Here, it was applied to the pathogenic flavivirus, tick-borne encephalitis virus (TBEV) which causes febrile illness and encephalitis in humans in forested regions of Europe and Asia. Using an infectious clone of the Oshima 5–10 strain ("wild-type virus"), a cassette of 1.4kb located in the NS5 coding region, was modified by randomly introducing 273 synonymous mutations ("re-encoded virus"). Whilst the in cellulo replicative fitness of the re-encoded virus was only slightly reduced, the re-encoded virus displayed an attenuated phenotype in a laboratory mouse model of non-lethal encephalitis. Following intra-peritoneal inoculation of either 2.105 or 2.106 TCID50 of virus, the frequency of viraemia, neurovirulence (measured using weight loss and appearance of symptoms) and neuroinvasiveness (detection of virus in the brain) were significantly decreased when compared with the wild-type virus. Mice infected by wild-type or re-encoded viruses produced comparable amounts of neutralising antibodies and results of challenge experiments demonstrated that mice previously infected with the re-encoded virus were protected against subsequent infection by the wild-type virus. This constitutes evidence that a mammalian species can be protected against infection by a virulent wild-type positive-stranded RNA virus following immunisation with a derived randomly re-encoded strain. Our results demonstrate that random codon re-encoding is potentially a simple and effective method of generating live-attenuated vaccine candidates against pathogenic flaviviruses.Large-scale codon re-encoding ( Flavivirus) is transmitted by ticks of the Ixodes genus. TBEV causes febrile illness and encephalitis in humans in forested regions of Europe and Asia. The incidence of TBE is increasing across Central and Eastern European countries despite the availability of several licensed inactivated vaccines and appropriate vaccination programmes. Large-scale codon re-encoding, a recently developed attenuation method that modifies viral RNA nucleotide composition of large coding regions without alteration of the encoded proteins, has been successfully applied to a variety of RNA viruses. In contrast with previous empirical methods of generating live attenuated vaccines, large-scale codon re-encoding facilitates rapid generation of vaccine candidates using reverse genetics methods, by direct control of the attenuation phenotype. Additional benefits include reduced costs and induction of long-term immunity. Here, we have applied the large-scale codon re-encoding method to the TBEV to demonstrate the principle of developing a live attenuated virus vaccine which protects mice against subsequent infection with the wild type virulent virus. This study therefore illustrates that codon re-encoding is potentially an easily derived and effective method of producing live attenuated vaccine candidates against positive-stranded RNA viruses.The arbovirus Tick-borne encephalitis virus (TBEV; genus  Flavivirus (family Flaviviridae) includes important human pathogens such as yellow fever virus (YFV), dengue virus (DENV), Japanese encephalitis virus (JEV), West Nile virus and tick-borne encephalitis virus (TBEV). Flaviviruses are enveloped, single-stranded positive-sense RNA viruses with virions, close to 50nm in diameter, and a viral genome of ca. 11 kb which includes a unique open reading frame (ORF) encoding structural (C-prM-E) and non-structural proteins (NS1–2A-2B-3–4A-4B-5)  or 2.106 TCID50 [high dose] of virus. Clinical monitoring included (i) the clinical manifestation of the disease  and (ii) the body weight curve  of either WT_IC virus or NS5_Reenc_IC virus. A control group of 16 mice was inoculated with PBS. Mice were weighed daily and monitored for the appearance of symptoms during the 20 day period of observation. Six mice per group were periodically euthanized to perform virus detection in sera and brains ).Four groups of 60 mice were inoculated with two doses  or WT groups of mice . In all groups, the highest proportion of viraemic animals was observed 3 to 7 days p.i. Viral RNA yields found in sera were heterogeneous and no significant difference was found in term of inoculation dose or strain of virus (WT_IC or NS5_Reenc_IC viruses) inoculated . TBEV-specific immunoglobulin G (IgG) antibodies were detected using a commercial diagnostic ELISA kit. High antibody titres were detected in mice infected by both the WT and the re-encoded viruses. Mice in the control group were negative   . Sero-neol group .6 TCID50 of WT_IC virus, and the protection was evaluated by determining for each group the proportion of mice (i) viraemic at day 2 post-challenge, (ii) with brains positive for the presence of TBEV at day 12 post-challenge and (iii) with a weight loss of more than 6% of the weight at days 11 and 12 post-challenge. Two control groups of mice were used: one group of 8 mice inoculated with PBS and then challenged with virus as described above (mock group), and one group of 4 mice inoculated and challenged with PBS .Forty days after the inoculation of two groups of mice with the NS5_Reenc_IC virus and two groups of mice with the WT_IC virus (at both high and low doses for both viruses), 8 mice per group were challenged with 2.10p = 0.03 for each dose). Amongst all the weighing realised at days 11 and 12 post-challenge . We found similar results with the challenged mice previously inoculated with the WT_IC virus: only one mouse inoculated with 2.105 TCID50 of WT_IC virus was below the detection threshold, only at day 12 post-challenge. In contrast, 87.5% of the weighing realised with the mice of the mock group were under this threshold .Protection was 100% effective in terms of viraemia, for challenged mice immunised with the NS5_Reenc_IC virus or the WT_IC virus (both doses). In contrast, all mice in the mock group were viraemic  and 75% (3/4) respectively of the challenged mice previously vaccinated with high and low doses of NS5_Reenc_IC virus. To determine whether or not the detected virus was the result of persistence in the brain, we quantified viral RNA yields with our two specific quantitative RT-PCR assays that were used previously in competition experiments (see above). For the challenged mice previously infected by the NS5_Reenc_IC virus, we found that the viral RNA present in the brain at day 12 post-challenge was exclusively the NS5_Reenc_IC virus but we failed to isolate the virus from these brain samples. Therefore, we obtained a protection of 100% in terms of neuroinvasion by the virus used for the protection experiments . However, for the mice vaccinated with the WT_IC virus, because the same virus was used to infect and challenge these mice, we could not draw valid conclusions in this instance.Surprisingly, we detected viral RNA in the brains of 100% of the challenged mice previously inoculated with the WT_IC virus and 100% of mice of the mock group. Viral RNA yields found in brains ranged from 10In cellulo competition experiments had shown that WT_IC virus had a higher replicative fitness than NS5_Reenc_IC virus (see above). We therefore conducted in vivo competition experiments to determine whether or not the loss of replicative fitness due to re-encoding had an impact on viraemia and viral yields in the brain. Two initial TCID50 ratios  were used to inoculate two groups of six mice. At days 3 and 5 p.i., one group of mice per initial TCID50 ratio was sacrificed and the relative proportions of each viral genome in sera and brains was estimated using RT-PCR as reported in the earlier example of in cellulo competition assays , a pathogenic TBFV that causes febrile illness and encephalitis in humans. Encephalitic flavivirus infections provoke CNS pathology which can be correlated with the observed morbidity and mortality: viruses replicating in the CNS induce direct neuronal damage causing severe CNS dysfunction often involving long-term neurological sequelae in non-fatal cases [We have evaluated al cases ,40,41. Ial cases ,43,44,45in vivo studies in a mouse model that faithfully mirrors many aspects of the infection in humans. Our experiments showed that decreased replicative fitness (as determined by in cellulo competition assays and in vivo viraemia measurement) was associated with reduced neuroinvasiveness as previously described [In vivo competition experiments shed further light on the mechanisms of pathogenesis: when a 50/50 (WT/Reenc) TCID50 initial ratio was used, as expected, the wild-type virus rapidly out-paced the re-encoded virus in blood and was the only virus detected in mouse brains. However, when a 10/90 (WT/Reenc) TCID50 initial ratio was used, the re-encoded virus was the only one detected in brain, suggesting an early and selective neuroinvasion process: only the majority virus present in blood during the first hours of the viraemia invades the central nervous system. Of course, more experiments are needed to confirm these findings and the potential role of viral interferences have to be assessed [Here, an infectious clone of a neurovirulent strain of TBEV (Far Eastern subtype) was used to perform escribed . In vivoassessed ,48.In this model, TBEV infection is frequently associated with asymptomatic persistence of the virus in mouse brain as previously described ,39 and c(i) to analyse the effect of genome random re-encoding on the fitness and clinical phenotype of a virulent TBEV strain and (ii) to perform a complete set of in vivo experiments including immunisation with a re-encoded virus and follow-up challenge experiments with an infectious wild-type virus. It has previously been demonstrated that large-scale re-encoding generates attenuated viruses and the studies support the proposal that relative degree of attenuation or replicative fitness can be regulated by modulating the number of introduced synonymous mutations [in cellulo was highly attenuated when tested in a mouse model and showed potential for use as a vaccine candidate [in cellulo attenuation [in cellulo and thus provide a relevant candidate to study the relative degree of in vivo attenuation. Accordingly, we introduced 273 random