Bunyaviridae) is due to its ability to adapt to changing conditions through intramolecular genetic changes and segment reassortment. Vertical transmission of LACV in mosquitoes increases the potential for segment reassortment. Studies were conducted to determine if segment reassortment was occurring in naturally infected Aedes triseriatus from Wisconsin and Minnesota in 2000, 2004, 2006 and 2007. Mosquito eggs were collected from various sites in Wisconsin and Minnesota. They were reared in the laboratory and adults were tested for LACV antigen by immunofluorescence assay. RNA was isolated from the abdomen of infected mosquitoes and portions of the small (S), medium (M) and large (L) viral genome segments were amplified by RT-PCR and sequenced. Overall, the viral sequences from 40 infected mosquitoes and 5 virus isolates were analyzed. Phylogenetic and linkage disequilibrium analyses revealed that approximately 25% of infected mosquitoes and viruses contained reassorted genome segments, suggesting that LACV segment reassortment is frequent in nature.The evolutionary success of La Crosse virus (LACV, family  Bunyaviridae, genus Orthobunyavirus) emerged as a significant human pathogen in the upper Midwestern United States, and it is now the most common cause of pediatric arboviral encephalitis in the U.S [Aedes triseriatus and small mammals . Aedes triseriatus develop a life-long infection, and infected females can transovarially transmit (TOT) the virus to their progeny [In the 1970s, La Crosse virus , medium (M), and small (S). The L segment encodes the RNA-dependent RNA polymerase , the M sBunyaviridae is attributed in part to their ability to adapt to varying conditions through genetic drift (intramolecular genetic changes) and genetic shift (segment reassortment).LACV exhibits considerable evolutionary potential in nature. There are distinct geographic genotypes of the virus in different areas of the United States -14, and Genetic drift occurs during genome replication and can result in viral diversity and altered fitness . RNA virAe. triseriatus and the newly generated viruses can be transmitted. The potential for segment reassortment increases when a transovarially-infected mosquito takes a blood meal from a viremic host [Laboratory studies have demonstrated the occurrence of genetic shift (segment reassortment) in mosquitoes that have become dually infected by ingesting viruses of two different LACV genotypes, either simultaneously or within two days of each other . LACV remic host . These mmic host . Segmentmic host -22.Evidence has also been presented for reassortment between LACV genotypes in nature. For example, the genomes of 23 isolates of LACV were analyzed by oligonucleotide fingerprinting and categorized in terms of the degree of their RNA sequence relatedness . One genOrthobunyaviruses and in other Bunyaviridae genera. Ngari virus is a newly emerged reassortant virus associated with severe disease epidemics in Africa [Orthobunyavirus that was first detected in Malaysia [Orthobunyaviruses also reassort [Hantavirus) have been detected in rodents in nature [Nairovirus) have also been detected [Genome segment reassortment has also been demonstrated among other n Africa . SequencMalaysia . Group Creassort . Phylogen nature  and reasdetected .Bunyaviridae family, the epidemiologic consequences of these evolutionary events are poorly understood. In this study molecular epidemiological techniques were used to investigate the evolutionary and reassortment potential of LACV in field-infected mosquitoes from the upper Midwest of the United States.Although genome reassortment appears to occur frequently in the A total of 6,791 mosquitoes collected as eggs at 151 study sites in Wisconsin, Minnesota, and Iowa  was actually 3.24 times larger than the diversity among synonymous substitutions (πs). The location and amino acid replacements are listed in Table The numbers of sequences analyzed and the number of segregating sites in each segment are shown in Table The program Tipdate  estimateThe haplotype grouping system was determined through a conservative phylogenetic analysis. The system identified three S haplotypes based on seven polymorphic sites, five of which were nonsynonymous mutations. The three haplotypes identified in the M segment were based on twelve polymorphic sites, seven of which were nonsynonymous. For the L segment, two haplotypes were identified based on thirteen polymorphic sites, twelve of which were nonsynonymous substitutions Figure .Ae. triseriatus  the genome sequence obtained by direct amplification of the viral RNA from the mosquito is the dominant viral sequence in the mosquito as well as in infectious virus and 2) that the estimation of reassortment frequency was not confounded by potential RNA quasispecies in the mosquitoes. Estimating the frequency of reassortment of LACV in nature would be improved by analysis of plaque-purified viruses isolated from the mosquitoes, preferably from their saliva or ovaries, which are the epidemiologically significant organs of transmission.In this regard, we were unsuccessful in isolating LACV from most field mosquitoes. The reasons for this are unknown; however, there are several potential explanations for this. Eggs were collected in the field and stored in a hot warehouse for variable periods of time awaiting shipment to Colorado. As soon as the eggs reached AIDL, they were placed in the insectary, hatched and reared. Environmental factors in the collection and shipping process could contribute to loss of virus titer. An additional complication could have been the isolation method. In previous studies, virus was isolated by inoculation of samples into suckling mouse brains. Cell culture assays are likely not as sensitive. Low virus titer, titer loss during processing, and insensitive isolation methods, likely contributed to the inability to isolate virus from mosquitoes.Ae. triseriatus in the field. LACV reassortants could be more virulent and could have altered vector species and vertebrate host ranges. New viruses could create new arbovirus cycles with potentially significant epidemiological consequences [Ae. triseriatus and chipmunks and tree squirrels. If a new virus established a transmission cycle that involved a mosquito species that fed more aggressively on humans, increased human infections could occur. If a new reassortant virus was more virulent or exhibited different tissue tropisms, infections could become clinically significant in both adults and children. For example, a new reassortant virus could replicate more efficiently in humans, resulting in greater viremia titers and more efficient infection of the central nervous system. Determination of the evolutionary potential of LACV through genetic shift may permit prediction of the epidemiologic consequences of these events.There are important public health implications of reassortment in LACV-infected equences . For exaThese studies illustrate the significant evolutionary and epidemic potential of viruses in the family Bunyaviridae. Viruses in this family have contributed inordinately to the list of newly emerged viruses , and theAedes triseriatus eggs were collected from five oviposition traps in each of 151 sites in Minnesota (n = 37), Wisconsin (n = 108) and Iowa (n = 6). Sites were established in areas where LACV encephalitis cases occurred or areas that contained clusters of people judged by the La Crosse County Public Health Department to be at risk for infection . Mosquito eggs that had entered diapause in fall 2000 were collected in the spring of 2001. Mosquito eggs were also collected between mid-June and August of 2004, 2006 and 2007. Eggs were collected in Crawford, La Crosse, Monroe, Vernon, Lafayette and Iowa counties in Wisconsin; Winona, Houston, and Grant counties in Minnesota; and Clayton and Allamakee counties in Iowa . A 1978 LACV (78V-8853) was isolated from an The LACV-positive mosquitoes were triturated with a pellet pestle (Fisher Scientific) in a 1.5 ml microcentrifuge tube containing 1 ml of minimum essential medium (MEM) (Gibco), 2% fetal bovine serum, 200 μg/ml penicillin/streptomycin, 200 μg/ml fungicide, 7.1 mM sodium bicarbonate, and 1× nonessential amino acids. The homogenate was centrifuged for 10 minutes at 500 × g to form a pellet.2. Supernatant from the centrifuged mosquito homogenate (0.2 ml) was added to one well in a six-well plate, incubated at 37°C for one hour. Following the incubation, 5 ml of medium were added to each well.Cell monolayers of Vero cells were grown in six-well plates at 37°C in an atmosphere of 5% CO-1 to 10-6 and 200 μl of each virus dilution was added to individual wells and incubated at 37°C for 1 hour. The virus inoculum was removed and 5 ml of overlay was added to the well. After six days of incubation at 37°C in 5% CO2, 200 μl of the detection solution, methylthiazolyldiphenyl-tetrazolium bromide (MTT) (5 mg/ml in PBS), was added to each well. The plates were incubated overnight and visible plaques were