Cryptococcus gattii is a basidiomycetous yeast that causes life-threatening disease in humans and animals. Within C. gattii, four molecular types are recognized (VGI to VGIV). The Australian VGII population has been in the spotlight since 2005, when it was suggested as the possible origin for the ongoing outbreak at Vancouver Island , with same-sex mating being suggested as the driving force behind the emergence of this outbreak, and is nowadays hypothesized as a widespread phenomenon in C. gattii. However, an in-depth characterization of the Australian VGII population is still lacking. The present work aimed to define the genetic variability within the Australian VGII population and determine processes shaping its population structure.CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and IGS1. Despite the overall clonality observed, the presence of MATa VGII isolates in Australia was demonstrated for the first time in association with recombination in MATα-MATa populations. Our results also support the hypothesis of a “smouldering” outbreak throughout the Australian continent, involving a limited number of VGII genotypes, which is possibly caused by a founder effect followed by a clonal expansion.A total of 54 clinical, veterinary and environmental VGII isolates from different parts of the Australian continent were studied. To place the Australian population in a global context, 17 isolates from North America, Europe, Asia and South America were included. Genetic variability was assessed using the newly adopted international consensus multi-locus sequence typing (MLST) scheme, including seven genetic loci: MATα-MATa population in Australia is in accordance with the natural life cycle of C. gattii involving opposite mating types and presents an alternative to the same-sex mating strategy suggested elsewhere. The potential for an Australian wide outbreak highlights the crucial issue to develop active surveillance procedures.The detection of sexual recombination in  Life-threatening infections due to fungi have increased significantly over recent decades, posing new challenges for public health Cryptococcus neoformans and Cryptococcus gattii are causative agents of cryptococcosis, a serious disease that manifests as meningitis and meningoencephalitis in humans C. neoformans has a worldwide distribution and infects predominantly patients with impaired immunity. In contrast, C. gattii infection has been mostly associated with immunocompetent hosts and was originally designated as a tropical and subtropical pathogen C. gattii have been reported from human and a wide range of animal species C. gattii is of great importance to better understand its life cycle and thereby determine exposure risks and implement preventive strategies, as required.The two basidiomycetous haploid yeasts, C. gattii VGI and eucalyptus trees, particularly with the native species Eucalyptus camaldulensis (river red gum) C. gattii and trees have been reported from other host plants in various countries C. gattii in Australia and its association with native eucalypt trees and the extensive exportation of those trees led to the hypothesis that C. gattii originated from Australia and was subsequently dispersed into other parts of the world through man-made horticulture In Australia, numerous studies have revealed an association between versus asexual reproductions in the life cycle of a fungus is a crucial biological issue. Sex allows for new genetic recombination and increases the potential for adaptation to new environments Cryptococcus can reproduce both sexually and asexually. Sexual reproduction involves a bipolar mating system with two mating type alleles, MATa and MATα. Mating occurs between opposite mating types, resulting in the formation of basidiospores. Asexual reproduction occurs via budding C. gattiiC. neoformans, this has only been observed under laboratory conditions Besides studying the environmental niche of a potential human pathogen, the determination of the relative importance of sexual C. gattii, four molecular types are recognized: VGI, VGII, VGIII and VGIV C. gattii, in concert with the large-scale exportation of these trees to other parts of the world over the last century supports the notion of an Australian origin for this fungus MATa VGII isolates Within C. gattii VGII population on a large geographical scale investigating 54 clinical, veterinary and environmental isolates from Queensland (QLD), New South Wales (NSW), Northern Territory (NT) and Western Australia (WA), using multilocus sequence typing (MLST); and (ii) determining the processes shaping its population structure, in particular the reproductive modes .To shed further light on the low virulent VGIIb Vancouver Island outbreak strain and its relationship with Australian isolates, the current study focused on (i) characterizing the genetic variability within the Australian CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and IGS1) of the Cryptococcus consensus multilocus sequence typing (MLST) scheme adopted by the International Society of Human and Animal Mycology (ISHAM), resulted in 4166 bp nucleotide positions when aligned with the two reference strains of the Vancouver Island outbreak, CDC R265 (VGIIa) and CDC R272 (VGIIb), from which 47 polymorphic sites were identified , GPD1 and LAC1  and URA5   .The allele combinations  and the The majority of the Australian isolates (∼72%) belonged to a single sequence type (ST7), which was identical to the allelic profile of the reference strain CDC R272 corresponding to the VGIIb low virulent genotype from Vancouver Island  . None ofTo determine whether scoring more loci would or wouldn't have increased the genetic diversity, the detected genotypic diversity was plotted against the number of loci analysed . This anTo place the Australian population in a global context additional isolates from North America, Europe, Asia and South America were studied . This anLooking at the geographical regions within Australia, WA showed the highest genetic diversity, with five sequence types detected out of the six present throughout Australia . Among tThe ST7 was present in all regions investigated and was by far the most common sequence type . Indeed,C. gattii VGII isolates in Australia.The distribution of the genetic diversity was also investigated in relation to the source of isolation . AmongstC. gattii VGII isolates studied were mating type α , and overall high pairwise frequency comparisons were obtained from 0 to 2 nucleotide differences. This goodness-of-fit between observed and expected pairwise difference distributions is likely to indicate an historical population expansion. The three neutrality tests, Tajima's D, Fu & Li's F* and Fu's Fs, failed to reveal a departure from the null hypothesis of neutral selection and/or population at equilibrium for all MLST loci  of all isolates belonged to a single widely distributed sequence type, namely ST7, corresponding to VGIIb, the minor/less virulent genotype involved in the Vancouver Island outbreak C. gattii VGII population. Among the 75 C. gattii VGII isolates typed in a study by Fraser et al.C. gattii VGII population. Such a low genetic diversity is supported by comparisons with other regions, especially South America, where of the seven MLST loci investigated, each observed sequence type corresponded to a distinct genotype  from European patients, which had previously travelled to Vancouver Island and those had physically transposed this genotype to Europe  based largely on circumstantial evidence MATα parents, namely the low virulent genotype VGIIb (ST7 in the current study) and an unknown mating partner. This speculation draws on the observation that, to date, only α-isolates have been detected on Vancouver Island and the notion that recombination has been detected within Australian populations constituted exclusively of VGII MATα - isolates MATa isolates had never been detected in Australia et al.MATa strains may contribute critically to breeding, but are in such low overall abundance that they are difficult to isolate via routine environmental sampling.Sexual recombination has been suggested as a major force for the natural evolution of virulence a in Australia, more precisely in south-western WA. Two isolates belonging to the same MLST genotype (ST38) were characterized as MATa. One isolate was isolated from a veterinary case, a Dalmatian dog from the Geraldton area, WA, in 2001, and the second one from a eucalyptus tree trunk (species not determined) from the Caversham Wildlife Park to the north of Perth, WA, in 2009, approximately 400 km apart. This clearly indicates that MATa-strains are present in the Australian environment. The current study further suggests sexual recombination among VGII MATα and MATa strains in Australia, an observation in accordance with the natural life cycle of C. gattii. Sexuality among α- and a-mating partners has also been suggested in the related C. gattii molecular type VGI Population genetic studies generally require two main aspects, (i) the use of polymorphic molecular markers, such as the seven MLST-ISHAM-adopted loci and (ii) access to a sampling size as large as possible. The extensive molecular analyses realized in this study have demonstrated for the first time the presence of the mating type In this context, it is important to note that when a pattern of recombination is detected, it is hard to differentiate whether it corresponds to a past or a contemporary event C. gattii VGII population. Clonality and sexual reproduction are not mutually exclusive. Evidence of both modes has already been highlighted i.e. asexual versus sexual reproduction, has important evolutionary implications. Asexual reproduction promotes the colonization of new habitats and infections by one or a few clones Penicillium marneffei, an opportunistic fungus capable of infecting HIV/AIDS patients, may be clustered according to ecological conditions. This