synonymous mutations in the NS5 gene of a neurovirulent strain of TBEV , with limited modification of G+C content or codon bias of the genome. This re-encoding protocol produced a virus variant that displayed no fitness difference when growth kinetics were compared with WT TBEV in mammalian cell cultures. However, more sensitive in cellulo competition experiments revealed that the replicative fitness of the wild-type virus was indeed higher than that of the re-encoded virus. Moreover, in vivo experiments in immunocompetent mice fully validated our starting hypothesis: the re-encoded virus could reproduce efficiently in mice but in competition experiments the wild-type virus had a significantly higher fitness. In vivo experiments also revealed the attenuated characteristics of the re-encoded virus, namely reduced neurovirulence in terms of weight loss and appearance of neurological symptoms and reduced neuroinvasiveness (i.e. lower proportion of mice with virus in the brain). It can therefore be concluded that the re-encoding process, although restricted to the NS5 gene, decreased the pathogenicity and led to the production of an attenuated phenotype of the normally highly virulent strain of TBEV.The main objectives of this study were utations ,25,26,27i.e. infected by either the wild-type or the re-encoded virus. Moreover, no significant differences in antibody titres were observed between mice infected with either virus. Thus, mice “immunised” by the re-encoded virus were likely to have been protected against subsequent TBEV infection. This was verified at 40 days post-infection by challenging the “immunised” mice using a high dose of intraperitoneally administered wild-type virus. Protection was effective in terms of viraemia, neurovirulence and neuroinvasion. Likewise, inoculation of CD155 tg mice with re-encoded strains of poliovirus induced the production of neutralising antibodies and protects the mice against a subsequent challenge with a lethal dose of virus [Concerning the mouse protection experiments, the results showed that, at 40 days post-infection, neutralising antibodies were produced by 100% of mice, of virus .in vivo attenuated strains of TBEV and infection of mice by re-encoded viruses can induce neutralising and protective immune responses against challenge with virulent homologous viruses. This represents evidence that a positive-stranded randomly re-encoded RNA virus could be developed and trialled as a potential vaccine to protect humans and/or animals against viral pathogens.The results provide a robust proof of concept: large-scale random re-encoding can be used to produce Many current virus vaccines were derived empirically and carry an inherent risk of vaccine-associated complications. Our findings open up new perspectives for the development of new-generation custom-designed re-encoded live-attenuated vaccines which are potentially safe, induce high levels of protective immunity and are relatively easy to produce.in vivo modification of the clinical picture provided by genome re-encoding. The results strongly suggest that re-encoding could be used in the future for attenuation of highly pathogenic viral species. However, it would be wiser and more practical in the specific case of TBEV, to develop a live attenuated re-encoded vaccine using a strain known to have a naturally lower association with neurovirulence and by inserting additional synonymous mutations in coding regions with a view to reducing the encephalitic potential of the virus and thus produce a potentially safer vaccine candidate. Whilst effective inactivated vaccines are available to prevent TBEV infections, the use of a live attenuated vaccine may have specific advantages, e.g. long-term protection and reduced costs [The use of a highly neurovirulent strain of TBEV in our experimental model enabled us to identify the ed costs .2 in Minimum Essential Medium with 7% fetal calf serum (Life Technologies) and 1% Penicillin/Streptomycin . Five-week-old C57Bl/6J mice females were provided by Charles River laboratories.Baby hamster kidney BHK21 (BHK21) cells  and mouse (L929) cells  were grown at 37°C with 5% COAnimal protocols were reviewed and approved by the ethics committee “Comité d’éthique en expérimentation animale de Marseille—C2EA—14” (protocol number 2504). All animal experiments were performed in compliance with French national guidelines and in accordance with the European legislation covering the use of animals for scientific purposes (Directive 210/63/EU).i.e. CGU, CGC, CGA, CGG, UCG, CCG, GCG, ACG), and unique restriction sites were conserved  (We modified a previously described IC of the Oshima 5–10 strain  GenBank. The re-Biolabs) , gel purComplete open reading frames of TBEV (n = 85) and other TBFV (n = 56) were manually extracted from GenBank . After incubation for 6 hours, cells were washed twice with Hank’s Balanced Salt Solution  and incubated until appearance of complete cytopathic effect (CPE). Cell supernatant medium was harvested, clarified by centrifugation and stored at -80°C. Each virus was then passaged in BHK21 cells at a calculated moi of 0.5 in a 175cm2 culture flask: after adsorption of the virus for 2 hours, the cells were washed twice (HBSS) and 50mL of medium was added and the flasks were incubated at 37°C for 72 hours. Cell supernatant media were harvested, clarified by centrifugation, aliquoted, stored at -80°C and used to perform in cellulo experiments. A similar experimental procedure was also carried out using L929 cells and the resulting cell supernatant medium was aliquoted, stored and used to perform the in vivo experiments. The integrity of the genome of all the viruses produced to perform in cellulo and in vivo experiments was verified using sequencing methods (Sanger methods).ICs were transfected into a 12.5cm2 culture flask of confluent BHK21 cells. Cells were washed twice (HBSS) 30 minutes after the infection and 7mL of medium was added. 800μL of cell supernatants were sampled just before the washes and at 2, 5, 10, 15, 23, 31 and 48 hours post-infection. They were clarified by centrifugation, aliquoted and stored at −80°C. They were then analyzed using a TCID50 assay (see below).A calculated moi of 200 or 0.5 was used to infect a 25cm2 culture flask of confluent BHK21 cells at a calculated moi of 0.5. Cells were washed twice with HBSS and then incubated for 48h after addition of 7mL of medium. Recovered infectious cell supernatant was then sequentially passaged 10 times in the same manner with the clarified cell supernatant medium from the previous passage. At each passage, a calculated moi of 1 was used. Aliquots of cell supernatant from each passage were clarified by centrifugation and stored at -80°C. Viral RNA was extracted from clarified culture supernatant medium using the EZ1 Virus Mini Kit v2 on the EZ1 Biorobot (both from Qiagen). Using two specific quantitative real time RT-PCR assays targeting the re-encoded NS5 coding region , the amount of viral RNA was assessed for each virus (WT_IC and NS5_Reenc_IC) and the ratio of the two values (WT_IC/NS5_Reenc_IC) was calculated.As described previously for chikungunya virus WT_IC virus was competed with NS5_Reenc_IC virus : five in5 TCID50 or 2.106 TCID50 of virus. In some experiments .100μL of the filtrates collected from brain suspensions (see above) were used to inoculate a 12.5cmAll quantitative real-time PCR (qRT-PCR) assays were performed with SuperScript III Platinium One-Step qRT-PCR kit. The mix content for a final volume of 25μL per sample, was as follows: a standard quantity of 2x of PCR Mastermix and Enzymes, both primers , probe  and 4μL of extracted nucleic acids. qRT-PCR were performed on CFX96 Real-Time System/C1000 Touch Thermal Cycler (Biorad) with the following conditions: 15min at 50°C, 2min at 95°C, then 45 times 15sec at 95°C and 40sec at 60°C, data collection occurring during this last step. Primers and probe sequences are detailed in S1 Table in For each determination, a 96-well plate culture of confluent BHK21 cells was inoculated with 150μL/well of serial 10-fold dilutions of clarified (centrifugation) cell supernatant medium, mouse sera or mouse brain filtrates: each dilution was repeated 6 times. The plates were incubated for 7 days and read for absence or presence of CPE in each well. Determination of the TCID50/mL was performed using the method of Reed and Muench .Sera were incubated for 30min at 56°C prior to viral serology. TBEV-specific immunoglobulin G (IgG) antibodies were detected using the Anti-TBE Virus ELISA (IgG) kit (Euroimmun). Sera were diluted 1:64 and then 1:101 prior to the first incubation using the Sample Buffer of the kit. Goat anti-mouse IgG antibodies (Invitrogen) diluted 1:2000 in BSA 0.7% (KPL) as secondary antibodies were used. Plates were read using the Sunrise reader (Tecan) at a wavelength of 450nm.Sera were incubated for 30min at 56°C prior to viral serology. For each serum, a 96-well plate culture of confluent BHK21 cells was inoculated with 50μL/well of WT_IC virus  and 50μL/well of a serial 2-fold dilution (first dilution at 1:40) of serum. Each row included 5 wells of serum dilution, a positive control (virus only) and a negative control with neither virus nor serum. The plates were incubated for 7 days and read for the absence or presence of a CPE in each well. The 50% plaque reduction neutralization titre (PRNT50/mL) was determined using the method of Reed and Muench [p values below 0.05 were considered significant.Kaplan-Meier survival analysis with Mandel-Cox’s Logrank tests, Student’s t tests and Fisher’s exact tests were performed using SPSS software package (IBM). S1 TextS1 Fig. Comparison between appearance of at least one symptom and weight loss.S2 Fig. Comparative analysis of qRT-PCR and TCID50 assays.S1 Table. Primers and probes used for the real time RT-PCR assays.S2 Table. List of 85 TBEV sequences retrieved from GenBank.S3 Table. List of 56 TBFV sequences retrieved from GenBank.S4 Table. Results of weighing of challenged mice at days 11 and 12 post-challenge.S1 Note. Re-encoded sequence .(PDF)Click here for additional data file.


