Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache’s reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level. For instance, vaccinia virus free T7 polymerase rescue systems121314in vivo. So far, recombinant silver haired bat lyssavirusWhereas optimized recovery systems allowed easy and rapid generation of recombinant viruses, the availability of only a limited number of cloned infectious full-length cDNA genomes is still a bottleneck. Many of the broadly used cDNA clones have been generated from cell culture adapted or attenuated vaccine strains1171920In view of rapidly increasing information on sequence variability within virus populations from modern deep sequencing technologiesE. coli chromosomes through the Rac- or Lambda- phage protein pair RecE/RecT or Redα/Redβ respectively2526E. coli plasmid vectors  or even  vectors .E. coli for fast and efficient cloning of complete RABV genome cDNAs into reverse genetics plasmid vectors  in  vectors . EfficiePrior to full length cDNA amplification and recombinatorial insertion in plasmid vectors, the 3′ and 5′-terminal sequences of two different RABVs of dog and fox origin and of the attenuated lab strain SAD L16 as a control were determined by vRNA genome end-joining and RT-PCR sequencing . The terFor insertion of full length cDNA copies into a plasmid vector by LLHR a derivative of pHaHd MiniGFPE. coli GbDir-05  and dog  were determined by ion torrent sequencing. With 4 and 3 positions in the wt RABV-Fox and wt RABV-Dog isolates, the number of single nucleotide polymorphisms was remarkably low .Four of the fox and four of the dog RABV-derived cDNA clones were randomly selected and also analyzed by ion torrent sequencing. The pRABV-Fox clones 8, 9, 10 and 11 differed from the progenitor sequences by 3, 4, 3 and 5 single nucleotide exchanges, respectively  resultinOf the four pRABV-Dog clones 3, 5, 14 and 15 only two contained nucleotide alterations compared to the progenitor virus . WhereasA major benefit of the new method is the direct recovery of infectious virus clones from the individual vectors. This not only allows sequence analysis of individual genome molecules but also functional downstream analysis of selected mutants and further targeted modification of newly cloned virus isolates.We therefore checked whether infectious viruses could be recovered from the eight full genome cDNA encoding plasmids described above. Virus rescue was supported by expression plasmids for the N, P and L proteins of SAD L16After passage of cell culture supernatants from transfected BSR-T7/5 cells to Na42/13 cells, infection and subsequent virus spread was monitored by indirect immunofluorescence against RABV N protein (not shown). Detection of N positive infected foci for rRABV-Fox clones 9 and 10 as well as for rRABV-Dog clones 5, 14 and 15 confirmed rescue of recombinant virus from the cloned cDNAs. In contrast, rRABV-Fox clone 11 as well as rRABV-Dog clone 3 were not detected in the passage experiments, although staining for virus expressed G protein in the transfected BSR T7/5 cells indicated successful virus rescue . In rRABReporter protein expressing viruses represent important tools to investigate tropism and pathogenesis of viruses. Therefore, we tested whether non-fixed virulent RABV with limited virus replication in cell culture tolerate insertion and expression of a reporter gene from an extra cistron.After insertion of a multiple cloning site upstream of the G/L gene border, the red fluorescent Katushka reporter gene together with the conserved gene border sequence from SAD L16 was inserted in pRABV-Dog 14  and pRABThese data demonstrated that even in a non-cell culture adapted genetic background insertion of additional transcription units and successful recovery of replicating virus is possible.5 and 105.4 TCID50/ml for wt RABV-Dog and rRABV-Dog clone 14 on N42/13 neuroblastoma cells . Similar survival times of rRABV-Fox clone 9 and wt RABV-Fox after high dose infection indicated, that these viruses were comparable in peripheral infections. After low dose i.m. infections with both viruses, 33% of the animals survived , confirmIn contrast to the fox viruses, rRABV-Dog clone 14 and wt RABV-Dog differed in i.m. inoculations . Whereasin vivo data demonstrated that the virulence of the clonal recombinant virus rRABV-Fox clone 9 and rRABV-Dog clone 14 did not loose their ability to cause rabies disease in the mouse model after peripheral inoculation, even at low dose infections.Overall, the E. coli. After selection of 160 ampicillin resistant E. coli colonies, 16 clones were used for identification of full-length genome cDNA insertion by restriction endonuclease digest (not shown). Identification of 12 positive rRABV-Fox(rac) clones confirmed the high efficiency of the method . Sequencing of two randomly picked recombinants revealed that both BATV L recombinants were 100% identical to each other. Direct RT-PCR sequencing of two independent RT-PCR products from virus RNA perfectly confirmed the sequences obtained in the clones, and excluded any randomely introduced sequence errors in the course of recombinatorial cloning. Accordingly, our data showed that the L segment of the here used progenitor virus  differed from a closely related Italian BATV isolate (Accession: KC168048) by five silent nucleotide exchanges.After LLHR of the PCR-amplified L-segment with the pHaHdmin-derived linear vector in These data demonstrated that LLHR works independently of the complete virus genome sequence, and adaption of the functional elements in the vector backbone of pHaHdmin flanking the recombineered viral cDNA to other NSV families can be easily done.We present a novel strategy for fast cloning of complete infectious negative strand RNA virus genomes into reverse genetics plasmid vectors. Beyond cloning of genomic virus cDNAs in a restriction endonuclease independent manner, the novel system exhibits several benefits for cloning and generation of recombinant negative strand RNA viruses that could create a milestone in reverse genetics technology: (i) Cloned genomes in the full length cDNA plasmid are derived from genome copies that really exist in the authentic progenitor virus populations, artificial combinations of sub-fragments can be excluded. (ii) The high efficiency of full length cDNA cloning opens novel possibilities to combine modern NGS methods of sequence analysis with the assessment of sequence variation in individual genome copies. (iii) Direct recovery of individual virus mutants allows rapid experimental proof of concepts generated by sequence analysis. (iv) Customized recombinant viruses from relevant reservoir hosts are easy to generate and will advance future pathogenesis research. (v) The flexibility by using PCR primers in the system allows quick adaptation of reverse genetics vectors to phylogenetically distant viruses.E. coli has been used mainly for genetic modification of large DNA fragments such as BACs, YACs or even E. coli chromosomes26E. coli DH5α target the same aim of efficient recombinatorial cloning32Although RecE/T-mediated homologous recombination in One further example for the power of the system is given by the combined selection of two amino acid exchanges (D266N and K508T) in the glycoprotein G after one passage of a fox derived RABV isolate in a heterologous raccoon host . By comp30Notably, by comparing RABVs circulating in Namibian kudu and jackal population amino acid replacements in the glycoprotein of Kudu-derived RABVs have been predicted to cause adaptation to Kudu-hostsWith a maximum of 5 single nucleotide exchanges in 8 completely sequenced full length clones, the frequency of detected mutations was low. Although we cannot exclude introduction of mutations during reverse transcription and PCR amplification of full length cDNAs, pre-existence of silent and non-silent mutations in the progenitor fox and dog sequence populations indicates that at least part of the identified mutations represent minor variations within the virus population and were not generated during the cloning process.Because of the low degree of sequence variations only four clonal sequences were required to identify cDNA clones that were identical to the consensus sequence of the progenitor virus isolates . In caseAccordingly, recombinant wild-type virus like rRABV-Fox clone 9 and rRABV-Dog clone 14 exhibited growth kinetics comparable to their parental virus isolates , even th5 TCID50/mouse, whereas comparable infections with non-fixed isolates such as European Bat Lyssaviruses (EBLVs) or Bokeloh Bat Lyssavirus (BBLV) are 100% lethal in 3 week old BALB/c mouse at those doses3 TCID50/mouse strongly supported the wt virus character of those clones. Notably, a higher lethality of rRABV-Dog 14 when compared to the parental wt RABV Dog showed, that use of a clonal virus can be superior in pathogenesis, maybe by the absence of attenuating virus variants. In view of the low level of sequence variability in both, the original dog and fox virus isolates  virus is only partially lethal after intramuscular inoculation of 5 × 10isolates , furtherFrom growth kinetics and peripheral mouse inoculations we conclude that the described blueprint for cloning and generation of recombinant wild-type RABV indeed allows the recovery of non-fixed recombinant viruses for further analyses related to RABV pathogenesis in relevant hosts. This possibility is new since so far only a limited number of recombinant RABVs12in vivo and whether such reporter viruses are usable tools to investigate RABV pathogenesis by tracking reporter expressing virulent and non-fixed RABV in vivo.Although it has been shown that additional genes can be inserted in recombinant RABVs and other non-segmented negative strand RNA viruses without effects on the virus replicationtively, together with limited cell culture replication shown here  to destroy methylated plasmid DNA. The PCR-products were then gel purified  and subjected to LLHR.The recombination vector pHaHdmin was linearized with mers see , the terVirus RNA was extracted from supernatant virions after polyethylenglycol (PEG) precipitation  or from infected BsrT7/5 or Na42/13 cell cultures with PeqGOLD TriFast™ reagent (Peqlab) according to the supplier’s recommendations.2, 1 mM DTT, pH 7.9) and was then purified  according to the manufacturer’s recommendations. 400 ng of RppH digested or not digested genomic RNA were ligated with 10 units T4 RNA Ligase (New England Biolabs) in 20 μl reaction buffer  for 1 h at 37 °C. The reaction was stopped by adding EDTA to a final concentration of 5 mM and 15 min incubation at 65 °C. Five μl of the ligation sample was directly used as a template for RT-PCR with SuperscriptIII reverse transcriptase (Invitrogen) and GoTaqFlexi (Peqlab) according to the manufacturer’s protocols with gene specific oligonucleotides listed in To determine the terminal sequences of RABV genomes, the viral RNA genomes isolated from PEG precipitated supernatant virions were circularized by T4 RNA Ligase. To remove the 5′-triphosphate residues from genomic RNAs prior to RNA ligation, 1 μg RNA was digested with 5 units of 5′ Pyrophosphohydrolase  in 20 μl reaction buffer . Briefly, 1.4 ml Luria Broth medium was inoculated with 30 μl of an overnight GB05-dir culture. After reaching an OD600 of 0.3, the RecE/RecT recombinases were induced by adding L-Arabinose to a final concentration of 0.30–0.35% (w/v). A not induced culture was used as a negative control. 35 min after induction, the bacteria were pelleted at 11.000 rpm and 2 °C for 30 and washed three times with ice-cold water, supernatants were discarded. 