Office-based transnasal flexible endoscopic surgery under topical anesthesia has recently been developed as an alternative for transoral laryngopharyngeal surgery under general anesthesia. The aim of this study was to evaluate differences in health care costs between the two surgical settings.PubMed, EMBASE and Cochrane Library were searched for studies reporting on costs of laryngopharyngeal procedures that could either be performed in the office or operating room . Quality assessment of the included references was performed.Of 2953 identified studies, 13 were included. Quality assessment revealed that methodology differed significantly among the included studies. All studies reported lower costs for procedures performed in the office compared to those performed in the operating room. The variation within reported hospital and physician charges was substantial.Office-based laryngopharyngeal procedures under topical anesthesia result in lower costs compared to similar procedures performed under general anesthesia.The online version of this article (10.1007/s00405-019-05617-z) contains supplementary material, which is available to authorized users. Most patients with lesions in the pharynx or larynx are traditionally treated in the operating room (OR) under general anesthesia. Since flexible endoscopes with distal chip technology have been introduced, the imaging quality improved significantly compared to fiber optic images. As a result, it has become easier to assess lesions in the laryngopharyngeal region. The recent incorporation of a working channel into digital transnasal video-endoscopes facilitates diagnostic and treatment-related procedures. The transnasal approach used during flexible endoscopy results in more surgical control and better patient tolerability than traditional transoral techniques . By elimOver the past 2 decades, several reviews have been conducted on the development of procedures in the office , 3-7. OfReimbursement is largely dependent on price negotiations between insurer and provider or hospital, as well as the combined clinical and economic value of the treatment. Therefore, it is important to get a better understanding of cost savings from OR-based laryngopharyngeal procedures to OB procedures, to advice decision-makers regarding adequate reimbursement levels. Even though the actual costs associated with the implementation and practice of OB surgery are context-specific, there is growing evidence to support that OB procedures can be performed at lower costs. This study aims to clarify these cost differences by presenting a systematic review of the relevant literature comparing the costs of laryngopharyngeal surgery performed in the office and the OR.A literature search was conducted in three electronic databases  in December 2017, and updated in June 2019. A research protocol was developed prior to the start of the review, which was published on the PROSPERO website on May 1st, 2018 . KeywordFirst, duplicate references were removed from the search results Fig. . Two autIn any analysis of comparative costs of alternative treatments, study outcomes must be an accurate reflection of the economic comparison. Some studies in the past decades, particularly those performed in the United States, have focused on patient bills (charges) as a proxy for costs, but it is important to make a distinction between the economic cost, accounting cost, and charges to the patient, and actual resource consumption . ChargesMany studies use methods to adjust market prices or hospital charges, most often cost-to-charge ratios. But the way the ratios are being calculated differs widely, including converting hospital charges to cost by use of hospital-level cost-to-charge ratios  or deparIn case important information was missing in the included articles , the corresponding author was consulted. In some cases, additional data were sent and used in our cost calculations. Therefore, the amounts displayed in this review can differ from the amounts displayed by the original articles. Additionally, some authors indicated that costs were an equivalent of charges in their health care system. Hence, charges displayed in those studies were documented as costs, as those amounts still reflected the (approximate) actual value of the provided service.To eliminate currency variability and thereby improve the comparability of study outcomes, costs were converted into Euros  using the CCEMG-EPPI-Centre Cost Converter. This converter accounted for currency conversion as well as inflation factors . When a Two authors (AS and DW) independently assessed the methodological quality of the included references. The BMJ checklist for economical evaluations  and the The search strategy yielded a total of 2953 unique articles Fig. . After eTable The quality assessment Table  illustraAll included studies reported that hospital costs for OB procedures were reduced compared to similar procedures performed in the OR. The relative reduction in hospital costs varied from 22%  to 46%  All included studies revealed that hospital charges for OB procedures were reduced compared to similar procedures performed in the OR. The relative reduction in hospital charges varied from 62%  to 98%  to 89% . By compExcept for charges reported in one study , otorhinThis study identified and evaluated differences in health care costs between the OB and OR-performed laryngopharyngeal surgery. By reviewing the relevant literature between 1995 and 2019, it was demonstrated that significant cost savings can be realized by shifting laryngopharyngeal procedures from the OR to the outpatient clinic.In this review, clear distinctions were made between costs and charges, because costs do not correspond with charges. It is important to consider that health care providers in certain countries tend to inflate charges for their services, as third party payers cover only small proportions of the billed amounts . This phThis review revealed that procedures under topical anesthesia resulted in hospital cost reductions of up to 95% per procedure compared to similar procedures under GA. However, the total costs per procedure varied widely between studies. Several reasons for variations in cost outcomes can be outlined. First, the investigated procedures require different equipment or materials. For example, flexible endoscopic laser surgery requires an expensive laser fiber, while flexible biopsy forceps for FEB are significantly less costly. In addition, the cost items that were involved in the analyses differed between the included studies. Differences existed in whether pre-operative general examinations, purchase and use of expensive equipment, or overhead costs were incorporated. Costs for learning these new surgical techniques  were not considered in any of the studies. Third, two of the included studies described that awake surgery was performed in the endoscopy suite  or in thPossible predictors of costs per procedure were analyzed by one study. Chandran et al. described that time spent in the operating theatre was a significant predictor for total costs, but when time was excluded from the multiple linear regression model, type of anesthesia emerged as an independent predictor of total costs . These fThe opacity of financial data in current health care systems around the world is challenging in this type of research and leads to limitations. The costs of delivering health care are obscured in layers of jargon and complex accounting . Hence, There is an opportunity for cost-effectiveness studies comparing laryngopharyngeal surgery in the office to transoral laryngopharyngeal surgery under general anesthesia, as costs from societal or patient perspective are not yet available. Taking into account the reduced sedative effect when topical anesthesia is used instead of general anesthesia, allowing patients to return to work even the same day as the procedure, costs from patients’ perspectives can be significantly diminished. Based on our findings, indications exist that performing laryngopharyngeal surgery in the office under topical anesthesia instead of the OR under GA results in significant costs savings for the hospital and third party payer. Other fields of medicine have also shown cost reductions in shifting surgical interventions to the office -34. To gIn conclusion, the current review demonstrates that shifting laryngopharyngeal procedures from the OR to the office results in apparent economic benefits. From a physician’s point of view, deciding whether to start performing office-based procedures will not only depend on costs, but also on training of the surgeon and other involved staff and availability of materials, instruments and facilities. The results of this review contribute to a better understanding of cost savings from shifting OR-based laryngopharyngeal procedures to the office and can reinforce physicians who are planning to make this shift. Furthermore, the results can be used to guide decision-makers regarding adequate reimbursement levels.Supplementary file1 (DOCX 14 kb)Below is the link to the electronic supplementary material.


