To the Editor: The first evidence of chikungunya virus in the Western Hemisphere was its detection in December 2013 in the French West Indies (Two cases of chikungunya in siblings (an 8-year old girl and a 10-year old boy) were recently identified at Toulouse University Hospital in southwestern France. Two days after these children had returned to France from the island of Martinique (French West Indies), acute fever associated with an arthromyalgic syndrome developed in these children. The children had maculopapular, nonpruriginous rashes on their arms and legs and endobuccal petechiae. The boy had bilateral knee effusions, and the girl had a measles-like rash that became more extended. Both children also had many mosquito bites that they scratched. They were discharged on the day of their admission. These 2 cases reported in metropolitan France after the patients visited Martinique indicate rapid spread of chikungunya virus.Aedes albopictus mosquitoes  is also an efficient vector of chikungunya virus and is found in many areas, including southern Europe. This mosquito species was responsible for the extensive chikungunya outbreak on La Réunion Island in the Indian Ocean  of southern France in 2013, where ≈13 million persons live. This mosquito, which is highly efficient in transmitting chikungunya virus .The presence of an effective vector, its progressive spread, and the outbreak of chikungunya in the Western Hemisphere increase concerns of a chikungunya outbreak in Europe (These measures must be extremely efficient because an outbreak of chikungunya in the Western Hemisphere could spread rapidly. All countries in southern Europe are concerned by this public health challenge, and the battle against chikungunya requires rapid establishment of a supranational organization that should be able in real time to collect and return epidemiologic, virologic, and entomologic data. Although the usual movements of tourists around southern Europe during the summer will increase the number of persons at risk in this area, an even greater threat is the international movement of >600,000 persons expected to attend the next Soccer World Cup in Brazil in 2014 (


Several lists of potentially inappropriate medication (PIM) for elderly patients have been developed worldwide in recent years. Those lists intend to reduce prescriptions of drugs that carry an unnecessarily high risk of adverse drug events in elderly patients. In 2010, an expert panel published the PRISCUS list for the German drug market. This study calculates the amount of drug reimbursement for PIM in Germany and potential cost effects from the perspective of statutory health insurance when these are replaced by the substitutes recommended by the PRISCUS list.Register-based data for the 30 top-selling drugs on the PRISCUS list in 2009 for patients greater than or equal to 65 years of age were provided by the Scientific Institute of the German Local Health Care Fund. We calculated the percentage of sales and defined daily doses for patients greater than or equal to 65 years of age compared with the total statutory health insurance population. Reimbursement costs for the recommended substitutions were estimated by considering different scenarios.In 2009, drug reimbursement for the 30 top-selling PIM prescribed to patients greater than or equal to 65 years of age were calculated to be €305.7 million. Prescribing the recommended substitution medication instead of PIM would lead to an increased total reimbursement cost for the German health care system ranging between from €325.9 million to €810.0 million.The results show that the substitution of PIM by medication deemed to be more appropriate for the elderly comes along with additional costs. Consequently, there is no short-term incentive for doing so from a payer perspective. Future studies have to consider the long-term effects and other sectors. Potentially inappropriate medication (PIM) can influence the safety of elderly patients’ drug therapy. Several international studies have shown that PIM might lead to higher morbidity and mortality as well as it contributes to adverse drug events (ADE) and hospital admissions –3. ConseIn Germany, the Federal Ministry of Health initiated an evaluation of PIM in 2008/2009 considering the country-specific characteristics of the pharmaceutical market. Details of the development process have been described elsewhere . SeveralThe aim of this investigation was to analyse the reimbursement costs of PIM according to the PRISCUS list and to evaluate the economic effects of a recommended substitution from the perspective of SHI in Germany. As the PRISCUS list was explicitly developed for the German pharmaceutical market, we will use it to detect PIM in the German SHI population. We provide information about (a) the proportion of agents on the PRISCUS list prescribed to elderly people ≥65 years of age, (b) the reimbursement costs of PIM from the perspective of SHI and (c) the potential costs of the recommended substitution medication.The methodology of the study consists of three components. First, anonymised medication data on patients ≥65 years of age were obtained and enriched with information about the entire SHI population. Second, costs for the prescription of surrogates were calculated based on different scenarios. Third, we conducted a literature research to identify international studies for comparison with our results.The underlying anonymised aggregated prescription data were provided by the Scientific Institute (WIdO) of the German Local Health Care Fund (AOK). It was founded in 1976 and has the function to carry out research projects aimed at ensuring high quality and economic health care for the community of the Local Health Care Funds . The WIdThe data set was enhanced with additional information from the pharmaceutical prescription report from 2010 (PPR) . The PPRTo calculate the potential costs of the recommended drug substitutions, we first derived the costs per DDD for the recommended surrogates from the PPR. In the first case, if a specifically drug was named, the costs per DDD were directly taken from the PPR. In the second case, if a drug group was recommended as an adequate alternative, a weighted average based on the costs per DDD and the prescribed annual DDDs was calculated. For example, the recommended alternatives on the PRISCUS list for indometacin are (1) paracetamol, (2) weak opioids (tramadol and codeine), (3) metamizol (after careful consideration of the risks and the benefits) and (4) "weaker" NSAIDs  . For (1)In a second step, the reimbursement costs for an adjusted drug therapy was estimated under the consideration of the recommended surrogate according to the PRISCUS list. We assumed that the amount of DDDs for the recommended surrogates would not differ from the actual prescribed DDDs of PIM. To calculate the economic impact, the costs per DDD for the proposed substitution were multiplied by the prescribed amount of DDDs for patients ≥65 years of age. As several drugs or drug groups are recommended in some cases, a range of costs per DDD for surrogates had to be considered. Thus, we performed three different scenarios. Besides the cheapest (Scenario 1) and the most expensive surrogate (Scenario 2), we also calculated the mean costs per DDD of the proposed alternatives (Scenario 3) and the corresponding costs of the prescription.nd of November 2014. In order to identify the prevalence of PIM in Germany, we combined 18 synonyms for PIM, such as potentially inappropriate medication, inappropriate prescribing, and inappropriate practice in prescribing with “PRISCUS”. For the identification of studies of the costs of PIM, we combined the keywords cost OR costs OR cost analysis OR cost analyses OR drug reimbursement OR drug reimbursements OR economic burden OR economic evaluation and elder* OR old* OR geriatric* OR senior OR aged OR aging with the same words on PIM as in the search on prevalence. The complete search history for both searches can be obtained from the first author upon request. The search was not limited with regard to language and timeframe. Identified studies were searched by reviewing the references as well. For search 1, only studies of the prevalence of PIM in Germany were included. For search 2, papers were included if they examined the nationwide costs of PIM on an annual basis. Papers only examining the prevalence or the applicability of tools to identify PIM were excluded.Two systematic literature reviews were conducted to compare the results with national and international studies. The first one consisted of studies dealing with the prevalence of PIM in Germany measured by applying the PRISCUS list. The second one focused on international studies analysing reimbursement costs. We carried out two Medline searches on the 2In 2009, the total expenditure for all 83 agents on the PRISCUS list was €387.8 million and the reimbursement costs of the 30 top-selling agents amounted to €305.7 million (78.8 %). The highest reimbursement costs were caused by solifenacin, with €32.5 million, followed by etoricoxib (€30.6 million), and zopiclone (€21.5 million), as displayed in Table Focusing on the number of PIM prescriptions to the elderly, there were 13.75 million prescriptions of the 30 top-selling PIM. For all PRISCUS agents, there were 16.93 million prescriptions to the elderly in 2009. Compared with the entire SHI population with 26.15 million prescriptions of the 30 top-selling PRISCUS agents and 