Bunyaviridae, with two of its gene segments showing ambisense gene arrangement. The virus causes significant economic losses to numerous field and horticultural crops worldwide.Viral small RNAs (vsiRNAs) in the infected host can be generated from viral double-stranded RNA replicative intermediates, self-complementary regions of the viral genome or from the action of host RNA-dependent RNA polymerases on viral templates. The vsiRNA abundance and profile as well as the endogenous small RNA population can vary between different hosts infected by the same virus influencing viral pathogenicity and host response. There are no reports on the analysis of vsiRNAs of Tomato spotted wilt virus (TSWV), a segmented negative stranded RNA virus in the family Nicotiana benthamiana and a commercial, susceptible host tomato. The total small (s) RNA reads in TSWV-infected tomato sample showed relatively equal distribution of 21, 22 and 24 nt, whereas N. benthamiana sample was dominated by 24 nt total sRNAs. The number of vsiRNA reads detected in tomato was many a magnitude (~350:1) higher than those found in N. benthamiana, however the profile of vsiRNAs in terms of relative abundance 21, 22 and 24 nt class size was similar in both the hosts. Maximum vsiRNA reads were obtained for the M RNA segment of TSWV while the largest L RNA segment had the least number of vsiRNAs in both tomato and N. benthamiana. Only the silencing suppressor, NSs, of TSWV recorded higher antisense vsiRNA with respect to the coding frame among all the genes of TSWV.Tomato spotted wilt virus (TSWV)-specific vsiRNAs were characterized by deep sequencing in virus-infected experimental host Details of the origin, distribution and abundance of TSWV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. It also has major implications toward our understanding of the differential processing of vsiRNAs in antiviral defense and viral pathogenicity. Bunyaviridae, genus Tospovirus), infect a broad range of staple and horticultural crops, impacting agriculture in tropical and subtropical areas globally . A-7. AN anthamiana . This isoduction , and it ficiency . It coule plants ,45. MaxiN/GC, N and RdRp are organized in the negative sense, whereas NSm and NSs are in the positive orientation of M and S RNAs, respectively (N/GC segment of the M RNA that gets processed to the highest level (35% and 28%) compared to NSm (9% and 8%) in tomato and N. benthamiana, respectively  the number of antisense-derived viral small RNAs was less than those of sense siRNAs. Examination of the relative abundance of sense and antisense viral small RNAs with respect to the coding frame of the different RNA segments of TSWV showed very interesting results . Only inIn CLRDV, a single-stranded positive sense RNA virus containing 6 open reading frames, equivalent amounts of sense and antisense vsRNAs were found in CLRDV infected cotton library . PantaleThe preference of viral siRNAs for uredines and adenine and possible selective loading into multiple AGO complexes may provide sequence specificity of RNA target recognition ,74,76,77N. benthamiana were grouped by the first nucleotide. In both the hosts, the 5’ nucleotide on 21 nt TSWV vsiRNAs was most frequently a Uracil (U) (55%/45% respectively) followed by Adenine (A) at (30%/30%),  and Guanine (G) with majority of 21 and 22 nt vsiRNAs showing a strong bias of sequence beginning with a 5’U [N. benthamiana infected with Tomato yellow leaf curl china virus has also been reported [TSWV vsiRNAs in infected tomato and 0%/30%), . Similarth a 5’U ,45. A sireported . Our obsreported ,38,78. Treported . Donairereported  also repN. benthamiana. and tomato respectively. The GC % of (53.8%) of BaMV vsiRNAs was also slightly higher from that of BaMV genome (50.6%) in systemically infected leaves of N. benthamiana [There was no noticeable difference between the relative frequencies of the 5’ nucleotides in vsiRNAs targeting the different TSWV genes in both the hosts . No diffthamiana . DCLs arthamiana ,40,79. N. benthamiana mirrored each other indicating that the host has not made any difference to the processing of vsiRNAs and it seems to be entirely dependent on the virus sequence  platforms, including Roche 454, Illumina GA, and the ABI SOLiD technologies are strongly biased toward certain sequences. For the short-read platforms (Illumina GA and ABI SOLiD) read-depth coverage decreases with increasing AT content [Groundnut ringspot virus, Tomato chlorotic spot virus, and Watermelon silver mottle virus, in a single small chimeric hairpin (hp) RNA construct and obtained resistance to all four viruses in transgenic plants. The hotspot peaks for TSWV vsiRNAs were distributed throughout the gene fragments and no simple relationship was observed between GC distribution and vsiRNAs hot spots. Interesting observation was one particular segment giving a very prominent peak in NSs at positions 1101-1119 in the S segment (starting at 1). This was calculated from positions with over 50000x or 100x 21 nt reads in tomato and escribed ,53. The  content . Previou content . Subsequ content ,82 showe content  combinedN. benthamiana samples having similar virus titer in both hosts. It is inferred that mRNAs are targeted by corresponding vsiRNAs [Cucurbit leaf crumple virus-infected plants, vsiRNA levels positively correlated with virus titers [Our results provide an in-depth investigation and characterization of viral siRNAs from a three component RNA virus with an ambisense coding strategy, and contribute to the understanding of the role of RNAi with respect to the TSWV genome. The number of TSWV-specific 21-22 nt RNA reads detected in tomato was many magnitudes higher than in  vsiRNAs , therefos titers . Thus, eFigure S1The distribution of 5’ terminal nucleotides of Tomato spotted wilt virus (TSWV) vsiRNAs (17-26 nt) derived from TSWV infected tomato and N.benthamiana. The relative percentage of the 5’terminal nucleotide of vsiRNAs is represented for the individual gene segments of TSWV in both hosts.(TIF)Click here for additional data file.Figure S2RNAfold analysis of Tomato spotted wilt virus (TSWV) in Large (L), Medium (M) and Small (S) RNA segments derived using the thermodynamic prediction of minimal free energy (MFE) (Lorenz2011), a mountain plot representation of the MFE structure is shown.(TIF)Click here for additional data file.


Of 3,110 (29.3%) samples positive for pandemic (H1N1) 2009 virus, 28% contained  For previous and current influenza A pandemics, postmortem studies have established a strong link between secondary bacterial infections and increased deaths ,2. Numerwww.diatherix.com; Huntsville, AL, USA) and screened for multiple respiratory pathogen gene targets. Diatherix, a reference laboratory certified by the Clinical Laboratory Improvement Amendments, provides molecular differential detection services based on target-enriched multiplex PCR technology. The respiratory infections panel detects bacterial and viral pathogens associated with respiratory infections and includes targets for the following: adenovirus , coxsackievirus, echovirus, human metapneumovirus (types A and B), influenza virus (types A and B), parainfluenza virus (types 1–4), respiratory syncytial virus (types A and B), rhinovirus, Acinetobacter baumannii, Chlamydophila pneumoniae, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Mycoplasma pneumoniae, Neisseria meningitidis, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes (group A). Additionally, targets specific for pandemic (H1N1) 2009 virus were developed, validated, and approved by the US Food and Drug Administration under Emergency Use Authorization provisions for patient testing.From May through October 2009, a total of 10,624 clinical samples from 23 states throughout the United States were submitted to Diatherix Laboratories  and MagnetX chemistry  according to manufacturers’ specifications. Multiplex PCR amplification and Luminex  liquid suspension detection methods were based on internally validated protocols. Reactions were amplified by using ABI 9700 thermocyclers , and the resulting PCR products were detected by using the LiquiChip 200 Workstation (Luminex) according to previously described protocols , S. pneumoniae , and H. influenzae  , and rhinovirus   were negative for all pathogens detectable with the assay. Among the 7,514 (70.73%) samples negative for pandemic (H1N1) 2009 virus, 3 bacterial pathogens predominated: ; 5.47%) . The mos; 5.98%) .>1 other pathogen was co-detected for 28% , S. pneumoniae , and H. influenzae   were positive for pandemic (H1N1) 2009 virus, representing 52.4% of samples positive for any pathogen. Among pandemic (H1N1) 2009 virus–positive samples,  for 28% . The mos; 3.54%) .t = 25.6, p = 0.01) was found for the age distribution between patients with positive and negative pandemic (H1N1) 2009 virus results. The mean ± SD age was 19.64 (±14.45) years for those who were pandemic (H1N1) 2009 positive and 29.67 (±19.74) years for those who were negative. The median age of the 5 patients for whom 3 other pathogens were co-detected with pandemic (H1N1) 2009 virus  was detected. Of the 96 samples in which pandemic (H1N1) 2009 virus and 2 other pathogens were co-detected  2009 virus and multiple other pathogens are often detected during autopsy  2009 virus strain.The true value of a multiplex molecular method of screening for infectious respiratory agents depends on the clinical relevance. Among the samples with Our findings suggest that multiplex screening for respiratory pathogens is useful for providing rapid surveillance information to inform physicians who would otherwise base decisions on clinical signs and symptoms alone. Electronic reporting of empirical laboratory respiratory pathogen detection provided by a Clinical Laboratory Improvement Amendments–approved laboratory can greatly enhance surveillance data collection . Because


Employing the criterion of 4-fold greater titer, 78 (10.3%) equines were seropositive for Ilheus virus, 59 (7.8%) for Saint Louis encephalitis virus, 24 (3.2%) for WNV, two (0.3%) for Cacipacore virus and one (0.1%) for Rocio virus. No serological evidence was found linking the neurological disease that affected local equines to WNV. All caimans and sheep were negative by blocking ELISA for flaviviruses. There were no seropositive equines for Bussuquara, Iguape, Yellow fever and all four Dengue virus serotypes. The detection of WNV-seropositive equines in ten ranches and ILHV and SLEV-seropositive equines in fourteen ranches of two different sub-regions of Pantanal is strong evidence of widespread circulation of these flaviviruses in the region.A recent study reported neutralizing antibodies to West Nile virus (WNV) in horses from four ranches of southern Pantanal. To extend that study, a serosurvey for WNV and 11 Brazilian flaviviruses was conducted with 760 equines, 238 sheep and 61 caimans from 17 local cattle ranches. Among the tested equines, 32 were collected from a ranch where a neurologic disorder outbreak had been recently reported. The sera were initially screened by using a blocking ELISA and then titrated by 90% plaque-reduction neutralization test (PRNT West Nile virus is maintained in cycles between birds and mosquitoes and recently reemerged as a worldwide major public health and veterinarian concern as the cause of human and equine encephalitis outbreaks. Recent studies have reported serological evidence of West Nile virus circulation in Pantanal, west-central region of Brazil. However, considering the co-circulation of various cross-reactive flaviviruses in Brazil and that most of the flaviviruses isolated in Brazil are unknown or understudied in the Pantanal, serological results should be interpreted with caution. Therefore, we conducted a serosurvey for West Nile virus and the 11 Brazilian flaviviruses of potential medical importance, utilizing equines, sheep and caimans as indicators, including 32 equines