Transmission of avian influenza viruses (AIV) between different avian species may require genome mutations that allow efficient virus replication in a new species and could increase virulence. To study the role of domestic poultry in the evolution of AIV we compared replication of low pathogenic (LP) AIV of subtypes H9N2, H7N7 and H6N8 in tracheal organ cultures (TOC) and primary embryo fibroblast cultures of chicken, turkey, Pekin duck and homing pigeon. Virus strain-dependent and avian species-related differences between LPAIV were observed in growth kinetics and induction of ciliostasis in TOC. In particular, our data demonstrate high susceptibility to LPAIV of turkey TOC contrasted with low susceptibility of homing pigeon TOC. Serial virus passages in the cells of heterologous host species resulted in adaptive mutations in the AIV genome, especially in the receptor-binding site and protease cleavage site of the hemagglutinin. Our data highlight differences in susceptibility of different birds to AIV viruses and emphasizes potential role of poultry in the emergence of new virus variants. Anas platyrhynchos) Wild aquatic birds are generally considered as the primary natural reservoir for avian influenza viruses (AIV) Susceptibility to influenza virus infection, development of clinical disease and the potential to spread AIV by viral shedding is highly variable between bird species and may depend on the AIV HA subtype Gallus gallus domesticus), turkey , Pekin duck (Anas platyrhynchos domestica) and homing pigeon (Columba livia domestica). The results demonstrated that susceptibility to AIV varied significantly between cell cultures of different bird species tested, with respiratory cells of turkey being highly susceptible and those of pigeon showing the lowest LPAIV replication rates. Adaptation of LPAIV to TOC and EF of different bird species led to changes in viral growth kinetics, induction of cell death and the development of influenza virus genome mutations. These were found most prominently in the protease cleavage and receptor-binding site (RBS) regions of the hemagglutinin.Our objectives were to investigate the possible role of different avian species in the process of AIV evolution, and to compare the potential adaptation and increase in virulence between different AIV subtypes. To address this objective, we serially passaged LPAIV of subtypes H9N2, H7N7 and H6N8 in tracheal organ cultures (TOC) and primary embryo fibroblasts (EF) of chicken (All viruses replicated in the first passage in TOC of each bird species without addition of exogenous proteases . The repThe replication of AIV in the respiratory epithelium of TOC was confirmed by immunohistochemistry (IHC) against viral nucleoprotein. The third consecutive passage of each virus in TOC-Ch was investigated and IHC-staining was seen in the respiratory epithelium of virus-infected TOC at 8, 24 and 48 hpi, which increased over time . At 8 hpThe different LPAIV induced ciliostasis in the respiratory epithelium at different time points . DiffereDegeneration of the respiratory epithelium was confirmed by histology and IHC staining for apoptotic cells in TOC-Ch . All virIn contrast to TOC, which contain endogenous virus-activating protease(s), LPAIV could only replicate efficiently in EF when trypsin was added to the culture medium. In this case, the replication rates after inoculation of primary EF cultures of chicken, turkey, Pekin duck and homing pigeon in general agreed with the results obtained using corresponding TOC.In the first passage, inoculation of EF of homing pigeon (PEF) resulted in the lowest viral titers, whereas in EF of turkey (TEF) all viruses propagated to the highest titers compared to EF of other avian species . Inoculain vitro, three consecutive passages of each virus were performed in TOC and EF cultures of chicken, turkey, Pekin duck and homing pigeon. Growth characteristics of Du/H7N7, Ch/H9N2 and Tu/H6N8 changed during these passages and revealed avian species-specific differences  compared to the first passage in TOC of all tested bird species but with species-specific differences . Ch/H9N2Serial passaging of Tu/H6N8 showed statistically significantly increased titers in TOC-Tu and TOC-Ch in the second and third passage . In TOC-TOC-Pi were additionally analyzed for ciliostasis up to seven days pi. All tested LPAIV failed to induce ciliostasis in TOC of homing pigeon at any of the later time points of the three passages (data not shown).Statistically significant increased replication rates pi (p<0.05) were seen in EF of all avian species for Ch/H9N2, but for Tu/H6N8 and Du/H7N7 only in DEF in the third passage . Only ChARSSR*GLF>PVRSSR*GLF; Tu/H6N8 substitution E327V: PQAETR*GLF>PQAVTR*GLF). The majority of HA mutations were located either in the receptor binding pocket (amino acid positions 137 and 226) or in its vicinity (position 158). Plaque purification and subsequent sequencing of TOC-Tu-adapted Ch/H9N2 virus confirmed presence of all three mutations in eight out of ten randomly picked plaques. Silent mutations occurred in the HA of PEF-adapted Tu/H6N8 .HA, PB2 and NS genes of third passage viruses were sequenced and compared with the original virus sequences. We focused mainly on these genes because they are known to play a critical role in the virus host range and interspecies transmission The PB2 of the Ch/H9N2 acquired two amino acid substitutions (E472K and L571I) after three passages in TOC and EF of all tested bird species, one additional mutation was found after passaging of the virus in PEF . The polThe NS1 protein of Ch/H9N2 showed an identical aa substitution after three consecutive passages in TOC-Pi as well as in TEF . Single The complete viral genome of Ch/H9N2 was sequenced after three passages in TOC of all tested species and compared to the wild-type virus, since it showed the highest mutation rate in the HA, PB2 and NS1 proteins. No predicted aa substitutions were detected in the PB1, PA, NP, NA and M1/M2 proteins after three consecutive passages in TOC of all tested species. Silent mutations were found only in the Ch/H9N2 PA sequence after passaging in TOC-Pi .Furthermore, the PB1, PA and NA genome segments of Du/H7N7 and Tu/H6N8 LPAIV were analysed for mutations after passaging in TOC-Ch. No mutations were detected in these genome segments.in vitro infection of LPAIV isolates Ch/H9N2, Tu/H6N8 and Du/H7N7 in TOC and EF of chicken, turkey, Pekin duck and homing pigeon. TOC mimic the natural local AIV-infection site in the respiratory tract of avian hosts and allow investigations under controlled conditions In order to investigate the adaptation potential of LPAIV of different subtypes to different avian species, we studied an All tested LPAIV showed clear tropism for the respiratory epithelium of infected TOC, which was confirmed by virus-antigen staining. Infected TOC-Ch showed an increase of viral nucleoprotein staining at later time points. Since reliable quantification of viral-antigen positive cells of the respiratory epithelium was not possible at later time points, correlations to viral titers could not be drawn.The induction of ciliostasis in TOC upon LPAIV infection allowed speculating on the virulence of LPAIV in the primary target cells of the respiratory epithelium. Ciliostasis is the result of necrosis or apoptosis of ciliated cells of the respiratory epithelium in vitro system to characterize influenza viruses and growth behavior Earlier induction of ciliostasis was seen during the adaptation process of the tested LPAIV in TOC. This observation correlated positively with the increase of viral titers at the designated time points. The assessment of ciliostasis of avian TOC is a widely used tool to analyze the growth behavior and pathogenesis of influenza A viruses as well as other avian viruses, which target cells of the respiratory tract, such as the infectious bronchitis virus (IBV) Since all tested species possess different sialic acid receptor profiles in their tracheas in vivo LPAIV and HPAIV infection Domestic turkeys possess a high potential to alter and transmit AIV to new host species Chicken TOC were also susceptible to all tested strains, while Du/H7N7 replicated to the highest titers and Tu/H6N8 induced the most severe ciliostasis. All tested LPAIV subtypes also replicated in cells of Pekin duck but with lower rates compared to chicken and turkey.in vivo influenza infection studies with different AIV subtypes mostly failed to induce successful virus replication and disease Pigeons are generally considered to be relatively resistant to influenza virus infection and to play only a minor epidemiological role in influenza virus transmission In a comparative study, lectin staining of chicken, duck and turkey revealed the presence of both α2,3 Sias and α2,6 Sias in the respiratory epithelium of tracheas with 90% and 20–90% positive cells respectively, depending on the age and avian species Furthermore, differences in innate immune reactions may contribute to the difference in species related susceptibility. Recently it was demonstrated that the retinoic acid-inducible gene I (RIG-I) is present in ducks and plays a role in clearing an influenza virus infection During interspecies transmission, AIV need to adapt to the new host in order to overcome existing host-range barriers and increase replication. In our study, the increased viral replication rates up to the third passage were likely due to adaptive mutations which allowed more efficient virus replication in the host tissue. Single mutations or a combination of only few amino acid substitutions have been shown to improve virus propagation in new hosts and display an important prerequisite for interspecies transmission Comparing the investigated virus subtypes, Ch/H9N2 showed the highest mutation rate within the three consecutive passages in TOC and EF cultures. This virus developed HA mutations in TOC of turkey, Pekin duck and homing pigeon as well as in EF culture of turkey, but not in primary cultures of the original host species chicken. These results suggest that the observed mutations allowed the virus to adapt to new host species. Du/H7N7 acquired mutations only after propagation in EF of turkey and Pekin duck. Tu/H6N8 mutated only after passaging in TOC-Ch and TOC-Du.in vivo nor in vitro and their role in AIV-infection of the different hosts needs to be elucidated further in animal experiments. It may be speculated that the change in cleavage efficacy may also lead to a more vigorous virus replication in the host followed by more severe clinical disease. Substitution N244Y is located at the interface between