It is uncertain whether the extra acquisition costs of atypical antipsychotics over typical antipsychotics are offset by their other reduced resource use especially in hospital services in China. This study compared the psychiatric-related health care resource utilization and direct medical costs for patients with schizophrenia initiating atypical or typical antipsychotics in Tianjin, China.Data were obtained from the Tianjin Urban Employee Basic Medical Insurance database (2008–2010). Adult patients with schizophrenia with ≥1 prescription for antipsychotics after ≥90-day washout and 12-month continuous enrollment after first prescription was included. Psychiatric-related resource utilization and direct medical costs of the atypical and typical cohorts were estimated during the 12-month follow-up period. Logistic regressions, ordinary least square (OLS), and generalized linear models (GLM) were employed to estimate differences of resource utilization and costs between the two cohorts. One-to-one propensity score matching was conducted as a sensitivity analysis.1131 patients initiating either atypical (N = 648) or typical antipsychotics (N = 483) were identified. Compared with the typical cohort, the atypical cohort had a lower likelihood of hospitalization  over the follow-up period. Medication costs for the atypical cohort were higher than the typical cohort ; however, their non-medication medical costs were significantly lower . The total direct medical costs were similar between the atypical and typical cohorts before  and after matching , consistent with the results from OLS and GLM models for matched cohorts.The atypical cohort had similar total direct medical costs compared to the typical cohort. Higher medication costs associated with atypical antipsychotics were offset by a reduction in non-medication medical costs, driven by fewer hospitalizations. Schizophrenia is a complex neurobehavioral disorder that adversely affects a broad range of psychological functions including thinking, perception, ideation, concentration, motivation, and judgment ,2. It afA cure for schizophrenia has not yet been found, but most patients’ symptoms can be improved with pharmacotherapy. Antipsychotic medications are the main stay of treatment used to manage acute psychotic exacerbations and as maintenance therapy to prevent relapse among patients with schizophrenia . The firHowever, the utilization of branded atypical antipsychotics may have been restricted in the years following their launch due to the higher daily acquisition costs, compared to the typical antipsychotics, which are available as generic formulations . Some stThe present study was therefore carried out to evaluate the health service utilization and costs of treatment initiation with atypical antipsychotics in patients with schizophrenia in comparison with typical antipsychotics, as prescribed in the context of routine clinical practice using real-world claims data in China. The first objective was to explore whether initiation with atypical medications was associated with better treatment outcomes compared with typical medications, as measured by better medication adherence and reduced rates of hospitalization. Secondly, the study aimed to explore whether atypical initiators had lower non-medication related medical costs, and if they did, whether the reductions were enough to offset their higher medication costs — in other words, whether atypical antipsychotics represented a cost-effective alternative to typical antipsychotics, based on the principle that a better outcome was achieved for a similar or lower total cost.This retrospective study used the Urban Employee Basic Medical Insurance (UEBMI) Claims of Tianjin from 2008 through 2010, which was obtained from the Tianjin Municipal Human Resources and Social Security Bureau through a formal request for research purposes. The UEBMI system, which is one of three basic medical insurance systems in China, was established in 1998 and covers employees and retirees in both the public and private sectors. The enrollees of the UEBMI in Tianjin included almost 5.1 million unique members by 2010, representing 51.7% of the registered residences in Tianjin city . The anaAdult patients (≥18 years of age) who had a schizophrenia-relevant diagnosis (ICD-10 code F20 supplemented by the Chinese description) and an initial prescription for an antipsychotic between April 1, 2008 and December 31, 2009 were included. The antipsychotic medications investigated in the present study comprised all the typical  and atypical antipsychotics  reimbursed by UEBMI. The index date was defined as the date of patients’ first prescriptions following a 90-day washout period during which patients did not receive any antipsychotics. Atypical and typical cohorts were defined according to patients’ initial prescription regardless of their subsequent switching patterns. Patients who received both atypical and typical drugs at the index date or who were not continuously insured for 3 months before (baseline period) or for 12 months after the index date (follow-up period) were excluded.Psychiatric-related health care resource utilization and direct medical costs for the 12-month follow-up period were estimated. Medical claims under the primary diagnosis of ICD-10 of F00-F99 (supplemented by Chinese description) were identified as psychiatric related. Pharmacy resource utilization included the use of any antipsychotics and concomitant medications including anxiolytics, anticholinergics, antidepressants, sedative-hypnotics medications, and mood stabilizers. Adherence was assessed by measuring the days on antipsychotics, which were the total number of non-overlapped days covered by antipsychotics during follow-up period. Persistence with antipsychotics was defined as the time to discontinuation for any cause and calculated as the number of consecutive days from the index date to the first medication gap of >30 days . MedicalPsychiatric-related costs were calculated from the payer’s perspective including the payment paid by the insurance and the patients. Direct medical costs were calculated as the sum of medication costs and non-medication medical costs. Medication costs were composed of antipsychotic medications, concomitant medications, and other medications. Non-medication medical costs corresponded to the use of any other health care services except medications, including the costs of both inpatient and outpatient services. The costs of inpatient services were further broken down into non-medication treatment costs, laboratory and diagnostic costs, and the costs of other medical services. Specifically, non-medication treatment costs corresponded to the costs of any other treatment except medications, which included nursing, monitoring, psychotherapy, behavioral training and intervention, etc. Laboratory and diagnostic costs referred to the costs of physical examinations and biochemical tests. Costs of other medical services included blood transfusions, medical consumables, air conditioning and heating fees.Descriptive analyses were undertaken to evaluate differences in baseline characteristics and outcomes between the atypical and typical cohorts. Group comparisons were performed using two-sample t-tests for continuous variables, and chi-square tests or Fisher’s exact tests for categorical variables.Multiple regression procedures were further employed to test for differences of resource utilization and costs between the two cohorts. Logistic regression models predicting the presence of any medical or concomitant pharmacy utilization in the follow-up year were developed. Ordinary least square (OLS) models and generalized linear models (GLM) with a log link and a negative binomial distribution were both used to estimate the differences in total costs and cost components. Negative binomial distribution was employed to take into account the over-dispersion of the cost data. Modified Park tests indicated that this specification was adequate relative to other types of distributions such as the Gamma or Poisson distribution . The incAll regression models adjusted for patient characteristics and indicators of disease complexity at baseline. Specifically, the following types of patient-level variables were used as control variables in each model: demographics , mental comorbidities , concomitant medications , mean number of psychiatric-related hospitalizations and outpatient visits and the total psychiatric-related direct medical costs during the prior 3 months.Statistical analyses were conducted using STATA 12.0. A P-value of <0.05 was considered as statistically significant. All costs were converted to USD with an exchange rate in 2009 .In observational studies, selection bias can be a potential limitation in evaluating the outcomes of alternative interventions because patients are not randomly assigned to different therapies. Accordingly, a sensitivity analysis using propensity-score matching, which can control observable selection bias and render the two treatment cohorts more comparable, was conducted to test the robustness of the estimated outcomes. In the current study, the propensity score is a subject’s probability of receiving atypical antipsychotics on the observed covariates. It was calculated with a binary logistic regression which controlled for all independent variables in multivariate analyses. A greedy, one-to-one matching algorithm without replacement was employed to form pairs of atypical and typical cohorts. With the non-replacement technique, when an atypical patient is matched to a typical patient, both cases are removed from the pool. The caliper width was narrowed down progressively from 0.2*δ (0.2 of the standard deviation of the estimated propensity score) until all the baseline variables appeared with no significant differences . OutcomeA total of 1131 patients who initiated therapy with atypical (N = 648) or typical (N = 483) antipsychotics with a prior 3-month washout period were identified  may potentially affect the results. Thirdly, all atypical antipsychotics were considered as a homogenous group of drugs in our study, as were all typical antipsychotics. In reality, there are differences in the efficacy, effectiveness, and adverse-effect profiles between individual drugs within the same group ,42. LastThe present study estimated and compared the psychiatric-related health care resource utilization and direct medical costs associated with the initial prescription with atypical antipsychotics compared with typical antipsychotics in the treatment of schizophrenia in patients from Tianjin, China. Medication costs were higher for patients initiated with atypical antipsychotics during the 12-month follow-up. However, the atypical cohort was less likely to be hospitalized compared to the typical cohort, which in turn led to decreased medical costs. Initiation with atypical antipsychotics for patients with schizophrenia did not appear to increase the use of health care services as a whole, and it was associated with a reduction in medical costs that was sufficient to offset the higher medication costs for the atypical antipsychotics.