picked and placed in 1 ml of MEM with 0.2% FBS for 1 hr at 37°C. An aliquot of the medium from the wells was added to Vero cells, and the presence of virus confirmed by detection of cytopathic effect.The virus isolates from 2006 and 2007 were plaque purified using monolayers of Vero cells in six-well plates . Virus iThe posterior half of each mosquito abdomen was individually homogenized in 500 μl of Trizol , using a pellet pestle , and then total RNA was extracted according to manufacturer's instructions.The medium and cells from wells with plaque purified virus were removed and placed in a 15 ml conical tube and centrifuged at 3000 rpm for 10 minutes. The supernatant was removed and the cell pellet was resuspended in 500 μl of Trizol . Total RNA was extracted according to manufacturer's instructions.RNA from the 1960 and 1978 LACV isolates was prepared by infection of C6/36 cell cultures at a multiplicity of infection of 0.01. Three days post-infection, cells were scraped into the medium, centrifuged and cell pellets were resuspended in 500 μl of Trizol for RNA extraction.Taq DNA polymerase  according to manufacturer's instructions. The primers specific for the S segment  amplified a 462 nucleotide region (nucleotides 144 to 604) of the nucleocapsid and NSs genes. This region was selected because it was the most variable region of the published S sequences. The S segment is 984 nucleotides in length, so the amplified region encompasses almost half the entire segment. The primers specific for the M segment  amplified a highly variable 411 nucleotide region in the 5' half of the G1 gene (nucleotides 1585 to 1995) [Ae. triseriatus ribosomal protein RpL34 mRNA were used as a positive control. PCR was performed as follows: 94°C for 5 minutes, 35 cycles of 94°C for 1 minute, 55°C for 1 minute and 72°C for 1 minute followed by a final extension at 72°C for 8 minutes.Portions of the LACV S, M, and L RNA segments were transcribed to cDNA using Superscript II reverse transcriptase  and amplified by PCR using Ex to 1995) . The priE. coli cells . Cells were grown on LB agar containing ampicillin (50 μg/ml) and kanamycin (50 μg/ml). Colonies were screened for inserts by PCR amplification using the original primers and positive products were purified using QIAquick spin columns . Three to five cDNA clones per segment from each mosquito were sequenced in both directions using the ABI PRISM dye terminator cycle sequencing kit  and the ABI 310 DNA automated sequencer at Macromolecular Resources, CSU. A 415 nucleotide region of the S segment (nucleotides 190–604), a 358 nucleotide region of the M segment (nucleotides 1637–1994), and a 447 nucleotide region of the L segment (nucleotides 179–625) were sequenced.PCR products were separated by electrophoresis in 1% agarose gels with TAE buffer, visualized with ethidium bromide, excised and extracted using the Powerprep Express Gel Extraction kit  according to manufacturer's instructions. PCR products were inserted into the pCR4-TOPO cloning vector  and resulting plasmids were used to transform competent TOP10 Genetic haplotypes were established for each of the three segments through maximum parsimony analysis, sequence identity matrix, neighbor joining distance matrix, and ratio of synonymous to nonsynonymous substitutions.s) and replacement substitutions (πs). The rate of molecular evolution (substitutions/site/year) was estimated using the program TipDate [For each segment, the computer program DnaSP 4.5  estimate TipDate . TipdateLinkage disequilibrium is a measure of the degree to which substitutions in a segment occur independently of one another. Substitutions that occur together in a segment at a rate predicted by their independent frequencies are in linkage equilibrium. Substitutions that occur more or less often than expected by random chance are considered to be in linkage disequilibrium. Linkage disequilibrium also tests whether sampling a portion of a genome segment is representative of the whole segment. Linkage equilibrium is detected when different parts of a segment are evolving independently and sequencing a portion of the segment may not provide a representative sample of the whole.i, Mj, and Lk appear in the same mosquito, where for example, Si is a unique sequence of the segment.A linkage disequilibrium analysis was also performed to determine if entire segments assort randomly, thereby suggesting segment reassortment. Segment are in disequilibrium when some combinations of segments occur together in a mosquito more or less often than would be predicted by their independent frequencies. The first step is to determine the number of times segment, Sijk is the number of times haplotype i, j, and k are expected to occur in a mosquito.Ei is the frequency of Si in the mosquito population and N is the number of mosquitoes. Linkage disequilibrium (Dijk) was then estimated.where pijk was determined [A Hill and Robertson correlation coefficient Rtermined .ijk2) was used as a metric of disequilibrium because it ranges from zero (linkage equilibrium) to one (linkage disequilibrium). Linkage disequilibrium patterns among all polymorphic sites were plotted on a heat map using the LDheatmap program in R [2Link) and the corresponding level of significance were calculated for each combination of haplotypes to test the hypothesis that the individual haplotype combinations are in linkage equilibrium.The squared correlation coefficient  4.0b10 package . The phyThe authors declare that they have no competing interests.SR and BrB carried out virological and molecular characterization of viruses. SR and WB conducted phylogenetic analyses and statistical analyses. DG identified field collection sites and managed field collections and logistics. CB, BaB, and WB conceived, designed, and managed the study. All authors participated in the preparation of the MS and approve its submission to Virology Journal.


Ixodes ricinus ticks that had fed on goats in northern Greece. The strain was almost identical to the prototype strain isolated 35 years ago.A strain of Greek goat encephaltitis virus was isolated from engorged  Flavivirus, family Flaviviridae. According to the latest taxonomy on flaviviruses, TBE virus is a species in the mammalian tick-borne virus group and has 3 subtypes: European, Far Eastern, and Siberian  is a zoonotic infection of the central nervous system; it is transmitted by ticks from the family Ixodidae. In an ecologic sense the disease agent, TBE virus, is an arbovirus (arthropod-borne virus); taxonomically, it is a member of the genus Ixodes persulcatus and I. ricinus, respectively  of 56 serum samples , and the rest was stored at –70°C until further use. PCRs were performed by using 2 different pairs of primers: 1 pair of degenerated primers for the 5′ end of the envelope (E) gene  assays. The pool consisted of 1 female and 9 male ticks collected in November 2004 in Vavdos village. Ticks of all other species were negative. Assuming that 1 tick per pool was positive, the total frequency of infected I. ricinus ticks was 0.28%; annual tick infection rate was 0.78% for 2004.One pool of Of the stored supernatant, 70 μL was inoculated onto Vero E6 cells; flasks were incubated at 37°C and passaged to fresh cells every 5 days. Viral supernatants were tested by immunofluorescent assay and RT-PCR. After the fifth consecutive passage, fluorescence was present and 3 more passages of the virus (Vavdos strain) were performed.Nucleotide sequences of the viral genes encoding 1 NS protein (NS5) as well as the capsid (C), membrane (M), and E proteins were determined by using the above-mentioned and newly designed primers (GenBank accession nos. EF693938 and EF693939). A high degree of homology with the Vergina strain (DQ235153) was observed, although the 2 strains were isolated 35 years apart and the isolation sites were 140 km apart from each other. The 2 strains differed by 2 nucleotides (0.13%) in the E gene; 1 resulted in an amino acid change , but they were identical in the C, M, and NS5 genes. Similar stable phylogenetic relationships were observed in TBE virus strains of other subtypes, which indicates that the virus is remarkably stable and not subject to major antigenic variation  of TBE viruses, Greek strains cluster together with Turkish sheep encephalitis virus (nt homology 95.5%) and form an independent clade with high bootstrap value, which might represent the southern subtype . GeneticVavdos is a mountainous village 800 m above sea level . The area where goats were grazing was covered by typical Mediterranean low vegetation and was located at the edge of an oak forest . No signNatural foci of GGE virus are present in northern Greece. The strain that circulates in Greece resembles that isolated from sheep in Turkey, which has not yet been associated with disease in humans. Sequencing of the complete genome, including the more variable 3′-noncoding region, and neutralization tests that are in progress will give further insights into this group of viruses.