suggests that clonality has led to the evolution of niche-adapted genotypes Saccharomyces cerevisiae, Goddard et al.The findings presented here suggest that asexual reproduction and sexual recombination both contribute to the genetic diversity and structure of the Australian C. gattii VGII population having undergone a reduction of its population size resulting in a historic founder effect . This presumptive founder effect was subsequently followed by asexual population growth. It has been postulated that C. gattii originating from Australia was subsequently exported to other regions of the world by the transport of eucalyptus trees Mismatch distributions for four of the seven loci were consistent with the distribution obtained under the sudden expansion model and thus may indicate an historical population expansion. Furthermore, repartitions of pairwise differences were typically L-shaped, a pattern consistent with a bottleneck phenomenon followed by a demographic expansion C. gattii VGII occurred in 1983 and 1985, in the NT and WA, respectively (a mating type), with a high prevalence of asymptomatic nasal colonisation, subclinical infection and clinical disease in exhibited animals , which persists despite attempted environmental control measures . Overall, these observations are consistent with the potential for more widespread outbreaks due to C. gattii VGII strains in Australia, in either south-western WA and/or Arnhemland, NT. Despite the fact that this study contains the largest set of VGII isolates ever collected from Australia, the limited sample size can only point to the possibility of an outbreak at a continental scale in Australia. However, the existing data without doubt emphasizes the need for an on-going surveillance of environmental, clinical and veterinary cryptococcal isolates from Australia to identify the extent of clonal outbreaks that might account for cases in high incidence areas. A problem concerns the question of how to monitor regions where the population density of humans and domesticated animals is low Considering the behaviour of emerging and re-emerging infectious diseases ectively . The firectively  [9]. Recectively  [24]. Ine.g. MLST home page: http://www.mlst.net). As a result of the current study an online database for C. gattii has been established (http://mlst.mycologylab.org) on the basis of the seven loci adopted by the ISHAM Working group for genotyping of C. neofromans and C. gattiiC. gattii population diversity. Considering intra-MLST comparisons, this study demonstrated unambiguously that the seven chosen loci were sufficient to analyze the genetic variability within the Australian C. gattii population, with the genotypic diversity reaching a plateau for a total of only three loci  . These isolates reflect also all possible isolation sources   , represenmental) .C. gattii VGII isolates were studied.The strains CDC R265  and CDC R272  et al.Isolates were subcultured onto Sabouraud Dextrose Agar (SDA) at 37°C for 72 h prior to DNA extraction. High molecular weight DNA was than extracted according to Ferrer URA5 gene via double digestion with the enzymes HhaI and Sau96I was performed to determine the molecular types, as previously described Restriction fragment length polymorphism (RFLP) analysis of the a mating type specific primer pair JOHE9787 and JOHE9788 a  was carried out using the α mating type specific primer pair MFαU and MFαL 88 a see .C. neoformans/C. gattii species complex CAP59), glyceraldehydes-3-phosphate dehydrogenase (GPD1), laccase (LAC1), phospholipase B (PLB1), Cu, Zn superoxide dismutase (SOD1) and orotidine monophosphate pyrophosphorylase (URA5), and a non-coding region, the intergenic spacer region of the rDNA (IGS1).The genetic variation within the Australian VGII population was studied using the ISHAM consensus MLST scheme for the 2, 2.5 U of taq polymerase , together with the buffer recommended by the manufacturer , following the published amplification conditions Amplifications were carried out in a 50 µl reaction volume, containing: 100 ng of template DNA, 0.2 mM of deoxynucleoside triphosphate each, 7.5 pmol of the appropriate primers CAP59, GPD1, PLB1, LAC1 and IGS1, according to Fraser et al.et al.URA5 and SOD1 loci, allele identification was undertaken by comparison with our own global cryptococcal sequence database. Due to the lack of a C. gattii MLST database, an MLST database based on BioloMICS software  was constructed for the 7 ISHAM consensus loci at the Molecular Mycology Research Laboratory and can be accessed at http://mlst.mycologylab.org. For allele identification, sequences were aligned using CLUSTAL X version 2.0 CAP59-2, GPD1-6, LAC1-4, PLB1-2, SOD1-15, URA5-2, IGS1-10, which corresponds to the sequence type 7 (ST7). Each discrete sequence type was also assigned an arbitrary number in the order of detection. The sequence type numbers were given in accordance with a global study currently undertaken in our laboratory.Each sequence was assigned a unique MLST allele number. Allele numbers were assigned for the following five loci: p-distance and all positions containing alignment gaps were eliminated in the pairwise sequence comparisons. The significance of nodes was tested by bootstrapping with 1000 replications. The two strains CDC R265 and CDC R272 were included as reference strains in this analysis. An unrooted Neighbor-Joining tree has also been constructed considering all the sequence types delineated in Australia and the representative sequence types from other parts of the world. The isolates used to represent sequence types from other regions then Australia were selected for sequencing according to previous studies An unrooted Neighbor-Joining tree was constructed from the concatenated DNA sequences (a combination of the sequences from the 7 loci) of all Australian isolates using MEGA version 4 Nucleotide polymorphism positions for the 7 loci were determined using the software DnaSP version 5 C. gattii VGII population we plotted genotypic diversity against the number of loci using Multilocus version 1.3 software n/n – 1(1 – Σpi2) where n is the total number of individuals sampled and pi the relative frequency of the ith genotype To determine whether the number of loci used was sufficient to access the genetic diversity in the Australian i.e. non random association) among the 7 MLST loci the index of association AI and the slightly modified statistic rBard were computed using the Multilocus version 1.3 software rBard statistic has been performed in order to complement the AI index. Indeed, the AI value obtained is dependent of the number of loci included in the analyses, whereas rBard removes this dependency and would allow comparisons among studies i.e. infinite recombination. Thus, the null hypothesis of no linkage disequilibrium, consequently of recombination, will not be rejected if the observed values of both statistics are not significantly different from the distribution of the values obtained with the 1000 artificially recombining datasets. AI and rBard indexes were computed on (i) the complete dataset and (ii) the clone-corrected dataset from which replicates from the same sequence type were removed. Both complete and clone-corrected analyses were carried out on populations having a number of sequence types greater than 3, and consequently on the global and the WA populations.To test for multilocus linkage disequilibrium , specific source and date of isolation. Mating types, allele numbers for the seven MLST loci and the corresponding sequence type (ST) are also presented.(DOC)Click here for additional data file.Table S2List of primers used in this study.(DOC)Click here for additional data file.Table S3GenBank accession numbers for the alleles obtained in the current study of the seven MLST loci studied.(DOC)Click here for additional data file.


In vitro, human primary macrophages are derived from peripheral blood CD14+ monocytes in the presence of macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF). These are essential for developing tissue/resting macrophages (M-Mϕ) and inflammatory macrophages (GM-Mϕ), respectively. While IFN production is similar between M-Mϕ and GM-Mϕ, M-Mϕ cannot produce IL-1β after DV infection. In contrast, GM-Mϕ is more susceptible to DV infection and DV triggers CLEC5A in GM-Mϕ to activate NLRP3 inflammasomes, which in turn release IL-18 and IL-1β that are critical for Th17 activation and contribute to disease severity. Thus, GM-Mϕ is more representative than M-Mϕ for investigating inflammasome activation in dengue infection, and is invaluable for revealing the molecular mechanism of pathogen-induced inflammatory reaction. Distinct phenotypes of macrophage subsets under the influence of M-CSF and GM-CSF raise the question of optimal conditions for culturing primary macrophages to study host-pathogen interaction.Macrophages (Mϕ) are the major source of inflammatory cytokines and are target cells for dengue virus (DV) replication. However, Mϕ are heterogeneous and their phenotypic and functional diversities are influenced by cytokines that regulate their differentiation, tissue distribution, and defense against invading pathogens.  It is transmitted among humans by the Aedes mosquitoes and is prevalent in over 100 tropical and sub-tropical countries, with about 2.5 billion people at risk  ligand 5; CXCL10: C-X-C motif chemokine 10; TGF-β: Transforming growth factor beta; IP-10: Interferon gamma-induced protein 10; MCP-1: Monocyte chemotactic protein-1; NF-κB: Nuclear factor-κB; HSV-1: Herpes simplex virus 1; KSHV: Kaposi's sarcoma-associated herpes virus; MV: Measles virus; CPV: Cytoplasmic polyhedrosis virus.The authors declare that they have no competing interests.MFW and STC were involved in literature review, discussion, and drafting of the manuscript. SLH initiated the concept and wrote the draft of this manuscript. All of the authors read and approved the final manuscript.


