To the Editor: Arthropodborne viruses (arboviruses) cause substantial human disease worldwide and have a pronounced effect on public health throughout Asia. Zika virus, discovered in Uganda in 1947  occurred in Jambi Province, central Sumatra, Indonesia. We received samples from 103 case-patients with clinically diagnosed dengue; these samples had been negative for DENV by RT-PCR, NS1 antigen detection, or evidence of seroconversion by ELISA (data not shown). We tested the samples for other viruses using alphavirus and flavivirus RT-PCR . In parallel, we attempted virus isolation using Vero cells. One sample, JMB-185, came from a 27-year-old man who sought treatment at the Jambi city hospital 2 days after illness onset with a sudden high fever, headache, elbow and knee arthralgia, myalgia, and malaise. He did not exhibit some common clinical characteristics of Zika virus infection . An additional larger amplicon was generated (nt 9278–9808 of NS5 gene), and a phylogenetic tree was constructed based on the partial sequence of the NS5 region (530 bp) for JMB-185 (GenBank accession no. KU179098) and other Zika virus sequences, including those from Cambodia, Yap Island, Thailand, and the Philippines . PhylogeThe isolation and characterization of Zika virus from a resident with no travel history confirm that the virus is circulating in Indonesia and that, by mimicking mild dengue infection, this infection is likely contributing to the large number of undiagnosed cases of acute febrile illness. Although reported human cases of Zika virus infection have been rare in Southeast Asia (


Average CRs were 0.009 mm/yr for five central processing facility (CPF) waters, which had no APB or SRB due to weekly biocide treatment and 0.036 mm/yr for 2 CPF tank bottom sludges, which had high numbers of APB (106/ml) and SRB (108/ml). Hence, corrosion monitoring with carbon steel beads indicated that biocide treatment of CPF waters decreased the CR, except where biocide did not penetrate. The CR for incubations with 20 ml of a produced water decreased from 0.061 to 0.007 mm/yr when increasing the number of beads from 1 to 40. CRs determined with beads were higher than those with coupons, possibly also due to a higher weight of iron per unit volume used in incubations with coupons. Use of 1 ml syringe columns, containing carbon steel beads, and injected with 10 ml/day of SRB-containing medium for 256 days gave a CR of 0.11 mm/yr under flow conditions. The standard deviation of the distribution of residual bead weights, a measure for the unevenness of the corrosion, increased with increasing CR. The most heavily corroded beads showed significant pitting. Hence the use of uniformly sized carbon steel beads offers new opportunities for screening and monitoring of corrosion including determination of the distribution of corrosion rates, which allows estimation of the probability of high rate events that may lead to failure.Microbially-influenced corrosion (MIC) contributes to the general corrosion rate (CR), which is typically measured with carbon steel coupons. Here we explore the use of carbon steel ball bearings, referred to as beads , for determining CRs. CRs for samples from an oil field in Oceania incubated with beads were determined by the weight loss method, using acid treatment to remove corrosion products. The release of ferrous and ferric iron was also measured and CRs based on weight loss and iron determination were in good agreement. Average CRs were 0.022 mm/yr for eight produced waters with high numbers (10 Because pitting corrosion is unpredictable pipelines must be regularly inspected to identify sections of decreased wall thickness, which may have experienced pitting corrosion. The surrounding areas may then be subjected to excavation to replace the corroded sections and prevent a future leak event. In the laboratory and in the field flat metal coupons are typically used to evaluate general and pitting corrosion by metal weight loss and by surface examination of the coupons, respectively. This allows definition of the pitting factor (P) as the ratio of the corrosion rate of the deepest pit (mm/yr) divided by the general corrosion rate (mm/yr).Corrosion of carbon steel is caused by physical, chemical, and microbiological factors . Although a wide variety of microbes may contribute the sulfate-reducing bacteria (SRB) are considered the main culprits. SRB can contribute to chemical MIC (CMIC) by reducing sulfate to sulfide using organic electron donors , as shown for lactate in Equation 1. The produced sulfide can then react with carbon steel to form hydrogen (Equation 2), which is then also used by SRB as electron donor for sulfate reduction (Equation 3):0 (Equation 4) or using these through a H2 intermediate :0, as per Equation 4  offers advantages for measuring general corrosion rates, because of their small size and therefore small weight variation. These beads can be used during incubations in serum bottles with or without shaking or can be packed in tubing or small columns for study of corrosion under flow conditions. Methods and results for these applications are presented here.2). Coupons of ASTM a366 carbon steel (2.0 × 1.0 × 0.1 cm) were cut at the Engineering Workshop of the University of Calgary. Beads and coupons were pretreated according to National Association of Corrosion Engineers (NACE) protocol RP0775-2005. This involved briefly sanding the coupons with grit size 400 sand paper. Beads were rolled between two sheets of grit size 400 sand paper for 1–2 min. Coupons or beads were then treated for 2 min with inhibited HCl and then for 2 min with 1.2 M NaHCO3. The beads and coupons were washed with deionized water (dH2O), then with acetone and allowed to dry. Inhibited HCl was made by dissolving 10.6 g/L of dibutylthiourea in 37% (w/w) HCl and diluting with an equal volume of dH2O. The weight of the pretreated beads or coupons was then determined thrice and the average value was used as the starting weight. The combined weight of the pretreated beads was determined prior to incubation. Individual bead weights prior to incubation were assumed to be same as the average.Beads of a36 carbon steel  were purchased from Motion Canada in Calgary, Alberta, Canada. Because of their use as ball bearings these are very similarly sized  for 1 min to remove remaining loosely-associated corrosion product. The combined weight of coupons or beads was then determined thrice and the average used to calculate the weight loss (ΔW in g). The corrosion rate CR (mm/yr) was then calculated as:3), A is the surface area of the coupon or of the beads and T is the incubation time in h. The factor 87,600 converts the measured CR from cm/h to mm/yr. Weights of individual beads were also determined to characterize the change in weight distribution following corrosion.Following the use of beads or coupons in a corrosion experiment these were again treated with NACE protocol RP0775-2005 to remove adhering corrosion products. Following treatment with inhibited HCl and with NaHCOin situ temperature of 70°C and produces light oil with an American Petroleum Institute (API) gravity of 40°. Samples of liquids were collected in 1 L glass or polyethylene containers, filled to the brim to exclude air as much as possible, whereas samples of solids were collected in Ziploc bags. Samples were stored and shipped at ambient temperature. Average transport time from the date of collection to arrival at the University of Calgary (UofC) was 2 weeks. Samples were stored in a Coy anaerobic hood in an atmosphere of 90% (v/v) N2 and 10% (v/v) CO2 (N2-CO2) immediately following arrival. This included samples of produced water , samples of central processing facility (CPF) water and of injection water  and samples of solids and sludges (SS), retrieved from tank bottoms or from pipelines as pigging solids. Extracts from SS samples were made by contacting 15 g of SS with 15 ml of dH2O in the anaerobic hood, vortexing for 5 min and allowing the solids to settle by gravity. The supernatant was used as the SS extract.Samples were obtained from an oil field in Oceania in December 2013 and January 2014  were also obtained from two fields in NE British Columbia and NW Alberta in which coiled tubing (CT) was used for removal of fracturing plugs during completion of horizontal wells for shale gas production operations.2PO4 buffer at pH 2.5.Samples were subjected to general physical and chemical characterization. This included determination of the pH and of the salinity in molar equivalent of NaCl (Meq) with an Orion pH meter (model 370) and using the same meter with an Orion conductivity cell (model 013005MD), respectively. The concentration of dissolved sulfide was measured with the diamine method  using the same procedure as described for SRB. Growth of APB results in a color change of the medium from red to orange/yellow. MPN values were calculated by comparing the positive pattern to a probability table for triplicate MPN tests.Most probable numbers (MPNs) of lactate-utilizing SRB and of glucose-fermenting acid-producing bacteria (APB) were determined from triplicate dilution series using 48-well microtiter plates  were placed in 50 ml serum bottles together with 20 acid pre-treated iron beads . For samples OC11_SS and OC16_PS 20 ml of mixed solids, oil and water were used. Acid pretreatment was as per NACE protocol RP0775-2005. Sodium sulfide (20 μl of 1 M Na2S) was added and the serum bottles were then closed with butyl rubber stoppers. This procedure was done in a Forma anaerobic hood with an atmosphere of 85% N2, 10% CO2, and 5% H2. The samples were then incubated for 15–18 days at room temperature while lying flat on the platform of an orbital shaker, shaking at 150 rpm. At the end of the incubation each serum bottle was treated as follows. At t = 0 the stopper was removed, a stirring bar was added and a volume of 1.78 ml of 12 N HCl was injected, giving a final concentration of 1 N. At t = 1, 2 …10 min 50 μl of sample was removed and added to 450 μl of a solution of 0.7 M hydroxylamine in 1 N HCl and left to react for 15 min at room temperature. Following that 100 μl of this solution was added to 900 μl of ferrozine-HEPES solution at t = 16, 17, ….25 min incubated for a further 15 min after which the absorbance values were measured at 562 nm (A562) in 1 min intervals. The concentrations of dissolved iron were