To analyze the impact of multimorbidity (MM) on health care costs taking into account data heterogeneity.Data come from a multicenter prospective cohort study of 1,050 randomly selected primary care patients aged 65 to 85 years suffering from MM in Germany. MM was defined as co-occurrence of ≥3 conditions from a list of 29 chronic diseases. A conditional inference tree (CTREE) algorithm was used to detect the underlying structure and most influential variables on costs of inpatient care, outpatient care, medications as well as formal and informal nursing care.Irrespective of the number and combination of co-morbidities, a limited number of factors influential on costs were detected. Parkinson’s disease (PD) and cardiac insufficiency (CI) were the most influential variables for total costs. Compared to patients not suffering from any of the two conditions, PD increases predicted mean total costs 3.5-fold to approximately € 11,000 per 6 months, and CI two-fold to approximately € 6,100. The high total costs of PD are largely due to costs of nursing care. Costs of inpatient care were significantly influenced by cerebral ischemia/chronic stroke, whereas medication costs were associated with COPD, insomnia, PD and Diabetes. Except for costs of nursing care, socio-demographic variables did not significantly influence costs.Irrespective of any combination and number of co-occurring diseases, PD and CI appear to be most influential on total health care costs in elderly patients with MM, and only a limited number of factors significantly influenced cost.ISRCTN89818205Current Controlled Trials  The concept of multimorbidity  relates to the coexistence of several chronic diseases in an individual . EspeciaIndividuals with multiple chronic conditions consume a disproportionally large share of total health services. In a systematic literature review, Lehnert et al.  found amChi-square automatic interaction detectors (CHAID) and conditional inference trees (CTREE) have been developed. As a limitation, CHAID requires categorical data and responses, while CTREE can deal with arbitrary scaled variables ) We adopted a societal perspective; therefore all resources and services used were recorded, regardless of whether they were covered by health or nursing insurance or paid for out-of-pocket. The cost categories analyzed in this study are direct costs of illness arising from the use of resources. We did not evaluate indirect costs due to lost productivity because of the advanced age of the subjects. Healthcare costs were calculated for a 6-month period, multiplying resource use by two in sections which covered a 3-month period. Costs were calculated from resource use as recorded in the questionnaire by means of unit costs. Resource categories and sources of unit costs are listed in Table Cost were calculated in € at 2009 price levels. Unit costs that were unavailable at year 2009 values were inflated or deflated to year 2009 price levels by means of the consumer price index .For statistical analysis, we categorized cost data as follows: 1) Costs of inpatient care comprising inpatient treatment in general hospitals, specialized psychiatric and neurological hospitals or rehabilitation clinics; 2) costs of outpatient care comprising outpatient physician treatment, other outpatient treatment, and medical supplies and dental prostheses; 3) costs of medication comprising pharmaceuticals; 4) costs of nursing care comprising nursing home care, professional home care and informal care.Missing values in items of the resource use questionnaire  were imputed using the means of the observed data for the respective items . Dosage of medication was an exception, however, since medications and their dosage were too varied interindividually for mean imputation to be possible. Therefore costs for medication with missing values for dosage were calculated using a conservative rule, whereby the pharmacy retail price of one package of the drug per 3 months was applied. Missing values in items of the standard MultiCare assessment battery were imputed using the hot deck method, in which missing values are replaced using observed values from a responding unit that is as similar as possible to the non-responding unit ,24. The 2). A detailed list of the diseases taken into account is provided in the Results section. Additionally, female sex  and private health insurance  were added as dichotomous variables. Education was included as a categorical variable, taking low education as reference. Besides this, age and the logarithmic (log) transformed income were included as continuous covariates. Log-transformation was used to achieve a linear relationship of predictors and outcomes. No additional distributional assumptions concerning the error terms or outcomes were made.We used a conditional model based on a supervised learning tree-algorithm in order to visualize a hierarchical data partition and to detect the underlying structure and most influential variables on total costs and on costs of the four different cost sectors  separately. As covariates we used binary variables for those 41 of the 46 diseases which had a prevalence rate of ≥1% in the sample. In addition, we included a binary variable for obesity  attempt to discriminate data into homogenous subsets. Thus, node  xi (see ). As spl xi (see ).Y is defined as conditional on a function g of a set of k arbitrarily scaled covariates X as f).To overcome these disadvantages and as a superior approach to CART, we used in a first step a non-parametric conditional inference tree (CTREE)-algorithm predicated on recursive binary partitioning embedded in the framework of permutation tests introduced by Strasser and Weber . Thus thLn based on a random selection of n i.i.d. observations was used to fit the tree-structured regression model. A vector of dichotomous case weights w representing each node was used to create the disjunctive subsets wleft,i = wiI(Xi ∈ A) and wright,i = wiI(Xi ∉ A) with I(⋅) as indicator function. A discrepancy measure of the formA learning sample A with h(⋅) as influence function. vec is the vec-operator and (⋅)T the transpose.was used to establish a two sample statistic for all possible subsets of H0 : f(Y|Xj) = f(Y) was tested on a pre-specified α = 0.05 level of significance. To incorporate different scaled covariates, a maximization of the test statistic based on the conditional mean and conditional variance over all possible subsets was established. In case of acceptance, the tree-algorithm was interrupted and no further data-split was performed. Otherwise the covariate Xj with the strongest influence on Y was selected as node, and the null hypothesis was tested in each subset of the tree. This approach guarantees the optimal sized tree is grown )To verify our results and detected splitting variables, variable importance scores were calculated based on conditional random forests for each cost sector.When comparing CTREE results to CART, in all cost sectors CART lead to more splits on the one hand, while pruning lead to no suitable trees. On the other hand, CART verified the findings of CTREE by showing identical nodes and/or grown tree structures. Furthermore, CTREE lead to theoretically reasonable splitting variables. Based on these findings as well as on calculated error terms and results achieved from the conditional random forests, our study emphasizes the superiority of the CTREE algorithm.Our statistical analysis was based on a pre-imputed master data set provided by the data management of the MultiCare study group which had used the hot deck method and conditional means for imputation of missing values. Although we are aware of benefits resulting from multiple imputation algorithms, we agreed on using the master data set for the sake of consistency and because the proportion of missing values in the variables used for our analysis was very small. Nevertheless, tree-based algorithms can handle complete data as well as missing data usually assuming Missing Completely At Random (MCAR).Patients with response-limitations due to medical reasons  as well as nursing home residents were excluded from the study sample. Therefore the impact of respective chronic conditions on health care costs could not be analyzed. Yet conditions associated with response-difficulties may strongly influence health care costs. For example, dementia is a very important and prevalent condition in the elderly associated with high health care costs. Dementia is often present in late stages of different diseases, such as PD, CI, chronic stroke and others. Future studies analyzing the impact of multimorbidity on health care costs should therefore consider surrogate responders for data collection in such response-limiting conditions.In elderly patients suffering from multiple chronic conditions, PD and cardiac insufficiency appear to be the chronic diseases most influential on total health care costs irrespective of the number and combination of other co-existing chronic conditions. Irrespective of any combination and number of co-occurring diseases, costs are significantly influenced by only a limited number of factors.AID: Automatic interaction detection; CART: Classification and regression trees; CCI: Cardiac insufficiency; CHAID: Chi-square automatic interaction detectors; COPD: Chronic obstructive pulmonary disease; CTREE: Conditional inference trees; GP: General practitioner; PAOD: Peripheral artery occlusive disease; PD: Parkinson's disease.The authors declare that they have no competing interests.HHK, ME and HL conceived and designed the analysis. BW and GS prepared the data for analysis. ME analyzed the data. HHK drafted the manuscript. HB, AF, JG, HHK, HL, WM, KM, SRH, IS, SW, HvdB and MS participated in study design and implementation. All authors read and approved the final manuscript.* Principal investigatorsHendrik van den Bussche and Martin SchererThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/13/219/prepub