collected from a ranch where a neurologic disorder had been recently reported among the equines. We found serological evidence of Ilheus, Saint Louis encephalitis, West Nile, Cacipacore and Rocio viruses in Pantanal equines. West Nile virus infection was not associated with the neurological disease of equines. The detection of WNV-seropositive equines in ten ranches and ILHV- and SLEV-seropositive equines in fourteen ranches of two different sub-regions of Pantanal is strong evidence of widespread circulation of these flaviviruses in the region. Flaviviruses represent a group of mosquito-borne viruses in Brazil that are annually involved in a large number of human cases of dengue fever countrywide and sporadic local outbreaks of sylvatic yellow fever Thirteen flaviviruses have been reported in Brazil, listed here in chronological order of discovery: YFV, ILHV, BSQV, SLEV, ROCV, CPCV, Dengue virus 1 (DENV-1) and Dengue virus 4 (DENV-4), Dengue virus 2 (DENV-2), Iguape virus (IGUV), Naranjal-like virus (NJLV), Dengue virus 3 (DENV-3) and Culex flavivirus (CXFV) 2, the Pantanal is a vast sedimentary floodplain characterized by seasonal flooding which determines specific ecosystem processes, with the occurrence of plants and animals that are adapted to the annual shrinking and expansion of habitats due to the seasonal hydrological regime The Pantanal wetland is a subtropical region of great biodiversity with strong potential for maintenance and evolution of mosquito-borne viruses. Comprising approximately 140,000 kmMost of the flaviviruses isolated in Brazil are unknown or understudied in the Pantanal. A small number of investigations in the Nhecolândia Sub-region of the Pantanal, MS, have detected serological evidence for four flaviviruses, including ILHV, SLEV, WNV and CPCV In October 2009, April, September and October 2010, blood samples were taken from 724 equines, 238 sheep and 61 caimans from 16 beef cattle ranches comprising an area of approximately 2,700 square-km in the Nhecolândia Sub-region of Pantanal, MS, west-central Brazil .The collections for this study were approved by the Animal Ethics Committee of Fundação Oswaldo Cruz  in compliance with the requirements of Brazilian Law 11794/2008, which rules on the scientific use of animals, including the principles of the Brazilian society of Science in laboratory animals. The collections were also approved by the Brazilian Institute of Environment and Natural Resources (licenses IBAMA 18363-1/2009 and 18363-2/2010).Caimans were captured in two ranches from sites where a high concentration of these animals was observed. Caiman blood samples were obtained by puncture of the internal jugular vein between the 1st and 2nd cervical vertebrae as described previously Blood samples from equines, including horses, donkeys and mules, and from sheep were taken by puncture of the jugular vein in 14 and nine different ranches, respectively. Gender, age, tameness, breed, vaccination status, travel history outside of Pantanal and history of abnormalities, such as clinical signs involving the central or peripheral nervous system, were recorded for each equine sample. Information about gender, age, breed and travel history outside of Pantanal were also recorded for each sheep. All animals sampled appeared healthy except for one horse that presented with clinical signs suggestive of neurological disorder.Trypanosoma evansi, a common pathogen involved in incoordination and instability of hind limbs in Pantanal equines. These equines had not been vaccinated for equine encephalitis viruses at the time of the outbreak. After the first deaths, the herd was vaccinated for eastern and western equine encephalitis viruses.Additionally, 36 horse samples collected in February 2010 from a ranch comprising roughly a 900-square-km area in the Nabileque Sub-region of Pantanal, also located in MS  were tes90, as previously described 90 for WNV.All samples were initially screened for flavivirus-reactive antibodies by blocking ELISA as described previously All reference viruses used for PRNT were previously submitted to partial nucleotide sequencing of the N terminal region of NS5 gene to confirm viral identity. High identity scores were obtained with the following sequences deposited at GenBank: DENV-1 (FJ562106), DENV-2 (GQ398257), DENV-3 (recombinant ChimeriVax Dengue 3 virus) (JN811143), DENV-4 (GQ199880), ROCV (AY632542), SLEV (EF158048), ILHV (AY632539), YFV (JN628279), BSQV (AY632536), CPCV (AF013367), IGUV (AY632538), and WNV (JN819325).Serum was considered seropositive to a virus when it reduced at least 90% of the formation of plaques of this virus and its reciprocal neutralizing antibody titer was at least four-fold greater than what was observed for the other 11 tested flaviviruses. The PRNT is the most specific serological test for the differentiation of flavivirus infections in convalescent serum samples 90 for WNV, all caiman samples confirmed negative. Sixty caiman samples had PRNT90 titer <10 and one <40 for WNV (low sample volume of the latter required testing at a lowest dilution of 1∶40). From 238 sheep samples tested, 235 had PRNT90 titers <10, two showed low neutralizing antibodies titers of 20 and 10 and one <40. From 760 equine samples initially screened for flavivirus-reactive antibodies by blocking ELISA, 396 (52.1%) were positive. When this subset was tested using PRNT90, 332 (43.7%) had neutralizing reactivity (PRNT90 titer ≥10) for ILHV, 251 (33%) for SLEV, 172 (22.6%) for WNV, 139 (18.3%) for CPCV, 130 (17.1%) for ROCV, 62 (8.2%) for IGUV, 14 (1.8%) for YFV, 12 (1.6%) for BSQV, four (0.5%) for DENV-1, three (0.4%) for DENV-2, one (0.1%) for DENV-4 and none for DENV-3  equines were seropositive for ILHV, 59 (7.8%) for SLEV, 24 (3.2%) for WNV  were detected for ILHV (n = 27), SLEV (n = 11) and WNV (n = 6).90 titers: 80 for SLEV, 40 for ILHV, 20 for YFV, 10 for ROCV and <10 for all other flaviviruses tested.The only horse from Nhecolândia Sub-region that presented neurological signs was seropositive for undifferentiated flavivirus with the following PRNT90 titers for WNV ≥40 and among them 26 showed equal or higher titers for one of the other flaviviruses tested. For these 37 equines, the serology profile could not be interpreted to determine whether WNV reactivity was due to infection with WNV or cross-reactivity from other flavivirus infections. In fact, six of them were considered seropositive for one of the 11 other flavivirus tested  had date of birth available. Stratifying the serology results by age demonstrated that risk of exposure to flaviviruses increased with age for flaviviruses in general and for ILHV (1-tailed p<0.001 for both), but not for SLEV (p = 0.17) or WNV (p = 0.46) . SLEV anEquines seropositive for CPCV and ROCV were sampled in October 2009. The two CPCV-seropositive horses were five and seven years of age and were from ranch PQ, which comprises an area of approximately 140 square-km. The only horse that was seropositive for ROCV had no record of birth date and was from ranch PM that encompasses a 360-square-km area.90, 11 (30.5%) confirmed seropositive for SLEV, nine (25.0%) for undifferentiated flavivirus(es), two (5.5%) for WNV, and one (2.8%) for ILHV  were seropositive for flavivirus infection by blocking ELISA. When this subset was tested by PRNTfor ILHV . Of the for ILHV . In spitIn the absence of direct viral detection, diagnosis of arbovirus infections is performed by indirect serological tests. However, cross-reactive antibody responses to closely related flaviviruses can complicate interpretation of serologic test results In the present study, serological evidence of ILHV, SLEV, WNV, CPCV and ROCV circulation was detected in the Nhecolândia and Nabileque sub-regions of Pantanal. Nhecolândia is the second largest sub-region of Pantanal comprising roughly 27,000 square-km or 19% of the total Pantanal area and it is the world's largest and most diverse area of subtropical lakes The seroprevalence of ILHV and all flaviviruses combined increased with age in equines . This teAedes scapularis specimens captured in the Nhecolândia Sub-region in 2010 In the present study, the detection of SLEV- and ILHV-seropositive geldings both born in September 26, 2009 and sampled on September 30, 2010 with no history of travel outside of Pantanal indicates that the transmission of SLEV and ILHV had occurred as recently as late 2009 or 2010. The enzootic status of ILHV is corroborated by the concurrent isolation of ILHV from Regarding WNV, the youngest seropositive equine was a 2 year-old untamed mare with no history of travel. Because it was sampled on October 23, 2009, the infection occurred between 2007 and 2009.The confirmed WNV-seropositive equines were detected in ten ranches comprising an area of approximately 2,500 square-km in the Nhecolândia and Nabileque sub-regions of the Pantanal, located in MS. Monotypic neutralizing reactions for WNV were detected in six ranches encompassing roughly 1,300 square-km, which comprise approximately 20% of the Nhecolândia Sub-region area. WNV-seropositive equines were detected in Nhecolândia and Nabileque ranches located roughly 90 and 100 km distant from the city of Corumbá, MS respectively . ConsideThe two CPCV-seropositive equines were sampled October 20, 2009 at five years of age and no history of travel outside of Pantanal, which indicates that infection had occurred between 2004 and 2009.We report here for the first time the detection of ROCV-neutralizing antibodies in the Pantanal region. ROCV, which is an indigenous Brazilian flavivirus, was the causative agent of an extensive encephalitis epidemic in southeastern Brazil in the 1970s No seropositivity for BSQV, IGUV, YFV and DENVs was confirmed among the equines tested in the present study. However, except for DENV-3, neutralizing antibodies were found to all the flaviviruses tested. More investigation is needed to understand the potential circulation of these viruses in the Pantanal region, as they may be restricted to ecological cycles for which equines, sheep and caiman fail to serve as indicators. A serosurvey of non-human primates and/or local human residents would be more instructive for these flaviviruses. Testing only equines may provide a biased view of the relative amounts of flavivirus transmission because equines may not mount immune responses to all viruses equally, or they may not attract all vectors equally. For example, they failed to generate primary immune responses to DENV-2 and SLEV For future flavivirus serosurveys to be held in the Pantanal, not only ILHV, SLEV, WNV, CPCV and ROCV should be included as differential diagnosis, but also those flaviviruses for which neutralizing antibodies titers ≥20 were detected, including BSQV, IGUV and YFV, if a PRNT with a four-fold greater titer criterion is used.Regarding the 36 equines collected from ranch PV in the Nabileque Sub-region of Pantanal after a neurologic disorder outbreak occurred in 2009 and 2010, no serological evidence was found linking this epizootic to WNV, which is currently the only flavivirus known to be involved in outbreaks of equine neurological disorders in the New World. Three of four survivors of the recent neurologic syndrome had elevated titers for SLEV, and one was the highest SLEV titer detected in the herd, suggesting that the etiologic agent was a flavivirus that triggered a boost of pre-existing SLEV antibody or that SLEV infection may have been associated with the neurologic syndrome as a contributing risk factor perhaps through antibody enhancement. Equines experimentally infected with SLEV have not developed overt disease Alligator mississippiensis has been shown to develop sufficient WNV viremia to infect blood-feeding mosquitoes Crocodylus moreletii) in Mexico Amphibians and reptiles may play a larger role in the transmission cycle of arboviruses than previously assumed. Because of their high energy conversion efficiency, ectotherms