accuracy, and a quantitative model based on partial least squares regression analysis was evaluated. This novel SERS-based virus detection method shows that SERS can be used to identify spectral fingerprints of human rotaviruses, and suggests that this detection method can be used for pathogen detection central to human health care. Group A rotaviruses are the leading cause of acute severe gastroenteritis in infants and young children worldwide with approximately 130 million children infected each year. This accounts for approximately 1/3 of all hospital admissions each year for diarrheal disease and is estimated to be responsible for over 500,000 deaths, 2 million hospitalizations, and 25 million clinic visits each year Rotaviruses are non-enveloped icosahedral particles containing 11 segments of dsRNA Commercial immunochromatographic assays and enzyme immunoassays are available for routine laboratory diagnosis of rotavirus in a clinical setting. These assays capitalizes on the conserved nature of VP6 among all group A rotaviruses; however, these tests provide no information on the genotypes, i.e., G and P types, which is essential for monitoring epidemics, identifying novel strains, and in controlling disease. Typing of rotavirus strains is achieved using genotype specific monoclonal antibodies (mAbs) in an ELISA assay, and by hemi-nested multiplex RT-PCR 4 to 1014 with respect to normal Raman intensities. Importantly, SERS retains all of the benefits of normal Raman spectroscopy while providing a markedly improved sensitivity, and as a result, SERS has advanced as the spectroscopic tool of choice for whole-organism fingerprinting Surface enhanced Raman spectroscopy (SERS) provides the ability to rapidly detect analytes with chemical specificity intrinsic to vibrational spectroscopy and is emerging as an important tool in bioanalytical applications including identification and classification of pathogenic organisms Bacillus subtilis and Escherichia coli; however in each report an incongruent spectral fingerprint was indicated 8 with less than 15% variation in SERS intensity from batch-to-batch The majority of SERS-based detection assays have been developed for bacteria a priori knowledge of the calibration sample identities to aid in the discrimination of classes, i.e., pathogens The method of data analysis is also a critical aspect of any diagnostic assay, particularly for vibrational spectroscopy. It is important to analyze the entire spectrum, or use specialized feature selection algorithms, since discrete patterns of multiple bands, rather than a single peak, are important for identification. Principal component analysis (PCA) is the most frequently employed multivariate technique used to reduce the dimensionality of the spectral dataset, reduce noise, and maximize total spectral variance among spectral fingerprints for each infectious agent Previously we have demonstrated SERS-based detection and differentiation of influenza, adenovirus, and respiratory syncytial viruses (RSV) using these OAD substrates Eight laboratory strains representative of the most commonly identified G and P genotypes were propagated in MA104 cells purchased from ATCC (CRL-2378) in the presence of trypsin for SERS-based evaluation, these are detailed in 2(g) before loading into a custom-designed, high vacuum electron beam evaporation (E-beam) system. Thin films of Ti (20 nm) and Ag (500 nm) were evaporated onto the substrates at a rate of 0.2 nm/s and 0.3 nm/s, respectively, with the incident vapor normal to the substrate surface. The Ti served as an adhesion layer. The substrates were then rotated by 86° with respect to the incident vapor. Ag nanorods were grown at this oblique angle at a rate of 0.3 nm/s until a quartz crystal microbalance (QCM) registered 2000 nm. The QCM was used to monitor the thickness of the film growth in-situ, and was positioned such that it directly faced the incident vapor. As reported elsewhere 2, and tilt angle of 71° with respect to the substrate normal.The oblique angle deposition (OAD) of aligned silver nanorod arrays as SERS substrates has been previously described −1 in the ExtendedScan mode using three coadded 10 s accumulations. A 1.0-µL aliquot of intact virus was applied to the Ag nanorod array substrate and allowed to evaporate at room temperature prior to spectrum acquisition.SERS spectra were acquired using a Renishaw inVia confocal Raman microscope system  equipped with a 785 nm near-infrared diode laser as the excitation source. Light from the high power (300 mW) laser was attenuated to ∼7 mW at the sample surface using a series of neutral density filters and focused into a ∼115 µm ×11 µm spot using a 5× objective. SERS spectra were acquired from 400–1800 cmst derivative, 15 point, Savitzky-Golay). These spectral processing steps facilitate visual comparison of the Raman peak positions for spectra collected at different locations on a SERS substrate and for different substrates.Preliminary studies were designed to assess the utility of the Ag nanorod substrates to generate SERS spectra of rotaviruses and to evaluate the reproducibility of the method. For these studies, spectra of rotavirus samples were either baseline corrected using a concave rubber band algorithm  computed with 10 iterations and 64 points or derivatized , operating in a MATLAB environment . Multiple PLS-DA models were built to classify the samples according to (1) rotavirus-positive or -negative, (2) strain, (3) G-genotype, or (4) P-genotype. SERS spectra used to generate the PLS-DA classification models were first processed by taking the first derivative of each spectrum  and then normalizing to unit vector length 6 ffu/mL for SERS evaluation. Initially, a single strain, RV3, was applied to the OAD-fabricated SERS substrate to assess the SERS signal. While SERS bands were detected, the signal was weak, and a thick sample film was observed on the biosensing substrate. SERS is a surface sensitive technique in which only the signal for the viruses in close proximity to the nanorod substrate is enhanced. It is likely that the thick sample layer caused scattering of the laser light prior to reaching the surface thereby significantly reducing signal enhancement. Better signals were be obtained by diluting the rotavirus samples with water, thereby eliminating the sample film and exciting the virus adsorbed directly on the substrate. A 1∶10 dilution of the cell lysate was found to provide an optimum balance of minimizing detrimental multilayer effects and maximizing the sample concentration for increased sensitivity.At the outset, rotavirus was propagated in MA104 cells and harvested as cell lysates. Virus was diluted to a titer of 10−1. Many of these bands are characteristic of protein and nucleic acid vibrations. For example, the bands at 1572 and 1447 cm−1 can be assigned to protein amide vibrations −1 can be assigned to carbohydrates −1 can be assigned to a carbon-nitrogen stretch A more extensive study was performed to assess the reproducibility of the SERS measurement for RV3. An RV3 sample was applied to three independently prepared OAD-fabricated SERS substrates and five spectra were collected from different locations on each substrate for a total of 15 spectra. The spectra were baseline corrected using an automated algorithm in the OPUS software and are presented in −1), and offset to highlight differences in the relative intensities of each band. The spectra are similar for each strain; however, the relative intensities of each band are different and are a function specific to the strain. It is important to note that the cell lysate produces a SERS spectrum, and given that the rotavirus samples are harvested in a cell lysate, all specimens have this background signal in common. While several matrix bands are common to the rotavirus-positive samples, the overall spectral shape is markedly different between rotavirus-positive and rotavirus-negative samples.The SERS spectra for each of the eight rotavirus strains  were com−1 is the most intense negative control band and is reasonably constant for all samples, thus it is anticipated that this band is due only to a vibrational mode of a matrix component and that changes in band intensity relative to the 633 cm−1 band are due to viral vibrations. Therefore, each spectrum was normalized to the band at 633 cm−1 prior to spectral subtraction. The difference spectra are plotted in −1. The fact that several bands are common to all strains of rotavirus suggests that analysis based on SERS spectral fingerprints can classify samples as rotavirus-positive or rotavirus-negative. We attribute these bands to vibrations of the virus, although it is conceivable that the bands may reflect virus-induced host factors rather than spectra of the virus itself. However, the subsequent sections which accurately categorize the samples according to strain and genotype suggests that the SERS spectra are a direct measurement of the virus particles rather than virus-induced host factors which would not likely classify according to strain and/or genotype. Moreover, we have measured a single virus type propagated in two unique matrices and found consistent peaks among the infected samples (unpublished data), thus providing further evidence that the SERS spectra are a direct measurement of the virus rather than host-response factors.To extract the spectral contribution due to the virus, the MA104 spectrum was subtracted from each rotavirus spectrum. The band at 633 cma prior knowledge is required for a training dataset in order to build a classification model to test unknown samples. The advantage of this method is in its ability to minimize the contribution of spectral features which vary within a particular sample type and maximize the contribution of spectral features which vary among sample types. Fifteen SERS spectra were collected from three substrates for each of the eight rotavirus-positive samples and the mock-infected MA104 cell lysate negative control to serve as multivariate biological fingerprints. The spectra were vector normalized . Effectively this procedure builds a classification model with 90% of the spectra and then tests the remaining 10% to assess classification accuracy. The process is performed iteratively, for a total of 10 iterations, until each sample is withheld from the model and tested as an unknown. The cross-validated predictions for each collected spectrum are plotted in PLS-DA was used to establish statistically significant differences between SERS spectra for rotavirus-positive and rotavirus-negative samples. PLS-DA is a multivariate, full-spectrum calibration method that determines the best-fit mathematical relationship between a descriptor matix, i.e., sample spectra, and a class matrix, i.e., sample identities ized see  to ensurAs described above, notable differences in the relative intensity of the SERS