The Flavivirus genus is composed by viral serocomplexes with relevant global epidemiological impact. Many areas of the world present both, vector fauna and geographical conditions compatible with co-circulation, importing, emergence, and epidemics of flaviviruses of different serocomplexes. In this study, we aimed to identify both, immunological determinants and patterns of immune response possibly involved in flavivirus serocomplex cross-protection. We searched B and T cells epitopes which were thoroughly shown to be involved in flavivirus immunological control. Such epitopes were analyzed regarding their conservation, population coverage, and location along flavivirus polyprotein. We found that epitopes capable of eliciting flavivirus cross-protective immunity to a wide range of human populations are concentrated in proteins E, NS3, and NS5. Such identification of both, immunological determinants and patterns of immune response involved in flavivirus cross-protective immunity should be considered in future vaccine development. Moreover, cross-reactive epitopes presented in this work may be involved in dynamics of diseases caused by flaviviruses worldwide. The Flavivirus genus of the Flaviviridae family is composed by over than 70 viral species with relevant global epidemiological impact. Flaviviruses viral particles have icosahedral capsid, are enveloped and present a single stranded genomic RNA of positive sense . The entFlavivirus genus is involved in important chapters of both, World and Brazilian epidemiology histories. Yellow fever virus (YFV), Dengue virus (DENV) and Zika virus (ZIKV) were sequentially imported to Brazil and South America. Cases of yellow fever, caused by YFV, were first reported in Brazil in 1685. Thousands of yellow fever cases were noticed until the beginning of the twentieth century, when the Aedes aegypti mosquitoe was identified as the main vector of the disease. Vector eradication together with vaccination contributed to elimination of yellow fever from Brazilian urban areas (Guillain-Barré syndrome in the Americas (West Nile virus (WNV), Japanese encephalitis virus (JEV), Saint Louis encephalitis virus (SLEV) and Rocio virus (ROCV)  . Such pain vitro , is observed in some cases of DENV infection. Immunoglobulins produced in a first infection by a specific serotype cross-react with viral particles present in a second infection caused by a different serotype. Viral particles are targeted to Fc-γ receptors bearing cells by non-neutralizing antibodies, which facilitates penetration and replication of DENV , 11. Incin vitro , 13–15. in vitro , 13–16 a, Zika virus (ZIKV), Yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), Saint Louis encephalitis virus (SLEV), Rocio virus (ROCV), Cacipocore virus (CPCV), Ilhéus virus (ILHV), and Iguape virus (IGUV). Sequences in FASTA format were retrieved from the National Center for Biotechnology Information (NCBI) protein database from September-2017 to September-2018 (http://www.ncbi.nlm.nih.gov/protein/). We aimed to recover sequences from all listed flavivirus species, isolated in all continents, with a limit of three sequences per country of each viral species. We noticed that for some species there were few deposits. In these cases, all available sequences were selected. Additional criteria for selecting sequences were: (i) complete annotation of structural and non-structural proteins within deposited polyprotein sequence and (ii) absence of undefined amino acid into sequence. The database consisted of a total of 325 polyprotein amino acid sequences . Criteria for selecting research articles were: (i) evaluation of epitope binding to HLA; (ii) determination of cytokine production pattern for both, CD4+ and CD8+ T cells; (iii) use of at least two different methods for determining cytokine production pattern and (iv), clear association with control of flavivirus infection. This last criterion involves validation of epitopes associated with survival under challenge experiments using humanized animals (transgenic mice expressing HLA), control of viral load under challenge experiments using humanized animals, infection of humans without disease and identification of important epitopes in immune responses elicited by well-known protective vaccines. Words used in the search were: CD4+ T cell, CD8+ T cell, epitopes, protection, immunity, Dengue virus, Zika virus, Yellow Fever virus, West Nile virus, Japanese encephalitis virus, Saint Louis encephalitis virus, Rocio virus, Cacipocore virus, Ilheus virus, and Iguape virus.We carried out a literature search for reports of experimental characterization of protective epitopes. CD4http://www.iedb.org)  . The seahttp://tools.iedb.org/conservancy) was used to determine the conservancy of the selected epitopes among the flavivirus sequences in the database previously constructed, as previously described , as previously described , were used for fine localization. Pymol (http://www.pymol.org/) was used to highlight selected epitopes along the proteins 3D models, as previously described . Thus, we used NS5 and NS3 3D models to study the locations of the epitopes. From fifteen epitopes, six were located in Helicase domain of the NS3 protein (NS3H), and eight were located in the RNA-dependent RNA polymerases domain of the NS5 protein (NS5p) . These r+ T limphocytes.In this study, we aimed to identify both, immunological determinants and patterns of immune response possibly involved in flavivirus serocomplex cross-protection. Our premise was based on epidemiology of flaviviruses: most of them have both, vectoring and geographic conditions to co-circulate all together in the Americas, Africa, Asia and Oceania –49. HoweThe glycoprotein E is involved in binding of the viral particle to host cell receptors. In addition, such protein is also involved in membrane fusion. The E glycoprotein is arranged in three domains . Domains I (DI) is central, surrounded by DII and DIII . The hydThe role of DIII in the flaviviral cycle is still unclear, although frequently related to a receptor binding function , 58, 59.+ T cells (With regard to conservation of T cell-specific immunological determinants among flavivirus serocomplexes, the YVF lost the highest amount of 100% conserved T cell epitopes see . This is T cells . However+ T cells. There is not a high number of highly conserved class-II HLA restricted epitopes among all serocomplexes. Nevertheless, we identified two peptides capable of binding class-II HLA, involved in anti-YFV protective immune response which are highly conserved among all flavivirus serocomplexes. Thus, it seems achieving multivalent protection among serocomplexes depends on overcoming immunodominance. Highly conserved class-II HLA restricted epitopes do not seem to be immunodominant in order to promote recall of serocomplex cross-reactive memory CD4+ T cells in a multivalent way.Cross-protective immunity into a given serocomplex seems to be more easily achieved , 64, 65.+ T cells. Some of the listed studies report generation os memory cells, which could also be recalled in order to favor cross-protective immunity.With regard to class-I HLA restricted epitopes, we found fifteen highly conserved peptides which were thoroughly shown to be involved in flavivirus infection control. The peptides APTRVVAAEM, DPASIAARGY, DISEMGANF, KAKGSRAIW, and RFLEFEALGF are located in the NS3 or NS5 proteins. They bind to a subset of multifunctional CD8+ T limphocytes capable of producing IFN-γ and TNF-α against DENV infection , 27. In + and T CD8+ lymphocytes. It is important to stress that approved vaccines represent three serocomplexes: DENV, JEV and YFV. However, most of those epitopes which are important for recalling cross-reactive T cells, are concentrated in non-structural proteins. The only anti-DENV vaccine currently approved for use in humans is a chimera between DENV and YFV. Its structural antigens are representative of DENV proteins, while non-structural antigens are representative of YFV proteins. In addition, the JEV vaccine is based on inactivated viruses. Enhancing the recall of cross-protective memory T cells would ideally happen with the use of live attenuated viruses or other vaccine approach capable of providing epitopes of non-structural proteins.Conserved epitopes could be enriched in artificial or natural immunizations. For example, sequential infection or administration of different anti-flavivirus vaccines would recall cross-reactive memory T CD4All datasets generated for this study are included in the manuscript/JA conceived the study and wrote the paper. LS, LG, and WL analyzed data and wrote the paper.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.


Shewanella oneidensis is one of the best-understood model organisms for extracellular electron transfer. Endogenously produced and exported flavin molecules seem to play an important role in this process and mediate the connection between respiratory enzymes on the cell surface and the insoluble substrate by acting as electron shuttle and cytochrome-bound cofactor. Consequently, the addition of riboflavin to a bioelectrochemical system (BES) containing S. oneidensis cells as biocatalyst leads to a strong current increase. Still, an external application of riboflavin to increase current production in continuously operating BESs does not seem to be applicable due to the constant washout of the soluble flavin compound. In this study, we developed a recyclable electron shuttle to overcome the limitation of mediator addition to BES. Riboflavin was coupled to magnetic beads that can easily be recycled from the medium. The effect on current production and cell distribution in a BES as well as the recovery rate and the stability of the beads was investigated. The addition of synthesized beads leads to a more than twofold higher current production, which was likely caused by increased biofilm production. Moreover, 90% of the flavin-coupled beads could be recovered from the BESs using a magnetic separator. In microbial fuel cells, microorganisms catalyze the direct conversion of chemical energy into an electrical current. This ability can be used in a variety of biotechnological processes like wastewater treatment or the production of platform chemicals. The biocatalytic microorganisms of this process are characterized by the ability to transfer respiratory electrons to the cell surface and subsequently onto solid state electron acceptors like for instance ferric iron minerals or, as a synthetic variant, an anode surface .Shewanella oneidensis (MR-1) and Geobacter sulfurreducens (PCA) are the two main model microbes for exploring molecular mechanisms of extracellular electron transfer (EET)  . The pater (EET) . Both orer (EET) . C-type  complex . Several complex , reducti complex  or media complex .2) and flavin mononucleotide (FMN)  . Howeverde (FMN) . Presumade (FMN) .In experiments based on the reduction of insoluble electron acceptors like metals, redox shuttles are often added in micromolar concentrations. However, natural secretion of flavins by MR-1 is much lower and seems to be dependent on various factors like growth phase, medium, electron acceptors, the BES setup and other experimental conditions. Already in The aim of this research was to study the application of a recyclable electron shuttle and its effect on current production in a BES in order to overcome disadvantages and limitations of flavin addition such as losses by washing out or high process costs. Therefore, riboflavin was coupled to magnetic beads and the effects on current production in MR-1 as well as their stability and recyclability were investigated.S. oneidensis barcode is a strain that was developed by gfp strain constitutively expresses a gfp gene and was used for fluorescence microcopy 2SO4, and 8.77 g NaCl supplemented with 1 mM MgSO4, 0.1 mM CaCl2, 1 g casamino acids, and trace elements . The pH was adjusted to 7.4 and all bottles were sealed with rubber stoppers. Prior to autoclaving, oxygen was removed from the medium via the repeated application of a vacuum and following sparging of the headspace with N2. Before inoculation into bioelectrochemical reactors, the cells were harvested by centrifugation  and washed twice with medium containing neither electron donor nor electron acceptor. Thereafter, the cells