100 ng of gel-purified linear minimal vector and 100 ng of genomic virus cDNA in a maximal volume of 15 μl H2O were added to the pelleted bacteria. Electroporation was performed in a 0.1 mm gapped cuvette at 1.35 kV . Subsequently, the bacteria were resuspended in 1 ml LB-medium and were incubated for 90 min at 37 °C. Recombination events were selected by over-night cultivation of the bacteria on LB Agar plates containing 100 μg/ml ampicillin. Ampicillin resistant colonies from induced and not-induced approaches were compared to estimate the efficiency of the recombination process. Insertion of cDNA genome copies into the vector was checked after plasmid preparation from individual clones by SacI digest.Recombination of linear long range RT-PCR products and linear plasmid vector derived PCR products was performed by electroporation of both DNAs in the Recombinant RABVs (rRABVs) were rescued by co-transfection of full length genome cDNA and expression plasmids into BSR T7/5 cells as described previously11For sequencing of the parental RABV isolates from fox (Germany 1998) and dog , total RNA was extracted from cell culture supernatant using peqGOLD TriFast  and RNeasy mini kit (Qiagen) as describedIndirect immunofluorescence with mouse monoclonal antibody E559EcoRI-rev, SacII digestion of both fragments, ligation and second round PCR amplification of a 0.82 kb DNA-fragment with the primers Dog-KpnI-for/Dog-EcoRI-rev. A 0.80 kb KpnI/EcoRI fragment from the product was inserted into KpnI/EcoRI digested pRABV-Dog. The resultant pRABV-Dog MCS was NheI/NotI digested and ligated with a 0.74 kb NheI/NotI fragment digested from pSADL16 Katushka (unpublished) containing the ORF of the red fluorescent marker. pRABV-Fox Katushka was constructed by a similar strategy. After PCR amplification of DNA fragments from pRABV-Fox clone 9 with primer pairs Dog-KpnI-for/Dog-EcoRI-rev and Dog-MCS-for/ Fox-Eco72I-rev  and gained governmental approval (registration number LALLF M-VTSD7221.3-2.1-00211). General care and methods used in the animal experiments were carried out according to the approved guidelines. The animals were checked daily for clinical signs and were euthanized after development of severe clinical signs of rabies indicated by . Three to four week old BALB/c mice were inoculated by the intracranial (i.c.) route of infection with 30 μl virus suspension at a dose of 50 focus forming units (ffu) per mouse (6 mice per group). Intramuscular (i.m.) inoculations were performed by injection of 30 μl of virus suspension (50 ffu/mouse or 5000 ffu/mouse) in the thigh of one hind leg. All inoculations were performed under anaesthesia (Isofluran).How to cite this article: Nolden, T. et al. Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof. Sci. Rep.6, 23887; doi: 10.1038/srep23887 (2016).


Nassella (Stipa) pulchra, the exotic annual grass Bromus hordeaceus, and three virus species, Barley yellow dwarf virus-PAV, Barley yellow dwarf virus-MAV, and Cereal yellow dwarf virus-RPV. In terms of virus transmission, the native host was less susceptible than the exotic host to MAV. Coinfection of PAV and MAV did not occur in any of the 157 co-inoculated native host plants. In the exotic host, PAV infection most strongly reduced root and shoot biomass, and coinfections that included PAV severely reduced biomass. Infection with single or multiple viruses did not affect biomass in the native host. However, in this species the most potentially pathogenic coinfections (PAV + MAV and PAV + MAV + RPV) did not occur. Together, these results suggest that interactions among multiple pathogens can have important consequences for host health, which may not be predictable from interactions between hosts and individual pathogens. This work addresses a key empirical gap in understanding the impact of multiple generalist pathogens on competing host species, with potential implications for population and community dynamics of native and exotic species. It also demonstrates how pathogens with relatively mild impacts independently can more substantially reduce host performance in coinfection.Pathogens are common and diverse in natural communities and have been implicated in the success of host invasions. Yet few studies have experimentally measured how pathogens impact native versus exotic hosts, particularly when individual hosts are simultaneously coinfected by diverse pathogens. To estimate effects of interactions among multiple pathogens within host individuals on both transmission of pathogens and fitness consequences for hosts, we conducted a greenhouse experiment using California grassland species: the native perennial grass  Pathogens are ubiquitous in wild plant communities ,2. DespiRecent work has shown that complex interactions between vector and virus species shape the community composition of a group of plant viruses, the barley yellow dwarf viruses (BYDVs) –10. HoweNassella (Stipa) pulchra, a perennial bunchgrass native to California, and Bromus hordeaceus, an invasive annual grass now ubiquitous in much of the California grassland habitat. The virus species are three common barley yellow dwarf viruses: Barley yellow dwarf virus-PAV, Barley yellow dwarf virus-MAV, and Cereal yellow dwarf virus-RPV . These viruses are systemic through phloem tissue, and are obligately transmitted by aphids, chiefly two species: Rhopalosiphum padi transmits PAV and RPV, and Sitobion avenae transmits PAV and MAV.Here, we address a key empirical gap that will inform how virus diversity and composition may impact plant communities. To our knowledge, no published studies have inoculated individual wild plants with more than two virus species, and research on fungal pathogens of plants suggests that the joint effect of three pathogens may not be simply predicted from the effects of two pathogens, and may vary among host species . We use Bromus hordeaceus biomass, basal area, and seed production, as well as perennial grass survival in the field [Nassella, (2) PAV and MAV would each have lower inoculation success when the other virus was present in a plant, (3) PAV would increase RPV inoculation success, and (4) both Nassella and Bromus would experience biomass reductions when infected that would increase with within-host virus diversity.Previous work has shown that California native perennial grasses tend to have lower susceptibility to PAV infection than exotic annual grasses , that PAhe field –19. BaseN. pulchra, and the exotic grass, B. hordeaceus. Plants were grown in 938 mL D60 Deepots  filled with Fafard 3B potting mix. Plants were inoculated via aphids with all possible combinations of three viruses, and control plants were mock-inoculated with uninfected aphids to account for the effect of aphid feeding. As a further control, three plants of each species received no aphids. In total, the experiment contained 600 plants divided into three blocks of 200 plants each (133 N. pulchra and 67 B. hordeaceus per block). The sample sizes were doubled for Nassella due to lower expected inoculation success from previous work [The primary purpose of this study was to examine the rates and impacts of infection and coinfection of different BYDV species in the California native bunchgrass, ous work . All treAvena sativa L. cv. ‘Coast Black’, undergoing approximately three transmission cycles per year. These isolates are the type specimens for the three virus species, and using them facilitates comparison of our results to the extensive published experiments on these same isolates, including both comparisons among virus species, and two-way interactions among virus species ..Avena saR. padi aphids that had been exposed to both viruses (these viruses are not known to interact within shared aphids). Co-inoculation of PAV and MAV using doubly exposed aphids was not possible because these viruses strongly interfere with each other within their shared aphid vector, S. avenae [R. padi for R. padi controls, PAV, RPV, and PAV+RPV; 5 S. avenae for S. avenae controls, MAV) or ten aphids per plant . The aphids were caged onto individual plants for a 72-hour inoculation access period, and then removed using es-fenvalerate  non-systemic insecticide.Plants were inoculated with viruses at the three-leaf stage. Before inoculation, the vectors acquired the virus by feeding on infected plant tissue in dishes for 48 hours. Plants were inoculated with five aphids for each viral species they received, as outlined in Table A in . avenae . The resNassella and 3 seeds per pot for Bromus (based on the higher expected germination fraction of Bromus). Two weeks prior to inoculations, pots were thinned to one plant per pot. Five weeks after inoculations, all above- and below-ground tissue was harvested from each plant. Between 0.2 and 0.3 grams (fresh weight) of above-ground plant tissue was collected for ELISA virus assays (described below). The remaining above-ground tissue was weighed fresh, then dried for at least 72 hours at 60°C and weighed dry. We estimated the additional dry mass of tissue collected for ELISAs based on the ratio of dry mass to wet mass in the main tissue samples, and added this amount to the above-ground biomass. We estimated below-ground biomass by hand-washing roots to remove all soil and debris, drying them for at least 72 hours at 60°C, and weighing them. Due to time constraints, roots were washed from a subset of plants representing each infection status category.Pots were initially seeded with 10 seeds per pot for In order to determine the infection status of each plant, we performed double-antibody sandwich indirect ELISA using monoclonal antibodies from Agdia, Inc., Elkhart IN, USA, as detailed in previous work ,18,19,25Infection is the outcome for a plant individual for a given virus, Virus 1 status and Virus 2 status represent the status of the plant with respect to the other two viruses , and Plant is the species identity of the focal individual .We used statistical models to test how the probability of becoming infected with a particular virus when inoculated  depended on the plant species identity and infection status with respect to the other two viruses. For each virus species, we fit a binomial generalized linear model (GLM) with a logit link, using the formula:To compare the inoculation success of the viruses, we also fit an overall model of inoculation success as a function of plant species and virus species, irrespective of infection status with respect to the other viruses:Because some plants were inoculated with multiple viruses, multiple data points could come from the same plant individual. We controlled for this non-independence by fitting the model as a binomial generalized linear mixed model (GLMM) with a random effect of plant individual. We additionally tested for overdispersion, but found little evidence of it (dispersion parameter = 1.005), and therefore did not correct for it.m0) to the above-ground biomass data and a second model to the below-ground biomass data.Although we initially intended to analyze just the data from plants in which treatments were fully successful , this subset of the data had low sample size for some treatments. As a result, we performed most of our analyses on the full dataset, using observed infection status (as opposed to treatment) as a predictor variable. However, we also fit one model to only the plants in which treatments were successful. For this, and all other model structures described below, we fit one model (R. padi), Con-5-Sa (received 5 S. avenae), and Con-5-Rp-5-Sa (received 5 R. padi and 5 S. avenae).The four control treatments are: No treatment (received no aphids), Con-5-Rp , (ii) infection class, treating each possible combination of the three viruses as a separate class , (iii) the number of viruses infecting a plant, assuming a linear effect of virus number (m7), and (iv) whether or not a plant was infected with any virus (m8). We used these different ways of describing infection as predictors in order to explore which provided the best model fit, penalized for model complexity, via AIC. All data analysis was performed in R version 2.15.1 [These models analyzed the impact of infection on biomass of each plant species in several ways: (i) infection status with respect to each of the viruses independently (n 2.15.1  using thn 2.15.1 .Bromus than in Nassella (p<0.001). Although PAV and MAV each tended to have lower inoculation success in the presence of the other, the differences were not significant (p>0.4), nor were there effects (p>0.4) of RPV infection on PAV or MAV