typically represent a large portion of the vertebrate biomass in many ecosystems, and may therefore greatly influence transmission dynamics, either as virus amplifiers or dilution hosts Caiman crocodilus yacare) in the Brazilian Pantanal, about 3.9 million non-hatchling caimans in 1993 90 for WNV. The absence of serological evidence of flavivirus circulation in caimans was unexpected. Possible explanations for these findings are: (i) low circulation of flavivirus in caimans, (ii) caimans may not mount immune responses to flaviviruses and, finally, (iii) caimans may not attract local mosquito vectors of flaviviruses.Because of widespread, high prevalence of the yacare caiman (Culex (Melanoconion) theobaldi and Mansonia spp. Aedes scapularis, which was the most prevalent species caught landing or blood-feeding on caimans from the Nhecolândia Sub-region In a study conducted also in the Pantanal, but in a different sub-region, 80% of mosquitoes caught blood-feeding on caimans were The absence of serological evidence of flavivirus circulation in sheep by blocking ELISA and the detection of only two samples with neutralizing antibodies for WNV in very low titers was also an unexpected result, considering that the tested sheep were temporally and spatially associated with equines that were seropositive for various flaviviruses. However, reports of serological evidence of WNV infection in sheep are scarce and have been showing low prevalence In the present study, serological evidence of ILHV, SLEV, WNV, CPCV and ROCV circulation was detected in equines of the Pantanal. Severe disease has been linked to these viruses in humans Considering the highly conservative criteria used in the present study, the detection of seropositive equines for ILHV, SLEV, WNV, CPCV and ROCV is strong evidence for local circulation of these flaviviruses in the Pantanal of MS, Brazil. These flaviviruses should be included as differential diagnosis in future flavivirus serosurveys to be held in the region. For PRNT studies, not only these flaviviruses, but also YFV, BSQV and IGUV should be included.It appears that WNV circulated recently in the Pantanal. The detection of WNV-seropositive equines in at least ten ranches is strong evidence of recent widespread circulation, but not necessarily establishment of WNV in the region. Circulation of WNV in this area has not yet been confirmed by virus isolation. Because detection of antibodies is indirect evidence of flavivirus circulation and because novel flaviviruses may exist in the region, we encourage efforts to isolate viruses to confirm the circulation of these flaviviruses. In future investigations of equine epizootics, efforts should be concentrated on the collection of samples suitable for viral isolation.


Km (180.27±6 µM) compared to the soluble enzyme using azocasein as substrate. Further, immobilized procerain B retains 38.6% activity till the 10th use, which strongly represents its industrial candidature.Proteases are involved in several crucial biological processes and reported to have important physiological functions. They also have multifarious applications in different industries. The immobilized form of the enzyme further improves its industrial applicability. Here, we report covalent immobilization of a novel cysteine endopeptidase (procerain B) on amberlite MB-150 beads through glutaraldehyde by Schiff base linkage. The immobilized product was examined extensively by Fourier Transform Infrared Spectroscopy (FTIR), Scanning electron microscopy (SEM) and Energy Dispersive X-ray (EDX) analysis. The characterization of the immobilized product showed broader pH and thermal optima compared to the soluble form of the enzyme. The immobilized form of procerain B also showed lower  Enzyme immobilization is a process of physical localization of enzymes to a defined surface which helps to improve several enzymatic properties and enhance their operational performance without disturbing their catalytic activity Carica papaya used for meat tenderization Proteases have several applications in food, dairy and detergent industries. It also has a wide range of applicability in medicines, energy production and environmental control Calotropis proceraProteases from different plants, animals and microbial sources are known. Plant cysteine proteases are well known for their high thermal stability which makes them a potential candidate for several industries where high temperature is required at certain stages Calotropis procera by the method of Singh et al., 2010The enzyme was purified from the latex of The protein concentration was determined at different steps of immobilization by the method of Bradford with BSA as standard The proteolytic activity of procerain B was determined as described earlier Procerain B was immobilized on glutaraldehyde activated amberlite MB-150 beads (100–200 mg) of diameter 0.5 mm. Before activation with glutaraldehyde, the beads were equilibrated for overnight at different pH in the range of pH 4 to 10. The buffers used for equilibration at different pH were, acetate buffer (50 mM) for pH 4, phosphate buffer (50 mM) for pH 6, Tris-HCl buffer (50 mM) for pH 8 and pH 10-. The equilibrated beads were treated with different glutaraldehyde concentrations in the range of 1% to 5%v/v at room temperature for different time intervals and then washed extensively with respective buffers to remove the excess of glutaraldehyde. The complete removal of unreacted glutaraldehyde was confirmed spectrophotometrically by monitoring the absorbance of wash, till the absorbance was lower than 0.01 at 280 nm. In order to determine the appropriate time for coupling reaction, the activated beads were incubated with enzyme for different time intervals and the coupling time which corresponds to maximum immobilization was used for further experiments. The activated beads were incubated with different concentrations of purified enzyme and the optimum concentration at which maximum immobilization was achieved was considered for further experiments.The immobilization (percentage enzyme activity on beads) was calculated as follows:Note: Total activity of immobilized protease was determined by subtracting total activity of unbound protease from total activity of soluble enzyme.The activation of amberlite beads with glutaraldehyde was confirmed by comparing the FTIR spectra of normal and glutaraldehyde activated beads. The FTIR spectra were taken with UNICAM Mattson1000 FTIR Spectrophotometer and compared with each other. In order to prepare the sample for FTIR analysis the beads were crushed with potassium bromide (KBr) and compressed in form of thin pellet which was used directly for the analysis.The detailed surface morphology and micro structural details of normal, activated and immobilized amberlite MB-150 beads were analyzed by SEM. The samples were coated with gold to make the surface conducting and then imaged and photographed by secondary electron imaging by SEM (LEO 1430 VP) at an acceleration voltage of 10.00 kV. The EDX spectra were also collected to analyze the elemental composition of bead surface.The activity of immobilized procerain B was studied as a function of pH to determine the optimum pH of immobilized enzyme. In all activity assay experiments the amount of amberlite beads were kept constant (50 mg). The buffers used for different pH were, 50 mM glycine-HCl (pH 2–3.5), 50 mM acetate buffer (4–5.5), 50 mM phosphate buffer (pH 6–7.5), 50 mM Tris-HCl buffer (pH 8–10), 50 mM Na-carbonate buffer (pH 10.5–12). The substrate solutions (1% azocasein or hemoglobin (w/v) were prepared in respective buffers. The beads with immobilized procerain B were incubated in respective buffers for 15 min and then the activity assay was performed as described earlier. Hemoglobin was used as substrate at pH less than 4.0 due to insolubility of azocasein at lower pH The effect of temperature on the activity of immobilized procerain B on amberlite beads were studied in the range of 10–95°C. Immobilized amberlite beads (50 mg) were incubated at different temperatures for 15 min in Tris-HCl buffer pH 8 and then assayed for proteolytic activity at corresponding temperatures as described earlier. The 1% azocasein solutions in same buffer were already incubated at respective temperatures and used as substrate during experiment. A control assay without enzyme was performed at each temperature and used as blank.The effect of pH (2–12) and temperature (10–95°C) on the stability of amberlite immobilized procerain B were studied in terms of residual activity. Immobilized amberlite beads (50 mg) were incubated at different pH for overnight at room temperature and the residual activity was tested by activity assay as described earlier with azocasein as substrate.Similarly the effect of temperature was also studied by incubating 50 mg of immobilized amberlite beads at different temperatures for 15 min and then testing the residual activity with azocasein as substrate at 37°C.m) and reaction velocity (Vmax) were calculated by Lineweaver-Burk plot.Kinetic parameters for immobilized procerain B were tested at pH 8 and 37°C with azocasein as substrate by increasing the concentration from 10 to 400 µM. For each concentration, a control reaction was done without enzyme. The Michaelis-Menten constant  for 6 h. The activated beads were washed extensively with respective buffers and then incubated for 24 hr with procerain B for immobilization. The maximum immobilization (52.65%) was observed at pH 8 . The time for activation with 4% glutaraldehyde concentration was also optimized in the range of 1–8 h and 4 h was found to be most effective (55.86±0.58% immobilization) for amberlite activation . However, the activation for more than 4 h did not show any further increase in immobilization. The next parameter to be optimized was coupling time for attachment of enzyme through free amino group with aldehyde of glutaraldehyde activated amberlite beads. The coupling time was varied from 8–32 h and 24 h was the most effective with 56.98±0.72% immobilization . For economic use of enzyme in immobilization the enzyme concentration was also optimized by varying the concentration from 0.2 mg/ml to 1.0 mg/ml. Maximum immobilization (62.07±1.13%) was achieved with 0.8 mg/ml enzyme concentration .The immobilization of procerain B on amberlite MB-150 beads was optimized in terms of glutaraldehyde concentration, activation time, coupling time and enzyme (Procerain B) concentration. Different glutaraldehyde concentrations (1–5%) were used to activate the beads for immobilization of enzyme and 4% was found to be optimum with 54.51±0.62% immobilization . The tim. The tim. The tim−1, 2925 cm−1 and 1121 cm−1 in normal amberlite beads  was due to amberlite composition and the corresponding peaks were also present in glutaraldehyde activated beads at 3411 cm−1, 2921 cm−1 and 1124 cm−1 respectively . The peak at 3427 cm−1 corresponds to O-H stretch of alcohol, 2925 cm−1 represents alkane C-H stretch and 1121 cm−1 reflects the C-N stretch of amine. The increased peak intensity at 1633 cm−1 in graph B might be due to activation of beads with glutaraldehyde. Other peaks at 1453 cm−1 in graph-A, 1413 cm−1 and 1475 cm−1 in graph-B were due to aromatic C = C stretch whereas the peak at 1220 cm−1 in graph B represents C-O stretch of acid. The SEM analysis of beads represents the changed topology of activated and immobilized beads in comparison to normal amberlite beads . The Energy Dispersive X-ray (EDX) analysis by area scan suggests increased nitrogen percentage on the surface of immobilized beads  in comparison of normal and activated one, which may be due to immobilization of procerain B on the surface of amberlite beads.The activation of amberlite MB-150 beads with 4% (v/v) glutaraldehyde concentration was confirmed by Fourier Transform Infrared Spectra (FTIR). The spectra of normal and activated beads were compared. The peaks at 3427 