were resuspended to an OD600 of 0.07 in medium containing 70 mM lactate but no electron acceptor. The gfp-strain was cultivated with the addition of kanamycin (50 mg L-1) and L-rhamnose (2.2 g L-1) for plasmid maintenance and continuous eGFP expression, respectively.All strains used in this study are listed in icrocopy . All str2, SGL Group, Germany) and platinum mesh  were used as anode and cathode material, respectively, and an Ag/AgCl electrode  was used as reference electrode. Before use, the anode was first rinsed with isopropanol, followed by deionized water. The complete bioelectrochemical setup was sterilized by autoclaving. During chronoamperometric experiments, the working electrode was poised to 0 mV vs. SHE using a potentiostat  and current was monitored for 46 h. BES were constantly flushed with N2 gas in order to provide anoxic conditions and constant mixing of the liquid phase.All bioelectrochemical experiments were conducted in triplicates using a single chamber MFC with a working volume of 270 ml . Graphitglycine-coupled beads. The beads had an average particle diameter of 25 μm and were stored in a 25% (w/v) suspension in 100% isopropanol. Prior to usage, the beads were rinsed three times with sterile medium without electron donor and acceptor as stated above.The magnetic beads  used in this work are spherical magnetic agarose beads, consisting of 6% cross-linked agarose. The beads were synthesized by the company as a solid phase-based material via EDC/NHS-mediated coupling for covalent modification. A riboflavin-modified epoxide function was coupled to the magnetic agarose with three different alkane carbon chains with a respective chain length of 4 (C4), 11 (C11), and 40 (C40) carbon atoms. An alternative, glycine-modified epoxide function was used as a control and is from here on referred to as The number of beads was quantified using a Neubauer chamber . The recovering process from the anolyte of the BES was performed using a magnetic separator and the recovery efficiency was calculated based on the difference between particle number added before the bioelectrochemical experiments and after the recovering process at the end of the experiments.Riboflavin was decoupled from the beads chemically by adding 100 mM NaOH for 30 min. The beads were then separated from the riboflavin solution using a magnetic separator. Riboflavin was quantified using a plate reader  at 440 nm . The ribGenomic DNA isolation as well qPCR based cell quantification were conducted according to 600 of 1. 1 ml of cells was mixed with 100 μl of beads (coupled to either riboflavin or glycine) and cells were incubated overnight under anoxic conditions. Subsequently, the beads were washed twice in anoxic medium lacking electron donor and acceptor using a magnetic separator. The binding of cells was assessed qualitatively under the microscope.Wild type and ΔOMC cells were pre-grown, harvested and centrifuged for 5 min at 6000 g. The optical density was adjusted in minimal medium with 50 mM lactate as electron donor and 10 mM fumarate as electron acceptor to ODT-test and F-test. All data sets were normally distributed. The level of significance was set to 5%.Significance of the data was determined via Welch Two Sample 2SO4 [electrolyte counter electrode (CE)] and lactate containing medium  were filled into the respective half-cell chambers. The flow cells were tempered under an incubation hood to 30°C and the solutions were pumped through the flow cell by a peristaltic pump. The WE solution was flushed with N2 with a flow rate of 5 mL min-1 in order to ensure anoxic conditions in the WE chamber. The polarization and EIS routines were subsequently started.Experiments in the impedance flow cell were performed according to A potential (E) of -199 mV vs. Ag/AgCl (0 mV vs. SHE) was applied to the indium tin oxide (ITO) WE and EIS was measured with a potentiostat .600 of 0.1. Beads were added simultaneously to the cells to the WE chamber. Similar to the bacteria the beads were circulated between the flow cell and WE chamber reservoir. EIS measurements were performed after 24 h of incubation.The frequency (f) range varied between 100 kHz and 50 mHz with an amplitude (U) of 10 mV rms and 10 points per decade. Polarization of the WE was started prior to the addition of bacteria to an ODMicroscopic images of cells growing on the ITO WE were taken 24 h after inoculation. Samples were viewed on a Leica DM 5500B and images were taken with a Leica DFC 360 FX camera and the corresponding Leica LAS AF Lite software. Pumping was paused during image acquisition to prevent image disturbance by the peristaltic pump.2. The addition of 37 nM riboflavin enhanced current density 6.5-fold and resulted in 20.1 (±2.05) μA/cm2  on current production in a BES inoculated with MR-1 was tested first. Current was monitored over time and the average current density was compared after 46 h. Without the addition of riboflavin, the cells produced an average current density of 3.1 (±0.28) μA/cm) μA/cm2 . A 50-fo2]. With increasing number of C-atoms between riboflavin and the magnetic beads, the current production dropped. Medium spacers exhibited a current of 4.6 (±0.22) μA/cm2 and long spacers of 3.1 ± 0.31 μA/cm2. Glycine-coupled beads served as control and produced a slightly higher amount of current compared to the experiment without addition of riboflavin or magnetic beads (4.6 ± 0.47 μA/cm2). With short spacers, addition of riboflavin-coupled beads to the anodic chamber resulted in a 2.4-fold increase of the average current density compared to experiment without magnetic beads, whereas the glycine-coupled control only led to a minor increase (1.4-fold).The next step was to transfer the effect of soluble riboflavin on the current density to a recyclable shuttle that can be extracted easily from the medium. Therefore, riboflavin was coupled to magnetic beads via organic spacers of different chain lengths: short (C4), medium (C11), and long (C40). The number of C-atoms in the spacer had a strong impact on the current density in the BES . Short sWe also analyzed the recovery rate and riboflavin concentration of all beads after the experiment and could measure for all setups very good recovery efficiencies of up to 90% . The staFrom the previous results, we were interested, if riboflavin coupled to the beads binds to OMCs as cofactor. We compared MR-1 cells with a mutant, which is lacking all OMCs (ΔOMC) and added beads coupled to riboflavin and glycine, respectively. Cells were transferred from an oxic to an anoxic environment and beads were harvested after 15 h of incubation. In OMCs, the binding pocket for riboflavin is oxygen sensitive and closed under anoxic conditions , so thatp = 0.021) and 6.3-fold more cells could be detected compared to BES without the addition of riboflavin. Of note, this correlates well with the measured current increase. Adding riboflavin-coupled beads to the BES also influences the distribution of the cells. As seen with riboflavin as soluble compound, significantly more cells attached to the anode (p = 0.042), whereas with glycine-coupled beads cell adhesion was positively affected but with rather high standard deviations which rendered this effect to be not significant (p = 0.277). Again, the increase in anode attached cells correlates roughly with the stimulating effect of the beads on current production. Therefore, it can be assumed that there is a correlation of cells on the anode and current production and that this correlation is driven by riboflavin (either coupled to beads or as free compound), whereas the beads per se affect biofilm growth at least not as robustly.We compared total cell numbers in the different experiments and quantified the number of planktonic vs. anode-attached cells to investigate the effect of riboflavin and magnetic beads on the cell distribution in the anode chamber in further detail. Therefore, qPCR analysis was conducted to quantify the number of planktonic and biofilm-attached cells in the BES containing soluble riboflavin, riboflavin coupled to C4-beads and glycine-coupled control beads . In BES CT) from 164.7 kΩ (±19.1) in the control BES (without beads addition) to 74.8 kΩ (±11.6) and a relatively similar decrease in the RCT was also observed after the addition of riboflavin-coupled beads [78.4 kΩ (±21.4)]. Fluorescence imaging was performed in order to illustrate the influence of the beads on the biofilm formation of MR-1 in the flow cell. Microscopic images taken in the flow cell  or glycine  did not show any significant difference in the charge transfer resistance. Since the beads are made out of iron oxide and covered by a presumably permeable agarose coating, it is possible that they are conductive themselves and can reduce the resistance of the anode by acting as an extension of the electrode, thereby extending the electroactive surface area and lowering the overall impedance. In this case, the covalently attached compound  does not seem to affect the electrochemical properties of the bioanode. However, the microscopic images indicate an increased biofilm production caused by the addition of beads to the system and regarding biofilm density, riboflavin-coupled beads clearly outperform glycine-coupled ones. This enhanced cell density is reflected in the qPCR data, where riboflavin-coupled beads also exhibits a boosting effect on the number of anode-attached cells.It is possible that the effect of the beads on a BES is dual: an improved conductivity leads to decreased resistance and by this more effective EET and current production, and (as stated above) riboflavin itself not only influences the electron transfer to the anode but also plays a role in biofilm production and/or formation.TA, L-AP, DR, ME, JC, and MS carried out the experiments. KS-R wrote the manuscript with support from DR and JG. RU and DH supervised the electrochemistry part. JG supervised the project.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.


Orthohantavirus, family Hantaviridae, we have employed cryo-electron microscopy (cryo-EM) for three New World hantaviruses: Andes (ANDV), Sin Nombre (SNV), and Black Creek Canal (BCCV). Building upon our prior cryo-EM and cryo-tomography study of the Old World hantavirus, Hantaan virus (HTNV), we have expanded our studies to examine the entire virion population present in cell culture supernatant. Hence, in contrast to the prior cryo-EM/ET studies in which we used a polyethylene precipitation, a sucrose gradient, and a sucrose cushion, we used two sucrose cushions. We inactivated the material after the first cushion. We tested the method using HTNV which has a known cryo-EM structure and observed equivalent results. Therefore, we used this method to assess the particle distribution of the New World hantaviruses by cryo-EM. Cryo-EM images showed a diverse range of sizes and morphologies for the New World viruses that we classified as round, tubular, and irregular. Strikingly, BCCV virions were mostly tubular. These first cryo-EM images of the New World Orthohantavirus confirm prior EM observations that noted tubular projections of SNV at the plasma membrane during virion morphogenesis but were not confirmed. These findings underscore the need for further investigation of virion morphogenesis of the Orthohantavirus. To further understanding of the structure and morphology of the  Orthohantavirus, Family Hantaviridae, Order Bunyavirales, are widely distributed in nature in rodents, bats, shrews, and moles [Hantaviruses, genus nd moles . Speciesnd moles . There and moles . Bunyavirales, such as Rift valley fever virus [Hantaviruses are negative-sense, single-stranded RNA viruses with three gene segments, small (S), medium (M), and large (L), which encode the nucleocapsid, Gn and Gc glycoproteins and the RNA-dependent RNA polymerase, respectively . Cryo-eler virus  or Uukuner virus , in whicer virus , deriveder virus .Orthohantavirus. Currently, there are no cryo-EM studies reporting the ultrastructure of New World hantaviruses. This may be due to the scarcity of cryo-EM microscopes in biosafety level-3 (BSL-3) facilities, or conversely, the lack of established methods to inactivate virions for removal from BSL-3 for imaging at BSL-1/2. To enable such studies, we have developed and verified a method for inactivation and