This extremely high protective capacity of pDCs secures the spleen's capability to function as a ‘sink’ for the virus produced in peripheral organs such as the liver. Furthermore, our results suggest that the pDC population in spleen ensures a robust protection against virus variants which substantially down-modulate IFN secretion. However, the ability of pDCs to protect against severe disease caused by virus variants exhibiting an enhanced liver tropism and higher replication rates appears to be rather limited. Taken together, this systems immunology analysis suggests that antiviral therapy against cytopathic viruses should primarily limit viral replication within peripheral target organs.Plasmacytoid dendritic cell (pDC)-mediated protection against cytopathic virus infection involves various molecular, cellular, tissue-scale, and organism-scale events. In order to better understand such multiscale interactions, we have implemented a systems immunology approach focusing on the analysis of the structure, dynamics and operating principles of virus-host interactions which constrain the initial spread of the pathogen. Using high-resolution experimental data sets coming from the well-described mouse hepatitis virus (MHV) model, we first calibrated basic modules including MHV infection of its primary target cells, i.e. pDCs and macrophages (Mφs). These basic building blocks were used to generate and validate an integrative mathematical model for in vivo infection dynamics. Parameter estimation for the system indicated that on a per capita basis, one infected pDC secretes sufficient type I IFN to protect 10 Human infections with highly virulent viruses, such as 1918 influenza or SARS-coronavirus, represent major threats to public health. The initial innate immune responses to such viruses have to restrict virus spread before the adaptive immune responses fully develop. Therefore, it is of fundamental practical importance to understand the robustness and fragility of the early protection against such virus infections mediated by the type I interferon (IFN) response. Because of the inherent complexity of the virus-host system, we have used mathematical modeling to predict the sensitivity of the kinetics and severity of infection to variations in virus and host parameters. Our results suggest that the spleen represents a robust sink system for systemic virus infection and that this system is able to cope with substantial variations in IFN secretion and virus production. However, the system is very fragile to only minor increases in the virus growth rate in peripheral tissues. Collectively, the mathematical approach described in this study allows us to identify the most robust virus and host parameters during early cytopathic virus infection and can serve as a paradigm for systems immunology analyses of multiscale virus-host interaction of many life-threatening cytopathic virus infections. Protection against life-threatening infections is a major function of the immune system. The systems biology view of the induction of the protective immune responses suggests that the kinetics of innate immune responses critically impinge on the development of pathogen-specific adaptive immune responses low DC subset that is characterized by a particular set of phenotypic markers and special functional properties Plasmacytoid dendritic cells (pDCs) are a CD11cCoronaviridae family that harbor a number of viruses causing severe diseases in animals and humans, such as acute hepatitis, encephalitis, infectious bronchitis, lethal infectious peritonitis, and the severe acute respiratory syndrome (SARS) The mouse hepatitis virus (MHV) infection represents a well-understood paradigmatic system for the analysis of type I IFN responses. MHV is a member of the Our studies on the role of pDCs in establishing the type I IFN-mediated protection of Mφs against cytopathic MHV infection suggested that the spleen may function as a ‘sink’ contributing to the elimination of the virus from the system. Under the condition of pDC-deficiency or lack of type I IFN responsiveness of Mφs, a severe disease is observed To describe quantitatively the structure, dynamics and the operating principles that permit pDCs to initially shield the host against an overwhelming spread of the cytopathic MHV infection, we followed a systems biology approach as outlined previously ifnar−/−) were used  the IFN-α level required for 2-fold inhibition of MHV production is about 46 pg/ml, (iii) the average secretion rate of IFN-α per infected pDC is ∼4.4*10−4 pg h−1 or, equivalently, ∼15586 molecules h−1. The latter estimate takes into account that the molecular weight of IFN-α is about 17000 atomic mass units (a.u.) and 1 a.u. = 1.67×10−24g.To estimate the kinetic parameters of MHV-pDC interaction, we used in vitro data on MHV infection of bone marrow-derived pDCs as described previously ere used . In addiIUnits (1 IU≅8.333 pg) of recombinant IFN-α  the concentration of IFN-α required for 2-fold inhibition of MHV production is about 0.1 pg/ml, and (iii) the per cell secretion rate of IFN-α is about 100-times smaller in Mφs  compared to pDCs. The calibrated modules for the in vitro infection of pDCs and Mφs thus provide valuable basic building blocks that allowed to proceed with the modeling of early kinetics (0–48 hours) of MHV growth and the IFN response in vivo.To identify the parameters of MHV infection and type I IFN-mediated protection of Mφs, we considered experimental data sets that were generated using a broad spectrum of IFN treatment conditions nt IFN-α  and , and increased about two-fold by 36 hours following infection. Here, we considered intermediate values, i.e. the ones observed 18 hours post infection, so that 7×105 pDCs and 6×106 Mφs were used to model the infection dynamics in spleen. To evaluate the qualitative consistency of the in vitro parameter estimates with the actually observed phenotype of MHV infection in vivo, we modeled the infections of the mixture of the above numbers of pDCs and Mφs with increasing virus doses . As shown in To further validate the calibrated modules, we considered an experimental in vitro system mimicking ‘in vitro spleen infection’. To this end, we first determined the cellular composition of spleen in terms of pDC and Mφ population sizes during early MHV infection. At the beginning of infection with 5×103 pfu and 5×105 pfu as shown in To set up the mathematical model for systemic MHV infection in mice, we proceeded in stages. First, we estimated the virus transfer rates between blood, spleen and liver together with the rate of alanine aminotransferase (ALT) increase using the compartmental model described in the  for Mφs . Taken t6 Mφs, which leads to the estimate of 3 IU/L, then the host is protected against infection with physiological doses when the number of pDCs in spleen is unchanged (7×105) or 10-fold reduced. The protection is lost if spleen contains only 7×103 pDCs and the dose of infection is larger than 100 pfu. This suggests that the protection unit of pDCs  under the permission numbers SG07/62 and SG07/63 granted by the Veterinary Office of the Canton of St. Gallen.−/−ifnar) C57BL/6 (B6) mice were obtained from Charles River Laboratories . Type I IFN receptor deficient mice (Vd) and IFNα (Id) were estimated by a linear regression procedure for the log-transformed values of the virus titer and IFN concentration using GraphPad Prism v.4 software (http://www.graphpad.com). The parameter estimates and their 95% Confidence Intervals (CIs) are presented in The persistence of virus and type I IFN in medium displays an exponential kinetics. The corresponding decay rate constants for MHV (C(t)) display kinetics which differs for some cell types or under certain conditions from the exponential (denoted by E) behavior G) as described by equationThe data on target cell persistence  is best approximated by the exponential decay with a half-life of 131 (hours), whereasMφ survival over the time of the experiment (2 to 3 hours) which reduces by a factor of 8.5 (to 14.7 hours) by day 1 of the experiment.pDCs follow the Gompertz kinetics with an initial half-life of 125 (AIC), defined as dn is the total number of scalar observations, L is the number of optimized parameters, I(p) is the Fisher information matrix, and The best-fit estimates for the death rate parameters for pDCs and Mφs are given in the V(t); the amount of type I IFN per ml, I(t); the density of uninfected target cells (pDC or Mφ), C(t); the density of infected target cells, V(t)C. The rate of change of the virus population is determined by virus secretion from infected cells, which starts after some latent period (time-delay) θ.To describe the antiviral IFN-α response in vitro, we reduced the complexity of the IFN system to four principal constituents: the virus titer/ml, The rate of change of the amount of IFN-α in the system results from the IFN production by virus infected target cells, which occurs with some secretion delay, and the decay of the free interferon molecules:The parameters of the equation, i.e. the average per cell secretion rate The rate of change of the number of infected target cells is modeled by the following equationThe first term in the right-hand side describes the emergence of the infected cells due to the virus infection of uninfected target cells whereas the second one accounts for the death of infected cells. The per capita death rate Finally, the rate equation for the density of uninfected target cells readsThe infection of mice with MHV leads to virus spread and growth in different organs. Virus population dynamics in