cmte beads , A was dte beads , A was d. It can be used in the reactions where alkaline pH is required. The functional thermal optima of procerain B immobilized on the surface of amberlite beads was found to be in the range of 45–65°C  which is higher than the soluble form In comparison to soluble procerain B . It can . Procerain B immobilized on amberlite beads follows Michaelis-Mentan kinetics with increasing substrate concentration. The Lineweaver-Burk plot showed an apparent Km of 180.27±6 µM for azocasein as substrate  which is less than soluble form of enzyme (210 µM). Decrease in Km reflects the enhanced affinity of amberlite immobilized enzyme for substrate in comparison to soluble form. This might be due to conformational change in enzyme after immobilization or increased localized concentration of substrate around beads due to particular ionic state of beads at that pH.Temperature stability of an enzyme is a prime concern especially for enzymes with industrial importance as same enzyme is going to be used for different batches of reactions. In terms of thermal stability, the immobilized procerain B retains its maximum activity (more than 80%) up to 60°C . Procerath batch of reaction .Reusability of immobilized enzyme is one of the important advantages which makes the immobilized enzyme a preferred choice over the soluble form. The procerain B immobilized on amberlite beads showed 38.60% activity till 10Amberlie MB-150 is a robust ionic resin for immobilization of procerain B. After glutaraldehyde activation nearly 62% immobilization of procerain B can be achieved with optimized parameters. The immobilized form of enzyme showed comparatively broad pH and temperature optima with better stability in alkaline pH range which proves its candidature as a potential candidate for different industrial purpose.Material S1EDX analysis of bead surface. A. Normal Amberlite MB-150 beads, B. Glutaraldehyde activated Amberlite MB-150 beads, C. Immobilized Amberlite MB-150 beads.(DOC)Click here for additional data file.


In 2012, an unprecedented large-scale outbreak of disease in pigs in China caused great economic losses to the swine industry. Isolates from pseudorabies virus epidemics in swine herds were characterized. Evidence confirmed that the pathogenic pseudorabies virus was the etiologic agent of this epidemic. Alphaherpesvirinae subfamily within the family Herpesviridae. This pathogen has major economic consequences in pig husbandry (Pseudorabies virus (PRV), also called Aujeszky disease virus or suid herpesvirus type 1, is a member of the Vaccination of pigs with attenuated live or inactivated vaccines is widely performed to reduce the huge economic losses caused by PRV infection , anorexia, coughing, respiratory distress, conjunctival serous and mucinous secretion, and posterior paralysis. The disease was first observed in older pigs and spread within 2–3 days to younger pigs. Duration of disease was 5–7 days. Rate of illness reached 50%, and mortality was 3%–5%. Most pig deaths were recorded on the third day after monitoring began. Abortion was observed in ≈35% of sows that were 70–90 days pregnant. Viscera  and serum samples were collected from dead pigs from different provinces. Pathologic examination showed the most striking gross lesions were consolidated in the lungs  and caused considerable economic losses among local pig farms.PRV has been recognized as a source of infection for pigs and continues to circulate globally among herds (Even though the PRV isolates showed nucleotide insertions in the gC, gD, and gE genes, the molecular mechanisms underlying their high pathogenesis have yet to be elucidated. The origin of these lethal isolates within China is still obscure, although phylogenetic trees based on the gE gene here indicated that the 3 isolates are more closely related to the Asia PRV isolates, especially the China isolates, than to isolates from other countries. Because the virulence and origin of PRV is thought to be associated with multiple factors, whether such insertions are related to the virulence of PRV remains an issue and requires further investigation.In summary, our study indicates that an outbreak of disease in pigs in China, which was of unprecedented scale, was caused by PRV infection. Other pathogens were ruled out. Our findings highlight the need to prevent and control the spread of this virus.Pseudorabies virus target genes and primers; sequences obtained from GenBank used in this study; immunohistochemical staining of lung and brain specimens of clinically sick pigs; PCR of infected tissues with specific primers for pseudorabies virus glycoprotein D–specific primers; and phylogenetic trees of glycoprotein E sequence.


Endogenous retrovirus (ERV) genomes integrated into the chromosomal DNA of the host were first detected in chickens and mice as Mendelian determinants of Gag and Env proteins and of the release of infectious virus particles. The presence of ERV was confirmed by DNA hybridization. With complete host genomes available for analysis, we can now see the great extent of viral invasion into the genomes of numerous vertebrate species, including humans. ERVs are found at many loci in host DNA and also in the genomes of large DNA viruses, such as herpesviruses and poxviruses. The evolution of xenotropism and cross-species infection is discussed in the light of the dynamic relationship between exogenous and endogenous retroviruses. Conveenv-defective Rous sarcoma virus (RSV) during my doctoral studies in 1966. That was the year that Peyton Rous was awarded the Nobel Prize for Medicine or Physiology for the isolation of his eponymous virus published in 1911, representing a record 55 year incubation period between reporting a discovery and the award [I first came across an endogenous factor which functionally complemented he award  in 1958 Integration of viral DNA into host DNA was first discerned for the prophage of the temperate bacteriophage lambda by Andre Lwoff in 1950 and for the simian DNA virus SV40 in cultured mammalian cells in 1968 .2.gag, pol and env. More recent evidence shows that the ‘complex’ spumaviruses and lentiviruses also have endogenous representatives in host species ranging from sloths to rabbits [I have previously described how endogenous retroviruses (ERV) were discovered , so a su rabbits –14. TherThe evidence for ERV was accrued first through virological and immunological observations, followed by molecular biological evidence of viral DNA hybridization to host DNA. The replacement of liquid DNA hybridization by Southern blotting revealed multiple endogenous insertions at many chromosomal loci. Since complete sequencing of host genomes was introduced, data mining and bio-informatic searches show how much DNA is of retroviral origin, ranging from single LTR inserts to complete genomes. For example, approximately 8% of human DNA represents fossil retroviral germ-line insertions .(a)env-defective Bryan strain of RSV was observed to release infectious virus from certain chick cells [env and gag markers were then shown to be inherited as a single host locus [In chickens, an immunological assay for Gag antigen was used to screen flocks to determine whether they harboured the alpha-retrovirus, avian leukosis virus (ALV). However, the specificity of this test was confounded by the presence of a cross-reacting antigen in some birds. This Gag-like antigen was shown to be inherited as a simple dominant allele in certain breeds of fowl . Meanwhick cells ,18. Thisck cells . The envst locus . In addist locus . The relst locus .Gallus [With the introduction of DNA hybridization methods, similar amounts of ALV DNA were found to be present in chicken DNA from phenotypically negative breeds as well as positive breeds . Later, Gallus . The smaGallus .(b)wnt and fgf families result in mammary carcinogenesis [Murine mammary tumour virus (MMTV) is the prototype beta-retrovirus. Breast cancer susceptibility in mice has taken a full circle in being viewed first as a genetic disease, then as an infection and then back to inheritance as a Mendelian provirus in certain mouse strains. In the early twentieth century, different inbred strains of mice were selected for having either a high incidence (C3H) or a low incidence (C57Bl) of breast cancer. In 1936, Bittner demonstrated that the incidence of breast cancer was determined by a transmissible factor in the milk so that a low strain mouse would develop breast cancer if suckled on a high strain foster-mother, and vice versa . The milogenesis .(c)Murine leukemia virus (MLV) is the protype gamma-retrovirus. There was evidence throughout the 1960s that latent MLV could be activated by radiation of apparently uninfected mice and that host genetics played an important role in MLV control and expression, but germ-line transmission of MLV was not conceptualized at that time. The Akr strain of inbred mice has a high incidence of thymic lymphoma associated with MLV, and it became a favoured model for viral leukaemogenesis. The true endogenous nature of the provirus was revealed when virus was induced in uninfected embryonic fibroblast cultures by treatment with bromodeoxyuridine ,35, whicEmv-11 and -12. They are examples of ‘ecotropic’ viruses, which can replicate to high titre in the species in which they are inherited; others are ‘xenotropic’ as they can infect human and other mammalian cells but not reinfect cells of other common inbred mouse strains, and others are ‘polytropic’ as they can infect a broader range of hosts. Jay Levy coined these terms of tropism and host range when he first discovered xenotropic virus in New Zealand black/white hybrid mice [In the genome of Akr mice, replication-competent MLV is integrated at two loci, rid mice . Furtherrid mice . The hosenv [During the 1970s, ERVs were found in numerous other mammalian species. For instance, infectious viruses are produced in the placenta of baboons , which renv . A relatenv  give a f3.Endogenous retroviral genomes were originally derived from exogenous infections of the germ-line. While some integration sites are ancient, others arise through activation and reinfection, as well as entry of new infectious viruses into a species throughout evolution. This has been mapped in the primate lineage leading to humans, where successive waves of HERV-K genomes have recolonized the host DNA . When acenv. A high-frequency genetic recombination occurs with endogenous env, provided that it is expressed and packaged into retroviral particles [env sequences occurs to generate a virus, which uses the polytopic receptor [Recombination between exogenous and endogenous retroviruses was first demonstrated using a non-defective strain of exogenous RSV with a host range marker determined by articles . Recombireceptor . The higreceptor .envs to give rise to leukaemogenic FeLV-B [env sequences [in vitro [In cats, the exogenous feline leukaemia virus (FeLV) with an A envelope recombines with endogenous c FeLV-B ,44. On tequences . In bothin vitro .onc-bearing retroviruses are replication defective but can be experimentally transmitted in vitro or by in vivo inoculation when replication-competent helper virus is present in excess to provide the missing replicative or structural proteins [The recombinant retroviruses that encode oncogenes are seldom, if ever, naturally transmitted. Most proteins . It is n4.in vivo spread of retroviruses [Generally speaking, endogenous retroviruses, which remain at their inherited loci, do not cause malignant disease until they become activated and undergo multiple rounds of replication in somatic cells. The generation of such numerous integration events by newly synthesized proviruses results in rare insertional activation of cellular oncogenes due to the regulatory signals contained within the LTRs . It folloviruses . RestricI view xenotropism as an example of the host evolving restriction to a retrovirus by mutating its