Cherry rusty mottle is a disease of sweet cherries first described in 1940 in western North America. Because of the graft-transmissible nature of the disease, a viral nature of the disease was assumed. Here, the complete genomic nucleotide sequences of virus isolates from two trees expressing cherry rusty mottle disease symptoms are characterized; the virus is designated cherry rusty mottle associated virus (CRMaV). The biological and molecular characteristics of this virus in comparison to those of cherry necrotic rusty mottle virus (CNRMV) and cherry green ring mottle virus (CGRMV) are described. CRMaV was subsequently detected in additional sweet cherry trees expressing symptoms of cherry rusty mottle disease.The online version of this article (doi:10.1007/s00705-013-1668-9) contains supplementary material, which is available to authorized users. Prunus avium) first described in 1940 in Washington State, USA ) were reported previously , 5. CNRMiviridae , 7. Reveiviridae , and a civiridae . At the To better understand virus-like agents of sweet cherry, the complete genomic sequence of a virus associated with CRMD was determined. Since Koch’s postulates for the etiology of CRMD have not yet been established, the virus sequenced in this study is designated cherry rusty mottle associated virus (CRMaV).P. avium ‘Bing’ and ‘Sam’, respectively, in screenhouses of the Clean Plant Center Northwest (CPCNW), located at Washington State University, Prosser, WA. Graft inoculation of indicator trees with bark chips from CRMD reference sources 95CI192R3 or B48-C resulted in the induction of typical rusty mottle symptoms consisting of chlorotic yellow leaf mottling on ‘Bing’ and chlorotic yellow mottle overlaid with reddish brown mottling on ‘Sam’  following the manufacturer’s recommendation with the addition of 14 μg non-acetylated bovine serum albumin per 25 μl reaction. Prune dwarf virus was present in both CRMD sources 95CI192R3, B48-C and in CNRMD source 103-13. CRMD 95CI192R3 was also infected with both Prunus necrotic ringspot virus and cherry virus A. RT-PCR tests for the presence of other cherry viruses, viroids and two bacteria were negative in all CRMD and CNRMD reference samples.The presence of common viruses and viroids of cherry was investigated by RT-PCR (Supplementary table S1 for primers). A one-tube RT-PCR system was used (SuperScript® III One-Step RT-PCR System with Platinum® 17) [Taq DNA Polymerase; Life Technologies). The 1.1-kbp products were ligated into pCR-2.1 (Life Technologies) and three clones of each amplification reaction were sequenced in both directions .Preliminary analysis of double-stranded RNA from affected trees yielded sequences closely related to CNRMV and CGRMV (unpublished); therefore, a forward primer (Fovea2: CTCA-TTC-ACA-TAG-CTT-AGGT) was designed from conserved regions located upstream of the coat protein region of full genomic sequences of CGRMV  and CNRMV . First-strand cDNA synthesis was initiated from total RNA with FRSTD primer (TATG-ACA-CGC-GTC-GAC-TAGC(T)17)  and reveThe sequence at the 5’-terminus was obtained by designing primers  from the conserved regions in the multiple sequence alignments of CGRMV and CNRMV. NGRM 6161R was used for first-strand cDNA synthesis, and NGRM 2996R was employed in 5’ rapid amplification of cDNA ends (5’-RACE) using a commercial kit (GeneRacer®: Life Technologies). Five clones of the resulting amplification products of 3 kbp were sequenced.® HS DNA Polymerase: Takara Bio, Inc., Otsu, Shiga, Japan). An A-overhang was added to the product with DNA polymerase , and the resulting product of 8.4 kbp was ligated into pCR®-XL-TOPO (Life Technologies). Three different clones from a single RT-PCR reaction were selected for sequencing by primer walking. Assembly of contigs and pairwise alignment of sequences were performed with BioEdit [Primers were designed from the sequences obtained above to amplify the full-length genomes in a two-step RT-PCR reaction. First-strand cDNA was initiated with a 3’-terminal, CRMaV-specific primer (CRM 95CI192R3 3’-end R1: GCA-TTG-CAA-ACT-AAA-GGA-AAA-T-ATAT); cDNA was used for PCR with the 5’-terminal primer (CRM 5’-end F1: GAAA-ACAA-ACAG-ACCA-AAAC-TAGC) and the 3’-terminal primer CRM 95CI192R3 3’-end R1 with DNA polymerase  sequences from CRMD reference trees 95CI192R3 and B48-C consist of 8,397 and 8,398 nt, respectively, excluding the poly A-tail . The viruses represented by these sequences are designated cherry rusty mottle associated virus (CRMaV), and the sequences were compared with those of known isolates of CGRMV and CNRMV, including three other unassigned members of the family Overall, the genome organization of CRMaV 95CI192R3 and B48-C is similar to that of CGRMV and CNRMV, with five presumed ORFs plus two ORFs nested within ORFs 2 and 5 and designated ORF 2a and 5a, respectively  of 230 kDa containing the conserved domains for viral methyltransferase (Met), helicase (Hel) and RNA-dependent RNA polymerase (RdRp) associated with the replicases of plant viruses; a papain-like cysteine protease (P-Pro) that processes the replicase polyprotein; an AlkB homologue; and an ovarian tumor (OTU)-like cysteine protease. These domains are also found in the replicase regions of CGRMV and CNRMV.ORF1 of CRMaV encodes a protein with a relative molecular mass  proteins . ORF2  of CRMaV is 230 kDa and shares a high degree of nt and aa sequence identity with CGRMV and CNRMV across all ORFs, it is evident that it is a member of the family Betaflexiviridae. The comparative values of aa sequences of replication proteins and CP between these viruses are above 45%, which is the minimum criterion for inclusion of the viruses within a single genus.Phylogenetic analysis of sequences of the large replication proteins (ORF1) of members of the family iviridae . MembersBetaflexiviridae is that members of distinct species have less than 72% nt sequence identity or 80% aa sequence identity in the whole CP or replication protein gene [Betaflexiviridae from the now defunct “Flexiviridae” was based on the phylogenetic reassessment of the replicase coding region, the assignment of CNRMV, CGRMV and CRMaV to three separate virus species is favored. This assessment is supported by examination of the biological data. The distinction between CNRMV, CGRMV and CRMaV is exemplified by distinctly different symptoms induced on different P. avium cultivars. As additional virus sequences are reported, the species demarcation criteria for the family Betaflexiviridae must be revised to address contradictory interpretations when CP versus replicase coding regions products are considered.The proposed criterion for species demarcation in the family ein gene . This crBetaflexiviridae. The process of genetic recombination could explain the relationships between the CP and replicase sequences of these three viruses. However, preliminary analyses using the Recombination Detection Program version 3 (RDP3) [Potexvirus [The results of this study reveal a newly identified virus, CRMaV, which is closely related to but distinct from CGRMV and CNRMV, both of which are unclassified members of the family 3 (RDP3)  for the texvirus .Supplementary material 1 (DOC 62 kb)Below is the link to the electronic supplementary material.


Chaetosiphon fragaefolii (Cockerell) (Homoptera: Aphididae), was collected on a native strawberry, Fragaria chiloensis (L.) Duchesne  from different sites in Chile. The presence of this aphid was recorded during two consecutive years. F. chiloensis plants were collected from seven natural and cultivated growing areas in central and southern Chile. Aphids were genotyped by cross-species amplification of four microsatellite loci from other aphid species. In addition, the aphid borne virus Strawberry mild yellow edge virus was confirmed in F. chiloensis plants by double-antibody sandwich ELISA and RT-PCR. Genetic variability and structure of the aphid populations was assessed from the geo-referenced individuals through AMOVA and a Bayesian assignment test. The presence of C. fragaefolii, during the two-year study was detected in only four of the seven sites . Genetic variation among these populations reached 19% of the total variance. When assigning the individuals to groups, these were separated in three genetic clusters geographically disjunct. Of the seven sampled sites, six were positive for the virus by RT-PCR, and five by double-antibody sandwich ELISA . The incidence of the virus ranged from 0–100%. Presence of the virus corresponded with the presence of the aphid in all but two sites (Chilian and Vilches). The greatest incidence of Strawberry mild yellow edge virus was related to the abundance of aphids. On the other hand, sequences of the coat protein gene of the different virus samples did not show correspondence with either the genetic groups of the aphids or the sampling sites. The genetic structure of aphids could suggest that dispersal is mainly through human activities, and the spread to natural areas has not yet occurred on a great scale.The monoecious anholocyclical aphid,  Chaetosiphon fragaefolii (Cockerell) (Homoptera: Aphididae) is an important pest of strawberry worldwide, presumed to originate from North America. Parthenogenetic forms occur all year round. Although in laboratory cultures and greenhouses male and oviparae female forms occur, this is rarely found in the field  (Fragaria chiloensis (L.) Duchesne   , althougosaceae) . F. chil7° 33′ S , and was7° 33′ S . HoweverC. fragaefolii persistently transmits several viruses, such as the Strawberry crinkle virus, Strawberry mottle virus, Strawberry mild yellow edge virus (SMYEV), and Strawberry vein banding virus , Vilches , Chovellen , Chilian , Contulmo , Petrohue , and Cucao   were done to avoid aphid cross-transmission between plants.During two consecutive years, seven areas in the central-south of Chile were sampled for 1° 7′ W) . When prGenomic DNA was obtained following the ‘salting out’ procedure from Sunnucks and Hales . The tisSm10, Sm11, and Sm17 (M62 and M37 (http://www.eppendorf.com), and performed in a 10 µl reaction mixture containing: 1 ng/µl DNA template, 2.5mM MgCl, 0.2 mM dNTP, 0.5 U Taq DNA polymerase , 0.5 µM of each primer, 20 mM Tris-HCl, pH 8.4, 50 mM KCl. PCR followed a program of 3 min of initial denaturation at 94° C and then 40 cycles of a 1 min denaturation step at 94° C, 1 min of annealing , a 45 sec extension at 72° C, and a final extension at 72° C for 4 min. Amplicons were separated in 6% Polyacrylamide denaturing gels using a BIO-RAD Sequi-Gen GT Electrophoresis Cell. After electrophoresis, gels were silver-stained to visualize the PCR products using the procedure described by Promega (http://www.promega.com)loaded in the same gel.A total of ten heterologous microsatellite loci were tested, but only five amplified successfully. These were and Sm17 , as well and M37 . These l and M37 . Polymer Promega . Variatihttp://www.bioreba.ch). Anti-viral inmunoglobulins were used in polystyrene microtitre plates according to the manufacturer's instructions.Double-antibody sandwich ELISA was performed as described by Clark and Adams  using a F. chiloensis leaf tissue was triturated in 3 ml extraction buffer  and centrifuged. Aliquots of 100 µl of prepared samples were added to each well duplicated, and