bands are observed for each rotavirus sample. Given the extreme surface sensitivity inherent to SERS, differences in the relative intensities of each of these bands likely reflect the structural differences in capsid proteins presented to the SERS substrate and reflect antigenic variation, i.e., genotypes. SERS bands from the two major outer capsid proteins, VP7 (G-type) and VP4 (P-type), are expected to dominate the SERS spectra. Based on this premise, it is expected that spectral features resulting from VP7 proteins will be consistent among those viruses belonging to the same G-type. Likewise, it is anticipated that spectral features resulting from VP4 proteins will be consistent among those viruses belonging to the same P-type.The same spectra evaluated above for the detection of rotavirus were analyzed to assess the ability of SERS to classify the samples according to virus subtypes, i.e., genotypes. First, a PLS-DA model was generated with a classification matrix assigning each spectrum to its specific P-genotype. For example, spectra collected for strains F45, RV4, WA, and YO were assigned to the same class, i.e., PAn additional independent PLS-DA model was built to classify the SERS spectra according to the rotavirus G-type. The rotavirus strains analyzed represented genotypes G1-G4 and G9; thus, these five types, in addition to a negative control group, were defined as classes in the PLS-DA model. A cross-validated model was generated with nine latent variables  regression analysis was used to explore the quantitative capability of SERS-based molecular fingerprinting. Two concentration ranges were investigated, one dataset covered a higher virus concentration range of 105 and 106 ffu/mL and SERS spectra were acquired for each concentration. The root mean square error for cross-validation (RMSECV) was analyzed to determine the optimum number of latent variable to include in the PLS model. The RMSECV decreases with the inclusion of each additional initial factor, reaching a minimum value with seven latent variables. Inclusion of additional factors increases the RMSECV due to overfitting of the data. A plot of the predicted rotavirus concentration for cross-validated samples versus the true concentration is presented in Rotavirus strain ST-3 was selected to assess the high concentration range. Ten calibration samples were prepared between 103–106 ffu/mL. SERS spectra were collected for each concentration and a PLS model was built. The RMSECV was minimized and the quantitative model was optimized with six latent variables. The predicted rotavirus concentration for cross-validated samples versus the true concentration is presented as a logarithmic plot in 4 ffu/mL. The plot positively deviates at concentrations lower than 104 ffu/mL and the predicted concentrations are elevated with respect to the actual sample concentration. The predicted rotavirus concentration for the negative lysate control is 1.4×104 ffu/mL. Complications from the complex background matrix caused the poor predictive value at these lower titers, and spectral features due to the cell lysate dominate the SERS signature.After conducting the high concentration range, rotavirus strain YO was selected to assess a more extensive concentration range. Seven test samples were prepared spanning the concentration range of 104 ffu/mL for cell lysate preparations of rotavirus. To date only two previous studies have addressed quantitative detection of viruses via intrinsic SERS fingerprinting. The first of these studies was conducted in our laboratory for the detection of respiratory syncytial virus (RSV) 2 pfu/mL. An important difference between our previous and current work is the sample type that was analyzed. The RSV samples were purified in water whereas the rotavirus samples were suspended in a complex cellular matrix. In a recent report, a commercial off-the-shelf SERS substrate was employed to detect and quantify bovine papular stomatitis virus, pseudocowpox virus, and Yaba monkey tumor virus 2 pfu/mL. The apparent decrease in sensitivity for the rotavirus samples in this study is attributed to interference in the SERS signal due to the background matrix medium. While the detection limit for rotavirus in a biological matrix is less sensitive than those reported limits for purified virus samples, this report is a first assessment of biological samples and aids in identifying challenges associated with the current protocol to direct future research to unlock the potential of SERS-based detection. It is important to note that in addition to affecting the detection limit, the sample matrix significantly impacts the classification model. For example, a classification model built with training samples from cell lysate can only be used accurately to classify unknown cell lysate samples. With the current methodology, not only is a calibration model necessary for each virus, but a model is also needed for each sample type, i.e., cell lysate, nasal wash, fecal, etc. Moreover, donor and/or day-to-day variations in these complex clinical specimens will cause the signal to fluctuate due to the matrix itself. Nonetheless, this study demonstrates the power of SERS to differentiate individual strains of viruses in less than one minute when coupled to chemometric methods for data analysis, and clearly demonstrates the tremendous advantage of intrinsic SERS-based detection of viruses compared to more traditional methods with respect to the detection speed and the ease of genotyping.There is an unmet need for the development of a rapid, sensitive test for the identification of viruses and classification of viral strains. The development of a SERS-based biosensor and its application to the rapid detection and differentiation of rotavirus genotypes and rotavirus strains is presented. The OAD fabrication method is capable of producing robust, reproducible biosensing SERS substrates which provide extremely high enhancement factors. Virus samples supplied in a biological matrix were directly applied to the OAD prepared substrates without pretreatment and a SERS viral fingerprint was collected in 30 s. Chemometric methods of data analysis, such as PLS-DA, facilitated the classification of the virus samples based on spectral differences. Four classification models based on different criteria are presented in which >96% of the samples were correctly classified in each model. Moreover, a quantitative model based on PLS resulted in a detection limit of ∼10The data also define the critical next steps in pursuit of a robust method of viral fingerprinting, namely, overcoming the challenges imposed by a complex and dynamic sample matrix. There are three approaches that can potentially address these challenges by discriminating against the matrix itself, or its signal, in favor of the virus or virus signal. First, there is the potential to include a simple sample filtration step to isolate the virus thereby removing the background matrix from the analysis. Commercial filters are currently available from Millipore for isolating adenovirus and lentivirus, with potential application to other viruses. Second, strategies to increase sensor selectivity can be explored. More traditional recognition elements such as an antibody or aptamer are potential candidates, although they may not be effective for emerging or mutant isolates. Alternatively, more novel perm-selective barriers are being explored for species selectivity Additional information as noted in text. This material is available free of charge via the Internet.Figure S1Cross validation results for PLS-DA P genotype classification of RV samples and negative control based on SERS spectra. P8 , P6 , P4 , .(0.67 MB TIF)Click here for additional data file.Figure S2Cross validation results for PLS-DA G genotype classification of RV samples and negative control based on SERS spectra. G9 , G3 , G1 , G2 , G4 , negative control (black circles).(0.89 MB TIF)Click here for additional data file.Figure S3Cross validation results for PLS-DA strain classification of RV samples and negative control based on SERS spectra.(1.30 MB TIF)Click here for additional data file.


One question that is well known to persons working with food virology, and especially noroviruses, is “How important are these viruses, actually?” Frequently you feel somewhat uneasy when you start to reply, talking about the “trivial illness” of gastroenteritis, continuing on to the economic impact caused by the huge number of cases, and finally ending up admitting that you really do not know the answer. This book does not provide an answer to this difficult question either, but it does give you, among other things, an introduction to the pitfalls in trying to “estimate the burden of an underreported disease.” One conclusion is that we probably underestimate its importance.The book consists of 10 chapters written by persons well known to food virologists as experts in their fields. It is well organized, well written, and presents the history of foodborne viruses, the state of the art, and a glance into the future. The history of food virology is given by Dean O. Cliver, one of the pioneers in this area, who spices up his contribution by sharing interesting personal experiences with the reader. Most of the focus in the book is, naturally, directed toward norovirus and hepatitis A virus. These viruses are covered by up-to-date presentations on molecular biology, clinical disease and diagnostics, pathogenesis and immunity, epidemiology, and detection in food matrixes. Recommendations are also given about which foods should be tested and under which circumstances. Although protocols are not presented, many references are listed.The molecular revolution has improved the detection of viruses that are impossible or difficult to propagate in cell culture, such as norovirus and hepatitis A virus, and reverse transcription–PCR has become the standard method for virus detection in food. However, sensitive detection methods are still lacking. This is one of the recurrent topics in the book because it affects outbreak investigation, risk assessment, and risk management.The need for standardization for virus detection in food is stated, as is the proper use of controls. Nobody will argue against detection of false-negative and false-positive results, but some thoughts on the cost-benefit aspects regarding accurate quantification of virus genomes in foods would have been interesting.The book provides a broad overview of the present situation and gives a good introduction to the topic. Factors that may contribute to the emergence of new viral diseases are also discussed, including demographic and pathogen-related changes. This is interesting reading because virus families and viral evolution are discussed within the context of foodborne transmission. The editors encourage the reader to enjoy the book, and, most of the time, I did.