any anatomical compartment results from a superposition of intra-compartmental production-elimination and inter-compartmental transfer of the virus. The compartmental model in order to describe the pathological consequences of MHV infection requires consideration of the virus population dynamics in spleen ml) for spleen, liver and blood were used: The virus populations in spleen and liver are assumed to follow a logistic growth with the outflow-inflow rates depending in a linear way on the virus concentration in the corresponding compartments. The parameters The infection of target cells with MHV induces a cytopathic effect leading to an earlier cell death. The primary cell targets of MHV in the liver compartment are hepatocytes. The severity of the virus-induced liver disease is characterized by the liver enzyme ALT concentration in serum The compartmental model of virus growth and ALT kinetics provides a tool to quantify the transfer coefficients of the virus for the spleen-blood-liver system (5)–(7) as well as the disease severity due to the virus presence in the liver.An integrative mathematical model to study the protective function of pDCs in MHV infection is assembled from the building block models described above. The model considers spleen, liver and blood compartments, in which MHV replication is described at different resolution levels. For spleen, a detailed description of the virus-target cells interaction is considered which includes MHV, IFN-α, pDCs, Mφs dynamics as modeled by the subset of equations (9):5 pDCs and 6×106 Mφs and there is a continuous turnover of these cell populations. Therefore, at any given moment the infected target cells represent a heterogeneous population with respect to the infection time and live expectancy rather than a cohort of cells with synchronous kinetics. This is taken into account by assuming exponential death kinetics for cell populations in spleen, with the rate constants corresponding to the in vitro derived estimates. The influx-elimination processes are described by the homeostasis terms for uninfected pDCs and Mφs (see the last two terms in the last two equations). The equation for virus in spleen considers the virus transfer between spleen and blood. The subset of equations for virus dynamics in blood, liver and the serum ALT level remains the same as equations (6)–(8). The following initial data for the delay differential equation system (6)–(9) specifying the intravenous infection were usedhttp://www.mathworks.com) or our original solver for stiff systems of delay equations Eighteen h post MHV infection, spleen contains about 7×10http://www.mathworks.com) or Absoft Pro Fortran Developer (http://www.absoft.com) and IMSL function minimization code ZXMIN based on a quasi-Newton method.The process through which the available information is used in order to estimate as accurately as possible the systems' dynamics is known as data assimilation. To calibrate the mathematical model using the sampled data, we optimally combined heterogeneous observations (coming from distinct series of experiments) with model predictions by optimizing a “cost function”, which expresses the distance between observations and the corresponding model values. The available data vary essentially in terms of the sample sizes, which is common for studies of virus and cell population dynamics. The most numerous data sets are the virus kinetics data. The corresponding samples followed a log-normal distribution. Assuming further that the errors in observations at successive times are independent and the variance of observation errors is constant for all observation times, we applied the maximum likelihood (ML) approach to parameter estimation as we described in detail in The number of reliably identifiable parameters in the mathematical models is limited by the amount and quality of the corresponding sets of experimental data which are available and also by the model structure. As the ‘large-scale’ systemic model of MHV infection was built using a modular (rather than monolithic) approach broadly accepted in systems biology The algebraic identifiability analysis of the basic IFN response module (equations 1–4) showed that if virus titer, IFN concentration and the fraction of live cells are the observable state characteristics, then the time-lag of virus production, the 50% inhibition threshold and the virus secretion rate represent a group of functionally related parameters, i.e. their estimates depend on each other with the statistical uncertainty further to be characterized by the confidence intervals. This dependence is consistent with them entering together the virus production term in equation (1) of the basic module. The other implication is that the structure permits the other model parameters to be identified. The identifiability test of the compartmental model (equations 5–8) suggests that it is identifiable, provided that the observable variables are the virus in spleen, blood and liver and the serum ALT blood, which is the current case. Furthermore, a global sensitivity analysis was performed which allowed to rank the influence of random variations in the model parameters on the variation in the serum ALT level. The methodology and the results are presented in Figure S15 pDCs predicted by the mathematical model is compared to experimentally observed values. pDCs from wt and −/−ifnar mice. At MOI = 0.1  the data represent the geometric mean ± SD from 5 experiments. At MOI = 0.01  the data represent the geometric mean ± SD from 2–4 experiments. Taking into account that the variability between experimental series is about 0.5 at the log10 - scale, the model calibrated on independent sets of data provides a valid description of the system.Viral replication in MHV-infected pDCs in vitro at MOI = 0.1 and 0.01. Virus produced at 24 hours after MHV infection of 10(0.07 MB DOC)Click here for additional data file.Figure S25 macrophages are depicted.The kinetics of viral replication in wt macrophages in vitro at MOI = 0.0001. The virus (red line), type I IFN (green line) and infected cell (blue line) kinetics are shown. The measurements of the virus titer (circles) observed at 12 and 24 hours after MHV infection of 10(0.06 MB DOC)Click here for additional data file.Figure S35 cells/well of the volume 500 µl. The data represent the geometric mean ± SD from 4 to 8 independent experiments.The effect of pDCs derived supernatant on MHV replication in wt macrophages in vitro at MOI = 1. The virus production in vitro was measured in wt macrophages pre-treated with the indicated amounts of IFNα-containing supernatant . Macrophages from B6 mice were plated at 5×10(0.05 MB DOC)Click here for additional data file.Figure S45 pDCs and 6×106 Mφs. Low (5×101 pfu), intermediate- (5×103 pfu) and high (5×105 pfu) dose infections are considered. The model consistently predicts the efficient containment of virus replication (left panel) and the dose dependent activation of the type I IFN synthesis (right panel).Dynamics of MHV infection as described by the ‘in vitro spleen’ approach. The virus kinetics predicted by the mathematical model for in vitro infection of 7×10(0.13 MB DOC)Click here for additional data file.Figure S55 pfu MHV. For depletion of pDCs, mice were injected i.p. with 0.5 mg of α-mPDCA-1 (Miltenyi Biotec) 12 h prior to infection or left untreated (n = 3 mice per group). (A) Fluorescence microscopic analysis of spleen sections at 48 h post infection using antibodies against B220 (blue), MHV-N (green) and F4/80 (red). Original magnification (×400). (B) Quantitative evaluation of macrophage infection. Values indicate numbers of MHV-N+F4/80+ per high power field (mean ± SEM). Three sections from each mouse were analyzed.Infection of macrophages assessed by in situ analysis. C57BL/6 mice were infected i.p. with 5×10(0.25 MB DOC)Click here for additional data file.Table S1(0.05 MB DOC)Click here for additional data file.Table S2(0.10 MB DOC)Click here for additional data file.


Paramecium bursaria  in Lake Biwa, the largest lake in Japan. The viruses detected were all Chlorella variabilis virus (CvV = NC64A virus). One of them, designated CvV-BW1, was subjected to further characterization.We performed an environmental study of viruses infecting the symbiotic single-celled algae of dnapol gene, CvV was classified into two clades, A and B. CvV-BW1 belonged to clade B, in contrast to all previously identified virus strains of group H that belonged to clade A.CvV-BW1 formed small plaques and had a linear DNA genome of 370 kb, as judged by pulsed-field gel electrophoresis. Restriction analysis indicated that CvV-BW1 DNA belongs to group H, one of the most resistant groups among CvV DNAs. Based on a phylogenetic tree constructed using the C. variabilis virus.We conclude that CvV-BW1 composes a distinct species within  Chlorella virus that infects Chlorella-like algae symbiotic with coelenterate Hydra viridis was first discovered in 1981 and designated HVCV (Hydra viridis Chlorella virus) [Chlorella virus that infects Chlorella-like algae symbiotic with ciliate Paramecium bursaria was described (Paramecium bursaria Chlorella virus [PBCV]) ) . Studies [PBCV])  and refeP. bursaria symbionts indicated that the algal group "American" containing strain NC64A and the algal group "European" containing strain Pbi are genetically distinct from each other, as well as from any known free-living algae and other symbiotic algal species [Chlorella variabilis ("American") and Micractinium reisseri ("European") [Hydra viridis Chlorella virus [HVCV], Paramecium bursaria Chlorella  [PBCV], and Acanthocystis