measured from a standard line for samples of ferric chloride in 1 N HCl, which were treated identically. The data  were used to extrapolate the dissolved iron concentration at t = 0 and this value was used to calculate the weight of iron remaining at time zero (W0) and the general corrosion rate. The period from 10 to 15 min was used to remove the beads from the solution and washing these briefly with water, 1 M NaHCO3 (2 min) and then again briefly with water. They were then immersed in acetone (1 min) and placed on Kimwipes to dry. They were rolled briefly between Kimwipes to remove any remaining loose corrosion product. The beads were then weighed to determine weight loss. The determined weight loss for beads incubated in acid for 10 min (ΔW10) was corrected for weight loss during the 10 min acid treatment, by using the formula: ΔW0 = ΔW10 × CFe, 0/CFe, 10. The corrosion rate (mm/yr) was then calculated as CR = ΔW0/ATD, where A was the surface area of the beads (3.55 cm2), T was the incubation time and D was the density of iron (7.85 g/cm3).In addition to measuring corrosion rate by weight loss, it was also measured as the release of ferrous iron measured spectrophotometrically with the ferrozine assay. Acid (1 N HCl) was used to dissolve ferrous iron from corrosion products, typically FeS and FeCO2-CO2 headspace and contained 10 mg of sodium bisulfite as oxygen scavenger. Sterile plastic syringes (60 ml), needles, gloves and sampling instructions were sent together with the serum bottles to assure aseptic sampling. Field personnel added 50 ml of sample to each of the 120 ml serum bottles and these were sent to the UofC. Once received the samples were placed on a shaker at 30°C and incubated for a total of 45 days, including shipping time. At the end of the incubation, coupons and beads were treated with NACE protocol RP0775-2005.Eight 120 ml serum bottles were sent to the field with either five carbon steel beads or two carbon steel coupons (2.0 × 1.0 × 0.1 cm) each. The beads and coupons were pre-treated as per NACE protocol RP0775-2005 and weighed. The bottles had an NFollowing treatment with NACE protocol RP0775-2005, the carbon steel beads were viewed under a dissecting microscope. LED lights were used to illuminate the beads in order to increase contrast and improve detail. An Olympus SZ61 microscope fitted with an Infinity 2 Camera was used to take digital pictures of the carbon steel beads in various positions.7 APB/ml. These samples had no significant MPNs of SRB with the exception of sample OC14_PW, which had 2.4 × 105 SRB/ml. Water samples from the CPF and injection waters had no significant MPNs of APB or SRB. The elimination of planktonic APB from CPF and injection waters was likely the result of biocide addition to the CPF. However, this did not appear to affect the MPNs of extracts from solids and sludge samples OC11_CPSS and OC16_CPSS, which had 105–106 APB/ml and 107–108 SRB/ml . A typical spreadsheet for collection and calculation of corrosion data for sample OC6 is shown in Table 10 = 5.50 mg was measured at 10 min  were measured by incubating 20 ml of sample with 20 pre-treated carbon steel beads (55.0 mg each) in 50 ml serum bottles under anaerobic conditions, while shaking at room temperature. The incubations were terminated by addition of 12 N HCl to a final concentration of 1 N. CRs were determined both from the measured concentration of dissolved and dissolving iron and from the measured weight loss, extrapolated to in Table . This waThe average weight loss CR for 8 produced waters was 0.022 ± 0.006 mm/yr, whereas that for 5 CPF and injection waters was lower at 0.009 ± 0.009 mm/yr Table . The higSerum bottles containing 20 ml of sample OC2_PW and either 1, 2, 5, 10, 20, 30, or 40 carbon steel beads were incubated and corrosion rates were determined by weight loss. The corrected weight loss CRs obtained following treatment with 1 N HCl for 10 min are indicated in Figure 2-CO2 were shipped to the field, where they were filled with 50 ml sample. When the serum bottles were received at the UofC a backpressure of 32–51 ml was measured (Table Capped and crimped serum bottles 120 ml) with either 2 coupons or 5 beads, 10 mg of sodium bisulfite and a headspace of N0 ml with2/g, respectively. This small difference was corrected for in the calculation of CR by Equation (6). A higher weight of carbon steel per unit volume for the incubation with coupons, as compared to beads  was 0.1185 mm/yr  were incubated with either five beads for 39 days or with three coupons for 32 days. Coupons (1.3 × 0.8 × 0.4 cm) were cut from CT carbon steel. The average weight loss corrosion rate for all 21 samples was 0.054 ± 0.049 mm/yr (range 0.008–0.185 mm/yr) for coupons and 0.214 ± 0.261 mm/yr (range 0.09–1.31 mm/yr) for beads. Images of highly and moderately corroded beads are shown in Figure Higher corrosion rates for beads as opposed to coupons were also observed with 21 samples obtained from coiled tubing (CT) operations in shale gas fields in NW British Columbia and NE Alberta. These samples had MPNs of APB, which varied from zero to 9.3 × 102-CO2 headspace for 51 h gave an average residual bead weight of 41.4 ± 8.9 mg (average ± SD for 50 beads), indicating highly uneven corrosion with an average CR of 16.7 ± 3.1 mm/yr. Indeed, extensive pitting was evident on the surface of highly corroded beads  each containing 15 ml of 1 N anoxic HCl and an N2 and reduction of sulfate to sulfide as per Equation (1). The medium had excess sulfate allowing in principle reduction of sulfate with Fe0 as the electron donor in the syringe columns as per Equation (4). Up-flow injection of chemostat effluent into a carbon steel- and glass-bead containing column for 256 days gave an overall general corrosion rate of 0.11 mm/yr. The residual weight of the beads ranged from 36.5 to 51.4 mg, with an average of 44.0 ± 3.3 mg. The average weight of beads prior to corrosion was 55.0 ± 0.3 mg. The significant 11-fold increase in SD from 0.3 to 3.3 mg indicated uneven (pitting) corrosion. The most heavily corroded beads had deep pits (not shown). Short-term injection (46–60 days) of the same chemostat effluent into columns containing only carbon steel beads gave general corrosion rates of 0.016–0.030 mm/yr. We are currently trying to find conditions giving increased corrosion to allow rapid determination of the effect of treatment with biocides or corrosion inhibitors on corrosion under flow conditions.Carbon steel beads  were packed in 1 ml syringe columns, which were subjected to injection of 10 ml/day of the effluent of a chemostat, containing actively growing SRB in defined medium with 10 mM lactate and 10 mM sulfate. The chemostat effluent had 10 mM acetate, 5 mM of residual sulfate, and 5 mM sulfide, indicating effective oxidation of lactate to acetate and CO0) as electron donor for their metabolism.The study of MIC involves determining (i) the contribution of microorganisms to general and pitting corrosion rates in the laboratory and in the field (ii) the structure and function of multiple species biofilms in catalyzing corrosion, and (iii) the mechanisms through which pure cultures or consortia of microorganisms are able to use metallic iron  or other methods  as electron donor for microbial metabolism is facilitated by using forms of iron with a large surface-to-volume ratio and a reactive surface. Early studies on the co-metabolism of Fe0 by SRB growing on lactate and sulfate used steel wool as the substrate with a surface to volume ratio of 100 cm3/g (Cord-Ruwisch and Widdel, 0 corrosion by methanogens used iron powder of which the properties were not stated (Daniels et al., 0 effectively as electron donor for the reduction of sulfate to sulfide, of CO2 to methane or of CO2 to acetate, have made use of Fe0 granules with a diameter of 0.1–0.2 cm (Dinh et al., The study of the effective use of metallic iron (FeIn conclusion, the use of carbon steel beads offers additional opportunities for screening and monitoring of corrosion, including MIC, in oil field samples. This includes determination of the distribution of corrosion rates, characterized by a mean and an SD allowing estimation of the probability of occurrence of rates high enough to lead to failure.GV—planned experiments, monitored progress and suggested revisions to protocols, drafted the final manuscript. PM—water chemistry analysis of oilfield samples from Oceania, conducted corrosion experiments with these samples. TP—conducted the carbon bead experiments in upflow bioreactors and associated water analysis and corrosion rate measurements. MS—did experiments with coiled tubing samples including water analysis, corrosion experiments, and data interpretation. YS—conducted all MPN assays for all samples in the study. AV—did experiments on corrosion of carbon steel beads in varied concentrations of acids. JV—assisted with upflow bioreactor carbon bead experiments and acid experiments with carbon steel beads. AS—coordinated collection and shipment of Oceania field samples to the lab. Provided facility operational details to assist with data interpretation. Contributed to manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HIV-1 envelope glycoproteins (Env) are the only viral antigens present on the virus surface and serve as the key targets for virus-neutralizing antibodies. However, HIV-1 deploys multiple strategies to shield the vulnerable sites on its Env from neutralizing antibodies. The V1V2 domain located at the apex of the HIV-1 Env spike is known to encompass highly variable loops, but V1V2 also contains immunogenic conserved elements recognized by cross-reactive antibodies. This study evaluates human monoclonal antibodies (mAbs) against V2 epitopes which overlap with the conserved integrin α4β7-binding LDV/I motif, designated as the V2i (integrin) epitopes. We postulate that the V2i Abs have weak or no neutralizing activities because the V2i epitopes are often occluded from antibody recognition. To gain insights into the mechanisms of the V2i occlusion, we evaluated three elements at the distal end of the V1V2 domain shown in the structure of V2i epitope complexed with mAb 830A to be important for antibody recognition of the V2i epitope. Amino-acid substitutions at position 179 that restore the LDV/I motif had minimal effects on virus sensitivity to neutralization by most V2i mAbs. However, a charge change at