West Nile virus (WNV) has become the principal cause of viral encephalitis in North America since its introduction in New York in 1999. This emerging virus is transmitted to humans via the bite of an infected mosquito. While there have been several candidates in clinical trials, there are no approved vaccines or WNV-specific therapies for the treatment of WNV disease in humans. From studies with small animal models and convalescent human patients, a great deal has been learned concerning the immune response to infection with WNV. Here, we provide an overview of a subset of that information regarding the humoral and antibody response generated during WNV infection. West Nile virus (WNV) is a neurotropic flavivirus that has seen an emergence into new geographical regions in the last decade. Originally isolated from a patient in Uganda in 1937 . WNV nowFlaviviradae family, WNV is classified within the Japanese Encephalitis virus serocomplex. WNV exists in an enzootic cycle between mosquitos and birds, but humans and horses can become infected when bitten by an infected mosquito. While ~80% of infections are asymptomatic, WNV infection can cause a range of symptoms from a mild febrile disease to flaccid paralysis to lethal encephalitis. While the most severe symptoms generally manifest in the elderly and immunocompromised, healthy individuals can also experience severe disease. A member of the T = 3 quasi-icosahedral symmetry [vβ3 [WNV has a positive, single-stranded ~11-kilobase RNA genome. The genome is encapsidated within multiple copies of the capsid (C) structural protein and enveloped in a lipid bilayer decorated by the two other structural proteins, membrane (M) and envelope (E). The infectious mature WNV particle is approximately 50 nm in diameter and has 180 copies of the E glycoprotein arranged in a try [vβ3 ,5, DC-SItry [vβ3 ,7, and otry [vβ3 . As the try [vβ3 ,11,12. TAnimal models have aided our understanding of WNV pathogenesis in the absence of data for human pathogenesis. From these studies, WNV pathogenesis has been classified into three stages: initial infection and spread, peripheral viral spread, and neuroinvasion. Upon transmission of WNV from the bite of an infected mosquito, the virus is believed to infect and replicate within keratinocytes and skin-resident dendritic cells. It is thought that DC migration to the draining lymph node leads to the next phase of infection as the virus replicates and is disseminated into peripheral organs. It is currently unclear what the major cellular reservoir of viral infection and replication WNV uses, but subsets of DCs, macrophages, and neutrophils have been suggested. The final stage of WNV pathogenesis involves neuroinvasion and infection of the brain and spinal cord. The mechanism(s) by which WNV gains entry into the CNS is incompletely understood, but it is the translocation of the virus into the CNS that leads to lethal disease. The E glycoprotein is the major flavivirus structural protein present on the viral surface, as well as the dominant target of neutralizing antibodies. The E glycoprotein is responsible for binding the host cellular receptor as well as endosomal fusion. The crystallographic structure of the E protein ectodomain of multiple flaviviruses has been determined ,19,20,21T = 3 symmetry discussed above. Although cleavage of prM is required for the production of infectious particles [The mature WNV virion is ~50 nm in diameter with 180 copies of the E glycoprotein smoothly arranged in a head-to-tail homodimeric fashion . The homarticles , this clarticles ,31,32. Aarticles . The varAs the endosomal compartment matures during WNV entry, an acid-induced dissociation of the E homodimers occurs, facilitating the irreversible formation of a fusogenic E trimer. While the exact structural arrangements on the surface of the virion are unknown, crystallographic structures inform us that the formation of the trimer requires a considerable movement at the DI-III linker as DIII rotates ~36 degrees relative to DI/II ,18,19. TThe structural flexibility of the E protein during maturation and fusion has been well established. However, studies over the last few years have revealed that the conformation of the virus, relative to antibody epitope exposure, is influenced by increasing the incubation time and temperature  show higher viral titers in the CNS and 100% mortality , presumaortality . Passivenfection . Beyond nfection . This stnfection . While anti-WNV IgG clearly protects mice from rechallenge, the role of IgG in primary WNV infection is unknown. Based on the known kinetics of production of anti-WNV IgG isotypes (appearing between days 6–8), the virus enters the CNS (day 3) and is mostly cleared in the periphery . In evalFlavivirus infection has been shown to induce life-long humoral protection from future infection with the homologous virus . The anti.e., linear peptides, soluble or yeast displayed forms of the E ectodomain or truncated E subdomains, intact virions or sub-viral particles) [The E protein, which comprises the majority of the virion surface, represents the major target of neutralizing antibodies to WNV infection. Antibody epitopes have been identified on all three domains (DI-DIII) ,62,63,64rticles) . While irticles) . In a serticles) ,62. Inderticles) . Both ofin vivo [in vivo have been described [Antibodies against WNV proteins other than E have been identified. As introduced above, a subset of infectious WNV virions retain varying levels of uncleaved prM. Antibodies that bind to prM have been identified in WNV immune sera ,70, and in vivo . This liescribed . Prophylescribed . Finallyescribed , as wellescribed  have beeStudies with WNV indicate that neutralization is governed by a stoichiometric threshold ,66,76,77Two critical factors determine whether the required threshold for antibody binding is met. Antibody affinity controls the fraction of epitopes occupied by antibody at a given concentration . DiffereEpitope accessibility has emerged as a critical factor that governs WNV neutralization. Mechanisms by which the virus can increase the number of epitopes docked by antibody increase the chances that neutralization requirements are met. Characteristics of WNV that modulate epitope accessibility, and their effects on neutralization potency will be discussed in detail below, using the mAbs E16 and E53 as examples. In the context of this review, an “inaccessible” epitope is one that cannot be engaged by antibody; this applies to cryptic epitopes that are not displayed on the surface of the virion, as well as those that are solvent accessible but precluded from binding by steric constraints.in vitro [Mapping studies identified a subset of WNV-specific antibodies with the potential to neutralize infection that bound epitopes not predicted to be accessible on the mature form of the virus . One expin vitro ,31,38,85in vitro . Using pin vitro . A hallmin vitro . Thus, aAs introduced in Ongoing studies indicate a correlation between flavivirus structural dynamics and the intrinsic stability of virus in solution. Some pathways of virus breathing may result in a virus that is “stuck” in a non-infectious state, which manifests as a loss of infectivity. Interestingly, the extent of WNV maturation impacts the intrinsic decay rate of the virus; fully mature virus decays (loses infectivity) at a slower rate than populations of WNV that retain high levels of prM . WNV virin vivo. Through the classical pathway of complement activation, C1q molecules that consist of six globular heads form multivalent interactions with the Fc-portions of either single pentameric IgM or multiple IgG antibodies docked on the surface of a virus. C1q opsonization triggers a cascade of cleavage events that result in the formation of the membrane attack complex (MAC) capable of viral lysis [in vitro, this does not seem to be dependent on MAC induced viral lysis. Neutralization assays performed in the presence of serum from mice deficient in various complement components demonstrated that C1q, but not C3 or C5 (which act downstream from C1q in the complement cascade), was necessary for the increase in neutralization [in vitro results in increased neutralization potency. Mechanistically, binding of C1q to antibody-virus complexes effectively lowers the stoichiometric threshold required for neutralization, and involves the ability of C1q to crosslink antibodies docked on the virion surface. Mannose binding lectin (MBL), a component of the non-classical lectin pathway of complement activation, has also been implicated in WNV neutralization. Similar to the mechanism of C1q, this inhibition does not require downstream activation of the complement cascade. However, the activity of MBL in limiting WNV infection is antibody independent; MBL binding to the virion surface may directly neutralize infection by inhibiting fusion [While engagement of the virion by a sufficient number of WNV-specific antibody molecules can directly neutralize infection by blocking binding or fusion, the non-antigen binding Fc-portion of an antibody is capable of modulating infection by interacting with additional proteins present al lysis . Howeverlization . Additiog fusion ,90.in vivo protective effects of antibodies that are poorly neutralizing in vitro [in vivo protective effects of DII-FL specific antibodies were greatly inferior to DIII-LR specific antibodies, which resulted in survival of even the FcγR/C1q deficient mice, this study highlights a mechanism by which poorly neutralizing antibodies may be protective in vivo. This is of particular importance based on the finding that the human antibody response is overwhelmingly directed at epitopes within the DII-FL, as discussed in A recent study demonstrated that Fc effector functions may enhance the in vitro . The abiin vivo, primarily during secondary infections, is a constant concern in the development of a much-needed vaccine [in vitro, clinical manifestations in human infections are not readily apparent [in vitro studies with WNV indicate that ADE and neutralization are related to one another by the number of antibodies bound to the virus. ADE is governed on the lower end by the number of antibody molecules required to mediate cell attachment, and on the upper end by the neutralization threshold. For DIII-LR antibodies, ADE is estimated to occur when between 15 and 30 antibody molecules are bound to the virus [in vivo [The Fc-region of antibody molecules has also been implicated in antibody-dependent enhancement of infection (ADE), a phenomenon that describes an increase in virus infection in the presence of sub-neutralizing antibody concentrations. In this process, antibody-virus complexes infect cells through uptake by Fc receptor interactions . A presu vaccine . While Aapparent ,93,94. Rhe virus . Since C[in vivo . Antibod[in vivo ,96,97. H[in vivo . RegardlMost data concerning the antibody response to WNV are from mouse studies, which indicated that a large portion of the humoral response was directed at epitopes within the DIII-LR ,99,100. There are no approved human vaccines against WNV. Both subunit and attenuated vaccine candidates have entered into phase I and phase II clinical trials , yet nonAs there is currently not an approved vaccine for the prevention of WNV, one possible treatment is passive administration of neutralizing antibody to patients affected by WNV disease. In studies with mice and hamsters, the E16 antibody was capable of protecting the majority of these animals up to five days post-infection with WNV . Indeed,in vivo; (iv) why does the human response preferentially target non-DIII-LR epitopes and what are the immunodominant epitopes; and (v) what are the early activating signals for B cells. The ability to understand WNV neutralization in quantitative terms and apply this knowledge towards dissecting the polyclonal response to WNV will aid in the design of future vaccine candidates.The need for WNV therapeutics and vaccines remains a high priority, as WNV continues to pose a significant public health threat in various regions of the world. The critical role of the WNV-specific humoral response and antibodies in controlling infection underscores the need for a greater understanding of this arm of the immune response. Considerable progress has been made towards mapping WNV epitopes within the E protein, and has led to the identification of potent neutralizing antibodies that have been adapted into candidates for therapeutic use. However, many questions remain unanswered and new questions have arisen from recent discoveries, including: (i) what is the role, if any, of IgG in primary infection; (ii) what is the role of anti-prM antibodies in infection; (iii) how does the structural heterogeneity of WNV  impact neutralization


