Mimivirus is the largest known virus whose genome and physical size are comparable to some small bacteria, blurring the boundary between a virus and a cell. Structural studies of Mimivirus have been difficult because of its size and long surface fibers. Here we report the use of enzymatic digestions to remove the surface fibers of Mimivirus in order to expose the surface of the viral capsid. Cryo-electron microscopy (cryoEM) and atomic force microscopy were able to show that the 20 icosahedral faces of Mimivirus capsids have hexagonal arrays of depressions. Each depression is surrounded by six trimeric capsomers that are similar in structure to those in many other large, icosahedral double-stranded DNA viruses. Whereas in most viruses these capsomers are hexagonally close-packed with the same orientation in each face, in Mimivirus there are vacancies at the systematic depressions with neighboring capsomers differing in orientation by 60°. The previously observed starfish-shaped feature is well-resolved and found to be on each virus particle and is associated with a special pentameric vertex. The arms of the starfish fit into the gaps between the five faces surrounding the unique vertex, acting as a seal. Furthermore, the enveloped nucleocapsid is accurately positioned and oriented within the capsid with a concave surface facing the unique vertex. Thus, the starfish-shaped feature and the organization of the nucleocapsid might regulate the delivery of the genome to the host. The structure of Mimivirus, as well as the various fiber components observed in the virus, suggests that the Mimivirus genome includes genes derived from both eukaryotic and prokaryotic organisms. The three-dimensional cryoEM reconstruction reported here is of a virus with a volume that is one order of magnitude larger than any previously reported molecular assembly studied at a resolution of equal to or better than 65 Å. Mimiviruses are larger than any other known virus, yet despite their size, the capsid has been shown to be a regular icosahedron. Using cryo-electron microscopy and atomic force microscopy, we show that the icosahedral symmetry is only approximate, in part because one of the 5-fold vertices has a unique “starfish-shaped” feature and because a better three-dimensional reconstruction was obtained by assuming only 5-fold symmetry. Contrary to expectations, the arrangement of the capsomers on the Mimivirus surface is not as that in many other large icosahedral dsDNA viruses. Instead, the faces of Mimivirus have systematic vacant sites that are surrounded by six capsomers with alternative orientations which differ by about 60°. The structure of Mimivirus was examined with cryo-electron microscopy and atomic force microscopy. The quasi-icosahedral virus has a unique vertex decorated by a starfish-like feature. The capsomers form hexagonal arrays on each face. Acanthamoeba polyphaga Mimivirus, is the largest known virus -2-aminoethanesulfonic acid, pH 7.5, 0.01% NaN3) at room temperature for at least one day. The sample was washed twice with TES and digested with five volumes of 14 mg/ml bromelain from pineapple stem (Sigma) in TES buffer  at room temperature for at least one day.The Mimivirus fibers were digested by sequential application of lysozyme and bromelain. The Mimivirus was pelleted by centrifuging at 1,000CryoEM data of untreated and defibered Mimivirus were collected as described previously . MicrogrMimivirus particles, both native and those treated with enzymes, were spread on freshly cleaved mica that was coated with poly-l-lysine and scanned under buffer. Capsids, which were pretreated with lysozyme and bromelain, were, in some experiments, further exposed to 1 mg/ml solutions of proteinase K and 1% SDS at 37 °C for 30 min to 2 h, washed with water, and then imaged. No fixation of any kind was used. Two methods were used to expel the DNA and other internal structures from the virus. In the first method, virus solution was dried on mica, rehydrated with a small amount of water, and then pressed between two surfaces of mica. In the second method, very concentrated virus solution was placed in small wells, crushed with a glass stick, diluted in water, and then deposited on mica. The Mimivirus DNA was recognized by comparing the AFM images with DNA extracted from PBCV1 , T4 phagAFM analysis was carried out using a Nanoscope III multimode instrument (Veeco Instruments). Samples were scanned at 25 °C using oxide-sharpened silicon nitride tips in a 75-μl fluid cell containing buffer or in air. For scanning in air, silicon tips were used. The images were collected in tapping mode  with an


Aedes albopictus mosquitoes collected in Dallas County, Texas, USA, in August 2009. The presence of this virus in an invasive vector species within a region that lies outside the virus’s historically recognized geographic range is of public health concern.We report the arthropod-borne pediatric encephalitic agent La Crosse virus in  Bunyaviridae and the genus Orthobunyavirus, LACV is enveloped and contains a negative-sense, tripartite genome with segments designated small (S), medium (M), and large (L). Cases of LACV-associated encephalitis, which can be fatal, occur within the geographic range of its principal vector, Aedes triseriatus mosquitoes. This native tree-hole breeding mosquito is distributed throughout wooded regions east of the Rocky Mountains within the United States. Historically, most LACV-associated encephalitis cases have occurred in upper midwestern states, including Wisconsin, Illinois, Minnesota, Indiana, and Ohio (La Crosse virus (LACV) is the most common cause of arthropod-borne, pediatric encephalitis in North America. A member of the California serogroup within the family and Ohio . In receand Ohio  (1). In and Ohio .Ae. albopictus is an invasive mosquito species that was first discovered in Houston, Texas, in 1985  demonstrated marked CPE. This condition represented a preliminary virus isolation-positive result. No CPE was observed in the BHK cells. Infected cells were then subjected to immunofluorescent antibody assays with antibodies directed against various arboviruses, followed by the use of fluorescein isothiocyanate–conjugated antimouse antibodies for detection. From these analyses, the isolate derived from pool no. AR6319  was determined to be a California serogroup virus. Furthermore, pool no. 6318 (Cx. quinquefasciatus) tested positive for West Nile virus, and pool no. 6320 (Ae. triseriatus) was negative for virus by the above described methods.Generated pools were macerated in 1.5 mL of bovine albumin diluent arbovirus medium followed by 2 rounds of centrifugation at 10,000 rpm for 5 min each. Between each round of centrifugation, a rest period of 15 min was used to facilitate pellet formation. After centrifugation, 50 µL of the resultant supernatant was injected onto BHK and Vero cells. These cells were incubated at 37°C and examined for cytopathic effect (CPE) over the next 10 days. At day 5 postinoculation, Vero cells inoculated with the supernatant derived from pool no. AR6319 , the pool and the Vero cell–derived isolate were sent to the Centers for Disease Control and Prevention in Fort Collins, CO, USA, for additional testing. Upon receipt of the samples at Fort Collins, a reverse transcription–PCR was performed to amplify cDNAs from all 3 segments of the orthobunyavirus genome by using the consensus oligonucleotide primers shown in the www.ncbi.nlm.nih.gov/BLAST) analyses; the results indicated that the pool and the isolate were positive for LACV S, M, and L segment RNAs.To further identify the California serogroup virus identified in pool no. AR6319  were generated for LACV RNAs extracted from LACV-positive pools and Vero cell isolates by using oligonucleotide primers specific for the previously published LACV prototype genome  and methods previously described  of 29 Phylogenetic analyses of partial LACV M segment sequences  indicateAe. albopictus mosquitoes in Dallas County, Texas, in late summer 2009 represents the possible expansion of the geographic range of an endemic pathogen within this invasive mosquito species in the United States. The subsequent occurrence of LACV in Fort Bend County in October 2009 should be of concern to public health practitioners who have been alerted to the presence of this pathogen near 2 major urban centers, Dallas and Houston. Of interest, San Angelo virus, which is serologically related to LACV, is known to occur in Texas and has been shown to replicate in and be transovarially transmitted by Ae. albopictus mosquitoes (Ae. albopictus mosquitoes (The presence of LACV in


To the Editor: In 2009, Kapoor et al. and Arthur et al. published reports on the prevalence of the newly identified parvovirus, human bocavirus 2 (HBoV-2), in fecal samples  hospitalized with acute LRTIs at Sanggyepaik Hospital in Seoul, South Korea. Previously, during January 2002–June 2006, a total of 173 serum samples had been obtained from children (age range 1 month–15 years) with hepatitis , Kawasaki disease (12 samples), and Henoch-Schönlein purpura (18 samples) and from healthy children   and a QIAamp DNA Blood Mini Kit (QIAGEN GmbH), respectively. All nasopharyngeal aspirates were tested by PCR for common respiratory viruses such as respiratory syncytial virus, influenza viruses A and B, parainfluenza virus, and adenovirus, as described previously  and presented in a topology tree, prepared by using MEGA 4.1 (www.megasoftware.net).HBoV-2 was detected by performing first-round PCR with primers based on the Of the 212 samples tested, the following viruses were detected: human respiratory syncytial virus (in 124 [58.4%] samples), human rhinovirus (24 [11.3%]), influenza virus A (18 [8.4%]), adenovirus (10 [4.7%]), and parainfluenza virus (8 [3.7%]). HBoV-1 was not detected in the study population. HBoV-2 DNA was found in 5 (2.3%) of the 212 samples collected; all positive samples had been obtained in October 2008. The age range of the children with HBoV-2–positive samples was 4–34 months (median 24 months), and all were male. The diagnoses were bronchiolitis for 3 children and bronchopneumonia for 2. The most frequently codetected virus was human respiratory syncytial virus, found in 4 (80%) of 5 samples. One sample that was negative for respiratory syncytial virus and positive for HBoV-2 was negative for all other respiratory viruses.Nucleotide sequences were determined for the NS-1 gene, and phylogenetic analyses, which included HBoV-3, a new lineage designated by Arthur et al. .,Recent studies have detected HBoV-1 in serum samples of children with Kawasaki disease and of an immunocompromised child with hepatitis (The absence of HBoV-1 in the samples examined was unexpected because HBoV-1 was detected in >10% of 558 respiratory samples collected from a demographically similar study population during the winter 2 years earlier (We demonstrated HBoV-2 DNA in the respiratory tract secretions of children with acute LRTIs. In most positive samples, the virus was found in addition to other respiratory viruses. A limitation is that the study did not consider health control measures and other clinical disease such as gastroenteritis and was conducted for a short time. The role of HBoV-2 in LRTIs remains unclear; further studies are needed to clarify whether this virus is only shed from the respiratory tract or whether it replicates in the gastrointestinal tract.