the HA monomers and could affect a fusion-promotion activity and stability of the molecule Mutations of functional HA regions have been shown to be responsible for successful adaptation to new host species Substitution V18I, which is located in the RNA binding domain, may affect binding of different RNA target structures by the NS1 which elicits the NS1 protein in countering the host cell antiviral defence http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html).The PB2 forms together with PB1, PA and NP the influenza virus polymerase complex, which is necessary for virus replication in the host cell in vivo in the different avian species. This needs to be further investigated.No additional mutations were observed in the PB1, PA, NP, NA and M1/M2 proteins of Ch/H9N2 after three consecutive passages in TOC of all four bird species. Also Tu/H6N8 and Du/H7N7 showed no additional mutations in the PB1, PA and NA proteins after adaptation in TOC-Ch. We cannot rule out that mutations in these proteins may have occurred in the adaptation process after further passages in TOC or may have occurred in vitro model. The LPAIV strain Ch/H9N2 showed particularly high potential to adapt to new avian species within a low number of serial passages. Mutations of the HA receptor binding site and cleavage site in cells of avian species other than the originated host provide circumstantial evidence that the HA plays a significant role in the AIV host range and transmission between avian species. In vitro studies in organ cultures such as TOC are an excellent system to provide the necessary data on possible adaptive mechanisms under controlled conditions. They are a prerequisite for possible further in vivo studies and can not be supplemented by in vivo studies, which anyway do not allow testing for different viruses in four different species under comparable experimental conditions. Further studies are needed to determine if the adapted viruses show increased potential to infect and replicate in mammalian cells, which would possibly enhance the risk of interspecies transmission.Our study demonstrated different susceptibilities of the analyzed bird species to LPAIV using TOC as an A/chicken/Saudi Arabia/CP7/1998 (H9N2) (Ch/H9N2), a field isolate from a meat-type chicken flock, was kindly provided by Hans-Christian Philipp from Lohmann Tierzucht . A/turkey/Canada/1963 (H6N8) (Tu/H6N8) was kindly provided by Klaus Peter Behr from AniCon Labor . Tu/H6N8 has a 23 amino-acid deletion in the neuraminidase stalk region 2, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS and stained with 1% crystal violet . Plaques were counted, and infectious titers were calculated from the mean of duplicate wells as plaque-forming units (PFU) per ml using the formula:Infectious virus titers were determined using plaque assay in Madin-Darby canine kidney  at incubation day 20 (TOC-Ch), turkey  at incubation day 26 (TOC-Tu), Pekin duck  at incubation day 25 (TOC-Du), and homing pigeon  at incubation day 17 (TOC-Pi) Briefly, embryos were sacrificed, and the tracheae were removed under sterile conditions. Each trachea was cut manually into approximately 0.8 mm thick rings using a microtome blade. Individual rings were transferred to 5 ml tubes  with 0.8 ml prewarmed Medium 199 with Hanks' salts (Biochrom) including 1% penicillin/streptomycin . TOC were cultured at 37.5°C in a rotating shaker  at lowest rotation speed.ex vivo cultures After 24 h, the ciliary activity of the respiratory epithelium of each TOC was assessed using an inverted microscope. Only rings with 100% ciliary activity were used for the experiments. TOC were infected 5 days after preparation to avoid negative effects due to early inflammatory responses of the tissue 2.Primary embryo fibroblast (EF) cultures were prepared according to standard protocols 2.MDCK cells were cultured in Dulbecco's MEM (Biochrom) with 10% FBS, 1% L-Glutamine and 1% P/S at 37.5°C and 5% COhttp://www.bmelv.de/SharedDocs/Rechtsgrundlagen/T/Tierschutzgesetz.html), as confirmed by the animal welfare official of the University of Veterinary Medicine Hannover. Protocols for working with embryonated eggs were in accordance with the European Union Legislation (http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=CELEX:32005L0094:EN:NOT). Embryos used for tracheal organ culture or embryonic fibroblast preparation did not undergo any procedures prior to humane sacrifice by decapitation. Virus propagation was carried out in embryonated chicken eggs inoculated at incubation day 10, maintained for three days and subsequently chilled at 4°C for 24 h.A study approval from an ethics committee was not required, since working with avian embryos is currently not regulated by legislation as animal experimentation in Germany  The notification shall indicate:the purpose of the planned experiment;the species and, in the case of vertebrates, also the number of animals to be used for the planned experiment;the type of animal experiments planned and the procedures to be used, including anaesthetization;the place, beginning and likely duration of the planned experiment;the name, address and expertise of the head of the experiment in charge, of his deputy and of the person performing the experiment as well as of the eligible persons for after-treatment;in the case of planned experiments pursuant to Article 8, paragraph 7 (1) the legal basis for exemption from authorization.”http://www.animallaw.info/nonus/statutes/stdeawa1998.htmThe viral genome segments encoding hemagglutinin (HA), polymerase basic protein 2 (PB2) and nonstructural protein (NS) of all three tested LPAIV were analyzed for mutations after three consecutive passages in EF and TOC of all four bird species. Based on the obtained results, Ch/H9N2 was selected as the most adaptive virus and all other AIV genome segments, namely polymerase basic protein 1 (PB1), polymerase acidic protein (PA), nucleoprotein (NP), neuraminidase (NA) and matrix protein (M1/M2) were sequenced after the third passage in TOC of either of the different species. Additionally, the PB1, PA and NA genome segments of Tu/H6N8 and Du/H7N7 were sequenced after three consecutive passages in TOC-Ch. The viral sequences obtained after the third passage were compared to those of the virus stocks to detect nucleotide and amino acid changes. The H3 numbering system Briefly, total RNA was extracted from EF and TOC culture supernatants and allantoic fluids of the virus stocks by QIAamp Viral RNA Mini Kit . RT-PCR amplification was carried out with universal primers described by Hoffmann et al. Mutations observed in the HA and PB2 of Ch/H9N2 were confirmed also in plaque purified virus preparations of the supernatant of the third passage in TOC-Tu. Briefly, after incubation of MDCK cells with 50 PFU virus for 1 h, cells were overlayed with DMEM (Biochrom) supplemented with 0.9% Biozym Plaque Agarose , 0.2% BSA and 1 µg/ml acetylated trypsin. Cells were incubated at 37.5°C and 5% CO2 for 72 h. Ten individual plaques were picked, each propagated in MDCK cell monolayers for 24 h, and subsequently investigated for mutations in the HA and PB2 as described above.4 PFU of the virus per individual culture using PBS-BSA as dilution medium. Virus-negative controls were incubated with PBS-BSA instead of the virus. After 1 h of incubation, TOC and EF were washed with PBS and subsequently cultured in 1 ml basal maintenance media used for cultivation of corresponding cultures including 0.2% BSA. 1 µg/ml acetylated trypsin was added to EF cultures. In the first passage, TOC and EF cultures were inoculated with virus stocks from embryonated chicken eggs. In the consecutive passages, cultures were inoculated with titrated culture supernatants, which had been collected in the previous passage 24 h postinfection (hpi). At eight, 24 and 48 hpi, supernatants of triplicate EF cultures and four randomly selected TOC were collected and pooled for each virus. Each virus pool was titrated in a plaque assay as described above. Cytopathic effects (CPE) were assessed in EF monolayers (n = 3). TOC were analyzed under the microscope for ciliostasis at the indicated time points for percentage of remaining ciliary activity as described previously Three consecutive passages of LPAIV Ch/H9N2, Tu/H6N8 and Du/H7N7 were made under identical conditions in TOC and EF cultures of chicken, turkey, Pekin duck and homing pigeon. Multicyclic replication kinetics was studied by inoculating EF cultures with 0.01 PFU of the virus per cell; TOC were inoculated with 10Immunohistochemistry was performed to detect influenza virus nucleoprotein (NP) antigen in TOC. Briefly, TOC cryosections (5 µm) were fixed in ice-cold acetone and blocked against endogenous peroxidase with 0.3% hydrogen peroxide in methanol. Normal horse serum and the Avidin/Biotin Blocking kit  were used to block nonspecific staining. Sections were incubated with mouse anti influenza A nucleoprotein antibody  diluted 1∶1000 in PBS followed by the Vectastain Elite ABC kit  according to the manufacturer's instructions. Peroxidase activity was developed using the DAB Peroxidase Substrate kit (Vector Laboratories). Sections were counterstained with hematoxylin and mounted with Aquatex .in situ cell death detection kit POD  according to manufacturer's instructions. Peroxidase activity was developed using the DAB Peroxidase Substrate Kit (Vector Laboratories) according to manufacturer's instructions. TOC sections were counterstained with hematoxylin and mounted with Aquatex. The presence of apoptotic cells was evaluated microscopically in the respiratory epithelium of Ch/H9N2, Tu/H6N8 and Du/H7N7 infected TOC-Ch at 8, 24 and 48 hpi and scored semiquantitatively .Detection of apoptotic cells was performed in TOC cryocections with the TUNEL  assay using the P value of <0.05.Statistically significant differences between viral titers were evaluated with the Randomized Complete Block Analysis of Variance (ANOVA) and Tukey HSD using Statistix 9.0 . Randomized Complete Block ANOVA allows the comparison of different growth curves using the data of multiple timepoints. Significant differences in induction of ciliostasis were assessed with the Kruskal-Wallis test and Wilcoxon Rank sum test (Statistix 9.0). Differences were considered significant at a The nucleotide sequences of the LPAIV obtained in this study can be found under GenBank accession numbers CY081259, CY081261, CY081263, CY081264, CY120060, CY081266, CY120061, CY081268 (Ch/H9N2); CY081270, CY081272, CY081273, CY081275, CY081277, CY081279 (Du/H7N7); and CY081280, CY081282, CY081284, CY081286, CY081288, CY081290 (Tu/H6N8).


