The large 2014/2015 Ebola virus outbreak in West Africa points out the urgent need to develop new preventive and therapeutic approaches that are effective against Ebola viruses and  can be rapidly utilized. Recently, a simple theoretical criterion for the virtual screening of molecular libraries for candidate inhibitors of Ebola virus infection was proposed. Using this method the ‘drug space’ was screened and 267 approved and 382 experimental drugs as candidates for treatment of the Ebola virus disease (EVD) have been selected. Detailed analysis of these drugs revealed the non-steroidal anti-inflammatory drug ibuprofen as an inexpensive, widely accessible and minimally toxic candidate for prevention and treatment of EVD. Furthermore, the molecular mechanism underlying this possible protective effect of ibuprofen against EVD is suggested in this article. World Health Organization, Ebola data and statistics). Although reports of new, confirmed cases of Ebola seemed to decrease in April of 2015 to approximately 30 cases per week, medical and aid organizations are warning that the current crisis is not over. Furthermore, there remains a risk for future epidemics. Therefore, there is an urgent need to develop new preventive and therapeutic approaches that can be rapidly utilized. New drugs for the treatment of EVD should be safe, efficacious, easy to manufacture and inexpensive in order to be successfully deployed in African countries. In contrast to the significant progress recently achieved in development of an effective Ebola virus vaccine1, therapeutic options are still limited2. The main obstacle represents identification of an appropriate therapeutic target, which has been largely hampered by the time and money-consuming development of new drugs, especially for neglected diseases. Furthermore, the registration of newly identified drugs, like favipiravir, for potential usage in EVD patients takes a relatively long period of time, even though it has been shown to be effective in non-human primate models. In order to avoid these obstacles, several approaches for repurposing of approved drugs for the treatment of EVD patients have been proposed in literature7.The recent Ebola virus outbreak in West Africa caused  a total of 25,556 confirmed cases, including 10,587 deaths  and the electron-ion interaction potential (EIIP) - parameters determining long-range interaction between biological molecules13 - we selected 267 approved and 382 experimental drugs as candidates for treatment of EVD. Further detailed analysis of these drugs, including molecular docking, revealed ibuprofen as an inexpensive, widely accessible and minimally toxic candidate for potential prevention and treatment of EVD. The molecular mechanism underlying the possible protective effect of ibuprofen against EVD is suggested.Recently, we proposed a relatively simple theoretical criterion for the fast virtual screening of molecular libraries for candidate inhibitors of Ebola virus infection8. This criterion is based on the calculation of EIIP and AQVN which are determined by following equations:Recently, we proposed a theoretical criterion for selection of candidate inhibitors of Ebola virus infectionwhere:i - Type of the chemical elementi - Valence of thei-th chemical elementZi - Number of thei-th chemical element atoms in the compoundnm - Number of types of chemical elements in the compondN - Total number of atomsThe EIIP values calculated according to the14. This approach showed that molecules which potentially block Ebola virus infection are placed within AQVN range (2.3–2.7) and EIIP range (0.829–0.954 Ry), respectively. Using this theoretical criterion the drug library encompassing 267 approved drugs selected as candidate inhibitors of the Ebola virus infection  was established15. This drug library was used for selection of an approved drug, which represents the optimal candidate for prevention and treatment of EVD.The AQVN and EIIP molecular descriptors determine the long-distance (>5Å) intermolecular interactions in biological systemsThe modelling of glycoprotein GP1 from Ebola virus was previously described inVEGA ZZ17, protonated according to physiological conditions and optimized on semi empirical PM6 level of theory usingMOPAC 2009.Ligands were built in17. The docking was carried out withAutodock Vina (version 1.1.2.)18. In both cases the whole receptor conformational space was searched, using grid boxes with dimensions 60×60×60 and 30×30×30Å3. The docking was carried out with weighting of hydrophilic interactions, and the corresponding parameter value in the docking configuration was set to -1.20 (compared to default weight: hydrogen = -0.587439). The exhaustiveness was set to 250. The conformations with lowest binding energy values were chosen. The calculations were carried out on thePARADOX Cluster computer.Ligand and receptor were prepared in VEGA ZZ15 for optimal candidates for prevention and treatment of EVD. To achieve this end, we used the following criteria: a) high efficacy of binding of drug to the Ebola virus glycoprotein GP1; b) low toxicity and accessibility (nonprescription drugs); and c) low cost. By mining of CEDD using these criteria, we selected ibuprofen as the best candidate. Ibuprofen is a nonsteroidal anti-inflammatory drug (NSAID). Its mode of action, as for other NSAIDs, is not completely understood, but may be related to prostaglandin synthetase inhibition. It achieves this effect on prostaglandin synthesis by inhibiting cyclooxygenase (COX I and II isotypes), an enzyme that is present in at least one isoform most of the tissues of the body and which is responsible for production of not only prostaglandins but also prostacyclins and tromboxane.We screened the CEDD library19. Similar results were obtained in a prospective study of ibuprofen use in healthy volunteers taking the maximum permitted, non-prescription dose of ibuprofen (1200 mg/day) for 10 days20. It was demonstrated that administration of ibuprofen in children is safe. A randomized, community-based study on the safety of ibuprofen in febrile children aged 2 years old (5 or 10 mg/kg) showed that the risk of hospitalization for gastrointestinal bleeding associated with this drug was 17 per 100,00021. It was also shown that fecal blood loss associated with application of prescription doses of ibuprofen (2400 mg/day) did not exceed the normal range22. Even in the case of post-surgical usage for pain control in children undergoing tonsillectomy, ibuprofen administration is considered to be safe and does not increase risk of hemorrhages23. Accordingly, we believe that it would be crucial to better understand the potential risk and benefits of ibuprofen usage in experimental models of EVD. Taken together, these data indicate that ibuprofen could be safely used in nonprescription doses in EVD patients, with potential antiviral effects as well as to alleviate the symptoms.Generally, ibuprofen is minimally toxic drug. A meta-analysis of eight placebo-controlled studies showed that application of non-prescription doses of ibuprofen (800–1200 mg/day) over 10 days caused no significant adverse events8. This suggests that ibuprofen and dexibuprofen are the only drugs from the NSAID group that potentially have anti-Ebolavirus effects, which should be tested bothin vitro andin vivo.To assess specificity of ibuprofen as a potential inhibitor of the Ebola virus infection, distribution of all approved NSAIDs in AQVN/EIIP space was analyzed. Results presented in  show thain silico study suggested that Elastin Microfibril Interface Located Proteins (EMILINs) are involved in interaction between GP1 and endothelial extracellular matrix (ECM)16. Docking of ibuprofen to the GP1 model gave conformations which bound to EMILINs binding domain on GP116. The binding site of ibuprofen on GP1 spans the edge of this region and consists of Thr 338, Ser 340, Gln 344 and Ala 41516. The intermolecular interactions between ibuprofen and binding site amino-acids include hydrogen bonds of carboxyl group with Thr 338, Ser 340 and Gln 334. Additional stabilization is provided through aromatic and hydrophobic interaction with Ala 415. This binding site is placed between two loops, which provide the possibility of stabilizing a particular conformation, and therefore possibly blocking receptors. The appropriately high binding energy of -9.0 kcal/mol favors this assumption. The binding conformation is presented in24. It was shown that Emilin-1 knockout mice display increased TGF-β1 signaling in the walls of their blood vessels, leading to peripheral vasoconstriction and arterial hypertension25. These matrix-dependent changes in the vascular hemodynamics caused by TGF-β1 and EMILIN-1 are important because they ultimately affect the cardiovascular morbidity and mortality rates. Recently, it was shown that activation of the TGF-β1 signaling pathway by Ebola virus plays an important role in pathogenesis of EVD26. These findings suggest the possibility that binding of GP1 to EMILIN-1 prevents its interaction with TGF-β1, which results in activation of TGF-β1 signaling pathway. Binding of ibuprofen to GP1 could prevent GP1/EMILIN-1 interaction allowing EMILIN-1 to keep control of TGF-β1 signaling pathway.A previousIn conclusion, presented results should encourage further investigation of ibuprofen and ibuprofen-inspired drugs as inexpensive, low-toxic and wide-accessible candidates for prevention and its usage in the treatment of EVD.10.5256/f1000research.6110.d4287715F1000Research: Dataset 2. Approved and experimental drugs selected as candidate for treatment of EVD, et al., deals with the identification of inhibitors against Ebola virus infection. Authors took advantage of existing approved and experimental drugs and, based onin silico analysis, proposed ibuprofen as a potential candidate for prevention and treartment of Ebola virus infection. EIIP and AQVN calculation, previously described by authors, was employed to define ibuprofen as the best candidate against Ebola virus infection.Current manuscript by VeljkovicThe manuscript is adequately presented and can be considered as acceptable for indexation.Minor points:Ebola virus infection is initiated by interaction between the virus glycoprotein GP1 and its cognete receptor(s). Putative receptor binding domain of GP1, key residues involved in GP1 protein folding or structure and a putative receptor binding pocket has been proposed and mapped to the N-terminal 150 amino acids of GP1 (33-185 residues).Authors should include in the discussion as how interaction of ibuprofen with GP1  will influence the receptor binding domain.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. et al., in silico methods were used with the aim of identifying inhibitors of Ebola virus infection. Particularly, authors focused their investigation on approved and experimental drugs and proposed ibuprofen as a candidate for prevention and treatment of Ebola virus. It is well known that Ebola virus represents an international health emergency and that there is an urgent need for therapeutics and prophylactic drugs.In the work of VelijkovicIn the present manuscript, authors applied a previously described and well-established criterion, based on EIIP and AQVN calculation, to select ibuprofen as the best candidate for Ebola virus disease treatment. Moreover, a mechanism of action consisting on GP1 inhibition has been proposed.Since molecular docking of ibuprofen was performed against the putative target GP1, it would be important to report the predicted binding energy  or score, and to compare this value with estimated or experimental affinity of reference GP1 ligands. Moreover, in a repurposing perspective, it would be interesting if authors could comment on the approximated dosage of ibuprofen they expect for providing anti-Ebola activity.Ibuprofen has a serum half-life of 1.8 to 2.0 hours. Did authors consider this aspect when claiming that ibuprofen could be also administered to patients to prevent Ebola virus disease.Liver and gastrointestinal tissues are among the most injured by Ebola virus infection. Moreover, it is well known that ibuprofen can cause serious gastrointestinal toxicity and should be used with caution in person with coagulation defects. Did authors considered ibuprofen side effects in the context of Ebola infection treatment? Following the question 1., a comment on the expected dosage would help to clarify most of these issues.It is not clear how ibuprofen can alleviate the symptoms of Ebola virus disease, also considering the proposed mechanism of action.It is the opinion of the present referee that some minor issues should be fixed before the manuscript may be considered as acceptable for indexation:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


