The design and implementation of a high-speed direct digital frequency synthesizer are presented. A modified Brent-Kung parallel adder is combined with pipelining technique to improve the speed of the system. A gated clock technique is proposed to reduce the number of registers in the phase accumulator design. The quarter wave symmetry technique is used to store only one quarter of the sine wave. The ROM lookup table (LUT) is partitioned into three 4-bit sub-ROMs based on angular decomposition technique and trigonometric identity. Exploiting the advantages of sine-cosine symmetrical attributes together with XOR logic gates, one sub-ROM block can be removed from the design. These techniques, compressed the ROM into 368 bits. The ROM compressed ratio is 534.2 : 1, with only two adders, two multipliers, and XOR-gates with high frequency resolution of 0.029 Hz. These techniques make the direct digital frequency synthesizer an attractive candidate for wireless communication applications.  Fout) is given byFclk is clock frequency, FCW is a frequency control word, and N is the phase accumulator width. Pipelining technique is defined as a technique that partitioned the given task into a number of subtasks that need to be performed in a sequence. The pipelining technique is used in  and fourth output [X(T + 4)], the N bit is shifted up by removing one and two bits of the FCW input and replacing them with the one and two bits from the lower pipelining stage, respectively, before they are added. The X(T + 2) output and X(T) output are used to generate X(T + 3).Clk/4) make the multiplexers choose one of the results at the output of the PA to overcome the holding time on the parallel adders as illustrated in This operation made the frequency tuning word held constant for four clock cycles without causing any imperfections in the PA output. The partitioned clock cycles .The 32-bit FCW input is sufficient to obtain 0.029 Hz frequency resolution from the 125 MHz clock frequency of Cyclone III FPGA kit board  and g is a generate function (gi = xi · yi).BK cells compute the carry for two or more of full adder (FA) cells, and they are calculated as a sum of  P cells , 24. TheCin in the prefix structure with some modifications, the prefix structure can be used in pipelining-based adder design. This approach is used in BK adder fast carry computation. However, in this paper, a modification is proposed to the BK adder so that it can be used in pipelining architecture. The proposed modification is by removing the operation of the g0, and the carry out of the first bit C1 can be achieved by a 2-1 multiplexer. The input to this multiplexer is x0 and Cin while p0 is the select input and the output is C1. The operation of the multiplexer is given byC1 is the carry out, p0 is the propagate function, Cin is the carry input, and x0 is the first bit input. The proposed modification of the 8-bit BK adder is shown in The general prefix addition algorithm is explained by Zimmermann in . By addiAn adder is a key element of the pipelining PA design, and a fast adder improves PA performance. Parallel-prefix adder tree structures such as Sklansky , Kogge-SA comparison has been made between conventional adder and several parallel-prefix adders for 12-bit, 18-bit, 24-bit, and 32-bit operations. The PA designs were coded in Verilog HDL and verified in Cyclone III FPGA kit board. Prior to that, all the designs were simulated by using ALTERA Quartus II. The comparison result is shown in π/2) of the sine waveform and two most significant bits (MSB) from the phase accumulator are used to reconstruct the full sine wave. From these two MSBs, one of them is used to determine if the sine amplitude is increasing or decreasing and the other one is used to determine its sign.ROM LUT or phase-to-amplitude converter (PAC) is a memory storage address for DDFS, which is used to convert the phase signal into an amplitude sine wave signal. High-accuracy output signal for DDFS requires a large number of LUT. The ROM size exponentially increases with increasing number of bit inputs. The designer's challenge is a tradeoff between reducing the ROM size while maintaining high performance . Quarter-wave symmetry technique is used to resize the ROM in the proposed DDFS design to store only one quarter (0 : π : 2π) and is achieved by adding full adder at the output gate to accomplish the full sine wave value. To save power and achieve a smaller area design, the 1/2 LSB offset is added to the stored memory address of sub-ROMs. This offset removes the full adder component from the 2's complement of the proposed design.The phase output is directly used in the first and third quarter while the inverse values of the phase output are used in the second and fourth quarters. This requires 2's complement when the phase is between , such that A < (π/2), B < (π/2)∗(1/2A), and C < (π/2)∗(1/2A+B), with the same approximations based on trigonometric identity.The angular decomposition technique based on trigonometric identity is one of the best techniques to reduce the quarter ROM LUT size. The quarter ROM LUT was partitioned into three (A and 2B) for sin and cosA and B. The calculated results of (sin-cosA) show that they are inversely symmetrical. Based on this, cos(A) can be obtained by complementing the sin(A) values and this is achieved by connecting the sin(A) output and the high Vcc to the XOR logic gate inputs. In this way, only one addressing sub-ROM is needed for sinA and cosA values. The equation for cosB calculation is given byB values with 4-bit input are fed into the sub-ROM B(B = 4); therefore cosB = cos(0.046019) = 0.999999677≅1. Thus, the (cosB) block ROM was also removed from the proposed design. Therefore, =C,wheresin(A+B)=sin(A+B+Cπ/214), which is approximately 3.83 × 10−4. The required ROM size for 12-bit input is 214 × 12 = 196608 bits. Applying the sine wave approximation based on trigonometric identity with three 4-bit ROM requires only 368 bits [(24 × 11 = 176) + (24 × 8 = 128) + (24 × 4 = 64)] for ROM A, ROM B, and ROM C, respectively. The compressed ratio (196608/368) is 534.2 : 1, with only two adders, two multiplayer adders, and XOR gate as additional hardware equipment.The error that results from the approximation in  is . The full amplitude waveform at the output of the ROM lookup table is shown using the Signal Tap logic analyzer in 14 × 12) bits were required to draw the full sine wave signal with 12-bit output. Quarter-wave symmetry and an angular decomposition technique based on trigonometric identity were applied. The ROM was compressed to only 368 bits, with 534.2 : 1 ratio. The measured DDFS output waveforms and spectra for different clock frequencies illustrate the purity of the sine wave output as shown in Fclk = 125 MHz (Cyclone III kit board clock frequency). The output frequency that was calculated is Fout = (FCW/2N) × Fclk = (229 − 1/232) × 125∗106 = 15.624999 MHz. The measured output frequency from oscilloscope is 15.63 MHz and this is closely matched with the calculation result.The 32-bit DDFS has been implemented on the Cyclone III FPGA kit board. The 32-bit phase accumulator output was truncated into 14 bits. Approximately 196608  was approximated based on dB(see ), where The measured DDFS output from the spectrum analyzer shows SNR of approximately 68 dB as shown in Figures A 32-bit high-speed DDFS system was designed with 0.029 Hz frequency resolution. The system was successfully simulated in ALTERA Quartus II software. The phase accumulator together with the ROM lookup table has been implemented on the Cyclone III FPGA kit and verified with a Signal Tap logic analyzer, and the complete DDFS system with digital to analog convertor has been implemented on the Cyclone III FPGA kit board and the performance has been measured using oscilloscope and spectrum analyzer. Parallel pipelining with clock gating has been applied on a modified BK adder for the proposed phase accumulator design. Wave symmetry and an angular decomposition technique based on trigonometric identity were used to reduce the ROM size. The ROM was compressed to only 368 bits, with a 534.2 : 1 ratio. The proposed design has demonstrated attractive results that improve the operation speed and reduce the ROM size significantly.