inoculation success (Table B in Nassella (n = 156 inoculations), and only occurred seven times in Bromus (n = 70 inoculations).We fit models of inoculation success for each virus separately and for all three viruses together. In the individual virus models, MAV had higher inoculation success in la p<0.00. AlthougNassella than in Bromus (p = 0.26) but MAV had lower success in Nassella , but MAV infections had significantly lower shoot biomass . In addition, MAV, PAV-MAV, PAV-RPV, and PAV-MAV-RPV coinfections had significantly (p<0.02) lower root biomass than other infected and uninfected plants  included plant species by infection status interactions. In that model, plants in all infection classes except RPV and MAV had significantly lower root biomass in Bromus , but not in Nassella  PAV and MAV would each have lower inoculation success when the other virus was present in a plant, (3) PAV would increase RPV inoculation success, and (4) both Nassella and Bromus would experience biomass reductions when infected that would increase with within-host virus diversity. Each of these predictions was partially, but not fully, supported. Nassella had lower susceptibility to MAV than Bromus, but host species did not significantly differ in susceptibility to PAV or RPV (prediction 1). Although the infection data qualitatively fit our predictions about the directional effects of virus interactions on inoculation success—that PAV and MAV would suppress each other and that PAV would facilitate RPV (predictions 2–3)—the effects were not statistically significant .Based on previous work with BYDVs, we predicted that (1) the viruses would have lower inoculation success in nificant . FinallyBromus and by up to 21% and 18% in Nassella above- and below-ground, respectively. Infected Bromus plants had lower above- and below-ground biomass than uninfected Bromus, and biomass declined with the number of viruses present; however, these patterns did not hold for Nassella, which had lower biomass than Bromus regardless of infection status but no significant effect of infection or virus number , we might expect viruses to play a relatively minor role for Nassella populations in the field. However, since Nassella is a perennial, any effects of infection may compound year after year by reducing biomass, survival, and fecundity in the long term [Nassella to coexist with exotic annual grasses like Bromus in California grasslands. Ideally, we would measure fitness effects on inoculated plants in the field, but such experiments are logistically challenging because they require access to local virus, aphid, and plant populations and control over aphid exposure throughout the growing season.Because ong term ,17. As aNassella) and favor the invasion of exotic annual grasses (like Bromus) [By measuring the effect of single- and multiple- infection on above- and below-ground biomass for two common plant species, this study highlights individual-level consequences of infection. A major future direction is to translate these individual effects to population- and community-level outcomes, using models parameterized based on the focal species. Pathogens that differentially affect the fitness of multiple host species can influence the outcome of competition between hosts , and pre Bromus) . Yet it  Bromus) . This stWith growing recognition that humans, plants, and wildlife host many interacting infectious agents, recent work has focused on understanding the factors controlling coinfection –32, and S1 Appendix(PDF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


Rhabdoviridae family, was able to evade the protective immune response induced by the DNA vaccination of rainbow trout. The experiments comprised repeated passages of a highly pathogenic VHSV isolate in a fish cell line in the presence of neutralizing fish serum (in vitro approach), and in rainbow trout immunized with the VHS DNA vaccine (in vivo approach). For the in vitro approach, the virus collected from the last passage (passaged virus) was as sensitive as the parental virus to serum neutralization, suggesting that the passaging did not promote the selection of virus populations able to bypass the neutralization by serum antibodies. Also, in the in vivo approach, where virus was passaged several times in vaccinated fish, no increased virulence nor increased persistence in vaccinated fish was observed in comparison with the parental virus. However, some of the vaccinated fish did get infected and could transmit the infection to naïve cohabitant fish. The results demonstrated that the DNA vaccine induced a robust protection, but also that the immunity was non-sterile. It is consequently important not to consider vaccinated fish as virus free in veterinary terms.DNA vaccines encoding viral glycoproteins have been very successful for induction of protective immunity against diseases caused by rhabdoviruses in cultured fish species. However, the vaccine concept is based on a single viral gene and since RNA viruses are known to possess high variability and adaptation capacity, this work aimed at evaluating whether viral haemorrhagic septicaemia virus (VHSV), an RNA virus and member of  Novirhabdovirus genus within the Rhabdoviridae family [Oncorhynchus mykiss) in Europe, causing high mortalities in all fish stages [Viral haemorrhagic septicaemia virus (VHSV) is a negative-sense, single-stranded RNA virus, which belongs to the e family . VHSV ish stages .Currently, no commercial vaccine against VHS is available. Several vaccination strategies have been tested to control this disease, among them live attenuated vaccines, inactivated vaccines, and recombinant protein vaccines, but with limited efficacy or compromised safety aspects , 4. By cThe traditional DNA vaccine against VHS consists of a plasmid designed for expression of the viral surface glycoprotein (G) in eukaryotic cells. The immune protection induced by this vaccine start as early as 4 days after intramuscular injection, when an early, non-specific, and short-lasting antiviral protection is triggered. This early protection has been related to interferon-associated mechanisms, which are characterized by the overexpression of multiples interferon-stimulated genes with antiviral functions such as Mx, Vig-1, Vig-2, and Vig-8 . After 4Although the high efficacy of the DNA vaccine against VHSV has been consistent under experimental conditions, its protective effect might be threatened following repeated use under field conditions, due to the high variability of RNA viruses. Whether this variability might allow generation and selection of VHSV mutants, capable of evading the immunological protection induced by the DNA vaccine remains to be addressed.Evidence of how the genetic variability of VHSV allows adaptation to selective conditions was shown when rainbow trout were injected with a plasmid encoding a neutralizing single chain antibody (scAb) against the G protein of VHSV. In this case, a neutralization escape mutant was isolated from the survivors of the infection . SimilarWhile mutations in the G protein can affect the efficacy of the adaptive protection, mutations in e.g. the non-structural-protein (NV) may potentially affect the ability of the virus to bypass the innate protection induced by the DNA vaccine. This viral protein has been suggested to inhibit the apoptotic signal in virus infected cells at an early stage of virus infection, thus affecting the virulence of the VHSV .The aim of this work was to determine whether VHSV within repetitive passages under the selective pressure of DNA vaccine-induced immunity, would be able to develop mutants that could escape from the early and late protective mechanisms induced by the vaccine, and from neutralizing serum antibodies induced in DNA-vaccinated rainbow trout. Knowledge of these aspects is essential for the design of robust and safe DNA vaccination strategies for protection of rainbow trout against VHS under field conditions.The fish cell lines used in this study were EPC (epithelioma papulosum cyprini)  and BF2 50) per ml, in BF2 cells [To propagate VHSV, BF2 cell cultures were inoculated with low multiplicity of infection of the virus and maintained at 15°C until a complete cytopathic effect (CPE) was observed. The supernatant was collected and centrifuged at 4500 x g for 15 min at 4°C to eliminate cellular debris. The virus was stored at -80°C. The titer was determined using the method of 50% tissue culture infective doses  in the left epaxial muscle below the dorsal fin with 25 μl of purified DNA plasmid in saline solution , as described earlier [For  earlier . This st earlier . Non-vacRepeated passaging of VHSV was performed in fish vaccinated 1 week before inoculation with virus as well as in fish vaccinated 6 weeks before inoculation with virus. The fish were vaccinated with either 0,1 or 1,0 μg of the vaccine. Non-vaccinated fish were included as controls to confirm virulence the passaged virus.5 TCID50 ml-1 of the VHSV isolate DK3592b, hereafter called parental virus. After this, water flow was restored. Moribund fish, with clinical signs of VHS, were euthanized with an overdose of benzocaine and stored at −20°C until further analysis. At 21 days post infection, the surviving fish were euthanized with an overdose of benzocaine. The sampled moribund fish were dissected, and spleen, liver, heart, head kidney, and brain were collected and pooled per fish in MEM. Organs were homogenized in a TissueLyser (Qiagen) for 2 min at 20 Hz. The homogenate was centrifuged at 4500 x g for 15 min, and the supernatant was collected to be treated with gentamicin overnight at 4°C. After the antibiotic treatment, the virus content was titrated on BF2 cells and the samples stored at -80°C. These first-passage homogenates were used to infect new batches of fish vaccinated 1 or 6 weeks earlier (the second passage).In the first passage, each treatment group included two aquaria with 25 fish in each. The infection was carried out by immersion in 8 l water for 3 h in static water with a virus concentration of 1 x 10Due to the low amount of virus recovered from the first passage, the second passage was performed by I.P injection of 50 μl tissue homogenate supernatant into 2x10 fish. Subsequent passages were performed as the second passage with the exception that the tissue homogenates included organs from both survivors and moribund fish. The procedure for monitoring and sampling was performed as explained in the first passage .A challenge trial with vaccinated fish was performed to compare the performance of the parental virus with the virus from the last passage in vaccinated fish, hereafter called passaged virus, under the selective pressure induced by DNA vaccination. To obtain sufficient virus for immersion challenge, the passaged virus, was passaged once in BF2 cells and titrated as outlined above.4 TCID50 ml-1. After this, the water flow was restored. Two weeks post-infection, 10 non-vaccinated (naïve) fish were transferred into each aquarium to cohabitate with the vaccinated fish. The naïve fish were tagged by cutting a part of the tail fin. After another 2 weeks, all surviving fish were euthanized with an overdose of anesthetics. The spleen, heart, liver, kidney and brain were collected and pooled from each fish individually. The tissues were homogenated and used for virological examination. The persistence in vaccinated fish and the spread of the virus to naïve fish were calculated as the percentages of virus-positive fish.The comparative analyses included accumulated mortality, the virus carrier state of the vaccinated fish, as well as the ability of the virus to spread to co-habitant naïve fish. The fish were vaccinated with 1,0 μg of the DNA vaccine and challenged at 1 or 6 weeks post-vaccination, using virus passaged at similar times post-vaccination. Fish challenged with the parental virus served as controls. As an example, when the fish were challenged at 1 week post-vaccination the passaged virus used was VHSV-1W0,1 and VHSV-1W1,0. With each virus, the infection was performed in duplicates with 20 fish