Economic theory provides the philosophical foundation for valuing costs in judging medical and public health interventions. When evaluating smoking cessation interventions, accurate data on costs are essential for understanding resource consumption. Smoking cessation interventions, for which prior data on resource costs are typically not available, present special challenges. We develop a micro-costing methodology for estimating the real resource costs of outreach motivational interviewing (MI) for smoking cessation and relapse prevention among low-income pregnant women and report results from a randomized controlled trial (RCT) employing the methodology. Methodological standards in cost analysis are necessary for comparison and uniformity in analysis across interventions. Estimating the costs of outreach programs is critical for understanding the economics of reaching underserved and hard-to-reach populations.Randomized controlled trial (1997-2000) collecting primary cost data for intervention. A sample of 302 low-income pregnant women was recruited from multiple obstetrical sites in the Boston metropolitan area. MI delivered by outreach health nurses vs. usual care (UC), with economic costs as the main outcome measures.The total cost of the MI intervention for 156 participants was $48,672 or $312 per participant. The total cost of $311.8 per participant for the MI intervention compared with a cost of $4.82 per participant for usual care, a difference of $307 . The total fixed costs of the MI were $3,930 and the total variable costs of the MI were $44,710. The total expected program costs for delivering MI to 500 participants would be 147,430, assuming no economies of scale in program delivery. The main cost components of outreach MI were intervention delivery, travel time, scheduling, and training.Grounded in economic theory, this methodology systematically identifies and measures resource utilization, using a process tracking system and calculates both component-specific and total costs of outreach MI. The methodology could help improve collection of accurate data on costs and estimates of the real resource costs of interventions alongside clinical trials and improve the validity and reliability of estimates of resource costs for interventions targeted at underserved and hard-to-reach populations. Economic theory provides the philosophical foundation for valuing costs in judging medical and public health interventions. Resources used in, and saved as a result of, medical and public health programs must be measured in terms of their monetary costs to understand the overall implications of such interventions. Under economic theory, the opportunity cost of a resource - or its value in its next best or alternative use - is the real cost of that resource to society. Under ideal circumstances  the price of a resource can be taken as its opportunity cost. However, medical and public health programs rarely operate under ideal market conditions. Thus, we cannot always rely solely on prices to value all resources in the economic evaluation of medical and public health interventions, although prices are almost always used for cost estimates. Rather, we need a systematic methodology for measuring costs grounded in the theory and process of identifying, estimating and valuing resource costs. This method can be described as micro-costing.Despite recent work on economic evaluation in clinical trials , which fCurrently available economic evaluations of behavioral counseling services for smoking cessation provide limited evidence on costs to determine optimal resource allocation strategies. With some notable exceptions, few studies have been performed in accordance with U.S. Panel on Cost-Effectiveness in Health and Medicine recommendations grounded in economic theory. While such limitations hinder comparisons among programs, they also represent an opportunity for future analyses to better adhere to sound and consistent methodologies. A lack of economic studies is common in public health and medicine, however, especially in the area of prevention services . DespiteSmoking imposes a significant economic burden on society, and smoking cessation can improve both the quantity and quality of life at low costs ,5. When In a randomized controlled trial (1997-2000), we compared MI on an outreach basis (N = 156) with Usual Care (UC) (N = 146) among low-income pregnant women in Boston. During an average of three home visits, a nurse delivered the MI intervention and built a relationship with the participant. The MI components were tailored to each participant's stage of readiness.Because this program consumed a considerable amount of professional time , it was important to precisely estimate its true costs. Therefore, we used micro-costing, which directly enumerates and subsequently values each input consumed for each particular participant. This approach's level of detail is especially important when personnel time or training account for a major portion of an intervention's costs. Because it is difficult to retrospectively create records of personnel time expended during a behavioral intervention, these data must be tracked during program delivery. For outreach programs, travel time and mileage must also be collected prospectively. To capture these details, we developed a process tracking system.When assessing the full costs of smoking cessation and relapse prevention programs, it is also important to account for potential future resource use or savings that might result from the intervention. Published estimates of net lifetime additional medical costs for smokers -10 and nWhen the Panel on Cost-Effectiveness in Health and Medicine recommended micro-costing in the late 1990s , it suggPotential study participants were recruited from a large number of community-based health care practices and community health care centers that provided prenatal care. To be eligible, women had to be less than 28 weeks pregnant, speak English or Spanish, and be either current smokers or recent quitters . They also could not be in drug addiction treatment. During routine prenatal visits, health care providers described the study and assessed women's interest in participating. Study personnel then received contact information and called interested women for further explanation of the study. Trained research assistants visited the women in their homes to answer questions, obtain informed consent, conduct the baseline survey, and complete other study assessments. The health care providers referred 549 women to the study. Of those, 65 did not meet eligibility requirements, and 68 could not be located. Of the remaining 416 women, 114 refused participation. The final sample at baseline was 302 pregnant women (73% of known eligible women).After the 302 women gave their consent and completed the baseline assessment, they were randomly assigned to either UC or MI. Follow-up assessments were conducted 10 weeks after baseline  and 4 to 6 months after the baby's birth . At each assessment visit, passive sampling dosimeters were placed in the home (kitchen and living room) for 7 days to assess air nicotine concentrations. Feedback about nicotine levels was provided as part of the MI intervention.After UC participants completed the baseline survey, a letter was sent to their prenatal care providers indicating the patient's study participation and recommending that the provider discuss the patient's smoking with her. A description of the study, a tip sheet for providers based on the Agency for Healthcare Research and Quality (AHRQ) guidelines  and a paIn addition to their usual care, MI participants received monthly visits from a Public Health Nurse until one month post-partum. The intervention was designed to address social contextual factors that might influence responses to the smoking intervention. The nurse was available to help with all aspects of pregnancy, including prenatal education, gaining needed social services, smoking cessation and secondhand smoke reduction.The intervention was based on motivational interviewing , which eThe first counseling session focused on increasing motivation, in part by addressing ambivalence associated with the decision to quit smoking. The nurses began the counseling session by presenting results from household nicotine assessments. If the woman was interested, the nurse discussed ways to either reduce the nicotine levels in the home or, if levels were low, ways to prevent them from increasing. Smoking cessation was discussed with women who were interested. Additionally, the nurses worked with the participants on setting goals in other areas of their lives . When appropriate, the relationship between these other goals and smoking was explored. The first counseling session placed a strong emphasis on goal setting. Progress toward those goals was the focus of the subsequent intervention visits.The theory and process of valuing costs through a micro-costing methodology rests on a three-step approach: identification, measurement, and valuation of resources used. While the Panel recommended following this theory and process, it provided no guidelines on implementing a micro-costing study, in general, and no procedures on how to do so in specific studies. Because micro-costing uses primary data on the exact number and type of resources consumed by each participant, it is most accurate when it tracks resource consumption as it occurs, thereby enhancing the validity and reliability of cost estimates. Once resource utilization is measured, the quantity of each type of resource consumed is multiplied by unit costs, and the results are summed to obtain total component-specific costs and overall cost. Total and component-specific costs can then be divided by the number of participants to determine expected costs per participant.The Panel also recommended that future costs and savings attributable to an intervention be included in cost analyses from the societal perspective, in addition to micro-costing of intervention costs. Many interventions, especially preventive ones, for example, can result in future savings that offset initial costs. Analyses performed from the perspective of a provider or payer would include only those costs and savings that they bear or realize. Thus, in this micro-costing study, we include the patient's costs as well as the providers' costs and we conceptualize future savings that could offset initial costs. We conclude with sensitivity analyses of the robustness of these estimates to changes in key cost components.In step 1, it is important to thoroughly delineate all inputs that might be affected by the intervention so as to account for the cost of each component. This first step involves the identification of resources used in the "production process" of the intervention. Each "work step" in the production process is then described in conjunction with the relevant cost components. This step may also be called component enumeration, whereby each factor involved in the "production" of the intervention is identified and fixed costs (that do not vary with volume) are separated from variable costs (that do vary with volume). The Panel recommended that incremental costs  rather than average costs  be collected and analyzed.As shown in Table Table To determine the amount of personnel time involved in the intervention, we had to describe the work-steps involved. They included scheduling and follow-up, preparation, travel, and intervention delivery (counseling sessions). The process tracking form served as a generic flow chart for these steps.We did not include the clinics' overhead costs  because they were similar in the intervention (MI) and control (UC) groups and as an outreach program, whereby the set-up and delivery of the intervention occurred "off-site," the similar overhead costs would likely be minimal. Because the participants were interviewed at home, clinic and hospital operating expenses (facilities costs) were not relevant. Neither were productivity costs   the intervention costs described above; (2) cost savings for neonatal intensive care, chronic medical conditions, and acute conditions during the first year of life; and (3) cost savings for maternal health care .To enhance the reliability and validity of our cost data, we measured and valued all inputs consumed as the program proceeded (as part of the RCT), rather than trying to reconstruct them retrospectively. To collect utilization data in the RCT, we developed a process tracking form for manually recording information on: (1) components delivered; 2) time spent with each participant to conduct the intervention or follow-up; (3) materials provided; and (4) travel times and distances. The costs collected were those necessary for reproducing the intervention in a non-research setting [ time speTime spent scheduling and contacting participants is typically an administrative cost, but we decided that nurses, rather than clerical staff, should have phone contact with participants before visiting their homes. The only additional support required was from a nurse supervisor, who was trained in delivering the MI intervention but spent little effort on the intervention itself.Invoices for photocopying and other production expenses were filed by research administrators and used as the bases for cost estimates and to confirm quantities. Facilities costs  were deemed similar in both groups, and therefore excluded. No new or existing equipment was required.A significant category of costs was, however, transportation costs. Participants did not incur travel costs because they received the intervention at home, but nurses did and they logged their time spent during travel and the number of miles to and from participants' homes on the process tracking form. We did not assume "stacking" of travel , although a program like this could involve "stacked travel," with potentially less travel time for outreach nurses. While participants did not spend time traveling to and from the intervention, they did spend time participating in it. Nurses logged participant time spent in the intervention, again through the process tracking system.For our outreach MI program, mean values for staff time were: (1) scheduling, 17 min; (2) preparation, 11 min; 3) intervention delivery, 66 min per session; and (4) travel to and from the delivery site, 40 min and 29 miles. Average participant time spent during MI was 66 minutes per session  to assign a monetary value to personnel time; thus, these rates would have to be adjusted for interventions outside the Boston area. The average nurse's salary plus benefits was $31.45 per hour .p = P*Q) were the product of price  and quantity .The calculations for determining the cost per unit of staff time are shown in Table