Intrinsic excitability is one of the pillars of neuronal behaviour . Combined experimental and modelling studies of neuronal excitability often provide an important insight into the brain functions. In this work we analyse a unified model that we derived for CA1/3 pyramidal neurons in Hodgkin-Huxley formalism [The analysis shows that the outward currents have a considerable influence on both excitability and the number of action potentials. An increase of high-voltage activated inward currents often decreases excitability, whereas an increase of low-voltage activated inward currents results in a large increase of it. Moreover, the outward currents in our model have a profound impact on the number of action potentials. Counter-intuitively, we find that either a decrease or increase of total Na+ current can result in an increase of excitability, as shown in Fig. 1


Chikungunya is a self-limited, systemic viral infection that has been a major health problem since the past few years. Ocular manifestations of the disease have become more prevalent in the recent years. Currently, there is neither a specific treatment nor vaccine available for chikungunya fever. This review highlights the current understanding on the pathogenesis, systemic changes with an emphasis on ocular findings, laboratory investigations, and prevention and treatment of this disease. Chikungunya fever is an arthropod-borne viral (arbovirus) disease -3 that hThere have been several accounts of epidemics of fever with arthralgia resembling present day chikungunya fever dating as far back as 1824 from India . HoweverFollowing the report from Tanganyika in 1952 ,9, chikuChikungunya fever re-emerged in India after nearly 32 years in October 2005 ,5,12. ThChikungunya fever is characterized by explosive outbreaks of epidemics followed by periods of disappearance sometimes lasting up to several decades. This behavior has been attributed to several reasons including the susceptibility of humans and the mosquito vectors of the virus- conditions facilitating mosquito breeding result in high vector density and ability of the vector to efficiently transmit the virus ,4,5.Chikungunya virus reservoirs; during interepidemic periods, several vertebrates, such as monkeys, rodents, and birds, have been implied as the reservoir. Vertical maternal-fetal transmission has been documented in pregnant women affected by chikungunya fever [The natural cycle of the virus is human-mosquito-human. There is, however, evidence of the existence of epizootic cycles that may retain the virus during the interepidemic period ,4,5,17. ya fever .Chikunguya fever .Alphavirus of the family Togaviridae whose genome consists of a linear, positive-sense, single-stranded RNA molecule, a 60- to 70-nm diameter capsid, and a phospholipid envelope [Chikungunya virus isolates collected from various geographical areas. These include the West-African phylogroup, the East, Central, and Southern African phylogroup, and the Asian phylogroup [The virus causing chikungunya fever is an envelope ,5. Threeylogroup ,20,21.Chikungunya virus isolates from Reunion Islands in the 2005 outbreak. This mutation is postulated to have facilitated the replication and transmission of the virus by reducing the cholesterol dependence of the virus [A mutation at residue 226 of the membrane fusion glycoprotein E1 (E1-A226V) was detected in more than 90% of he virus .Aedes in the Asian region. Aedes aegypti is considered to be the principal vector, and Aedes albopictus (Asian tiger mosquito) has also recently emerged as an important vector. A. aegypti breeds in stored fresh water, such as that in coolers, flower vases, water tanks, and discarded household junk items like vehicle tires, coconut shells, pots, cans, and bins in urban and semiurban environments [Chikungunya fever is transmitted by the bite of mosquitoes of the genus ronments ,24. AdulThe principal cell types infected by chikungunya are fibroblasts, epithelial cells, and lymphoid cells . In humaChikungunya virus leads to apoptosis through both the intrinsic and extrinsic pathway [Interferon gamma and IL-12 levels have been observed to rise dramatically during the acute phase of chikungunya fever. The level of IL-12 returns to normalcy in patients who recover. In contrast, patients who develop chronic arthritis show persistently high IL-12 levels. Histologic examination of synovia from patients with chronic arthritis following chikungunya fever has revealed joint inflammation due to macrophages containing viral material. Metalloprotease (MMP2) also contributes to tissue damage.  pathway .Chikungunya fever is known to affect all age groups. Both males and females are equally affected. The incubation period ranges between 2 to 7 days ,28.Chikungunya virus infection is characterized by the sudden onset of high-grade fever with chills, headache, malaise, arthralgia or arthritis, vomiting, myalgia, skin rash, and low back pain  over a 10- to 12-week period, with only subtle retinal pigment epithelial changes remaining to suggest an earlier infection. Macular ischemia and optic disk changes account for the poor visual outcome seen in a minority of these patients . Acute-oPrompt visual recovery is usually the norm with immediate administration of systemic steroid therapy ,53,54. AChikungunya virus antigens and normal or altered host tissue proteins, immediate hypersensitivity reactions, and stimulations of a pathogenic lymphocytic reaction may be responsible for this delayed immune response [The systemic manifestations of the fever are related to viremia, while joint involvement is believed to be an immune-mediated reaction to the viral antigen . The exaresponse ,30.Laboratory tests used to diagnose chikungunya infection include virus isolation, serological tests, and molecular techniques. Virus isolation and real-time polymerase chain reaction (RT-PCR) are useful during the initial viremic phase of the illness, whereas antibody demonstration from the serum is of use in the later phase of the disease.Chikungunya virus can be performed using RT-PCR by amplifying a fragment of the E-2 gene [Chikungunya virus by targeting the nsP1 and E1 genes has been developed by Hasebe et al. [Viral culture is the gold standard test for the diagnosis of chikungunya fever, and it is based on inoculation of mosquito cell cultures or mammalian cell cultures ,5,28. SpE-2 gene ,58. Combe et al. . Anothere et al. . RT-PCR e et al. .Enzyme-linked immunosorbent assay (ELISA), immunochromatographic test (ICT), indirect immunofluorescent method, hemagglutination inhibition, or neutralization techniques may be used to detect chikungunya IgM and IgG antibodies in the serum. The IgM antibodies are detectable after a mean period of 2 days from the onset of the fever, and they persist for several weeks, sometimes even up to 3 months, whereas IgG antibodies can be detected in the convalescent samples and persist for years . The senBlood counts may be normal, or patients may have leukopenia with relative lymphocytosis. Erythrocyte sedimentation rate is significantly elevated, and low rise in C-reactive protein is positive in acute cases .Chikungunya virus using RT-PCR was reported to be positive in a case of Fuchs' heterochromic iridocyclitis [Nearly all the studies reporting ocular manifestations of chikungunya fever have utilized either virus detection from the serum using RT-PCR or antibody detection from the serum of patients. Ocular fluid (aqueous humor) analysis for cyclitis  and anotcyclitis  RT-PCR scyclitis . Unfortucyclitis -68 or ancyclitis  technoloChikungunya virus, an RNA virus, is unstable over time, and there is no specific antiviral drug available to date for the treatment of Chikungunya virus infection. Treatment at the acute stage of the disease is symptomatic with antipyretics (acetaminophen is preferred over aspirin so as to avoid bleeding complications) and nonsteroidal anti-inflammatory drugs [The ry drugs ,70. Chrory drugs ,71, cortry drugs .Chikungunya virus is still on.Anterior uveitis has been treated with topical steroids and cycloplegic agents ,49. AssoChikungunya virus, prevention of disease spread by vector control still remains the most important mode of control for chikungunya infection. Elimination of breeding sites and source reduction are effective methods. The vector A. aegypti is a container habitat species and breeds primarily in artificial containers and receptacles. Measures to control mosquito breeding include covering of water tanks, cisterns, and other water storage equipment; removal of tires and coconut shells that may collect water; regular emptying of bird baths and pet water bowls; trimming of tall grass/weeds; and the introduction of larvivorous fish such as the guppy in ornamental water tanks [In the absence of a specific treatment or vaccines against er tanks .NN-diethyl-m-toluamide) on the exposed skin, and even insecticide spraying to kill the mosquitoes [Persons living in or traveling to mosquito-infested areas must use protective measures to prevent mosquito bites. These maybe in the form of insecticide-treated nets, use of mosquito repellant creams (such as those containing squitoes .Chikungunya virus immunoglobulins. Human polyvalent immunoglobulins have been purified from plasma samples obtained from donors in the convalescent phase of chikungunya infection, and the preventive and curative effects of these immunoglobulins have been investigated in mouse models of Chikungunya virus infection [Chikungunya virus and are at risk of severe infection, such as neonates born to viremic mothers [Another strategy in its developmental stages is specific immunoprophylaxis with human anti-nfection . This ma mothers  and adulTo this day, there is no commercial vaccine for chikungunya. Live chikungunya vaccines have been abandoned to avoid the possible risk of viral persistence .Ocular manifestations of chikungunya infection can be present at the time of fever or may manifest after many weeks. Anterior uveitis, optic neuritis, and retinitis are the commonest manifestations. Diagnosis of chikungunya can be made either by RT-PCR from the serum or ocular fluids or by demonstration of antibodies in the serum. In the absence of a specific antiviral regimen, the treatment of ocular disease is supportive. Exact pathogenesis of the ocular manifestations, development of specific antiviral therapy, and vaccination against chikungunya are fields that require further research.Written informed consent was obtained from the patient for publication.The authors declare that they have no competing interests.PM designed the article outline, collected the data, and drafted the manuscript. KA collected the data and drafted the manuscript. RS prepared the manuscript. All authors read and approved the final manuscript.