negative and positive commercial controls were added to each plate. A total of 20 randomly chosen plants were tested for each site. Samples were read 30 and 60 min after ELISA at 405 nm in a microtitre plate reader VICTOR X3 . ELISA readings were considered positive when the absorbance of sample wells was at least two times greater than the mean absorbance of the negative controls.F. chiloensis plants was also confirmed with RT-PCR analysis with coat protein specific primers following the protocol of Thompson et al. , 0.05% v/v Tween-20, 2% w/v PVP-40, 0.2% w/v ovalbumin, 0.5% w/v bovine serum albumin, 0.05% w/v NaNs) and transferred to a 1.5 mL plastic tube. Then, 100 µl 10% Nlauryl sarcosyl and 5 µl 2-meraptoethanol were added to the tube. From this mixture, a total of 200 µl was taken, and 400 µl of grinding buffer was added and incubated at 70° C with intermittent shaking for 10 min. Then, tubes were placed on ice for 5 min, and centrifuged at 13,000 rpm for 10 min. Next, 150 µl of EtOH, 300 µl 6 M NaI solution, and 25 µl of re-suspended silica were added to the supernatant. Subsequently, the pellet was resuspended in 500 µl of wash buffer and dried. After drying, the pellet was re-suspended in 100 µl of sterilized distilled water, incubated at 70° C for 5 min, and centrifuged at 13,000 rpm for 3 min. RNA integrity was checked by electrophoresis on 1 % agarose gels, and by the A260/A280 ratio using a spectrophotometer .As recombinant strains of virus could be undetected using strain specific monoclonal antibodies , the presence of SMYEV in Complementary DNAs were prepared using SuperScript III Reverse Transcriptase (RT) (Invitrogen) as reported previously by Chang et al. . The RT http://www.genscript.com) from 3 ml overnight cultures containing ampicillin. Recombinant plasmids were verified by EcoR1 digestion. Sequencing reactions were performed at the Macrogen Inc. facilities (http://www.macrogen.com) in an ABI3730 XL automatic DNA sequencer.The resulting DNA fragments were cloned in the TOPO® TA vector (Invitrogen). Two µl of the ligation solution was used to transform One Shot Mach1-T1 chemically competent cells (Invitrogen). Plasmids were isolated using QuickClean 5M miniprep kit . PhiPT is analogous to Fst when the data are haploid, or when assumptions of HWE are not met (PhiPT) and the geographical distance between sites, using zt version 1.0 with 10,000 permutations  and Contulmo (0.31). The number of effective alleles varied from 3 and 9 . Therefore HWE was estimated excluding M37, with no significant difference with the estimations including M37. Genetic differentiation in the data was modest, with PhiPT values reaching 0.19, although PhiPT values between pairs of sites varied from 0.03 to 0.33 (p = 0.008). The number of clusters in optimal partition assignment with BAPS determined K = 3, with a log marginal likelihood of optimal partition of 757.4084, and the posterior probability reaching its highest value (∼1). Aphids from sites Chovellen and Curepto conformed one cluster, whereas aphids from Contulmo alone formed a second cluster. Aphids from Cucao formed a third cluster (As derived from  3 and 9 . All loclgorithm  implemenlgorithm  was high to 0.33 . The gre to 0.33 . There w cluster . This saC. fragaefolii). SMYEV, however, was detected at six of these seven sites. Mean aphid abundance per leaf per site ranged from 0.8 to 14.5, with the highest numbers occurring at Contulmo  when compared with the remaining samples . In addiC. fragaefolii populations was highest at Cucao and Contulmo. Such values are lower than those reported in studies from other aphid species (M37) presented high values of null alleles, but was consistent over all populations, and did not affect the overall results. Genetic variability was modest,showing geographic structure especially with the most southern population (Cucao). Bayesian analysis showed three clear clusters, which was in agreement with the AMOVA results, as sites in each cluster shared low PhiPT values, and the highest PhiPT values were between sites that were assigned to other clusters , suggests little migration between sites. It is important to mention that Cucao is a population located on the Chiloé island, a rather big island in the south of Chile. This may explain the genetic distance of the southern C. fragaefolii from the northern populations.Clusters agreed with geographical distance between sampling sites, as areas that were close to each other formed a single cluster, and sites further away formed separated genetic clusters . In factC. fragaefolii on F. chiloensis , the virus had a low incidence (0.05%). At both sites, however, Neuquenaphis edwardsi (Laing) (Hemiptera: Aphididae), a native species of aphid frequently found on southern beech Nothofagus sp. Blume , was found developing on wild F. chiloensis. It is common to find (as is the case for both sites) F. chiloensis associated to Nothofagus forests . Further studies are needed to better understand these findings and to confirm F. chiloensis as an alternative host for N. edwardsi, or for other native aphid species. It has been reported that introduced aphids can be very harmful to the native flora (F. chiloensis cultivation area (Contulmo)  . Most orC. fragaefolii aphids have been introduced and spread recently, the low incidence of the virus in the native F. chiloensis could be explained. However, sequence data of the virus found on F. chiloensis suggest that the virus is not different between cultivated and wild F. chiloensis. Thus, the presence of the aphid clearly increases the incidence of the virus, even though it is already present in natural and cultivated F. chiloensis with no clear grouping of the viral strains, with the exception of Cucao. Cucao is the most southern group, and phylogenic trees could suggest isolation.As


Serum samples were obtained from 24 horses in the State of Coahuila, Mexico, in December 2002. Antibodies to West Nile virus were detected by epitope-blocking enzyme-linked immunosorbent assay and confirmed by plaque reduction neutralization test in 15 (62.5%) horses. We report the first West Nile virus activity in northern Mexico. Flaviviridae, genus Flavivirus) is a member of the Japanese encephalitis virus complex, which also includes Japanese encephalitis virus, Saint Louis encephalitis virus (SLEV), and Murray Valley encephalitis virus  of 254 Texas counties, including most counties that border Coahuila State . In the present study, blood samples were taken from 24 domestic horses at study sites located in Ciudad Acuña, Jiménez, and Saltillo . The CiuThe climatic conditions of the three study sites are similar and can be described as hot, dry, and arid. The average annual temperature ranges from 18°C to 22°C. The average rainfall is from 100 to 300 mm per year. The sites in Ciudad Acuña and Jiménez are approximately 300 m above sea level. The Saltillo site is situated at an elevation of approximately 1500 m.A horse from the Ciudad Acuña ranch died October 17, 3 days after being observed with neurologic signs. The case was not reported immediately, and we were unable to obtain a tissue specimen postmortem. On December 19, with the assistance of a local veterinary practitioner, we sampled 14 horses at this site, 5 of which had developed neurologic disease in mid- to late October. Clinical symptoms included ataxia, weakness of limbs, trembling, and anxiety. All five horses survived. The horses with clinical signs were from 1 to 5 years of age; three were male, and two were female. Ages and sexes of horses without clinical symptoms were not documented. The veterinarian reported a great abundance of mosquitoes in the area. Another six horses were sampled in Jiménez and four in Saltillo, none of which had signs of illness. According to the owners, none of the horses had ever been outside the State of Coahuila, and none of the horses had been vaccinated against WNV.>30% to indicate the presence of viral antibodies (All serum samples were tested for antibodies to WNV by epitope-blocking enzyme-linked immunosorbent assay (ELISA). Blocking ELISAs were performed by using the WNV-specific monoclonal antibody (MAb) 3.1112G, as previously described .To validate the above assays, we tested serum samples for neutralizing antibodies to WNV and SLEV by plaque reduction neutralization assay (PRNT). Testing for neutralizing antibody to SLEV was important because this virus is enzootic in the Americas and antibodies to WNV and SLEV often cross-react. Furthermore, horses are susceptible to SLEV infection, although clinical manifestations have not been reported  of 5 horses with clinical symptoms, and 10 (52.6%) of 19 horses without clinical symptoms. Therefore, the rate of asymptomatic seropositivity was high, with 10 (66.7%) of 15 WNV-infected horses showing no signs of illness. Similarly, 21 (58.3%) of 36 WNV-infected horses sampled during a serosurvey in New York in 1999 showed no clinical signs  horses were considered to be seropositive for WNV by PRNT because the antibody titers for WNV were less than fourfold higher that the corresponding SLEV titer . These 1We were unable to detect RNA in any horse serum by reverse-transcription polymerase chain reaction with WNV-specific primers . The birds were trapped in February 2003, 2 months after we obtained samples from the horses in Coahuila State. However, equine cases often precede the detection of seropositive birds. For example, an equine case was the first indication of WNV activity in 29%  of the United States counties to report virus activity in 2002 (In summary, we have obtained serologic evidence for antibodies to WNV in horses in the State of Coahuila, Mexico. In the accompanying manuscript, we report the detection of antibodies to WNV in horses in the State of Yucatan (