Costs of severe sepsis care from middle-income countries are lacking. This study investigated direct ICU costs and factors that could affect the financial outcomes.A prospective cohort study was conducted in the medical ICU of a tertiary referral university teaching hospital in Thailand over a 4-year period.A total of 897 patients, with 683 76.1%) having septic shock. Overall ICU mortality was 38.3%. The median (interquartile range) ICU length of stay (LOS) was 4 (2 to 9) days. Community, nosocomial and ICU-acquired infection were documented in 574, 282 and 41 patients, respectively. The median ICU costs were €2,067.2 (986.3 to 4.084.6) per patient and €456.6 (315.3 to 721.8) per day. The ICU costs accounted for 64.7% of the hospital costs. In 2008 to 2011, the ICU costs significantly decreased by 40% from €2,695.7 to €1,617, whereas the daily ICU costs decreased only 3.3% from €463.9 to €448.7 (Figure % having The ICU costs of severe sepsis management significantly declined in Thailand. However, the ICU costs were a financial burden accounting for two-thirds of the hospital costs. It is essential for intensivists to contribute a high standard of care within a restricted budget. The cost-effectiveness analysis should be evaluated in sepsis care cases.


A wide range of viruses from different virus families in different geographical areas, may cause immediate or delayed neuropathological changes and neurological manifestations in humans and animals. Infection by neurotropic viruses as well as the resulting immune response can irreversibly disrupt the complex structural and functional architecture of the central nervous system, frequently leaving the patient or affected animal with a poor or fatal prognosis. Mechanisms that govern neuropathogenesis and immunopathogenesis of viral infections are highlighted, using examples of well-studied virus infections that are associated with these alterations in different populations throughout the world. A better understanding of the molecular, epidemiological and biological characteristics of these infections and in particular of mechanisms that underlie their clinical manifestations may be expected to provide tools for the development of more effective intervention strategies and treatment regimens.The online version of this article (doi:10.1007/s00401-015-1511-3) contains supplementary material, which is available to authorized users. Neurotropic virus infections continue to cause major disease and economic burdens on society, and pose a major challenge to human and animal health care systems due to the associated morbidity and mortality worldwide, and to the unique problems in providing treatment to the patients involved. This is largely due to unique features of the central nervous system (CNS), with a plethora of interconnected and interdependent cell types, complex structures and functions, reduced immune surveillance and limited regeneration capacity. Infection by neurotropic viruses as well as the local induced immune responses can irreversibly disrupt the complex structural and functional architecture of the CNS, frequently leaving the patient or affected animal with a poor or fatal prognosis. Besides immediate and direct effects, there are several neurological disorders often associated with autoimmune mechanisms that are assumed to be delayed virus-induced disorders: multiple sclerosis, Guillain–Barré syndrome, narcolepsy and encephalitis lethargica. Neurotropic pathogens can access the brain by various routes including retrograde axonal transport along motor and olfactory neurons, haematogenous spread across the blood–brain barrier (BBB), blood–cerebrospinal fluid barrier, meningeal–cerebrospinal fluid barrier, via direct infection of endothelial cells or via spread of infected leukocytes across the BBB into the brain parenchyma Fig. . There iAlphavirus of the Togaviridae family comprises a group of enveloped, single-strand positive-sense RNA viruses, most of which are transmitted by mosquitoes. Typically, human infections with old-world alphaviruses such as O’nyong-nyong and Ross River virus manifest as fever, maculopapular rash and polyarthritis, whereas infection with the new-world equine encephalitis viruses (EEVs) can result in potentially fatal encephalitis. Encephalitic alphaviruses of public health concern include the new-world alphaviruses Eastern equine encephalitis virus (EEEV), Venezuelan equine encephalitis viruses (VEEV) and Western equine encephalitis virus (WEEV). More recently, the old-world Chikungunya virus (CHIKV) was also associated with encephalitis. Considering that WEEV cases have become infrequent and sporadic, this chapter focuses on neurologic manifestations of EEEV, VEEV and CHIKV infections.The genus Culiseta melanura. Several Aedes, Coquillettidia and Culex mosquitoes function as bridge vectors, transmitting the virus to equines and humans, which can suffer from severe disease. The related VEE viruses circulate in Central and South America. These viruses are grouped into six antigenic subtypes (I–VI). Human epidemics and equine epizootics are almost exclusively caused by viruses of antigenic variants A/B and C of subtype I. The remaining antigenic variants circulate in enzootic sylvatic cycles between rodents and Culex (Melanoconion) mosquitoes. Subtypes I A/B and C, which are highly virulent for horses, are believed to result from adaptation of enzootic strains to equines . This adaptation allows the virus to replicate to extremely high levels, resulting in 20–80 % mortality [Aedes (Ochlerotatus) taeniorhynchus is considered the major bridge vector of epidemic/epizootic strains. The geographic distribution of CHIKV includes sub-Saharan Africa, India, Southeast Asia, the Western Pacific with recent spread to the Caribbean and South America. The virus circulates in a sylvatic cycle between nonhuman primates and forest-dwelling Aedes mosquitoes. In the urban cycle, Aedes aegypti and Aedes albopictus mosquitoes are responsible for virus transmission to humans.EEEV is enzootic to the eastern United States, the Great Lakes region and the Gulf Coast where it circulates between birds and the ornithophilic mosquito ortality . Aedes  EEEV strains can lead to a case fatality ratio of up to 40 %, which makes NA-EEEV the most deadly encephalitic alphavirus. Patients that survive the infection may suffer from serious sequelae such as mental retardation and paralysis.Gross pathological examinations of human cases have revealed diffuse cerebral edema, vascular congestion and occasionally hemorrhages. Due to the hematogenous route of infection in the acute stage of the disease, a predominant panencephalitis and meningitis with vasculitis, occasional thrombosis and perivascular to widespread edema are found. Neuronophagia, microglial nodules and lympho-histiocytic, predominantly perivascular infiltrations are encountered in various regions of the brain. However, a predilection of damage exists for basal ganglia and brain stem. Alterations to the spinal cord are very limited with occasional lesions in the upper cervical region , 74, 117Symptoms of VEEV infection generally manifest 2–5 days after a mosquito bite. Symptoms of VEEV infection include fever with chills, severe retro-orbital and occipital headache, nausea with vomiting, sore throat, diarrhea, tachycardia and myalgia; typically centering in the thighs and lumbar region of the back . ChildreHistopathology studies on autopsied patients have identified spleen, lymph nodes, intestinal lymphoid tissues, liver, lungs and the CNS as the principle target organs of VEEV. Moderate to marked diffuse congestion and edema with hemorrhage can be observed in brain, gastrointestinal tract and lungs. Mild or focal mixed inflammatory cell infiltrates can be detected in the leptomeninges and perivascular spaces. Depletion of lymphocytes with vascular thrombosis and necrosis of follicles can be observed in lymph nodes, spleen and gastrointestinal tract. Hepatocellular degeneration with necrosis of individual cells is observed in the majority of cases. A high percentage of individuals may present interstitial pneumonia. Although the major target cells of VEEV in humans have yet to be identified, pathological findings have suggested that hepatocytes, vascular endothelial cells, B cells, and cortical neurons are affected during the course of the disease. Importantly, lesions were proposed to result from direct injury or immune-mediated clearance of infected cells .After an incubation period of typically 3–7 days, CHIKV infection manifests with high fever, headache, maculopapular rash and painful arthralgia. Other symptoms include retro-orbital pain, myocarditis and hemorrhage. Most patients recover within 1–2 weeks, but joint pain may persist for several months to years. Sporadic CHIKV neurological manifestations have been described since the 1960s and 1970s, but cases were not well documented. More recently, a series of serious