Orthobunyavirus of the Simbu serogroup named Schmallenberg virus (SBV) emerged in continental Europe. The virus is transmitted by hematophagous arthropods, with the Culicoides species as, so far known, main vectors. Infection with the virus can cause clinical signs in adult ruminants including diarrhea, fever and reduced milk production. Transplacental infection of the developing fetus can lead to malformations of varying severity. To assess seroprevalence of SBV in Sweden an indirect enzyme-linked immunosorbent assay (ELISA) was established in connection with the surveys. Here, we describe the development and evaluation of the indirect ELISA, based on whole virus as the coating antigen and a monoclonal antibody for the detection of antibodies to SBV in ruminant sera. The evaluation includes comparison between the in-house ELISA, virus neutralization test and an indirect commercial ELISA.In late 2011, a new The optimal working dilutions of antigens and conjugate were estimated with checkerboard titrations. Comparative studies, including ROC analyses, were used for the selection of an optimal cut-off . With an estimated S/P value of 15% the whole virus ELISA showed a specificity of 100% and a sensitivity of 99.19% compared to virus neutralization test (VNT) and with a good consistency as shown in reproducibility and variability experiments. Furthermore, the comparison of our whole virus indirect ELISA to an indirect ELISA with a SBV nucleoprotein antigen, demonstrated a higher sensitivity of our test.The indirect whole virus ELISA described in this paper is a readily available test for serological analysis of SBV antibodies. Since this in-house ELISA demonstrates a specificity and sensitivity comparable to virus neutralization test and also shows a higher sensitivity compared to commercially available indirect ELISA, it is a useful alternative for surveillance and screening purposes of SBV. Since Schmallenberg virus (SBV) was identified in October 2011 . In cattOrthobunyavirus within the family Bunyaviridae and related to the Simbu serogroup [Sathuperi virus and is not a reassortant but rather likely one of the ancestors of Shamonda virus [The Schmallenberg virus is a member of the genus erogroup . The virerogroup . The enverogroup  and is cda virus .In the spring of 2012, before the vector season started, several serosurveys were performed in Sweden . At thatThe present study describes the establishment and evaluation of an indirect ELISA for the detection of antibodies to SBV in cattle, sheep and goat sera.4.25 TCID50/ml was prepared, aliquoted and stored in −80°C until used.The Schmallenberg virus isolate BH80/11-4, kindly provided by the Friedrich-Loeffler-Institut, Germany, was used for the ELISA antigen preparation and in virus neutralization test (VNT) . After aSBV positive and negative ruminant sera, as confirmed by virus neutralizing test (VNT), were used for determining cut-off values, specificity and sensitivity of the in-house ELISA and for comparative studies between the in-house ELISA, VNT and a commercial ELISA.The sera included three hundred negative sera collected from Swedish holdings before any introduction of SBV to Sweden was confirmed as well as positive sera from naturally infected animals, including 64 bovine, 48 ovine and 11 caprine sera from France, The Netherlands, Finland and Sweden (see Table 50 of virus in a volume of 50 μl per well was then added to the microtitreplates with the exception of the first row with 1:2 serum dilutions where only medium was added (serum controls). After preincubation at 37°C for 1 h approximately 20000 cells in a volume of 50 μl in EMEM supplemented with 20% FBS were added to each well. The plates were then incubated for 3–4 days at 37°C under 5% CO2. After incubation, the plates were examined in a light microscope for the presence of virus specific cytopathogenic effects (cpe). The neutralizing titer of a serum was determined as the highest dilution in which the cell monolayer was intact. In each run of the VNT, positive and negative control sera were included.Serum samples were analyzed for neutralizing antibodies in a virus neutralizing test (VNT) designed for SBV at our laboratory. The virus isolate used was BH80/11-4, and passaged in BHK-21 cells cultivated in Eagle’s minimal essential medium (EMEM) complete  with 2% FBS. Before analyzing, the sera were heated for 30 min at 56°C. The VNT was performed in 96-well microtitreplates in which sera were 2-fold diluted in EMEM in volumes of 50 μl in duplicate starting from 1:2 up to 1:256. Between 30 and 300 TCIDsample)/ODpositive control × 100). A S/P value of >60% was considered as positive.A commercial indirect ELISA based on a recombinant SBV nucleocapside protein antigen and a multi-species conjugate  was usedSBV was cultivated in Vero cells for the preparation of virus antigen. Non infected Vero cells were used for the preparation of control antigen.2 tissue culture bottles . The medium was removed and the cell monolayer was rinsed with phosphate-buffered saline (PBS) and then inoculated with 10 ml of virus diluted 1:10 in EMEM without FBS. After one hour of adsorption at 37°C, 40 ml of EMEM with 2% FBS was added to the virus infected cells. The cultures were then incubated at 37°C under daily observation. When a cytopathogenic effect of 80-90% was seen, usually after 2 days, the cultures were frozen at −80°C. Non-infected Vero cells grown and handled under similar conditions were used for the preparation of control antigen. The cell bottles were freeze-thawed consecutively two times and the material was centrifuged at 1500 x g for 15 min. The pelleted cell debris were dissolved in 1/100 of the starting volume in PBS and stored in −80°C until used. The supernatants were precipitated and concentrated with 10% polyethylene glycol (PEG) as described by Simard et al. [Vero cells were grown at 37°C to 80-90% confluence in 162 cmd et al. .1 also binding to ovine and caprine IgG  was used as conjugate. The antigens were initially titrated in two-fold dilutions from 1:1000 to 8000, and thereafter evaluated in the dilution range of 1:1500 to 1:4000. The sera were diluted 1:100 and the conjugate was diluted 1:5000, 1:10 000, 1:20 000 and 1:40 000.Checkerboard titrations were performed to find optimal working dilutions of antigens and conjugate. Three variants of virus and control antigens were tested; the antigen concentrated by PEG saturation (A), antigen from the cell debris fraction (B) and a combination of PEG precipitatedted antigen and cell debris antigen (A + B). Bovine sera with a VNT titer 1:128 and <1:4 was used as positive and negative controls. A HRP conjugated monoclonal antibody anti-bovine IgG1  diluted 1:10 000 in TBS-T. After one hour incubation at RT the strips were washed and the bound secondary antibodies were detected by using ECL substrate and Hyper film ECL .The antigen consisting of the A + B combination were separated on 10% SDS-PAGE and transferred onto a 0.45 μm nitrocellulose membrane (Bio-Rad) at 100 V for 1 h with a Criterion™ Blotter (Bio-Rad). The membrane was blocked with 5% skimmed milk powder in Tris-buffered saline pH 7.6 with 0.05% Tween-20 (TBS-T) and cut into stripes. The SBV proteins on the blot were detected by SBV positive sheep and cattle sera. Sera from uninfected animals were used as controls. All sera were diluted 1:100 in TBS-T and incubated for 1 h at room temperature (RT). The stripes were washed with TBS-T and then incubated with a HRP conjugated mouse anti-bovine IgGAccording to the results from checkerboard titrations of the different preparations of ELISA reagents the following basic protocol was implemented:2SO4. The optical density (OD) was measured at 450 nm with a Multiskan EX microplate photometer (Thermo Labsystems). Positive, low positive and negative control sera were included on each plate for the evaluation of each assay. The OD value of each sample and control sera was calculated as the difference between the OD values of the virus antigen well and the control antigen well . The results of the serum samples were then expressed as percentage of the positive control serum (S/P%).Microtitreplates  where coated with virus antigen and control antigen diluted 1:3000 in 0.05 M carbonate buffer, pH 9.6 and incubated overnight at 4°C. The plates were washed three times with 0.01 M PBS, pH 7.4 containing 0.05% Tween-20 (PBST). Serum samples diluted 1:100 in PBST with 3% FBS were added in an antigen and a control antigen coated well. After incubation for one hour at 37°C the plates were washed three times with PBST and the conjugate at a 1:20 000 dilution in PBST was added. The plates were then incubated for one hour at 37°C. After washing four times in PBST a TMB  substrate solution was added and incubated for 10 min at room temperature thereafter the reaction was stopped by adding 2 M HThree hundred VNT negative and 123 positive cattle, sheep and goat sera  was calculated between plates for the inter-assay variation (reproducibility) and within the plates for the intra-assay variation (variability).The relative specificity and sensitivity of the in-house ELISA and a commercial ELISA  were comMedCalc® v2.3.0 software was used for the TG-ROC analysis and dot plot graph.After cultivating SBV on Vero cells the cultures were freeze-thawed and then centrifuged to separate the cell debris from the cultivating material. Virus in the supernatant was concentrated by PEG precipitation. Both the pelleted cell debris fraction and the PEG concentrated virus were then treated with Triton x-100 at three different concentrations of 0.1%, 0.5%, 1.0%, to explore the concentration required for an optimal solubilization and virus inactivation. The preparations were inoculated on to Vero cells to detect any presence of infectious virus by its CPE. Virus prevalence was found already at the first passage when a concentration of 0.1% of Triton x-100 was used. At a Triton x-100 concentration of 0.5% virus could be demonstrated after 2 passages on cell cultures. When a Triton x-100 concentration of 1.0% was used no virus could be detected even after three passages on cell cultures.The virus content of the cell debris fraction and the PEG concentrated virus was preliminary evaluated in an ELISA by serially dilutions. Both fractions were treated with 1.0% Triton x-100, in addition the cell debris fraction was centrifuged to remove residual cell debris, before the evaluation. Since it was found that the amount of cell bound virus was significant, three different preparations were finally evaluated, one from the PEG concentrated virus (A), one from the original cell debris (B) and a combination of both (A + B).Checkerboard titrations were performed to select the optimal dilution and preparation of the three different antigen variants and the optimal dilution of conjugate. Figure The reproducibility of the antigen preparation method was confirmed by the comparison of three separately manufactured batches (data not shown).To determine the cut-off value and to calculate the relative sensitivity and specificity a TG-ROC analysis was performed with the use of all VNT positive and VNT negative sera  of 5.0%. Analysis of the intra plate variation, including 12 replicates of the four sera on one plate, gave a mean CV of 4.5%. These data suggest that the in-house ELISA has the capacity to generate reproducible results with low variation.Table The SBV is now established throughout Europe. The use of serological surveys makes large scale testing possible to map spread of infection and to better understand disease transmission dynamics. For serosurveillance and seromonitoring purposes in Sweden, an indirect ELISA was developed using whole SBV as antigen. In this work, we present the development and the evaluation of an indirect in-house ELISA to detect antibodies from cattle, sheep and goat against the full SB virus. Evaluation of the in-house ELISA was done by comparing results with VNT and a commercial ELISA based on a recombinant SBV nucleoprotein.During the preparation of the ELISA SBV antigen from infected cell cultures it was found that the obtained cell material fraction contained considerable amount of viral proteins. This has previously been reported for virus belonging to the Simbu serogroup ,19. By uTriton x-100 was used for virus inactivation. This solvent/detergent works by disrupting the lipid layer of enveloped viruses. The method is effective, simple and rapid with a confirmed capacity to inactivate model enveloped viruses by >4 to >6 log after 30 min at 22°C ,21. The In assessing the cut-off value of the in-house ELISA, not only high sensitivity with a maximized specificity was taken into account, but also the varying distribution of the S/P values and the degree of separation between positive and negative sera of each group of cattle, sheep and goat sera. Based on TG-ROC analysis of positive and negative sera as well as comparative analysis of in-house ELISA and VNT of serially diluted bovine sera in negative bovine serum, the cut-off value of S/P =15 was finally selected giving an overall sensitivity of 99.19% and a specificity of 100%. The prevalence of both nucleocapside protein (N) and the glycoprotein (Gc) of the ELISA antigen were confirmed by western blot analysis. Along with the glycoprotein Gn, the Gc glycoprotein is involved in viral attachment and cell fusion and is considered to be the target for neutralizing antibodies -24. The 1, is developed and evaluated to detect cattle, sheep and goat antibodies in sera. Since IgG1 represent the major Ig class in milk throughout the lactation period, this conjugate was thought to be particularly useful in the detection of milk antibodies [The conjugate, a monoclonal antibody against bovine IgGtibodies ,27. FurtAn indirect ELISA was established with the use of whole virus as coating antigen and a monoclonal antibody for the detection of antibodies to SBV in bovine, ovine and caprine sera. The in-house ELISA showed good reproducibility and repeatability with high correlation to virus neutralization test. In a comparative study between the in-house ELISA, based on whole virus, and an ELISA based on recombinant nucleoprotein, the former showed a higher sensitivity indicating that the use of whole virus as antigen in this case, can be of advantage. The assay provides a rapid serological detection method that would be suitable for serosurveillance of SBV infection on a large scale.