Dynamic expansions of toxic polyglutamine (polyQ)-encoding CAG repeats in ubiquitously expressed, but otherwise unrelated, genes cause a number of late-onset progressive neurodegenerative disorders, including Huntington disease and the spinocerebellar ataxias. As polyQ toxicity in these disorders increases with repeat length, the intergenerational expansion of unstable CAG repeats leads to anticipation, an earlier age-at-onset in successive generations. Crucially, disease associated alleles are also somatically unstable and continue to expand throughout the lifetime of the individual. Interestingly, the inherited polyQ length mediating a specific age-at-onset of symptoms varies markedly between disorders. It is widely assumed that these inter-locus differences in polyQ toxicity are mediated by protein context effects. Previously, we demonstrated that the tendency of expanded CAG•CTG repeats to undergo further intergenerational expansion (their ‘expandability’) also differs between disorders and these effects are strongly correlated with the GC content of the genomic flanking DNA. Here we show that the inter-locus toxicity of the expanded polyQ tracts of these disorders also correlates with both the expandability of the underlying CAG repeat and the GC content of the genomic DNA flanking sequences. Inter-locus polyQ toxicity does not correlate with properties of the mRNA or protein sequences, with polyQ location within the gene or protein, or steady state transcript levels in the brain. These data suggest that the observed inter-locus differences in polyQ toxicity are not mediated solely by protein context effects, but that genomic context is also important, an effect that may be mediated by modifying the rate at which somatic expansion of the DNA delivers proteins to their cytotoxic state. The second class is defined by a polyglutamine (polyQ)-encoding CAG repeat. The unstable CAG polyQ repeat disorders include Huntington disease (HD), spinal and bulbar muscular atrophy, X-linked , dentatorubral-pallidoluysian atrophy (DRPLA), Machado-Joseph disease , and the spinocerebellar ataxias, 1 (SCA1), 2 (SCA2), 7 (SCA7) and 17 (SCA17). Each of these polyQ disorders is a late-onset neurodegenerative disease associated with the loss of specific neuronal populations A growing number of inherited human diseases have been associated with DNA tandem repeat instability, trinucleotide repeats of the motif CAG•CTG comprising the largest class of such repetitive elements Expanded trinucleotide repeat instability is described as a ‘dynamic mutation’, as the frequency and magnitude of length changes vary as the repeat number changes Significantly, at expanded trinucleotide repeats toxicity increases with length, longer repeat tracts resulting in greater levels of cell death and dysfunction in affected tissues, and a more severe phenotype. Thus, intergenerational increases in expanded triplet repeat length are consistent with ‘anticipation’, a clinical characteristic common to these disorders, whereby an earlier age of disease onset and increased severity of symptoms is observed in successive generations Msh2Msh3Pms2Ogg1Dnmt1DNA ligase 1Xpatrans-acting factors involved in governing expanded repeat behaviour such as the mismatch repair system, sex of the transmitting parent and tissue type cis-acting factors in CAG•CTG instability. Expanded CAG•CTG instability is locus-specific, not genome-wide indicating that factors local to the repeat influence its mutability. The most obvious factors are those internal to the array such as number of repeats and sequence purity cis-elements in the DNA sequences flanking the repeat unit cis-acting modifiers of repeat stability flanking the repeat The precise mechanism(s) underlying the dynamic mutation of CAG•CTG repeats remains unknown. Indeed, transgenic mouse studies have variously implicated a range of DNA repair genes including Hprt gene can reproduce features of the associated human disorders including a late onset neurological phenotype and neuronal intranuclear inclusions DrosophilaAs all the dynamic repeat disorders that possess an expanded polyQ tract are dominant, display a similar inverse relationship between polyQ length and age-at-onset, and lead to progressive neuronal degeneration e.g. by modifying aggregation dynamics), or by effects on the normal function of the protein.Despite shared components of the pathogenic pathway, and although all expanded polyQ disorders show a similar inverse relationship between polyQ number and age-at-onset of symptoms, the absolute number of polyQ repeats associated with a given age-at-onset of symptoms varies considerably between the disorders HTT exon 1 fragment, by the simple addition of a FLAG-tag, caused a previously non-toxic fragment of HTT exon 1 to induce characteristic length-dependent polyQ toxicity As the size of the native expanded-polyQ containing proteins varies greatly (41 kDa–347 kDa), their primary sequences are not similar, and the position of the tract relative to the translation start site differs, the polyQ tracts clearly do have very different protein contexts. Indeed, there is ample evidence that changing the flanking amino acid sequence of an expanded polyQ tract can change its pathogenic potential. For instance, studies in yeast showed that altering the flanking sequence of an expanded HD It is our primary hypothesis that ongoing somatic expansion contributes toward disease progression in the repeat expansion disorders In order to investigate the factors mediating differences in polyQ toxicity, we sought to develop a robust quantitative measure of inter-locus polyQ toxicity. Previously, Gusella and MacDonald analysed published studies to collate measured repeat length versus age-at-onset data from large numbers of patients with the polyQ disorders rho = 0.75; P = 0.03; N = 7) , 40  or 50  years was used to determine inter-locus polyQ toxicity, suggesting that the inter-locus polyQ toxicity values as determined at 32 years age-at-onset are broadly representative of the relationship between the variables throughout the dataset as a whole. Again, a similarly significant relationship between inter-locus polyQ toxicity and locus expandability  was obtained when using inter-locus toxicity values derived from a linear model of the relationship between repeat length and age-at-onset  . Similarat-onset .e.g.MJD = 11; NSCA1 = 7; Mann-Whitney U = 0; P<0.0001) and cerebral white matter ; similar to the relative levels of germ line expandability observed in these disorders . Again, the levels of somatic expandability in HD and SCA7 were comparable to the levels of germ line expandability observed in these disorders  from a distance of 140 bp to 850 bp from the loci, whereas a more punctuated profile was found 3′ of the repeat array  was performed. A statistically significant correlation between inter-locus polyQ toxicity and flanking genomic DNA GC content was observed from 100 bp    to appro= 0.015) . A simil= 0.015) .r = −0.28; P = 0.58; N = 7). Employing the sliding window approach as before, a significant correlation between flanking mRNA sequence and GC content was only found immediately proximal (<100 bp) to the repeat tract and dropped rapidly thereafter  or translation start site .We considered it possible that the observed CAG repeat genomic DNA flanking sequence GC content correlation with inter-locus polyQ toxicity could reflect effects mediated at the level of the mRNA. Employing manually curated RefSeq mRNA sequences for each gene, we investigated the association between inter-locus polyQ toxicity and mRNA GC content. No significant correlation between inter-locus polyQ toxicity and total mRNA GC content was found  or normal human cerebellum ,  data of human brain  N = 7), . Similar N = 7), .e.g.i.e. that the age-at-onset of an individual with a polyQ disorder is a function of the inherited allele length coupled with the rate at which it expands somatically, and that the somatic expansion rate is mediated by the GC content of the flanking DNA.Although the inverse relationship between age-at-onset and repeat length is broadly similar in the dynamic DNA polyQ disorders, the inherited number of repeats required to cause a given age-at-onset varies dramatically between disorders  [2]. NotWe previously detailed a significant association between flanking GC content and locus expandability CACNA1A calcium channel gene cause the highly overlapping episodic ataxia type 2A phenotype N = 9) for inclusion in the main analyses presented in this study, we decided to test whether the relationship between expandability, inter-locus polyQ toxicity and flanking DNA GC content of unstable SCA17 alleles fitted with our model of somatic mosaicism mediated inter-locus polyQ toxicity. SCA17 data was obtained by meta-analysis of published cases of unstable SCA17 alleles  , and there is some debate as to whether SCA6 represents a true polyQ repeat disorder. Although the carboxy terminus can form polyglutamine aggregates  alleles . Interes = 0.02) . Moreove = 0.02) .ATXN2 do not induce SCA2, but instead produce a Parkinsonian phenotype, despite the fact that both the pure and interrupted repeat tracts encode pure polyQ The data we have presented here further support a role for somatic expansion in the dynamic DNA disorders. The hypothesis that somatic expansion of repeats contributes towards age-at-onset of symptoms and disease progression is further supported by a number of observations in patients. For instance, individuals with expanded yet stable SCA1 alleles exhibit significantly delayed onset of symptoms Our model of expansion-mediated disease pathogenesis is further supported by a recent computational study which predicted that repeat expansion in somatic tissue determines both age-at-onset and the rate of disease progression Msh2 null allele in a knock-in HD mouse model resulted in a 5-month delay in the appearance of aggregates Msh2 would be expected to lead to a cancer predisposition phenotype Msh3 knockouts are not cancer prone These data further support the concept that somatic expansion makes a substantial contribution to disease progression. As such, treatments that resulted in a suppression of repeat expansion would be expected to be therapeutically beneficial in vitro, it would be a major surprise if they did not. Likewise, gene expression levels and the tissue-specificity of gene expression patterns would also be expected to contribute. Nonetheless, the coefficient of correlation between GC content and inter-locus polyQ toxicity, r = −0.76, suggests that flanking GC content accounts for ∼57% of the considerable inter-locus variation in polyQ toxicity. Assuming that a sizeable proportion of this effect is mediated via effects on repeat stability, then these data provide the first quantitative insights into how effective therapies that stabilised the repeat tract might be.We have revealed a significant association between inter-locus polyQ toxicity and both repeat expandability and the GC content of the flanking DNA. These data provide the first quantitative insights into how to rationalise the observed dramatic differences in inter-locus polyQ toxicity. Of course, these data do not preclude a role for protein context in also contributing toward inter-locus polyQ toxicity and, given the dramatic effects on polyQ toxicity observed http://www.ensembl.org/index.html). The accession numbers of the mRNA sequences employed for each disorder were; NM_001007026 (ATN1), NM_000332 (ATXN1), NM_002973 (ATXN2), NM_000333 (ATXN7), NM_004993 (ATXN3), NM_000044 (AR) and NM_002111 (HTT). The accession numbers of the protein sequences employed were; NP_001007027 (ATN1), NP_000323.2 (ATXN1), NP_002964.2 (ATXN2), NP_000324 (ATXN7), NP_004984 (ATXN3), NP_000035 (AR) and NP_002102 (HTT). Repeat length versus age-at-onset data for each locus was previously collated from published studies http://www.expasy.ch/). All GC content analyses were performed with custom written software implemented in the Perl programming language. STRIDE, DSSP, and STR secondary structure predictions were performed via the SAM server (url: http://www.soe.ucsc.edu/research/compbio/sam.html). SPSS (version 13) and GraphPad Prism® (version 5) were used for statistical analyses.All genomic DNA analyses used the NCBI 36 (November 2005) assembly of the human genome, obtained from the Ensembl web server  of regression line describing the relationship between age-at-onset and repeat number for each disorder. Confidence limits of regression line were determined using GraphPad Prism® (version 5).