Previously, we reported two distinct types of virus infecting thraustochytrids  suggesting they have wide distributions in the host-virus systems of coastal environments. Here we conducted a field survey from 2004 through 2005 to show the fluctuation pattern of thraustochytrids and their viruses in Hiroshima Bay, Japan. During the field survey, we monitored the dynamics of the two types of thraustochytrid-infecting virus: small viruses causing lysis of Aurantiochytrium sp. NIBH N1-27 (identified as AuRNAV) and the large viruses of Sicyoidochytrium minutum NBRC 102975 (similar to SmDNAV in physiology and morphology). Fluctuation patterns of the two distinct types of virus were different from each other. This may reflect the difference in the preference of organic substrates; i.e., it may be likely the host of AuRNAV (Aurantiochytrium sp.) increases utilizing algal dead bodies or feeble cells as the virus shows a large increase in abundance following raphidophyte blooms; whereas, the trophic nutrient supply for S. minutum may primarily depend on other constantly-supplied organic compounds because it did not show any significant change in abundance throughout the survey. Further study concerning the population composition of thraustochytrids and their viruses may demonstrate the microbial ecology  of marine environments.Thraustochytrids are cosmopolitan osmotrophic or heterotrophic microorganisms that are considered as important decomposers in coastal ecosystems. However, because of a lack of estimation method for each genus or systematic group of them, relatively little is known about their ecology  Their bHeterosigma akashiwo-HaV(Heterosigma akashiwo virus) and Heterocapsa circularisquama-HcRNAV (Heterocapsa circularisquama RNA virus) are well known host-virus systems [On the other hand, viruses are highly abundant in marine environments and are recognized as important pathogens in controlling bacterial and algal biomass , 14, nut systems , 19. In  systems , 21. ConAurantiochytrium RNA virus: reported as SssRNAV) and SmDNAV (Sicyoidochytrium minutum DNA virus) [Aurantiochytrium sp.  , 23. AuRd Honda, ); and SmWater samples from the surface layer (0 m) and 0.2 m above the sediment-water interface (B-0.2 m) were collected in 2004 (May—Dec) and 2005 (Apr—Aug) at a semi-enclosed basin  located in Hiroshima Bay, Japan . No specDuring the survey, the water temperature and salinity (psu) of the water column ranged at 16.4–28.1°C and 16.6–30.3, respectively.Phytoplankton abundance was enumerated by direct count using light microscopy. During the 2005 survey, the abundance of thraustochytrids was estimated using a modified MPN (most probable number) technique with pine pollen .Virus titration of seawater samples (0 m and B-0.2 m) and sediment samples was conducted using the extinction dilution method . Six thrOne clonal virus was isolated from each MPN assay. Clonal pathogens were obtained using two cycles of the extinction dilution procedure  with theTEM observation of the isolated viral clones was performed to show their morphology. Negatively stained viruses were prepared according to the method of Takao et al. ; and obsAurantiochytrium sp. NIBH N1-27 was AuRNAV . An AuRNAV-specific probe was synthesized using the AlkPhos Direct Labelling and Detection System with CDP-Star  and the cDNA of AuRNAV including the non-coding and ORF2 region  as the template [Aurantiochytrium sp. NIBH N1-27) and incubated for 48 h. This is long enough for the tested-viruses to complete one replication cycle as previously evaluated [+ (Amersham Biosciences); then, hybridization was performed according to the manufacturer's protocol using the above mentioned AuRNAV-specific probe; and detected with chemical illumination using a LAS-3000 Northern dot-blot analysis was performed to identify whether the viral isolate causing lysis of template . Each vivaluated . Two-μl Aurantiochytrium sp. NIBH N1-27 and S. minutum NBRC 102975, respectively. TEM observation revealed the former were small icosahedral viruses similar to AuRNAV  and the latter were large ellipse viruses resembling SmDNAV in morphology (ca. 150 nm in diameter and lacking a tail) (Aurantiochytrium sp. NIBH N1-27 were identified as AuRNAV [S. minutum NBRC 102975 were considered to be (or closely related to) SmDNAV [We succeeded in isolating 118 viral isolates infecting thraustochytrids during the field surveys conducted in 2004 and 2005. Among the clones, 52 and 66 were infectious to  a tail) . Norther a tail) . Integras AuRNAV ; whereas) SmDNAV .Chaetoceros and Skeletonema dominated in the water column almost throughout the survey period except during a Heterosigma akashiwo bloom where the highest cell density was 9.7 × 104 cells ml-1 on Jun 11  and Sicyoidochytrium minutum NBRC 102975 (SmDNAV-like viruses) were detected and successfully isolated (3 infectious units ml-1) at ten days after the H. akashiwo bloom termination. Then, they rapidly decreased to below the detection limit at 3.0 infectious units ml-1 after 23 Jul . Therefore, we thought that the spike peak of thraustochytrids existed after the H. akashiwo bloom but before the peak of AuRNAV. To confirm the hypothesis, we also estimated the abundance of thraustochytrids during the field survey in 2005.Considering the fact that viruses can’t reproduce without their specific host, fluctuations in abundance of certain virus may reflect the host dynamics. In addition, our preliminary inoculation experiments showed that H. akashiwo bloom and short period dominance of Thalassiosira spp. (14–19 Jul)  was detected with a maximum cell density of 8.8 × 103 cells ml-1 during Jun 23 to 27  . H. akas23 to 27 .2 infectious units ml-1) at the end of the H. akashiwo bloom, and one month later  (2 infectious units ml-1) on 26 Jul  . SmDNAV-n 26 Jul .1 to 2.3 × 105 cells L-1. An increase accompanied with an increase of AuRNAV was observed at the end of the H. akashiwo bloom (from 23 Jun through 5 Jul) and was followed by a drastic decrease; then, they showed a moderate increase . The spike peak of AuRNAV detected in 2004 was considered as the result of virus infection to the dominant thraustochytrids. On the other hand, in 2005, land water supply was less than 2004 before the H. akashiwo bloom. Then, the bloom scale was not as large as in 2004  dominated (14–19 Jul). In this case, it is considered that Aurantiochytrium sp. NIBH N1-27-type thraustochytrids increased by utilizing Thalassiosira sp. cells. Gaertner [Aurantiochytrium sp. NIBH N1-27 attached and propagated on the surface of diatom cells, as was observed in the case of H. akashiwo cells (data not shown). These results suggest that Aurantiochytrium sp. NIBH N1-27-type thraustochytrids may have the ability to effectively utilize the dead cells or feeble cells of phytoplankton .Peaks of AuRNAV and thraustochytrids were also detected from 19 Jul through 2 Aug 2005  followinGaertner  reportedH. akashiwo bloom (S. minutum may depend on constantly supplied organic matter (ex. DOM/POM from land). In 2005, the dynamics of SmDNAV-like viruses showed a moderate increase in abundance at the surface layer after a drastic inflow of huge amount of river water , and large viruses infecting Sicyoidochytrium minutum NBRC 102975 (D and F).AuRNAV (A), SmDNAV (B), small viruses infecting (EPS)Click here for additional data file.S2 FigRhizosolenia setigera RNA virus (A2), SmDNAV(A3), AuRNAV strains  isolated from other part of Japan coast area (A4–8), Viruses infecting Aurantiochytrium sp. NIBH N1-27 isolated in this study (A9—F10).AuRNAV (A1), (EPS)Click here for additional data file.


Here, we report the complete polyprotein sequence of a dengue virus 2 strain isolated in Brazil. This virus belongs to the American genotype and has the ability to cause neurovirulence in immunocompetent adult mice. The data presented here may help understand the genetic determinants responsible for neurovirulence. Dengue virus (DENV), which is composed of four distinct serotypes (DENV1 to -4), is responsible for the most prevalent arthropod-borne viral infection of humans in tropical and subtropical countries. The mechanisms involved in dengue pathogenesis and severity of the symptoms vary according to host and viral factors. In fact, natural DENV variants may express distinct molecular and physiological features that can impact viral fitness and virulence.We report here the near-full-length genome sequence of an American strain of DENV2 JHA1 strain). This virus was isolated from a patient in Belém do Pará, Brazil, presenting with dengue fever  genes of the JHA1 strain showed that the virus belongs to the serotype 2 American genotype. Since the capacity to kill immunocompetent adult mice after intracranial inoculation is an atypical virulence property of this virus, we decided to determine the full-length sequence encoding the viral polyprotein.The virus was PCR amplified using a previously described protocol  with thrn = 29; GenBank accession numbers HM582099 to HM582117, GQ868588, GQ868589, GQ868599, EU056811, EU0568112, FJ898449, JX966380, AY702040, and AY744147). Genetic distance was estimated for the American viruses (excluding JHA1) and also between JHA1 and other American strains. The average distance (p-distance) among American viruses (without JHA1) was 0.015, which is in accordance with the genetic distance observed for viruses belonging to the same genotype. However, JHA1 presented about 6.0% divergence (p-distance = 0.058 to 0.064) from other American viruses. Intriguingly, according to the envelope-based tree, JHA1 was very closely related to Trin53, which was isolated in 1953 from a female patient in Trinidad and Tobago  neurovirulent mouse-adapted strain revealed that both viruses have a lysine (K) at position 126, identified as a major determinant of neurovirulence in NGC .In sum, we described here the complete polyprotein sequence of a DENV2 isolated in Brazil, which is atypically neurovirulent to adult immunocompetent mice and belongs to the American genotype.JQ686088. The version described in this paper is the updated version, JQ686088.2.The genome described in this work has been deposited in GenBank under the accession no.