The monetary value of the time the participants spent receiving the MI was estimated by their hourly wage. For low-income women, we used the minimum wage in 1997 obtained from the Bureau of Labor Statistics (BLS) .Costs of purchasing consumables used in the intervention were obtained from research administrators. Costs of laboratory tests used to analyze nicotine samples were recorded, as was the cost of photocopying the self-help materials.Future cost savings associated with smoking cessation and relapse prevention cannot be determined while a clinical trial is in progress and therefore must be gross-costed, using rough estimates from the literature and sensitivity analysis. These costs were assumed to be zero in the base case. Published estimates of net lifetime additional medical costs for smokers are $6,239 (discounted 1990 dollars) -10. PublSensitivity analyses were conducted to analyze the variation in total costs according to uncertainties in key cost categories, such as intervention delivery, staff travel, and analysis of nicotine samples.Table The total fixed costs of the MI were $3,930 and the total variable costs of the MI were $44,710. The total cost of the MI intervention for 156 participants was $48,672 or $312 per participant. The costs and costs per participant of the individual components were :• Eight-hour training session for the nurses and their supervisor = $3,930 or $25.2 per MI participant• MI Scheduling - 17 minutes on average; @ $0.52/minute = $8.8 per intervention session or $26.5 per participant• MI Preparation - 11 minutes on average; @ $0.52/minute = $5.7 per intervention session or $17.2 per participant• MI Delivery - 66 minutes on average; @ $0.52/minute = $34.3 per session or $103 per participant• Travel to and from the intervention delivery site - 40 minutes on average; @ $0.52/minute = $20.8 per visit or $62.4 per participant• Participant time spent in MI - 66 minutes on average; @ a minimum wage of $5.15 per hour = $17.0 per participant• Transportation to and from the MI delivery site for mileage (29 miles) @$0.25 per mile = $21.8 per participant• Printed self-help materials (7 items for the MI group and 2 for UC group) = $4.7 (MI) per participant and $1.8 (UC) per participant, a difference of $2.90 per MI participant• Cost of analyzing a nicotine sample - $17 per sample = $34 per participant The total cost of $311.8 per participant for MI intervention compared with a cost of $4.82 per participant for usual care, a difference of $307   its systematic nature; 2) standardization across cost components; 3) reliability and validity resulting from cost collection during actual program delivery; and 4) ability to account for the most significant inputs . It is akin to detailed cost-accounting using the principles of industrial engineering and time-motion studies to estimate and compare actual production costs -22. One There are other substance use treatment costing methods, such as the use of the Drug Abuse Treatment Cost Analysis Program (DATCAP) , and SubThus, despite the noted limitations, the detailed cost information collected by this methodology is essential for understanding the levels and types of resources necessary for effective implementation of smoking cessation and relapse prevention programs. As an input to cost-effectiveness analyses  and costThe authors declare that they have no competing interests.JR, MW, and KE conceived of the study concept and design, drafted the manuscript and supervised the study. All authors participated in data acquisition, analysis and interpretation of the data, and in critical revision of the manuscript for important intellectual content.The pre-publication history for this paper can be accessed here:


The vaccinia virus (VV) F2L gene encodes a functional deoxyuridine triphosphatase (dUTPase) that catalyzes the conversion of dUTP to dUMP and is thought to minimize the incorporation of deoxyuridine residues into the viral genome. Previous studies with with a complex, multigene deletion in this virus suggested that the gene was not required for viral replication, but the impact of deleting this gene alone has not been determined in vitro or in vivo. Although the crystal structure for this enzyme has been determined, its potential as a target for antiviral therapy is unclear.The F2L gene was replaced with GFP in the WR strain of VV to assess its effect on viral replication. The resulting virus replicated well in cell culture and its replication kinetics were almost indistinguishable from those of the wt virus and attained similar titers. The virus also appeared to be as pathogenic as the WR strain suggesting that it also replicated well in mice. Cells infected with the dUTPase mutant would be predicted to affect pyrimidine deoxynucleotide pools and might be expected to exhibit altered susceptibility to pyrimidine analogs. The antiviral activity of cidofovir and four thymidine analogs were evaluated both in the mutant and the parent strain of this virus. The dUTPase knockout remained fully susceptible to cidofovir and idoxuridine, but was hypersensitive to the drug (N)-methanocarbathymidine, suggesting that pyrimidine metabolism was altered in cells infected with the mutant virus. The absence of dUTPase should reduce cellular dUMP pools and may result in a reduced conversion to dTMP by thymidylate synthetase or an increased reliance on the salvage of thymidine by the viral thymidine kinase.We confirmed that F2L was not required for replication in cell culture and determined that it does not play a significant role on virulence of the virus in intranasally infected mice. The recombinant virus is hypersensitive to (N)-methanocarbathymidine and may reflect metabolic differences in the mutant virus. All free-living organisms have mechanisms to minimize the incorporation of uracil in their genomes. These residues in DNA can arise either through misincorporation of dUTP by DNA polymerase or the spontaneous deamination of cytosine and can result in A:T transition mutations in one of the nascent strands . MinimizKm for dUTP was 1 μM and that it was competitively inhibited by 8-azido-ATP [These protective enzymes are also present in many viruses including retroviruses, herpesviruses and orthopoxviruses . Proteinzido-ATP . RecentlIf the dUTPase fulfills an essential role in viral replication then inhibitors of this enzyme might have the potential to be used in the treatment of orthopoxvirus infections. One previous report described a recombinant virus with a large deletion resulting in the elimination of 55 open reading frames including F2L . This rewt) parental virus. The insertion of the gfp resulted in the deletion of most of the F2L open reading frame from amino acids 11–129 . Two isolates of wt VV WR, and VV Δ F2L-gfp were evaluated in this experiment to assess the virulence characteristic of these strains. Mice were observed daily for 21 days and were evaluated for clinical signs of infection and mortality. Infection with 1.6 × 10Deletion of the dUTPase is predicted to effect pyrimidine metabolism in infected cells and may alter the susceptibility of the mutant virus to some antiviral drugs. A set of thymidine analogs was selected and a standard plaque reduction assay was used to evaluate the susceptibility of the dUTPase mutant and the parent virus. The mutant remained fully sensitive to all of the drugs tested including cidofovir (CDV), idoxuridine (IDU), and two thymidine analogs reported to require phosphorylation by the VV thymidine kinase (TK) . The onl50 from approximately 103 PFU to more than 106 PFU of the mutant virus. Thus, the observed virulence of the HSV mutant was dependent on the route of administration and it is unclear if reduced virulence would be observed following intranasal inoculation.Results presented here are consistent with a previous report that showed that the dUTPase was not required for replication in cell culture using a virus containing a multigene deletion . They alWe show here that there is little if any attenuation when mice are intranasally infected with VV in which the dUTPase has been deleted. Additional experiments are required to resolve this issue and the rabbit model of VV infection and might be a more sensitive indicator of reduced virulence associated with the mutant virus . It is aDifferences in pyrimidine metabolism were predicted to occur in the absence of the dUTPase so a set of selected thymidine analogs were used as potential indicators of metabolic differences. The efficacy of the CDV control virus was unchanged in the mutant, as was the activity of IDU and two thymidine analogs reported previously . HoweverData presented here suggest that VV dUTPase is not required for viral replication in cell culture. The deletion of F2L also does not appear to impact the virulence of the virus in mice following intranasal infection. These studies suggest that dUTPase is not a particularly good target for the development of antiviral therapies, although it remains possible that other animal models may identify an important function of this enzyme. The mutation does not affect the efficacy of most antiviral drugs including CDV although it appears to confer a modest hypersensitivity to N-MCT and may reflect metabolic differences in the mutant virus.wt VV strain WR (Turner) were received from Dr. Pete Turner, University of Florida, Gainesville, FL. VV strain WR (UAB) used in the animal studies was obtained from the American Type Culture Collection (ATCC), Manassas, VA. Working stocks of both VV-WR isolates and VV ΔF2L-gfp were propagated in Vero cells obtained from ATCC. Human foreskin fibroblasts were prepared as primary cultures from freshly obtained newborn human foreskins as soon as possible after circumcision. Culture medium for both cell lines was minimum essential medium (MEM) with Earle's salts containing 10% fetal bovine serum and standard concentrations of L-glutamine, penicillin and gentamicin.Recombinant virus designated VV ΔF2L-gfp and the parental The drugs tested included CDV, IDU, N-MCT, 5--1-(2-deoxypento-furanosyl)-pyrimidine-2,4-dione (PFT3), and 1-(2-deoxypentofuranosyl)-5- [pyrimidine-2,4-dione (PFT4). Compounds designated PFT3 and PFT4 were synthesized by Paul Torrence (Northern Arizona University) and were described previously ; N-MCT wAAGCTTAACTGGTGAGTTAATATTCATGTTGAAC, HindIII site underlined) against the complement of F2L residues 3–31 were used to PCR amplify a 487-bp fragment upstream from F2L. A 531-bp PCR product consisting of the downstream flank from F2L was made using primers IDT 717  against F2L residues 417–436 and IDT 718 (CATACATCGTCTACCCAATTCGG) against F1L. HindIII-digested, dephosphorylated IDT 715+716 PCR product and XhoI-digested, dephosphorylated IDT 717+718 PCR product were ligated to a HindIII-XhoI restriction fragment consisting of the PE/L promoter linked to the gfp gene. The ligation mix was PCR amplified with primers IDT 715 and IDT 718 to generate a 1.8-kb product of gfp flanked by portions of F3L and F1L. CV-1 cells were infected with wt VV-WR, transfected with the F3L-gfp-F1L DNA, and plaques expressing gfp were isolated by fluorescence. The resulting virus was designated VV ΔF2L-gfp and contained a deletion in F2L corresponding to amino acids 11–129 of the open reading frame. The genomic structure and purity of this virus was confirmed by PCR using primers IDT 715 + 718. No fragment of 1.4 kb corresponding to wt F2L plus flanks was detected, but a 1.8 kb fragment of F3L-gfp-F1L was present.A recombinant was constructed from VV strain WR with most of F2L replaced with the gfp gene driven by the synthetic E/L promoter by methods similar to those described previously . Primers2 and 90% humidity. Triplicate wells were infected with wt VV-WR or VV Δ F2L-gfp at an MOI of 0.001. Infected plates were frozen at -80°C at 2, 8, 12, 24, 36 and 48 h post infection. Duplicate titrations of each of the triplicate wells were conducted in HFF cells in 6 well plates. Plaques were enumerated and titers were determined for each time point and virus.To determine the in vitro replication of the viruses, HFF cells were incubated in 6 well plates for 24 h prior to infection at 37°C with 5% CO50) values were calculated by standard methods.Plaque reduction assays were performed using HFF cells added to six well plates and incubated 48 h prior to infection. On the day of assay, drug at two times the final desired concentration was diluted serially 1:5 in 2X MEM with 10% FBS to provide six concentrations. Aspiration of culture medium from triplicate wells for each drug concentration was followed by addition of 0.2 ml/well of diluted virus which would give 20–30 plaques per well in MEM containing 10% FBS or 0.2 ml medium for drug toxicity wells. The plates were incubated for one h with shaking every 15 minutes. An equal amount of 1% agarose was added to an equal volume of each drug dilution and this mixture was added to each well in 2 ml volumes and the plates incubated for three days. The cells were stained with a 0.02% solution of neutral red  in PBS and incubated for 5–6 h. The stain was aspirated, and plaques counted using a stereomicroscope at 10× magnification and 50% effective concentration . Two isolates of WR, and VV Δ F2L-gfp were evaluated in this experiment to assess the virulence characteristic of these strains. Mice were observed daily for 21 days and were evaluated for clinical signs of infection and mortality.50), effective concentration (EC50), American type culture collection (ATCC), 5--1-(2-deoxypento-furanosyl)-pyrimidine-2,4-dione (PFT3), 1-(2-deoxypentofuranosyl)-5-[pyrimidine-2,4-dione (PFT4), minimum essential medium (MEM), central nervous system (CNS).Hour (h), wild type (wt), plaque forming unit (PFU), vaccinia virus (VV), herpes simplex virus (HSV), (N)-methanocarbathymidine (N-MCT), cidofovir (CDV), idoxuridine (IDU), N-MCT monophosphate (N-MCT-MP), deoxyuridine triphosphatase (dUTPase), deoxyuridine triphosphate (dUTP), deoxyuridine diphosphate (dUDP), deoxyuridine monophosphate (dUMP), green fluorescent protein (GFP), deoxythymidine monophosphated (TMP), uracil DNA glycosylase (UNG), human foreskin fibroblast (HFF), lethal dose 50% (LDThe author(s) declare that they have no competing interests.MNP contributed to the design of experiments, analysis of the data and the drafted the manuscript. ERK contributed to the conception of the studies and the critical review of the manuscript. DCQ contributed to the design of the experiments and analysis of the data. KAK contributed to the acquisition and interpretation of data. RWM contributed to the conception of the studies and the critical review of the manuscript. PCT contributed to the design of experiments, the acquisition and analysis of data and the critical review of the manuscript.


