Hantavirus, the hemorrhagic causative agent of two clinical diseases, is found worldwide with variation in severity, incidence and mortality. The most lethal hantaviruses are found on the American continent where the most prevalent viruses like Andes virus and Sin Nombre virus are known to cause hantavirus pulmonary syndrome. New World hantavirus infection of immunocompetent hamsters results in an asymptomatic infection except for Andes virus and Maporal virus; the only hantaviruses causing a lethal disease in immunocompetent Syrian hamsters mimicking hantavirus pulmonary syndrome in humans.Hamsters, immunosuppressed with dexamethasone and cyclophosphamide, were infected intramuscularly with different New World hantavirus strains . In the present study, we show that immunosuppression of hamsters followed by infection with a New World hantavirus results in an acute disease that precisely mimics both hantavirus disease in humans and Andes virus infection of hamsters.Infected hamsters showed specific clinical signs of disease and moreover, histological analysis of lung tissue showed signs of pulmonary edema and inflammation within alveolar septa. In this study, we were able to infect immunosuppressed hamsters with different New World hantaviruses reaching a lethal outcome with signs of disease mimicking human disease. Hemorrhagic fever viruses have become of much greater importance in recent years. The more prominent presence of these viruses in a day-to-day setting and their impact on global health has had a positive impact on studying these viruses. Resolving their disease mechanisms and ways to intervene in their replication and associated health burden will stay a challenge for the upcoming decades. This challenge also counts for hantavirus, an emerging zoonosis. No vaccine or antiviral treatment is WHO prequalified or has shown to elicit a good protective or therapeutic antiviral response in humans. One of the main reasons is the lack of good animal disease models and the research focusing on this topic. Without a representative model, it is difficult to investigate new potential drugs. Within this setting we focused on establishing a disease model for the most prominent New World hantaviruses. Hantaviridae, a virus family within the order Bunyavirales in which the genus Orthohantavirus harbors 41 species. These viruses are enveloped, negative sense single-stranded RNA viruses that are not transmitted by arthropods as other families within the order Bunyavirales but by rodents and insectivores. Hantavirus is a segmented virus consisting of 3 segments; S segment, M segment and L segment. The segments encode the nucleocapsid, the glycoproteins (Gn and Gc) and the RNA-dependent RNA polymerase, respectively.Hantaviruses are part of the Rodents are the natural hosts for hantaviruses and the primary source for human infections, although new hantaviruses were recently discovered or isolated from bats, moles and shrews–3. EvideHantaviruses have a clinical impact as they are the source for two diseases; hemorrhagic fever with renal syndrome (HFRS) in the Old World/Eurasia and hantavirus pulmonary syndrome (HPS) in the New World/the Americas. In Western Europe, Puumala virus is responsible for a, mostly milder form of HFRS, named nephropathia epidemica. The complexity and the presence of overlapping symptoms seen in illness caused by several distinct hantaviruses raised the idea of ‘hantavirus disease’ as a more general name .HPS onset is characterized by typical viral symptoms: headache, fever, myalgia, nausea, diarrhea and even abdominal pain are frequently present. This onset of disease is called the prodromal phase and is followed by the cardiopulmonary phase, the diuretic phase and the convalescent phase. The second phase starts rather abrupt and mortality is mostly seen within 24 hours after the onset of respiratory symptoms . When patients survive this phase the diuretic phase starts, which is characterized by high urine flow rates and improvement of the respiratory symptoms. After entering this phase patients are believed to have a relatively good prognosis of survival. The convalescent phase following diuresis is the start of recovery, which can take several months and even years –7.Since its discovery in 1978, . One of Building further on this model, the use of immunosuppressive agents was tested by Brocato et al. with the goal of adapting the model for Sin Nombre virus (SNV) . SNV wasThe scope of the current study is to further explore the use of immunosuppressive treatment to achieve an animal disease model for New World hantaviruses. Several New World hantaviruses were tested, based on the results from the model adaptation by Brocato et al., who saw the development of an HPS-like disease when using immunosuppressed Syrian hamsters to study.All animal work was done in a biosafety level 3+ setting. This study was approved and supervised by the KU Leuven Animal Welfare Body (KU Leuven 206/2012—LA1210186) in compliance with Belgian and European statutes and regulations relating to animals and experiments involving animals.ad libitum. The animals were maintained in an approved biosafety level 3+ facility under conditions that met all requirements for animal care. Animals were sacrificed by lethal pentobarbital injection upon sedation with 2.5% isoflurane.Female Syrian golden hamsters, with a minimum weight of 120 g, were purchased from Janvier Labs . All hamsters were housed per pair in an individually ventilated cage (IVC) system provided with high absorbent nesting material. Food and water were available 2 incubator (5%) on 37°C. Ten days post infection, cells were disrupted by 3 consecutive freeze/thaw cycles followed by sucrose cushion ultracentrifugation to purify virus preparations. Viral stocks were titrated by plaque assay. Female Syrian hamsters were, upon sedation with isoflurane (IMPAC 6 veterinary anesthesia machine), subjected to an intramuscular injection of 200 μL virus suspension . Sterile PBS was used to dilute the virus. Hamsters were monitored continuously during the entire procedure and upon recovery rehoused in the IVC system.Different hantaviruses were used to challenge hamsters: Andes virus strain Chile-9717869, Bayou virus , Black CHantavirus focus forming plaque assays were performed as described elsewhere . Brieflyet al [Hamsters were treated for 11 days with dexamethasone  and cyclophosphamide  starting 3 days prior to virus infection and ending 7 days post infection using 25G x5/8 needles . The used dosing scheme was composed of daily administrations of dexamethasone and cyclophosphamide based on the dosing scheme described by Brocato et al . Boost dHamsters were anesthetized as described above, and 10,000 neutralizing antibody units of convalescent heat-inactivated serum from C57BL/6 mice  diluted in sterile PBS (pH 7.4), were injected subcutaneously with a 1-ml syringe with a 25-gauge needle one day before infection with Bayou virus (BAYV) or Black Creek Canal virus (BCCV) .Organs were extracted using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. The tissue homogenizing step was performed on a Minilys homogenizer  using zirconium oxide beads . RT-PCR was performed using strain specific primers for BCCV and BAYV  targeting the S-segment of these viruses. The One step RT-PCR kit (Qiagen) was used to perform the reaction according to the manufacturer’s instructions. Positive bands were confirmed with chain-terminating sequencing as previously described .Lung tissue samples were fixed in 10% buffered formalin and embedded in paraffin. Tissue sections (4 μm) were stained with hematoxylin and eosin for routine histological examination. Histopathological grading of lung tissue was performed using a standardized scoring system between 0 (absent), 0.5 , 1 (few spots), 2 , and 3 (diffuse confluent areas) .P value) was set at 0.05. P values were calculated by both a log-rank test and a Gehan-Breslow-Wilcoxon test for all groups.Kaplan-Meier survival curves were made and illustrated using GraphPad Prism 6. The significance threshold  according to the treatment scheme in The onset of disease was abrupt and mortality occurred within 24–36 hours after the first signs. For ANDV, 100% mortality was observed between 12 and 15 days post infection (dpi), for Bayou virus (BAYV) between 14 and 18 dpi, for Black Creek Canal virus (BCCV) between 12 and 15 dpi, for Caño Delgadito virus (CDV) between 13 and 20 dpi and for SNV between 11 and 14 dpi. For Choclo virus (CHOV), Laguna Negra virus (LNV), and Maporal virus (MAPV), respectively 75%, 50% and 50% mortality was observed 35 days post infection, with onset of mortality starting 14 dpi for CHOV and MAPV and 17 dpi for LNV. All hamsters of the negative control group  and the PUUV group were still alive at the termination of the experiment 35 days post infection with no noted signs of disease .P values were calculated for all groups in comparison with the DEX-CYP control group . The null hypothesis states that both groups are not showing significant differences in survival. For this experiment, the observed differences in survival are all significant  except for the PUUV strain CG1820, implying that there is a difference in survival between the virus-infected groups and the DEX-CYP control group. P values were respectively calculated with the Log-rank test and Gehan-Breslow-Wilcoxon test for each virus group: BAYV, CDV, BCCV, ANDV and SNV, Log-rank: p value <0.0001, Gehan-Breslow-Wilcoxon: p value = 0.0002; LNV and MAPV, Log-rank: p value = 0.0251, Gehan-Breslow-Wilcoxon: p value = 0.0265; and CHOV; Log-rank: p value = 0.0024, Gehan-Breslow-Wilcoxon: p value = 0.0032.To observe differences in survival between the virus-infected groups and the DEX-CYP control group, Three groups of 20 female golden Syrian hamsters were challenged with BCCV, BAYV and sterile phosphate-buffered saline. Hamsters were treated with DEX/CYP as described in Hematoxylin and eosin staining (H&E) was used to check the lung tissue for pulmonary abnormalities. Passive immunization was carried out to confirm that the virus was responsible for the observed disease and mortality. For this experiment 4 groups of 8 hamsters were treated with dexamethasone and cyclophosphamide according to the dosing scheme explained in Several attempts have been made to establish an animal model for hantavirus-like diseases that reflects a human-like disease . The firA previous study tested the pathogenicity of CHOV in immunocompetent Syrian golden hamsters and saw no mortality . It was Recently a hamster model using Turkish hamsters was described for LNV. In this model 5 different hamster species were infected with SNV, ANDV, MAPV and LNV. Only when LNV was used for infection in Turkish hamsters mortality was observed (43%) . In the Bunyavirales. The role of lipid concentrations in the cell and plasma membrane could be of importance for the establishment of viral infections as well as viral entry when mediated through raft-dependent endocytosis [The fact that immunosuppressive agents are able to make Syrian hamsters vulnerable for hantaviruses other than Andes virus indicates that an interesting and probably complicated interplay between the virus and the immune system is responsible for the occurrence of hantavirus disease and its associated mortality. Even more interesting is the question what differentiates Andes virus from other New World hantaviruses, since they have high phylogenetic similarity. Currently, several hypotheses have been proposed to explain the observed discrepancies in pathogenicity between the different hantavirus strains. Thus far, Andes virus is the only known strain that gives 100% mortality in immunocompetent hamsters . In a reocytosis , 38.Besides ANDV there are other examples of hantavirus strains showing a different infection potential depending on the genotype. Dobrava-Belgrade (DOBV) is an Old World hantavirus causing hemorrhagic fever with renal syndrome in Europe, with case fatality rates going up to 15% depending on the genotype . DiffereHistology of selected tissues showed a clear difference between the control group and the virus-infected groups. The control group received immunosuppression but did not show any signs of disease. This indicates that the observed histological findings in the virus-infected groups are the result of the viral infection and not due to the treatment. This was also supported by the passive immunization, which clearly indicated that the virus is responsible for the signs of disease.The observed clinical signs and onset of disease are alike with what has been described for HPS in humans and the ANDV model in Syrian hamsters. This is remarkable since it has been suggested that hantavirus pathogenesis is the result of an overactive immune response, which is being suppressed here by dexamethasone and cyclophosphamide treatment.Future research should focus more on understanding the mechanisms of disease after infection with specific hantaviruses to find effective countermeasures. Why are some virus strains more lethal and how to they induce mortality? How do they interact with the immune system and how is this related to disease development?+ and CD8+ T cells in Syrian hamster before infection did not lead to lethality when infecting with ANDV [Several New World virus strains were able to induce mortality up to 100% indicating that disease and mortality can be induced by using immune suppression. For the Old World viruses this was not seen, indicating that the viral aspect in the disease development is an important feature. It has been suggested several times that the immune system or the modulation of the immune system could be responsible for disease development. WBC counts in the SNV immunosuppressed model showed a 3- to 4-fold reduction compared to the control group indicating that the adaptive immune response is significantly depleted by a combination of cyclophosphamide and dexamethasone . This inith ANDV , 41.in vivo testing is limited to the ANDV model or animal models lacking the human-like disease [A good working animal model is a necessity for the development of good antiviral products. Several deadly New World hantaviruses have been identified as the causative agent of HPS. A broad-spectrum antiviral compound active against several hantaviruses would be the best outcome from an economic and scientific point of view. Novel antivirals are being tested, some showing promising results but  disease , 42. TheS1 TableCorresponding p-values were calculated for each of the groups. For hamster groups with a 100% mortality rate a significant weight loss was observed.(DOCX)Click here for additional data file.