N-octadecylchitosan, which consists of 70 mol% of 2-(octadecylamino)-2-deoxy-d-glucopyranose, 17 mol% of 2-amino-2-deoxy-d-glucopyranose, and 13 mol% of 2-acetamido-2-deoxy-d-glucopyranose, were covalently immobilized to carboxylated porous supports composed of chitosan and used for the adsorption of pyrogenic lipopolysaccharide. When human serum albumin solution, including 5 mg mL-1 of albumin and 5.6 ng mL-1 of lipopolysaccharide, was passed through a column packed with the resulting porous supports bearing lipid membranes assembled in nanoscale, lipopolysaccharide was removed to as low as a detection limit of 0.020 ng mL-1 with a quantitative recovery of protein. On the other hand, in the case of directly N-octadecylated porous supports having cationic and hydrophobic ligands which are not assembled as lipid membranes, lipopolysaccharide could not be removed to the detection limit and protein recovery was lower than the porous supports bearing lipid membranes. The difference above as well as difference from conventional adsorbents suggested that the selectivity was attributable to an interaction between the cationic lipid membranes of N-octadecylchitosan and lipopolysaccharide as well as protein. The porous supports bearing lipid membranes were stable in 0.5 M NaOH and 0.1 M HCl at ambient temperature. Considering the confirmed excellent selectivity and chemical stability, their practical use as separation media in the pharmaceutical manufacturing can be expected.Polymeric lipid membranes of  Lipid membranes have been receiving considerable attention as models for various chemical investigations. Densely packed alkyl chains with hydrophilic head groups can have both a hydrophobic interaction and a hydrophilic one with guest substrates. This specific environment provided by lipid membranes is essential for molecular recognition in biological membranes and has been studied in the areas of chemical sensing and separation ,2.-1 because of its strong pyrogenicity [Proteins or other biologically active substances, especially those produced by recombinant microorganisms, are usually contaminated with lipopolysaccharide (LPS) . LPS, whgenicity . Intensigenicity . For exagenicity  and affigenicity , polymyxgenicity , and polgenicity . Howevergenicity  and has N-octadecylchitosan consisting of 2-deoxy-2-octadecylamino-d-glucopyranose , and 2-acetamido-2-deoxy-d-glucopyranose (GlcNAc) to carboxylated porous supports composed of chitosan [-1 from human serum albumin (HSA) solution with a quantitative recovery of protein showing the possibility of their practical use.We have reported the covalent immobilization of polymeric lipid membranes of chitosan , and thechitosan . In this-1  which corresponded to 2.67 × 104 of molecular weight relative to poly(ethylene glycol). A cross-linked porous chitosan having a particle size of 45 to 420 μm and an average pore diameter of 2 μm, a product of Kurita Water Industries, Ltd. , was used as obtained. 1-Bromooctadecane, succinic anhydride , 1-ethyl-3-(3-(dimethylamino)propyl)-carbodiimide hydrochloride (water-soluble carbodiimide (WSC), Dojindo Laboratories, Kumamoto, Japan), N-hydroxysuccinimide (HOSu), and d-(+)-glucosamine hydrochloride  were used as obtained. A volume of adsorbent was determined after standing for 24 h in a measuring cylinder with water. Infrared (IR) spectra were recorded using IR-810 . Total organic carbon content analysis and differential scanning calorimetry (DSC) were carried out using TOC-5000A  and DSC220C , respectively. Scanning electron micrographs (SEM) and transmission electron micrographs (TEM) were taken at JEOL DATUM  using JSM-6400 F (JEOL) and JEM-1200EX (JEOL), respectively. DEAE-Sepharose CL-6B and Pyrosep  were obtained from manufacturers. HSA (20% w/v) and LPS (Escherichia coli serotype O127:B8) were products of Nihon Pharmaceutical Co., Ltd.  and Difco Laboratories , respectively, and used as obtained. Toxicolor , which is a chromogenic Limulus amebocyte lysate test, was used as an assay method for LPS. Samples containing LPS were diluted with Tris–HCl buffer (pH 8.0) to lower than 0.085 ng mL-1 of LPS and assayed by the method recommended by the manufacturer. The detection limit of LPS in this test was as low as 0.020 ng mL-1, which corresponded to 0.06 endotoxin unit. HSA concentration was measured by UV at 236 nm to avoid interference of a stabilizer N-acetyltryptophan showing adsorption at 280 nm.Chitosan was purchased from Dai-ichi Kogyo Seiyaku Co., Ltd. . The degree of deacetylation was determined as 87 mol% by colloidal titration. Intrinsic viscosity was 1.42 dL gN,N-dimethylacetamide to yield N-octadecylchitosan consisting 70 mol% of GlcNC18, 17 mol% of GlcN, and 13 mol% of GlcNAc. In DSC of N-octadecylchitosan, an endothermic peak was observed (Tc = 46°C) indicating a gel to liquid-crystalline phase transition. Dispersion liquid was prepared by suspending N-octadecylchitosan in water including hydrochloric acid and successive sonication. Electron microscopic observation of the dispersion liquid revealed the existence of unilamellar vesicles having diameters of 10 to 150 nm [N-octadecylchitosan was reacted with the carboxylated porous supports in the presence of WSC and HOSu to form amide bonds from primary amino groups of N-octadecylchitosan and carboxyl groups of the porous supports. The resulting materials were further reacted with N-acetylglucosamine to block the remaining carboxyl groups by amidation [Preparation of porous supports bearing lipid membranes is described briefly with the conceptual scheme Figure . Chitosao 150 nm . Carboxy2) was reacted with mild stirring using 1-bromooctadecane  in the presence of Na2CO3  at 70°C for 69 h in dimethylacetamide under nitrogen atmosphere. Particles were recovered by filtration and washed with water, warm ethyl acetate, ethanol, and water successively. Changes in the appearance and porous structure were not observed by SEM. In the IR spectra (KBr pellet), νCH of CH2 2,925 and 2,850 cm-1 was observed. Ion-exchange capacity was 2.3 meq g-1 dry particles. Using this value, the colloidal equivalent of chitosan  used for the preparation of the cross-linked porous chitosan and the ion-exchange capacity of the cross-linked porous chitosan (3.8 meq g-1) which was cross-linked by 1,6-diisocyanatohexane, GlcN of the cross-linked porous chitosan, and GlcNC18 of the resulting directly alkylated porous supports were calculated as 69 and 47 mol%, respectively. Therefore, about half of the monosaccharide units of the support particles are deemed to be octadecylated.After being washed by dimethylacetamide, the cross-linked porous chitosan .Purified water and buffer solutions were sterilized using an autoclave at 115°C to 121°C for 15 min. Glass wares were also sterilized using the autoclave at 250°C for 2 h. LPS aqueous solution, which was prepared by vortex mixing and dilution with water, was added to HSA preparation. The solution was filtered with a filter disk having 0.2-μm-diameter pores and diluted with a buffer solution to a desired concentration for column-wise experiments. Phosphate buffer was used for experiments at pH 7.0 and 8.0, and acetic acid buffer was used for those at pH 4.3 and 5.3. Buffer solutions were prepared by adding NaOH solution of a predetermined concentration to phosphoric acid or acetic acid to obtain the desired ionic strength . After washing with water for 3 h, 0.1 M acetic acid was passed through for 1.5 h to convert amino groups of N-octadecylchitosan immobilized on the supports to their ammonium forms. After the buffer solution was passed through for 6.5 h, HSA solution was passed through at 5 mL h-1 for 15 to 16 h. The eluted solution was collected immediately as ten fractions of 7.5 mL each. Fractions of 2, 4, 6, 8, and 10 were analyzed for the concentrations of LPS and HSA.Column-wise adsorption was done at 20°C. Adsorbents were suspended in water and fed into a glass column (8 mm i.d. × 100 mm length) using a LC-6A pump . The length of the gel bed was between 930 and 980 mm. The resulting column was washed with 0.5 M NaOH, at 10 mL hN-octadecylchitosan were immersed in 0.5 M NaOH or 0.1 M HCl at ambient temperature overnight and then washed with water and subjected to IR spectroscopic analysis. The supports (20 mL) were packed into a column . The packed column was filled with 0.5 M NaOH and allowed to stand overnight at 20°C. After washing with 200 mL of water, 50 mL of water was circulated in the column for 24 h at a flow rate of 1 BV h-1. The water was recovered and subjected to an analysis of total organic carbon content [Porous supports bearing lipid membranes of -1 (νC=O of COOH) and an increase of intensity at 1,655 and 1,560 cm-1 (νC=O of NHCO) indicating selective N-succinylation. After further reaction with the vesicular dispersion of N-octadecylchitosan, a small but distinct increase of νCH at 2,925 cm-1 and a disappearance of νC=O of COOH at 1,720 cm-1 were observed. The difference spectrum, N-octadecylchitosan-immobilized supports minus carboxylated ones, demonstrated νCH of N-octadecylchitosan methylenes at 2,925 and 2,850 cm-1 and νC=O of NHCO at 1,655 and 1,560 cm-1. These results supported the covalent immobilization of N-octadecylchitosan to the carboxylated supports by amide bonds. A rougher surface was observed at the scanning electron micrograph of N-octadecylchitosan-immobilized supports compared to carboxylated ones. Furthermore, threadlike materials in order of tens of angstrom thickness were observed around the fibrous support in TEM of ultrathin sections of the N-octadecylchitosan-immobilized supports  consisting of chitosan and N-octadecylchitosan. These values are considerably lower than that of the diethylaminoethyl (DEAE) group  used for usual anion-exchange chromatography and lead to a weak anion-exchange property. The difficulty of hydrophobic adsorption of albumin to lipid membranes in rigid gel phase also seems to be preferable for a good recovery of HSA [-1 with 96% of HSA recovery. It seems that LPS molecules which interacted with protein could be removed by the porous supports bearing lipid membranes by a strong interaction between LPS and cationic lipid membranes. The structural similarity between LPS and N-octadecylchitosan lipid membrane seemed to enhance the interaction too [Based on above information, the removal of LPS to a lower concentration by the porous supports bearing lipid membranes can be attributable to both an electrostatic interaction and hydrophobic one between the cationic lipid membranes of y of HSA . It is ooup pKa, 1.5 used tion too . On the E. coli[E. coli leads to a further requirement of selective removal of LPS. Considering the selectivity for LPS of the porous supports bearing lipid membranes, their application for purification of antibody drugs is interesting.Specific interaction for LPS of the porous supports bearing lipid membranes seems to be useful in a forthcoming issue. Antibody drugs with superior efficacy have been developed intensively in the last few decades. However, since they are produced by mammalian cells such as Chinese hamster ovary, their cost is very expensive and needs to be reduced. For this issue, intensive researches have been continued for production of antibody drugs using microorganisms such as E. coli. AlthougN-octadecylchitosan was evaluated by measuring the total organic carbon content in an eluent from a column packed with 20 mL of supports. The total organic carbon in the recovered water described in the experimental section was 400 μg L-1[N-octadecylchitosan, the eluted N-octadecylchitosan  is calculated as 3.0 × 10-5 g. This amount is 0.038% of the N-octadecylchitosan immobilized on the 20-mL supports. This stability for alkali is attributable to the stability of the amide linkage used for the immobilization of N-octadecylchitosan as well as the stability of the support material. Any substantial change was not observed in the IR spectra of the porous supports bearing lipid membranes after immersion in 0.5 M NaOH or 0.1 M HCl overnight at ambient temperature. This chemical stability, especially to 0.5 M NaOH immersion which is used as a standard depyrogenation procedure, is robust enough for a practical application in the pharmaceutical industry.The elution property of separation medium is a key issue in liquid purification for the pharmaceutical industry. Elution from porous supports bearing lipid membranes of 00 μg L-1. Even asN-octadecylchitosan assembled in nanoscale adsorb LPS selectively from HSA solution at pH 4.3 to 8.0 with the ionic strength of 0.05 to 0.1. LPS was removed to as low as a detection limit of 0.020 ng mL-1 by a column-wise adsorption with a quantitative recovery of HSA. Since LPS includes a terminal diglucosamine which is negatively charged and highly substituted with long-chain fatty acids, LPS is adsorbed by both an ionic interaction and a hydrophobic one. In addition, the low pKa of the chitosan-based material as well as the rigid gel phase of lipid membranes leads to a relatively weak interaction between HSA and results in the selective adsorption of LPS. For a practical use in separation, especially such as liquid purification for the pharmaceutical industry, a lower elution property of the separation medium is a key issue. Considering the excellent selectivity and the chemical stability of the supports bearing cationic lipid membranes of N-octadecylchitosan, their practical use as separation media in pharmaceutical manufacturing can be expected.Porous supports bearing cationic lipid membranes of The author declares that he has no competing interests.