The rate and timing of reversion is, however, highly variable. We quantified reversion to WT of a series of SIV and SHIV viruses containing minor amounts of WT virus in pigtail macaques using a sensitive PCR assay. Infection with mixes of EM and WT virus containing ≥10% WT virus results in immediate and rapid outgrowth of WT virus at SIV Gag CD8 T cell epitopes within 7 days of infection of pigtail macaques with SHIV or SIV. In contrast, infection with biologically passaged SHIVmn229 viruses with much smaller proportions of WT sequence, or a molecular clone of pure EM SIVmac239, demonstrated a delayed or slow pattern of reversion. WT virus was not detectable until ≥8 days after inoculation and took ≥8 weeks to become the dominant quasispecies. A delayed pattern of reversion was associated with significantly lower viral loads. The diversity of the infecting inoculum determines the timing of reversion to WT virus, which in turn predicts the outcome of infection. The delay in reversion of fitness-reducing CD8 T cell escape mutations in some scenarios suggests opportunities to reduce the pathogenicity of HIV during very early infection.Escape mutant (EM) virus that evades CD8+ T cell recognition is frequently observed following infection with HIV-1 or SIV. This EM virus is often less replicatively “fit” compared to wild-type (WT) virus, as demonstrated by reversion to WT upon transmission of HIV to a naïve host and the association of EM virus with lower viral load  Understanding how to contain HIV replication by the immune system is a key goal of vaccine strategies. HIV frequently mutates to avoid immune recognition, but this may come at a “fitness cost”, weakening the virus. When HIV is transmitted to a new host, the mutations often revert back to wild-type, allowing the virus to regain a fitter state. We found that when multiple HIV-like viruses are transmitted to monkeys, containing both mutant and wild-type, reversion to wild-type is very rapid and the fitter virus results in higher viral levels. In contrast, when only escape mutant virus initiates the infection, reversion to wild-type is delayed to later during early infection, and lower levels of virus result. Our results suggest that the composition of the infecting virus plays a role in determining the outcome of HIV infections. Strategies to maintain weakened virus strains during the early HIV infection may help the host control virus replication. The high mutation rate of HIV coupled with the strong immune selection pressure exerted by CD8+ T lymphocytes leads to the frequent selection of ‘escape mutant’ (EM) virus that contains mutations within CD8+ T cell epitopes that abrogates their recognition and killing by T cells. Although these mutations confer an advantage on EM virus, they also incur a ‘fitness cost’, as EM virus often has lower replicative capacity than the original wild-type (WT) virus, particularly when mutations occur in conserved regions such as Gag in vivo is however highly variable. Factors that impact the rate of immune escape and reversion include the particular CD8 T cell epitope, target cell availability, prior vaccination, the presence of compensatory mutations, the MHC of the donor and recipient, and timing of appearance of the mutation in vivo competition between EM and WT variants under different conditions.The rate of reversion from EM to WT in vivo and the impact of this on viral load in acute and chronic infection.A considerable technical hurdle in evaluating the evolution of immune escape variants is sensitively and specifically quantifying minor variants. Cloning and sequencing is poorly quantitative for variants of <10% of the total unless prohibitively large numbers of clones are analyzed. We recently developed quantitative real-time PCR assays (qRT-PCRs) to study escape mutations that emerge at 2 different SIV Gag CD8 T cell epitopes (KP9 and AF9) in pigtail macaques Macaca nemestrina) were approved by the University of Melbourne and CSIRO livestock industries Animal Ethics Committees. Pigtail macaques were typed by reference strand-mediated conformational analysis for the MHC-I alleles Mane-A*10 and Mane-A*17 allele which present SIV Gag164–172 KP9 epitope and SIV Gag371–379 AF9 epitope respectively Experiments on outbred pigtail macaques (mn229 viruses (K165R) from 3 different viral inocula derived from pigtail macaques expressing the restricting Mane-A*10 allele were assessed following passage of each virus to 2–4 naïve Mane-A*10 negative pigtail macaques. We studied reversion of the original SHIVmn229 stock in 4 animals as previously described mn229 stock was derived following passage of SHIVHXB2 in Mane-A*10 positive pigtail macaques and is 89% EM and 11% WT at the K165 position within the Gag KP9 epitope Reversion of KP9 EM SHIVmn229, directly from the serum / cells of two infected animals that showed escape at the KP9 epitope. This passaged virus contained a much lower level of WT virus than the original SHIVmn229 stock, and was inoculated into 4 additional Mane-A*10 negative macaques. Two macaques (6274 and 6366) received 1 ml of plasma and 3×106 peripheral blood mononuclear cells (PBMC) from the donor Mane-A*10+ animal 4296 8 weeks after SHIVmn229 infection. Two additional macaques received plasma and serum samples from Mane-A*10 positive animal 6279 11 weeks after SHIVmn229 infection. Animals 4296 and 6279 were previously described as part of a DNA and Fowlpoxvirus vaccine studies in macaques mn229 challenge Reversion of the K165R mutation back to WT was also studied by transmission of further passages of the SHIVmn229 with 21 unvaccinated control pigtail macaques previously infected with the original SHIVmn229 stock (89∶11 EM∶WT) previously used across several vaccine studies To assess the pathogenicity of the EM viruses, we compared viral loads of the passaged low WT SHIVMane-A*17 negative macaques infected with SHIVSF162P3 passaged in vivo in a Mane-A*17+ pigtail macaque. This passaged SHIV acquired a 6-nucleotide deletion within the AF9 epitope Reversion of AF9 was studied as previously described mac239 proviral plasmid GATTTGGAGCAGAAGTAGTGCC-3′5′ -GGTAAAATTGATAGAGGAAAAGA and CTCTTTTCCTCTATCAATTTTACC-3′5′- GGCACTACTTCTGCTCCAAAT. The mutated 5′ vector and 3′ vector of SIVmac239mac239 DNA was infectious in vitro by transfecting plasmids into HeLa cells and testing the supernatants for their ability to infect CEMX174 in vitro (not shown).To examine the effect of infection with pure pathogenic R5-tropic KP9 EM virus, we used PCR mutagenesis techniques to mutate the translated amino acid from lysine to arginine at Gag position 165 within our previously described SIVin vivo, we injected animals with 150 µg of the K165R SIVmac239 proviral DNA intramuscularly, a technique we previously used to initiate infections with both WT and nef/LTR-deleted SIVmac239 strains mn229 stock containing 11% WT virus at KP9), we also inoculated 2 animals IM with 150 ug of a 90∶10 mix of K165R EM and WT SIVmac239 proviral plasmids.To initiate infections 4 cycler. Analysis was performed using SDS applications version 1.9 (ABI) or Eppendorf Realplex4 software. Baselines were set 2 cycles earlier than real reported fluorescence and threshold value was determined by setting threshold bar within the linear data phase. Samples amplifying after 40 cycles were regarded as negative, and corresponds to <1.5-Log10 SHIV/SIV RNA copies/ml of plasma .To quantify virus levels of WT or EM quasispecies at the KP9 and AF9 epitopes we employed recently published novel real-time PCR assays Wf(t) is the fraction of wild type and Mf(t) is the fraction of escape mutant clones at time t, we calculate the time-dependent reversion rate R from the proportions of clones of each type at the end points st and et of the time interval ,In order to compare the dynamics of reversion in different animals we analysed reversion rates as previously described W) and escape mutant (M) virus with different, but constant in time, replicative capacities, infect the same pool of target cells T , generating infected cells WI and MI respectively. This model is described by the set of equations:T are replaced from an external source at the rate λ and in the absence of infection are lost at the rate Td. The difference in replicative capacities of WT and EM is the result of different rates of infection of target cells . Cells infected by both strains die at the same rate δ (since there is no immune response specific to one strain). Both strains are produced from infected cells at the same rate p and are cleared at the same rate c.In order to explain the experimental results, we used a standard model of viral dynamics applied to reversion st and et,Wr = Wp/cβ and Mr = Mp/cβ are the replicative capacities of WT and EM respectively, and st, et). The model gives the reversion rate directly proportional to target cell number.This model predicts the reversion rate in the time interval between Mane-A*10 negative macaques infected with our SHIVmn229 viral stock  is almost identical and very rapid in all Mane-A*10 negative animals studied . For these virus transfer experiments, we selected donor Mane-A*10+ animals that were previously vaccinated with SIV Gag-expressing DNA and recombinant Fowlpoxvirus vaccines The rapid outgrowth of WT virus in the previous experiments suggested sufficient quantities of WT virus were present in the inoculum to co-infect the host and then rapidly out-compete the EM virus. We therefore sought to elucidate the impact of much smaller amounts of WT KP9 on reversion at KP9. We first chose to transfer plasma and cells from in vivo passaged SHIVmn229, which contained lower levels of WT virus  and R5-tropic SHIVSF162P3 (for the AF9 epitope) strongly suggested that the levels of WT virus in the inoculum have a major bearing on the time needed for outgrowth of the WT virus. However, it is difficult to completely exclude that these uncloned viral stocks contain quasi-species with mutations at other sites or compensatory mutations . To avoid these potential confounders we constructed a molecular clone of the K165R KP9 EM virus within SIVmac239. This enabled us to evaluate rates of reversion using a separate R5-tropic virus in a very tightly controlled manner. We chose to infect naïve Mane-A*10 negative pigtail macaques using plasmid DNA, an approach we and others have previously used successfully, both with attenuated and WT viruses in vitro prior to in vivo inoculation.Our analyses of biologic isolates of X4-tropic SHIVmac239 conforms to the same general principles observed with SHIVmn229 and SHIVSF162P3 we inoculated 2 naïve Mane-A*10 negative pigtail macaques using a 90∶10 mix of EM∶WT plasmid DNA. Using our qRT-PCR assay to detect WT or EM virus, both WT and EM virus grew readily for the first 2 weeks but WT virus subsequently rapidly outgrew the EM virus  is important to viral growth and the rate of reversion One prediction of this model is that not only will it take longer to reach 50% WT when target cell dynamics are taken into account, but that the reversion rate itself at 50% WT will be slower with lower WT fraction in the inoculum. Indeed, the reversion rate observed at 50% WT is significantly correlated to the fraction of WT in the inoculum  from previous infection studies mn229 with less WT virus at KP9 (that reverts slowly), we compared