Staphylococcus epidermidis, S. aureus, Escherichia coli, Enterococcus faecalis, Candida albicans) commonly causing surgical site bone infection, by microcalorimetry. Calcium sulphate beads containing gentamicin were incubated in microcalorimetry ampoules containing different concentrations of the corresponding microorganism. Growth medium with each germ and unloaded beads was used as positive control, growth medium with loaded beads alone as negative control. Bacterial growth-related heat production at 37 °C was measured for 24 h. Cultures without gentamicin-loaded beads produced heat-flow peaks corresponding to the exponential growth of the corresponding microorganisms in nutrient-rich medium. In contrast, cultures with gentamicin-loaded beads completely suppressed heat production during 24 h, demonstrating their antibiotic activity. Gentamicin-loaded beads effectively inhibited growth of susceptible microorganisms, under the described in vitro conditions.Osteomyelitis is responsible for high treatment costs, long hospital stays, and results in substantial morbidity. Treatment with surgical debridement and antibiotic-impregnated Polymethylmetacrylate (PMMA) beads is the standard of care, providing high local but low serum antibiotic concentrations, thereby avoiding systemic toxicity. However, for several reasons, the beads require surgical removal. Alternative antibiotic delivery systems should improve the treatment of bone infection, actively encourage bone healing and require no additional surgery for removal. We investigated the activity of gentamicin-loaded bioabsorbable beads against different microorganisms ( Perioperative antimicrobial prophylaxis, improved surgical technique and laminar airflows in the operating rooms have diminished the infection rate after internal fixation. However, it still remains approximately 0.5%–2% in closed fractures , and up et al. [et al. [The standard treatment of osteomyelitis is a combination of surgical debridement and antimicrobial therapy . Conventet al.  proved t [et al.  showed tUnfortunately, several concerns about the use of PMMA as a delivery vehicle for depot antibiotics have risen. Retained PMMA acts as a foreign body once the release of the antibiotic has fallen below therapeutic levels, and the presence of prolonged subtherapeutic levels increases the risk of emergence of bacteria resistant to the antibiotic . In addi4) has been used since 1892 as a bone defect filler [4 while forming new bone [in vitro antibacterial activity of gentamicin-loaded plaster of Paris beads on bacterial cultures [in vitro and in vivo studies have been performed with commercially available tobramycin-laden calcium sulphate beads [Alternative materials to PMMA for the delivery of depot antibiotics should therefore exhibit a faster and more complete release of the contained antibiotic to diminish the risk of resistance development . In addit filler ,20,21, pnew bone ,23. It icultures ,25. In acultures  gentamicte beads ,28,29, wte beads ,31,32.in vitro activity of gentamicin-loaded bioabsorbable beads against different microorganisms commonly causing surgical site bone infection, by microcalorimetry. We expect that these beads will exhibit a strong antibacterial local effect against microorganisms susceptible to gentamicin, whereas the growth of germs not susceptible to gentamicin should not be affected.We investigated the S. aureus (A), S. epidermidis (B), E. faecalis (C), E. coli (D) and C. albicans (E) in the presence of calcium-sulphate beads with or without (control) gentamicin for 24 h.5, 106 and 107 CFU/mL. An initial heat-flow was detected for all microorganisms (heat flow exceeding 20 μW) within 1 h independently of the presence of gentamicin. Nevertheless, as shown in Beads were challenged with exponentially growing bacteria in concentrations of 10C. albicans, heat produced by microbial growth could be distinguished from the background heat produced from the chemical reaction between the bead and the media. As shown in C. albicans continued to produce heat over 24 h with a stable heat signal of ~80–100 μW. In contrast, for the different gentamicin-susceptible bacterial species tested, the heat-production decreased during 24 h, indicating that the bacteria were not able to grow (7 CFU/mL) for all bacterial species tested. Control beads exhibited no antibacterial effect. In the presence of an unloaded control bead the bacterial heat-flow continued to rise exponentially until the heat-flow peak was reached. The heat-flow peak is reached when the microorganisms stop to actively divide and enter the stationary growth-phase, due to the lack of nutrients or oxygen.By including a gentamicin non-susceptible microorganism in the study,  to grow A–D. The S. epidermidis, S. aureus, E. faecalis and E. coli, respectively. Only in the presence of the highest bacterial inoculum some heat was produced. In contrast, for C. albicans heat-flow peaks ranging from 51 to 53 μW were observed The heat-flow peak of the negative controls was deduced for the control and the gentamicin beads, respectively. For control beads, a proportional delay in time-to-peak was observed with decreasing bacterial concentrations. When comparing the heat-flow peaks, corresponding to the total number of active cells, a nearly 100% reduction was observed in the presence of gentamicin in comparison to the control beads, for Microcalorimetry is an innovative method based on measuring heat from replicating microorganisms in culture. It was first evaluated as a rapid and accurate screening method for contaminated platelet concentrates . The heaet al. [et al. [in vitro antibacterial activity of gentamicin-loaded plaster of Paris beads on bacterial cultures.In our experiment measures obtained by microcalorimetry indicated a nearly 100% reduction of the heat-flow peaks for all the bacterial species in the presence of gentamicin, in comparison to the control beads, which proved that the gentamicin-loaded beads effectively inhibited even the highest bacterial load tested for all the gentamicin-susceptible bacterial species they were challenged with, clearly demonstrating their antibiotic activity. Our findings correlate thus with the results of Mackey et al.  and Mous [et al. , who alrC. albicans reached in the presence or absence of gentamicin, a clear reduction in growth-related heat was observed in the presence of the antibiotic-loaded bead, although C. albicans is a gentamicin non-susceptible microorganism. As shown by Wichelhaus et al. [et al. [in vitro burst-release, mostly caused by diffusion, of gentamicin within the first 24 h (approximately 80%), followed by a more gradual release over a period of at least 10 days. Considering the gentamicin loading of 2.5 mg per bead, gentamicin concentrations above 500 µg/mL could be expected in the calorimetric ampoule. Short-duration high-concentration levels at the infection site are desirable for antibiotics such as aminoglycosides that increase their effectiveness with increasing concentration (dose-dependent killing of bacteria) [C. albicans is most likely induced by the high gentamicin concentration in the growth media. This could become a major issue in the clinical setting, as local antibiotic delivery vehicles diminish systemic toxicity at the price of higher local toxicity [et al. [3H-thymidine incorporation, while concentrations above 700 µg/mL also decreased total DNA in a study on human osteoblast-like cells. Hanssen et al. [Interestingly, when comparing the heat-flow peak of s et al.  and by K [et al. , calciumacteria) . As gentacteria)  , implant size, concentration of contained antibiotic, etc., to determine the expected clinical performance before clinical studies can be initiated.Subsequently, standardized ® beads G , biconvex rounded cylindrical beads  consisting essentially of calcium sulphate dihydrate, and containing gentamicin , were used. They are indicated to fill bone voids that result from surgical debridement of bone infections, and are supposed to enhance antibiotic protection and encourage bone growth.Herafill® beads, obtained from an Osteoset Resorbable Mini-Bead Kit-Fast Cure 5cc , cylindrical beads  consisting essentially of calcium sulphate hemihydrate and containing no antibiotics, were used in the positive control group (unloaded bead plus microorganism). Osteoset® beads are indicated to be used as osteoconductive material for the ingrowth of blood vessels and osteoprogenitor cells from the graft bed in the treatment of bone voids.OsteosetStaphylococcus epidermidis RP62A (ATCC 35984), S. aureus (ATCC 29213), Escherichia coli (DH5α), Enterococcus faecalis (ATCC 19433) and Candida albicans (ATCC 90028). Strains were stored at −80 °C and cultured overnight on human blood agar plates before each experiment.Laboratory strains of different microorganisms causing bone infection were studied, including 6–108 colony-forming units (CFU)/mL depending on the microorganism. 4 mL sterile microcalorimetric glass ampoules were filled with 3 mL of Müller-Hinton broth  and inoculated with three different inocula corresponding to final bacterial concentrations of 105, 106 and 107 CFU/mL. C. albicans was tested in concentrations of 103, 104 and 105 CFU/mL. Microorganisms were incubated 2 h at 37 °C in order to initiate the exponential growth phase.A bacterial or fungal suspension of McFarland 0.5 was prepared, corresponding to a density of 10After the incubation, one bead, with or without gentamicin, was transferred to each ampoule. An ampoule containing a bead and media only served as negative control. All ampoules were closed with a rubber cap, sealed by manual crimping and sequentially introduced into the calorimetry instrument. After 15 min in the thermal equilibration position, ampoules were lowered into the measurement position. Heat flow was measured for up to 24 h at 10 s intervals. A 48-channel batch calorimeter  was used to measure the heat flow at 37 °C controlled at 0.0001 °C and a sensitivity of 0.2 μW. Heat was measured continuously and expressed as heat flow over time (in microwatts (μW)). Calorimetric detection time was defined as the time from insertion of the ampoule into the calorimeter until an exponentially rising heat flow signal exceeding 20 μW was detected.Data collection was performed using the calorimeter manufacturer’s software. Data was processed in Prism software, version 5.0a .in vitro conditions. Standardized animal studies are mandatory to determine the elution kinetics and the antibacterial effect of gentamicin under in vivo conditions. Finally, clinical studies are needed to demonstrate that treatment against bone infection with the use of commercially available gentamicin-loaded bioabsorbable calcium sulphate beads is a good therapeutic option.The gentamicin-loaded bioabsorbable calcium sulphate beads investigated in this study effectively inhibited growth of gentamicin-susceptible microorganisms, under the described