Virus is known to resonate in the confined-acoustic dipolar mode with microwave of the same frequency. However this effect was not considered in previous virus-microwave interaction studies and microwave-based virus epidemic prevention. Here we show that this structure-resonant energy transfer effect from microwaves to virus can be efficient enough so that airborne virus was inactivated with reasonable microwave power density safe for the open public. We demonstrate this effect by measuring the residual viral infectivity of influenza A virus after illuminating microwaves with different frequencies and powers. We also established a theoretical model to estimate the microwaves power threshold for virus inactivation and good agreement with experiments was obtained. Such structure-resonant energy transfer induced inactivation is mainly through physically fracturing the virus structure, which was confirmed by real-time reverse transcription polymerase chain reaction. These results provide a pathway toward establishing a new epidemic prevention strategy in open public for airborne virus. Current airborne virus epidemic prevention to be used in public space includes strong chemical inactivation, UV irradiation, and microwave thermal heating. All these methods affect the open public. In 1980s, Robach In this article, we show that SRET from microwave to virus can be efficient enough so that airborne virus was inactivated with reasonable microwave power density safe for the open public. To investigate the SRET efficiency from EM waves to CAVs in viruses, we first developed a theoretical model to describe the relation between the induced stress and the field magnitude of the illuminating microwave. Since the viruses could be inactivated when the induced stress fractures the structure of viruses, we propose to explore the SRET efficiency from microwaves to viruses through measuring the virus inactivation threshold. Based on the proposed model, we studied the inactivation ratio of influenza A (H3N2) virus at dipolar-mode-resonance and off-resonance microwave frequencies as well as with different microwave powers. Plaque assayFrom the transmission electron microscope images, people knew that the virions of influenza viruses are basically spherical balls packing genomes inside. Since the protein and genome have similar mechanical propertiesDue to the spatial confinement, not only electronic but also acoustic energy quantization has been observed in low dimensional systems such as quantum dots and nano-wires. In 1882, Lamb studied the torsional (TOR) and spheroidal (SPH) modes of a homogeneous free sphere by considering the stress-free boundary condition on the surfaceω is the angular frequency of the vibrational mode, R is the radius of the nano-sphere, cland ctare longitudinal and transverse sound velocities respectively. A comparison between the commonly observed wherem*) of core and shell in the analysis, the relative motion of the displacement can be shown in the following equation:In this work, microwaves were applied to excite the dipolar resonance of the whole virus structure. By exciting the dipolar mode of the nanosphere, core and shell with opposite charge distributions would move in opposite directions and will resonate like a damped mass-spring systemz is the relative displacement between the core and shell; b is the damping coefficient, which is related to the surrounding environment; k is the effective spring constant of this system. By assuming z(t) proportional to exp(iωt), one can solve the complex angular frequency of the resonator as:where b/2m*), which corresponds to ω0/2QTherefore the decay rate of the oscillation equals to the imaginary part of the frequency (ω0) of this system is , where q is the total amount of charge distributed in the core and shell region of a virus:The intrinsic resonance angular frequency  due to the location of the maximum distortions, as illustrated in If the required stress threshold Q based on equation Q plays a major role. By changing the pH value of the solution, charge status of viral surface can be modulated, which affects the Q of the vibration. For example, previous studies indicated that the cavity Q of spherical viruses ranges between 2–10 by raising the pH value of the solution from 5.4 to 7.4Q, more energy can be confined inside the resonator, which leads to much lower microwave field threshold magnitude at the resonant frequency.m*) of virus is thus 14.5 MDa. From the literatureq, Q and ω0 of the studied H3N2 virus has to be obtained by measuring the microwave absorption spectrum of viruses.To experimentally study the efficiency of the SRET from microwaves to CAVs of spherical viruses, influenza A virus subtype H3N2 was used. H3N2 is a subtype of influenza A virus that causes flu. Such viruses can infect birds and mammals and are increasingly abundant in seasonal influenza, which kills an estimated 6309 people in the United States each year, including pneumonia and influenza causesL) in order to measure the microwave absorption spectrum of viruses. This microwave microfluidic channel can provide a microwave bandwidth over 40 GHz. The measured results were summarized in Q was only 1.95 by measuring the full width at half maximum of the spectrum. Since the density of viruses (N) in the solution was 7.51014 m−3, experimental absorption cross section of the virus at the resonant frequency can be calculated by the equation below:As shown in From equation By setting Pstress = 0.141 MPa, m* = 14.5 MDa, Q = 1.95 and ω0 = 2π × 8.22 GHz into equation So far, all parameters for estimating electric field threshold in equation f/3)1/5 W/m2 at frequencies between 3 and 96 GHz (f is in GHz)2 at 6 GHz, 122 W/m2 at 8 GHz, and 127 W/m2 at 10 GHz for averaged values of the power densities in air. Assuming all the microwave power in air 100% transmitted into a specimen, and by taking the dielectric constant of water 71.92 (6 GHz), 67.4 (8 GHz), and 63.04 (10 GHz)Based on the IEEE Microwave Safety Standard, the spatial averaged value of the power density in air in open public space shall not exceed the equivalent power density of 100(Ntest) and the titer of unilluminated control sets (Ncontrol) to calculate the inactivation ratio (1 − Ntest/Ncontrol) at different frequencies between 6–12 GHz.To investigate the resonant effect, we first measured the residual viral infectivity of influenza A virus after illuminating microwave of different frequencies. As shown in 2, for microwaves between 6 and 12 GHz. Since the aperture size of our horn antenna was 9.8 cm × 7.1 cm. The required threshold power input ranges from 0.57 W to 3.92 W for 6–12 GHz microwaves. We thus first applied 6.3 W (38 dBm) fixed microwave power, which is higher than all the threshold power input, into the horn antenna for the frequency dependency studies. After considering the transmission coefficient of our horn antenna, this experimental condition corresponded to 765 – 882 W/m2 average illuminated power density on the sample surface, corresponding to the field intensity inside the specimen of 260–296 V/m respectively. For 8–8.4 GHz microwave at the resonant frequency, the average illuminated power density was about 810 W/m2, equivalent to 273 V/m effective field intensity inside the sample. We thus expect to observe the inactivation effect throughout the studied spectral range. As been summarized in Based on 2 in specimen. Since we assume all power can transmit from air to specimen, power density in air is also 82.3 W/m2, which is 1.48 times lower than the IEEE safety standard. 2 in air, corresponding to an effective field intensity inside samples of 274, 171, 87, and 68 V/m, respectively. It is noted that the experiment with 82 W/m2 in air was performed in a different experimental setup, as shown in To further investigate the efficiency of this SRET effect from microwave to virus and the threshold effect, we further measured the inactivation ratio of H3N2 virus with different power densities at the resonant frequency ~8 GHz of the confined acoustic dipolar mode. Our theoretical model predicted an inactivation threshold field intensity of 86.9 V/m, corresponding to an average microwave power density of 82.3 W/m2 (lower than the IEEE safety standard in public space) 8 GHz microwaves on our viral solution, corresponding to an average 87 V/m electric field intensity inside the solution, confirming that our proposed simple model to estimate the field threshold (86.9 V/m) to structurally fracture the virus is quantitatively correct, especially combining the observed threshold effect as discussed above. With a low resonator quality factor (around and less than 2 for H3N2), we also observed virus inactivation in off-resonant frequencies (6-12GHz), following a trend predicted with our model. However for off-resonant frequencies, the simple harmonic oscillator model seems to always estimate a lower threshold in the Stoke-side (lower-frequency-side) of the resonant frequency than the anti-Stoke-side (high-frequency-side). For example, at DC (0 frequency) it still predicts a relatively low threshold field magnitude to fracture the virus. This is different from our observation. We observe that the anti-Stoke-side is with a better inactivation ratio than the Stoke-side, and the source for disagreement should be the over-simplification of the adopted model.Compared with the simple theoretical model for threshold estimation as summarized in Our result regarding the efficient SRET to inactivate virus with a low microwave power has a profound meaning. As we introduced, in the past few decades, tremendous efforts have been made to kill airborne viruses such as SARS or influenza A, which have caused catastrophic illness worldwide. Active airborne viruses are always transported inside tiny water droplets, thus similar to our experimental condition. A strategy for airborne virus epidemic prevention in open public is thus highly desired. Our finding represents the first possible mechanism to inactivate airborne viruses without affecting the open public, since the required microwave power could be within the IEEE safety standard. Comparing this work with traditional microwave thermal inactivation, previous