preservation of virion structure. The method eliminates two steps we have used to purify hantaviral particles for cryo-EM in our prior publication , overnig2 for 7 or 10 days, concentrated and inactivated at BSL-3 at the Regional Biocontainment Laboratory at the University of Louisville or University of Tennessee Health Science Center prior to removal. Briefly, four T175 flasks containing Vero E6 cells were infected at a MOI of 0.1. Viruses were inactivated and purified as described in [g. The supernatant was transferred to a new 50 mL conical and centrifuged again for 15 min at 3000× g. The clarified supernatant was layered on top of a 30% sucrose cushion and centrifuged for 8 h at 10,000× g in a fixed angle rotor at 4 °C. Pelleted material was resuspended in a total volume of 1.54 mL of HNE pH 7.2  and allowed to resuspend on ice for 15 min to overnight. Virus-containing supernatant was inactivated by bringing the suspension to a final v/v of 1% glutaraldehyde and placed on ice, in closed tubes wrapped in aluminum foil. The material was shipped overnight on ice blocks to Purdue University. On arrival, the samples were pelleted through a second 30% sucrose cushion. The pellet was resuspended in 100 µL TNE buffer and processed for cryo-EM. Virus strains used in these studies were HTNV 76-118, ANDV isolate Chile-9717869, BCCV isolate 807040, SNV strain Muerto Canyon (MCV), and SNV strain Convict Creek 107 (CC107). Hantaviruses were amplified in Vero E6 cells in Minimal Essential medium (EMEM) supplemented with 10% fetal bovine serum (FBS) and 5 mM penicillin/streptomycin, incubated at 37 °C, 5% CO5 were observed.To verify inactivation, supernatant from HTNV-infected Vero E6 cells that were grown on 6 well plates for 7 days was harvested. Samples were treated with a final volume of 1% glutaraldehyde, or vehicle, and incubated for 30 min. Inactivated or active virus culture was diluted serially, 10-fold in the DMEM cell culture media followed by inoculation and cultivation in Vero E6 cells in a 96-well plate. A week later, inoculated cells were fixed with acetone-methanol (1:1) mixture and air-dried. Cells were probed for virus by ELISA using polyclonal immune serum to the glycoprotein of HTNV. As compared to the virus-infected Vero E6 cells, no virus was detected. In the untreated samples, titers of greater than 10For cryo-EM analysis, 3.5 µL of the virus samples were loaded onto glow discharged Holey Carbon grids . The grids were blotted for 6 s prior to plunging into liquid ethane using Gatan Cryoplunge™ 3 . The grids were stored under liquid nitrogen until further use. Blotting times and ice thickness for all viruses were optimized to suit the different sizes of all observed particles. Grids were loaded on to a CM200 (FEG Phillips) electron microscope or a FEI Titan Krios to screen for the presence of virus particles. Ice thickness was estimated for every grid as per the method by Yan et al. . Project−/Å2/s. Tomograms were collected in super resolution mode at a pixel size of 0.65 Å, at a defocus of −3 to −4 um, using a dose symmetric collection scheme. A python script for motion correction was run in parallel, which also sorted the images from negative to positive tilts. Tilt series were binned 4× before processing. Reconstruction was carried out in IMOD using both SIRT and weighted back projection method. The SIRT reconstruction was further processed by Non-Linear Anisotropic Diffusion filtering to enhance contrast.Virus samples were mixed with 10 nm gold bead solution at a 5:1 ratio. About 3.5 µL of the virus sample was loaded on glow discharged, 200 mesh Holey Carbon grid, blotted for 6 s and plunge frozen. For tomography, tilt series were collected on an FEI Titan Krios using Leginon at a nominal magnification of 11,000×, on a K2 Summit camera, from −60 °C to +60 °C in 2° increments, with a total dose varying between 60–100 e5 to 106 PFU of per ml for HTNV-infected Vero E6 cells supernatant. Hence, we estimated a final yield of 2 × 107 to 108 virions from 200 mL media harvested. We typically observed an average of 1–2 virions observed per hole on the cryo-EM grids. Our prior studies reported the first cryo-EM and cryo-ET of HTNV , in whicWe analyzed the morphologies of at least 100 HTNV particles by cryo-EM. We estimated and optimized ice thickness for each virus keeping in mind the diverse range of sizes Yan et al. . About 6We prepared inactivated ANDV particles using our purification strategy described above and in  and examIn previous cryo-EM studies, Old World hantaviruses HTNV and TULV were reported to have locally ordered arrays of glycoprotein spikes ,6,11. ToHantaviruses have internal ribonucleoprotein complexes (RNPs), formed by copies of the nucleoprotein polymerized on the three segments of the RNA genome. The RNPs have been previously observed for HTNV as 10 nm thick parallel rods . TomograTwo strains of Sin Nombre virus, CC107 and MCV, were prepared using the same purification and inactivation strategy. Viruses belonging to both strains also adopted a range of round, tubular and irregular morphologies, but CC107 was more often round A and MCVOrthohantavirus morphology reported to date. BCCV had the longest average length among all hantaviruses analyzed and is the only hantavirus in this study observed to be mostly tubular.The most extreme distribution of morphology was observed for BCCV in which 72% of the particles were tubular . ElongatNew World hantaviruses have been largely unexplored structurally because of the containment required and the lack of microscopes within BSL-3 facilities. In the present study, we prepared inactivated BSL-3 viruses for cryo-EM imaging using a FEI Titan Krios microscope, outside of containment. The strategy uses two gentle, low-speed centrifugation steps of the virion preparation to concentrate the virus and remove glutaraldehyde . GlutaraThis study is the first report of a cryo-EM analysis of New World hantaviruses. Our cryo-EM images and analyses suggest that there are differences in the proportions of round, elongated and irregular shapes among hantaviruses and even between strains within a single species (CC107 versus MCV). HTNV and ANDV had twice as many round particles as the North American New World hantaviruses, BCCV and SNV strains. BCCV particles had the greatest abundance of tubular morphologies of all viruses examined and the greatest lengths of any hantavirus (Two observations that we made from the tomograms of ANDV were the large size of some particles and the empty patches on the virion surface. Regarding the large size of some particles, an obvious question is whether these particles contain additional copies of the genomes. A second observation of our examination of the ANDV tomograms showed that several particles were observed to have large areas of the membrane completely devoid of spikes. This has been reported previously for HTNV  and TULVThe first glimpse into New World hantavirus morphology raises many questions and new hypotheses to be tested in future studies. Since very little is known regarding how hantaviruses assemble and bud at the membrane, future research that seeks to discover how genome segments are incorporated and what determines the morphology of these pleotropic hantaviruses in the absence of a matrix protein will be critical. The 2D images showed beautiful arrays of intact spikes on all the viruses; however, these were mostly asymmetric particles not suitable for single particle averaging. Future efforts will focus on collection of larger tomography datasets for subvolume averaging of glycoprotein spikes and the internal RNPs. The RNP segments of ANDV were often observed to appear as parallel straight rods in multiple particles, with some bent and curved RNPs as well, with portions that may be in contact with membrane or cytoplasmic domains of viral glycoproteins. Cross sectional and parallel views of multiple RNPs were observed in each virion and may reflect more than one copy of the three segments in the genome. How the RNP is structured within the virion to promote assembly is an open question that may be revealed by subvolume averaging. Lastly, future research that examines the process of New World viruses budding from cells will provide important new information on the biology of these important human pathogens.


Epigenetics is defined as the science that studies the modifications of gene expression that are not owed to mutations or changes in the genetic sequence. Recently, strong evidences are pinpointing toward a solid interplay between such epigenetic alterations and the outcome of human cytomegalovirus (HCMV) infection. Guided by the previous possibly promising experimental trials of human immunodeficiency virus (HIV) epigenetic reprogramming, the latter is paving the road toward two major approaches to control viral gene expression or latency. Reactivating HCMV from the latent phase (“shock and kill” paradigm) or alternatively repressing the virus lytic and reactivation phases (“block and lock” paradigm) by epigenetic-targeted therapy represent encouraging options to overcome latency and viral shedding or otherwise replication and infectivity, which could lead eventually to control the infection and its complications. Not limited to HIV and HCMV, this concept is similarly studied in the context of hepatitis B and C virus, herpes simplex virus, and Epstein-Barr virus. Therefore, epigenetic manipulations stand as a pioneering research area in modern biology and could constitute a curative methodology by potentially consenting the development of broad-spectrum antivirals to control viral infections in vivo. Since its emergence for the first time in 1940, the epigenetic field is witnessing a continuous surge over the last decades , recent Since the introduction of combination antiretroviral therapy (cART), survival and quality of life among HIV-infected patients significantly improved , with a This strategy is grounded on the concept that the latent HIV provirus could be switch on from latency (shock) into an active form prone to eradication (kill) through the humoral immune response, CD8+ T cells-mediated lysis, virus-induced apoptosis, or activation-induced cell death . SeveralIn contrast to LRA, chemical agents could “block” the ongoing viremia during cART, by “locking” the HIV promoter in a super latency state resistant to reactivation stimuli. In fact, the “block and lock” strategy is emerging as a new approach to functionally cure HIV. Didehydro-cortistatin A (dCA), a specific and potent Tat inhibitor  has beenHerpesviridae family [Human cytomegalovirus (HCMV) is a ubiquitous pathogen also denoted as human herpesvirus 5 (HHV5). It is a member of betaherpesvirinae, a subfamily of the e family . HCMV ine family . Howevere family . Converse family , 53, whee family . Added te family . HCMV exe family . The lyte family . During e family  via the e family . This ise family  and latee family . Recent e family . In facte family . Such moe family .Although the few available antiviral drugs have granted chief advances in HCMV disease treatment and prophylaxis, their clinical applicability and utility confront several barriers . First, In contrast to the virus’ wide tropism during productive infection , severalHCMV lytic cycle and reactivation from latency are under the control of several epigenetic mechanisms. For example, histone demethylases (HDMs), due to their ability to remove the repressive marks, will promote productive infection. HDMs constitute a large family of more than 20 demethylases that are divided into two functional enzymatic families: the Lys-specific demethylase (LSD), also known as KDM1A and the Jumonji C (JMJC) protein families . Two HDMBesides infection with HIV and HCMV, any viral infection might be potentially treated by new therapeutics targeting the epigenetic mechanisms Table . We presHepatitis B virus (HBV) is a highly transmissible double-stranded DNA virus responsible of acute and chronic hepatitis B (CHB) in humans. Worldwide, about 240 million people are chronically infected, which could ultimately lead to liver fibrosis, cirrhosis, and the development of hepatocellular carcinoma (HCC) . DespiteInfection with hepatitis C virus (HCV), a small enveloped RNA virus , is consHerpesviridae family, HSV-1 conserves its ability to remain