To determine resistance of highly pathogenic avian influenza (H5N1) virus to chlorination, we exposed allantoic fluid containing 2 virus strains to chlorinated buffer at pH 7 and 8, at 5°C. Free chlorine concentrations typically used in drinking water treatment are sufficient to inactivate the virus by >3 orders of magnitude. Growing concerns about the public health threat posed by highly pathogenic avian influenza (HPAI) subtype H5N1 has prompted interest in evaluating environmental control measures for this virus. The World Health Organization has noted that more information is needed on the effectiveness of inactivation of subtype H5N1 in water  virus were used in this study /mL (oC under 5% CO2 for 96 h and examined by light microscopy for cytopathic effect (CPE). Culture plates were stained with 1% (w/v) crystal violet in 10% (v/v) neutral-buffered formalin for further examination. Failure to produce CPE indicated that the virus was not capable of infecting the cells. The neutralized buffer control without virus did not cause CPE. All experiments were conducted in duplicate under Biosafety Level 3 agricultural conditions.The infectivity of the samples was quantified by using microtiter endpoint titration  were used to determine the rate of inactivation for the 2 pH levels. Ct values are commonly used to make disinfection recommendations for water treatment and provide a means for comparing biocidal activity for various microorganisms  required to achieve 1, 2, and 3 orders of magnitude inactivation for both strains at the 2 pH levels. Covariance analysis of the decay coefficients indicated no significant difference in the inactivation of the 2 virus strains at pH 8.0 (p = 0.10). Rapid inactivation at pH 7.0 did not allow for statistical evaluation.The results of the chlorination experiments  represenCt values of 6 and 8 mg-min/L to achieve enteric virus inactivation of 3 and 4 orders of magnitude, respectively  in the water environment. The information on chlorine disinfection presented here should be helpful for developing risk management procedures regarding the role of water in the transmission of the virus to humans and poultry.The results of this study confirm that avian influenza (H5N1) is readily inactivated by chlorination. Although the viral inoculum exerted a considerable initial chlorine demand, the maintenance of a free chlorine residual (0.52–1.08 mg/L) was sufficient to inactivate the virus by >3 orders of magnitude within an exposure time of 1 minute. Chlorine demand would also be anticipated when the virus is associated with fecal material. These findings indicate that the ability to compensate for an initial chlorine demand followed by exposure to a relatively low level of free chlorine for a short time is sufficient to inactivate the virus by chlorination. For drinking water disinfection at conditions similar to those used in this study, the US Environmental Protection Agency specifies free chlorine


Social anxiety disorder (SAD) is associated with substantial reduction in health-related quality of life (HRQoL). Escitalopram has proven efficacy in the short-term treatment of SAD and prevention of relapse.To determine whether the clinical effects of treatment translated into HRQoL benefits and to investigate costs of SAD treatment.Data on HRQoL and resource utilisation were collected in a previously published clinical trial of escitalopram in relapse prevention. Among 517 patients, 371 responded to 12 weeks of open-label treatment with escitalopram and were randomised to escitalopram or placebo for 24 weeks. HRQoL was assessed using the short form (SF)-36 instrument and SF-6D utilities  were calculated. Costs were calculated for responders over the acute phase and for non-relapsed patients over the continuation phase, applying UK unit costs.Health-related quality of life was significantly improved after the acute phase when compared with baseline. The SF-6D utility increased by 0.047 in responders (p < 0.0001) and 0.021 in non-responders (p = 0.0005). Healthcare costs were non-significantly lower in acute phase than during prestudy phase (p = 0.0587 from NHS perspective), as were productivity costs (p = 0.1440). HRQoL at last visit was lower in relapsed than non-relapsed patients. The difference in utility was −0.026 (p = 0.0007). Healthcare and productivity costs were non-significantly lower in the escitalopram group than in the placebo group.Both effective acute treatment of SAD and prevention of relapse with escitalopram are associated with significant HRQoL benefits. Despite some limitations, the cost analysis suggests that savings in physician-visits and inpatient care may offset drug acquisition costs. Escitalopram is effective in the treatment of patients with generalised social anxiety disorder (SAD) and the prevention of relapse.Health-related quality of life is substantially impaired in patients with SAD.Acute treatment of SAD and prevention of relapse with escitalopram have positive effects on HRQOL.Drug acquisition costs associated with escitalopram were offset by savings in physician-visits and inpatient care in the studied sample.Social phobia is a commonly occurring anxiety disorder often associated with serious role impairment . Social Generalised SAD, also known as generalised social phobia, is defined as a persistent fear of most social or performance situations in which one is exposed to unfamiliar people or to possible scrutiny by others . In the Individuals with SAD have a high risk of developing additional anxiety and mood disorders, including suicidal behaviour . AdditioEstablished treatments for SAD include cognitive behaviour therapy and selective serotonin reuptake inhibitors (SSRIs). A number of SSRIs, including paroxetine, sertraline and fluvoxamine, have been found to be effective in the treatment of generalised SAD, based on randomised, placebo-controlled, clinical trials –14. FurtMontgomery et al.  reportedn = 190) at a fixed dose of 10 or 20 mg, or placebo (n = 181). Escitalopram and placebo tablets were identical in smell and appearance, and packed identically. Treatment was continued for 24 weeks unless the patient relapsed or was withdrawn for other reasons. Response was defined as a Clinical Global Impression-Improvement (CGI-I) score of one or two . It was concluded that escitalopram is effective and well tolerated for long-term treatment of generalised SAD.Health-related quality of life was assessed using the Medical Outcome Study Short Form (SF-36) . This isPatients completed the SF-36 questionnaire at initiation of the acute phase (baseline), the end of the acute phase, and at 12 and 24 weeks after randomisation. Patients who did not complete the study attended an early discontinuation visit, at which the SF-36 was administered.Health state utilities are preference-based index measures of well-being that can be used to estimate quality-adjusted life-years (QALYs) for use in economic evaluations . ConventBrazier and colleagues ,25 derivA resource utilisation questionnaire, specifically developed for the purpose of this study, was administered at 12-week intervals (i.e. at the same visits as SF-36). In this questionnaire, SAD-related resources used over the past 12 weeks were recorded, including physician consultations (excluding consultations carried out for the study), visits to other healthcare professionals and social workers, and hospitalisations. In addition, patients were asked for details of sick leave days related to SAD occurring during the study or the 12 weeks preceding baseline. Resources used after relapse or withdrawals from the study were not recorded. Cost estimates also included acquisition of escitalopram. Counts of returned tablets were recorded at all visits. These data were used to calculate the cost of escitalopram during the acute phase and over the two trimesters (12-week periods) of the continuation phase.Healthcare resources were valued using unit costs prevailing during 2006 in the UK, from the perspective of the NHS and Personal Social Services (abbreviated as the NHS perspective below), and from the perspective of society. Costs associated with non-conventional medicine, such as consultations with acupuncture specialists or chiropractors, were accounted for from the societal perspective but not from the NHS perspective. Productivity costs were reported separately from other costs. The cost of a lost work day was based on mean gross UK daily earnings in 2006. A value of £107.46 per sick leave day was applied .As resources used after discontinuation of the study treatment were not systematically collected, cost estimates for each study trimester are based on patients who completed the trimester. Therefore, this analysis only reports costs in responders over the acute phase and in non-relapsed patients over the continuation phase.t-tests. Similarly, HRQoL scores were compared between patients who subsequently relapsed and other patients who continued to receive long-term treatment. Data from questionnaires completed at time of relapse were used for patients who relapsed, and data collected at the end of the continuation phase or at withdrawal were used for the remaining patients. Analysis of covariance was used to compare HRQoL scores at the end of the continuation phase, at relapse or upon withdrawal between the two treatment groups, adjusting for scores at randomisation. This adjustment was necessary as HRQoL at randomisation might differ between patients who subsequently relapsed and those who did not, eventhough all were in remission.Descriptive statistical analyses were performed. Mean and standard deviations were calculated for all HRQoL scores and costs by trimester (12-week period). HRQoL scores at the end of acute phase or discontinuation were compared between responders and non-responders using t-tests were performed to compare costs over the 12 weeks preceding baseline with costs over the acute phase. In addition, cost measurements were compared between the escitalopram and placebo patients for each trimester of the continuation phase using t-tests. Healthcare costs were estimated with and without the inclusion of hospitalisations as a small number of costly hospitalisations introduced substantial variability around overall cost estimates.Paired A level of significance of 5% was used in interpreting results of statistical tests. The hypothesis of equality in quality of life between the two groups of patients or between two visits was rejected when there was a p-value under 0.00625 for at least one SF-36 domain score, based on the Bonferroni method. All analyses were performed using SAS version 9.1 .The numbers of patients at different stages of the study are shown in Short form-36 domain scores at baseline and at the end of acute phase are shown in t-tests comparing costs between prestudy and acute phases lead to different interpretations according to the perspective. From the societal perspective, estimated costs were significantly lower in the acute phase compared with those in prestudy phase (p = 0.0410). From the NHS perspective, the difference between periods was negative but not statistically significant (p = 0.0587). The analyses in There was substantial variability in the cost of lost workdays because many patients took no time off work and a few patients were absent over long periods . 