position 153 in the V1 region significantly increased sensitivity of subtype C virus ZM109 to most V2i mAbs. Separately, a disulfide bond introduced to stabilize the hypervariable region of V2 loop also enhanced virus neutralization by some V2i mAbs, but the effects varied depending on the virus. These data demonstrate that multiple elements within the V1V2 domain act independently and in a virus-dependent fashion to govern the antibody recognition and accessibility of V2i epitopes, suggesting the need for multi-pronged strategies to counter the escape and the shielding mechanisms obstructing the V2i Abs from neutralizing HIV-1. Vaccines are urgently needed to control HIV-1 infection worldwide, but the development of efficacious vaccines against HIV-1 remains an unsolved challenge. The RV144 prime-boost vaccine regimen tested in a phase III clinical trial in Thailand is the only candidate vaccine showing an efficacy that reaches 60% after 1 year but declines to ~30% after 3.5 years of follow up , furtherA number of monoclonal antibodies (mAbs) against V1V2 have been isolated from HIV-infected individuals and from RV144 vaccine recipients –12. ThusIn this study we evaluated the specific elements in the distal V1V2 domain elucidated in the crystal structure of the V2i epitope bound by mAb 830A  and asseenv was constructed in our laboratory [Plasmids expressing HIV-1 Env of 6535 (clone 3 (SVPB5)), BaL (clone BaL.01), REJO (pREJO4541 clone 67 (SVPB16)), and ZM109  were used for generating HIV-1 pseudoviruses. These plasmids were obtained from Drs. David Montefiori, Feng Gao, Ming Li, John Mascola, B.H. Hahn, J. F. Salazar-Gonzalez, C.A. Derdeyn and E. Hunter through the NIH AIDS Research and Reference Reagent Program (ARRRP). The plasmid bearing SF162 (pIRESSF162) boratory . The 293rev- and env- expressing plasmids and the pNL4-3Δenv R-E- plasmid using the jetPEI transfection reagent (Polyplus-transfect SA). Supernatants were harvested after 48 hrs and clarified by centrifugation and 0.45 μm filtration. Some virus stocks were concentrated with a 100kDa Amicon filter (Millipore). Single-use aliquots were stored at −80°C. Virus titration was performed on TZM-bl cells with the Beta-Glo Assay System (Promega) as described previously [Pseudoviruses were produced by co-transfecting 293T/17 cells with HIV-1 eviously . Virus tSeven V2i mAbs, a control mAb 1418 (specific for human parvovirus B19), V3 crown mAbs , and the CD4-binding site (CD4bs) mAbs 654-D and 1331 were used in this study and produced as described previously . Both 65Single or double mutations were introduced in the HIV-1 Env V1V2 region using the QuikChange II XL Site-Directed Mutagenesis Kit , according to the manufacturer's instructions. All mutant constructs were sequenced to verify that the correct amino acid changes were made.Structural modeling and analysis were carried out using ICM and figures were generated using PyMol . The gp120 trimer structure data was obtained from the PDB access number 4TVP.Virus neutralization was measured using a β-galactosidase-based assay  with TZM-bl target cells . BrieflyThe relative binding affinity of mAbs to gp120s from 6535, REJO, ZM109 and its mutant pseudoviruses were measured by a sandwich ELISA. The ELISA plates were coated with sheep anti-gp120 Abs , blocked with DMEM containing 10% FBS, and incubated with 1% Triton X100-treated pseudoviruses. Serially diluted mAbs (0.01–10μg/ml) were then added for 2 hrs, and the bound mAbs were detected with alkaline phosphatase-conjugated goat anti-human IgG Fc and p-nitrophenyl phosphate substrate. HIV+ serum pool obtained anonymously was used for normalization of the gp120 input and CD4-IgG2 binding was also assessed for control.Comparisons of virus neutralization and mAb binding were performed using GraphPad Prism 6. Statistical analyses were performed on neutralization data which reached ≥50%. Comparison of neutralization data was done on the mAb titration curves with a two-way ANOVA test.179LDV/I181 integrin-binding motif, 2) mutations at residue 153 that reinstate the highly conserved E in V1, and 3) introduction of a disulfide bond to stabilize the hypervariable region of V2 loop.This study sought to determine the elements within the distal region of the V1V2 domain that influence mAb recognition of the V2i epitopes in order to exert virus neutralization. To this end, HIV-1 pseudoviruses displaying varying degrees of neutralization sensitivity to V2i mAbs were selected . The stu179LDV/I181 motif, especially at positions 179 and 180 (HxB2 numbering) abrogates binding of most V2i mAbs to SF162 gp120. The x-ray crystallographic analyses of V2i mAb 830A in complex with its V1V2 epitope further showed residue 179L to be one of the key contacts for the 830A-V1V2 interaction [An earlier mutagenesis study to map V2i epitopes revealed the importance of the α4β7 integrin binding motif for the reactivity of all seven human V2i mAbs tested , 14, 15.eraction , sequencTo determine whether the changes observed with 6535 (T179L) neutralization by mAbs 830A and 697 are due to alterations in the mAb binding, ELISA was performed to test the binding activity of V2i mAbs to gp120 solubilized from the 6535 (T179L) mutant versus WT viruses. MAb 830A bound poorly to soluble gp120 from 6535 WT. The T179L mutation resulted in significant gain of 830A reactivity, and this correlated with enhanced neutralization of the 6535 (T179L) mutant by 830A. gp120 binding activities of V2i mAbs 697 and 1393A, V3 mAb 447-52D, the CD4bs mAbs 654-D and NIH45-46 were unchanged . Hence, 153E residue on their Envs, but these viruses are resistant to neutralization by V2i mAbs . Viruses pseudotyped with the SF162 and BaL.01 mutants were infectious , allowin179LDV/I181 motif have by and large minimal effects (This study sought to evaluate the elements within the distal end of the V1V2 domain of the HIV-1 Env for their contribution to the escape or shielding of the V2i epitopes from Ab recognition. Of the three elements studied here, amino acid substitutions at position 179 that restore the  effects . In cont effects –5. The e179L is one of the key contact points for 830A [179L is not critical for the other V2i epitopes as is for 830A. The importance of 179L residue is also virus dependent. An earlier study by Mayr et al. [179Q, the Q179L mutation did not affect 830A and all other V2i mAbs tested (As observed with mAbs targeting the other Env epitopes , 29–35, r et al.  showed ts tested . REJO al153R residue (153R residue is another important contact site revealed in the crystal structures of mAb 830A and its epitope [179L mutations, these data support the idea that unique single-point mutations offer survival benefits for the different HIV-1 strains; however, this escape mechanism enables virus evasion from a particular Ab or a small subset of Abs, leaving the virus susceptible to many other Abs targeting even nearby overlapping epitopes on the HIV-1 Env.Another example of a single amino acid variation that influences a V2i mAb is demonstrated by the ZM109 virus which is not recognized by the V2i mAb 1357 due to an unusual  residue . The 153 epitope . In most epitope . HoweverIn addition to epitope escape by amino acid variations, the V2i mAbs may be blocked from binding their epitopes and neutralizing the virus by epitope masking, without alterations on the epitopes themselves. Epitope masking has the potential to mediate a more pervasive impact, because multiple epitopes may be shielded simultaneously, enabling virus escape from a larger set of Abs. However, the epitopes are not perpetually masked. As shown by Munro et al. , the natIndependent of the R153E mutation, the addition of a disulfide bond to the highly flexible V2 loop also augmented ZM109 sensitivity to neutralization by the V2i mAbs . A pair The epitope masking has been extensively studied in the context of neutralizing epitopes at the crown of the V3 loop , 39–43,


Hospital based delivery has been an expensive experience for poor households because of hidden costs which are usually unaccounted in hospital costs. The main aim of this study was to estimate the hidden costs of hospital based delivery and determine the factors associated with the hidden costs.A hospital based cross-sectional study was conducted among 384 post-partum mothers with their husbands/house heads during the discharge time in Manipal Teaching Hospital and Western Regional Hospital, Pokhara, Nepal. A face to face interview with each respondent was conducted using a structured questionnaire. Hidden costs were calculated based on the price rate of the market during the time of the study.The total hidden costs for normal delivery and C-section delivery were 243.4 USD (US Dollar) and 321.6 USD respectively. Of the total maternity care expenditures; higher mean expenditures were found for food & drinking (53.07%), clothes (9.8%) and transport (7.3%). For postpartum women with their husband or house head, the total mean opportunity cost of “days of work loss” were 84.1 USD and 81.9 USD for normal delivery and C-section respectively. Factors such as literate mother (p = 0.007), employed house head (p = 0.011), monthly family income more than 25,000 NRs  (p = 0.014), private hospital as a place of delivery (p = 0.0001), C-section as a mode of delivery (p = 0.0001), longer duration (>5days) of stay in hospital (p = 0.0001), longer distance (>15km) from house to hospital (p = 0.0001) and longer travel time (>240 minutes) from house to hospital (p = 0.007) showed a significant association with the higher hidden costs (>25000 NRs).Experiences of hidden costs on hospital based delivery and opportunity costs of days of work loss were found high. Several socio-demographic factors, delivery related factors  were associated with hidden costs. Hidden costs can be a critical factor for many poor and remote households who attend the hospital for delivery. Current remuneration  for maternity incentive needs to account the hidden costs by increasing it to 250 USD for normal delivery and 350 USD for C-section. Decentralization of the obstetric care to remote and under-privileged population might reduce the economic burden of pregnant women and can facilitate their attendance at the health care centers. In Nepal, a poor household woman faces a significant economic impediment in accessing health services during delivery due to hidden costs. A normal