SVlm21 is a mutant of Sindbis virus which was isolated by serial passage of virus in mosquito cells maintained in low-methionine medium; it therefore has a low-methionine-resistant (LMR) phenotype. This phenotype requires mutations at nts 319 and 321; these mutations result in Arg to Leu and Ser to Cys changes at positions 87 and 88 respectively in the viral methyl transferase, nsP1. To better understand the genesis of SVlm21, we carried out serial passages of viruses having only one of these amino acid changes, but in mosquito cells maintained in normal methionine-medium. Whether the passage was begun with SV319 or with SV321, the dominant virus population which emerged always acquired the second SVlm21 amino acid change. However, when the passage was begun with virus having neither the nt 319 or the nt321 mutation, even after many passages neither of these mutations was seen in the passaged virus population. Virus with the LMR phenotype emerged earlier when the virus encoded a wild-type RDRP (passage 4) rather than the mutant RDRP encoded by SVpzf (passage 7). When the methionine concentration in the medium of mosquito cells was increased to 250 µM, more than 20 passages were required until the LMR phenotype predominated. Competition experiments were carried out to compare the relative fitness of SVlm21, SVwt, SV319 and SV321 to each other. Our results indicated that SVlm21 was dominant to SVwt, as well as to both SV319 and SV321. However, SV319 and SV321 were able to co-exist with SVwt implying that in these mixed infection the presence of SVwt inhibited the emergence of SVlm21. Finally, our experiments highlight how a virus population by mutation and selection can adapt to the intracellular concentration of a simple metabolite, S-adenosylmethionine. Changes in the properties of a virus can enhance its virulence, lead to resistance to antiviral therapy, and necessitate the modification of vaccines. Like all other replicating biological entities, viruses change and evolve by random mutation followed by selection. Indeed they provide excellent model systems for the study of these processes 1].−5 to as high as 10−3 substitutions per nucleotide copied 2]. Thus, it may be expected that every time the genome of a virus such as Sindbis virus (SV)  (GenBank Accession number: NC_001547) is copied there would be on the average one mutation somewhere in the genome.RNA viruses have, relative to DNA viruses, a high mutation rate, estimated to range from 10The high mutation rate of RNA viruses has led to the concept of “quasispecies” in describing populations of RNA viruses 2,3]. This concept implies that in any given population of an RNA virus, there is a dominant viral genotype (and phenotype) and a swarm of mutant genotypes each present in much lower numbers. Which viral genotype becomes and remains dominant in a population depends on the selective pressures exerted at any given time on the viral population. Given the high mutation rate of RNA viruses, it is likely that in any large viral population there are a small number of viral particles with a mutation at any given site in the genome. Some of these mutations would, of course, be lethal.In the work described below, we made use of two Sindbis virus mutants isolated in our laboratory. SVlm21, is a mutant which was isolated by serial passage of SV in mosquito cells maintained in media with decreasing levels of methionine, and which has a low methionine-resistant (LMR) phenotype, i.e. it is able to replicate in mosquito cells maintained in a methionine-deficient medium 4]. Our explanation for the phenotype of SVlm21 is as follows: methionine deprivation results in a decrease in the level of S-adenosylmethionine (AdoMet); since AdoMet is a substrate for the Sindbis virus GTP methyltransferase (nsP1) 5,6], and is required for formation of the 5′ methylated G cap structures on the viral genomic and subgenomic RNAs, therefore in methionine-deprived mosquito cells, the 5′ termini of these RNAs are not capped and methylated and virus replication is inhibited. SVlm21, by making an altered GTP methyltransferase with a lower Km for AdoMet, is able to grow mosquito cells with low levels of AdoMet 7].Two mutations are required for the LMR phenotype of SVlm21: G319U and A321U . These mSVpzf, is a mutant of Sindbis virus we selected in mosquito cells on the basis of its resistance to pyrazofurin (PZF), a nucleoside analog which blocks an early step in the pyrimidine biosynthetic pathway, and thereby lowers the levels of UTP and CTP in treated cells 8]. Three mutations, all in the region encoding nsP4, the viral RNA-dependent RNA polymerase (RDRP), are required for the resistance of SVpzf to PZF, and thus for the ability to replicate in cells with low levels of UTP/CTP 8]. Since all three SVpzf mutations are in the sequence encoding nsP4, , and the resultant amino acid changes likely affect the affinity of nsP4 for at least two of its substrates, UTP and CTP, it is probable that these mutations affect the fidelity of viral RNA synthesis by SVpzf.If we consider a Sindbis virus population as a quasispecies, it is expected that in a population of wild type virus there would be a small number of viruses with the SVlm21 mutation at nt 319 (SV319) and a small number with the SVlm21 mutation at nt 321(SV321). Under the strong selection pressure exerted by serial passage in mosquito cells maintained in a low methionine (LM) medium, it is not surprising that a dominant virus population eventually emerged with both of the SVlm21 mutations and thus the ability to replicate in methionine-deprived cells 6].To further clarify the genesis of SVlm21, we wished to know what kind of virus population would result if we began a serial passage in mosquito cells of virus having just one of the SVlm21mutations(SV319 or SV321), but maintained the cells in normal methionine (NM) medium instead of LM medium, thus supposedly eliminating the selective pressure favoring SVlm21.There were three possibilities. 1) The virus with only one of the SVlm21 mutations might continue as the major virus population; 2) the mutation at nt 319 or nt 321 might be lost so that the virus population would revert to the wild type; or 3) the virus might acquire the second SVlm21 mutation and the LMR phenotype. Our results indicate that the passaged virus inevitably acquired the second of the SVlm21 mutations. Furthermore, we observed that if the virus encoded the SVpzf form of nsP4 the emergence of the population with the LMR phenotype occurred several passages later than was the case with virus encoding the wt form of nsP4. We also carried out competition experiments maintaining infected cells in NM medium in order to compare the fitness of SVwt, SV319, SV321, and SVlm21 relative to each other. Our findings indicate that under these conditions SVlm21 out-competed both SVwt as well as SV319, and SV321.Aedes albopictus mosquito cells (C7–10) were grown at 28 degrees with 5% CO2 in E medium , containing 5% fetal bovine serum 9] . BSC40 cells  were grown at 37 degrees with 5% CO2 in Eagle’s MEM supplemented with 10% fetal bovine serum.Aedes albopictus mosquito cells, and after forty-eight hours at 34 degrees , virus was harvested. This virus was passage 0. The six passage 0 viruses were titered by plaque assay, adjusted to similar titers, and serially passaged in mosquito cells (35 mm plates) without dilution twelve times; virus was harvested at 24 hours following each passage. For each passage, mosquito cell cultures were infected with 0.5 ml of medium from the previous passage. The mosquito cells were maintained at 34 degrees after infection in 2 ml of E medium (but with 0.2% BSA in place of serum) containing a normal concentration of methionine (100 µM). Virus harvested at each passage was assayed for the ability to form plaques on a monolayer of mosquito cells maintained under an agarose overlay containing 100 µM methionine (NM), and under an overlay containing only 5 µM methionine (LM).Beginning with the plasmid pToto, an infectious clone of Sindbis virus 11], five derivatives of pToto were generated giving a total of six different versions of pToto: 1) no mutations in nsP1 and wt nsP4, 2) no mutations in nsP1 and SV pzf nsP4, 3) nt 319 mutation in nsP1 and wt nsP4, 4) nt 319 mutation in nsp1 and SVpzf nsP4, 5) nt 321 mutation in nsP1, and wt nsP4, and 6) nt 321 mutation in nsP1 and SVpzf nsP4. Each of these plasmids was linearized by digestion with Xho I and incubated with SP6 RNA polymerase 11]. The resulting transcripts were transfected into The initiating viruses for these passage series were derived from the infectious clone, pToto-luc 12] ; derivatives of pToto-luc were prepared with and without the SVpzf mutations in nsP4, and with the nsP1 mutations at nt 319 or nt 321 as indicated. Viruses were passaged as described above. To test for the LMR phenotype, mosquito cells in 24 well plates were infected with virus from each passage and maintained in NM medium or LM medium for 24 hr. Cells were lysed in 30 µl of Reporter Lysis Buffer. Fifty µl of LAR (Luciferase Assay Reagent) was added to 10 µl of cell lysate and assayed for luciferase activity using the luciferase assay system according to the manufacturer’s instructions (Promega).5′-GAATCAAACAGCCGACC; Reverse primer, W4: 5′-CTTTGGTTGCAATGCCAG) were used for RT-PCR to amplify the nsP1 coding region using the SuperScript III One-Step RT-PCR kit from Invitrogen. The cDNA was sequenced by Macrogen Sequencing Service Center using the primer SV25.Twenty hours after infection total RNA was extracted from infected cells using Trizol reagent (Invitrogen). One µg of total RNA and primers specific to the Sindbis virus genome  14]. The fluorescent intensity was read by a FLx 800 Fluorescence Microplate Reader (BioTek).Six different versions of Sindbis virus were prepared and passaged as described under 9 pfu/ml. The titers were similar in all six passage series. In contrast, the titers of LMR virus varied not only with the passage number, but also with the genotype of the starting virus. When the starting virus encoded wt nsP4, and had the nt 319 mutation, the LMR phenotype began to emerge after four passages, and by the fifth passage the titers under the NM and LM overlays were equal, indicating that the dominant virus population had the LMR phenotype. Similar findings