in each aquarium. The infection was carried out by immersion in static water in the 8 l aquaria for 3 h with a final concentration of the virus at 1x10Supernatants from tissue homogenates treated as described above were used to prepare 10-fold serial dilutions, which were inoculated onto BF2 monolayers with 60–80% confluency in 24-well tissue culture plates. The cultures were maintained at 15°C for 7 days when each well was examined. The wells with CPE were considered positive. The identity of the CPE-causing virus was confirmed by PCR as outlined below.An RT-PCR assay with primers specific for the VHSV nucleoprotein (N)-gene was used to confirm the identity of the passaged virus as well the virus re-isolated from sampled fish in the comparative challenge trial. Total RNA was isolated from 100 μl of each supernatant using the RNeasy mini kit (Qiagen). Subsequently, the cDNA was synthesized using the iScript kit (BioRad) following the manufacturer's recommendation. The qPCR reaction contained 1 μl of the cDNA, 7,5 μl of SuperMix, 0,03 μl of Rox, 0.3 μl of each primer VHSV-N-For 5’-AGG TCT CAG ATG TCA TCA AGG AG-3’ and VHSV-N-Rev 5’-CGG TGG AGC TCC TGA AGT T-3’, and 5.87 μl of free-nuclease water. PCR amplification program involved an initial step at 50°C for 2 min, and then a denaturation cycle 95°C for 2 min. These steps were followed by 40 cycles 95°C for 15 sec and 60°C for 1 min. Amplification was performed in MX Pro-Mx3005P thermocycler (Stratagene). Samples giving a signal within 25 cycles and an amplicon product with a melting temperature corresponding that obtained with the positive VHSV isolate DK3592b control, were considered as positive.For the first passage, serial 10-fold dilutions of VHSV isolate DK3592b 30 μl) were mixed in wells of 96-well plates with 15 μl of a 1/40 dilution of the heat treated (30 min at 45°C) neutralizing trout serum obtained after repeated immunization with the DNA vaccine (pcDNA3-vhsG) . Followi μl were in vitro passaged virus and the parental virus to the neutralizing effect of the immune serum was compared using the plaque neutralization assay (PNT), in which the titer of the serum is defined as the reciprocal value of the highest serum dilution reducing the number of plaques by 50% compared to a normal trout serum control [The susceptibility of the  control . To evalEach replicate in both the virulence and the persistence analyses was considered as an individual sample to evaluate intergroup variability. The statistical analysis for difference between treatments was performed by the Kruskal-Wallis test . Differences were considered significant at p<0.05.nd passage. This might suggest that the protection induced by 1,0 μg of the DNA vaccine was able to clear the virus in the immunized fish more efficiently than in fish vaccinated with 0,1 μg DNA, although the latter dose was still highly protective (data not shown).To test the ability of the virus to bypass the early protection induced by the DNA vaccine, we passaged VHSV in rainbow trout vaccinated 1 week before inoculation with virus. When the fish used for passaging had been vaccinated with a dose of 0,1 μg of the DNA vaccine, we were able to isolate virus from dead and vaccinated surviving fish in each of the successive 5 passages. When fish given 1,0 μg of the DNA vaccine were used, we were able to isolate virus only until the 2To test the ability of the virus to bypass the late protection induced by the DNA vaccine, the passaging was performed in rainbow trout vaccinated 6 weeks before inoculation. In this setup, VHSV was re-isolated from dead and vaccinated survivor fish in all 4 passages using both vaccine doses .After successive passages, the virus obtained from the last passage under selective conditions (passaged virus), was amplified by 1 passage on BF2 cells and later compared with the parental VHSV isolate.in vivo approach included three factors; mortalities induced in vaccinated and non-vaccinated fish, the capacity of the virus to infect and persist in vaccinated fish, and the ability of the virus to spread from vaccinated carriers to cohabitant naïve fish.The comparison for the The mortality rates caused by the passaged virus and the parental virus in vaccinated fish were both low, ranging between 0–12%. Only one of the duplicate aquaria with fish inoculated with the VHSV-1W1,0, and one of the duplicates inoculated with the VHSV-6W0,1, showed higher mortalities (about 20%) than the aquaria inoculated with the parental virus . Since tThe evaluation of the ability of the virus to infect vaccinated fish showed that both the parental and passaged virus were able to infect and persist in some of the vaccinated fish for at least 4 weeks post-challenge . The higThe analysis of whether vaccinated carriers infected with either the passaged or the parental virus were capable of transmitting the infection to naïve fish showed that even though only a few fish could be detected as carriers, transmission did take place for the passaged as well as for the parental virus, in at least in one of the replicates . The setAfter 11 successive passage in cell culture in the presence of neutralizing trout serum, we compared the ability of the passaged virus and the parental virus to escape from the neutralization by a trout immune serum. As shown in The glycoprotein gene of the 3 preparations of passaged virus and the parental virus was sequenced and compared to determine the presence of mutations. The 4 sequences had 100% nucleotide identity (not shown).This work focused on analyzing whether the fish rhabdovirus VHSV was able to mutate and escape from the protective mechanisms induced by the VHS DNA vaccine. Such mutants would represent a potential risk of reemergence of the disease in vaccinated fish populations, reducing the applied potential of the DNA vaccine. To our knowledge, this important aspect remains to be addressed for the otherwise extensively analyzed and highly protective fish DNA vaccines. Our results suggest that the immune response triggered by the vaccine is rather robust and not easily bypassed by the virus.The high genetic variability of RNA viruses is due to the high replication rates, large population size, and high mutation rates, which together generate a diverse spectrum of virus variants in every replication cycle and result in a population of one dominant virus variant along with multiple low-frequency virus variants. This phenomenon is known as the quasispecies theory and allows the virus to adapt rapidly to new environments, and to new hosts . When RNin vivo approach was used to evaluate the ability of VHSV to evade the early and late protection induced by the DNA vaccine in rainbow trout fingerlings, associated with innate and adaptive immunity, respectively [in vitro approach was applied, to evaluate the ability of the virus to evade the neutralizing effect of serum from rainbow trout immunized with the DNA vaccine.The experimental design included two approaches. First, an ectively , 33, 34.in vivo passaging, the initial inoculation of the fish was done by immersion. i.e. the natural route of infection. But from the second passage, the inoculation was performed by I.P injection due to the low amounts of virus recovered from each passage, and to avoid propagation of the virus in cell culture, which could compromise the selection procedure. In order to obtain enough virus to perform the infection in the final evaluation trial by immersion, one cell culture passage was required. Although this was a compromise, the risk of this significantly changing the virus population after several passages under selective conditions in vivo was considered to be small. Sequence analysis by next generation sequencing would be required to confirm this, but was beyond the scope of the present study.In the in vivo approach took into account that an escape mutant could have diverse strategies to bypass the immune protection induced by the DNA vaccine. Among these strategies, escape mutants could have increased virulence in vaccinated fish, causing higher mortality rates as a result of a more efficient viral replication. Alternatively, decreased virulence might reduce clearance, thereby allowing the virus to persist in the host and spread to cohabitant fish. However, the in vivo approach evaluation showed no advantage of any of the batches of passaged virus in comparison with the parental virus, neither considering virulence, persistence in vaccinated fish, nor the capacity of vaccinated fish carriers to transmit the infection to naïve cohabitant fish.The analysis of the According to Read et al. 2015, infected vaccinated chickens were associated with a higher risk of spread of highly virulent virus than the infected non-vaccinated chickens. The rationale behind this theory was that non-vaccinated chickens would die out rapidly and thereby allow the infection to be kept under control, while the infected vaccinated chicken would survive the infection and allow the virus to persist, replicate and spread in the host population for an extended period. Furthermore, the persistence in vaccinated chickens could promote the selection of hyperpathogenic virus strains that could cause a more severe disease .While this scenario might be true for some highly lethal viruses, our results suggest that it does not count for VHSV infections in rainbow trout. Although the virus causes high mortality, some individuals often survive and clearance from such fish is slower compared to DNA vaccinated individuals . Therefoin vitro aim was look at the adaptive humoral immune response to DNA vaccination alone and evaluate whether serial passages of VHSV in the presence of serum from rainbow trout immunized with the DNA vaccine, would favour propagation of neutralization escape mutants. The trout immune serum used here was obtained from a hyperimmunized fish [The zed fish .Previous works showed that viruses like VHSV and IHNV were able to generate escape mutants resistant to neutralization by monoclonal antibodies after a few passages in cell culture , 25. Anoin vitro analysis suggested that VHSV cannot easily escape even from the neutralizing serum antibodies, which is only one mechanism of the adaptive responses induced by DNA vaccination. However, while a single vaccination did not induce an antibody response in all individuals, particularly at lower temperatures [in vivo suggested that the broad nature of the immune response triggered by the vaccine, involving a range of both innate and adaptive mechanisms, made escape mutation incompatible with maintaining the viability and the infectious capacity of the virus.However, after 11 passages, there was no evidence of mutants escaping the neutralizing effect of the trout immune serum. This was confirmed by the fact that the passaged and the parental virus had a 100% identical glycoprotein gene sequence. In contrast to this, mutants escaping neutralization by mouse monoclonal antibodies carried one or few amino acid substitutions in the translated G gene . The experatures , our faiOur setup only included the most stringent selective condition by challenging vaccinated fish with virus carrying a G gene identical to the vaccine gene. It can therefore not be excluded that viral escape from DNA vaccine induced immunity might arise under less selective conditions, such as when the vaccine G gene is heterologous to that of the infecting virus, resulting in a less complete protection . The safIn conclusion, our results suggest a low probability of occurrence of escape mutants under optimal DNA vaccination conditions. In applied terms, this represents an additional advantage of this rather efficient vaccine against VHSV, making it a safe prophylactic tool. However, we also observed that some of the vaccinated fish can get subclinically infected and that the infection can be transmitted to naïve cohabitants if these are stocked with the vaccinated fish shortly after their exposure to the virus. Vaccinated fish from endemically infected zones should therefore be considered to be potential carriers in terms of trade regulations.