delivery for a woman from low to middle income family costs about 3 months of a household income compared to just over a month in a high income family . MaternaThe Maternal Mortality Rate in Nepal was estimated to be 258 deaths per 100,000 live births . Under-uNepal has formulated the national policies and plans to enhance the delivery of basic and essential health services to the general public. In 2008/9, the Government of Nepal (GoN) introduced the provision of free health service program to the general public through all governmental health care institutions and health workers. Under free health service scheme, GoN provides up to 40 essential medicines for free, through district level health facilities .The drug availability in health facilities, especially the contraceptives, maternal and child health commodities, and selected essential drugs have improved the overall health care. However, there are several challenges for its implementation, of those are the dependence of the budget to donors , insufficient services to ensure the universal access with desired quality, limited human resources and lack of co-ordination between central and peripheral health facilities .The government of Nepal started a Maternal Incentive Scheme in 25 least developed districts to encourage women to deliver babies at hospitals in 2005 which was later revised as “Safe motherhood program” in 2007 . This prIn Nepal, bulk of core costs  are officially free of charge but the overall maternity care has been found to be expensive for families because of hidden costs which includes the purchase of drugs from private pharmacies, food costs, transportation costs, communication expenses (accessory communication need while being away from home), loss of days at work by both the patient and the patient’s accompaniers, unaccounted ancillary treatments, wasted drugs and laboratory tests –14. HiddAmong many other reasons, economic constraint faced by rural women attending the urban health centers possesses a significant impact on utilization of health services . It is nThis study was a cross-sectional study estimating the hidden costs of hospital based delivery from the patient’s perspective. The duration of the study was from August 2014 to December 2014.Aamma Surukshya Karyakram, literally meaning “Safer Maternity Program” which is operating under the Ministry of Health and Population. The Safer Maternity Program provides both free childbirth services and incentives to women giving birth in a health facility in addition to the incentives to health care worker for each delivery attended [Participants for the study were recruited from two tertiary referral hospitals  and Western Regional Hospital (WRH)) in Western Nepal. Both of these hospitals had attended , 8.To estimate the hidden costs of hospital based delivery, 384 post-partum mothers with their husband or house head (284 samples from WRH and 100 samples from MTH) who attended the hospital were interviewed. The interview took place from September 23, 2014 until November 25, 2014. A representative sample size from each health institution was calculated through probability proportionate to size sampling technique. A convenient sampling technique was applied to select the participants (women with their husband or house head). Post-partum mothers were excluded if they did not deliver at the hospital.Aama Surakhshya) was implemented. All questions were asked in Nepali language.The study questionnaire  was deveAll costs were calculated in Nepalese Rupees . From the patient’s perspective, replacement cost method was used to estimate the hidden costs of hospital based delivery. The cost was calculated based on the market rate for that particular date with the unit cost of a postpartum mother with their husband or house head as a proxy value (or ‘shadow price’) . CaretakRespondents were also asked about their loss of wages during the period of hospital delivery in order to calculate the opportunity cost for days of work loss generated by the condition, assuming that the respondent’s level of earnings reflected productivity  . In thisDescriptive statistics were presented as frequency, percentage and mean. Chi-square test was used to analyze the association between various variables with hidden costs. P-value less than or equal to 0.05 was taken for statistical significance.The hidden costs of delivery services included transportation expenses, food and drinking expenses, communication expenses, cost for laundry, cost for fuel, cost for child care, cost for clothes (sleep wear for baby and women), accessories expenses  and loss of wage (opportunity costs of days of work loss) during the hospital stay. A total hidden cost was measured in US dollars to the same year. We dichotomized it into two categories: <25000 NRs and >25000 NRs, which represented 44.8% and 55.2% of participants respectively. Since the data was normally distributed thus absolute mean was selected as cut off.Socio-demographic characteristics , delivery related characteristics  were analyzed to determine the association with total hidden costs.This study obtained an ethical approval from the Department of Public Health of La Grandee International College. The study was a part of the thesis for the completion of “Bachelor Public Health” Program in La Grandee International College under Pokhara University. The college has an independent research supervisory department which examines the various issues including ethical issues of research. All research studies are endorsed by college only after the approval from academic committee and research supervisory department. Further approval from both hospitals  was obtained before the start of the study. Written informed consent was obtained from all participants who were 18 years or above. For respondents (post-partum mothers) who were below 18 years, written informed consent was obtained from their husbands or household heads with oral consent from respondents. The consent procedure was approved both by the college and the hospitals where study was conducted. The lowest age of post-partum mother in this study was 15 years. No incentives were provided to participants.The mean age was 23.69±4.47 years (range 15 years to 43 years). Majority of respondents 69% (265) had normal vaginal delivery in the study period followed by 31% (119) who underwent C-section delivery as shown in Hidden expenditures in normal and C-section delivery were 24,817.1 NRs (243.4 USD) and 32,792.2 NRs (321.6 USD) respectively as shown in To our knowledge, this is the first study in Nepal assessing the hidden costs during hospital delivery. In this study, the average total hidden costs was 27,288.5 NRs (267.6 USD) which was 87.5% of total average hospital based delivery expenditures . These findings suggest that hospital based delivery can be an expensive experience for women from lower socio-economic status as this study shows that the median patient’s expenditure on hospital based delivery  was 13% in proportion of median annual family income . Postpartum mothers who came with their husband or house head in this study were economically vulnerable than in the study conducted in the public hospital of Bangladesh  where meThe patient’s mean expenditure on food and drinking (53.07%) was higher than for clothes (9.8%) and transportation (7.3%). The finding in this study is unique as (other) studies from different parts of the world showed different lists of expenses. In a study conducted at government facilities in Bangladesh , costs fThe mean opportunity cost of days of work loss (loss of wages) for normal delivery and C-section were 8,582.2 NRs 84.1 USD) and 8,354.04 NRs (81.9 USD) respectively which showed that loss of wages for pregnant women with their husband or house head during delivery was one of the major contributors to the hidden costs. A previous study in Nepal [ USD and To reduce negative externalities from hospital based delivery, this study showed that hidden costs need to be reduced by focusing on several socio-demographic factors  and delivery related factors . The findings in this study are consistent with studies conducted in Lao People's Democratic Republic , Greece Furthermore, this study found that none of the factors like age of respondents, religion, educational status of husband, educational status of house head, occupation of husband, occupation of house head and number of pregnancies affected the hidden costs on the delivery services (p>0.05).This study may not represent the more rural communities as the study was conducted in tertiary health facilities in an urban area, however, tertiary health care centers selected in this study are attended by participants from various regions including rural regions. The generalizability of this study is therefore, partially restricted to urban settings. Further studies are recommended to conduct in rural settings such as district hospital in hilly and mountainous regions that might better reveal the rural hospital attendance. Besides hidden costs estimation, intangible costs which reflect the pregnant women and the relatives’ psychosocial stresses to travel to the hospital and stay at hospital were beyond the scope of this study.Experiences of hidden costs on hospital based delivery and opportunity cost of days of work loss were high and postpartum mothers with their husband or house head were economically affected. Half of the hidden costs were attributed to food and drinking. Hidden costs and opportunity cost could be a significant barrier to hospital delivery for women from poor household and women from remote regions. Education of mothers, occupation of house head, family monthly income, mode of delivery, place of delivery, length of stay in the hospital, distance from house to hospital, travel time from house to hospital were factors associated with the hidden cost on hospital based delivery. Current delivery services need to take in account hidden costs/expenditures for pregnant women and their relatives. Current remuneration (10–70 USD) needs substantial revision to account the hidden costs of hospital based delivery by increasing it to 250 USD for normal delivery and 350 USD for C-section. Decentralization of the obstetric care to remote and under-privileged population might reduce the economic burden of pregnant women attending health care facilities.S1 File(PDF)Click here for additional data file.


