Pomegranate is a source of some very potent antioxidants  which are considered to be also potent anti-atherogenic agents. The combination of the above unique various types of pomegranate polyphenols provides a much wider spectrum of action against several types of free radicals. Indeed, pomegranate is superior in comparison to other antioxidants in protecting low-density lipoprotein  and high-density lipoprotein  from oxidation, and as a result it attenuates atherosclerosis development and its consequent cardiovascular events. Pomegranate antioxidants are not free, but are attached to the pomegranate sugars, and hence were shown to be beneficial even in diabetic patients. Furthermore, pomegranate antioxidants are unique in their ability to increase the activity of the HDL-associated paraoxonase 1 (PON1), which breaks down harmful oxidized lipids in lipoproteins, in macrophages, and in atherosclerotic plaques. Finally, unique pomegranate antioxidants beneficially decrease blood pressure. All the above beneficial characteristics make the pomegranate a uniquely healthy fruit. The pomegranate tree, which is said to have flourished in the Garden of Eden, has been extensively used as a folk medicine in many cultures.4In vivo studies were conducted in order to evaluate whether the active antioxidant components of PJ are absorbed. Recent studies examined the bioavailability and metabolism of punicalagin in the rat as an animal model.n = 5), and the second one with the same diet plus 6% punicalagin (n = 5). The daily intake of punicalagin ranged from 0.6 g to 1.2 g. Glucuronides of methyl ether derivatives of ellagic acid and punicalagin were detected in plasma. 6H-Dibenzo  pyran-6-one derivatives were also observed in the plasma, especially during the last few weeks of the study. In urine, the metabolite urolithin was observed along with 6H-dibenzo  pyran-6-one derivatives, and they were present as aglycones or as glucuronides. It was concluded that since only 3%–6% of the ingested punicalagin was detected as such, or as metabolites in urine and feces, the majority of this ellagitannin has to be converted to undetectable metabolites or accumulated in non-analyzed tissues. Only traces of punicalagin metabolites were detected in liver or kidney. In humans, following consumption of PJ (180mL) containing 25 mg of ellagic acid and 318 mg of hydrolyzable ellagitannins , ellagic acid was detected in human plasma 1 hour post-ingestion at a maximum concentration of 32 ng/mL, and by 4 hours it was completely eliminated.7in vitro availability method,Upon analyzing the influence of the physiological conditions in the stomach and small intestine on pomegranate bioactive compounds bioavailability using an PJ is suggested as the “heart-healthy” fruit juice,The effect of a daily consumption of PJ for 3 months on myocardial perfusion in 45 patients who had coronary heart disease (CHD) was also studied.12n = 146), or a control beverage (n = 143) for up to 18 months. No significant difference in overall CIMT progression rate was observed between PJ and control treatments. In exploratory analyses, however, of subjects in the most adverse tertiles for baseline serum lipid peroxides, triglycerides (TGs), high-density lipoprotein (HDL) cholesterol, TGs/HDL cholesterol, total cholesterol/HDL cholesterol, and apolipoprotein-B100, those in the PJ group had significantly less anterior wall and/or composite CIMT progression versus control subjects. These results suggest that, in subjects at moderate CHD risk, PJ consumption had no significant effect on overall CIMT progression rate, but slowed CIMT progression in subjects with increased oxidative stress and disturbances in the TG-rich lipoprotein/HDL axis.13A randomized, double-blind trial assessed the influence of PJ consumption on anterior and posterior CIMT progression rates in subjects at moderate risk for coronary heart disease. Subjects were men (45–74 years old) and women (55–74 years old) with one or more major CHD risk factors and baseline posterior wall CIMT of 0.7–2.0 mm, without any significant stenosis. Participants consumed 240 mL/day of PJ (P<0.01) 16% decreased susceptibility to free radical-induced lipid peroxidation, in comparison to plasma obtained prior to PJ consumption, as measured by lipid peroxides formation, or by total antioxidant status (TAS) in serum.18The oxidative modification hypothesis of atherosclerosis proposes that low-density lipoprotein (LDL) oxidation plays a pivotal role in early atherogenesis. This hypothesis is supported by evidence that oxidized LDL (Ox-LDL) is present in atherosclerotic lesions, and in human plasma from patients with cardiovascular diseases, and it correlates with the presence of angiographically documented complicated plaques,P<0.01) reduction in the concentration of antibodies against Ox-LDL by 24% and 19% was observed after 1 and 3 months of PJ consumption, respectively . TAS in serum from these patients was substantially increased 2.3-fold . These results indicate that PJ administration to patients with CAS substantially reduced their serum oxidative status and could thus inhibit plasma lipid peroxidation. The susceptibility of the patients’ serum to free radical-induced oxidation decreased after 12 months of PJ consumption by up to 62% , for 2 hours at 37°C. Then, HDL was isolated from the serum by ultracentrifugation, and Western blot analysis was performed. After serum incubation with PJ /mL) or with punicalagin, the protein content of HDL-bound PON1 significantly increased by 36% and by 14%, respectively, as compared to control serum. Upon increasing the concentration of PJ or punicalagin up to 36μg GAE/mL, HDL-bound PON1 protein further increased, and it was 62% or 83% higher than that observed in control patients’ serum (no PJ), respectively.The increment in PON1 protein could result from the direct effect of PJ on PON1 expression in the liver.We thus conclude that PJ as well as POMxl consumption by diabetic patients contributes to PON1 stabilization by increasing its association with HDL and therefore enhancing PON1 catalytic activities. The ratio between HDL-associated PON1 and free PON1 gradually decreased as the extent of HDL oxidation increased. The antioxidants vitamin E or PJ inhibited the oxidation-mediated redistribution of PON1 in serum. Indeed, PJ and its purified major polyphenols punicalagin, gallic acid, and ellagic acid all increased PON1 binding also to HDL.33in vitro. As reduction in serum ACE activity, even with no decrement in blood pressure BP, was previously shown to attenuate atherosclerosis, PJ can offer a wide protection against cardiovascular diseases which could be related to its inhibitory effect on oxidative stress and on serum ACE activity.As some antioxidants were recently shown to reduce blood pressure (BP), we studied the effect of PJ consumption  by hypertensive patients on their BP and on serum angiotensin-converting enzyme (ACE) activity.P<0.05) reduced by 7%, 11%,10%, 10%, and 12% after 1, 3, 6, 9, and 12 months of PJ consumption, respectively, compared to values obtained before treatment. In contrast, PJ consumption had no significant effect on the patient’s diastolic blood pressure.12In CAS patients the systolic BP was significantly (P < 0.001), diastolic BP, and mean arterial pressure . The fall in BP was not paralleled by changes in concentration of serum ACE.In another study, healthy participants consumed 330mL/day of PJ or control drink for 4 weeks.P = 0.041). There were no treatment effects for DVP-RI, DVP-SI, or diastolic BP. In conclusion, consumption of a single drink containing ellagitannin-rich pomegranate extract did not decrease postprandial plasma triglyceride concentrations, but suppressed the postprandial increase in systolic BP following the high-fat meal.37The effect of PJ supplementation for a short term was also analyzed.in vivo by arterial wall cells.45Macrophage cholesterol, triglyceride, and oxidized lipids accumulation and foam cell formation are the hallmarks of early atherogenesis.in vitro in J774A.1 macrophages.46PJ consumption by patients with type 2 diabetes mellitus significantly decreased the extent of Ox-LDL cellular uptake by their HMDM by 36%, , as was in vitro to inhibit macrophage cholesterol biosynthesis.48The carotid lesions from CAS patients who consumed PJ contained also less cholesterol . This coStatins therapy made a significant health benefit, mainly in cardiovascular protection,53Phytosterols, which encompass plant sterols and stanols, are steroid compounds similar in their structure to cholesterol.in vitro the anti-atherogenic effects on macrophage cholesterol biosynthesis rate, and on cellular oxidative stress, of the combination of simvastatin with punicalagin, or with a phytosterol (β-sitosterol), or with PJ , as well as its catalytic activities, resulting in the hydrolysis of lipid peroxides. Moreover, PJ has a remarkable effect on macrophage and lesion atherogenicity. Pomegranate juice consumption decreased oxidative stress in macrophages and in atherosclerotic lesions, and the extent of Ox-LDL uptake by macrophages. This could be a direct antioxidant effect of PJ, or an indirect effect, by increasing HDL-associated PON1 as well as cellular PON2. Interestingly, the lesion cholesterol levels were decreased after PJ consumption. This could be related to the reduction in Ox-LDL uptake by macrophages, to PJ/PON1-induced inhibition of cholesterol biosynthesis, and to PON1 stimulation of HDL-mediated cholesterol efflux from arterial macrophages.in vivo, in humans .The preferred pomegranate product in terms of biological potency and consequent health benefits is PJ from the whole fruit (including the peel). Since the combination of antioxidants that exists in PJ can provide a wider range of free radical scavenging capacities than an individual antioxidant, clinical and nutritional studies in humans should be directed towards the use of combinations of several types of dietary antioxidants, as well as combinations of flavonoids together with other nutritional antioxidants, such as vitamin E or carotenoids. In addition, PJ can be beneficially used in combination with low-dose statins in hypercholesterolemic patients.All these antioxidative and anti-atherogenic effects of pomegranate polyphenols were clearly demonstrated Finally, it is also important to identify populations suitable for antioxidant treatment, as antioxidants treatment may be beneficial only in subjects who are under excess oxidative stress.