31.71 million prescriptions of all 83 PRISCUS agents, 52.6 % and 44.9 % of the prescriptions were allocated to the elderly, respectively. Regarding all prescriptions to the entire SHI population (626.3 million in 2009), the elderly obtained 56 % (350.7 million) of them [The sum of DDDs for the 30 top-selling PRISCUS agents was 502.9 million DDDs and 598.3 million DDDs for all 83 agents. The leading agents were doxazosin (51.4 million DDDs), amitriptyline (45.7 million DDDs), and sotalol (34.0 million DDDs). In the entire SHI population, 864.40 million DDDs of the 30 top-selling PIM and 1,124.0 million DDDs of all PIM were prescribed. Thus, the majority of prescribed DDDs of the 30 top-selling PIM (58.2 %) may be assigned to patients for whom the drugs are potentially inadequate. Regarding all PIM on the PRISCUS list, 53.3 % of the DDDs for the entire SHI population were prescribed to the elderly.Table Considering the results for Scenario 1, only 10 surrogates lead to increased reimbursement costs, whereas the substitution of the remaining 20 agents leads to a cost decrease. However, these 10 surrogates increase the costs by +200.0 % on average, whereas the decrease is -53.3 % on average. The cost driver in Scenario 1 is memantine (€3.86/DDD), a proposed surrogate for piracetam (€0.45/DDD), pentoxifyllin (€0.54/DDD), and naftidrofuryl (€1.11/DDD). The substitution of these three agents by memantine causes costs of €70.6 million, €57.9 million and €29.0 million, respectively. This results in a potential cost increase of €133.9 million (+467.0 %), from €23.6 million to €157.5 million.In Scenario 2, 22 agents increase the costs by +289.0 % on average. The most cost-intensive surrogates are acetycholinesterase inhibitors (€4.25/DDD) – as proposed alternatives for piracetam, pentoxifyllin, and naftidrofuryl – and this would lead to increased reimbursement costs of €173.4 million (+635.0 %), from €23.6 million to €197.0 million. The other cost drivers in Scenario 2 are the recommended substitutes for etoricoxib . This substitution would raise reimbursement costs from €30.6 million to €101.56 million (+232.0 %). If indomethacin (0.46€/DDD) was substituted by codeine (2.65€/DDD), the cost of the medication would rise by +476.4 %. Only the substitution of eight agents would lead to a decrease in total reimbursement costs, by -27.5 %.Considering the mean surrogate costs in Scenario 3, in 14 cases the substitution of the 30 top-selling agents would lead to a decrease in reimbursement costs by -37.9 % on average. However, these savings would be compensated for by the 16 remaining agents, which lead to an average increase of +160.7 %.To our knowledge, this is the first study to assess the reimbursement costs of PIM according to the PRISCUS list in Germany from a SHI perspective. We conducted a comprehensive analysis based on prescription data from 2009 in order to depict the prevalence of PIM in the elderly in Germany and to quantify the economic impact of prescriptions as well as the substitution costs for the recommended medication.Our search yielded eight studies dealing with the prevalence of PIM in Germany, according to the PRISCUS list , 17–21. The six remaining studies calculated prevalence based on smaller patient samples and in different study settings. The results lay between 16.6 % in an emergency department  and 43 %A search of economic analyses showed that limited information is available. In total, 12 publications dealing with the costs of PIM were identified, but none of them considered German SHI. The studies were conducted in the US –26, IrelBradley et al. analysed the PIM of patients >70 years of age according to the STOPP criteria in Northern Ireland, using data from the enhanced prescribing database of the National Health Service . In totaIn our study, the reimbursement costs of PIM were €387.8 million. Thus, the total reimbursement costs for PIM prescriptions in Germany are significantly higher than those in Finland, Ireland, and Northern Ireland, but lower than those in the US. The total sales of all proprietary mediclinical products in SHI were €28,499 million . Thus, tIn general, the comparability of our results with other international studies is limited. First, different tools were used to detect PIM: Bradley et al. and Cahir et al. applied the STOPP criteria, which contain 65 criteria systematised according to the physiological systems to identify PIM , 28, 32.Regarding PIM substitution, reimbursement costs are about to rise, independent of the scenario. If all prescriptions of PIM were substituted by the low-cost alternative, the total costs would be €325.9 million. Substitution by the most expensive alternative would lead to a total cost of €810.0 million. This gap can be explained by the enormous difference in the costs of the recommended therapeutic alternatives. For example, there are several different alternatives for etoricoxib , metamizol, anticonvulsants ). The cheapest alternative to etoricoxib (€1.21/DDD) is paracetamol (€0.49/DDD). The most expensive alternative is newer anti-epileptics at €4.03/DDD. The difference in costs per DDD might result in enormous cost differences in each scenario. By contrast, for solifenacin, which costs €1.43/DDD according to the PPR, the only alternative according to the PRISCUS list is trospium, with a cost of €1.05/DDD. Consequently, in both scenarios the substitution of solifenacin will decrease the total costs.From a short-term economic perspective, there would be little incentive for SHI to support the substitution of PRISCUS agents. However, it has been shown that PIM leads to ADE which in turn can cause inpatient costs , 34, 35.Our research revealed several aspects to be considered in future research on PIM. The PRISCUS list was published in 2010. Our study is based on data from 2009. Thus, our study does not analyse the effect of the PRISCUS list and possible changes in the prescription behaviour of physicians in Germany, but serves as a reference for future comparisons. Further research should investigate if and to what extent prescription patterns and PIM costs have developed due to the PRISCUS list in order to show the influence and relevance for practical use.Our investigation demonstrated that PIM is a relevant topic in drug therapy in the elderly in Germany. The elderly account for approximately 55 % of the sales of the 30 top-selling PRISCUS agents. Similar percentages were calculated for DDDs and prescriptions. The proportion of sales, DDDs, and prescriptions to the elderly is lower in all 83 agents of the PRISCUS list. The decrease underlines that only some of the 83 PRISCUS agents are really relevant. Thus, from an economic perspective and in terms of feasibility, a shorter PRISCUS list might be more applicable for the daily health service. However, the medical perspective has to be taken into consideration as well. A short-list should also include those agents with the worst risk/benefit ratios for the elderly.There are limitations to this study as a result of the methods used. First, for several PIM the recommended substitutions may not be clinically adequate in a majority of clinical cases, while in other cases the recommended substitution would lead to off-label use. For example, herbal products, such as valerian, are often named as surrogates for psycholeptics . From a medical perspective, however, it is questionable if a highly effective sedative can be completely substituted by herbal alternatives. Furthermore, valerian is a non-refundable OTC and no costs occur by substituting it. Consequently, in the low-cost scenario, approximately €42 million can be "saved" by applying valerian. Second, some PRISCUS agents are only potentially inappropriate, if a certain dose is exceeded or a certain release form is applied. This was not taken into account, as the data set of WIdO consisted of total prescriptions. Third, we only analysed the 30 top-selling drugs.Another limitation is related to our applied scenarios. In general, the scenarios assumed that a PIM would be fully substituted by the cheapest, most expensive, or mean costs. However, a complete substitution might be unlikely in daily practice, which highlights the need for a calculation based on individual patient data. In general, the proposed substitutions have to be evaluated carefully by physicians. In some cases, groups of potential surrogates include other medication form the PRISCUS list. For example, the recommended substitution of amitriptyline are SSRI. However, the group of SSRI comprises PIM as well e.g. fluoxetine. It remains unclear if in those cases PIM might be substituted by another PIM as well. For our study, we included all recommended surrogates.This is the first study to assess the economic relevance of PIM on the PRISCUS list in Germany from a SHI perspective. It was shown that PIM is present in Germany and approximately 5 % of all prescriptions made to people aged ≥65 years are potentially inappropriate. Moreover, the reimbursement costs of PIM in the elderly account for 1.36 % of total sales to patients ≥65 years of age. The substitution of PRISCUS agents would lead to an increase in costs independent of the chosen scenario. Thus, from a short-term perspective, there is no incentive for SHI to promote substitution. However, in the long-term, hospitalisation and recurrent physician visits should be addressed.Not applicable.Not applicable.The datasets supporting the conclusions of this article are included within the article.