(TIF)Click here for additional data file.Figure S2The correlation between expandability and toxicity is maintained when using a linear function to describe the relationship between age-at-onset and repeat length. (A) The graph shows the linear regression lines fitted to the age-at-onset and inherited repeat length distributions in the seven polyQ disorders. The inter-locus polyQ toxicities were derived from the parameters of the regression line of each disorder for the modal age-at-onset of 32 years (dashed lines). (B) Plot of ranked expandability and ranked inter-locus polyQ toxicity at the modal age-at-onset (32 years) with the regression line. .(TIF)Click here for additional data file.Figure S3Correlation of flanking primary amino acid sequence properties with inter-locus polyQ toxicity. Using a window size of 21 amino acids and a step size of one, locus toxicity was correlated (Spearman's rank) with various physiochemical and compositional characteristics of the primary protein sequence at every amino-acid position flanking the polyQ repeat. Repeat size was normalised to 21 glutamines. The dashed lines represent the threshold for statistical significance (P<0.05). As the 3′ sequence of ATXN3 extends just 83 amino acids away from the repeat, all correlations beyond this point involve the remaining six sequences with a correspondingly higher 5% significance threshold. Amino-acid properties were derived from the sources indicated in (TIF)Click here for additional data file.Figure S4Correlation of predicted flanking secondary protein structure with inter-locus polyQ toxicity. Using a window size of four amino-acids and a step size of one, inter-locus polyQ toxicity was correlated (Spearman's rank) with the predicted secondary structure as determined from scales of secondary structure formation potential at every amino acid position flanking the polyQ repeat. Repeat size was normalised to 21 glutamines. The dashed lines represent the threshold for statistical significance (P<0.05). As the 3′ sequence of ATXN3 extends just 83 amino acids away from the repeat, all correlations beyond this point involve the remaining six sequences with a correspondingly higher 5% significance threshold. Amino-acid properties were derived from the sources indicated in (TIF)Click here for additional data file.Figure S5Inter-locus polyQ toxicity does not correlate with 1st and 2nd base GC content. Inter-locus polyQ toxicity does not correlate with 1st and 2nd base GC content, but does correlate with the 3rd base GC content. GC content was sampled using a sliding window of 30 bp (10 codons) and a step size of 3 bp (1 codon). The threshold for statistical significance (dashed lines) is also shown. Only sequences 3′ of the CAG repeat tract were analysed as insufficient sequence is present 5′ of the repeat due to proximity of the repeat tract to the transcription start site at many loci .(TIF)Click here for additional data file.Figure S6Inclusion of unstable SCA17 alleles strengthens correlation between inter-locus toxicity and expandability. (A) The graph shows the exponential decay regression lines fitted to the age-at-onset and inherited repeat length distributions in the eight polyQ disorders including SCA17. The inter-locus polyQ toxicities were derived from the parameters of the regression line of each disorder for the modal age-at-onset of 30.5 years (dashed lines). (B) Plot of ranked expandability and ranked inter-locus polyQ toxicity at the modal age-at-onset (30.5 years) with the regression line . (C) The graph shows the regression analysis between inter-locus polyQ toxicity and the GC content of the genomic DNA flanking sequences at a distance of 100 bp .(TIF)Click here for additional data file.Table S1A. Age-at-death and somatic expandability in MJD patients. B. Age-at-death and somatic expandability in SCA1 patients. C. Levels of somatic expandability are greater in SCA1 than MJD.(DOC)Click here for additional data file.Table S21. B. Somatic expandability* in buccal cells of SCA7 patients1. C. Levels of somatic expandability are greater in SCA7 than HD.A. Somatic expandability in buccal cells of HD patients(DOC)Click here for additional data file.Table S3Inter-locus polyQ toxicity and expandability of the dynamic DNA polyQ loci.(DOC)Click here for additional data file.Table S4Age-at-onset and expandability of unstable SCA17 alleles.(DOC)Click here for additional data file.


Virus-infected fruit bats showed no signs of clinical infection. Alphacoronavirus) and group 2 (genus Betacoronavirus) CoVs. Phylogenetic analysis of the RNA-dependent RNA polymerase gene showed that groups 1 and 2 CoVs were similar to Bat-CoV/China/A515/2005 (95% nt sequence identity) and Bat-CoV/HKU9–1/China/2007 (83% identity), respectively. To propagate group 2 CoVs obtained from a lesser dog-faced fruit bat (Cynopterus brachyotis), we administered intestine samples orally to Leschenault rousette bats (Rousettus leschenaulti) maintained in our laboratory. After virus replication in the bats was confirmed, an additional passage of the virus was made in Leschenault rousette bats, and bat pathogenesis was investigated. Fruit bats infected with virus did not show clinical signs of infection.Fifty-two bats captured during July 2008 in the Philippines were tested by reverse transcription–PCR to detect bat coronavirus (CoV) RNA. The overall prevalence of virus RNA was 55.8%. We found 2 groups of sequences that belonged to group 1 (genus  Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) is a newly emerged zoonotic CoV that caused an international epidemic in 2003. Epidemiologic studies have demonstrated that the first human cases of SARS were caused by CoVs closely related to those found in Himalayan palm civets and raccoon dogs in wildlife markets  orally infected with intestinal tissues and contents from a lesser dog-faced fruit bat (Cynopterus brachyotis). After infection, clinical signs of infected bats were examined, and pathogenesis in bats was investigated.In this study, we detected bat CoVs in the Philippines. We attempted to isolate bat CoVs and virus RNA from cell cultures and from Leschenault rousette bats  of tiletamine and zolazepam  and killed by cardiac exsanguination. The experiment was conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals, Graduate School of Agriculture and Life Sciences, University of Tokyo.Virus RNA was extracted from samples obtained from field bats and from experimentally infected bats by using an SV Total RNA Isolation System Kit  according to the manufacturer’s instructions. Extracted RNA was eluted in 50 µL of RNase-free water. For cDNA synthesis, RNA (5 μL), a random hexamer, and a SuperScript III Kit  were used.All cDNA samples obtained from field bats and experimentally infected bats were tested by using conventional and nested PCR. On the basis of previous reports, we used a PCR and a pair of consensus primers specific for a highly conserved region of the RNA-dependent RNA polymerase (RdRp) gene (Two microliters of cDNA was added to a 25-μL reaction mixture containing 2× GoTaq PCR Master Mix (Promega) and 0.2 μM of 5′-GGTTGGGACTATCCTAAGTGTGA-3′ (primer 1) and 5′-CCATCATCAGATAGAATCATCATA-3′ (primer 2). The PCR conditions were 2 min at 94°C; 35 cycles for 20 s at 94°C, 30 s at 50°C, and 30 s at 72°C; and 1 min at 72°C. PCR amplicons were gel purified by using NucleoSpin Extract II  and cloned by using a TOPO-TA pCR2.1 Cloning Kit (Invitrogen). Sequencing was performed in an ABI 3130 XL DNA analyzer .On the basis of the sequences obtained, we designed new specific primer pairs for the Bat-CoV/Philippines/Diliman1552G1/2008 sequence (5′-TGATTTCTGCAATGATACTTGGTTC-3′ and 5′-ACTTGATGATCTGTAACAACAATCG-3′) and for the Bat-CoV/Philippines/Diliman1525G2/2008 sequence (5′-TACAACCTACGCTGCAACTC-3′ and 5′-ATGAGTGTGCACAAGTGCTTAG-3′). These primers were used as the inner primer set for the nested PCR after the first PCR was performed with primers 1 and 2. Aliquots (2 µL) of cDNA for primary amplification were added to 2× GoTaq Master Mix (Promega) and primers 1 and 2. Amplification was performed by using 15 cycles at conditions described above. Aliquots (2 µL) of primary amplification products were used for the second PCR with GoTaq Master Mix and the inner primers. The second PCR was performed by using 35 cycles at the conditions described above. PCR products were extracted from gels by using NucleoSpin Extract II and subjected to direct sequencing or TA cloning.C. brachyotis) was homogenized in a sterile mortar. After low-speed centrifugation , the supernatant was used for oral infection. Experimentally infected bats were examined daily for clinical signs of infection. Fecal specimens were obtained from a clean translucent plastic sheet spread along the bottom of the cage. All bats were killed after being anesthetized with diethyl ether, and organs  and serum samples were obtained.Leschenault rousette bats were obtained from zoos in Japan. Seven bats were randomly selected for the experiments. In each experiment, 2 bats were placed in a negative-pressure isolator. One additional bat was kept in a separate isolator as a control. A sample of large intestine from a lesser dog-faced fruit bat 7a, Ns7b, and Ns7c protein nucleotide sequences, we conducted PCR and DNA sequencing in the same manner as for determination of partial RdRp nucleotide sequence described above by using the HKU9-Leader42–64 primer (5′-CCGTTTCGTCTTGTACGAATCAC-3′) and the 3siteAd20T primer (5′-CTGATCTAGAGGTACCGGATCCTTTTTTTTTTTTTTTTTTTT-3′). To detect virus mRNA, we conducted reverse transcription–PCR (RT-PCR) by using 2 primer sets: HKU9-Leader42–64 and N468–448r (5′-GTTACGTGTGCCCATGTCACC-3′) and HKU9-Leader42–64 and Ns7a440–420r (5′-CAAGCCACAACAACATTAGG-3′).cDNA synthesis was performed by using 0.5 μL total RNA and the PrimeScript RT Reagent Kit  according to the manufacturer’s protocol. Virus RNA was quantified by using Power SYBR Green PCR Master Mix (Applied Biosystems) with 2 μL of reverse-transcribed cDNA. Quantitative real-time PCR was performed by using the Thermal Cycler Dice System (TaKaRa). The temperature program consisted of an initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. The primer pair for the real-time PCR was designed on the basis of the partial RdRp sequences of Bat-CoV/Philippines/Diliman1525G2/2008; primers used were 5′-TCCTAAGTGTGATAGAGCTATGCC-3′ and 5′-GTGCACACTCATTTGCTAACCG-3′. In each experiment, 10-fold serial dilutions of plasmid DNA containing the partial RdRp gene were tested in duplicate to establish a standard curve for calculating the relative amount of RNA in each sample. All samples were analyzed at least 3 times. To confirm the specificity of each PCR product, we conducted a melting curve analysis immediately after the amplification phase of each PCR. The amount of RNA in each sample was expressed as the average value (copy number per weight [milligrams] of sample).Alphacoronavirus) (n = 4) and group 2 CoV (genus Betacoronavirus) (n = 5).During July 2008, a total of 52 bats were obtained at 3 locations in Diliman and 1 location in Los Baños, the Philippines . RT-PCRswww.ncbi.nlm.nih.gov/BLAST) search findings of GenBank indicated that the partial RdRp sequence was most similar to that of Bat-CoV/China/A515/2005 (95% nt identity).A 440-bp consensus nt sequence of the group 1 CoV was obtained on the basis of alignment of 4 group 1 CoV sequences detected (>98% nt identity with each other) and deposited in GenBank as Bat-CoV/Philippines/Diliman1552G1/2008 (DNA Database