were observed when the starting virus encoded the wt nsP4, but had the SVlm21 mutation at nt 321.When assayed under an NM overlay , the virus titers in the six different passage series gradually rose in the first four passages, and then from passages five to twelve exceeded 10When the starting virus encoded the SVpzf form of nsP4, and had the SVlm21 mutation at either nt 319 or nt 321, a similar phenomenon was noted, but the LMR phenotype did not begin to emerge until passage 7 or 8, and virus with this phenotype did not become the dominant population until passage ten or eleven. When the starting virus had neither the nt 319 nor the nt 321 mutation, there was no emergence of virus with the LMR phenotype even after twelve passages. This was independent of whether the virus encoded the wt or the SVpzf nsP4. The emergence of the LMR virus during the passage series is depicted in These passages series were repeated three times, each time beginning with passage 0 virus; similar results were obtained each time, even to the passage numbers at which the LMR virus emerged.Sequencing of the viral RNA from infected cells indicated that the appearance of the LMR phenotype correlated with the appearance of the second SVlm21 mutation in nsP1. Thus when the starting virus had the SVlm21 mutation either at nt 319 or nt 321 and encoded the wt nsP4, the second mutation appeared first in infected cells producing passage 4 virus. However, when the starting virus had only one of the two SVlm21 mutations, but encoded the SVpzf nsP4, the second mutation was not seen until the cells were making the passage 7 virus, or three passages later than when the virus contained the wt nsP4. When virus with neither the nt 319 nor the nt 321 mutation was passaged, no mutation was seen during the twelve passages at either of these two sites. This was so whether the virus contained the wt or the SVpzf nsP4. The nt sequences were read from about position 70–75 for about 800–900 nts. Apart from the mutations at nt 319 and nt 321 no other mutations were seen. Thus there was no evidence of a general hypermutability, at least in this region of the viral genome.To confirm these findings we repeated the experiment just described , but used a different method to assay for the LMR phenotype. This was done by using viruses derived from the different versions of pToto already described, but with a luciferase gene inserted into the nsP3 coding sequence 12]. To detect the LMR phenotype with these viruses, virus from each passage was used to infect mosquito cells which were then maintained in LM medium or in NM medium. Twenty four hours later, the mosquito cells were lysed and luciferase activity measured. Virus with the LMR phenotype should grow equally well in cells maintained in LM medium and NM medium, and thus should generate roughly equal levels of luciferase activity in cells kept in the two types of media. Virus lacking the LMR phenotype should generate much less luciferase when infected cells are maintained in LM medium than when cells are maintained in NM medium.The results of these and the preceding experiments showed that under the conditions used, the presence of one of the two SVlm21 mutations strongly favored the emergence of the second SVlm21 mutation in the virus population, even without the selective pressure of LM medium, and that when virus encoded the wt nsP4, the second mutation emerged more quickly in the passage series than when the virus encoded the SVpzf form of nsP4.In the experiments just carried out, the virus with the nt 319 mutation had a CUU codon which encoded Leu 87 . We nextWe next asked whether the LMR phenotype would be generated so readily if two nt changes were needed to encode one of the amino acids required for the SVlm21 phenotype. Thus, a serial passage was begun as before, with virus derived from a version of pToto that encoded an Arg at position 87 of nsP1 (as in SVwt), and a Cys at position 88 (as in SVlm21); but in this case, the codon for Arg 87 was AGA, not CGU as in the wt virus (the Cys codon was UGU). This meant that two nt changes would be needed to change the Arg 87 codon to a Leu codon and give rise to the LMR phenotype.Another very similar experiment was carried out; however, this time the virus to be passaged had a CUU codon for Leu 87 as in SVlm21, but a UCG codon for Ser 88, instead of an AGU codon as in SVwt. To generate the LMR phenotype Ser 88 would have to be changed to a Cys residue, which would require two nt changes. Viruses having the UCG or the AGU codon for Ser 88 were passaged in mosquito cells maintained in NM medium, along with control viruses having the wt codons for both amino acids 87 and 88. The appearance of the LMR phenotype was monitored as above using the luciferase assay.The results (not shown) were very similar to those shown in Sequencing of the viral RNA made by the virus which originally had the UCG codon for Ser 88, but had acquired the LMR phenotype, revealed that the codon for Cys 88 was now UGC, not UGU as in SVlm21. This result showed that the two nt changes needed for the Ser to Cys change at position 88 of nsP1 had indeed occurred, and confirmed that even when two nt changes were needed to generate the LMR phenotype, this phenotype occurred as rapidly in the passage series as when only one nt change was required.The preceding experiments were done with the assumption that NM medium (100 µM methionine) represented a non-selective medium. However, the consistent emergence of the LMR phenotype when the passage series were initiated with virus containing one of the SVlm21 mutations and carried out in mosquito cells maintained after infection in NM medium, led us to question this assumption. Perhaps if infected cells were maintained in medium with a higher level of methionine, presumably leading to higher levels of intracellular AdoMet, there would be much less selective pressure favoring the emergence of LMR virus which as noted above encodes a GTP methyltransferase with a higher affinity for AdoMet.Accordingly, passage series were initiated as before with viruses containing the luciferase gene, but this time infected mosquito cells were maintained in a high-methionine (HM) medium containing 250 µM methionine for each of ten passages. In contrast to what was observed in the earlier experiments, virus with the LMR phenotype did not become the dominant population in any of the passage series. However, when the passage series was extended, LMR virus did eventually emerge, but as before only in the passages initiated with virus having either the nt 319 or the nt 321 mutation. Once again, the LMR virus appeared earlier when virus encoded the wt nsP4 rather than the SVpzf nsP4. Thus with the viruses encoding wt nsP4, the LMR phenotype was seen at passage 21, whereas with virus encoding the mutant nsP4, not until passage 24. When the passages were initiated with virus having neither the nt 319 nor the nt 321 mutation, the LMR phenotype was not seen even after 28 passages.When we first described SVlm21 4] we observed that although the replication of Sindbis virus in mosquito cells was sensitive to methionine-deprivation, such was not the case for replication in vertebrate cells. This might be explained if the AdoMet synthetase (the substrates for which are methionine and ATP) of mammalian cells were more efficient in maintaining adequate levels of AdoMet even in the face of a low intracellular level of methionine. If such were the case, there would be little selective advantage for the LMR phenotype in virus passaged in mammalian cells maintained after infection in LM medium, let alone NM medium.To determine whether the LMR virus would emerge in mammalian cells a passage series was carried out with the six viruses as already described for To compare the levels of AdoMet in C7–10 cells and BSC40 cells, we measured the concentrations of this metabolite in both cell types maintained in medium containing different concentrations of methionine. As shown in To shed more light on the emergence of virus with the LMR phenotype, we wished to determine how the various viruses we have been studying competed with each other. The preceding experiments showed that when passaged in mosquito cells maintained in NM medium, the SVlm21 virus always out-competed both the virus with only the nt 319 mutation and the virus with only the nt 321 mutation. However, it was also important to know which virus would prove the more fit during serial passages when we co-infected cells with 1) wild type virus and SVlm21, 2) wild type virus and virus with only the nt 319 mutation, or 3) wild type virus and virus with only the nt 321 mutation. In all these experiments cells were maintained in NM medium.These experiments were set up by co-infecting cells, with two different viruses at an MOI of one, serially passaging the viruses and then assessing the outcomes by determining the nts at positions 319 and 321.A similar result was seen when cells were co-infected with virus having only the nt 319 mutation and wt virus. A different situation was seen when cells were co-infected with wt virus and SVlm21. Most notable among our findings is that even in cells maintained in NM medium, the serial passage in mosquito cells of virus having either of the two SVlm21 mutations led to the acquisition of the second of the SVlm21 mutations and thus the LMR phenotype. This would not be surprising when virus was passaged in cells in an LM medium where there would be strong selective pressure for this phenotype. However, it was surprising that this also occurred when the serial passage was done in cells in NM medium or even in HM medium, although in the latter case the appearance of the second mutation and the LMR phenotype was much delayed.The observation that an increased methionine concentration in the medium and the resulting increase in the intracellular concentration of AdoMet delayed the appearance of virus with the LMR phenotype is consistent with the idea that these conditions reduce the selective pressure favoring that phenotype. Conversely, reducing the AdoMet in the infected cells would increase the selective pressure favoring the LMR phenotype as was illustrated by our initial selection of SVlm21 4].Given that the various Sindbis virus populations represent in each case a quasispecies, then even when a passage is