latent in the sensory neurons of the trigeminal ganglion as a nucleosome-associated episome in the nucleus of the host cell [Herpes simplex virus 1 (HSV-1), a double-stranded DNA virus , is a ubost cell , which most cell . A transost cell . In the ost cell . Interesost cell . Also, Host cell , ATAC [1ost cell , and CLOost cell . In addiost cell . Not onlost cell . Given tost cell . Howeverost cell . This isost cell . Same waost cell , 168. Suost cell . This waost cell . Attractost cell , 171. Thost cell , 173, reHerpesviridae family, subfamily Gammaherpesvirinae [Epstein-Barr virus (EBV), or human herpesvirus 4 (HH4), is a DNA virus  that belsvirinae . Like otsvirinae . It has svirinae . During svirinae , 179. Thsvirinae . Not limsvirinae . In factsvirinae . Other tsvirinae , nasophasvirinae , Hodgkinsvirinae , and othsvirinae , 187. Insvirinae . BIM is svirinae . During svirinae . Besidessvirinae . For exasvirinae . In addisvirinae . A comprsvirinae .Powered by the supportive results in cell cultures and mouse models, epigenetic drug candidates are recently being clinically evaluated as potential antiviral drugs. In this section, some completed and ongoing clinical trials are cited, as an attempt to present preliminary data about the use of epigenetic drugs to manipulate viral infections or viral infection-related malignancies Table . As mostOver the past years, epigenetic studies have revealed novel principles and profoundly broadened our knowledge about the interplay between viruses, cellular transcription factors, histones, and nonhistones modifying enzymes. As most of those modifications are reversible, manipulating this complex machinery could have a critical role in determining an active lytic or latent viral infection and subsequent viral reactivation from latency. This diverts the end goal to permanently silence the virus in latent reservoirs so that the possibility of reactivation is diminished, or to eradicate it through purging the viral reservoirs after reactivating it. Advantageously, and in contrast to the conventional antivirals, it is hypothesized that the emergence of resistant strains is minimized, as those chromatin modulation components target the host, rather than viral-encoded factors. Furthermore, epigenetic therapy could exemplify the “two birds, one stone” concept in the scenario of viral co-infection, e.g., with HCMV-HSV, HIV-HCV, and HIV-HBV. However, this tremendous array for new targets is a double-edged sword. In fact, the available epigenetics therapies lack specificity, which raises questions about their cytotoxic side effects due to unintended global epigenetic modifications and complicates the achievement of a therapeutic index within the acceptable toxicity levels. Intriguingly, designing and testing target-specific inhibitors (specific HDAC inhibitors rather than pan-inhibitors for example) could improve therapeutic outcomes through dropping the off-target undesired effects. This could be partly achieved through studying structure-activity relationship (SAR) to select a potent and selective compound for further mechanistic studies. In addition, advancement in epigenetic analysis tools such as epigenome microarray and combining chromatin immune-precipitation (Chip) to next-generation sequencing (NGS) could provide a useful tool to decipher the multiprotein complexes involved in the epigenetic control of viral infections. In addition, addressing the role of the less studied post-translational modifications such as phosphorylation or sumoylation can shed light on new aspects of the dynamic host-viral interplay. Altogether, new therapeutic approaches are actively needed to fight viral infections and drugs targeting epigenetic players could lead to major therapeutic breakthroughs in the future.


Aedes aegypti (L.) is the primary vector of chikungunya, dengue, yellow fever, and Zika viruses. The leucine-rich repeats (LRR)-containing domain is evolutionarily conserved in many proteins associated with innate immunity in invertebrates and vertebrates, as well as plants. We focused on the AaeLRIM1 and AaeAPL1 gene expressions in response to Zika virus (ZIKV) and chikungunya virus (CHIKV) infection using a time course study, as well as the developmental expressions in the eggs, larvae, pupae, and adults. RNA-seq analysis data provided 60 leucine-rich repeat related transcriptions in Ae. aegypti in response to Zika virus . RNA-seq analysis data showed that AaeLRIM1 (AAEL012086-RA) and AaeAPL1 (AAEL009520-RA) were significantly upregulated 2.5 and 3-fold during infection by ZIKV 7-days post infection (dpi) of an Ae. aegypti Key West strain compared to an Orlando strain. The qPCR data showed that LRR-containing proteins related genes, AaeLRIM1 and AaeAPL1, and five paralogues were expressed 100-fold lower than other nuclear genes, such as defensin, during all developmental stages examined. Together, these data provide insights into the transcription profiles of LRR proteins of Ae. aegypti during its development and in response to infection with emergent arboviruses. The leucine-rich repeats (LRR)-containing domain is noted to be evolutionarily conserved among many proteins correlated with innate immunity in an array of organisms, including invertebrates, vertebrates, and plants . Innate Anopheles mosquitoes, LRIM1, APL1C, and LRR-containing proteins activate complement-like defense responses against malaria by forming a disulphide-bridge that interacts with thioester-containing protein 1 (TEP1), a complement C3-like protein  ,48. Relage ΔΔCT] ,50,51.p-value (p-adj), log2 fold-change (log2FC), or both for downstream analysis. The RNA-seq data have been deposited to NCBI (https://www.ncbi.nlm.nih.gov/gds/?term=GSE118858). RNA-seq analysis data provided 60 leucine-rich repeat related transcriptions in the Ae. aegypti in response to Zika virus -containing domain as well as the relationship between gene expressions. So, we used a multivariate analysis of variance approach (MANOVA), which enabled us to simultaneously measure various treatment effects  on the expression of multiple genes. Further, the MANOVA is associated with standardized canonical coefficients, which describe the relative contribution of each of the variables (genes) to a treatment effect and their direction to one another (positive or negative) .AaeLRIM1 and AaeAPL1. The relative contribution and relationship of AaeLRIM1 and AaeAPL1 to developmental treatment effects were assessed using standardized canonical coefficients (SCC) . When significant effects were detected, we used univariate comparisons among treatment least-squares means for the developmental stages (Tukey-Kramer method). Separate analyses were performed for the immature stages and adult stages. Similarly, we used MANOVA to measure the expression of AaeLRIM1 or AaeAPL1 following ingestion of CHIKV and ZIKV infected blood. We tested for all main treatment factors and interactions.Multivariate analysis of variance (MANOVA) and ANOVA were used to measure developmentally regulated gene expression of


Oryctolagus cuniculus), which is caused by a Myxoma virus (MYXV) infection—a leporipoxvirus that is found naturally in some Sylvilagus rabbit species in South America and California. The introduction of MYXV into feral European rabbit populations of Australia and Europe, in the early 1950s, demonstrated the best-documented field example of host–virus coevolution, following a cross-species transmission. Recently, a new cross-species jump of MYXV has been suggested in both Great Britain and Spain, where European brown hares (Lepus europaeus) and Iberian hares (Lepus granatensis) were found dead with lesions consistent with those observed in myxomatosis. To investigate the possibility of a new cross-species transmission event by MYXV, tissue samples collected from a wild Iberian hare found dead in Spain (Toledo region) were analyzed and deep sequenced. Our results reported a new MYXV isolate (MYXV Toledo) in the tissues of this species. The genome of this new virus was found to encode three disruptive genes  and a novel ~2.8 kb recombinant region, which resulted from an insertion of four novel poxviral genes towards the 3’ end of the negative strand of its genome. From the open reading frames inserted into the MYXV Toledo virus, a new orthologue of a poxvirus host range gene family member was identified, which was related to the MYXV gene M064R. Overall, we confirmed the identity of a new MYXV isolate in Iberian hares, which, we hypothesized, was able to more effectively counteract the host defenses in hares and start an infectious process in this new host. Myxomatosis is a lethal disease in wild European and domestic rabbits ( Leporipoxvirus genus, is the etiological agent of myxomatosis, which is a highly lethal viral disease in wild and domestic European rabbits (Oryctolagus cuniculus) ) was used as a biological agent ,6,7. It Sylvilagus species  developed tumors, following mosquito transfers, but these failed to be mosquito-infective lesions [Sylvilagus species  when infected with the Brazilian Lau strain also developed prominent tumors, however, in this case the South American strain produced mosquito-infective lesions [Lepus californicus) inoculated with Californian MYXV did not form tumors [Lepus europaeus), cases of myxomatosis have been reported sporadically and in small numbers. In the past, the confirmation of the disease arose from injecting rabbits with tissues from dead hares and replicating its typical clinical symptoms [The susceptibility of other leporids species to MYXV has been tested in controlled experiments, while evidence of myxomatosis in wild leporid populations have been seldom reported. Using a California MYXV strain, four different North American  lesions . Three o lesions . On the m tumors . In wildsymptoms . Most resymptoms .Lepus granatensis) populations, mainly in the Andalusia and Castilla-La Mancha regions. The Spanish Ministry of Agriculture, Fisheries, and Food, and the Institute for Game and Wildlife Research identified what appeared to be a cross-species transmission into a new leporid species. Iberian hares were found in a moribund state, with signs of blindness, weakness, and disorientation, and were consequently analyzed in different laboratories. Here, using culturing and deep sequencing, we genetically characterized, for the first time, a recombinant MYXV isolated from an Iberian hare carcass exhibiting classical symptoms of myxomatosis collected in the Toledo province of Spain, during the 2018 outbreak (referred to as MYXV Toledo).Recently, in late summer–fall of 2018, the first cases of myxomatosis were reported in the wild Spanish Iberian hare (L. granatensis) female, was found dead on 21st of August 2018 in La Villa de Don Fabrique municipality in the Toledo province of Spain. The hare manifested lesions compatible with myxomatosis in European rabbits  were maintained in Dulbecco’s modified Eagle medium , supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 100 U/mL of penicillin/streptomycin. The Cells were maintained at 37 °C in a humidified 5% COL. granatensis) specimen were manually homogenized. A small volume (5–10 μL) of the processed tissues was used to inoculate confluent RK13 cells monolayers in a 6-well plate, and was allowed to incubate at 37 °C. Two days after infection, distinctive MYXV foci were visualized using a Leica DMI6000 B inverted microscope (−5) was used for inoculating a new 150 mm dish. After 2 days of infection, a last round of cell harvest, freeze–thaw cycles, and sonication was done, before proceeding to virus amplification into twenty 150 mm dishes. Purification of the virus through a 36% sucrose cushion was performed, as described before [Samples from the lesions of the eyelid of an Iberian hare , coupled with a local MYXV ORF database generated from the MYXV genomes. ORFs that did not have any similarity to MYXV ORFs were analyzed using BLASTn and BLASTx sequence queries [All MYXV and poxvirus RefSeqs were downloaded from GenBank on 9 April, 2019. Global alignments of the MYXV with the genome determined in this study were carried out using MAFFT . ORFs in queries . All paiNucleotide sequence and protein sequence aligns were undertaken using MAFFT . The nucOryctolagus cuniculis), a successful cross-species transmission can occur. Indeed, when