turfacea Chlorella virus [ATCV]) and strange names based on host strains  have been used. In this report, viruses infecting C. variabilis and M. reisseri are referred to as C. variabilis virus (CvV) and M. reisseri virus (MrV), respectively  and NC64A (ATCC 50258) were cultured in C liquid medium . Exc. Exc-1. P. bursaria population and the probability of its burst . In 2003 and 2004, a major outbreak of koi herpes virus (KHV) occurred in Japan. Populations of koi (common carp) in Lake Biwa were attacked by the virus from May to June 2004, which caused mass death of the fish. Large numbers of koi carcasses washed ashore onto the coastal area of a sampling point, Shin-Asahi Windmill Village (site 8). At this time, shallow water around this point seemed to be under low-oxygen conditions caused by decomposition of fish carcasses. We detected the highest virus concentrations at this sampling point at these times. In contrast, lower densities of viruses were common in July 2004 at all sampling points , had smaller plaques (about 1 mm in diameter) in addition to the normal-sized plaques  in GC, whereas enzymes II were rich (75%) in AT. This result can be rationalized in two ways: CvV-BW1 DNA is rich in AT and poor in GC or CvV-BW1 DNA is highly modified at G and/or C. Nucleotide sequence analysis of clones in the CvV-BW1 genome library did not reveal any evidence that CvV-BW1 DNA was AT-rich; according to our preliminary genome analysis, the GC content of CvV-BW1 is in the vicinity of 41.3%. Therefore, we suspected that CvV-BW1 DNA would have a high incidence of G and/or C modification. To confirm this speculation, we examined the frequencies of modified nucleotides in CvV-BW1 DNA; the results revealed 33.2% 5 mC relative to 5 mC+C and 31.0% 6 mA relative to 6 mA+A.The best characterized CvV, PBCV-1, encodes hyaluronan synthase (HAS), which functions in the production of hyaluronan, a polysaccharide covering the outside of the algal cell wall . Graves dnapol, of viruses appear to have evolved from a common ancestral gene, and are highly conserved within the viral family Phycodnaviridae [dnapol sequences indicated that this 2060-bp fragment contained an intron of 86 bp. Introns of the same length are present in dnapol of AR-158, NY-2A, NY-2B, and NYs-1 [dnapol, CvV was found to be divided into two clades, A and B, with a minimum distance of 0.237 between these clades. As shown in Fig. dnapol genes.The DNA polymerase genes, aviridae ,29. Thernd NYs-1 . In the dnapol clade A (101-bp intron group)  differ from CvV-BW1 in some respects. NY-2A belongs to restriction group I, NYs-1 belongs to group F, and NY-2B belongs to group G. Although the restriction group of AR158 has not been determined, AR158 does not encode HAS [Van Etten et al.  reportedcode HAS . Taken tC. variabilis virus (NC64A virus) but not M. reisseri virus (Pbi virus) in the water of Lake Biwa, Japan. The highest virus density was recorded in water under low-oxygen conditions, whereas lower virus densities were commonly found in the seasons when the lake waters reached up to around 30°C. These results suggest that viral density is affected by the population density of P. bursaria and its burst ratio.We detected C. variabilis virus.The viral strain CvV-BW1 found in Lake Biwa was examined in detail with regard to plaque size, electron microscopic features, protein composition, genome size, restriction enzyme digestion, level of DNA methylation, production of hyaluronan, and phylogeny of the DNA polymerase gene. Taken together, all of these observations indicate that CvV-BW1 is likely to be a new species of Chlorella variabilis virus; MrV: Micractinium reisseri virus; PBCV: Paramecium bursaria Chlorella virus.CvV: The authors declare that they have no competing interests.MS screened and isolated the viral strains, and then tested hyaluronan productivity. YK observed viruses by electron microscopy. SiU carried out the protein analysis. YM examined the viral genome sizes, and then MS and YM confirmed the results of restriction enzyme digestion. YH examined viral DNA modification. RH contributed to DNA polymerase gene analyses. RH and BiO prepared the manuscript. NI initially conceived of this study and RH, MK, BiO, NI finalized the experimental design. All authors have read and approved the final manuscript.1 Department of Biomedical Science, College of Life Sciences, Ritsumeikan University, Noji Higashi 1-1-1, Kusatsu, 525-8577 Japan.2 Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, Noji Higashi 1-1-1, Kusatsu, 525-8577 Japan.3 Department of Biotechnology, College of Life Sciences, Ritsumeikan University, Noji Higashi 1-1-1, Kusatsu, 525-8577 Japan.4 Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Noji Higashi 1-1-1, Kusatsu, 525-8577 Japan.


We know what causes the disease—a virus belonging to the family Flaviviridae. Although an effective vaccine is available, the WHO still estimates an annual occurrence of 200000 yellow fever cases with 30000 related deaths. These statistics are even more alarming when we consider the fact that no disease can be considered as strictly endemic today. Airplanes unwittingly transport infected travelers, vectors, viruses, bacteria, and their disease to all corners of the earth. Mosquitoes can carry the YF virus globally with the present ease in international travel. Americans, Europeans, and especially, Asians are vulnerable as they have the competent vector mosquitoes and nonhuman primates. or C6/36 A. albopictus cells (the latter insect cell lines have the advantage of demonstrating a clear cytopathic effect) is the choice for diagnosis. Rapid virus identification will permit the timely activation of control systems for prevention of further cases and epidemic situations.This clinical and seroepidemiological study has used virus isolation by conventional cell culture and mouse brain inoculation methods, followed by IF. Serum IgM Mac-ELISA and IgG ELISA were used at the nearest US regional medical research institute for the confirmation of clinical cases. An antibody titer of 200 was declared positive though no repeat tests were done to demonstrate a four-fold rise in titer. It was not deemed necessary in this case as all soldiers who were infected with YF virus, had not been previously immunized. A cross-reactive flaviviral antibody, such as dengue, is a challenge in diagnosis. However, the study in Amazon has ruled this out by using various cross-reacting antigens to YF virus as control and also as appropriate negative controls. This study had not done a confirmatory RT-PCR that is available at the WHO regional reference laboratory for the YF virus. It is known that daignosis of arboviral infections like yellow fever is done by serum or cerebrospinal fluid (CSF) analysis for virus-specific IgM and neutralizing antibodies.[A. aegypti might exist, there would have been disastrous civilian infections. Also significant is the observation that bed nets were not protective, which suggests an exposure to vector bites occurring in the daytime. Army personnel, travelers, and infants below six months of age in the YF endemic zone would need to be immunized and active seroprevalence of adult civilians checked out during epidemics to assess their immune status.If these infected soldiers (in the viremic phase) had traveled to their urban homes where YF virus is an enveloped, single-stranded, positive sense RNA virus. Despite several different genotypes, there is a single serotype that is antigenically conserved. Therefore, the 17D YF vaccine protects against all strains of the virus. YF can be substantially controlled through a single dose (unlike the avian and swine influenza which show antigenic drift).One out of seven persons infected with the YF virus develops illness during an epidemic, but only one adverse event occurs for every 200,000 to 400,000 immunized with the YF vaccine. The WHO's International Health Regulations (2005) recommend that all individuals should obtain an International Certification of Vaccination or Prophylaxis (ICVP) if visiting endemic zones (or even in transit) and during outbreaks.The live-attenuated strain of YF virus known as 17D, has been used creating a Yellow Fever vaccine used in humans since more than 50 years. The GlobThe live vaccine dose given subcutaneously has a highly favorable benefit-risk profile (with 99.9% protection within a month) and sustains immunity for over 35 years (ten years is the rule for revaccination). A single vaccination to cover 80% of the susceptible population is recommended for protection. Children should be immunized at nine months through EPI in YF endemic countries. However, at present, the stockpile of vaccine available at the WHO may not be adequate if a large outbreak occurs. Newer vaccine preparations are envisaged and safe vaccines need to be developed for use in pregnant women and those below six months of age.Over the last 20 years, the numbers of YF epidemics have risen and more countries are reporting cases. There is still a large susceptible, unvaccinated population in both Africa and the Americas. Changes in the world's environment, such as deforestation, global warming, and urbanization, have increased mosquito numbers and habitats, enabling frequent contact of humans, mosquito, and virus. It is true that YF remains the only disease for which proof of vaccination is required as a condition on entry. Widespread international travel could play a role in spreading the disease, an event that may constitute a “public health emergency of international concern”. Therefore, mass vaccination campaigns and surveillance should continue.