begun with virus having only one of the SVlm21 mutations, there may already be in each stock very small numbers of virions with both of the SVlm21 mutations. What we were assessing in our experiments, however, were the properties of the dominant virus populations. The point at which a dominant virus population emerges that has both of the SVlm21 mutations is clearly dependent on the selective pressures exerted, the most important of which must surely be the concentration of AdoMet in the infected cells.. To compare the relative fitness of the other viruses, we carried out competition experiments as described above. , the presence of SVwt along with SV319 or SV321 appeared to inhibit the emergence of SVlm21 as the dominant virus population. Why this is so is not known. The results of the competition experiments are also consistent with the observations that when a passage was begun with either SV319 or SV321, the result was always a virus population that had acquired the second SVlm21 mutation rather than a population that had lost the first SVlm21 mutation.In considering the factors influencing the emergence of virus with the LMR phenotype, it was important to determine the fitness of SVwt, SVlm21, SV319, and SV321 relative to each other. The results shown in In carrying out these experiments we also wished to see whether the SVpzf mutations in nsP4 would influence the emergence of a virus population with the second SVlm21 mutation. We found consistently that in every passage series, virus with the SVpzf form of nsP4 acquired the LMR phenotype several passages later than did virus with the wt form of nsP4. By itself this does not permit the conclusion that the SVpzf RDRP has a higher fidelity, i.e. is less mutagenic than the SVwt RDRP, but it does recall the observations of Pfeiffer and Kirkegaard 15] who reported that a poliovirus mutant resistant to the nucleoside analog, ribavirin, was slower to develop resistance to guanidinium than was wild type virus. Similarily, Coffey et al. 16] observed that a single mutation in the Chikungunya virus nsP4, that was associated with resistance to ribavirin and 5-fluorouracil, increased replication fidelity and reduced genetic diversity.E. coli cultured for many thousands of generations, in which the occurrence of a specific mutation making the organism able to utilize citrate appeared to be dependent on a prior mutation(s) in that population. They referred to this phenomenon as “historical contingency”.Our findings 1) that when a passage was begun with virus that had neither of the SVlm21 mutations, neither of these mutations appeared even after 12 passages and 2) that if a passage was begun with virus that had one of the SVlm21 mutations, then inevitably the second mutation appeared during the passage, calls to mind the paper by Blount et al., 17] describing work with Although much work has been done describing mutations in viral genomes that relate to resistance to antiviral drugs, little has been reported concerning how particular viruses, by mutation or selection, have adapted to the concentrations of intracellular metabolites so as to replicate optimally in the host cells that they infect. This is an especially interesting question for arthropod-borne viruses which must replicate in two different host types that are widely separated phylogenetically, and have significant differences in their biochemistry and metabolism. It is likely that the recently developing field of metabolomics will have much to teach us in this regard. For example, global profiling of changes in metabolites in Sindbis virus- infected vertebrate and insect cells will indicate how virus infection influences the metabolic pathways in both cell types and identification of key metabolites in infected cells will be useful for biomarker discovery 18] 19].


All viruses need to bind to specific receptor molecules on the surface of target cells to initiate infection. Virus–receptor binding is highly specific, and this specificity determines both the species and the cell type that can be infected by a given virus. In some well-studied cases, the virus-binding region on the receptor has been found to be unrelated to the receptor's normal cellular function. Resistance to virus infection can thus evolve by selection of mutations that alter amino acids in the binding region with minimal effect on normal function. This sort of positive selection can be used to infer the history of the host–virus “arms race” during their coevolution. In a new study, Demogines et al. use a combination of phylogenetic, structural, and virological analysis to infer the history and significance of positive selection on the transferrin receptor TfR1, a housekeeping protein required for iron uptake and the cell surface receptor for at least three different types of virus. The authors show that only two parts of the rodent TfR1 molecule have been subject to positive selection and that these correspond to the binding sites for two of these viruses—the mouse mammary tumor virus (a retrovirus) and Machupo virus (an arenavirus). They confirmed this result by introducing the inferred binding site mutations into the wild-type protein and testing for receptor function. Related arenaviruses are beginning to spread in human populations in South America as the cause of often fatal hemorrhagic fevers, and, although Demogines et al. could find no evidence of TfR1 mutations in this region that might have been selected as a consequence of human infection, the authors identified one such mutation in Asian populations that affects infection with these viruses. Recent research on a number of fronts is making clear the remarkable extent to which interactions with infectious agents have shaped the evolution of their hosts. In particular, the survival, replication, and spread of viruses depend on interaction with many normal cell components, sometimes referred to as “host dependency factors.” As a rule, these factors are proteins that the cell requires for some normal function, but that have been co-opted by a virus for the same or another function. A large number of such factors have recently been identified by screening small interfering RNA (siRNA) libraries for effects on HIV replication A characteristic of restriction factor genes—unlike genes encoding housekeeping functions, which usually evolve under purifying selection—is that they often exhibit periods of positive selection in their evolutionary history One might imagine that such positive selection could also play out in the opposite direction in dependency factors, but, with the exceptions discussed below, there are few examples. One reason for the absence of an obvious arms race involving such factors may be that the virus interacts with them in the same way that normal cell components do, so there is no room for change without disrupting normal function. Factors involved in intracellular transport of viral components, gene expression, nucleic acid replication, and other functions may exhibit low dN/dS ratios for this reason. In addition, factors that provide indirect support for viral replication, such as enzymes of nucleotide metabolism, could also be expected to fall into this category.PLOS BiologyAn exception to the apparent lack of positive selection in dependency factors is the interaction with viruses and their cellular receptors, in which signs of an evolutionary arms race have been apparent from classical genetic studies for many years, and for which a paper published in this issue of The virus–receptor interaction is exquisitely specific, and a single amino acid change in the receptor can completely abrogate this activity. Thus, the presence of a specific receptor is often a critical determinant in the species specificity of virus infection, and the cell specificity of receptor expression can be a decisive factor in the disease specificity of a virus. For example, HIV uses the CD4 protein as a receptor. CD4 is a key molecule in immune signaling and is expressed on, among others, helper T cells in all mammals. AIDS is a result of a slow loss of these cells due to the effects of virus infection. Despite the widespread distribution of CD4 in mammals, HIV-1 can use only the homolog found in primates for entry.Tvb, comes in at least four flavors, encoding resistance or sensitivity to subgroups B, D, and E in various combinations Tvb alleles, and then by evolution of viruses capable of using either the resistant forms of Tvb or another protein as receptor. A similar sort of evolutionary back and forth is also apparent in the endogenous murine leukemia viruses In well-studied cases, such as HIV-1, the binding site used by a virus is in a different portion of the receptor molecule than the site of ligand binding, and mutations in the virus-binding site are likely to be of little consequence to the normal host function. These properties can set the stage for the same sort of arms race as occurs with restriction factors. Indeed, the first host genetic elements found to affect retrovirus infection were receptor polymorphisms in chickens that prevent infection with avian leukosis viruses (ALVs) Mus musculus). Remarkably, only six residues exhibited ratios significantly greater than 1, and these mapped exactly to the MMTV and Machupo binding sites previously determined (M. musculus, and has not been described in any other rodent. Based on the evidence of positive selection for its binding sites, the authors speculate that it may once have had a much larger host range, but has become extinct in most lineages, perhaps by being unable to keep up with the resistant mutations in TfR1. Consistent with this idea, Demogines et al. identified closely related, but noninfectious, endogenous MMTV-like proviruses in a number of related rodent species, likely fossil remnants of the now extinct viruses.Demogines et al. termined . FurtherThe paper concludes with an intriguing observation and an interesting evolutionary puzzle. Although most South American arenavirus infections occur in rodents, several such viruses are spreading into humans in the same region Given the evident importance of “new” viral diseases as they emerge into the human population, multifaceted approaches such as the one used in this paper promise to provide a valuable tool kit for understanding and preparing for their appearance.