Objectives To assess the frequency of fatal recrudescence from Ebola virus disease after discharge from treatment centres, and explore the influence of infecting dose on case fatality rates.Design Retrospective cohort study.Setting Western Area, Sierra Leone.Participants 151 survivors treated for Ebola virus disease at the Kerry Town treatment centre and discharged. Survivors were followed up for a vital status check at four to nine months after discharge, and again at six to 13 months after discharge. Verbal autopsies were conducted for four survivors who had died since discharge . Survivors still living in Western Area were interviewed together with their household members. Exposure level to Ebola virus disease was ascertained as a proxy of infecting dose, including for those who died.Main outcome measures Risks and causes of late death; case fatality rates; odds ratios of death from Ebola virus disease by age, sex, exposure level, date, occupation, and household risk factors.Results Follow-up information was obtained on all 151 survivors of Ebola virus disease, a mean of 10 months after discharge. Four deaths occurred after discharge, all within six weeks: two probably due to late complications, one to prior tuberculosis, and only one after apparent full recovery, giving a maximum estimate of recrudescence leading to death of 0.7%. In these households, 395 people were reported to have had Ebola virus disease, of whom 227 died. A further 53 people fulfilled the case definition for probable disease, of whom 11 died. Therefore, the case fatality rate was 57.5% (227/395) for reported Ebola virus disease, or 53.1% (238/448) including probable disease. Case fatality rates were higher in children aged under 2 years and adults older than 30 years, in larger households, and in infections occurring earlier in the epidemic in Sierra Leone. There was no consistent trend of case fatality rate with exposure level, although increasing exposure increased the risk of Ebola virus disease.Conclusions In this study of survivors in Western Area, Sierra Leone, late recrudescence of severe Ebola virus disease appears to be rare. There was no evidence for an effect of infecting dose (as measured by exposure level) on the severity of disease. Sudan and Bundibugyo ebolavirus species than for Zaire ebolavirus.Zaire ebolavirus, the case fatality rate based on the notification data for certain and probable cases was 65%, slightly lower in Guinea and higher in Liberia than in Sierra Leone, but some cases may not have been notified.Understanding who dies from Ebola virus disease is crucial for determining the effect of interventions and planning the public health response. The case fatality rate for the disease is high but estimates have varied between outbreaks and in reports describing the west African outbreak. In previous outbreaks, case fatality rates have been between 34% and 88%, with generally lower rates for the Late deaths due to Ebola complications or recrudescence of the virus would also be excluded from estimated case fatality rates. The recrudescence of Ebola virus disease in a nurse in the United Kingdom, nine months after the original episode, raised the possibility that similar events are occurring but are being missed in west Africa, where they might be fatal.Several studies have looked at risk factors for death from Ebola virus disease. There is a clear association with age in the larger studies, with the lowest case fatality rates in children aged over 4 years and high rates in children aged under 2 years and older adults, and little difference by sex.16In the first known Ebola outbreak, in Yambuku, Democratic Republic of Congo, case fatality rates were higher in patients who acquired the infection following injection  than by contact .In this retrospective cohort study, we assess risk factors for death from Ebola virus disease, including level of exposure to individuals with Ebola virus disease and their body fluids as a proxy of infecting dose. We also assess the frequency of late deaths in those patients discharged as survivors.As part of a retrospective cohort study of transmission patterns, all survivors (or their parents or guardians) who were discharged from the Kerry Town Ebola treatment centre between November 2014 and March 2015 were sought and asked to attend an interview, together with anyone who was living with them at the time that anyone in their household had Ebola virus disease. All the people living in the household at that time were enumerated, and their age, sex, and whether they had had or died from Ebola virus disease was recorded. For those household members who were not said to have had Ebola virus disease, we asked about symptoms at that time. For those discharged as survivors following negative PCR tests for the virus, but who were subsequently found to have died, a verbal autopsy with family members was conducted by a physician, and we examined medical notes and sought information from the treating physicians, where available. The verbal autopsies used a modified version of the World Health Organization’s 2014 verbal autopsy instrument. Households were sought for interview between June and September 2015, and again between December 2015 and January 2016 to confirm vital status and conduct verbal autopsies. Individual, written informed consent was sought before interviews, with consent from parents or guardians for those aged under 18 years.To estimate the level of exposure to Ebola virus, we asked household members to describe in their own words what happened when the Ebola infection struck. For each person with Ebola virus disease, we asked what symptoms they had had, who had taken care of them, who helped them with different activities, who shared a bed with them, among other details. We also asked about any external contacts. The aim was to identify the extent of contact with possibly infective body fluids. Using an eight level scale, we assigned the maximum contact level for each person in the household. We predefined this scale on the basis of the available literature and in discussion with frontline health workers working with individuals with Ebola virus disease. Exposure, from the highest to lowest levels, was defined as follows:Direct contact with, or touching, the body of a person who died of Ebola virus diseaseDirect contact with the body fluids of a patient who has Ebola virus disease with wet symptoms Direct contact with a patient with wet symptoms Direct contact with a patient with dry symptoms Indirect contact with a patient with wet symptoms Indirect contact with a patient with dry symptomsMinimal contact No known contactWe defined individuals with Ebola virus disease as those already known as survivors from the Kerry Town treatment centre, reported by their families to be survivors from other treatment centres, or reported to have died of the disease. In addition, we included individuals (living or dead) not reported as having had Ebola virus disease but who had symptoms fitting the Sierra Leone case definition of probable disease,Analyses of risk factors for death used multiple logistic regression, adjusting for clustering by household using random effects. In addition to age, sex, and exposure level, we assessed other risk factors for associations with the outcome of Ebola virus disease. These factors included first or subsequent case in the household, date of Ebola virus infection in household, position in household (head or member), occupation, number of people in the household, and household living conditions ). Age, sex, and exposure level were kept in the multivariable model a priori. We added other variables one by one and retained them in the model if they were associated with mortality. We repeated the analyses excluding those cases and deaths classified as probable Ebola virus disease but not reported as Ebola virus disease by the family. We used Stata 14 for analysis.Two survivors of Ebola virus disease were involved in the development of the questionnaire and the implementation of the study and were asked to advise on interpretation and writing up of results. There are no plans to disseminate the results of the research directly to the study participants or the relevant patient community, but we will disseminate results to the Ministry of Health and Sanitation and the Ministry of Social Welfare, Gender and Children’s Affairs in Sierra Leone, who have responsibility for Ebola survivors.We obtained follow-up information on all 151 survivors who had been discharged from the Kerry Town Ebola treatment centre with negative blood tests, either from direct contact, or from families or other informants. Four of these survivors had died. The others were known to be alive for a mean of 10 months (range six to 13) after discharge. Details of the four late deaths are as follows:Patient A: a 25 year old woman who died 15 days after discharge. During admission, she showed signs of hepatitis. Her liver function tests had greatly improved before discharge but her amylase level was very high. At discharge, her family reported that she was unable to walk but could crawl. She “felt fine” for two days but then developed abdominal swelling, diarrhoea, and swelling of the legs and face, and she looked pale and jaundiced. A postmortem swab by the burial team was found to be negative for Ebola virus by PCR.Patient B: a 32 year old woman who died one day after discharge. She was very confused on admission, then improved but continued to act strangely. She had high blood pressure on some days but not consistently. Her platelet count was normal. At discharge, she was unable to walk. The following day, she had a sudden severe headache and was unable to talk or use her limbs. She died that evening. A postmortem swab by the burial team was found to be negative for Ebola virus by PCR.Patient C: a 17 year old boy who died five weeks after discharge. His health was reported to have returned to normal after discharge. He then developed weight loss, night sweats, and a productive cough that started after discharge. One week before death, he had pain and difficulty swallowing solids but no other specific symptoms. He died in his sleep. A postmortem swab by the burial team was found to be negative for Ebola virus by PCR.Patient D: a 6 year old boy who died one week after discharge. He had had a cough for several months before having Ebola virus disease. On recovery, he remained short of breath with a productive cough and fluctuating pyrexia that did not respond to antibiotics. He was transferred to a paediatric hospital for investigation of possible tuberculosis. A chest radiograph was compatible with miliary tuberculosis. A postmortem PCR test was borderline positive for Ebola virus.Of the 151 Kerry Town survivors sought for interview in June to September 2015, eight were living outside Western Area. We did not seek to interview households of survivors known to have died after discharge, except for patient A (because there was another survivor in the household). One survivor refused to take part and 16 were unavailable or not contactable at that time. Therefore, the remaining 123 survivors (including patient A) were included in the study. The 123 survivors lived in 94 households with 816 household members. We excluded four household members whose cause of death was unclear . Excluding one index survivor per household would give a case fatality rate of 75% (227/301) for reported Ebola virus disease, or a rate of 67% (238/354) including probable disease. We did not include households in which all individuals with Ebola virus disease died; therefore, small households could have been under-represented in our sample. This exclusion might partly explain the association found between case fatality rate and household size, but it is also possible that large households with many affected members found it particularly difficult to provide care. Associations between the other risk factors and death should not be biased. We were able to include probable cases and deaths that might have been missed from notification data, and assess their influence on the results. In our study, inclusion of probable disease lowered the case fatality rate, but had little effect on the associations with mortality. We did not know which of the deaths occurred in treatment centres, so could not assess the benefit of admission directly.To our knowledge, this is the first cohort study of Ebola virus disease with active follow-up for late deaths, and the first large community based study to look at risk factors for death from the disease. Previous studies of sequelae have reported on patients seen in clinics so would have missed any deaths.13The U shaped pattern of death by age was similar to that seen in the WHO notification data23This study also looks at the association between exposure level (as a proxy of dose) and case fatality rate in Ebola virus disease. A lack of association between infecting dose and severity of disease suggests that symptomatic illness can be established by one or very few organisms.30The age pattern of the case fatality rate suggests that differences in susceptibility are important in determining the outcome of Ebola virus disease. However, the associations with time period, occupation, and household size suggest that care given was crucial in reducing mortality, emphasising the importance of Ebola treatment centres. Infecting dose of the virus did not appear to have a role. All deaths after discharge occurred within a few weeks, and we have follow-up information six to 13 months later on all survivors. Recrudescence of severe active disease leading to death appears to be rare, which should be reassuring for Ebola survivors and their contacts, but does not remove the need for continued monitoring of survivors’ health.Understanding who dies from Ebola virus disease, and when, is crucial for determining the effect of interventions and planning the public health responseCase fatality rates vary by age and viral load on admission to treatment centres, but it is not known if they vary by infecting doseFrequency of recrudescence and late deaths from Ebola virus disease after discharge from treatment centres is unknownThis is the first cohort study of Ebola virus disease with active follow-up for late deaths, and the first large community based study to investigate risk factors for death from the diseaseRecrudescence of severe Ebola virus disease appears to be rare up to 10 months after dischargeInfecting dose, as measured by extent of exposure to body fluids, strongly correlated with risk of developing the disease, but there was no consistent trend with case fatality rate