Although it has been reported that DGL has a significant pain relief effect in treating osteoarthritis, little is known about the characteristics of the effects of this compound on dental pain. The present study was undertaken to evaluate the applicability of DGL as a medicament to control pulpalgia. Using an  Chitin is a polysaccharide mainly located in the outer skeletal tissue of crustacean such as crabs, shrimps, and lobsters  and is t2-, and IL-8-production and phosphorylation of p38 MAPK) [DGL has a variety of biological activities. DGL suppress the functions of the neutrophils, thereby possibly exhibiting anti-inflammatory actions , and DGL38 MAPK) .DGL has been clinically used as an effective medicament , 7. For We have shown that DGL accelerates cell proliferation and differentiation and promotes tissue regeneration on dental pulp wounds . DGL supPreviously, we used bradykinin as a chemical nociceptive stimulant and evaluated the possibility of DGL as a medicament to control dental pain. The results suggested that DGL have a pain relief effect on patients with dental pain.However, the pain relief mechanism of DGL in the setting of dental pain is poorly understood. In this study, we revaluated whether DGL exhibits relief of dental pain caused by the pain-producing substance, 5-hydroxytryptamine (5-HT), instead of bradykinin in order to elucidate the pain relief of mechanism of DGL.The methods described here follow the ethical guidelines of and received approval from the Animal Welfare Committee of Nagasaki University . in vitro jaw-nerve preparation, as described elsewhere [ in vitro jaw-nerve preparation was slightly modified in order to enable easy direct access to the dental pulp  were used in the present study. The animals were deeply anesthetized with thiamylal sodium . We used anlsewhere . An origtal pulp . The manThe inferior alveolar nerve was identified at the foramen mandibulae on one side and isolated from the surrounding tissue. The mandible on one side was removed together with the inferior alveolar nerve by cutting the temporomandibular joint. Finally, approximately 20 mm of the inferior alveolar nerve trunk was obtained from the foramen mandibulae and the proximal end of the nerve was ligated with cotton thread. A small hole was carefully made in the center of the incisor tooth in order to expose the dental pulp using a dental bur .2, 1.2 mM of MgSO4, 1.2 mM of KH2PO4, 22.4 mM of NaHCO3, and 20 mM of glucose was used for perfusion.Modified Krebs-Henseleit solution consisting of 110.9 mM of NaCl, 4.8 mM of KCl, 2.5 mM of CaCl10H12N2O)  was dissolved in the sterile physiological saline . 10−5 M 5-HT solution was used to chemically stimulate the tooth pulpal nociceptors.5-HT . The molecular weight of the DGL used in this experiment was approximately 215 Da. A 10% (w/v) solusion was prepared by dissolving DGL powder in sterile physiological saline.As shown in 2 : CO2 (95% : 5%). The fluid was heated to maintain the temperature at 31°C with a feedback-controller.The mandible was placed in the test pool, and the inferior alveolar nerve was passed through a hole at the center of the partition plate and placed in the recording chamber (oil pool), where it was fixed to the wall of the chamber with cotton thread. A moderate amount of tension was applied so as to easily insert the recording electrode. The hole at the center of the partition plate was capped with Vaseline. The oil pool was filled with liquid paraffin in order to immerse the inferior alveolar nerve. The test pool was perfused (0.3 mL/s) with the modified Krebs-Henseleit solution. The solution was saturated with a gas mixture of OThe inferior alveolar nerve in the oil pool was desheathed by slipping off the epineurium. A tungsten microelectrode  was inserted into the inferior alveolar nerve trunk using a micromanipulator  in order to record single fiber responses. The action potential was fed into a high-impedance, low-noise amplifier  and displayed on a computer through the CED 1401 interface . A spike analysis was carried out using the Spike 2 software program for Windows (Version 2.01).It is shown that C-fibers terminals are located in the center core of the pulp . In orde5-HT was used as a chemical nociceptive stimulant and poured into the test chamber with application near the exposed tooth pulp at a speed of 0.1 mL/s. Sixty seconds after 5-HT application, the surface of the exposed pulp was treated with DGL solution in the DGL group and physiological saline in the control group.t-test. A P value of less than 0.05 was considered to be significant.The obtained data were evaluated statistically using the StatView software program, Version 5.0 . The values were expressed as the mean ± SE, and the differences between the control and DGL groups were compared using unpaired Student's n = 10), while that in the DGL group was 0.39 ± 0.10 Hz (n = 17). There were no significant differences in the initial firing rate before 5-HT application between the control and DGL groups  five minutes after saline application, while that in the DGL group was 0.15 ± 0.05 Hz (n = 17) five minutes after DGL application. The DGL group exhibited a significantly lower frequency than the control groups (asterisk) during 300–420 s (P < 0.05).In vitro preparations have advantages in quantitatively controlling all environmental variables surrounding the oral tissue and directly applying chemical solutions to the receptive field [ve field . Therefove field , 25, 26.We have investigated analgesic effect using 1, 5, 10, and 15% of DGL in preliminary study. In the present study, we used a 10% (w/v) solution prepared by dissolving DGL powder in sterile physiological saline. As a result, 10% DGL has been determined as an optimal concentration. At this concentration, there appeared to be no inflammatory reactions, as no enhancement of spike responses was observed after DGL application as shown in The effects of DGL on dental pain have been little reported. Previously, we used bradykinin as a chemical nociceptive stimulant and to assess whether DGL exerts a pain relief effect in patients with dental pain .The present study was undertaken in order to evaluate the effects of directly applying DGL to exposed dental pulp and demonstrated that DGL suppresses the nociceptive responses evoked by 5-HT application on the tooth pulp. These results indicate that DGL suppresses the nociceptive responses evoked by two different chemical nociceptive stimulants, namely, bradykinin and 5-HT.TRPV1 is a cation channel that is activated by a variety of chemical stimulants. The best-known activator of TRPV1 is capsaicin. The activation of TRPV1 results in painful sensations. Bradykinin can sensitize TRPV1 via the G protein coupled receptor (GPCR) , 29. FurIn addition, it has been reported that DGL reduces the elementary current amplitude and increases the mean channel open time . Because2) [The precise mechanisms underlying the antinociceptive actions of DGL have not yet been elucidated; however, glucosamine-induced antinociception is presumed to be caused by anti-inflammatory effects. DGL suppresses neutrophil functions   and inhi2) . TherefoAs stated before, DGL has a significant pain relief effect in patients with osteoarthritis , 35. OstThe results of the present study show that DGL may have a pain relief effect in patients with dental pain. These findings led us to examine the application of DGL as a dental analgesic agent. The use of glucosamine to treat dental disorders is thought to be a possible alternative with a promising future.