Myotis daubentonii (Daubenton's bat), was measured with a novel quantitative reverse transcription–polymerase chain reaction assay. High levels of genomic RNA were found in the brain and were also detectable in the tongue, bladder, and stomach.Organ distribution of European bat lyssavirus type 2 viral RNA in its reservoir host,  Eptesicus serotinus) (Myotis bats (Myotis daubentonii [Daubenton's bat] and Myotis dasycneme) and has been identified in 3 locations in Europe: the Netherlands, the United Kingdom, and Switzerland  and quantified. Dilutions were made to either 0.25 μg/μL (bat 603/04) or 1 μg/μL (bat 696/04) to standardize the quantity of RNA used for reverse transcription. Primer EBLVNa was used for cDNA synthesis from the genomic (negative) sense strand as previously described , panel AA representative plot of amplification curves from organ samples taken from bat 696/04 is shown in the Figure, panel B, with 10 μL of product separated by electrophoresis on a 1% agarose gel included for comparison. The quantitative results for viral RNA load for both bats are shown in The detection and quantification of EBLV-2 RNA in bat organs by real-time PCR show the potential distribution of this virus. The choice of organ tested in both cases was severely limited by degradation of the carcass before investigation. Furthermore, live virus could not be recovered from many organs because of cytotoxicity of the samples and virus degradation caused by repeated freezing and thawing.Viable virus was recovered from the brain of bat 603/04. Since the brain is the main site of EBLV-2 replication, this finding suggests that the virus displays a similar neurotropism to classical rabies virus. Rabies virus, especially in the late stages of disease, disseminates from the brain to other innervated sites within the host (Dissemination of rabies virus to the salivary glands and subsequent virus shedding enables transmission through biting. Detection of EBLV-2 RNA in the tongue of infected bats leads us to conclude that transmission of EBLV-2 may occur through biting. However, since EBLV-2 genome was detected in a bladder sample, we cannot exclude the possibility of virus release from urine. In future cases, where possible, organs such as the salivary glands and lungs should be examined to provide further evidence for the route of virus transmission between bats.


Increasing infections with Monkeypox and Cowpox viruses pose a continuous and growing threat to human health. The standard method for detecting poxvirus neutralizing antibodies is the plaque-reduction neutralization test that is specific but also time-consuming and laborious. Therefore, a rapid and reliable method was developed to determine neutralizing antibody titers within twelve hours. The new assay measures viral mRNA transcription as a marker for actively replicating virus after incomplete neutralization using real-time PCR. The increasing number of humans infected with zoonotic orthopox viruses (OPV) such as Cowpox and Monkeypox virus poses a continuous and increasing threat to human health . Thus, eRecently, four alternative methods were described to determine neutralizing anti-Vacinia virus (VACV) antibodies using either a beta-galactosidase expressing VACV Western Reserve strain (WR)  or recomThe assay presented here uses VACV LE and human VACV immunoglobulin (HIVIG) as a model system and quantifies neutralizing anti-VACV antibodies by combining the classic PRNT with a OPV-specific real-time PCR (designated NT-PCR) allowing quantification of replicating virus within a few hours after infection of the host cell.12/13 assay targets the gene for the VACV LE DNA-binding phosphoprotein involved in DNA replication and nucleotide metabolism. The other two OPV-specific real-time PCR assays, D8L and Rpo18 [6 to 10 copies per reaction with an overall R2 of ≥ 0.98 and PCR efficiencies ≥ 95% , 1:320  and 1:640 . The PRNT titer for Omrigam was always 1:320 . As both HVIG showed the same neutralizing activity in the PRNT, VIG was used for establishment of the NT-PCR assay for reason of availability.Neutralizing antibody titers of HVIG (VIG and Omrigam) were first determined with the standard PRNT. The PRNT titer is defined as the antibody dilution resulting in 50% plaque reduction. HVIG preparations were tested using 4.4 × 106 copies of the cellular reference gene transcript of c-myc. The c-myc gene is expressed constitutively and independently from experimental conditions and sample treatment, different cell types and developmental stages. It is not affected by the infection with different OPVs . After pre-incubation of VACV LE with VIG fewer cells became infected compared to VACV LE alone resulting in a decreased virus mRNA copy number due to neutralization of infectious virus. For VACV LE incubated with negative human serum virus mRNA copy numbers were set to 100%. Based on the mRNA copy numbers determined for each antibody dilution the percentage of replicating virus was calculated in comparison to the 100% expression in virus control sample.The NT-PCR assay principle is based on measuring actively replicating VACV LE by quantifying virus mRNA levels in infected Vero E6 cells. Since cell numbers per well, RNA integrity and quantity can vary all virus copy numbers were normalized against 101, 2.2 × 102 and 4.4 × 102 pfu/well were tested using experimental conditions similar to the standard PRNT with 1 h neutralization time and 24 h for infection and replication in order to compare both assays. Using 4.4 × 101 and 2.2 × 102 pfu/well of VACV LE, levels of viral mRNA were below detection threshold of the OPV12/13 real-time PCR assay (< 101 copies) for antibody dilutions 1:40-1:80. For antibody dilutions ≥ 1:160 copy numbers varied between 101 and 103 copies. No correlation between virus replication and antibody dilution was observed. In contrast, when using a virus dose of 4.4 × 102 pfu/well, ≥ 101 OPV12/13 copies were detected for all antibody dilutions tested. A significant increase of virus replication could be detected for an antibody dilution of ≥ 1:640. Therefore, an infectious dose of 4.4 × 102 pfu was selected for further experiments.For the NT-PCR assay virus concentrations of 4.4 × 102 OPV12/13 copies for antibody dilutions < 1:640 whereas copy numbers for dilutions ≥ 1:640 increased at least twofold (data not shown). A visual cut-off was set for a dilution of 1:640 and neutralization time of 2 h was chosen for further validation.To reduce handling time, neutralization efficacy was evaluated after 1 h, 2 h and 3 h of co-incubation of VACV LE with the VIG antibodies. For 1 h and 3 h of co-incubation no significant correlation was detected due to high variability of virus replication for all antibody dilutions. After 2 h a significant increase of virus replication was observed correlating with increasing antibody dilution. Virus mRNA copy numbers revealed ≤ 3x1012/13 assay, two other real-time PCR assays  were tested simultaneously. While all three assays target genes highly conserved between OPV species only OPV12/13 and Rpo18 target genes directly involved in virus replication. Although all tested OPV-specific real-time PCR assays have similar characteristics in terms of sensitivity, amplification efficiency and specificity no significant correlation between antibody dilution and virus neutralization could be seen with the D8L real-time PCR assay (data not shown). Thus, the D8L assay