Homo sapiens from the curse of this ancestral disease was neither easy nor without controversy Now that the 20th century has passed into the domain of history books, we can retrospectively begin to assess the relative contributions that the many advances in the realm of infectious disease have actually made to public health in general. At the top of this virtuous list will surely be the discovery of antibiotics in the 1930s and the use of vaccination to eradicate smallpox as an extant human disease in the 1960s and 1970s. As clearly pointed out in a recent book by D. A. Henderson, one of the leaders of the global smallpox eradication program, this task of ridding pas de trois with the WHO that continues to this day. In the case of the US, input was sought from the Institute of Medicine (IOM), which has struck two expert committees , Atlanta, United States of America, and at Vector, Novosobirsk, Russia. WHO convened a standing committee to oversee these repositories and issue regulatory approval for any research studies that utilized the live virus stocks at the two sites, with the tacit assumption that the only justifiable long-term fate for these stocks was an autoclave. Then, the revelation that variola virus had been covertly weaponized and stockpiled by the Soviet military http://www.poxvirus.org), derived from independent isolates collected throughout the world at various times in the 20th century, new clues as to the origin, spread, and evolution of variola clades within the human population have been deduced In the meantime, particularly in the past decade, some genuinely intriguing science has been conducted with variola virus and closely related pathogenic orthopoxviruses. Many diverse scientific fields that impinge directly on the issue of variola virus research potential  have been moving forward at breakneck speed, and so has the technical ability to query related issues like viral pathogenesis and host tropism. The nearly complete genomic sequences of two variola isolates were first published in the early 1990s, but now that almost 50 distinct genome sequences are available on the Web   or safer for immunocompromised individuals  have been developed and stockpiled. These drugs and vaccines have the dual benefit that they are likely also efficacious for other related zoonotic orthopoxvirus infections of humans, particularly monkeypox and cowpox viruses. Additionally, the new anti-poxviral drugs have been used to treat rare cases of runaway infections with the live vaccinia vaccine itself. Newer diagnostics based on PCR techniques or directed sequencing have refined the ability to distinguish bona fide variola infections from those caused by closely related orthopoxviruses. In fact, these advances can be counted among the genuine success stories made possible by the increased biodefense funding in the US since the terrorism events of 2001.But despite these advances, there is far more that we simply do not understand about smallpox disease or its causative virus. The smallpox vaccine, vaccinia virus, remains the poster-child for human vaccines, but we have only begun to understand how vaccinia-induced immune responses protect vaccinees from orthopoxvirus infections In the 1980s, the debate focused on whether the destruction of the declared variola stocks would actually free the planet forever from the specter of smallpox re-emergence, or whether destruction would simply make the world a more dangerous place where suspected covert stocks of virus would assume greater danger as potential bioweapons or agents of bioterrorism. Even now in the first decade of the 21st century, we still do not know if any live variola virus stocks exist outside of the two WHO-approved repositories, but the combination of an extensive public literature on variola virus genomic sequences coupled with the rapidly advancing technologies of DNA gene synthesis and synthetic biology have now made the possibility of creating live variola virus  from scratch readily achievable.Although variola virus remains the first and only human pathogen to sit in the gallows, waiting potential execution, it will not be the last. Polio stands a reasonable chance of being eradicated as a human disease in our lifetime (and Rinderpest as a cattle disease), but as Wimmer's lab showed in 2002, any scientist with access to a gene synthesizer can now construct live polio virus using relatively standard laboratory reagents http://www.who.int/csr/disease/smallpox/research/en/index.html).Given the above conundrum, the obvious question is whether the destruction of the declared live variola virus stocks would be a genuine victory for humankind or merely be a symbolic gesture that provides only an illusion of security. Should the message to the scientific community of the future be that no further scientific queries will be tolerated that require live variola virus? Presently, the WHO-mandated restrictions on labs working even with noninfectious plasmid DNAs containing variola gene sequences are very stringent Similar ethical issues remain when we consider the likelihood of whether a new recombinant orthopoxvirus, derived from poxviruses that alone are nonpathogenic to humans, might be capable of causing smallpox-like disease in humans. It is already known that zoonotic infections with monkeypox virus resemble clinical smallpox closely, but these infections are only poorly transmissible from human to human Ultimately, the reason this debate remains contentious is that variola virus has killed more human beings in the civilized era than any other known pathogen. Even though the disease itself has not been seen for over 30 years, pictures of its victims still have the power to remind us of why this viral pathogen is still feared . No civiPLoS Pathogens are invited to comment on this issue online via the Comments tab, which is located underneath the article title .The debate about the potential destruction of variola virus, for better or worse, is returning to the front page. Now, however, the emergence of open access publishing and open source technology allows for more input and dialogue from a wider spectrum of people who may wish their views to be registered. The member states of the World Health Assembly will soon be polled for their vote on whether the existing declared stocks of variola virus should be destroyed or not. So, until then, the readers of The debate may also prove to be instructive when the next human microbial pathogen lands on death row, awaiting our collective verdict.


Oncorhynchus mykiss) has been without any outbreaks of VHS for many years until the disease emerged in a farm in western Norway in November 2007. The fish were, in addition to VHS virus, positive for gill chlamydia-like bacteria, Flavobacterium psychrophilum, and a microsporidian. A new VHS virus genotype III was isolated from the fish in RTgill-W1 cells and the complete coding region  was sequenced. This virus was also used in a challenge experiment to see if it could cause any mortality in rainbow trout in sea water.Norwegian production of rainbow trout (This is the first time a nearly complete sequence of a genotype III virus isolate has been presented. The organization of the genes is the same as in the other VHS virus genotypes studied (GI and GIV). Between the ORFs are nontranslated regions that contain highly conserved sequences encompassing the polyadenylation signal for one gene, and the putative transcription initiation site of the next gene. The intergenic regions vary in length from 74 nt to 128 nt. The nucleotide sequence is more similar to genotype I isolates compared to isolates from genotype II and IV. Analyses of the sequences of the N and G protein genes show that this new isolate is distinct from other VHS virus isolates and groups closely together with isolates from genotype III. In a challenge experiment, using intraperitoneal (ip) injection of the isolate, co-habitation with infected fish, and bath challenge, mortalities slightly above 40% were obtained. There was no significant difference in mortality between the bath challenged group and the ip injected group, while the mortality in the co-habitation group was as low as 30%.All VHS virus isolates in genotype III are from marine fish in the North East Atlantic. Unlike the other known genotype III isolates, which are of low virulence, this new isolate is moderately virulent. It was not possible to detect any changes in the virus genome that could explain the higher virulence. A major problem for the study of virulence factors is the lack of information about other genotype III isolates. Novirhabdovirus, family Rhabdoviridae .,8,11.NovAccording to existing literature challenge of rainbow trout with the VHS virus, genotype III, should not result in any significant mortality . HoweverThe aim of the present study is to see if this VHS virus genotype III with the first completely characterized coding region may cause any mortality when challenging rainbow trout in full sea water. The genome of this isolate will be compared with partly sequenced VHS virus, genotype III, and completely sequenced coding regions and intergenic regions of other VHS virus, genotypes I and IV. Such a comparison may also give clues as to which changes in the genome may influence the virulence or ability to cause mortality in rainbow trout populations.EU481506). The sequence is 11,065 nucleotides (nt) long and contains six open reading frames (ORF) in the order 3'-N-P-M-G-NV-L-5'. This arrangement is identical to what has been found for other, fully sequenced, VHS virus isolates in genotype I [AB490792). Between the ORFs are nontranslated regions that include highly conserved sequences encompassing the polyadenylation signal for one gene, and the putative transcription initiation site of the next gene. An alignment of the conserved nontranslated sequences is shown in Figure The first genome of a VHS virus in genotype III, strain FA28.11.07, containing all protein coding sequences (CDS) and intergenic regions (ITRs), has been sequenced  from France. The conserved domains III and IV containing the four major motifs A, B, C and D localized between aa 566 and 790 [cf [The last ORF (1984 aa) in the VHS virus genome is the large (L) protein that encodes the RNA-dependent RNA polymerase. The L protein from