Apis mellifera) from colonies selected for high (HMP) and low (LMP) Varroa destructor mite population growth. Two viruses, ABPV and SBV, were never detected. For adults without mite infestation, DWV, IAPV, BQCV and KBV were detected in the HMP colony; however, only BQCV was detected in the LMP colony but at similar levels as in the HMP colony. With mite infestation, the four viruses were detected in adults of the HMP colony but all at higher amounts than in the LMP colony. For brood without mite infestation, DWV and IAPV were detected in the HMP colony, but no viruses were detected in the LMP colony. With mite infestation of brood, the four viruses were detected in the HMP colony, but only DWV and IAPV were detected and at lower amounts in the LMP colony. An epidemiological explanation for these results is that pre-experiment differences in virus presence and levels existed between the HMP and LMP colonies. It is also possible that low V. destructor population growth in the LMP colony resulted in the bees being less exposed to the mite and thus less likely to have virus infections. LMP and HMP bees may have also differed in susceptibility to virus infection.A comparison was made of the prevalence and relative quantification of deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), Kashmir bee virus (KBV), acute bee paralysis virus (ABPV) and sac brood virus (SBV) in brood and adult honey bees ( Varroa destructor has become the most serious health problem of the western honey bee, Apis mellifera, worldwide. This mite is one of the factors associated with the unprecedented loss of honey bee colonies recently experienced in parts of Europe and North America [V. destructor may be so harmful to honey bees not only because of its feeding on the bee's haemolymph, but also because it transmits and favors the multiplication of honey bee viruses. Increases in the incidence and levels of several honey bee viruses have been observed with V. destructor as an inducer/or vector of several viruses [The parasitic mite  America –4. Recen viruses .The viruses most commonly found in surveys of honey bee colonies worldwide, are deformed wing virus (DWV), acute bee paralysis virus (ABPV), sac brood virus (SBV), black queen cell virus (BQCV), Kashmir bee virus (KBV) and Israeli acute paralysis virus (IAPV) –8. DWV, V. destructor [It is well known that honey bee genotypes vary in their resistance to structor –17. Howestructor , 19.V. destructor population growth. These colonies had previously bee shown to differ in grooming behavior and V. destructor reproduction rates [The objective of this study was to determine and compare the prevalence and relative amounts of DWV, IAPV, BQCV, ABPV, SBV and KBV in brood and adult honey bees between colonies with either low or high on rates , 21.Experiments were conducted at the Honey Bee Research Centre (HBRC) of the University of Guelph, in Guelph, Ontario, Canada. Full description of the methods used to screen for mite population growth in colonies is described elsewhere . BrieflyOne colony each from the HMP and LMP groups was selected as source colonies for brood and adult worker bees. The mite infestation rates of these colonies were 96, 10 and 17 for daily mite drop, number of mites/100 bees and number of mites/100 cells, respectively for the HMP colony, whereas for the LMP colony, these rates corresponded to 6, 1 and 0.7, respectively (20). Frames containing newly capped cells were used for brood experiments, whereas newly emerged bees were utilized for adult bee experiments. To obtain adult bees, frames containing emerging brood were collected from honey bee source colonies and incubated overnight inside screened cages (5 x 28 x 25.5 cm) at 33–35°C, and 60% RH.V. destructor were obtained from a heavily infested honey bee colony that had not been treated with miticides for at least six months. Mites were harvested by opening capped brood cells and by collecting them using a fine paintbrush. The harvested mites were either used for virus analysis or infestation. For virus analysis, three samples of approx. 100 V. destructor mites were placed in 1.5 mL microfuge tubes and kept frozen at -70°C until analyzed. For infestation, mites were kept in a Petri dish lined with moist filter paper and allowed to feed upon two white-eyed bee pupae from a non-infested colony. The mites were used within 2 h after collection.Adults V. destructor, groups of 15 newly-capped brood cells from combs of either a LMP or HMP colony were opened by cutting a thin slit approximately 2 mm long using a sterile blade, and then a single mite was transferred into each cell using a fine paintbrush [To infest brood with intbrush . The sliV. destructor, ten newly emerged bees were introduced into a Benton queen cage and three cages were used for a total of 30 bees. Two mites were placed on the body of each bee through the screen of the cages using a fine brush. In these cages, the bees were fed queen candy ad libitum and given water by spreading drops of water on the screen of the cages twice a day. Bees were incubated at 33–35°C and 60% RH. For the control, newly emerged bees were placed in Benton queen cages and incubated as above without the addition of mites. Surviving workers were collected at 7 dpi and stored individually at -70°C in 1.5 mL microfuge tubes until analysis. This experiment was replicated three times.To infest adult bees with 18 as primer, following the instructions of the manufacturer.Total RNA was extracted after homogenizing five bees (brood or adults) or 100 mites as per Chen et al. . The conTaq polymerase (New England BioLabs), 1 μl of the cDNA sample, and 7.8 μl of dd H2O. For IAPV, ABPV, SBV and KBV, the PCR conditions were 94°C for 3 min, followed by 35 cycles of 30 s at 94°C, 60 s at 55°C and 60 s at 72°C, and a final extension step at 72°C for 10 min. Amplification conditions for DWV and BQCV were the same, except that the annealing temperature was 58°C.Multiplex simultaneous reactions were done combining one set of virus-specific primers with the RpS5 primers. All PCR reactions were done with a Mastercycler . Each 15 μl of reaction contained 1.5 μl of 10x PCR buffer , 0.5 μl 10 mM of dNTPs , 1 μl each of 10 μM forward and reverse primers for RpS5 and 10 μM forward and reverse primers for ABPV, KBV, DWV, BQCV, SBV or IAPV, 0.2 μl 5U/μl of The amounts of ABPV, KBV, DWV, BQCV, SBV and IAPV relative to a bee constitutive control gene were determined by a multiplex reverse transcription-PCR (RT-PCR). Primers for the constitutive honey bee gene were for the ribosomal protein RpS5 gene described by Thompson et al. . PrimersM Imaging System, Upland, CA, USA). To confirm the identity of each PCR product, randomly selected PCR products were purified using the EZ-10 Spin Column DNA Gel Extraction Kit (Bio Basic Canada Inc.) and sequenced at the Laboratory Services of the University of Guelph. The sequences were used to search the GenBank nr database of NCBI by BLASTn, and all of them showed ≥ 96% identity to their respective target virus sequence.PCR products were separated on 1% TAE agarose gels and stained with ethidium bromide. A 100 bp DNA ladder (Bio Basic Inc.) was included in each gel. Images of the gels were captured using a digital camera with a Benchtop UV Transilluminator  of viral RNA. To determine whether quantification at 35 amplification cycles was not affected by signal saturation of the band intensities, randomly selected samples with high, medium and low RQUs of DWV were also quantified in the same manner with 25 and 30 amplification cycles, and the RQU values were not significantly different for those samples whether 25, 30 and 35 amplification cycles were used .The intensity of the amplified bands was quantified in pixels using the Scion Image  as per Dean et al. . Semi-qut-tests. Data on the number of mites per 100 brood or adults were square root-arcsine transformed, whereas the data for daily mite fall counts were subjected to log transformations because they were not normally distributed. t tests were also used to compare HMP and LMP bees of the different treatments for differences in relative amounts of viral RNA. Values presented are means ± standard error of the mean. All statistical analyses were performed with the R-Statistical Program [Data were visually examined for normality prior to statistical analyses using box plots. Paired comparisons of LMP and HMP colonies for daily mite fall counts, number of mites per 100 brood cells and number of mites per 100 adult bees were done using  Program .P >0.05). In contrast, significant differences were detected in summer (P <0.001) with mean daily mite fall counts of 7.8 ±1.4 for LMP colonies compared with 88.8 ±17.4 for HMP colonies.Among 152 honey bee colonies, the ten LMP colonies chosen with a low difference between spring and summer in mean daily mite fall counts (approx. 4) was over 20 times less than the ten HMP colonies chosen with a high difference in mean daily mite fall counts for the same period (approx. 86). However, the mean daily mite fall counts between the LMP and HMP colonies in the spring  showed no significant differences (P <0.01) for both the mean number of V. destructor per 100 cells  and the mean number of V. destructor per 100 worker bees .Differences between LMP and HMP colonies in mean daily mite fall counts between spring and summer correlated well with the levels of infestation of brood (number of mites per 100 cells) and infestation of the adults (number of mites per 100 bees) in summer. There were significant differences between LMP and HMP colonies . DWV occurred in significantly higher amounts than IAPV, BQCV and KBV in adults from the HMP colony, but that difference was much less for adults from the LMP colony, being only significantly higher than IAPV and KBV.For non-infested adult bees, which were caged and treated like the infested adults but without the addition of the mite, DWV, IAPV, BQCV and KBV were detected from the selected HMP colony . Howevercolonies . HoweverV. destructor, DWV, IAPV, BQCV and KBV were found in the HMP colony, but only DWV and IAPV were detected in the LMP colony (P <0.05). DWV was much more common than the other viruses for brood from both the mite infested LMP and HMP colonies.For non-infested brood, where the cells were opened and resealed as per mite infestation but without the addition of the mite, DWV and IAPV were detected in the HMP colony, whereas no viruses were detected in the LMP colony . Seven dP colony . HoweverV. destructor population growth between HMP and LMP colonies was quite large (approx. 20 fold) from spring to summer. For these same colonies, Guzman-Novoa et al. [Varroa sensitive hygienic behavior of adult bees because foundress mites that escape the removal process from cells by hygienic bees have a lower reproductive potential therefore reducing V. destructor population growth [V. destructor.The difference in a et al.  showed t approx.  fold fro approx.  