In this review we aimed to determine the economic impact of epilepsy and factors associated with costs to individuals and health systems.A narrative systematic review of incidence and case series studies with prospective consecutive patient recruitment and economic outcomes published before July 2014 were retrieved from Medline, Embase and PsycInfo.Of 322 studies reviewed, 22 studies met the inclusion criteria and 14 were from high income country settings. The total costs associated with epilepsy varied significantly in relation to the duration and severity of the condition, response to treatment, and health care setting. Where assessed, ‘out of pocket’ costs and productivity losses were found to create substantial burden on households which may be offset by health insurance. However, populations covered ostensibly for the upfront costs of care can still bear a significant economic burden.Epilepsy poses a substantial economic burden for health systems and individuals and their families. There is uncertainty over the degree to which private health insurance or social health insurance coverage provides adequate protection from the costs of epilepsy. Future research is required to examine the role of different models of care and insurance programs in protecting against economic hardship for this condition, particularly in low and middle income settings.The online version of this article (doi:10.1186/s12883-015-0494-y) contains supplementary material, which is available to authorized users. Epilepsy is one of the most common neurological conditions which occurs in about 5 to 8 cases per 1000 people per annum in developed countries . EpilepsA previous review of the economic impact of epilepsy in high and low and middle income countries was undertaken in 2008 and does not incorporate more recent studies  Most othData were identified from published research articles and abstracts using a manual search in MEDLINE, Embase and PsycINFO databases, searched from inception to July 2014. We included incidence studies and case series with prospective consecutive patient recruitment within clearly defined geographical and time-limited boundaries. There were no restrictions on the basis of language, sample size, or duration of follow-up. Where articles were published in a language other than English, assistance was sought in translating the articles. Studies excluded were those limited to specific patient characteristics such as sex, where the recruitment strategy used convenience sampling with retrospective recruitment, were limited to unstructured assessment of psychosocial outcomes and focussed solely on clinical outcomes. If several articles reported outcomes from the same study population, data were taken from the first publication that referred to each follow-up period.Two authors  developed the search strategy using relevant terms that included common keywords for ‘epilepsy’  combined with common keywords for ‘costs’  , costs to individuals and households (out- of-pocket costs and patient co-payments associated with treatment), and indirect costs in terms of lost income and production. The latter could be measured either in monetary units or some other measure such as time off work.KP, MH and JM identified and reviewed papers for inclusion based on title and abstract in line with the inclusion criteria. The reference lists of all full text articles were reviewed to identify further articles of relevance that required retrieval of the full text. For all included studies (published full texts), a data extraction form was used to collect information on study design, setting, and outcome measures  . A cost-Nine of the eighteen studies that reported direct costs compared different items of expenditures and found that drugs and hospital services were the major sources of costs , 27, 32.Two studies found the health care setting was a significant determinant in the variation of direct costs , 17. ForAs previously reported, six studies from the Netherlands, US, Nigeria, Cameroon, Cote d’Ivoire and India reported out-of-pocket costs. A study from the US  and the In the US, out-of-pocket costs for outpatient visits, hospitals stays, emergency department visits and prescription medications were compared between individuals with epilepsy who were uninsured, had Medicaid coverage, had Medicare (<65 and ≥ 65) or private insurance. The uninsured reported the highest out-of-pocket expenditure US $ 1018 for hospital stays. Uninsured individuals also experienced significantly higher per-visit and total costs for emergency department care compared with patients in all other insurance groups. They also paid the most out-of-pocket for prescription medication .In Nigeria and Cote d’Ivoire patients with epilepsy were found to incur substantial out of pocket burdens – for instance, in Nigeria, 50 % of a cohort of children attending a tertiary centre incurred out of pocket costs of over 20 % annual family income , 32. TheTen studies from nine countries  estimated the indirect costs related to epilepsy. These studies used a variety of ways to identify indirect costs. Three studies measured the costs in monetary units , 23, 32.Five of the studies evaluated the employment circumstances of patients with epilepsy , 31, 33 The Spanish study found that the costs varied significantly between different patients  46 % of The Dutch study found that patients from an epilepsy centre reported the highest productivity changes and unemployment rates compared with patients from the university hospital and from the general practices .A German study found that the unemployment rate due to epilepsy was higher for non-surgically treated patients with drug-resistant temporal lobe epilepsy compared to surgically treated patients. Freedom from seizures was found to be a significant determinant for socioeconomic outcomes - 64 % of the surgical patients became seizure-free whereas 23 % of the non-surgically treated group achieved freedom from seizures due to modifications in antiepileptic drug treatment .Seven studies assessed direct costs through questionnaires,, 24, 32 The method of ascertainment was similar amongst the ten studies estimating indirect costs. All of them measured their outcome using self-reported data. Seven studies used a questionnaire , 28, 32,Overall there was a low risk of bias in the studies reviewed Table . The incMost studies of the economic impact of epilepsy have focused primarily on the direct costs of treatment and have been conducted in high income country settings. In the small number of studies where patients were followed up from initial diagnosis, such costs were found to peak in the initial year of diagnosis, due mainly to surgery or vagus nerve stimulation (VNS). All studies generally reported significant ongoing costs incurred from medications and outpatient medical consultations, with substantially higher costs associated with ongoing seizures.Out-of-pocket costs were assessed in six studies. One study from the Netherlands assessed out-of-pocket alongside total costs of treatment and found a significantly large component of direct costs  based mainly on the costs of unpaid care and transportation . This fiIn Cote d’Ivoire such costs were found to be a major contributor to non-adherence to medications . In suchOne study that examined the role of private health insurance from the US found that lack of health insurance coverage was associated with fewer visits to neurologists and greater out-of-pockets costs of medicines, compared to those with insurance. Medicaid coverage was found to offer financial protection in terms of lower out-of-pocket payments despite the same health care utilisation as those with private insurance .Where assessed, indirect costs associated with loss of productivity and employment, were shown to have been major sources of burden associated with this condition. These were reflected in reduced employment/productivity, school attendance and income; as very few of these studies provided monetary estimates of these effects, it is not possible to draw conclusions about the relative burden of indirect from direct costs. The conclusions within individual studies were that indirect costs were overwhelming and constituted a significant burden to individuals and societies, and tended to be greater when there were ongoing seizures particularly among those in lower socioeconomic groups. More data are required to confirm the robustness of these findings in other settings.We recognise that as the majority of the studies relied exclusively on self-reported costs, there is the potential for recall bias to affect the findings. While prospective diaries may mitigate some of the problems associated with self-report by reducing reliance on patient recall, these are often unreliable when patients lack information about the nature of procedures and tests being carried out. Ideally, future research would involve the use of linked administrative data sets for the assessment of direct health care costs. However, where self-reported is likely to be the only feasible means of collecting data on out-of-pocket and indirect costs, the use of prospective diaries and minimising the time between follow-up interviews may improve the reliability of such data.Nineteen of the twenty-two studies we identified were cohort studies; the others were a case–control and quasi-experimental studies. The length of follow-up in these studies ranged from 3 months to 6 years; most were 12 to 24 months. Whilst the evidence suggests that long-term direct costs of treatment tends to remain steady (at least for those with control over recurrent seizures), data from patients with longer follow-up will provide a better understanding of treatment compliance and its relationship with costs over time.A major limitation of this review is the poor comparability of findings across studies due to differences in methods and scope. This limited our ability to make direct comparisons of the size of the burden of epilepsy across different populations and health care settings. On the other hand, the merits of such an exercise may be questionable as it may be more relevant to examine the factors associated with variations in cost, such as insurance coverage and category of illness, as a means of providing guidance for policy and the development and targeting of interventions. It was not possible to provide conclusive assessment of risk of bias due to variation in study questions and study designs. Given that the studies included in this review were largely observational and the objective was not to generate an estimate of a pooled treatment effect, such concerns over potential bias can to a large extent be discounted.A further limitation of this review is that the majority of epilepsy sufferers are found in low and middle income countries in Africa, Central and South America (>80 %) however most of the published research on this topic and so included in this review have been conducted in higher income countries where epilepsy may be a comparably lower burden. It is likely that this review underestimates the true economic burden faced by households in settings where there are fewer resources and a weaker health system capacity to support people with epilepsy and their households. This misalignment between the regions with the greatest disease burden and populations most researched highlights an urgent need for more research in low and middle income countries to guide policy and planning initiatives to address the economic burden of epilepsy in these settings.Our review uncovered a small number of varied studies that have examined the costs associated with treatment of epilepsy. The focus of most of the papers was on the direct costs to health systems but a small number also addressed out-of-pocket and indirect costs associated with loss of income and employment. The main findings are that the key drivers of cost are costs of surgery/VNS, and severity and degree of seizure control. In the Netherlands, where patients are ostensibly protected by universal health care programs, significant out-of-pocket costs were evident due to unpaid care and patient transportation. While in the US, health insurance may offset the costs of treatment and enabling access but does not liberate individuals from bearing a significant burden of out-of-pocket costs associated with ongoing costs of managing illness. Given that the magnitude of such costs is inherently context-specific, there is considerable scope for future research in this aspect of epilepsy, particularly in low and middle income countries.As an appendix, we have provided the search strategy used to obtained the results of this systematic review. As additional files, we have included completed widely accepted systematic review guidelines: the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Guidelines for Meta-Analyses and Systematic Reviews of Observational Studies (MOOSE). In addition, we have provided the data extraction form employed by the authors to undertake data extraction for each included paper.