Influenza virus infection causes global shutdown of host protein synthesis in the infected cells, while viral proteins are efficiently produced. This host shutoff activity is considered to allow the virus to escape host innate and acquired immune recognition, which otherwise restricts viral replication and spread. It had been thought that cap snatching activity and degradation of host RNA polymerase II induced by viral RNA polymerase cause host shutoff ,2. HowevPA-X encodes N-terminal 191 amino acids of PA protein and unique C-terminal sequences (41 or 61 amino acid residues) produced by +1 reading frame of PA mRNA via ribosomal frameshifting . PA-X coin vitro and in vivo. The pH1N1 PA-XFS-infected mice cleared the virus and recovered faster than wild type-infected mice [To analyze the effect of PA-X of currently circulating viruses on virus growth and pathogenicity, we recently rescued a mutant 2009 pandemic H1N1 virus (pH1N1) expressing reduced amount of PA-X through mutations at the frameshift motif (PA-XFS) . In agreted mice . Given tted mice , it is pHighly pathogenic influenza viruses, such as 1918 virus or avian H5N1 viruses induce an extensive host cytokine response, known as cytokine storm, causing tissue injury and virus pathogenicity. Reduced PA-X expression of 1918 virus enhanced virus pathogenicity due to the accelerated cytokine responses and severe tissue injury . Avian HImportantly, pH1N1 PA-XFS induced a greater humoral response than wild type virus in mice, even though virus replication was attenuated in their lungs . This re


In the frame of active lyssavirus surveillance in bats, oropharyngeal swabs from German (N = 2297) and Danish (N = 134) insectivorous bats were investigated using a newly developed generic pan-lyssavirus real-time reverse transcriptase PCR (RT-qPCR).In total, 15 RT-qPCR positive swabs were detected. Remarkably, sequencing of positive samples did not confirm the presence of bat associated lyssaviruses but revealed nine distinct novel rhabdovirus-related sequences.Rhabdoviridae.Several novel rhabdovirus-related sequences were detected both in German and Danish insectivorous bats. The results also prove that the novel generic pan-lyssavirus RT-qPCR offers a very broad detection range that allows the collection of further valuable data concerning the broad and complex diversity within the family  European bat lyssavirus type 1 and 2 as well as Bokeloh bat lyssavirus, in total, 15 swabs from bats originating from the Northeast of Germany (12) and Central Jutland in Denmark (3) had Cq values between 33.5 and 44.6 when using the assay targeting a conserved region on the polymerase (L) gene , 27676  and RV-DK2  however, showed the highest homology with less than 80% with Isfahan virus [AJ810084], Kotonkan virus [HM474855] and Long island tick rhabdovirus [KJ396935]. These rhabdoviruses were isolated from the sandfly Phlebotomus papatasi, from Culicoides sp. and from Amblyomma americanum, respectively. The sequences of the German samples 26530 , 26855 , 26873 , 26876  and 29130  as well as the Danish sample RV-DK1  showed the highest identities with less than 75% for different strains of Wongabel virus, Mossuril virus or Bovine ephemeral fever virus.The partial L gene sequences from virus samples 26907 [KJ614425, Rhabdoviridae in GenBank confirmed the affinity of all nine distinct rhabdovirus-related sequences among the dimarhabdovirus supergroup  values of 26–36 (Table For the German samples, based on the generated sequence data, specific real-time RT-PCRs were developed and applied Table , using t36 Table  indicatiRecently, highly sensitive sophisticated molecular techniques have been used to elucidate the role bats play in the epidemiology of rhabdoviruses. Our results corroborate previous studies that have revealed the presence of novel rhabdoviruses or rhabdoviral sequences in bats from Africa, America and Europe -13. SimiRhabdoviridae.In conclusion, we were able to detect novel rhabdovirus-related sequences in oropharyngeal swabs obtained from German and Danish bats, using a generic pan-lyssavirus L-gene RT-qPCR. As indicated previously , this sy


The genome of a Newcastle disease virus isolated from a Japanese quail in 2003 is reported here. The genome is 15,192 nucleotides (nt) long, as found in the recent genotypes, and grouped as genotype VIIb, with a 6-nt insertion. This is the first report on the sequence of a genotype VII Newcastle disease virus (NDV) from India. Paramyxoviridae family and genus Avulavirus, has been shown to be present on six of the seven continents, and reports indicate the susceptibility of 241 different species, which includes 27 of the 50 orders of birds, to this virus. There has been a greater understanding of the diversity of ND virus (NDV) as a result of different vaccines that have been in use and also due to the availability of the whole-genome sequence information from different countries. An analysis of the genome had enabled the classification of the virus into 11 different genotypes (Newcastle disease (ND) virus, a member of the enotypes  and suggCoturnix coturnix japonica) with clinical signs of diarrhea, lethargy, and nervous symptoms. This virus is a mesogenic strain, since it had an intracerebral pathogenicity index (ICPI) value of 1.98 and a mean death time of 67 h. Genotyping based on the 374-bp region of the fusion protein gene sequence confirmed it to belong to genotype VIIb. The whole-genome information was generated following assembly of the chromatograms  from three different clones using 36 different primer pairs, as reported in our earlier study  isolated in 2003 in 10-day-old specific-pathogen-free chicken eggs from a pooled homogenate of spleen and brain samples from a Japanese quail  downstream of the untranslated region of the nucleoprotein gene, as seen in the recent genotypes. The fusion protein cleavage site of this strain was All the residues that contribute to the sialic acid binding site and neutralizing epitopes as already reported were invariant in the Indian genotype VII strain. A comparison revealed isoleucine at position 69 of the fusion protein to be unique; however, this needs confirmation with more isolates . The preKF740478.The whole-genome sequence has been submitted to GenBank under the accession no.


Scaptotrigona postica propolis remain unknown, the little information about this product is concentrated in antibacterial and anti fungi actions, but don't has related of antiviral action.Studies about viral infections have a great importance in human and veterinary health, and the number of medications available to treatment these diseases is very reduced, making the search for antiviral molecules an important focus for scientific research. Propolis is a bee material manufactured by the mix of exudate of plants, saliva and bee wax. This product is used to seal the hive and involve dead invaders. Their chemical profile is very variable, and depending on the geographic origin and plant conditions of growth. The use of propolis (bee-glue) for various purposes has reports at before Christ. In Egypt, propolis was used in the preservation of bodies, performing a function of balsam, and its use persists to today in folk medicine to treat various pathologies, being widely used around the world. It is known that propolis of Apis Mellifera has compounds with antiviral activity on virus like Influenza A and B, Vaccinia virus, Hepatitis virus, HIV, Herpes virus, HIV and Poliovirus. In Brazil exists a subfamily of bees named Meliponinae, the stingless bees mixing the resins collected of plants with wax and ground, producing a different type of propolis, named geopropolis, but not all bees of this family produce this type of propolis, like Scaptotrigona postica. The biological activities of Scaptotrigona postica.Purify, isolate, and characterize substances with antiviral activity of propolis from The propolis collected in Barra do Corda, city of Maranhão state, was partitioned with hexane, ethyl acetate and a solution of water/methanol (1:1). The propolis crude, as well its purified fractions, were tested by viral titer reduction technique, and determination of viral mRNA against measles, picornavirus, influenza virus and rubella virus.3 times the replication of rubella virus. At the moment, we are performed the optimization of the purification process.Experiments with the purified fraction led to a 64-fold reduction of picornavirus production, 32-fold reduction in influenza virus production and 8-fold reduction of measles virus. Assays using RT-PCR, to determine viral mRNA present in the treated and infected cells, also was performed. The purified antiviral fraction was able to reduce at 10


I came to the United States in 1973 because the then Chair of Bacteriology and Immunology at the University of North Carolina at Chapel Hill (UNC), the late Philip Manire, did a sabbatical at the Lister Institute in London where I was working on my Ph.D. “What are you going to do when you finish?” he asked. I was one of those students that my current Chair berates for not having thought about life after graduation and had to confess that I didn’t really know. I had been working as a research assistant and had more or less assumed that I would keep doing the same thing; no one had suggested anything to the contrary. “Why not come to the States for two years and see what it is like?” he said. “There is someone in my department working on this new virus, Epstein–Barr virus [EBV], maybe you can work with him.” It seemed like a good idea, and 42 years later, I am just closing a career working on that “new virus,” EBV, and I am still in the US.EBV appealed to me because I liked immunology, I thought viruses were really interesting, and a virus that infected the immune system seemed to represent an ideal combination for study. In Joe Pagano’s lab at UNC, I had pretty much free range in what I did. I was in a supportive environment with wonderful colleagues in what eventually became the Lineberger Comprehensive Cancer Center. Looking back, it is amazing to me how, even as a very junior postdoc, busy, established investigators would take time to talk with me and discuss ideas. At UNC, I was also encouraged to write grants and got my first chance to fly independently.That new—actually quite old—virus, EBV, infects just about all of us. We carry it in our B lymphocytes and in our epithelial cells, and generally speaking, it does not seem to do most of us much harm. If we are first infected after the age of about ten or 11, there is a good chance that we will get infectious mononucleosis—temporarily debilitating, but only rarely any worse. A bout of infectious mononucleosis does, however, slightly increase the chance of subsequently developing Hodgkin’s lymphoma or multiple sclerosis, and as we age, an enormous part of our immune system is devoted to keeping EBV in check, probably reducing our ability to respond to other pathogens. If you lose immune control of the virus, B cell lymphomas can develop, and such EBV-driven tumors are a scourge of transplant recipients and AIDS patients. More difficult to explain, and the subject of considerable research effort over the years, has been the association of EBV with nasopharyngeal carcinomas, a subset of gastric cancers, and a variety of rarer cancers such as natural killer/T cell lymphomas and leiomyosarcomas. It has been estimated that EBV is responsible for about 200,000 new cases of cancer worldwide, every year.As my own research on EBV continued, my lab began to focus on the envelope proteins of the virus that allow the virus to attach to a cell, fuse its envelope with the cell membrane, and get inside. After all, no virus can survive or cause disease, including cancer, unless it can penetrate a cell. So we studied virus proteins involved in attachment and entry into its two major targets, B lymphocytes and epithelial cells, and we sought to identify partners on the cell surface that collaborated with the virus. Over the years, we discovered that the virus uses HLA class II to trigger its entry into a B cell and a subset of alpha v integrins to trigger its entry into epithelial cells. We found that a complex of three virus proteins, called gH, gL, and gp42, regulates B cell fusion and that a subset of only two of them, gH and gL, regulates epithelial cell fusion. To our delight, this allowed us to uncover a mechanism that the virus uses to switch its tropism alternately from B cell to epithelial cell and back again to maintain a cycle of low level replication in all who have been infected. From study of virus glycoproteins, perhaps a bit esoteric, we ended up learning something important about pathogenesis and finding new targets to limit the ability of the virus to spread.Why did I stay in the US? Because with a good idea and a plausible way to test a hypothesis, anyone could get research support. The US was the scientific mecca for investigators wanting to test new ideas and come up with new understanding—often in unexpected ways. Is it still that mecca? Not unless we continue to support the basic research that opens up surprising new horizons and unanticipated opportunities and keeps the US at the forefront of discovery.