68% of patients were in paid employment or self-employed. The mean number of workdays lost decreased from 2.54 over the prestudy phase to 1.96 over the acute phase among responders; this difference was not statistically significant (p = 0.1440).Short form-36 scores 24 weeks after randomisation or at last visit, displayed in Health-related quality of life scores at the last assessment visit were similar to those at randomisation among patients who received long-term treatment with escitalopram see . HoweverFor those patients who had not relapsed and completed the SF-36 questionnaire 12 weeks after randomisation , all mean domain scores were higher in the escitalopram arm, but no difference reached the 0.00625 significance level. The SF domain score was higher by 6.93 in the escitalopram arm (p = 0.0194), the RE domain score by 10.15 (p = 0.0153) and the MH domain score by 6.29 (p = 0.0072); other differences were smaller. The overall difference in SF-6D utility at 12 weeks after randomisation was not statistically significant .Mean costs among non-relapsed patients were non-significantly lower in the escitalopram group than in the placebo group over the two trimesters of the continuation phase, from the NHS and societal perspectives see . As noteEstimates of productivity costs were lower over each trimester of the randomisation phase than over the open-label phase across the two treatment arms see . HoweverThis study examined HRQoL and healthcare costs in patients with SAD treated with escitalopram over 12 or 36 weeks. One of the findings of this study was that HRQoL, as measured by the SF-36 instrument, was improved after 12 weeks of treatment with escitalopram. Unsurprisingly, the improvement was greater for treatment responders [i.e. those with a CGI-I of one or two ] than noQuantitatively, the results suggested that the effect of escitalopram on HRQoL was somewhat more modest in patients with generalised SAD than in those with generalised anxiety disorder (GAD). A study of escitalopram using a similar design among patients with GAD had shown that the SF-6D score increased from 0.64 at baseline to 0.77 at the end of the open-label period (compared with an increase from 0.66 to 0.71 in responders only in this study). Furthermore, in the GAD study, the decrement in SF-6D utility associated with relapse was 0.06 (compared with 0.03 in SAD).. Similarly, a non-significant trend towards lower productivity costs in the escitalopram arm, compared with the placebo arm, was found over the continuation phase.Total healthcare costs were significantly lower over the acute phase than over the 12 weeks preceding the study from the societal perspective (p = 0.0410). This suggested that the acquisition cost of escitalopram was more than offset by savings in physician-visits and inpatient care. However, the difference in costs between the prestudy and acute phases was not statistically significant from the NHS perspective (p = 0.0587), although costs from the NHS perspective were close to those from the societal perspective. Furthermore, when hospitalisation costs were excluded, healthcare costs were very similar between the two periods. Likewise, productivity costs were not significantly different between the prestudy and acute phases. The comparison of healthcare costs between the acute and continuation phases suggests that costs increased among non-relapsed patients in the placebo arm but not in the escitalopram arm. However, healthcare costs in non-relapsed patients were not significantly different between treatment arms, neither from NHS perspective nor from societal perspectiveThis study has several limitations, especially concerning the evaluation of the extent to which costs are influenced by response to treatment. The comparison of costs during the acute phase with costs prior to baseline should be interpreted with caution, as study-related visits occurring over the acute phase were not counted, although a proportion of them might have occurred independently of the study. This might account for a part of the difference in societal costs between the prestudy and acute phases, as the difference in costs of physician-visits represented 73% of the difference in societal costs. Thus, if costs of physician-visits were not counted, the total costs in the prestudy and acute phases would be roughly similar, although still slightly lower in the acute phase. Furthermore, the difference in hospitalisation costs, which represented 85% of the difference in total healthcare costs from the societal perspective, is subject to uncertainty. Only 10 patients were hospitalised in the prestudy phase and two in the acute phase . However, the hypothesis that effective treatment is associated with a reduction in hospitalisation costs does seem plausible, as anxiety disorders are known to cause hospitalisations (psychiatric or non-psychiatric) . CompareCosts were estimated by applying unit prices prevailing in the UK for patients from all countries participating in the trial. Among 371 patients who were included in the analysis for the continuation phase, 35 were from the UK. The comparison of costs between treatment groups assumes that there was no interaction between treatment and country on resource utilisation. This assumption has been made in a number of other economic evaluations . HoweverIt was not possible to distinguish between cases that did not use any resource during a trimester and cases where resource use was not reported. This might have led to an underestimation of costs, irrespective of treatment group or period. However, the extent of this underestimation would have been relatively small if completion rates for the resource utilisation questionnaire were comparable to those for the SF-36 questionnaire. Approximately 95% of patients completed the SF-36 at end of the study or after discontinuation, irrespective of treatment group or presence of relapse. Although approximately one-quarter of those who discontinued did not complete the HRQoL assessment at the time of discontinuing the study treatment, SF-36 data were collected at a later visit in most of these cases.This study compared costs and HRQoL between treatment groups, but no cost-effectiveness analysis (CEA) was actually performed. The comparison of costs between treatment groups was restricted to non-relapsed patients. Relapses are likely to generate costs as a new therapy will be initiated in many cases and physician-visits will occur at shorter intervals following initiation of new therapy. For example, according to guidelines of the National Institute for Health and Clinical Excellence for the management of GAD in the UK, efficacy and side effects should be reviewed at 2, 4, 6 and 12 weeks following prescription of a new therapy, and subsequently at 8- to 12-week intervals . In ordeAlthough no CEA was performed in this study, the study provides data that could be useful for future economic evaluations of treatment of SAD, in particular health state utilities. The utility improvement associated with response was estimated at 0.047 (95% CI: 0.040–0.055), by comparison with baseline value, or 0.040 (95% CI: 0.026–0.054) by comparison with the value in non-responders. Furthermore, the utility decrement associated with relapse was −0.026 (95% CI: −0.042 to −0.011). These are probably conservative estimates, as they are based on the SF-6D instrument. The SF-6D utility index has a smaller range than the EQ-5D, another widely used generic HRQoL instrument providing health state utilities. Also, differences in utility between health states based on SF-6D are often smaller than the differences based on the EQ-5D ,32.The literature relating to HRQoL in SAD is not very abundant, but some previous studies have shown that SAD is associated with a marked reduction in HRQoL, for example Stein et al. , who useIn conclusion, changes in the severity of SAD translate into changes in overall HRQoL. Patients with SAD responding to acute treatment report improved HRQoL, and those who subsequently relapse have reduced scores. Furthermore, long-term treatment with escitalopram, which is effective in preventing relapse, is associated with a significant benefit in terms of HRQoL. The evidence for treatment-related changes in healthcare costs is limited, but suggests that additional drug acquisition costs may be offset by reductions in costs because of physician-visits and hospitalisation.


Cost comparisons included comprehensive data of hospital costs, diagnostic interventions and extramural care. We acquired detailed information on health care consumption from a case record form and from monthly home visits by a specialised nurse. The initial costs of stent placement were higher than the costs of brachytherapy . Total medical costs were, however, similar . Total hospital stay during follow-up was 11.5 days after stent placement vs 12.4 days after brachytherapy, which was responsible for the high intramural costs in both treatment groups . Costs for medical procedures during follow-up were higher after stent placement , while the costs of extramural care were similar . In conclusion, there are only small differences between the total medical costs of both palliative treatment modalities, despite the fact that the initial costs of stent placement are much higher than those of brachytherapy. Therefore, cost considerations should not play an important role in decision making on the appropriate palliative treatment strategy for patients with malignant dysphagia.Self-expanding metal stent placement and single-dose brachytherapy are commonly used for the palliation of oesophageal obstruction due to inoperable oesophagogastric cancer. We randomised 209 patients to the placement of an Ultraflex stent ( The incidence of oesophageal cancer has risen rapidly, due to a marked increase in the incidence of adenocarcinoma  or single-dose (12 Gy) brachytherapy (n=101). For brachytherapy, a flexible applicator  with a diameter of 10 mm was passed down the oesophagus. A single dose of 12 Gy was administered with the radioactive source 192Iridium at 1 cm from the source axis of the applicator. The study was approved by the Central Committee on Research Involving Human Subjects in the Netherlands.We performed a prospective study in three university hospitals and six general hospitals in the Netherlands. Between December 1999 and July 2002, 209 consecutive patients with dysphagia from inoperable carcinoma of the oesophagus or gastric cardia due to metastases and/or a poor medical condition were randomised to placement of a covered Ultraflex stent  during control visits, re-treatments and admissions.Real medical costs were calculated by multiplying the volumes of health care use with the corresponding unit prices. We made a distinction between the full cost price of the interventions of brachytherapy and stent placement by itself and the total medical costs per patient during follow-up.The calculation of the full cost price of brachytherapy and stent placement consisted of detailed measurement of investments in manpower, equipment, materials, housing and overhead. The salary schemes of hospitals and other health care suppliers were used to estimate costs per hour for each caregiver. Taxes, social securities and vacations were included, as well as the costs of the time that could not be assigned to other patients. The costs of equipment included those of depreciation, interest and maintenance.For the calculation of the total medical costs per patient, we distinguished intramural medical costs  and extramural medical costs . Costs caused by loss of production due to absence from work were not taken into account, because the majority of patients were retired from work.For the most important cost items, unit prices were determined by following the micro-costing method (U test. Since cost data per patient (but not per day care) are typically highly skewed, we used nonparametric bootstrap techniques to derive a 95% confidence interval for the differences in distributions of the direct medical costs.All analyses were performed on an intention-to-treat basis. The cost differences between brachytherapy and stent placement were analysed using the Mann–Whitney In a sensitivity analysis, the effect of