outbreaks occurred in countries with modern clinical facilities that facilitated more detailed studies leading to novel insights into CHIKV-mediated neurological disease. During the well-documented outbreak in 2005–2006 on Réunion Island, neurological signs were reported in 12 % of patients . In chilBornaviridae, of the order Mononegavirales and are single-stranded negative-sensed RNA viruses. These viruses display a number of unique features that are not typical for non-segmented negative-sense RNA viruses such as nuclear transcription and replication and their ability to establish a non-cytolytic persistent CNS infection. The classical mammalian Bornavirus BoDV-1 , the prototype of this family, causes a typically fatal neurological disease  in horses and sheep in endemic areas of central Europe with an incubation period of 2–4 months before the onset of clinical neurological signs of infection (reviewed in [Crocidura leucodon) in endemic areas of Germany and Switzerland [Bornaviruses belong to the family iewed in ). Howevetzerland , 56.Bornaviridae until the last decade. Since then, knowledge on Bornaviruses has expanded tremendously. Endogenous genome-integrated Bornavirus-like sequences have been detected in various vertebrate species, including snakes, bats, elephants, fish, lemurs, rodents, squirrels, primates and humans [BoDV-1 was considered the only member of the family d humans , 59. Mord humans , 67. A nd humans . In contd humans , 60. It d humans . The incCrocidura leucodon, BoDV-1 disseminates to both neural and extra-neural tissues and is continuously shed via secretions, excretions and skin scaling [In the natural host,  scaling , 92. In  scaling . PatientExperimental Bornavirus infections have provided evidence for intranasal virus transmission via olfactory nerve endings with retrograde transaxonal spread to the olfactory bulb and subsequently the brain. Primary target cells are neurons and astrocytes, but oligodendrocytes and ependymal cells can also be infected. Pathogenesis is best studied in the rat model where remarkable differences in clinical course and virus distribution occur depending on the state of the immune system and time point of infection (reviewed in ). InfectBunyaviridae comprises over 350 named viruses, most of which are transmitted by insects or ticks. Bunyaviruses are enveloped, spherical particles of about 100 nm in diameter, containing a three-segmented negative-strand RNA genome. The Orthobunyavirus and Phlebovirus genera include members that are associated with human CNS disease. The Orthobunyavirus genus is the largest of the Bunyaviridae family, comprising more than 170 viruses. Important human pathogens include Oropouche virus, Ngari virus and La Crosse virus (LACV); whereas the former two are associated with acute, self-limiting febrile illness or hemorrhagic fever, respectively, LACV is associated with neurological disease and is the major cause of arboviral (arthropod-borne virus) neuroinvasive disease in the United States. LACV is present throughout the mid-western and eastern United States and is also emerging in Appalachia, where it circulates between the primary mosquito vector Aedes (Ochleratatus) triseriatus (eastern treehole mosquito) and small mammals, mostly chipmunks and tree squirrels. A member of the phlebovirus genus associated with encephalitis is Rift Valley fever virus (RVFV). RVFV is transmitted by Aedine, Culicine and Anopheline mosquitoes among mammals, of which domesticated ruminants are the most susceptible to disease. Currently, the virus is largely confined to the African continent, but global distribution of potential mosquito vectors explains the fear for future “virgin soil” epizootics and epidemics. The sandfly-transmitted Phlebovirus, Toscana virus (TOSV), is associated with neurological disease and is a major cause of acute, generally mild aseptic meningitis in the Mediterranean region. In these areas, the virus is transmitted by Phlebotomus perniciosus and Phlebotomus perfiliewi flies.The family Symptoms resulting from LACV infection develop after an incubation period of 5–15 days. Like most arbovirus infections, LACV infections are generally subclinical or present as a self-limiting febrile disease with headache, fever, nausea and vomiting with minimal neurological involvement. Children under 15 years of age are at risk of developing severe neurological disease, which may resemble herpes simplex virus (HSV) encephalitis, particularly when focal signs are present , 148. ReCallithrix jacchus). In these monkeys, disease following peripheral infection manifests as either severe hepatic/hemorrhagic disease or encephalitis, whereas intranasal inoculation results in fatal encephalitis [Human RVFV infections result from mosquito bites or contact with contaminated animal products. Most infections manifest typically after 2–6 days as a self-limited, benign illness with flu-like symptoms. In <1 % of cases, severe complications may develop including fulminant hepatitis, renal failure, retinopathy resulting in temporary or permanent blindness, hemorrhagic fever and encephalitis. The latter presents 1–4 weeks after onset of initial symptoms. The first fatal RVF cases, associated with hemorrhagic fever and encephalitis, occurred in South Africa in 1975 . Encephaphalitis . In a suphalitis . PrimaryThe high seroprevalence of TOSV antibodies in the Mediterranean region with limited reports of clinical cases makes clear that TOSV infections are generally asymptomatic or manifest as a self-limited benign illness. This so-called “sandfly fever” has an incubation period of 3–6 days and may be associated with fever, myalgia, malaise and abnormalities in liver and hematological values. A substantial number of patients develop skin rash. Neurological manifestations may present with Kernig sign, nuchal rigidity, photophobia, consciousness troubles, tremors, nystagmus and paresis. In some patients, the disease progresses from mild acute lymphocytic meningitis to meningoencephalitis or even encephalitis . AlthougFlaviviridae consists of four genera; Hepacivirus which includes Hepatitis C virus, Pestivirus which includes Bovine viral diarrhea virus, Pegivirus which includes GB virus C  and the genus Flavivirus. The Flavivirus genus includes viruses that are etiological agents of arboviral encephalitides and more systemic disease spectra including hemorrhagic fevers . Flaviviruses are single-strand positive-sense RNA viruses.The family Ixodes ricinus and Ixodes persulcatus in Europe, whereas Ixodes ovatus transmits the virus in Japan. Other virus transmission modes include consumption of infected unpasteurized milk or milk products from infected livestock, particularly goats. Phylogenetic analysis of several TBEV strains indicated the existence of three subtypes: (1) European (2) Siberian and (3) Far-Eastern subtype. The differences between the subtypes constitute 5–6 % on amino acid level, but it is unclear whether these subtypes differ in virulence.Tick-borne encephalitis virus (TBEV) is an important cause of CNS infection in Europe and Asia . The disThe incubation period of TBE depends on the route of infection, which ranges from 7 to 14 days after a tick bite and 3–5 days after alimentary transmission . About 7tegmentum of medulla oblongata , pontine nuclei and tegmentum of pons  and caudate nucleus [The frequency of abnormal electroencephalogram findings appeared similar between patients with meningoencephalitis (91 %) and meningoencephalomyelitis (96 %) . MRI dat nucleus .Fig. 4NeCulex mosquitoes, in an enzootic cycle. Humans become infected when bitten by a mosquito carrying the virus. Since the first isolation of WNV in 1937 there have been sporadic cases and outbreaks worldwide. A number of large outbreaks have occurred in North America since 1999 including a particularly large outbreak of encephalitis in 2002 with 2942 cases [West Nile virus (WNV) is transmitted between birds by The incubation period for WNV is normally 3–14 days. Patients with WN fever develop a sudden onset of an acute non-specific flu-like illness . Some paCulex mosquitoes to vertebrate hosts including birds and bats [St. Louis Encephalitis virus (SLEV) is found in the Americas with eight recognized lineages . The virand bats , 69. SLEand bats . Seizureand bats , 137, a and bats , 137. Aland bats , few stuCulex mosquitoes, most commonly Culex tritaeniorhynchus, which breed in rice paddy fields and other water sources. Virus transmission occurs in an enzootic  cycle with a natural transmission cycle between birds and mosquitoes and an ‘amplifying cycle’ between pigs and mosquitoes. JEV can be transmitted to humans via the bite of an infected mosquito which subsequently becomes the ‘dead-end’ host with no further transmission of the virus. It is estimated that <1 % infected individuals develop clinical features following an incubation period of 5–15 days, which may range from a flu-like illness, to