the virologic outcome of infection with both viruses. Both viruses grew effectively and exponentially during acute infection . The increased proportion of WT virus at peak viral load in animals infected with SHIVmn229 stock is associated with an increased peak viral load in the animals , and for passaged SHIVmn229 it was 2.08×106 . Set point viral load was also significantly higher in animals infected with stock virus (mn229 stock it was 9.4×105 (95%CI from 7.3×105 to 2.6×106), and for the passaged virus it was 2.3×104 . Thus, the slow reversion of WT virus in animals infected with a low proportion of WT virus has direct implications for the virological outcome of infection.Slow reversion to WT virus implies that the infecting virus population is dominated by the less fit EM virus for longer periods of time in comparison to animals with fast reversion. In theory, this could result in lower viral loads and less pathogenic infections. Goepfert and colleagues recently showed HIV-1 strains transmitted with multiple Gag CD8 T cell escape mutations resulted in overall lower viral loads nfection . However animals : median ck virus : for SHIWe conducted a large series of infections with combinations of WT and CD8 T cell EM SHIV and SIV viruses. Using a sensitive real-time PCR assay to simultaneously quantify levels of EM and WT viremia, we found levels of WT virus in transmitted EM viruses had a marked influence on the timing and rate of reversion to WT. Where substantial amount of WT virus are present in the inoculum, “reversion” (outgrowth of WT virus) is rapid, but if WT virus is minimal or absent in the inoculum, there is a delay in reversion and when reversion begins, it takes much longer to reach the same levels as the EM virus. The consistency of these findings across multiple SHIV and SIV isolates suggest this is a general phenomenon of CD8 T cell EM reversion. This has important consequences for HIV pathogenesis, since we found a delayed and slow pattern of reversion of biologic SHIV quasispecies (where the less fit EM virus dominates the infection for longer periods) is associated with significantly reduced viral loads through to chronic infection. Since virtually every HIV infection involves the selection of EM viruses, transmission of EM viruses or mixes of EM and WT virus will be the norm. Our studies suggest that in the subset of around one quarter of new HIV infections, where 2 or more viruses are transmitted mn229-infected pigtail macaques suggests that the KP9-specific CD8 T cell response is highly immunodominant and the rapidly pathogenic nature of the virus rarely permits generation of detectable responses to other epitopes, suggesting the much lower proportions of WT virus in the passaged SHIVmn229 viruses were primarily responsible for the lower viral loads. None-the-less, larger studies of cloned SIV viruses would assist in further defining the role of individual mutations or combinations of CD8 T cell escape mutations in reducing viral pathogenicity.A potential limitation to our findings of reduced viral load following infection with EM SHIV quasispecies with very low levels of WT virus is that we cannot exclude that mutations other that the KP9 K165R EM present, including distant compensatory mutations, in the passaged virus inocula could also be contributing to the reduced VL upon transmission. Indeed, human HIV-1 transmission studies by Goepfert and colleagues suggest that multiple Gag CD8 T cell epitope mutations may result in reduced VLs in the recipients The obvious question arises as to why outgrowth of WT virus is so slow when it does not proceed during very early acute infection, since the intrinsic fitness cost of the mutation should be the same regardless of the timing of infection. Modelling reveals that reversion rate is proportional to target cell number A caveat to these studies is the study of CXCR4-utilizing SHIV viruses for these analyses. Since these viruses deplete all naïve CXCR4-expressing CD4 T cells, the depletion of peripheral CD4 T cells should approximate total CD4 T cell loss, and thus frequent monitoring of peripheral CD4 T cells allows us to model CD4 T cell depletion and reversion. Typical CCR5-utilizing HIV-1 and SIV strains target memory CD4 T cells which are most abundant in the gastrointestinal tract and rapidly depleted during acute infection. These cells are less amenable to the frequent monitoring required to correlate of the levels of CD4 T cell depletion with the rate of reversion of viral escape mutants. However, previous analysis comparing the dynamics of CD4+ T cell depletion in the blood during SHIV infection and in the gut following SIV infection suggest that the dynamics are remarkably similar mn229, SHIVSF162P3 and SIVmac239 infection models. When WT virus was <4% of the inoculum, there was a consistent delay of 8–11 days until WT virus is first detected even at very low levels. This occurs during the critical window of early virus dissemination, the so-called “eclipse” phase of very early acute HIV-1 infection of humans Although we studied a modest number of animals within each group, our findings were consistent across both SHIV and SIV infection models using both biological isolates (for the SHIV studies) and molecular clones (for the SIV studies). When WT virus comprised ≥10% of the inoculum, expansion of WT virus during acute infection was exponential and uniform across SHIV


Chikungunya virus (CHIKV), a mosquitoborne alphavirus; is endemic in Africa and Asia. In 2005–2006, CHIKV epidemics were reported in islands in the Indian Ocean and in southern India. We present data on laboratory-confirmed CHIKV infections among travelers returning from India to the United States during 2006. Alphavirus, family Togaviridae) usually associated with acute epidemic polyarthralgia. The virus is serologically and genetically most closely related to o’nyong-nyong, Igbo Ora, and, to a lesser extent, Mayaro and Ross River viruses, all of which are associated with acute polyarthralgia (Chikungunya virus (CHIKV) is a mosquito-transmitted virus . A typical serologic algorithm involves testing acute- and convalescent-phase serum specimens for immunoglobulin M (IgM) and IgG antibody, followed by a plaque reduction neutralization test (PRNT). Virus isolation and RT-PCR are normally used with early acute-phase specimens (before day 5 post-onset) because duration of viremia is typically 2–4 days.Recent CHIKV outbreaks have been reported in several islands in the Indian Ocean as well as in southern India, where >1 million cases were reported in 2006 .Serum samples were received by the Centers for Disease Control and Prevention  from April 2006 to December 2006 as part of routine diagnostic and reference services available to public health laboratories. A total of 106 serum samples were received from persons returning from regions with epidemics or where CHIKV is endemic (79 from India and the Indian Ocean islands and 27 from Africa) with compatible CHIKV illness and submitted by state public health laboratories. Serum samples were tested for antibodies to several viruses known to occur in the region of travel and residence by IgM capture ELISA and a standard IgG ELISA .<6. Viral titers of these specimens were estimated by quantitative RT-PCR that used CHIKV quantity standards (determined by plaque assay) to generate a standard curve. Titers of 8 specimens ranged from 103.9 PFU/mL to 106.8 PFU/mL.All serum specimens were tested by a quantitative, real-time, fluorescent probed–based RT-PCR assay for CHIKV RNA. Two primer probe sets were designed in unique regions of the viral genome and reacted specifically with CHIKV RNA and not with related or unrelated viruses . Both seAll acute-phase specimens (on or before day 8 postonset) were also tested for CHIKV by virus isolation in Vero cells. Isolation was performed by using a recently developed protocol in which cells were grown in glass shell vials and centrifuged to enhance viral infectivity . Five serum specimens displayed prominent and characteristic cytopathic effect on day 2 postinfection, and virus was identified as CHIKV by RT-PCR. All virus isolates were obtained from acute-phase specimens that also were positive by RT-PCR. Three serum specimens  showed positive RT-PCR results, but CHIKV was not isolated from these specimens. In these 3 specimens, inability to isolate virus may have been related to viral titers, which were lower than most of the virus isolation–positive samples, or to handling or storage of these samples. All 8 virus-positive specimens  were collected <7 days postonset and were negative for IgM and IgG antibodies to CHIKV. Nearly all of the specimens collected <7 days postonset were positive by 1 of the virus-based tests. The 2 exceptions, samples 1 and 9, were positive for IgM and IgG antibodies to CHIKV and had high PRNT titers. These findings indicate that these samples were not true acute-phase specimens; the true onset or collection date had likely been reported incorrectly.www.ncbi.nlm.nih.gov/blast) of the 8 sequences showed that the highest percentage identity was to CHIKV strains recently isolated from travelers returning from Indian Ocean islands . Percentage identity matches between the 8 viruses and Indian Ocean CHIKV strains were >99.5%, with 5 to 8 mismatches occurring randomly. In comparison, percentage identities of the 8 viruses to CHIKV prototype S27 or to a strain previously isolated from India (Nagpur/653496) were 95.1% and 94.4%, respectively.To identify the strain of CHIKV in these specimens, a 2,122-bp fragment from the structural region of the genome  was amplified from all 8 virus-positive specimens by RT-PCR and subjected to nucleic acid sequencing with previously described primers  returned to US regions (South Carolina and Louisiana) known to have populations of Ae. albopictus.The data reported confirm that the widespread CHIKV epidemic in southern India has infected US travelers. CHIKV infections among international travelers are not unexpected; in 2005–2006, ≈800 CHIKV infections were reported in France, primarily in travelers returning from Réunion Island  from patients returning from regions endemic for CHIKV or regions with epidemics and take appropriate safety precautions.Nevertheless, returning travelers with high viremia levels, who live in areas with established