receptor. The non-functional receptor prevents further spread between hosts and also restricts an activated endogenous retrovirus propagating to high virus load and multiple new integration sites. Certain mutations in cell surface receptors which no longer allow endogenous retroviruses to bind or enter cells would not disable the normal, physiological functions of the receptors . These wIn hosts which have not been exposed to or invaded by a particular retrovirus, receptor mutations will not be subject to selection. Hence the virus will appear to be xenotropic in that it can potentially infect cells of naive species. It is interesting that exogenous, infectious gamma-retroviruses have not been found in humans, which is likely due to restrictions other than receptors in primary cells . XenotroMus caroli and M. cervicolor) [Felis such as the Egyptian sand cat (probably the ancestor of the domestic cat) and the European wild cat [It is remarkable how certain ERV have jumped by infection to colonize phylogenetically distant hosts. ERV of South East Asian species of mice (vicolor)  are closvicolor) , and alsvicolor) . Similarwild cat . It seemwild cat . These e5.ERV can be put to physiological use by their hosts, either at the gene regulatory level or as proteins. Tissue-specific enhancers in the ERV LTR are an example of transcriptional control. All mammals express amylase in the pancreas, but rodents and Old World primates also express amylase in salivary glands. In both the cases, ERV elements play a role in salivary expression in remarkably convergent evolution. The activation of salivary amylase in the human parotid gland is controlled by a retroviral insertion, which occurred during Old World primate evolution . Then geMany years ago, it was noted that MLV is not transcriptionally active in murine embryonal carcinoma stem cells, but that during differentiation into somatic cells MLV genes begin to be expressed . This suThe advantage to the host of ERV protein expression is most dramatic in the evolution of the mammalian placenta. We suggested that the ERV-3 envelope might function in placental trophoblast cell fusion and immunological protection of the fetus , but it ERV glycoproteins might also be useful in blocking receptors against exogenous retrovirus infection or against activated replicating ERV. This phenomenon used to be termed retroviral interference but is now usually called super-infection resistance . Cells tFv-4 restriction factor for ecotropic MLV [If an ERV expresses Env, even in the absence of whole virus particles, the endogenous glycoprotein can block the receptor. Super-infection resistance by ERV was first observed with the endogenous ALV glycoprotein  and the opic MLV ,64. The opic MLV . By beco6.Retroviruses are typically not particularly choosy about sites of proviral integration while generally preferring active chromatin, although some such as ALV have distinct target site preferences. Therefore, it is not too surprising that retroviral genomes have been detected in the genomes of large DNA viruses. The first example was of sequences of avian reticulendotheliosis virus (REV) in the genome of the avian alpha-herpesvirus, Marek's disease virus  and the 7.Human herpesvirus 6 (HHV-6) is transmitted as a Mendelian element in certain human families ,10. HoweThe presence of cDNA of non-retroviral RNA viruses is discussed in this theme issue. It was first reported for measles virus by Zhdanov in 1975 in Moscow  and was env sequences appear to accumulate more rapidly in greater abundance as endogenous genomes [These RNA viruses do not, of course, possess reverse transcriptase. One effector agent appears to be the reverse transcriptase of LINE elements and the DNA fragments of RNA viruses may be regarded as pseudogenes. However, the reverse transcriptase of endogenous intracisternal A particles (IAP) may also play a role as LCMV, and bornavirus sequences are found within the IAP provirus . It is i genomes  because,Overall, these examples show how a wide variety of viruses can invade host genomes upon which host natural selection can then exert its effects.


To identify the immediate economic impact of maternal death on rural Chinese households.Results are reported from a study that matched 195 households who had suffered a maternal death to 384 households that experienced a childbirth without maternal death in rural areas of three provinces in China, using quantitative questionnaire to compare differences of direct and indirect costs between two groups.p<0.001). More than 40% of the direct costs were attributed to funeral expenses. Hospitalization and emergency care expenses were the largest proportion of non-funeral direct costs and were higher in households with maternal death than the comparison group . To cover most of the high direct costs, 44.1% of affected households utilized compensation from hospitals, and the rest affected households (55.9%) utilized borrowing money or taking loans as major source of money to offset direct costs. The median economic burden of the direct (and non-reimbursed) costs of a maternal death was quite high - 37.0% of the household’s annual income, which was approximately 4 times as high as the threshold for an expense being considered catastrophic.The direct costs of a maternal death were significantly higher than the costs of a childbirth without a maternal death (US$4,119 vs. $370, The immediate direct costs of maternal deaths are extremely catastrophic for the rural Chinese households in three provinces studied. The priority assigned to reducing the approximately 358,000 global maternal deaths per year is arguably at its highest levels since the 1994 International Conference on Population and Development and the establishment of the Millennium Development Goals (MDGs) The imperative to reduce risk due to childbirth is a fundamental human right, yet the economic aspects of maternal mortality are important to understand. However, that topic has not been widely explored in the literature on maternal health There is much debate about whether maternal deaths are a symptom of poverty or whether they can push families into poverty. There is evidence on the co-existence of high maternal mortality ratios (MMR) and low level of economic development The study matched eligible households experiencing maternal death with those of childbirth without maternal death (using selection criteria described below) to compare differences in indicators of direct and indirect costs as collected by direct interview within one to three months after maternal death or childbirth, . The results in this paper therefore describe the immediate economic shock of the maternal death. The study protocol and data collection instruments were approved by the ethical reviews boards of Peking University, and the World Health Organization. Signed informed consent statements were obtained prior to any interview. All interviews were entirely voluntary (no incentives were provided) and were timed to occur after the funeral period was completed.Three provinces  were purposively selected to represent settings with low, moderate, and high MMRs, with consideration given to comparable geographic area, population size, and transportation routes. The 2009 rural institutional delivery rate in Hebei, Henan, and Yunnan are all quite high  Households with maternal death (the affected group) were each matched to two households of childbirth without maternal death (the comparison group) according to the inclusion criteria as follows.Having maternal death within 3 months of the interview; Maternal death defined as the death of a woman while pregnant  or within 42 days of termination of pregnancy, not for accidental causes unrelated to the pregnancy.Having childbirth within 3 months of the interview, and;Living in the same administrative village, and;Similar economic status evaluated by the administrative village cadres  and;The household type is the same as that of the affected family before maternal death .There were a total of 530 maternal deaths in Hebei, Henan, and Yunnan provinces between June 2009 and October 2010 (the study period). Of these cases, 40.9% (217 out of 530) were determined to be ineligible/did not meet the inclusion criteria and an additional 22.3% of the cases (118 out of 530) were eligible but refused to be interviewed. The principle reasons for refusing to be interviewed were unresolved medical malpractice legal suits, or extreme grief among the surviving family members. Overall, 36.8% of the total number of maternal deaths (195 out of 530) were eligible and accepted the interview. We compared the age and gestational week of maternal deaths between those accepted and refused to be interviewed. Results showed that no significant difference was observed (data not shown).We purposively selected 384 matching households of childbirth without maternal death (using the selection criteria). There was no refusal from these families, i.e., 100% of the contacted families consented to take part in the study . The comparison households were interviewed within two days of the affected family’s intake interview. It’s noted that in Yunnan province six of the affected households were matched with only one comparison due to difficulties in identifying a second eligible comparison family in the remote and under-populated areas.Potentially eligible families were identified by the County Maternal and Child Health Office in the selected provinces. Preliminary contact was made by one member of the provincial study team within three months (but not under three weeks) of the maternal death or childbirth. After eligibility was confirmed and informed consent was made, the person in charge of the household economy was invited to a face-to-face interview. Each interview was conducted in Chinese or local dialect by members of the study team who had received standardized training on interviewing techniques. Additional training was provided to interviewers by a psychological expert with experience in bereavement, in order to further reduce the potential harm to the study’s subjects. During each interview, we collected data with quantitative questionnaires, which was designed after a literature review of cost-of-illness studies The questionnaire included questions on the household demographic status , whether the family was nuclear family, etc.), household economic status , direct and indirect costs of maternal death or childbirth, and coping strategy. Direct costs, i.e. out-of-pocket expenditures, included hospitalization and emergency costs, transportation costs, extra costs, and funeral costs, those were being listed as four items in the questionnaire. To estimate indirect costs, we collected self-reported lost work-day during the hospitalization and medical treatment, funeral, and prolonged bereavement, then the total lost work-days were multiplied by estimated daily income per capita. Coping strategy, defined as sources of money used to offset direct costs, were also obtained, which included positive sources and negative sources .2 or Fisher’s exact tests were used for comparison. For continuous variables, difference in means for normally distributed data was tested by t-test, and other continuous variable, such as household annual expenditure, time and costs data which had positively skewed distributions, were tested for differences by rank-sum test. P<0.05 was considered to be statistically significant. The exchange rate (US$1 =  ¥6.7) was used to convert the payment from Chinese RMB (¥) to US$.The database was developed using the Epidata 3.1 software. Statistical analyses were conducted with SPSS Version 10.0. The demographic characteristics , direct and indirect costs, and coping strategies were compared between the affected and comparison group. For categorical variables and proportion comparison, χp = 0.803), proportion of nuclear families (p = 0.846), proportion of minorities (p = 0.290), and