We report West Nile virus (WNV) activity in the Dominican Republic for the first time. Specific anti-WNV antibodies were detected in 5 (15%) of 33 resident birds sampled at one location in November 2002. One seropositive bird was <4 months old, indicating a recent infection. Flaviviridae, genus Flavivirus) in the Western Hemisphere took place in New York in 1999, with deaths observed in humans, horses, and numerous species of wild birds  according to standard methods  with the WNV-specific monoclonal antibody 3.1112G, which discriminates between WNV and SLEV infections in birds  that was <4 months old, suggesting that virus transmission was recent. Virus was not isolated from any of the tissues tested from 118 resident and 29 migratory birds , nor did we detect WNV RNA in any of the tissue homogenates from flavivirus-seropositive birds.Serum samples from nine resident birds tested positive for flavivirus-neutralizing antibodies . Of thesOur finding of WNV-neutralizing antibodies in five resident birds represents the first evidence of WNV activity in the Dominican Republic. No cases of WNV infection in humans, horses, or birds were known at the time of sampling. The birds in this study could have been infected with WNV in the Dominican Republic as recently as early November 2002; nonetheless, the virus probably arrived earlier in the Caribbean region. Because no current infections were detected, our results reflect past virus transmission activity. Although we cannot determine when this activity began, the seropositive immature quail-dove presumably was infected after mid-July 2002, when it was born, and before early November 2002, in order to have stimulated detectable antibody production by mid-November.The earliest evidence of WNV transmission in the West Indies is a human case from the Cayman Islands in 2001  at Los Haitises were found to be seropositive, the results suggest that transmission of WNV among bird populations at that site was widespread. For comparison, seroprevalence of WNV-neutralizing antibodies in resident birds was 50% (CI 44% to 57%) in Queens, New York City, after the 1999 outbreak (The evidence for local WNV transmission in the Dominican Republic indicates risk for West Nile fever and meningoencephalitis in the human, equine, and avian populations of Hispaniola. We suggest that WNV be considered in the differential diagnosis of humans and other vertebrates with central nervous system disease in Hispaniola.


Little is known about the costs of devices explanted during anterior cervical diskectomy and fusion surgery. This retrospective study analyzes the costs to a single hospital of plates, screws and spacers used in all single-level anterior diskectomy and fusion (single-ADF) operations performed during a 1-year period.Our objective was to determine the costs of instrumentation explanted (i.e. implanted during surgery but removed prior to closure) during 87 single-ADF procedures performed at a single institution within a single year, 2009. All 87 single-ADF procedures were analyzed to determine the frequency and costs (without overhead) to the hospital for both permanently implanted and explanted anterior cervical screws, plates, and spacers . All patients undergoing single-ADF were included in this study irrespective of the diagnosis related group (DRG) category.The costs, without overhead to the hospital, for the permanently implanted instrumentation were: screws ; plates ; allograft spacers ; cages ; and autografts , for a total of $355,863. The additional costs to the hospital for explanted instrumentation were: 37 screws ; 7 plates ; and 8 allograft spacers ($9093: 7 patients); there were no explanted cages. The total cost of the explanted devices was $32,850.During 87 single-ADF procedures, a total of 37 screws, 7 plates, and 8 spacers were explanted in 24 (27.6%) patients, resulting in an additional $32,850, 9.2%, to the cost of the implanted devices. The multiple plate/screw systems and bone spacers which have been developed for anterior cervical surgery can be combined resulting in a large number of choices. Although select studies have evaluated the costs of these combined implanted devices, little is known about the frequency and costs associated with explantation . The purpose here was to look more closely at the instrumentation used during 87 single-level anterior cervical diskectomy/fusion (single-ADF) operations performed in 2009 at a single institution. In particular, we evaluated the frequency and costs of implanted and explanted devices used to perform these 87 single-ADF procedures.During the calendar year 2009, 87 single-ADF procedures were performed at a single institution. Patients from all DRG categories undergoing single-ADF were included in this study.We evaluated the cost to the hospital, without overhead, for all instrumentation ) utilized in each case. In particular, we not only evaluated the costs of permanently implanted devices, but also calculated the incidence of, and costs associated with, explanted instrumentation. In all cases, the costs incurred by the hospital to the vendors were recorded; associated overhead costs were not included in this study.The total cost incurred by the hospital for permanently implanted plates, screws, and spacers utilized to perform the 87 single-ADF was $355,863. The total cost of the explanted instrumentation, including screws, plates, and spacers, was $32,850 or 9.2% of the cost of the implanted instrumentation. The total cost for seven plates, explanted in five cases, was $12,743 . A totalIndividual Surgeons and Explantation The 87 single-ADF operations were performed by 11 surgeons, with five of these surgeons performing four or fewer operations. The other six surgeons performed between 7 and 25 single-ADF, and accounted for 76 of the 87 operations. All six of these surgeons explanted devices, and explantation occurred in 12.5–46.2% of their cases.Devices were explanted in 24, or 27.6%, of the 87 single-ADF performed at one institution during 2009. Explantation was common among the surgeons performing the larger number of cases. For the six surgeons performing 76 of the cases (range 7–25 cases per surgeon), devices were explanted in between 12.5% and 46.2% of their cases. The cost, without overhead, to the hospital for all explanted instrumentation, in all 87 cases, came to $32,850 or 9.2% of the total cost of $355,863 of the permanently implanted instrumentation.What is an acceptable explantation rate? We were not able to find literature that addresses this important question. In the senior author’s experience, 5–10% of the cases might require explantation of instrumentation. If we assume a target of 10% frequency for explantation, as opposed to the 27.6% observed here, then it should be possible to at least reduce the costs associated with explantation by 50%.Because of the escalating costs for health care delivery, it is essential to identify opportunities for cost savings. In fact, the costs of explanted devices in single-ADF may underestimate the potential for savings associated with explantation in general. For example, much higher explantation costs might be anticipated for the more extensive cervical, thoracic, and lumbar spinal procedures.Given the range of costs associated with implanted devices, insurance carriers may soon seek to cap reimbursements to hospitals. Therefore, the benefits of costly implanted spinal devices must be weighed. Unfortunately, this study did not include outcome data. However, a few series of single-ADF have focused on cost-effectiveness. One study examined the relative costs for four treatment groups: the cage alone group was the least expensive, the disc arthroplasty and cage/plate/bone substitute groups showed comparable intermediate costs, while the autograft group was the most expensive; pain at the hip graft site for the latter group required a longer average 5 day) length of hospital stay (LOS). A second day leng2In summary, we found that explantation occurred in 27.6% of the 87 cases in this series. This added 9.2% to the cost of implanted devices in single-ADF procedures. Identification of these additional costs has prompted measures to examine and reduce costs associated with explantation in general at our institution.