fold fron growth . Thus, bAdults of the HMP colony appeared to be more susceptible to bee viruses than those of the LMP colony, even without exposure to mites. Four viruses were found in the control adults from the HMP colony versus only one virus in the control adults of the LMP colony. However, the relative amount of that virus (BQCV) did not differ between the adults of the HMP and LMP colonies. In addition, control brood of the HMP colony without exposure to mites appeared to be more susceptible to bee viruses than control brood of the LMP colony. Two viruses were detected in HMP brood compared to no viruses detected in LMP brood.V. destructor, the same four viruses were detected in adults from both the HMP and LMP colonies. However, the relative amounts of those viruses were always significantly higher in adults from the HMP versus those of the LMP colony. There was also a difference in the occurrence of viruses in brood from HMP and LMP colonies infested with V. destructor. Four viruses were detected in brood from the HMP but only two from the LMP colonies. For the two viruses found in brood of both the HMP and LMP colonies, relative levels were always significantly higher in brood of the HMP colony. Thus, both adults and brood of the HMP colony with V. destructor appeared to be more susceptible to several bee viruses than those of the LMP colony.Following infestation with V. destructor and thus less parasitism by the mite with a correspondingly lessened chance for latent virus infections activated by parasitism by V. destructor. However, these results may also have an genotypic explanation; the LMP brood and adults may have had higher resistance to viral replication following transmission and/or activation of inapparent viruses.The results with non-infested and mite-infested bees suggest that the lower virus levels in LMP brood and adults may have been due to epidemiological causes as bees from the LMP colony would have had less exposure to V. destructor [V. destructor parasitizing newly emerged honey bees using similar methods as in this study [V. destructor secreted during parasitism have been proposed to activate DWV infections in honey bees through suppression of the bee's immune system [V. destructor were shown to damage haemocytes [V. destructor-transmitted viruses in the LMP colony for both infested and non-infested bees as a consequence of low mite parasitism. However, this possible explanation is less convincing for some viruses, such as IAPV, where its prevalence and titers have not been highly correlated with V. destructor levels [Several honey bee viruses appear to be directly linked to the level of mite feeding. For example, there was a significant and rapid increase in DWV levels after brood or adult honey bees were exposed to structor , 35, andis study . Becausee system . Suppresemocytes . Thus, iemocytes , 21, thir levels .V. destructor. They showed that DWV levels increased similarly between MR and MS colonies from summer to autumn, except for slightly higher DWV levels in MR colonies in July. In contrast, BQCV and SBV levels decreased by several orders of magnitude in adults in MR versus MS colonies. Although KBV levels did not differ between adults of the MR and MS colonies, this was believed to be likely due to it being always at very low levels . While the study by Locke et al. [V. destructor population growth. One possible explanation for the different results is that there was natural selection for mite resistance in the Gotland populations for over a decade versus a one-time artificial selection for mite resistance in the Guelph populations.By comparison, Locke et al.  took hone et al.  and thisV. destructor infestation and prevalence and levels of several viruses in bees from colonies selected for low and high mite population growth that also differ in the expression of traits related to mite resistance [Although we show a strong link between levels of sistance , 21, the


A modifiable spreadsheet tool will enable health officials to plan for thesebirths. The marked increase in infants born with microcephaly in Brazil after a 2015 outbreakof Zika virus (Zika virus) disease suggests an association between maternal Zikavirus infection and congenital microcephaly. To project the timing of delivery ofinfants born to mothers infected during early pregnancy in 1 city in Bahia State,Brazil, we incorporated data on reported Zika virus disease cases and microcephalycases into a graphical schematic of weekly birth cohorts. We projected that thesebirths would occur through February 2016. Applying similar projections to ahypothetical location at which Zika virus transmission started in November, weprojected that full-term infants at risk for Zika virus infection would be bornduring April–September 2016. We also developed a modifiable spreadsheet toolthat public health officials and researchers can use for their countries to plan fordeliveries of infants to women who were infected with Zika virus during differentpregnancy trimesters.  Aedes aegyptimosquitoes; maternal–fetal transmission of Zika virus has been reported  reported an outbreak of Zika virus (Zikavirus) disease in Brazil , we created figures demonstrating cohorts of pregnant women by week ofdelivery and then extrapolated to the beginning of pregnancy. Live-birth data fromBrazil showed small differences in the proportions of infants born at full term(37–41 weeks) with microcephaly (76.7%) compared with those born at full termwithout birth defects (83.6%) . We alsoIn Bahia, ≈4,000 infants are born each week . DuThe increased number of reported cases of microcephaly in Bahia State began with Octoberbirths; reported cases rose sharply during November 2015–January 2016. For thecity of Salvador, these November 2015–January 2016 births corresponded to thehighest likelihood of infection occurring in the first trimester or early in the secondtrimester of pregnancy, assuming that the date of report approximates the date of birth.There are no reports to indicate whether the city of Salvador experienced the Zika virusdisease outbreak earlier or later than the rest of Bahia State.In Country A , for theTo enable readers to project months when births with exposure in different trimesterscan be expected, we developed a modifiable spreadsheet tool . Users mOur projections, based on ecologic data, indicate that in Bahia State, Brazil, Zikavirus infection during the first trimester or early in the second trimester of pregnancyis temporally associated with the observed increase in infants born with microcephaly;this projection is consistent with the observed reported decline for January andFebruary 2016. This finding adds to pathologic findings documenting Zika virus infectionin several infants with microcephaly  and loca,Understanding the timing of Zika virus infection of pregnant women is key because theeffects of infection on pregnancy and fetal and infant outcomes is likely to vary bygestational timing, as has been demonstrated for other congenital infections such asrubella and cytomegalovirus; transmission risk may also vary according to gestationaltiming  (Some of the reported cases of microcephaly included in the graph are still beingassessed, and some might not meet the final case definition for microcephaly in Brazil(i.e., head circumference We assumed that the birth rates in these models remain constant throughout the year,which is not true for all locations. The data for Zika virus infection and infants withmicrocephaly are based on dates of report, which are probably later than actualoccurrence.Despite these limitations, our assessments provide some indication that the period ofhighest risk might be during the first trimester or early in the second trimester ofpregnancy. This assessment can help inform public health officials about risks formicrocephaly and help them plan for deliveries in areas where Zika virus diseaseoutbreaks occur. Conducting surveillance for microcephaly but also other pregnancyoutcomes such as pregnancy loss and other birth defects will enable continued evaluationof any effects of Zika virus disease might have on pregnancy. These data also emphasizethe role of arboviral disease–tracking activities for informing public healthplanning. The US Centers for Disease Control and Prevention has prepared interimguidelines for US healthcare providers who care for women who are pregnant during a Zikaoutbreak (The consequences of Zika virus infection during pregnancy are not fully understood.Given the growing evidence of an association with microcephaly (Modifiable spreadsheet for projecting periods of delivery of at-risk infants afterZika virus disease outbreaks.


Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by SFTS virus and characterized by a high case fatality rate. Currently, there is no effective therapy for the disease. While the administration of ribavirin does not improve the case fatality rate or viral load in patient blood, it can inhibit viral infection in vitro.Vero cells were pre-treated with interferons (IFNs) α, β, and γ alone and in combination with ribavirin drugs and inoculated with SFTS virus. Three days later, supernatants were harvested and subjected to virus titration. An unpaired t-test was used for statistical analysis of the drugs’ effects.10 reduction in viral titers.While the effects of IFNγ at high concentrations were slightly weaker than those of the other IFNs, all IFNs showed dose-dependent inhibitory effects. The combined usage of IFNs with ribavirin at 90 % effective concentrations showed large inhibitory effects, with over a 3 logThe combined usage of one of type-I/II IFNs with ribavirin drastically reduced SFTS virus infection and therefore may be useful in the treatment of SFTS. Bunyaviridae, genus Phlebovirus), the discovery of which was reported in 2011 [Severe fever with thrombocytopenia syndrome (SFTS) is a recently-identified tick-born infectious disease characterized by fever, gastrointestinal symptoms, thrombocytopaenia, leukopaenia, and elevated levels of liver enzymes in the peripheral blood , 2. MultIt has been reported that SFTS patients treated with ribavirin, plasma exchange, antibiotics, or steroids have recovered –11; howeIn the present study, we examined the effects of interferons (IFNs) alone and in combination with ribavirin on SFTS virus infection in vitro. The combined usage of IFNs with ribavirin showed large inhibitory effects, suggesting their usability in SFTS treatment.90 and EC99), the drug concentrations at which viral titers were 1 and 2 log-reduced, respectively. The EC90 values of IFNα, β, and γ were 29 U/ml, 24 U/ml, and 12 ng/ml, respectively. The EC99 values of IFNα, β, and γ were 210 U/ml, 160 U/ml, and >2,000 ng/ml, respectively.Viral titers obtained from Vero cells, which were inoculated in the absence/presence of indicated concentrations of IFNs, are shown in Fig. Cytotoxicity was examined by measuring cell viability after cell culture in the presence of drugs without virus inoculation. As shown in Fig. 90 of ribavirin in the inhibition of SFTS virus infection was calculated as 43 μg/ml. The combination effects of each of the IFNs with ribavirin were examined at concentrations of their respective EC90 values. At the same time, the single effects of each drug were also examined . Viral titers were greatly reduced by the combined usage of IFNα-ribavirin (3.6 log), IFNβ-ribavirin (3.2 log), and IFNγ-ribavirin (3.4 log). Differences between the effects of the combined usage and those of the single usage were statistically significant . The combination effects in the reduction of viral titers were also examined among the three IFNs . The results of triple or quadruple combinations of IFNs and ribavirin, which showed greater inhibitory effects on SFTS virus infection than single- or double-combination-usage of the drugs, are indicated in Fig. Using its reduction curve   Fig. 1d1d, the Ened Fig. . As expeThe examination of cytotoxicity in the combination effect study revealed that >70 % viability was maintained in all combinations Fig. , while sThe combined usage of two or more drugs drastically reduces growth of hepatitis C virus and human immunodeficiency virus (HIV) in vitro and has improved the treatment of both diseases , 16. In 7 U of human-use-approved IFNs [90 of the SFTS virus in vitro. Thus, the combined usage of ribavirin and IFNs at acceptable dosages might result in a large reduction of SFTS virus infection in vivo and could therefore be an effective therapy for the treatment of SFTS patients. Because SFTS virus has been shown to suppress cellular IFN responses [In the present study, the single usage of ribavirin  or IFN wved IFNs , 28, whiesponses , 30, the90, the combination effects of IFNs without ribavirin on SFTS virus infection, especially the effects of IFNα and β, were small. However, such small combination effects among the IFNs had been expected due to the following reasons: (i) although precise mechanism (s) by which ribavirin affects SFTS virus replication is unclear, ribavirin and IFNs likely work independently of each other [In comparison with the combination effects of a single IFN with ribavirin at their respective ECch other ; (ii) IFch other . The resch other  for SFTSThe combined usage of one of types I/II IFNs with ribavirin drastically reduced SFTS virus infection and therefore may be useful in the treatment of SFTS.Monkey kidney-derived Vero cells  were cultured at 37 °C in Dulbecco’s modified Eagle’s medium  supplemented with 5 % heat-inactivated fetal calf serum (FCS) (Sigma) and antibiotics . Isolation of the Japanese SFTS virus strain SPL030 has been reported  and viru50 of SFTS virus was added to the culture without removal of the drugs, and then cultured for 3 days. Culture supernatants were harvested and subjected to virus titration [Ribavirin, provided by Yamasa-Shouyu Co., Ltd. , was dissolved in phosphate-buffered saline at a concentration of 100 mg/ml. IFNα , β , and γ  were dissolved in DMEM supplemented with 2 % FCS at 500,000 U/ml, 500,000 U/ml, and 200 μg/ml, respectively. For treatment of cells, these drugs were further diluted with cell culture medium at indicated concentration (s), alone or in indicated combinations. Cells were pre-treated with drugs at indicated concentrations for 1 h. One hundred TCIDitration .Cytotoxicity of drugs against Vero cells was measured as described previously . Cells wAn unpaired t-test was used for statistical analysis of the drugs’ effects.