O,O′-diisopropyl (3-(L-1-(benzylamino)-1-oxo-3- phenylpropan-2-yl)thioureido)(phenyl)methyl phosphonate (2009104) was studied for its activity on tobacco mosaic virus (TMV). It showed good curative activity in vivo and the curative activity at 500 μg/mL was found to be 53.3%. In vivo treatment with the control agent Ningnanmycin at 500 μg/mL resulted in 51.2% inhibition and curative inhibition rates respectively. Dot-ELISA test was employed to verify the efficacy of activity of compound 200910 for anti-TMV activity. The mechanism of action of compound 2009104 to resist TMV was also studied. The results showed that the resistance enzymes PAL, POD, SOD activity and chlorophyll content after TMV inoculation K326 (Nicotiana tabacum K326) of tobacco plants followed by treatment with compound 2009104 were significantly enhanced. The study of the effect of compound 2009104 on TMV capsid protein (CP) showed that it inhibited the polymerization process of TMV-CP in vitro.Using half-leaf method  Plant virus is a type of plant disease, known as “plants cancer”. In recent years, the impact of climate anomalies and the areas of crops affected by plant virus disease are on the rise resulting in tremendous economic losses in the world in general and China’s agriculture in particular. Plant virus is an obligate parasite in plant cells. Even in the cell nucleus, nucleic acid (DNA or RNA) replication occurs and coat protein is synthesized in the host cell. Since plants do not have the same immune system as animals, and the virus is absolutely dependent on their hosts, to control plant virus disease and develop plant virus inhibitors appears to be extremely difficult. Tobacco mosaic virus (TMV) disease is an important class of common disease occurring in tobacco plants growing all over the world. This virus seriously affects the quality and yield of tobacco. It is estimated that each year an economic loss worth one billion U.S. dollars  is encouetc. In recent years, certain thiourea inhibitors of plant viruses have also aroused widespread interest in both biological and chemical sectors [O,O′-diisopropyl (3-(L-1- (benzylamino)-1-oxo-3-phenylpropan-2-yl)thioureido) (phenyl)-methylphosphonate (2009104) (molecular structure shown in 326 (Nicotiana tabacum K326) of tobacco plants indicated that the induced resistance was significantly related to enhancement of activity of enzymes PAL, POD, SOD and decrease of chlorophyll content. The influence of TMV-CP showed that the compound 2009104 inhibited its polymerization process in vitro.Thiourea derivatives have a wide range of biological activity on the pesticides which include plant growth regulatory , herbici sectors . In ordeDevelopment of antiviral agents acting in plants seems to be far more economic when compared to similar research cost for anti-viral agents that act on animals. Nevertheless, mechanistic studies with anti-viral agents for plants require more painstaking research and extensive screening of drugs. The agent being screened can, not only inhibit the proliferation of virus, but also harm the host or even improve other symptoms in the host making the current study a truly challenging one. Mode of action of antiviral agents concerns mainly on the effect of agents on susceptible plants. These include, but are not limited to, whether the agents are susceptible to regulate endogenous hormones; whether the use of biological and chemical factors of treatment plants has any role in viral response of the plants or development of anti-viral factors for local or systemic resistance. To systematically study the mechanism of action with compound 2009104, tests were conducted with tobacco as the main plant. A systematic study on the role of compound 2009104 offering resistance to TMV by using physiology, biochemistry and molecular biology experiment was carried out.in vivo; infection curative inhibition rate was 53.3% in vivo, the protective inhibition rate was 58.9%. The results showed that compound 2009104 possessed better inhibitory activity against TMV.The results of curative, protective and inactivation efficacies of compound 2009104 at a dose of 500 ug/mL against TMV are given in in vivo concentrations, there are large differences in the control effect of each treatment. This may be seen from the view point of the mode of action of compound 2009104 which is not single, but multifaceted. It incapicates the virus from invading outside the body and can also repress the virus to breed within the body, while playing a protective role on the host plants. However, the exact pathway through which these roles are performed is still unclear and needs further study.The results fully demonstrated that the effect of compound 2009104 for viral replication and proliferation was strongly inhibited, but its mode of action and role of the locus are not clear. The inhibitory effect of the compound on the performance of viral RNA in blocking viral replication or protein synthesis, or the virus shell strip or assembly process, needs further investigation. It is worth mentioning that, although with different pharmaceutical treatments, compound 2009104 can reduce TMV According to the results shown in It can be seen from It can be seen from It can again be seen from Photosynthesis is a special, and the most basic, life process of green plants providing them necessary growth and energy. Chlorophyll is the photosynthetic organelle of plants whose content is closely related to photosynthesis, extent of viral infection in plants leading to proliferation, expansion and destruction of the plant chloroplasts and factors retarding the synthesis of chlorophyll causing leaf chlorosis and mosaic. Studies have shown that the photosynthetic pigment content is significantly reduced in TMV infected leaves and can lead to severe influenza illness . It can The experimental results of TMV-CP extracts identified by SDS-PAGE electrophoresis, are shown in Plant virus coat protein plays a very important role throughout the life of the virus. Antiviral agents acting on the virus coat protein can interfere with any one of the steps of virus reproduction in the plant. Previous studies have shown that infection of free nucleic acid of TMV to host, accounts only up to 1% of a complete virus particle in terms of infection activity, while the viral nucleic acid which is not assembled cannot effectively spread over long distances in the host body. Therefore, the virus nucleic acid interaction with the viral coat protein assembled into a complete virus particle is an important part of virus to maintain normal infection activity and long-distance transmission in the host body. As a first step in virus assembly, viral coat protein must be a certain type of polymer that can further fit to the assembly of viral RNA to make a complete virus particle. If the antiviral drugs interfere with this process, there may be an impact on subsequent assembly steps. Consequently, the virus cannot spread in the host body thus inhibiting the virus infection activity. This eventually leads to the desired antiviral effect . Therefoin vitro polymerization process. TMV-CP combines the compound 2009104 in its UV absorption at 320 nm. With the increase in temperature, the trend in the change of value was far less pronounced than the control. According to Smith, KM and other researchers, the observed absorbance of TMV-CP at 320 nm is directly proportional to the degree of polymerization with itself, whereas the degree of change of TMV-CP in vitro polymerization is proportional to the temperature. Thus, it may be derived that the compound 2009104 possesses, to a certain extent, an inhibition effect of TMV-CP in vitro polymerization, in comparison with TMV-CP treated with ribavirin. The absorbance at 320 nm of TMV-CP treated by compound 2009104 displayed a moderate upward trend which is indicative of the fact that the efficacy of the compound 2009104 to TMV-CP was less than the control agent ribavirin.It can be seen from 326 spare.In general, tobacco mosaic virus (TMV) was stored in the laboratory after purification of tobacco propagation KNicotiana glutinosa), dry spot for the TMV host and seeds were purchased from the Institute of Chinese Academy of Agricultural Sciences tobacco. General Tobacco K326 (Nicotiana tabacum K326) for the greenhouse propagation and the seeds were purchased from the Institute of Chinese Academy of Agricultural Sciences tobacco.Heart leaf tobacco -1-oxo-3-phenylpropan-2-yl)thioureido) (phenyl)methyl phosphonate (2009104) was provided by our laboratory; 2% Ningnanmycin AS was provided by Heilongjiang Qiangr Biochemical Technology Development Co., Ltd.The 0.45 μm nitrocellulose membrane was purchased from PALL Gelman Laboratory Co., Ltd. While Horseradish peroxidase labeled goat anti-rabbit secondary antibody, NBT and BCIP was procured from Beijing Solarbio Science & Technology Co., Ltd. and TMV antibody was supplied by Agdia Co., Ltd.Gooding method was used for the purification of test virus , stored Five to six leaf spots of blight host of similar growth were selected. First, sap fraction treated leaves inoculated with the virus were painted, applied 500 μg/mL of the test compound and Ningnanmycin in the left lobe after 2 h, right half of the leaf was treated with the solvent as control. Three samples from each treatment group  were repeated thrice and investigated the number of host dry spots after the spots were completely dry. Finally, lesion inhibition was calculated.Five to six leaf spots of blight host of similar growth were selected; a coating was gently applied with a brush in the left lobe at 500 μg/mL for the test compound and Ningnanmycin. The solvent coating was applied in the right lobe as control. The handing of virus inoculated leaf of sap fraction was done after 24 h, each dealing with three  and being repeated 3 times. The number of host dry spots was investigated after the spots were completely dry, and then lesion inhibition was calculated.Five to six leaf spots of blight host of similar growth were selected; a dose of 500 μg/mL for each test compound and Ningnanmycin was mixed with the virus sap at 1:1 (v/v) of passivized sample for 0.5 h. Mechanical inoculation was used to handle left leaves, the virus sap and solvent 1:1 (v/v) fraction in the right half of the leaves were inoculated in each of the three treatment groups  and repeated 3 times. The number of host dry spots was investigated after the spots were completely dry, and then lesion inhibition was calculated.By modifying the work of Wu  and otheBy modifying the work of Xue  and otheL-phenylalanine as the substrate to 3 mL 0.05 M pH 8.8 by sodium borate buffer and 0.5 mL enzyme solution as the blank control. The instrument was set at zero, UV spectrophotometer was operated at 290 nm and OD1 value was measured. The reaction was conducted at 40 °C on a water bath for 5 h, quenched on an ice bath to stop the reaction. At 290 nm, OD2 value was measured by subtracting OD1 as the net value added for activity. An increase of 0.01 per hour to OD290 amount of enzyme activity as a unit of U was obtained, repeated 3 times for each sample.By modifying the work of Zhu , the cruThe work of Wang  was modi2O2) solution and mixed evenly to start enzyme reaction with the same volume of distilled water instead of hydrogen peroxide as the blank control, as a zero instrument. After