severe and often fatal encephalitis in ~20 % of patients. Whereas children are more commonly affected, JEV-induced disease is also seen in adults, especially in areas where there is no pre-existing immunity. Seizures are a frequent clinical manifestation. However, AFP, extra-pyramidal features and focal neurological deficits have also been described. Those with a reduced conscious level or abnormal tone, signs of brainstem herniation, raised intracranial pressure, seizures and fever tend to have a worse prognosis [Among the many arthropod-borne viruses that cause brain infections, Japanese encephalitis virus (JEV) is the most important, being responsible for large outbreaks across Asia. JEV is endemic in South and Southeast Asia Fig. d. It is rognosis . Disabilrognosis . Howeverrognosis , 94.Upon skin inoculation, the virus spreads hematogenously to the liver, kidneys, heart and spleen. The virus eventually crosses the blood–brain barrier (BBB) to enter the CNS. There is still uncertainty as to how the virus crosses the BBB. One theory is a ‘Trojan horse’ mechanism, whereby the virus is transported across the BBB via macrophages or monocytes. Other suggested theories include the passive transport of the virus across the endothelium, , 123 andHerpesviridae is an ancient family of enveloped double-stranded DNA viruses which are widely disseminated in the animal world. Based on genomic and biological properties herpesviruses are divided into three subfamilies: α, β and γ viruses. Alpha-herpesviruses have a short reproductive cycle and establish latency in sensory ganglion neurons. Beta- and gamma-herpesviruses have a more restricted cell type range and replicate relatively slow in cell culture. Whereas beta-herpesviruses can establish latency in multiple cell types and organs , gamma-herpesviruses are restricted to specific lymphocyte subsets including B and T cells. Among the eight human herpesviruses (HHV), the alpha subfamily includes herpes simplex viruses type 1 (HSV-1) and -2 (HSV-2) and varicella zoster virus (VZV). Human cytomegalovirus (HCMV) and the roseola viruses HHV-6 and -7 belong to the beta subfamily and the oncogenic gamma subfamily consists of Epstein–Barr virus (EBV) and HHV-8. Primary HHV infection, commonly during early childhood via the orofacial route, is typically asymptomatic or mild  and does not warrant therapy [The  therapy .A hallmark of herpesviruses is the establishment of life-long latency in specific cell types and intermittent reactivation leading to asymptomatic virus shedding or recrudescent disease. Whereas innate immunity prevents virus dissemination during primary infection, adaptive immunity is pivotal to control latency. Consequently, severe HHV infections are more common in HHV-naïve adults and immunocompromised patients, but also associated with the anatomic site of infection. HHV infections of immune-privileged sites, like the CNS and eye, may lead to potential sight- or even life-threatening diseases, respectively. Whereas HSV and VZV enter the CNS via the transaxonal route, the remaining six HHVs spread to the brain via the hematogenous route within lymphocytes. The high prevalence of HHV infections worldwide argues for their potential etiopathogenic role in CNS diseases that can be monophasic, recrudescent or even chronic. The clinical and pathological features of the key HHVs associated with CNS dysfunction will be discussed.HSV infection, either due to primary or reactivation of latent virus, is the most common cause of infectious encephalitis in humans . The incClinical outcomes are variable and largely depend on prompt initiation of antiviral therapy. Prior to use of the antiviral acyclovir (ACV), mortality of HSE patients was >70 % with a high rate of severe sequelae among survivors. Sequelae are still found and include focal or generalized seizures, personality changes, impaired memory and/or cognition, motor deficits or aphasia. Predictors for a prolonged clinical course are the initial clinical presentation with coma and high lesion load on brain imaging, but not duration of antiviral therapy or age at onset of disease . HSE is VZV, a neurotropic HHV that establishes life-long latency in almost all sensory ganglia, is the only HHV that causes two different diseases during primary (chickenpox: varicella) and reactivation (shingles: herpes zoster) . VaricelHuman cytomegalovirus (HCMV) has the largest genome amongst HHV and causes a variety of diseases, ranging from self-limiting to fatal  with cliEpstein–Barr virus is the causative agent of infectious mononucleosis (IM) in naïve children and young adults, with clinical symptoms evident 4–6 weeks following infection. Less than 5 % of primary EBV infections cause CNS disease that present as meningitis, encephalitis, cerebellitis, cranial or peripheral neuropathies and polyradiculomyelitis. Neurological involvement may occur shortly before, during or after IM or even in the absence thereof. Polyradiculomyelitis is most often as a post-infectious autoimmune-mediated syndrome and patients respond well to steroid therapy often given in combination with antivirals . EBV-relThe two HHV-6 variants, HHV-6A and -B, have recently been re-classified as separate HHVs based on differences in epidemiology, cell tropism and disease association . WhereasNeurological complications associated with human orthomyxovirus infections are most commonly associated with Influenza A viruses which are enveloped segmented negative-strand RNA viruses.Influenza A viruses are subtyped based on their surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Many host species have their own strains , although cross-species transmission occurs frequently. Humans can be infected with seasonal, pandemic and zoonotic influenza A viruses. Seasonal influenza A viruses (H3N2 and H1N1) circulate within the population, causing yearly epidemics. Pandemic influenza viruses are the result of cross-species transmission, after which they adapt to humans and spread worldwide. Four pandemics have occurred in the last century, the 1918 H1N1 ‘Spanish flu’, the 1957 H2N2 ‘Asian flu’, the 1968 ‘Hong Kong flu’ and the 2009 H1N1 ‘Mexican flu’ or ‘swine flu’. Zoonotic influenza virus infections in humans are the result of interspecies transmission, without subsequent efficient transmission among humans. The best known examples are avian influenza H7N9 virus and highly pathogenic avian influenza (HPAI) H5N1 virus, which intermittently are transmitted from their poultry reservoirs to humans.The most common extra-respiratory complication of influenza is the development of CNS disease . InfluenMaternal influenza has been associated with schizophrenia and bipolar disorder (BD) in the offspring. Early studies of prenatal influenza and schizophrenia were ecologic, associating influenza epidemics in populations to patients who would have been in utero during these epidemics . HoweverThe pathogenesis of influenza virus-induced CNS disease in humans is largely unknown. Influenza virus-associated CNS disease could be an indirect effect of the infection, due to systemic cytokines, a direct effect of virus entry into the CNS, or a combination of both . SeveralThe route of entry into the CNS is not completely understood, but recent data indicate that influenza viruses may enter the CNS directly from the nasal cavity via the olfactory nerve in humans . Virus eParamyxoviridae is comprised of a broad group of enveloped single-stranded negative-sensed RNA viruses of animals and humans. In recent years, our understanding of the true diversity of paramyxoviruses has undergone a renaissance with the discovery of novel paramyxovirus sequences in bats, rodents and birds [The family nd birds . HoweverMeasles virus is the prototype member of the genus morbillivirus and remains a leading cause of morbidity and mortality in the developing world with an estimated 122,000 deaths in 2012 . ClinicaAcute demyelinating encephalomyelitis (ADME) typically occurs in 1:1000 measles patients. Few studies have definitively linked MV infection to this condition. Instead, ADME is considered an autoimmune disease in which an acute virus infection leads to inflammation of the brain with concomitant myelin damage. Acute lesions are disseminated throughout the CNS and are characterised by perivenous infiltration and demyelination . In mostSpecific mechanisms of MV spread within the CNS are unknown but detailed pathological and ultrastructural studies of SSPE brain tissue have indicated that the mechanism of MV spread in the CNS is profoundly different from that observed in non-neural cells. MV infection of the CNS is characterised by a lack of virus budding and an absence of giant cell formation. This has led to the suggestion that measles virus spread between neural cells occurs through lateral cell–cell contacts in the absence of appreciable levels of cell-to-cell fusion . In spitRubulavirus and causes a disease that was first accurately