Giardia intestinalis, a highly prevalent protozoon that impacts children’s nutritional status in developing countries. The objective of this study was to explore spatial and molecular epidemiology of Giardia intestinalis in children of Amerindian descent in the Brazilian Amazon.Current control policies for intestinal parasitosis focuses on soil-transmitted helminths, being ineffective against G. intestinalis infection was 16.9% (73/433), reaching 22.2% (35/158) among children aged 2–5 years, and a wide distribution throughout the city with some hot spots. Positivity-rate was similar among children living in distinct socioeconomic strata . Sequencing of the β-giardin gene revealed 52.2% (n = 12) of assemblage A and 47.8% (n = 11) of assemblage B with high haplotype diversity for the latter. The isolates clustered into two well-supported G. intestinalis clades. A total of 38 haplotypes were obtained, with the following subassemblages distribution: 5.3% (n = 2) AII, 26.3% (n = 10) AIII, 7.9% (n = 3) BIII, and 60.5% (n = 23) new B genotypes not previously described.A cross sectional survey was performed in the Brazilian Amazon with 433 children aged 1 to 14 years. Fecal samples were processed through parasitological techniques and molecular characterization. Prevalence of Giardia intestinalis infection presents a high prevalence rate among Amerindian descended children living in Santa Isabel do Rio Negro/Amazon. The wide distribution observed in a small city suggests the presence of multiple sources of infection, which could be related to environmental contamination with feces, possibly of human and animal origin, highlighting the need of improving sanitation, safe water supply and access to diagnosis and adequate treatment of infections. Giardia intestinalis stands out for its high frequency in different socioenvironmental scenarios and its prevalence in both developed and developing countries [G. intestinalis presents high levels of genetic diversity, which have been classified into eight assemblages (A-H). Parasites isolated from humans belong to the globally distributed assemblages A and B, which also have other animals as hosts, being potentially zoonotic [Among the intestinal parasites, ountries –5. G. inzoonotic . Genotypzoonotic .G. intestinalis occur after the ingestion of cysts in contaminated water, directly from person to person by fecal-oral contamination or, occasionally, from food [G. intestinalis [Infections with rom food . Low-incstinalis . Giardiastinalis ,8.G. intestinalis is an important cause of diarrhea, most infections have chronic and asymptomatic character [G. intestinalis includes apoptosis of enterocytes, epithelial cell damage, and consequent malabsorption [G. intestinalis infection has been shown to impact the nutritional status of children, with the potential of seriously compromising their physical development [Although haracter . The patsorption . Importaelopment –15.G. intestinalis infection in children of Amerindian descent that live in a remote municipality in the Brazilian Amazon.While control policies for intestinal parasitoses have been successful against soil-transmitted helminths, these same policies are ineffective against protozoan parasites as the treatment for the diseases they cause requires different drugs and more complex ministration schedules –18. HereThis study was a cross-sectional survey performed in 433 children from Santa Isabel do Rio Negro in 2011 . This sm® software . Maps were generated using the kernel density estimation method, and only first order effects were evaluated. The maps were made using data provided by OpenStreetMap® available under the Open Database License (https://www.openstreetmap.org/copyright).Georeferencing was performed with a Global Position System in the SAD-69 geodetic datum. Spatial data were analyzed in a GIS platform using ArcGis 9.3G. intestinalis-positive stool samples using the ZR Fungal/Bacterial DNA kit . For the amplification of the 753-bp β-giardin (βG) gene fragment we utilized the G7-G759 primers, as described by Cacciò et al. [® T-Easy . Briefly, the inserts were amplified by PCR using the M13 primer and sequenced [DNA was extracted, in a field laboratory, only from parasitologicaly confirmed ò et al. . Productequenced . We usedβG sequences were inferred with BEAST-1.8 and PhyML-3.0, respectively. The Akaike and Bayesian Information Criteria of jMODELTEST-2 were used to elect Tamura-Nei with four gamma categories as the best-fit evolutionary model for the dataset. Eighteen orthologous sequences representing the diversity of G. intestinalis (six of the eight known assemblages) were retrieved from GenBank and added to the analyses. Genealogies were reconstructed with Network-4.6  using the median-joining method with maximum-parsimony post-processing.Bayesian and maximum-likelihood phylogenetic trees based on 657-bp This study was approved by the Evandro Chagas Research Institute Committee for Ethics on Research of FIOCRUZ (0011.0.009.000–3). The parent or legal guardian of all children included in this study provided written informed consent on their behalf.G. intestinalis infection was 16.9% (73/433). Infection was more frequent among children aged 2–5 years old and among males  positive stool samples. From these, 23 (46.0%) were good-quality sequences (fragment of 657-bp). The isolates clustered into two well-supported s clades . The assG. intestinalis assemblages A and B are widely distributed in the studied region  AII, 26.3% (n = 10) AIII, 7.9% (n = 3) BIII, and 60.5% (n = 23) new B genotypes not previously described . Two disG. intestinalis infection was distributed throughout the city, with some hotspots of higher frequency. Interestingly, G. intestinalis positivity is not associated with income stratum. The wide distribution observed in a small city suggests the presence of multiple sources of infection, which could be related to environmental contamination with feces, possibly of human and animal origin [G. intestinalis. The observed hotspots on the margins of the Rio Negro suggest that people who live closer to the river are at greater risk of becoming infected.l origin . Many hoG. intestinalis and Giardia enterica [The high genetic divergence between A and B (5.5–6.3%) supports previous proposal for their separation in two taxa, enterica , 24. Assenterica , evidencGiardia cysts [The Amazon region is the largest drainage basin in the world and harbors one-fifth of the fresh water reserves on the planet. Paradoxically, the living conditions of many people who inhabit this basin are substandard, favoring the transmission of fecal-oral diseases such as giardiasis. It has been proposed that the routine water treatment practices usually employ concentrations of chlorine able to inactivate only bacterial and viral pathogens, but not ia cysts –26. Thusia cysts . Despiteia cysts .


We have sequenced the complete coding region of a chikungunya virus isolate (R91064) used in the formulation of CBER/FDA RNA reference reagents for nucleic acid technology testing. Aedes aegypti and Aedes albopictus that has caused explosive epidemics in Africa, Asia, and the Indian and Pacific Ocean islands. There are three recognized CHIKV genotypes: West African, East/Central/South African (ECSA), and Asian (–Chikungunya virus (CHIKV) is an alphavirus transmitted by the mosquitoes nd Asian . By Decend Asian –4. Most d Asian –. Human Cd Asian –. There ad Asian –. To date20 primer (Invitrogen). Overlapping PCR products were produced with TaKaRa LA polymerase (TaKaRa) and sequenced by the Sanger method. The sequences were assembled and examined using Sequencher version 5.0 (GeneCodes Corp.). The total length of the CR of the virus is 11,237 nucleotides (nt). Partial 5′- and 3′-untranslated region sequences were obtained of 60/76 and 362/513 nucleotides, respectively  (We describe here the complete coding region sequence (CR) of CHIKV strain R91064, for which only a partial genome  was available . Cell cuKJ941050.The complete coding region sequence of the CHIKV strain R91064 has been submitted to the GenBank under the accession no.