being reacted for 5 minutes, the OD values were measured at 470 nm, per mg protein per minute with the added value of OD470 as activity. The experiment was repeated 3 times with each sample.In accordance with the work reported by Wang , 5 times326 was selected. At first, the 7th leaf was identified and a brush was dipped in viral juice. The whole leaf was first inoculated with the virus followed by with a sterile water rinse. Leaves were dried and coated for pharmaceutical facilities. The samples were measured on every second day for their chlorophyll contents. Determination of chlorophyll content: the test samples were cut, weighed and put into 5 mL of 2:1 mixture of acetone and ethanol in the refrigerator at 4 ×C, the dark extraction was carried out for 24 h. The chlorophyll after extraction was placed in a 1 cm thick cuvette, with a 2:1 mixture as reference. The UV spectrophotometer was employed at 645 nm and 663 nm to measure absorbance, chlorophyll content (mg g−1 FW) with Ca + Cb.According to the work conducted by Peng , similar645 and A663 are the absorbance values at the corresponding wavelengths, V is the volume of extraction, W is the leaf fresh weight and chlorophyll content is described in mg·g−1.In the above equation, APreparation of TMV-CP was executed by dialysis, as described by Fu .By following the methods of Jiang  and otheCompound 2009104 is an antiviral agent, has better control effect against TMV in a variety of plant viruses. It can be seen that compound 2009104 plays a direct role in virus particles. It can eradicate the TMV infection thereby protecting tobacco leaves from the control experiments on the dry spots of host of the compound 2009104. In addition to showing good protective and curative effect, curative effect better than antiviral agent Ningnanmycin and protective and inhibition effects close to Ningnanmycin were noted.in vivo. The best inoculation was obtained after compound 2009104 was mixed with TMV for 0.5 h. On the other hand, inhibition rate of 84.92%, the lowest control effect with compound 2009104 was found after being inoculated for 2 h.Biological method  and immunological method (Dot-ELISA) were used in this study to determine the virus concentration in the host treated by 2009104. It may be observed from the results that irrespective of the treatment, compound 2009104 could reduce tobacco mosaic virus concentration Most of the preparation and application of plant virus inhibitors generally deal with how resistance to plant disease and its corresponding activity are developed. The present work investigates the role of compound 2009104 towards host plant defense enzymes. The effects were measured by colorimetry and the results are described as follows: Treatment of tobacco leaves infected with TMV by 2009104 showed enhancement of PAL, POD, SOD and other related activities of defensive enzymes to different extents. The increase in PAL, POD and SOD activities in tobacco leaves brought about by the action of the compound enhances resistance against plant disease. The results showed that tobacco leaves treated by compound 2009104 exhibit a variety of physiological and biochemical indicators which are comparable to those of healthy plants. Furthermore, studies were also conducted to understand the effect of compound 2009104 in terms of host chlorophyll content and the outcomes are described below: chlorophyll content was significantly lowered in TMV infected tobacco leaves. Nevertheless, after being treated with compound 2009104, chlorophyll content of the leaves started to rise but remained lower than the control throughout the study. The results reflect that treatment of tobacco leaves by 2009104 can considerably reduce the amount of damage caused by the virus and improve the chlorophyll content, thereby enhancing the host resistance.in vitro. The decrease in absorbance may be attributed to the dissociation of capsid protein oligomers by the interaction of compound 2009104 with TMV-CP during the polymerization in vitro. Therefore, it may be presumed that compound 2009104 can interfere with the TMV-CP polymerization in vitro thereby not only affecting virus assembly but also restricting long-distance transmission of the virus in the host and the extent of viral infection in vivo.The study showed that after treatment of the leaves by compound 2009104, the absorbance values of TMV-CP at different temperatures were much lower than the control suggesting that 2009104 may strongly interfere with polymerization process of TMV-CP The work demonstrates the first use of Dot-ELISA method to validate high antiviral activity of compound 2009104 by half-leaf method. The results of verification measured by the half-leaf method are fairly consistent. Compound 2009104 has been found to induce resistance against plant disease for the first time. Another noticeable discovery with this compound was that it could interfere with the assembly process of TMV-CP.


During outbreaks of infectious diseases or in cases of severely ill patients, it is imperative to identify the causative agent. This report describes several events in which virus isolation and identification by electron microscopy were critical to initial recognition of the etiologic agent, which was further analyzed by additional laboratory diagnostic assays. Examples include severe acute respiratory syndrome coronavirus, and Nipah, lymphocytic choriomeningitis, West Nile, Cache Valley, and Heartland viruses. These cases illustrate the importance of the techniques of cell culture and electron microscopy in pathogen identification and recognition of emerging diseases. Filoviridae, now known as Zaire ebolavirus (Paramyxoviridae by EM (Bunyaviridae (Thin section and negative stain electron microscopy (EM) examination of viruses grown in cultured cells have been instrumental in determining an etiologic agent in numerous disease outbreaks caused by previously unknown viruses. Many examples have been reported. In 1976, EM of cell culture isolates identified the causative virus of an outbreak of hemorrhagic fever in Zaire as a member of the family Inoculation of patient specimens onto cultured cells or into laboratory animals enables biologic amplification of virus particles to levels where they can be detected by EM and identified to a virus family because, with a few exceptions  enabled initial recognition and identification of a cause of the viral illness.Of the variety of tissue culture cells available, many are maintained in minimal essential medium at 37°C. Once cells have become confluent, they can be inoculated with suspensions of ground tissues , incubated for 1 hour, and grown until there is an ≈3+ cytopathic effect. Cells are then removed from the growth vessel by scraping or with glass beads, rinsed with 0.1 mol/L phosphate buffer, centrifuged, and fixed in buffered 2.5% glutaraldehyde for 1 hour.Specimens are postfixed in 1% osmium tetroxide, en bloc stained with 4% uranyl acetate, dehydrated through a graded series of alcohol and propylene oxide, and embedded in a mixture of Epon substitute and Araldite. Thin sections are stained with 4% uranyl acetate and Reynolds’s lead citrate.Rhinolophus sinicus) because SARS corona-like viruses were identified and characterized in these animals  , an arenavirus transmitted by rodents  by ELISA and identified as Cache Valley virus by PCR and sequencing  and at CDC from blood, serum, and cerebrospinal fluid, and EM examination recognized a virus belonging to the family Ehrlichia sp. was initially suspected, and leukocytes from the patients were inoculated onto DH82 cells, a canine monocyte cell line. When cytologic changes were seen in the culture, cells were processed for EM examination. Rather than a bacteria, a bunyavirus was recognized. It was identified as a new member of the genus Phlebovirus by deep sequencing and is now known as Heartland virus (In a recent second instance, a bunyavirus was isolated from 2 patients in northwestern Missouri, USA, who had a history of tick bites. Both patients had fever, fatigue, diarrhea, thrombocytopenia, and leukopenia, but recovered from their illnesses and were released after 10–12 days of hospitalization. An Virus particles in bunyavirus-infected cells bud upon smooth cytoplasmic membranes and are found in vesicles and also extracellularly (For decades, the combination of the classical techniques of virus isolation in tissue culture and examination by EM has been critical in detection of previously unrecognized viruses. Cell culture is a fundamental procedure that can be accomplished in most hospital microbiology laboratories and should be considered if an infectious viral agent is suspected. Although some examples provided in this report were handled in biosafety level 3 laboratories at CDC, other examples were handled as routine microbiological isolations in hospitals or public health departments equipped to perform routine virus isolation.Other useful laboratory methods for diagnosis of an unknown virus include serologic testing; IFA, histopathologic, and IHC assays; PCR; and sequencing. Metagenomics with deep sequencing is a recent advancement of a molecular technique that enables genomic analysis of microorganisms without the need to isolate and culture pathogens. High-throughput sequencing uses random amplified DNA products and compares the obtained product sequences with available extensive banks of sequences for final identification of the agent detected. Because random primers are used to nonspecifically amplify templates for sequencing, there is no need for prior knowledge of the suspected target. This technology is advancing rapidly and improvements in the field will undoubtedly solidify its use in the field of pathogen discovery. Previously unknown viruses that have been recognized by using this technique include Schmallenberg virus (As molecular diagnostic techniques progress in scope and magnitude, it is critical to retain and use classical techniques, including cell culture and EM, that complement the advances in molecular methods. There is a continuing need to train younger scientists in these traditional methods to maintain an underlying expertise. In particular, with EM, the electron microscopist needs to be able to differentiate between infectious agents and artifacts or look-alike structures. The combination of cell culture and EM is an unbiased approach to identification of a previously unrecognized pathogen. Similarly, unbiased thinking and collaboration among clinicians, epidemiologists, microbiologists, and laboratorians who use different technologies are critical for successful investigations of diseases of unknown origin.