Accipiter gentiles) resulted in the isolation of WNV lineage 2. During the summer of 2004, and in subsequent years, the virus appeared to spread locally throughout Hungary and into neighboring Austria. Subsequently, WNV lineage 2 emerged in Greece in 2010 and in Italy in 2011, involving outbreaks on the Italian mainland and Sardinia. Further spread through the Balkan countries is also suspected. Whole genome sequencing has confirmed that the virus responsible for the outbreaks in Greece and Italy was almost identical to that isolated in Hungary. However, unlike the outbreaks in Hungary, the burden of disease in Mediterranean countries has fallen upon the human population with numerous cases of West Nile fever and a relatively higher mortality rate than in previous outbreaks. The emergence of WNV lineage 2 in Europe, its over-wintering and subsequent spread over large distances illustrates the repeated threat of emerging mosquito-borne diseases. This article will review the emergence of WNV lineage 2 in Europe; consider the pathways for virus spread and the public health implications for the continent.West Nile virus (WNV) is transmitted by mosquitoes and causes fever and encephalitis in humans, equines, and occasionally wild birds. The virus was first isolated in sub-Saharan Africa where it is endemic. WNV lineage 1 has been responsible for repeated disease outbreaks in the countries of the Mediterranean basin over the past 50 years. This lineage was also introduced into North America in 1999 causing widespread human, equine, and avian mortality. WNV lineage 2, the first WNV lineage to be isolated, was believed to be restricted to sub-Saharan Africa causing a relatively mild fever in humans. However, in 2004, an investigation in Hungary of a case of encephalitis in a wild goshawk ( In recent years, arthropod-borne viruses have shown an increasing ability to spread beyond the areas, which had been considered to be their established geographic ranges. A number of factors are driving this process including bird migration, increasing global trade, and the movement of vector species . This raFlavivirus, family Flaviviridae, and is phylogenetically and antigenically related to Japanese encephalitis virus. The virus was first isolated from the blood of a woman suffering a febrile illness in the West Nile district of Uganda  in Hungary  in northern Italy, where lineage 1 has been endemic since 2008  substitution within the NS3 gene, which was present in WNV isolates responsible for the Greek outbreak but not the Hungarian or Italian isolates , 51. HowAccipiter gentiles) in Hungary, and the genomic sequence of this isolate demonstrated closest homology with a group of southern African strains  migrate from breeding areas in central Europe to wintering areas in Africa, and serological studies on individual birds on their arrival in resting areas of Greece suggest that they were exposed to WNV lineage 2 in the area of origin in central Europe, and may constitute a candidate for introduction of lineage 2 strains from central Europe to Greece  of Greek lineage 2 strains is similar to that observed in neuroinvasive lineage 1 isolates , while aCulex, particularly species in the Cx. pipiens complex  and Cx. quinquefasciatus   . In the  pipiens , which in the UK . These sn the UK . However marshes , 61.The occurrence and abundance of potential vector species are a prerequisite for enzootic transmission of mosquito-borne viruses in the UK. Thirty-four species of mosquitoes have been recorded in the British Isles, of which nine species have been implicated in WNV transmission elsewhere . ThirteeCx. pipiens sensu stricto (s.s.) bite both humans and birds; the two forms within this species are morphologically indistinguishable, but they are physiologically different. Cx. pipiens f. pipiens is mostly ornithophagic and rarely bite humans, the immature stages are found in permanent water, it overwinters in the adult stage and is multivoltine (Cx. pipiens f. molestus is highly anthropophilic (though it may bite birds), the immature stages live underground , and all life stages occur throughout the year . Although the risk of WNV transmission to humans in the UK is still low due to limited human exposure to bridge vectors, the risk for transmission was higher in Kent because of the presence of other bridge vector species such as Cx. pipiens s.l. and both migratory and resident birds. The authors highlighted the potential risk for horses, which are often grazed in this part of country. At present, screening of specimens of Cx. modestus for WNV and other flaviviruses such as Usutu virus is being undertaken by cross-governmental groups in the UK under a “One Health” initiative.Golding et al.  showed tThe increase in human population, expansion of trading routes and tourism, deforestation, and climate change are some of the factors that have facilitated the rapid dispersal of vector species into new geographical areas . Recent Aedes aegypti, Ae. albopictus, Ae. atropalpus, Ae. japonicus, Ae. koreicus, and Ae. triseriatus). These have mainly been imported through the international trade in used tires and Lucky bamboo, although public and/or private ground transport have also been implicated. Of these, Ae. albopictus presents the greatest threat. Female Ae. albopictus lay desiccation-resistant eggs above the surface of the water in tree holes, tires, or other water-holding containers (Ae. albopictus is considered one of the top 100 invasive species of mosquitoes worldwide (For invasive mosquitoes in Europe, six exotic species have been identified (ntainers . Its abiorldwide .Aedes albopictus is a known vector of CHIKV, and laboratory infectious studies have shown that this species is a competent vector of WNV (r of WNV . It has r of WNV , althougCulex species, are critical for the spread of disease. Further study of factors such as mosquito distribution, host biting preference, and species hybridization will improve understanding of WNV persistence and assessment of the risk to human populations.West Nile virus lineage 2 was introduced into Europe in 2004 and subsequently emerged in a number of countries. It has overwintered in these countries and in the case of Italy is co-circulating with WNV lineage 1 and Usutu virus. Migratory birds are likely to be the main vehicle of movement for the virus, both as a source of introduction and the means by which it has spread within Europe. However, there is little direct evidence to support this. The disease manifestations appear to vary in different countries. In Hungary, raptors appear to be affected while in Greece the main burden of disease has fallen on the human population. The reasons for this are not clear, but the variation could be caused by mutations within particular virus strains that persist in different locations. The abundance and composition of mosquito populations, particularly Following detection of WNV, there is a need to implement public health measures to protect at risk populations, particularly the elderly. This includes measures to reduce mosquito biting such as destruction of larval habitats and applications of larvicides. Measures that protect the individual include the application of mosquito repellents and clothing that reduces the exposure of bare skin. Currently, there is no human vaccine available. The risk of further spread of WNV lineage 2 to countries around the Mediterranean Sea is high. Countries in northern Europe appear to be at lower risk due to reduced mosquito abundance and lower winter temperatures. However, constant vigilance is needed to monitor for any change in environmental or ecological conditions that could make the introduction and persistence of WNV in northerly latitudes possible.Nicholas Johnson conceived the idea for this review. Luis M. Hernández-Triana, Claire Jeffries, Karen Mansfield, George Carnell, Anthony Fooks, and Nicholas Johnson prepared the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TNF-α, OPG and IL-1β expression levels in otosclerotic stape footplates to the occurrence of measles virus infection. 61 patients with otosclerosis were treated surgically. Thirty-one stapes obtained from cadavers of people, who had died from a sudden cause were used as a control group. The presence of measles virus RNA and the expression levels of TNF-α, IL-1β and OPG in otosclerotic foci were assessed using one-step RT-PCR. The presence of measles virus RNA was noted in 80.3 % of otosclerotic stapes (49 out of 61) and 9.7 % of normal tissues (3 out of 31). Transcript of TNF-α, IL-1β and OPG was detected in 40, 46 and 18 virus-positive stapes, respectively. The transcript level of TNF-α and IL-1β was significantly higher in otosclerotic tissues comparing to normal tissue. The OPG expression level was significantly lower in otosclerotic tissues comparing to controls. The presence of measles virus RNA in the stapes may indicate its role in the pathogenesis of otosclerosis. The presence of TNF-α and  IL-1β mRNA in the virus-positive stapes could be the result of viral antigen stimulation and may be a marker of inflammation the otosclerotic focus. The lack of OPG mRNA and the presence of TNF-α and IL-1β  mRNA in the majority of otosclerotic tissues reflect the bone remodeling process occurring in the stapes.Persistent measles virus infections play a crucial role in the pathomechanism of otosclerosis. The study was undertaken to investigate the role of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and osteoprotegerin (OPG) in otosclerotic bone remodeling and to assess the relation of  Hearing loss