Background and purpose Commercial gentamicin-loaded bone cement beads  constitute an effective delivery system for local antibiotic therapy. These beads are not available in all parts of the world, and are too expensive for frequent use in others. Thus, orthopedic surgeons worldwide make antibiotic-loaded beads themselves. However, these beads are usually not as effective as the commercial beads because of inadequate release kinetics. Our purpose was to develop a simple, cheap, and effective formulation to prepare gentamicin-loaded beads with release properties and antibacterial efficacy similar to the commercially ones.Methods Acrylic beads were prepared with variable monomer content: 100% (500 μL/g polymer), 75%, and 50% to increase gentamicin release through creation of a less dense polymer matrix. Using the optimal monomer content, different gel-forming polymeric fillers were added to enhance the permeation of fluids into the beads. Polyvinylpyrrolidone (PVP) 17 was selected as a suitable filler; its concentration was varied and the antibiotic release and antibacterial efficacy of these beads were compared with the corresponding properties of the commercial ones.Results Gentamicin release rate and the extent of release from beads prepared with 50% monomer increased when the PVP17 content was increased. Beads with 15 w/w% PVP17 released 87% of their antibiotic content. This is substantially more than the gentamicin release from Septopal beads (59%). Acrylic beads with 15 w/w% PVP17 reduced bacterial growth by up to 93%, which is similar to the antibacterial properties of the commercial ones.Interpretation A simple, cheap, and effective formulation and preparation process has been described for hand-made gentamicin-releasing acrylic beads, with better release kinetics and with antibacterial efficacy similar to that of the commercial ones. In Europe, commercially available gentamicin-loaded polymethylmethacrylate beads  constitute an effective delivery system for local antibiotic therapy for osteomyelitis  . Thus, tGentamicin-loaded PMMA beads, which are commercially available under the name Septopal, were obtained from Biomet Europe . One bead (7.0 mm in diameter) contains 7.5 mg of gentamicin sulfate.Simplex-P bone cement powder  was mixed with powdered gentamicin sulfate  for 2 min in a ceramic bowl using a spatula. One gram of gentamicin sulfate was added to 40 g polymer powder. The resulting mixture was subsequently combined with 20 mL of monomer in a ceramic bowl and mixed for 2 min with a spatula according to the manufacturer's instructions. Beads thus prepared (500 μL/g polymer) will be termed “100% monomer”. In addition, beads were prepared with 75% and 50% of the prescribed amount of monomer . The material was mixed until a doughy paste was obtained, and the gentamicin-PMMA-MMA mixture was hand-rolled into beads.Powdered PMMA and gentamicin sulfate (1 g gentamicin sulfate and 40 g PMMA powder) were mixed. Then one of the polymeric fillers was added to this powder mixture. 3 biocompatible gel-forming polymeric fillers were used: polyvinylpyrrolidone with a molecular weight of 360 kDa (PVP 90K) , polyvinylpyrrolidone with a molecular weight of 7–11 kDa , and hydroxypropylmethylcellulose . Polymeric fillers were mixed at a concentration of 10 w/w% with respect to the amount of polymer powder. The resulting mixtures were finally combined with 50% (250 μL/g polymer) of the prescribed amount of monomer and the beads were prepared as described above.Powdered PMMA and gentamicin sulfate (1 g gentamicin sulfate and 40 g PMMA powder) were subsequently mixed with different amounts of PVP 17  and beads were prepared with 50% monomer (250 μL/g polymer) as described above.A gentamicin-loaded acrylic bead was immersed in 10 mL sterile phosphate-buffered saline (PBS) at pH 7.4 and incubated at 37°C. At designated time intervals , 500-μL aliquots of the gentamicin solution in PBS were taken and the total amount of buffer was restored to 10 mL.Gentamicin concentrations were measured using a procedure described by The release experiments with beads prepared with different concentrations of PVP 17 were performed in triplicate and a statistical analysis (Student's t-test for independent samples) was done in order to compare the gentamicin release rates from the handmade beads with those from commercial Septopal beads. A 95%  confidence interval was applied for statistical significance.To compare the polymer matrix of our hand-rolled beads with that of Septopal beads, scanning electron microscopy (SEM) was performed. Examination was done at 2.0 kV in a JEOL field emission scanning electron microscope type 6301F. Beads were sputter-coated with a 3-nm thick conductive layer of gold/ palladium (80/20).3 hand-rolled or Septopal beads were each immersed in 5 mL tryptone soya broth  and incubated at 37°C. Each bead was transferred on a daily basis to 5 mL fresh TSB and again incubated at 37°C, yielding broth containing antibiotic released over a 24-h period for biofilm growth studies. Only broth collected after 1, 2, 3, 7, and 14 days was used for further evaluation. Elution media were stored in a refrigerator at 4°C until further use.Staphylococcus aureus is a common cause of osteomyelitis. Thus, biofilms were grown using a clinical strain, S. aureus 5298, isolated from a patient with an implant-related osteomyelitis at the University Medical Center Groningen, the Netherlands. This strain was gentamicin-sensitive, with an MIC value of 0.75 μg/mL. A preculture of the strain was used to fill 96-well plates with 200 μL bacterial suspension . Biofilms were grown for 24 h at 37°C. Subsequently, the wells were flushed with 200 μL PBS to remove free-floating bacteria. Then, the wells were stained with 200 μL 1% crystal violet for 30 min, washed with 200 μL demineralized water to remove the excess stain, and the crystal violet was solubilized in 200 μL of ethanol-acetone . The absorbance at 575 nm was subsequently determined using a microtiter plate reader and expressed as a percentage with respect to the absorbance of control biofilms grown in the absence of released antibiotics. All experiments were repeated 3 times with separately grown bacteria.Biofilm formation is a key process in the development and persistence of osteomyelitis, and Statistical analysis was done in order to compare the antibacterial efficacy of the hand-rolled beads with that of Septopal beads. Differences were analyzed pair-wise for significance using Student's t-test, with significance assumed at p-values ≤ 0.05.The different hand-rolled beads had an average diameter of 13 mm and their average weight was 1.45 g, 1.34 g, and 1.23 g for beads prepared with 100%, 75%, and 50% monomer, respectively.Release of gentamicin from beads prepared with 100% monomer leveled off within the time interval of the experiment, and was confined to about 8% of the total amount of gentamicin included. Reduction of the amount of monomer caused incomplete polymerization and sintering of the polymer beads, which resulted in an almost 2-fold increase in gentamicin release when 50% of monomer was employed, as compared to beads made with 100% monomer .Inclusion of a biodegradable filler almost tripled the amount of gentamicin release compared to its release in the absence of fillers. There was little difference in release rates between the different fillers .Based on the observation that the dissolution of HPMC created a sticky and viscous medium, which was difficult to handle, and the fact that PVP 17 had the highest purity, we decided to vary the amount of PVP 17  and to cBeads prepared with 100% monomer formed a dense material with much mass and little porosity. Reduction of the amount of monomer relative to the prescribed amount caused a major increase in porosity, and the polymer particles appeared more “sintered” rather than “fused” together .Both bead systems showed high reductions in biofilm growth for up to 14 days after immersion . There wWe have described a simple, cheap, and effective formulation and preparation process for gentamicin-releasing PMMA beads, with release kinetics that are somewhat better than those observed for commercially available Septopal beads. The improved release kinetics are mainly the result of increased porosity of the beads, due to the use of only 50% of the prescribed amount of monomer, which causes sintering rather than polymerization fusion of polymer particles, leaving a porous matrix. Secondly, the addition of a gel-forming, water-soluble polymeric filler, PVP 17, ensures penetration of fluids into all parts of the matrix (full percolation), thereby increasing the total amount of drug that is released.The efficiency of the polymerization reaction became less when the proportion of liquid monomer added to the polymeric bone cement powder was reduced. If the amount of monomer is insufficient for the reaction to occur at all possible contact sites of the polymer particles, a more porous matrix will be formed, allowing penetration of water to most sites in the beads. This will lead to a higher amount of gentamicin being released . On the The use of gel-forming, water-soluble polymeric fillers turned out to be indispensable to increase the gentamicin release to levels comparable to or higher than the release of gentamicin by the commercially available Septopal beads. Although we chose to use PVP 17 for this purpose, other biodegradable fillers might also have served the purpose. PVP 17-filled beads showed excellent gentamicin release profiles, and the Kollidon PVP 17 PF grade is considered safe for parenteral use in humans (Böhler 1999). Moreover, the PVP 17 could easily be combined with the acrylic polymer in the production process.Several studies have been done to investigate the effect of biodegradable fillers on the drug release of antibiotic-loaded bone cement. The gentamicin-loaded beads based on the use of half the prescribed amount of monomer and the addition of gel-forming polymeric filler reduced biofilm formation as effectively as commercially available Septopal beads. The  reduction in efficacy of our hand-rolled beads is presumably due to a less favorable area-to-volume ratio, as the hand-rolled beads were larger than the commercially available beads. Moreover, hand-rolled beads are usually not uniform in size, giving much larger standard deviations than obtained with commercially available beads and attesting to the greater reproducibility of Septopal beads. To solve this problem, a beads template system could be used, in which antibiotic-loaded beads of uniform size and small diameter could be produced. A template system should also be considered because of the short time available to prepare beads with half of the prescribed amount of monomer.To verify the significance of our in vitro observations, in vivo studies will be required that take into account both complete eradication of the infection and the time required to achieve this outcome, but we believe the results reported here support our hypothesis that acrylic beads containing half the prescribed amount of monomer and PVP may offer a therapeutic efficacy similar to that of commercially available Septopal beads. Application of this novel formulation of acrylic beads in combination with a bead-template system would be useful for three different groups of orthopedic surgeons, to make chains of antibiotic-loaded bone cement beads: (1) surgeons in countries where they are not allowed to import the Septopal beads, such as the USA; (2) surgeons in developing countries, since these beads are approximately 3 times cheaper than the commercial ones (apart from the costs of the template system); and (3) surgeons who are familiar with the use of the Septopal beads but who need to treat their patients with another antibiotic because of the increasing number of patients infected with gentamicin-resistent bacteria. The method we describe here also makes it possible to create bone cement beads loaded with vancomycin, clindamycin, or other antibiotics that are not yet available commercially.HR: concept, study design, data collection, analysis, and manuscript preparation. HvdM, HF, SS, JvH, and HB: study design, analysis, and revision of manuscript. DN: concept, study design, analysis, and revision of manuscript.No competing interests declared.