Brevicoryne brassicae virus (BrBV) is a positive-strand genomic RNA virus which is unassigned tentative member of the genus Iflavirus. BrBv was first identified and characterized in the late 90’s in the cabbage aphid in the United Kingdom (UK)  and was fully sequenced, using random amplification of encapsidated RNA. No other reports have been published demonstrating detection of this virus outside the UK.Brevicoryne brassicae virus was identified from Brevicoryne brassicae aphids growing on wild mustard plants (Sinapis arvensis) in northern Israel. The virus genome was partially assembled from purified siRNA using the Illumina MiSeq Sequencing System with limited success. Combining classical viral RNA purification and RT-PCR amplification followed by traditional Sanger sequencing enabled obtaining the complete genomic sequence. The Israeli strain of BrBV shared 95 % nucleotide sequence identity with the BrBV found in the United Kingdom.A new isolate of the cabbage aphid virus The detection of BrBV in Israel indicates a broader geographical distribution of the virus”.The online version of this article (doi:10.1186/s12985-016-0500-z) contains supplementary material, which is available to authorized users. Cripavirus genus (family Dicistroviridae) includes two aphid viruses: Rhopalosiphum padi virus (RhPV) [Aphid lethal paralysis virus (ALPV). ALPV was first isolated from the bird cherry-oat aphid, Rhopalosiphum padi [Aphis nerii [Acyrthosiphon pisum [Acyrthosipon pisum virus (APV) [Brevicoryne brassicae virus (BrBV) [Iflavirus [http://www.ictvonline.org/virusTaxonomy.asp).In the 1990s, aphid-infecting viruses were discovered and characterized; most of them contained an RNA genome. The s (RhPV)  and Aphihum padi , 5, thenis nerii , and recon pisum . In addius (APV)  and Brevs (BrBV) . Interess (BrBV)  No others (BrBV) . CurrentBrevicoryne brassicae that was highly infested by the parasitic wasp Aphidius colemani  growing in the northern coastal area of Israel and were further investigated in the current study. Later on, BrBV-IL was identified in two additional locations, Bet-Dagan and Modi’in, out of 12 B. brassicae samples of aphid populations across the country.From 2009 to 2012, a survey aimed at identifying aphid-pathogenic viruses was conducted in Israel to find viruses that might be integrated into future biological aphid control. In the current study, BrBV-IL was found in a dense population of the cabbage aphid ani Fig. –c. The aB. brassicae aphids using the Viral RNA Extraction Kit  and served as the template for a reverse-transcription (RT) reaction carried out using Maxima Reverse Transcriptase  and the complementary primer R-5′-TTTAAAGAAAGAGCACTGTCCT-3′, followed by PCR amplification using JMR PCR mix  and primer set no. 5 , followed by mirVana miRNA Isolation Kit  for the enrichment of small RNA molecules. Recently, using NGS has become more and more advantageous for the discovery of new viruses and microorganisms. Moreover, the ability of whole viral assembly sequencing from small RNAs using rapid, accurate and high throughput technology allow to detect the presence of low concentration of new and cryptic viruses [Viral RNA was purified from  5 Table , designe viruses , 11.TablThe small RNA was used for the construction of a miRNA library using TruSeq Small RNA Sample Preparation Kit . The library was sequenced by the Illumina MiSeq Sequencing System (Volcani Center) and provided 217,324 reads , of which only 46 reads matched the BrBV genome (accession number NC_009530.1)  analysis forced us to combine the traditional sequencing method based on RT-PCR amplification to retrieve the complete genome sequence.B. brassicae [Viral purification was carried out on 100 g brassica leaves carrying a dense population of BrBV-IL-infected rassicae . The prerassicae  and the http://blast.ncbi.nlm.nih.gov/Blast.cgi) revealed 95 % nucleotide sequence identity and 98 % deduced amino acid sequence identity to the UK isolate of BrBV (accession number NC_009530.1).Sequence homology of BrBV-IL determined using the Basic Local Alignment Search Tool  enabled locating the 46 reads  within the genome, enabling a first determination of the viral agent with only preliminary information on nucleotide sequence. The complete genome sequence of BrBV-IL was obtained by traditional RT-PCR amplification followed by nucleotide sequencing  in the family Dicistroviridae, genus Aparavirus , type II and type III (Fig. Based on a rooted phylogenetic tree analysis of the long ORF encoding the polyprotein of the viruses belonging to the rus Fig. , it is sIII Fig. . As showIflaviridae and further studies should shed additional light on the taxonomic affiliation among the genera. In addition, pathogenicity experiments aimed at testing the infectivity potential of BrBV-IL on different aphid species could potentially lead to future application as a biopesticide.Future discovery of new members of the family BrBV-IL genome sequence was submitted to genbank under accession number KP777548.


Our study showed that influenza whole virus vaccines of major seasonal A and B strains activated DC more efficiently than those of pandemic swine-origin H1N1 and pandemic-like avian H5N1 strains. In contrast, influenza split virus vaccines had a low ability to activate DC, regardless which strain was investigated. We also observed that whole virus vaccines stimulated virus-specific CD8+ memory T cells much stronger compared to split virus counterparts, whereas both vaccine formats activated CD4+ Th cell responses similarly. Moreover, our data showed that whole virus vaccine material is delivered into the cytosolic pathway of DC for effective activation of virus-specific CD8+ T cells. We conclude that vaccines against seasonal and pandemic (-like) influenza strains that aim to stimulate cross-reacting CD8+ T cells should include whole virus rather than split virus formulations.Pandemic and seasonal influenza viruses cause considerable morbidity and mortality in the general human population. Protection from severe disease may result from vaccines that activate antigen-presenting DC for effective stimulation of influenza-specific memory T cells. Special attention is paid to vaccine-induced CD8 In addition, the swine-origin H1N1 virus, which has provoked the latest influenza pandemic in 2009, and the pandemic-like H5N1 virus, which was recently reported to require as few as 5 amino acid substitutions to become airborne transmissible between ferrets, create common fears of a pandemic with a highly pathogenic influenza virus + and CD8+ memory T cells in vitro. Vaccines were derived from major seasonal influenza A and B strains as well as from pandemic swine-origin H1N1 and pandemic-like avian H5N1 strains, respectively. Due to the double-inactivated non-infectious nature of the vaccines, CD8+ T-cell stimulation required that viral antigens present in the vaccines were cross-presented onto MHC class I molecules in APC, as has been successfully demonstrated for mature DC + T cells that are cross-reactive against multiple seasonal and non-seasonal influenza strains.The current study directly compared influenza whole virus and split virus vaccine formulations prepared from the same individual virus stocks for the ability to stimulate influenza-specific CD4Donors were randomly selected healthy volunteers who participated in this study after written informed consent in accordance with the Helsinki Protocol and after receiving permission by the local ethics committee . Median age was 26 years .Influenza vaccines were prepared from recent pandemic (-like) and seasonal virus strains and are listed in Optimal results in flow cytometry and ELISpot assays were obtained with 10 µg/mL (referring to HA content) of vaccines, as determined in preceding dose-titration experiments.+ and CD8+ T cells were positively selected from PBMC by immuno-magnetic MicroBeads . DC were generated from CD14 MicroBeads-selected monocytes and matured by exogenous cytokines PBMC were isolated from buffy coats by Ficoll centrifugation and stored in liquid nitrogen. CD45 cells for 4 h with 6.5×103 hemagglutination units (4×105 EID50) of Puerto Rico 8 virus . After final washing, epoxomicin-treated (or DMSO-treated) antigen-pulsed or infected DC were used in ELISpot assays.In some experiments, d5-old DC were pre-treated with irreversible proteasome inhibitor epoxomicin  for 1 h, then washed and loaded for 4 h in epoxomicin (2 µM)-containing medium with whole virus UR-w (10 µg/mL), split virus UR-s (10 µg/mL) or influenza A H3N2 nucleoprotein oligopeptide mix , respectively. For infection with live influenza virus, DC received same epoxomicin treatment but were incubated at 4×104 or more events were evaluated using BD FACSDiva software (BD Biosciences). Dead cells were excluded by standard 7-AAD staining (BD Biosciences). Statistical data analysis was conducted with SPSS Statistics software .Cells were stained with FITC-, PE-, APC- or Horizon V450-conjugated mAb  and analyzed on BD FACSCanto II flow cytometer (BD Biosciences). After gating on viable cells, 10+ and CD8+ cells, immuno-magnetically purified T cell subsets were incubated with autologous vaccine-pulsed DC for 20 h. Then intracellular cytokine staining (ICS) of stimulated T cell subsets was performed using Cytofix/Cytoperm Plus Kit with BD GolgiStop, following the manufacturer's instructions (BD Biosciences). Analyses of extracellular or intracellular cytokines were performed on BD FACSCanto II flow cytometer and evaluated using BD FACSDiva and FCAP Array software . IFN-α levels in DC supernatants were measured by ELISA .IL-6, IL-12, and TNF-α secretion of DC was measured by cytometric bead array (BD Biosciences) in culture supernatants of DC upon incubation with vaccine preparations for 48 h. To measure intracellular IFN-γ production of CD45/well and 1×104/well, respectively. Wells containing T cells plus DC plus TBS vaccine buffer and those with T cells only served as controls. ELISpot plates were incubated for 40 h, except for 20 h assays including proteasome-inhibited DC. Spots were counted using automated ELISpot readers ImmunoSpot Series 5 Versa  and Zeiss KS ELISpot 4.9 . Data are means ± standard deviation (SD) of duplicates or triplicates, respectively.IFN-γ ELISpot assays were performed as previously described Intracellular localization of influenza virus vaccine material was analyzed using a Zeiss LSM 510-UV confocal laser scanning microscope equipped with Zeiss LSM Image Examiner software . Briefly, d5-old immature DC were pre-treated for 4 h with epoxomicin (1 µg/mL) followed by 4 h incubation with A/H3N2-Uruguay whole virus vaccine (50 µg/mL). Pulsed cells were then washed twice in Saponin buffer and stained with mouse-anti-human influenza A H3N2-specific mAb against nucleoprotein  together with rabbit polyclonal antibodies against human endosomal proteins Lamp1 and Rab5 . Cells were washed twice and stained with fluorochrome-conjugated goat-anti-mouse and goat-anti-rabbit secondary antibodies . Isotype-matched IgG antibodies were used as controls. Nuclear co-staining was performed with Hoechst 33342 dye (Gibco/Life Technologies). Permeabilization and fixation were performed in Cytoperm/Cytofix Buffer (BD Biosciences).5 per well in DC medium in 24-well plates containing carbon-coated sapphire discs  for 4 h or overnight to allow cell adherence to discs. Afterwards, virus vaccines were added at 50 µg/mL for 2 h. In some experiments DC were pre-incubated with 1 µg/mL epoxomicin for 4 h before vaccines were added. After vaccine treatment, DC were fixed and sliced with a microtome prior to TEM analysis.Transmission electron microscopy (TEM) analysis was performed with a Zeiss 912 Omega microscope  at 120 kV. A preparation protocol based on high-pressure freezing and freeze substitution was used Whole virus vaccines were analyzed for the ability to induce maturation in monocyte-derived immature DC of healthy individuals. Vaccines were available from seasonal strains A/H1N1-Brisbane, A/H3N2-Uruguay, and B/Brisbane, as well as from recent pandemic-like avian and pandemic swine-origin strains A/H5N1-Indonesia, A/H5N1-Vietnam, and A/H1N1-California, respectively . Flow cySupernatants of DC cultures were measured for IFN-α, IL-6, and TNF-α  usually Split virus vaccines that had been prepared from the same individual virus stocks as whole virus vaccines were also analyzed for impact on DC phenotype by flow cytometry. The direct comparison with whole virus vaccines consistently showed that split virus preparations had a much lower ability to up-regulate the expression of DC maturation markers . In addiin vitro pre-pulsing with vaccine preparations, DC received a maturation cytokine cocktail consisting of IL-6, TNF-α, IL-1β, and PGE2 to ensure optimal APC function. This appeared necessary, because the preceding experiments had shown a lower DC maturation ability of pandemic (-like) versus seasonal vaccines as well as of split virus versus whole virus preparations. However, DC maturation cytokines were not essentially required for recognition, as they generated only a slight albeit non-significant increase of vaccine-reactive CD4+ and CD8+ T cells in IFN-γ ELISpot assay  and seasonal influenza vaccines in healthy volunteers. During ot assay .