Nursing care is a major portion of routine hospital care, especially when compared to more ancillary services such as labs, drugs, radiology, and other diagnostic and therapeutic procedures performed by doctors at the hospital. Although the nursing profession had been developed in Malaysia since the 1900s, the current hospital billing system does not reflect what nurses provide for their patients. To date, nursing service costs are poorly estimated in Malaysian economic studies. Nursing services are commonly lumped together into the patient’s total charge per day in the hospital, and billed as a flat daily rate, based on the type of room they occupy. The primary objective of this study was to calculate the inpatient nursing costs in six medical wards, which provide varied specialty services for UKMMC’s inpatients.A descriptive cross-sectional design was applied and a top down costing form was used to calculate the nursing costs (unit/day). The data comprise of: total number of staff nurses and their grades; salary received per year ; patient days per year; and average length of hospital stay (ALOS) in 2011. These data were analysed using SPSS version 21.The nursing unit costs per day for inpatients were identified: medical (RM101); surgical (RM151); paediatric (RM199); obstetrics and gynaecology (RM219); orthopaedic (RM115); psychiatric (RM151); ENT (RM130) and ophthalmology (RM164). The top five highest mean costs for nursing components with MY-DRG for medical inpatients were: A-4-10-III (septicaemia severe) ; A-4-14-III  ; A-4-10-II (moderate septicaemia) ; A-4-14-II  and finally A-4-13-III  .The present study only took nurses’ salaries into account. However, further justifications are needed to calculate the nursing costs using top down costing, in order to achieve a more accurate and cohesive result.


Bunyaviridae family and is associated with an acute febrile illness in several central Asian countries. Using next-generation sequencing technologies, we report here the full-genome sequence for this novel unclassified arboviral pathogen circulating in central Asia.Issyk-Kul virus (ISKV) is an ungrouped virus tentatively assigned to the  Nyctalus noctula bat trapped near Lake Issyk-Kul, Kyrgyzstan; the virus was subsequently identified in Tajikistan and Kazakhstan, with sporadic outbreaks of human disease reported in all 3 countries  was first isolated in 1970 from a ountries . Clinicaountries . The virde novo assembly of unmapped reads  using Velvet version 1.2.03  were identified as viral with near-complete coverage of S, M, and L segments. Initial data identified the virus as belonging to the Nairovirus genus; unresolved terminal regions were confirmed using bespoke pan-nairovirus terminal region primers in combination with internal primers based on our next-generation sequencing (NGS) data. PCR amplicons were Sanger sequenced and aligned with the NGS de novo assembled sequences, resulting in complete S, M, and L segments. The NGS data were remapped against these reference sequences using previously described methods , and host rRNA was depleted using Terminator 5′-phosphate-dependent exonuclease (Epicentre Biotechnologies), as described previously . The depn 1.2.03 , with a KR709219 to KR709221.The complete genomic sequence of ISKV has been deposited in GenBank under the accession numbers


Sensitive molecular techniques are needed for rapid detection of the R292K oseltamivir-resistant mutant of influenza A(H7/N9) virus strain to monitor its transmission and guide antiviral treatment. We developed a real-time reverse transcription PCR and single nucleotide polymorphism probes to differentiate this mutant strain in mixed virus populations in human specimens. An unusually high proportion of severe cases and a high case-fatality rate have been observed for patients infected with this virus (An outbreak of human infections with a novel reassortant avian-origin influenza A(H7N9) virus occurred in several provinces of China during March 2013  gene (R292K) and its association with severe clinical outcome in infected persons (The NA R292K assay has 2 reactions with 1 pair of primers. One reaction contained a FAM-labeled SNP probe specific for the 292K mutant strain and a second reaction contained a VIC-labeled probe specific for the R292 wild-type strain .1–1011 copies) in sterile water and used to test the assay. The linear range of sensitivity was 102–108 copies. The lower limit of detection was 100 copies/reaction (3/3 reactions detected) for wild-type and mutant virus  virus and confirmed by using Sanger sequencing . The 2 pnt virus . HoweverOf 35 respiratory samples tested, 6 were infected with influenza A(H3N2), 2 with influenza A(H1N1) virus, 6 with influenza A(H1N1)pdm09 virus, 4 with parainfluenza virus, 4 with human rhinovirus, 4 with human coronavirus, 5 with influenza B virus, and 4 with respiratory syncytial virus. In addition, 6 other respiratory samples were virus negative. Cross-amplification was not observed during sample testing. Thus, the assay is highly specific for detecting the mutant NA gene of influenza A(H7N9) virus.4 copies/reaction were prepared at the following ratios of mutant virus to wild-type virus: 2:98, 5:95, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, and 98:2. The ΔCtK – R of the mixture a ratio of 50:50 was used as the assay-specific normalization value in determination of the percentage of 292K mutant in mixed population as described by Liu et al.  virus who had various disease outcomes . The timThe 7 samples that contained the 292K mutant were obtained from 4 patients: 2 patients who died (patients 2 and 3) and 2 patients who recovered (patients 8 and 10). In our previous study, sequencing of the NA gene was not successful for the first throat swab specimens from patients 8 and 10 because of low viral load  binding preference, which favor greater replication in the lower respiratory tract. A similar phenomenon was observed in patients infected with the HA D222G mutant of influenza A(H1N1)pdm09 virus; this virus showed preferential replication in the lower respiratory tract and this infection was correlated with severe outcomes or deaths  virus. The sensitivity of the assay is lower than that of an HA7-specific real-time RT-PCR  virus were not detected by this RT-PCR). However, as a screening tool, this assay is sensitive, specific, fast, and inexpensive.This assay had a detection threshold of 10% for a mutant strain in a mixed viral population, which is more sensitive than Sanger sequencing  (PCR for detection of oseltamivir resistance in influenza A(H7N9) virus.


Ornithodoros parkeri, O. sonrai, and O. tartakovskyi for >2,900 days after they had been exposed to Karshi virus, a member of the mammalian tick-borne flavivirus group.Members of the mammalian tick-borne flavivirus group, including tick-borne encephalitis virus, are responsible for at least 10,000 clinical cases of tick-borne encephalitis each year. To attempt to explain the long-term maintenance of members of this group, we followed Ticks were exposed to Karshi virus either by allowing them to feed on viremic suckling mice or by intracoelomic inoculation. The ticks were then allowed to feed individually on suckling mice after various periods of extrinsic incubation to determine their ability to transmit virus by bite and to determine how long the ticks would remain infectious. The ticks remained efficient vectors of Karshi virus, even when tested >2,900 d after their initial exposure to virus, including those ticks exposed to Karshi virus either orally or by inoculation.Ornithodoros spp. ticks were able to transmit Karshi virus for >2,900 days (nearly 8 years) after a single exposure to a viremic mouse. Therefore, these ticks may serve as a long-term maintenance mechanism for Karshi virus and potentially other members of the mammalian tick-borne flavivirus group. Ornithodoros parkeri, O. sonrai, and O. tartakovskyi ticks to Karshi virus, a member of the mammalian tick-borne flavivirus group. Ticks were exposed to Karshi virus either by allowing them to feed on viremic suckling mice or by intracoelomic inoculation. To determine their ability to maintain the virus for an extended period of time and to transmit Karshi virus, ticks were allowed to feed individually on suckling mice after various periods of extrinsic incubation. Ticks exposed to Karshi virus, either orally or by inoculation, remained efficient vectors of Karshi virus, even when tested >2,900 days (approximately 8 years) after their initial exposure to virus. Therefore, these ticks may serve as a long-term maintenance mechanism for Karshi virus and potentially other members of the mammalian tick-borne flavivirus group.Members of the mammalian tick-borne flavivirus group, including tick-borne encephalitis virus, remain a significant cause of human disease and are responsible for at least 10,000 clinical cases of tick-borne encephalitis each year. One of the principal questions in their epidemiology is how they persist from year to year in a given area. To attempt to explain the long-term maintenance of members of this group, we exposed  Flavivirus, family Flaviviridae) [Karshi virus is a member of the mammalian tick-borne flavivirus group (genus viridae) . Membersviridae) ,2. This viridae) ,3, are rviridae) . A seconviridae) . Althougviridae) . Howeverviridae) ,2.Ixodes ricinus and I. persulcatus being the principal vectors of CEEV and RSSEV viruses, respectively [Borrelia burgdorferi, in I. scapularis. In the Lyme disease cycle, the mouse, Peromyscus leucopus, remains infectious for several months [Ornithodoros attach and complete feeding usually within 10–30 min and most complete feeding within an hour [Ornithodoros spp. ticks are able to become infected and transmit members of the mammalian tick-borne flavivirus group [Ornithodoros tholozani were shown to transmit Borrelia persica (a causative agent of relapsing fever) for at least 13 years after a single exposure [O. turicata were able to transmit B. recurrentis (repsorted as Spirochaeta recurrentis) for at least 6.5 years [Ornithodoros spp. ticks are considered to be nidicolous, i.e., living in close association with their vertebrate hosts such as living in rodent burrows [Onithodoros sonrai are found in burrows of many rodent genera in Senegal and