Flaviviridae, genus Flavivirus, holds many of the world’s most prevalent arboviral diseases that are also considered the most important travel related arboviral infections. In most cases, flavivirus diagnosis in travelers is primarily based on serology as viremia is often low and typically has already been reduced to undetectable levels when symptoms set in and patients seek medical attention. Serological differentiation between flaviviruses and the false-positive results caused by vaccination and cross-reactivity among the different species, are problematic for surveillance and diagnostics of flaviviruses. Their partially overlapping geographic distribution and symptoms, combined with increase in travel, and preexisting antibodies due to flavivirus vaccinations, expand the need for rapid and reliable multiplex diagnostic tests to supplement currently used methods.The family Flavivirus. Sera from clinical flavivirus patients were used for primary development of the protein microarray.We describe the development of a multiplex serological protein microarray using recombinant NS1 proteins for detection of medically important viruses within the genus Results show a high IgG and IgM sensitivity and specificity for individual NS1 antigens, and limited cross reactivity, even within serocomplexes. In addition, the serology based on this array allows for discrimination between infection and vaccination response for JEV vaccine, and no cross-reactivity with TBEV and YFV vaccine induced antibodies when testing for antibodies to other flaviviruses.Based on these data, multiplex NS1-based protein microarray is a promising tool for surveillance and diagnosis of flaviviruses. The number of international travelers has increased dramatically in recent decades. This has contributed to the increase in infectious diseases in travelers which are not present in their countries of origin and so may cause a threat to the public health. Viruses transmitted by biting insects (vector-borne viruses) are an important group within these travel-related diseases. They are found across the world and can cause debilitating and life-threatening symptoms, like inflammation of the brain or excessive bleeding. Many of these diseases are difficult to distinguish from each other. They cause comparable symptoms and are genetically closely related. Testing for long lists of diseases is time consuming and expensive. Here we develop a novel testing tool that allows doctors and researchers to test for multiple viruses with just one test. The method, which uses a specific part of the virus that makes distinguishing between infections with these closely related viruses possible, requires only one drop of blood. This allows us to test for multiple viruses simultaneously with the same amount of blood previously used to test for only one virus, while distinguishing between genetically closely related viruses. Flaviviridae, genus Flavivirus, holds many of the world’s most prevalent arboviral diseases that are also considered the most important travel related arboviral infections.[The family fections. As the gfections. Increasefections., 4Flaviviruses are single stranded enveloped viruses with an RNA genome of about 11 kb length. The genome is composed of three structural  and seven non-structural proteins .The genus is divided into serocomplexes that are distinguished based on neutralizing antibody reactivity . The amiTo overcome flavivirus cross-reactivity in diagnostics the use of recombinant antigens in ELISA is to be preferred over whole virus as it increases specificity.–16 EnvelFlavivirus genus.To enable fast, syndrome based laboratory testing that focuses on multiple rather than individual viruses, we developed a protein microarray, using recombinant NS1 proteins, as a serological test for medically important viruses within the Sera from anonymized patients were used for primary development of the protein microarray. Patients were diagnosed according to international accepted criteria combining clinical symptoms, epidemiological data, and standard serological methods  and laboratory confirmed by either VNT or PCR with the exception of 10 patients suspected of JEV. Information on each patient group used is presented in FJ687432.1), Dengue virus 2 (genbank:FJ744720.1), Dengue virus 3, (genbank:FJ744738.1), Dengue virus 4 (genbank:EU854300.1), Japanese encephalitis virus (genbank:NC_001437.1), St. Louis encephalitis virus (genbank:ACB58159.1), Yellow fever virus (genbank:JN620362.1) and West Nile virus (genbank:EU081844.1)Custom-made NS1 proteins produced in human embryonic kidney 293 (HEK293) cells to ensure proper folding, glycosylation and dimerization were used . A V5-epitope and Histag were added to the C-terminus for protein quantification and filtration. Proteins were expressed for Dengue virus 1 (genbank:NC006551.1) was produced in-house in a HEK293 cell-line. The NS1 gene was produced by Genscript  with an additional V5-epitope and Histag on the C-terminus and cloned into a pcDNA-DEST40 vector  that contained a neomycine resistance gene. The vector which contained the NS1 gene was transfected into HEK293 cells. Neomycine resistant clones were selected and tested for protein expression by immune fluorescent assay using anti-V5 monoclonal antibody. Selected clones were grown in flasks and secreted NS1 protein into the medium . The secreted protein was purified from the medium by FPLC using a Ni-NTA column  according to the manufacturer’s instructions.Usutu virus NS1  and spotted in triplicate as a within-test control per pad. Antigens were spotted onto a nitrocellulose pad coated glass slide  using a non-contact protein array spotter  as previously described. Per spotPatient sera were tested on dried slides as previously described. In shortVirus antigens were spotted in serial two-fold dilutions ranging from 1:2 to 1:16 for initial checkerboard titrations to determine optimum protein concentration as previously described. AntigensA script was written in R using adFJ687432.1), Dengue virus 2 (genbank:FJ744720.1), Dengue virus 3, (genbank:FJ744738.1), Dengue virus 4 (genbank:EU854300.1), Japanese encephalitis virus (genbank:NC_001437.1), St. Louis encephalitis virus (genbank:ACB58159.1), Yellow fever virus (genbank:JN620362.1) and West Nile virus (genbank:EU081844.1), Usutu virus NS1 (genbank:NC006551.1).Dengue virus 1  positive control sera and low in negative control sera and in sera from individuals vaccinated for JEV, TBEV or YFV (p<0.01) with the exception of YFV NS1 antigen with YFV vaccinees . Only soAt low serum dilutions, some patients showed antibody IgG reactivity to multiple antigens, and therefore ROC curves were calculated in multiple dilutions and the signals for the 1:20 dilutions were used for signal cut-off calculations. The 1:10 and 1:20 serum dilutions produced comparable results in sensitivity and specificity, but with significantly lower background for the 1:20 dilutions. At 1:40 serum dilutions, the sensitivity started to decrease.Only 13 DENV positive patients (travelers) had known primary DENV infections with a PCR confirmed serotype (DENV1–3). All other patients with PCR confirmed DENV (serotype 1–4) were from DENV endemic countries and could not be confirmed as primary infections. As not all DENV infections were known to be primary, the highest signal to DENV1–4 NS1 was used for calculation of the DENV cut-offs. The optimal cut-off for all proteins was around a fluorescent signal of 15,000 for IgG and 4,000 for IgM, producing sensitivity and specificity of 86% to 100% .For USUV, SLEV and YFV only one or two positive patient samples were available so that proper ROC curves could not be calculated, but background signals were in the same range as for the other antigens . Serum sIn order to study cross-reactivity within and between serocomplexes, serum samples were serially diluted and titers were calculated in R. Typical individual patient profiles are shown in IgG profiles from individual patients were combined into a heatmap  to confiFor 13 known primary DENV cases, the serotype had been determined by RT-PCR. All but one serum had highest IgG antibody levels to the infecting serotype, but IgM antibody reactivity was lower and less discriminatory.We developed a first generation protein microarray for rapid, multiplex and virus-specific IgM and IgG tests for diagnosis of flavivirus infections that could differentiate between virus species, even within flavivirus serocomplexes, as well as between vaccinated and infected individuals, with the exception of YFV vaccinated individuals. Initial validation shows that this microarray can be used for flavivirus surveillance in travelers and potentially in regions with co-circulation of multiple flaviviruses. To establish this we first investigated the sensitivity of NS1 antigens to their homologous sera on a protein microarray. The results show good IgG and IgM sensitivity for both JEV and DENV serocomplex viruses. The sensitivity is comparable to current IFA and ELISA commercial kits. However, our sample selection was tested with a multitude of standard serological assays. Comparing our results to commercial ELISA or IFA kits should therefore be done with caution. Our results confirm that NS1 provides a good sensitive and specific antigen tool for serological diagnosis.For DENV, the sensitivity of the IgM assay was lower than for IgG. There are several possible explanations for this finding. First, lower DENV IgM sensitivity may have resulted because five to ten days post onset of symptoms may be too early for detecting seroconversion in serum samples. EvaluatiSecond, a study of antibody reactivity to individual DENV proteins has found that the measured mean OD is lower for NS1 compared to E-protein with many samples clustering closer to the cut-off compared to E-protein antibodies. This makThird, patient-to-patient variation in antibody responses to individual viral antigens may cause discrepancies of test results. NS1 based protein assays could potentially pick up infections missed by prM-E based front-line serological tests. AntibodiFinally, a number of the DENV cases may have been secondary, tertiary or quaternary infections. Several of the samples originated from DENV hyperendemic countries and showed high titers to multiple DENV and other flaviviruses, further supporting this assumption. Literature shows that IgM antibody titers produced during a secondary DENV infection are absent or lower and produced during a shorter period. This reduces the sensitivity of IgM antibodies in non-primary infections significantly. Despite The biggest challenge of current flavivirus serodiagnostics is virus specific differentiation., 4 We anThe cause of the outliers shown in While we show excellent discrimination in antibody responses to viruses within the JEV complex, this is less straightforward for DENV. The protein microarray IgG antibody reactivity to individual DENV NS1 antigens shows capability to distinguish serotypes (as can be seen in Finally, we looked at the ability to rule out false positive reactions due to vaccination. This was not possible for YFV vaccination when testing against YFV NS1. YFV vaccine is a live attenuated vaccine and causes mild infection resulting in NS1 antigen and antibody production. We do, hSerological differentiation between flaviviruses and the false-positive results caused by vaccination are a serious problem for surveillance and diagnostics of flaviviruses. Analysis of our NS1-based protein microarray results showed a high IgG and IgM sensitivity and specificity for individual antigens even within the same serocomplex, and limited cross reactivity. In addition, the serology based on this array allowed discrimination between infection and vaccination response for JEV vaccine, and no cross-reactivity with TBEV and YFV vaccine induced antibodies when testing for antibodies to other flaviviruses. Based on this data, our multiplex NS1-based protein microarray is a promising tool for surveillance and diagnosis of flaviviruses.