The HIV-1 virus can enter a dormant state and become inactive, which reduces accessibility by antiviral drugs. We approach this latency problem from an unconventional point of view, with the focus on understanding how intrinsic chemical noise (copy number fluctuations of the Tat protein) can be used to assist the activation process of the latent virus. Several phase diagrams have been constructed in order to visualize in which regions of the parameter space noise can drive the activation process. Essential to the study is the use of a hyperbolic coordinate system, which greatly facilitates quantification of how the various reaction rate combinations shape the noise behavior of the Tat protein feedback system. We have designed a mathematical manual of how to approach the problem of activation quantitatively, and introduce the notion of an “operating point” of the virus. For both noise-free and noise-based strategies we show how operating point off-sets induce changes in the number of Tat molecules. The major result of the analysis is that for every noise-free strategy there is a noise-based strategy that requires lower dosage, but achieves the same anti-latency effect. It appears that the noise-based activation is advantageous for every operating point. Human immunodeficiency virus 1 (HIV-1), initially reported nearly 30 years ago, represents a major global health problem with millions of people infected . One of There are many reservoirs, e.g., cell types, in the body that can harbor the latent virus. CD4+ T-cells have been identified as one of the largest pools of the viral DNA. This is also one of the best characterized reservoirs . Once a 6, though the frequency can be lower . Accordingly, a noise-based treatment designed to move the operating point into a noisier region will work uniformly on all points in this set. This would always result in a relatively large dose reduction; Figure We now briefly discuss possible effects of chromatin on the operating point of a virus. The transcription rate We have designed a generic mathematical manual of how to approach the problem of the HIV latency in a quantitative manner, accompanied by an example of how to use this manual. We formalised several concepts that are vaguely defined or understood only intuitively. The first key concept is the notion of an “operating point” of the virus and how it is affected by the action of a drug. The second key concepts of the mathematical formalism is the notion of a particular observable that strongly affects the activation probability. We suggested a mathematical way of describing how each therapy affects an operating point and how this in turn influences the observable that controls the activation probability. The third key ingredient is a dose reduction coefficient, which can be used to quantitatively compare various therapies.We have suggested and investigated two rather general strategies for the virus activation, the noise-free and the noise-based strategy. In the first approach, anti-latency agents are administered such that the activation happens with almost absolute certainty. In order to achieve such certainty, possibly unreasonable quantities of drugs need to be administered. In the second approach, drugs are administered in such a way that the activation is less certain but still happens with a relatively large probability. The idea behind the noise-based strategy is to reduce the quantity of drugs that need to be administered in order to achieve activation.Tat protein feedback loop, the most important part of the the HIV latency control. Based on the structure of the loop we suggested a class of noise-based activation strategies. We envision such an activation strategy in a procedure where one constantly supplies exogenous Tat at a very small rate, and adds a combination of anti-latency drugs that would off-set the operating point of the virus towards noisier regions.The mathematical manual is rather generic. To demonstrate how the mathematical manual can be used, we have focused on the simplest possible model of the In this context we considered two observables and could compute the dose reduction coefficient for both cases to answer the fundamental question: for which operating points the noise-base therapy is advantageous over the noise-free therapy in the sense of possible dosage reduction? This addresses the practical problem of reducing effects of toxicity during the anti-latent treatment. Three phase diagrams were constructed to explain what controls the noise, and how this control can be used to battle latency. Our analysis of the phase diagrams indicates that the noise-based therapy is always advantageous, no matter which operating point the virus adopts. This results holds regardless of which observable is targeted. This is the major result of our analysis.The mathematical manual is currently based on rather qualitative assumptions, but it is very generic and can be easily extended and refined. For example, we have used the assumption of small operating point offsets in order to linearize the theory. One can easily consider non-infinitesimal off-sets, as demonstrated in the example. We discussed several possible extensions which are left for future work.feature benefiting treatment. We showed that such strategy should be efficient for any latent cell.In summary, we suggest an activation principle where intrinsic noise is considered a Both authors declare that they have no competing interests.ZK has defined the research theme, developed the model, and performed computations. ZK and AJ analysed the results, discussed, and prepared the manuscript. Both authors read and approved the final manuscript.


Renal cell carcinoma (RCC) is the third most common genitourinary cancer and the most common primary renal neoplasm. Estimates of the economic burden of RCC in the United States range from approximately $400 million (in year 2000 dollars) to $4.4 billion (in year 2005 dollars). Actual costs associated with RCC, particularly for elderly Medicare patients who account for 46% of U.S. patients hospitalized for RCC, are poorly understood.To estimate all-cause health care costs associated with RCC using the combined Surveillance Epidemiology and End Results (SEER)-Medicare database.The sample was limited to non-HMO patients aged 65 years or older who were diagnosed with a first primary RCC  between 1995 and 2002. Our final sample included 4,938 patients with RCC and 9,876 non-HMO noncancer comparison group cases without chronic renal disease drawn from the SEER 5% Medicare sample and matched by a propensity score calculated from age, gender, race/ethnicity, and comorbidities. Costs were defined as payments made by Medicare for all-cause medical treatments including inpatient stays, emergency room visits, outpatient procedures, office visits, home health visits, durable medical equipment, and hospice care, but excluding out-patient prescription drugs. Using the method of Bang and Tsiatis (2000), we estimated cumulative costs at 1 and 5 years by estimating average costs for each patient in each month up to 60 months following diagnosis. Total costs were weighted sums of monthly costs, where weights were the inverse probability that the patient was not censored, and inverse probabilities were estimated by Kaplan-Meier estimates of time to censoring. Using the method of Lin (2000), we performed multivariate analyses of costs by fitting each of the 60 monthly costs to linear models that controlled for demographic characteristics and comorbidities. Marginal effects of covariates on 1- and 5-year costs were obtained by summing the coefficients for months 1 through 12 and months 1 through 60, respectively. Confidence intervals were obtained by bootstrapping.Patients with RCC and matched comparison group cases had similar demographic characteristics, comorbidities, and chronic conditions. At the start of the fifth year post-diagnosis, there were 1,208 Medicare RCC cases of the original 4,938 (20.8%). Mean costs per patient per month (PPPM) in the first year were $3,673 for patients with RCC and $793 for comparison group patients. PPPM costs were higher for RCC patients with more advanced stage  disease. Average cumulative total costs for RCC patients were $33,605 per patient in the first year following diagnosis and $59,397 per patient in the first 5 years following diagnosis. Several patient-specific factors were associated with 1- and 5-year costs in multivariate analyses, including age, race/ethnicity, and comorbidities. Among RCC patients, treatment with surgery and radiation was associated with higher costs per patient than treatment with surgery alone at 1 year  and 5 years . RCC patients who received chemotherapy as part of their treatment regimen also had significantly higher costs per patient than those who received surgery alone at 1 year  and 5 years .Newly diagnosed RCC is associated with a significant economic burden, which is largely determined by several patient characteristics, disease stage, and treatment choice.


To the Editor: The ≈20 recognized arenaviruses in the Americas are hosted by rodents of the family Cricetidae; 1 exception may be hosted by a bat  . The landscape at the study sites is dominated by tropical savanna, small patches of forest, and cultivated land. Live-capture traps were set in a variety of habitats at each locality, and captured rodents were processed according to methods by Mills et al. (Blood samples were tested for arenavirus immunoglobulin (Ig) G by indirect immunofluorescent antibody assays. Guanarito and Pichindé virus–infected Vero E6 cells were used as antigens on spot slides. The secondary antibody was a fluorescein-conjugated goat, antimouse IgG. Serum samples were screened at a dilution of 1:10 and endpoint titers were measured by using serial 2-fold dilutions (1:10–1:320)  or known to be hosted by species that we captured (Guanarito virus). Among the 10 to virus , suggestZ. brevicauda rodents (1.1%) had antibody only to Guanarito virus or had a 4-fold greater titer to Guanarito virus, much lower than the 15% antibody prevalence in the same species in the Venezuelan hemorrhagic fever–endemic area, Portuguesa State, Venezuela (Surprisingly, only 2 O. fulvescens rodent had a low antibody titer only to Pichindé virus. This apparent 4% antibody prevalence is based on only 28 mice. The significance of this finding is not clear but may represent spillover or an undescribed arenavirus specific to the species O. fulvescens. Again, additional studies are needed.The single antibody-positive >1 arenaviruses circulating among common rodent hosts in Caribbean Colombia. We emphasize that many New World arenaviruses are likely cross-reactive to the antigens we used; recovery and sequencing of viral RNA will be essential to fully characterize these viruses. Hemorrhagic fever of arenaviral origin should be included in the differential diagnosis of tropical fevers, at least in our study region. As the human population of the rural Department of Córdoba and adjacent areas of the Caribbean coast of Colombia continues to increase, the potential for arenavirus-related disease could become a public health concern.Our results demonstrate the presence of