excluding ‘palliation-related costs’ was assessed by leaving these costs out of consideration. We performed calculations assuming that nursing home admissions and nursing care at home were not directly related to both treatments, but could be attributed to the advanced stage of the disease.The two patient groups were comparable with respect to patient and tumour characteristics. Both treatment groups consisted predominantly of males, with a mean age of 69 years . Major complications within 7 days after treatment included perforation (n=3), fever (n=2), severe pain (n=2) and aspiration pneumonia (n=2). Late major complications consisted predominantly of haemorrhage (n=19) occurring more frequently after stent placement (14 vs 5 after brachytherapy). The need for re-intervention for persistent or recurrent dysphagia was not significantly different for both groups . Recurrent dysphagia after stent placement was predominantly caused by tumour overgrowth (n=16), stent migration (n=18) or food bolus obstruction (n=16), and was treated by placement of a second stent, endoscopic stent clearance or a variety of other treatments. The majority of re-interventions after brachytherapy were caused by tumour persistence (n=18) or tumour recurrence (n=26), both most frequently treated with placement of a stent.Dysphagia improved more rapidly after stent placement than after brachytherapy. However, the overall improvement of dysphagia was better after brachytherapy. More complications occurred after stent placement . There was an overall long-term benefit in general (EORTC QLQ C-30 and EuroQol-5D) and disease-specific quality of life scores (EORTC OES-23) in the brachytherapy group during follow-up.The median survival was similar for both treatment groups  than for brachytherapy (€570). The main cause for this difference was the high purchase costs of the Ultraflex stent (€1100) (vs 16.3 days). The main reason for this was the longer period patients randomised to brachytherapy were admitted to nursing homes (11.0 vs 4.6 days). The average time spent in hospital was similar for both treatments (12.4 for brachytherapy and 11.5 for stent placement). The costs for intramural care were by far the highest cost category for both treatments, but differences were not statistically significant . Costs of medical procedures during follow-up were significantly higher for stent placement (€249) than for brachytherapy (€168) (P=0.002), since major complications and re-interventions occurred more often after stent placement than brachytherapy. The average costs for extramural care were €1278 for brachytherapy and €1046 for stent placement. For both treatments, this could largely be attributed to home visits by the general practitioner and specialised nursing care at home. The costs for medications were similar for brachytherapy and stent placement (€350 and €325).P>0.20). If the ‘palliation-related’ health care was not taken into consideration, then the costs of intramural and extramural care for brachytherapy and stent placement decreased. This resulted in total medical costs for brachytherapy of €8490 and for stent placement of €8538 (P>0.20).The total average costs per patient for both treatments were similar at €11 195 for brachytherapy and €10 078 for stent placement  subsequently received a stent, while 24 out of 108 (22%) of the patients randomised to stent placement received a second stent during follow-up. Since our analysis was based on the intention-to-treat principle, costs of non-assigned treatment were accounted to the randomised treatment group. Total treatment costs, which included the average costs of additional treatment plus re-intervention, were higher for stent placement. However, if the intramural and extramural health care costs were also taken into account, then these high initial costs were only a small part of the total medical costs, which resulted in similar total medical costs.Cost comparisons between medical treatments are often based only on the initial costs. This would imply that stent placement, with the high purchase costs of the device, would be less attractive than brachytherapy. In this study, we clearly demonstrated that the total medical costs of stent placement and brachytherapy were similar when the full follow-up period was considered. This illustrates that cost comparisons between interventions may vary substantially, depending on which, and how many, components are included in a total cost equation .n<35) in these studies (Few studies have been published on costs of brachytherapy or stent placement in the palliative treatment of oesophageal cancer. Three studies compared stent placement with plastic endoprostheses . FarndonA common problem when using clinical trials for any kind of cost assessment arises from the fact that the clinical protocol mandates more visits, consultations and examinations than otherwise used in clinical practice (Despite the high costs involved, detailed cost studies in the treatment of malignant disease and palliative therapy have received little attention. This may be due to the inherent difficulties in performing such studies. Follow-up of patients with malignant disease is sometimes difficult since the mortality rate is high, particularly among patients receiving palliative therapy . In a paThis study focused on costs and not on efficiency. The primary aim of both treatments is to palliate symptoms rather than to improve the survival of oesophageal cancer. Both treatments resulted in an improvement of an important symptom of inoperable oesophageal cancer, that is, dysphagia. As survival of the two treatment groups was comparable, we did not perform a formal cost-effectiveness analysis. Despite a less rapid relief of dysphagia and a higher initial failure rate, brachytherapy was found to be an attractive alternative to stent placement in the palliation of malignant dysphagia, as brachytherapy was safer with fewer procedures needed for recurrent dysphagia (In conclusion, our study provides detailed insight into the total medical costs of two frequently used palliative treatments of dysphagia due to oesophageal cancer, that is, stent placement and brachytherapy. In spite of the higher initial costs for stent placement than for brachytherapy, total medical costs were similar. Therefore, cost considerations should not play an important role in decision making on the appropriate treatment strategy.


In cell culture-based influenza vaccine production the monitoring of virus titres and cell physiology during infection is of great importance for process characterisation and optimisation. While conventional virus quantification methods give only virus titres in the culture broth, data obtained by fluorescence labelling of intracellular virus proteins provide additional information on infection dynamics. Flow cytometry represents a valuable tool to investigate the influences of cultivation conditions and process variations on virus replication and virus yields.In this study, fluorescein-labelled monoclonal antibodies against influenza A virus matrix protein 1 and nucleoprotein were used for monitoring the infection status of adherent Madin-Darby canine kidney cells from bioreactor samples. Monoclonal antibody binding was shown for influenza A virus strains of different subtypes  and host specificity . At high multiplicity of infection in a bioreactor, the onset of viral protein accumulation in adherent cells on microcarriers was detected at about 2 to 4 h post infection by flow cytometry. In contrast, a significant increase in titre by hemagglutination assay was detected at the earliest 4 to 6 h post infection.It is shown that flow cytometry is a sensitive and robust method for the monitoring of viral infection in fixed cells from bioreactor samples. Therefore, it is a valuable addition to other detection methods of influenza virus infection such as immunotitration and RNA hybridisation. Thousands of individual cells are measured per sample. Thus, the presented method is believed to be quite independent of the concentration of infected cells  in bioreactors. This allows to perform detailed studies on factors relevant for optimization of virus yields in cell cultures. The method could also be used for process characterisation and investigations concerning reproducibility in vaccine manufacturing. Today, human influenza vaccines are still mainly produced in embryonated hen's eggs. This production system has certain disadvantages. The amount of vaccine produced is limited to the availability of embryonated eggs, which might be a problem in case of increased demand for vaccination, e.g. during a pandemic ,2. Furthin vitro studies in virological and medical research.Additionally to biochemical engineering approaches, investigation of cellular processes during viral infection is of great importance for process optimisation. For this purpose, monitoring of influenza virus production and spread of the infection on a cellular level could provide essential information. Furthermore, qualitative and quantitative monitoring of influenza virus infections is of interest for Monitoring of influenza virus production and spread of the infection can also be useful for established vaccine production processes. There, it might be used to characterize variations in between process batches with regard to reproducibility and standardisation as recommended in the process analytical technology (PAT) guidelines by the Food and Drug Administration (FDA) .vitro have been established over the years. Widespread classical methods are based on titrations of virus particles in tissue-culture supernatant [Numerous methods for the assessment of influenza A virus infection in ernatant ,10.50) [The hemagglutination assay quantifies the concentration of infectious and non-infectious virions via binding to erythrocytes ,11. In i50) . Titrati50) ,22. The 50) .The translation of viral mRNA can be detected via immunofluorescence microscopy of virus proteins. This can be done either using polyclonal  or monocThe monitoring and quantification of host-cell infection during cell culture-based influenza A virus production needs to meet several goals: Preferably, it should be sensitive, quantitative and robust enough to handle bioprocess modifications such as differences in multiplicity of infection (moi) or cell concentration at time of infection. The assay should be applicable to influenza A virus strains of different host species to cover human and veterinary influenza vaccine manufacturing. Furthermore, the assay should allow monitoring of different virus subtypes to comply with the annual recommendations of the World Health Organization for human vaccines . FinallyThe use of flow cytometry for indirect detection of virus-infected cells with polyclonal antibodies against herpes simplex virus type 1 (HSV-1) and influenza C virus was presented by Steele-Mortimer et al. . The metIn the following, we present a sensitive and quantitative method for the flow cytometric detection of influenza A virus infection with monoclonal antibodies in adherent MDCK cells fixed in ethanol and PFA/ethanol. This method is based on a ready-to-use mixture of fluorochrome-labelled murine monoclonal antibodies against human influenza virus strain A (H1N1) NP and matrix protein 1 (M1). Both M1 and NP are synthesized in the cytoplasm of the host cells soon after infection and are transferred into the nucleus for ribonucleoprotein packaging . The mixAccording to the recommendations of WHO, influenza virus strains used for vaccine manufacturing are adapted each year depending on the expected prevalent virus strains. For a broad application range, detection of influenza A virus strains covering a broad subtype spectrum is desirable. In addition, detection of strains derived from various