Mycobacterium tuberculosis Beijing lineage. However, amplification of QUB11a occasionally produces large fragments  that are not easily measured by capillary electrophoresis because of a lack of the typical stutter peak patterns that are used for counting repeat numbers. IS6110 insertion may complicate VNTR analysis of large QUB11a fragments in M. tuberculosis. We established a method for determining both tandem repeat numbers and IS6110 insertion in the QUB11a locus of M. tuberculosis using capillary electrophoresis analysis and BsmBI digestion. All 29 large QUB11a fragments  investigated contained IS6110 insertions and varied in the number of repeats (18 patterns) and location of IS6110 insertions. This method allows VNTR analysis with high discrimination.QUB11a is used as a locus for variable number of tandem repeats (VNTR) analysis of Mycobacterium tuberculosis [ M. tuberculosis [QUB11a, or variable number of tandem repeats (VNTR) locus 2163a, is an analytical locus that has been useful for molecular epidemiological analysis of the Beijing lineage ofrculosis  and Russrculosis  and the rculosis  that werrculosis . Despiterculosis . Howeverrculosis  and Mill6110  [ Mycobacterium caprae [ M. tuberculosis. Insertion would likely complicate VNTR analysis of large QUB11a fragments in M. tuberculosis, indicating that a method for analyzing these large QUB11a fragments may increase the discriminatory power of M. tuberculosis typing by VNTR. Therefore, the aims of the present study were to determine the presence or absence, frequency, location, and stability of IS6110 insertions in the QUB11a locus of M. tuberculosis isolates and to develop a VNTR typing method for the QUB11a locus.IS M. tuberculosis isolates collected from humans in Fukuoka Prefecture (n = 306) and neighboring prefectures (n = 6), Japan, between 2012 and 2013 was performed as described previously [VNTR analysis of QUB11a from 312Amplification products corresponding to the QUB11a locus were analyzed by capillary electrophoresis  and GeneMapper software (Life Technologies) to determine the fragment sizes . GeneScan 1200 LIZ Size Standard (Life Technologies) was used as an internal size marker. Analysis showed that 29 (9.3%) of the isolates produced large fragments  . These l6110 insertion in QUB11a from the 29 isolates were determined by Sanger analysis . The large fragment was amplified from all 29 isolates using primers iwamoto-F and iwamoto-R as described previously [VNTR numbers and presence or absence and location of the ISeviously  and theneviously , INS1, Ieviously , Nandagoeviously , ISseq-F6110 in the QUB11a locus, 26 of the 29 isolates were passaged in 2% Ogawa's medium  every 2 weeks for 20 weeks to give a total of 10 passages (stability test).To confirm the stability of IS6110 insertion, along with tandem repeat number(s), in the QUB11a locus based on capillary electrophoresis analysis of BsmBI-digested fragments  but not cut the QUB11a repeat region. The QUB11a locus was amplified from genomic DNA from the 29 isolates using primers iwamoto-F (forward) and iwamoto-R (reverse), which were labeled with fluorescent dyes fluorescein (FAM) and NED, respectively. The amplified fragments were treated with BsmBI  at 37°C for 1 h. The BsmBI-treated fragments were then analyzed simultaneously by capillary electrophoresis, as described above.We also aimed to establish a simple method for detection of the ISragments . BsmBI wThis study was carried out in strict accordance with the guidelines of the Ethics Regulations Related to Epidemiological Research at the Fukuoka Institute of Health and Environmental Sciences, which is based on domestic standards , and approved by the Ethics Committee of Fukuoka Institute of Health and Environmental Sciences under permit number 26-9. The strains tested were anonymized and donated by the hospitals.6110 insertions and varied in the number of repeats (18 patterns) and the locations of IS6110 insertions  contained ISsertions . IS6110 cept one , in whic6110 in QUB11a did not change in any of the 26 isolates during the 10 passages.The number of repeats and the presence and location of IS BsmBI, two of the expected fragments were detected by capillary electrophoresis –(c) shown in  BsmBI. Therefore, the size of the PCR product is calculated as follows: 604 + 339 + 925 = 1,868 (bp), or detected fragment length with labeled forward PCR primer (bp) + detected fragment length with labeled reverse PCR primer (bp) + theoretical length of IS6110 portion (bp) = estimated length of the amplified QUB11a locus (bp). Next, the number of repeats was determined as follows: )/69 = 4.898. In the calculation, the figures used were obtained as follows: 1,868: detected length (bp) of the amplified QUB11a locus; 1,359: length of IS6110 (bp); 171: length of flanking sequence (bp); and 69: repeat unit length (bp) of tandem repeats in the QUB11a fragment (bp). The capillary electrophoresis method used detects repeats (including the IS6110 region) as well as contiguous sequences  in the QUB11a locus.When the large fragment of QUB11a  was dige Figures , and aga Figures . Subsequ Figures . Therefoy = 0.0814 + 1.0016x, R = 0.9998 . Using this regression equation, the isolate that produced fragments (a)–(c) in  BsmBI-digested PCR products from eight amplified QUB11a loci. Incidentally, the amplified QUB11a fragments from four isolates were much smaller  than the rest of the isolates and were not cleaved by BsmBI  and increases the accuracy and discriminatory power of VNTR typing at the QUB11a locus of M. tuberculosis.In the present study, we established a method to detect IS6110 sequences vary [ in silico analysis of 229 elements from 17 M. tuberculosis strains available from GenBank  and showed that all (229/229) of these regions contained the two BsmBI restriction sites (data not shown). Moreover, the present study showed that IS6110 in the QUB11a locus is relatively stable, which is consistent with a previous report [ M. tuberculosis isolates.Although ISces vary , we also6110) (data not shown). The present method typed QUB11a regions correctly, and the 24-locus typing assay revealed the possibility of epidemiological linkage of these isolates. Although our method might not be applicable for all other strains that harbor large QUB11a fragments, it may contribute to expanding the discriminatory power of VNTR for M. tuberculosis Beijing lineage.Discrimination between strains of Beijing lineage is important for countries and areas where Beijing lineage is dominant , 2. Usin


Ebola Virus Disease (EVD) is caused by Ebola virus, which is often accompanied by fatal hemorrhagic fever upon infection in humans. This virus has caused the majority of deaths in human. There are no proper vaccinations and medications available for EVD. It is pivoting the attraction of scientist to develop the potent vaccination or novel lead to inhibit Ebola virus.In the present study, we developed 3D-QSAR and the pharmacophoric model from the previous reported potent compounds for the Ebola virus.Results & Discussion: The pharmacophoric model AAAP.116 was generated with better survival value and selectivity. Moreover, the 3D-QSAR model also showed the best r2 value 0.99 using PLS factor. Thereby, we found the higher F value, which demonstrated the statistical significance of both the models. Furthermore, homological modeling and molecular docking study were performed to analyze the affinity of the potent lead. This showed the best binding energy and bond formation with targeted protein.Finally, all the results of this study concluded that 3D-QSAR and Pharmacophore models may be helpful to search potent lead for EVD treatment in future. The EVD is more prevalent in Africa except other countries. There are mostly found in species such as Sudan ebolavirus, Zaire ebolavirus, Budibugyo ebolavirus and Tai Forest ebolavirus . These vThe previous literature regarding the adenine-sugar containing derivatives has the potent inhibitor for Ebola Virus . The aimThis above study parameter revealed and provided the potent model for the development of novel compounds for the Ebola virus. However, this study would be a milestone in the path, who is working for the search of the lead as a potent Ebola virus inhibitor.22.11). The chemical structure was drawn using by ChemDraw 12.0 and their geometry was optimized with the Gauss View 5.0. software. On the other hand, the energy minimization was evaluated by the ChemPro3D. Finally, bond length and Angle of atoms were optimized via Argus Lab. (http://autodock.scripps.edu/) along with its Lamarckian Genetic Algorithm (LGA).In the present study, we have searched literature on Ebola virus inhibitor, where we observed that a few data is available on the inhibitor of Ebola virus. Thereby, we used the series of 9 potent compounds containing adenine derivatives shown in Fig. (50) towards Ebola virus. In the present study, The IC50 was used for the pharmacophore generation and QSAR analysis. Further, the IC50converted into the corresponding pIC50 [−log(IC50)] and used as dependent variables QSAR and pharmacophore calculations (Table 1).Adenine derivatives were used 50% inhibition concentration . In which, phase tool was applied to find the common pharmacophoric feature of this series compounds. Whereas, Ligprep tool was used for prepare ligands in order to get stable conformational of structures and attaches hydrogen’s which neutralize the charges at a user-defined pH. The most stable conformation was obtained via conversion of these structures into 3D structures.The development of pharmacophoric of Adenine derivatives was carried out 50 (Table 2).The activity threshold was assigned 5 for the active and 4 for inactive. This activity threshold was preferred on the basis of ICvia a tree-based partitioning. However, the atoms of all ligands were assigned by pharmacophore H-bond acceptor (A), features aromatic ring (R), Hydrophobic group (H), and negative charge group (N), H-bond donor (D), positively charge group (P)  were used as chemical sources to obtain the reputable one homological Ebola virus sequence and further performed the Run blast and get the sequences of amino acid which is used to the homology of Ebolavirus MRRVILPTAPPEYMEAIYPARSNSTIARGGNSNTGFLTPESVNGDTPSNPLRPIADDTIDHASHTPGSVSSAFILEAMVNVISGPKVLMKQIPIWLPLGVADQKTYSFDSTTAAIMLASYTITHFGKATNPLVRVNRLGPGIPDHPLRLLRIGNQAFLQEFVLPPVQLPQYFTFDLTALKLITQPLPAATWTDDTPTGSNGALRPGISFHPKLRPILLPNKSGKKGNSADLTSPEKIQAIMTSLQDFKIVPIDPTKNIMGIEVPETLVHKLTGKKVTSKNGQPIIPVLLPKYIGLDPVAPGDLTMVITQDCDTCHSPASLPAVVEK33.1The main aim of present study was to investigate the 3D atoms base features and develop the potent pharmacophoric model the screening and searching potent lead towards the Ebola virus.5), which depends on the IC50 activity against Ebola virus replication in Vero E6 cells and phase predicted activity .Common pharmacophore hypothesis was selected and the tree-based partition algorithm was used to generate the four variants of probable common hypotheses . Further3). The alignment hypothesis of inactive and active scoring was shown in Fig. (3). Moreover, the 3D-QSAR model was generated after the choice of best hypothesis scores. The fitness of both models was shown the higher degree of robustness. One PLS factors were used to generate the best QSAR model and the LOO method was also applied to determine the coefficient value (r2) of 0.98 and cross-validated correlation coefficient (q2) of -0.058. In this regards, we were found the higher F value, which demonstrates that the statistical significance in both models and highly supported via lower variance ratio (p) values which intensify a greater degree of confidence. However, a small standard deviation showed the best fitness for the QSAR model. The root mean square error and Pearson’s (r2) display the predictive ability of the test set of both models. The significance of the productive model was obtained where all significant values of data plotted around the best fit lines. Finally, the similar trend was found in the observed and predicted value which exhibited that similar predictive values show the better prediction of the model.The pharmacophoric hypothesis consisted of the three acceptors (A) and also one has the positive ionic (P) features. This hypothesis was measured on the base of regular performance of multiple runs and precious statistical analysis. On the other hand, the alignment of the pharmacophoric hypothesis with the fitness score and the better fit ligands are shown in Table (3.24). The cubes were based on the view effect of acceptor and ionic positive effect with the positive coefficient and negative coefficient, which by characterize dark blue for the positive coefficient and dark red for the negative coefficient. Moreover, the positive coefficient shows an increasing in activity, whereas a negative coefficient demonstrates the decreasing activity. The 3D-QSAR represents the coefficient of three pink cubes containing H-bond acceptor  