In a transmission experiment we showed that social interactions may affect the disease progression. Queens with forced contact to symptomatic worker bees acquired an overt infection with up to 1011 virus copies per head in six days. In contrast, queens in contact with symptomatic worker bees, but with a chance to receive food from healthy bees outside the cage appeared healthy. The virus loads did not exceed 107 in the majority of these queens after nine days. Symptomatic worker bees may transmit sufficient active CBPV particles to the queen through trophallaxis, to cause an overt infection. Chronic bee paralysis virus (CBPV) is known as a disease of worker honey bees. To investigate pathogenesis of the CBPV on the queen, the sole reproductive individual in a colony, we conducted experiments regarding the susceptibility of queens to CBPV. Results from susceptibility experiment showed a similar disease progress in the queens compared to worker bees after infection. Infected queens exhibit symptoms by Day 6 post infection and virus levels reach 10 Apis mellifera) is host of more than a dozen viruses [The honey bee  [Nosema ceranae and CBPV may act synergistically to kill caged bees [4 virus particles were identified in individual mites [Formica rufa and Camponotus vagus) as a potential secondary host and reservoir of CBPV around apiaries [Several viruses of honey bees are known to be associated with, and vectored by, parasites. Black Queen Cell Virus (BQCV) has been linked to an infection with the gut parasite sema spp ,20. The us (DWV) ,22 and Aus (DWV) . Experimged bees , howeverged bees . Early sged bees ,25, but al mites . The samapiaries , althougUsing real-time PCR, CBPV can be detected in all developmental stages of the bee from egg to adult , but onlTwo separate experiments were performed to test the susceptibility to CBPV and the possible transmission pathway of CBPV between workers and queen. In the first experiment, we investigated whether queens have a different susceptibility to CBPV than that described for worker bees in the colony. Queens (n = 39) that were infected as described in Experimental Symptoms were also observed in the two control groups shaved+water and shaved only, starting on day 7 p.i., see methods for details. Shaving is the accepted method for infection of bees with CBPV ,12, as i7 copies (7.0 × 108–2.3 × 1011 copies per queen head), while 5 queens showed a moderate CBPV load of 104–106 (range 1.5 × 104–2.4 × 106). Some queens were also found to be infected with a low level of DWV (range 2 × 102–4.9 × 106), but we did not detect viruses of the ABPV complex in any of the queens. Out of 28 queens in the three control groups, symptoms and mortality were observed in 11 and 9 individuals, respectively. However, the virus loads of the 11 symptomatic queens remained comparatively low, ranging from 1.7 ×104 to 2.7 ×107. An analysis of variance based on rank transformed data showed that the virus loads between the infection group and the three control groups were significantly different . Furthermore, a comparison of the mean values by a LSD test showed no significant differences between the three control groups . The vir8 to above 1011 copies per head, and thus fell in the range of virus levels published for symptomatic workers [4 and 106 copies per head. It is possible that not the entire volume of virus suspension was absorbed, or the shaving of the thorax was not effective enough to open the queens’ cuticle. The lower viral titer in these five queens may thus be the result of a low level virus infection and viral replication within the queen, rather than a reduced susceptibility. In spite of these lower CBPV titers all five queens developed CBPV symptoms and died. We can exclude DWV and ABPV complex infections as cause of death, due to our testing of infection with RT-qPCR.The results of this experiment clearly show that there is no difference between queens and workers in susceptibility to CBPV. The post mortem virus titers in the majority of the infected queens reached levels from 10 workers ,17. AlthAll queens in this experiment were taken out of production hives and approximately one year old. Thus, they already may have carried an unapparent infection before the experiment started, as this possibility is known from previous published reports ,27,28. SIn the second experiment, three contact options between workers and queens were compared to evaluate possible transmission routes of CBPV between worker bees and the queen in the colony. In group A (queens kept in cages together with symptomatic worker bees in the incubator) 23 of 29 individuals started showing symptoms 5–6 days p.i.. Similarly, 15 of 32 queens in group B  showed symptoms by day 6. In contrast, only three of the 29 queens in group C (queens kept together with symptomatic bees in shipping cages inside mating nucs) started to show symptoms by day 9 after introduction. However, recognition of symptoms inside the queen shipping cages was more difficult and early symptoms may have been missed.9–4.4 × 1011 virus copies, while the four remaining queens showed a moderate level (4.4 × 104–1.4 × 106 virus copies per head). Among these four, three died even before onset of symptoms. From 32 queens with a topical application of CBPV (Group B), 15 queens developed symptoms and had high virus level (2.9 × 109–3.6 × 1011), but for 17 individuals no symptoms were observed and they showed no virus or only a moderate virus load (1.4 × 104–1.5 × 107). Among these, four queens died before onset of symptoms, maybe for reasons not related to viral infection, such as handling or experimental stress. Comparatively low viral titers were quantified in the majority of queens kept with symptomatic workers in queen shipping cages inside mating nucs (Group C). Only six of these queens had more than 108 viral copies per individual, and among these four died before termination of the experiment without symptoms having been noticed. The remaining 23 queens had fewer than 107 copies per queen. Analysis of variance from the rank transformed viral loads resulted in significant differences among the three experimental groups A, B and C  had high viral loads. Twenty-five of 29 queens showed a high range between 3.0 × 10 B and C .9 virus copies in the majority of these queens. This is similar to the result we obtained in experiment 1 and also to virus levels found in workers after experimental inoculation [7 copies in the head is a good indicator of disease outbreak also in queens [A LSD test was used to compare the mean rank transformed viral load among these three groups. Significant differences were observed among all pairs of groups . Paralysculation . Howevern queens ,17,26. IOur research thus demonstrates that forced contact of queens with symptomatic worker bees can lead to an overt infection at least as effectively as the topical application of virus suspension, similar to what is known for contact between workers . Onset oper os has been reported as less effective than other routes of virus transmission [We therefore suggest that in the context of the colony a transmission of virus through trophallactic food exchange with symptomatic bees may play a more significant role than previously estimated, since infection smission , but seesmission . The queApis mellifera [8 copies/µL (for virus quantification see below). Naturally infected trembling and hairless honey bees were sampled from colonies suffering from chronic bee paralysis in an apiary of the Bieneninstitut Kirchhain, Germany. The virus was partially purified according to the COLOSS BEEBOOK chapter on virus research in ellifera . Brieflyellifera . This coellifera , BQCV [3ellifera , DWV [36ellifera , and theellifera . FollowiIn the first experiment, we tested the susceptibility of queens to CBPV. Using one year old queens we investigated the development of symptoms and mortality after topical application of CBPV solution or abrasion of thoracic hair without virus application. Finally, we determined the virus load of each queen after they succumbed to the infection. In the second experiment, using young and unmated queens, we compared the efficiency of direct virus application to infection via contact to symptomatic worker bees. In addition, we collected data on the effect of the queen’s environment and food supply on the course of infection. 