was not used for further experiments. In contrast, after 7 h but not after 5 h of virus replication, the Rpo18 and OPV12/13 real-time PCR assays were successfully and consistently detecting increased viral mRNA levels for antibody dilutions ≥ 1:1280 and 1:640, respectively, indicating reduced virus neutralization. Using the OPV12/13 and Rpo18 assay a significant correlation between antibody dilution and virus replication was detected. The results for OPV12/13 and Rpo 18 suggest that it is important to use a real-time PCR that targets a gene directly involved in virus replication. As the OPV12/13 assay was originally designed to detect the DNA binding phosphoprotein of VACV the OPV12/13 and an infection time of 7 h was selected for further validation of the NT-PCR.While a complete replication cycle of OPV takes between 9 and 24 h ,12, earlTo ensure a qualitative and repetitive readout, the neutralizing antibody titer (= NT-PCR titer) was calculated based on a plot of the antibody dilution versus percentage of virus replication: the point at which the curve becomes near-horizontally  defines the NT-PCR titer Figure . This poIntra-assay variation of the NT-PCR was determined based on four runs performed in parallel. To evaluate inter-assay variability the NT-PCR assay was performed on four different days. Calculation of the NT-PCR titer was performed for a total of seven assays table . The NT-To explore the NT-PCR assay's application for clinical use, serum samples of six individuals recently vaccinated with VACV LE were tested using both, PRNT and NT-PCR. As only small serum volumes were available, serial dilution of serum was started at 1:40. For two of the six serum samples the outcome was identical in both tests table . HoweverAlthough further evaluation with clinical samples is needed this preliminary evaluation indicates that it will be suitable for routine diagnostic use especially if results are needed rapidly, if plasma components interfere with the cell culture or if neutralizing antibodies to viruses with no or poor cytopathic effect in cell culture (no need for the detection of plaques) need to be analyzed. It should be easily adaptable to other DNA viruses as soon as a suitable real-time PCR assay is available to detect viral mRNA. For DNA viruses the detection of viral mRNA indicates the presence of actively replicating virus in the cell limiting the potential effect of contaminating DNA from the genome of the inoculated virus just sticking to the cell surface. Also, the assay should be adaptable to RNA viruses if active virus replication could be determined and distinguished from mere contamination from viral genomic RNA. Assay conditions need to be adapted for each new virus, however, this is also true for standard PRNT assays.2 pfu/well VACV LE, 2 h of neutralization time, 7 h of infection and replication time and final quantitative examination of virus mRNA copies in cells by quantitative OPV12/13 real-time PCR.Taken together, the new and rapid NT-PCR assay can be completed within 12 h while the turn-around time of the standard PRNT is at least 4 days. Final NT-PCR conditions are: 4.4 × 10The NT-PCR assay is a fast, reliable and sensitive tool for the detection of VACV neutralizing antibodies. It will be suitable for routine diagnostic and could pose a valuable alternative to the standard PRNT assay. The low detection limit and the high degree of accuracy for the detection of replicating virus by real-time PCR resulted in high reproducibility and short assay turn-around time. This assay can be performed with all wild-type OPV strains without the need of particular VACV reporter strains. It may also be beneficial for viruses with no or poor cytopathic effect in cell culture.VACV LE was propagated in Hep2 cells (ECACC: 86030501). Vero E6 cells (ECACC: 85020205) were used for virus stock titrations, for standard PRNT and NT-PCR. Overlay medium was 1.6% carboxymethylcellulose (CMC). All cells were maintained according to ECACC recommendations.8 pfu/mL), human Vaccinia virus immunoglobulin (HVIG)  and sera from six individuals vaccinated with VACV LE were used. All sera were tested for absence of cytotoxicity.VACV LE   and virus was allowed to adsorb for 1 h at 37°C. Next, cells were overlaid with 400 μL overlay medium and incubated for 4 days. Medium was aspirated and cells were fixed with 4% formaldehyde for 30 min, stained with naphthalen blue black and washed once with tap water. Plaques were counted and the titer for 50% plaque reduction was calculated referred to VACV-negative serum.For standard PRNT 8.5 × 102, 2.2 × 102 and 4.4 × 101 pfu/well), a neutralization time between 1 h, 2 h and 3 h, total incubation time for virus entry and initial replication of 24 h, 7 h or 5 h and three different viral target sequences for OPV-specific real-time PCR were investigated. All other preceding steps were performed according to the standard PRNT. After incubation, medium was aspirated and cells were washed with PBS, trypsinized, washed twice with PBS and centrifuged . Cell pellet was immediately resuspended in 700 μL RLT buffer and RNA was extracted using the RNeasy Mini Kit  according to the manufacturer's protocol. Residual DNA was eliminated by DNase treatment . cDNA was synthesized in a total volume of 20 μL containing 500 ng Oligo dT(18) primer, 500 ng calf thymus DNA and 10 μL RNA. No RNase inhibitors were added as all RNA samples were processed immediately after extraction. After 5 min at 65°C a mix consisting of 4 μL 5 × buffer, 2 μL 0.1 M DTT, 0.4 μL 25 mM dNTP and 200 U Superscript II reverse transcriptase  was added.To optimize the NT-PCR assay, three VACV LE concentrations . The 25 μL reaction volume included 2 μL cDNA or plasmid DNA, 2.5 μL 10 × buffer, 2.0 μL MgCl2 (50 mM), 1 μL dNTP (25 mM each), 0.75 μL of forward and reverse primers (10 μM), 0.25 μL probe (10 μM), 0.25 μL ROX (100 mM), 1 U Platinum Taq polymerase  and 15.3 μL water. Cycling conditions were 2 min at 50°C, 10 min at 95°C, followed by 45 cycles of 15 sec at 95°C and 30 sec at 60°C .Samples were incubated at 37°C for 50 min and at 70°C for 15 min. For each RNA sample a reverse transcription (RT) control reaction was performed replacing the reverse transcriptase water to verify absence of contaminating DNA. Quality of cDNAs was tested using the c-myc real-time PCR assay which simultaneously served as control of RNA extraction and cDNA synthesis. Only cDNA with CStatistical analyses were done using GraphPad's Prism software (Version 5.0a) and a repeated measures ANOVA test and "Tukey's Multiple Comparison Test" was used for statistical analysis and p-value ≤ 0.05 was regarded as significant.HVIG: human Vaccina virus immunoglobulin, OPV: orthopox virus, PRNT: plaque-reduction neutralization test, VACV: Vaccinia virus, VACV LE: Vaccinia virus Lister ElstreeThe authors declare that they have no competing interests.MK made substantial contributions to the study design, participated in data acquisition, analysis and interpretation and drafted and revised the manuscript critically. AD was mainly responsible for data acquisition and analysis and for interpretation of data. GFL was involved in data analysis and in discussing the manuscript. PWD participated in data analysis and interpretation. HE made substantial contributions to conception and design of the experiment and in revising the manuscript. All authors read and approved the final manuscript.