Prodiplosis longifila is a severe pest, mainly of Solanaceae, in South America and some years ago it damaged Tahiti lime crops in the United States. It is a potential invasive pest. Despite its presence in Colombia, nothing is known regarding the taxonomic identification of P. longifila or the characteristics of the damage it produces. Moreover, the current and potential distributions of this pest are unknown. To determine these factors, P. longifila was sampled in several Solanaceae- and Citrusxlatifolia (Tahiti lime)-producing areas in Colombia. The larvae consumed tender foliage, flowers and fruits in tomato, fruits in sweet pepper, and buds in Tahiti lime. P. longifila was not found in asparagus or in potatoes. Its presence in Tahiti lime was previously unknown in Colombia. Adults recovered in the laboratory were taxonomically identified using male morphological characteristics such as the shapes of the genitalia, antenna and wing. P. longifila was found in the Andean region of Colombia. The ecological niche model for populations found in tomato suggests that P. longifila is limited in its distribution by altitude and variables associated with temperature and precipitation. The highest probability of occurrence is in areas where tomato, sweet pepper and the new host, Tahiti lime, are grown. Therefore, it is necessary to implement preventive measures, such as planting tomato materials free of P. longifila larvae, in areas where the pest is not yet present but where there is the potential for its development.The Dipteran  Prodiplosis longifila Gagné is one of the most important pests of Solanaceae and asparagus in the Neotropics. In tomato, it can cause up to 100% loss in Colombia and up to 60% loss in Ecuador , where an infestation reaches up to 16% of buds  in 1984 , sweet pepper (Capsicum annuum L.) and other companion plants  that produce approximately 259,104 t/year with an average yield of 38.2 t/ha per cropping season  and small fruits of tomato plants, fruits of sweet pepper and flower buds and flowers of Tahiti lime. This is the first report of the insect consuming Tahiti lime (flower buds) in Colombia. P. longifila was found between 739 and 2168 m.a.s.l in The Andes of Colombia and it is limited in its distribution by altitude and variables associated with temperature and precipitation.P. longifila infestation, while in sweet pepper and Tahiti lime, P. longifila was found in 31% and 75% of the crops visited, respectively. P. longifila was not found in crops of hot peppers, potatoes or asparagus. The morphological identification determined that the adult from tomato, paprika and Tahiti lime are morphologically similar and correspond to P. longifila.During the study, 167 batches of crops of peppers, asparagus, Tahiti lime, potato, paprika and tomato were sampled in Colombia, and one batch of Tahiti lime was sampled in the United States. Of the 167 plots sampled in Colombia, 28% were located in the Valle del Cauca, followed by Boyacá (16%), Antioquia (15%), Huila (13%), Santander (9%), Caldas (5%), Cundinamarca (4%), Cauca (3%), Nariño (3%), Quindío (2%) and, finally, Risaralda (2%). As for the presence/absence of Cecidomyiidae, it was found that in Colombia, 53% of the crops visited presented at least one species. In the tomato crop, it was found that 65% of the sites visited had P.longifila, and the rest corresponded to other species of Cecidomyiidae. Table P. longifila by department and sampled crops.In total, 540 microscope slides were prepared; 485 corresponded to P. longifila males have a soft and thin body and the R5 vein is slightly curved beyond the wing apex. The antennae have 14 flagellomeres, each consisting of two nodes  or scab, and the fruit loses its commercial value  that are affected changed from green to fuchsia colour and stop their growth , bio_10 (mean temperature of the warmest trimester), bio_11 (mean temperature of the coldest trimester), bio_19 (precipitation of the coldest trimester), bio_16 (precipitation of the wettest trimester) and bio_15 . Altitude was the variable that had the most relevance. The map of the predictive model of the potential distribution of P. longifila in Colombia is presented in Figure The insect was found between 739 and 2168 m.a.s.l. Figure P. longifila, an increase in the distribution of P. longifila to Nariño, Meta and Caquetá is predicted. The values with a higher probability of occurrence are in Huila, Tolima and Valle del Cauca, where tomato, Tahiti lime and sweet pepper are grown.Based on the distribution prediction map generated for P. longifila feeds on tomato  and sweet pepper . Here, it is reported for the first time consuming Tahiti lime in Colombia (flower buds). P. longifila damage is easily detected in tomato and sweet pepper, but in flower buds of Tahiti lime, it may be confused with fungal infections that can occur after larval feeding  in 1941  , in part because increasing temperature decreases the development time in some species of Cecidomyiidae , the altitude is inversely correlated with temperature. This could be the reason why the pest was not found in potatoes in Colombia, given that optimal production of that crop occurs between 2500 and 3000 m.a.s.l. in the Andes , and even in the departments at risk, as in Meta and Caquetá, the species would be on the eastern slope of the Andes. Because the foothills of Meta have 1000 ha cultivated with Tahiti lime  and are seen as an area to increase Tahiti lime production for exportation from Colombia , which is responsible for the partial regulation of P. longifila in Tahiti lime in the USA , which could influence its distribution.Although the model reflects the great expansion of Cisneros . P. longP. longifila larvae could spread  with wet paper towels to prevent drying and transported to the Laboratory of Entomology and Acarology of the National University of Colombia at Palmira. To obtain adults, recovery chambers were used, as described by Gagné , and kepP. longifila were collected in Tahiti lime of Florida (USA) and sent by JE Peña (University of Florida); these samples were also processed for identification.Taxonomic identification was based on the morphology of males Gagné ; the abdP. longifila, georeferenced points identified with a GPS (Garmin GPSMAP 60CSx) were mapped using the DIVA-GIS program.To build the current distribution map of P. longifila in tomato in Colombia. To build the model, 90 sampling points corresponding to tomato crops were used, and for its validation, 39 points were used. At each sampling point, the altitude was determined. Climatic data obtained from the WorldClim database  was evaluated. To do this, a Pearson correlation was performed, and its significance was estimated by Student’s t test. Once the variables with the greatest effect on P. longifila were selected, MAXENT 3.3.3 k  value, which is an independent measure of the threshold model performance, was used; AUC = 1.0 optimum; AUC = 0.5 weak (Araújo et al. The Jackknife Maxent test was used to determine the importance of each variable and its relationship with


The use of Routine Outcome Monitoring (ROM) in mental health care has increased widely during the past decade. Little is known, however, on the implementation and applicability of ROM outcome in daily clinical practice. In the Netherlands, an extensive ROM-protocol for patients with psychotic disorders has been implemented over the last years (ROM-Phamous). The current study investigated to what extent ROM results translate to daily clinical practice. Therefore, we investigated whether clinical problems as identified with ROM were detected and used in the treatment of patients with psychotic disorders.n = 1040), a random sample of 100 patients diagnosed with a psychotic disorder was drawn. ROM-data used in this study included a physical examination, laboratory tests, interviews and self-report questionnaires. Based on these data, the prevalence of positive and negative symptoms, psychosocial problems and cardiovascular risk factors was determined. Next, we investigated whether these problems, as identified with ROM, were reflected in the treatment plans of patients, as an indication of the use of ROM in clinical practice.Out of the ROM database of 2010 (The sample consisted of 63 males and 37 females. The mean age was 44 and the mean duration of illness was 17.7 years. The prevalence of positive and negative symptoms, psychosocial problems and cardiovascular risk factors ranged from 11 to 86 %. In the majority of cases, problems as identified with ROM were not reflected in the treatment plans of patients.We found a substantial discrepancy between the ROM measurements and the treatment plans, i.e. low rates of detection of symptoms, psychosocial problems and cardiovascular risk factors in the treatment plans, even though these problems were identified with ROM. The opposite occurred as well, where problems were reflected in the treatment plans but not identified with ROM. Thus, ROM and daily clinical practice appear to be two separate processes, whereas ideally they should be integrated. Strong efforts should be made to integrate ROM and consequent treatment activities. Such integration may help to provide patients with adequate and customized care and simultaneously minimize under- and over-treatment. Psychiatric disorders with a psychotic component include schizophrenia, schizophreniform disorder, schizoaffective disorder, delusional disorder, brief psychotic disorder, psychotic disorder not otherwise specified, and psychosis associated with substance use or medical conditions, according to the DSM-IV-TR classification system . Core feBoth positive symptoms and negative symptoms greatly interfere with daily life functioning . MoreoveRoutine Outcome Monitoring (ROM) in adult patients may improve the diagnostics and treatment of psychiatric problems, according to a review of Carlier et al. . HoweverOver the last years, a protocol called the Routine Outcome Monitoring Pharmacotherapy Monitoring and Outcome Survey (ROM-Phamous) has been developed in the Northern Netherlands  , to optiThe present study investigated to what extent ROM results of patients with a psychotic disorder translate to daily clinical practice. To this end, we investigated whether clinical problems as identified with ROM were reflected in the treatment plans of patients. The frequently occurring clinical problems that we chose to investigate, were positive and negative symptoms, problems with social functioning, problems with daily activities and cardiovascular risk factors. As previous studies have shown that clinicians do not always use ROM outcome in their day-to-day work , 16 and In 2010, the ROM-Phamous database contained information of 1040 patients who received care at Lentis Psychiatric Institute or at the University Center of Psychiatry of the University Medical Center Groningen (UMCG) in the Netherlands. The current study could be performed without extra assessments of the patients, since the ROM-data were available at the start of the study. The Medical Ethical Committee of the UMCG has confirmed that anonymized ROM-Phamous data may be used for scientific research and, therefore, this study did not require additional ethics approval. The study was executed in line with local legislation and the Declaration of Helsinki.The ROM-Phamous screening protocol is extensive and contains a battery of instruments, including a physical examination , laboratory tests (including glucose