of Japan [DDBJ] accession no. AB539080). BLAST  and deposited in GenBank as Bat-CoV/Philippines/Diliman1525G2/2008 (DDBJ accession no. AB539081). A BLAST search suggested that the partial RdRp sequence was novel but most similar to that of Bat-CoV/HKU9–1/China/2007 (83% nt identity). A phylogenetic tree was constructed with the partial RdRp-deduced amino acid sequence (120 aa) and available sequences of known CoVs . Data inSpecific and nested primer pairs for group 1b bat CoV and group 2d bat CoV sequences were designed, and nested PCR was performed by using cDNAs of all samples. Twenty additional amplicons (≈200-bp sequences) were obtained by using primers specific for group 2d bat CoVs. After direct sequencing or TA cloning, partial sequences of all amplicons obtained were found to be nearly identical to group 2d bat CoVs (>98% nt identity) and resulted in a total CoV prevalence of 55.8% . All seqCytopathic effects were not observed in any of the cells  tested with bat intestinal specimens positive for both detected viruses by RT-PCR. Results of RT-PCR for cell lysates to detect viral replication also were negative.6 copies of viral genome, was given orally to 2 fruit bats (bats A and B). After confirmation that these bats showed no clinical signs of infection, they were killed 6 days after infection.To obtain bat CoVs from field samples, we administered virus orally to 2 Leschenault rousette bats maintained in the Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, University of Tokyo. The volume of intestine samples collected from insectivorous bats was less than that from fruit bats because of their body size, and all positive samples for the group 1b bat CoV genome were derived from small insectivorous bats. Oral infection was conducted only with samples positive for the group 2d bat CoV genome. A homogenized large intestine sample (60 mg) derived from a lesser dog-faced fruit bat, which contained 7.8 × 10Virus genome was detected only in the small and large intestines of both bats by RT-PCR . Virus wFor murine hepatitis virus and several CoVs, an ≈70-bp leader sequence is added to the 5′ end of the transcription regulatory sequence of each nested mRNA during mRNA processing . The HKUTo confirm presence of transcribed virus mRNA in bats A and B , RT-PCR R. leschenaulti fruit bats. A 60-mg sample of the small intestine from bat A was given orally to 2 bats (bats C and D), and 500 μL of phosphate-buffered saline was given orally to 1 bat (bat E) as a control. These bats were killed 6 days after infection. Clinical signs were not observed in the experimentally infected bats. Virus genome amplification was not detected by RT-PCR in any samples . However, virus RNA was detected in the small intestine  from the small intestine of 2 bats (A and B) were given orally to 2 other bats (F and G), which were killed 3 days after infection. Six intestinal samples were obtained from each bat to determine site specificity of virus growth. The entire intestine (duodenum to the large intestine) was divided into 6 equal parts (1–6). Virus RNA was detected only by RT-nested PCR in the small and large intestines , and no After the SARS epidemic in 2003, bats were identified as carriers of CoVs in China. Recently, bat CoVs have also been detected in several other regions, including Germany, North and South America, and Africa. In the current study, we confirmed the presence of 2 CoVs in bats in the Philippines. Our findings suggest that CoV circulation in bats is worldwide. Although only 52 bats were tested, CoV RNA was present in 55.8% large intestine samples from these bats. Moreover, all bats tested seemed to be healthy. Thus, bats may be persistently infected carriers of CoVs. These data are consistent with results of previous reports of CoV detection in bats (Scotophilus kuhlii). However, the partial RdRp sequence of the virus was most similar to that of Bat-CoV/China/A515/2005 (95% nt sequence identity), which was also detected in bats of the same species in the southern China on Hainan Island (RNA of group 1b bat CoV was detected in 4 (57.1%) of 7 insectivorous bats. All bats positive for group 1b bat CoV RNA belonged to the same species, the Lesser Asiatic yellow bat . This finding suggests that replication of group 2d bat CoV in the intestine is low.The complete N sequence of group 2d bat CoV suggests that it is a novel virus and most similar to that of Bat-CoV/HKU9–1/China/2007 (77% aa sequence identity). Woo et al.  suggest <100 mg. Therefore, most samples were inadequate for virus isolation, especially virus-positive samples for group 1 bat CoVs from insectivorous bats.We attempted to isolate bat CoVs from several cell lines and primary cultured cells. However, virus replication was not observed, which is consistent with results of a previous report (To obtain sufficient tissue to isolate virus RNA, we attempted to infect fruit bats with bat CoV. Although we could not obtain bats of the species from which group 2d bat CoV was detected in the field survey, we obtained Leschenault rousette bats from zoos in Japan. In addition, Bat-CoV/HKU9–1/China/2007, which was most similar to group 2d bat CoVs by phylogenetic analysis, was identified in this species in Hong Kong , and theC. brachyotis, the level of viral genome amplification was low in experimental infection. This finding may have been caused by a difference in viral replication in bats of different species. In the field survey, partial nucleotide sequences of group 2d bat CoVs, were detected in 4 bat species. A high prevalence of virus RNA was observed in each bat species (Experimental infection was conducted in fruit bats by using tissues from virus-infected bats to determine virus pathogenicity. However, infected bats showed no signs of a pathologic effect, although low levels of virus RNAs were detected in the small and large intestines of these bats. These findings suggest that fruit bats can be infected with bat CoV without showing any signs of infection. However, compared with primary infection by field samples obtained from  species . These fFurther investigation of bat CoV ecology is needed to better understand the risk for infection with this virus. Knowing this risk could help elucidate emergence of SARS. Although we demonstrated in vivo propagation of a bat CoV, a bat CoV culture system is needed to obtain additional information about this virus.


Mycobacterium cultures, from patients suspected of tuberculosis or nontuberculous mycobacteria (NTM) infection, need to be identified. It is most critical to identify cultures belonging to the Mycobacterium tuberculosis complex, but also important to recognize clinically irrelevant or important NTM to allow appropriate patient management. Identification of M. tuberculosis can be achieved by a simple and cheap lateral flow assay, but identification of other Mycobacterium spp. generally requires more complex molecular methods. Here we demonstrate that a paramagnetic liquid bead array method can be used to capture mycobacterial rRNA in crude lysates of positive cultures and use a robust reader to identify the species in a direct and sensitive manner. We developed an array composed of paramagnetic beads coupled to oligonucleotides to capture 16 rRNA from eight specific Mycobacterium species and a single secondary biotinilated reporter probe to allow the captured rRNA to be detected. A ninth less specific bead and its associated reporter probe, designed to capture 23S rRNA from mycobacteria and related genera, is included as an internal control to confirm the presence of bacterial rRNA from a GC rich Gram variable genera. Using this rRNA capture assay (rCapA) with the array developed we were already able to confirm the presence of members of the M. tuberculosis complex and to discriminate a range of NTM species. This approach is not based on DNA amplification and therefore does not require precautions to avoid amplicon contamination. Moreover, the new generation of stable and cost effective liquid bead readers provides the necessary multiplexing potential to develop a robust and highly discriminatory assay. Mycobacterium tuberculosis. Critically liquid culture is also more effective at recovering other members of the Mycobacterium genus M. tuberculosis complex species, the so called non tuberculous mycobacteria (NTM), when cultured may represent environmental contaminants, colonization or, particularly in immunosuppressed or aging populations, represent genuine pathogens Mycobacterium species identification from clinical cultures increasingly important. Also, the treatment for M. tuberculosis complex infection requires at least 6 months and is specifically tailored to this organism. Disease caused by other (NTM) species requires different drug combinations Automated liquid culture is faster than culture on solid media and in fact the gold standard in the diagnosis of tuberculosis (TB). For these reasons liquid culture is widely used to detect and confirm infection with M. tuberculosis complex. Alternatively, mycobacterial DNA amplification may be used, followed by a “macro array” hybridization assay, which can specifically identify a wide range of Mycobacterium species Currently, speciation of cultured mycobacterial species is most effectively achieved by either a simple lateral flow test Mycobacterium species and a secondary biotinilated reporter probe. A ninth bead, designed to non-specifically capture 23S rRNA from mycobacteria and related genera and its associated reporter probe, is also included as an internal control to confirm the presence of bacterial rRNA from a GC rich Gram variable genus. After hybridization the beads and captured RNA are magnetically concentrated, the lysis buffer is removed, replaced with analysis buffer and the signal developed. The processed beads are subsequently analysed using a MAGPIX™  device and the hybridization profile used to identify the species present in the culture. The MAGPIX platform, which unlike previous Luminex readers is based on a CCD camera and LED illuminated flow cell rather that a flow cytometer, is a compact robust devise capable of identifying and quantifying the signal collected on an suspension of up to 50 different bead species. Beads can either be purchased pre coupled to assay reagents or, as in this report, coupled by the end user to develop novel assays.Here we propose an alternative concept with the potential to be simpler than an DNA amplification/macro array approach, but like the AccuProbe method not susceptible to the risk of PCR contamination and associated requirements for amplicon clean and exposed rooms, while providing much more information on species identification than a lateral flow assay Mycobacterium genus consists of more than 100 recognized species. So in order to demonstrate the concept and develop and optimize protocols presented in this paper we developed a pilot test bead array to identify nine species. None the less this method successfully discriminated a useful range of species previously recovered from patient material and associated with different clinical significance The M. marinum, or 36°C for other Mycobacterium species to early log phase in 10 mL of Middlebrook 7H9 liquid media supplemented with OADC. Subsequently, we tested a series of 14 Mycobacteria