The virus may contaminate live canine vaccines based on cultured feline cells. The Nine specific pathogen free dogs were divided into three groups, with each group consisting of one female and two male dogs. Dogs were subcutaneously inoculated in the neck with either 1 ml RD114 stock virus (group A), inactivated RD114 virus suspension (group B), or cell culture medium (group C) as a negative control. To assess blood cell counts and biochemical properties, blood samples from each group were collected 5 days before inoculation, just prior to inoculation, and 1, 3, 7 and 10 days post-inoculation.During the experimental period of 51 days, none of the dogs inoculated with RD114 virus showed any clinical signs, significant increases in rectal temperature or abnormal blood biochemical characteristics including C-reactive protein when compared with the negative controls. We were not able to re-isolate the RD114 virus from buffy coat cells of group A dogs. Additionally, we could not detect RD114 provirus in the genomic DNA isolated from peripheral blood leukocytes, lymph node, spleen and sternal bone marrow cells.Signs of RD114 virus proliferation were not found after subcutaneous infection of dogs. Although the potential risk caused by infection with RD114 virus in dogs could not be assessed in this study, we suspect that RD114 virus has little or no virulence in dogs. It is k5 infectious units/ml. To prepare inactivated RD114 virus as inocula, the stock virus was added to an equal volume of diethylether, then vigorously and intermittently mixed at room temperature (around 20°C) for 3 min. Absence of infectious RD114 virus in the inactivated viral preparation was confirmed by the LacZ marker rescue assay.A LacZ marker rescue assay was used to detect and titrate infectious RD114 virus ,6. The pNine 10-months-old SPF beagles  were divided into three groups . Each group consisted of one female and two male dogs with individual dogs confined to cages. The dogs were inoculated subcutaneously in the neck with either 1 ml of RD114 stock virus (group A), inactivated RD114 virus suspension (group B) or cell culture medium (group C) as a negative control. The inoculation route was chosen according to the manufacturer's instructions for the live canine vaccines. All animal studies were conducted in accordance with the National Veterinary Assay Laboratory Guide for the Care and Use of Laboratory Animals, and the relevant Animal Welfare Acts. The dogs were euthanized 51 days post inoculation (PI) and autopsied. The thoracic organs, abdominal organs and bone marrow were macroscopically examined. The axillary lymph nodes, spleen and sternal bone marrow were collected for further virological examinations.Re-isolation of the RD114 virus was attempted by co-culturing TE671(LacZ) cells with buffy coat cells. To assess the blood cell counts and biochemical properties, blood samples from each group were collected 5 days before inoculation, just prior to inoculation (day 0), and at 1, 3, 7 and 10 days PI. White blood cells (WBC), red blood cells, haemoglobin, haematocrit, mean cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, and platelet numbers were determined in the blood cell counts. The biochemical analyses included total protein, albumin, total bilirubin, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, alkaline phosphatase, lactic dehydrogenase, amylase, lipase, blood urea nitrogen, creatinine, total cholesterol, triglyceride, sodium, potassium, chlorine, calcium, inorganic phosphorous, glucose and total bile acid levels. The C-reactive protein (CRP) concentration, a sensitive indicator of inflammation in dogs, was analysed at days 0, 7, and 51 PI. Additionally, during the experimental period, clinical signs were recorded daily, and rectal temperature and body weight were measured at least once a week.et al. [env and pol genes of RD114 virus. The single step PCR assay was performed on genomic DNA extracted from TE671(LacZ) cells infected with ten-fold serially diluted (100 to 10-10) stock virus. The limit of detection was 10-5 and 10-6 for the env and pol genes, respectively. Then, the LacZ pseudotype virus positive cells were detected 17, 2 and 0 cells for 10-4, 10-5 and 10-6 diluted points, respectively.Genomic DNA was extracted from ethylenediaminetetraacetic acid (EDTA) stabilized whole blood, axillary lymph nodes, spleen and bone marrow, and subjected to polymerase chain reaction (PCR) analysis. In addition, genomic DNA was also extracted from TE671(LacZ) cells, 12 days after co-culture with buffy coat cells. The PCR was conducted as described by Sakaguchi P values of <0.05 considered to be significant.The mean values of all measured variables of each group were calculated. Statistical analyses were performed using SPSS software (version 13). The differences between the three groups at each time point were compared with During the experimental period, none of the dogs in groups A and B showed any clinical signs, including significant increases in rectal temperature, or distinct abnormal biochemical blood characteristics including CRP when compared with group C dogs. In addition, the WBC counts of group A and B dogs were nearly equal to those of group C, and body weight increased in all animals after inoculation Figure . StatistInfectious RD114 virus was not detected from TE671(LacZ) cells co-cultured with buffy coat cells using the LacZ marker rescue assay. This finding was also confirmed by PCR assays using genomic DNA from the co-cultured cells as a template. Additionally, RD114 provirus was not detected in genomic DNA extracted from peripheral blood, lymph nodes, spleen and sternal bone marrow, using the one-step PCR assays Figure. .in vitro [5 infectious units of RD114 virus. These results suggest that RD114 did not proliferate and disseminate in the dogs, or that RD114 virus did not proliferate efficiently in the blood cells and hematopoietic system of dogs. The virus stock, neutralized with the sera of each dog, was inoculated onto TE671(LacZ) cells, and the number of lacZ positive foci among the three groups were counted. In the present study, the antibody titres remained unchanged in all dogs following inoculation (data not shown), implying that RD114 virus did not proliferate in dogs.It has been reported that RD114 virus infects a variety of canine cell lines, such as the Mardin-Darby canine kidney line . RD114 vin vitro . TherefoThe findings indicate that the RD114 virus has little or no virulence in dogs. The potential risk caused by infection with RD114 virus in dogs cannot be accurately assessed because a longer PI period is required for the verification or exclusion of retroviral infection. It is also possible that dogs younger than 10-months might be more susceptible to RD114 virus infection. Millions of puppies are vaccinated worldwide on an annual basis. Therefore it is impossible to completely rule out adverse effects of the infection. Many exogenous retroviruses cause leukaemia and tumours in a wide variety of animal species, and further research is required to isolate or detect RD114 virus in dogs that have developed leukaemia and tumours following use of live canine vaccines manufactured using feline cells. Hopefully, such research would clarify the possible relationship between the occurrence of diseases in dogs and vaccination.Signs of RD114 virus proliferation were not found after subcutaneous infection of dogs. Although the potential risk caused by infection with RD114 virus in dogs could not be assessed in this study because the minimum infectious dose and pathogenicity remain unknown, we suspect that RD114 virus has little or no virulence in dogs.SPF: Specific pathogen free. The dogs were free from infections with canine adenovirus (CAdV-2), canine coronavirus, canine distempervirus, canine parainfluenzavirus, canine parvovirus, leptospira and rabies virus.The authors declare that they have no competing interests.RN participated in the design of the study and performed the statistical analysis. NH performed autopsy. TU organised basic equipment. TO participated in collecting blood samples. TT performed finalisation of the manuscript. TS conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Unnoticed and unpainted, these vectors nurture the dark underpinning of untamed nature, including arthropod-borne disease: in this issue of Emerging Infectious Diseases alone, sleeping sickness in Uganda; dengue in Cuba, French Guiana, Bangladesh, and Myanmar; cutaneous leishmaniasis in Colombia; malaria in Western Kenya; West Nile virus in Guadeloupe; murine virus in the Canary Islands.


To rapidly and specifically identify highly virulent influenza virus strains, we prepared an azobenzene-tethered hairpin-type peptide nucleic acid, bisPNA-AZO, which has a complementary sequence against a highly conserved genomic RNA sequence within the ribonucleoprotein complex of the 2009 pandemic influenza A virus, H1N1 subtype. bisPNA-AZO recognizes the conserved virus genome sequence in a sequence-specific manner. Immobilization of bisPNA-AZO on a plate allowed capture of the target virus gene and the generation of a visual colour signal. The threat of an influenza virus pandemic is increasing worldwide, highlighting the need for a diagnostic test kit that can identify the viral strain and its drug resistance. Current methods for identifying influenza virus strains are mainly based on real-time polymerase chain reaction (RT-PCR) technology Peptide nucleic acids (PNAs) are DNA/RNA analogues in which the phosphate backbone has been replaced by a neutral amide backbone composed of N-(2-aminoethyl)glycine linkages. The advantages of PNAs for molecular recognition are their high binding affinity In this study, we prepared a newly synthesized bisPNA-AZO which contains an azobenzene amino acid linker virus required priming with DNA oligonucleotide primer 1∶5′-GGCGTCATGGAAAAGAACAT-3′ (nt 364–383) or primer 2∶5′-CATGACCCTCGAGGAAATGT-3′ (nt 276–295). First, 90 µl of the influenza A virus (6.6 × 103 pfu/ml) and 10 µl of lysis buffer solution were incubated on ice for 30 min. Prior to the extension reaction, 27 µl of the virus solution and 3 µl of PNAs  were annealed at 25°C for 30 min, then 1 µl of virus-PNA solution was mixed with a solution containing RT buffer, 5 mM MgCl2, 0.5 U RNase inhibitor, 1 mM dNTP and 1 µl of Moloney murine leukemia virus (MuLV) reverse transcriptase  in a final volume of 20 µl. Reverse transcription was performed at 30°C for 10 min, 42°C for 20 min, and stopped by inactivation of the enzymes at 95°C for 5 min.Synthesis of the first strand cDNA for the NS gene of influenza A/Osaka/180/2009 (H1N1pdm) 4)2SO4, 0.1% Triton X-100, 1.5 mM MgCl2, 0.2 mM dNTP, 0.2 mM forward and reverse primers, and 1U of KOD-Plus  in a final volume of 50 µl. After 2 min incubation at 94°C, 40 cycles with a temperature profile of 10 s at 98°C, 30 s at 50°C, and 2 min at 68°C were performed in a thermal cycler. DNA amplified at each reaction condition was dissolved in loading buffer containing 30% glycerol, 0.025% bromophenol blue and 0.025% xylencyanol. The mixture was analyzed on a 2.0% agarose gel by electrophoresis. The gel was stained with ethidium bromide and the gel image was observed under UV irradiation.PCR amplification of influenza A virus genes was carried out using the above-mentioned cDNA samples as templates employing the NS specific primer sets (primer 1 and primer 3∶5′-ACCCCAACTGCATTTTTGAC-3′ (nt 519–539) or primer 2 and primer 4∶5′-ATTGCTCCCTCCTCAGTGAA-3′ (nt 447–466)). This