MYXV first entered the European rabbit host, it was immediately pathogenic and caused close to 100% mortality. After the use of MYXV in the 1950s to control feral rabbit populations in Australia and Europe, rapid co-evolutionary changes occurred in both the rabbit host and the virus, due to an increased resistance in rabbit populations and the appearance of less virulent virus strains [Lepus europaeus) [The natural host for MYXV is the South American tapeti (South American strains) ,4. As ex strains ,24. In 2ropaeus) . HoweverIn this study, a new MYXV virus (MYXV Toledo) was isolated and sequenced from an Iberian hare found in the Toledo province, which presented the classical lesions of myxomatosis, including a bilateral blepharitis, conjunctivitis and a swollen vulvar and anal region A,B. The M0005.1L to M011L), and 23.1 kb extending from the right TIR (M143R to M000.5L), which mostly contain genes involved in the MYXV virulence and host subversion, while the central 124.5 kb region (M012L to M142R) includes a mixture of virulence genes and essential viral genes conserved across all poxviruses [M009L, M152R, and M036L. Phylogenetically, MYXV-Tol clusters with the majority of the MYXV genomes ’. The de novo assembled genome was 164,579 bps . This gexviruses . Around  genomes . FurtherM009L encoded a putative E3 ubiquitin (Ub) ligase of 509 aa with a N-terminal BTB-BACK domain, followed by 4 Kelch motifs [M009L of MYXV-Tol was disrupted by an insertion of four nucleotides , causing a frameshift mutation. This indel resulted in a smaller truncated M009L predicted protein of 148 aa. Several reports have shown that this same gene was also disrupted in multiple Australian MYXV strains [M036L gene , thereby creating a premature stop codon in the frame, within this gene. M036L is an orthologue of the O1 protein that is found in the orthopoxvirus vaccinia virus (VACV) [M152R contributes to the observed virulence of MYXV-Tol in Iberian hares.As previously reported for the MYXV isolates from feral rabbits in Australia and Great Britain, single or multiple indels that result in the disruption of ORFs are relatively common ,28,29. Ih motifs . Our gen strains , which ss (VACV) . Howevers (VACV) . In the s (VACV) . In the s (VACV) , both ofs (VACV) ,35,36. PM060R, M061R, M064R, and M065R genes from MYXV. We exploited the sequence similarity searches to predict the functions of these new MYXV-Tol proteins. According to the obtained results, the recombinant region encodes a known virion protein (rPox-virion protein), followed by a thymidine kinase , a C7L-like host range protein (rPox-host range protein), and a poly A polymerase subunit (rPox-poly(A) Pol subunit) (M060R to M065R) [M062R and the M063R genes that are present in all MYXV virus are not present in the new recombinant insertion region at the left end (encoded on the minus strand) of MYXV-Tol  . In the o M065R) ,37. The MYXV-Tol . A BLASTMYXV-Tol  sharing MYXV-Tol . These rOccurrences of recombination between leporipoxviruses have been described before. In fact, it has been established that the malignant rabbit fibroma virus (RFV) is a result of a recombination event between two other leporipoxviruses, the Shope rabbit fibroma virus (SRFV), and MYXV ,25. The M062R, M063R, and M064R [The rPox-thymidine kinase predicted protein sequence was found to share ~70% of its identity with its homologous protein from leporipoxviruses and 60–65% of identity with that of capripoxviruses and cervidpoxviruses . The rPond M064R . It has nd M064R . In our M009L, M036L, and M152R, the MYXV-Tol was found to have a full complement of genes present in other MYXV isolates and strains. So the question arises as to how MYXV-Tol , likely became pathogenic in Iberian hares. Examination was carried out on the four new poxvirus genes found in the recombinant “cassette” at the left end of the MYXV-Tol genome (encoded on the negative strand), which might induce factor(s) that mediate host range or immunosuppression in hares, allowing an increased infection and propagation of this new virus in hares. Regarding virulence in hares, it was likely that the acquisition of new genes involved in immunosuppression or host-range functions in specific cell types might have a preponderant role in this apparent species leaping of MYXV-Tol [M064R-like gene of MYXV-Tol shares relatively low similarity (~40%) to its orthologous proteins found in other leporipoxviruses, it is likely that this new protein has acquired new roles, perhaps reflected by alternative host targets, compared to those in the known MYXV strains. Host range proteins are defined as a group of virus-produced proteins that important for the capacity of the virus to infect cells or tissues of certain species [Other than the disruption of MYXV-Tol . From thMYXV-Tol ,40,41. H species . The cap species ,43. In fThe data presented in this paper report that MYXV-Tol is a result of a recombinant event between a MYXV virus and a still-unreported poxvirus that shares common ancestral sequences to that of capripoxviruses, cervidpoxviruses, suipoxviruses, yatapoxviruses, and three unclassified poxviruses . Recent reports genetically characterized a large number of European and Australian strain MYXV genomic sequences ,27 and h


Detection of waterborne enteric viruses is an essential tool in assessing the risk of waterborne transmission. Cell culture is considered a gold standard for detection of these viruses. However, it is important to recognize the uncertainty and limitations of enteric virus detection in cell culture. Cell culture cannot support replication of all virus types and strains, and numerous factors control the efficacy of specific virus detection assays, including chemical additives, cell culture passage number, and sequential passage of a sample in cell culture. These factors can result in a 2- to 100-fold underestimation of virus infectivity. Molecular methods reduce the time for detection of viruses and are useful for detection of those that do not produce cytopathogenic effects. The usefulness of polymerase chain reaction (PCR) to access virus infectivity has been demonstrated for only a limited number of enteric viruses and is limited by an understanding of the mechanism of virus inactivation. All of these issues are important to consider when assessing waterborne infectious viruses and expected goals on virus reductions needed for recycled water. The use of safety factors to account for this may be useful to ensure that the risks in drinking water and recycled water for potable reuse are minimized.  Forications . To achiBefore the development and application of molecular methods for the assessment of virus occurrence in water, animal cell culture was the only practical method available. Virus growth in cell culture indicates the potential for the virus to replicate in humans and cause disease. Enteroviruses were found to readily grow in cell culture from the earliest days of techniques for maintaining animal cells in the laboratory. Because they were so easily cultivated, most of our historic knowledge on enteric virus behavior in water and removal by water/wastewater treatment processes is based on enteroviruses. The safety associated with vaccine poliovirus strains allowed for bench- and pilot-scale testing of treatment processes under controlled conditions. However, enteroviruses have rarely been associated with waterborne disease, and today, we know they are only a small fraction of the viral community found in wastewater that is capable of causing illness in humans ,5. This Two types of cell culture have been used for the detection of viruses in water. Primary cell cultures originate directly from the organs of animals and humans. The most commonly used cell cultures in virology derive from primates, including humans and monkeys; rodents, such as hamsters, rats, and mice; and birds, most notably chickens ,16. MoreThe number of times a cell line has been passaged in the laboratory may also affect the ability of the virus to replicate . CertainOver time cell cultures become less efficient for replication of certain types of viruses . PreviouIn the case of the double-stranded DNA adenovirus, it has been found that replication of the virus after ultraviolet light exposure is dependent upon the ability of the cell line to repair damage to the DNA . UV lighVirus concentrates from different water matrices  can contain compounds toxic to cell cultures used for the detection of infectious viruses. The cytotoxicity may be associated with metals, complex mixtures of compounds associated with microalgae or plants as well as the reagents used for virus concentration and recovery ,33,34. NViral growth in cell culture is limited by the ability of virions to attach to specific receptors on the surface of animal cells and their ability to replicate within the cells. For the enteroviruses and many of the enteric viruses, this results in morphological changes induced in individual cells or groups of cells by virus infection that can be easily recognized by light microscopy and collectively called cytopathic or cytopathogenic effect (CPE). However, there are viruses whose replication may be limited to one or a few adjacent cells with no obvious cytopathogenic effects . AlternaPicornaviridae family [Cell culture is also less permissive for the growth of naturally occurring viruses than laboratory-grown viruses. Viruses grown in the laboratory have been selected for their rapid growth in cell culture and the number of virions observed under an electron microscope versus number observed by CPE or plaque-forming units (PFU) is usually 1:2 to 1:100 depending on the virus and method of assay ,41. In te family . Passagee family  as mutane family . The rate family . Using te family . The rese family . In a sie family .The degree of viral aggregation may also influence the underestimation of infective viruses in a sample. Aggregated viruses in cell culture are often only counted as one infectious virus as a result of only one countable plaque . HoweverAnother complicating factor is that one group of viruses may grow faster than another or interfere with the replication of another group of viruses , which a50) assay that quantifies the amount of virus required to produce CPE in, or kill 50% of virus-inoculated cultured cells in a multi-welled plate [2 of cell monolayer [The three most common methods for quantitative detection of virus replication in cell culture are the total culturable virus quantal assay (TCVQA) which requires computation of a most probable number (MPN), the plaque assay which quantifies the number of plaque-forming units in a virus sample as plaque-forming units (PFU), and the 50% tissue culture infective dose  and ethidium monoazide (EMA) in conjunction with qPCR , have been used to determine the potential infectivity of enteric viruses in water ,70,71. TAnother qPCR-based framework has been described and used to estimate virus infectivity . The fraDetermining the concentration of infectious enteric viruses in water reuse systems will likely be problematic into the near future. No one cell culture system can detect all of the infectious viruses that may be present in an environmental sample. However, advances in molecular biology which allow us to detect the genome of viruses known to infect humans and animals in environmental samples have revealed that the number of viruses may be 100 to 1000 greater than that detected by cell culture ,76. ThisAnother approach to consider is the use of a safety factor when estimating the true concentration of an infection virus in an environmental sample. This might be useful since no one method can detect all of the likely infectious virus present in environmental samples. When estimating risk from chemicals, it is common to take into consideration the uncertainty of using data on toxicity developed in animals to humans and the lack of data. Usually, safety factors of 10 to 100 are used to estimate acceptable levels of risk. While this may be useful for estimating levels of infectious virus in raw wastewaters, it becomes more problematic when dealing with treated wastewater and environmental waters. However, considering the factors outlined in this review affecting assays for enteric viruses that a safety factor of 10 would not be unreasonable.