Growth Indicator (MGIT) Tubes , one uninoculated, cultured at the RIVM that were previously identified using reverse line blot tests  for identification, which we used to check the suitability of our method for use in combination with the MGIT culture system . Once prepared coupled microspheres were re-suspended in 200 ul of 1×TE buffer pH 8 and stored at 4°C in the dark until needed. These solutions of labeled beads were then combined to produce bead mixtures (liquid arrays) and suspended in 2×SSPE  as required to a final concentration 45,000 of each bead species per ml. Aliquots of these mixtures were used in the assay with approximately 1,500 beads of each species used per reaction.n spacer  and fiveMycobacterial cultures were heated to 80°C for 30 min prior to lysis. Bacteria (0.5 ml for Middlebrook culture and 4 ml for MGIT cultures) were then pelleted by spinning at 10,000 g for 5 min in a micro-centrifuge and the supernatant discarded. The pelleted cells were then re-suspended, at 1 to 2× original concentration for Middlebrook cultures and 20× concentration for MGIT cultures, in 5 M GTC buffer . Then to 150 ul aliquots of the concentrated cells 0.2 g of 0.1 mm diameter Zirconium/Silica beads  was added and the cells mechanically lysed by shaking in a Retch shaker  at max speed (30 Hz) typically for 10 min but up to 60 min to investigate the effect of increased shaking.To 100 µl aliquots of the bacterial lysate prepared above 5 µl of a 5 µM solution of each labeled detector ologonucleotide and 33 µl of a bead mixture  was added. The mixture was then heated to 95°C for 3 min and held at 50°C with gentle shaking to allow the hybridization to proceed. For method development hybridization was allowed to continue overnight, but hybridization kinetics were studied by removing aliquots of selected samples at 1, 3, 5, and 18 hrs. After hybridization the samples were placed on a magnetic separator to concentrate the beads and the supernatant carefully removed. The beads were then immediately re-suspended in 40 µl of 2×SSPE at 50°C and 5 ul of 5 µM of each detector oligonucleotide. The beads were then held at 50°C for a further 30 min with gentle shaking.After this secondary hybridization/wash the beads were magnetically concentrated on a magnetic separator and washed twice with 100 ul of 2×SSPE at 50°C. The beads were then re-suspended in 40 µl 1×TMAC buffer  at 50°C along with 12 µl reporter mix (10 µg/ml streptavidin phycoerythrin conjugate in TMAC). Labeling was allowed to continue for 5 min in the MAGPIX instrument at 50°C then the signals were measured and the data exported to Excel 2010 .5′GRGRTACTCGAGTGGCGAAC3′ and 16SR-5′GACGACGGGTAGCCGGCC.In order to resolve unexpected results we PCR amplified and cycle sequenced a portion of the 16S rRNA gene which contained the region targeted in our assay, using primers 16SF-M. marinum probe but this did not result in misidentification as the M. marinum strain tested gave only a weak signal with the MTB complex probe  ranged from 827 to 15,047 resulting in a characteristic profile in each case . During ex probe .Effective lysis was essential to obtain good signal and complete lysis of the MTBC strains tested was not achieved using our lysis protocol . ReducedStaphylococcus aureus and Escherichia coli, gave no detectable signal with the array. We also tested two mycolic acid containing, GC rich bacteria, identified based on partial 16S rRNA sequence as Nocadia asteroides and Gordonia bronchialis, which were originally obtained from sputum culture. These strains gave no detectable signal with the 16S rRNA probes, but yielded a clear signal with the 23S positive control bead  gave a signal with the M. malmoense beads and the 23S positive control but no signal with the other beads including the M. avium probe. Because of this unexplained result we amplified and sequenced the respective part of the 16S rRNA of this strain and confirmed it had been misidentified in our collection M. avium, M. xenopi, M. abscessus, M. kansasii, and M. marinum probes exclusively reacted with members of their respective species  stimulated us to explore the possibility of using this system to identify The samples from MGIT cultures tested gave higher signals  but alsoM. avium culture demonstrated lysis of this species was almost complete after 10 min . Thus, in common with most molecular assays not all probes designed in silico function in this assay even if the sequence is correct and probes must be experimentally screened for functionality.The nctional  and 15 MBacterial speciation is required when liquid culture is performed for tuberculosis diagnostics Mycobacterium isolates associated with disease and colonization or contamination should be undertaken. The analysis system used in this study is in principle capable of detecting 50 unique bead species although systems with even higher multiplexing capacity are available Mycobacterium species from clinical material using the bead array detection system, although, other detection methods are being developed and it is not totally inconceivable that the rapid developments in DNA and nano technology relating to detection and signal amplification may ultimately be able to achieve direct identification of bacteria in clinical material using this approach. These developments in molecular detection As a next step, extension and optimization of the panel of beads/probes and testing the performance on a larger collection of The use of an amplification free system is potentially very robust utilizing mainly stable chemical reagents and is intrinsically resistant to amplicon contamination problems that have complicated the provision of amplification assays for infectious disease diagnostics where they are most needed. Finally, even moderate increases in sensitivity would allow RNA species other than the very highly expressed rRNA to be targeted e.g. highly expressed mRNA species such as rpoB mRNA


Filoviruses, including Ebola virus, are unusual in being filamentous animal viruses. Structural data on the arrangement, stoichiometry and organisation of the component molecules of filoviruses has until now been lacking, partially due to the need to work under level 4 biological containment. The present study provides unique insights into the structure of this deadly pathogen.We have investigated the structure of Ebola virus using a combination of cryo-electron microscopy, cryo-electron tomography, sub-tomogram averaging, and single particle image processing. Here we report the three-dimensional structure and architecture of Ebola virus and establish that multiple copies of the RNA genome can be packaged to produce polyploid virus particles, through an extreme degree of length polymorphism. We show that the helical Ebola virus inner nucleocapsid containing RNA and nucleoprotein is stabilized by an outer layer of VP24-VP35 bridges. Elucidation of the structure of the membrane-associated glycoprotein in its native state indicates that the putative receptor-binding site is occluded within the molecule, while a major neutralizing epitope is exposed on its surface proximal to the viral envelope. The matrix protein VP40 forms a regular lattice within the envelope, although its contacts with the nucleocapsid are irregular.The results of this study demonstrate a modular organization in Ebola virus that accommodates a well-ordered, symmetrical nucleocapsid within a flexible, tubular membrane envelope. Paramyxoviridae and the Birnaviridae where particles may contain up to four copies of the RNA genomes Viruses have evolved as genome packaging machines to efficiently transfer nucleic acids between susceptible host cells, ensuring replication. The majority of viruses have hollow, quasi-spherical shells rather than tubular structures, perhaps because this gives the most efficient packaging of nucleic acid with a fixed copy number of coat protein subunits. In non-enveloped viruses, the volume enclosed by the  icosahedral structure is a constraint on the size of the genome, giving a limited capacity to encode capsid proteins, and usually restricts the genome copy number, or ploidy of the virion, to one Filoviridae family, including the Ebolavirus and Marburgvirus genera, cause haemorrhagic fevers with high mortality in humans, and no effective treatments are currently approved The Research on filoviruses has been hampered by their status as biosafety level 4 pathogens. Previous investigations of filovirus structures within embedded, sectioned and metal-stained cells by electron tomography revealed few details of the high resolution oligomeric structure We identified filamentous EBOV particles 20 microns or longer, with a well ordered internal structure, and a helical nucleocapsid giving an internal “herring-bone” appearance using cryo-EM and cryo-ET , S5. TheWe also observed “empty” filaments that lack nucleocapsids, with a random length and a smaller diameter . These eThe Ebola nucleocapsid structure was solved to 19 Å resolution (FSC 0.5 criteria) using linear regions of 34,605 images  are situated at the midpoint of the structure just above the stalk  software . Automated eucentricity determination, and focusing were performed using the Xplore3D data acquisition software . For cryo-ET, data collection was done using the Xplore3D data acquisition software, the low-dose unit, and the TIA software .Samples for cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) were mixed with BSA coated 10 nm gold particles  at a ratio of 2∶1 (virus∶gold) for cryo-ET, and (9∶1) for cryo-EM. Specimens (4 µl) were then applied to glow-discharged quantifoil grids with 2 µm holes spaced at 1 µm intervals . Grids were subsequently plunge cooled in liquid ethane using a Vitrobot Mark IV . Specimens were transferred to a Tecnai 20 G2 transmission electron microscope (FEI) operated at 200 kV, equipped with a Gatan CT3500TR single tilt rotation low-temperature specimen holder. For cryo-EM imaging was conducted at temperatures of ∼−185°C. Images were recorded using an Eagle 4K CCD camera . For single particle image analysis, images were taken at 50,000× or 80,000× magnification at 2–4 µm defocus, with a dose of 10 electrons/ÅThe exact magnification in the microscope at the CCD detector was determined using a calibration grid . Ebola virus length measurements (n = 2090) were made using the Image J software package Tomographic image analysis of cryo-ET data was carried out with the Inspect3D Xpress software package . The tomographic images were aligned to each other by a two-step process. The first step involved alignment of adjacent images by cross correlation. This process was repeated several times until the shift between adjacent images was below one pixel in either the X or Y plane. The second step involved the alignment of the entire image stack using 10 nm colloidal gold particles as fiducial markers. In this instance the term “image stack” refers to all the images collected in a single tomographic tilt. This procedure involves the selection of ten to twenty of the 10 nm gold particles and the subsequent identification and tracking of these particles in all of the images in the image stack. The locations of these particles are then used in conjecture with the tilt angles of each image to globally align all of the images to each