resulted in the amplification of 175 bp (nt 364–539) in the NS gene and 191 bp (nt 276–467) in the HA gene. Amplification of each DNA was performed with a MJ Mini™ thermal cycler  in a volume of 50 µl in 8-well plates. The reaction contained 1 µl of cDNA sample from the above-described reverse transcription, 120 mM Tris–HCl (pH 8.3), 10 mM KCl, 6 mM  and loaded with 20 µl of the manufacturer’s recommended dilution of the anti-influenza A virus nucleoprotein and 20 µl of goat anti-mouse IgG secondary antibody conjugated with HPR in 2% BSA-PBS at a final dilution of 1/20. The wells were placed on an orbital shaker at r.t. for 2 h. After washing the wells with 0.05% Triton-X100-PBS (300 µl × 3), the wells were incubated with an ECL assay solution for 10 or 60 min. HPR conjugated with the anti-mouse IgG secondary antibody produces a stable fluorescent intermediate from TMB in ECL assay solution. This intermediate has an excitation maximum of 430 nm and an emission maximum of 503 nm. This fluorescence spectrum was recorded using a Shimadzu RF-S300PC  and fluorescence quartz cuvettes (8.5 × 10 mm).In each well of the ELISA plate, 0.5 µg of PNA was immobilized via primary amine crosslinking. Prior to detection of the influenza A virus genome, the well was pre-warmed with 0.05% Triton-X100-PBS solution at 50°C for 15 min to prevent inter- and intra-molecular aggregation of PNA. At the same time, 90 µl of influenza A or B virus containing 6.6 × 102, 6.6 × 103, 6.6 × 104 or 6.6 × 105 pfu/ml were mixed with 10 µl of 10% lysis buffer and incubated on ice for 30 min. After removing the Triton-PBS solution from the wells, 100 µl of the lysis buffer-treated virus were added to each well and incubated for 1 h at r.t. Then, the wells were washed with 0.05% Triton-X100-PBS (100 µl × 3) and were loaded with 20 µl of the manufacturer’s recommended dilution of the anti-influenza A virus nucleoprotein and 20 µl of goat anti-mouse IgG secondary antibody conjugated with alkaline phosphatase in 2% BSA-PBS at a final dilution of 1/20. The wells were placed on an orbital shaker at r.t. for 2 h. After washing the wells with 0.05% Triton-X100-PBS (300 µl × 3), the wells were incubated with a BICP/NBT assay solution for 1 h. Alkaline phosphatase conjugated with the anti-mouse IgG secondary antibody produces a stable purple dye. Images of the plates were taken using a digital camera.In each well of the ELISA plate, 0.5 µg of PNA was immobilized via primary amine crosslinking. Prior to detecting the influenza A virus genome, the well was pre-warmed with 0.05% Triton-X100-PBS solution at 50°C for 15 min to prevent inter- and intra-molecular aggregation of PNA. At the same time, 90 µl of influenza virus containing 6.6 × 102–4) complexed with ssDNA containing the conserved sequence of the NS gene of influenza A/Osaka/180/2009(H1N1pdm) were analysed on a UV1700 spectrophotometer (Shimadzu) using a microcell (8 cell×1 mm) at 260 nm. BisPNA and ssDNA were suspended in Na2HPO4 buffer  at 5 µM each. The temperature was ramped down from 95 to 10°C at a rate of −1°C/min.Melting profiles of bisPNA  gene  for influenza A/H3N2 viruses   and their molecular weights were confirmed by MALDI-TOF MS analysis  that form ribonucleoprotein (RNP) complexes together with a viral RNA (vRNA)-dependent RNA polymerase complex and many nucleoprotein (NP) molecules  NS gene . PNA 2 cd linker . Based oQ508865) . PNA 5 h1–6 in 10 mM Na2PO4 buffer (pH 6.9) containing 10% lysis solution for 30 min at 25°C. The affinities of PNA 1–6 for the vRNAs were evaluated from their inhibitory effects on cDNA synthesis by MuLV reverse transcriptase. The NS gene of the H1N1pdm virus was reverse-transcribed into cDNA by primer 1 and primer 2, designed for the upper region of the PNA binding site. Thus, cDNA synthesis would terminate if the PNA strongly bound to the vRNA. The resulting cDNA was diluted 50-fold with distilled water and then amplified by polymerase chain reaction (PCR) using either primer 1 and 3 (which contains the PNA binding site) or primer 2 and 4 (which does not contain the PNA binding site)  virus and contain poly-pyrimidine bases, also did not inhibit reverse transcription of the NS gene of the H1N1pdm virus  virus was propagated in chicken embryonated eggs and purified by sucrose gradient ultracentrifugation. For the PNA binding assay, the viruses were incubated with PNA ng site) . The PCRng site) . Below 4dm virus . PNA 2 scription . The subdm virus . Thus, P1–3 on an enzyme-linked immunosorbent assay (ELISA) plate . Briefly, primary amine groups of the lysine at the end of the PNA sequences were covalently linked to the surface of a 96-well plate. Each influenza virus solution in 10% lysis buffer was added to the PNA-immobilized plate for 1 h at ambient temperature. Since influenza A virus RNA forms a stable RNP complex with the nucleoprotein (NP) To evaluate whether these PNAs can capture the NS gene, we immobilized 0.5 µg of PNA 1 did not capture the NS gene of A/H1N1pdm under these conditions , A/Panama/2007/99 (H3N2) or B/Yamanashi/166/98 virus. A/H1N1pdm was also incubated in PNA non-modified wells to evaluate non-specific binding of the NS gene to the well. We confirmed that PNA 3 selectively captured the NS gene of the influenza A/H1N1pdm virus  as substrates. Purple dye production increased in a virus concentration-dependent manner . The dettients 3 , while ntients 3 .The binding of oligonucleotides to influenza A virus genes is often hindered by the RNA tertiary structure and by bound proteins File S1Includes figures S1–S13.(DOC)Click here for additional data file.


Osteoporotic fractures impose a huge economic burden on society. Though several cost of illness studies from other countries exist, no equivalent study has been conducted in Austria. Our study aims at assessing costs resulting from osteoporotic fractures in Austria in the year 2008 from a societal perspective.We took both direct and indirect costs into consideration. Direct costs encompass medical costs such as expenses for pharmaceuticals, inpatient and outpatient medical care costs, as well as other medical services . Non-medical direct costs include transportation costs and medical devices . Indirect costs refer to costs of productivity losses due to absence of work. Moreover, we included costs for early retirement and opportunity costs of informal care provided by family members. For our analysis, we combined data of official statistics, expert estimates as well as unique patient surveys that are currently conducted in the course of an international osteoporotic fracture study in Austria.For the year 2008, the total annual financial burden incurred by osteoporotic fractures in Austria amounted to approx. €685.2 million, the largest fraction of which was due to the opportunity cost of family care (30.2%), followed by costs for hospitalization (26.6%).The financial burden of osteoporotic fractures in Austria is substantial. Our findings may have implications for future economic analyses, and also support health care authorities in their decision making. Osteoporosis is defined as a „systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture” . The aveAssessment of the national financial burden of osteoporosis is usually undertaken by means of cost-of-illness studies , and sevAustria counted some 8.3 million inhabitants at the beginning of 2008. Similar to many other European countries, Austria’s age pyramid at the beginning of the twenty-first century shows a narrow base due to a reduction in birth-rates, whereas the percentage of the senior population 50 years of age and older, is increasing. Within the elderly Austrian population, the number of women clearly exceeds the number of men which is due not only to the higher life expectancy of women, but also to the large number of men who died in World War II.The incidence of osteoporotic fractures in the Austrian population has been estimated to be among the highest worldwide . For exaThe aim of the present study was to estimate the financial burden of osteoporotic fractures in the Austrian population, including fractures of the hip, vertebrae, distal forearm, humerus, and ribs.To estimate the financial burden, both direct and indirect costs related to osteoporotic fractures have been considered, except costs for rehabilitation. Direct costs in general encompass medical as well as non-medical costs directly related to the causative disease. Accordingly, we included costs for pharmacotherapy, hospital and nursing home, as well as costs for (follow-up) radiographs, wheel chairs, crutches, transport to outpatient settings etc. In contrast, indirect costs, sometimes also referred to as productivity costs, are a measure of present and/or future productivity losses caused by a disease. Indirect costs were calculated according to the human capital approach . Hence, http://www.medscinet.com/icuros/project.aspx]. To assess sources and quantities of costs for the prevailing study, an interim analysis of the Austrian study arm of the ICUROS was performed. Data were extracted from a total of 916 patients who had finished phase II of the study, i.e. who had at least one follow-up interview within the first year after the fracture. Of these patients, 488 had a hip fracture, 158 had a clinical vertebral fracture, 140 had distal forearm fracture, and 122 had a fracture of the humerus. Information on following parameters was obtained: Number of outpatient visits at the primary care level and/or hospital, number of home visits, number of phone counselling, use and dosage of drugs, number and type of community services and other services (e.g. nursing), information on working situation, type of employment , number of sick days, informal care, information on investments directly or indirectly caused by fracture and its consequences .Source and quantity of costs following the first year after fracture have been obtained from the Austrian branch of the International Costs and Utilities Related to Osteoporotic Fractures Study (ICUROS). The ICUROS is an ongoing international study supported by the International Osteoporosis Foundation (IOF) which aims at assessing the consequences of osteoporotic fractures in terms of costs and health related quality of life (QOL) in a standardized form, making results comparable among the different participating countries worldwide [data unpublished, Depending on the source of costs, costs themselves where then obtained from institutions and services which most likely would have readily accessible the according information.The number of patients with osteoporotic fractures was calculated for the entire Austrian population. The following types of fracture were included: hip fracture, humeral fracture, clinical (symptomatic) vertebral fracture, distal forearm fracture, and rib fracture. Since most but not all of the fractures may have been due to osteoporosis per se, specific weighting factors have been applied .The number of patients with hip fractures in Austria in 2008 was obtained from the Austrian Hospital Discharge Register (AHDR) for the entire population. As one hundred percent of people who sustain a hip fracture are admitted to hospitals, the number obtained from the AHDR is equivalent to the total number of patients who sustained a hip fracture in 2008. To correct for possible multiple registrations (within a year after admission) for the same diagnosis, a correction factor (0.9) has been applied. These data have been published recently .The number of patients with humeral fractures