Pandemic (H1N1) 2009 influenza virus spread throughout the world since most people did not have immunity against the virus. In the post pandemic phase when many humans might possess immunity against the pandemic virus, one of the concerns is infection in immunocompromised people. Therefore, we used an immunosuppressed macaque model to examine pathogenicity of the pandemic (H1N1) 2009 virus under an immunocompromised condition. The virus in nasal samples of immunosuppressed macaques infected with the pandemic (H1N1) 2009 virus was detected longer after infection than was the virus in nasal samples of immunocompetent macaques. As expected, not only virus amounts but also virus propagation sites in the immunosuppressed macaques were larger than those in lungs of the immunocompetent macaques when they were infected with the pandemic virus. Immunosuppressed macaques possessed low levels of immune cells producing cytokines and chemokines, but levels of inflammatory cytokines/chemokine interleukin (IL)-6, IL-18, and monocyte chemotactic protein (MCP)-1 in lungs of the immunosuppressed macaques were higher than those in lungs of the immunocompetent macaques, though the differences were not statistically significant. Therefore, under an immunosuppressive condition, the pandemic influenza (H1N1) 2009 virus might cause more severe morbidity with high cytokine/chemokine production by the host innate immune system than that seen in macaques under the immunocompetent condition. Therefore, antigenicity of the pandemic (H1N1) 2009 virus seems to have been maintained even after it became a seasonal influenza virus. Since many people might possess immunity against the pandemic strain due to infection and vaccination after 2009, we need to pay further attention to immunocompromised people as well as elderly and young people who have low levels of immune responses against the virus.A pandemic H1N1 influenza virus emerged and spread throughout the world in 2009 . After tex.html) , the panex.html)  (http://microsporidia infection might contribute to morbidity [Staphylococcus [Non-human primates have been used to extrapolate pathogenicity of various pathogens in humans. We reported that the pandemic (H1N1) 2009 virus propagated in the lungs of immunologically naïve macaques more vigorously than did the seasonal Russian flu virus [orbidity  as repororbidity . Irradiaococcus  as was rococcus . After tococcus  as reporococcus ,18. In aococcus  as was hococcus . TherefoTo reveal the pathogenicity of influenza virus in immunocompromised hosts compared to that in immunocompetent hosts, we used an immunosuppressed macaque model and examined symptoms and virus propagation. We also compared pathogenicity of pandemic (H1N1) 2009 influenza virus to that of the seasonal H1N1 influenza virus isolated in 2007 (Russian flu virus) in immunosuppressed macaques. Our immunosuppressive protocol decreased the concentration of white blood cells, especially lymphocytes in peripheral blood, and inhibited interleukin-2 (IL-2) responses of lymphocytes. Under the immunosuppressed condition, propagation of pandemic (H1N1) 2009 and Russian flu viruses in the respiratory tract was prolonged compared to that under the immunocompetent condition. Furthermore, Russian flu virus was detected in the lungs of immunosuppressed macaques but not in the lungs of immunocompetent macaques. Even under the immunocompromised condition, levels of cytokine production 7 days after infection in the lungs of immunosuppressed macaques were higher than those in the lungs of immunocompetent macaques. These results suggest that immunosuppressive treatment to inhibit inflammation might not necessarily ameliorate hypercytokinemia and symptoms in influenza virus infection.To establish a protocol for suppressing the immune system in cynomolgus macaques, we firstly counted white blood cell numbers in blood as a biomarker for effective immunosuppression . According to previous studies -25, we c+ cells, was decreased instead of increase of granulocyte ratios and the percentage of lymphocytes was recovered on day 10 (3 days after the end of treatment)  were decreased on day 6 , monocytes (R2), and granulocytes (R3) were identified on the basis of forward and side scatters . In the eatment) . In the on day 6 . The decon day 6 .Since CA inhibits the function of calcineurin, resulting in induction of anergy in T lymphocytes ,27, we mTo examine the pathogenicity of influenza virus in immunosuppressed macaques, we administered CP and CA in the high dose regimen as determined above  and thenThe virus was detected in nasal samples of all three macaques without immunosuppression challenged with seasonal virus YOK91 until up to day 5 after infection, whereas the virus was detected in nasal samples of two immunosuppressed macaques  until day 7 and in tracheal and bronchial samples until day 6 in one of the immunosuppressed macaques (Y5) . A compaWe calculated the areas under the virus titer curves (virus titer AUCs) to compare overall virus propagation. Significant differences on average virus titer AUCs in nasal, tracheal, and bronchial samples were observed between the macaques inoculated with YOK91 and those inoculated with NRT1 without immunosuppression .10TCID50/g), ileum (1.67 log10TCID50/g), colon (1.67 log10TCID50/g), and urinary bladder (1.67 log10TCID50/g) samples in the N4 macaque, and colon (1.67 log10TCID50/g) in the N6 macaque when we collected two pieces of tissue samples from each organ. The presence of virus was confirmed by immunohistochemical staining of nucleoprotein (NP) in a small number of type I and type II alveolar epithelial cells in the lung of an immunosuppressed macaque infected with YOK1 (Y4), but not in the lung of an immunocompetent macaque infected with YOK1 (Y3)  . NP was OK1 (Y3) , whereasOK1 (Y3) . The numOK1 (Y3) . These rWe examined inflammatory responses in the macaques with or without immunosuppression after infection. Cytokine production was examined in lung tissues excised 7 days after virus infection . Levels We histologically examined lung tissues 7 days after infection. Without immunosuppression, congestion in lungs of the macaques infected with NRT1 and YOK91 was macroscopically observed . MicroscIn the present study, we revealed that the pandemic (H1N1) 2009 virus and the seasonal Russian flu virus propagated in immunosuppressed macaques and that inflammatory cytokines and chemokines were produced in lungs of macaques treated with CP and CA. The Russian flu virus was detected not only in the upper airway but also in the lower airway under the immunosuppressed condition. The pandemic (H1N1) 2009 virus was detected in both the upper and lower airways without immunosuppression, and higher virus titers in the lungs were detected in immunosuppressed macaques than in immunocompetent macaques. In addition, the pandemic (H1N1) 2009 virus was detected in non-respiratory organs of immunosuppressed macaques. These results indicated that a high level of virus propagation inducing cytokine production was related to severe morbidity in immunosuppressed macaques, that immunosuppression allowed the virus to spread to organs other than the main propagation sites in immunocompetent macaques and that non-highly pathogenic influenza virus systemically replicated under the immunocompromised condition.In immunosuppressed macaques infected with either virus, infiltration of leukocytes was minimal in the lungs and congestive edema was observed in alveoli probably due to cytokines and chemokines. Therefore, we speculated that virus in the lungs of immunosuppressed macaques might induce inflammatory cytokine/chemokine production by non-leukocytes, i.e., alveolar epithelial cells or stromal cells -31, thouAlthough virus titers in nasal samples of the immunosuppressed macaques on day 7 after pandemic (H1N1) 2009 virus inoculation were significantly higher than those in nasal samples of the immunocompetent macaques, the virus titer AUCs in the trachea and bronchus of macaques infected with the pandemic virus under the immunosuppressive condition were lower than those in the trachea and bronchus of macaques without immunosuppression as shown in CA and CP are widely used for prevention of rejection in organ transplantations and treatments for cancers . In patiBased on these features, the immunosuppressed macaque model would be also useful for evaluating the emergence of drug-resistant strains during treatment, such strains having been reported in immunocompromised patients ,49. Sinc2, suggesting that fever dependent on toll-like receptor (TLR) signals, e.g. TLR7, having been shown to be a receptor for virus RNA [Inflammatory responses observed in immunosuppressed macaques infected with the virus appeared to be contradictory to the immunocompromised condition. It has been reported that lipopolysaccharide (LPS)-induced fever was independent of prostaglandin Eirus RNA , might nirus RNA . Furtherirus RNA , suggestirus RNA . These rirus RNA . Therefoirus RNA ,56. We wThis study was carried out in strict accordance with the Guidelines for the Husbandry and Management of Laboratory Animals of the Research Center for Animal Life Science at Shiga University of Medical Science and Standards Relating to the Care and Management, etc. of Experimental Animals . The protocol was approved by the Shiga University of Medical Science Animal Experiment Committee (Permit numbers: 2009-8-6H and 2010-10-1). The Research Center for Animal Life Science at Shiga University of Medical Science has a permit for importation of cynomolgus macaques. Regular veterinary care and monitoring, balanced nutrition and environmental enrichment were provided by the Research Center for Animal Life Science at Shiga University of Medical Science. The macaques were euthanized at endpoint on 7 days after virus inoculation using ketamine/xylazine followed by intravenous injection of pentobarbital (200 mg/kg). Animals were monitored every day during the study to be clinically scored as described in ad libitum. Animals were singly housed in the cages equipping bars to climb up and puzzle feeders for environmental enrichment under controlled conditions of humidity (40 ± 5%), temperature (25 ± 1°C), and light . In the text and figures, individual macaques are distinguished by the following abbreviations: Y1, Y2, Y3 for macaques inoculated with YOK91 without immunosuppression, Y4, Y5, Y6 for macaques inoculated with YOK91 with immunosuppression, N1, N2, N3 for macaques inoculated with NRT1 without immunosuppression, and N4, N5, N6 for macaques inoculated with NRT1 with immunosuppression. The absence of influenza A virus nucleoprotein-specific antibodies in their sera was confirmed before experiments using an antigen-specific enzyme-linked immunosorbent assay (ELISA), AniGen AIV Ab ELISA , for currently circulating influenza virus. Two weeks before virus inoculation, a telemetry probe  was implanted in the peritoneal cavity of each macaque under ketamine/xylazine anesthesia followed by isoflurane inhalation to monitor body temperature. Antibiotics and analgesics were used after the surgery. The macaques used in this study were free from B virus, hepatitis E virus, Mycobacterium tuberculosis, Shigellaspp., Salmonella spp., and Entamoeba histolytica. All experiments using viruses were performed in the biosafety level 3 facility of the Research Center for Animal Life Science, Shiga University of Medical Science. Virus (3 × 106TCID50/7ml) was inoculated