for the ether linkage is essential for biological activity. A light blue color around the Nitrogen into the aromatic ring contained positively charge group (P), which is responsible for increasing the activity towards the Ebola virus. Moreover, the red color near the methyl alcohol group was shown the decreased activity.3D structure characteristics of atomic cubes exhibit the color according to the coefficient values shown in Fig.  using Autodoc 4.1 beside by LGA algorithm parameter for computerized flexible ligand docking [6). The most active compound EB_5 was shown the best docking poses along with bonds and its distance with the assigned target, shown in Fig. (5). The docking study of the potent compounds was performed to explore the essential amino acids which are responsible for activity and also support our pharmacophoric model on the basis of atomic bond formation of the ligand with the receptor. docking . The binThe most active compound of this series showed the better  binding affinity along with bonds formation with target. There were a number of aminoacid residues involved for the formation of bonds with lead. These vital amino acids may be used as potential binding pocket for further drug development towards EV.The observation of result concluded that the common pharmacophore model of adenine derivatives responsible for inhibitory activity in order to Ebolavirus. Thereby, common pharmacophore alignment and 3D-QSAR models were created. This provided the significant information about the 3D chemical structure feature requirements for the target related to Ebola virus. The statistical analysis assessment indicates robustness and productivity, which ensure the reliability. Whenever, the pharmacophore models display the significant optimal feature for development or researching of novel lead toward of Ebola virus. Moreover, 3D-QSAR model explored the effect substituted of the chemical feature such as A light blue color around the Nitrogen into the aromatic ring contained positively charge group (P), which is responsible for increasing the activity towards the Ebola virus. Moreover, the red color near the methyl alcohol group was shown the decreased activity. On the other hand, pharmacophore patern of titled compunds resposive for the inhibitory activity towards the Ebola virus-like as the substitution at H-bond acceptor and positively charge group (P) into the adenine ring (Nitrogen). The substitution of the hydroxyl group of the sugar moiety of H-bond acceptor region and substitution of methyl alcohol group at the sugar moiety into another H-bond acceptor region show the increasing activity. Besides, the substitution of methyl or any other group near the sugar moiety may decrease the activity. On the other hand, the molecular docking was provided the potent binding pocket into the protein structure of Ebola virus and also provided the significant information in order to ligand-protein affinity along with bond formation with specific amino acid into the target protein. Finally, the model was developed from the QSAR and pharmacophore (hypothesis AAAP.116) for the Ebola virus inhibitor might provide the essential atomic structural requirements to the researcher for development of novel potent lead for the inhibition of Ebola virus.


There is growing concern about the economic impact of cardiovascular diseases(CVD) in Brazil and worldwide.To estimate the economic impact of CVD in Brazil in the last five years.The information to estimate CVD costs was taken from national databases,adding the direct costs with hospitalizations, outpatient visits andbenefits granted by social security. Indirect costs were added to thecalculation, such as loss of income caused by CVD morbidity ormortality.CVD mortality accounts for 28% of all deaths in Brazil in the last five yearsand for 38% of deaths in the productive age range (18 to 65 years). Theestimated costs of CVD were R$ 37.1 billion in 2015, a 17% increase in theperiod from 2010 to 2015. The estimated costs of premature death due to CVDrepresent 61% of the total cost of CVD, Direct costs with hospitalizationsand consultations were 22%, and costs related to the loss of productivityrelated to the disease were 15% of the total. Health expenditures in Brazilare estimated at 9.5% of GDP and the average cost of CVD was estimated at0.7% of GDP.CVD costs have increased significantly in the last five years. It isestimated that CVD costs increase as the Brazilian population ages and theprevalence of CVD increases. Statistics show that approximately half of these deaths occurduring the productive life of individuals2 and CVD accounts for most of them, accounting for37%.1Non-communicable chronic diseases (CDNs) - mainly cardiovascular disease (CVD),cancer, chronic respiratory diseases - are the leading cause of death, causingapproximately 38 million deaths annually worldwide.4The socioeconomic impact of chronic diseases is increasing and is considered aproblem for the world public health. In addition to premature deaths, NCDs areresponsible for incapacity for work, reduction of family incomes and reduction ofproductivity.5 In this same period, developed countries spend anaverage of 4632 dollars per capita, that correspond to the average of 16,8% of thegovernmental expenses with health.Current health spending in Brazil is approximately 9.5% of GDP per capita. Data fromthe World Health Organization indicate health expenditures of $ 1078 per capita in2012 in Brazil. Of these 47.5% are financed by the government, which corresponds to7.9% of the total expenditure of the Brazilian government.6 and recent IBGE data show that Brazil is changing its agestructure very rapidly, increasing the proportion of elderly people and lifeexpectancy of the Brazilian.7 Agingtends to increase the incidence of CVD and, consequently, its costsexponentially.8The cost of hospitalizations for cardiovascular diseases is considered the largestcause of hospital admissions in BrazilThis study was designed to estimate the economic impact of CVD based on Braziliandata. Our estimate of socioeconomic impact is based on government expenditures inBrazil, once the information from the public database of the health system was used.The methodology proposed in the present study includes the direct and indirect costsrelated to CVD.Health costs can be divided into:medical care to the patient,such as medical services rendered and treatments performed, andnon-medical costs ;9Direct costs: costs of direct Morbiditycosts are defined as expenses for the temporary or permanentloss of work activities due to the disease studied. Mortalitycosts are the costs estimated for premature death as aconsequence of illness.9Indirect costs: costs of morbidity and mortality. 10 the hospitalmorbidity was obtained at the approved hospital admission events of the DATASUSHospital Information System (SIH) and outpatient information system(SIA),11 in additionto information on social security expenses for temporary and / or permanentremoval - DATAPREV.12Data sources are publicly available information on the Hospital Mortality System(SIM),13 was collected, as well as from the access to the WorldHealth Organization (WHO) database.14 To estimate the costs of private care, the sources ofinformation in the National Health Agency (ANS) were used. In order to evaluatethe impact of the cost of mortality, estimates from the Brazilian Institute ofGeography and Statistics (IBGE) were used, such as: population estimates, lifeexpectancy by sex and age group, average salary of the Brazilian population, andrate of unemployment.7 Aproductive range was considered from 18 to 65 years.In order to estimate the total cost of diseases in Brazil, information fromprevious observational studies11 andcorresponds to all events registered in Chapter IDC-10: IX Circulatory diseasesin the SUS.15 To estimate thenumber of hospital admissions in private care, public data banks (ANS)16 were used, which show thecoverage rate of the beneficiaries of the private health plans, that is, thepercentage of beneficiaries who use private plans each year. The formula used toestimate the number of admissions to supplementary care was:The number of hospital admissions for CVD is available in the billing dataapproved in the SIH-SUS,Number of hospitalizations of the supplementary care11 We extractedthe information of quantities approved in the Subgroup procedure: 0301Consultations / Attendance / Monitoring. As the number of consultationsperformed by ICD of illness is not available in the health information system,in the present study the percentage of hospitalizations for CVD was calculatedon the total hospitalizations. This percentage value was applied in the totalnumber of consultations performed in the SUS , since it may beunderestimated. To estimate the number of outpatient visits performed in theprivate sector, the coverage rate of the ANS was applied.The number of medical consultations performed by CVD was estimated through theOutpatient Production of the SUS - Brazil - by location of care, available inthe SIA / SUS.16 and reimbursement amounts forhospitalizations and/or outpatient visits were accounted for the direct costscalculations.SUS referrals to private sector beneficiaries are accounted by the ANSThe direct costs were calculated in Brazilian currency (reais) for the year2016.The hospital costs related to CVD events were separated in didactic form in:Clinical treatments of CVD - subgroup 0303, titled "Clinicaltreatments ";11Surgical treatments of CVD - subgroup 0406, entitled "CirculatorySystem Surgery" - which include: cardiological surgical procedures,arrhythmia procedures , coronary angioplasty, andvascular .The costs of surgical hospitalizations were increased by estimates of costs oforthotics, prostheses or special materials (OPME). In order to estimate costsincurred with OPME, is used the price base available at ANVISA, with the lowestprice practiced in Brazil for each type of OPME.17 The costsof morbidity can be defined as time and economic production lost by thepatient's absence from their usual activities and work as a direct result of CVDor their treatment.18 Thecalculations were grouped into two components:Indirect costs are calculated through the costs of morbidity (loss ofproductivity caused by CVD) together with the costs of mortality, cost of CVDpremature death.Costs for temporary removals from the work of the patients employed(absenteeism): in this component are included the removals forhospitalizations and medical consultations, and added the valuesspent in the displacement, that is, the transportation for eachconsultation carried out;Costs of patients who are no longer in working condition as aconsequence of CVD . Costs of pensions and sickness-help caused by CVD.12The information available on the Social Security website was used to estimate theimpact of temporary (sickness) and permanent (retired) removal. An analysis wasmade of the number of benefits granted because of CVD excluding benefits fromother diseases outside the ICD-10 Chapter IX. Diseases of the circulatorysystem.19 Thebenefits granted because of CVD correspond to 25% of the total expenses grantedby the Social Security.Mortality costs are estimated by estimating the years ofproductive life lost due to premature death as a consequence of CVD. Thecalculation is done by multiplying the number of deaths because of CVD accordingto the age group by the number of days lost (difference between the lifeexpectancy of the Brazilian population and the age of prematuredeath).17 This data isexpressed in economic value multiplying the days lost by the estimated income ofthe Brazilian until the age of 65 years. 