admitted to hospitals in Austria in 2008 has been drawn from the AHDR. In contrast to patients who sustain a fracture of the hip, only a part of those who sustain a humeral fracture are admitted to hospitals. The proportion of patients who have been treated in an outpatient setting was assessed by extracting databases from seven large Trauma Units in Austria who covered about 20% of all humeral fractures that occurred in 2008 nationwide. Based on the data obtained from these Trauma Units, it has been estimated that the proportion of humeral fracture patients who have been admitted to hospitals (and hence captured by the AHDR) is 57%. An according correction factor was applied.The number of patients with clinical vertebral fractures who have been admitted to hospitals in Austria in 2008 was derived from the AHDR. However, since not all patients with clinical vertebral fractures are admitted to hospitals, this number would reflect only part of the total number of patients. According to data derived from a recently published Swedish study, it was estimated that the number of patients with clinical vertebral fractures admitted to hospitals account for approximately 10% of all female patients with clinical vertebral fractures, and 15% of all male patients nationwide . To estiThe number of patients with forearm fractures who have been admitted to hospitals in Austria in 2008 was derived from the AHDR. Again, this number would reflect only part of the total number of patients who had sustained a forearm fracture, since many of these patients may be treated in an outpatient setting. Several studies have indicated that in general only approximately 20% of patients with distal forearm fractures are admitted to hospitals and treated in an inpatient setting, e.g. for the purpose of operative intervention -19. To eThe number of patients with rib fractures who have been admitted to hospitals in 2008 in Austria was derived from the AHDR. Again, this number would reflect only part of the total number of patients who had sustained a rib fracture that came to clinical attention . To estiData on the average duration of stay in hospital per fracture type  was obtained from the ICUROS, and/or provided by Statistics Austria. To estimate the total number of days of stay in hospital, the number of days of stay in hospitals per fracture type was multiplied by the number of patients who had sustained an according fracture under consideration of correction factors where applicable (see above), and results obtained from the different fracture types were then summarized.Data on costs per day of stay in hospital were provided by the Austrian Federal Ministry of Health. In average, in 2008 the cost of one day spent in an orthopaedic or trauma department of an Austrian Hospital was € 592 . This value was then multiplied by the total calculated hospital days of stay which have been caused by osteoporotic fractures.. In average, costs for consultation of an orthopaedist in a hospital outpatient clinic were € 53.55 per consultation, and costs for consultation of an orthopaedist in the primary care setting were € 73.10 per consultation. These costs were then multiplied by the according number of consultations.Costs incurred through consultations of practicing and/or hospital employed specialists (e.g. orthopaedists) were calculated using data provided by the Central Association of Austrian Social Security InstitutionsNumber of radiographs, physical therapies and transportation as consequences of a fracture were obtained from the ICUROS database. Unit costs for radiographs, bone mineral density measurement  and physical therapy were provided by the Central Association of Austrian Social Security Institutions. Average unit cost of BMD-measurement (lumbar spine and hip) was estimated at € 35.00. Average unit cost of a radiograph was estimated at € 80.40, and the average unit cost of a standard physical therapy was estimated at € 30.00. Information on costs of non-emergency transportation was provided by a common Austrian Ambulance Service (Samariterbund). Mean cost per transportation was estimated at € 57.00. Unit cost for wheelchairs, wheeled walkers, crutches, ramps, cranes etc. were obtained through information provided by the according retail stores.Costs of pharmacotherapy were calculated based on the estimated number of incident osteoporotic fractures in Austria in 2008. However, since data from the Austrian study arm of the ICUROS indicates that only about 22% of patients with recent osteoporotic fracture would receive adequate osteoporosis treatment (data unpublished), estimation of costs of pharmacotherapy only took into account this proportion of patients. Furthermore, to further improve accuracy of estimation, we also considered the proportional distribution of osteoporosis drugs as derived from the ICUROS database. For estimation of costs incurred by analgesics, calcium and vitamin D, again data from the ICUROS were extracted which indicated that ~37% of patients following an osteoporotic fracture would receive analgesic therapy, and 40% would receive calcium and vitamin D. Drug prices were based on the 2008 version of the Austrian EKO (Erstattungskodex) [Number of hours of nursing care and informal care as a consequence of osteoporotic fracture was obtained from the Austrian ICUROS database. Information on costs of nursing was provided by Statistics Austria, and the average cost of one hour of nursing was estimated at € 30. Information on number of hours of informal (unpaid) care provided by family members or other persons as a consequence of osteoporotic fracture was obtained from the Austrian ICUROS database. Cost of one hour of informal care was estimated at € 27.50, reflecting the average hourly wage of an Austrian employee .Data regarding number and costs of sick-days due to osteoporotic fractures in the Austrian population were provided by Statistics Austria and the Austrian Pension Insurance Authority. In total, 3.490 men and 2.336 women had been on sick-leave due to such conditions in 2008. The average number of sick-days in these patients was 16.3 in men, and 18.9 in women. To estimate the costs of absenteeism, the mean annual gross salary per capita of Austrian employees was divided by 220 working days, and this number was then multiplied by the average number of osteoporotic-fracture associated sick-days. However, it should be considered that the costs estimated as described above, would rather underestimate the true costs, as wages alone would not include for example loss of profit or the employer’s contribution to the Austrian Social Security institutions. Therefore, to estimate the total loss of added value incurred through absenteeism, data from the official Input–output Table (IOT) were extracted. Data were provided by Statistics Austria. By means of this analysis, information on so called second round effects was obtained. Second round effects typically occur due to reduced productivity in one company or institution (e.g. caused by absenteeism of employees), with the consequence of decreasing output in supplying companies or institutions .According to data provided by the Federal Ministry of Labour, Social Affairs and Consumer Protection, as well as Statistics Austria, a total of 338.462 Austrians were registered as in early retirement due to disability  in 2008 , and 25.The total cost of osteoporotic fractures in Austria from a societal perspective is € 685.6 million Table . The twoIn 2008, an estimated total of 119,911 patients with incident osteoporosis-related fractures were treated in Austria  to private practice offices and hospital outpatient clinics for all fractures was 252,229 and 306,127, respectively , risedronate 3.6%), ibandronate (3.6%), zoledronate (1.4%), parathyroid hormone , Raloxifene (0.4%), strontium ranelate (0.4%), or calcitonin nasal-spray (0.4%)  were spent on hospitalisation, followed by ambulatory care , and pharmacotherapy . 34% of the total costs comprise indirect costs with estimates of informal care being the biggest part . Osteoporosis is also associated with costs due to lost economic value-added which amounts to approx. €40 million.Although costs of osteoporotic fractures have also been estimated in other cost-of-illness studies, these estimates cannot be directly compared because of differences in resource use and patterns of care as well as different price levels of the treatment of fractures. However, these studies confirm that with respect to direct costs, the most costly fractures are hip fractures followed by vertebral fractures -28.Our study, which also took into account nursing and informal care costs derived from the ICUROS data on utilisation of these resources, shows that vertebral fractures are associated with the highest proportion of these cost factors, which is in line with the results from the KOFOR study .Most published studies on costs of osteoporosis used only hospitalization rates and applied internationally accepted attribution factors ,10,28-32There are, however, several methodological issues to be considered in interpreting the results of our study. Although it is mandatory for all hospitals in Austria to record discharge diagnoses by using the code classes of the International Classification of Diseases (ICD-10), it is possible that patients have multiple registrations for the same diagnosis. Based on this, Dimai et al. used a correction factor of 0.9 to adjust the incidence calculations for hip fractures in Austria . Since tAnother limitation of our study may be the method used to estimate those fractures, which are only partially treated in hospitals. For instance, it has been estimated that only one in three of those who experience a vertebral fracture will come to medical attention . From thUnfortunately, our study has not been designed to include costs for rehab. However, we believe that this limitation of our study would not impose a significant bias, since e.g. in Germany only 4% of total expenses are incurred by rehabilitation costs .In contrast to the high expenditures, significant progress in the treatment and prevention of osteoporosis has been achieved in the past two decades with the development of different compounds with a proven effect on bone mineral density and reduction of fracture risk -36.Despite of this, approximately 50% of patients do not follow their prescribed treatment regimen and/or discontinue treatment within one year . Poor coThe results of our cost-of-illness study show that osteoporosis is a costly disease with a significant financial burden to the Austrian society. To improve the awareness of osteoporosis being not only an economic problem, but also a determinant of quality of life in patients affected, appropriate public health strategies need to be applied. In this regard, the substantial socio-economic burden resulting from osteoporotic fractures underlines the importance of pharmacological treatment in those who are at risk of osteoporosis, to prevent first and subsequent fractures.The research project was financially supported by Amgen Austria.HD carried out the epidemiological studies, and drafted the manuscript. KR participated in the epidemiological studies. MP participated in the coordination of the study, and contributed to the draft. FB performed statistical analyses and participated in the design of the study. US participated in the design and coordination of the study. RM participated in the coordination of the study, participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.