The application of new techniques and materials in total knee arthroplasty (TKA) continue to be a primary focus in orthopedic surgery. The primary aim of the present study is to evaluate post TKA total range of motion (ROM) among a group of patients who received a gender specific high-flexion design modification implant compared to a control group of patients who received non-gender specific implants.The control group was comprised of 39 TKAs that were recruited pre-operatively and received the non-gender specific implant while the study group consisted of 39 TKAs who received gender specific implants. The study group yielded an improvement in mean post-operative ROM of 21° at 12 months, whereas the mean improvement in ROM among the control group was 11°. Thus, the study group had a 10° increased ROM improvement (91%) over the control group (p = 0.00060). In addition, 100% of the subjects with gender specific high-flexion implants achieved greater or equal ROM post-operatively compared to 82% for the control cohort. Lastly, women who exhibited greater pre-operative ROM and lower body mass index (BMI) were found to benefit the most with the gender specific prosthesis.Our study demonstrates that among subjects with a normal BMI, the gender specific high-flexion knee implant is associated with increased ROM as compared to the non-gender specific non-high-flexion implant designs. For the past forty years, advancements in approaches to total knee arthroplasty (TKA) have remained a primary focus in the field of orthopedic surgery. The development of intramedullary and extramedullary cutting instruments and enhanced fixation techniques in the 1970’s led to advancements in prosthesis design and surgical expertise in the 1980’s that have facilitated long-term implant survival. Few chaIn efforts to produce a knee prosthetic with optimal fit, researchers have compared current prosthetic dimensions to morphological knee measurements of large patient populations undergoing TKA. A critical study that analyzed 337 knee surgeries for distal femur size, revealed a wide aspect ratio  dimension divided by the anterior-posterior (A/P) dimension) variation between the male and female populations. ResultsFor a given femoral A/P size, males have a larger or broader femoral M/L dimension; therefore, traditional femoral prosthetics in women tend to be oversized. In addition, women have less prominent anterior condyles. Poilvache and Insall reportedThe literature is limited regarding the potential benefits of gender specific knee implants for TKA. The primary aim of the study was to evaluate the performance of a newer TKA design that takes into account the noted high-flexion and anatomical differences in male and female knees and evaluate if those modifications truly make a difference in post-operative ROM.The study protocol was approved by The Methodist Hospital Research Institute in Houston, Texas. A consecutive series of 77 women with a total of 97 TKAs were recruited pre-operatively in an IRB-approved study from the principal investigator’s clinical practice to make up the control group. Each patient underwent primary TKA between November 2005 and October 2006, receiving the Zimmer  NexGen CR implant without a high-flexion modification. Of the 77 women, 33 completed their 12-month follow-up visit, yielding 39 total TKAs that were utilized in the analysis. The relatively high rate of attrition can be attributed to loss to follow-up and subjects being excluded if they were unable to comply with the pre-set time points of the post-operative follow-up visits. The study group consisted of 82 women (97 TKAs) who were recruited between October 2006 and February 2008. These subjects received the Zimmer NexGen High-Flex Gender Solutions knee prosthesis. Of the 97 knees, the first 39 TKAs with follow-up data through one year were included in the analysis to yield equal group sizes. A total of 28 of these gender specific implants were NexGen CR-Flex, while the remaining 11 were NexGen LPS-Flex. Additional fileAll surgeries were performed by the principal investigator utilizing computer-assisted navigation via a minimally invasive (quad sparing) technique with the same gap settings and extension passive ranges in order to normalize surgical technique mismatch. This approach ensured that early patients did not have an advantage due to differences in range under anesthesia or restrictive tightness on ligament tensioning. The control and study cohorts received identical pre- and post-operative care.2 respectively, while the mean age and BMI for the study cohort was 67.9 years and 29.9 kg/m2. Also, pre-operative extension and flexion did not prove statistically significant, thereby minimizing the possibility of confounding variables in this study.Exclusion criteria for participation included: under 20 years of age, cancer, major anatomical compromise, and bone deformity or contradiction. The two cohorts revealed no statistically-significant differences in any demographic feature. The mean age and BMI for the control cohort was 68.3 years and 30.0 kg/mPatients were evaluated at three designated post-operative time intervals: 2 months, 6 months and 12 months. All patients within the balanced cohorts of 39 TKAs returned for their first and last post-operative visits. At each post-operative visit, the principal investigator determined extension and flexion using a goniometer. Post-operative complications and all occurrences of post-operative manipulation under anesthesia or implant revision were recorded.t-test with α = 0.05 (twin-tailed) to accommodate unequal variance and sample size. The correlation between post-operative ROM and continuous variables  was determined using scatter plots and least squares best-fit trend lines. Statistical differences in the occurrences of complications, manipulation under anesthesia, and revisions for each group were determined using Pearson’s chi-squared test.Pre- and post-operative extension, flexion, and ROM and net change (improvement) in ROM between pre-operative and each post-operative visit were analyzed using Welch’s Summary statistics are shown in TableThe progression of ROM improvement from pre-operative values for both cohorts is illustrated in FigureThe post-operative (12 months) was analyzed with respect to pre-operative ROM for both cohorts to reveal the class of subjects for which greatest post-operative ROM was achieved. FigureAlso, as indicated in Figure2, ROM improvement increased by 0.8° for the gender specific implant. Based on this analysis, women in the normal range (BMI approximately 20–26) can expect the full gain of the gender specific implant, while obese women (BMI approximately 30–40 kg/m2) will likely achieve less than 10° improvement over the unisex implant.The ROM improvement was further analyzed with regard to BMI for each cohort as illustrated in FigureThe ROM improvement was also analyzed with respect to femoral implant A/P size to assess if physical size could play a role in improving range when using more modern designs in condylar dimensions. FigureFinally, a total of 184 knee surgeries were retrospectively analyzed with regard to occurrences of post-operative complications and revisions to compare with the same time frame of one year in the study group. With the exception of revisions, no relationship existed between complications  and type of prosthetic. However, revisions were 5 times more likely  with the unisex implant than the gender specific implant. The revisions consisted of 4 mechanical failures and one infection.This study provides evidence to support the notion that newer designs of dimensional matching by aspect ratio and high-flexion modifications may yield early recovery advantages over conventional unisex designs of the past. Specifically, upon examining 78 TKAs, the 39 gender-specific high-flexion knee prosthetics improved pre-operative ROM an average 21° at 12 months, while the 39 conventional designed implants improved ROM by 11°, resulting in a 10° ROM improvement differential  attributable to the gender-specific implant (p = 0.00060). Computer-assisted surgery was employed in an attempt to hold surgeon related variables constant throughout the study minimizing surgeon bias. Moreover, all subjects with gender-specific high-flexion implants achieved post-operative ROM at least equivalent to their pre-operative value, whereas 82% of subjects with the unisex implant reached or exceeded their pre-operative ROM. Revisions were 5 times more likely (p = 0.00015) with the unisex implant.2) achieved the full benefit of the gender specific implant, whereas obese women (30–40 kg/m2) achieved on average less than the 10° ROM gain possible with respect to unisex implants. As a coarse approximation, for each unit of decreasing BMI, an additional degree of ROM improvement can be expected with the gender specific implant. These BMI-associated results reinforce the notion that non-obese patients fare better in TKA outcome than obese patients as reported by others[Regardless of the choice of implant, subjects with greater pre-operative ROM posted the greatest postoperative ROM. However, those women who exhibited greater pre-operative ROM and lower BMI benefited the most with the gender specific prosthesis as compared to the unisex implant. Specifically, women in the normal BMI range  ROM improvement of a gender-matched high-flexion designknee prosthetic was 10° (or 91%) superior to the conventional unisex prosthesis. Also, women who exhibited greater pre-operative ROM and lower BMI were found to benefit the most with the gender-specific prosthesis. For each decreasing unit of BMI, an additional degree of ROM improvement can be expected with the gender-specific implant. These modest improvements suggest the optimal knee of the future may well be a David R. Lionberger, MD had competing interests as he had consultancies with the following companies: Pfizer, Zimmer, and Proctor & Gamble. All other authors declare that they have no competing interests.DRL: data generation, manuscript preparation. MDE: manuscript preparation, statistical analysis and results. KB: data collection. LF: manuscript revision. All authors read and approved the final manuscript.Sample size calculations for continuous response variables.Click here for file


Aedes albopictus (Skuse) reproductive parameters and the possibility of vertical transmission. Two strains were collected in the area where the epidemic occurred in 2007, one isolated from mosquitoes, the other one isolated from a viraemic patient. Different types of blood meals, either infected or non-infected, were offered to Ae. albopictus females, that were then analyzed at increasing time post infection. The virus titre, measured by two RT-PCR methods in the blood meals, influenced the rate of infection and the rate of dissemination of CHIKV in Ae. albopictus body. We found individual variability with respect to the infection/dissemination rates and their latency both considering the female's body and appendages. The hatching rate was significantly lower for the eggs laid by the infected females than for the control eggs, while the mortality during the larval development (from first instar larva to adult emergence) was similar among the progeny of infected and non-infected female groups. Our findings seem to support the hypothesis that the vertical transmission is a rare event under our conditions, and that a certain time period is required in order to get the ovarioles infected. Field observations conducted during the Spring 2008 showed no evidence of the presence of infected overwintering progeny produced by Ae. albopictus females infected during the 2007 outbreak.We investigated the impact of CHIKV strains on some  Chikungunya virus (CHIKV) is an arbovirus of the family Togaviridae, which is a human pathogen and causes symptoms such as high fever, severe joint pain , marked weakness and may be accompanied by skin manifestations The first known outbreak of CHIKV was described in 1952 in Tanzania The epidemic index case was identified as a person travelling from India  in June 2007. After the first outbreak, four secondary minor outbreaks originated in Cervia, Ravenna, Cesena and Rimini, totalling 248 cases confirmed by laboratory investigations, 142 of which were residents in the initial affected area.Aedes albopictus (Skuse) The vector was identified as the mosquito Aedes albopictus can easily adapt to urban environments, exploiting all kinds of artificial containers as larval habitats. It feeds on many vertebrates, including birds, reptiles and amphibians, but prefers mammals, and in particular humans Aedes albopictus is well established in several European countries Ae. albopictus is active all year-round in Southern Italy Although Ae. aegypti L. and Ae. albopictus, while Hailin et al. Ae. albopictus strains from La Réunion, were unable to detect vertical transmission.Data on vertical transmission under laboratory conditions are scarce and inconsistent. Mourya Ae. albopictus collected in Italy in the area where the epidemic occurred in 2007. The search for the virus presence was conducted in parallel by two Laboratories that applied different PCR methods. According to Turell et al. The purpose of this study was to investigate the possible vertical transmission of CHIKV in a strain of Aedes albopictus females used in the experiment originated from eggs collected in the area of the 2007 CHIKV epidemic in Autumn and kept in laboratory at 28±2°C, 80% RH, 16∶8 photoperiod. Larvae (F0) were fed on a diet consisting of dry cat food (Friskies®), yeast and dry fish food (Tetramin®). Once at the adult stage they were given a 10% sucrose solution and females were offered a defibrinated rabbit blood meal, provided through a thermostatically controlled device. A filter paper was placed into a beaker as an egg deposition substrate for females. A sample of the F1 adults was analyzed by RT-PCR to check for the absence of CHIKV. The experimental infection was then performed using adult females of the F1 generation.7.8 and 106 TCID50/mL, respectively. The sequence obtained from the mosquito isolate (GB EU244823) had a 99% identity with 2006 and 2007 Indian isolates , and showed the E1-Ala226Val mutation.Two CHIKV isolates obtained during the 2007 epidemic were tested in parallel: the strain A (ITA07-RA1) was isolated by the Zooprofilactic Institute of Lombardia and Emilia-Romagna (from now on IZSLER) from adult mosquitoes collected in Castiglione di Ravenna during the epidemic. The strain B (ITA7-BIO 07) was isolated at the Department of Microbiology, University Hospital S. Orsola-Malpighi of Bologna (from now on DMUNIBO), from a viremic patient. Both strains were passaged 5 times in Vero cells before being used in the study. The titre of the strains A and B were 10The infectious meal consisted of 2/3 washed human erythrocytes and 1/3 viral suspension (volume) in DMEM cell culture medium  mixed by a magnetic stirrer for 1 min just before the experiment.For both the virus isolates, three suspensions of 6 mL each were prepared, containing three different virus titres:7.3 TCID50/ml (A1), 106.3 TCID50/mL (A2), 105.3 TCID50/ml (A3)isolate A: 105.5 TCID50/ml (B1), 105.0 TCID50/mL (B2), 104.5 TCID50/ml (B3)isolate B: 10The oral infection was performed at the BL3 laboratory of the Agriculture and Environment Centre “G. Nicoli” (from now on CAA). Twenty-four hours before the infection, groups of 60 females aged 4–5 days were isolated in Plexiglas cages (18×18×18 cm) without sugar supply. Three non-infected blood meals were prepared as control: mechanically defibrinated rabbit blood (R), human washed erythrocytes (HE), human washed erythrocytes mixed with the culture medium used for the virus suspension (HE-M). Two infected blood meals were prepared by adding to the HE-M the isolate A (HE-sA) and the isolate B (HE-sB). The control blood meals R, HE, and HE-M were offered to two groups of females, while the blood meals HE-sA and HE-sB were offered to three groups of females.The blood meals were maintained at 37°C by means of a thermostatically controlled device, and were offered to the females for 30 minutes. After that period, all the non-engorged females were removed from the cages, and samples of 1–3 engorged females were collected from the cages with infected blood to determine the amount of virus ingested. The residual blood was recovered from the feeding devices to check for the virus presence by means of RT-PCR.The remaining engorged females were left in cages provisioned with an oviposition substrate and had free access to a 10% sucrose solution, at 28.0±1.0°C, 80% RH and 16∶8 photoperiod. They were blood fed again 6 and 14 days after the first blood meal, with non-infected rabbit blood meals in order to obtain a total of three oviposition cycles.Before each blood meal, three females were collected from each cage, anaesthetized on ice, and their legs were separated from the body. Legs and body of each mosquito were then separately analyzed by RT-PCR, to check for the viral infection (presence of the virus in the abdomen) and virus dissemination (presence of the virus in the legs). At the end of the experiment  all females were collected from the cages, and the body and legs of each mosquito were separately analyzed by RT-PCR.1G1, F1G2 and F1G3) were treated for hatching The eggs obtained from the three gonotrophic cycles . Females were individually homogenized in 2 mL of Lysis Buffer and mixed. After 10 min incubation at room temperature, the lysate was centrifuged at 3,000 rpm for 5 min to eliminate mosquito debris, and processed by the NucliSENS easyMAG instrument.The extracted RNA was processed following two different protocols: the two steps RT-PCR method plus HybProbe reaction mix  according to the manufacturer instructions with a final volume of 20 µL, containing 1 pmol/µL of each of the primers CHIK-F 5′-AAGCTYCGCGTCCTTTACCAAG-3′ and CHIK-R 5′- CCAAATTGTCCYGGTCTTCCT-3′, plus 0.2 pmol/µL of CHIK-P labelled probe FAM-5′-CCAATGTCYTCMGCCTGGACACCTTT-3′TAMRA. RT-PCR assay was carried out in a LightCycler 1.5 instrument  with the following cycling parameters: 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 30 s, and 72°C for 45 s then a cooling step of 40°C for 30 s. A standard calibration curve was constructed using a 10-fold dilution series of a plasmid containing the PCR amplicon as the insert . The assay showed linear results for 8 logs of CHIKV plasmid dilutions. The analytical sensitivity was of 100 copies per reaction. In each assay a sample of CHIKV RNA was amplified as positive control.The amplification target was 208 bp of the E1 gene, which codes for the structural envelope protein E1. cDNA synthesis was achieved by using SuperScript® II Reverse Transcriptase  according to the manufacturer instructions and random primer (examer). A total of 3 µL of cDNA was amplified by using LightCycler® FastStart DNA Master5′-TCGACGCGCCCTCTTTAA-3′, CHIK E1R 5′-ATCGAATGCACCGCACACT-3′), 1 µM labelled probe (CHIK E1P 5′-fam-ACCAGCCTGCACCCATTCCTCAGAC-3′tamra) and 5 µL of extracted RNA. RT-PCR assay was carried out in a LightCycler 1.5 instrument  with the following cycling parameters: 55°C for 10 min, 95°C for 2 min, 50 cycles of 95°C for 10 s and 60°C for 35 s, then a cooling step of 40°C for 30 s. We obtained a quantification standard curve using a 10-fold dilution series of a plasmid, containing the PCR amplicon as the insert. This was obtained by cloning the PCR amplicon (using CHIK E1F and CHIK E1R primers) into the pCR 8/GW/TOPO TA . The assay showed linear results for 8 logs of CHIKV plasmid dilutions. The analytical sensitivity was of 50 copies per reaction. In each assay a sample of CHIKV RNA was amplified as positive control.The amplification target was 127 bp of the E1 gene, which codes for the structural envelope protein E1. A 20 µl reaction volume contained 10 µL 2× reaction mix, 6.75 mM MgSO4, 0.8 µl RT/Taq  0.44 µM of each primer  was used for means comparison. The percent data were angularly transformed before analysis. Differences in infectivity and dissemination capability of the two tested strains were compared by means of the Chi-square test. Means are presented with their standard deviations (SD). = 5.08 and P<0.03) than either the washed erythrocytes alone (HE) or the washed erythrocytes mixed with the virus suspension B (HE-sB)  was more attractive (F  (HE-sB) . = 15.20 and P<0.001), among which no statistically significant difference was found  = 65.70 and P<0.001)  .Fourteen females fed with infected blood were tested immediately after the meal (DPI = 0) and tested positive , while aP<0.05). The strain B showed also higher dissemination rate than the strain A. In fact, at the end of the experiment, the percentages of females with PCR positive legs was 64.4% (N = 30) for females infected with strain B, and 36.1% for the females infected with strain A (N = 54) (P<0.05).The virus strain B showed a higher capability to infect the mosquito females  in comparison to the strain A  (The highest virus titre of the strain A (A1) produced an intermediate level of infection and dissemination at 6 and 14 DPI, while it reached the 100% of infection and dissemination at 19 DPI. All the three titres of the strain B showed intermediate to low levels of infection and dissemination at 6 DPI, while the highest and medium virus titres  produced 100% rate of infection and dissemination at 14 and 19 DPI. The dissemination rate of both strains was higher at day 19  than at day 6 . = 6.54 and P = 0.02), the fecundity did not show any difference among the five groups  = 1.93 and P = 0.21) while the fertility of the eggs was lower in the two groups that had the first blood meal on blood infected with both strains  = 12.8 and P<0.001)  . = 0.90 and P = 0.51). The females of the HE group showed significantly higher fecundity in comparison to the other four groups  = 8.0 and P<0.001). No statistically significant difference was found between the egg fertility of the HE-sA and HE-sB females . The HE-sB females  showed lower egg fertility with respect to the R, HE and HE-M females  = 9.99 and P<0.001)  .Among the 101 females fed with infected blood we collected 14 females at DPI 0 for immediate analysis. From the 87 remaining females we obtained 689 adults  and the adult production ranged from the 25.9% of the females blood fed on HE-sA1 to the 6.2% of the females blood fed on HE-sB3 . On aver1 to 41.2% for the females fed with HE-M2  and the percent production of adults ranged from 70.5% for the females fed with Rth HE-M2 .2 = 0.76).Both the body and the legs of each mosquito were analyzed in parallel by the two RT-PCR methods, and the quantitative results, expressed in terms of number of viral copies obtained per microliter, showed a high positive correlation between the two methods of analysis  detected.The one-step PCR was more sensitive than the two-step PCR, with differences in the range of 10–100 viral copies per microliter , but similar to the findings of Vazeille et al. 7 pfu/mL and tested also the CHIKV A226V mutated strain).In our experiment, the virus titre in the blood meals influenced the rate of infection as well as the rate of dissemination of CHIKV in bopictus . The valStrain B, despite the lower viral titres used in comparison to those used for strain A, was able to infect most of the females analyzed and to disseminate in the peripheral body parts, showing significantly higher infectivity capability. This finding is somewhat surprising, as both the viral isolates were obtained during the 2007 epidemic in Emilia-Romagna when a single index case was identified Ae. albopictus, but we could observe a high rate of dissemination at 6 DPI, in particular at the highest titres tested, and a trend of increase of the dissemination rate at 14 and 19 DPI , qRT-PCR analysis found that the number of viral copies tend to increase in the female body at 6 DPI when compared with the values found at 0 DPI . HoweverAnopheles gambiae Giles and An. stephensi Liston, infected by the rodent malaria agent Plasmodium yoelii nigeriensis, showed reduced fecundity and fertility levels Culex tarsalis Coquillett experimentally infected with West Nile virus Culiseta melanura (Coquillett) infected with the Alphavirus Eastern equine encephalitis Some studies showed that pathogen infection may have detrimental impact on the vector reproductive capacity: In agreement with these findings, in our experiment the hatching rate was significantly lower for the eggs laid by the infected females than for the control eggs , while tvs non-infected eggs Two possible explanations of the impact on the eggs fertility have been proposed: 1. a pathogenic action on the females, causing a decreased capacity to produce fertile eggs with a similar impact on infected The depressive effect of the virus on the fertility of infected females , describWe did not find any clear relationship between the occurrence of transovarial transmission and virus titres used to infect the females during the first blood meal. The virus titres tested in this study may be considered lower than the maximum viremic titre observed in humans albopictus female's ovarioles infected.Interestingly, in our study all of the three infected mosquitoes  were obtained during the second gonotrophic cycle, which followed a non-infected blood meal. This finding support the hypothesis that the virus requires quite a long time period to get Ae. The occurrence of such a low number of cases of vertical transmission  indicates that this is a rare event under our conditions, in agreement with the results of field observations conducted during the autumn 2007 and spring 2008, when about 8,000 larvae, collected in the Chikungunya affected area, were analyzed and tested negative, thus confirming that the vertical transmission in our scenario has to be considered a very rare phenomenon .