7The benefits granted by Social Security were analyzed by the frequency of eventsin the sample in the CVD disease group in the period from 2008 to 2013. For theestimation of removals occurred in the years 2014 and 2015, a projection wasmade based on the time series of the period from 2008 to 2013 from a model(ARIMA). The ARIMA model is a type of moving average model. A methodology widelyused for the elaboration of forecasting studies, applied in the spss softwareforecasting module.There was a decrease in the number of clinical admissions due to CVD from 874,949 in2010 to 807,304 in 2015. However, there was an increase in CVD surgicalhospitalizations from 246,038 to 279,010, with a 55% increase in hospitalizationsfor procedures related to vascular surgeries, 35% for coronary angioplasty, and 34%for electrophysiological studies (FES).The estimated direct costs of public sector with CVD in Brazil from 2010 to 2015are shown in 20 that use eachtype of material. The estimated values are shown in The expenses related to the OPMEs were included in the amounts spent on thesurgical hospitalizations in CVD in the SUS. The average values in Brazil spenton OPME were multiplied by the number of procedures with codes in the proceduretable (TABNET)The amounts reimbursed by the ANS to the public sector for Hospital AdmissionsAuthorizations (AIH) and/or High Complexity Procedure Authorization (APAC) forvisits to the SUS are added to the direct cost calculation. 21 For the calculation of thepercentage of total expenditure with CVD, the same percentage of 10% was usedyear by year.The costs of the drugs used for CVD were estimated through the informationcollected in the transparency portal of Brazil. All direct expenditures of thefederal government with pharmaceutical medicines were added.12 The data available refer to the years 2008 to2013, without updates until the completion of the present study. The valuesspent in the years 2010 to 2013 in Brazil by CVD are detailed in Social Security spending on pensions and sickness-help because of CVD isavailable with data open on the DATAPREV portal.The costs of morbidity (absenteeism) are shown in Mortality costs are shown in The total costs estimated with CVD in Brazil in the period from 2010 to 2015 isshown in The costs estimated for premature death with CVD represent 61% of total cost forCVD, direct costs were 22% and costs for morbidity were 15% of total CVDcosts.The percentage of GDP with CVD by the study estimate was 0.8% in 2010, 0.7% inthe years 2011 to 2014 and 0.6% of GDP in 2015, with an average of 0.7% of GDPover the last five years.22 The present study has a methodology that proposes to usethe data sources with greater reliability and with greater speed in obtaining theinformation. The sources of billing expenses in Brazil occur with a maximum delay ofabout 2 months after the current month, for which reason they were prioritized. Itis important to point out that the present study was based on the use of the largestpossible amount of information available in public databases. As the nationallybased information system is still settling in Brazil, billing data on SIH and SIAmay be underestimated, and this is a limitation of the study.It is essential today, with scarce resources, to discuss health costs based onsources of secure and real-time information.23Another limitation of the study is information related to mortality, pensions andsickness-help, as well as the amounts of direct reimbursements from ANS to SUS.These data were not available for the years 2014 and 2015 in their fullness, in thisway, it was chosen to estimate these values through linear progression. Expenseswith drugs for treatment of cardiovascular disease are not available in Brazilianpublic information systems. The present study estimated expenditures for CVD drugs,considering that 10% of the pharmaceutical care expenses in Brazil are for CVDtreatment. This strategy can be considered conservative. The criteria adopted forthe selection of sources was to use as much information as possible in publicdatabases, and to strengthen this information as a basis for the future of thecountry's public policies.The direct costs of supplementary health should be underestimated, since using thecoverage rate available in the ANS assumes that the costs of supplementary medicineare at least similar to those spent in the SUS, which is known not to be areality.The number of CVD pensions has been decreasing in the country in the analyzed period,and the number of sickness-help has increased. This can be considered an indirectindication that the Brazilian population is living with CVD, without interruption oflabor activities. This may have been due to improved health, or changes in pensionlegislation.24With the epidemiological transition in Brazil, associated with population aging, itis vehement that several studies estimate health costs, especially CVD, as the maincause of death due to illness in the country. As shown in the last five years, CVDcosts are growing, and it is essential that health promotion measures occur toreduce premature deaths.25and in line with the global targets for the reduction of CVD, the SociedadeBrasileira de Cardiologia (SBC) launched in 2013 an importantpublication aimed at increasing CVD prevention in Brazil.24 In order to achieve a reduction in the impact ofCVD in Brazil, it is essential to involve everyone in society. The fight to preventand improve the quality of life of the population is urgent, especially in adeveloping country such as Brazil where resources are scarce.The direct and indirect costs of CVD in Brazil have increased in the last five yearsin Brazil. This increase was more significant in drug costs (88%), followed bysocial security costs (66%) and morbidity costs (33%). These data are indirectindications that there is an increase in the population living with CVD. The numberof social security benefits has increased in the last five years in proportion tothe sickness-help, since it is possible to visualize the fall of the pensionsoccurred by CVD. When analyzed as a percentage of GDP, CVD costs are stable,probably due to the lower number of years of life lost. The goal of the WHO was toreduce 25% of CVDNCs by 2025,


The virus to microbial cell ratio ranged from 1.1 to 8.1 and averaged 3.0 ± 1.6 with virus abundance most strongly correlated to cell abundance . Both viruses and cells were positively correlated to dissolved organic carbon (DOC) with cells having a slightly stronger correlation . Groundwater uranium was also strongly correlated with DOC and virus and cell abundance . Together the data indicate that microbial cell and virus abundance are correlated to the geochemical conditions in the aquifer. As such local geochemical conditions likely control microbial host cell abundance which in turn controls viral abundance. Given the potential impacts of viral-mediated cell lysis such as liberation of labile organic matter from lysed cells and changes in microbial community structure, viral interactions with the microbiota should be considered in an effort to understand subsurface biogeochemical cycling and contaminant mobility.Viruses are the most abundant biological entity on Earth and their interactions with microbial communities are recognized to influence microbial ecology and impact biogeochemical cycling in various ecosystems. While the factors that control the distribution of viruses in surface aquatic environments are well-characterized, the abundance and distribution of continental subsurface viruses with respect to microbial abundance and biogeochemical parameters have not yet been established. In order to begin to understand the factors governing virus distribution in subsurface environments, we assessed microbial cell and virus abundance in groundwater concurrent with groundwater chemistry in a uranium impacted alluvial aquifer adjoining the Colorado River near Rifle, CO. Virus abundance ranged from 8.0 × 10 Groundw−1. An elevated groundwater U concentration (171 μg L−1) was measured in well 656 in the eastern portion of the site, but groundwater in this region is geochemically distinct from the major plume with significantly lower groundwater DOC, DIC, and sulfate concentrations. Accordingly, well 656 is excluded from assignment to the major contaminant plume that is located at the center of the field site. Eleven monitoring wells within the uranium contaminated plume and nine monitoring wells outside the plume region were sampled in this study  for geochemical analyses and enumeration of cells and viruses were collected from purged  wells at a 5 m depth using a peristaltic pump. Groundwater was filtered through 0.45 μm PVDF filters to remove sediment particles for cell enumeration, and through 0.1 μm PVDF filters to remove cells for virus enumeration. In order to reduce background noise and improve filter clarity, samples for virus enumeration were treated with a nuclease, DNase I (10 U mLp-phenylenediamine). Background fluoresence in groundwater samples was minimal and did not interfere with enumeration , while cells were collected on black polycarbonate filters (0.2 μm). Between 0.5 and 1 mL of sample was passed through each filter. SYBR Green I  was used to stain the filters (15 min) for enumeration by epifluorescence microscopy. We note that because SYBR Green I binds to dsDNA more efficiently than ssDNA and RNA, the total virus count may be underestimated. After staining and drying, filters were mounted on slides with an anti-fading solution  into the well. Groundwater samples were directly filtered (0.45 μm PTFE) into glass vials for analysis of DOC/DIC while samples for anion analysis were filtered directly into HDPE vials. Vials were capped, leaving no headspace, and stored at 4°C prior to analyses  concentrations were measured Spatial interpolation  was used to geographically represent the distribution of viruses and cells as well as geochemical parameters  across the alluvial aquifer . Significance level was defined at 4 to 1.0 × 106 viruses mL−1   Figure  and is c) Figure , marine ) Figure , marine ) Figure , marine ) Figure , and dee) Figure . The virt-test, p < 0.01) within the uranium plume  compared to cell abundance in groundwater outside of the plume , U , and sulfate concentrations . However, it should be noted that groundwater DOC concentrations were also higher within the uranium plume , relative to the concentrations measured outside  of the uranium plume , 5.2 × 105 ± 2.4 × 105 viruses mL−1 , relative to the abundance of viruses outside of the region, 2.5 × 105 ± 1.4 × 105 viruses mL−1  and virus abundance  strongly suggest that DOC was sufficiently bioavailable to stimulate microbial activity. Microbial activity in groundwater is often stimulated by inputs of DOC . While we did not test lability of the DOC, correlations with cell  predominated throughout most of the plume, oxic conditions were identified in two wells within the center of the plume region: LQ105 (4.3 mg L−1) and U01 (2.6 mg L−1)   within the plume and 451.5 ± 125.8 mg L−1 outside of the contaminant plume   compared to outside   Table . Dissolv1) Table . Thus lo1) Table . Groundwe Figure . Nitratey) Table . This may) Table . The oxi9) Table . Sulfate9) Table  within t9) Table . As suchHere we demonstrate that viruses, cells, and DOC are enriched in the major plume region of the Rifle aquifer. The widespread distribution of viruses at the Rifle aquifer is consistent with prior suggestions that viruses may play a potential role in influencing biogeochemical cycling at the site , lytic kill-the-winner dynamics are suggested to be favored (Knowles et al., Within the Rifle aquifer, virus mediated cell lysis has been suggested to contribute biologically available organic carbon or to suppress certain taxa responsible for biogeochemically important reactions at the site (Wrighton et al., DP and KAW contributed to laboratory and statistical analyses of data. KHW and MR contributed to field sample collection and geochemical analyses. JN contributed to the spatial interpolation of data. DP and KAW contributed to the experimental design and data interpretation. All authors contributed to writing of the manuscript.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.