+ and CD8+ T cells of 10 healthy individuals in IFN-γ ELISpot assay. As shown in detail for a single representative donor, IFN-γ secreting CD4+ and CD8+ T cells were observed upon stimulation with every influenza whole virus vaccine preparation tested  strains (5 CD4+ T cells were 105 (30–217), 110 (23–324), 112 (23–300), 87 (23–204), 135 (13–245), and 126 (5–255) for swine-origin A/H1N1-California, avian A/H5N1-Indonesia, avian A/H5N1-Vietnam, A/H1N1-Brisbane, A/H3N2-Uruguay, and B/Brisbane, respectively. In contrast, frequencies of whole virus vaccine reactive CD8+ T cells were approximately 2- to 10-fold lower than that of CD4+ counterparts, as determined in the same donor cohort  vaccines were of lower magnitude. Median values (and ranges) per 1×105 CD8+ T cells were 10 (5–54), 24 (6–173), 30 (7–173), 50 (13–211), 40 (15–223), and 18 (2–170) for A/H1N1-California, A/H5N1-Indonesia, A/H5N1-Vietnam, A/H1N1-Brisbane, A/H3N2-Uruguay, and B/Brisbane, respectively.Vaccine-loaded matured DC were subsequently used as APC to stimulate autologous CD4n tested . Data su strains . Median r cohort . Highest+ T cells to whole virus and split virus vaccine preparations was very similar for most influenza strains  strains  was completely unable to compensate for the poor effects of split virus vaccine preparations to stimulate influenza-specific memory CD8+ T cells in vitro.In the same donor cohort (n = 10), IFN-γ ELISpot reactivity of CD4 strains . In cont strains . In dire+ T cells but not to CD4+ T cells was confirmed using flow cytometry staining for intracellular IFN-γ , effector memory (CD45RA+CCR7−) and late effector (CD45RA+CCR7−) T-cell subsets, respectively (data not shown).The improved immunostimulatory capacity of influenza whole virus versus split virus vaccines to CD8ar IFN-γ . The infin vitro ability to mature DC and to stimulate IFN-γ-secreting memory CD8+ T cells compared to whole virus counterparts. In contrast, split virus and whole virus vaccines showed a similar efficiency in inducing IFN-γ secretion by pre-existing virus-specific CD4+ T cells. These data suggested that the structural integrity of whole virus vaccines is of crucial importance for eliciting antiviral CD8+ (but not CD4+) memory T-cell responses, but the exact mechanism behind this hypothesis was unclear. One possible explanation could be that whole virus vaccine production preserved the ability of virus material to fuse with endolysosomal membranes inside the DC. Subsequently, viral proteins could gain access to the cytosol, where they are degraded by proteasomal enzymes and enter the MHC class I pathway for successful stimulation of influenza-specific CD8+ T cells.Split virus vaccines prepared from whole virus vaccines by single Triton X-100 detergent incubation showed a similar distribution and concentration of proteins as whole virus formulations in SDS-PAGE analysis, but had lost structural integrity of virus particles according to electron microscopy pictures . They haTo investigate this hypothesis confocal laser scanning microscopy (LSM) was performed in order to localize influenza virus material inside DC. After loading with whole virus vaccine of the seasonal A/H3N2-Uruguay strain, viral nucleoprotein (NP) was clearly observed at the outer cell membrane of DC as well as intracellularly . Post-prVaccine-loaded DC were also analyzed by TEM. The pandemic-like avian A/H5N1-Vietnam whole virus vaccine consisted of filamentous particles that located at the periphery of endolysosomal structures . At high+ T-cell reactivity to whole virus A/H3N2-Uruguay vaccine and live Puerto Rico 8 virus by 99% and 97%, respectively. Epoxomicin-induced inhibition was also observed at significant levels, when CD8+ T-cell reactivity was measured to the split virus A/H3N2-Uruguay vaccine (i.e. 90%) as well as to an oligopeptide mix derived from the influenza A/H3N2 nucleoprotein (i.e. 66%). As epoxomicin pre-treatment did not decrease viability of DC according to trypan blue staining controls, nearly complete loss of CD8+ T-cell reactivity to whole virus vaccine in proteasome-inhibited DC argued against a mere toxic effect but suggested that whole virus vaccine material effectively enters the cytosol for proteasomal digestion and processing by the MHC class I pathway.To obtain additional evidence that whole virus vaccine material enters the cytosolic compartment of DC, proteasomes were irreversibly inhibited by epoxomicin before loading cells with the whole virus A/H3N2-Uruguay vaccine. DC infected with live influenza virus of the Puerto Rico 8 laboratory strain was included as positive control. in vitro ability to mature human monocyte-derived DC. Thus whole virus vaccine production including double-inactivation by formalin and ultraviolet treatment can preserve important pathogen-associated molecular patterns (PAMP) of seasonal influenza viruses for binding pattern recognition receptors (PRR) to induce DC maturation The study shows that whole virus influenza vaccines prepared from major seasonal A and B strains have a strong Pandemic (-like) swine-origin and avian whole virus vaccines had an overall lower ability to induce DC maturation. This observation was somewhat surprising because the major immune response triggering receptors TLR 7 and TLR 8 expressed by DC are known to bind a broad pattern of viral single-stranded RNA (ssRNA) In contrast to whole virus vaccines, split virus counterparts produced by single Triton X-100 treatment completely lost the ability to induce DC maturation in all tested seasonal and pandemic-like strains. Most likely reasons for this observation are the detergent-induced structural disintegration and the loss of functionally active RNA components of influenza viruses  [25]. It+ T cells of every study participant in vitro. Median CD8+ T-cell reactivity was highest to both seasonal influenza A vaccines, and was slightly lower to the seasonal influenza B as well as to the pandemic (-like) avian and swine-origin influenza A vaccines. This difference in pre-existing immunity level of CD8+ T cells could be caused by the exposure history of donors to individual influenza virus strains. Notably, the immune-stimulatory effect on CD8+ T cells was also found if DC did not receive exogenous maturation cytokines during vaccine loading and if the pandemic (-like) whole virus vaccines with the observed deficiency to promote DC maturation were used. This observation clearly demonstrates that whole virus vaccine particles itself do not need to possess significant DC maturation capability in order to stimulate IFN-γ production in virus-specific CD8+ T cells. In contrast, split virus vaccine counterparts showed a considerably lower (>1-log) ability to activate virus-specific memory CD8+ T cells. The results suggested that whole virus vaccine material effectively enters the cytosol after uptake into DC, where influenza proteins are digested by the proteasome complex to fragments that move into the MHC class I pathway + T-cell reactivity. Because DC viability was not impaired directly after epoxomicin treatment, reduced immune-stimulatory activity might be explained by other reasons, e.g. drug-induced NF-κB inactivation or delayed toxicity in DC + T cells. Altogether this suggested that the production of CD8+ T-cell epitopes from whole virus vaccine material essentially requires proteasome function, similarly to what is known for live virus + T-cell reactivity to split virus vaccines was found to be much lower compared to that against whole virus vaccines, it was also sensitive to epoxomicin treatment at reduced but still significant degree. These data suggest that split virus material may also escape the endolysosome and rely on proteosomal degradation. As an alternative explanation, epoxomicin may not only block the proteasome complex but also impact other processes involved in MHC class I presentation of split virus material.A most striking observation was that influenza whole virus vaccines pulsed on DC effectively stimulated IFN-γ secreting memory CD8+ T cells + T cells by the natural route. In contrast, our data show that influenza NP derived from a double-inactivated whole virus vaccine could clearly be detected inside the cells but outside the endolysosomal compartment by confocal LSM. Additionally, TEM analysis providing much higher resolution suggested ongoing fusion of elongated and round-shaped whole virus vaccine particles with endolysosomal membranes and localization of filamentous particles in the cytosol in immediate proximity to endolysosomes. In summary, confocal LSM and TEM data suggest that a fraction of whole virus vaccine particles is indeed capable of escaping endolysosomal compartments. However, as it was not possible to visualize the structurally disintegrated components of split virus vaccines inside DC using confocal LSM or TEM, these data do not allow a direct comparison of whole and split virus vaccines.There are contradictory reports whether inactivation of live influenza virus by heat and formalin abrogates the membrane fusion activity and other important features that can influence the immunogenicity for CD8in vitro immune response of memory CD4+ T cells against seasonal and pandemic (-like) influenza vaccines showed major differences compared to that of the CD8+ subset. First, the level of reactivity was approximately 2- to 10-fold higher in CD4+ versus CD8+ T cells and did not vary considerably between seasonal and pandemic (-like) strains. In addition, split virus and whole virus vaccines were nearly equally efficient in stimulating virus-specific memory CD4+ T cells, indicating that split virus material gained sufficient access to the endolysosomal MHC class II pathway The + and CD8+ T-cell reactivity to seasonal and pandemic (-like) influenza viruses is common in the general human population The study confirms previous reports showing that heterosubtypic CD4+ as well as CD8+ memory T cells in vitro. The (re)-activation of virus-specific CD8+ T cells did not depend on DC with fully mature phenotype, as observed for the pandemic (-like) strain vaccines. Directly compared split virus vaccines were much less potent in stimulating CD8+ T cells, either due to abrogated membrane fusion ability or inability to activate DC for DC-intrinsic mechanisms of cross-presentation. As most CD8+ CTL are directed against conserved internal influenza virus proteins + responses is of major importance to establish broad cross-protection against seasonal as well as new pandemic (-like) strains. In this regard the study provides a clear rationale to use whole virus vaccines instead of split virus counterparts, if in vivo stimulation of coordinate CD4+ as well as CD8+ T-cell immunity against influenza viruses represents a main aim of vaccination. Prospective clinical trials directly comparing split virus and whole virus vaccines are required to find out if the presented in vitro data can be confirmed in vivo. Such trials should include a careful monitoring of the full repertoire of influenza-specific CD4+ and CD8+ T cells, for which the current data are certainly helpful. The presumably improved immunogenicity of influenza whole virus vaccines would then be calculated on the risk of vaccine-induced side effects, which has been reported by some investigators to be higher than that of split virus and subunit seasonal influenza vaccines In summary, seasonal and pandemic (-like) influenza whole virus vaccines loaded on monocyte-derived DC effectively stimulated heterosubtypic virus-specific CD4Figure S1Treatment of vaccine-loaded DC with maturation cytokines can improve detection of H5N1 influenza-specific T cells. CD4+ and CD8+ T cells purified from PBMC of 5 randomly selected healthy donors were analyzed for reactivity to the avian A/H5N1-Vietnam whole virus vaccine in IFN-γ ELISpot assay. APC were autologous DC that were pre-incubated for 48 h with 10 µg/mL vaccine preparation. During antigen pulsing, DC were either treated with maturation cytokines IL-6, TNF-α, IL-1β, and PGE2 (‘cocktail’) or were left untreated (w/o ‘cocktail’). (A) Data obtained in healthy individual HD26 with 1×105 CD8+ T cells (black columns) or 1×105 CD4+ T cells (grey columns) plated per well.  Box plot diagrams include data of CD8+ (B) and CD4+ (C) responses in 5 healthy volunteers. P-values were calculated using the two-tailed paired-sample Wilcoxon signed-rank test. SFC, spot-forming cells; w/o, without.