into nostrils (0.5 ml for each nostril), oral cavity (0.5 ml for the surface of each tonsil), and trachea (5 ml). Under ketamine/xylazine anesthesia, 2 cotton sticks  were used to collect fluid samples in nasal cavities and tracheas, and the sticks were subsequently immersed in 1 ml of phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) and antibiotics. A bronchoscope  and cytology brushes  were used to obtain bronchial samples. The brushes were immersed in 1 ml PBS with BSA.Five- to seven-year-old female cynomolgus macaques from Vietnam and the Philippines  were used. The cynomolgus macaques used in the present study were healthy young adults. All procedures were performed under ketamine and xylazine anesthesia, and all efforts were made to minimize suffering. Food pellets of CMK-2  were provided once a day after recovery from anesthesia and drinking water was available The seasonal influenza virus A/Yokohama/91/2007 (H1N1)  taxonomy database ID: 568510) was propagated in Madin-Darby canine kidney (MDCK) cells twice at Yokohama City Institute and once in MDCK cells at Shiga University of Medical Science . The panIn order to assess virus replication, serial dilutions of swab samples and homogenized tissue samples were inoculated onto confluent MDCK cells as described previously . CytopatCyclophosphamide  was dissolved in saline  at 40 mg/ml, stored at 4 °C after filtration, and used within 2 days of preparation. CP was intravenously administered by bolus injection. Cyclosporine A  was orally administered by catheters. The concentration of CA in plasma was measured by SRL Inc., Tokyo, Japan, with a double-antibody radioimmunoassay method.The numbers of leukocytes in blood were counted using a hemocytometer and a microscope. For flow cytometry, whole blood cells were stained with phycoerythrin (PE)-conjugated anti-CD8 , allophycocyanin (APC)-conjugated anti-CD4 , FITC-conjugated anti-CD14 , and PE-conjugated anti-CD20 . Dead cells were excluded by staining with ethidium monoacetate . Red blood cells were lysed with tris-ammonium chloride. Cells were fixed with PBS containing 4% paraformaldehyde before flow cytometer analysis.5 cells were cultured with anti-CD3 (clone: SP34), anti-CD28 (clone: CD28.2) and anti-CD49d (clone: 9F10) antibodies  or with phorbol 12-myristate 13-acetate (PMA)  and ionomycin  in 96-well U-bottom plates for 24 h and supernatants were collected. The concentrations of IL-2 in culture supernatants were determined using MAX Deluxe SET Human IL-2 (BioLegend).In the experiment for which results are shown in TM Human Interferon Beta ELISA kit  and the human TGF-β1 ELISA kit , respectively. The concentrations of TNF-α, IL-1β, IL-6, IL-18, and MCP-1 in homogenates were measured using the Milliplex MAP non-human primate cytokine panel and Luminex200 .In the experiment for which results are shown in After autopsy, lung tissue samples were fixed with 10% formalin, and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E). Influenza virus nucleoprotein (NP) was stained with anti-NP antibody, HB65, after treatment of sections with pronase . After iFigure S1Flow cytometric analysis of peripheral blood cells in immunosuppressed macaques.Cynomolgus macaques were administered CP and CA as indicated in (TIF)Click here for additional data file.Figure S2Body temperature after immunosuppression and virus infection.Cynomolgus macaques were administered a high dose of CP and CA as shown in (TIF)Click here for additional data file.Figure S3Blood cell populations in immunosuppressed macaques infected with influenza virus.Macaques were administered CP and CA from day -7 to day 0 and then inoculated with influenza virus YOK91, Y1-Y6, or NRT1, N1-N6, on day 0. Blood was collected on the indicated days. Blood cells were stained with fluorescence-conjugated antibodies specific for CD4, CD8, CD14, and CD20. The concentration of each population was calculated using white blood cell counts shown in +, CD8+, and CD20+ cells were calculated in R1 (low FSC/low SSC) as shown in Figure S1. The concentrations of CD14+ cells were calculated in R2 (high FSC/low SSC). The concentrations of granulocytes were calculated in R3 (high FSC/high SSC). The left y-axis indicates numbers of CD4+, CD8+, CD14+, and CD20+ cells. The right y-axis indicates numbers of granulocytes.(TIF)Click here for additional data file.Figure S4Immunohistochemical staining for influenza virus NP in organs of immunocompetent and immunocompromised macaques.Tissues were collected as described in the legend for (TIF)Click here for additional data file.Table S1Clinical scoring used in this study.Animals were monitored every day during the study to be clinically scored. Animals would be euthanized if their clinical scores reached 15 (a humane endpoint).(DOCX)Click here for additional data file.


After the 2011 declaration of rinderpest disease eradication, we surveyed 150 countries about rinderpest virus stocks. Forty-four laboratories in 35 countries held laboratory-attenuated strains, field strains, or diagnostic samples. Vaccine and reagent production and laboratory experiments continued. Rigorous standards are necessary to ensure that stocks are kept under safe conditions. Morbillivirus; the disease has had a devastating effect on livestock health, productivity, and welfare. It became the second disease to be eradicated, 30 years after smallpox eradication. The last reported outbreak of rinderpest occurred during 2001 in Kenya  and the World Organisation for Animal Health (OIE). Rinderpest is a disease caused by rinderpest virus, a paramyxovirus of the genus Unintentional or deliberate virus release remains a serious concern as long as virus-containing material remains in the possession of vaccine manufacturers or research and diagnostic laboratories. An example to substantiate this concern is the last reported case of smallpox in a human, which originated from a laboratory . It was complemented with the results of a previous questionnaire survey undertaken during 2010 by the FAO and OIE to identify countries holding rinderpest virus stocks and with information obtained from unpublished, or “grey,” literature and direct discussions with laboratory staff. As a result, information was available for 150 (76%) of the 198 OIE or FAO member and associate member countries .Thirty-five countries held rinderpest virus–containing material: 37% in Asia, 29% in Africa, and 26% in Europe. Extrapolating these results for countries for which information was not available, we estimated a global tally of 41  countries that held rinderpest virus .A susceptible host infected by a laboratory-attenuated strain cannot become infectious unless mutations facilitating virus reversion to transmissibility occur during infection. Laboratory-attenuated strains were the most commonly held virus type, kept in 34 countries, mainly as live vaccines or virus seed for vaccine production. Live vaccine stocks could be quantified for 11 countries, 4 of which had 100,000–4,000,000 doses. Field strains, which included mild to virulent strains that are transmissible between hosts, were held in 11 countries. We also received incomplete information; for example, several laboratory personnel reported holding virus in the past but did not specify whether their laboratories still held it. When we accounted for this incomplete information, we determined that 14 countries could be holding field strains . DiagnosRinderpest virus manipulations were being performed by some countries. A total of 115,000 live vaccine doses were produced in 1 country in 2011, and diagnostic reagent tests have been produced by using live virus in 3 countries since 2010. Basic research was still being conducted in 3 countries, of which 1 was conducting in vivo experiments on rabbits using laboratory-attenuated strains. Laboratories in some countries stated that they planned to develop marker and recombinant vaccines and to maintain and upgrade diagnostic reagent stocks. The sequencing of field strains was also being considered.We identified 44 laboratories holding viruses. Among the 36 laboratories for which biosafety level (BSL) was known, 11 (31%) of 36 laboratories holding attenuated strains were classified as BSL-2 or lower, as were 2 (17%) of 12 laboratories holding field strains and 3 (38%) of 8 laboratories holding diagnostic samples. Nine (39%) of 23 laboratories reported providing veterinary authorities with an inventory of the material containing rinderpest virus that were held, and 3 (13%) laboratories reported that authorization from veterinary authorities was required before they could manipulate rinderpest viruses.Fourteen national veterinary authorities reported holding a complete and up-to-date inventory of all rinderpest virus for all laboratories within the country. However, 43% of them did not report all of the virus types that were reported as being held by the laboratories themselves.Veterinary authorities of the 66 countries where rinderpest vaccines were used during the past 30 years were invited to forward a separate online questionnaire to field veterinarians, to assess their awareness of rinderpest and vaccine use. Of 70 respondents from 21 countries, none reported any use of rinderpest vaccines after 2003; however, 36% of respondents from 9 countries estimated that the likelihood of using rinderpest vaccines for routine vaccination against rinderpest or peste des petits ruminants, another morbillivirus disease, in their region within the next 5 years was “possible” to “very likely.”Because data were not available for all countries or all laboratories within countries, the number of virus stocks was probably underestimated. Some laboratory staff could retain material containing rinderpest virus without having ever conducted research on rinderpest or reported their stocks to their national authority. These laboratories might have been missed by our sampling approach. The number of respondents might have been greater if we had made questionnaires available in other languages. However, despite the sampling limitations, we can conclude that a large number of countries hold material containing rinderpest virus.Some veterinary authorities did not report all the virus types held in their countries, despite most of them claiming to maintain an inventory. Unless all virus stocks in all countries can be assessed, virus sources could be missed by future relocation plans.The reasons given for conducting basic research on rinderpest included that rinderpest viruses were a good model for studying aspects of morbilliviruses, such as mechanisms of replication, host pathogenicity, and interactions between host cell factors and viruses. Although such research might still be relevant and the production and management of vaccine and diagnostic reagent stocks could be key components of contingency plans, modifications to laboratory activities and protocols are needed to meet compliance standards recently recommended by FAO and OIE (Some field veterinarians reported believing that rinderpest vaccines might be used in their region in the near future. We did not investigate the reasons behind these opinions, but awareness clearly needs to be raised in the veterinary sector about risks for rinderpest reintroduction despite eradication and, more specifically, about the restricted use of rinderpest vaccines solely for the management of confirmed rinderpest outbreaks .Estimation of the total number of countries holding rinderpest viruses.