The World Health Organization (WHO) currently recommends pre-screening for past infection prior to administration of the only licensed dengue vaccine, CYD-TDV. Using a threshold modelling analysis, we identify settings where this guidance prohibits positive net-benefits, and are thus unfavourable. Generally, however, our model shows test-then-vaccinate strategies can improve CYD-TDV economic viability: effective testing reduces unnecessary vaccination costs while increasing health benefits. With sufficiently low testing cost, those trends outweigh additional screening costs, expanding the range of settings with positive net-benefits. This work highlights two aspects for further analysis of test-then-vaccinate strategies. We found that starting routine testing at younger ages could increase benefits; if real tests are shown to sufficiently address safety concerns, the manufacturer, regulators and WHO should revisit guidance restricting use to 9-years-and-older recipients. We also found that repeat testing could improve return-on-investment (ROI), despite increasing intervention costs. Thus, more detailed analyses should address questions on repeat testing and testing periodicity, in addition to real test sensitivity and specificity. Our results follow from a mathematical model relating ROI to epidemiology, intervention strategy, and costs for testing, vaccination and dengue infections. We applied this model to a range of strategies, costs and epidemiological settings pertinent to CYD-TDV. However, general trends may not apply locally, so we provide our model and analyses as an R package available via CRAN, denvax. To apply to their setting, decision-makers need only local estimates of age-specific seroprevalence and costs for secondary infections. While mDengue infections elicit complex immune responses, particularly in regions where people typically experience multiple infections. There are four known dengue serotypes; infection by one confers apparently lifelong immunity to it, and temporary immunity to others. Disease threat varies substantially by infection number: primary infections are generally asymptomatic, and when symptomatic are rarely severe; secondary infections are more often symptomatic, and more often severe; and post-secondary infections are almost always asymptomatic. This pathogenicity pattern is caused by antibody-dependent enhancement ,4. To avA multi-model comparison study estimated that using CYD-TDV in high-burden settings would reduce both moderate and severe cases overall . These fContinuing observation in trial populations confirmed increased risk of severe outcomes in seronegative recipients . The StrAt US$78 per vaccinated individual, CYD-TDV is a marginal investment in many settings , so addiUsing this model, we found that test-then-vaccinate strategies generally provide health benefits compared with both non-vaccination and vaccination without testing, but not necessarily outweighing the additional costs. We evaluate the balance of benefits and costs using return-on-investment , which also enables comparison against other development options.We found that test-then-vaccinate strategies typically yield their highest ROI when testing starts younger than the currently recommended 9 years. We also found ROI improves with periodic re-testing of initially seronegative individuals.2.We derive ROIs using two limiting assumptions. We ignore transmission, and thus indirect vaccination benefits, and assume interventions work deterministically. Treating dengue incidence like an environmental risk is justified in endemic settings, given the predicted limited impact on transmission from CYD-TDV . AssuminThe electronic supplementary material provides derivations; the R package, denvax, implements the analyses .2.1.We represent dengue disease with three infection outcomes—primary like, secondary like and post-secondary like—each with cost reflecting their respective disease risk and severity. Note that these average costs include all outcomes, from asymptomatic infections to death. We assume secondary infections have the highest cost, and post-secondary infections have zero cost. We assume CYD-TDV acts like a silent natural infection, preventing one of these outcomes, and testing reveals an individual’s infection history. 2.2.F, S) weighted by relevant life history probabilities denoted N out of X lifetime infections by testing age A). Costs also include testing and, potentially, vaccination  during the intervention window. Strategies are defined by initial testing age and maximum allowed tests (L) at a rate of one per year. The model denotes the seroconversion probability between age A and A + 1 as binary, detecting only the presence of past infection, and ordinal, detecting the number of past infections. Because the ordinal test also eliminates vaccination costs for people with multiple past infections, it is always more effective (ignoring cost). Though ordinal tests are theoretically better, we expect binary tests are more realistic, and assumed them for our general results.We calculate individual lifetime costs, using average primary and secondary infection costs 〉 , and prequation ), for a ROI is the net benefits  per intervention cost,F,To identify circumstances where adding testing increases intervention benefits, we compared vaccination with and without testing, which produces a similar equation, but which depends on 2.3.pH, population fraction at high risk; and the annual avoidance probability for low (sL) and high (sH) risk groups. The probability of being seropositive at age A is thusWe model dengue exposure in annual increments: each year, an individual is potentially exposed. We divide the population into risk groups, low and high. Thus, the exposure model has three parameters: We use a maximum likelihood approach to fit this model to age-seroprevalence data, then use the parameters to simulate exposure histories. For each life-year, we uniformly select one of the four serotypes to cause exposures, then probabilistically expose individuals based on their risk. Exposure leads to infection, unless the individual was (i) previously infected by this serotype or (ii) infected in the previous year. We estimate lifetime outcome probabilities by aggregating many simulated individual histories.3.In these sections, we demonstrate the types of analyses available to the decision-makers using the models described above. Because each region will differ, in costs, epidemiology and decision criteria, we cannot present a singular recommendation. We illustrate detailed application of the framework with two examples including particular epidemiology and costs, but these are demonstrations, not conclusive results.3.1.We applied our model across a range of epidemiological settings and potential relative costs. 3.2.While the general results suggest trends, they also identify the need for region-specific analyses. To that end, we implemented the model in an R package, denvax, which can be used with local epidemiological and economic data.We demonstrate applying the approach with two practical examples for Malaysia and Peru, using epidemiological data from CYD14  and a loτ, ν} = {0.06, 0.91} for Malaysia and {0.02, 0.35} for Peru.Here we assume that vaccination (i.e. full dose regimen and service delivery) costs US$78 per fully vaccinated child in both locations . We assuSince we are assuming costs, we focus on ROI for varying initial and maximum testing ages in 4.We demonstrated an approach that identifies CYD-TDV vaccination scenarios worthy of further investigation, and implemented a framework for local authorities to assess their potential return-on-investment using region-specific epidemiology and costs. It is impossible to provide universal answers to how or whether CYD-TDV should be used, but it is possible to describe a limiting relationship among a small set of factors and ascertain general trends.We identified two such trends that should inform work on test-then-vaccinate strategies. First, repeat testing may improve cost-effectiveness over single testing; for some settings, this occurs even with relatively high testing costs. Second, we found routine test-then-vaccinate programmes often provide better ROI when targeting younger recipients, including those below the currently recommended 9 years old.Since our model uses several optimistic assumptions, it is only appropriate for ruling out CYD-TDV in unfavourable settings, not for conclusively supporting its use. Justifying CYD-TDV requires more model realism. Future work should refine vaccine performance, which we know to be imperfect even in seropositive recipients and which has uncertain durability. Likewise, even the gold-standard tests for detecting prior dengue infection provide imperfect classification, particularly in the presence of other circulating arboviruses. Should a point-of-care RDT be licensed, it is unlikely to have better performance than the current gold standard . IncludiHowever, incorporating insights from our model may help recover benefits. For example, we found annually repeating testing generally has superior performance, and under our assumptions biennial testing would be even better. This suggests repeat testing with lower frequency may be more cost-effective in more realistic models as well. Similarly, the substantial ROI improvement for younger intervention ages suggests that the current age guidelines for vaccination should be revisited if safety can be guaranteed by a highly specific test. Despite current licensing, CYD-TDV trials included younger participants and, after controlling for seropositivity, found safe and efficacious outcomes ; both thAccounting for these potential advantages properly will require more detailed data. Repeat testing may enable economies of scale (e.g. cheaper per unit tests), but will impose additional costs (e.g. record-keeping and service logistics). A model with repeat tests will also need to represent how individual test results are correlated. A lower routine intervention age will also affect test performance and vaccine efficacy. These concerns should be addressed in test development, so that data are available to inform future modelling work.Multiple dengue vaccine candidates are currently in trials ,24. Whil