The ongoing Ebola outbreak in West Africa has resulted in fast-track development of vaccine candidates. We tested a vesicular stomatitis virus vector expressing Ebola virus glycoprotein for safety in pigs. Inoculation did not cause disease and vaccine virus shedding was minimal, which indicated that the vaccine virus does not pose a risk of dissemination in pigs. The current Ebola virus (EBOV) outbreak in West Africa has shown the need for an effective vaccine against this virus. As a result, clinical trials to test several vaccine candidates have been expedited  were obtained from the Washington State University College of Veterinary Medicine . One group of 5 pigs and 1 group of 6 pigs were inoculated with rVSVwt and rVSV∆G/EBOVGP, respectively, as controls; 2 animals were mock inoculated with culture medium (Dulbecco modified Eagle medium). Animals were inoculated with 10At regular intervals after inoculation, clinical examinations were performed to determine the health status of the animals and to collect nasal, throat, and rectal swab samples for virologic analysis; blood was collected to determine the humoral immune response. Three animals inoculated with rVSVwt and rVSV∆G/EBOVGP were euthanized at 3 days postinoculation (dpi) as per protocol; the remaining animals were euthanized at 21 dpi.0.83 50% tissue culture infectious dose [TCID50]/mL)  .Three animals in each group were euthanized at 3 dpi. Tissue samples from lip, tongue, snout, footpad, coronary band, interdigital skin, tonsil, oronasopharynx, inguinal lymph node, axillary lymph node, cervical lymph node, mesenteric lymph node, bronchial lymph node, nasal mucosa, trachea, bronchus, lungs, heart, liver, spleen, kidney, adrenal gland, pancreas, jejunum, colon, urinary bladder, cervical spinal cord, frontal brain, cerebellum, and brain stem of these animals were collected for virus titration and histologic analysis.2.3 and 102.6 TCID50/g), but virus was not detected in any of the other tissues collected at 3 dpi. In 1 of the pigs inoculated with rVSV∆G/EBOVGP, virus was detected in the snout (virus titer 102.5 TCID50/g) and in another pig in an inguinal lymph node (virus titer 105.1 TCID50/g). This high titer in the inguinal lymph node might have been the result of a change in cell tropism caused by use of EBOVGP rather than VSV glycoprotein (VSV-G). Histologic analysis did not identify lesions consistent with VSV infection in the rVSVwt– or rVSV∆G/EBOVGP–inoculated animals. At the end of the experiment at 21 dpi, the same tissues were collected for virologic and histologic analysis; virus could no longer be detected in any of the tissues derived from the inoculated animals, and no histologic lesions were present.In 2 of 3 animals inoculated with rVSVwt, virus was detected in the snout (virus titers 10Serum samples collected at 21 dpi were analyzed for IgG against VSV-G or EBOVGP by using an IgG ELISA and secreted forms of these glycoproteins as antigens (Our data indicate that, although rVSV∆G/EBOVGP can replicate in pigs, this vaccine virus does not result in overt clinical disease, and virus shedding is minimal. Because a high dose of the vaccine was directly injected intradermally into the snouts of the animals in this study and yet did not cause disease, it is unlikely that vaccination of humans with the rVSV∆G/EBOVGP vector would result in a productive infection with clinical disease in domestic pigs during a spillover event. Moreover, even if this spillover were to occur, the near absence of virus shedding in the rVSV∆G/EBOVGP–infected animals suggests that spillover would not result in maintenance of rVSV∆G/EBOVGP within a pig herd. This study provides data to support the safety of the live-attenuated VSV∆G/EBOVGP vaccine in a relevant livestock species. Should exposure/infection of pigs occur during a vaccination trial in humans, it is highly unlikely that signs of disease would develop in pigs or that the vaccine virus would be disseminated by interspecies or intraspecies transmission.


Flavivirus is a genus of RNA viruses that includes multiple long known human, animal, and zoonotic pathogens such as Dengue virus, yellow fever virus, West Nile virus, or Japanese encephalitis virus, as well as other less known viruses that represent potential threats for human and animal health such as Usutu or Zika viruses. Flavivirus replication is based on endoplasmic reticulum-derived structures. Membrane remodeling and accumulation of viral factors induce endoplasmic reticulum stress that results in activation of a cellular signaling response termed unfolded protein response (UPR), which can be modulated by the viruses for their own benefit. Concomitant with the activation of the UPR, an upregulation of the autophagic pathway in cells infected with different flaviviruses has also been described. This review addresses the current knowledge of the relationship between endoplasmic reticulum stress, UPR, and autophagy in flavivirus-infected cells and the growing evidences for an involvement of these cellular pathways in the replication and pathogenesis of these viruses.The  In recent years, the knowledge of virus–host interactions has unveiled multiple connections between virus life cycle steps and a variety of cellular organelles and signaling pathways. Deciphering the complexity of these interactions will provide key information for the control of viral pathogens. This mini-review addresses the current knowledge and challenges for a deep understanding of the interactions of flaviviruses with the endoplasmic reticulum (ER) and two related cellular pathways: the unfolded protein response (UPR) and autophagy.Flavivirus genus comprises more than 50 distinct species of enveloped positive single strand RNA viruses. This genus is classified into the Flaviviridae family together with Pestivirus, Hepacivirus, and Pegivirus (http://www.ictvonline.org/virusTaxonomy.asp). Flaviviruses include multiple well known human, animal, and zoonotic pathogens such as yellow fever virus (YFV), dengue virus (DENV), tick-borne encephalitis virus (TBEV), Japanese encephalitis virus (JEV), St. Louis encephalitis virus (SLEV), or West Nile virus (WNV), as well as other emerging or re-emerging pathogens such as Usutu virus (USUV) or Zika virus, which are now being considered as potential threats for human and animal health (arthropod-borne viruses), most flaviviruses are transmitted by mosquitoes or ticks and maintained in nature through complex infectious cycles that involve different hosts. The variety of symptoms caused by flaviviruses includes jaundice (YFV), febrile illnesses , hemorrhagic fevers (DENV), or encephalitis . As a result of different factors, including globalization of travel and trade, climate warming, or changes in land use and vector behavior, different flaviviruses are currently becoming global health threats with DENV being amongst the most prominent human pathogens. In fact, DENV is responsible for up to 50 million infections each year, including 22,000 deaths, mostly among children (http://www.who.int/csr/disease/dengue/impact/en/). There are several vaccines against flaviviruses currently licensed for use in humans  or animals . As other positive-strand RNA viruses, flaviviruses rearrange host cell membranes to build organelle-like structures in order to establish the appropriate environment for viral replication . These characteristic structures usually referred to as vesicle packets (VPs) or double membrane vesicles (DMVs) have been associated with viral genome replication , mitogen-activated extracellular signal-regulated protein kinase (ERK1/2), and protein-serine kinase B alpha  leads to the activation of the three UPR signaling pathways , the stimulation of proapoptotic effectors with high-level signaling through PERK and eIF2α phosphorylation resulting in CHOP activation and induction of apoptotic effectors caspase 12 and poly ADP ribose polymerase (PARP) has been described . By facilitating the removal of damaged organelles and cytoplasmic protein aggregates, autophagy has been proven to be essential for the maintenance of cellular homeostasis  or lysosomes (resulting in autolysosomes). Different roles for multiple cellular proteins involved in autophagy have been reported to date. One of the most widely used indicators of upregulation of autophagy is the cytoplasmic aggregation of microtubule-associated protein 1 light chain 3 (LC3), that is modified by its conjugation to phosphatidylethanolamine and targeted to autophagic membranes labeling autophagic vacuoles . An upreEV, USUV , the hepEV, USUV , and theEV, USUV . InteresEV, USUV . This maEV, USUV . The rolEV, USUV , apoptosEV, USUV , innate EV, USUV , or adeqEV, USUV . Even moEV, USUV , suggest viruses . However viruses . Neverth viruses , thus suFlaviviridae , although relationships between activation of the UPR, membrane remodeling, and autophagy induction have not been addressed in most cases or remain controversial. For instance, the induction of autophagy and UPR has been shown for HCV, but the mechanistic link between the induction of these two cellular processes remains unclear. Some authors have addressed the relationship between both mechanisms, reporting that down-regulation of a variety of UPR modulators inhibits HCV-induced LC3-phosphatidylethanolamine conjugation, a hallmark of autophagic vesicle accumulation (An induction or manipulation of the UPR has also been described for a wide variety of members of the mulation , or suggmulation . Conversmulation . Supportmulation . In the mulation , thus sumulation . This prin vivo using animal models, of course, having in mind the complex biology of these pathogens that include infection of different host cells within their infectious cycle, which could complicate the interpretation of these studies. In fact, autophagy and UPR currently represent druggable pathways under evaluation for the treatment of multiple human disorders (in vivo against flavivirus infection (The detailed knowledge of the interaction of flaviviruses with the ER is attractive to refine current antiviral strategies against these viruses and to explore novel therapeutic approaches. The view of the ER as a mere replication platform in flavivirus infection should be changed and more emphasis should be given to its profound remodeling of its architecture and composition induced by the infection, including the activation/rearrangement of cellular pathways related to this organelle which are connected with other relevant pathways as apoptosis and innate immunity. In this way, deciphering the puzzle between autophagy, the UPR, and their potential connections could help to build a more complete picture of flavivirus interactions with host cells. An important challenge will be the analysis of autophagy and UPR during flavivirus infection isorders , and recnfection .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Motivation: An accurate genome assembly from short read sequencing data is critical for downstream analysis, for example allowing investigation of variants within a sequenced population. However, assembling sequencing data from virus samples, especially RNA viruses, into a genome sequence is challenging due to the combination of viral population diversity and extremely uneven read depth caused by amplification bias in the inevitable reverse transcription and polymerase chain reaction amplification process of current methods.Results: We developed a new de novo assembler called IVA (Iterative Virus Assembler) designed specifically for read pairs sequenced at highly variable depth from RNA virus samples. We tested IVA on datasets from 140 sequenced samples from human immunodeficiency virus-1 or influenza-virus-infected people and demonstrated that IVA outperforms all other virus de novo assemblers.Availability and implementation: The software runs under Linux, has the GPLv3 licence and is freely available from http://sanger-pathogens.github.io/ivaContact:iva@sanger.ac.ukSupplementary information:Supplementary data are available at Bioinformatics online. The main challenge of assembling sequence data from an RNA virus sample into a consensus sequence lies in the extremely variable read depth from current sequencing approaches combined with the extensive viral population diversity. An example is shown in http://en.wikipedia.org/wiki/Sequence_assembly), VICUNA , is a completely Supplementary Figure S1 and full details are in the Supplementary Material. Before assembling, adapter sequences are removed from the reads using Trimmomatic . During mapping, IVA also uses SAMtools  nucmer to compare contigs with a reference genome, (ii) GAGE  samples and 98 Influenza A and B virus samples. See the g Kraken . For theSupplementary Tables S1 and S2), significantly more than the other assemblers. These low numbers are generally due to contigs of incorrect length . IVA had the smallest variation in these results, especially for the Influenza virus samples . The proportion of each reference genome assembled into contigs was 89.8-98.3% for HIV-1. The corresponding values for Influenza virus ranged from 89.8 (PRICE) to 98.8% (IVA). The mean per cent of HIV-1 annotation features transferred by RATT from IVA assemblies was 99.0% on both HIV-1 and Influenza virus samples. This was more than the other assemblers, except VICUNA with alternative settings that achieved 99.2% mean annotation transfer, at the expense of a duplication rate more than double that of IVA (Supplementary Table S1). There were few assembly errors—Trinity produced none, and IVA and VICUNA made one error each. The typical run time was under 10 h and none of the assemblers had excessive memory requirements . IVA was slightly slower on the HIV-1 samples but was comparable to PRICE and faster than VICUNA on the Influenza virus data.The ideal assembler output is defined as one contig for HIV-1, or exactly one contig for each Influenza virus genome segment, with the expected length compared to the closest reference and no duplication. IVA generated ideal assemblies for 57% of the HIV samples and 21% of the Influenza virus samples (t length a or duplConsidering the number of ideal assemblies produced by the available tools, it can be seen that assembling RNA virus genomes is challenging. However, IVA was consistently better at producing single sequences representing the consensus sequence of each virus population, especially on the Influenza virus data. In contrast, the other tools tended to either produce multiple copies of parts of each genome or miss entire regions from their output. In summary, we developed IVA specifically to assemble short read sequencing data from RNA virus samples and have shown that it produces significantly higher quality assemblies than existing approaches.