works27272 radiated microwave power density (0.63 W required power input) in our resonant inactivation case, the current microwave thermal heating method to inactivate virus usually requires more than 100 W microwave power at 2.45 GHz272 at a frequency of 6 GHz by using an infrared thermal imaging camera with a temperature accuracy of 0.05 °C . The temperature rise after 15 minutes radiation was 7 °C, from 27.5 °C up to 34.5 °C. We thus exclude the possible contribution of microwave thermal heating effect to inactivation H3N2 viruses under our experimental condition.A previous study2, the microwave frequency was tuned to the dipolar mode resonant frequency of 8.35 GHz, and the illumination time was 15 minutes. To avoid possible existing viral RNA in solution before the microwave illumination, we pretreated the virus samples by adding RNase to degrade the existing RNA outside the viral particles. As can be revealed in To double-confirm our proposed mechanism that the inactivation was through physically fracturing the structure of viruses, we established a fractured virus model by freezing the virus samples with liquid nitrogen and thawed immediately and repeated several times. We then preformed real-time RT-PCR (Reverse Transcription Polymerase Chain Reaction) experiment for RNA amplification in order to compare the results after microwave illumination with the established fractured-virus model. Without protein denature, the established fractured-virus model allowed the viral RNA content to be extracted after fracturing. We then performed the RT-PCR experiments to amplify the extracted viral RNA after fracturing either through the frozen fracturing model or after microwave resonant irradiation. Since all the RNAs outside the virus surface in the solution were eliminated before the microwave illumination, for the pretreat samples the only way to detect the RNA signal after the illumination was to fracture the virus so that the RNA can be released. This result was also in good agreement on the amount of amplified RNA in the control cases. These facts confirmed that the inactivation of viruses by illuminating microwaves at the dipolar mode resonant frequency was through physically fracturing the lipid-envelope of the influenza virus without denaturing the viral RNA.2, corresponding to an effective electric field of 167 V/m inside the specimens. The rest of the experimental conditions were the same as that of To show that the investigated SRET effect can be applied to deactivate viruses other than H3N2, we have also performed the real-time RT-PCR experiment on H1N1 virus. The result was shown in In summary, we investigated the structure resonance energy transfer from microwave to CAVs of H3N2 virus in water-based solution. The efficiency of such energy transfer was investigated through exploring the virus inactivation ratio. Based on the proposed damped mass-spring model and the experimentally measured microwave absorption cross-section of a single virus, threshold magnitude of electric field to fracture viruses at different illuminated frequencies can be estimated. After the illumination by the microwave, the plaque assay experiment indicated that the inactivation ratio reaches its maximum at the resonant frequency of the dipolar resonance. The real-time RT-PCR experiment double confirmed that the main inactivation mechanism was through physically fracturing viruses without degrading viral RNA genome. This work not only theoretically and experimentally demonstrates a new energy transfer mechanism between EM waves and viruses, but also indicates an efficient SRET effect. Our results have important implications for the interaction between microwaves and biological tissues, which is a highly concerned public issue. With an observed inactivation threshold with a microwave power density within the IEEE safety standard, the demonstrated SRET mechanism also provides a pathway toward establishing a new epidemic prevention strategy in open public for airborne viruses.2 flasks, inoculated by influenza A virus H3N2  or H1N1  with multiplicity of infection (MOI) of 0.01 and incubated at 37 °C in a 5% CO2 incubator for 2–3 days. When the cytopathic effect (CPE) of the inoculated cells reached to 3 + (75%), we harvested the viral supernatant. The viruses were aliquot and stored at −80 °C for further use.To prepare the H3N2 and H1N1 virus samples, Madin-Darby canine kidney (MDCK) cells were confluent grown in 75 cmL). The gap between the signal electrode and the ground electrode of the CPW was 25 μm. In order to decrease microwave loss on the electrodes, the gold layer thickness of the electrode was 1.2 μm. On the surface of electrodes, we grew a thermal isolator layer of silicon dioxide on the sensing zone by Plasma Enhanced Chemical Vapor Deposition (PECVD) to lower the temperature rise of fluids due to microwave dielectric heating14 m−3. The spectrum of the solution without viruses was first measured as a reference; solution with viruses was later measured. By removing the solution background, the microwave absorption spectrum of H3N2 viruses in solution was thus obtained (The microwave absorption spectrum measurement was performed by combining the coplanar waveguide (CPW) circuit with a microfluidic channel. As shown in obtained  followin14 m−3 particle density) under illumination was drop on the slide and covered with a cover glass or was contained inside the cuvettes. Under such an experimental geometry, we illuminated viruses for 15 minutes at various microwave frequencies or at various microwave powers. After illumination, we used buffer to wash-down and collect the viruses, which introduced a 10-fold dilution of virus concentration. Then the illuminated viral solutions, together with the control sets were sent for plaque assay.In our experiments, we used two different microwave sources, depending on the utilized microwave power: a network analyzer  or an Yttrium iron garnet (YIG) oscillator. Then the microwave signal was amplified by a power amplifier (QPJ-06183640) and radiated from the horn antenna . The aperture size of the antenna was 9.8 cm × 7.1 cm. To avoid damage on oscillator and amplifier caused by back reflection, we added an isolator and a directional coupler. All the microwave systems were put in a P2 class flow hood. The antenna was directed toward the bottom  or the s5/mL) to individual well. After confluent growth of MDCK cells in plates, the cells were successively washed with phosphate buffer saline (pH 7.2) and Eagles’ MEM with 2 μg/mL TPCK trypsin. After washed, 100 μL of the ten-fold serial dilution of viruses were added into each well. For better virus adsorption, the inoculated cells were incubated at 37 °C in a 5% CO2 incubator for one hour. After then, the virus inoculums were removed and the Eagles’ minimal essential medium with 2 μg/mL TPCK trypsin and 0.5% agarose was added. After the gel formation, the plates were put in a 5% CO2 incubator for at least 42–48 hrs. After incubation, the plates were fixed with 10% formalin for 1 hour. After pour off agarose, the fixed cells were stained with crystal violet for 15 min and washed with tap water. Below certain virus concentration, the plaques wouldn’t overlap each other seriously and can be counted unambiguously. Considering the corresponding dilution factor, the plaque forming unit per mL (pfu/mL) of the original virus can thus be quantified. The titer measurement will be performed three times on the same sample to reduce quantization error. Quantified by the plaque assay, the concentration of active viruses in our prepared viral solutions was around 107/ml for H1N1 and H3N2.To measure the activity of viruses, we employed a quantitative plaque assay. The MDCK cells used for plaque assay were grown in 6 wells plates by adding 3 mL of cells  with primer AMF (sequence: 5′-GAGTCTTCTAACCGAGGTCGAAACGTA-3′), primer flu-AR (sequence: 5′-CAAAGCGTCTACGCTGCAGTCC-3′) and flu A (5′-FAM-tttgtgtttacgctcaccgt-TAMRA-3′) probe. For control group, RNase was added for 10 minutes digestion and stopped with RNase inhibitor. Then viruses were fractured artificially by freeze-thaw treatment. We thus were able to observe that the RNA signal of virus rose after 15 cycles of the amplification. For pre-treated groups, the RNase treatment were done and stopped before 8 GHz microwave illumination in order to make sure that RNA outside the viral envelope was eliminated in the first place. For the post-treat groups, RNase was added right after the illumination. If the virus particles were fractured, the released RNA would be degraded by RNase.How to cite this article: Yang, S.-C. et al. Efficient Structure Resonance Energy Transfer from Microwaves to Confined Acoustic Vibrations in Viruses. Sci. Rep. 5, 18030; doi: 10.1038/srep18030 (2015).


Rhipicephalus spp. tick pools  collected from the Menglian district of Yunnan, China. The sequencing run resulted in 3,641,088, 3,106,733, and 3,871,851 reads in each tick pool after trimming. Reads and assembled contiguous sequences (contigs) were subject to basic local alignment search tool analysis against the GenBank database. Large numbers of reads and contigs related to known viral sequences corresponding to a broad range of viral families were identified. Some of the sequences originated from viruses that have not been described previously in ticks. Our findings will facilitate better understanding of the tick virome, and add to our current knowledge of disease-causing viruses in ticks living under natural conditions.Besides mosquitoes, ticks are regarded as the primary source of vector-borne infectious diseases. Indeed, a wide variety of severe infectious human diseases, including those involving viruses, are transmitted by ticks in many parts of the world. To date, there are no published reports on the use of next-generation sequencing for studying viral diversity in ticks or discovering new viruses in these arthropods from China. Here, Ion-torrent sequencing was used to investigate the presence of viruses in three  Ixodes, Haemaphysalis, Hyalomma, Amblyomma, Dermacentor, Rhipicephalus, and Boophilus [Ticks are second only to mosquitoes as important arthropod vectors for spreading viruses from wildlife to domestic animals and humans. They are also a source of unknown viruses. To date, at least 38 known viral species are transmitted by ticks, and some of them are a significant threat to human health . Such vioophilus .Phlebovirus genus of the family Bunyaviridae, was found to be the causative agent of severe fever with thrombocytopenia syndrome. Later on, Haemaphysalis longicornis was identified as the primary vector of SFTSV [Amblyomma americanum ticks have been suggested as potential vectors of this disease [In recent years, novel tick-borne viral diseases have emerged worldwide. From 2009 to 2011, an acute febrile illness of tick-borne origin was noted in several Chinese provinces and killed about 30% of the people infected. The novel virus, SFTSV, which belongs to the of SFTSV . HRTV, a disease , 9.Amblyomma americanum, Dermacentor variablilits, and Ixodes scapularis ticks in the United States was published. Their results reveal novel highly divergent viruses in ticks, which include nairoviruses, phleboviruses, monoegavirusand viruses with similarity to plant and insect viruses [Next generation sequencing (NGS) technologies provide a powerful means of studying viral metagenomics, and can help us to gain better understanding of viral populations and discover