Glossina austeni population from Unguja Island (Zanzibar) using an area-wide integrated pest management approach with a sterile insect technique (SIT) component (1994–1997) encouraged several African countries, including Ethiopia, to incorporate the SIT in their national tsetse control programs. A large facility to produce tsetse flies for SIT application in Ethiopia was inaugurated in 2007. To support this project, a Glossina pallidipes colony originating from Ethiopia was successfully established in 1996, but later up to 85% of adult flies displayed symptoms of SGH. As a result, the colony declined and became extinct by 2002. The difficulties experienced with the rearing of G. pallidipes, epitomized by the collapse of the G. pallidipes colony originating from Ethiopia, prompted the urgent need to develop management strategies for the salivary gland hypertrophy virus (SGHV) for this species. As a first step to identify suitable management strategies, the virus isolated from G. pallidipes (GpSGHV) was recently sequenced and research was initiated on virus transmission and pathology. Different approaches to prevent virus replication and its horizontal transmission during blood feeding have been proposed. These include the use of antiviral drugs such as acyclovir and valacyclovir added to the blood for feeding or the use of antibodies against SGHV virion proteins. In addition, preliminary attempts to silence the expression of an essential viral protein using RNA interference will be discussed.Many species of tsetse flies (Diptera: Glossinidae) are infected with a virus that causes salivary gland hypertrophy (SGH), and flies with SGH symptoms have a reduced fecundity and fertility. The prevalence of SGH in wild tsetse populations is usually very low (0.2%–5%), but higher prevalence rates (15.2%) have been observed occasionally. The successful eradication of a  Glossina spp.) are the only cyclical vectors of two debilitating diseases in Africa, sleeping sickness in humans (human African trypanosomosis [HAT] caused by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense) and the cattle disease nagana  Tsetse flies , has been sequenced Successful eradication of This paper reviews data on the biology, epidemiology, transmission, and dynamics of the GpSGHV in field populations and laboratory colonies and describes potential strategies to manage the virus' impact in tsetse laboratory colonies. The limitations that hinder the use of this virus as a biological control agent for tsetse control are likewise discussed.Articles were identified by searching Medline through PubMed using various combinations of terms, including “Salivary gland hypertrophy virus”, “tsetse”, “SIT”, “RNAi”, “Antibodies neutralization”, and “Antiviral drugs”. Research papers and case reports from African countries were retrieved. Additional articles were obtained by citation tracking of review and original articles. The review also drew on conference proceedings and original research conducted by the authors.Since the first record of SGH in 1934 by Whitnall Based on these previous observations, we constructed a model of the vertical transmission of the virus in tsetse populations . In eachWe have recently demonstrated that the virus released by infected flies during feeding on an in vitro membrane feeding system is an important source of contamination under laboratory conditions G. pallidipes colony established in 1983 at the Insect Pest Control Laboratory collected from a population in the Lambwe Valley, Kenya, and in 2002 of a colony established in 1996 from Arba Minch, Ethiopia. The collapse of these colonies prompted research to understand the modalities of virus transmission under laboratory conditions and to develop potential virus management strategies In contrast to field conditions, the prevalence of SGH in tsetse laboratory colonies can increase up to 85% in certain cases. This high prevalence was responsible for the collapse in 1987 of the Many insect pathogenic viruses are currently used as biological control agents, e.g., baculoviruses in lepidopteran hosts From a purely practical point of view, the development of a biopesticide to control tsetse flies based on SGHV faces several technical challenges, a major one being the mass production of the virus for field application and its formulation. Artificial infection of healthy flies by feeding on contaminated blood is efficient, but the symptoms of SGH corresponding to highest virus loads are observed only in the next generation. Attempts to multiply the SGHV in an alternative host  were not successful. Furthermore, no in vitro system to multiply the virus in cell culture is presently available. Finally, the formulation of a virus suspension allowing the virus particles to retain their infectivity under field conditions appears insuperable. Unlike baculoviruses, SGHV does not produce occlusion bodies and the virus envelope is extremely fragile. More than 90% of a purified virus suspension looses its infectivity after 3 days at 4°C (unpublished data). These difficulties make the use of this virus as a biological control agent impractical.In view of our current understanding of the epidemiology of the virus under laboratory conditions, two possible strategies to manage the infection in a colony emerged: one based on the reduction or inhibition of horizontal virus transmission, the other on the reduction or inhibition of virus replication to avoid the development of symptomatic infections in a colony. To mitigate horizontal transmission in laboratory colonies, two approaches are being explored: (a) changing the feeding protocol currently used in colony rearing and/or (b) neutralizing the virus released during blood feeding by adding virus-specific antibodies to the blood. To reduce virus replication, two approaches have been tested: (c) inhibiting replication with commercially available antiviral drugs used to inhibit the DNA polymerase of similar viruses and (d) developing RNA interference to silence essential virus specific gene(s).6 to 102 virus particles per fly) in three generations As mentioned above, the in vitro membrane feeding protocol using successive feeds on the same membrane favors horizontal virus transmission from infected to healthy colony flies. However, feeding the flies on fresh, unused  blood and membrane at each meal (referred to as “clean feeding”) resulted in a four log reduction of the average virus load per fly . Several other antiviral drugs are currently available for DNA viruses that could be screened to assess their impact on GpSGHV DNA replication. Several parameters, such as (i) absence of any negative effect on fly survival and productivity, (ii) significant reduction of virus loads, (iii) suitable bioavailability when offered to the flies mixed with blood, and (iv) affordable price will have to be considered for selecting appropriate antiviral drugs for use in large-scale tsetse rearing facilities. Administration of one or more antiviral drugs could be combined with other methods of virus management.The discovery in the late 1970s that acyclic nucleoside analogs, in particular acyclovir, could inhibit DNA replication of herpes simplex virus (HSV) at concentrations far below those that affect cellular DNA synthesis sparked a new era in antiviral chemotherapy per os infectivity factors to mitigate the SGHV infection is worth evaluating.RNA interference (RNAi) has recently emerged as a powerful tool for specific gene silencing in gene therapy G. pallidipes that incorporate the release of sterile males because it reduces the productivity of G. pallidipes colonies, which in certain circumstance has resulted in the collapse of the colony. The virus cannot be used as a biological control agent due to several limitations. So far the virus has been studied from the point of view of its impact on G. pallidipes colonies in a rearing facility, but the impact of the virus on release programs in terms of competiveness, performance, and mating behavior of symptomatic and asymptomatic infected male flies still requires further study.In conclusion, the GpSGHV represents a threat to integrated control programs against G. pallidipes laboratory colonies prevalence can reach 85%.Tsetse salivary gland hypertrophy syndrome was reported in wild tsetse populations from several species in different countries with a prevalence of 0.5%–15%, while in Tsetse salivary gland hypertrophy virus (SGHV) cannot be used as a biological control agent in tsetse control program due to the virus characteristics, i.e., fragile structure of virus particles and limited role of horizontal virus transmission.G. pallidipes in large-scale facilities by reducing fly productivity, which hinders SIT programs for tsetse control.GpSGHV seriously impedes rearing of A virus management strategy based on reducing horizontal virus transmission by changing the blood feeding system currently used, neutralizing virus infection using virus-specific antibodies, and reducing virus replication by administration of antiviral drugs or RNAi technology is being developed.Glossina pallidipes in the Umfolosi Game Reserve, Zululand. Onderstepoort J Vet Sci Anim Ind 2: 7–21.Whitnall ABM (1934) The Trypanosome infections of Glossina pallidipes. Trans R Soc Trop Med Hyg 72: 234–238.Jaenson TGT (1978) Virus-like rods associated with salivary gland hyperplasia in tsetse, Glossina pallidipes (Diptera: Glossinidae). J Invertebr Pathol 52: 1–8.Jura WGZO, Odhiambo TR, Otieno LH, Tabu NO (1988) Gonadal lesions in virus-infected male and female tsetse, Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) reveals a novel large double-stranded circular DNA virus. J Virol 82: 4595–4611. doi:10.1128/JVI.02588-07.Abd-Alla AMM, Cousserans F, Parker AG, Jehle JA, Parker NJ, et al. (2008) Genome analysis of a Glossina pallidipes (Diptera: Glossinidae). Virus Res 150: 103–110.Abd-Alla AMM, Kariithi H, Parker AG, Robinson AS, Kiflom M, et al. (2010) Dynamics of the salivary gland hypertrophy virus in laboratory colonies of