annual household expenditure(p = 0.117). The women in affected group tended to be older than those in comparison group (p<0.001). Although women in the affected group were more likely to have delivered at home than in the comparison group , there are approximately 65% of women in both study groups delivered in county or upper-level hospitals. A higher proportion of women in the affected group had a cesarean section than in the comparison group .The sample’s demographic characteristics reveal a successful matching of the two study groups . The twop<0.001). Funeral costs made up the largest category of the direct costs for the affected group . The non-funeral direct costs were quite substantial in affected group  and were more than six times higher than in comparison group (US$370)(p<0.001), reflecting the difference in hospitalization and emergency costs . Excluding 12 outliers (8 in affected group and 4 in unaffected group) higher than 3 SDs of mean, direct costs were still significantly higher in affected group compared with unaffected group . We conducted multi-variable linear regression to control for possible confounders, including province, age of the mother, whether the mother was literate, whether the mother had pregnancy complications etc. The results showed that after controlling these characteristics, the non-funeral direct costs were still significantly higher in affected group, and the ratio between two groups increased slightly (3.32 vs. 3.46)  see . The non.46) see .p = 0.004). However the families with a maternal death experienced a substantial number of lost working days during the funeral and bereavement period. Results from self-reported lost work-days were summed and multiplied by the estimated daily income per capita  to estimate total lost economic productivity. The total lost economic productivity of households with maternal death was significantly higher than that in comparison group . Excluding 9 outliers (3 in affected group and 6 in unaffected group) higher than 3 SDs of mean, indirect costs were still significantly higher in affected group compared with unaffected group .Indirect costs included lost household economic productivity during the hospitalization and medical treatment, funeral, and prolonged bereavement . FamilieRespondents identified several sources of income which were used to raise funds paid out as direct costs of the maternal death or childbirth without maternal death . These dSlightly less than one-half (44.1%) of the affected households received compensation from the hospital, and 29.2% of the affected households had all of the direct costs for the maternal death paid by the hospital where the woman died. Among the approximately one-half (55.9%) of the affected households that did not receive any payment from the hospital, their largest source of revenue for paying the direct costs of the maternal death came from borrowing money or taking loans (65.1% of the direct costs).In contrast, the households of childbirth without maternal death utilized a narrower range of financial support mechanisms, with the majority reporting supports that came from gifts from relatives or friends (32.9%) or by drawing upon the household savings (24.8%).The expenditure of a disease is considered to be catastrophic for the household economy if its economic burden crosses certain threshold, which has different calculation methods by different researchers. We employed two more frequently used methods in this study.·0% for households without maternal death (the means were 101.2% vs.8.8%) (p<0.001). Without excluding the amounts reimbursed by hospitals, insurance, government or neighbors, the median of economic burden was equal to 128.8% of the household’s annual income, compared to 8.0% of the household’s annual income for childbirth without maternal death (the means were 244.4% vs.17.3%) (p<0.001).One method is direct costs expressed as a percentage of the household’s total annual income, and the threshold is 10.0% p<0.001).An alternative method for determining catastrophic health expenditures defines the direct costs excluding reimbursed money higher than 40% of household expenditures after excluding food costs The economic burden of a maternal death observed in this study is much higher than that associated with other priority diseases. For example, cost-of-illness studies that used the 10% threshold definition on malaria reported a direct economic burden from 2.0% to 2.9% for malaria The findings that the direct medical costs of a maternal death  were significantly higher than the costs of childbirth without maternal death are not unexpected. However the high proportion of families who had catastrophic health expenditures as a result of a maternal death draws attention. The affected families were more vulnerable to catastrophic health expenditures than the comparison group, due to their lower levels of household income, and the overall magnitude of the economic shock is not trival and should be a focus of serious attention by local and provincial authorities.The costs associated with a maternal death (direct and indirect) will clearly vary by setting and should not be overly generalized. For example, the direct non-funeral costs observed in our study are higher than the cost of treatment of life-threatening obstetric complications reported in Burkina Faso (US$48) Although not formal policy, the common practice of hospitals to provide cash payments to the families who suffered a maternal death does work to alleviate some of the immediate economic shock of the high medical care costs. Approximately one-third of the affected families reported that these payments covered all of their direct costs for the maternal death. The terms of the payments were not explored by this study, but credible anecdotal evidence indicates that it can be linked to an agreement not to pursue legal suit for malpractice against the hospital or physician. Often the payments were offered only after family members protested angrily in the hospital compounds. These payments are largely ad hoc with little record established on the condition and criteria for determining the amount or source of budget used for the payments. Given the frequency of these types of mal-practice payments encountered in our study, it seems reasonable to suggest that this is a priority area for government regulation. Additionally, little is known about the likelihood of a hospital to turn away complicated or near death maternal patients for fear of being liable to pay out compensation to the surviving family members. More research is needed on this troubling new trend in rural China.The households with a maternal death who did not have all direct costs paid by the hospitals managed the expenses by borrowing money or taking loans, drawing down their savings and selling assets – thereby pushing poor households even deeper into poverty. Local government did provide some measure of financial relief, amounting to only 0.5% of direct costs of these households, but at too low a level to make any appreciable difference on the economic burden caused by paying for the end of life medical procedures surrounding the maternal death. In addition to regulating the informal payments made by hospitals in the case of maternal deaths, local governments need to reconsider the amounts made available to families who have experienced catastrophic health expenditures. Financial assistance such as new types of insurance for pregnant women, increased action from government and NGOs programs associated with financial aid to the poor, children schooling, or elderly pension are suggested as avenues for providing emergency financial relief.Households with catastrophic economic burden coupled with fewer assets, reduced savings and increased debt are likely to suffer consequences that affect all the surviving family members, including reduced expenditures for food and education. Clearly the high economic burden that the maternal death causes has resulted in a severe economic shock in the short term. The resilience of the affected families to this shock over time is unknown and future research that tracks the longer term effects over time are needed to fully understand the implications of the immediate catastrophic health expenditures associated with a maternal death.To our knowledge, the present study is the first to compare the direct costs, indirect costs and economic burden between the households which experienced childbirth with and without maternal death in rural areas of China, or elsewhere in the world. Results from our study are generalizable only within China, but can inform understanding of the multiple consequences of maternal death in other settings, and provide the first reference on the economic implications of this tragedy. The consequence of maternal mortality is extremely difficult to measure, because it is a relatively rare event. We had overcome this difficulty to achieve the relatively large sample by utilizing a national surveillance network of maternal health in China. The field work for gathering this evidence was quite laborious as extra effort was made to recruit subjects, as most fatalities occurred in under-populated areas with inconvenient transportation. We hope that our first study on this topic will encourage others to undertake similar research, and create evidence for successful advocacy to increase the financial support provided to poor families who have suffered a maternal death.Table S1Impact of possible confounders on non-funeral direct costs: multi-variable linear regression.(DOC)Click here for additional data file.


We report 2 clinically characteristic and serologically positive cases of Ross River virus infection in Canadian tourists who visited Fiji in late 2003 and early 2004. This report suggests that Ross River virus is once again circulating in Fiji, where it apparently disappeared after causing an epidemic in 1979 to 1980. The growing appreciation of travelers as sentinels for the emergence of infectious diseases is based on the immunologic naiveté of travelers, their defined exposure in time and space, and sufficient diagnostic resources after their return to an industrialized country. Reports of infectious diseases in travelers in unusual numbers or from new geographic locations can inform a public health response. We report 2 recent apparent cases of Ross River virus disease ("epidemic polyarthritis") in Canadian travelers to Fiji, ≈1,000 miles from the region  where the virus is endemic, enzootic, and often epidemic  are the most important vertebrate amplifying hosts  in neutralization tests was also observed. The subsequent decrease in this patient's Ross River virus–specific IgM reactivity and neutralizing antibody titer within a few months also argues for a recent Ross River virus infection. In the second case, the high but stable anti-Ross River virus neutralizing antibody titer may reflect the fact that the earliest sample available for testing was obtained >2 weeks after illness onset when the patient's anti–Ross River virus neutralizing antibody titer may have already peaked.If Ross River virus was circulating in Fiji in 2003 and 2004, at least 2 basic hypotheses may explain its reemergence there. The first of these, which seems the most plausible, involves occasional reintroduction of this virus from the known disease-endemic region , sometimes resulting in local transmission, ultimately followed by local extinction. Circumstantial evidence to support this hypothesis includes the fact that, during the same period that the 2 patients described here traveled to Fiji, Australia was experiencing its usual summer surge in Ross River virus incidence  should be familiar with the clinical features of Ross River virus disease, as well as its potential public health importance, and realize that diagnostic tests for this infection currently are available at only a few public health reference laboratories .