unknown viruses in a number of environments , 11. NGS viruses . HoweverRhipicephalus is known to associate with many viral pathogens, such as Thogoto virus , Wad Medani virus , CCHFV and NSDV , Kismayo virus and Chim virus , and Kandam virus , all of which can cause disease in livestock and humans [Rhipicephalus is the one of the most common tick genus in Menglian and generally throughout southwest China [Rhipicephalus microplus, R. haemaphysaloides and R. sanguineus are very common tick species in this region.Recent reports indicate that many tick-borne diseases exist in the Yunnan Province of China, such as Kyasanur forest disease, Crimean-Congo hemorrhagic fever, Colorado tick fever, and severe fever with thrombocytopenia syndrome –20. The d humans . Rhipicest China , 23, whiRhipicephalus spp. ticks collected from the field in the Menglian district of Yunnan, China. The viral communities from three tick pools from two collection sites were analyzed and compared, and numerous virus-related sequences were identified.In this study, Ion-torrent sequencing was used to investigate the presence of viruses in A total of 387 ticks were collected in the Menglian district of Yunnan, China in 2011 and 2013 . One-hunRhipicephalus spp. ticks as a pool for Ion-torrent sequencing , 30 cycles/sec with 1 ml of phosphate-buffered saline and 5 mm stainless steel beads, and then then centrifuged at 5,000 × g for 10 min. The supernatant was transferred to a fresh tube and then centrifuged at 13,000 × g for 15 min. The supernatant was filtered through a 0.45 μM Millex filter  to remove eukaryotic and bacterial cell-sized particles. Total viral nucleic acids (DNA and RNA) were extracted from a 140-μl sample (from the above step) using a QIAamp Viral RNA Mini kit  according to the manufacturer’s instructions. Nucleic acids were eluted into 40 μl of AVE buffer [2, 2 μl of 0.1 M DTT, 1 μl of RNaseOUT(40 U/μl) and 1 μl of SuperScript III RT (200 U/μl) was added to each nucleic acid–primer mixture. The reaction mixture was incubated at 25°C for 10 min and 50°C for 50 min, followed by enzyme inactivation at 85°C for 5 min and chilling on ice for 1 min. For the second-strand cDNA synthesis, 0.5 μl (20 pmol) of Brs primer, 2.5 μl of 10 × Klenow fragment buffer, and 2 μl of Klenow fragment  were added. The reaction mixture was incubated at 25°C for 10 min and 37°C for 60 min, followed by enzyme inactivation at 75°C for 10 min [First-strand cDNA synthesis was performed with a SuperScript III First-Strand System Kit . An 8 μl volume of purified viral nucleic acids from each tick pool was mixed with 1 μl of 10 mM deoxynucleoside triphosphate and 1 μl (50 ng/μl) of Brs primer (a random primer with the tag sequence at the 5’ end) , after wr 10 min .PCR assays were performed using a primer identical to the tag sequence of the Bra primer and using Phusion High-Fidelity DNA polymerase , and lower cycles (≤20 cycles) were used to reducing amplification bias. Next, the products were purified to acquire three cDNA libraries. Each cDNA library was loaded onto one single Ion-Torrent 318 chip, and Ion-Torrent sequencing  was performed by the Beijing Institute of Genomics at the Chinese Academy of Sciences.http://edwards.sdsu.edu/cgi-bin/prinseq/prinseq.cgi) and Mothur v.1.33.0 (http://www.mothur.org/). The metagenomic reads were subjected to a basic local alignment search tool (BLASTx) comparison against the National Center for Biotechnology Information  non-redundant database (nr) and its viral database by using an E-value cutoff of 1e-04. The taxonomy of the viral community was analyzed and compared using the MEGAN5 analysis toolset (http://ab.inf.uni-tuebingen.de/software/megan/). The reads that hit viral database sequences were assembled by a metagenomic de novo assembly for the Ion-Torrent model of SeqMan NGen 11.2.1 , and the contiguous sequences (contigs) obtained were subject to BLAST against GenBank using BLASTn and BLASTx.Raw data were filtered into short (<100 bp) and long (>350 bp) reads, low quality scores (<20), exact duplicate reads and trimmed primer sequences by PRINSEQ 0.20.4 . Gaps were regarded as a pairwise deletion unless specifically noted.The nucleotide sequences and translated amino acid sequences of contigs with high similarity to known viral nucleic acids and proteins in GenBank were used for phylogenetic analysis. Alignments were performed by ClustalW and phylogenetic trees were constructed by the Maximum Likelihood method with a bootstrap of 1,000 replicates through MEGA 6.06 , about 12.2%  of reads from NY-11, 3.0%  of reads from NY-13, and 10.3%  of reads from MM-13 were corresponded to known viral sequences.The reads identified from the three tick viromes were classified into bacteria, invertebrates, vertebrates, plants, algae, and protozoan viruses  in three viromes were identified . Next, t−6, −6; From the BLASTn results  which encoding RNA polymerase, pre-glycoprotein and nucleoprotein, respectively [−6) relationships with Nairovirus (Nairovirus nucleoprotein (40.66% identity). The contigs corresponding to the Nairovirus L protein cover most of the L protein ORF. Additionally, the cross-blastn results revealed overlapping nucleic acid regions between NY-11 and NY-13 contigs (related to Nairovirus) that had almost the same sequences (99.8% identity). The specific primers  with 90% coverage of the Nairovirus L protein.ectively . Numerouirovirus . Contig  primers  based onNairovirus genus, but was distantly related to the known nairoviruses (Nairovirus genus. This virus was named as Nayun tick nairovirus (NTNV).The tick nairovirus-related nucleoprotein amino acid sequence was used to determine phylogenetic relationships. The result showed it clustered with the oviruses , suggestThetatorquevirus, a recently discovered genus of the Anelloviridae family, is a small, non-enveloped DNA virus with a circular single-stranded DNA genome (∼2–3 kb) containing three or four overlapping ORFs  [Anelloviridae in sample NY-11 is a novel virus cluster with TTcV (nd ORF3) . The knond ORF3) , and ticith TTcV .Rhabdoviridae family presents in vertebrates, invertebrates and plants. The viral genome is a negative-sense single-stranded RNA containing at least five ORFs that encode its structural protein . Currently, Rhabdoviridae consist of nine named genera [Lyssavirus) is the best-known member of this family which can cause lethal disease of human. Many viruses from genus Vesiculovirus are typical arboviruses. There were many reports about rhabdovirus from ticks. Such as Isfahan virus which belongs to the genus Vesiculovirus has been isolated from Hyalomma asiaticm ticks in Turkmenia [Amblyomma variegatum ticks in the Republic of Guinea [Amblyomma americanum ticks in New York State of American [Vesiculovirus. A partial sequence from the large polymerase protein (L) region (Genbank Accession Number: KP141757) was used for determining phylogenetic relationships. However the phylogenetic tree revealed that tick rhabdovirus from NY-13 represents a distinct and divergent lineage which shows no clear relationship to Vesiculovirus and any other named genus  is the best characterized member of this family, with a genome size of about 6.4 kb in length [Tobamovirus. All of them relating to TMV had 89–98% sequence identity scores and comprised ∼52% coverage of the TMV genome.n length . We idenMyoviridae, Podoviridae and Siphoviridae as well as unclassified phages  and Salmonella (the host for the phage FSL SP-031) bacteria have both been reported in ticks previously [The phages identified in the tick virome may have originated from the bacterial flora of the tick or that of the hosts they had fed upon. eviously , 35.Sclerotinia sclerotiorum, is a single-stranded DNA virus with a circular genome (∼ 2.2 kb) containing two major ORFs, a 312 amino acid coat protein and a replication-associated protein of 324 amino acids. This virus is the only known example of a DNA virus that infects a fungus. In seven contigs, five of them  were related to the coat protein, and two of them (contig637 and 846) to the replication-associated protein of SSHSDV-1.Two large contigs (contigs 16 and 839) in NY-11 share amino acid sequence similarities with the major capsid protein of unclassified ssDNA viruses denoted as Dragonfly-associated microphage 1. This microphage is a single-stranded, circular DNA virus with a 4.5-kb genome encoding five ORFs . Furthermore, seven contigs in NY-11 shared amino acid sequence similarities with unclassified ssDNA viruses including the fungal virus Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SSHSDV-1) . SSHSDV-Rhipicephalus spp. tick is well suited as a vector of zoonotic disease because it feeds on a wide assortment of animal hosts in the wild, as well as humans. Moreover, it is a vector for many pathogenic agents, such as CCHFV, NSDV, Chim virus, Thogoto virus, and Kandam virus [Rhipicephalus spp. is extensive in southwest China. Therefore, it is important to use highly sensitivity methods to identify or monitor medically important viruses in Rhipicephalus spp. The present study is the first where NGS was used to explore the variety of viral communities that exist in Rhipicephalus spp. ticks in China. High-throughput sequencing was performed by the Ion-torrent technique (Invitrogen). It can result in higher speed, lower cost, and smaller instrument size. Compared to the Hiseq 2000 technique, the sequencing quality of Ion Torrent is more stable, has a higher map rate, and the GC depth distribution is better [am virus , 37, 38.s better . The IonThrough BLASTx and BLASTn analyses, a large number of viral sequences were found in ticks, some of which had a close relationship with known viral sequences, while some additional sequences potentially corresponded to novel viruses.Nairovirus), followed by phages in NY-11 and NY-13. However, the most abundant sequences in MM-13 were phages, followed by invertebrate viruses, while there were only a few sequences corresponding to animal viruses  scores between the contigs relating to Nairovirus from NY-11 and NY-13. These findings indicated that the same nairovirus-related agent was present in the NY-11 and NY-13 tick samples. The phylogenetic analysis indicated that the NTNV clustered with Nairovirus, but had a distant relationship with known nairoviruses. Unfortunately, we did not obtain the large, assembled contigs corresponding to the Nairovirus M protein (glycoprotein). The lack of recognizable M segment is consistent with the reports about the tick virome in the U.S. [Bunyavirdae M segments were unable to identify. This may be due to the complicated secondary structure of M segments which inhibit efficient cDNA synthesis and interfere with amplification. Or because the glycoprotein has greater variation than nucleoprotein and L polymerase, we can’t get the related sequences through the alignment with the known database.Almost all known nairoviruses are transmitted by ticks, and some of them, such as CCHFV and NSDV, have been isolated from or identified in ivestock . In the the U.S. . They reOur analysis uncovered the anellovirus and phages in ticks, which is very similar to what’s found and proposed in mosquito . The aneCaulimoviridae, Nanoviridae, Geminiviridae, Virgaviridae and Sobemovirus Click here for additional data file.S2 TableTable shows the viral genus, family and host information of the reads from three tick viromes.(DOCX)Click here for additional data file.S3 Table(DOC)Click here for additional data file.