Sodalis glossinidius and Wigglesworthia glossinidia) of the tsetse fly (Diptera: Glossinidae) are known to supplement dietary deficiencies and modulate the reproductive fitness and the defense system of the fly. Some tsetse fly species are also infected with the bacterium, Wolbachia and with the Glossina hytrosavirus (GpSGHV). Laboratory-bred G. pallidipes exhibit chronic asymptomatic and acute symptomatic GpSGHV infection, with the former being the most common in these colonies. However, under as yet undefined conditions, the asymptomatic state can convert to the symptomatic state, leading to detectable salivary gland hypertrophy (SGH+) syndrome. In this study, we investigated the interplay between the bacterial symbiome and GpSGHV during development of G. pallidipes by knocking down the symbionts with antibiotic. Intrahaemocoelic injection of GpSGHV led to high virus titre (109 virus copies), but was not accompanied by either the onset of detectable SGH+, or release of detectable virus particles into the blood meals during feeding events. When the F1 generations of GpSGHV-challenged mothers were dissected within 24 h post-eclosion, SGH+ was observed to increase from 4.5% in the first larviposition cycle to >95% in the fourth cycle. Despite being sterile, these F1 SGH+ progeny mated readily. Removal of the tsetse symbiome, however, suppressed transgenerational transfer of the virus via milk secretions and blocked the ability of GpSGHV to infect salivary glands of the F1 progeny. Whereas GpSGHV infects and replicates in salivary glands of developing pupa, the virus is unable to induce SGH+ within fully differentiated adult salivary glands. The F1 SGH+ adults are responsible for the GpSGHV-induced colony collapse in tsetse factories. Our data suggest that GpSGHV has co-evolved with the tsetse symbiome and that the symbionts play key roles in the virus transmission from mother to progeny.The vertically transmitted endosymbionts ( Glossina spp.), obligatory blood-feeders, are distributed throughout tropical sub-Saharan Africa and are vectors of Trypanosoma spp. that cause African animal trypanosomosis (AAT) or nagana in livestock and human African trypanosomosis (HAT) or sleeping sickness in humans Tsetse flies , a rod-shaped dsDNA virus, replicates in the nucleus and acquires a fragile outer envelope in the cytoplasm +) syndrome. Symptomatic infections in tsetse flies directly impact various host fitness parameters + can be transmitted per os to asymptomatic flies thus boosting infection levels Many of the tsetse species, including Wigglesworthia glossinidia (family Enterobacteriaceae), the obligate primary mutualist that is harboured in bacteriocytes localized in the anterior midgut, provides metabolic supplementation to this hematophagous insect, producing vitamins that are absent in blood meals Sodalis glossinidius (family Enterobacteriaceae) that is found both as extracellular and intracellular secondary symbiont in tsetse flies Wigglesworthia and Sodalis complement each other in thiamine biosynthesis Wigglesworthia and Sodalis are maternally transmitted; in these adenotrophic viviparous females both bacteria have the ability to enter the milk glands, from which they are transmitted to developing larva Wolbachia pipientis, detected in certain tsetse fly populations, is an intracellular alpha-proteobacterium that is transmitted from the mother to the egg cytoplasm. In insects, Wolbachia can affect life history traits, including the host reproduction, behaviour, immune competence, development and longevity In addition to the hytrosavirus, tsetse fly species harbour a complex symbiome comprised of obligate and facultative symbiotic bacteria that are critical to their nutritional and reproductive fitness G. morsitans morsitans, the primary symbiont Wigglesworthia produces metabolites in the midgut bacteriome that down-regulate auto-activation of the innate defence systems that subsequently can impact fly fitness Wigglesworthia to the F1 progeny via milk gland secretions up-regulates the cell defence system; removal of symbionts with antibiotics results in the F1 being immuno-compromised This study was initiated in response to numerous reports on the role that bacterial symbionts play in maintaining the “immune status” in various insects with disruption of host symbiont homeostasis in these hosts resulting in increased susceptibility to disease Sodalis, Wigglesworthia, and/or Wolbachia) would impact either the levels of GpSGHV or expression of SGH+ symptoms in adult G. pallidipes. Therefore, we set up a series of bioassays to examine the effects of antibiotic treatment on both asymptomatic flies and flies injected with infected gland extracts. Our working hypothesis was that the antibiotic-induced immune activation/suppression would alter virus titre in adults or in the F1 progeny and trigger changes in the expression of SGH+ symptoms. Using quantitative PCR (qPCR) we measured the impact of the treatments on the relative levels of the symbiome in adult G. pallidipes. In the light of the observed delayed effects of virus injections on tsetse flies, we followed all treatments through to the emergence of the F1 progeny.Hence, we speculated that alterations in the symbiome  for 10–15 minute intervals, three times per week, using a membrane-feeding technique All experiments were carried on a G. pallidipes exhibiting overt SGH+ symptoms selected from the colony odv-e66 gene) amplicons as previously described 6 virus copies were estimated to be present in a 2 µl aliquot of gland filtrate used as inoculum in the experiments described below.In this study, the virus inoculum was prepared from male G. pallidipes, teneral  flies were pre-screened for GpSGHV infection by a non-destructive PCR as previously described with slight modification To investigate GpSGHV replication in −4 in filter-sterilized phosphate buffered saline (PBS) and the same number of flies were mock-injected with PBS. Each treatment was replicated 3 times. Immediately after injection, flies  from one group were frozen at −20°C for subsequent quantitation of virus copy numbers by qPCR analysis. The remaining 5 groups were maintained on normal blood meal for 1, 3, 5, 7 and 9 feeds. Forty-eight hours after the end of the respective number of feeds, the flies were frozen at −20°C for later qPCR analysis.For injections, 6 groups of flies, each consisting of 4 males and 12 females, were injected with 2 µl of the virus suspension  was fed on normal blood; the second cage was fed blood supplemented with 40 µg/ml of the penicillin-based antibiotic ampicillin (Sigma) three times per week for the duration of the experiment. Based on prior research on The ability of both asymptomatic and symptomatic infected flies to produce and release GpSGHV in salivary secretions was examined. Groups of 24 flies  were either mock-injected with PBS- or virus-challenged as described above. The flies were kept in individual cells and subsequently fed on 250 µl of normal blood each for 21 days post infection (dpi). After the last blood meal, approximately 100 µl of the remaining blood was collected to estimate copies of virus particles released into the blood meals during the feeding event. DNA was extracted from both the collected blood and whole flies bodies using DNeasy Blood and Tissue Kit  according to manufactures instruction. The virus copy number released into the blood was estimated by qPCR 1 adults using the DNeasy 96 DNA Blood Kit (Qiagen). For quality control of the DNA extraction step, samples were diluted in TE buffer, PCR amplified with primers designed for the tsetse housekeeping β-tubulin gene (Table S1), and visualized on ethidium bromide-stained 1% agarose gels. The few extractions that produced weak bands (or no product) were excluded from the subsequent analyses. Subsets of samples producing homogenous and strong positive amplicons with the housekeeping gene primers 1 adults, (Table S1).DNA was extracted from the entire fly from both the sampled parents and FS. glossinidius isolated from G. morsitans morsitans were used to quantitate the Sodalis levels in the G. pallipides adults (Table S1). Initial attempts to use the primers designed from the thiamine biosynthesis protein (thiC) gene of W. glossinidia (40) failed to amplify product from the genomic G. pallipides DNA samples. Using degenerate primers designed from the open-reading frames of thiC genes  with the CODEHOP program thiC sequence of the W. glossinidia of G. pallipides (GenBank Acc KC470073). Primers designed from the conserved ThiC domain were used to quantitate the levels of W. glossinidia in the G. pallipides adults (Table S1). To prepare qPCR standard curves for both the viral and bacterial amplicons, regions flanking the target genes were cloned, and the PCR amplified products were purified and quantified using Nanodrop spectrophotometry providing an estimate of equivalent copy number. Serial dilutions from each standard were run in triplicate to produce a standard curve used to estimate titres in experimental samples.Primers designed from the flagellin C (FliCF) gene of Wolbachia in G. pallidipes was assessed initially with the Wolbachia surface protein Wsp gene primer set and cycling conditions of Jeyaprakash and Hoy ISNew PCR was performed with only one primer targeting the flanking terminal inverted repeats (TIR) of the insertion sequence element wsp PCR-blot technique to detect Wolbachia in the G. pallidipes sample set according to the protocols outlined by Schneider et al., The presence of Treatments were analysed by Analysis of variance (ANOVA) and individual treatment means compared with the Tukey-Kramer HSD test G. pallidipes with virus preparation did not cause any initial perturbations in adult activity or fitness. Virus-challenged adults mated, imbibed blood meals and produced numbers of male and female F1 progeny during the initial five weeks that mirrored those produced by control (PBS-injected) females (Table S2). Surprisingly, haemocoelic injection of the GpSGHV into adults did not increase the incidence of SGH+ in those flies (+ in virus-injected adults. Time-course assays demonstrated that virus-injected flies had a virus titre (∼109 virus copy number) after 21 dpi that was significantly greater than levels (∼104) detected at 0 dpi  (4 to 107 copies in the PBS(control)-injected non-injected flies, values that agreed with the virus loads detected previously in asymptomatic colonized G. pallidipes1 adults displaying SGH also did not display SGH symptoms (data not shown). Unlike the PBS-control flies, the titres in the virus-injected flies (excluding those diagnosed as symptomatic) increased to >109 copies per fly over the seven-week sampling interval and were significantly greater (P<0.0001) than the levels associated with the PBS-injected asymptomatic flies  of virus copies into the blood during a single feeding event . In contrast, flies displaying SGH+ symptoms produce and release large numbers of enveloped virus in the lumen of the salivary gland, depositing up to 107 virus copies into the blood per feeding event +, induced a chronic disease resulting in a marked reduction in fly survival at the late stages (>35 dpi); the increased mortality rates resulted in reductions in pupal production . In replicated assays, the frequency of SGH+ symptoms in the F1 progeny produced at different larviposition cycles was determined (1 adults (0.7%) displayed detectable SGH+ symptoms whereas 59% of the F1 adults produced by virus-injected females exhibited SGH+ symptoms. It should be noted that the F1 adults were dissected within 0–24 h post-eclosion, suggesting that the GpSGHV infected and replicated in salivary glands during the differentiation of imaginal discs in pupae. The prevalence of SGH+ in these newly emerged F1 adults was associated with their larviposition cycle, SGH+ symptoms increased from 4.5% in adults from the first larviposition cycle to 100% in adults from the fourth larviposition cycle  from virus-injected parents, presumed to be SGH-positive at emergence, produced no progeny.Ten pairs of FG. pallidipes lab colony used in our studies, like other tsetse flies Sodalis and Wigglesworthia and possibly Wolbachia. Initial comparisons among samples from PBS (control)- and virus-injected G. pallidipes, using both conventional PCR and qPCR, demonstrated that lab colony flies retained a similar titre (1–5×105 copies/fly) of Sodalis and Wigglesworthia throughout the adult lifespan  and the second targeting a high copy number transposon locus, the insertion sequence element New (ISNew) Wolbachia in the G. pallidipes colony . Subsequently we employed a highly sensitive wsp PCR-blot technique to reveal potential low titre Wolbachia infections that might have been overlooked with wsp and ISNew PCR. Negative results suggested that the lab colony is either void of Wolbachia or harbours the symbiont at levels far below the detection limit of even our high-sensitivity screening approach. In light of negative results with control G. pallidipes additional qPCR screening of Wolbachia in virus-injected and ampicillin treated samples were not conducted.Different primers were employed to screen for G. pallidipes injected with either PBS or virus gland suspension were fed blood three times per week supplemented with 40 µg ampicillin/ml of blood throughout adulthood. Ampicillin treatments caused a significant 10-fold reduction in Sodalis titres in G. pallidipes . Flies fed ampicillin treated blood readily mated and produced a normal complement of F1 offspring. Antibiotic treatment caused a minor increase in pupal mortality in the F1 from both the PBS-controls (5%) and the virus-injected adults (8%). However, these impacts were much less than ∼20% reduction in emergence observed in an earlier study with the ampicillin treatments of G. m. morsitansP = 0.598; The ampicillin therapy, although dramatically lowering the bacterial symbiont levels, did not cause drastic impacts on the survival, reproductive behaviour, or fertility of treated + symptoms in the F1 progeny of the virus-injected parents (4 copies/fly) of the F1 progeny from the antibiotic treated virus-injected parents were similar to the virus titres (1×104 copies/fly) detected in the F1 progeny from the symbiotic PBS-injected controls (7 copies/fly) detected in the F1 progeny from virus-injected parents fed unamended blood. Therefore, removal of the symbiome either suppressed the trans-generational transfer of the virus via the milk glands to the F1 or blocked the ability of the SGHV to infect and replicate in the salivary gland of the F1 adults.Most notable is our finding that antibiotic treatment negated the expression of SGH parents Fig. 3B.G. pallidipes colony universally harbours an asymptomatic GpSGHV infection. The virus levels and the low incidence of SGH+ detected in the control treatments are similar to the background levels observed routinely in this G. pallidipes colony + symptoms. Evidently, the virus was able to infect and undergo limited replication in cells that were not infected by the virus present in asymptomatic flies. It is likely that haemocoelic injection provided the virus access to tissues and cells that did not harbour the latent virus present in the asymptomatic fly. Potentially, the asymptomatic infection may provide cross-protection limiting the development of SGH+ symptoms in virus-injected flies. Previously, Jura et al G. m. morsitans and G. m. centralis with GpSGHV-infected gland homogenate resulted in the production of 100% symptomatic adults. These reports, in combination with our observations on G. pallidipes, suggest that the GpSGHV is capable of infecting and replicating during the ontogeny of the adult salivary gland occurring during pupation, but it is unable to develop SGH+ symptoms in the differentiated adult salivary gland cells in a host-dependent manner. It should be noted that, the results obtained with GpSGHV in G. pallidipes differed markedly from those achieved by injecting adult house flies with the MdSGHV. In house flies, haemocoelic injection with very low levels of virus induced both a 100% incidence of SGH+ symptoms and a total shutdown of egg production within 72–96 h post-injection In this study, we confirmed that the G. pallidipes suggest that the asymptomatic or latent infection is restricted spatially and temporally to select cells or undergoes only partial replication in adult cells to maintain titres at steady-state levels throughout the one- to three-month adult stage. Presently, nothing is known about the replicative pathway of GpSGHV in these asymptomatic tsetse flies. Potentially, observed changes in viral copy number represent only DNA replication and may not involve the production of infectious viral particles. Unlike the situation with GpSGHV, the housefly-MdSGHV system does not harbour asymptomatic infections, rather, the virus, once present in the host rapidly induces SGH+ symptoms leading to a massive increase in viral titre The variable but low virus titres in control G. pallidipes flies differ from MdSGHV injected into house flies 10 copies/fly in its homologous host (house fly) and 109 copies/fly in a heterologous host , respectively, by two dpi + due to massive viral replication S. calcitrans challenged with MdSGHV did not develop SGH+ symptoms, yet still supported relatively rapid replication of MdSGHV. The subsequent consequence of the MdSGHV replication in both hosts was virus-induced sterility in female flies. Injection of GpSGHV into G. pallidipes did not block oogenesis or intra-uterine larval development.The dynamics of GpSGHV replication in virus-challenged 1 SGH+ adults prepared as single pairs were observed to mate readily in small cages. These observations differed from the mating behaviour exhibited by MdSGHV-infected female house flies; these females refused to copulate and rejected both SGH+ and healthy males 1 SGH+G. pallidipes did not produce any progeny; such virus-induced sterility is believed to be responsible for virus-induced colony collapse in tsetse factories and agrees with prior reports conducted on symptomatic G. pallidipes and G. m. centralisThe FSodalis or Wigglesworthia in the parent flies or the subsequent transfer of these bacteria to F1 progeny. The titres detected in adult G. pallidipes are similar to bacterial levels detected in G. m. morsitansSodalis detected in the newly emerged F1 adults suggest that replication of the free-living Sodalis during pupation is suppressed; bacteria deposited in utero were incubated for approximately thirty days at 24°C after larviposition yet they attained titres of less than 104 cells/adult at eclosion. The intracellular Wigglesworthia symbiont also displayed lower titres in teneral F1 adults.The additional viral replication, induced by injection with the infected gland preparation, did not hinder either the maintenance of either G. pallidipes did not impact their fecundity or the rate of F1 adult eclosion. Similar results have been observed with G. m. morsitans fed ampicillin-treated blood meals 1 progeny; virus levels in these insects were comparable to the F1 progeny from PBS-injected adults fed normal blood. These findings conflict somewhat with Wang et al., G. m. morsitans resulted in a significant suppression of viral copy numbers in the F1 and F2 generation. It should be noted that analyses on G. m. moristans were conducted on flies collected at forty days post-eclosion; in our study the virus titration was conducted on teneral F1 flies. Secondly, the G. m. moristans colony does not express the symptomatic SGH+ phenotype that is associated, albeit at low levels, with the G. pallidipes colony.Antibiotic therapy of Wolbachia in G. pallidipes is in agreement with past reports. In their large-scale screen on diverse Glossina species, Doudoumis et al., G. pallidipes were positive for Wolbachia infection. In this study, more cases of infection were found in field strains than in lab colonies. In accordance with our findings they reported that samples from the G. pallidipes lab colony in Seibersdorf, Austria, were all negative for Wolbachia. Whether the lack of Wolbachia in the G. pallidipes is linked to the asymptomatic maintenance or the harmful impact of symptomatic infection on GpSGHV in this colony is unknown. In the light of the recently published report demonstrating the antiviral activity induced by Wolbachia in infected hosts, Wolbachia in the G. pallidipes colony may explain the severe negative impact of the GpSGHV on large scale colonies. Although the virus was reported in other tsetse fly species harbouring Wolbachia infection G. fuscipes fuscipes populations suggested that the levels of PCR-detectable GpSGHV is influenced by the tsetse fly genotype and is inversely correlated with the prevalence of WolbachiaThe lack of detectable G. pallidipes to ampicillin-treated blood was expected to cause a partially decrease the Sodalis titres without impacting Wigglesworthia1 progeny. Antibiotic treatment and concomitant reduction of bacterial symbiont levels in the F1 of the virus-injected parents was correlated with suppression of the SGH+ phenotype in the F1. This finding was surprising as microbial symbionts have been reported frequently to prime the insect immune system increasing host resistance to infection by various viruses, protozoans, and nematodes Wigglesworthia associated with G. m. morsitans adults stimulates the production of the catalytically active tsetse fly peptidoglycan recognition protein (PGRP-LB) in the midgut bacteriome Wigglesworthia preventing activation of the innate defence systems that can impact tsetse fly fitness and reduce fecundity 1 that are highly sensitive to trypanosome infection G. pallidipes, suppression of the symbionts did not impact virus titres in the parental generation suggesting that GpSGHV is unresponsive to ampicillin-induced alterations of the innate defence system.Continuous exposure of et al., Bemisia tabaci involved endosymbiont-produced GroEL homologues. Feeding white flies anti-GroEL antibodies caused an 80% reduction in TYLC transmission and reduced haemolymph virus titres below detection levels. More recently, Gottlieb et al., Hamiltonella is responsible for the efficient TYLCV transmission by the whitefly B biotype; the Q-biotype lacking the Hamiltonella is a less efficient TYLCV transmitter. The interaction of the GroEL with PLRV has been proposed to protect haemolymph-borne virus from host defences allowing for virus transfer to the salivary gland lumen Wigglesworthia and secondary Sodalis symbionts of G. m. morsitans also produce 60 kDa GroEL chaperones Wigglesworthia GroEL is the most highly expressed protein, yet remains localized within the bacteriome that harbours the primary symbionts. To date, nothing is known about the distribution and function of the Sodalis GroEL. Notably, this secondary symbiont is found in both extracellular and intracellular locations and is not restricted to a bacteriome-type structure The concept that symbionts may modulate viral biology in insects is not novel; endosymbionts have been shown to mediate the vector competence of both aphids and whiteflies Sodalis proteins including the major outer membrane lipoprotein, outer membrane protein A, outer membrane protein F, hypothetical phage protein, peptidoglycan-associated lipoprotein, type-III secretion apparatus, and the GroEL peptide were detected in the envelope fraction. These peptides were not detected in the extracted nucleocapsid or asymptomatic salivary gland homogenate fractions. The virus was purified from dissected hypertrophied salivary glands from symptomatic flies and may not represent the phenotype present in the haemolymph or in the milk glands. Regardless, the selective sequestration of these bacterial peptides in the viral envelope suggests a functional role in possible immune evasion or in selective transmission of the virus from infected to healthy flies. Antibiotic treatment would, most likely, reduce the levels of bacterial products available for decorating the viral envelope.Recent proteomic analysis of GpSGHV revealed that the viral envelope, in addition to containing peptides associated with viral proteins, contained many peptides that displayed homology to host proteins and to bacterial symbionts G. pallidipes is found typically as a latent infection causing little or no fitness cost to the host fly. The symptomatic state is normally activated in a small percentage of the colony. Attempts to induce SGH+ by injecting flies with a high dosage of virus failed but caused increased viral titres. These females after their third larviposition produced F1 adults that frequently displayed SGH+. Treating parents with antibiotics blocked the trans-generational transmission of the virus that resulted in SGH+. The antibiotic therapy suppressed the primary and secondary bacterial symbionts levels in both parent and their subsequent transfer to the F1 flies. These findings suggest that this dsDNA insect virus has evolved in close association with the symbiome to mediate the switch from the asymptomatic (latent) to symptomatic state.In summary, the GpSGHV in Table S1Primers. A list of the primers used to conduct both end-point and quantitative PCR reactions.(DOCX)Click here for additional data file.Table S21 progeny from the various treatments.Fitness data. Summary of fitness data on the pregnancies and resulting F(DOCX)Click here for additional data file.Figure S1GpSGHV copies released during feeding. Graph depicting the average number virus copies estimated by qPCR deposited by the G. pallidipes adults that have been injected with GpSGHV or with PBS (control) into the blood during a single membrane feeding event. Panel A represents the level of virus in the host fly at the time of feeding (28 dpi) and Panel B the number of virus copies released into the blood during the feeding event from adults harboring low and high levels of GpSGHV copy numbers. It should be noted that unlike these symptomatic flies harboring symptomatic SHG+ flies have been reported to release 107 virus copies per feeding event .(DOCX)Click here for additional data file.Figure S2Lack of Wolbachia in G. pallipides females.Wolbachia-specific PCR on G. pallidipes females. Presence of Wolbachia was tested using two primer sets targeting (A) the Wolbachia outer surface protein gene (wsp), and (B) the insertion sequence element ISNew. (C) Quality of DNA was assessed by using Glossina-specific tubulin primer . Wsp and ISNew PCR do not produce amplicons in all samples except for the positive control . Tubulin PCR, however, shows bright signals in all samples (C). Positive controls for PCR are Wolbachia high titer G. morsitans centralis denoted with (+), not that both gene targets were detected with this DNA template. The (M) designates the 1 kb DNA ladder used as size reference.(DOCX)Click here for additional data file.