(TIF)Click here for additional data file.Figure S2+ but similar or lower CD4+ T cell responses in comparison to their split virus counterparts.Whole virus vaccines induce stronger CD8 IFN-γ production of CD8+ and CD4+ T cells purified from PBMC of healthy individuals was measured by intracellular cytokine staining and ELISpot after stimulation with autologous monocyte-derived DC pre-loaded with influenza whole virus and split virus vaccine formulations. DC had been treated with maturation cytokines during pulsing with seasonal A/H3N2-Uruguay or A/H1N1-Brisbane vaccines. (A) In intracellular cytokine staining, cells were gated on CD3+CD4+ (or CD3+CD8+) and analyzed for intracellular IFN-γ. Fractions (%) of IFN-γ positive CD8+ or CD4+ T cells are added. (B) IFN-γ ELISpot data obtained from the same donor with 1×105 CD4+ T cells (grey columns) or 1×105 CD8+ T cells (black columns) plated per well are shown.(TIF)Click here for additional data file.Figure S3Protein composition, RNA content, and physical structure of whole virus and split virus vaccines. Shown data are from vaccine formulations of seasonal strain B/Brisbane (BB). (A) Semiquantitative SDS-PAGE analysis indicated similar distribution and concentration of proteins in split virus (s) and whole virus (w) preparations. (B) Electropherograms showed clearly different size distribution of RNA from split virus versus whole virus preparations. RNA of split virus preparation was considerably more degraded and of much smaller size. Split virus and whole virus preparations contained 7 pg and 627 pg RNA per µL per dose (500 µL), respectively (not shown). (C) TEM analysis of vaccine samples using a FEI Tecnai 10 microscope operated at 100 kV. Pictures were taken with an Olympus Veleta camera. The size scale is indicated. FU, fluorescence units; nt, nucleotides; kDa, kiloDalton.(TIF)Click here for additional data file.Figure S4Confocal LSM analysis of DC loaded with whole and split virus vaccines. Immature DC were analyzed after 4 h incubation with seasonal A/H3N2-Uruguay whole virus vaccine (URw), its corresponding split virus vaccine (URs), or PBS as negative control. The nucleus (Hoechst 33342) is stained in blue color, whereas endolysosomal compartments (Lamp1/Rab5) are marked in red color, and nucleoprotein (NP) of H3N2 in green color, respectively.(TIF)Click here for additional data file.Figure S5Particles from avian A/H5N1-Vietnam whole virus vaccine can be detected in the cytosol. Similar to (TIF)Click here for additional data file.


Mumps virus is a negative-sense, single stranded RNA virus consisting of a ribonucleocapsid core enveloped by a lipid membrane derived from host cell, which causes mumps disease preventable by vaccination. Since virus lipid envelope and glycosylation pattern are not encoded by the virus but dependent on the host cell at least to some extent, the aim of this work was to analyse L-Zagreb (L-Zg) mumps virus lipids and proteins derived from two cell types; Vero and chicken embryo fibroblasts (CEF). Jeryl Lynn 5 (JL5) mumps strain lipids were also analysed.in-gel trypsinized and identified by MS as well as tandem MS.Virus lipids were isolated by organic phase extraction and subjected to 2D-high performance thin layer chromatography followed by lipid extraction and identification by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Virus samples were also subjected to gel electrophoresis under denaturating conditions and protein bands were excised, Results showed that lipids of both mumps virus strains derived from Vero cells contained complex glycolipids with up to five monosaccharide units whereas the lipid pattern of mumps virus derived from CEF was less complex. Mumps virus was found to contain expected structural proteins with exception of fusion (F) protein which was not detected but on the other hand, V protein was detected. Most interesting finding related to the mumps proteins is the detection of several forms of nucleoprotein (NP), some of which appear to be C-terminally truncated.Differences found in lipid and protein content of mumps virus demonstrated the importance of detailed biochemical characterization of mumps virus and the methodology described here could provide a means for a more comprehensive quality control in vaccine production.The online version of this article (doi:10.1186/s12985-016-0463-0) contains supplementary material, which is available to authorized users. Paramyxoviridae, subfamily Paramyxovirinae, genus Rubulavirus that causes mumps disease. Mumps virions are pleomorphic particles ranging from 100 to 800 nm in size [e.g. human immunodeficiency virus type-1 (HIV-1) [Mumps virus is a nonsegmented, negative stranded RNA virus of the family  in size . Due to  in size . Nucleop in size , 5. V pr in size –8. SH pr in size  with pos in size . Virus e (HIV-1) , 13. Lip (HIV-1) –17, muri (HIV-1) , hepatit (HIV-1) , Singapo (HIV-1) , semliki (HIV-1) –23, and  (HIV-1) , 24. The (HIV-1) –32.Mass spectrometry (MS) is a powerful tool in the field of biochemistry and is emerging as a core component of fundamental discoveries also in the field of virology . CombinaVirus proteome and lipidome are expressed virus features that to some extent depend on the host cell and these must have an impact on virus characteristics such as infectivity and stability. Considering live virus vaccines, these features possibly affect vaccine performance. Therefore, methods characterizing both virus proteins and lipids could be a tool in raising the level of viral vaccines quality control. The aim of this research was to analyse lipid and protein pattern of the mumps virus derived from two cell lines (Vero and chicken embryo fibroblasts (CEF)) by means of mass spectrometry. Genome of L-Zagreb (L-Zg) mumps virus has been fully characterized  and hereInvestigation of the most abundant mumps virus proteins was performed on L-Zg mumps virus grown on two cell substrates, CEF and Vero cells. Purified mumps virus was subjected to electrophoresis under denaturating conditions. Results show that proteins of mumps virus derived from two cell types, CEF and Vero, are similar  and also multiple NP forms were confirmed [Proteins not detected and identified in any of our tested samples in purified virus preparations were F, SH and I protein. I protein is not considered as a structural protein so its presence in purified virus preparations is not expected. SH protein was detected in virus infected cells and is considered to be a membrane protein , 10 but peptides . Processpeptides  and in cpeptides . Howeveronfirmed  same as Paramyxovirinae is largely unstructured varying widely in length and sequence composition [In mumps virus derived from CEF, NP has been detected in three bands and the band with the highest apparent molecular mass is most intensive and corresponds to theoretical  NP molecular mass around 61 kDa, whereas the other two have lower molecular mass, around 55 and 48 kDa Fig. . PresencM protein was detected in two very closely neighbouring gel bands under non-reducing conditions around 40 kDa corresponding nicely to the theoretical molecular mass of M protein. One band of a slightly higher apparent molecular mass was detected under reducing conditions. MS sequence analysis of M protein-derived peptides confirmed a protein modification, the acetylation of the N-terminus. M protein is one of the three most intensive proteins of mumps virus detected under experimental conditions used here, in addition to HN and NP, which is expected since it is a structural protein linking nucleocapsid to the lipid envelope.According to literature HN protein is a disulphide bonded oligomer , thoughtIn addition to P protein that was detected in protein band together with smallest NP form (in both CEF and Vero samples), L protein was also detected (in Vero samples), both at apparent molecular masses corresponding to the theoretical ones. V protein was also detected (in Vero samples) but as a faint band. V protein has not been shown as a part of mumps virion so far but this could be a result of limitation of the techniques used, it was only detected in cell lysate .m/z 500.Lipid classes present in two mumps virus strains, L-Zg and Jeryl Lynn 5 (JL5), grown in two cell lines, CEF and Vero, were analysed as well as lipids present in non-infected cells. Lipids were isolated by organic extraction, separated by two-dimensional high-performance thin layer chromatography (2D-HPTLC), visualised by primuline staining, extracted from the excised chromatographic plate material and analysed by MALDI MS. This methodology is relatively straightforward and provides wealth of data, however it is only a qualitative approach providing a global view on present lipid classes which does not detect lipids with masses below e.g. CEF do not contain glycolipids in contrast to Vero cells. One could presume that such complex glycolipids on the viral surface result in significant differences during e.g. cell infection and this we plan to investigate in our future work.Representative 2D-HPTLC separations of mumps virus lipids and lipids isolated from cells are given in Fig. Presented data on mumps virus lipids and proteins indicate the presence of host cell proteins in mumps virions as found for other viruses. Interestingly, three NP forms were detected, two of which appear to be C-terminally truncated NP proteins of yet unknown function. Lipid analysis of mumps virus showed lipid pattern to be host dependent and viruses derived from Vero cells were found to contain glycolipids with up to five monosaccharide units. Presented results demonstrate virus variability and impact of these differences on virus characteristics remains to be determined.Vero cells (African green monkey kidney cells) were purchased from ATCC and maintained in Minimum Essential Medium with Hank’s salts (MEM-H) (AppliChem) supplemented with 10 % foetal calf serum (FCS) (Moregate) and 50 μL/mL neomycin (Gibco-Life Technologies). Chicken embryo fibroblasts (CEF) were prepared from the 11-day old embryonated chicken eggs as described . L-Zg mug to remove large-sized contaminants, and after that viruses were ultracentrifuged at 142,000 × g for 2 h and the pellet was resuspended in phosphate-buffered saline.Both viruses, L-Zg and JL5, were grown in Vero cells and CEFs in MEM-(H) with 2 % FCS at m.o.i. 0.0001. After 24 h the medium was replaced with medium without serum and virus was further grown until the cytopathic effect was observed. Then the medium was collected and centrifuged at 3000 × Electrophoresis under denaturating conditions was performed using 8 cm 4–12 % Bis-Tris precast gels, using MES running buffer and Novex Sharp Standard, in an XCell Sure Lock system from Invitrogen (Carlsbad) according to manufacturer’s instructions. Western blot detection was performed using guinea pig serum against mumps virus which was cultured in Vero cells and purified by ultracentrifugation. ECL Prime Western Blotting Detection Reagent was used for detection, according to the manufacturer’s instructions . Detection of protein bands was performed using acidic Coomassie Brilliant Blue R250 solution. Protein bands were excised from the gel, trypsinized and peptides isolated and purified for MS analysis as described . Experimin vacuo and separated by 2D-HPTLC  using chloroform/methanol/conc. ammonia 10:5:1 (V/V/V) for first dimension and after air-drying by means of chloroform/methanol/acetic acid/water 15:3:2:0.6 (V/V/V) for second dimension. Lipids were detected using primuline solution (0.05 % solution of primuline in acetone/water 8:2 (V/V)) and 365 nm UV light. Lipid spots were scratched from plates and 250 μL of chloroform/methanol/water 10:10:0.1 (V/V/V) was added in order to extract the lipids and extraction procedure was repeated two times. Obtained extracts were pooled and dried in vacuo. Lipids were dissolved in 4 μL of 20 mg/mL 2,4,6-trihydroxyacetophenone solution in methanol and applied to a stainless steel MALDI (matrix-assisted laser desorption/ionization) -target. Ricinus oil  was used for mass calibration of the MS instruments. Experiments were performed at least with three separate samples.Lipid isolation was performed according to Bligh and Dyer . Lipids 2 instrument  or an UltrafleXtreme  in positive, reflectron ion mode. AXIMA TOF2 is equipped with a 20 Hz nitrogen laser (337 nm) and was operated in the positive ion mode applying an accelerating voltage of 20 keV. Analyses by means of an UltrafleXtreme (with a 2 kHz SmartBeam solid state laser (355 nm)) were performed at an acceleration voltage of 8 kV in the positive mode.Measurements were performed either on an AXIMA TOF2 instruments were converted into mzXML files and processed using mMass [2, ± 200 ppm and ± 200 ppm for UltrafleXtreme (respectively), two missed trypsin cleavages, carbamidomethylation of Cys and with variable modification: oxidation of Met, ammonia loss from N-terminal Cys, N-acetylation, deamidation, and phosphorylation. Data on lipids were analysed using LIPID MAPS Structure Database (LMSD) [Raw data generated on AXIMA TOFng mMass . Data gee (LMSD) .