Economic evaluations are a form of applied welfare economics and aim to inform policy makers about the impacts on social welfare of particular policies. It is clear that in light of this aim, economic evaluations need to include all relevant societal costs and benefits related to a policy. Only then, a full welfare economic trade-off between costs and benefits can be made to facilitate welfare improving decision making.It is clear that current economic evaluations in the field of health care commonly do not include all relevant costs and benefits related to interventions. In many jurisdictions, a health care perspective is prescribed, which precludes the inclusion of relevant societal impacts, such as productivity costs, costs of informal care and costs in other sectors.Also in countries prescribing or allowing broader perspectives, the inclusion of broader costs and benefits related to health care interventions are often ignored. This partly is related to a lack of methodological development and consensus.In this presentation, I will highlight some of the main, often ignored, categories of societal costs and benefits, also touching on the methodological development in measuring and valuing these. I will also briefly address the need for investigating the monetary value of health and for inclusion of equity considerations in economic evaluations.Only by enriching the evidence base provided by health economic evaluations can these ultimately contribute to welfare improving decisions.


This paper is focused on examining the number of deaths’ increases participation in the propagating the Ebola virus during the period ranging from March to October 2014. An application of the MGARCH-DCC model regressions on four countries has led to discover that the finding that human contact play a significant role in transmitting the Ebola virus. Our findings also reveal that Guinea has already suffered from a spread-like virus originating from Sierra Lione and Liberia. Noteworthy also, other countries are now liable to such a risk; for instance, Nigeria is a country vulnerable to the propagation of this virus. Consequently, we undertake to conduct our forecasts for EGARCH model estimates implements; which has estimated a decrease in the Ebola virus incurred number of deadly Ebola virus over the two months following the November and December. Discovered in 1976 in the western equatorial province of Sudan and in a nearby region of northern Zaire (now Democratic Republic of Congo (DRC)) a, commono (DRC)) -16 and 31] to eqyt} with an appropriate estimator This exact LW procedure as frequency labelled, implies replacing 2dIλj in  by \docuμ = 0 in . Since tyt} is given by a process similar to equation  are used to compute the feasible estimator as follows:For the purpose of attaining, a feasible procedure, he considers two necessary steps, the first of which serves to determine an estimator of d < 2. Similarly, and as indicated as shown by ’. Each variable constitutes a constant function along with its own past values. Thus, the autoregressive process reduced form is written as:In this section, the dynamic conditional correlations’ two-stage model will be treated as proposed by . Let us A(L) is the polynomial delay, and εt = [εt1εt2]’is a vector of residuals training from the estimation auto regression process for each variable whose variance-covariance matrix is described by Ht = {hi}t with s.where: Ht such as:Actually, the DCC-MGARCH model can be easily apprehended by rewriting the matrix of variance-covariance Rt = {ρij,t} representing the conditional correlation coefficients matrix. The Dt contained elements are generated into a GARCH  process, which can be formulated as:where: In addition,  considerQt = {qij,t} represents the unconditional variance-covariance matrix, which are temporally invariant. The parameters  are supposed to respectively intercept the shock effects and delay the dynamic correlations at the level of recent contemporary. As for Q*t, it stands for a diagonal matrix containing the square root of the main diagonal elements of Qt. Regarding to our example, this matrix is written as:where: Rt whose main diagonal consists of 1.with: The model parameters are estimated via the maximum likelihood DCC method. In this regard,  have shoRt within the log-likelihood the function. The advantage of such a method is that it allows for getting the likelihood function sum of the GARCH univariate models. In other words, this initial step, series to achieve we obtain the parameters of equation . In addition, if the Ebola dynamics were to be modeled in real time, it would be critical to consider possible delays while report some cases and outcomes.In fact, the Ebola virus can persist in the patient’s body for about 2 months and, the sick and dead people are contagious. Following the incubation period ranging between 2 and 21 days (a period with no symptoms and no contagious), the first symptomsThe Ebola virus transmission is marked by two major phases. The first started in July 2014, when Guinea, Liberia and Nigeria become affected with the shocks. As for, the second phase, it started in August 2014, when all the four countries become affected by at least a single shock during that month. Concerning Guinea and Nigeria, there were structural changes for two months, September  and August . This fact indicates an approximation of the Ebola virus quiet periods boundaries and times. This denotes that the number of fatal Ebola viruses appears to be is more dispersed during these two months owing to the increasing number of Ebola virus incurred deaths. As a matter of fact, 494 deaths were recorded in August reaching 739 deaths in late September in Guinea.In Liberia, one might well note that the four points marking structural changes through the ICSS algorithm are sited during August and early September. During the end of the period, the number of deaths increased remarkably from 260 to 953. In regard of Nigeria, only two structural change points have been highlighted occurring between late August and early September. This might well has its explanation in the increase noticed in the number of deaths growing up from 1 to eight due to the Ebola virus. Unless it is imperative, the studied countries are recommended to suspend all their flights towards the countries where the Ebola haemorrhagic fever cases have proven to be remarkably serious, such as Guinea, Sierra Leone, Liberia and Nigeria. Indeed, for five months, the Ebola outbreak was the worst since the emergence of the initially high contagious hemorrhagic fever in 1976. Actually, it was at the origin of 1,145 reported deaths according to the World Health Organization (WHO), reporting 413 cases in Liberia, 380 in Guinea, Sierra Leone and 348 in Nigeria.0 is shown to be negative and significant except for Liberia and Nigeria. This may well further evidence in favor of variance reduction after a positive shock and vice versa.Such interesting findings appear to be unable to capture the asymmetry of volatility reaction to external shocks. For this reason, an EGARCH  model is estimated whose empirical results are reported in Table 0 appears to differ greatly between (Guinea and Sierra Leone) on the one hand, and (Liberia and Nigeria) on the other hand. This suggests that the volatile countries’ exposure and liability to risks varies not only among (Guinea and Sierra Leone) and (Liberia and Nigeria), but also among the countries under review. Obviously, this might have an association with the number of deaths which seemed to be lower with respect to (Liberia and Nigeria) than to (Guinea and Sierra Leone), mainly on the onset of the Ebola virus disease. As for parameter δ1 which serves to measure the conditional volatility persistence, its estimators prove to be significant. The highest value related to this parameter has been recorded for Guinea and Liberia exceeding 0.9, in respect of its value registered in Sierra Leone and Nigeria. It also reveals volatility dynamics which appear to be more persistent with regard to Guinea. Besides, our results appear also to confirm the persistence of asymmetry in the volatility reaction to shock size. Indeed, the estimated value of γ1 is positive for all the countries except for Liberia, scoring its highest points in (Guinea and Sierra Leone), suggesting that the volatility dependency to market events is more important for both Guinea and Sierra Leone.Noteworthy, however, the estimated value of γRegarding the number of deaths registered in Guinea, Sierra Leone Liberia and Nigeria, 260 daily data were pointed during the entire period from: 2014-03-22 until 2014-12-30. The in-sample period: from 2014-03-22 until 2014-10-14 consisted of 206 daily data points while the out-of-sample period from: 2011-01-03 until 2014-04-15 consisted of 54 daily data points. In Figure A decrease in the number of Ebola virus incurred deaths was noticed during the following two months (November and December 2014). This is mainly due to a drastic reduction in the number of animals natural reservoirs of the virus. Indeed, the upper and lower figures appear to have a similar shape to the graph in blue. This indicates well a significant decrease in the number of the Ebola virus caused fatalities in Guinea. The virus transmission remains intense in Guinea with a clear exponential in the daily deaths number time series. In Sierra Leone, for instance, one may notice that the number of daily Ebola deaths remained constant during the following two months (November and December). This number marked some peaks from one day to another. As for Liberia, the number of the Ebola viruses is expected to increase over the next 60 days (November and December). Liberia contributed strongly in the high number of the whole outbreak cases registered during the last two months (November and December). Regarding, Nigeria, the number of forecast deaths registered via EGARCH process highlights this figure constancy over the coming months (November and December). For the sake of determining the causal relationship between Ebola and the studied countries, the Granger causality test is applied as introduced by .This part is interested to test the presence of Granger causality relationship in a bivariate model among different series  on the studied countries. To note, the Granger causality test is a statistical hypothesis test helpful in determining whether a certain time series is useful for forecasting another (see ). OrdinaGroup 1: it is composed of four countries; Guineag is the origin source of Ebola virus, i.e. the African countries are recently affected with the Ebola virus through Guinea. This group includes Guinea, Sierra Leone, Liberia and Nigeria.Group2: is composed of such countries as Guinea, Liberia and Nigeria which are affected by Sierra Leoneh where a high number of deaths predominate.Group 3: consists of such countries high as Guinea, Sierra Leone and Nigeria affected by the Ebola virus as transmitted from Liberiai.Group 4: includes Guinea, Sierra Leone and Liberia in which the virus is transmuted from Nigeriaj.In this section, the Granger causality tests are going to be administered, using. We use the unrestricted model through which causality will be investigated between the number of deaths respective to Guinea, Sierra Leone, Liberia and Nigeria. The relevant results are summarized in Table The number of causal relations hips is depicted in the following table. A causal relationship is persistent in each of the first three groups, but no relationship has been noticed to exist in the fourth group.According to Table Moreover, it is clear that the disease in Sierra Leone has resulted in the same disease in Liberia . Accordingly, one might well infer that the Ebola virus may have been spread from Guinea through Sierra Leone to Liberia. It has also been shown that the Ebola virus has been spread from Liberia to Sierra Leone. These two findings several that the Ebola virus native countries are discovered to be Guinea and Sierra Leone. For the purpose checking the right transmission of the Ebola virus among these countries, we have made appeal to the DCC-MGARCH technique. In fact, this framework enables us to estimate two main parameters, the first of which helps determine the persistence of the Ebola virus, while the second serves to demonstrate the Ebola virus shock contamination effect through the considered countries.am and bn, respectively, underlining the persistence of shock effect and contamination. Based on Table am proves to be high and significant (0.583) highlighting the continuity of the Ebola virus impact. Consequently, a high probability remains as to the virus transmission from Guinea to Nigeria. As matter of fact, coefficient am has been demonstrated and deemed important because the study period has covered only 8 months (March-October 2014). During this fairly short period, several structural changes have been noticed to take place  as figured in Table