and cholesterol levels), an interview (PANSS ), and cl2), diabetes mellitus (fasting plasma glucose levels >7.0 mmol/L), hypertension (blood pressure levels >140/90 mmHg (without diabetes) or >130/80 mmHg (with diabetes)) and dyslipidaemia  or >4.5 mmol/L (with diabetes) and/or LDL-cholesterol >3 mmol/L (without diabetes) or >2.5 mmol/L (with diabetes)). These criteria were established by De Hert and others, in collaboration with the European Psychiatric Association [Demographic ROM-data were used, including gender, age and duration of illness. Demographic information of patients in the sample was compared to all patients in the ROM-database. Severity of psychiatric symptoms was assessed using the Positive and Negative Syndrome Scale Remission (PANSS-R) . To asseThe anonymized data were analysed by an independent researcher (MT). Descriptive statistics were performed in IBM SPSS Statistics 20 . For eacX2 (1) = 0.026; p = 0.872), age  and duration of illness  between the selected sample and the other patients in the database.Patient characteristics and the prevalence of the investigated problem areas, based on the ROM-data, are displayed in Table mean = 6.7; sd = 4.9). Figure mean = 5; ranging from 1 patient to 16 patients between the different problem areas). In many cases, problems were detected with ROM, but not reflected in the treatment plan. This was especially striking for positive and negative symptoms, overweight, dyslipidaemia and smoking . The opposite occurred as well, where problems were not detected with ROM, but were reflected in the treatment plans. This occurred most frequently in problems with social functioning and daily activities .The time between the ROM-screening and the draft of the treatment plan ranged from 0 to 18 months (This study investigated to what extent ROM results of patients with a psychotic disorder translate to daily clinical practice. We found a substantial discrepancy between the ROM measurements and the treatment plans of patients, i.e. low rates of detection of symptoms, psychosocial problems and cardiovascular risk factors in the treatment plans, even though these problems were identified with ROM. The opposite occurred as well, where problems were reflected in the treatment plans but not identified with ROM. These exploratory results suggest that ROM and daily clinical practice are two separate processes, whereas ideally they should be integrated. An effort should be made to improve this integration, as this may help to provide patients with more adequate and customized care and simultaneously minimize under- and over-treatment.Positive and negative symptoms, psychosocial problems and cardiovascular risk factors still occur relatively often in this population suffering from psychotic disorders. Our findings are in line with many other studies indicating that a high proportion of patients with psychotic disorders have little or no social contact or friends and experience difficulties getting involved in social activities or work . Also, tHowever, the most striking finding is that clinicians do not adjust the treatment according to the ROM results. Our findings illustrate that ROM is not implemented to its full benefit in the healthcare provided for these patients. These findings also underline the notion that merely introducing a ROM-system in mental health care is not enough , 27, as Clearly, the implementation of ROM results in the treatment of patients must be improved. A first step should be to have the interpretation of ROM-results and the updating of the treatment plan within a limited time frame. This would help to reduce the long time between the ROM-screening and the draft of the treatment plan that we found in our study. Also, to improve the standard of care, the decision making process of clinicians could be made more explicit and transparent. To this end, all identified problems must be described in the treatment plan. Next, considerations should be described about whether or not treatment will be given. This will help to avoid certain problems to be discussed repeatedly and, more importantly, will avoid that problems are not taken into consideration. In addition, it should be clearly stated when treatment is not given and whether this is because the available treatment options did not work or whether it is due to lack of availability of care. The first issue should be addressed with treatment research and strict adherence to treatment guidelines, whereas the latter should be addressed with health care policy adjustments.An interesting result of our study was the finding that in some cases problems were reflected in the treatment plan, but were not identified with ROM. This was mostly seen in problems with social functioning and problems with daily activities. Presumably, our ROM measures did not pick up these signals or patients may not have reported these problems due to lack of insight or an adjustment of expectations to their chronic situation. Unexpectedly, we found this discrepancy for positive symptoms as well. Possibly, positive symptoms are still mentioned as treatment goals, even when symptoms do not interfere with daily life functioning anymore and therefore are not detected with ROM. In general, it is possible that positive symptoms and psychosocial problems already receive much attention in clinical practice. This could be because these problems usually stand out and are difficult to overlook. Apparently, the clinician’s focus of treatment seems to be directed most strongly on psychotic symptoms and psychosocial symptoms and less on negative symptoms and physical health.Strength of the study is the broad scope, as multiple areas of functioning were investigated with ROM. Moreover, the sample is an epidemiologically representative sample from the majority of patients with schizophrenia in the province of Groningen, the Netherlands. Also, the sample is representative for all patients in the ROM database. There are some limitations of the study. Firstly, our data was only retrieved from clinical records and we did not explicitly ask clinicians about their decision making process. It is therefore possible that certain problems that were identified with ROM, were not reflected in the treatment plan for specific reasons we are not aware of. Lastly, the data was collected and scored by one independent researcher. In future studies, data collection should be done by at least two independent researchers to improve reliability.This study prompts our mental health care organization as well as others to actively investigate the current state of affairs. We expect to see improvements in the implementation of ROM within the next years. All stakeholders should aim to bridge the gap between ROM and the treatment of patients as this may help to improve the quality of care, especially in patients needing long term care. To achieve this, it would be helpful to develop software applications that can reduce time burdens and make ROM outcome assessment more user-friendly for clinicians and patients . We are Our study investigated to what extent ROM results of patients with a psychotic disorder translate to daily clinical practice. We found a substantial discrepancy between the ROM measurements and the treatment plans of patients. These exploratory results suggest that the connection between ROM and the treatment that patients receive is far from optimal yet. Strong efforts should be made to integrate routine outcome monitoring and consequent treatment activities. Such integration may help to provide patients with the most adequate and customized care and simultaneously minimize under- and over-treatment.


Ebola virus is back, this time in West Africa, with over 350 cases and a 69% case fatality ratio at the time of this writing Ebolavirus genus is comprised of five species, Zaire, Sudan, Taï Forest, Bundibugyo, and Reston, each associated with a consistent case fatality and more or less well-identified endemic area , Franquet's epauletted fruit bat (Epomops franqueti), and the little collared fruit bat (Myonycteris torquata) are among the leading candidates If Ebola virus was introduced into Guinea from afar, the more likely traveler was a bat. Although a virus has not yet been isolated, PCR and serologic evidence accumulated over the past decade suggests that fruit bats are the likely reservoir for Ebola virus. The hammer-headed fruit bat . More than half of Guineans live below the national poverty line and about 20% live in extreme poverty. The Guinea forest region, traditionally comprised of small and isolated populations of diverse ethnic groups who hold little power and pose little threat to the larger groups closer to the capital, has been habitually neglected, receiving little attention or capital investment. Rather, the region was systematically plundered and the forest decimated by clear-cut logging, leaving the “Guinea Forest Region” largely deforested .The forest region also shares borders with Sierra Leone, Liberia, and Cote d'Ivoire, three countries suffering civil war in recent decades. Consequently, the region has found itself home to tens of thousands of refugees fleeing these conflicts, adding to both the ecologic and economic burden. A United Nations High Commission for Refugees census of camps in the forest region in 2004 registered 59,000 refugees. Although the formal refugee camps have now been dismantled with improved political stability in the surrounding countries, the impact on the region is long lasting. Having worked in Guinea for a decade (1998–2008) on research projects based very close to the epicenter of the current Ebola virus outbreak, one of the authors (DGB) witnessed this “de-development” first-hand; on every trip back to Guinea, on every long drive from Conakry to the forest region, the infrastructure seemed to be further deteriorated—the once-paved road was worse, the public services less, the prices higher, the forest thinner  and 4.Guinea fell further into governmental and civil disarray after former president Lansana Conté's death in 2008 left a power vacuum, with a series of coup d'états and periods of violence. Although the political situation has now somewhat stabilized, the country struggles to progress; socioeconomic indicators such as life expectancy (56 years) and growth national income (GNI) per capita ($440) have crept up in the past few years, but still remain disparagingly low. Despite a wealth of mineral and other natural resources, Guinea still possesses the eighth lowest GNI per capita in the world, and the incidence of poverty has been steadily increasing since 2003.Lastly, why is this outbreak of Ebola virus happening now? As best as can be determined, the first case of Ebola virus disease in Guinea occurred in December 2013, at the beginning of the dry season, a finding consistent with observations from other countries that outbreaks often begin during the transition from the rainy to dry seasons The precise factors that result in an Ebola virus outbreak remain unknown, but a broad examination of the complex and interwoven ecology and socioeconomics may help us better understand what has already happened and be on the lookout for what might happen next, including determining regions and populations at risk. Although the focus is often on the rapidity and efficacy of the short-term international response, attention to these admittedly challenging underlying factors will be required for long-term prevention and control.