in humans significantly decreases quality of life and often leads to social isolation. One of the causes of acquired hearing loss is otosclerosis. The unexplained etiology and pathomechanism of this disorder generates great interest within the international scientific community.Otosclerosis occurs only in humans and is the cause of 5–9 % of all hearing losses and 18–22 % of those with conductive hearing losses. Approximately 10 % of patients with otosclerosis develop sensorineural hearing loss . Women aAccording to our current knowledge, interacting environmental and genetic factors are the underlining causes of the disease. Measles virus elements have been frequently detected in otosclerotic tissues, so there is a reliable hypothesis about the viral origin of the disorder , 14–20. Osteoprotegerin (OPG) is a member of the TNF superfamily and also participates in bone homeostasis. OPG is also known as TNFRSF11B (tumor necrosis factor receptor superfamily member 11B) and its main function is the inhibition of bone resorption. This glycoprotein inhibits maturation and activation of osteoclasts and induces their apoptosis –26. ThusIn our study, we analyzed the expression level of proinflammatory cytokines , OPG and the presence of measles virus RNA in fragments of the stapes (superstructure and part of the otosclerotic changed footplate) removed during stapedotomy performed in patients with otosclerosis. Our findings support the assumption on primary role of measles virus infection in pathogenesis of otosclerotic bone remodeling in the stapes.N = 61, male = 21, female = 40), treated surgically in the Department of Otolaryngology, Medical University of Gdańsk, were analyzed prospectively. The otolaryngological preoperative examination consisted of detailed anamnesis and audiological studies. Each patient underwent surgery on the stapes to improve their hearing. During the stapedotomy, the superstructure and part of the stapes footplate were removed and subjected to further molecular analysis. The diagnosis of otosclerosis was based on clinical, audiometric and surgical findings. The assessment of otosclerotic lesions during the surgery was based on the Portmann Classification eviously  and are TNF-α, IL-1β, and OPG transcripts were analyzed via real-time RT-PCR with the use LightCycler RNA Master HybProbe . The reaction was performed in a Light Cycler 2.0 . The β-actin transcript was used as an internal standard and was amplified together with each target gene transcript in the same tubes using primers and probes as shown in Table The levels of 2, 0.5 μM of each PCR primer, 0.2 μM of each hybridization probe and ~50 ng of total RNA isolated from analyzed tissue. The concentration of FL probe for β-actin was 0.4 μM . Analysis of the data was performed using Light Cycler software.Thermocycling was conducted at a final volume of 20 μl medium containing: 7.5 μl of Master HybProbe, 3.25 mM Mn[OAc]μM Table . As a neV was used to assess effect size of a relationship between variables in this design. It was assumed that the results between 0.50 and 0.75 indicate moderate effect size. In order to compare two independent groups, a t test was performed followed by effect size assessment with Cohen’s d. It was assumed that d between 0.20 and 0.50 indicate modest effect size, but d between 0.50 and 0.80 demonstrate medium effect size.To run quantitative analyses several statistical test were used. Chi-squared statistic for 2 × 2 design was applied. In addition, Cramer’s The mean age of patients was 43.5 years (range 24–64 years). Female to male ratio was 1.9:1. The air-bone gap at the 1,000 Hz was at least 30 dB, conductive (59 % of patients) or mixed hearing loss (41 % of patients) was associated with the absence of the stapedial reflex on pre-operative audiometry. The average patient’s symptom duration (mainly hearing loss) was 9 years (range 0.5–40). More than half of all cases were classified as type 4 according to the Portmann Classification.χ2(1.88) = 41.75; p < 0.001; V = 0.67].Measles virus RNA was detected in 49 out of 61 (80.3 %) stapes obtained from patients with otosclerosis. In the stapes from the control group, the presence of measles virus RNA was noted in three cases out of 31 (9.6 %)  and in 11 virus-negative (91.7 %) bone samples from the clinical group. IL-1β transcript was detected in 46 virus-positive (93.8 %) and in 10 virus-negative (83.3 %) stapes obtained from patients with otosclerosis OPG transcript was detectable in only 18 virus-positive (36.7 %) and in 5 virus-negative (41.7 %) patient’s stapes. In the specimens from cadavers, OPG mRNA was detectable in 23 (74.1 %) stapes, TNF-α mRNA in 8 (25.8 %) stapes and IL-1β mRNA in 6 (19.3 %) stapes. However, we observed that the TNF-α and IL-1β expression levels were significantly higher in tissue samples obtained from patients with otosclerosis as compared to the control, t (<1.96) ; p < 0.001.Conversely, the transcript level of the d statistics indicates that the extent of these differences was either small (d > 20) or medium (d > 50). In the measles virus-positive samples, there was no correlation between the transcript levels for genes TNF-α and OPG and between the transcript levels for genes IL-1β and OPG. It was estimated that the level of TNF-α, IL-1β and OPG gene expression in the stapes taken from people suffering from otosclerosis is not dependent on the presence of measles virus RNA (t < 1).The analysis of the value of Cohen’s TNF-α, IL-1β and OPG expression.We observed the presence of measles virus RNA in 49 stapes taken from patients with otosclerosis. 19.7 % of specimens were free from virus particles. According to Iyer and Gristwood, the histopathological image corresponds to the image of the operating microscope . Owing tThe results of our research can be compared with the reports of Karosi et al. , 31, 32.It should be stressed that although several reports employing various methodologies documented some relations of measles virus infection with development of otosclerosis, there are also studies showing lack of viral RNA in samples from patients with otosclerosis. Grayeli et al.  did not TNF-α in 88 out of 99 measles virus RNA positive otosclerotic samples (88.8 %) and in 8 without viral RNA.The pro-inflammatory cytokines TNF-α, and IL-1β cause increased inflow of immature forms of osteoclasts from bone marrow into the blood and then into the middle ear, where the inflammation occurs. In contrast to OPG, TNF-α and IL-1β have an anti-apoptotic effect on osteoclasts and thus prolong osteoclast life and increase bone resorption . Karosi IL-1β. The presence of the IL-1β transcript was recorded in 93.8 % of the virus-positive otosclerotic samples. We used the real-time PCR method, which, apart from the qualitative analysis of the genetic material, allows the determination of the quantity of the analyzed material initially present in the tested sample. Thus, we checked if there is a connection between the levels of TNF-α, IL-1β, OPG expression and the presence of measles virus RNA in the otosclerotic stapes. It transpired that the level of expression for these genes in the stapes removed from people suffering from otosclerosis does not depend on the presence of the viral RNA. However, more essential seems to be the presence or absence of their mRNAs in the otosclerotic focus.We achieved results similar to Karosi et al. , 19 (81.OPG and the presence of the TNF-α and IL-1β in the otosclerotic stapes simultaneously indicates the suppression of OPG due to increased levels of the pro-inflammatory cytokines. The presence of OPG mRNA and the lack of IL-1β and TNF-α mRNA in most of the stapes removed from healthy people who had died a sudden death might confirm our assumption.We assume that the lack of


Our group has previously described that indirect and intangible costs substantially burden households with a food allergic adult. We now extend our investigation to children and adolescents.To estimate the total, direct, indirect and intangible costs of food allergy, in households with at least one child or adolescent with objectively-diagnosed allergy to staple foods , and to compare these costs with age- and sex- matched controls.Participants included 84 children and 60 adolescents (cases), and 94 children and 56 adolescents (controls). Direct and indirect cost data collected via the Food Allergy Socio-Economic Questionnaire  from parents of children and adolescents with objectively-diagnosed allergy to staple foods (cases) were compared to data from age- and sex-matched controls, and calculated as annual household costs. Total costs were defined as direct + indirect costs. Direct and indirect costs were also considered independently. Intangible costs included measures of self-reported health, standard of living and losses of well-being.Annual total household costs were significantly higher for cases than controls, for children  and adolescents , and were driven by direct (e.g. medicines) and indirect  costs. Children, but not adolescents, with a history of anaphylaxis had higher annual direct costs vs. those without anaphylaxis . Intangible costs were greater amongst cases than controls for both age groups .Households with children and adolescents with objectively-diagnosed allergy to staple foods have higher total household costs than controls. Direct and indirect household costs were significantly higher for food allergic children, but not food allergic adolescents, compared to controls. Among both children and adolescents, objectively-diagnosed allergy to staple foods adversely impacts intangible costs.