To better understand the natural history of Hendra virus infection and its tendency to relapse, 2 humans infected with this virus were monitored after acute infection. Virus was not detected in blood samples when patients were followed-up at 2 and 6 years. Thus, no evidence was found for prolonged virus shedding. Paramyxoviridae, genus Henipavirus) can cause respiratory disease and encephalitis in humans. Hendra virus infection is acquired by close contact with horses infected by spillover infection from fruit bats, which are the natural reservoir for these viruses .Of 7 recognized humans infected with Hendra virus, 3 survived. Of these 3 persons, clinical samples were available for 2 survivors. Patient 1 was a 25-year-old woman (veterinarian) in whom a self-limited influenza-like illness (ILI) developed in November 2004 , blood, urine, and cerebrospinal fluid were tested for viral RNA by using qRT-PCR.>1.5 years after initial examination. The prolonged IgM response in patient 2 might have been caused by persistent infection or associated with more severe acute encephalitic manifestations. A prolonged IgM response has been observed in persons with West Nile virus meningitis, and encephalitis and may be a general aspect of central nervous system involvement  were tested. Patient 1 was not included because virus shedding was not detected at any time during her infection. Times chosen were from the earliest to the latest time point at which a positive result was obtained by qRT-PCR .Hendra virus RNA was detected in blood, NPA (or swab specimen), and urine, confirming the suitability of these sample types. Frequency of detection varied for 3 patients. None of the 3 sample types was consistently better than another, and all were suitable choices for diagnostic purposes. Hendra virus RNA was also detected in cerebrospinal fluid, but there were too few samples to determine whether this material was a reliable sample type.Serologic and PCR results were reported for the 2 patients during the early phase of infection (However, because of the small number of samples tested in this study, prolonged shedding after acute Hendra virus infection cannot be ruled out. In 1995, a patient who had an episode of aseptic meningitis after caring for 2 Hendra virus–infected horses had a relapse 13 months after the initial episode (The mechanism by which henipaviruses remain dormant is unknown. Virus has not been isolated from persons with relapse cases (It would be desirable to use PCR to test a variety of sample types for diagnosis of infection with Hendra virus. All sample types tested were suitable in terms of sensitivity. However, obtaining an NPA specimen during the acute phase of infection has a potential risk for aerosol contamination, and needle stick injury is a risk if a blood sample is obtained. Collection of a urine sample may be an acceptable risk without reducing the sensitivity of detection and risk to health care staff. This procedure should assist clinicians in management of henipavirus infections.


Community-based peer support has been shown to be effective in improving exclusive breastfeeding rates in a variety of settings.We conducted a cost analysis of a community cluster randomised-controlled trial (Promise-EBF), aimed at promoting exclusive infant feeding in three sites in South Africa. The costs were considered from the perspective of health service providers. Peer supporters in this trial visited women to support exclusive infant feeding, once antenatally and four times postpartum.The total economic cost of the Promise-EBF intervention was US$393 656, with average costs per woman and per visit of US$228 and US$52, respectively. The average costs per woman and visit in an operational ‘non research’ scenario were US$137 and US$32 per woman and visit, respectively. Investing in the promotion of exclusive infant feeding requires substantial financial commitment from policy makers. Extending the tasks of multi-skilled community health workers (CHWs) to include promoting exclusive infant feeding is a potential option for reducing these costs. In order to avoid efficiency losses, we recommend that the time requirements for delivering the promotion of exclusive infant feeding are considered when integrating it within the existing activities of CHWs.This paper focuses on interventions for exclusive infant feeding, but its findings more generally illustrate the importance of documenting and quantifying factors that affect the feasibility and sustainability of community-based interventions, which are receiving increased focus in low income settings. Suboptimal breastfeeding has been estimated to be responsible for 1.4 million child deaths worldwide, which represents 12% of deaths in children under 5 years of age and 44 million disability adjusted life years (DALYs) Evidence from a systematic review suggests that Community health workers (CHW) can be effective in improving exclusive breastfeeding (EBF) rates. This international experience is confirmed in the South African context. However, the extent to which CHWs can improve EBF rates is varied. An intervention cohort study from Kwa-Zulu Natal (VTS) reported EBF rates of 76.5% and 66.7% at 5 months for HIV negative and positive women, respectively following intensive home visit support Community-based interventions and task shifting are now high on the Millennium Development Goals (MDG) policy agenda. Large investments are being made in CHW programmes through disease specific channels; this is evidenced by the use of CHWs in HIV, TB, child health and malaria programmes Economic evaluations from South Africa and Uganda have shown that stand alone individual peer support is not inexpensive In this paper we analyse the costs of providing peer counselling through five visits at home to promote EBF up to 3 months after delivery in three South African communities. We also assessed the potential affordability of the intervention in an operational setting. This study provides evidence from an upper middle-income country in sub-Saharan Africa on costs of promoting EBF through a low intensity intervention (five visits), in a high antenatal HIV prevalence setting, where the national exclusive breast feeding prevalence is low.Written informed consent was obtained, and all participants were informed that they could refuse participation or withdraw from the discussions at any time. Ethical approval was granted by the University of the Western Cape.http://clinicaltrials.gov/ct2/show/NCT00397150)], was designed to improve the rates of exclusive infant feeding (i.e. exclusive breastfeeding (EBF) at 12 weeks through the assistance of community peer supporters. PROMISE-EBF A cluster randomised controlled trial (RCT), known as PROMISE-EBF [Promoting infant health and nutrition in sub-Saharan Africa: Safety and efficacy of exclusive breastfeeding promotion in the era of HIV , expectant mothers are ideally offered voluntary counselling and testing for HIV, and are also counselled on infant feeding choices. Optimally, at the end of routine antenatal care, HIV positive mothers were to be in a position to choose between EBF and EFF based on WHO AFASS recommendations. In the intervention clusters, the peer supporters' tasks were to recruit pregnant women, establish the mother's feeding choice and thereafter support the mother in carrying out her choice of EBF or EFF. Mixed feeding was discouraged. The intervention included at least one antenatal home visits by peer supporter, plus four visits after delivery at 1, 4, 7 and 10 weeks, with the possibility of an extra visit when necessary. In the control clusters, mothers also received visits; however, the content of their visits was not on feeding. It was on accessing social grants.A total of 18 female peer supporters were employed in the infant feeding support intervention arm: five in Paarl, six in Rietvlei and seven in Umlazi. Each peer supporter had a designated geographic area. Peer supporters had completed at least 12 years of schooling, had an interest in child health, prior experience of community involvement and lived within the selected trial clusters. There was neither an age limit nor a requirement for them to have personally breast fed. Peer supporters had to successfully complete a literacy and basic counselling skills assessment. They received five training sessions. The first session was a five-day WHO/United Nations Children's Fund (UNICEF) HIV and Infant Feeding Counselling Course The cluster size was determined by the estimated local fertility rates and appropriate number of women needed for the trial. The research team identified rational distinct geographic units containing 3000 women of childbearing age in both Paarl and Umlazi, and 1500 women in Rietvlei. Peer supporters commenced work in September 2005 and completed follow-up of all women recruited for peer support in December 2007.One supervisor per site was appointed to manage and support 10–14 peer supporters. The supervisors had varied skills, ages and backgrounds. They were all experienced field researchers. Their role was to help the peer supporters, and to encourage them to give high quality and consistent counselling. Peer supporters had monthly group meetings with a supervisor, at least one individual contact session each week  and were observed counselling a mother during a home visit at least once a month. In addition, supervisors visited a random sample of mothers to verify that peer support had taken place according to schedule. The supervisor workload varied across the three sites.Supervisors were themselves supervised telephonically or in person by a qualified social worker from the research team, who liaised directly with senior research staff. This person visited the site once a month and served as an intermediary between peer supporter supervisors and the research project manager. The senior research staff had training in nursing and maternal and child health.Peer supporter supervisors received six training courses. They received two sessions of trainers courses: a five-day WHO/United Nations Children's Fund (UNICEF) HIV and Infant Feeding Counselling Course, Full economic evaluation is defined as a comparison of two or more interventions in terms of their costs and consequences The costing was conducted from a provider's perspective. Promise-EBF costs were measured prospectively throughout the duration of the trial. In addition, we reviewed the projects financial records – budget and expenditure reports from all three  institutions. We estimated economic costs approximating the actual opportunity value of the resources Promise-EBF was a research project, and therefore some resources were used for both research activities and the intervention. Our primary concern was to estimate the costs of establishing and running a peer-counselling intervention to promote exclusive infant feeding, and not to estimate research costs. Therefore, we allocated joint costs (research and intervention) by interviewing project managers about the proportion of time that the cost item was used for during the intervention, and based on this, excluded the research-related costs. Each salary included reflecting the proportion of time spent on the intervention (50% of the site supervisor and 25% of the project manager's time). The use of drivers varied between sites. Umlazi had two drivers, each of whom spent 30% of their time on the intervention. In Rietvlei, the driver spent 30% of his time on the intervention, while in Paarl the driver spent 85% of his time. Vehicles, office equipment and supplies were included under supervision since they were used for the supervision of peer supporters.The study utilised a combination of activity-based costing and an ingredients approach Within the set-up costs, some costs were categorised as once-off (i.e. costs incurred only once for the program) and repeatable costs (i.e. to be repeated if the program is rolled out to other districts). Set-up costs are expected to yield benefits for several years after project initiation, and were therefore annuitised Capital costs were calculated using the replacement value of each item, the estimated number of useful life years and were annuitised using a discount rate of 8% (the interest rate of South African long-term government bonds), which is consistent with other studies in South Africa The purchasing power of money diminishes with inflation, and costs were adjusted for time using the consumer price index, excluding mortgage bonds Data sources included time-use logs filled by peer supporters and data from the accounting systems of the three institutions that jointly implemented the intervention. The project manager, supervisor and peer supporter supervisors were interviewed to validate the expenditure information.The costs of implementing peer support included four main categories. First was the monthly peer supporter stipend of US$127, which increased to US$152 in the second year of the trial. Secondly, each peer supporter had a mobile phone and was provided with airtime vouchers worth US$14 per month to communicate with the mothers. Thirdly, peer supporters in two sites (Rietvlei and Umlazi) used public transport to reach some of their clients due to long travel distances and were reimbursed for these costs. The final cost was for a once-off household incentive of one food parcel worth US$5 per household receiving peer support.The costs of peer supporter supervision included the salaries of supervisors, project managers and drivers.The post set-up training included training of new peer supporters due to staff turnover and continuing education for existing peer supporters. Overhead costs included all the inputs