other. The last step in this process was to calculate the three dimensional reconstruction of the tomogram from the aligned images. In this study we used the simultaneous iterative reconstruction technique (SIRT) algorithm with 10 iterations to calculate the final three-dimensional reconstruction (tomogram).3 pixel sub-tomogram was extracted from a tomogram using the Chimera 3 pixel sub-tomogram contained a single linear segment. This template was then used by the ARGOS software to conduct an exhaustive search of the original tomogram for similar structures. This analysis involved a six dimensional search matrix . The entire search process was sped up by the ARGOS software by utilizing parallel processing on the computer's graphics processing unit (GPU). Once individual sub-tomograms were selected based on correlation, they were inspected and compared to the initial template sub-tomogram. The extracted and aligned sub-tomograms were subsequently averaged with a filter that minimized the missing wedge artifact. This average structure then was used as the reference and the entire procedure was repeated several times.Sub-tomogram image analysis of cryo-ET data was carried out with the Automated Recognition of Geometries, Objects, and Segmentations (ARGOS) software package . For this analysis an 805, which estimates defocus and corrects for ctf by phase-flipping. Images of the spike  and nucleocapsid  were selected for image analysis. The resolution of the cryo-EM reconstruction was estimated by Fourier shell correlation using the FSC 0.5 criteria. In all subsequent sections image analysis procedures were conducted using the SPIDER software package unless otherwise stated.Single particle cryo-EM image processing was carried out using the EMAN/EMAN2 and SPIDER/WEB image processing program packages Analysis of initial images of the “straight” linear segments of the Ebola virus using Fourier transformation indicated that there was sufficient bending of the helical nucleocapsid to make standard helical analysis problematic. Therefore, an initial reference free single particle 2D analysis was conducted in EMAN using the “startnrclasses” program to identify any potentially recurring motifs within linear regions of the Ebola virus.In order to further investigate the nucleocapsid repeat identified in the 2D analysis the iterative helical real space reconstruction method (IHRSR) was implemented For the spike dataset two image populations were combined composed of side, and end-on perspectives. For the side view perspective images were subfield directly off of the Ebola virus cryo-EM images. For the end-on perspectives sub-tomographic volumes were extracted from the tomographic reconstructions of the Ebola VLP. The 3D volumes were then added in the “Z” plane to generate 2D projection averages which were then used for the subsequent single particle image analysis. The data were the processed using EMAN to generate an initial 3D reconstruction, which was then refined in SPIDER using the projection matching technique as previously described The 3D cryo-EM reconstructions, cryo-ET reconstructions, 3D models of the Ebola virus, and the atomic resolution structure 3CSY.pdb were visualized using UCSF Chimera software package  Figure S1High magnification images showing linear regions of Ebola virus. The diameter of EBOV is constant in linear regions of the virus containing a nucleocapsid (A). The viral filaments are not perfectly straight, and are often curved, complicating helical image processing. Glycoprotein spikes (GP), envelope (E), and nucleocapsid (NC) are all clearly visible. The black spherical objects (shown by black arrows) are 10 nm colloidal gold particles which are used for automated focusing and tomography alignment. (B) Images of “comma-shaped” Ebola virus. Each of the comma-shaped viruses has a single copy of the genome which can be seen running through the center of the virus and curving at one end of the virus to form the globular head. In some of the heads there is a low-density region (LD) devoid of nucleocapsid. In others, the nucleocapsid is sharply bent where it folds back on itself and looks like a check mark (right panel). (C) Ebola virus structures with and without nucleocapsid. Empty tubular filaments (E), and viral filaments containing a nucleocapsid (NC) are shown. Constrictions at the transition points where the nucleocapsid ends and the viral membrane continues as an empty tubular structure are indicated by red arrow heads. The diameters are as follows: nucleocapsid, 41 nm; virus with nucleocapsid, 96–98 nm; empty filaments, 48–52 nm. (D) Ebola virus with interior vesicles. Viral particles containing additional membrane vesicles within the envelope are shown by a green “V”. Although present in a minority of virus particles, when present they are usually at the ends of the virus with a globular head. The image in the left hand column shows an example where the vesicles are in the middle of the virus.(TIF)Click here for additional data file.Figure S2Tomographic slices of Ebola virus. Slices in “Z” of Ebola virus tomogram reveal the components of the virus and nucleocapsid (A). The insets show 2D averages of the nucleocapsid. The slice through the top of the virus reveals the envelope and glycoprotein spikes. The next slice cuts through the top of the nucleocapsid (NC) revealing the banding pattern which represents VP24–VP35 bridge. The third slice cuts through the middle of the NC. The tube-like component of the NC is primarily composed of NP. The last panel shows the average of 53 slices which make up the volume encompassing the entire NC. The image and the average in the inset contain all structural components of the NC. (B) The linear region of the tomographic nucleocapsid reconstruction (A) was translated in Z and rotated 360° in plane. At each shift/rotation point the volume was correlated to the initial (un-shifted) volume. The correlation plot is shown as a grey scale image. Eight regions have been highlighted demonstrating a characteristic right-handed helical pattern. (C). For comparison, both left and right handed helical correlation plots are shown. (D), Region three is shown, with the locations of correlation maxima shown with an X. The angular distance between each maximum was calculated from several plots. A total of 71 measurements gave an average angular distance of 33.6°+/−8.5° between helical repeats, resulting in 10.7 repeats per turn. Using the 6.96 nm pitch  the step in Z per helical repeat was calculated as 0.65 nm. These helical symmetry values were then imposed on the nucleocapsid tomogram and this structure was used as the initial reference volume for refinement using the iterative helical real space reconstruction method.(TIF)Click here for additional data file.Figure S3Surface spike distribution in the Ebola VLP. Longitudinal Z-slices through the top and middle of the particle are shown, as well as the end-on view (A). The tomogram is shown as a shaded surface at a density threshold that indicates the spikes (B). The volume from one side of the tomogram has been extracted, and a red-blue color scheme shows the depth at which the spikes are located. This region of the envelope has a surface area of 15,651 nm2. Selected spikes have been identified by red circles, the single particle reconstruction of the spike is shown at the same scale to the right in a red square for comparison. The same region in (B) is shown in (C) with a solid orange cylinder to provide a visual cue for the viral envelope. Eighty-six individual spikes were counted (white spheres) and have a patchy distribution (D), each spike would occupy an average area of 182 nm2, giving an average spacing between spikes of 15.2 nm. The reconstruction of the spike (blue) with the docked KZ52 Fab (purple) has been included to show that there is ample room for antibody attachment.(TIF)Click here for additional data file.Figure S4Extraction of Ebola nucleocapsid structure for sub-tomographic analysis. The tomogram of a linear region of the Ebola virus was used as the first reference for sub-tomogram analysis (A). When viewed along the helical axis (Y) or from the end perspectives  the basic components are visible. The tomographic volume was also cylindrically masked along the X-axis, selecting only the density containing the nucleocapsid, to highlight the components of the nucleocapsid in the tomogram (B). Two-dimensional single particle image analysis was carried out with cryo-images (C) (not tomographic data sets), for comparison to the 3D tomographic data. The average shown in this panel was generated by reference free classification, using the “startnrclasses” program in EMAN (TIF)Click here for additional data file.Figure S5Representative low-magnification images of Ebola virus. Frozen hydrated virus is clearly visible with sections of the filamentous virus over both the support film and across the holes in the quantifoil film. Individual G1 (single genome copy) virus is circled in red, several sections containing a nucleocapsid are indicated by a blue arrowhead, and regions without a nucleocapsid are indicated by a magenta arrowhead. Globular heads are identified by yellow arrowheads. In this image the circles  are filled with frozen hydrated virus in a thin aqueous layer, and the quantifoil support film appears as darker grey.(TIF)Click here for additional data file.Table S1Length analysis of “continuous” Ebola virus particles. The length of 2090 EBOV particles were measured using ImageJ (TIF)Click here for additional data file.Table S2Modeling of RNA in the Ebola nucleocapsid. Two previously determined atomic resolution structures of negative stranded RNA viruses (VSV (2GIC.pdb) (TIF)Click here for additional data file.Movie S13-D reconstruction of Ebola virus nucleocapsid. This movie shows the three-dimensional structure of the of the Ebola virus nucleocapsid as shaded surface representation. The surface is set at a density threshold which would include one copy of NP, VP24,VP30, VP35, and the RNA. The nucleocapsid rotates, and then is sliced through the Z-axis to show the internal components of the structure.(MOV)Click here for additional data file.Movie S2Tomogram of Ebola virus. This movie shows slices in “Z” through a cryo-electron tomogram of a linear region of Ebola virus. The slices which are in the Z-plane pass back and forth through the virus showing various components such as the surface glycoprotein spikes and the nucleocapsid in the interior of the virus.(MOV)Click here for additional data file.Movie S3Ebola virus spike distribution. This movie shows one surface of a cryo-electron tomogram of an Ebola virus-like particle, generated by expressing the VP40 and GP proteins. The structure rotates showing the distribution of spikes. The locations of individual spikes are identified by white spheres, and the reconstruction is replaced by a cylinder to show the patchy distribution of spikes on the surface of the virus-like particle.(MOV)Click here for additional data file.Movie S43-D reconstruction of Ebola virus spike. This movie shows the three-dimensional structure of the of the Ebola virus GP spike. The structure rotates showing views from different angles, and indicates the location of the docked 3CSY.pdb (MOV)Click here for additional data file.