FA28.11.07 is highly similar to the other L protein genes sequenced from other VHS virus isolates, with the exception of one isolate of genotype I, isolate FR-07-71  by Dale et al [Analyses of the relationship of the VHS virus isolate FA28.11.07, based on nucleotides of the complete open reading frame (ORF) of the N (1215 nt) and G 1524 nt) proteins, show that this isolate belong to genotype III Figures  and 4. T nt protele et al . The VHSVibrio spp. and Aliivibrio spp.) were isolated from a few fish  and IPN virus was present in all of the fish. The dominating bacteria were Vibrio splendidus-like. The mortality stopped one week before the start of the experiment. However, the fish were still positive for IPN virus at the time of challenge, i.e. they were carriers of the virus . The fish remained positive for IPN virus throughout the experimental period and a few fish were also positive for Vibrio spp and Aliivibrio spp. All fish tested before the start of the experiment were negative for VHS virus.The rainbow trout used in the challenge experiment, came from fresh water and were put directly in full sea water, where they suffered some mortality (8.8%) during the acclimatization period. Most of the mortality seemed to be due to poor smoltification, but bacteria , and up 48.4% in the i.p. challenged group, V (N = 31) Figure . The morNot all fish that died in the different groups were positive for presence of VHS virus. If the fish that were negative for presence of VHS virus are removed from the mortalities the pattern of mortality remains, however, more or less the same Figure . The morNone of the fish in groups V (N = 11) and KV (N = 16) were positive for VHS virus at the termination of the experiment 53 days post challenge, while 2 and 3 fish out of 33 and 37 fish examined were positive in groups BV and H, respectively. These five fish were carriers of VHS virus  and did not show any signs of disease. The virus was present in kidney, heart and spleen tissues, while the brain from one fish only was positive. Of the 97 fish sampled at the termination of the experiment 53 days post challenge 5.2% were carriers of the VHS virus.The amount of VHS virus template in the kidney and brain of fish in the two challenge groups V and BV have been quantified using the elongation factor alpha as a standard. The kidney tissue from 13 individuals in group V was positive for presence of VHS virus template while only 10 individuals had positive brain tissue (CNS). In the BV group 28 and 27 individuals had positive kidney and CNS, respectively. Only one fish was found to be positive for VHS virus 20 days after injection of the virus (group V), while in the bath challenged group nine fish were positive. An individual sampled 12 days after challenge in group V had the highest expression of VHS virus genome/mRNA. This expression was 6.7 million times higher compared to the lowest expression  of these templates in positive kidney tissue. The individual, in the V group, with the highest expression of VHS virus template in the CNS was sampled 7 days post challenge. None of the fish in this group had positive CNS after 18 days post challenge. In the bath challenged group, BV, the highest expression of VHS virus templates in kidney tissue was found 11 days post challenge while the highest expression in the CNS was seen 42 days post challenge. The latter specimen had negative kidney tissue. Of the nine fish that were positive after day 20 post challenge five had positive kidneys and 8 had positive CNS.FJ362510, FJ362511, H group: FJ362512, FJ362513, KV group: FJ362514, V group: FJ362515).VHS viruses were isolated from all challenged groups except the control group and the CH group. Partial sequences of the genome showed that the reisolated viruses were identical to the FA28.11.07 isolate  and 62.2 (N = 23), respectively. One moribund fish in group BK, collected 32 days after start of the experiment, had a haematocrit value = eight. The haematocrit values in all groups at the termination of the experiment are presented in table AB490792) have already been published and this study presents the first complete sequence of the coding region of a genotype III isolate (FA28.11.07). Like all other members of the genus Novirhabdovirus the VHS virus genotype III has the same gene arrangement and similar intergenic regions (ITRs) with polyadenylation signals and transcription initiation sites [The complete genome of VHS virus isolates belonging to genotypes I ,20 and Ion sites ,20,23-25on sites .(C. herrengus),Norway pout (T. esmarkii) and predators of these species, like cod (G. morhua) and haddock (M. aeglefinus) could carry the virus close to aquaculture facilities. Virulence factors for the VHS virus have not been identified, but it has been shown that the genetic difference between virulent freshwater strains and avirulent marine strains can be very small [The distribution of VHS virus genotype III in the North Sea and the North Atlantic ocean has been well documented ,26-28. Sry small . VHS virry small . Hence, ry small . The virThe fish used in the challenge study suffered a low mortality the first two weeks after arrival, but the mortality ceased one week prior to the challenge. A single cause of these mortalities was not identified, but several factors could have played a role. The fish were taken from a fresh water site, and put directly in full seawater and this stressful event was probably the main cause for the mortalities. However, among the mortalities were fish positive for bacteria and IPN virus. These agents may have affected the mortality observed in the period before start of the challenge and during the experimental period. The IPN virus detected before challenge was also present in the fish throughout the experimental period, but at a low level (carrier state). IPN viruses are very common in the production of salmonids in Norway and it is not known if these virus infections may interfere with infections with VHS virus.in vitro infectivity of VHS virus isolates in primary cell cultures. The present study shows that rainbow trout in full sea water suffers a moderate mortality (about 40%) after exposure to the marine VHS virus genotype III isolate, FA28.11. 07, irrespective of challenge method (immersion or injection). However, the mortalities obtained in this study are relatively high (regardless of infection route), compared to previous studies where rainbow trout has been challenged with genotype III isolates of VHS virus [It has been shown in virulence studies of VHS viruses that the challenge method is important for the resulting mortality . Using mHS virus ,31, but HS virus . In the HS virus  10.1 graHS virus  is not a50 = 0.5 × 108 TCID50/fish and bath TCID50 = 0.8 × 108 TCID50/ml), which is exceedingly higher than what has been reported by other studies infecting rainbow trout with marine genotypes [The fish in the V and BV group in this experiment, were challenged with a high dose , was isolated from rainbow trout (Oncorhychus mykiss) suffering mortality in a marine farm in Storfjorden at the northern west coast of Norway [GU121099, GU121100 and isolate FA28.02.08V, Accession nos: GU121101, GU121102), and in July 2008 this virus was also found in a more distant farm in the same fjord. All farms are owned by the same company.In November 2007 a viral haemorrhagic septicaemia virus (VHSV), genotype III , only 2 out of 30 moribund rainbow trout were found to be positive for presence of VHS virus on the collection date 28 November 2007. The number of VHS virus positive fish increased to 50% in the middle of December 2007, while it was not possible to detect the virus in kidney and brain tissues from fish (N = 60) sampled in March 2008 In addition to VHS virus type III, the rainbow trout in the first farm were infected by Clupea harengus) collected at the outlet of Storfjorden, but this isolate (CH18.03.08) belonged to genogroup Ib .A VHS virus isolate was also obtained from herring  were checked for presence of VHS virus and IPN virus, using real time RT PCR assays (see below), before the start of the experiment. None of the fish that died during the acclimatization period were positive for VHS virus, but they were all carriers of the IPN virus and a few were positive for different bacteria (Vibrio spp. and Moritella viscosa). The fish were kept in 5 tanks (0.15 m3) with running sea water (34‰) at mean temperature of 10°C.Rainbow trout, with a mean weight and length of 47.4 gram and 15.7 cm, were taken into the research facility, Industrilaboratoriet, at the University of Bergen in January 2008. The fish came from a fresh water site in western Norway and were put directly in full sea water (34‰). A low mortality was registered during the first two weeks after arrival and some of the fish had skin ulcers. Bacteria, The challenge experiment was designed to see if the VHS virus isolate, FA28.11.07, may cause mortality and if different challenge methods may influence mortality. The wild type VHS virus from herring (CH15.02.08) was used as a control.9/ml, ie. giving a final challenge dose of TCID50 = 1.1 × 106/gram fish tissue (or TCID50 = 0.5 × 108/fish). In addition to the ip challenged fish 30 rainbow trout (code = KV) were added as co-habitants. A total of 61 rainbow trout were kept in tank 1.This tank was used to test the effect of intraperitoneal injection (ip) of the VHS virus isolate FA28.11.07 and the effect of transmission from infected to non-infected rainbow trout (co-habitation effect). Rainbow trout, N = 31 (code = V), were injected intraperitoneal (ip) with 0.2 ml of supernatant from cell culture with TCID50 of 1.0 × 109 VHS virions/ml) in 20 liter sea water for 30 minutes, i.e. at a concentration of TCID50 = 0.8 × 106/ml.This tank contained 68 rainbow trout (code = BV) that were challenged by a bath consisting of 15 ml of the VHS virus isolate FA28.11.07 (TCID50 = 1.0 × 10This tank contained 68 rainbow trout (code = BK) that were bathed in 15 ml cell culture media in 20 liter sea water for 30 minutes. This group constituted the control group for the bath challenged rainbow trout.This tank was used to test if VHS virus from rainbow trout carriers could be transmitted by bath challenge. Rainbow trout, N = 61 (code = H), were bathed for one hour in 20 liters of sea water added 45 ml of