Luteoviridae, such as cereal yellow dwarf virus (CYDV), requires a series of precisely orchestrated interactions between virus, plant, and aphid proteins. Natural selection has favored these viruses to be retained in the phloem to facilitate acquisition and transmission by aphids. We show that treatment of infected oat tissue homogenate with sodium sulfite reduces transmission of the purified virus by aphids. Transmission electron microscopy data indicated no gross change in virion morphology due to treatments. However, treated virions were not acquired by aphids through the hindgut epithelial cells and were not transmitted when injected directly into the hemocoel. Analysis of virus preparations using nanoflow liquid chromatography coupled to tandem mass spectrometry revealed a number of host plant proteins co-purifying with viruses, some of which were lost following sodium sulfite treatment. Using targeted mass spectrometry, we show data suggesting that several of the virus-associated host plant proteins accumulated to higher levels in aphids that were fed on CYDV-infected plants compared to healthy plants. We propose two hypotheses to explain these observations, and these are not mutually exclusive: (a) that sodium sulfite treatment disrupts critical virion-host protein interactions required for aphid transmission, or (b) that host infection with CYDV modulates phloem protein expression in a way that is favorable for virus uptake by aphids. Importantly, the genes coding for the plant proteins associated with virus may be examined as targets in breeding cereal crops for new modes of virus resistance that disrupt phloem-virus or aphid-virus interactions.Circulative transmission of viruses in the  Luteoviridae, including Cereal yellow dwarf virus (CYDV)-RPV, referred to herein as luteovirids, are each exclusively transmitted by one or a few species of aphids in a persistent, circulative, non-propagative manner. Aphids can transmit acquired virus for days or weeks and even after molting Virus species in the The virus encodes two structural proteins that make up the capsid. These proteins orchestrate systemic movement within host plants and transcytosis within aphids Sitobion avenae, four 2-D gel spots were differentially expressed between parthenogenetically-reproducing F1 clones that differed in their vectoring capacity for two isolates of the species Barley yellow dwarf virus (BYDV)-PAV S. avenae were identified as potential receptors for BYDV-MAV based on virus overlay and 2-D immunoblot assays Beet western yellows virus (BWYV). Actin and GAPDH-3, but not rack-1, also interacted with two BWYV RTP mutants displaying a reduction in aphid transmission ability in vivo functions of these interactions in aphids, or (b), that these RTP mutants still retain the interaction interface for binding with these aphid proteins. Cyclophilin and a luciferase homologue were identified from the greenbug aphid Schizaphis graminum as binding to CYDV-RPV. These proteins were also differentially expressed among different S. graminum F2 genotypes with different CYDV-RPV vectoring capacity Although a number of aphid proteins that bind luteovirids have been identified, the molecular details of virus movement in aphids are not well understood. Due to the paucity of molecular tools for the study of aphids, most virus-binding proteins have been identified using proteomics approaches. In In plants, luteovirids move from cell-to-cell and long distance as virions. However, in plants luteovirids must also replicate, assemble into virions, thwart the plant immune response and exhibit phloem-tissue tropism for host-to-host transmission by aphids, processes which likely involve the recruitment of a wide range of host factors to accomplish 2SO3) and ethylenediaminetetraacetic acid (EDTA) are commonly added to infected plant homogenate during virus purification to minimize the effects of polyphenolic compounds and virion aggregation, respectively Sodium sulfite . Total RNA was also extracted from those aphids immediately following the 3-day IAP. Viral RNA (vRNA) was detected within aphids after a 24-h AAP for each virus treatment preparation  using RTTo determine if either additive affected the ability of virus to move into gut cells, we used TEM to visualize virion attachment and virus penetration across the gut membrane . Aphids After virus egress from the HG, virions must remain stable in the hemolymph and pass through the ASG for transmission to occur. To determine whether the treatments had an effect on these latter steps in the circulative transmission pathway, treated and non-treated virions were microinjected into aphid hemolymph to bypass the HG barrier. Transmission efficiency was reduced when aphids were injected with sodium sulfite-treated or sodium sulfite plus EDTA-treated virions, 8% and 0%, respectively, relative to aphids microinjected with EDTA-treated or untreated virions, 78% and 91%, respectively . These dTo test the hypothesis that sodium sulfite treatment changes the host-virus interactome, LC-MS/MS was used to analyze tryptic digests of non-transmissible and transmissible virion preparations purified from infected oat plants. Although the genome of oat is not available, we were able to use predicted proteins from available genomes of related cereals for homology-based protein identification of the plant proteins found in the virus preparations. Twenty proteins were identified in the transmissible, but not the non-transmissible virus preparations . SixteenTo rule out co-sedimentation of plant proteins with purified virus based on density alone, we used LC-MS/MS to thoroughly characterize sucrose gradient fractions of healthy plant homogenates . HealthyAnalysis of purified sodium sulfite and EDTA-treated and non-treated CYDV-RPV using 1-D gel electrophoresis often showed distinct protein profiles . These din silico predictions of their mass:charge ratios. In contrast to the discovery approach that enabled us to ask “What host proteins are in the different virus preparations?”  mass spectrometry ations?” , SRM enaRhopalosiphum padi  fed on healthy or infected plants for 21 days was digested with trypsin and analyzed by SRM. Peptides from five of the plant proteins listed in R. padi homogenate  from Coast black oats was first reported by Rochow and Brakke 32− ↔ RS− + RS.SO3−. Above pH 9, the equilibrium constant does not favor the total cleavage of the disulfide bond, and large excesses of sulfite are required to drive the reaction to completion. However, at lower pH values (e. g. pH 7 of the homogenization buffer used in these virus preparations) the thiol predominates over the thiolate ion resulting in more complex reaction kinetics and more favorable equilibriums −1M−1 as compared to one having two neutral neighbors, 367 s−1M−1, a 106-fold range in rate constants The action of sulfite on disulfides (e.g. cystines) can be represented by the equilibrium equation: R.S.S.R + SOin vivo interactions between divalent cations and the virus capsid may be required for long-term virion stability. Indeed, other icosahedral plant viruses such as rice yellow mottle virus, tomato bushy stunt virus, southern bean mosaic virus and cowpea chlorotic mottle virus are stabilized by divalent cations such as Ca2+ and Mg2+, With these thoughts in mind, the three cysteines in the CP or RTP of CYDV-RPV may provide some clues  as to whPhysalis floridana, but not in Solanum tuberosom, Nicotiana benthamiana, or Nicotiana clevelandiiP. floridanaThese data support the hypothesis that transmission of CYDV-RPV requires the formation of a critical disulfide bond pairing either intramolecular, within the CP or RTP, or intermolecular with a specific host protein, and that treatment with sodium sulfite promotes a random process of thiol-disulfide exchange that creates structures that interfere with normal virus transmission. Sodium sulfite-treated virions did not enter into the HG or into the ASG and thus, a partial overlap in the biochemical mechanisms for virus entry may exist in these two aphid tissues. Previous work using infectious mutants of other luteovirids supports the role of cysteine residues within the capsid as contributing to virus-host specificity and even aphid transmission. PLRV mutants with cysteine deletions or modification of residues flanking cysteines residues show phenotypes in a host-dependent manner Two mutants D-P-K  and H-C-K (which deletes the cysteine residue) showed defects in systemic infection in the host Diuraphis noxia, the mRNA encoding for cellulose synthase is up-regulated 4 to7-fold during aphid infestation LC-MS/MS enabled us to detect numerous host proteins co-purifying with virion particles in the sucrose gradients. The SRM data indicate a complex picture on the roles of these plant proteins in luteovirid transmission by aphids. How might host proteins also participate in aphid transmission? As a result of natural selection, aphid acquisition of CYDV-RPV by aphids may have been favored from phloem cells with higher protein expression, a phenotype that could have evolved as a response from virus infection, the act of aphid feeding, or both in combination. Co-ingestion of soluble plant proteins that associate with virions may help stimulate endocytosis into the epithelial cells of the aphid HG in a host-dependent manner. These data are consistent with previous observations of host proteins as associated with insect-transmitted viruses in planta. Most plant viruses, including CYDV-RPV, move from cell-to-cell in host plants via plasmodesmata  inoculated 7 to 8 weeks previously with viruliferous R. padi as described 2HPO4) buffer (pH adjusted to 7 using 0.1 M KH2PO4) at 2.5 ml g−1 tissue. Tissue was homogenized with phosphate buffer containing 1% cellulase with and without 0.01 M EDTA and 0.5% sodium sulfite together or individually. Sucrose gradients were fractionated using a density-gradient fractionator (Teledyne-ISCO) at sensitivity 0.5 and chart speed set at 60 cm/h. Two milliliter fractions were collected along the entire gradient for each gradient. The virus fractions were collected as 4 ml gradient fractions and were concentrated by centrifugation for 4 h at 113,613×g in a type 70Ti rotor (Beckman Coulter). The remaining fractions were stored at −80°C until needed. Supernatant was discarded and the pellet was resuspended overnight in 0.01 M phosphate buffer, pH 7. Purified virus was equally divided among several tubes and stored at −80°C. As a control, 500 g of healthy tissue was homogenized in phosphate buffer containing 1% cellulase and purified following the above protocol.CYDV-RPV was purified from oat plants . Excess antibody was wicked off with a wedge of filter paper and grids were rinsed in 3 drops of PBS, wicking off excess with filter paper after each rinse. Antibody-coated grids were incubated for 1 h on a 10 µl drop of virus. Virus was wicked off using filter paper and grids were rinsed in 2 drops of PBS, followed by 3 drops of sterile distilled water, wicking excess after each rinse. Grids were stained by incubating for 3 min on a 20 µl drop of 2% aqueous uranyl acetate, excess stain was wicked off and grids were stored dry in a grid box. Grids were viewed on a Jeol 1200 TEM at the Electron Microscope Facility for The Huck Institute of the Life Sciences at The Pennsylvania State University.R. padi aphids were allowed a 24–48 h AAP on stretched Parafilm® membranes made by standard protocol by stretching in two directions until very thin. Approximately 75 µl of the virus preparation, at 20–60 µg/ml concentration containing 15% sucrose was used in each membrane. After 24 h, aphids were allowed a 3–4 day IAP on 7 day old oat seedlings (Coast black), 5 aphids per plant, 12–16 plants per treatment. Plants were fumigated with Orthene to kill the aphids. The fumigated plants were placed in the greenhouse and observed for symptom expression 3–4 weeks later. Infected plants were evaluated by obvious yellowing and reddening of the leaves, and dwarfing of the plant. In addition, a randomly selected subset of the leaves (symptomatic and asymptomatic) was tested by double-antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) using anti-CYDV-RPV antibodies. Each type of virus preparation was tested 2–4 times.For each purified virus preparation , healthy R. padi, we performed a log-linear analysis for a three-way contingency table on the transmission results following