9 virus copies.Infection group (n = 39): a small area on the thorax was carefully shaved and 4 µL of virus suspension was topically applied. Thus, each queens received approximately 2 × 10Control group 1 (n = 10): the queens were immobilized, a small area on the thorax was shaved and 4 µL of distilled water was applied.Control group 2 (n = 8): the queens were immobilized, and a small area was shaved, but nothing was applied.Control group 3 (n = 10): the queens were immobilized, but no treatment was applied.The queens in this experiment were mated and approximately one year old. They originated from apparently healthy colonies of the Kirchhain institute, which were routinely re-queened in the late summer 2012. The queens themselves did not show obvious disease symptoms. They were divided into three control groups and one infection group. After immobilizing by cooling on ice, the queens received the following treatments:The queens were kept with about 30 healthy worker bees in metal cages supplied with sucrose syrup, sugar candy and pollen in an incubator at 28 °C and 65% RH. Symptoms and mortality were recorded several times per day until day 15 post infection (p.i.), at which time all experimentally infected queens had died. Dead queens and workers were collected immediately to be frozen at −80 °C until RNA extraction. On day 15 all remaining live queens were also collected and frozen at −80 °C. 7 virus copies [Group A (n = 29): to simulate a natural infection via feeding and contact, queens were kept in metal cages together with about 30 workers each who all suffered from a natural infection with CBPV. These workers were symptomatic and most likely each contained more than 10s copies . To mini8 copies/µL) was applied to a small shaved area on the thorax of the queens. After the virus suspension was absorbed, each queen was returned to her cage. All cages were supplied with sucrose syrup, sugar candy and pollen cake.Group B (n = 32): queens were kept with about 30 healthy bees each in metal cages. Following immobilization on ice, 4 µL of virus suspension  were divided into three groups which received the following infection treatments:Group C (n = 29): queens were kept in plastic queen shipping cages  togetherThese two groups of queens were kept together in the same incubator at 28 °C and 65% RH. Symptoms and mortality were recorded daily. After observing symptoms in the first queens on day 6 p.i., all queens from both groups were collected in individual tubes and immediately frozen at −80 °C for virus quantification.The head of each queen was placed in a 2 mL tube together with a metal bearing ball and freeze-dried on a Heto Lyopro 6000 for three days at 0.009 hPa and −93 °C. Samples were homogenized on a Geno Grinder 2000 for 30 s at 500 Hz. The homogenized head was used for total RNA extraction using NucleoMag 96 RNA kit  on a Kingfisher Magnetic Extractor following the manufacturer’s guidelines. The extracted RNA was stored at −80 °C until further use. A two-step real-time RT-PCR assay was used to quantify CBPV in the samples. First, cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit . Thereafter, the synthesized cDNA was diluted 10 fold in RNase/DNase-free water to use for quantitative PCR amplifications. ® Green DNA binding dye (Applied Biosystems). A final volume of 12 µL for qRT‑PCR was loaded on an optical 384 well PCR plate (ABgene) with a primer concentration of 0.4 µM. RNase-free water was used as template for two negative controls (No Target Control (NTC) and No Reverse Transcriptase (NRT)) [N-glycosylase (UDG)), 10 min at 95 °C, followed by 40 cycles consisting of denaturing at 95 °C for 15 s, annealing/extension at 60 °C for 35 s. Virus loads in each sample were quantified using absolute quantification methods based on standard curves obtained through serial dilutions of known amounts of the amplicons as described before [Quantitative PCR amplifications were carried out on a vii7 apparatus (Applied Biosystems) in duplicate for each sample using SYBRe (NRT)) . The thed before . The refd before  . The mean values of different groups were compared using Least Significant Difference (LSD) test and the term significant has been used to indicate differences for which one by R . The results of this study reveal that CBPV successfully replicates in queens and the disease progresses in much the same way as is known for worker bees ,17. SympThe results from the transmission experiment suggest that behavioral patterns are in place to protect the queen from infection by symptomatic bees. We cannot decide from this experiment if queens avoid contact with or uptake of food from diseased workers, or if diseased workers try to avoid interaction with the queen. In this experiment, queens were exposed to virus both via the epidermal cytoplasm and trophallactic feeding. Confining queens into cages with exclusively symptomatic bees resulted in infective infection, similar to those queens with virus suspensions topically applied. In contrast, the onset of symptoms was considerably delayed in queens caged with symptomatic bees, but with access to healthy workers on the outside of the cage. These queens were exposed to virus via the epidermal cytoplasm, but they had the chance to be fed by healthy worker bees through the netted cage.The results of this study suggest that behavioral strategies, such as avoidance of contact between infected worker bees and queen, or even infected workers leaving the hive altogether, play an important role in keeping the queen safe from exposure to CBPV inside a colony.


Testing for cerebrospinal fluid (CSF)-restricted oligoclonal bands (OCB) by isoelectric focusing is used to detect intrathecally produced total IgG. By contrast, antibody indices (AI) are assessed to test for intrathecally produced antigen-specific IgG. A number of previous cases reports have suggested that AI testing might be more sensitive than OCB testing in detecting intrathecal IgG synthesis.Here we report on 21 patients with positive AI for either herpes simplex virus, varicella zoster virus, cytomegalovirus, measles virus, rubella virus, or Borrelia burgdorferi in the absence of total-IgG OCB and, accordingly, in the presence of a normal total-IgG CSF/serum ratio.Our findings indicate that AI testing should not generally be omitted in OCB-negative patients and provide a rationale for systematic and prospective studies on the comparative sensitivity and specificity of AI and total-IgG OCB testing in infectious and other diseases of the CNS. Testing for cerebrospinal fluid (CSF)-restricted total IgG oligoclonal bands (OCB) by isoelectric focusing is a highly sensitive method for detecting intrathecal IgG synthesis, but does not take into account antibody specificity. By contrast, antibody indices (AI) are assessed to test for antigen-specific intrathecal IgG synthesis . It is uBack in 1992, however, Felgenhauer and Reiber reported a positive varicella zoster virus (VZV)-specific AI in 6 total-IgG OCB-negative patients with VZV ganglionitis as well as in 4 total-IgG OCB-negative patients with VZV meningitis, suggesting that AI calculation might be more sensitive than total-IgG OCB testing /QIgG[total], if QIgG[total]<Qlim, and AI=QIgG[spec]/Qlim, if QIgG[total]>Qlim, with QIgG[spec]=IgGspec[CSF]/IgGspec[serum], and QIgG[total]=IgGtotal[CSF]/IgGtotal[serum],3. The uTo assess the plausibility of the negative total-IgG OCB results, we analysed each patients’ total-IgG CSF/serum ratio (QIgG). Elevated QIgG in the absence of total-IgG OCB positivity would suggest a false-negative OCB result. However, QIgG was found to be normal in all patients (i.e. QIgG<Qlim) as shown in FigureThese cases confirm that negative total-IgG OCBs in CSF samples might not always predict the absence of positive virus- or bacterium-specific IgG-AIs in patients with suspected CNS infection. Omitting AI testing in OCB negative patients might thus be a possible diagnostic pitfall. In addition to VZV infection , positivAround 2-5 % of patients with MS are negative for OCBs. This subgroup, which is small in relative numbers but not so small in absolute numbers due to the high prevalence of MS, is particularly challenging from a diagnostic point of view. A higher sensitivity of antigen-specific AI calculation compared to total-IgG OCB testing might therefore also be of potential relevance for the laboratory diagnosis of multiple sclerosis (MS), since a majority of patients with MS display a polyspecific humoral antibody response to a broad variety of viral and bacterial antigens as determined by AI calculation, with intrathecal antibodies to MV, RV and VZV as its most common constituents (termed MRZ reaction or MRZR), as demonstrated by Reiber and others -9. ApartOn the other hand, the fact that a subset of patients with MS presents with a monospecific AI elevation rather than the typical polyspecific, oligoclonal reaction, at least at first presentation , and thaThe reason for the higher sensitivity of AI detection compared to total-IgG detection in the patients reported here is unknown. Infections are often associated with severe blood-CSF barrier dysfunction; the presence of OCB may become undetectable in those cases because of a relative increase of the blood-derived polyclonal IgG fraction. In such cases OCBs may possibly become positive after normalization of barrier dysfunction. However, high CSF/serum albumin ratios were present only in a subset of our patients (see FigurePossibly, AIs might become positive earlier than total-IgG OCBs. If so, AI assessment might be particularly useful at disease onset. However, as OCBs remained negative in the few patients with follow-up LPs in our study, our study provides no direct evidence for this hypothesis.There are some caveats. First, as it is the case with IgG responses in general, there might be a delay in AIs becoming positive as indicated by case 1 in TableIn summary, the cases reported here indicate that AI testing should not generally be omitted in total-IgG OCB-negative patients and provides a rationale for systematic and prospective studies on the comparative sensitivity and specificity of AI and total-IgG OCB testing in infectious diseases of the CNS as well as in other indications.AI: antibody index; Bb: Borrelia burgdorferi; CSF, cerebrospinal fluid; CMV: cytomegalovirus; CNS: central nervous system; HSV: herpes simplex virus; IgG: immunoglobulin G; LP: lumbar puncture; MRZR: measles virus, rubella virus, and varicella zoster virus reaction; MS: multiple sclerosis; MV: measles virus; NB: neuroborreliosis; OCB: oligoclonal IgG bands; PCR: polymerase chain reaction; PNS: paraneoplastic neurological syndromes; Q: ratio; RV: rubella virus; VZV: varicella zoster virus; WCC: white cell count.The authors declare that they have no competing interests.S.J. conceived the study, analysed the data, and wrote the initial draft; P.E. and M.W. conducted and interpreted the laboratory tests; S.J., P.E., B.W., and M.E. revised the manuscript for important intellectual content. All authors have read and approved the final version of the manuscript.