Progenitors of Arctic-like rabies viruses, which now circulate extensively in India, may have been responsible for the emergence of the Arctic rabies lineage. A collection of 37 rabies-infected samples, 10 human saliva and 27 animal brain, were recovered during 2001–2004 from the cities of Bangalore and Hyderabad in southern India and from Kasauli, a mountainous region in Himachal Pradesh, northern India. Phylogenetic analysis of partial N gene nucleotide sequences of these 37 specimens and 1 archival specimen identified 2 groups, divided according to their geographic (north or south) origins. Comparison of selected Indian viruses with representative rabies viruses recovered worldwide showed a close association of all Indian isolates with the circumpolar Arctic rabies lineage distributed throughout northern latitudes of North America and Europe and other viruses recovered from several Asian countries. Lyssavirus genus; various rabies virus variants, which circulate widely in many dog populations throughout Asia  test  recovereAntemortem saliva samples were obtained from 37 human patients with clinical signs consistent with a diagnosis of rabies. All patients were located at 1 of 3 hospitals in Bangalore or at 1 hospital in Hyderabad. The molecular methods described below confirmed 10 of these samples  as rabieTotal RNA was recovered from each specimen by using TRIzol reagent  or TRIzol LS reagent  as recommended by the supplier . Standard reverse transcription–PCR (RT-PCR) was used to amplify the complete N gene of rabies virus as previously described , specimens from Greenland, and 2 specimens from the former Soviet Union (from Yakutia in the north and Tuva in the south). A prior analysis of Arctic specimens , and Chabarovsk (857r). Two members of the northern Indian group (IN-1) of this study formed an outlying branch closely associated with this group.This study benefited from an initiative to explore the utility of PCR technology for antemortem diagnosis of rabies in human saliva samples. Of 37 suspected rabies cases, 10 were confirmed positive by this technique. Unfortunately, no subsequent follow-up of these patients or postmortem analysis of brain material by DFA was possible. At least some of these patients for whom the saliva test was negative for rabies had likely contracted rabies but had no detectable shedding of virus in saliva during the period of saliva collection. Thus, using these data to infer rabies incidence in humans is difficult. Further application of this method, together with improved follow-up of patient outcome, is needed.,,Previous reports  circulate extensively in northern areas of Russia but have also been found in the Tuva region just north of western Mongolia. These viruses are closely related to all the Arctic strain viruses recovered from the Americas. With the exception of the sample from Madras, all Indian isolates recovered from the south of the country, including 1 from a tourist visiting Goa (,,,The evolutionary mechanisms underlying these phylogenetic patterns can only be speculated upon at this time. Observations made in Canada throughout the 20th century (A single Indian rabies specimen, INDIA-dog recovered from Madras on the southeastern Indian coast (We hope that this report will encourage further studies that apply these molecular approaches to the diagnosis of additional rabies cases and the characterization of viruses recovered from other parts of India. Increased knowledge of the complexity of the rabies situation in India should spur efforts to improve public awareness and to better control this disease. Moreover, the data presented here promise to alter current paradigms about the emergence of Arctic rabies.Additional virus samples from other countries included in the phylogenetic analysis


Marburg virus nucleic acid was found in 12 bats, antibodies were found in 2 species of these bats, but no live virus was isolated. To determine reservoir hosts for Marburg virus (MARV), we examined the fauna of a mine in northeastern Democratic Republic of the Congo. The mine was associated with a protracted outbreak of Marburg hemorrhagic fever during 1998–2000. We found MARV nucleic acid in 12 bats, comprising 3.0%–3.6% of 2 species of insectivorous bat and 1 species of fruit bat. We found antibody to the virus in the serum of 9.7% of 1 of the insectivorous species and in 20.5% of the fruit bat species, but attempts to isolate virus were unsuccessful. Filoviridae, cause outbreaks of severe hemorrhagic fever in Africa. Although humans have on occasion acquired infection from contact with tissues of diseased nonhuman primates and other mammals, the reservoir hosts of the viruses in nature remain unknown.Marburg virus (MARV) and Ebola virus, members of the family syndrome hémorragique de Durba [hemorrhagic syndrome of Durba] had been associated with the mine since at least 1987, and a survivor of a 1994 outbreak was found to have antibodies against MARV. The fauna of Goroumbwa Mine included bats, rodents, shrews, frogs, snakes, cockroaches, crickets, spiders, wasps, and moth flies ; the case-fatality ratio was 83%  were collected by hand or with sweepnets. Vertebrates were euthanized and dissected on site. Blood samples were collected; and samples of liver, lung, spleen, kidney, testes, brain, salivary glands, and fetuses of pregnant females were preserved along with the arthropods in liquid nitrogen dry-shipping containers for transport to the National Institute for Communicable Diseases in South Africa. Extra liver samples were collected for phylogenetic studies on bats and rodents, and formalin-fixed tissue samples were kept for possible histopathologic and immunohistochemical examination. Carcasses were fixed in formalin for α-taxonomy purposes.Megachiroptera) and 12 species of insectivorous bats (Microchiroptera), including samples from 56 Chaerephon pumila, 32 Rousettus aegyptiacus, 27 Mops condylurus, 16 Hipposideros caffer, plus 57 samples from 11 other species.Vertebrate tissue and arthropod suspensions were processed and tested for filovirus nucleic acids by reverse transcription–PCR (RT-PCR) and nested PCR by using filovirus-specific large (L) protein gene primers and nested MARV-specific viral protein 35 (VP35) primers as described for samples from human patients during the outbreak  roosted in the mine, clustered within the upper galleries. Although the numbers of insectivorous bats were difficult to estimate because these bats roosted mainly in the deeper recesses of the mine, the catch rates indicated substantial numbers of the eloquent horseshoe bat (Rhinolophus eloquens) and the greater long-fingered bat (Miniopterus inflatus). Few microchiropterans were caught in May, but catch rates improved in October after adjustment of trapping hours and the gauge of mist nets used. Pregnancy was recorded in 12 (24%) of 50 R. aegyptiacus females in May and in 2 (4.2%) of 47 females in October; descended testes were found in 2 (6%) of 33 males in May and 19 (25%) of 76 in October. The only indication of breeding activity observed in microchiropterans was that 1/7 Rh. eloquens females was pregnant in May.The numbers of specimens collected, plus the results of RT-PCR, nested PCR, attempts to isolate virus in cell culture, and ELISA antibody determinations, are summarized in the M. inflatus, 7 (3.6%) of 197 Rh. eloquens, and 4 (3.1%) of 127 R. aegyptiacus. Nested VP35 PCR on individual tissues of the positive bats produced positive results for liver, spleen, kidney, lung, salivary gland (3/5 bats), and heart (2/5 bats). Attempts to isolate virus in cell cultures from pooled organs were uniformly negative. Applying an ELISA cutoff value of 16.4 PP, determined as 3 × (mean + 3 SD) of values recorded for 188 bat serum samples from Kruger National Park, antibody activity to MARV was detected by ELISA in 20 (9.7%) of 206 Rh. eloquens and in 32 (20.5%) of 156 R. aegyptiacus serum specimens from Durba  of 33 om Durba . PrevalePhylogenetic analysis of the sequences determined for the twelve 302-nt MARV VP35 gene fragments amplified from bat specimens (GenBank accession nos. EU11794–EU118805) showed that 6 corresponded to sequences previously determined for virus isolates from humans during the epidemic . AlthougThe history of filovirus outbreaks shows several instances from which it can be inferred that bats may have served as the source of infection. Anecdotal evidence indicates that during shipment from Uganda, the monkeys associated with the first outbreak of Marburg hemorrhagic fever in Europe in 1967 were kept in a holding facility on a Lake Victoria island that had large numbers of fruit bats. In the second filovirus outbreak in 1975, Marburg hemorrhagic fever developed in 2 tourists who had slept in rooms with insectivorous bats at 2 locations in Zimbabwe (The circumstantial evidence in the Marburg hemorrhagic fever outbreak in Durba strongly implicates Goroumbwa Mine as the source of human infection. At least 9 genetic variants of MARV circulated in humans during the outbreak. And because laboratory testing was limited to a few patients, additional variants could have been undetected, as substantiated by our evidence of 6 more variants in bats. The evolution and perpetuation of multiple genetic variants of virus in a fixed location would require a suitably large reservoir host population with constant recruitment through reproduction or migration of susceptible individuals, as generally occurs in small vertebrate and invertebrate populations such as the bat population of Goroumbwa Mine. Failure to isolate live virus may be because it was present in very low concentrations, either early or late in the course of infection. This was the first detection of filovirus nucleic acid and antibody in bats, a phenomenon which was subsequently demonstrated with Ebola virus and MARV nucleic acids and antibodies in fruit bats collected in 2002 and 2005 in Gabon, where it again proved impossible to isolate live virus (The nature of filovirus infection in bats may vary with age and reproductive status. A seasonal pattern in the occurrence of human disease was noted over the 2 years of the epidemic in Durba; transmission began in October–November and peaked in January–February (


Three outbreaks of Ebola hemorrhagic fever have recently occurred in Gabon. Virus has been isolated from clinical materials from all three outbreaks, and nucleotide sequence analysis of the glycoprotein gene of the isolates and virus present in clinical samples has been carried out. These data indicate that each of the three outbreaks should be considered an independent emergence of a different Ebola virus of the Zaire subtype. As in earlier Ebola virus outbreaks, no genetic variability was detected between virus samples taken during an individual outbreak.