Cryptococcus gattii is one of the causative agents of human cryptococcosis. Highly virulent strains of serotype B C. gattii have been studied in detail, but little information is available on the pathogenic properties of serotype C isolates. In this study, we analyzed pathogenic determinants in three serotype C C. gattii isolates . Isolate ATCC 24066 (molecular type VGIII) differed from isolates WM 779 and 106.97 (both VGIV) in capsule dimensions, expression of CAP genes, chitooligomer distribution, and induction of host chitinase activity. Isolate WM 779 was more efficient than the others in producing pigments and all three isolates had distinct patterns of reactivity with antibodies to glucuronoxylomannan. This great phenotypic diversity reflected in differential pathogenicity. VGIV isolates WM 779 and 106.97 were similar in their ability to cause lethality and produced higher pulmonary fungal burden in a murine model of cryptococcosis, while isolate ATCC 24066 (VGIII) was unable to reach the brain and caused reduced lethality in intranasally infected mice. These results demonstrate a high diversity in the pathogenic potential of isolates of C. gattii belonging to the molecular types VGIII and VGIV. Cryptococcus neoformans and C. gattii, the etiological agents of this mycosisC. neoformans species complexC. neoformans is cosmopolitan and commonly causes disease in immunocompromised individualsC. gattii preferentially affects immunocompetent individuals particularly in tropical and sub-tropical regionsC. gattii outbreaks have also been reported in the Pacific Northwest Region of the United States and in the Vancouver island4678Cryptococcosis is a fungal disease that kills approximately 650,000 individuals every yearC. gattii is classified into four different molecular types 10C. gattii1314C. gattii VGII outbreak lineages derived from mating events in the Brazilian Amazon rainforest and then dispersed to temperate regions1617C. gattii, but rather with still unknown individual strain attributesC. gattii and C. neoformans produce capsules mainly composed of polysaccharides, which are considered to be essential for cryptococcal pathogenesisC. neoformans to colonize the brainC. gattii has stimulated a number of studies focusing on how the fungus interacts with host cells27C. gattii of the molecular types VGIII and VGIV. Our results suggest an important phenotypic diversity among these three C. gattii isolates, including differences in pigmentation, capsular serology, gene expression in response to capsule-inducing conditions, chitooligomer production in vivo and induction of chitinase activity in mice. These differences were correlated with the ability of C. gattii to kill infected mice.The clinical relevance of VGII isolates of C. neoformans/C. gattii species complex has recently been proposedC. gattii classification into four molecular types, as the acceptance of the new taxonomic proposal in the Cryptococcus community is still uncertainAlthough a revision of the C. gattii were not known. That is why, the molecular types of those two isolates were determined via comparative URA5-RFLP analysis with the restriction enzymes HhaI and Sau96I with previously defined molecular types of the reference isolates of the major molecular types of C. gattii  was used, as it is a simple and reproducible method allowing the separation of the all major molecular types within the C. neoformans/C. gattii species complex29323332C. gattii isolates by MALDI-TOF generates consistency score values in the range of 2.3 to 3 for highly probable species identification, 2 to 2.29 for secure genus identification associated with probable species identification, 1.7 to 1.99 for probable genus identification and zero to 1.69 for not reliable identificationC. gattii C. gattii cells grown in vitro revealed that all three isolates had the staining profile that was previously observed in C. gattii serotype B and C. neoformans serotype A isolatesin vivo varies depending on the induction of pulmonary chitinasesC. gattii 106.97 and WM 779 (annular detection of chitooligomers) induced chitinase activity in a time-dependent fashion. In contrast, C. gattii ATCC 24066 isolate (no chitooligomer detection) induced chitinase activity at the lowest levels.Chitooligomers are readily detectable components of the cell wall-capsule interface in both C. gattii isolates studied herein differed in key determinants of the pathogenic potential observed within C. gattii. In summary, isolate ATCC 24066 differed from WM 779 and 106.97 cells in molecular type, capsule dimensions, expression of CAP genes, chitooligomer distribution, and induction of host chitinase, but additional diversity among the three isolates was also detected. For instance, isolate WM 779 was more efficient than the others in producing pigments, as already reportedP = 0.0003). Isolates WM 779 and 106.97 were similar in their ability to cause lethality and, in fact, they produced higher pulmonary fungal burden  suggesting that the traditional classification of the C. neoformans species complex into four different serotypes may include still unknown capsular, serological determinants. In our study, carbohydrate analysis of capsular extracts revealed similar GXM composition, but the presence of secondary monosaccharides was different in each isolate. Therefore, the possibility that capsular components associate with other surface components resulting in modified serological reactivity cannot be ruled out. This hypothesis is supported by the recent demonstration that glycan association in the surface of C. neoformans resulted in hybrid polysaccharides with diverse immunological functionsAntibody reactivity with capsular components is essential for the classification of C. neoformans to the central nervous systemin vivo and elevated chitinase activity in the lung of infected mice. Pulmonary chitinase activity was linked with cryptococcal infection52The presence of fungal cells in brain tissue also varied in a murine model of cryptococcosis after infection with each isolate studied here. Only isolates 106.97 and WM 779, both VGIV isolates, disseminated to the brain, an observation that was linked to their ability to kill mice. A recent study by our group suggested that recognition of chitooligomers by macrophages is involved in dissemination of C. gattii isolates has been suggested in a number of studies31620C. gattii, but rather with still undetermined attributes of each isolate analyzed9C. gattii have been reported in MexicoVariable virulence in C. gattii isolates belonging to molecular types VGIII and VGIV are highly diverse in pathogenicity and phenotype. Although the molecular regulators of this potential diversity are still obscure, we propose that differences in melanin production, expression of CAP genes, capsular structure and surface architecture are involved in pathogenic diversity. These results reinforce the notion that even the serotype C VGIII and VGIV C. gattii isolates manifest hypervirulent phenotypes and open new venues for the investigation on how C. gattii cells cause damage to the host.Our work supports the notion that http://www.nature.com/srep/policies/index.html.Methods described in this section were carried out in accordance with the approved guidelines available in C. gattii serotype C isolates used in this study included 106.97 , KH2PO4 (29.4 mM), glycine (13 mM), and thiamine-HCl (3 μM). Growth rates were determined by counting the cell number in a Neubauer chamber in 24 h intervals. Yeast cells were harvested by centrifugation and washed in phosphate-buffered saline (PBS). Molecular typing was performed as described previously32http://imagej.nih.gov). For determination of urease activity, the cells were grown for 24 h in liquid Sabouraud medium, washed three times with PBS and tested as described beforeSaccharomyces cerevisae (strain RSY113) was used as a negative control in these assays. Melanin production was determined visually after the cells were spotted on solid minimal medium supplemented with 1 mM l-dopa and cultivated for 120 h either at 30 oC or 37 oC60The three −ΔCT method described by Livak & Schmittgen, 2001Each isolate was cultivated in liquid Sabouraud and in minimal media for 48 h as described above. Cells were collected by centrifugation for RNA extraction using the Trizol reagent (Invitrogen) according to the manufacturer’s protocol. Briefly, after DNAse treatment, RNA preparations were submitted to cDNA synthesis using M-MLV reverse transcriptase enzyme (Promega). Real-time PCR reactions were performed in an Applied Biosystems StepOnePlus Real-Time PCR System. Thermal cycling conditions consisted of an annealing step at 95 °C for 15 s followed by an elongation step at 60 °C for 1 min along for 40 cycles. Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen) was used with 4 pmol of each primer and 2 μl of the cDNA template in a final reaction volume of 20 μl. Each sample was analyzed in triplicate. Melting curve analyses were performed at the end of each reaction. Results were normalized by using yeast ß-actin gene amplification. Relative expression was determined by the 2C. neoformans cellsO-trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis according to previously described methodsCellular polysaccharides were extracted with dimethyl sulfoxide (DMSO), using protocols previously established for efficient removal of GXM from C. gattii were analyzed by fluorescence microscopy. Yeast cells (107) were fixed in 4% paraformaldehyde , blocked with 1% (w/v) BSA in PBS  and subsequently incubated with four different monoclonal antibodies (mAbs) raised to GXM, which were kindly donated by Dr. Arturo Casadevall . The mAbs used in this study were: IgG1 18B744C. gattii at a final concentration of 10 μg/ml . The cells were subsequently incubated with Alexa Fluor™ 488-conjugated secondary antibodies  and washed with PBS. Stained cells were analyzed by flow cytometry using a FACScan apparatus. Data was analyzed using the FlowJo7 software . Alternatively, stained cells were probed with Calcofluor White at 5 μg/ml for 30 min (37 °C). The cells were finally counter-stained with India ink for analysis of capsular dimensions. After further washing with PBS, the cells were observed under a fluorescence optical microscope Axioplan 2 . Images were processed using the analysis software (Zen 2.0) and Adobe Photoshop CS5 was used for preparing figure panels.General properties of the cell surface of C. gattii. The animals were anesthetized with ketamine (10 mg/kg) and xylazine (4 mg/kg) and inoculated intranasally with a fungal suspension of 105 yeast cells in a final volume of 50 μl PBS. Groups were observed twice a day for 70 days for determination of mortality rates. Alternatively, groups of 12 mice were infected as described above with each of the isolates studied here. After 4, 14 and 21 days post infection, animals (n = 4) infected with each isolate were sacrificed and the lungs and the brains were excised and homogenized. These macerates were plated on Sabouraud medium for colony forming units (CFU) determination as previously describedad libitum. Animals were maintained following the rules of the National Council of Animal Experimentation Control (CONCEA) and the Brazilian College of Animal Experimentation (COBEA).BALB/c  mice (n = 7) approximately 4–6 weeks old were used for determination of mortality curves, fungal burden, pulmonary microscopic examination and chitinase activity after infection with t test for comparison of sample data and the Kaplan-Meier survival statistics were carried out using the GraphPad Prism 6.0 software (GraphPad Software).Student’s How to cite this article: Rodrigues, J. et al. Pathogenic diversity amongst serotype C VGIII and VGIV Cryptococcus gattii isolates. Sci. Rep.5, 11717; doi: 10.1038/srep11717 (2015).