Danio rerio) and summarize current knowledge concerning the phylogenetic evolution of kindred proteins. We identified a cobalamin binding capacity in zebrafish protein extracts (8.2 pmol/fish) and ambient water (13.5 pmol/fish) associated with a single protein. The protein showed resistance toward degradation by trypsin and chymotrypsin . The cobalamin analogue, cobinamide, bound weaker to the zebrafish cobalamin binder than to human HC, but stronger than to human TC and IF. Affinity for another analogue, adenosyl-pseudo-cobalamin was low compared with human HC and TC, but high compared with human IF. The absorbance spectrum of the purified protein in complex with hydroxo-cobalamin resembled those of human HC and IF, but not TC. We searched available databases to further explore the phylogenies of the three cobalamin-binding proteins in higher vertebrates. Apparently, TC-like proteins are the oldest evolutionary derivatives followed by IF and HC . Our findings suggest that the only cobalamin-binding protein in zebrafish is an intermediate between the three human cobalamin binders. These findings support the hypothesis about a common ancestral gene for all cobalamin-binding proteins in higher vertebrates.In humans, three soluble extracellular cobalamin-binding proteins; transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are involved in the uptake and transport of cobalamin. In this study, we investigate a cobalamin-binding protein from zebrafish ( The proteins TC and IF  is an important vertebrate model organism, which has an advantage of rapid embryonic development if compared with higher vertebrate models, such as rats and mice. As mammals and other vertebrates, the zebrafish have two intracellular cobalamin-dependent enzymes, methylmalonyl coenzyme A mutase (NCBI GeneID 569581) and 5-methyl tetrahydrofolate homocysteine methyl transferase (NCBI GeneID 378847) Zebrafish (after divergence of the bony fish (Osteichthyes).In this study, we searched for soluble cobalamin-binding proteins in zebrafish and found only one resembling an intermediate between the cobalamin-binding proteins found in humans. Analysis of the sequence databases indicated that the cobalamin-binding proteins in higher vertebrates might have descended from a common ancestral gene 2, 6H2O , 400 mM NaCl, and two tablets per 50 ml buffer of protease inhibitor cocktail  using a tissue rupture . For studies investigating the effect of trypsin and chymotrypsin on protein stability, no protease inhibitors were added to the buffer before homogenization. To ensure cell rupture, the homogenates were subjected to three freeze-thaw cycles followed by three times 10 seconds of ultra sonication . Finally, protein extracts were centrifuged at 4°C for 40 minutes at 20.000×g, and the supernatants were stored at −20°C until analysis. According to the "European convention for the protection of vertebrate animals used for experimental and other scientific purpose" no approval is necessary for the humane sacrifice of the zebrafish used in this study.Zebrafish were kept in tap water with 0.02% Aquarium START Plus Solution  and were fed Flakes Complete Fish food  according to the manufactures' instructions. On the day of sacrifice, zebrafish were homogenized on ice in 10 mM PIPES pH 7.4 , 1 mM EDTA , 3 mM MgClHuman spermal fluid and gastric juice were collected by medical staff as part of diagnostic tests for acidity  and infertility  12BC) was measured as described by Gottlieb et al57[Co]-cobalamin) (5 nM)  in 200 µl 0.1% phosphate-buffered bovine albumin (PBA) for 15 minutes, and 500 µl hemoglobin-coated charcoal solution was added to precipitate unbound cobalamin. The charcoal solution was prepared by mixing equal volumes of a 5% aqueous suspension of activated charcoal  with a 0.5% aqueous solution of bovine hemoglobin . After additional 10 minutes of incubation, charcoal-bound cobalamin was precipitated by centrifugation for 10 minutes at 2600×g. Finally, the supernatants were measured for radioactivity in the Wizard Automatic Gamma Counter (Perkin Elmer).Unsaturated cobalamin binding capacity  with a detection range of 55–1476 pM. Zebrafish protein extracts were diluted five times with 0.9% NaCl solution prior to analysis.2PO4 pH 7.5 and subjected to measurement of unsaturated cobalamin binding capacity.Five zebrafish were kept in 100 ml tap water for six hours to allow secretion of cobalamin-binding protein into the medium. The water was collected and stored at −20°C until further analysis. For each collection, 1 ml water sample was taken out before storage, and sample solvents were evaporated in a Hetovac vacuum centrifuge . Pellets were resuspended in 100 µl NaH12BC) or 100 µl collected zebrafish-ambient-water (∼ 700 pmol/L UB12BC) were incubated with 50 µl 57[Co]-cobalamin (5 nM) for 30 minutes at room temperature. Then, the samples were applied to a Superdex 200 column  attached to a Dionex ICS-3000 HPLC system. Collected fractions were measured for radioactivity in the Wizard Automatic Gamma Counter and for cobalamin content in the Cobas 6000 E immunoassay system. Blue Dextran  and Na22  was used for determination of void volume (V0) and total volume (Vt), respectively. For comparison, size exclusion chromatography of the human cobalamin-binding proteins, TC, HC, and IF, was performed.For size exclusion chromatography, 100 µl zebrafish protein extract  was incubated with 25 µl 57[Co]-cobalamin (5 nM) in 0.1 M Tris-HCl pH 7.4 in a total volume of 800 µl for 30 minutes at room temperature. Increasing concentrations (0 units to 500 units) of trypsin  or chymotrypsin  in a total volume of 100 µl 0.1 M Tris-HCl pH 7.4 were added, and the mixture was incubated for 18 hours at 37°C. Unbound cobalamin was absorbed on hemoglobin-coated charcoal  and precipitated by centrifugation for 10 minutes at 2600×g. The radioactivity of supernatants was measured for radioactivity in a Wizard Automatic Gamma Counter.The sensitivity of the cobalamin-binding protein from zebrafish toward enzymatic cleavage was explored using increasing concentrations of trypsin and chymotrypsin. For each protein extract , the UB12BC was adjusted to 2 nM by dilution with binding buffer , and 600 µl of each sample was incubated with 10 µl 57[Co]-cobalamin (5 nM) for 30 minutes at room temperature. A 100 µl suspension of Con A Sepharose (prepared accordingly to the manufacturer's instructions) was mixed with 200 µl of the binding buffer, and the samples were incubated for 1 hour at room temperature before centrifugation (5 minutes at 8000×g). The 57[Co]-cobalamin present in the supernatant and precipitate was measured in a Wizard Automatic Gamma Counter.The presence of carbohydrates in the cobalamin-binding protein from zebrafish was examined by precipitation with Con A Sepharose , a lectin known to bind α-D-mannopyranosyl, α-D-glucopyranosyl, and kindred compounds. For each protein extract , the UB57[Co]-cobalamin (0.4 nM) tracer solution prior to addition of protein extract (UB12BC adjusted to 0.3 nM). The mixture was incubated for 18 hours at 4°C, whereupon the unbound cobalamin was precipitated, and the radioactivity of supernatant was measured in a Wizard Automatic Gamma Counter. For a comparison, the human cobalamin-binding proteins, TC, HC, and IF, were also examined.The ability of zebrafish extract to bind different corrinoids was explored using a competitive assay 2PO4 pH 7.5 and incubated at 57 °C for five hours, while gently mixing the solution every 20–30 minutes. Sodium azide was added to a final concentration of 0.02%, and the suspension was placed at 4°C for 20 hours to stabilize the thermo-labile bond between the cobalt atom of cobalamin and the amino group of the sepharose. Prior to use, the cobalamin-coupled sepharose was extensively washed with 50 volumes of cold 0.1 M NaH2PO4 pH 7.5 followed by 30 volumes of demineralized water and six volumes of 0.1 M NaH2PO4 pH 7.5 to eliminate the excess of free cobalamin. The collected zebrafish-ambient-water (100 ml) was applied to the column