Cassava ranks fifth among the starch producing crops of the world, its annual bioethanol yield is higher than for any other crop. Cassava cultivar KU50, the most widely grown cultivar for non-food purposes is susceptible to Sri Lankan cassava mosaic virus (SLCMV). The objective of this work was to engineer resistance to SLCMV by RNA interference (RNAi) in order to increase biomass yield, an important aspect for bioethanol production. Here, we produced transgenic KU50 lines expressing dsRNA homologous to the region between the AV2 and AV1 of DNA A of SLCMV. High level expression of dsRNA of SLCMV did not induce any growth abnormality in the transgenic plants. Transgenic lines displayed high levels of resistance to SLCMV compared to the wild-type plants and no virus load could be detected in uninoculated new leaves of the infected resistant lines after PCR amplification and RT-PCR analysis. The agronomic performance of the transgenic lines was unimpaired after inoculation with the virus as the plants presented similar growth when compared to the mock inoculated control plants and revealed no apparent reduction in the amount and weight of tubers produced. We show that the resistance is correlated with post-transcriptional gene silencing because of the production of transgene specific siRNA. The results demonstrate that transgenic lines exhibited high levels of resistance to SLCMV. This resistance coupled with the desirable yield components in the transgenic lines makes them better candidates for exploitation in the production of biomass as well as bioethanol. Manihot esculenta) is an important root crop in Africa, Asia and South America, providing energy for about one billion people. It is the 3rd largest source of carbohydrates for human food in the world, a staple or subsidiary food for about a fifth of the world’s population and raw material for starch based industries. Its flexible harvesting time, tolerance to adverse environmental conditions such as drought and poor soils, little requirement to agricultural fertilizers and high starch content make cassava one of the most attractive plants for starch production in future. In recent years, cassava has emerged as a potential biofuel plant, ranking among other high energy crops such as maize, sugarcane and sweet sorghum. Indeed, the annual yield of cassava-derived bioethanol was found to be higher than for any other crop, including sugarcane [Cassava  known as the common region (CR). The CR harbours the viral promoters, origin of replication and sequences involved in binding of DNA A-encoded replication associated protein (Rep), the only virus encoded product required for viral DNA replication [CMD is caused by several cassava mosaic begomoviruses, which are transmitted, plant-to-plant, exclusively by the whitefly lication . DNA A elication .African cassava mosaic virus (ACMV) was the first virus species found to be associated with CMD in Africa, although no fewer than seven begomovirus species are now recognized in Africa [CMD is widespread in Africa and the Indian subcontinent . Africann Africa . In the n Africa . Anothern Africa . ICMV ann Africa .To date, a number of strategies to engineer CMD resistance in cassava have been reported mostly for ACMV . For exaAfrican cassava mosaic virus [Cucumber mosaic virus, Zucchini yellow mosaic virus and Watermelon mosaic virus [Bean golden mosaic virus [Potato leaf roll virus, Potato virus Y and Potato virus X [Papaya ring spot virus [Plum pox virus [CMD is currently managed by multiplication and distribution of disease-free stem cuttings. It has been difficult to produce SLCMV-resistant cassava by conventional breeding because of high heterozygosity and inbreeding depression of elite cultivars or farmer-preferred landraces. As a result, new strategies to control SLCMV are desirable. Post-transcriptional gene silencing (PTGS) or RNA interference (RNAi) is a technology that offers significant potential to control plant viral pathogens. RNAi has been applied to generate resistance to ic virus , Cucumbeic virus –19, Beanic virus , Potato  virus X , 22, Papot virus  and Plumox virus , 25.RNA interference (RNAi) is a conserved mechanism that recognizes double-stranded RNA (dsRNA) as a signal to trigger sequence-specific degradation of homologous mRNA. The key feature of RNAi is short dsRNA fragments known as “short interfering RNAs (siRNAs)” of 21–25 bp in length, which are produced by the cleavage of dsRNA by a ds-specific ribonuclease termed ‘Dicer’. Once generated, the siRNA are then recognized by a ribonuclease complex known as the RNA-induced silencing complex (RISC) and used as a guide for the recognition and sequence-specific degradation of homologous mRNAs –29, resuCurrently, the elite cassava variety, Thai cassava cultivar Kasetsart University 50 (KU50) one of the most important cassava cultivar in the world is grown by many farmers and biofuel industries in Asia under different names because of its high root yield and high root starch content with good germination and vigorous plant growth with wide adaptation . TherefoIn this article, we report a simple, efficient, quick and reproducible regeneration protocol for cassava cultivar KU50 through somatic embryogenesis. This protocol allowed us to produce transgenic cassava lines that express dsRNA homologous to the region between the AV2 and AV1 of SLCMV. Transgenic lines obtained displayed high levels of resistance to SLCMV and agronomic performance in the transgenic lines was not affected in the presence of the virus. We show that the resistance is correlated with post-transcriptional gene silencing because of the production of transgenic specific siRNA.Enlarged axillary buds , plate 2Escherichia coli, the two DNA fragments were separated by a 278 bp cat 1 1st intron spacer derived from castor bean catalase gene [In order to express virus-derived dsRNA in cassava, we produced a construct containing inverted repeat of 527 bp DNA fragment of SLCMV. The 527 bp fragment, which includes part of the AV2 (137–493 bp) and half of the AV1 (297–534 bp) spans from nucleotide 8–534. To ensure stability of the inverted repeat in ase gene . The entAgrobacterium and plants were regenerated as described above. In order to avoid possible chimeric plants, the shoots produced were subcultured in selective rooting medium. After 3 rounds of subculture, the chimeric shoots completely bleached and died or produced shoots on the surface of the medium while only 10 produced roots in the medium. Eight plantlets out of the 10 rooted shoots were transgenic as the 527 bp fragment of the SLCMV transgene  to 3  . Based oAs shown in Next we determined whether agronomic performance of these plants might be affected in the presence of the virus. First, we compared the plant height of the inoculated transgenic plants and the mock inoculated plant at 60 dpi . There wWe assessed the level of transmission of SLCMV to cassava by mechanical inoculation with infectious clone. Six out of the 7 Wild-type plants inoculated presented symptoms, which ranged from leaf curling, stunted growth and chlorosis, showing 85.7 percent transmission across two independent experiments . High trBefore challenging the plants with SLCMV clone pSL7, northern analysis was carried out to detect SLCMV-specific siRNAs in transgenic and wild-type plants. The presence of siRNA is characteristic of PTGS and would indicate whether the PTGS response had been activated, especially in lines K4-K10 where transcript levels of the hairpin were either low or not detected. First, we screened the plants by PCR and RT-PCR to exclude a potential latent infection with SLCMV, which might result in virus-derived siRNA that are not of transgene origin. No SLCMV signal was detected in any of the lines tested (data not shown), indicating that no pre-induced PTGS effects are active in the transgenic plants. When northern blot was performed in the uninoculated transgenic plants, all transgenic lines, except line K3 were found to accumulate siRNAs signals of about 21–23 nt, homologous to the sense and antisense SLCMV sequences . No siRNPCR amplification  and RT-PRegeneration of whole plants from cell culture systems is often the limiting step in the application of various biotechnological techniques to crop improvement. A prerequisite for genetic modification of cassava is a reliable regeneration system. Establishment of a regeneration system for elite cassava cultivar such as KU50 will open up the possibility for genetic manipulation of the cultivar and provide the information needed for handling and introduction of useful genes. Adventitious shoot regeneration via somatic embryogenesis is highly desirable as the process affords high multiplication rates and results in propagules which possess both root and shoot axes. Somatic embryos may develop from single cell and, dependent on the genetic stability of the regeneration system, plants with novel genotypes may be recovered .In several cassava cultivars, apical meristem and young leaf lobes have been frequently used for regeneration –36. HoweEast African cassava mosaic Zanzibar virus (EACMZV) was necessary for infection in tobacco and cassava. The AV2 of Indian cassava mosaic virus (ICMV) has been shown to be involved in the cell-to-cell movement [tomato leaf curl virus (ToLCV) and tomato yellow leaf curl virus (TYLCV) is shown to act as a potent suppressor of gene silencing activity. Silencing of AV2 gene of ToLCV by antisense RNA [AV1/AV2 genes of ToLCV by artificial micro RNA [AV1/AV2 genes would disrupt the encapsidation and the next round of virus infection, and would be a useful strategy to develop broad-spectrum resistance against CMD in cassava, and other begomoviruses where numerous distinct virus species/strain may cause disease in a particular crop. Using this construct, we demonstrated that transgenic cassava expressing 527 bp fragment of SLCMV in the form of an intermolecular intron-hairpin RNA displayed high levels of resistance to SLCMV infection compared to the wild-type plant. We show that resistance was acquired by RNA silencing through the production of transgene-specific siRNA. The present study and work reported elsewhere [AV1/AV2 could be targeted to confer resistance to geminiviruses.Cassava mosaic diseases can result in massive economic losses for industries using it for biofuel production, and also cause food and economic insecurity in countries where cassava is used as a staple food and cash crop. Therefore, attempts should be made to bring CMD under control in these regions. Unfortunately, control measures presently employed in infected regions are based on the chemical control of the vector population, with partial efficacy, environmental negative effect, elimination of natural enemies, and appearance of pesticide-resistant whitefly. Thus, producing disease resistant or tolerant varieties of cassava by genetic engineering is the best way to control the disease in the field. To date, considerable effort has been made by scientists to engineer cassava for resistance to African cassava mosaic virus by RNA silencing , 14, 35 movement . Furtherense RNA  and AV1/icro RNA  resultedicro RNA , indicatlsewhere , 41 suggThe use of siRNAs, an intermediate in the gene-silencing pathway, has become a