Passatempo virus was isolated during a zoonotic outbreak. Biologic features and molecular characterization of hemagglutinin, thymidine kinase, and vaccinia growth factor genes suggested a vaccinia virus infection, which strengthens the idea of the reemergence and circulation of vaccinia virus in Brazil. Molecular polymorphisms indicated that Passatempo virus is a different isolate. Since aha, tk, and vgf genes were amplified by polymerase chain reaction with Taq polymerase   . Regarding ha sequences, PSTV was clustered to ARAV and CTGV (The alignments were used to construct phylogenetic trees by the neighbor-joining method using the Tamura Nei model implemented in MEGA3 (and CTGV .vgf and ha gene analysis of PSTV, ARAV, and CTGV indicated that genetic heterogeneity exists among these viruses, which suggests that the ha gene deletion found in PSTV, ARAV, CTGV, and VACV-IOC could be a signature of New World or Brazilian VACV strains.The phylogenetic tree analysis suggested a strong phylogenetic relationship between PSTV and other Brazilian VACV strains. However, the Additionally, that RFLP analysis showed a pattern identical with other Brazilian strains, similar to VACV-WR and different from CPXV, suggests that a cladogenesis event may have occurred. This conclusion is feasible considering that these viruses could be circulating in the wild since smallpox vaccination or even before, going back to the colonization of South America, when cattle and other animals were brought to the New World without quarantine or inspection. The VACV variants buffalopox and rabbitpox have originated from VACV subspeciation (That humans were also infected and that these persons were all milkers, phenomena that had been observed during other Brazilian VACV outbreaks, points to an occupational zoonosis. Although parapoxvirus infection has been placed in the category of occupational zoonosis, to our knowledge no other orthopoxviruses have been reported to cause an occupational hazard. Economic losses are also a matter of concern. In addition to the reduction in milk production, extra veterinary costs are due to the usual occurrence of secondary infections on cows' teats leading to mastitis. The reduction in milk production is a concern because Brazil is a major milk exporter. Therefore, the spread of these viruses could severely impact the country's economy. In this regard, the clinical features, widespread dissemination, and epidemiology of the etiologic agent of these outbreaks must be understood.,,,Since 1963, all Brazilian orthopoxvirus isolates have been characterized as VACV strains ,,, indicate


To evaluate the importance of human exposure to Rift Valley fever virus in Chad, investigations were carried out to determine specific antibody prevalence in domestic ruminants during the 2002 rainy season. Results highlighted recent, substantial, active transmission of this virus. The virology laboratory of the unit of the Tropical Medicine Institute of the French Defense Medical Service (IMTSSA) investigated some cases of self-limiting nonmalarious febrile syndromes occurring among soldiers stationed in Chad. By using C6/36 and Vero cell lines, peripheral blood lymphocytes collected from two soldiers on duty in Chad during the 2001 rainy season were cocultured. Two strains of Rift Valley fever virus (RVFV) were isolated and identified by using indirect immunofluorescence, reverse transcriptase–polymerase chain reaction, and sequencing , within the slaughterhouses of the cities of N’Djamena  and Abéché (220 km east of N’Djamena), blood samples were collected from randomly selected sheep, goats, and horned cattle . AccordiEach sample was accompanied by information on the age of the animal (teeth examination), species, sex, and origin. Blood was centrifuged within 24 hours of collection. The serum was transferred into cryotubes and frozen at –80°C so that samples would arrive at IMTSSA for analysis still frozen (–20°C). Each sample was systematically tested for RVFV-specific immunoglobulin (Ig) G by using an enzyme-linked immunosorbent assay (ELISA). First, ELISA screening was done by using antigen capture (by mouse hyperimmune ascitic fluid) and detecting specific IgG in the diluted serum (1/500). The antigen used was a precipitate (polyethylene glycol 6000) of the supernatant of Vero cells infected with the RVFV clone 13.  and IgM by using the M-antibody capture method. The most frequently used techniques for detecting anti-RVFV antibodies are immunofluorescence, plaque reduction neutralization assay, and immunoenzymatic assays  with a high threshold of positivity. Only serum samples containing both specific antibodies against the envelope glycoproteins (G1 and G2) and the nucleocapsid (NC) protein were considered positive. Comparative results between the two techniques confirm high specificity of ELISA (97% of the serum samples positive by ELISA were confirmed by Western blot). This high specificity has been described previously by crosschecking results with those of virus neutralization assay  could not be collected for animals led to the N’Djamena slaughterhouse  and are imprecise for those received at the Abéché slaughterhouse . Thus, charting the distribution of RVFV-positive animals and the geographic distribution of the virus is not possible. Nevertheless, the weak antibody prevalence in animals killed in the Abéché slaughterhouse should be noted, which allows us to conclude that this particular area is still isolated from RVFV.


CypA participates in protein folding, cell signaling, inflammation and tumorigenesis. Further, CypA plays critical roles in the replication of several viruses. Upon influenza virus infection, CypA inhibits viral replication by interacting with the M1 protein. In addition, CypA is incorporated into the influenza virus virions. Finally, Cyclosporin A (CsA), the main inhibitor of CypA, inhibits influenza virus replication through CypA-dependent and -independent pathways. This review briefly summarizes recent advances in understanding the roles of CypA during influenza virus infection. Cyclophilin A (CypA) is the main member of the immunophilin superfamily that has peptidyl-prolyl  CypA haactivity . Furtheractivity , which iactivity ,24. Extractivity ,27,28,29Accumulating evidence indicates that CypA is an important host factor for successful viral infection. CypA is involved in the lifecycle of several viruses, such as human immunodeficiency virus type 1 (HIV-1), influenza virus, hepatitis C virus (HCV), hepatitis B virus (HBV), vesicular stomatitis virus (VSV), vaccinia virus (VV), severe acute respiratory syndrome coronavirus (SARS-CoV) and Rotavirus (RV) ,19,30,31PPIA) gene, and regulatory PPIA polymorphisms are a component of genetic susceptibility to HIV-1 infection or disease progression [CypA has been extensively studied from the gene to protein level during HIV-1 infection. CypA is encoded by the peptidyl prolyl isomerase A  Tax oncoprotein and promote malignant transformation [In 2008, CypA was shown to be in the core of the inﬂuenza virion  and is uormation . This suThe well-known immunosuppressive drug CsA inhibits influenza virus replication through CypA-dependent and -independent pathways. In detail, CsA inhibits influenza virus replication at a post-transcriptional level and impairs the nuclear export of viral mRNA in the absence of CypA . In addiAs was reported, CypA is also incorporated into influenza virions ,32. HoweUpon influenza virus infection, CypA acts at multiple steps of the viral lifecycle, including uncoating, viral protein entry into the nucleus, viral protein stability and assembly . In someViral infection usually triggers host antiviral defenses, such as the interferon-I (IFN-I)-mediated antiviral response. Recently, CypA was found to play important roles in host antiviral innate immunity. For example, during HIV-1 infection, the interaction of CypA and newly synthesized HIV-1 CA assists HIV-1 to induce DC maturation, an antiviral type I interferon response, and the activation of T-cells. In addition, CypA inhibits Rotavirus replication by facilitating host IFN-β production. Whether CypA mediates host antiviral defenses during influenza virus infection is an interesting question and requires further study. In addition, CypA is secreted into the extracellular space, and the function of extracellular CypA during influenza virus replication deserves further study.


Culex pipiens mosquitoes during a severe outbreak of human West Nile disease in Greece 2010. The virus showed closest genetic relationship to the lineage 2 strain that emerged in Hungary in 2004; increased virulence may be associated with amino acid substitution H249P.We conducted a complete genome analysis of a West Nile virus detected in  Culex species; humans, horses, and other mammals are incidental hosts. Although most human WNV infections remain subclinical, febrile illness develops in ≈20% of infected persons and neuroinvasive disease in <1%. Severe disease is more frequent among the elderly and immunocompromised (West Nile virus (WNV) is a flavivirus maintained in an enzootic cycle between bird amplifying hosts and ornithophilic mosquito vectors, mainly Culex and Aedes species mosquitoes in southern Moravia, Czech Republic, close to the Austrian border, without recognized pathogenicity for mammals  of 392 persons exhibited neutralizing WNV antibodies, 2 with high titers. These findings suggested that WNV, or an antigenically closely related flavivirus, circulates, at least locally, in rural areas in Greece , a village 30 km northeast of Thessaloniki, was found to be positive for WNV RNA by reverse transcription–PCR. Sequencing of a 146-nt fragment of the nonstructural protein 5 (NS5) gene gave the first evidence that the virus belongs to lineage 2, and that it is highly similar to the strain that emerged in Hungary in 2004 and to isolates from fatal human and animal cases in South Africa . Amplification products were directly sequenced in both directions; sequences were aligned and compiled, and an 11,028-nt continuous sequence was obtained, representing the nearly complete genome sequence of the virus, designated Nea Santa-Greece-2010. Because of the limited amount of sample material, the last 20 nt of the 5′ and 3′ ends were not determined by rapid amplification of cDNA ends. The nucleotide sequence was deposited in GenBank database under accession no. HQ537483.To amplify overlapping PCR products of the WNV from the Greek outbreak, we used RNA from the above-mentioned mosquito pool and reverse transcription–PCRs designed for the amplification of the complete genome of lineage 2 WNV, which had emerged in Hungary in 2004  was translated to a 3,434-aa polypeptide sequence. The nucleotide and putative amino acid sequences were aligned with all complete lineage 2 WNV nt and aa sequences available in GenBank, as well as with representatives of other WNV lineages.The highest (99.6%) nucleotide identity of the Nea Santa-Greece-2010 sequence was found to the goshawk-Hungary-2004 strain. Only single nucleotide substitutions (n = 44) were detected, equally distributed over the genome. The putative aa sequences of the polyprotein precursor of the 2 viruses were 99.7% identical. The alignment of the putative precursor polypeptide sequences showed aa substitutions unique for the European lineage 2 WNVs. These substitutions were found in the nonstructural proteins .30P substitution in the NS2A protein reduced the ability of the virus to inhibit interferon induction , NS4A protein (A79T), and NS5 protein  were predicted to be possible virulence markers (Several studies have focused on the determination of genetic markers for pathogenicity and virulence of WNV strains. In the New York strain of WNV, envelope protein glycosylation proved to be a molecular determinant of neuroinvasiveness (Viruses of lineages 3, 4, and 5 contain aspargine, threonine, and threonine at this locus, respectively . The infCx. pipiens mosquitoes during the 2010 outbreak in Greece, indicate that, most likely, descendants of the goshawk-Hungary-2004 strain spread southward to the Balkan Peninsula and reached northern Greece. Our data indicate that an independent introduction of a highly similar lineage 2 WNV from Africa to northern Greece is unlikely. The importance of the H249P aa change in the NS3 protein of the Nea Santa-Greece-2010 virus should be verified in experimental studies. This aa change also emphasizes the value of close genetic monitoring of strains involved in additional European WNV outbreaks.Epidemiologic observations of the lineage 2 WNV in Europe during 2004–2010, and the close genetic relatedness of the WNV circulating in Hungary and eastern Austria to the virus identified in