with a flow rate that guaranteed five minutes residence time of the solution inside the matrix. Afterwards, the column was washed with 15 volumes of 0.1 M Tris pH 7.5 with 1 M NaCl and with three volumes of 0.1 M NaH2PO4 pH 7.5. Absorbed cobalamin-binding protein was detached from the column after incubation at 37°C for 24 hours, and it was eluted with two volumes of 0.1 M NaH2PO4 pH 7.5 warmed to 37°C. The eluate was subjected to six hours dialysis in 14 kDa cut-off dialysis tubes  against 0.1 M NaH2PO4 pH 7.5 followed by 18 hours dialysis against demineralized water. Finally, the solvents were evaporated in a Hetovac vacuum centrifuge, and the pellet was resuspended in 0.1 M NaH2PO4 pH 7.5. Recovery was judged by comparison of the collected cobalamin with the amount of UB12BC loaded into the column.Unsaturated cobalamin-binding protein secreted from the zebrafish into the ambient water was purified on an affinity column with hydroxy-cobalamin coupled to a sepharose matrix Purity of the protein sample was evaluated by 12% SDS-PAGE, adding 25 µl (12 µg) of the protein per lane of a precast Tris-HCl gel  stained by Coomassie Brilliant Blue according to the standard procedure.The absorbance spectrum of the purified zebrafish cobalamin binder in complex with hydroxo-cobalamin was recorded on a Varian Cary 50 spectrophotometer . Spectral transition upon addition of exogenous 15 mM histidine was examined as a test on the coordination of the endogenous His-residue to the cobalt ion The National Center for Biotechnology Information (NCBI) database Protein extracts of homogenized zebrafish (n = 5) contained 11.3 [6.3–14.2] pmol cobalamin/fish (mean and [range]) and had an unsaturated cobalamin binding capacity of 8.2 [6.3–10.1] pmol/fish . ProvideFive zebrafish were kept in 100 ml tap water for six hours before the concentration of cobalamin and the unsaturated cobalamin binding capacity of the ambient water were determined. The water was found to contain 112 [98–135] pmol/l cobalamin  . Also, aWe investigated sensitivity of the zebrafish cobalamin binder, as compared to the human cobalamin binders, toward proteolytic degradation caused by addition of increasing concentrations of trypsin and chymotrypsin. While human TC and HC were degraded by trypsin and chymotrypsin; the zebrafish cobalamin binder and human IF remained intact under the experimental conditions .The enzymatic resistance of the cobalamin-binding protein from zebrafish was not caused by a carbohydrate coating, since the protein did not interact with Con A (data not shown).d<<1 pM)Human TC, HC, and IF have comparable high affinities for the physiological active forms of cobalamin . This source was chosen because a lower amount of contaminants was present in comparison to the protein extract (not shown). As judged by the reduced SDS-PAGE, the purified protein had an approximate molecular mass of 46 kDa . Similar spectral changes were observed earlier in human HC and IF but not in human and bovine TCs, where the endogenous His-residue of the binder is already coordinated to cobalt ion and hinders interaction with the exogenous His The absorbance spectrum of the purified cobalamin binder from zebrafish in complex with hydroxo-cobalamin (≈ 40 µM) was examined and compared to those of human TC, HC, and IF, described earlier 372-residue  not found in TC 385 in HC, Tyr380 in TC) is a characteristic trait of IF According to the NCBI database The occurrence of the cobalamin-binding proteins, TC, HC, and IF, in different species within the subphylum vertebrata was investigated using NCBI and UCSC search tools Danio rerio) and compared it with the three known soluble extracellular cobalamin-binding proteins present in humans and other mammals. Those are IF, the protein facilitating intestinal uptake of cobalamin; TC, the protein mediating the uptake of cobalamin by the cells of the body; and HC, a protein of unknown function present in plasma and most extracellular fluids In the present study, we investigated the soluble cobalamin-binding protein from zebrafish . Yet, it did not bind to Con A Sepharose and seemingly lacks carbohydrates (like TC). In addition, the zebrafish protein displayed intermediate affinities for the cobalamin analogues cobinamide and adenosyl-pseudo-cobalamin, which was not consistent with any of the human type proteins .In order to further characterize the protein, we purified it by affinity chromatography. The characterization covered only a few basic features, because of a limited amount of available material . The zebrafish cobalamin binder had a molecular mass of approximately 46 kDa, which is similar to human TC and expected from the amino acid sequence (429 aa) found in the NCBI database (GeneID 407646). In contrast, the spectral properties of the zebrafish binder resembled those of HC and IF. Absence of protection of the cobalt ion in bound hydroxo-cobalamin clearly distanced the zebrafish protein from human and bovine TCs (both exhibiting this protection).In summary, we found the cobalamin-binding protein from zebrafish to be a structural hybrid between the human cobalamin-binding proteins. Based on the sequence collected from the databases, the zebrafish cobalamin binder has the highest overall identity with TC, but the amino acid composition in the cobalamin-binding site resembles mostly IF.after divergence of the bony fish (Osteichthyes). This is supported by the in situ phylogenetic studies presented in These findings in zebrafish suggest that the three known cobalamin-binding proteins in higher vertebrates have descended from a common ancestral gene Danio rerio). The protein behaves like a structural intermediate between the three known extracellular cobalamin binders, IF, TC, and HC, suggesting the zebrafish protein to be a common ancestor for the cobalamin binders found in higher vertebrates.In conclusion, we report that only one soluble extracellular cobalamin binder is present in zebrafish Click here for additional data file.


Immunity elicited by live attenuated vaccines confers protection against viral pathogens causing measles, yellow fever, smallpox and others, but this approach is not well suited to HIV vaccine development. Accordingly, to develop a vaccine that incorporates features of a live virus that make it immunogenic without the inherent safety issues associated with attenuated lentiviruses, we are developing replication-competent, recombinant vesicular stomatitis virus (rVSV) vectors for delivery of SIV and HIV vaccines.An rVSV vector was constructed in which the natural glycoprotein (G) was replaced with SIVmac239 Env, and an additional transcription unit was introduced to encode SIVmac239 Gag. The chimeric VSV deltaG-SIV virus vaccine was rescued and evaluated in vitro and in vivo.8 PFU, no adverse reactions were observed and serum antibody responses against Gag and Env were elicited, which were boosted by IM immunization with Ad5-SIV Gag and Ad5-SIV Env vectors.Infection of susceptible cells with this chimeric rVSV-SIV GagEnv virus produced infectious VSV particles containing functional SIV Env, and lentivirus-like particles containing Gag and Env. Serial passage of rVSV-SIV GagEnv vector in CD4+/CCR5+ cells resulted in improved replicative fitness evident by a 2-log increase in infectious virus titers. This adapted virus retained CD4/CCR5 dependence, infected primary rhesus PBMCs and cells isolated from lymph node and duodenum tissues ex vivo. Primary human monocyte-derived dendritic cells (MDDCs) also were susceptible to infection with the rVSV-SIV GagEnv vector. Indian rhesus macaques were immunized with increasing doses of rVSV-SIV GagEnv intramuscularly (IM) to observe reactions to vaccination with this new vector and to quantify immune responses. At the highest dose of 1x10A prototype chimeric VSV-SIV live virus vaccine was developed and evaluated in the NHP model. Chimeric VSV-SIV and VSV-HIV vaccine candidates designed for improved replication and immunogenicity currently are being evaluated in vitro and in vivo.