microinjection using +/− EDTA, +/− sodium sulfite and +/− transmission as the three variables. Simulations and the experiment were run using the statistical software package JMP (SAS).In addition to membrane feeding, virus was directly injected into the hemocoel of the aphid to bypass the HG. For each purified virus preparation, 10 nl of a 20–60 µg/ml virus preparation was injected into an aphid using a Microinjector IM300 (Narishige). Three aphids were placed onto a 7 day old oat seedling (Coast black), 12–16 plants per virus treatment, for a 4 day IAP, after which plants were fumigated to terminate feeding. Plants were evaluated for infection 3–4 weeks later as described above. To test for synergistic effects of EDTA and sodium sulfite treatment on CYDV-RPV transmission by R. padi aphids were allowed a 24 h AAP on membranes, as described previously, which contained 40 µg/ml of purified virus in 15% sucrose buffered with 0.01 M phosphate buffer, pH 7. The following virus treatments used were preparations containing 1) phosphate buffer only, 2) phosphate buffer including EDTA only, and 3) phosphate buffer including Na2SO3 only. To examine an aphid’s ability to acquire virus, after 24 h, six aphids were collected, divided randomly into three groups of two for each treatment and stored at −80°C in 25 µl nuclease-free water in an RNase-free microcentrifuge tube. The remaining aphids were transferred to healthy oat seedlings (Coast black), placing five aphids per seedling. Aphids were allowed a 3 day IAP to test for transmissibility of the virus, and aphids were collected to examine the treatment effects on virus uptake into the hemocoel. For each virus treatment, six aphids were collected, divided randomly into three groups of two for each treatment and stored at −80°C in 25 µl nuclease-free water in an RNase-free microcentrifuge tube. Total RNA was isolated, five µl of RNA was used in an RT-PCR as described 5′-ATGAGTACGGTCGTCCTTAGATCC-3′; RPV-CP Rev: 5′-CTATTTTGGGTTTTGTAGCTGGAC-3′) were used to amplify a 614 bp fragment.Healthy R. padi were allowed a 48 h AAP on membranes containing 40 µg/ml per virus preparation. For each virus preparation, ten aphids were collected after feeding, the head and cauda were removed using a razor blade, and heads and abdomens were fixed for TEM in 1% formaldehyde-2% gluteraldehyde in 0.02 M sodium cacodylate (pH 7.2) containing 10 mM calcium chloride and 0.05% sodium azide for 24 h. Aphids were subsequently prepared for TEM accordingly TEM was also used to detect virus within the aphid. 25 µg of virus preparations were separated on 10–20% Novex tricine gels (Invitrogen) according to the manufacturer’s instructions, at 125 V for 2 hr at room temperature in the SureLock XCell minicell (Invitrogen). Gels were fixed for 30 min in 100 ml of a solution containing 50% methanol and 7% acetic acid. After 30 min, the fixing solution was replaced with fresh fixing solution. The gels were stained overnight with a 1∶1 dilution of Sypro Ruby stain (Invitrogen) in nanopure water at room temperature in the dark. The gels were then washed for 30 min in a solution containing 10% methanol and 7% acetic acid. Gels were scanned on the Typhoon Variable Mode Imager  at 100 dpi and visualized with the 532 nm laser using the 610BP30 filter. The bands were excised from the gel using a bench top UV transilluminator at a wavelength of 302 nm.18 5 µm, 20 mm×180 µm trapping column and a UPLC BEH C18 1.7 µm, 15 cm×75 µm analytical column (Waters). The samples were transferred to the trapping column using a 5 µL partial loop injection with a 0.1% solution of formic acid (FA) in water at a flow rate of 7 µL/min for 3 min. Mobile phase A consisted of 0.1% FA in water and mobile phase B consisted of 0.1% FA in ACN. Following desalting and concentration, the trapping column was subjected to a reverse flush to the analytical column and separated with a gradient of 2–40% mobile phase B over 60 min at a flow rate of 300 nL/min, followed by a 5 min rinse with 95% of mobile phase B. The column was re-equilibrated at initial conditions for 20 min. Column temperature was maintained at 35°C. 100 fmol/µL [Glu1]-fibrinopeptide B in 25% ACN with 0.1% FA was used as the lock mass compound and was delivered via the auxiliary pump of the LC system at a flow rate of 300 nL/min to the reference sprayer of the NanoLockSpray source of the mass spectrometer. The eluent from the analytical column was delivered to the analytical sprayer of the same source through a PicoTip emitter  with 10 µm tip diameter.Each gel band was subjected to an in gel tryptic digestion and extraction as described Mass spectrometric analysis of tryptic peptides was performed using a Synapt HDMS mass spectrometer  or a 4700 Proteomics Analyzer (Applied Biosystems) as described in For analysis of purified CYDV-RPV virions in solution, disulfide bonds were reduced with 5 mM dithiothreitol (DTT) for 30 min, followed by alkylation with 10 mM iodoacetamide (IAA) for 30 min, in the dark. Reduction and alkylation were performed at 25°C. CYDV-RPV virions were then digested with trypsin  at 35°C for 16 h. The peptide mixture was desalted using a C18 sep-pak (Waters) and stored at −80°C for 1 week prior to MS analysis.For nLC-MS/MS analysis on the LTQ-Orbitrap Velos , the tryptic digest was reconstituted in 10 µL of 2% ACN with 0.5% formic acid (FA). The mass spectrometer was equipped with a “Plug and Play” nano ion source device . The nanoLC was performed using a Dionex UltiMate3000 MDLC system . The gel extracted peptides (5–10 µL) were injected using a “User Defined Program” onto a PepMap C18 trap column  at a 20 µL/min flow rate for on-line desalting and then separated on a PepMap C18 reverse phase (RP) nano column  which was installed in the “Plug and Play” device with a 10-µm spray emitter  mounted in front of the Orbitrap ion transfer tube. The peptides were then eluted in a 60 min gradient of 10% to 40% ACN in 0.1% FA at 300 nL/min. The Orbitrap Velos was operated as described previously Tandem mass spectra from purified CYDV-RPV were converted mascot generic format (MGF) peak list files using Proteome Discovery 1.1. An in-house FASTA protein database was created from all NCBI entries, including all the translations of all the available cereal genomes, common contaminants, and viruses, was downloaded on January 31, 2012. This strategy was used as opposed to restricting the search to green plants to minimize false matches due to the presence of virus gene products or to the presence of other unanticipated sources of proteins (such as from plant-infecting bacteria of fungi) in the samples. All data were searched against this database using Mascot v2.3.02  as follows. Fixed carbamidomethyl and variable methionine oxidation were used as modifications. Precursor ion tolerances were set to 30 parts per million (ppm), and fragment tolerance was 0.8 Dalton (Da). ESI-Trap was selected as the instrument type. The enzyme selected was trypsin with 1 missed tryptic cleavage permitted.Mascot *.dat files were created in Mascot and loaded into Scaffold (version 3_00_05). Peptide and protein probabilities were calculated using PeptideProphet and ProteinProphet algorithms R. padi were maintained on CYDV-RPV infected or healthy caged oats at 20°C with an 18-h photo-period for 21 days. Aphids were harvested from plants for protein extraction as described Parthenogenetically reproducing aphid colonies of Protein pellets were prepared for mass spectrometry as previously described 2 laser puller , and then packed to a length of 15 cm with 4 µm C12 reverse phase particles . Two µL of the 1 µg/µL digested aphid protein extracts were injected directly on the column and eluted with a flow-rate of 300 nL/mn. The gradient ramped from 2% B (80∶20 ACN/H20) to 37% B across 50 min, and then increased to 80% B and held constant for 5 min. Initial conditions were restored for the final 15 min of the run. Electrospray ionization (ESI) was initiated by applying 2.2 kV via a liquid junction distally from the ESI tip. The capillary voltage and temperature were 42 V and 275°C, respectively. MS analyses were performed using a TSQ Vantage  operating in SRM mode. For SRM-mass spectrometry, the doubly charged precursor ions were monitored in Q1 with a resolution of 0.7 full width at half-maximum (FWHM) and three to four singly charged y-ions for each peptide were monitored in Q3 at 0.7 FWHM. Each transition was monitored for 25 ms (dwell time) enabling a maximum duty cycle of 2.5 s.Nano-flow liquid chromatography was performed using an Eksigent 1D nanoLC system  with direct column injection. Tips were pulled from silica capillary (75 µm I.D. × 360 µm O.D.) in-house using a commercial COR. padi fed on CYDV-RPV infected tissues. From these samples, MS/MS data were collected for Skyline-predicted tryptic peptide ions from host proteins of interest. These data were imported back into Skyline for refinement of the SRM method. During refinement, we selected proteotypic peptides that ionized well (3–4 abundant y-ions for each peptide) and showed reproducible chromatographic retention properties and made a new SRM method. The new, data-driven, refined SRM method was exported to the mass spectrometer. Three biological replicates were analyzed using the refined SRM method, and a Student’s T-test was used to compare total peak areas. A normalization factor of 0.92 was calculated by monitoring for peptides derived from two different proteins that were not differentially expressed between treatments and applied to the peptides derived from healthy samples. Both raw and normalized peak areas for each transition are reported  1-D PAGE of proteins from purified virus showing bands that were excised and digested with trypsin. Lane 1, Broad range molecular weight standards (Biorad) in kDa; Lane 2, transmissible virus; Lane 3; non-transmissible virus. The band containing the RTP is encircled in red. Other bands subjected to LC- MS/MS analysis are indicated with a red *. Multiple proteins were found in each lane. These proteins were abundant contaminants (reported in the text and (TIF)Click here for additional data file.Figure S2Clustal W alignment showing CYDV-PRV cysteine residues in the RTP in the context of a multiple alignment of twelve luteovirid species. C136 and C373 are highly conserved among luteovirids whereas C112 is unique to CYDV-RPV. In CYDV-RPV, all three cysteine residues within the RTP are flanked by at least one basic amino acid, making them especially reactive and likely to be involved in disulfide bonding.(TIF)Click here for additional data file.Figure S3SRM transitions of the peptides from host plant proteins that were detected in tryptic digests of pooled, whole-body R. padi protein samples (from data shown in ). One replicate per peptide is shown. Two peptides from cellulose synthase show no statistically significant fold-change in aphids reared on CYDV-RPV infected or healthy plants (A) SQTGDFDHNR detected aphids fed on CYDV-RPV infected plants or (B) healthy plants, (C) IPMFAYVSR detected in aphids fed on CYDV-RPV infected plants or (D) healthy plants. The peptide FGGDTYCCR from thaumatin-like protein 5 detected in aphids fed on CYDV-RPV infected plants in (E) or healthy plants in (F). The peptide VLYSSCYVR from 33-kD secretory protein detected in aphids fed on CYDV-RPV infected plants (G) or healthy plants (H). The peptide VLYSSCYVR was at the lower limit of detection in the samples derived from aphids reared on healthy plants. The peptide SDSIITAYR from pyruvate dehydrogenase E1 derived from samples of aphids collected from CYDV-RPV infected plants (I) or healthy plants (J). One peptide was detected from pyruvate dehydrogenase E2: GLGMIAEEVK was only detected in samples of aphids reared on CYDV-RPV infected tissues (K), and not in aphids reared on healthy tissue (L). The next step to confirm these differences in aphids fed on healthy or infected plants is validation and absolute quantification of these peptides via the use of stable isotope labeled peptides.(TIF)Click here for additional data file.Table S1Plant proteins that were identified in the sucrose gradient fractions from healthy oat plants.(PDF)Click here for additional data file.Table S2Raw and normalized peak areas, T-test results, and retention time coefficient of variation for plant peptides detected using SRM in aphids fed on CRDV-RPV infected or healthy plants.(PDF)Click here for additional data file.