western Africa [O. tartakovskyi, which is widely distributed in central Asia from Iran to the Xinjiang Province in western China are found in burrows of various rodent species, but primarily the great gerbil, Rhombomys opimus, [O. parkeri found in the western portions of the United States and Canada, is associated with numerous rodent species, but primarily prairie dogs [O. sonrai, O. parkeri, and O. tartakovskyi ticks to transmit Karshi virus over an extended period of time.The natural transmission cycle of the mammalian tick-borne flavivirus group involves ixodid ticks and rodents, with ectively ,7. This l months . Therefol months ,10, the l months , members an hour . Also, t an hour ,13. Prevus group –16 as weus group . Because.5 years . In addi burrows . Onithodn Africa ; O. tart opimus, ,21; and rie dogs ,23. To dOrnithodoros ticks. These included a laboratory colony of O. sonrai derived from wild-caught specimens excavated from mammal burrows in the Bandia Forest of Senegal in 1989 [O. parkeri derived from specimens captured in Spicer City, CA, in 1965. The National Institute of Allergy and Infectious Diseases provided a laboratory colony of O. tartakovskyi. All three colonies were maintained as described by Durden et al. [We used three species of  in 1989 . No virun et al. .We used the U2-2247 strain of Karshi virus. It had been passaged once in Vero cells and once in suckling mice before use in these experiments. Serial dilutions of blood, brain, and tick samples were tested for virus by plaque assay on confluent monolayers of 2- to 3-d-old primary chicken embryo cells or by subcutaneous inoculation into 2- to 4-d-old suckling mice. The identity of the original virus, and virus recovered from ticks and mice, was confirmed by a Karshi-specific quantitative real-time Real Time- polymerase chain reaction (PCR) assay and by direct sequencing of the PCR products ,25.6.3 suckling mouse lethal dose50 (SMLD50) units of Karshi virus. Two or 3 days after inoculation, a Karshi virus-inoculated mouse was placed in a cage containing ~50 O. sonrai, O. parkeri, or O. tartakovskyi ticks at various stages of development . After the ticks had been allowed to attach to the mouse for about 5 min, the mouse was removed and a second virus-inoculated mouse was added to the cage. This was repeated for up to three mice for each species of tick used in this study. The ticks were allowed to feed on the virus-inoculated mouse for about 2 h. At that time, those ticks that had attached and did not feed were removed and discarded. Each mouse was then euthanized with CO2 and blood was collected by cardiac puncture. Blood was mixed 1:10 in diluent  and frozen at -70°C until tested to determine the viremia at the time of tick feeding. The engorged ticks were placed in a cage maintained at room temperature (~20°C) until tested for either infection or for the ability to transmit virus by bite. For each species, some of the ticks that had not attached to a virus-inoculated mouse were inoculated intracoelomically with 104 SMLD50 (107.5 SMLD50/ml) of the same virus strain that had been used to infect the mice [O. parkeri were maintained in an incubator maintained at 26°C rather than at ambient air temperature.One-day-old suckling mice (BALB/c strain) were inoculated intraperitoneally with 10the mice . These i2, and brain samples were obtained from a subset of them and then triturated (1:10) in diluent and frozen at -70°C until tested for virus. In most of the tick transmission trials, ticks were caged individually in plastic vials  after feeding on the mice. Many of these same ticks were tested multiple times over the following 8 years for their ability to transmit virus by bite.To determine transmission rates, virus-exposed ticks were allowed to feed for up to 2 hours on naive suckling mice (either BALBc or Swiss Webster) individually, i.e., one tick per mouse. These suckling mice were marked by subcutaneous inoculation of India ink, returned to their dam, and then monitored daily over the next 21 d for signs of viral infection. Each litter contained one or two suckling mice that were either unexposed to ticks or were fed upon by a tick from the uninfected colony to serve as negative controls. Moribund mice were euthanized with COResearch was conducted under an IACUC approved protocol in compliance with the Animal Welfare Act, PHS Policy, and other Federal statutes and regulations relating to animals and experiments involving animals. The facility where this research was conducted is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 2011. The USAMRIID IACUC approved these studies.6.5 to 106.7 SMLD50/ml. When allowed to feed on a susceptible mouse ≤94 days after the initial blood meal, transmission was very inefficient, with none of 17 ticks transmitting virus to the mice  transmitted each time they were allowed to feed. Each of the species transmitted virus the last time it was tested, and all species transmitted virus for at least 2,000 days  tested at ≥64 days after inoculation transmitted Karshi virus by bite. Data for each transmission attempt is provided in For both hi virus . HoweverThese 34 ticks took a total of 43 blood meals from susceptible mice and transmitted virus in each case . IndividOrnithodoros spp. ticks were able to transmit Karshi virus for >2,900 days (nearly 8 years) after a single exposure to a viremic mouse. Therefore, these ticks may serve as a long-term maintenance mechanism for Karshi virus and potentially other members of the mammalian tick-borne flavivirus group. This study was a continuation of a study [Ornithodoros ticks to transmit Karshi virus, but that original study only followed the tick for 3 years. a study  that exaI. ricinis and I. persulcatus in Europe and Asia, respectively [I. cookei and I. scapularis in the Americas [Ornithodoros ticks living in that burrow. When that rodent died, or was killed by a predator, the burrow would remain vacant until discovered by a new rodent. Individual Ornithodoros ticks can remain viable for up to 4 years between feedings [Ornithodoros ticks, the rodent would become infected and all the ixodid ticks present on that rodent exposed to virus. These ixodid ticks could then spread the virus to other rodents and to larger mammals including humans.Traditionally, viruses in the mammalian tick-borne flavivirus group have been associated with ixodid ticks, ectively ,7 and wiAmericas . Larval Americas , regardlAmericas . HoweverAmericas . Given tAmericas ,10, one feedings ,30,31 anfeedings ,32–34. IOrnithodoros ticks have a wide distribution, with species found in much of the range of the mammalian tick-borne flaviviruses [Ornithodoros have not been described, i.e., the northeastern US for Powasson virus and deer tick virus, and parts of the northern range of the mammalian tick-borne flaviviruses in Eurasia. Therefore, other methods must exist for the perpetuation of these viruses in those areas.iviruses ,36. HoweOrnithodoros ticks. These include Karshi virus [Experimental studies on members of the mammalian tick-borne flavivirus group have focused on ixodid ticks. However, several members of this and the closely related seabird tick-borne flaviviruses group have been isolated from naturally occurring hi virus , KFDV [3hi virus , Alkhurmhi virus , Meaban hi virus , and Sauhi virus .O. parkeri, O. sonrai, and O. tartakovskyi to infection with Karshi virus; their ability to transmit this virus for extended periods ; their long life span; and the isolation of several members of both the mammalian and seabird tick-borne flavivirus groups from Ornithodoros ticks indicate that Ornithodoros species should be studied as potential long-term reservoir hosts for members of the tick-borne flavivirus groups.Therefore, the susceptibility of S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Background. Five years since the successful introduction of MenAfriVac in a mass vaccination campaign targeting 1- to 29-year-olds in Burkina Faso, consideration must be given to the optimal strategies for sustaining population protection. This study aims to estimate the economic impact of a range of vaccination strategies in Burkina Faso.Methods. We performed a cost-of-illness study, comparing different vaccination scenarios in terms of costs to both households and health systems over a 26-year time horizon. These scenarios are (1) reactive vaccination campaign (baseline comparator); (2) preventive vaccination campaign; (3) routine immunization at 9 months; and (4) a combination of routine and an initial catchup campaign of children under 5. Costs were estimated from a literature review, which included unpublished programmatic documents and peer-reviewed publications. The future disease burden for each vaccination strategy was predicted using a dynamic transmission model of group A Neisseria meningitidis.Results. From 2010 to 2014, the total costs associated with the preventive campaign targeting 1- to 29-year-olds with MenAfriVac were similar to the estimated costs of the reactive vaccination strategy (approximately 10 million US dollars [USD]). Between 2015 and 2035, routine immunization with or without a catch-up campaign of 1- to 4-year-olds is cost saving compared with the reactive strategy, both with and without discounting costs and cases. Most of the savings are accrued from lower costs of case management and household costs resulting from a lower burden of disease. After the initial investment in the preventive strategy, 1 USD invested in the routine strategy saves an additional 1.3 USD compared to the reactive strategy.Conclusions. Prevention strategies using MenAfriVac will be significantly cost saving in Burkina Faso, both for the health system and for households, compared with the reactive strategy. This will protect households from catastrophic expenditures and increase the development capacity of the population. Neisseria meningitidis group A (MenA) [Burkina Faso is one of the few countries whose boundaries lie wholly within the African “meningitis belt,” and thus experiences a particularly high incidence of meningococcal meningitis, with epidemics occurring regularly , 2. In 2A (MenA) , 4. TherA (MenA) , and a sA (MenA) , althougA (MenA) . To sustA (MenA) .Prior to MenAfriVac introduction in 2010, the public health response to MenA relied on the