described by Hippocrates in the first book of the Epidemics. Alongside the more common complication orchitis, the neurological complication of mumps was first noted by Robert Hamilton in 1758. In spite of the ubiquitous nature of MuV as a common childhood disease in the pre-vaccine era, surprisingly little is known about the pathogenesis of mumps. MuV infects a susceptible host via the respiratory tract and then spreads to local lymph nodes where amplification of the virus results in systemic virus spread. Following an incubation period of 7–21 days, the classical signs of mumps develop, such as bilateral swelling of the parotid glands (parotitis). MuV also infect the CNS and although estimates vary, it has been suggested that this can occur in up to 30 % of cases with approximately 1:6000 cases complicated by more severe MuV encephalitis [Mumps virus (MuV) is a member of the genus phalitis . In the phalitis . The morphalitis . Patholophalitis .Henipavirus are zoonotic viruses which are capable of inducing severe neurological disease in humans. This occurs following virus spread via intermediate animal hosts from flying foxes of the genus Pteropus which serve as the reservoir host for these viruses. HeV was first detected in Queensland in 1994 following a disease outbreak in 21 racehorses and two humans and was rapidly identified as a novel paramyxovirus [Nipah virus (NiV) and Hendra virus (HeV) virus, both members of the genus yxovirus . NiV wasyxovirus . Human Nyxovirus . Prodromyxovirus , 147. Suyxovirus , 147. HoPicornaviridae is one of the largest virus families consisting of small, non-enveloped single-stranded RNA viruses. The family is classified into 29 genera including many human pathogens such as poliovirus (PV) and the non-polio enteroviruses (EVs) of the Enterovirus genus, human parechovirus (HPeV) and hepatitis A virus. Animal pathogens include foot-and-mouth disease virus and Theiler’s murine encephalomyelitis virus. Clinical symptoms caused by picornaviruses in humans vary greatly, ranging from the common cold to life-threatening infections such as encephalitis and myocarditis. Hereafter, picornaviruses that can cause neurological disease in humans will be discussed. CNS infections are most often caused by EVs and are estimated to cause 10–15 million infections in the United States with at least 30,000–50,000 hospitalizations per year, that are mainly due to aseptic meningitis [Parechovirus genus. The HPeV species are only found in primates and now consists of 16 types [The family ningitis . Based oningitis . Followiningitis , which b16 types . The disEnterovirus infections of the brain are caused by a variety of EV genotypes and occur most often in children under 10 years of age. EVs account for more than 90 % of viral meningitis cases, while encephalitis only occurs in 3 % of neurological EV infections , 129. MePicornaviruses are transmitted via the oral–fecal route. Following transmission, the sites of primary replication are considered the respiratory and gastrointestinal tract. From there, the virus spreads via the blood to a variety of target organs. EVs can infect many different target cells including epithelial cells, neurons and cardiomyocytes . There iThe local immune response against picornavirus CNS infections is largely unknown and is mainly deducted from immunological evaluations in EV infection. Undoubtedly an adequate humoral response with release of neutralizing antibodies is crucial as a defence mechanism. Indeed, patients with antibody deficiencies, such as X-linked agammaglobulinemia, are at increased risk for chronic enteroviral meningoencephalitis and long-term neurological symptoms . Lack ofOther EVs have emerged more recently. From the late 1990s, EV71 has caused massive outbreaks in the Asian Pacific region associated with brain stem encephalitis causing hundreds of deaths mainly in children . In addiHuman Parechovirus infections are usually associated with (upper) respiratory tract and gastrointestinal infections in young children, although incidentally severe neurologic disease such as flaccid paralysis, meningitis, encephalitis and encephalomyelitis has been reported. The clinical picture is clearly different from the other HPeV infections. HPeV3 infection in adults is often asymptomatic or elicits minor disease such as myalgia , whereasRhabdoviridae comprises 11 genera, of which viruses within the genus Lyssavirus and Vesiculovirus are of medical and veterinary importance. The genus Lyssavirus contains seven genotypes: rabies virus , Lagos bat virus (genotype 2), Mokola virus (genotype 3), Duvenhage virus (genotype 4), European Bat lyssaviruses type 1 and 2 (genotypes 5 and 6), and Australian bat lyssavirus (genotype 7). RABV is prevalent throughout the world with only few countries being rabies free. The virus infects mammalian species, most frequently carnivores and bats. Genotypes 2 through 7 are geographically restricted and have bat reservoirs. The genus Vesiculovirus contains the vesicular stomatitis virus (VSV), an arthropod-borne virus that infects primarily rodents, swine, horses, and cattle. Humans only experience mild symptoms upon VSV infection. Currently there is no effective treatment for clinical rabies and a better understanding of the pathogenesis is needed to develop better post-exposure treatment regimens and curative treatments for this invariably lethal disease.The family Rabies is a neurological disease characterized by a fatal encephalitis, caused by RABV or one of the other lyssaviruses, which are usually transmitted through a bite or scratch of infected carnivores  or bats (bat rabies). Rabies presents as either furious or paralytic rabies . Approximately 55,000 to 60,000 people are reported to die annually as a result of rabies. Clinical manifestations of terrestrial and bat rabies are different . This diIt is generally believed that low titer virus exposure will first lead to one or more rounds of replication in muscle cells before motor neurons at the neuromuscular junctions are infected. In contrast, exposure to a high virus titer, leads to direct infection of motor neurons whereas sensory neurons are probably not infected. Nevertheless, several cases of rabies following skin lesions have been reported , 133, alAir-borne infection with RABV or infection via transplantation of infected tissues or organs represents uncommon modes of transmission , 140. A Infections with a wide range of viruses from different virus families may cause immediate or delayed neuropathological changes and neurological manifestations in humans and animals worldwide. The ability of a virus to cause an acute or more long-term infection of the CNS is undoubtedly due to a complex milieu of many factors including host genetics, specific interaction with the host immune system, capacity to spread rapidly to immunoprivileged sites such as the brain and unique modalities of cell-to-cell spread within the CNS. Collectively these interactions determine the speed and severity of the disease course. A better understanding of the molecular, epidemiological and biological characteristics of these infections and in particular of the mechanisms that underlie neuropathological or immune-pathological alterations associated with their clinical manifestations are expected to provide tools for the development of more effective intervention strategies and treatment regimens. This will be of critical importance in the event of future novel neurotropic infections caused by zoonotic viruses.Supplementary material 1 (DOCX 93 kb)


Reverse genetics can be used in the bovine leukemia virus (BLV) system to characterize mechanisms of viral persistence and pathogenesis. The question addressed here pertains to the role of glycans bound to the BLV envelope glycoprotein (SU). A commonly accepted hypothesis is that addition of carbohydrates to the SU protein potentially creates a structure called « glycan shield » that confers resistance to the virus against the host immune response. On the other hand, glycosylation can also modulate attachment of the virus to the cell membrane. To unravel the role of SU glycosylation, three complementary strategies were developed: pharmacological inhibition of different glycosylation pathways, interference with glycan attachment and site-directed mutagenesis of N-glycosylation sites in an infectious BLV provirus. The different approaches show that glycosylation is required for cell fusion, as expected. Simultaneous mutation of all 8 potential N-glycosylation sites destroys infectivity. Surprisingly, mutation of the asparagine residue at position 230 creates a virus having an increased capacity to form syncytia in vitro. Compared to wild-type BLV, mutant N230 also replicates at accelerated rates in vivo. Collectively, this data thus illustrates an example of a N-glycosylation site that restricts viral replication, contrasting with the hypothesis supported by glycan shield model.