bn, the Ebola virus spread transmission is observed to occur between Guinea-Liberia, Guinea-Sierra Leone and Sierra Leone-Liberia. Yet, parameter bn appears to be not significant owing mainly to of the lack of data (only 206 observations) and the GARCH family. Indeed, the latter entails the availability of a large amount of data, parameters’ cruciality. For this reason, we have reckoned it more useful to apply the DCC-MGARCH rather than the BEKK-MGARCH which admits further parameters than does the DCC version. As results, we consider it’s rather effective to study the Ebola virus contagion via DCC figures.The two major main parameters considered are Figure k.On August 26, the African Development Bank estimated that Ebola outbreak could cost between 1 and 1.5 percentage points of the GDP in Liberia, Sierra Leone and Guinea, starting it a “country which began to recover difficult years of crisis, civil wars of the 60s, 80s and 90s”l.On September 17th, a report released from the World Bank warned against a “catastrophic” economic impact the epidemic may have on Liberia, Sierra Leone and Guinea; due to the virus associated “fear factor” paralyze activities. According to the World Bank orientations, the combined GDPs of Liberia, Guinea and Sierra Leone could be cut by $ 359 million in 2014 and $ 809 million by 2015 if the epidemic were to remain uncontained. As a result, economic growth would then fall, in the following year, from 11.7 points to 8.9 points in Liberia and Sierra Leone, with a risk of plunging the two poorest countries in recessionCriticized in an internal report for the delay to take the necessary measure relevant to curb Ebola outbreak, the World Health Organization announced that the two anti-Ebola vaccines would be tested as soon as possible in Switzerland, subject to authorization of the medical authorities. French researchers beginning to the Commission for Atomic Energy and Alternative Energies (CEA) have also announced that a rapid test has been developed to diagnose the virus within fifteen minutes. Since its introduction in early March, the virus has been responsible for killing more than 4,500 people, mainly in Liberia, Sierra Leone and Guinea, according to the latest WHO released report (October 2014).Throughout, the scone of this study paper attempts have been made to test whether the Ebola virus can stand as an origin source of contagion during 2014 transmission process using the number of deaths data available from the World Health Organization. More particularly, we have undertaken to examine whether the Ebola virus can lead to contagious effects implicated on both conditional means and volatilities of its relevant number of deaths occurring during the recent period (March-October 2014). In fact previous studies have failed to take account of the important distinction both of the interdependence and contagion concepts. In this paper, contagion is defined as being the significant spillover of the asset-specific idiosyncratic virus during the crisis and as well following the first affection by the Ebola virus . For an The empirical findings have revealed that contagion-in-mean effects turn out to be multidirectional given the fact that the Ebola virus chocks emanating from any of the four studied countries can sweep across throughout the entirety of investigated countries; however, contagion-in-volatility effects appear to be driven mainly by the Ebola epidemic stemming negative return shocks. This empirical result indicates well that shocks to countries’ return can eventually become contagious not only at the volatility level, but also at the mean level, significantly implying that the number of deaths communication can actually constitute a major cause of contagion throughout the recent period.aThe virus family of which Ebola is a member, “Filoviruses” are far more ancient than previously thought.bEbola-Reston virus was recognized in monkey export ability in the Philippines. No human virus was identified.cSpecifically, contagion is defined in this paper as being significant spillovers of asset-specific idiosyncratic shocks during the crisis, after the Ebola virus or systematic risks have been accounted for [nted for .dWeekly data are used here to get meaningful statistical generalizations and obtain a better picture of the movements of Ebola virus cases’ number.eAs a first step, stationarity in the time series is checked by applying the Augmented Dickey Fuller (ADF) test. The results allow us to reject the null hypothesis stipulating that the Ebola virus returns have a unit root in favour of the alternative hypothesis .fFever, weakness, muscle and joint pain, diarrhea, internal and external bleeding.gIn Guinea, there are 1298 cases, of which 1062 are confirmed, with 768 deaths. The epidemic epicenter is Guéckédou, Guinea, but other affected provinces include also’: Beyla Conakry, Coyah, Dabola Dalaba, Forecariah Macenta Nzérékoré Kindia Kérouané, Kissidougou Dubréka and Lola Yomou. The conakry isolation center was set up by MSF. It seems that appearence of new cases in the Guéckédfou region is decreasing .hIn Sierra Leone, there are 2948 cases with 880 deaths, among 2596 confirmed cases. All regions are discovered to be affected. The isolation and diagnosis center is sited in Kenema. The MSF has also implemented an isolation Kailahun and Lakka structures  on the outskirts of Freetown. The Choitrams Hospital can no longer accept hospitalization.iIn Liberia, there are 3924 cases in total, 941 of which were confirmed, in 1795 with 1188 and 2210 suspicious deaths. It is likely that these figures are far below reality. The Diagnostic Center is located in the Liberian Institute of Biomedical Research (LIBR) near Monrovia. The hospitals fully devoted to the Ebola case are: ELWA, ELWA3, JFK in Monrovia, are hospitals which cannot cope with the influx of new patients.jIn Nigeria, the last new case dates back to September 5 in Lagos and Port Harcourt September 1 (15 in Lagos and 4 in Port Harcourt). The epidemic appears under control in Nigeria but there are still 25 subject contacts under observation. The US CDC reduced its recommendation travel Level 1 (can proceed with caution).kEbola could cost “a lot” to the African economy, Le Monde, August 26, 2014.lEbola: an economic impact “catastrophic” envisaged by the World Bank, Le Monde, 17 October 2014.


There has been significant attention paid in recent years to the economic costs of health service provision for people with palliative care needs. However, little is known about the costs incurred by family caregivers who typically provide the bulk of care for people at the end of life.To explore the nature and range of financial costs incurred by family caregiving within a palliative care context.In-depth qualitative interviews were conducted with 30 family/whānau caregivers who were currently caring for someone with a life-limiting illness or had done so within the preceding year. Narrative analysis was used to identify impacts and costs at the personal, interpersonal, sociocultural and structural levels.Auckland, New Zealand.Costs of caregiving were significant and, for participants, resulted in debt or even bankruptcy. A range of direct  and indirect costs  were reported. A multi-level qualitative analysis revealed how costs operated at a number of levels . The palliative care context increased costs, as meeting needs were prioritised over cost. In addition, support from statutory service providers to access sources of financial support was limited.Families incur significant financial costs when caring for someone at the end of life. Research is now needed to quantify the financial contribution of family and whānau caregiving within a palliative care context, particularly given attempts in many countries to shift more palliative care provision into community settings. What is already known about the topic?Family members provide the majority of care for people with palliative care needs.Research conducted with older people indicates that the financial costs associated with family caregiving can be significant.Little is known about the nature and extent of these costs within a palliative care context.What this paper adds?This study identifies that family caregivers experience a range of direct and indirect costs associated with caregiving.The palliative care context exacerbates many of these costs.Support from statutory service providers to access sources of financial support was limited.Implications for practice, theory or policyThis study indicates an urgent need for policy makers to consider the financial costs of family caregiving, particularly within the context of drives to increase community-based palliative care provision.Future research is needed to quantify these costs.Culturally appropriate methodological approaches must be used to ensure the inclusion of research participants from indigenous and minority cultural and ethnic groups who are often disproportionately affected by the costs incurred.2 One perceived benefit of this approach is a reduction in acute hospital costs for people in the last year of life; it is argued that this money would be better spent providing community health services which could enable people to remain in their own home up until death.3 However, one issue that has been neglected within this debate is that reducing hospital admissions would have implications not only for community service provision but also for family caregivers who, it has been estimated, already provide 75%–90% of home-based care for people who are near the end of life.4A key policy priority in many developed countries is shifting the provision of palliative and end-of-life care from acute hospital settings into the community.5 Indeed, studies were identified which demonstrated that economic costs associated with caregiving have negative implications for the well-being and health of family caregivers,7 and financial strain is even associated with family preference for comfort care over life-extending treatment.8 Since the review was conducted, one article has been published which does quantify the costs of caregiving for family caregivers in Canada. Drawing on data from the family caregivers of patients with cancer accessing a home-based palliative care programme, Chai et al.9 concluded that ‘unpaid care costs accounted for the largest portion of total palliative care costs, averaging 76.7% over the last year of life’ (p. 34). This study therefore confirms that, within the Canadian context, family caregivers of people with cancer utilising specialist palliative care services are incurring a far greater proportion of caring costs at the end of life when compared with those incurred by health services. Research is now needed which extends these findings beyond cancer to other diagnostic groups and national contexts.A recent systematic review conducted to explore the financial costs incurred by family caregivers identified no previous studies focusing specifically upon the financial costs of family caregiving at the end of life, although there was some evidence that these costs were significant.10 The failure to adopt a societal approach to costing by including family caregiving costs runs the risk of decision-making at a policy level which shifts even more of the costs of caregiving onto families, with significant implications for both their well-being and their capacity to care.In the absence of further research, there is a danger that economic evaluations conducted within a palliative care context will only capture those costs which can be easily measured, namely, those incurred by statutory service providers. For example, the recent UK palliative care funding review to inform policy making and commissioning decisions in relation to palliative care focused only upon those costs incurred by the state.http://www.teara.govt.nz/en/whanau-maori-and-family/page-1)).To explore family and whānau carers’ experiences of the financial impact of caring within a palliative care context  were recruited through a tertiary hospital palliative care service. Clinical staff identified eligible carers and invited them to talk to a research nurse if interested in participating. Only three of the carers approached declined participation in the study, all for reasons unconnected with the research itself. A second recruitment strategy was adopted to ensure appropriate representation of Māori caregivers. A total of 13 participants (9 Māori and 4 non-Māori) were recruited via community newspaper articles about the research and Māori radio and TV coverage. Māori, the indigenous people of Aotearoa, New Zealand, tend to have poorer health than non-Māori and a higher level of unmet need for health care generally.15 Māori who live in more deprived areas are more likely to have greater unmet primary health care needs and therefore face a potentially higher cost burden relative to more affluent carers, so were an important target group for this