powerful tool for specifically down regulating gene expression, and has been demonstrated successfully in a wide variety of cells and organisms . It is oApart from transgenic line K3, which was susceptible to the virus , the remRNA-mediated resistance has advantages for environmental biosafety over protein mediated resistance as the potential risks of heterologous encapsidation and recombination of virus are diminished. The major drawback in RNA silencing is that it is usually highly sequence specific, and viruses having between 10–15% nucleotide diversity are mostly insensitive to the technology . SequencIn cassava, up to 90% of its starch is stored in the tubers, which makes them suitable for biofuel production. Therefore, high tuber yield is an important factor for bioethanol production in cassava. When cassava is attacked by CMD, there is reduced tuber yield in terms of number and size, many of them rotting, making them unsuitable for use for biofuel production. In this regard, we performed another round of inoculation and checked the agronomic performance of the plants in the presence of the virus. It was encouraging to see that, besides the absence of disease symptoms and virus load in the resistant transgenic plants, the agronomic performance of these plants was not affected by infection with the virus and the expression of SLCMV dsRNA did not intrinsically cause yield depression, as the plants presented similar growth when compared to the mock inoculated control plants and revealed no apparent reduction in the amount of tubers produced as well as weight of tubers . This deIn this study, since the number of replicates was low, we used mechanical inoculation with infectious clone to introduce SLCMV DNA into cassava. Cloned SLCMV DNA has been shown to be infectious to cassava by agroinoculation  and biol4), solidified with 0.8% plant agar at 28°C with a 16-h photoperiod and subcultured at 8 week-intervals. All media supplements were added prior to adjusting the pH to 5.8 and autoclaving at 121°C for 15 min. For dark cultures, all plate cultures were sealed with parafilm and wrapped in aluminum foil. All cultures were maintained at a temperature of 28 ±2°C.Commercially important cassava cultivar KU50, which was provided from Genetic Resource Unit of CIAT, Cali, Colombia, was tested for its regeneration ability. The plant material was maintained as shoot cultures on MS2  for axillary bud enlargement. Six days later, enlarged axillary buds were isolated and transferred to plates containing MS2 supplemented with 12 g/l picloram (MSP). After 2 weeks in culture, somatic embryo structures (SEs) were produced on the axillary buds. Once the SE developed to torpedo stage, they were transferred to the same fresh medium (MSP) at 2 weeks interval for 6 weeks (3 cycles) for further growth. At each transfer, the soft non embryogenic tissues were removed. Four weeks later, the somatic embryos were transferred to MS2 supplemented with 0.5 mg/l BA and 0.5 mg/IBA for cotyledon production. To produce shoots and roots, the cotyledons were transferred to MS2 medium but without 2 μM CuSO4 and exposed to light. Roots and shoots developed within 4 weeks in culture.The regeneration protocol is according to the method described by Bull et al. , but witattL1 and attL2 recombination sites. The transformants were subjected to sequencing the targeted DNA fragment by using M13 primers and analyzed. The correct transformant containing the 527 bp fragment was subcloned in the sense orientation between the attB2 and attB1 recombination sites and in the antisense orientation between the attB1 and attB2 recombination sites on either ends of a 278 bp fragment of cat1-intron in the binary vector pEKH2IN2  by eLR clonase  recombination reaction. The product was transformed into TOP10 chemical competent cells  and selected on kanamycin-containing LB plates. Clones were verified by digestion with EcoRV. The plasmid, which contains marker genes for neomycin phosphotransferase (nptII) and hygromycin phosphotransferase (hpt), and the SLCMV gene being driven by cauliflower mosaic virus (CaMV) 35S promoter, was introduced into Agrobacterium tumefaciens strain EHA105 by triparental mating.A DNA fragment (527 bp) of SLCMV segment A (KC424490) was isolated by RT-PCR using a pair of Deng’s primers  Table 4Table 4. Agrobacterium tumefaciens strain EHA105 carrying the binary vector pEKH2IN2SLCMV was cultured overnight on a reciprocal shaker (120 cycles/min) at 28°C in 50 ml liquid LB medium containing 50 mg/l kanamycin and 25 mg/l chloramphenicol. The bacterial suspension was centrifuged and then resuspended to final density of OD600 = 0.5 in inoculation medium, which was liquid MS2 containing 200 μM acetosyringone and incubated at room temperature for 1 h before inoculation.Agrobacterium. These tissues were transferred to Erlenmeyer flasks containing Agrobacterium suspension for inoculation. The flasks were wrapped with aluminum foil and incubated on rotary shaker (R-20 min) at 80 cycles/min for 30 min. The tissues were retrieved by filtering, and placed on filter papers to remove excess bacteria. The SEs were then cultured onto plates containing MSP supplemented with 200 μM acetosyringone. The plates were wrapped with aluminum foil and co-cultivated at 24°C for 2 days and then exposed to light for another 2 days. Thereafter, the SEs were transferred to the MSP containing 20 mg/l meropenem and maintained at 28°C, dark for 1 week (pre-selection). After 1 week, the SEs were cultured on MSP containing 50 mg/l kanamycin and 20 mg/l meropenem for 2 weeks for maturation. If bacteria overgrowth was noted after co-cultivation, the tissues were washed three times with sterile distilled water containing 10 mg/l meropenem. For production of cotyledons, SEs were transferred to MS2 supplemented with 0.5 mg/l BA and 0.5 mg/l IBA and containing the same concentrations of antibiotics and exposed to light. Cotyledons were transferred to MS2 but without 2 μM CuSO4 for shoot and root development. Regenerated plantlets having shoots and roots were excised and cultured in selective rooting medium, which was MS medium containing 3 g/l gelrite and 100 mg/l kanamycin. Putative transgenic shoots formed roots in the medium, while non-transgenic shoots developed roots on the surface of the medium. Therefore, shoots which produced roots in the medium were selected and micropropagated in vitro for subsequent molecular analysis and transfer to soil.For axillary bud enlargement, nodal stem cuttings were cultured on MSB for 6 days. Thereafter, the enlarged axillary buds were isolated and cultured on MSP for 6 weeks with subcultures at 2 week-intervals for somatic embryo (SE) induction. The SEs were cycled again in the same medium for 4 weeks before being used for infection with in vitro plants using a modified cetyltrimethylammonium bromide (CTAB) protocol [nptII gene, the primers used were NPTII-5P and NPTII-3P  and probed with SLCMV PCR-DIG labelled probe. The probe DNA fragment was generated from plasmid DNA pEKH2IN2SLCMV and labelled by PCR using DIG-2’-deoxyuridine 5’-triphosphate (DIG-dUTP) in the sense and antisense direction according to the supplier’s instruction . Prehybridization (3 h) and hybridization (overnight) were carried out at 38°C and 42°C respectively, using high-SDS hybridization buffer containing 50% deionized formamide, 5 × SSC, 50 mM sodium phosphate (pH 7.0), 2% blocking solution, 0.1% N-lauroylsarcosine and 10% SDS. Post hybridization washes and detection using CDP star were performed according to the instruction manual of the DIG labelling and Detection System . For northern blot, 15 μg of the total RNA was denatured and separated on 1.5% agarose gel. Separated RNAs were transferred to nylon membrane, probed with a DNA probe of SLCMV gene and detected following the same procedure used in Southern blot analysis, with slight modification where necessary. Pre-hybridization (2 h), hybridization (overnight) and washing were performed at 50°C.Genomic DNAs were extracted from young leaves of protocol . Total Rprotocol  with sliprotocol . PCR ampNPTII-3P . For Sou8000) as described by Smith and Eamens [in vitro transcription of the SLCMV gene in the antisense and sense orientation using T7 RNA polymerase from DIG RNA labelling Kit SP6/T7, Roche Diagnostic GmbH, Boehringer Mannheim, Germany. Briefly, 10 μg of plasmid DNA extracted from pCR8/GW/TOPO, was linearized by digesting with ApaI at 37°C for 3 h. The linearized plasmid was purified via phenol/chloroform extraction and ethanol precipitation. Then, 1 μg of the purified template DNA was mixed with 12 μl of DMPC water, 2 μl of 10x NTP labelling mix, 2 μl of 10x Transcription buffer, 1 μl of Protector RNase inhibitor and 2 μl of RNA polymerase T7. The mixture was incubated at 37°C for 2 h and 2 μl of 0.2 M EDTA (pH8.0) was added to stop the reaction. In order to improve the signal, the probe was hydrolyzed with carbonate buffer (60 mM sodium carbonate and 40 mM sodium bicarbonate), and incubating at 60°C for 5 h [Total RNA was isolated as described above, and small RNAs were enriched from the total RNA by polyethylene glycol  to create wounds. The leaves were rinsed with 50 mM sodium phosphate buffer containing 0.4% sodium sulphite, pH 7.5. Then, SLCMV clone pSL7 was applied by rubbing each leaf several times back and forth using a cotton bud. Second, three days after the first inoculation, another young leaf was cut off at the node. The cut surface on the stem was rinsed with sodium phosphate buffer and the same concentration of viral clone was applied by rubbing several times. In parallel, mock control plants were inoculated with 50 mM sodium phosphate buffer only. The experiment was performed twice.Four week-old hardened transgenic and wild-type plants lines obtained from 3 generations of Symptom development was assessed every alternate day but the final classification was recorded at 45 days post inoculation (dpi). The number of leaves per plant showing mosaic symptoms was recorded at 45 and 60 dpi. Also, the plant height, number, length and weight of tubers were recorded at 60 dpi. Disease symptom severity on fully expanded leaves was recorded at 45 dpi on a scale of 0–3 . Disease indices were calculated using the method described by  as folloWhere n is the number of leaves in each grade (0–3) with respect to the symptom severity score. Plants with a disease index of 0% were considered as immune, those with a disease index <25% as having high levels of resistance (HR), those with a disease index of 25.1–50.0% as being moderately resistant (MR), those with a disease index of 50.1–75.0% as susceptible (S), and those with a disease index of 75.1–100% as highly susceptible (HS).Sixty days after infection, PCR and RT-PCR analyses were carried out to detect SLCMV cassava leaves. DNAs were extracted from new leaves that emerged from infected transgenic and wild-type plants using CTAB. Primers SLCMV-A-5P and SLCMV-A-3P  were useData on yield (length and weight of tubers) were subjected to analysis of variance (ANOVA) test using a completely randomized design (CRD). Means were separated by least significant difference (LSD) test at the 5% probability level. All computations were performed using SPSS 17.0 statistical package for Windows .