Orthobunyavirus, family Bunyaviridae) isolated in the Brazilian Amazon region: Mirim virus  and Ananindeua virus .This is the first announcement of two nearly complete viral genome sequences belonging to the Guama serogroup (genus  Bunyaviridae is composed of five genera, Orthobunyavirus, Nairovirus, Phlebovirus, Hantavirus, and Tospovirus. The Guama serogroup belongs to the Orthobunyavirus genus, composed of tripartite, single-stranded, and negative-sense RNA genomes, namely, large (L), medium (M), and small (S) genomes. The L-RNA encodes the RNA-dependent RNA polymerase. The M-RNA encodes two glycoproteins, Gn and Gc, and a nonstructural protein, NSm. The S-RNA encodes the nucleocapsid protein (N) and usually a nonstructural protein, NSs  and Catu virus (CATUV). Nowadays, this group of viruses has other members identified: Mirim virus (MIRV), Ananindeua virus (ANUV), Timboteua virus (TBTV), Bertioga virus (BERV), Cananeia virus (CNAV), Itimirim virus (ITIV), Guaratuba virus (GTBV), and Mahogany hammock virus (MHV). Two of these viruses, GMAV and CATUV, present relevance for public health because they cause acute febrile disease in humans characterized by fever of sudden onset, headache, arthralgia, myalgia, photophobia, asthenia, and other symptoms  in a forest area of the Agronômico do Norte Institute , in Belém, Pará, Brazil. The ANUV BEAN109303 was isolated in 1966 from a marsupial  in Brazil  (The MIRV BEAN7722 was isolated in 1957 from a sentinel monkey (á State) .Brain tissues of newborn mice infected with prototype strains of MIRV and ANUV were propagated into Vero cells, and after 2 to 6 days postinfection, when the cells were showing approximately 90% cytopathic effect (CPE), the supernatant was harvested and treated with DNase to digest cellular DNAs. The RNA was extracted by MagNA Pure LC 2.0 equipment (Roche Life Science) using the MagNA Pure LC total nucleic acid isolation kit (Roche Life Science). Afterward, we followed all steps of the 454 pyrosequencing protocols (Roche): cDNA library construction, emulsion PCR-titration, emulsion PCR, and sequencing (de novo Assembler (Newbler version 2.6), and analyses were realized using the software Geneious version 6.1.4. Here, we announced the first report of complete genome sequence (coding region) of the orthobunyaviruses MIRV and ANUV.The sequences were assembled using the GS KY013487 to KY013489, and ANUV, KY013484 to KY013486.This whole-genome shotgun project has been deposited in GenBank under the following accession numbers: MIRV,


WT), mutant peripherin-2 (perMT), or Rom-1 complexes, which can be formed in patients heterozygous for peripherin-2 mutations. Both mutants are misfolded and lead to decreased binding to perWT and Rom-1. Furthermore, both mutants are preferentially forming non-covalent perMT-perMT, perWT-perMT, and Rom-1-perMT dimers. However, only perWT-perMT, but not perMT-perMT or Rom-1-perMT complexes could be targeted to murine rod OS. Our study provides first evidence that non-covalent perWT-perMT dimers can be targeted to rod OS. Finally, our study unravels unexpected opposing roles of perWT and Rom-1 in rod OS targeting of adRP-linked peripherin-2 mutants and suggests a new treatment strategy for the affected individuals.Mutations in the photoreceptor outer segment (OS) specific peripherin-2 lead to autosomal dominant retinitis pigmentosa (adRP). By contrast, mutations in the peripherin-2 homolog Rom-1 cause digenic RP in combination with certain heterozygous mutations in peripherin-2. The mechanisms underlying the differential role of peripherin-2 and Rom-1 in RP pathophysiology remained elusive so far. Here, focusing on two adRP-linked peripherin-2 mutants, P210L and C214S, we analyzed the binding characteristics, protein assembly, and rod OS targeting of wild type (per These homomeric peripherin-2 complexes are crucial for proper OS morphology and architecture4. Peripherin-2 also forms heteromeric complexes with its homolog Rom-1, however, the functional importance of these complexes remained unclear6. Autosomal dominant retinitis pigmentosa (adRP) is the most frequent hereditary retinal disorder characterized by progressive loss of vision due to rod photoreceptor degeneration. Mutations in the peripherin-2 gene (PRPH2) are among the most common causes for adRP8. Interestingly, despite the high sequence identity (approx. 35%) and the high structural conservation of Rom-1 and peripherin-29, mutations in Rom-1 are not clearly linked to monogenic adRP. Some homozygous Rom-1 mutations, however, cause digenic RP in combination with a heterozygous mutation in PRPH2, suggesting that Rom-1 could function as genetic modifier that shapes the disease progression11. Nevertheless, the molecular mechanisms underlying the postulated modifier function of Rom-1 have not been determined yet12. The majority of adRP-linked peripherin-2 mutants are located within the large loop domain connecting the transmembrane domain 3 and 4, also known as EC2 or D2 loop domain14. Some adRP-linked peripherin-2 mutants in EC2 were shown to influence the subunit assembly16. In this context, it was postulated that peripherin-2 tetramerization is crucial for rod OS targeting. However, this conclusion was drawn from experiments addressing pure homotypic perMT complexes16. Nevertheless, due to the autosomal dominant fashion of PRPH2-linked adRP, different combinations of peripherin-2 protein complexes  can be formed in heterozygous patients carrying wild type and mutant PRPH2 alleles. In addition, each of these complexes can exist in different equilibria of mono-, di- and tetramers and higher-order oligomers, respectively. This high complexity precluded a more accurate investigation of how subunit assembly might influence the rod OS targeting of the single peripherin-2 protein complexes.The tetraspanin peripherin-2 is exclusively expressed in outer segments (OS), specific light detecting compartments of photoreceptors. Peripherin-2 forms homo- and heteromeric protein complexes in OS. The core homomeric peripherin-2 unit is the non-covalent tetramer, which can also assemble into covalently linked octamers and higher-order oligomers19. Recently, we also reported that the two adRP-linked mutations (perP210L and perC214S) located within the highly conserved tetraspanin PxxCC motif are mislocalized to rod inner segments (IS) when expressed in wild type mice using adeno-associated virus (AAV) vectors20. Other studies demonstrated that perC214S affects homotypic mutant interactions and the heteromerization with Rom-122. Despite these findings, little is known about the specific binding characteristics of this and other peripherin-2 mutants to perWT or Rom-1 in their native environment (e.g. rod OS). Moreover, the precise contribution of perWT and Rom-1 on binding properties, subunit assembly, and rod OS targeting of the respective perWT-perMT and Rom-1-perMT complexes is not clear. Nevertheless, such information is crucial to understand the full complexity of PRPH2-linked adRP and for developing successful treatments for the disease. Here, combining different methods we provide novel insights into the binding characteristics, subunit assembly, and rod OS targeting of perP210L and perC214S mutants. We show that perWT and Rom-1 form heterodimers with both peripherin-2 mutants. However, only perWT-perMT but not Rom1-perMT complexes could be targeted to rod OS suggesting unexpected opposing roles of peripherin-2 and Rom-1 in rod OS targeting of adRP-linked peripherin-2 mutants.In previous studies, we provided a proof-of-principle for FRET-based quantitative analysis of protein-protein interactions in isolated rod and cone OSC214S and perP210L, eight additional disease-linked peripherin-2 D2 loop domain mutants did not affect rod OS targeting20  experiments from HEK293T cells co-transfected with the respective peripherin-2 mutants and perWT or Rom-1, respectively. Only perC214S and perP210L attenuated the binding to perWT, while binding of other peripherin-2 mutants to perWT remained unaffected  be crucial for proper folding of peripherin-2 or ii) represent the protein-protein interaction interface. Using CD spectroscopy, a recent study suggested that C214S impedes protein folding by increasing the percentage of ß-sheets concomitant with a decrease in helices in the D2 loop23. However, it remained unclear which part(s) of the D2 loop is (are) affected by this mutation. To address this issue more directly, we established a protease digestion assay on membrane preparations from HEK293T cells expressing citrine-tagged WT and mutant peripherin-2 transgenes. For this purpose, we took advantage of the fact that endogenously expressed proteases are regularly present in these preparations. In addition, membrane preparations are expected to retain the native protein structure as they are isolated without detergents or reducing agents. Protein cleavage occurs on the exposed domain regions, which are accessible for endogenous proteases. Accordingly, improper protein folding could lead to masking of existing or to an exposure of hidden protease cleavage sites. Using a GFP antibody that recognizes the N-terminal citrine tag of our peripherin-2 constructs, we could detect three peripherin-2 bands at 61.5 kDa, 52.4 kDa and 42.5 kDa  and the cysteine at position 214 (C214) are part of the highly conserved tetraspanin PxxCC motif, whose function has not been clearly determined yet. The reduced binding of perkDa Fig. . The caltif Fig. . This ban 4 Fig. . Hence, 24. However, it remained unclear whether the perC214S and perP210L residues or flanking regions might also contribute to this type of interactions. To test this possibility, we performed a peptide competition assay using a peptide corresponding to the residues 201–220 of native human peripherin-2. Provided that the positions P210 and C214 participate in peripherin-2 homomerization, this peptide should compete with the binding and, hence, result in a reduction of homomeric protein-protein interactions. However, in subsequent co-IP experiments even in the presence of a very high peptide concentration (10 mM), we could not detect any decrease in peripherin-2 binding compared to the peptide-free approach  varies between the single FRET measurements. By plotting the FRET efficiency (EA) values against the corresponding cerulean/citrine MR, we observed a sufficient variability of the FRET fluorophore expression for each combination. The maximal FRET efficiency (EAmax) was calculated from the binding curve for saturable binding as described previously17 and is proportional to the binding strength of the respective protein complex in the equilibrium. Analysis of EAmax for the different perWT and perMT combinations allowed us to draw several important conclusions. First, robust and unusually high EAmax values could be observed for the perWT only FRET pair  and perC214S only (26.47%) FRET pairs when compared to perWT only  and the perWT-perC214S (8.58%) combinations, led to a robust decrease in EAmax  indicating that heteromeric Rom-1-perWT complexes bind less tight than their homomeric perWT only counterparts  transiently expressed in HEK293T cells. In line with previous work, we found the perWT complexes only in sucrose gradient fractions containing the non-covalent tetramers as well as in fractions containing