Progressive supranuclear palsy (PSP) and multiple system atrophy (MSA) are progressive disabling neurological conditions usually fatal within 10 years of onset. Little is known about the economic costs of these conditions. This paper reports service use and costs from France, Germany and the UK and identifies patient characteristics that are associated with cost. 767 patients were recruited, and 760 included in the study, from 44 centres as part of the NNIPPS trial. Service use during the previous six months was measured at entry to the study and costs calculated. Mean six-month costs were calculated for 742 patients. Data on patient sociodemographic and clinical characteristics were recorded and used in regression models to identify predictors of service costs and unpaid care costs . The mean six-month service costs of PSP were €24,491 in France, €30,643 in Germany and €25,655 in the UK. The costs for MSA were €28,924, €25,645 and €19,103 respectively. Unpaid care accounted for 68–76%. Formal and unpaid costs were significantly higher the more severe the illness, as indicated by the Parkinson's Plus Symptom scale. There was a significant inverse relationship between service and unpaid care costs. Progressive supranuclear palsy (PSP) and multiple system atrophy (MSA) are rare progressive neurodegenerative disorders, usually presenting as akinetic-rigid syndromes (‘parkinson plus syndromes’). Age at onset is typically between 55 and 65, and the average life expectancy from onset is 5–10 years for both diseases Patients with MSA and PSP require care from a range of services, and it is likely that such inputs will need to increase as the disease progresses. However, to date there have been no attempts to comprehensively measure and cost service use for patients. Measures of costs for a representative sample of patients allow us to determine relationships between care inputs and patient needs, assess the cost-effectiveness of specific treatments and highlight the impact that these conditions have on society as a whole.As part of the NNIPPS study Patients gave written consent prior to participating in the study. The protocol and amendments were reviewed and approved by the Comité de Protection des Personnes of Pitié-Salpêtrière Hospital (France), the UK Multicentre Research Ethics Committee (MREC), (UK), Ethikkommission of the University of Ulm, (Germany), and by local Institutional Review Boards (Ethics Committees) where appropriate . These include, UK: Ethics committees of Belfast, Birmingham - City Hospital, Birmingham - Queen Elizabeth Hospital, Cambridge, Liverpool, King's College London, NHNN & Queen Square Hospital, Newcastle upon Tyne, Stafford, Aberdeen, Guernsey, Swansea. Germany: Aachen, Berlin, Bochum, Dresden, Freiburg, Halle, Hannover, Magdeburg, München, Regensburg, Rostock, Tübingen, Ulm.The broad aims of the NNIPPS project were to evaluate the use of riluzole, a potential neuroprotector and to assess the natural history of PSP and MSA. Details of the inclusion criteria and study design are published elsewhere Patients were to be followed-up 3-monthly for three years for safety assessment. For efficacy and natural history assessment, the following measures were completed at entry to the study (between 2000 and 2002) and used in the subsequent economic analyses: (i) demographic characteristics , (ii) vital signs (including weight and height), (iii) Hoehn and Yahr disease staging scale (HYS) It is important in any economic study to include information on all services that may be used because of the specific condition under investigation. Service use during the six-month period prior to interview was measured using the Client Service Receipt Inventory (CSRI) adapted for these disorders . ParticiUnit costs were attached to the service use measures in order to generate service costs and were obtained from a number of sources. UK unit costs were taken from a recognised national source The study recorded personal care input from family members and friends. Whilst care from friends or family members is rarely paid for, it still represents an important economic cost as the time spent caring could have been used in alternative activities. We made the assumption that if the family member or friend was not providing care then this would have to be provided by services and consequently we valued unpaid care at the cost of a home care worker. .Data on medications received were recorded for each patient in the study. Medication names were entered as free-text variables and approximately 1000 different names were recorded (some being the same medications with slightly different spellings or brand names). A pragmatic approach was taken whereby only medication taken by at least 1.5% of the sample was costed. The costs of drugs were obtained where possible from the British National Formulary (the most common source for such data in the UK) and combined with medication levels and period of receipt. Medication costs were subsequently converted to Euros by dividing by 0.663 (the exchange rate at the time of the study) Costs were reported for each country and strata (PSP/MSA) separately. Tests for significant differences between MSA and PSP were not conducted at this stage, as there could be differences between the samples in terms of patient characteristics that might also influence costs. The existence of significant differences in costs was, rather, explored using multivariate analyses. Two regression models were produced. In the first model the dependent variable was the total service costs excluding unpaid care costs. The second model used unpaid care costs as the dependent variable. Variables for inclusion in these two models were chosen because they described features that pre-disposed clients to use services , features that enabled them to access services  and features that described their illness  Cost data are typically positively skewed and this can lead to similarly skewed residuals representing a violation of one of the assumptions on which ordinary least squares models are based. One option for addressing this is to use bootstrapping methods which make no assumption about the underlying data distribution. However, the need for modelling the actual data distribution has been advocated Of the 760 patients in the ITT cohort usable data on service use was available at baseline for 742 participants . Population characteristics are shown in There were some similarities across the three countries in the use of services by patients with PSP . The majThere were very similar patterns of service use for patients with MSA . Again, The distribution of service costs (excluding unpaid care from family/friends) shows that in France, social care accounts for around one-third of PSP costs whilst in Germany and the UK the main contributor to PSP costs is inpatient care . For MSAThe regression analyses for formal care costs (i.e. excluding unpaid care from family/friends) reveals that costs were significantly associated with the NNIPPS-PPS scale, and this relationship was very similar in each country. In France, a one-unit increase in the PPS was associated with a 1.9% increase in costs, whilst in Germany and the UK the figure was 1.7%. In each country there was also an inverse relationship between formal care and unpaid care costs. If the latter increase by €1000 then there is a reduction in formal care costs of 1.2% in France and 1.1% in both Germany and the UK. In France women had formal care costs that were 35% higher on average than for men and those with further education had 63% higher costs than those with the most limited education. In Germany participants with MMSE scores between 28 and 30 had costs that were 48% higher than those with lower scores.Unpaid care costs were also significantly associated with NNIPPS-PPS scores, with a slightly greater impact of a one-unit increase in Germany (2.2%) compared to France (1.6%) and the UK (1.3%). The inverse relationship with formal care costs was again apparent with similar impacts one a €1000 increase in France (3.7%), Germany (4.1%) and the UK (3.5%). Secondary education was associated with significantly lower unpaid care costs in both France and the UK, with the latter also having significantly lower costs for patients with a university education. In France, patients with PSP had significantly lower unpaid care costs than those with MSA whilst in the UK women had significantly lower costs than men.There was no relation between costs, formal or unpaid, and age or disease duration.This is the first study of the costs of providing care for people with PSP and MSA, and we have found that patients with PSP and MSA use a wide range of services and that the mean six-month costs reported are substantial. Most of the costs are related to unpaid care. The costs detailed here are for only three countries and any extrapolation needs to be made with caution. However, using the lowest and highest costs for PSP (€24491 and €30643) and for MSA (€19103 and €28924) and the prevalence figures reported in the Service and unpaid family costs were higher for patients with higher scores on the NNIPPS-PPS which is what we would expect if resources are more likely to be required by those with greater levels of symptom severity. This suggests that interventions which successfully modify disease progression may reduce the economic burden of these conditions. Data on changes in the NNIPPS-PPS have been reported elsewhere The costs of unpaid care were significantly lower for women then men in the UK. Life expectancy and caring roles help to explain this finding, although it is surprising that it was not observed in France or Germany. There was an inverse relationship between unpaid family care costs and service care costs, indicating that one substitutes for the other . Costs are significantly associated with symptom severity and if this can be modified then substantial reductions may be realised.Appendix S1Client Service Receipt Inventory.(DOC)Click here for additional data file.Appendix S2Unit costs used in cost calculations (Euro's).(DOC)Click here for additional data file.


Betaflexiviridae. The genome consists of 7,987 nucleotides, excluding the poly-A tail, and has four open reading frames (ORFs). Analysis of the whole genome and putative proteins encoded by each ORF revealed greatest sequence similarity to a cherry isolate of cherry mottle leaf virus (CMLV). The two isolates have similar genome organizations and share 88 and 93 % homology in their corresponding products of the replicase and coat protein genes, respectively. CMLV has been reported from several Prunus spp. and may be associated with peach wart-like disease symptoms on peach fruit.The complete nucleotide sequence and genome organization of a peach virus isolate from a naturally infected peach tree showing typical peach wart-like symptoms on the fruit surface was determined and compared to sequences of members of the family The online version of this article (doi:10.1007/s00705-013-1698-3) contains supplementary material, which is available to authorized users. Prunus persica cv. Elberta) exhibiting peach wart symptoms . CMLV infects several species of Prunus and can be vectored by eriophyid mites, Eriophyes inaequalis [Peach wart disease (PWD) is characterized by hard, wart-like outgrowths on the fruit surface . Becauseoms Fig.  was testllovirus ; the tesaequalis . RT-PCR ® 2.1 (Invitrogen) and sequenced. Rapid amplification of cDNA ends was used to obtain the 5′ terminus of the RNA  with primers designed from the most upstream sequence (Table S1). Sequences were edited and assembled from at least eight overlapping fragments using CLC Main Workbench version 6 . Multiple sequence alignments were performed with Clustal W [To obtain additional sequence information, sequences from the 3′ region were amplified as described previously with an adapter primer [lustal W .de novo contigs generated were 26,187, with an average coverage per nucleotide of 1,186. Any contigs greater than 300 nt were compared to virus sequences in GenBank using the BLAST algorithm, and no signature sequences of viruses or virus-like agents other than CMLV were evident. A 7,998-nt fragment homologous to CMLV was assembled from 190,571 reads, and this virus sequence was 99 % identical to the sequence obtained from primer walking.It is possible that a single agent or mixture of agents could cause PWD. In order to ascertain the presence of additional viruses in the original PWD-affected peach, total RNA from this source was subjected to deep sequencing. Multiplexed 50-bp single-end Illumina sequencing generated a total of 15,702,158 reads from this sample. The total Trichovirus . Phylogenetic relationships to members of the family Betaflexiviridae were determined by the maximum-likelihood method in MEGA5 [The complete genome of the virus isolate from the PWD-affected tree  is 8,004 nt excluding the poly (A) tract and contains four putative open reading frames (ORFs). Its genome organization is typical of members of the genus in MEGA5 . The PWDIn pairwise comparisons between CMLV-SA1162-21 and the PWD-associated strain, the putative amino acid sequence identities of the four ORFs were 88, 83, 93 and 82 %, respectively, suggesting that the virus sequences represent the same virus species [The 5′UTR of CMLV-95CI215 shows highest similarity to that of peach mosaic virus (PcMoV) (83 %) rather than CMLV-SA1162-21 (80 %). CMLV is taxonomically and serologically related to PcMoV . In contThe association of CMLV-95CI215 with PWD and the absence of additional virus signature sequences suggest that CMLV-95CI215 is the causal agent of PWD. The current study lays the groundwork for developing full-length infectious clones of the peach isolate to verify the role of CMLV-95CI215 in PWD of peach and to investigate the role ORF3 as a host range determinant.Supplementary material 1 (DOC 132 kb)Supplementary material 2 (DOC 361 kb)Supplementary material 3 (DOC 52 kb)Supplementary material 4 (DOC 123 kb)Below is the link to the electronic supplementary material.