Anopheles maculipennis s.l. mosquitoes the apparently nearly complete genome of one previously undescribed virus related to chronic bee paralysis virus, and, from a pool of Ochlerotatus caspius and Oc. detritus mosquitoes, a nearly complete entomobirnavirus genome. We also reconstructed long sequences (1503-6557 nt) related to at least nine other viruses. Crucially, several of the sequences detected were reconstructed from host organisms highly divergent from those in which related viruses have been previously isolated or discovered. It is clear that viral transmission and maintenance cycles in nature are likely to be significantly more complex and taxonomically diverse than previously expected. We investigated whether small RNA (sRNA) sequenced from field-collected mosquitoes and chironomids (Diptera) can be used as a proxy signature of viral prevalence within a range of species and viral groups, using sRNAs sequenced from wild-caught specimens, to inform total RNA deep sequencing of samples of particular interest. Using this strategy, we sequenced from adult  The emergence of new infectious diseases, many of which are caused by RNA viruses, is a major threat to human, animal and plant health and agriculture. RNA viruses demonstrate remarkable capacity to evolve due to large population size, short generation times and high mutation and recombination rates. Many are also vector-borne, potentially increasing the prevalence and range of a given virus in natural ecosystems. Understanding the mechanisms underlying viral emergence is key for the rational design of antiviral therapies and control strategies. A first step in this process is the characterisation of the true distribution of virus genetic variability, both spatially and taxonomically across different host species. Recent research has demonstrated that there exists a vast diversity of previously undiscovered viruses in the natural environment . For exaStegomyia aegypti [=Aedes aegypti] mosquitoes infected with dengue virus (DENV) or Sindbis virus (SINV), Culex quinquefasciatus mosquitoes orally exposed to West Nile virus (WNV) and Drosophila infected with flock house virus (FHV) . The first isolations of ISFs from the natural environment include CFAV from Puerto Rico and Kamiti River virus (KRV) from Kenya [Culex flavivirus (CxFV) has been isolated and characterised from Culex mosquitoes from Asia, the Americas and Africa [Aedes flavivirus (AeFV) from Stegomyia species in Japan [Culex tritaeniorhynchus in Vietnam [Mansonia africana nigerrima in Uganda [Culex, Aedimorphus, Ochlerotatus and/or Stegomyia that may represent DNA integrations into genomes of mosquitoes [Culex mosquitoes [Culex genome will eventually be found to contain some integrated flaviviral-like sequence in common with Aedimorphus, Ochlerotatus and/or Stegomyia species [We identified a partial flavivirus-related sequence in adult chironomids. The sequence was most closely related to Culex flavivirus and other ISFs. Cell fusing agent virus (CFAV) was the first ISF to be discovered over 30 years ago during laboratory studies, in the supernatant medium from a om Kenya ,2. More d Africa ,62,62,64in Japan , Quang B Vietnam  and Nakin Uganda , which an Uganda . Our stusquitoes ,72,73,74squitoes . They sc species . The ISFCulex pipiens from a laboratory colony [bona fide viruses. Such controls must be incorporated in future studies, namely testing via (i) lack of reverse transcriptase and (ii) DNase-treatment of samples.Regardless of whether the ISF-like sequences detected in the current study originate from virus infection or genome integrations, either would likely result from a long history of contact and interaction between ISF-like viruses and a range of insect taxa. We may hypothesise a common source of virus association or infection for all three dipteran groups , potentially related to a shared environment and/or transmission and maintenance cycles. Previous studies have presented evidence for vertical transmission of ISFs: CFAV was isolated from both sexes of a number of mosquito species  and the y colony . Chironoy colony  and sharBunyaviridae in adult culicine mosquitoes. The contig contained a single long ORF that showed greatest similarity to members of the genus Phlebovirus, and the currently unclassified GOUV which was originally detected in specimens of the mosquito genera Anopheles (anophelines), Culex and Uranotaenia (culicines) in West Africa. Here, we provide evidence for a related novel virus from mosquitoes collected in France. Further field studies are necessary to allow the potential full isolation of this strain, which considering its divergence from GOUV, may constitute a novel genus within the family Bunyaviridae. In addition, we detected sequences in adult anopheline mosquitoes and adult culicine mosquitoes with similarity to the orbivirus VP1/RdRp-encoding segment (segment 1). Orbiviruses infect a range of hosts, including cattle, goats and sheep. The three economically most important orbiviruses are bluetongue virus, African horse sickness virus and epizootic hemorrhagic disease virus, all of which are transmitted by Culicoides species (midges of the dipteran family Ceratopogonidae). The detection of novel orbivirus sequences in both culicine and anopheline mosquitoes may warrant further study, with the aim of viral isolation and full characterisation. From adult chironomids and adult culicine mosquitoes, we also identified sequences related to Spissistilus festinus virus 1 (SpFV1) and Circulifer tenellus virus 1 (CiTV1). SpFV1 and CiTV1 were originally isolated from hemipteran insects  whereas the current study provides evidence for related sequences present in dipteran insects (both chironomids and culicine mosquitoes). We also detected a sequence related to the large segment of members of the family Culex flavivirus CxFV, Nakiwogo virus NAKV and Kamiti River virus KRV (flaviviruses), related to isolates first detected in the laboratory, e.g. DXV and CFAV. First, these novel naturally occurring strains provide useful comparisons for research on cell culture adapted strains such as DXV, and tools to examine the response of multi-passaged insect cell lines such as C6/36. In the wider picture, it is becoming clear in an increasingly large number of viral families that there is a diverse and highly prevalent background of transmission and maintenance of numerous viral strains across a range of host taxa against which known pathogens are evolving. The relevance of this genetic diversity in terms of potential competitive exclusion, superinfection or potential recombination events requires extensive study.We see parallels among many of the viral families encountered in this study, particularly the entomobirnaviruses and flaviviruses, which include recently discovered potential strains of highly divergent viruses in a range of species, e.g. ESV, CYV and MXV (entomobirnaviruses) and bona fide viruses from RNA transcripts of genome-integrated virus sequences. In the cases of the CBPV-like virus AACV and the entomobirnavirus CAZV, the presence of both segments, the high coverage, the presence of multiple well-supported (and often synonymous) polymorphisms and the presence of terminal motifs strongly suggest that these sequences derive from bona fide replicating viruses. The origin of the other sequences identified is less certain. Further investigations aimed at the isolation and full characterisation of viruses from a wide range of insects is a priority. The narnavirus-, mitovirus- and partitivirus-like sequences obtained in the current study may derive from fungal (or other) contaminants of the insect preparations, either external or internal (e.g. insect gut contents). Similarly, a number of plant-virus-like sequences (not reported) were detected in samples 7 and 9. During the analysis, we found a number of other virus-related sequences (not reported) that appeared to have fragmented ORFs , and some of these likely represent transcribed genome-integrated sequences. A large number of rhabdovirus-like sequence fragments were identified but were not further analysed as genome integration of rhabdovirus-like sequences has been shown to be particularly common in arthropod genomes [Although the sequences identified in the present study are certainly of viral origin, our methods do not allow us to definitively distinguish sequences of  genomes ,45. bona fide (non-integrated) virus sequence, e.g. if the host species sample preparation is not genetically uniform or if related sequences have been integrated at multiple sites within the same genome. Once integrated, such sequences evolve at a rate many orders of magnitude more slowly than replicating RNA viruses, and thus such sequences may provide a window on the time-scale of evolution of RNA viruses, which appears to occur at a much slower rate than what one would infer from extrapolating short-term evolutionary rates [In some cases, genome-integrated sequences appear to provide a functional benefit to the host, and are retained (exaptation) and may be subjected to purifying selection ,76,77. TMost of the sequences we identified contained the viral RdRp. This may be because the RdRp tends to evolve more slowly than other regions of an RNA virus genome; hence, it is easier to identify the RdRp in highly divergent viruses based on blastp analysis. This is also true for integrated sequences: whereas the RdRp of ancient RNA viruses, e.g. 10-100 mya, may resemble the RdRp of extant RNA viruses, other proteins of integrated ancient RNA viruses may not be detectable by blastp comparison to extant viruses. It is possible that applying structure-based homology search tools, such as hhpred , to our a priori knowledge. Except for the entomorbirnavirus-related sequences, all exhibited low amino acid sequence identities (24-42%) to known viruses.In this study, we report the discovery of viruses in wild-caught dipteran insects using deep sequencing of RNA. In our approach, total small RNAs were isolated from a field collection, sequenced via Illumina and assembled into contigs via Velvet and analysed using a custom-built bioinformatics workflow process. Results were used to inform total RNA sequencing of samples of particular interest . This strategy enabled the construction of two complete or nearly complete virus genomes and numerous other novel virus sequences with minimal