detection of localized epidemics through surveillance and subsequent reactive immunization campaigns with polysaccharide vaccines   and prospective from 2015 onward, where future costs are projected. New cases are included during the entire study period. We therefore differentiate the time horizon into 2 periods: 2010–2014 and 2015–2035. The 2035 cutoff is based on an expected 10-year duration of protection of MenAfriVac against MenA and an expected waning of the effects of the 2010 campaign around 15 years after vaccine introduction, in the absence of further immunization .The study compares the costs of reactive campaigns for individuals aged 2–30 years with a polysaccharide A + C meningococcal vaccine (reactive strategy) to each of the 3 new vaccination strategies aimed at preventing MenA using MenAfriVac while targeting different age groups : (1) a single preventive campaign in 2010 for individuals aged 1–29 years (preventive strategy); (2) routine immunization at 9 months of age 5 years after the preventive campaign (routine strategy); and (3) routine immunization at 9 months of age 5 years after the preventive campaign, and a catch-up of children born since the preventive campaign (combination strategy).The incidence of MenA in each of the vaccination strategies is predicted from a transmission dynamic model of MenA and MenAfriVac immunization . The modCosts and cases are primarily undiscounted, but we also explored the sensitivity of results to 2 alternative discounting scenarios: discounting of 3% for both cases and costs and discounting of costs at 3% with no discounting of cases. All costs were adjusted to 2012 US dollars (USD). For the period 2015–2035, the price of vaccines is an average of the expected price during that period.Different types of costs are calculated: (1) the costs of case management and of vaccination for the health system, and (2) the direct nonmedical costs (DNMCs) and indirect costs (ICs) for households. We assume that case management of meningitis is free for households and is all supported by the health system, according to the policy promoted by the international community during epidemics of meningitis in Burkina Faso . We furtThe case management costs are calculated by multiplying the estimated number of cases of MenA under each strategy by an average cost of case management calculated using a multilinear regression analysis including data from 27 countries on the direct medical costs (DMCs) of meningitis cases in low- and middle-income countries . OriginaThe costs of reactive campaigns are based on the total costs of reactive vaccination estimated in a study performed in Burkina Faso in 2007 , dividedAs an alternative, we estimated the cost of reactive vaccination based on the number of affected districts in Burkina Faso that were immunized in the last MenA epidemic in 2006–2007. Here we assumed that a similar epidemic would occur every 10 years, requiring reactive campaigns in 46 districts with 1 district per year requiring a reactive campaign in interepidemic years. The median district population size in Burkina Faso in 2010 was 233 315, and we assumed that 75% of the district population would be targeted (as a proxy for those aged 2–30 years) at a cost of 1.45 USD per person vaccinated .The cost of the preventive campaign was derived from the WHO report on the 2010 preventive campaign in Burkina Faso and Gavi financial documents and includes total vaccine costs and total delivery costs. The costs of vaccination of the other strategies is based on (1) the unit delivery costs derived from a review of national program documents; (2) the projection of fully loaded price for vaccines and injection supplies per dose; and (3) an estimate of the number of doses required for each strategy given the target population estimated from United Nations population data . The aveThe number of doses of vaccines required is based on the target population, the expected coverage rate, and wastage rates of each vaccination strategy Table .Table 3Two types of costs for households are taken into account: DNMCs and ICs.r = 0.61) [DNMCs are nonmedical costs incurred by the patient and/or the family carer because of the illness episode. For instance, in Burkina Faso, hospitals do not provide certain services  and there is hardly any transport or ambulance service. Consequently, households have to bear these transport costs, even for seriously ill patients. The family carers also have to pay for food for the patient and themselves, and buy soap and other personal hygiene items. Additionally, DNMCs include phone calls to family and costs for visitors Table . This DN = 0.61) .ICs are estimated as the loss of income due to a temporary work interruption, calculated by multiplying the average number of days of illness (from ) by the The economic impact is estimated from the costs saved by both households and the health system. Savings are calculated as the difference between the costs of the baseline strategy (reactive strategy) and each alternative strategy over the same period of time; if the differential is positive, the alternative strategy is cost saving. The savings are subdivided by cost category .The number of cases of MenA expected in Burkina Faso varies from one strategy to the other (more detail is given in ). In theThe reactive strategy led to higher total costs both for the health system and for households, regardless of the comparison strategy Table .From 2010 to 2014, the costs of the preventive and the reactive strategy are almost the same, with 9.7 and 10.0 million USD, respectively (3.1% difference). However, the structure of the costs are very different: the cost of the preventive campaigns is 1.7 times that of the reactive campaigns, but there are no costs associated with cases under the preventive strategy as no MenA cases occurred during this time period.Between 2015 and 2035, the cost of the reactive strategy is calculated as 59.9 million USD. By comparison, from 2015 to 2035 the routine and the combination strategies would cost a total of 27.4 to 24.6 million USD, respectively. De facto, the total cost for the reactive strategy is 2.2 times higher than the routine and the combination strategies. This is explained both by a higher number of cases of MenA, resulting in higher costs of case management and of DNMC and IC for households, and the higher costs of vaccination. The costs of the routine and the combination strategies are very similar due both to the low costs of the catch-up campaign in 2015 and to the lowest incidence of MenA under the combination strategy yielding lower case management costs, DNMCs, and ICs.Within each strategy, total vaccination costs are higher than those linked to care of cases , due to vaccination leading to a decreased number of cases—and thus to a reduction of the costs linked to illness.A huge decrease in the costs of case management is observed for all preventive strategies compared with the reactive strategy, as many fewer cases are predicted to occur Table . With roBetween 2010 and 2014, the health system costs associated with vaccination were higher for the preventive strategy, as all 1- to 29-year-olds in Burkina Faso were targeted nationwide, with costs concentrated in one single month in a single year (December 2010). By contrast, reactive campaigns selectively target districts experiencing epidemics and not the national population.Between 2015 and 2035, vaccination costs of the reactive strategy are 1.4 to 1.3 times higher than those of the routine and the combination strategies, respectively ; there are no cases of MenA thanks to the preventive campaign, but this mass campaign was costly in terms of vaccination costs.Ultimately, each dollar invested in routine immunization generates savings of an additional 1.3 USD, and each dollar invested in the combination strategy saves 1.2 USD.If discounting the costs at 3%, all the alternative strategies are still cost-saving both for the health system and for the households. However, the savings are lower than in the undiscounted scenario and amount to 17.8–18 million USD for the combination and the routine strategies, respectively Figure . The onlWhen discounting both cases and costs at 3%, the preventive strategy costs 1.2 million USD more than the reactive strategy between 2010 and 2014. The long-term strategies remain cost-saving, with savings amounting to a maximum of 3.9 million USD for the routine strategy between 2015 and 2035. However, in this scenario vaccination costs are higher for each of the alternative strategies than for the reactive strategies (Figure We find that the introduction and sustained use of MenAfriVac to prevent MenA has a substantial positive economic impact in Burkina Faso. Each of the preventive strategies considered generates considerable savings compared to the reactive strategy using a polysaccharide vaccine between 2015 and 2035, both with and without discounting costs at 3%. Indeed, each dollar invested in the combination strategy recommended by SAGE saves an additional 1.2 USD; in total, up to 32.3 million USD can be saved. Savings to the health system are accrued by lower costs of case management, and in the long term, also through reduced vaccination costs. By preventing MenA, MenAfriVac will reduce the economic burden on households and save thousands of households from catastrophic expenditures and pauperization . MeningiOur methodological choices were conservative and, thus, we may have underestimated the economic benefits of MenAfriVac. The costs to households may be underestimated for several reasons. We assumed that costs of case management are entirely supported by the health system; however, although care is supposed to be free of charge during an epidemic, an earlier study in Burkina Faso  estimateOur estimates of the epidemiological impact of MenAfriVac from 2015 through 2035 are based on a transmission dynamic model of MenA . DiffereThe introduction of MenAfriVac across the African meningitis belt has dramatically reduced the burden of MenA disease. With appropriate long-term immunization strategies as recommended by SAGE, this remarkable success promises to continue. The economic impact of MenAfriVac is illustrated here for Burkina Faso, and adds to the evidence on the remarkable public health success of this vaccine.