We report a case of Barmah Forest virus infection in a child from Central Province, Papua New Guinea, who had no previous travel history. Genomic characterization of the virus showed divergent origin compared with viruses previously detected, supporting the hypothesis that the range of Barmah Forest virus extends beyond Australia. Togaviridae (genus Alphavirus) that comprises several human arboviruses, including Ross River virus (RRV), Sindbis virus, and chikungunya virus. BFV was first isolated in 1974 from Culex annulirostris mosquitoes collected in the Barmah Forest within the state of Victoria and simultaneously from mosquitoes collected in southwest Queensland, Australia  and Ae. camptorhynchus (Victoria), found in salt marshes and from the midge Culicoides marksi in Northern Territory (––Barmah Forest virus (BFV) is an arbovirus that is pathogenic to humans and is traditionally considered to be endemic only to Australia  who had no history of international travel came to an outpatient health clinic in a coastal village northwest of Port Moresby, Central Province, Papua New Guinea, because of an undifferentiated fever. Rash, muscle pain, and polyarthralgia were not noted at that time. Blood samples (containing EDTA anticoagulant) were collected as part of ongoing febrile illness surveillance and transferred to the Port Moresby laboratory of the Papua New Guinea Institute of Medical Research, where extraction of nucleic acids was performed.Orientia tsutsugamushi, Leptospira sp., and Rickettsia sp. All test results were negative, except for a BFV TaqMan RT-PCR, which showed a positive result.We screened eluates by using a real-time reverse transcription PCR (RT-PCR) for a range of pathogens known to cause febrile illnesses, including BFV, chikungunya virus, dengue virus, Japanese encephalitis virus, RRV, West Nile (Kunjin) virus, Zika virus, We isolated BFV by inoculating 100 μL of patient serum onto cultured Vero cells (strain PNG_BFV) and extracting and assessing the nucleic acid content of the harvested cell culture material by using a BFV-specific real-time RT-PCR. The result was positive, suggesting viral replication in culture and confirming the presence of BFV within the specimen of the patient.http://www.epibio.com), and subjected this library to whole-genome sequencing by using the MiSeq System (https://www.illumina.com). We obtained 32 million paired-end reads and mapped them to the only available full-length  BFV reference genome sequence   and an isolate from Victoria (M4208_16/17) with the newly generated PNG_BFV envelope (E2) protein gene sequence of 1,263 nt.To determine the evolutionary relationship of the PNG_BFV strain with those detected in Australia, we estimated their phylogenetic relationships by using the maximum-likelihood method and the time to most recent common ancestor of each node by using Bayesian methods. We aligned the complete envelope (E2) sequences of all currently available BFV strains (n = 7) in the Virus Pathogen Resource database . The wide CIs suggest that sampling was inadequate to provide a precise estimate of the time of divergence and evolutionary rate, which would be greatly improved with access to additional BFV whole-genome sequences and full-length E2 gene sequences, which are currently not available for public access.Subsequently, we estimated the molecular clock for the final dataset of 8 complete E2 protein sequences with a sampling range of 1974–2016, under a stick clock model; a constant coalescent population size and the Hasegawa, Kishino, and Yano substitution model by using the Bayesian Markov chain Monte Carlo method in BEAST version 1.8 , panel BWe report a case of infection with BFV in a child who had no travel history from the Central Province of Papua New Guinea. BFV has been traditionally believed to be endemic only to Australia. Whole-genome sequencing, followed by phylogenetic analysis, showed that this strain was highly divergent from known strains from Australia. These findings placed the Papua New Guinea virus strain within its own clade and supported the hypothesis that the range of BFV extends beyond Australia. Molecular clock analysis indicates that the virus strains from Papua New Guinea and Australia probably diverged during or before the early 1900s, raising questions on the origins and the overall genetic diversity of BFV. On the basis of currently available data, the probable origins of these viruses, either from Australia or neighboring northern countries, such as Papua New Guinea, are inconclusive.The timeline of divergence suggests that this divergence could have occurred by movement of humans, livestock, or mosquitoes from or to Australia during the early 1900s by trade routes or movement of troops during World War I. Increased mosquito surveillance and serosurveys of the population in Papua New Guinea is needed to determine the endemic nature of BFV, which is likely to extend beyond the single detection noted within the Central Province.


We report here the whole-genome sequence of 11 Zika virus (ZIKV) samples from six pediatric patients in Nicaragua. Serum samples were collected, and ZIKV was isolated in tissue culture. Both serum and virus isolates were sequenced. The consensus ZIKV genomes are greater than 99% identical to each other. Flaviviridae family and can be transmitted to humans via a mosquito vector or other routes, such as sexual transmission. The ZIKV genome is a 10.8-kb single-stranded positive-sense RNA molecule. Although Zika is typically asymptomatic or presents a mild illness, it can cause severe neurological defects, particularly in fetuses of women infected during pregnancy. After the 2007 outbreak in the Federated States of Micronesia (Zika virus (ZIKV) belongs to the Aedes albopictus C6/36 mosquito cells. Consensus genome sequence data were generated from the serum samples and the passaged virus isolates. The GenBank accession numbers for five pairs of samples  are as follows: KY765318 and KY765321, KY765317 and KY765322, KY765323 and KY765326, KY765325 and KY765327, and KY765319 and KY765320. The sequence from a serum sample from another patient is also reported (KY765324).Serum samples were collected from six pediatric patients between April and July 2016 from Managua, Nicaragua. The patients, aged 2 to 11 years old, were symptomatic and recovered from the infection. Serum samples were collected 1 to 2 days after the onset of symptoms, and the virus was isolated in The QiaAMP viral RNA mini extraction kit (Qiagen) was used to extract RNA from both serum samples and single-passage virus isolates. RNA sequence-independent single-primer amplification (RNA-SISPA) was performed , 4 priorde novo algorithm. The optimal ZIKV reference genome was identified by using a BLASTn  with an average coverage of 254×. The coding regions for these assembled genomes, except for the noncontiguous ZIKV/Homo sapiens/NIC/6406_13A1/2016 sequence (GenBank accession number KY765319), were annotated with the VIGOR software . This is somewhat expected since these samples were collected from patients within the same geographical and temporal distribution and since there was no selective pressure during single passaging. We expect that the sequence data and associated metadata reported here will provide additional statistical power to future metanalyses.KY765317 to KY765327.These sequences reported here were deposited in GenBank under the accession numbers


We sequenced the virus genomes from 3 pregnant women in Thailand with Zika virus diagnoses. All had infections with the Asian lineage. The woman infected at gestational week 9, and not those infected at weeks 20 and 24, had a fetus with microcephaly. Asian lineage Zika viruses can cause microcephaly. Although Zika virus has circulated in Asia longer than in the Americas, only 3 confirmed cases of congenital Zika virus infection with microcephaly have been reported in Asia  (. proposed that a serine to asparagine substitution (S17N) in the premembrane protein (stably conserved in the American lineage but not in the Asian) contributes to the onset of microcephaly  were detectable by real-time PCR. PW2 had detectable hepatitis B viral surface antigen but no concurrent medical conditions. These findings suggest that Zika virus was the causative agent of this case of microcephaly. PW3 had a maculopapular rash without fever or conjunctivitis and received a Zika virus diagnosis at 20 weeks’ gestation. Her urine sample was positive for Zika virus , and she gave birth to a healthy infant at full term. The last 2 samples were from a 6-year-old child with mild fever and maculopapular rash  and a 64-year-old man with fever and maculopapular rash .We sequenced 7 Zika virus genomes obtained from 5 patients, including 3 pregnant women (PW1–3), in 2016 and 2017. PW1 had fever, maculopapular rash, and mild conjunctivitis at 24 weeks of gestation. Her urine sample was positive for Zika virus , and she gave birth to an infant without birth defects at full term. PW2 had a suspected Zika virus infection at 9 weeks’ gestation with high fever, maculopapular rash, and mild conjunctivitis. At 16 weeks, a sample of the amniotic fluid was positive for Zika virus . The pregnancy was terminated at 17 weeks. Autopsy of the fetus demonstrated a head circumference of 12.5 cm ; Zika virus was detected in the brain  and placenta . No other etiologic agents associated with birth defects  from GenBank to compare these isolates by phylogenetic analysis. All 7 BKK Zika virus isolates grouped within the Asian lineage . Virus f–The 3 cases in pregnant women described here support the hypothesis that the timing of Zika virus infection during pregnancy might be a key contributor to the development of microcephaly during congenital Zika virus infection. PW2 was infected around week 9 of gestation, during embryonic neurulation and cortical neurogenesis, which lay the foundation for the developing brain. Infection during week 20 (for PW3) and 24 (for PW1) of gestation did not led to microcephaly. Our observations are in agreement with reports involving American lineage Zika viruses that show a high risk for microcephaly when infection occurs before week 21 (