Southern rice black-streaked dwarf virus (SRBSDV) have led to severe economic losses in South China in recent years. The distribution and diversity of SRBSDV have not been investigated in the main rice and maize growing areas in China. In this study, the distribution of SRBSDV in China was determined by using reverse transcription-polymerase chain reaction (RT-PCR).Rice and maize dwarf diseases caused by the newly introduced Between 2009 and 2010, 2404 plant samples  with dwarf symptoms were collected from fields in 194 counties of 17 provinces in China and SRBSDV was detected. The results indicated that 1545 (64.27%) of samples (both rice and maize) were infected with SRBSDV. SRBSDV was detected widely in Hainan, Guangdong, Guangxi, Yunnan, Guizhou, Chongqing, Fujian, Jiangxi, Hunan, Hubei, Anhui, Jiangsu, and Zhejiang provinces, which suggests SRBSDV is an important pathogen causing rice dwarfing diseases in South China. Phylogenetic analysis of 15 representative virus isolates revealed that SRBSDV isolates in China had high levels of nucleotide and amino acid sequence identities (>97.8%).SRBSDV spreads naturally in Yangtze River basin and south region, the location of the major rice production areas. In comparison, the virus rarely spreads north of Yangtze River in North China. Distribution of SRBSDV is consistent with the migrating and existing ranges of its vector WBPH, suggesting that SRBSDV might be introduced into South China along with the migration of viruliferous WBPH. Southern rice black-streaked dwarf virus (SRBSDV) was first identified in Yangjiang, Guangdong province in China in 2001, and has been proposed as a new member of the genus Fijivirus in the family Reoviridae[Zea mays), Coix chinensis, Echinochloa, Juncellus and Pennisetum[Sogatella furcifera Horváth) in a persistent, circulative-propagative manner. The virus, however, cannot be transmitted from WBPH female adults to their progeny via eggs [Laodelphax striatellus Fallén), a major vector of the Rice black-streaked dwarf virus (RBSDV) [ennisetum. SRBSDV  (RBSDV) , which i (RBSDV) . Availab (RBSDV) .Fijivirus. RBSDV was widespread and prevalent in most parts of East, North, Northwest, and Northeast China in the last century [SRBSDV shares major similarities of symptoms, host ranges, virion morphology, and serology with RBSDV ,2, anoth century ,7. The t century . Due to  century -10, faciSRBSDV causes a devastating disease that threatens rice production in many provinces in China. In 2009, the rice black-streaked dwarf disease caused by SRBSDV resulted in significant losses in late rice production in South China, especially in Hunan, Hubei, Guangxi, and Jiangxi provinces ,12. In 2A novel rice black-streaked dwarf disease caused by SRBSDV, exhibiting similar symptoms with another rice dwarfing disease caused by RBSDV in the field, was investigated and identified in China. A duplex RT-PCR was performed to distinguish accurately the two viruses. The 569-bp fragments were produced from SRBSDV-infected plant samples, whereas the 1119-bp products were amplified from samples infected by RBSDV. No amplified products were detected from healthy control plants  from partial virus isolates were amplified with the specific primers S10-F/S10-R Figure . FurtherBased on the outer CP gene sequences of SRBSDV isolates detected in the experiment, together with two additional sequences from GenBank (EU784840 and EU523360), the phylogenetic tree was constructed with a RBSDV isolate (AY050489) as the outgroup  hybrid combinations were cultivated and grown to increase rice yield in South China. A previous study indicated that these japonica-indica hybrids were more favorable for WBPH than japonica rice, and the phenomenon was termed as “super-susceptibility” . Other sThough limited maize plants were infected by SRBSDV in our investigation, SRBSDV has the potential to cause severe losses in maize. If SRBSDV spreads to North China, where maize is planted widely, through the migration of WBPH, the virus might threaten maize production as RBSDV has done ,7. NotabRice ragged stunt virus (RRSV), Rice gall dwarf virus (RGDV), Rice dwarf virus (RDV) and Rice grassy stunt virus (RGSV). These viruses are common on rice plants in South China, and can also produce dwarf-like symptoms. Of course, positive samples for SRBSDV might be mixed-infected by these viruses. An expanded surveying for all rice virus categories and distribution is needed in the future, which will be helpful for understanding viral epidemiology and controlling disease.In our investigation, all samples had dwarf symptoms, but detection rates of SRBSDV and RBSDV were 64.27% and 13.52% respectively, without co-infection of the two viruses. The remaining samples might be infected by other viruses, such as With increasing global temperature and more frequent warm winters, the attacking period of WBPH might be prolonged, and the vector might advance across Vietnam and across China. In addition to the susceptible varieties and cultivation systems, warmer winters will further aggravate the SRBSDV disease occurrence in rice in South China. Therefore, it is urgent to cultivate resistant rice and maize varieties and develop other integrated control strategies for reducing the possibility of damage by SRBSDV in China.Field surveys were done between 2009 and 2010 in 194 counties of 17 provinces in China, including Guangdong, Guangxi, Hunan, Hubei, Jiangxi, Hainan, Yunnan, Guizhou, Sichuan, Chongqing, Fujian, Zhejiang, Shanghai, Jiangsu, Anhui, Shandong, and Hebei provinces. 2404 cereal crop samples (2294 rice and the rest maize samples) with typical stunting or dwarf symptoms were collected from fields in the above mentioned provinces , and these samples covered more than 300 rice and maize cultivars. At least three rice or maize plants were sampled in each field. Leaf or stem tissues of samples were either used directly for total RNA extraction or frozen at −70°C for further study.Total RNAs from healthy and virus-infected control plants as well as field samples were extracted following the standard protocol of TRIzol® reagent . The concentration and quality of each RNA sample were determined with an Eppendorf Biophotometer plus . For the simultaneous detection and distinguishing of SRBSDV and RBSDV, a quick duplex RT-PCR analysis was developed as described by Ji et al. (2011) . Three sPfu DNA polymerase and designed specific primers. In the duplex RT-PCR, primer S9-SR-F, S9-RB-F and S9-R were mixed into the same reaction system to conduct PCR, and then amplified products were separated using 1.5% agarose gel electrophoresis to distinguish simultaneously SRBSDV and RBSDV. The PCR products from S10-F/S10-R primer were cloned individually using pMD18-T vector system  and sequenced using an automated dye terminator sequencing system  according to the manufacturer’s protocol. Sequence data was analyzed with DNAstar software. Phylogenetic analysis and molecular diversity among isolates were estimated by calculating the p-distance values for nucleotide and amino acid comparisons using the MEGA program (version 4.0). To determine the relationships between SRBSDV and RBSDV, a phylogenetic tree was constructed via the neighbor-joining (NJ) algorithm with Kimura 2-parameter model.First strand cDNA was synthesized with random 6 hexamer as primer by using 1st strand cDNA Synthesis Kit  according to the manufacturer’s protocols. Subsequent PCR amplification was performed by using The authors declare that they have no competing interests.YZ and WL designed the study. ZC, WL, FS, GZ, JW and YZ collected the samples. RG and ZC performed the RT-PCR tests. ZC, SL and YZ analyzed the data. SL, XZ and YZ wrote and finalized the manuscript. All authors read and approved the final manuscript.


In a recent commentary about “neural reuse,” a commentator noted that the implications of these theories for the treatment of brain injuries have been foreshadowed in the writings of the German-American neurologist Kurt Goldstein (1878–1965) . The relationship between mental performances and definite areas in the brain constitute a far more complicated problem than the so-called localization theory has assumed .Goldstein was imprisoned and tortured while his assistant (and later spouse) Eva Rothmann (1897–1960) petitioned the high-ranking Nazi, Matthias Heinrich Goering (1879–1945), who was a psychologist by training, for his release. This was granted on the condition that Goldstein would leave Germany forever—a demand with which he promptly complied. Taking up an ad hoc position at the University of Amsterdam from 1933 to 1934, made possible by a Rockefeller fellowship, he wrote his masterpiece, Subsequently, Goldstein immigrated to the USA, where he fulfilled a number of academic roles. He was professor of neurology at Columbia from 1936 to 1940, and in 1938 he delivered the William James lectures at Harvard. From 1940 to 1945 he had a position at Tufts in Boston together with a neuropsychiatry practice, while he continued to lecture at City University of New York from 1950 to 1955. In his late seventies he still taught courses at Brandeis University. Yet his last years were marred by tragedy: his wife committed suicide after a long illness and for Goldstein himself, America remained a foreign country. He died in 1965 after a fall that resulted in aphasia, a condition he had always closely researched .