hosts would be desirable. The antibodies used in this study originally targeted human influenza A virus (H1N1) M1 and NP. In order to verify if influenza virus strains from other hosts could also be detected with these antibodies, cells infected with equine influenza A virus (H3N8) were analyzed by flow cytometry and laser-scanning microscopy.Laser-scanning microscopy was used for qualitative assessment of the fluorescence-labelling of equine influenza virus-infected samples fig. . UninfecThe detection of influenza A virus infected cells was also tested with cells infected by viruses of other host specificity and subtype. Therefore, cells infected by human influenza A virus (H1N1) and porcine influenza A virus H1N2) were compared to equine influenza A virus (H3N8) infected cells , thus representing the treatment of infected cells more precisely than the use of cells harvested from growth medium. Two types of negative control were used: uninfected cells harvested from cultivation in cell growth medium (CGM) without medium exchange  of the cumulative fluorescence distribution (F) of mock infected cells. The fluorescence intensity at x0.99 did not only depend on the mean fluorescence intensity of the mock-infected cells (mean im) but also on the corresponding fluorescence distribution width.The fluorescence distributions measured by flow cytometry were used to quantify the status of infection . Cultivation conditions preceding infection  also had an impact on unspecific cell fluorescence, as shown by differences in fluorescence of uninfected compared to mock-infected cells see fig. . Mock-inm) taking into account the slope of the calibration curve (eq. 1).In order to compare measured fluorescence intensities of cell populations, a fluorescence calibration was done using Sphero FITC Calibration particles fig . Accordit = 0MEF,). The number of MEF bound specifically to virus proteins of an infected host cell (ci) was calculated by subtracting the number of unspecifically bound MEF  from the measured total number of MEF per cell .The fluorescence threshold of samples taken at time of infection (t = 0 h) was used to determine the number of MEF bound unspecifically to cells  caused by washing steps and medium exchange table . Equine 0.99 a minimum signal intensity (cMEF) of 3.58*105 MEF was necessary. Between 2 to 4 h p.i. the mean cMEF of all measured cells increased, indicating the onset of intracellular accumulation of synthesized virus proteins. Most of the expected fraction of infected cells  could be detected at 6 h post infection. Thus, this was considered the earliest time-point of quantification of cellular infection. The percentage of infected cells remained relatively constant (mean: 90.0% ± 4.9%) up to 22 h p.i. and quite unaffected by changes of the mean fluorescence, especially between 16 to 22 h post infection. During this time the measured fluorescence intensities showed large variations (mean cMEF: 2.18*106 ± 41.9%), probably indicating variations in virus protein content per cell over time of infection.To characterize the time course of the fraction of infected cells in a single-step infection experiment, MDCK cells were infected with equine influenza virus at a moi of 3.0 fig. . The cel50 assay . The cells were washed and infected with equine influenza A virus (H3N8) at a moi of 3. The time course of the cellular infection status is shown in fig. say fig. , respecti) at 4 h p.i. . This indicated that the antibody mixture could be used for an even broader range of virus strains and host species origins than previously reported for the antibody against NP only .The method was shown to be suitable for the measurement of fixed samples taken at different time-points during an experiment . In conThe measured fluorescence from samples of equine influenza virus-infected cultures taken at different time-points suggests a time-dependent behaviour of the immunofluorescence signal fig. . This isThe earliest time-point of detection of virus protein accumulation was between 2 to 4 h p.i., which is consistent with the findings for the microscopic analysis using an antibody against influenza A virus NP only . Compare7 virions/ml).In comparison with two titration methods routinely used in vaccine manufacturing, flow cytometry could detect viral infection at an earlier stage fig. . However50 assay, was not detected before 4 to 6 h p.i. , multiplicity of infection and cell concentrations – flow cytometry is a useful addition to standard titration methods for the monitoring of influenza A virus replication. Furthermore, it allows the generation of large data sets for statistical process analysis and validation of mathematical models for virus dynamics.The application of this monitoring method in vaccine manufacturing may give new insights on virus infection dynamics supporting process development and optimization. For example, characterisation of the influence of moi on virus spreading and virus yields for different virus subtypes. In established processes, the method could be used for process analysis according to the PAT initiative to complement analysis of production runs, to facilitate interpretation of the impact of process variations on product quality or to support measures for improving batch to batch reproducibility.Madin-Darby canine kidney cells were obtained from ECACC (No. 84121903) and used for viral infection studies as described previously . The adhEquine influenza strain A/Equine 2 (H3N8)/Newmarket/1/93 (NIBSC), human influenza strain A (H1N1)/Puerto Rico/8/34 (NIBSC) and porcine influenza A/Swine (H1N2)/Bakum 1832/00 . Mock-infected MDCK cells were cultivated accordingly, harvested and fixed 1 h after the medium exchange (mock-infected negative control).For positive controls, MDCK cells infected after 4 days (moi = 1.0) were used. Equine and human influenza A virus infected cells were harvested 18.0 h post infection (p.i.), whereas porcine influenza A virus infected cells were harvested 24.3 h post infection. All standard samples were stored in aliquots at -20°C until further analysis.Small-scale bioreactor cultivations of MDCK cells were performed in a bioreactor (1 L wv) equipped with a glass ball agitator (DasGip). Cytodex 1 microcarriers were used at a concentration of 2 g/l (about 8 000 particles/ml). After a cell growth phase of 95 h the cells were washed with PBS and CGM was replaced by the same volume of VMM. Then the cells were infected with equine influenza virus (moi = 3.0).RBC) per ml test volume was kept constant (1*107 RBC/ml) for all measurements (eq.3).Titration of total viral particles was based on the method by Mahy and Kangro  with mod7 virus particles/ml. Due to a 2n dilution of samples the standard error of the method was ± log 0.3.The lower detection limit of the assay was 2.0*1050) titrations based on [2 infectious virus particles/ml with a standard error of the method of ± log 0.3 due to 2n dilution of samples.Infectious virus particle concentrations were determined as tissue-culture infectious dose  to concentrate floating cells in a reduced volume. Adherent cells were washed with PBS and detached by trypsin/EDTA treatment  at 37°C. Subsequently, detached cells and cells from supernatant were pooled. Samples from microcarrier cultivations were separated into two fractions by sedimentation of the microcarriers. The culture supernatants were centrifuged to concentrate floating cells as described for tissue-culture flask supernatants. The settled microcarriers were washed with PBS. In a subsequent step the adherent cells were detached from the microcarriers with trypsin/EDTA . After cell detachment, the cells from culture supernatants were added again. Cells were separated from the empty microcarriers by sedimentation of the microcarriers.6 cells per aliquot were fixed in ethanol  and stored at -20°C for at least 2 h. Infectious virus particles were inactivated by ethanol fixation (data not shown). For additional PFA fixation, the cells were kept in PFA  for 20 min at room temperature. Afterwards, the samples were centrifuged  and washed once with PBS prior to ethanol fixation as described above.From these pooled cell suspensions, 1.0*10For the detection of influenza A virus infection, a mixture of murine monoclonal antibodies was used . The reaEthanol-fixed aliquots of suspended cells were centrifuged at 100 g for 20 min at 4°C. The supernatant was discarded and the pellet was washed with 5 ml PBS containing glycine (2% w/v) and BSA (0.1% w/v). Subsequently, the samples were centrifuged at 100 g for 30 min at 4°C. The pellet was resuspended in 2 ml PBS with glycine and BSA, transferred into 2 ml reaction tubes and centrifuged at 60 g for 15 min at 4°C. After removal of the supernatant the pellet was dissolved in 25 μl of the FITC-MAb solution and incubated for 1 h in the dark at 37°C on a tube roller. Following the incubation, unbound antibody was removed by addition of 1.8 ml PBS and subsequent centrifugation . Finally, the pellet was resuspended in 0.5 ml PBS for flow cytometry or in 0.1 ml PBS for immunofluorescence microscopy, respectively. The samples were stored in the dark at room temperature until analysis.In addition to immunostaining, the cell cycle distribution was determined via DNA content in bioreactor samples (data not shown). DNA content measurements were performed as previously described . BrieflyMeasured fluorescence intensities were standardised using a fluorescein-specific fluorescence calibration kit . MEF values of the calibration beads were determined in comparison to fluorescein-conjugated beads (NIST) by the supplier. Accordingly, the MEF values were taken from the supplier's certificate of analysis. The calibration beads were diluted in PBS in order to avoid pH-based differences in fluorescence per fluorescein molecule between calibration and sample measurements . The mea4 single cells analyzed. Cells were distinguished from debris via forward-light scattering (FSC) and side-light scattering (SSC). Single cells were discriminated from cell aggregates using plots of forward-light scattering signal area (FSC-A) against forward-light scattering signal height (FSC-H). FITC fluorescence acquisition was performed with logarithmic binning.Cytometric measurements were performed with a Beckman Coulter Epics XL cytometer (Beckman Coulter) equipped with a 488 nm argon laser using the Expo32 software (Beckman Coulter). Two aliquots of each sample were stained and 1.0 * 10Cells of bioreactor samples were stained with both propidium iodide and FITC-MAbs. Here, cell aggregates were distinguished from single cells via the signal ratio of red fluorescence signal area and signal peak.0.99) of the cumulative distribution (F). Mean x0.99 was determined from two measurements of an immunostained negative control, using mock-infected or infected cells (harvested 0 h p.i.), respectively.For the discrimination between infected and uninfected cells mock-infected negative control samples were stained and analyzed. The fluorescence intensity border between infected and uninfected cells was set to a quantile (mean x50: tissue-culture infectious dose; toi: time of infection.FITC-MAbs: fluorescein-conjugated monoclonal antibodies; HA: hemagglutinin; M1: influenza A virus matrix protein 1; MDCK: Madin-Darby canine kidney cells; MEF: molecules of equivalent fluorescein; MFI: mean fluorescence intensity; moi: multiplicity of infection; NP: influenza A virus nucleoprotein; PAT: process analytical technology; PFA: paraformaldehyde; p.i.: post infection; PMT: photomultiplier tube; TCIDJS designed and carried out the experimental study and drafted the manuscript. YG and UR originally conceived the study, supported interpretation of data and critically revised the manuscript. All authors read and approved the final version.