TOC summary line: This new virus may be another cause of encephalitis. For many encephalitis cases, the cause remains unidentified. After 2 children (from the same family) received a diagnosis of acute necrotizing encephalopathy at Centre Hospitalier Universitaire , we attempted to identify the etiologic agent. Because clinical samples from the 2 patients were negative for all pathogens tested, urine and throat swab specimens were added to epithelial cells, and virus isolates detected were characterized by molecular analysis and electron microscopy. We identified a novel reovirus strain (serotype 2), MRV2Tou05, which seems to be closely related to porcine and human strains. A specific antibody response directed against this new reovirus strain was observed in convalescent-phase serum specimens from the patients, whereas no response was observed in 38 serum specimens from 38 healthy adults. This novel reovirus is a new etiologic agent of encephalitis. Orthoreovirus, are nonenveloped double-stranded RNA viruses with a genome composed of 10 segments. These viruses have 3 major serotypes: type 1 Lang (T1L), type 2 Jones (T2J), and type 3 Dearing (T3D), which can be differentiated by neutralization and hemagglutination inhibition tests  and his 22-month-old cousin (patient 2) were hospitalized with the same ANE-specific symptoms a few days apart in March–April 2005 in Centre Hospitalier Universitaire, Clocheville . Serum, urine, CSF, and throat swab specimens were collected from both children. An influenza-like syndrome developed simultaneously in the mother of patient 2.To detect herpesviruses and enteroviruses, we used PCR or reverse transcription PCR (RT-PCR), respectively, with commercially available reagents . Nasopharyngeal aspirates were tested by indirect immunofluorescence, by using specific monoclonal antibodies (Argene), for influenza viruses A and B, respiratory syncytial virus, parainfluenza viruses 1–3, and adenoviruses. Serologic assays (ELISA) with commercially available reagents  were conducted to detect immunoglobulin (Ig) G and IgM against herpes simplex viruses, Epstein-Barr virus, measles virus, and mumps virus. Antibodies to influenza viruses were tested by complement fixation assays with antigens derived from influenza viruses A and B. Serologic assays were also conducted to detect IgG and IgM against hantavirus, tick-borne encephalitis virus, dengue virus, and chikungunya virus . In addition, because the 2 patients had been in contact with an uncle who had returned from Asia (Indonesia), the presence of Hendra virus and Nipah virus nucleic acid sequences in CSF was investigated.Urine and throat swab specimens from each patient were added to MRC5, MDCK, and Vero cells. Early viral stocks were made from urine and throat specimens added to MRC5 cells as soon as cytopathic effects (CPEs) were observed. Late viral stocks were obtained after inoculation of BGM cells. Cells were harvested as soon as CPEs were observed and fixed by incubation for 48 h in 4% paraformaldehyde and 1% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.2, as described  were constructed using ClustalW 1.74  obtained from 38 healthy blood donors were tested by Western blot for antibodies against the MRV2Tou05, Lang (T1L), Jones (T2J), and Dearing (T3D) strains.Viral RNA was extracted from infected cells and supernatant and from patients’ samples by using the Nuclisens EasyMAG Kit (bioMérieux). Mock cells and supernatant from uninfected cells were used for RNA extraction as negative controls. A molecular test was set up with the L3 segment as target with primers enabling detection of the 3 reovirus serotypes .3 on day 5. An electroencephalogram showed generalized slow-wave activity. Computed tomography of the brain showed a low-density change in the thalami. Brain magnetic resonance imaging (MRI) performed on the first day of hospitalization showed multiple symmetric lesions, with high signal intensity on T2-weighted images and low signal intensity on T1-weighted images, mostly involving the thalami bilaterally but also the brainstem tegmentum  on March 29, 2005, with fever, vomiting, and impaired consciousness. Results of his neurologic examination were normal, except for palsy of the left facial nerve. The Glasgow Coma Scale score was 7. He could respond to simple verbal commands, such as requests to open eyes or make some other movements, but he was lethargic (no articulated language) and had epilepsy-like abnormal movements of the face or distal muscles. For 2 days previously, he had experienced a prodromal illness with high temperature (40°) and headache. Results of an analysis of CSF on the first day of hospitalization were within normal limits and showed a protein level of 0.81 g/L and 4 leukocytes/mmegmentum , panel APatient 2, a 22-month-old girl, was the cousin of patient 1. She was admitted 4 days later, on April 3, 2005, with drowsiness and fever after 1 day of high fever, asthenia, and rhinorrhea. A neurologic examination showed brisk deep tendon reflexes and extensor plantar responses; Glasgow Coma Score was 8. CSF initially showed a slightly elevated protein level  with no cells, and findings were within normal limits on day 4 of illness . Radiologic examinations of the brain showed similar lesions to those of her cousin , panel BTwo other family members (the mother of patient 2 and the grandmother of both children) also had influenza-like symptoms  at the same period for a few days without neurologic signs. The past family history and medical history did not appear relevant.Results of molecular tests for herpesviruses and enteroviruses performed initially on the CSF specimens from both children were negative. The results were also negative for infection of respiratory viruses . Serologic test results for HIV were negative. Serologic assays for IgG and IgM against herpes simplex virus, Epstein-Barr virus, measles virus, and mumps virus did not show IgM. Serologic assays were negative for both IgG and IgM against hantavirus, tick-borne encephalitis virus, dengue virus, and chikungunya virus. The results were also negative for nucleic acid sequences of Hendra virus and Nipah virus . All serum specimens collected during the acute and convalescent phases were negative for antibodies against influenza viruses.Nonoriented virus isolation was attempted by inoculation of MRC5, MCDK, BGM, and Vero cell cultures with urine collected from each patient on April 7 and with a throat swab specimen from patient 2 on April 11. A CPE was observed on day 7 in MRC5 cells inoculated with urine from both patients, and on day 7 in MCR5 cells and day 10 in MDCK cells inoculated with a throat swab specimen from patient 2. A discrepant result for enterovirus was obtained when identification was attempted. Indeed, indirect immunofluorescence on fixed cells was positive for enterovirus , but results of RT-PCR for detection of enteroviruses, performed on the cell culture supernatant, were negative. The virus isolates were then identified as reovirus type 2 by seroneutralization at the National Reference Center for Enteroviruses . The discrepancy initially observed between immunofluoresence and RT-PCR results may be attributed to the cross-reactivity of the Pan-Entero Blend reagent toward reoviruses, as mentioned in the manufacturer’s description. Microscopic examination after staining of infected cells showed voluminous cytoplasmic inclusions characteristic of CPEs induced by reoviruses; electron microscopy showed the accumulation of virions in formation .Reovirus sequences were searched for retrospectively by RT-PCR in the patients’ available specimens. A specific 512-bp fragment corresponding to the L3 expected region was obtained from urine specimens from both patients and from 1 serum specimen that was obtained 21 days after the onset of symptoms from patient 1 (data not shown). All amplified fragments were sequenced and showed identical profiles.www.ncbi.nlm.nih.gov/Database/) to determine the percentage of identity with mammalian reoviruses. Sequence analysis of the 3 large segments, the 3 medium segments, and the S2, S3, and S4 segments indicated overall a close genetic relationship between MRV2Tou05 and swine SC-A viruses and a relationship with the 2 reovirus prototypes 1 and 3 , the etiologic agent remains unknown  were closely related to the swine reovirus strain (91%–97% identity), except for the S1 and S3 segments. The S1 segment determines the reovirus serotype and encodes the nonstructural protein sigma 1s and the viral cell attachment protein sigma 1. The S1 gene showed a high identity score (83%) with human type 2 strains isolated from fecal specimens of 2 children in China in 1982 (A specific antibody response against the MRV2Tou05 strain developed in the 2 patients and in the mother of patient 2, whereas no antibody response against MRV2Tou05 was detected in any of the 38 healthy blood donors. However, serum samples from 52% of these healthy adults contained antibodies directed against at least 1 of the 3 human reovirus prototype strains. In a study in Germany, similar seroprevalence was observed for reovirus type 3 antibodies in a healthy population (,,ANE predominantly affects infants and young children in eastern Asia, but sporadic cases are regularly diagnosed in other parts of the world. The most frequent pathogens involved in ANE are viruses, most commonly influenza A and B (The origin of several genome segments of this reassortant in swine in Asia and the relatedness of other segments to human serotype 2 reoviruses described in Asia are surprising. Possibly the MRV2Tou05 was imported by the uncle, who had just returned from Indonesia a few days before the onset of symptoms in the children and the mother. Searching for antibodies against MRV2Tou05 in serum specimens from the uncle would have been informative but no blood samples were available.The role of reoviruses as etiologic agents for symptomatic human diseases remains controversial. They are designated as orphan viruses, and more than half of the adult population possess antibodies directed against reoviruses, which suggests that infection occurs frequently without any specific effect on human health. However, reovirus strains have been isolated from persons with serious human diseases (This study describes the entire molecular characterization of a new reovirus strain isolated from 2 familial ANE patients. Its isolation and molecular detection from patients’ samples and the specific immune response toward this type 2 strain suggest an etiologic role for this reovirus in these unexplained ANE cases. The reproduction of symptoms in an animal model and in vitro studies of the cellular interactions and apoptosis of MRV2Tou05 are needed to help clarify the exact role of this novel reovirus strain. Identifying the MRV2Tou05 reovirus sequence could contribute to the improvement of ANE diagnosis and treatment, for example, by confirming susceptibility to viral infection and clarifying the possible role of other common viruses in its pathogenicity.


A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was rapidly used to detect serotype Asia 1 of foot-and-mouth disease virus (FMDV) within 45 min at 61°C. All FMDV serotype Asia 1 reference strains were positive by RT-LAMP, while other viruses such as FMDV serotypes O, C, A and classical swine fever virus, swine vesicular disease virus, porcine reproductive and respiratory syndrome virus and Japanese encephalitis virus remained negative. Furthermore, FMDV sreotype Asia 1 positive samples were able to detect by RT-LAMP assay. This RT-LAMP assay may be suitable particularly for diagnosis of FMDV serotype Asia 1 infection in field stations. Aphthovirus of the family Picornaviridae, which is divided into seven serotypes with no cross-protection conferred among the serotypes [Foot-and-mouth disease virus (FMDV) is a member of the genus erotypes . FMDV seerotypes ,3. Due terotypes -6. RapidConventional laboratory diagnosis of FMD was made by enzyme-linked immunosorbent assay (ELISA) detection of specific viral antigens and by observation of cytopathogenic effects in cell culture ,7-9. AltA novel nucleic acid amplification method, termed reverse transcription loop-mediated isothermal amplification (RT-LAMP), which was applied successfully to the detection of influenza A virus, Newcastle disease virus and classical swine fever virus, porcine reproductive and respiratory syndrome virus -22. In tFour primers including FIP, BIP, F, and B for RT-LAMP were designed by targeting the conserved regions of VP1 sequence from FMDV serotype Asia 1 , A , C (C/SU/1958), Asia 1 (Asia 1/JS/2005) and classical swine fever virus (CSFV), swine vesicular disease virus (SVDV), porcine reproductive and respiratory syndrome virus (PRRSV) and Japanese encephalitis virus (JEV) were tested. The results indicated that the RT-LAMP assay was able to type the FMDV serotype Asia 1 strain used in this experiment as each showed the characteristic ladder-like pattern in the gel (Figure To assess the applicability of this method, 125 clinical samples were used in the study (Table Taken together, the RT-LAMP assay was rapid, specific, and sensitive for typing FMDV serotype Asia 1 in clinical samples from the infected pigs. This method not only reduced the diagnosis time significantly but also may be a potential for wider use in field practice.The authors declare that they have no competing interests.HT C and J Z designed the research, then carried out most of the experiments. XT L supported experimets. HT C and YS L wrote and revised the manuscript. All of the authors approved the final version of the manuscript.