research.15 Previous research has indicated that targeting community media is a successful strategy for recruiting Māori, who are under-represented not only in research16 but also as users of specialist palliative care services.17 Sampling was purposive to include a mix of information-rich participants with diverse demographic characteristics  and involved in various types of carer relationships.18 Māori were over-sampled as they comprise 14.9% of the population but are over-represented in health care inequity. Sufficient diversity in terms of these characteristics was believed to have been achieved when we had recruited 30 participants, which is also considered an adequate sample size for an in-depth, exploratory study of this type.18 Interviews were conducted by T.M.M. (Māori participants) and R.A. (non-Māori participants). Participants were recruited and interviewed over 6 months from November 2012. All participants provided written informed consent prior to interview. Participant details are summarised in The choice of qualitative methods was guided by the research questionn = 25) by telephone (n = 2), at the hospital (n = 1) or in a café (n = 2) depending on participant preference. Interviews lasted between 30 and 90 min. All interviews conducted by the Māori researcher incorporated a Kaupapa Māori approach. Kanohi-ki-te-kanohi (face-to-face) interviews catered for the rangatira (chiefly status) of participants and Māori cultural research protocols were observed.20Interviews were conducted at participants’ homes or a relative’s home . Views were also sought about appropriate methods for conducting research in this area; these data will be reported elsewhere. Interviews were digitally recorded with participants’ consent and transcribed verbatim. Summaries of interview data were presented back to participants for their feedback to maximise methodological rigour and, in particular, the confirmability of findings.21The interview guide was informed by relevant literature13 cost areas and cultural concerns. A more focused analysis was then conducted using the narrative gerontology framework of personal, interpersonal, sociocultural and structural dimensions of experience.22 Analysis was led by T.M.M. (Māori) and R.A. (non-Māori); a selection of transcripts was independently reviewed by C.G. and coding was carried out by consensus to ensure rigour and trustworthiness. This framework supported analyses of financial costs which operate at all these levels. Verbatim quotes are presented with anonymised initials and the relationship to the person with life-limiting illness .Qualitative data software (NVivo 10) was used as the filing system for the initial categorising and overview of responses to interview questions, experience-centred narratives,Participants reported a wide range of experiences with relation to the costs of caring and described the significant implications of these costs. Participant data have been summarised under the following themes: the motivations for caring, the range and nature of costs, the impact of the ‘end of life’ phase on costs, the impact on wider family and the lack of support systems. These themes can best be understood within the context of a multi-level narrative gerontology model of personal, interpersonal, sociocultural and structural domains, as will be described below.[Money] never came into the equation … It was a lot of hard work but when I look at it I’m glad I was there and not strangers. While motivations and meanings of care were not the focus of this research, the sensitivities of talking about money ‘at a time like this’ (i.e. when someone had a life-limiting illness) meant participants always framed their talk of costs with comments on the importance and meaning of care to them. As one participant reported,The way I see it is a parent raises a child, their whole role is to look after the child until they become an adult and then I see it, once you’re an adult we should repay that back. You know, because your parent’s health and everything starts to fail as they get older. So my obligation is to them. Māori participants’ caregiving commitment was often informed by cultural values steeped in āroha (compassion) and manaakitanga (preservation of mana and dignity), which were prioritised over care costs. Many participants invoked notions of reciprocity in their discussions:Sometimes food, the children are fed and we adults just have the leftover, so we could make ends meet. But that’s, but we always think of it’s temporary, and whenever the day will come, it will end. Things will be back to what, to whatever we usually do. However, it was clear that the financial costs incurred by caregiving were significant for all participants, although the extent of the financial impact of caregiving was determined by participants’ current financial situation and therefore varied markedly within our sample. Participants who reported the highest levels of financial resource considered themselves ‘lucky’ and reported only having to cut back on planned expenditure, such as a trip overseas to visit a new grandchild, in order to meet their caring costs. However, others with fewer financial resources reported a range of more serious repercussions. These included incurring significant debt, moving to a smaller house and, in the most extreme cases, going without food because they could not afford to buy enough for everyone in the household:Participants reported that a broad range of direct and indirect costs were incurred by caregiving see .Direct costs were those involving direct outlays of money. Those most frequently mentioned were parking and transport costs related to their family member’s hospital appointments and admissions. Costs of clothing and bed linen were also mentioned by many participants; for example, some care recipients required new clothes following weight loss or required new bed linen because of increased frequency of bedding changes.Diverse costs associated with medical treatment were reported, including those related to paying for medication and general practitioner (GP) visits and paying for alternative or complementary therapies. All participants mentioned food costs as an expenditure that had risen significantly during their family member’s illness. The final section of our results explores these costs in more detail by demonstrating how a multi-level analysis is useful in understanding the complex ways in which these costs operate. Finally, costs of funerals and tangihanga  were discussed. Some participants’ family members had insurance to cover at least part of the cost; others reported getting estimates of costs prior to their family member’s death, so they could put financial plans in place or using government-assisted funeral subsidies. Several Māori participants anticipated funeral expenses and had taken out funeral insurance cover. Government-assisted funeral subsidies managed by Work and Income New Zealand were also utilised. Lack of resources led to several whānau being unable to conduct traditional tangihanga at ancestral homes.Indirect costs were those incurred by participants as a result of their caregiving role. These included costs relating to lost employment opportunities, cultural obligations and personal costs related to carer health and well-being. Participants who were in paid work were often forced to fit in caring tasks around work, negotiating to work from home, using up annual leave and sick leave or taking unpaid leave. Some participants had to give up paid work to care; others had their benefits cut because they were unable to actively look for work because of caring responsibilities. Self-employment allowed some flexibility and also meant there were no paid leave provisions. One self-employed participant, for example, reported that his income halved when caring for his wife who had to leave her full-time job, leaving him with the ‘balancing act’ of trying to work, care for two children and care for her. After her death, he was forced to sell the family home and buy a smaller, more affordable property. For Māori, the cultural obligation and preference to return to ancestral homes before death and/or post death (tangihanga) incurred additional transport costs and other expenses associated with meeting these cultural end-of-life needs. In several cases, customary funeral traditions were interrupted due to a lack of resources. Some participants also reported that caring had negatively affected their own health and well-being. Costs incurred as a result included physiotherapy for back injuries resulting from lifting their family member. Anxiety, depression and insomnia associated with caregiving could also incur financial costs in terms of GP visits and medications, both at the time of caregiving and into bereavement.Mum doesn’t eat, okay? When mum feels like eating, if mum said to me, ‘Oh I feel like scallops’, I’d hunt the earth down to find scallops for Mum because if she’s going to eat that, I’d rather her have that. While any type of caregiving can have financial costs associated with it, participants’ responses highlighted how the particular context of caring for someone with palliative care needs could exacerbate financial costs and constrain the mitigation of such costs. Indeed, carers had a sense of urgency about meeting the person’s needs, regardless of cost, as death approached. For example, within the Māori cohort, many carers reported purchasing kaimoana (seafood) to satisfy the cultural preferences of the dying individual, while participants in the non-Māori cohort reported sourcing very expensive food items to try and ‘tempt’ their family member to eat when appetite was affected by nausea and illness:We got the doctor to come and visit her […] it was just so much easier for him to come to her. Even though it cost 70 dollars instead of 15 or 20 or whatever it was. It was worth the extra costs. Similarly, participants reported paying for anything they could that would make life easier for their family member. For example, several talked about paying extra money to receive GP home visits because of the burden of attending the practice:So I gave up my [job]; I resigned from my job which, yeah, which I knew would put me in a position where I wouldn’t be able to cope financially. A patient’s desire to ‘die at home’ would often come at a cost, including a financial cost, in terms of family members having to provide nursing care. For example, a Māori participant terminated paid employment to provide full-time care for her older mother, knowing that it would incur financial hardship to nurse her at home:She prefers the family to care for her because obviously they know her better than any stranger from outside and it’s much better for her, so she knows who’s caring for her. Because she’d rather have family look after her than anybody else. A New Zealand European son reported that his mother with bowel cancer was ‘adamant’ she wanted ‘to die at home in her own bed’ which meant that he used up all his sick and annual leave as he and his wife took on the responsibility of caring, including hourly toileting, and was ‘completely shattered’ when it was over. A man who had recently been made redundant moved in to help care for his sister, so her husband could keep working, even though he was then penalised for not actively job-seeking:Financial costs also had an impact on the wider family or social network, including after the death of the patient. While in most cases, the bulk of the costs were incurred by one primary carer, typically there were multiple family/whānau members involved in providing care and sharing costs.Mum can’t look after Dad and Mum tries to look after Dad and then Dad tries to look after Mum and then Dad worries if Mum fell he wouldn’t be able to pick her up and so we’ve sort of taken those worries away from them both [by having Mum move in with us] … I cook meals at night for Dad and then I take them around at night for Dad and then [niece] will do everything else that needs to be doing, like the housework and the cleaning and things like that. Several Māori whānau identified they had two or more income streams flowing into their home. However, this did not always result in money being equitably dispersed to help off-set care costs. It is also important to recognise that some participants had multiple caring responsibilities, as exemplified in this daughter’s account of caring for her older parents:The complex care requirements across family networks, both in terms of time and money, could have an enduring and compounding impact on carers that many felt was not sufficiently recognised by health or social service providers, employers or wider social networks.They don’t come out and give you a brochure and say, ‘Here you go, here’s an application form, fill it in and you’ll get some money’. No, you’ve got to ask so that can sometimes be extremely frustrating because you don’t know how you’re going to survive this