To the Editor:Hendravirus (HeV) and Nipahvirus (NiV), the only known members of the genus Henipavirus, are 2 emerging paramyxoviruses that are highly pathogenic in a variety of vertebrate animals, including humans (Pteropus), and some insectivous bat species have been found to be reservoir hosts of henipaviruses in Australia and Asian countries . Reverse transcription–PCR (RT-PCR) was performed by using primers against the NiV N gene as described previously  by ELISA, and 33 were positive . These sMyotis species from Yunnan Province. Antibody was detected in samples from 3 of 4 Myotis species captured in the same location in 2006 and 2007. A relatively high prevalence of henipavirus antibody was also found among Rousettus leschenaultia samples from Hainan Province in 2007. Notably, Yunnan and Hainan are both located in southern China. Although pteropid bats are not found in China, these data suggest henipaviruses could be introduced into China by other susceptible bat species that overlap their habitat with pteropid bats in neighboring countries.This study systematically investigated NiV presence among bats in China. The detection of henipavirus antibody suggests that several bat species have been exposed to NiV or a closely related virus. The prevalence of antibody was especially prominent among >1 Nipah-like viruses could be circulating among the bat populations sampled in this study, producing antibodies that cross-react with, but do not neutralize, the prototype Malaysian NiV virus isolate. This phenomenon has been observed previously by our group for severe acute respiratory syndrome (SARS)–like viruses in horseshoe bats, whose sera cross-reacted with, but did not neutralize, the SARS virus in humans (Several possibilities may explain the failure to detect neutralizing antibodies. One might be the unique immune response among those nonpteropid bats, which results in a low level of neutralizing antibodies that are difficult to detect in the current assay systems. Alternatively, and perhaps more likely, Obtaining serologic evidence of viruses in bat populations is typically more successful as a screening tool than either nucleic acid based assays or virus isolation; this is likely attributable to the often low-level of virus replication, the transient nature of the infection in bats, or both. The inability to amplify NiV sequences may have been attributable to the viral RNA present among these samples being below the threshold of detection in our assay or to the absence of infection in the population at the time of sampling. In addition, the primers used in the PCR may target regions of the NiV N protein that exhibit substantial sequence divergence in a Nipah-like virus.Rousettus, Myotis, Miniopterus, and Hipposideros naturally reside in trees, buildings, and caves that can be in close proximity to human residential areas, which increases the potential of transmission of zoonotic pathogens from bats to humans. The increased risk for these zoonotic infections to spread from bats to humans in areas of cohabitation is best illustrated by the repeated spillover events involving NiV in Bangladesh (Bat species in the genera Detection of Nipah virus antibody among bat serum samples collected from 10 provinces in China, 2004-2007*


The high binding specificity of short 10 to 30 mer oligonucleotide probes enables single base mismatch (MM) discrimination and thus provides the basis for genotyping and resequencing microarray applications. Recent experiments indicate that the underlying principles governing DNA microarray hybridization – and in particular MM discrimination – are not completely understood. Microarrays usually address complex mixtures of DNA targets. In order to reduce the level of complexity and to study the problem of surface-based hybridization with point defects in more detail, we performed array based hybridization experiments in well controlled and simple situations.We performed microarray hybridization experiments with short 16 to 40 mer target and probe lengths (in situations without competitive hybridization) in order to systematically investigate the impact of point-mutations – varying defect type and position – on the oligonucleotide duplex binding affinity. The influence of single base bulges and single base MMs depends predominantly on position – it is largest in the middle of the strand. The position-dependent influence of base bulges is very similar to that of single base MMs, however certain bulges give rise to an unexpectedly high binding affinity. Besides the defect (MM or bulge) type, which is the second contribution in importance to hybridization affinity, there is also a sequence dependence, which extends beyond the defect next-neighbor and which is difficult to quantify. Direct comparison between binding affinities of DNA/DNA and RNA/DNA duplexes shows, that RNA/DNA purine-purine MMs are more discriminating than corresponding DNA/DNA MMs. In DNA/DNA MM discrimination the affected base pair (C·G vs. A·T) is the pertinent parameter. We attribute these differences to the different structures of the duplexes (A vs. B form).We have shown that DNA microarrays can resolve even subtle changes in hybridization affinity for simple target mixtures. We have further shown that the impact of point defects on oligonucleotide stability can be broken down to a hierarchy of effects. In order to explain our observations we propose DNA molecular dynamics – in form of zipping of the oligonucleotide duplex – to play an important role. DNA microarray technology relies on the highly specific binding affinity of surface-tethered DNA probe sequences to complementary target sequences. Nucleic acid hybridization, the sequential base pairing between complementary probe and target strands, results in the formation of stable double-stranded duplexes. In microarray hybridization assays single-stranded nucleic acid targets – contained in a complex mixture of diffierent target sequences in solution – freely diffuse over the surface-tethered probes until they are captured by a complementary probe. Target strands often carry fluorescent dye labels to enable quantitative detection of the individual target species. Hybridized targets can be identified by the position of the corresponding microarray features (each containing one particular species of surface-tethered probe strands) within the regular grid of the DNA microarray.In DNA microarray applications, along with a high binding affinity (providing sensitivity), a high specificity of probe-target hybridization is required to discriminate between sometimes very similar homologous sequences. Binding specificity is particularly important in genotyping applications where Single Nucleotide Polymorphisms (SNPs), genetic variations of single bases, are concerned. SNPs determine genetic individuality, but also predisposition to a variety of genetic diseases, response to drugs, pathogens, chemicals and other agents. SNPs are of great interest not only for genetic research but also for medical diagnostics and therapy ,2.SNPs and point-mutations can be detected by means of relatively short 10 to 30 mer probes: Already a single mismatched (MM) base pair can result in a significant decrease of the duplex binding affinity with respect to the corresponding perfect matching (PM) duplex [et al. [The binding affinity of mismatched duplexes – in bulk solution – is commonly predicted on the basis of the nearest-neighbor model -6. A recet al.  revealedet al. -10. Furtet al.  and variet al.  have a sin vitro transcripts [Our study is a comprehensive approach to understand how point defects affect the hybridization of fluorescently labeled oligonucleotide targets to surface-bound oligonucleotide probes. Rather than previous work on single base MMs, which has been conducted with complex target mixtures either from PCR products  or in vinscripts , we emplnscripts  we perfoin situ synthesis [DNA chips were fabricated by light-directed ynthesis ,15 with ynthesis -21 develynthesis . Sets ofynthesis ,9,10 andet al. [et al. [After the current article was submitted, we became aware of further related studies in this area. Suzuki et al.  performe [et al.  performeThe focus of the present paper is on the impact of various defect types (single base mismatches and single base bulges) on the hybridization signal.Our microarray hybridization experiments performed in this study provide quantitative information on the binding affinity of individual mismatched duplexes by means of the hybridization signal intensity (fluorescence of hybridized targets). Since the absolute hybridization signal intensities of the different sequence motifs employed in this study Tab.  are subjsolid black line in Fig. The "defect profile" plots . Since the relative hybridization signal intensities within the individual probe sets are largely unaffected by this variation, we normalize the "defect profiles" by division with their standard deviation. The resulting database comprising normalized hybridization signals from about 1000 different single MM probe sequences, enables categorization of the binding affinities according to the mismatch type.In the following we use the notation of the mismatch base pair For statistical analysis of MM type and nearest-neighbor influences the superposed positional influence needs to be eliminated by subtraction of the mean profile. The resulting position-independent defect profile (for simplicity we keep the term "defect profile") consisting of influences of defect type and defect neighborhood only is shown in Fig. δImp from the mean MM profiles , this is due to the reduced number of binding base pairs in the mismatched duplexes . In single base insertions no binding base pair is substituted, but we see that the influence of the inserted base clearly depends on its neighbor. The individual curves . Group II bulges located near the center of 16 mer probes often show hybridization signals with a similar intensity as the corresponding PM probe  result obe Fig. . A statiobe Fig.  comprisiGroup I bulges: For G-insertions next to a T  have also been observed for certain X and Y . Hybridization signals of MMs  originating from C·G base pairs are about 25% smaller (in the median) than for MMs from A·T base pairs. Single base deletions affecting C·G base pairs result in about 30% smaller hybridization signals than deletions affecting A·T base pairs. The deletion profile in Fig. We also observe a noticeable influence of the next-neighbor bases of the mismatch  NN pairs . We propGroup II bulges). This consistency suggests a common origin of the positional influence, independent of defect type.We observe a very similar position dependence for single base bulge defects as for single base mismatches. Also, the magnitudes of the impacts of the MMs and base bulges on the hybridization signal are very similar . Since there are no long range molecular forces, we infer that the molecular dynamics must play a role, effects like breathing bubbles or zipping could be at the origin. This influence of the duplex sequence and the observed symmetry of the defect positional influence with respect to the duplex ends suggest that end-domain opening  must be suspected to be a key mechanism for understanding the influence of defect position on duplex stability.δ2(PM/MM) hybridization signal values and δRemoving the positional influence in our data, we see that single-base MMs introduced at the site of a C·G base pair result in a larger decrease of the hybridization signal  than MM defects affecting A·T base pairs. The same applies for single base deletions see Fig. . These eously in .et al. [2(PM/MM) hybridization signal values in Fig. 5a in [et al. [We emphasize the good correlation between our DNA/DNA MM stability order Fig.  and the et al.  (the MM g. 5a in ). A majog. 5a in ) found G [et al.  – in accOur direct comparison between DNA/DNA and RNA/DNA hybridization on microarrays reveals – for RNA/DNA duplexes – an increased destabilization of purine-purine mismatches, with respect to other MM types. An explanatory approach for the observed differences between DNA/DNA and RNA/DNA binding affinities is, that purine-purine MMs cause larger steric hindrance in the A-form hybrid duplexes than in the B-form DNA/DNA duplexes.In contrast to  we did nGroup I and Group II binding affinities δIbulge  is distinctly larger than the defect-type related variation of binding affinities δIMM  span the range T = 37°C).We observe significantly increased hybridization signals of single- base insertion defects in which the insertion base is placed next to a like-base. Our investigation shows that (on the microarray) the difference between see Fig. . For comenergies  for T = Group II bulges in comparison with Group I bulges has been investigated previously in solution rather than on microarrays [Group II bulges originates from positional degeneracy of the base bulge. Additional conformational freedom, entailing higher entropy, results in lowered duplex free energy (thus in increased stability). According to Zhu et al. [R·T· ln 2 = -0.43 kcal/mol at 37°C) for a two-position degeneracy. Znosko et al. [Group II duplexes to be on average δΔG37 = -0.8 kcal/mol more stable than Group I duplexes. The latter value matches better our observation of Group II hybridization close to the perfect match hybridization signal.The increased stability of roarrays ,34,35. Au et al.  positionu et al.  of -R·T·Group II duplexes we propose the following mechanism  base must act as a kinetic barrier, interrupting the rapid zipping of the duplex. The 1-nt frameshift between the (largely) complementary strands, owing to the unpaired bulge base prevents closing beyond the defect and results in a partially zipped, and correspondingly weakly-bound, duplex. Duplex closure can only progress if the interfering surplus base is giving way (adopting a favorable looped-out or stacked conformation), thus allowing the subsequent base to form a Watson-Crick base pair with the complementary base in the target strand. From this point zipping can progress rapidly. Therefore compared to Watson-Crick nearest-neighbor pairs, a base bulge (similar to a mismatch) has a decreased ratio of zipping/unzipping-rates k+/k- and thus favors unzipping of the duplex (i.e. the duplex dissociation rate kdiss is increased with respect to the perfectly matching duplex). For Group II bulges the k+/k- ratio at the defect site is increased with respect to Group I bulges: in case of a Group II bulge there is an increased probability that any of the degenerate bases makes way  while simultaneously the subsequent base forms a base pair. This is due to the increased number of possible molecular conformations, which can lead to continuation of the zipping. Then, as the frameshift is compensated, the rapid zipping to complete the duplex occurs. Since the nucleation rate knuc of Group I and Group II duplexes may be assumed to be the same, the binding affinity of Group II duplexes must be increased.For explanation of the large binding affinity of in situ synthesis with the Geniom® One instrument . Similar as in our study, they compared normalized hybridization signal intensities.Tautz and coworkers  performein vitro transcribed RNA targets [However, an important difference between the experiments described in  and our  targets  originatet al. [et al. [G37 values of mismatched trinucleotide duplexes, is considering absolute stability parameters, whereas [guide strand (a single RNA strand which is bound to the RISC complex) and a complementary mRNA. Schwarz et al. (see Schwarz: table 5b) have shown that among all MM-types incorporated at position 10 of the guide strand (apart from the point mutations the sequence of the guide strand was preserved) purine-purine MMs resulted in the least silencing of gene activity , whereas U·G, C·U and U·U mismatches resulted in a very efficient gene silencing  and can therefore exclude that target secondary structure, steric hindrance, labeling or competitive effects are relevant for an explanation of the observed results.In our microarray-based hybridization assays the binding affinity of mispaired duplexes is dominated by the influence of defect position. The influence of the defect-type is about half in magnitude, when compared to defect-position.Group II bulges  have stronger hybridization signals than expected from previous data. Experimentally, it is not completely clear, whether the strong positional influence on oligonucleotide binding affinity is restricted to surface-hybridization or if it is also relevant for solution-phase hybridization . The comparison to other related work [There is also an influence of the neighboring sequence, which has farther reach than the defect next neighbor. Although this long reach interaction must somehow be related to the base stacking energies, we did not find a simple description. We attribute so far unexplained long range effects, in particular a trough-shaped position dependence, to molecular dynamics. We propose a molecular zipping mechanism as a suitable explanation. Zipping agrees well with the observation that ted work ,7,32, hoThe use of DNA microarrays enables a detailed investigation of oligonucleotide duplex binding affinities producing a wealth of data in simple experiments. We demonstrate that important aspects  about the impact of point defects on oligonucleotide duplex binding affinities are not yet understood. Our results from simple, controlled experiments agree well with results from extracting data from complex DNA target mixtures ,9. This All reagents were used as purchased without further purification. Unless specified otherwise aqueous solutions were prepared with nuclease-free Milli-Q water (18.2 MΩ cm).20 mm round cover glasses ; Deconex 11 UNIVERSAL ; (3-aminopropyl)-triethoxysilane (APTES) (Sigma-Aldrich); ethanol analytical grade ; 1,2-dichloroethane ; phosphorous dendrimers with aldehyde moieties cyclotriphosphazene- PMMH-96 ; potassium hydroxide (Carl Roth GmbH); sodium borohydride .RayDite™ photolabile 3'-nitrophenylpropyloxycarbonyl (NPPOC)-phosphoramidites (NPPOC-dA(tac), NPPOC-dC(ib), NPPOC-dG (ipac), NPPOC-dT) were purchased from Sigma-Proligo . Acetonitrile ; Activator 42 0.25 M (Sigma-Proligo); iodine based oxidizer . Photo-deprotection is carried out in a mildly basic solution of 25 mM piperidine  in anhydrous acetonitrile. Final base deprotection is performed in a 1:1 mixture of etylenediamine  and ethanol . UV glue  is employed to glue the chip after synthesis onto a stainless steel support.The hybridization buffer comprises 5 × SSPE pH 7.4, with either 0.1% SDS or 0.01% Tween 20; the initial target concentration in the hybridization solution was 1 nM in all experiments.Cy3-labeled target oligonucleotides (DNA and RNA) – see Tab. et al. [in situ synthesis process (coupling of phosphoramidite building blocks) the aldehyde moieties of the dendrimers are reduced to hydroxyl groups. Reduction is performed in an aqueous solution of 0.35% sodium borohydride . After rinsing with MilliQ-water the slides are ready for use. Long term storage for more than one year at 4°C (under air atmosphere) doesn't affect the substrates.Dendrimer-functionalized substrates were prepared according to LeBerre et al. . For comin situ synthesis [et al. [in situ on hydroxy-functionalized phosphorus dendrimer supports. The initial photoreactive monolayer is created by coupling of NPPOC-dT-phosphoramidite. Subsequent light-directed synthesis was performed with NPPOC-phosphoramidite chemistry [Oligonucleotide microarrays tailor-made for our experiments were fabricated in-house employing light-directed ynthesis ,15. The ynthesis -21,40 is [et al. . Microarhemistry .Probe sets for the experiments are derived from various 16–25 mer probe sequence motifs that are complementary to the set of fluorescently labeled target sequences Tab.  which duμm (6 × 6 DMD pixels) is used to minimize image analysis related quantification errors.DNA chips produced for this study typically comprise about 2000 to 3000 features. A relatively large feature size of 21 μl is formed by a cutout in a 1.5 mm sheet of PDMS silicone rubber. Temperature is controlled with a foil heater attached to a stainless steel plate composing the backside of the hybridization chamber.Hybridization of fluorescently labeled targets to surface-bound probes is carried out in a temperature-controlled hybridization chamber. The chip, synthesized on a 20 mm diameter cover glass (glue-fixed onto a stainless steel support), constitutes a window into the chamber. The chamber volume of 150 Relative intensities within the probe sets are largely independent of the hybridization time, chosen to be 10 minutes, typically. Probe sequence motifs with small hybridization affinities are hybridized for up to 30 minutes to achieve a sufficiently large hybridization signal/background ratio. Microarray hybridizations Hybridization temperature for 16 mer probes was typically 30°C. An increased hybridization temperature of 40°C has been applied for probes complementary to the target URA. At 30°C these, due to their large hybridization affinity, hybridize with reduced defect discrimination. Probes with a length of 20 and more bases are hybridized at 40°C. Hybridization is monitored in real-time on an Olympus IX81 fluorescence microscope. During acquisition of the hybridization signal the microarray is left in the hybridization solution. A 10 × 0.4NA UPlanApo objective provides a sufficiently large field of view. An electron multiplying CCD camera (Hamamatsu EM-CCD 9102) with a 1000 × 1000 pixel resolution is used for image acquisition. During image acquisition shade correction is performed to compensate for intensity inhomogeneities in fluorescence excitation.Image analysis software developed in-house is employed to read the intensities of hundreds of features simultaneously.Ifeat,meas is composed of the feature intensity Ifeat and the solution background intensity Iback (originating from fluorescent targets floating above the microarray in the hybridization solution). The overall intensity Ifeat,meas = f (x)·(Ifeat + Iback) is affected by the function f(x) which accounts for spatial variations of the fluorescence excitation and the light collection efficiency of the microscope system (e.g. due to vignetting). Apart from Ifeat,meas we also locally ·Iback. A solution-background correction is performed by subtraction of the background fluorescence intensity. Further, by division by the solution background intensity f(x)·Iback we cancel the feature-position related bias f(x).Hybridization signal measurements are performed with the microarray immersed in the hybridization solution. Thus, the measured hybridization intensity signal δIMM to the hybridization signal.In the further analysis we separate between the relatively strong defect positional influence and the defect-type related influence on the binding affinity. The positional influence is calculated as the moving average of mismatch hybridization signals  over a window of five consecutive MM-positions. By subtraction of the mean profile we obtain the MM-type