the disulfide-linked octamers under non-reducing conditions  allows for a separation of different covalently or non-covalently linked protein complexes across a sucrose gradient according to their specific molecular weight. Using SDGC, previous studies analyzed the impact of peripherin-2 mutants including perons Fig. . By conters Fig. . Similarone Fig. . In presons Fig. . Moreoveons Fig. . Given tcf. Fig. , we conctes Fig. . Moreoveons Fig. . Taking cf. Fig.  and thatcf. Fig. , we concely Fig. .Figure 3WT, Rom-1-perP210L, and Rom-1-perC214S complexes. As expected, in the Rom-1-perWT combination a robust Rom-1 signal was found in non-covalent tetramer fractions  induces retinal degeneration27. In line with the postulated dose-dependency, transgenic mice expressing the perC214S mutant show reduced amounts of mutant protein in rod OS  which results in haploinsufficiency22. Interestingly, genetic supplementation of perWT on the perC214S mutant background led to a noticeable rescue of the retinal function28. Although helpful in initial stages of therapy development, the application spectrum of genetic supplementation is restricted to model organisms. By contrast, rAAV-mediated gene delivery has proven to be a very promising technique for therapeutic intervention on retinal diseases like retinitis pigmentosa in mammals32. Our data suggest that rAAV-mediated gene supplementation of perWT could support a rescue of perMT targeting defects to rod OS in adRP patients carrying mislocalizing peripherin-2 mutations. We could not detect any detrimental effects on outer nuclear layer (ONL) thickness in fully developed retinas of WT animals co-injected with perWT and perMT transgenes six months post injection  did not change the accessibility of the proteases in our assay. This emphasizes the crucial role of the proline at position 210 and cysteine at position 214 for the proper folding of the D2 loop domain. Furthermore, our results suggest that the apparent reduction of perMT binding to perWT in co-IP and in FRET experiments from HEK293T cells is most likely caused by protein misfolding. Our FRET experiments on isolated rod OS indicate that this apparent decrease in binding to perWT does not result from reduced binding affinities, but rather seems to be a consequence of reduced initial binding kinetics of perP210L and perC214S to perWT. In other words, this suggests that the first step of perMT binding to perWT is impeded, however, once bound, the affinity of both mutants to perWT remains largely unchanged. These results are well matched on our peptide competition assay indicating that the PxxCC motif does not represent the interface for homomeric peripherin-2 interactions. EAmax values resulting from the transgenic perWT were higher in HEK293T cells compared to the rod OS. This difference could be caused by non-labeled endogenous peripherin-2 or Rom-1 in the retina, which are also expected to bind to transgenic perWT (incomplete labeling). However, the perWT EAmax values from isolated rod OS are still in a very high range highlighting the suitability of the FRET approach to addressing peripherin-2 oligomerization in these compartments.P210 and C214 are located within the PxxCC motif, which is conserved among most tetraspaninsWT-perMT dimers can be formed and targeted to rod OS. 2) Non-covalent Rom-1-perMT heterodimers are stacked in the inner segments. The first deduction is very surprising as non-covalent tetramerization was considered to be crucial for peripherin-2 targeting to rods16. This conclusion was drawn from different sets of experiments in previous studies: i) When analyzing perWT complexes isolated from OS, no dimers could be detected35. ii) Tetramerization-deficient peripherin-2 mutants could not be targeted to rod OS16. iii) Peripherin-2 mutants which preferentially form homodimers are not targeted to rod OS16. However, none of these experiments is sufficient to state if perWT-perWT or perWT-perMT dimers can also be targeted to rod OS. First, perWT does not form dimers in SDGC experiments when expressed alone, which explains its absence in rod OS under physiological conditions. Second, only rod OS targeting of pure perMT complexes was examined so far16. Consequently, to the best of our knowledge, this is the first study addressing the principle capability of perWT-perMT dimers to be targeted to rod OS. The finding that Rom-1-perMT heterodimers cannot be targeted to rod OS suggests that Rom-1 cannot fully compensate for the peripherin-2 function in rods under these conditions. Currently, one can only speculate about the physiological role of these opposing effects of peripherin-2 and Rom-1 in terms of binding and targeting of misfolded peripherin-2 mutants. A recent study revealed that Rom-1 promotes the trafficking of the Y141C peripherin-2 mutant in vitro and converts the cone-dominant pattern dystrophy to RP phenotype in vivo12. Similar effects are not expected to occur for the C214S or P210L mutation, as Rom-1 cannot promote targeting of these mutants to rod OS. Together, these findings indicate that Rom-1 might play a yet undefined role in pathophysiology of retinal diseases as a mutation-dependent disease modifier.When correlating our results from SDGC to those obtained from rod OS targeting experiments Fig. , two ver13. Despite some advances, the molecular mechanisms underpinning this differential penetrance of peripherin-2 mutants in rods and cones are still not fully understood. In light of our results, it is tempting to speculate that Rom-1 might be one of the key players shaping the penetrance of peripherin-2 mutants in rods and cones. Finally, in the context of gene therapy, the postulated novel function of Rom-1 raises the possibility that the efficiency of gene supplementation therapy of adRP-associated peripherin-2 point mutations could be improved by co-delivering peripherin-2 with Rom-1.Rom-1 and peripherin-2 are also expressed in cones and many mutations in peripherin-2 (but not in Rom-1) are linked to retinal diseases primarily affecting cone photoreceptorsThe studies were carried out in accordance with the approved guidelines of the local committee of laboratory animal care (District Government of Upper Bavaria) and German Laws on animal welfare (Tierschutzgesetz). Anesthesia was performed by intraperitoneal injection of ketamine (40 mg/kg body weight) and xylazine (20 mg/kg body weight). Euthanasia was performed by cervical dislocation.20 and Rom-1 transgenes were subcloned to the pAAV2/8 YF vector30 containing the human Rhodopsin (hRHO) promoter using standard cloning techniques. For expression in HEK293T cells , the peripherin-2 and Rom-1 transgenes were subcloned to the standard pcDNA3.1 vector . Peripherin-2 mutants were introduced by site directed mutagenesis . All transgenes were sequenced prior to use.For expression in rods, the single human peripherin-236. Co-IPs were performed with the anti-myc antibody  using Protein G magnetic beads  according to the manufacturer’s protocol.For co-immunoprecipitations (co-IPs) transiently transfected HEK293T cells were harvested 48 hours post transfection and membrane preparations were conducted as describedFor western blotting, injected retinas of light adapted wild type C57BL6/J animals were used four weeks post injection. The dissected retinas were homogenized in membrane preparation buffer using the Potter S homogenizer .36 of transfected HEK293T cells were incubated for 5 h at room temperature (RT) and processed for western blotting. No protease inhibitors were added to the membrane preparations prior to the incubation. For immunoblotting, antibodies were used in following dilutions: mouse anti-myc (see above), 1:2000; mouse anti-GFP , 1:2000; mouse anti-ATPase , 1:2000.For peptide competition assay, the peripherin-2 peptide (peptide sequence CDGRYLVDGVPFSCCNPSSPR) was obtained from jpt Innovative Peptide Solutions . The peptide was added to the respective membrane preparation and the solution was incubated with the myc-antibody-coupled magnetic beads for 30 min at room temperature. The co-IPs containing the peptide-free membrane preparations were processed under identical conditions. For the native protease cleavage assay, membrane preparations2 using the Potter S homogenizer (see above). Continuous density gradients of 5–20% (wt/vol) sucrose were prepared by underlayering 0.5 ml of 5%, 10%, 15% and 20% sucrose containing 0.1% Triton X-100 and 10 mM N-ethylmaleimide . The gradient was left at RT for 1 h to facilitate diffusion and chilled on ice for 30 min prior to sample application. To determine the weight of single peripherin-2 and Rom-1 protein complexes in each immunoblot, DNA standards of defined molecular weights ) were mixed to lysates containing 200 µg of the respective proteins. The mixture was carefully layered on top of the gradient and centrifuged at 46,700 rpm for 2 h at 4 °C in a Beckman Coulter Optima L-80K Ultracentrifuge . Afterwards, the centrifuge tube was punctured and fractions were collected dropwise (5 drops/tube). Fractions from each sucrose gradient were separately used for immunoblotting with myc- or flag-specific antibodies and for agarose gel electrophoresis to detect the DNA standards. The suitability of DNA standards was first validated for perWT only. As shown in Fig. WT gives rise to well-defined fractions of non-covalent tetramers, covalent octamers, and high-order oligomers. The molecular weight of the DNA in the same fractions very nicely correlated with the molecular weights of the corresponding perWT complexes. Therefore, for each of the remaining immunoblots, DNA standards were used to determine the fractions containing the different perWT, perMT, or Rom-1 complexes.For sucrose density gradient centrifugation, HEK293T cells were transiently transfected with the respective peripherin-2 constructs. Cells were harvested 48 h post transfection and homogenized in a lysis buffer comprising 0.5% Triton X-100, 150 mM NaCl and 2 mM CaClet al.37 with modifications published in Becirovic et al. and Nguyen et al.19. Briefly, transfected HEK293T cells and isolated rod OS were measured in a FRET imaging solution comprising 2 mM CaCl2, 10 mM glucose, 10 mM HEPES sodium salt, 5 mM KCl, 1 mM MgCl2, 140 mM NaCl, pH 7.4, at RT. 3 cube FRET measurements were conducted using a Leica DMI6000B inverted fluorescent microscope  in combination with a photomultiplier detection system including a photomultiplier tube . As excitation source, a DeltaRamX monochromator was used and FRET data were acquired with FelixGX software . FRET filter cubes were as follows : cerulean/CFP , FRET  and citrine/YFP  . The fluorescence intensities were measured in single cells co-expressing varying, but sufficient levels of cerulean- and citrine-tagged proteins with the respective filter cube. Subsequent FRET ratios (FR) were calculated from the signal intensities according to the 3 cube FRET equation described in Shaltiel et al.38. Apparent FRET efficiencies (EA) at given cerulean/citrine molar ratios (MR) were obtained from equation  and Excel .3 cube FRET was performed as described in detail in Butz Amax is the maximal FRET efficiency that can be calculated for saturated donor concentrations and K is an analogon of the dissociation constant. Ex vivo FRET experiments were conducted on single rod OS isolated from six pooled retinas for each FRET pair.Binding curves were calculated using equation :2\docume30. 