Ageratum conyzoides, one Sonchus oleraceus and one turnip (Brassica rapa var. rapa) exhibiting virus-like symptoms were collected from Pakistan and Nepal. Full-length begomovirus clones were obtained from the four plant samples and betasatellite clones from three of these. The begomovirus sequences were shown to be isolates of Ageratum enation virus (AEV) with greater than 89.1% nucleotide sequence identity to the 26 AEV sequences available in the databases. The three betasatellite sequences were shown to be isolates of Ageratum yellow leaf curl betasatellite (AYLCB) with greater than 90% identity to the 18 AYLCB sequences available in the databases. The AEV sequences were shown to fall into two distinct strains, for which the names Nepal  and India (isolates occurring only in India) strains are proposed. For the clones obtained from two AEV isolates, with their AYLCB, infectivity was shown by Agrobacterium-mediated inoculation to Nicotiana benthamiana, N. tabacum, Solanum lycopersicon and A. conyzoides. N. benthamiana plants infected with AEV alone or betasatellite alone showed no symptoms. N. benthamiana plants infected with AEV with its associated betasatellite showed leaf curl symptoms. The findings show that AEV is predominantly a virus of weeds that has the capacity to infect crops. AYLCB appears to be the common partner betasatellite of AEV and is associated with diseases with a range of very different symptoms in the same plant species. The inability to satisfy Koch’s postulates with the cloned components of isolate SOL in A. conyzoides suggests that the etiology may be more complex than a single virus with a single betasatellite.Samples of two  Geminiviridae are important pathogens of cultivated plants throughout the warmer parts of the World and are also increasingly spreading into more temperate areas. Mainly a problem in developing nations of the tropics, some of the viruses also cause significant problems in more developed countries. A good example is Tomato yellow leaf curl virus (TYLCV). TYLCV has its origins in the eastern Mediterranean/Middle East but has in the last 20 years spread across southern Europe, North Africa, the Caribbean, parts of the Americas, China, Japan and Australia ) ,11. The Rep is a rolling circle replication (RCR) initiator protein that interferes with host cell-cycle and is the only virus encoded protein required for virus replication ). To iniAgeratum enation virus (AEV), first identified in Nepal in the late 1990s. We show that AEV has two distinct strains, and to occur across a large geographic area. Although initially isolated from weeds, we show it is also now crossing into cultivated species and could become a more significant problem for agriculture in the future.The analysis presented here concerns a virus, Apa1) site, to PCR-amplify the complete begomovirus genome. The full-length begomovirus from isolate ABF was PCR-amplified with primer pair BF/BR [HindIII restriction site designed to a partial sequence obtained with the universal begomovirus primers described previously [Total nucleic acids were extracted from leaf samples as previously described . For isoir BF/BR . The fuleviously . Betasateviously . PotentiThe complete nucleotide sequences of potentially full-length clones were determined by dideoxynucleotide chain-termination sequencing using Genome Lab Dye Terminator Cycle Sequencing kits  with reaction products run on a Beckman Coulter automated sequencer (CEQ 8000) or commercially . Sequence information was stored, assembled and analyzed using the Lasergene sequence analysis package .Geminiviridae Study Group of the International Committee on Taxonomy of Viruses [Phylogenetic analyses were conducted on matrices of aligned sequences using the neighbor-joining and bootstrap options of Phylip (v. 3.5c) running on an IBM compatible personal computer. Sequence alignments were produced using CLUSTAL X . Phyloge Viruses .ApaI-XbaI fragment of ~1.6 kb, containing the entire intergenic region, was released from the full-length clone and ligated into the binary vector pGreen0029 [ApaI, was then ligated into the ApaI site of the pGreen vector containing the 1.6 kb fragment to yield a 1.6-mer partial tandem repeat construct. A similar strategy, but using restriction endonucleases KpnI and ClaI, was followed to produce partial repeat constructs of the betasatellites.Partial direct repeat constructs of the begomoviruses and betasatellites cloned from isolates SOL and ACL were produce for the analysis of infectivity. For the begomovirus clones an reen0029 . The fulAgrobacterium tumefaciens strain GV3101. Agrobacterium cultures for inoculation were grown at 28 °C for 48 h to an optical density at 600 nm of 0.6 with antibiotic selection. The bacterial cells were pelleted (5000 g for 15 min at 4 °C) and resuspended in 10 mM MgCl2 and 150 µg/mL of acetosyringone. Cells were incubated on ice for 3 h and then inoculated into plants as previously described [Plants for inoculation were grown in a glasshouse with a 16 h light/8 h dark cycle at 28 °C. The binary vector constructs were electroporated into escribed .Ageratum conyzoides (isolate ACL) and Sonchus oleraceous (SOL), showing yellow vein symptoms (Brassica rapa var. rapa), exhibiting foliar vein yellowing, was collected from Faisalabad, Pakistan during 2006 (isolate ABF). Additionally a leaf sample originating from an A. conyzoides plant, collected in Nepal during the 1990s (isolate ACN), showing a novel symptom was included. This plant appeared superficially healthy  and with a 10 year gap, the four sequences show high levels of sequence conservation (between 95.2%–98.7% nucleotide sequence identity). Based on the 89% nucleotide sequence identity demarcation threshold for identification of begomovirus species, this indicates that all four clones are isolates of a single species ,30.Begomovirus and given the name Ageratum enation virus [Trichosanthes dioica (unpublished), EU867513 isolated from Amaranthus cruentus (unpublished), KC795968 and KC818421 isolated from tomato (unpublished), JX436472 isolated from tomato (unpublished), HE861940 isolated from soybean (unpublished), JQ911765 and HM149260 isolated from Papaver somniferum (unpublished), JX436473 isolated from Fenugreek (unpublished), JQ911767, KJ488990 and KJ488991 isolated from Ageratum sp. (unpublished), JF728865 and JF728867 (isolated from carrot [A. conyzoides [Amaranthus (unpublished), FN794201 isolated from Crassocephalum crepidioides [Zinnia elegans [Cleome gynandra [A. conyzoides [Tobacco curly shoot virus (TbCSV). This confirms the 30 sequences as isolates of a distinct begomovirus species, which has previously been named AEV [The AEV clone obtained from Nepal was obtained some time ago and was at the time recognized as a distinct species within the genus on virus . The seqm carrot ), JF7288nyzoides , JF68224idioides , FN54309 elegans , FJ17703gynandra  and FN79nyzoides . Some ofA closer analysis of the AEV sequences shows them to fall into three groups. The first group consists of all the AEV sequences characterized here, as well as GQ268327, EU867513, KC818421, KC795968, and FJ177031. These nine sequences show between 93.9% and 100% nucleotide sequence identity . The seqPapaya leaf crumple virus (PaLCrV) as the donor of the ori for the India strain (results not shown). PaLCrV occurs in India and has IRD and iteron sequences identical to those of India strain AEV sequences.Interestingly the four AEV sequences have Rep IRD sequences predicted to be either FKIN  or LKIN (AJ437618). The other three Nepal strain isolates also have LKIN. In contrast, all India strain isolates have an IRD with the predicted sequence FQIY. The corresponding iteron sequences are GGT/AGT for all Nepal strain isolates and either GGTG/AC/A, or possibly GTACT, for all India strain isolates and both problematic isolates. The alignment of all AEV sequences suggested that the differences between the Nepal and India strain isolates may be due to recombination across the origin of replication, possibly with A phylogenetic dendrogram based upon an alignment of the four sequences obtained here, the 26 other AEV sequences available in the databases and selected other begomovirus sequences available in the databases is shown in Three full-length betasatellite clones were obtained. Unfortunately the satellite for isolate ACN was not obtained for comparison, although the presence of a betasatellite with this isolate has been shown previously . The thrComparison of the three betasatellite sequences obtained here to sequences available in the databases shows them to be most similar to Ageratum yellow leaf curl betasatellite . Six defective AYLCB are available in the databases  ranging in size from 891 bp to 1270 bp, which are included in the analysis but not included in the calculated identity values. To all other betasatellite sequences available in the databases the percentage nucleotide sequence identity was less than 62% (not shown) with the highest sequence identity levels (57.5%–61.3%) to an isolate of Tomato leaf curl Bangladesh betasatellite (AJ542489).A phylogenetic dendrogram, based upon an alignment of the full-length sequences of the 3 betasatellites obtained here, all available AYLCB sequences in the databases and selected other betasatellite sequences from the databases is shown in S. oleraceus (isolate SOL) and A. conyzoides (isolate ACL) were produced for Agrobacterium-mediated inoculation of plants. The results of the infectivity studies are summarized in N. benthamiana with the AEV and AYLCB clones isolated from S. oleraceus (AEVSOL and AYLCBSOL) led to the appearance of symptoms consisting of vein thickening and dimples .Partial repeat constructs of the AEV and betasatellite clones isolated from N. tabacum, inoculation with AEVSOL and AYLCBSOL initially resulted in mild vein yellowing  led to the first symptoms appearing within 12 dpi, consisting of narrow leaves with a pointed apex with some foliar chlorosis for leaves developing subsequent to inoculation. As plants developed, the leaves remained small and chlorotic with some twisting  plants inoculated with AEVACL and AYLCBACL the symptoms were not very distinct with older plants showing stunting, reduced leaf size and a mild foliar chlorosis , which is the type isolate of the species [The begomovirus species AEV was first established in 2003, when the presently applicable species demarcation criteria for the genus  species . Since t species ,33,34,35et al. [et al. [Tobacco curly shoot virus isolated from wild sunflower originating from India (for HQ407397) and Alternanthera yellow vein virus (AlYVV) in Sonchus arvensis originating from Pakistan (for FN432358) [S. arvensis was identified in co-infection with a second betasatellite, Cotton leaf curl Multan betasatellite (CLCuMB). The identification of AYLCB with several distinct begomoviruses suggests that, as has been shown for other betasatellites [The results obtained here, as well as the results of Kumar et al.  and Marw [et al. , suggestN432358) . It is itellites ,35,38, tACL/AYLCBACL, infectivity was shown to the plant species from which the clones were isolated (A. conyzoides). However, the symptoms  induced by the cloned virus/betasatellite in A. conyzoides did not resemble the symptoms exhibited by the plant from which the clones were obtained (vein yellowing). The reason for this is unclear. Possibly the plant contained additional viruses/betasatellites that were responsible for the yellow vein symptoms, although there was no evidence for this. Nevertheless, Koch’s postulates for this isolate inducing yellow vein disease of Ageratum have not been satisfied. Possibly the Ageratum plant from which AYLCBACL was isolated contained more than one “type” of AYLCB. Certainly this betasatellite species is associated with, and thus likely can induce, a range of distinct symptoms. Kumar et al. [A. conyzoides, induced typical yellow vein symptoms in A. conyzoides. AYLCB clones have also previously been shown to be infectious in the presence of Ageratum yellow vein virus, a virus that is not reported to be associated with this species of betasatellite, inducing typical yellow vein symptoms in A. conyzoides [The results presented here demonstrated the infectivity of two of the AEV clones and their respective AYLCB clones. Both pairs of clones were highly infectious to a range of plants and, for AEVr et al.  showed tnyzoides .A. conyzoides plant from which AEVACN was isolated and induced by AEVSOL/AYLCBSOL infection of N. benthamiana and N. tabacum are unusual. This symptom, as far as we are aware, has not been previously reported for any begomovirus infection of plants and was not noted by Kumar et al. [et al. [Cabbage leaf curl virus (a bipartite begomovirus originating from the New World which is only distantly related to AEV) from a Potato virus X vector induced a very similar dimple structure on leaves of N. benthamiana. For AEV however, infection of N. benthamiana in the absence of the betasatellite did not induce symptoms, suggesting that in this case the unusual symptom is due betasatellite rather than the virus.The “dimple” symptom, exhibited by the r et al.  for their et al. . However [et al.  have shoBrassicaceae (turnip), Asteraceae , Amaranthaceae (A. cruentus), Cucurbitaceae (T. dioica), Apiaceae (carrot) and Cleomaceae (C. gynandra). This indicates that AEV is predominantly a weed infecting begomovirus, although it apparently has the capacity to occasionally infect crop species and is reported to be causing losses of the minor grain crop A. cruentus [It is evident, both from the results presented here and from the work of others, that AEV has a large natural host range that includes species from the families cruentus  and carrcruentus .The availability of infectious AEV and AYLCB clones that induce distinct symptoms in common hosts opens up the possibility of investigating the molecular basis for these differences by, for example, exchanging betasatellite components. It will be particularly interesting to discover the basis for the unusual symptoms (dimples), which have not been reported for any other begomovirus. This will be the focus of our future studies.