Nosocomial outbreaks, especially with (multi-)resistant microorganisms, are a major problem for health care institutions. They can cause morbidity and mortality for patients and controlling these costs substantial amounts of funds and resources. However, how much is unclear. This study sets out to provide a comparable overview of the costs of multiple outbreaks in a single academic hospital in the Netherlands.Based on interviews with the involved staff, multiple databases and stored records from the Infection Prevention Division all actions undertaken, extra staff employment, use of resources, bed-occupancy rates, and other miscellaneous cost drivers during different outbreaks were scored and quantified into Euros. This led to total costs per outbreak and an estimated average cost per positive patient per outbreak day.Seven outbreaks that occurred between 2012 and 2014 in the hospital were evaluated. Total costs for the hospital ranged between €10,778 and €356,754. Costs per positive patient per outbreak day, ranged between €10 and €1,369 , with a mean of €546 and a median of €519. Majority of the costs (50%) were made because of closed beds.This analysis is the first to give a comparable overview of various outbreaks, caused by different microorganisms, in the same hospital and all analyzed with the same method. It shows a large variation within the average costs due to different factors . All outbreaks however cost considerable amounts of efforts and money , including missed revenue and control measures. Nosocomial outbreaks are a major problem for health care institutions due to increased morbidity and mortality for the affected patients. The containment and control of these outbreaks costs substantial amounts of funds and resources, especially when left unnoticed or untreated . Rising www.wip.nl). They provide over 130 different guidelines on infection prevention, stating all the actions health care institutions should perform and facilitate. The Search-and-Destroy policy for MRSA is one of the success stories of the Dutch infection prevention approach [Within the Netherlands there is proactive national infection prevention and control collaboration through the Working group Infection Prevention . An outbreak was defined as at least two patients who were tested positive as indicator for colonization or infection for the same microorganism , with some epidemiological link . The duration of an outbreak was counted in days and began on the day that the Infection Prevention Unit started measures to control the outbreak until the day that they decided the risk of transmission was over and additional control measures were not deemed necessary anymore. When an outbreak was suspected, the Infection Prevention Unit provided assistance to the affected ward and advised on extra surveillance cultures, extra cleaning, isolation of patients, and possible closure of the ward if necessary. Actions are based on the local and national infection prevention guidelines.www.cbs.nl).Counted costs were divided into: microbiological diagnostics/surveillance costs; missed revenue due to closed beds (based upon the difference in bed occupancy rates compared to the two months before); additional cleaning costs; additional personnel ; costs made for contact or strict isolation of patients and other costs . In order to take into account all possible costs that occurred, a wide range of different sources were used to ensure that no expenses were overlooked. Admission data came from the general hospital database. Numbers of cultures came from the Medical Microbiology Database and the prices for the diagnostics were internal cost prices (depending on type of diagnostic between €25 and €200 per sample). Extra personnel and possible other costs were calculated based on interviews with the, at that time, involved staff , together with detailed case reports from each outbreak made by the Infection Prevention Unit. Bed day cost prices  and personnel costs (ranging between €19.23 and €75.54 per hour) were based upon Dutch reference prices [http://www.ccmo.nl). Calculations were done with Microsoft Excel  and SPSS . When statistics were performed, a significance level of p < 0.05 was applied.This analysis followed the CHEERS guideline and included all applicable items as recommended when reporting economic evaluations . The stuPantoea transmission was treated as an outbreak because it occurred on a neonatal ICU.In total, seven different outbreaks could be financially evaluated. One outbreak caused by a virus and seven bacterial outbreaks . The mean of the total average costs per positive patient per day comes to €546 and the median to €519.Within the average costs per positive patient per outbreak day, the majority of the costs (50%) were made because there was the closure of (multiple) ward(s) leading to missed revenue; 17% of the costs were for extra microbiological diagnostics; 11% due to contact or strict isolation of patients; 10% for extra personnel; 7% for other costs; and 5% due to extra cleaning on the affected wards  were tested. Due to the short incubation time, chances of undetected transmission are small. Furthermore, in this case closure of the ward was deemed unnecessary and there was no excess morbidity or mortality for the patients due to their Noro infection. In almost all cases of the evaluated outbreaks, patients were colonized but not infected. During the Klebsiella outbreak in the rehabilitation center there was excess morbidity in the form of one sepsis episode and subsequently all costs made during this admission were taken into account and categorized under ‘Other’. For the S. marcescens outbreak there was excess length of stay observed for two patients by the treating medical specialist. Also in this case, these costs were taken into account and categorized under ‘Other’. Ergo, it seems that outbreak and microorganism specific characteristics cause large variation in the total costs. This variation together with the relatively small number of outbreaks also meant that correlation analyses on the data were impracticable. We therefore chose to give a descriptive overview. Based upon this cost-analysis we hypothesize that on average a viral outbreak is most likely to be less expensive than a bacterial one. This is mainly due to easier and quicker detection which reduces the duration and the microbiological costs. For bacterial outbreaks we observed large variation in the costs. One of the biggest cost drivers is the closure of wards and the subsequent drop in revenue, especially when this closure has consequences for scheduled procedures. Cleaning costs are dependent on the type of ward, with less additional costs on an ICU ward, because cleaning procedures are already strict and higher costs in the rehabilitation center due to extra rooms (e.g. physiotherapy exercise rooms) that had to be cleaned more strict than normal.Seven outbreaks were evaluated and costs varied substantially. On average, the additional costs due to an outbreak were €546 per positive patient per outbreak day. These average costs ranged between €10 and €1,369. The most expensive outbreak per patient per outbreak day was an ESBL producing P. aeruginosa, MRSA, Acinetobacter and VRE [Although there are numerous studies on the  burden of disease of resistant organisms and nosocomial infections , 10, the and VRE –19. AlthStrength of this study is the fact that multiple outbreaks caused by different microorganisms in a single hospital were evaluated. This gives a comparable overview of how cost categories differ between different outbreak situations. Limitations are that it is a retrospective analysis and it might be that data are missing because of this. However, by using multiple sources for data, this aspect is minimized. Still, preferable all data are collected prospectively. Depending on national health care systems it will differ who will be bear the costs. This makes comparison between different studies from different countries more difficult. By presenting the costs within different categories we tried to make interpretation of the data more flexible and adaptable to other settings. Finally, with only one viral outbreak, this category is under represented, there were however no more suitable viral outbreaks to include during the evaluated time-period.Concluding, we present a cost-analysis of multiple outbreaks in an academic center. Although costs differ between different outbreaks, due the microorganism or type of ward, the average costs per patient per day seem substantial. Especially with ever rising antimicrobial resistance levels, such outbreaks as described here are becoming continuously more difficult to treat and costs are expected to rise even further in the future. Average costs of an outbreak per patient per day as presented here can be used to further clarify costs and benefits within hospitals related to infection prevention. Ultimately this should help decision makers to justify the necessary investments in infection prevention and control measures. Our study may thus contribute to more transparency in health care budgets and improve the decision making processes concerning where to invest. Notably, extra argumentation can be found in these findings for investments into infection prevention and control measures to avert outbreaks or to contain them swiftly.