1010 rAAV particles were delivered subretinally to wild type C57Bl6/J mice by a single injection on postnatal day 14. Four weeks post injection, all injected retinas were analyzed for fluorescence using scanning laser ophthalmoscopy .The production of single-strand AAVs (rAAV 2/8 YF) containing the hRHO promoter and the subretinal injection technique were described previously19. Briefly, mice were sacrificed four weeks post injection and the isolated retinas were collected in a 1.5-ml reaction tube containing 100 µl of PBS. The OS were separated from the retina by vortexing for 15–30 s and centrifuging for 30 s at 500 × g. Afterwards the OS containing supernatant was collected and used for subsequent FRET measurements.A detailed procedure of rod OS preparation was described recently39. Retinal and OS images were obtained by the TCS SP8 confocal scan microscope , acquired with the LASX software , and processed with the ImageJ software . We used the rabbit anti-CNGB1a antibody (1:10.00040) as marker for rod outer segments. Citrine was excited with the 514 nm laser and detected with the Hybrid (HyD) detector using a 520–540 nm emission filter. Cerulean was excited using the 448 nm laser and detected via the HyD detector with a 460–480 nm emission filter. Images were taken using the 40x and 63x objectives.For immunohistology, wild type C57BL6/J mice were injected on P14 with the respective rAAV particles. Four weeks post injection, the retinas were dissected and processed for immunohistology as described41. Fluorescence signals were examined at 488 nm excitation for citrine/cerulean detection (BP 550/49 nm emission filter).Confocal scanning laser ophthalmoscopy (cSLO) and optical coherence tomography (OCT) were performed 6 months post injection using a modified Spectralis HRA + OCT system  as described recentlyn is the number of trials. For multiple comparisons one-way ANOVA followed by the Tukey’s test was used.All values are given as mean ± SEM, and All data generated or analyzed during this study are included in this published article (and its Supplementary Information files).Supplementary Information


Congenital Zika virus infection is associated with severe brain anomalies and impaired function. To determine outcomes, we followed 2 affected children for ≈30 months. For 1 who was symptomatic at birth, transient hepatitis developed. However, neurodevelopment for both children was age appropriate. Zika virus, a flavivirus, is a teratogenic and neurotropic infectious pathogen  and neonate (day 3) and in breast milk on day 2. The neonate was breastfed for 2 months. He remained asymptomatic, and his neurologic development followed a typical course. At 32 months of age, CDAS scores indicated a need to monitor motor development but overall did not indicate neurocognitive problems.9 thrombocytes/L), leukopenia (4.6 × 109 cells/L), cytolysis, and cholestasis . Signs of Zika virus infection (rash) appeared in the mother on day 3 and in the neonate on day 4. Reverse transcription PCRs for Zika virus of blood and urine were positive for the mother (day 1) and the neonate (days 4 and 7) and in breast milk on day 8. On day 2, laboratory testing of blood from the neonate indicated thrombocytopenia , was associated with neither marked illness at birth nor neurodevelopmental deficits by 30 months of age. However, assessment of late-onset cognitive or sensory deficits requires longer follow-up. Transmission is assumed unlikely to occur through breastfeeding; Zika virus isolated from milk is not replicative  infections. This milder pattern of peripartum Zika virus infection differs from the usually severe neonatal dengue virus infection (severe thrombocytopenia) and peripartum chikungunya virus infection .,Our findings, along with findings of mild brain lesions  after late intrauterine exposure to Zika virus  of the international community, which should enable harmonized follow-up and evaluation of developmental outcomes for children exposed to Zika virus.Example of the Child Development Assessment Scale for case-patient 2.


Here, we report the complete coding genome sequence of a novel reassortant and very virulent infectious bursal disease virus (IBDV), designated JBN2011. Characterization of the JBN2011 genome suggests that it is a rare recombinant virus having a very virulent IBDV segment A and a Bursine-2-like attenuated IBDV segment B. Infectious bursal disease virus (IBDV) is an important viral pathogen of poultry. Two serotypes of IBDV are recognized, serotype 1 viruses, which are pathogenic for chickens, and serotype 2 viruses, which are not. Serotype 1 viruses are further classified into the following four pathotypes: attenuated (at), classical virulent (cv), antigenic variant (av), and very virulent (vv)  from a dead 4-week-old broiler chicken suspected of having infectious bursal disease .Segment A has a coding region  containing two partially overlapping open reading frames that encode VP5 (159 amino acids [aa]) and a precursor polyprotein (pp) . During virus replication, the PP is cleaved to yield VP2, VP4, and VP3 by autocatalysis. The VP5 protein contains four extra N-terminal aa residues (MLSL), as found in vvIBDV and cvIBDV . PhylogeSegment B encoding VP1 (881 aa) harbored aa substitutions at positions I61V, D146E, N147G, E242D, A287T, M390L, D393E, P562S, P687S, and R695K, which are typical of non-vvIBDVs 710. In paMF188862 (segment A) and MF188863 (segment B).The sequence of JBN2011 has been deposited in GenBank under accession numbers


A Zika virus disease outbreak occurred in Roatán, Honduras, during September 2015–July 2016. Blood samples and clinical information were obtained from 183 patients given a clinical diagnosis of suspected dengue virus infection. A total of 79 patients were positive for Zika virus, 13 for chikungunya virus, and 6 for dengue virus. Zika virus is a mosquitoborne flavivirus that is clinically nonspecific and associated with severe congenital injury  and laboratory diagnostic findings for cases of presumptive dengue. As of September 2015, Zika virus had not been identified in Roatán. We report emergence of Zika virus on Roatán.We conducted a cross-sectional survey of patients who came to Public Hospital Roatán during September 2015–July 2016 and were given a clinical diagnosis of suspected dengue fever. This hospital, the only public hospital in Roatán, has 58 inpatient beds. Approximately 90% of dengue cases on Roatán are diagnosed at this hospital; most case-patients came to the emergency department, which had 18,578 visits in 2015 .We used a convenience sampling approach. All patients >2 years of age given a diagnosis of clinically suspected DENV infection at the hospital during the enrollment period were eligible. Suspected DENV infection was diagnosed by using criteria of the Honduran Ministry of Health, which are fever plus 2 of the following: nausea/vomiting, rash, headache/retroorbital pain, myalgias/arthralgias, petechiae/positive tourniquet test result, leukopenia, and bleeding.am–4:00 pm) Monday through Friday. Patients underwent a phlebotomy and completed a clinical questionnaire, administered verbally in English or Spanish that addressed demographics, migration history, employment, medical history, and symptoms and signs of arbovirus infection.Patients were enrolled after we obtained informed consent; minors were enrolled only after we obtained parental permission. Ethical approval was granted by institutional review boards of the University of California, San Francisco, and Universidad Nacional Autónoma de Honduras. Enrollment of participants and blood collection were restricted to workday hours (8:00 Samples of whole blood (5–9 mL) were collected in EDTA-treated vacutainers  within 1 h of diagnosis. Samples were centrifuged , which yielded sufficient plasma to prepare 5 equal aliquots . Aliquots were labeled with patient identification numbers and stored frozen at −30°C pending for testing.In July 2016, samples were shipped on dry ice to Blood Systems Research Institute  where initial testing was performed by using the Trioplex Assay  for detection of DENV, chikungunya virus (CHIKV), and Zika virus RNA. RNA was extracted from 140 μL of plasma and eluted in 60 μL of buffer .Multiplex real-time reverse transcription PCR (RT-PCR) was performed using the SuperScript III Platinum One-Step qRT-PCR Kit  with DENV, CHIKV, and Zika virus primers and probes developed at the Centers for Disease Control and Prevention. Each duplicate reaction contained 10 μL of sample RNA in a reaction volume of 25 µL. Samples were tested in a 96-well format in a real-time instrument . Results were considered positive if the cycle threshold was <38.A more sensitive Zika virus–only test based on transcription-mediated amplification , which processed 0.5-mL plasma into each amplification reaction, was performed in parallel for all samples to confirm Zika virus infections detected by the Trioplex Assay and detect low levels of Zika virus RNA , which generated global positioning system coordinates for their homes. The survey, laboratory test data, and global positioning system coordinates were uploaded into ArcGIS version 10.3.1 software . All potential predictors of Zika virus infection were analyzed by using univariate logistic regression models in Stata version 13 . Characteristics that were significant (p = 0.10) by univariate analysis were entered into a multivariable logistic regression model.A total of 183 patients participated and provided blood samples . Most  and headache  were independently associated with Zika virus infection. Fever  and vomiting  were associated with a decreased risk for Zika virus infection .This study had limitations. Our exclusive enrollment of patients who came to Public Hospital Roatán excluded patients who came to other healthcare facilities or outside daytime working hours who were not sufficiently symptomatic to seek care. Alternative body fluids  were not available for testing, and serologic testing for Zika virus was not performed because of cost constraints and cross-reactivity between Zika virus and DENV antibodies. We did not perform confirmatory testing of CHIKV- and DENV-positive samples. This study could be biased for detecting Zika virus and not DENV/CHIKV because of use of a more sensitive assay.We demonstrated the evolution of the Zika virus disease outbreak in Roatán, Honduras, in early 2016. Findings highlight challenges in case ascertainment based only on clinical diagnosis. In the absence of laboratory confirmation, clinical diagnosis has low specificity because of overlap of signs and symptoms between infections with Zika virus, DENV, CHIKV, and a host of other regionally endemic infections. Enrollment was contingent upon a suspected diagnosis of dengue. However, DENV was detected in <5% of case-patients, whereas 43% of case-patients had acute Zika virus infections.After a rapid peak in early February 2016, there was a slow decrease in reported cases through July 2016, when enrollment concluded. Access to laboratory testing remains a barrier to surveillance and clinical management in low-resource countries where Zika virus disease outbreaks are predominantly focused, but laboratory infrastructure is lacking (