dependent contributions δIMM from different defect profiles it is necessary to account for the fact that the mismatch discrimination depends on the binding affinity. Mismatch discrimination is stronger in weakly-binding short duplexes or duplexes with a large AT-content. Vice versa, in case of duplexes with larger binding affinities the differences between PM and MM duplexes and among different MMs, respectively, may be rather small. We performed normalization of δIMM by division by the standard deviation σprofile . Each microarray assay investigated single base MM and bulge defects for 6 different probe sequence motifs (obtained by shifting the 16 to 20 mer probe motif with respect to the longer target sequence). Two replicates of each feature block are employed to control for the reproducibility of the measurement.Three microarrays were fabricated, each one focussing on one particular target sequence  show basically the same trend of binding affinities which is, however, superposed by a strong sequence dependent bias.TN developed the experimental setup, performed the experiments, carried out the data analysis and drafted the manuscript. OE aided in DNA chip synthesis and data analysis. TM participated in the development of the DNA microarray synthesizer. WM and JK participated in data interpretation and helped to draft the manuscript. AO conceived of the study, and participated in its design and coordination and aided in drafting the manuscript.The raw hybridization signal intensities of the 16 mer probes in a microarray hybridization experiment on single base mismatch discrimination. The data was extracted from fluorescence micrographs  of the hybridized microarrays. The dataset comprises the hybridization signal raw data and probe/target sequences of 24 mismatch defect profiles.Click here for fileδImp and the predicted Gibbs free energy increments δδX·Y (in A) and according to the flanking base pairs (in B). Data points indicate the median δImp of the individual mismatch/flanking base pair categories. The small number of data within the individual categories  can result in outliers. The exact MM-category corresponding to each data point can be identified by looking up the symbols in the identical plots in (A) and (B). Part (A) shows a weak, approximately linear correlation between δImp and δδCorrelation between the MM-type related hybridization signal deviations from the mean profile rameters . HybridiClick here for fileThe raw hybridization signal intensities of the 22–26 mer probes in a microarray hybridization experiment on the binding affinity of bulged duplexes. Hybridization signal intensities were extracted from fluorescence micrographs  of the hybridized microarrays. The dataset comprises the hybridization signal data and probe/target sequences of 14 defect profiles.Click here for fileThe raw hybridization signal intensities of the 16 mer probes in a microarray hybridization experiment designed for a direct comparison between binding affinities of single base mismatches and single base bulges. The data was extracted from fluorescence micrographs  of the hybridized microarrays. The dataset comprises the hybridization signal raw data and probe/target sequences of 23 defect profiles.Click here for fileX-IY  reveal correlations between the hybridization signals of different insertion types. To exclude the impact of systematically increased intensities of Group II insertions only Group I insertions are regarded here. Between T- and G-insertions (and between C- and A- insertions) a correlation, as indicated by a narrow distribution with a pronounced peak near zero, is observed. The broad distribution of hybridization signals differences between G and A insertions doesn't show a distinct peak, indicating that there is no correlation but rather an anti-correlation for insertions of A and G.Histograms of hybridization signal differences IClick here for fileComparison of DNA/DNA and RNA/DNA mismatch hybridization signals – mismatch defect profiles. Parts A-D make a direct comparison of hybridization signals, obtained from subsequent hybridization of RNA targets (top) and DNA targets (bottom) on the same microarray. The defect positional influence is identical for DNA/DNA and RNA/DNA hybridization. However, the impact of MM-types reveals systematic differences. The sequences shown in the plots are the probe sequence motifs that have been modified by base substitution. The hybridization signal (in a.u.) is plotted against the defect position. Hybridization signal processing: solution-background correction (see Methods section). Substitution bases A (red cross), C (green circle), G (blue star) and T (cyan triangle) either result in 3 MM duplexes and one PM duplex at every defect position; Hybridization signals of duplexes with single base deletions (yellow line); moving average MM hybridization signal (black line).Click here for fileHybridization signal variation between pairs of mismatched RNA/DNA- and analog DNA/DNA-duplexes . The largest differences between RNA/DNA and DNA/DNA binding affinities were found for the MM-types T·G, G·A and A·G.Click here for fileThe raw hybridization signal intensities of this microarray hybridization experiment provide a direct comparison between RNA/DNA and DNA/DNA hybridization. Hybridization signal intensity raw data was extracted from fluorescence micrographs  of the hybridized microarrays. The dataset contains the data of 3 independent experiments (performed with 3 different microarrays). Each microarray dataset comprises the hybridization signal data and probe/target sequences of 24 defect profiles.Click here for fileμm) in the 16 mer mismatch experiment. The shading-corrected image shows two feature blocks corresponding to two different 16 mer probe sequence motifs  both hybridizing with the fluorescently labeled target sequence COM (5'-Cy3-AACTCGCTATAATGACCTGGACTG-3'). Each feature block comprises all single base mismatches of the particular probe sequence. Groups of four features (as indicated by the marked groups 1 and 2) correspond to each one of the 16 possible mismatch base positions. As indicated by the letters between the feature blocks the uppermost row of features in each group corresponds to an A base at the corresponding base position, followed by probes with C, G and T ) originating from the fluorescence microscope optics (e.g. due to inhomogeneous fluorescence excitation or vignetting) we apply a bias correction procedure on the raw intensity data: The f(x) component in the raw hybridization signal intensity is canceled by normalization with the local solution background fluorescence intensity f(x)·Iback. (B) Normalization of the MM-type dependent component δIMM of the hybridization signal is necessary since the magnitude of mismatch discrimination depends on the binding affinity of the sequence motif. The defect profile in a) shows a large MM discrimination , whereas the defect profile in b) shows a small MM discrimination . In the position-independent defect profiles (right) the positional influence  has been subtracted, to yield the MM-type dependent influence δIMM. For statistical analysis of the defect-type contribution, including comparable data from different defect profiles, normalization is performed by division through the standard deviation σprofile of the position-independent defect profile.Data analysis procedures. (A) To reduce intensity gradients on the microarray  for the single base mismatch/single base insertion experiments (compare with Additional file Click here for file


We determined the complete sequence of the nucleoprotein (N) gene and compared it with those of 26 representative lyssaviruses obtained from databases. The Aravan virus was distinguished from seven distinct genotypes on the basis of nucleotide and amino acid identity. Phylogenetic analysis based on both nucleotide and amino acid sequences showed that the Aravan virus was more closely related to genotypes 4, 5, and—to a lesser extent—6, which circulates among insectivorus bats in Europe and Africa. The Aravan virus does not belong to any of the seven known genotypes of lyssaviruses, namely, rabies, Lagos bat, Mokola, and Duvenhage viruses and European bat lyssavirus 1, European bat lyssavirus 2, and Australian bat lyssavirus. Based on these data, we propose a new genotype for the Lyssavirus genus.The Aravan virus was isolated from a Lesser Mouse-eared Bat  Lyssavirus genus includes seven genotypes: rabies virus , Lagos bat virus (genotype 2), Mokola virus (genotype 3), Duvenhage virus (genotype 4), European bat lyssavirus 1 , European bat lyssavirus 2 , and Australian bat lyssavirus  (Eidolon helvum) in Nigeria in 1956 ((Micropterus pusillus) in the Central Africa Republic (Crocidura sp.) and a child in Nigeria in 1968 (Miniopterus sp. bats in 1981 (Eptesicus serotinus) in Germany in 1968 (Myotis genus (Myotis dasycneme and M. daubentonii)  in the Philippines  in Kyrghyzstan in 1991. The antigenic profile of the virus was analyzed by using two panels of antinucleocapsid (N) gene monoclonal antibodies developed at the Wistar Institute of Anatomy and Biology (USA) and the Central Veterinary Laboratory of Great Britain   during a survey of 269 bats collected in the Osh region of Kyrghyzstan from 1988 to 1992 . cDNA was obtained with a T-Primed First-Strand kit . PCR amplification and sequencing of the N gene were performed by using the sense primer AraN-S01  and an antisense primer AraNC-1400 . The cDNA template (2 μL) was amplified by using primers (AraN-S01 and AraNC-1400), according to the manufacturer’s instruction . PCR reactions were incubated at 94°C for 2 min, subjected to 40 cycles of 94°C for 30 s, 48° for 20 s, and 68°C for 2 min, and a final extension at 68°C for 7 min in a DNA thermal cycler  (MEGA 2 (The 1350-nt and the deduced 450 amino acid (aa) sequences of the N gene of the Aravan virus were aligned with 26 lyssaviruses by using Clustal W program . The results confirmed that the Aravan virus is a lyssavirus.The 1350-nt and the deduced 450 aa sequences of the Aravan virus were compared with 26 representative lyssaviruses belonging to seven genotypes . We seleA phylogenetic tree of 27 lyssaviruses, including the Aravan virus, based on the 1350-nt sequence of the N gene was constructed by using the vesicular stomatitis Indiana virus  as an outgroup , a. The Lyssavirus genus.These results, along with those in In this article, we have reported the first lyssavirus distinct from rabies virus originating on the Asian continent. The Aravan virus was more closely related to genotypes 4, 5, and, to a lesser extent, 6, which circulates among insectivorus bats in Europe and Africa. The lesser mouse-eared bat, from which the Aravan virus was isolated, is widely distributed in northern Africa, the Mediterranean, southern Europe, Crimea, Caucasus, Palestine, southwest Asia, and parts of central and eastern Asia. This information should be considered in the discussion of lyssavirus classification and evolution, as it suggests the possibility of a broader geographic distribution of the Aravan virus. We have no information about human rabies caused by bat exposure from central Asia, and rabies surveillance in this area is not known well. Based on this information and the virus’ misdiagnoses as rabies, we consider that transmission of Aravan virus to humans is possible. Indeed, this finding stimulates interest in new genotypes of lyssaviruses and is important from the viewpoint of public health, necessitating further lyssavirus surveillance of bats on the Asian continent.