financially. There were variable accounts as to what statutory (government) help was available in the context of ‘being palliative’. New Zealand has an increasingly constrained, publicly funded health system with some provision for end-of-life care, but exactly what was available, and when, was often unclear to participants. Interviewees were appreciative of help and support they received but were frustrated at systems and bureaucracies, including the apparently random discovery of entitlements, as one participant explained,not a widespread expectation that ‘the state should provide’ or that people were entitled to a lot from the health and social system. The key issue was that if there were agreed provisions, it should be easy to find out about them and to use them effectively. One participant suggested that there should be a ‘palliative care needs-based assessment’, where needs were assessed across the person and the carer network, in order to identify resource constraints and provide clear information on costs and entitlements.There was 23 The way these dimensions interacted with one another in relation to financial costs is represented in personal and interpersonal costs sat alongside each other and could also overlap. For example, one participant reported using up all her paid leave but being supported financially by her brothers, so she could care for their father; another, by contrast, stated that her brother offered no financial or other help but wanted money from her mother’s house sale. The central relationships between carers and care-receivers were located within the context of the sociocultural realm, where community or wider social or financial resources may be available, and where cultural or gendered practices around care produced particular expectations and costs. As one participant explained,I’m the only girl, I’ve got three older brothers, they’ve all got families and other commitments. I, on the other hand (laughter) have not got any attachments at the moment, but of course being of Cook Island descent, it’s family first. Four dimensions of costs were identified in our analyses: personal, interpersonal, sociocultural and structural.structural systems within which care occurs were found to have cost implications at all levels. For example, many participants appreciated New Zealand’s publicly funded health emergency services but not the lack of ongoing or palliative care provision:I think our health system is fantastic in emergency situations and being very thorough, doing all the tests and the staff are wonderful, they are. But it’s the follow up, it’s what happens after the immediate emergency part has passed, it’s the follow up care that is inadequate … Finally, the An example of how costs operated across the four levels of the framework is outlined in 9 and can have serious and long-lasting repercussions. While significant costs have also been reported in the wider caregiving literature,23 it was clear that the context of a life-limiting illness was significant to the nature and extent of financial outlay.This study is one of the first to explore the range, and impact, of financial costs borne by family caregivers within a palliative care context. Our findings are consistent with the limited evidence base in this area which indicates that these costs are substantial24 as well as the wider caregiving literature. Within New Zealand, in line with other countries with a partly or fully privatised health sector, the costs of unsubsidised medications and GP home visits are acknowledged to place a significant burden upon patients and family caregivers. Similarly, the costs of travelling to health appointments and parking charges, both of which were frequently raised by our participants, have also been discussed within the wider literature.25 Our findings confirm that large regional rather than local hospitals , poor public transport infrastructure (Auckland lacks good public transport) and privatised contracts for revenue-generating parking and food provision in hospitals all compounded our participants’ costs. This highlights the interplay of structural and personal/interpersonal dimensions on financial costs.Our findings were interpreted within the context of a multi-dimensional model of the costs of caring. Within the personal and interpersonal domains, our findings indicate that a range of direct costs are incurred by family caregivers in many diverse areas. Many of these were consistent with the previous limited research in this area, for example, the work by Dumont et al.,26Our study also extends previous findings by identifying a range of direct costs that go beyond those related to a patient’s medical care. Food was a frequently mentioned expense, and our multi-level analysis enabled insight into the different ways in which these costs were incurred. In addition, indirect costs were reported, including those related to present and future paid employment and a carer’s own health and well-being. This finding is of particular concern given evidence which suggests that informal caregiving is often associated with poor physical and mental health, which may be exacerbated by the caring role.27 The financial costs of family caregiving can therefore be seen as exacerbating pre-existing social inequities.A range of costs was also identified within the sociocultural domain. Costs were experienced differently across the sample, with participants with limited financial means disproportionately impacted. Low-income Māori families accrued debt at a time when cultural and familial imperatives to care were at their greatest. For Māori participants, caring took place against a backdrop of economic and social disadvantage associated with the context of colonialism and disenfranchisement.29 This echoes the use of the four dimensions in other narrative research investigating how individual narratives are inevitably intertwined with collective effects.30The structural level can also be linked to the critique of neoliberal welfare and support systems where the rhetoric that ‘family care is best’ is underpinned by a less overt assertion that ‘family care is cheapest’ (to the state).This study was an initial exploratory project in New Zealand’s largest city, therefore findings may not be applicable to other contexts or settings. However, our study presents the first data of its kind and underlines the importance of further research to provide quantifiable data on the nature and extent of family caregiving costs.The multi-level analysis of costs offers opportunities for multi-level actions to mitigate costs from personal and family financial planning to better cultural awareness around customary practices and associated costs. The presence of statutory support for those caring for people in a palliative care context was valued; the absence of information and transparent eligibility and access to such support was not. While our findings show that financial costs were significant, there was also evidence of resourceful financial problem-solving and a strong commitment to care. However, this commitment to care cannot be realised without further research which quantifies the nature and extent of costs, the development of innovative solutions for supporting family members caring for those at the end of life and policy commitment to supporting those family caregivers who are key to palliative care provision.


The precise efficacy of nucleoside analogue reverse-transcriptase inhibitors (NRTIs) in preventing and inhibiting virus replication remains unknown in RT-SHIV infected Chinese-origin rhesus macaques (Ch RM).50 RT-SHIV and treated by gavage with NRTIs (20 mg AZT and 10 mg 3TC twice per day) for four consecutive weeks beginning at one hour, on day 217 or 297 post inoculation, respectively. Treatment with AZT/3TC inhibited transiently RT-SHIV replication during chronic infection, but did not significantly affect peripheral blood CD4+ T cells in macaques. Treatment with AZT/3TC at 1 hour post infection prevented RT-SHIV infection in two out of four animals during the 120-day observation period.Ch RM were inoculated intravenously with 200 TCIDTherefore, the Ch RM model with RT-SHIV infection can be used to evaluate the efficacy of new NRTIs. Due tmacaques-8. RT-SHmacaques,9. Up tomacaques, pigtailmacaques, and Chimacaques. An incrmacaques; howevermacaques,13. It i[in vitro. However50 (3.35 × 105 copies/mL) cell-free RT-SHIV, in which the RT gene of SIVmac239 was replaced with that of HIV-1 clone HXBc2[+ T cells were determined longitudinally by quantitative RT-PCR  were born and housed in a specific pathogen free (SPF) facility at Institute of Laboratory Animal Science (ILAS), Chinese Academy of Medical Sciences. All naïve animals were negative for tests of simian type D retroviruses (SRV), simian T cell leukemia virus-1 (STLV), simian immunodeficiency virus (SIV), monkey B virus (BV), and tubercle bacillus (TB). Animal care was compliant with the Institute of Laboratory Animal Science Guidelines for the Care and Use of Laboratory Animals (est. 2006), and the experimental protocol was approved by the Animal Research and Care Committee of ILAS , and the levels of viremia in those experimental monkeys were lower than that of the detection threshold (virus RNA load <100 copies/mL) at most time points post drug treatment  that received AZT/3TC on day 297 post inoculation with nearly 65% reduced numbers of peripheral blood CD4+ T cells (Figures + T cells in macaques.As expected, plasma RT-SHIV-specific RNA was detected in the control group of macaques and reached a peak of 0.69 ~ 6.30 × 10in vitro and in macaques[50 RT-SHIV and observed that treatment with both AZT and 3TC one hour post inoculation prevented RT-SHIV replication in two out of four macaques and treatment with the same drugs at peak infection inhibited virus replication in four macaques. These data indicated that RT-SHIV was sensitive to NRTIs in Ch RM. Conceivably, treatment with these medicines may effectively prevent HIV replication and AIDS development if a natural infection occurs with a dose of HIV. Our data are consistent with a previous report that prophylactic treatment with a single substance post exposure reduces the probability of an infection[This study provided evidence to demonstrate that treatment with AZT/3TC prevented and inhibited RT-SHIV replication in a Ch RM model of AIDS. A number of RT-SHIV strains, in which the RT was replaced with the RT from an HIV-1 clone, have been constructed to evaluate the efficacy and safety of HIV-specific NNRTIs and NRTIs both macaques,6,19. Thmacaques. Hence, macaques. The Ch nfection. Our resIn summary, our data indicate that AZT and 3TC treatment post inoculation of RT-SHIV can prevent and inhibit RT-SHIV replication in Ch RM. Therefore, the RT-SHIV/Ch RM model may be valuable to evaluate NRTIs.NRTIs: Nucleoside analogue reverse-transcriptase inhibitors; Ch RM: Chinese-origin rhesus macaque; NHP: Non-human primate; SIV: Simian immunodeficiency virus; NNRTIs: Non-nucleoside reverse transcriptase inhibitors; PEP: Post-exposure prophylaxis; RT: Reverse transcriptase; HIV: Human immunodeficiency virus; HAART: Highly active antiretroviral therapy; In RM: India origin rhesus macaque; AZT: Zidovudine; 3TC: Lamivudine; SPF: Specific pathogen free; ILAS: Institute of Laboratory Animal Science; SRV: Simian type D retroviruses; STLV: Simian T cell leukemia virus-1; BV: Monkey B virus; TB: Tubercle bacillus.The author declares that they have no competing interests.+ T cell count. ZC participated in the collection of data of plasma viral load and analyzed the data. HJ participated in the manipulation of animal. CQ and QW participated in the design of the study. All authors have read and approved the final manuscript.WW wrote the manuscript, designed the study and analyzed the data. NY participated in the collection of data of CD4Supplementary Materials and Methods.Click here for file


To validate the feasibility of using next-generation sequencing in an African context, the complete genome of a rabies virus isolated from a human patient was obtained by high-throughput sequencing after virus isolation in mice and random unbiased amplification. Phylogenetic analysis suggested that this virus belongs to the Africa II clade. Lyssavirus and family Rhabdoviridae. Humans get infected through close contact with secretions of rabid animals, mainly dogs. Rabies is almost always fatal once symptoms appear. Annually, 55,000 people die of rabies mostly in Asia and Africa (Although it is a fatal but preventable disease, rabies is still a widespread neglected zoonosis that represents an important public health threat in developing countries . Rabies d Africa . Howeverd Africa , 4. Reced Africa . Althougd Africa . High-thd Africa . Reads cd Africa . Final ad Africa . Each pod Africa . Sequencd Africa . To our KF977826.The complete genome sequence is available in the DDBJ/EMBL/GenBank database under accession no.