To the Editor: Phleboviruses  are arthropod-borne, single-stranded, RNA viruses. Their genome consists of 3 segments—small, medium, and large—which encode the nucleoprotein and nonstructural proteins, the envelope glycoproteins, and the viral polymerase, respectively  and had vomited 1 time while in nursery school. He had sudden onset of eye gaze, perioral cyanosis, masseter muscle spasm, generalized tonic convulsions of the body and extremities, and involuntary loss of urine. The episode lasted ≈3 minutes, after which the child became irritable and sleepy.At the time of hospital admission (30 minutes later), he was afebrile and conscious. The boy’s history contained no previous neurologic or developmental disabilities and no family history of epilepsy or febrile seizures. Clinical examination, which included a thorough general and detailed neurologic evaluation, revealed no abnormalities except mild rhinitis. Laboratory tests showed leukocytosis  with 85.7% neutrophils. Blood levels of electrolytes, urea nitrogen, creatinine, glucose, albumin, bilirubin, alkaline phosphatase, and aminotransferases; prothrombin time; and urinalysis results were within reference limits. Electroencephalogram showed no brain abnormalities. Lumbar puncture and neuroimaging were not considered necessary.After 2 days of hospitalization, the child recovered, was free of signs and symptoms, and was discharged from the hospital with a diagnosis of simple febrile seizure and mild upper respiratory infection. According to a report from his mother 1 year later, the child remains well without any recurrence of febrile or other type of seizures.Phlebovirus and clusters with phleboviruses of the Salehabad serocomplex, such as Salehabad virus and Arbia virus, differing by 21.6% and 29.6% with Salehabad virus and Arbia virus at the nucleotide level and by 3% and 17.7% at the amino acid level, respectively (Viral RNA was extracted from the patient’s blood sample taken at the time of hospital admission. Nested reverse transcription–PCR using degenerate primers was applied to amplify a 222-bp fragment of the large RNA segment of phleboviruses . The retectively .Detection of the Adria virus sequence in the patient’s blood suggests that this virus is pathogenic to humans. As expected, serologic testing of the sample taken at the time of admission produced negative results for phleboviruses; a convalescent-phase blood sample was not available. Although the course of the disease in the child was mild, further studies will show the role of this strain in public health.Because the duration of viremia in persons with phlebovirus infections is short, use of molecular methods for the laboratory diagnosis of phleboviral infections is limited; and even when a phleboviral infection is confirmed by serologic testing, the exact strain is difficult to determine. Physicians in Greece, as in other Mediterranean countries, should be aware of the circulation of phleboviruses and potential risk for phlebovirus-associated infections during the summer. Such infections, especially with neurologic signs, should be included in the differential diagnosis of summer febrile syndromes.


Drosophila melanogaster is an ideal candidate for providing a specific and acquired response to subsequent infection. We exposed D. melanogaster to two challenges of a virus known to produce an antiviral RNAi response, to examine whether any protective effects of prior exposure on survival were observed.Immune priming has been shown to occur in a wide array of invertebrate taxa, with individuals exposed to a pathogen showing increased protection upon subsequent exposure. However, the mechanisms underlying immune priming are poorly understood. The antiviral RNAi response in In this experiment we found no evidence that prior exposure to Drosophila C Virus (DCV) protects flies from a subsequent lethal challenge, with almost identical levels of mortality in flies previously exposed to DCV or a control.Our results confirm the finding that ‘acquired’ immune responses are not ubiquitous across all invertebrate-pathogen interactions. We discuss why we may have observed no effect in this study, with focus on the mechanistic basis of the RNAi pathway. Over the past decade, a number of studies have demonstrated that invertebrates that have previously encountered a pathogen/parasite appear to be protected upon secondary exposure, which has been termed “immune priming” Drosophila primed with Streptococcus pneumonia bacteria were protected against a secondary lethal challenge of homologous but not a taxonomically diverse range of other bacteria Tribolium castaneumDaphnia magnaMacrocyclops albidus infected with tapeworms Penaeus shrimp with previous exposure to white spot syndrome virus show increased survival on subsequent exposure to virus, with protection persisting for up to two months Plodia interpunctella exposed to a low dose of its natural DNA virus show increased survival on a subsequent challenge to a lethal dose, as do their offspring Dscam. By being alternatively spliced, Dscam may be able to produce sufficient receptor diversity to distinguish between different pathogen strains or species Despite the insect innate immune system recognising and killing invaders using receptors and effectors that target molecules conserved across a broad taxonomic range of pathogens, immune priming can sometimes be highly specific Drosophila from a number of positive and negative sense RNA viruses, as well as a DNA virus A promising candidate for acquired and specific immunity against viruses is RNAi Drosophila cells, persistent infections of RNA viruses result in viral RNA being reverse transcribed into chimeric DNA molecules containing retro-transposon and viral sequences Rather than the host always clearing a sub-lethal dose of a virus, acute viral infections can develop into persistent infections, where the virus can be present at a low level and cause little damage Drosophila melanogaster and its naturally occurring pathogen, Drosophila C Virus (DCV). DCV is a positive sense RNA virus in the family DicistroviridaeD. melanogaster and other Drosophila species in the wild Pastrel have been implicated in protecting flies against DCV To examine whether previous exposure to an insect RNA virus can protect against subsequent infection by the same pathogen, we carried out an experiment using Our approach was to expose flies to DCV in two challenges. The first challenge was a low dose that would replicate – and so produce an immune response – but cause minimal mortality. The second was a lethal dose of DCV to see whether the first challenge would protect flies. This is different to the approach in the past priming literature, where the primary infection is dead or rapidly cleared °C to remove any cellular components or bacteria. Virus was then aliquoted and frozen at −80°C. Uninfected cell culture for control solution was produced by growing DL2 cells as for virus production but DCV was replaced with Drosophila Ringer’s solution −1 to 10−12 were carried out in Schneider’s medium, and each dilution was added to 8 wells of a plate of DL2 cells. After 7 days the wells were examined and classed as “infected” when cell death and cytopathic effects were clearly visible. The Tissue Culture Infective Dose 50 (TCID50) was calculated by the Reed-Muench end-point method DCV was produced in Schneider Drosophila line 2 (DL2) cells 50 2.32×106) used was the maximum non-lethal dose, to try to ensure replication of the virus. The second challenge (TCID50 4.64×107) was a dose that killed ∼85% of flies in a pilot study . Virus extract or uninfected cell culture medium was diluted to the desired concentration using Drosophila Ringer’s solution To infect flies with DCV, a 0.0175 mm diameter anodized steel needle  was bent ∼0.25 mm from the end , dipped in DCV or control solution (uninfected cell culture medium), and the bent part of the needle pricked into the pleural suture on the thorax of flies. A pilot study was used to calculate a suitable sub-lethal dose of DCV . The priot study . The sec50 2.32×106) or control medium and then challenged again with the high virus dose (TCID50 4.64×107) or control medium, 3 days after the first challenge. Flies therefore fall into four treatments : control-control, control-virus, virus-virus, virus-control. The 3 day gap between the first and second challenge was designed to allow the first viral challenge to replicate and produce an immune response. Our aim was to produce an immune response that loitered and so conferred protection on subsequent exposure. DCV viral load has been shown to increase from day one post-infection, so we were confident the virus would be replicating and so producing dsRNA For the main experiment, 3–4 day old female flies were first challenged with the low virus dose . Flies were pricked on the opposite side of the thorax for the first and second challenge. As there is a systemic RNAi response in Drosophila, inoculating flies in a different location is not expected to alter RNAi-mediated protection The experiment was carried out over four days with all the treatments performed on each day and the treatment order randomised within each day. In total we challenged and measured the survival of 2310 flies. Challenged flies were kept in vials of cornmeal medium at 25w1118 isogenic line kindly provided by Luis Teixeira Wolbachia before the experiment by RT-PCR as in The flies used throughout the experiment were from the DrosDel 50 2.32×106), and were then either snap frozen immediately or three days later (7 replicates of 10 flies for each time point).To confirm that flies had not become infected with DCV through laboratory contamination during the experiment, and to see whether the first challenge had been cleared or persisted, viral titre was measured by quantitative reverse transcription (qRT-) PCR in flies from the control-control or virus-control treatment respectively on day 14 after the second challenge. Additionally, to confirm whether the first challenge dose replicated, 140 flies were stabbed with the first challenge dose (TCIDEF1α100E)) using a QuantiTect Virus qRT-PCR kit  and Taqman hydrolysis probes  on a BioRad iQ5 thermocycler. Reactions were carried out in duplex with probes using Cy5 (DCV) and FAM (Ef1α100E) reporter dyes, following manufactures instructions. Relative viral RNA copy number was then calculated relative to the endogenous control using the ΔCt  method, where the relative viral RNA load is given by 2Ct–Control Ct)−. RNA was then extracted using a chloroform-isopropanol extraction, as described in ith individual from vial j at time t was modelled as:The effect of treatment on survival rates was analysed using a Cox’s proportional hazards mixed effect model, which accounted for between-vial variation in survival rates. The hazard for the H0(t) is the baseline hazard at time t, iX is a vector of the fixed effects, β is the corresponding vector of coefficients, and jb is a random effect of vial j nested within day of the experiment. The fixed effects comprised treatment and day of infection. Flies alive at the end of the experiment were censored. The model was fitted by maximum likelihood using the coxme package t values for the qRT-PCR data on viral load were not normally distributed so were analysed using a Wilcoxon Rank Sum test.Where P = 0.73). By day 14, both control-virus and virus-virus treatments had 88% mortality. The initial low dose of virus itself caused some mortality, with 16% of the virus-control flies dead on day 14 compared to 4% of control-control flies (P<0.001).Exposing flies to a low dose of virus had no effect on the rate at which they die when subsequently infected with a larger lethal dose  from the control-control treatment contained no detectable viral RNA. This suggests no flies were accidentally contaminated with DCV during the experiment.W = 44, P = 0.01). By day 14 of the main experiment, 50% of virus-control treated vials contained detectable amounts of virus , suggesting the infection may have been persistent/maintained at low levels in some flies. In vials where DCV RNA was detectable at day 14, there was significantly greater mortality compared to vials where no DCV RNA was detected .The first challenge (low dose) of the virus replicated in almost all samples. Only two of seven samples (each sample consisted of 10 flies) contained detectable amounts of viral RNA immediately after infection, compared to six out of seven samples three days post-infection. There were significantly greater quantities of viral RNA in day three samples , and day3 = 2509.16 (SE = 1529.73), Wilcoxon rank sum test: We found that in this instance flies previously exposed to DCV were not protected against mortality on subsequent exposure. Given DCV is targeted by the RNAi pathway There are several possible explanations as to why primary exposure did not protect against mortality during secondary challenge in this experiment. Firstly, DCV is known to encode a viral suppressor of RNAi (VSR) that binds dsRNA and inhibits the production of siRNAs It may be the case that the immune response against the primary challenge does not persist for long enough to provide prophylaxis. Previous studies in other systems have shown priming can be long-lived D. melanogaster lacks two mechanisms found in other organisms that are important for generating a systemic RNAi response to viral infection  Immune priming is not a universal phenomenon in invertebrates, with previous studies finding variation in the degree of protection, with some pathogens not eliciting a response Immune priming has been demonstrated in a number of invertebrates. We hypothesised that the RNAi pathway could provide an acquired and sequence-specific antiviral immune response. Here, we have examined whether exposure of an insect to an RNA virus results in protection on subsequent exposure. We found no evidence that previous exposure to DCV protected flies from a subsequent lethal challenge in this experiment.Figure S1Dilution pilot survival data. Flies were stabbed with various doses of DCV (TCID50 of 4.64×109 to 4.64×104). Control flies were stabbed with uninfected cell culture medium. Each treatment consists of 3 vials of 20 flies with the exception of the 109 treatment where there were only 2 vials. Following this study, another pilot study was carried out using TCID50 of 2.32×106 and 9.28×105  to determine the maximum sub-lethal dose. With these doses there were no observable differences in control vs virus treatment at day 10 post-infection.(TIFF)Click here for additional data file.Table S1Primer and Probe sequences used for qRT-PCR.(DOCX)Click here for additional data file.Dataset S1Experimental mortality data, columns are: day of death, day of experiment, whether the primary and secondary challenge was control (C) or virus (V), whether the fly was censored , and the vial identity.(CSV)Click here for additional data file.