Geminiviridae comprises a group of plant-infecting circular ssDNA viruses that severely constrain agricultural production throughout the temperate regions of the world, and are a particularly serious threat to food security in sub-Saharan Africa. While geminiviruses exhibit considerable diversity in terms of their nucleotide sequences, genome structures, host ranges and insect vectors, the best characterised and economically most important of these viruses are those in the genus Begomovirus. Whereas begomoviruses are generally considered to be either monopartite (one ssDNA component) or bipartite , many apparently monopartite begomoviruses are associated with additional subviral ssDNA satellite components, called alpha- (DNA-αs) or betasatellites (DNA-βs). Additionally, subgenomic molecules, also known as defective interfering (DIs) DNAs that are usually derived from the parent helper virus through deletions of parts of its genome, are also associated with bipartite and monopartite begomoviruses. The past three decades have witnessed the emergence and diversification of various new begomoviral species and associated DI DNAs, in southern Africa, East Africa, and proximal Indian Ocean islands, which today threaten important vegetable and commercial crops such as, tobacco, cassava, tomato, sweet potato, and beans. This review aims to describe what is known about these viruses and their impacts on sustainable production in this sensitive region of the world.The family  Geminiviridae) in the genus Begomovirus are among the most devastating pathogens worldwide of a variety of cultivated crops, including cassava, sweet potato, beans, tomato, cotton and grain legumes   and ACMVs island . DNA-A as island . As ments island  likely hs island . Similars island ,111,112.s island . Togethes island ,114,115 s island .et al.  (ACMV-[KE]), in N. benthamiana [East African cassava mosaic virus (EACMV-[TZ15] (AY828226), a bipartite begomovirus in Tanzania [South African cassava mosaic virus-[South Africa:99] (accession number: JX233822)  DNA-A and B infectious clones. An investigation to determine the effect of DI on SACMV-[ZA] replication and symptom development in an experimental host N. benthamiana and the cassava, the natural host, was performed  infection, appeared to be reduced, with more leaf blistering and curling. This may be due to differences in viral-host factor interactions during viral replication between a model host (Nicotiana benthamiana) and a natural host (cassava)  infection alone, or co-inoculation with the DI dimer. Modulating increases and decreases in SACMV-[ZA] DNA-A and DNA-B titers in both plants systems may be a consequence of a cyclic replication pattern typical of a situation with bipartite viruses, such as SACMV-[ZA], where two DNA components are interdependent on each other for different functions at different times, such as replication (DNA-A) and movement proteins (DNA-B). Increases in DNA-A levels later on during the infection period indicate a recovery in DNA-A replication which may be predicted since DNA-A  is the rate-limiting factor for both its own autonomous replication and is required by both DNA-B and the DI for trans-replication  compete for Rep binding in the common region, there is an ‘interdependence’ between these two components, which is likely to result in a modular pattern of increases and decreases in DNA-A in relation to DNA-B. The presence of an additional DNA-B-derived DI DNA is also likely to influence this process. Modulation of genomic and defective DI DNA components has also been observed in RNA helper and satellites  in a naturally infected field cassava plant collected from the Mpumalanga Province in South Africa  Figure c. This Derformed . Viral lerformed , or inocerformed . A direccassava) . Cassava viruses . Anotherlication ,129. Symtellites . HoweverSolanum lycopersicum, formerly Lycopersicon esculentum) is an important vegetable crop with considerable nutritional and economic value. It belongs to the Solanaceae family, which in addition to S. lycopersicum L., contain more than 10 related wild species ) ) . At some time in the late 1980s or early 1990s, the Mld and IL strains of TYLCV spread from the western Mediterranean region (presumably human mediated means) to the islands of Reunion and Mauritius in the SWIO region ,135,142.Tomato curly stunt virus (ToCSV-[ZA:Ond:98]) was identified [Tomato curly stunt Mooketsi virus (ToCSMV-[ZA:Mks:07]), Tomato curly stunt Lanseria virus (ToCSLV-[ZA:Lan:08]) and Tomato curly stunt Noordoewer virus (ToCSNV-[ZA:Nwr06:08]) ) ,145. Whir06:08]) ,145. Tomato leaf curl Uganda virus) or clade VI, those from South Africa group within clade V and are most closely related to bipartite viruses in the Africa/Cassava bipartite group (clade IV). The remaining African tomato-infecting isolates from Tanzania and Zimbabwe, cluster with central/west/north African  and SWIO isolates (Madagascar and Comoros) in clade VI. The concordant phylogenetic and geographical clustering of South African and SWIO tomato-infecting viruses strongly suggests that they are all indigenous to their respective regions. This is further supported by the fact that recombination events detectable within the genomes of the South African and SWIO viruses display a marked tendency to have involved parental viruses from these same regions  was a natural recombinant of sweepoviruses consisting of two distinct parental genomic sequences from SPLCSPV and Sweetpotato leaf curl Georgia virus (SPLCGV). It is therefore likely that, as has been proposed for similar discoveries in other parts of the world such as in Sicily, Peru, US, China and India , namely ‘HG’ and ‘mild’ df DNAs, were detected in Zimbabwe tobacco cutlivar HG . These wN. tabacum cvs. HG and Burley showed downward leaf curl and puckering, and vein thickening, while the leaves of the other two cultivars (unknown) showed either severe leaf curl (with cup-shaped enations) or mild upward leaf curling. Sequencing of the intergenic region (IR) and coat protein (CP) demonstrated that the four TbLCZV-[ZW] isolates were 98% similar  isolates, it was suggested that the df DNAs may be playing a role in symptom amelioration. However, the potential role of betasatellites in disease symptom modification, and the possibility of recombinant viruses contributing to the different leaf curl phenotypes cannot be ruled out. This was not considered at the time of the study, since the extent of recombination and betasatellites were not well known. Sequencing of full-length DNA-As from a large number of infected tobacco samples may reveal greater genetic diversity, and the search for a betasatellite may prove useful in elucidation of the aetiology of tobacco leaf curl disease in southern Africa.A perplexing feature of tobacco leaf curl since its earliest reports has been the observed variation in symptom phenotype and severity. McClean  describe similar . Variati similar , with N. McClean , and N. symptoms . The sevPhaseolus vulgaris) are grown by both small-scale farmers and commercial farmers in many SADC and SWIO countries including, Lesotho, Madagascar, Malawi, Mauritius, Swaziland, Tanzania, Zimbabwe and South Africa [Bean common mosaic virus (BCMV) in many of these countries [Bean golden mosaic virus (BGMV) [Bean dwarf mosaic virus (BDMV) [Bean golden yellow mosaic virus (BGYMV), all of which are New World begomovirus species. To date, the only geminivirus known to infect beans in the SADC region is the mastrevirus Bean yellow dwarf virus (BeYDV) first isolated in South Africa [Tobacco yellow dwarf virus (TbYDV) [Cotton leaf curl Gezira virus (CLCuGV), a species originally found in cotton from Egypt and the Sudan, was reported to infect beans in Madagascar [French (green) beans  , Bean dws (BDMV) , and Beah Africa , which istralia) . In the dagascar . Nicotiana benthamiana, N. tabacum, Solanum lycopersicon, Datura stramonium and Arabidopsis thaliana [vice versa.BeYDV can cause yield losses of between 85 and 92% in infected plants  with symthaliana , it is aP. vulgaris [Centrosema pubescens and Puerariaavanica in the DRC [Bemisiatabaci, and facilitate survival of the viruses. An example is recombination among isolates of Tobacco leaf curl Japan virus and Honey suckle yellow vein virus from an ornamental plant [Finally, it is interesting to note both that the cassava-infecting begomovirus SACMV is also able to infect vulgaris , and tha the DRC . While t the DRC ,195, whial plant . Whiteflal plant , previoual plant ,61,197. B. tabaci, which exhibits considerable genetic and biological variation, has resulted in the explosion of new begomoviral species complexes in many tropical, subtropical and mild temperate climes in the past three decades. Genetic recombination due to sharing of common hosts, and expansion of host ranges for some begomoviral species, has resulted in diverse species complexes which pose a serious threat to agriculture not only in southern and eastern African countries, but globally. While cassava mosaic disease-associated begomovirus diversity and complexes are the only ones to have been extensively studied in many regions of Africa [Whitefly-transmitted geminiviruses are thought to have evolved in the Old World (Eastern Hemisphere), where both monopartite and bipartite viruses are extant . The glof Africa ,102, a rf Africa . This waf Africa ,114,145,f Africa , but thef Africa  is highlB. tabaci cryptic species complex (currently consisting of 24 distinct species and many haplotype variants) [The emergence of cassava, tobacco and tomato-infecting begomoviruses throughout the SADC and SWIO regions was likely due to introduction and intensive cultivation of exotic crop species having been introduced into environments harbouring indigenous begomoviruses. Furthermore, the ariants) , that trariants) ,28,43,77ariants) ,144,145 ariants) , demonst