The emergence of human and animal rabies in Bali since November 2008 has attracted local, national and international interest. The potential origin and time of introduction of rabies virus to Bali is described. The nucleoprotein (N) gene of rabies virus from dog brain and human clinical specimens was sequenced using an automated DNA sequencer. Phylogenetic inference with Bayesian Markov Chain Monte Carlo (MCMC) analysis using the Bayesian Evolutionary Analysis by Sampling Trees (BEAST) v. 1.7.5 software confirmed that the outbreak of rabies in Bali was caused by an Indonesian lineage virus following a single introduction. The ancestor of Bali viruses was the descendant of a virus from Kalimantan. Contact tracing showed that the event most likely occurred in early 2008. The introduction of rabies into a large unvaccinated dog population in Bali clearly demonstrates the risk of disease transmission for government agencies and should lead to an increased preparedness and efforts for sustained risk reduction to prevent such events from occurring in future. Lyssavirus genus of the Rhabdoviridae family . The tree also show that Kalimantan KL 00-18 was the ancestor for Sulawesi and Flores viruses.All viruses detected in animals and humans in Bali were closely related to each other with a genetic distance of 0·0004. The MCC tree of phylogenetic analysis of the 892 bp fragments of RABV nucleoprotein genes from animals and humans during the recent outbreak in Bali and street viruses from various countries is presented in A list of events linked to the introduction of rabies in Bali is presented in An understanding of how RABVs enter a rabies-free area may prove invaluable in preventing the future spread of RABV from one island or one country to another. It is especially pertinent for archipelagos such as Indonesia. Molecular epidemiology provides a more accurate and relatively simple tool to answer this question compared to serology or other conventional methods , 17. AltPhylogeography of RABVs from around the world shows distinct differences. RABVs from Asia, Africa and Europe have been divided into subgroups of Arctic, Europe, Africa, Middle East, South Asia, Malay and South East Asia . IndonesThese results also show that the outbreak was most likely caused by a single incursion. The samples analysed in this study represent human and dog viruses from four out of nine Bali districts collected during the peak of the outbreak between 2009 and 2010 . The anaet al. [Conducting rabies forensic tests to predict the origin of the causative outbreak is paramount in preventing such an event in the future. The advance of statistical inference brings a new insight for a virus forensic study. As the virus, especially RNA viruses such as influenza and rabies , undergoet al.  were ablUsing the protocol, it was discovered that the ancestor of Bali viruses was the descendant of Kalimantan 00-18 strain, but not significantly separated from those of Sulawesi and Flores. Multiple MCMC analysis have also been conducted to test TMRCA with a pre-defined taxon subset if the Bali viruses were closely related to Kalimantan 00-18, Sulawesi or Flores viruses. The valid estimate was found in Kalimantan 00-18 and the Bali subset. This result supports the tree analysis that the Bali outbreak was caused by a Kalimantan descendant, which is correspondingly the ancestor of Sulawesi and Flores viruses.P < 0·1 [However, the actual generation time, and therefore, geographical origin of the Bali outbreak remains a mystery. The lower bound of the HPD Kalimantan–Bali subset was around 18 years as shown by TMRCA analysis. This means that the Bali virus ancestor diverged a decade before the actual isolation year of the putative ancestor. The clade support for the divergence of Kalimantan to Bali and Sulawesi-Flores strains is indeed very strong (posterior probability of 98%, P < 0·1 , which dKalimantan and Sulawesi are indeed the most plausible hypothetical origin of the Bali strains. Kalimantan and Sulawesi are known to be rabies-endemic islands for decades , while FFrom an anthropological point of view, Kalimantan and Sulawesi appear to be a reasonable origin of Bali's RABV. There is a strong belief in the country that some tribes in those islands, especially the Bugis tribe, are real sea people . They usIf the Bali RABV was, indeed, introduced from Kalimantan or Sulawesi, this would be a repeat of events where rabies was introduced to Flores from Sulawesi in 1997. The mode of introduction was thought to be via fishermen who brought an infected dog to the East Flores Island .It is likely that the virus was transferred to Bali via a similar route. Legal and illegal transportation of dog and cats has been found to be a major risk of rabies introduction in island regions like Taiwan . HoweverThe transport of dogs from other islands to Bali for business purposes cannot be ignored. Transport of dogs, cats and monkeys to Bali has been banned for a long time to keep the province rabies free. However, the regulation has not been properly enforced and public participation has been lacking. Illegal transport occurs mostly at Gilimanuk Harbour, which is the entry point for ferries from Java. Given that the circulating virus in Bali was not closely related to viruses from Java, it is unlikely that rabies was introduced through dog trafficking for commercial purposes.Another possibility is the use of dogs for food. Balinese traditionally do not eat dog meat but there are some tribes in Indonesia that do consume dog meat. Some tribes, including tribes from Sulawesi, serve dog meat at parties as a special treat for guests and some people from dog meat-eating tribes have settled in Bali and introduced the dog-eating practice to the locals. Many small restaurants now offer dog meat in Bali, especially in Badung, the site of the index human case, Denpasar and Singaraja. Bali is the likely source of dogs for most restaurants. It is possible that some fishermen might have transported an infected dog to Bali as a gift for relatives for dog meat consumption at a party. Importation of dogs for the meat trade has been claimed as the source for the introduction of rabies in Maluku .Although it is not a listed bioterrorism agent , rabies RABV was most likely to have been introduced to Bali in early 2008. We found that there have been other deaths with clinical signs of rabies and a history of dog bite at the same time and area as the index case. The earliest dog bite incident causing clinical signs and death from rabies was recorded early in September 2008. However, after the index case, a 32-year-old male who was bitten around July 2008 and died on 16 January 2009 was also confirmed as positive [The likelihood that this represents a repeat of events where rabies-infected dogs have been transported from one island to another, as seen from Sulawesi to Flores , 25, andRisk reduction efforts should equally take place in endemic islands and districts not only in new outbreak areas as widely practised in Indonesia. Regarding the fact that dogs are the main carriers of rabies in the most parts of the world , 2, as sHaving a long stretch of beaches, quarantine control of animal transport at every beach is hardly possible in Indonesia. Adequate knowledge, awareness and behaviour regarding rabies risks should be made available in the free, at risk, as well as endemic areas in Indonesia. When it comes to inter-island transport, all dogs should have been vaccinated properly.In conclusion, the outbreak of rabies in Bali was caused by a virus from Indonesia following a single introduction, and most likely occurred in early 2008. The lessons learned from this introduction of rabies into a large unvaccinated dog population provide a clear demonstration of the risks for government agencies and call for an increased preparedness to prevent such events from occurring in the future. Movement of people and their pets throughout the archipelago mean that virus introduction may occur in other areas. The occurrence of rabies in Bali has shown that public and healthcare provider awareness, dog vaccination programmes and good stocks of human anti-rabies and immunoglobulin are all crucial in preventing human rabies deaths.Supplementary MaterialSupplementary information supplied by authors.Click here for additional data file.


Zaire ebolavirus strain that has been linked by epidemiological evidence to a potential “patient zero” who succumbed to infection in December, 2013  expressing Ebola virus glycoprotein(s) . A single dose of 10 review; . The use10 recombinant adenovirus particles expressing the glycoproteins of Z. ebolavirus conferred complete protection from a lethal challenge with Z. ebolavirus, whereas a lower vaccine dosage protected most infected animals , provided complete protection in infected nonhuman primates that were already showing signs of Ebola virus disease   . This suThe development of antiviral drugs against Ebola virus has also progressed. The nucleoside analogs BCX4430 and T-705 (favipiravir) inhibit viral RNA synthesis with a minimal effect on cellular RNA synthesis . StudiesSince the first Ebola outbreak, we have furthered our understanding of this virus and its life cycle, and advances have been made in the development of potential prophylactic and therapeutic treatments. However, we are not there yet, and more basic and translational research is needed. But limited funding has slowed the progress. Additional funding for basic research is essential to identify the virus reservoir, determine the pathogenesis, and identify viral and host targets for antiviral drug discovery. Similarly, more funding for translational research is needed to test novel vaccines and antiviral drugs in nonhuman primates  and to test promising candidates in clinical trials.The current outbreak is a glaring wake-up call of the devastation the Ebola virus can inflict on the human population. This outbreak has exposed the world's lack of preparedness to respond to and control such infectious disease outbreaks. Our society has concerned itself with the potential of biological terrorist attacks or laboratory-acquired outbreaks; while such scenarios cannot be dismissed, this Ebola virus outbreak makes plain that the true threat of infectious diseases comes from nature. Funding and research (including gain-of-function experiments) must be stable and steady, not dictated by the world's attention, to ensure that we are ready for the next crisis.