Cereal yellow dwarf virus-RPV were purified from infected oat tissue and analysed by MS. Two conserved residues, K147 and K181, in the virus coat protein, were confidently identified to contain epsilon-N-acetyl groups. While no functional data are available for K147, K181 lies within an interfacial region critical for virion assembly and stability. The signature immonium ion at m/z 126.0919 demonstrated the presence of N-acetyllysine, and the sequence fragment ions enabled an unambiguous assignment of the epsilon-N-acetyl modification on K181. We hypothesize that selection favours acetylation of K181 in a fraction of coat protein monomers to stabilize the capsid by promoting intermonomer salt bridge formation.Virions of the RPV strain of  Potato leafroll virus (PLRV) and the barley/cereal/maize yellow dwarf viruses, members of the Luteoviridae and referred to hereafter as luteovirids, are globally important viruses that infect most staple food crops and are model viruses for studies on the regulation of circulative plant virus transmission by aphid vectors  with peptide-N-glycosidase F was found to disrupt aphid transmission of the virus, suggesting N-linked glycosylation occurs on either the virus structural proteins  was purified from systemically infected oat tissue  in a two-step process  of 4.1e−05, an ion score of 44 and a mass measurement error of 0.68 p.p.m. No fragmentation was observed around the acetylated lysine residue, K147 , containing the acetylK181  in the datasets, indicating that these lysine residues exist in the capsid in both acetylated and positively charged (containing the ammonium ion) forms. To the best of our knowledge, these data are the first evidence of post-translational modifications of the luteovirid structural proteins and the structural proteins of any non-propagative plant virus. This finding is significant because virions are the vehicles for the movement of the virus genome in plant hosts and insect vectors  species PAV, PAS, MAV and GAV all have a serine at site K147, and Rose spring dwarf associated virus (RsDAV) and Pea enation mosaic virus (PEMV) have a threonine. These residues could be all modified in such a way as to make the site more or less charged . Acetylation of K181 may play a more critical role in luteovirid biology, as described below.To glean insights into the conservation of these lysine residues across multiple species of luteovirids, residues . The acein planta  technology. Mutations in the CP and RTP of PLRV, a polerovirus related to CYDV-RPV, can result in phenotypic defects in virus assembly, systemic infection, and aphid transmission . One of n planta . In otheWe hypothesize that the acetylation status of K181 regulates virion stability and protein–protein interactions at this critical KGNG interfacial region. For example, acetylation of K181 may serve to stabilize interactions between CP monomers, promoting a disorder-to-order transition that would have a major impact on protein topology in this region of the capsid. Not mutually exclusive to the potential impact on disorder in this region, lysine acetylation has been shown to promote stabilizing salt bridges in other higher-order polymeric proteins (Luteoviridae except PEMV (data not shown). Conservation across all these virus species suggests selection pressure on N183 deamidation and for the virus to maintain some flexibility to determine the charge at this site (it is intriguing to note that selection has not produced any luteovirids that simply encode an aspartic acid residue at that position).To provide further support for this hypothesis, residues in this region would have to be available for salt bridge formation. In RPV, the peptide that we found acetylated was also measured to be deamidated at N183 , which rm/z 10 wide selected ion monitoring (SIM) acquisition, followed by collision induced dissociation using the HCD cell on an Orbitrap Velos mass spectrometer and subsequent mass analysis in the Orbitrap analyser. The latter provided high mass accuracy and high-resolution measurement of fragment ions. As this peptide contains the NG sequence which can rapidly deamidate to DG (m/z 816–826) of the amidated and deamidated precursor ions are shown in AcKGDGASSVAGSFK.I, is shown in N-acetyllysine immonium ion at m/z 126.090. These data unambiguously assign an acetylation site to K181 (To unambiguously assign the deamidation of N183 and acetylation on K181, the sample was reanalysed in such a manner as to specifically target charge and acetylation state variants of this peptide. Each precursor mass was subjected to an  to K181 .Schizaphis graminum, a vector species for CYDV-RPV, is an acetyl-CoA ligase (S. graminum CoA ligase may play a central role in determining the availability of acetyl groups to viral or aphid protein substrates and that regulation of the acetylation helps to determine variation in vectoring capacity in this species. These new data suggest that further studies on the role of acetylation of luteovirids in virus assembly, host systemic infection and circulative transmission are pertinent.Protein acetylation has received widespread attention in animal biology . Acetyl-


Orthobunyavirus within the family Bunyaviridae belonging to the Simbu serogroup. Schmallenberg virus infects ruminants and has since its discovery in the autumn 2011 been detected/spread to large parts of Europe. Most bunyaviruses are arboviruses, and SBV has been detected in biting midges in different European countries, suggesting that they may play a role in the transmission of the virus. It is not known how SBV was introduced to Europe and if SBV is present in countries outside of Europe. Thus, in this study, we conducted a serological screening for SBV antibodies in cattle (no. 79), sheep (no. 145) and goat (no. 141) in the Zambezia Province in Mozambique during September 2013. The results show a high percentage of antibody-positive animals. All farms tested had seropositive animals; cattle displayed the highest prevalence with 100% positive animals. Sheep and goat also displayed high number of positive animals with a 43–97% and 72–100% within-herd seroprevalence, respectively. This initial serological screening suggests that SBV is present on the African continent. However, cross-reactivity with other members of the Simbu serogroup cannot be ruled out, and further studies are needed to identify and characterize the virus responsible for the antibody-positive results.Schmallenberg virus (SBV) is a novel  Orthobunyavirus belonging to the Simbu serogroup, and the virus was provisionally named Schmallenberg virus (SBV) after the town in Germany where the virus was first identified  in Denmark  – cattle, sheep and goat in the Zambezia Province in Mozambique  according to the manufacturer's instructions with undiluted serum. Positive serum samples were diluted 8× to confirm positivity. The choice of an 8× dilution was based on the analytic sensitivity tests performed by ID-vet . Optical reading was done at 450 nm, and the cut-off values suggested by the manufacturer were used, that is, S/N ≤ 40% were considered positive, 40-% < S/N ≤ 50% doubtful and S/N > 50% were considered negative.All farms investigated had animals that tested positive for SBV antibodies, as summarized in TableAlthough the results from this screening study suggest that SBV might be present in Mozambique, cross-reactivity with another member of the Simbu serogroup cannot be ruled out. Serological cross-reactions between members of the group have been previously described (Kinney and Calisher,