Mayaro virus is endemic in South America and sporadic outbreaks have been described. It causes a dengue-like febrile illness accompanied by severe and long-lasting polyarthralgias. Outside endemic regions, however, the disease is not well known and can be misdiagnosed as dengue. International travellers are at risk to acquire Mayaro virus and due to increased worldwide travel infectious disease specialists need to be aware of such rare clinical entities.We report the first Mayaro virus infection imported into Germany. A 20-year-old woman developed fever, myalgia, maculopapular rash, and polyarthralgias following a 10-day trip in the Rurrenabaque region of Bolivia. Severe polyarthralgias persisted for 5 months and were treated with non-steroidal anti-inflammatory drugs. Serological analysis demonstrated Mayaro virus-specific-IgM and -IgG antibodies two months after onset of symptoms. Except for CXCL8/IL-8 other proinflammatory chemokines and cytokines were unremarkable at this time.Dissemination of knowledge on rare disease might improve patient management. Understanding the inherent features of Mayaro virus infection and how the virus interacts with its host are essential for optimal patient care and therapy. Togaviridae and have a worldwide distribution. They can be associated with rheumatic disease in humans. The most important arthritogenic alphaviruses are Barmah Forest virus (BFV), chikungunya virus (CHIKV), Ross River virus (RRV), Sindbis virus (SINV), and Mayaro virus (MAYV). Occasionally, these arthritogenic alphaviruses can cause large and unpredictable outbreaks as seen in the 2004–2011 CHIKV epidemic in the Indian Ocean [Mosquito-borne alphaviruses belong to the family an Ocean . Of notean Ocean ,4. Diseaan Ocean . Here, wIn July 2012, a 20-year old woman presented to our outpatient clinic with symmetrical polyarthritis after a long-term travel to South America. At presentation, she complained about highly painful ankles and elbows, and she was barely able to walk without assistance. Between September 2011 and June 2012 she had travelled as a tourist through Peru, Bolivia, and Ecuador. In Mid-April 2012 she spent 10 days in the tropical rainforest of the Rurrenabaque region, which is situated in the Amazon basin about 400 km north of La Paz, Bolivia. There, she volunteered in a wildlife resort, taking care of a variety of monkeys and wild cats. One day after leaving the resort she developed spiking fevers, headache, myalgia followed by aphthous oral ulcers and a maculopapular rash of the whole body four days later. Around the same time she developed a diffuse arthralgia, which after initial improvement slowly worsened and became highly incapacitating. Her previous medical history was unremarkable. She did not take regular medication. Prior to her travel, she had received complete vaccinations for yellow fever, rabies, hepatitis A and B.On physical examination tenderness and mild swelling of both ankles was noted. Tenderness of the elbows, shoulders and interphalangeal joints and wrists without swelling was also observed.Borrelia burgdorferi, and Treponema pallidum were negative. Serology for Epstein-Barr virus and parvovirus B19 was indicative of past infection. A slightly raised dengue virus (DENV) IgG was interpreted as cross-reactivity after yellow fever vaccination while DENV IgM was negative. In addition, autoimmune serology including anti-citrullin peptide antibodies or anti-nuclear antibodies was negative.Results of a full blood count, liver and renal function tests were normal, as were levels of electrolytes, lactate dehydrogenase, and C-reactive protein. Blood cultures remained sterile. Blood smears for malaria were repeatedly negative. Serologic testing for cytomegalovirus, hepatitis C virus, human immunodeficiency virus, Due to her travel to the Amazon basin and prolonged course of arthralgia, MAYV infection was considered as a differential diagnosis. Serologic testing for mosquito-borne alphaviruses using the indirect immunofluorescence test (IIFT) and virus neutralization test (VNT) confirmed the diagnosis of MAYV infection Table . TestingSerum samples collected 83, 106, and 167 days after onset of symptoms, respectively, were analyzed for chemokine/cytokine levels using the Cytometric Bead Array (CBA) Human Chemokine Kit and Human Th1/Th2 Cytokine kit  according to the manufacturer’s instructions.Except for CXCL8/IL-8, which was elevated more than 15-fold on day 83 after onset of symptoms, chemokines  and cytokines  were not different to healthy controls (n = 5 samples) . During follow up visits the patient reported persistent arthalgias of both ankles and elbows, which slowly improved over time. The patient continued to take NSAID for over 2 months and her symptoms finally resolved after 5 months.To our knowledge, this MAYV case is the fourth of a European patient with a travel history to South America and in particular the first imported MAYV case into Germany ,6,7. MAYOur patient presented with a travel and exposure history typical for an infection with an arthritogenic alphavirus and displayed symptoms identical to reports published previously ,6,7. HowVNT is considered gold standard for the diagnosis of MAYV infection. However, the requirement for a biosafety level 3 laboratory to conduct these assays considerably limits their utility. IIFT and ELISA have been also described, but they are prone to cross-reactivity between alphaviruses ,6. DiagnBeyond pure case detection it should be noted that travellers can potentially act as sentinel for emerging infectious diseases . In lighHaemagogus janthinomys which lives in the forest canopy and propagates in a sylvatic cycle predominantly in monkeys [Among the arthritogenic alphaviruses MAYV is restricted to South America and infections have been reported from countries including Brazil, Bolivia, Colombia, the Guiana’s, and Venezuela. Sporadic outbreaks involving up to 100 cases have been described in several regions -14. Resi monkeys ,17. Henc monkeys ,13,18. C monkeys .From a clinical perspective arthritogenic alphaviruses cause a similar picture consisting of acute fever, malaise, headache, maculo-papular rash, myalgia lasting for 3 – 7 days, and a characteristic and often debilitating polyarthraglia/polyarthritis lasting up to 6 months . A rash Here, we report the first case of MAYV associated arthritis in a returning traveller from Germany. No inflammatory correlate was detectable despite long term sequelae. With increasing intercontinental travel, infectious disease specialists and rheumatologists need to be aware of alphavirus infection as a cause of febrile illness followed by persisting polyathralgia. Our clinical case illustrates that MAYV infection should be considered after travel to tropical South America, especially, if the travel itinerary included extended exposure in the Amazon rain forest and its wildlife.Written informed consent was obtained from the patient for publication of this case. A copy of the written consent is available for review by the Series Editor of this journal.The authors declare that they have no competing interests.CT and MP drafted and wrote the manuscript. CT and WVK took care of the patient. JH and MP carried out the cytokine assays. LA, PE, and JSC carried out the IIFT and VNT. WVK critically revised the manuscript. All authors have read the manuscript and approved its final version.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/369/prepub