not directly involved in delivering the intervention .The totaPROMISE EBF was a vertical stand-alone project. We present cost estimates that are as representative as possible for a real operational set-up of a peer support intervention. We do this by adjusting the PROMISE EBF estimates of resource use and values for start-up activities, peer supporter supervision and the actual peer supervision .Our focus was at the district level. Therefore, we excluded the costs of developing the intervention and a manual. These activities typically take place at national level, and thus were a one-off central activity, which will not be repeated when the intervention is rolled out.In an operational South African setting, the project manager would have lower levels of skills than in Promise-EBF and would typically be an assistant director at a district level. Each district already has an assistant director and implementing this project will therefore not necessitate employing new management. Instead we assume that 25% of existing assistant directors time will be spent on the intervention, and included the corresponding proportion of an assistant director salary package.Peer supporter supervisors salaries in the trial ranged from US$16 269 to US$29 457 per year across the three sites for being employed on a part time (50%) basis. It is difficult to estimate peer supporter supervisor salary scales in an operational scenario because in South Africa, CHWs exist in various forms with various types of supervision Travel costs were adjusted downward because of the fact that the study offices in all sites were far away from the communities. In a district setting, the offices will typically be much closer, and drivers' salaries were therefore excluded as they are not generally part of a district set up.In the second year of the Promise-EBF study, it was decided to provide households with a once-off incentive for receiving peer support in the form of a food parcel worth US$5. This was induced by trial conditions and would not be repeated in the operational setup. These costs were excluded, although we acknowledge that these incentives could have had an impact on the project uptake.The impact of the intervention was measured using the number of women who received peer support and who practiced exclusive breastfeeding up to three months after delivery. The two main outputs were total number of mothers' counselled and total number of counselling sessions or visits. These were combined with total costs to calculate average costs per visit and per mother counselled. We used these to approximate the cost per month of exclusive breast feeding (MEBF) which was defined as our primary health outcome of interest. MEBF were the sum of the duration, in months, that a child was exclusively breastfed. The cost per MEBF was expressed as the total cost divided by total MEBF at three months.During the project period of 2.5 years, a total of 1725 women were followed up, with 526, 611 and 588 from Paarl, Rietvlei and Umlazi, respectively. On average, women received four visits in Paarl and Rietvlei, which was one visit below the target. In Umlazi, women on average received the planned five visits.The total economic costs in the Promise-EBF scenario were US$393 656. The average costs per woman and per visit in the Promise-EBF scenario were US$228 and US$52 respectively . The MEBThe peer support activity was the second largest cost driver, representing 27% of the costs. Personnel costs accounted for 75% of peer support costs. In the second year of the study, the food parcels, valued at US$5 each accounted for 13% of the peer support costs. Communication in the form of mobile phone airtime amounted to 7% of peer support costs. Peer supporters in the Rietvlei and Umlazi sites resorted to public transport when the distances between households were not within walking distance. Although peer supporters had to reside in the same clusters as the pregnant women in order to avoid transport expenditure, distances between households proved to be more than envisaged, especially in the rural site. In Paarl, peer supporters were able to conduct all the visits on foot. Travel costs for peer supporters accounted for 3% of the total costs.Training accounted for only 6% of the total costs. In total, 25 training sessions were conducted. Of these, four were initial training courses during the set-up phase, 16 were refresher courses and five were for staff replacement.Overhead costs accounted for 11% of the total costs, of which office rental was the highest cost driver with a share of 72%.We report the attrition of peer supporters in both arms in order to capture the potential magnitude of this problem. This trial employed 38 peer supporters (counting both the control and intervention arms), of which, one-third resigned during the study period. Attrition varied by site: 60% in Paarl, 36% in Rietvlei and 14% in Umlazi. In Paarl, 6 of the 10 peer supporters resigned and had to be replaced, and in Rietvlei, 5 out of 14 resigned. In Umlazi, none of the peer supporters resigned, but four were lost due to illness. It was only in Umlazi that back-up peer supporters were trained at the start of the study, meaning that peer supporters could be replaced immediately. In both arms, peer supporters were required to conduct the same number of visits, their stipends were of the same value and they were supervised by the same person. Peer supporters left for better paying employment opportunities.The cost pattern in the individual sites was similar to that of the combined analysis, although the percentage share of costs for each activity varied somewhat . Peer suIn the operational scenario, the total costs were reduced by 38%, which amounted to US$ 236 914. The average costs per woman and per visit were US$137 and US$32 respectively . In the The operational cost of US$137 per woman is substantial compared to an average of US$38 per uninsured person per annum in 2007 (time of the study) for public sector expenditure on non-hospital primary health care (PHC) services in South Africa Several factors may have increased the costs of Promise-EBF. Firstly, Promise-EBF was a geographically limited intervention and could therefore not benefit from potential economies of scale. Secondly, peer supporters in Promise-EBF delivered a single intervention, promotion of exclusive breastfeeding. CHW programmes that are focused on a single intervention (either curative or preventive) have been found to be expensive compared to those that cover multiple interventions The cost per woman of US$ 220 for the Promise-EBF intervention in South Africa was nearly twice the cost per woman for a comparable intervention (US$139) in Uganda et al (discussed in the introduction of this paper) clearly demonstrated that the reduction in number of visits reduces total costs The numbers of home visits (i.e. the intensity of the intervention) have been shown to influence total costs. The simplified scenarios presented by Desmond The Promise-EBF trial had a total of five visits per mother: Women who received peer support were more likely to breast feed than those who did not receive the support In Promise-EBF, peer supporters were employed with the same stipend as those working in the public sector. In contrast, in the Bland et al et al, requires careful planning in South Africa. Firstly, it will only be feasible if the existing CHWs have spare time. Breastfeeding support requires scheduled counselling visits, and adherence to these is strongly associated with adherence to EBF The option of expanding the tasks of existing multi-purpose CHWs to include EBF promotion, as suggested by Desmond The South African National Department of Health is currently piloting primary healthcare (PHC) reengineering. The reengineering of PHC in South Africa has a strong focus on community based services through PHC outreach teams. These teams include multi skilled CHWs. Thus, there is now an opportunity for EBF promotion to be part of a broader integration and coordination of CHW services. If the promotion of EBF is to be part of PHC reengineering, both CHW's and their supervisors would have to receive sufficient training on supporting pregnant women on appropriate infant feeding choice while they are pregnant and EBF support after delivery. Effective support for EBF requires multiple visits. Thus, CHW supervision should ensure that CHW's work plan includes scheduling subsequent visits.CHWs have been shown to be effective when functioning within a limited scope This costing analysis was strengthened by prospective and rigorous process evaluations, which were qualitative studies. These studies captured experiences of peer supporters This costing exercise was conducted from a provider's perspective, meaning that only costs incurred by the provider were considered. This approach leaves out the value of mothers' time of receiving the intervention. Costs that largely affect recipients of interventions are travelling costs and the opportunity cost of their time (i.e. the time required to receive the intervention). Given that the intervention was delivered at home, it is unlikely that women incurred travelling costs. The intervention only required that women set aside time to receive support from a peer supporter. These visits did not exceed one hour. Therefore, women had to forgo a small portion of their time to receive the intervention. It is reasonable to assume that taking a provider's perspective did not leave out substantial costs from the women's point of view. The study also did not consider the cost-savings represented by reduced incidences of diarrhoea and other childhood diseases such as pneumonia. Our method of separating research and routine implementation could have been strengthened through careful observation in a time-motion study.Investing in the promotion of exclusive infant feeding as a stand-alone peer support service such as the Promise-EBF requires a substantial financial commitment from government, with a cost of US$228 per pregnant woman. It is therefore important to consider possible avenues of cost reduction, such as integrating infant feeding peer support into existing CHW programmes. We demonstrate how the cost per woman can be reduced by about 40% through integration with existing CHW services. Too little is known about how well breastfeeding can be promoted in an integrated set-up, and analysis of the impact of alternative implementation schemes, combined with economic evaluation, is essential in assessing whether the added costs are reasonable compared to improvements in child health.We believe the relevance of this paper extends beyond the particular intervention of promoting EBF, to document and quantify factors that in general affect the feasibility and sustainability of community-based interventions. This is important knowledge for sub Saharan Africa, where increasing focus is put on community-based interventions.


The second study, with a GM-CSF-expressing herpes simplex virus type 1 , was presented at the recent American Society of Clinical Oncology meeting. Four hundred thirty-six patients were randomized (2:1) to single-agent T-Vec or subcutaneous GM-CSF, resulting in a significantly greater durable response rate and median time to treatment failure for the virotherapy arm (http://chicago2013.asco.org/immunotherapies-offer-bright-prospects-advanced-melanoma). Again, both oncolytic and loco-regional/systemic immunotherapeutic activity was documented. In light of these studies, it is important to recognize that Pexa-Vec and T-Vec merely represent two regiments of a larger army of targeted biological agents that includes reovirus , various adenoviruses, coxsackie virus and other herpes and VV vectors. In that vein, VV appear to be particularly promising candidate anti-cancer agents. As we have shown, elucidation of intracellular mechanisms may uncover other avenues of attack, through which combination therapies can be exploited for greater anti-tumour efficacy. Further therapeutic alliances might be sought by genetic manipulation of the viral genome itself to deliver a therapeutic payload and form a ‘coalition of killing’. The potential of targeted, multi-modality therapy centred on the biology of viral oncolytics is an emerging, yet exciting, field of investigation and highlights the crucial role of understanding viral biology. In the words of Sun Tzu (“The Art of War”): “We cannot enter into alliances with neighbouring princes until we are acquainted with their designs.”In a recent study of combined vaccinia virus (VV) and radiation therapy (RT) in BRAF-mutant melanomas, we dissected the precise events by which RT enhances viral cytotoxicity. Our studies revealed that the complex interplay between RT, virus and cell signaling pathways caused a completely novel synthetically lethal form of tumor cell death. We initially sought to assess the effect of combining a VV GLV-1h68) and RT in melanoma cell lines with different genetic backgrounds . We hypothesised that RT would induce stress responses in all tumor genotypes and activate host survival pathways to create an environment favorable to viral replication and subsequent oncolysis. Contrary to our expectations, enhanced tumor cell kill with the combination of virus and RT only occurred in BRAF-mutant melanoma cells. In mechanistic studies, it was clear that VV triggered activation of the c-Jun N-terminal Kinase (JNK) pathway and tumor necrosis factor-α (TNF-α) signaling in all genotypes. In BRAF-mutant cells, virus-induced TNF-α production was associated with anti-apoptotic/pro-survival effects, as previously described with MEK inhibitor-induced cytotoxicity h68 and R and Akt