sterile filtered homogenate (0.2 μm) of gill and kidney tissues from rainbow trout that were asymptomatic carriers of VHS virus. It was not possible to culture the virus from this homogenate. The virus was only detected by real time RT PCR. The homogenate was obtained from carrier rainbow trout that came from the same marine farm as the VHS virus isolate FA28.11.07.This tank was used to test the possible effect on rainbow trout of a wild type VHS virus detected in herring (CH18.03.08) from the outlet of Storfjorden. Homogenate made from brain and kidney tissue of the herring was intraperitoneal injected into rainbow trout, N = 30 (code = CH). Each fish was injected with 0.2 ml of sterile filtered homogenate (0.2 μm).Dead and moribund fish were removed from the tanks two times a day, and weight and length were registered. All fish were examined for external and internal signs of disease. To rule out bacterial infections as a cause of mortality, bacterial samples were taken from a selection of dead/moribund fish and fish sampled randomly. Bacteria were isolated from kidney and grown on blood agar plates containing 1.5% NaCl and incubated at 15°C for at least two weeks. When growth was observed on the agar plates the bacteria were identified by sequencing of the 16S gene using a set of general prokaryotic primers targeting this gene (see below).The following tissues were sampled from all dead/moribund fish; gills, heart, kidney, spleen, and CNS. The tissues were stored at -20°C before being used for reisolation of the VHS virus or RNA extraction. All dead/moribund fish were tested for presence of VHS virus and IPN virus by real-time RT-PCR. To obtain information about tissue tropism the head-kidney, heart, spleen and CNS tissues were tested from 25 fish with respect to presence of VHS virus. Based on the results from the tissue tropism study the kidney and CNS from all fish in the different groups were tested for VHS virus, while only the kidneys were tested for presence of IPN virus. A blood sample, for measuring the haematocrit, was taken from all fish.Several tissues  were sampled from a few individuals in each group and fixed in Karnovsky for examination of histopathology.2 tissue culture flasks (Nunc) at 20°C in Eagles Minimum Essential Medium (EMEM) (Sigma) supplemented with 10% Foetal Bovine Serum (FBS) (10% v/v), L-glutamine (4 mM) and gentamicin (50 μg/ml). The cells were then subcultured for 7-10 days until the tissue flasks were covered with 60-80% confluent monolayer.RTgill-W1 cells  were culSupernatant containing VHS virus isolate FA28.11.08 (second passage) was diluted 1:100 in PBS and incubated for 1 hour at 15°C in cell culture flasks with the monolayer of RTgill-W1 cells. The inoculum was then removed and replaced by supplemented EMEM as described above, but with 1% FBS. The cells were incubated for five days until cytopathic effect (CPE) could be observed. RNA was extracted from infected cells and reverse transcribed into cDNA as previously described  and a re1 to 1017, was added. Each dilution was used in four parallel wells. The 96-well tray was incubated for 10 days, and TCID50 (The dilution in which 50% of the cells are dead) was determined by comparing with uninfected cells.RT-gill W-1 cells were seeded in a 96-well tray and allowed to form a monolayer. A 10-fold dilution series of the VHS virus isolate was made in 2% infection medium and added to the monolayers. To each well, 200 μl of each dilution, ranging from 10RTgill-W1 cells were also used for reisolation of VHS virus from the different fish groups challenged. Tissue homogenates were made from kidney or CNS tissues from selected fish  in the different groups and sterile filtered (0.2 μm) before inoculation on the cells. The cells were kept for three passages or until a CPE could be observed. Presence of VHS virus in cell cultures was confirmed by real time RT PCR and sequencing of the PCR products.4. Ultrathin sections were cut on Reichert-Jung Ultracut E. The ultrathin sections (30 - 40 nm) were stained for 1.5 hours in 2% aqueous uranyl acetate solution and then stained with lead citrate.Tissues  collected were fixed by immersion, at 6°C, in a modified Karnovsky fixative where the distilled water was replaced by a Ringers solution. The fixative contained 4% sucrose. Before embedding in Historesin the tissues were dehydrated through graded series of ethanol as recommended by the manufacturer. Semi sections, 1.5 μm thick, were cut on a Reichert-Jung 2050 microtome and stained in toluidine blue.3 Before embedding in EMBED-812 (Electron Microscopy Sciences) the tissues were stained/post-fixed in 1% OsOThe extractions of RNA from tissues were performed as described by Devold et al. . RNA-pel6 (random hexamers) and 4 μl RNA template, making a total of 10 μl, was incubated at 70°C for 5 min. A RT-mix was made from 5 μl 5 × RT-buffer 5, 1,25 μl DTT (200 mM DL-dithiothreitol), 2,5 μl 10 mM dNTP, 0,5 μl RNasin, 0,15 μl MMLV and 5,6 μl DEPC-water, making a total volume of 15 μl. This RT-mix was added to the 10 μl template/pd(N)6 mix making a total volume of 25 μl. This solution was incubated at 37°C for 60 min [cDNA synthesis was performed as follows; 5 μl DEPC-water, 1 μl pd(N)r 60 min  after whDNA was extracted from all isolated bacteria using the DNeasy DNA Tissue kit (Qiagen) following the manufactures description of protocol. Elution was performed twice in 50 μl 10 mM Tris-HCl, pH = 8.5 to increase the overall DNA yield, and the DNA was stored at -20°C.FJ362510, FJ362511, FJ362512, FJ362513, FJ362514, FJ362515). VG1 (5'-ATG GAA TGG AAC ACT TTT TTC-3') and VD3 (5'-TGT GAT CAT GGG TCC TGG TG-3') were used as PCR primers [The partial G gene (654 nucleotides) from VHS virus present in tissues of challenged rainbow trout and virus isolated from challenged fish in cell culture, was obtained by PCR using the cDNA synthesized as described above  as described by the manufacturer. Sequencing was then performed in both directions using ABI PRISM BigDye terminator chemistry (version 2) according to Applied biosystems (ABI). The PCR primers were used for sequencing.EU481506).In this experiment six real time RT-PCR assays were used table . PrimersParanucleospora theridion, Flavobacterium psychrophilum, Candidatus Piscichlamydia salmonis and the new chlamydia species, targeting the genes coding for the SSU, are presented in table The internal control assay El-1A targets the Atlantic salmon cellular elongation factor . The assVerso™ 1-step QRT-PCR ROX Kit and Absolute™ QPCR ROX Mix were used for the real time RT-PCR assays. Analysis was performed in a ABI 7500 sequence detection system (Applied Biosystems). The reaction was 15 min at 50°C (Reverse Transcriptase step), 15 min at 95°C (Polymerase activation step) followed by 45 cycles of 95°C for 15 seconds (DNA-dissociation) and 60°C for one minute .-06 ng/μl for VHS virus assay and 243.6 ng/μl to 2.436-06 ng/μl for the elongation factor assay. Template used in the efficiency test was as described above. The dilution series was analyzed in triplicates using one-step real-time RT PCR. The mean Ct value for each triplicate was calculated and a standard curve was made by plotting Ct values against the serial logarithmic dilutions. The amplification efficiency (E) for the VHSV08 and EF1A assays were calculated using the formula: (10-1/-slope)-1 and were found to be E = 0,98394 and E = 0,8952, respectively.Prior to real-time RT PCR analysis the VHS virus assay was optimized with regards to concentrations of primers and probe. An efficiency test was preformed to test the efficiency of the VHS virus and elongation factor assays in order to be able to perform relative quantification of the amount of VHS virus in the samples. This test was performed by using a tenfold dilution series. The dilution series was made in the concentration ranging from 477.6 ng/μl to 4.776To perform the sensitivity test a twofold dilution series was made from 0,4 ng/μl to 0,0625 ng/μl. 10 replicates were made from each dilution and analyzed using one-step real time PCR. The sensitivity limit of the VHSV08 assay was set as the highest dilution where all 10 replicates were positive. The detection limit, derived from this sensitivity test for the VHSV08 assay, was found to be Ct = 37.0. This implies that Ct values higher than this limit may not be reproducible.reference)Ctreference/(Etarget)Ct target, where E = amplification efficiency.The expression of the target VHS virus genome template was calculated using the formula for normalized expression (NE): NE = (EEU481506). The N and G protein genes were aligned with homologous gene sequences from a selected number of VHS viruses already available on the EMBL nucleotide database, including a Norwegian isolate collected from rainbow trout in 1968 (Accession no: AY546621). The VHS virus (CH18.03.08) from herring , collected in the outlet of the fjord, was also included in the analysis. To perform pairwise comparisons between the different viruses, the multiple sequence alignment editor GeneDoc (Available at: http://www.psc.edu/biomed/genedoc) was used. Polymorphic regions were manually aligned and compared for both genes. Gaps in the alignment were deleted .The nearly complete genome of the VHS virus isolate, FA28.11.07, from the farmed rainbow trout kept in Storfjorden western Norway, was sequenced (Accession no. http://www.tree-puzzle.de). TREE-PUZZLE reconstructs phylogenetic trees from molecular data by maximum likelihood, and computes maximum likelihood distances and branch lengths. A model corresponding to GTR+I was identified by the Akaike information criterion using the Modeltest 3.6 script [Phylogenetic analyses of the data sets were performed using PAUP* version 4.0  and TREE6 script  in PAUP 6 script , to be s6 script .The authors declare that they have no competing interests.AN conceived the study. AN, HD and SN planned the experimental design. AN, HD and SN carried out the challenge experiment and sampling. SN cultured the virus and did all the sequencing. HD analyzed all samples. KW did the processing for histology and AN and KW did the histological examinations. KFO sampled the field material and helped with the processing and analyzing of this material. AN and HD drafted the manuscript. All authors critically reviewed and approved the final manuscript.