Closteroviridae. Several factors have shaped the genetic structure and diversity of closteroviruses. (I) A strong negative selection seems to be responsible for the high genetic stability in space and time for some viruses. (2) Long distance migration, probably by human transport of infected propagative plant material, have caused that genetically similar virus isolates are found in distant geographical regions. (3) Recombination between divergent sequence variants have generated new genotypes and plays an important role for the evolution of some viruses of the family Closteroviridae. (4) Interaction between virus strains or between different viruses in mixed infections may alter accumulation of certain strains. (5) Host change or virus transmission by insect vectors induced changes in the viral population structure due to positive selection of sequence variants with higher fitness for host-virus or vector-virus interaction (adaptation) or by genetic drift due to random selection of sequence variants during the population bottleneck associated to the transmission process.RNA viruses have a great potential for genetic variation, rapid evolution and adaptation. Characterization of the genetic variation of viral populations provides relevant information on the processes involved in virus evolution and epidemiology and it is crucial for designing reliable diagnostic tools and developing efficient and durable disease control strategies. Here we performed an updated analysis of sequences available in Genbank and reviewed present knowledge on the genetic variability and evolutionary processes of viruses of the family  Whitefly-transmitted, bipartite or tripartite genome, and the CP gene is located upstream of the CPm gene. Viruses analyzed here are: Blackberry yellow vein-associated virus (BYVaV), Cucurbit yellow stunting disorder virus (CYSDV), Potato yellow vein virus (PYVV), Sweet potato chlorotic stunt virus (SPCSV), Tomato chlorosis virus (ToCV), and Tomato infectious chlorosis virus (TICV).Closterovirus. Mostly aphid-transmitted, monopartite genome, and the CP gene located downstream of the CPm gene. Viruses studied here are: Citrus tristeza virus (CTV) and Grapevine leafroll-associated virus 2 (GLRaV-2).The family ng up to  nm and fClosteroviridae by analysis of the coat protein genes using nucleotide sequences retrieved from Genbank and present an updated review on the genetic variability and evolutionary processes of the viral populations of members of the family Closteroviridae.In this work we performed an updated analysis of the genetic variation of viruses in the family Closteroviridae were retrieved from GenBank (http://www.ncbi.nlm.nih.gov and http://www.dpvweb.net). The coat protein genes (CP or CPm) were selected because they are present in all viruses and because it is the genomic region for which more sequences are available. Only those viruses with sequences of five or more different isolates were analyzed (Table Nucleotide sequences from worldwide isolates of members of the family ed Table . Multipled Table  implemened Table .The program MEGA 5.05 was used for: (1) inference of phylogenetic relationships between isolates of each viral species by the neighbor-joining method  loc33 Table . FinallyClosterovirus, the grape-infecting GLRaV-2 showed high genetic variation  origin region and likely initial dispersion area. GLRaV-11 had four genetic groups with isolates from Argentina and one group with one isolate from Greece, suggesting that Argentina could be a dispersion center for this virus. Finally, GLRaV-2 had six genetic groups with isolates from Italy distributed in five groups and isolates from USA in three groups which pointed at both countries as possible dispersion zones. Another explanation is that the locations with divergent isolates received them via importation of infected grapevine material from different areas. In most cases, the phylogenetic relationships among viral isolates were not geographically structured and often isolates from distant regions were genetically very close . The global gene flow was high for GLRaV-3, moderate for GRLaV-5 and GRLaV-2, and low for criniviruses and CTV (Table Fst = 0.000), albeit this could be due to a unique migration event given the genetic stability of this virus. SPCSV showed also a high geographical structure between the two genetic groups, but availability of only one isolate in one group precluded gene flow analysis between both groups. In group I, the comparison of three neighboring countries of East Africa showed a high gene flow between Uganda and Kenya (Fst = 0.027) but very low between them and Tanzania (Fst ≈ 0.450). In spite of its low genetic diversity, ToCV showed a low gene flow between Europe and North America (Fst = 0.560). GRLaV-2 with a moderate global flow showed a puzzling migration pattern which did not correspond to geographic proximity. Thus, extensive gene flow occurred between Poland and China and between Italy, Brazil and USA but this was very low between Italy and Poland. Finally, CTV, the best documented virus with isolates from 47 countries showed different degrees of gene flow which were not correlated with geographic distance. Some subpopulations had a high gene flow (Fst < 0.100), e.g., California, Mexico, Spain, Italy, and Portugal, or Brazil, Angola, China, and Portugal, whereas other subpopulations were almost isolated (Fst > 0.300) such as Cuba , Trinidad and Tobago, and Argentina. Several reports showed that genetically and biologically divergent isolates of CTV have been introduced one or several times in Iran, Sicily , Cyprus, and Dominican Republic  and allow interaction and/or recombination between different virus entities which can affect to pathogenicity and adaptability.Viruses of the family of Closteroviridae. It has been postulated that the ancestor of this family was a smaller filamentous virus composed of three genes encoding replication-associated proteins, the p6-like movement protein and a single coat protein , both with isolates differing in the number of genes   detected in the closterovirus CTV  also provides important information to understand viral evolution. This has been studied for GRLaV-1  in mixed infections  and increased symptom severity in mixed infections , Ipomovirus (Sweet potato mild mottle virus), Cucumovirus (Cucumber mosaic virus), and putative members of the genus Carlavirus (Sweet potato chlorotic fleck virus and C-6 virus) in double and triple infections, which was associated to an increase in the severity of symptoms. In some cases SPCSV titer was reduced indicating an antagonistic interaction  in tomato plants susceptible to both viruses resulted in rapid death of the plants, with a pronounced enhancement of ToCV accumulation, whereas TSWV accumulation was not altered. However, in tomato cultivars carrying the Sw-5 gene that confers resistance to TSWV, preinfection with ToCV resulted in TSWV resistance breakage, a phenomenon not observed when plants were simultaneously co-inoculated with both viruses. This suggested that a threshold level or a time lapse is needed for ToCV to interfere or downregulate the defense response in the TSWV-resistant plants  of one or both variants in comparison with single infections. This effect seems to be due to the supression of a host defense mechanism, e.g., gene silencing, by one of the viruses that inhibits accumulation of the other virus in single infections . This phenomenon has been exploited as a disease control strategy named cross protection consisting of preinoculation of the plant with a mild isolate to protect it against damage caused by infection with a virulent isolate. This interaction is most common between genetically related viruses and it has been hypothesized that it might be caused by competition for host resources or because previous infection would trigger the gene silencing antiviral defence of the plant that would impair infection by the second virus  likely reflects vector adaptation as a driving force in the diversification in this family (Karasev, Bemisia tabaci in comparison with Trialeurodes vaporariorum (Berdiales et al., B. tabaci by the biotype B, a very poor vector of LIYV (Cohen et al., T. abutilonea (Wintermantel, Aphis gossypii Glover was very low but this increased in 1960–1970 in Israel and California. (Bar-Joseph and Loebenstein, Toxoptera citricida in comparison with the mild components (Brlansky et al., Finally, interactions with the vector can also have an important effect in viral evolution. The association between phylogenetic relationship among members of the three genera of the Karasev, . In crinKarasev, , the speClosteroviridae. Analysis of the genetic variability and population structure shows a limited genetic variation as in other plant viruses. This seems to occur mainly by a strong negative selection, indicated by the low number of non-synonymous substutions with respect to the synonymous substitutions in the three genera of this family. Some viruses, had isolate groups with a great genetic divergence between groups but each group being very homogeneous. This suggests that their sequence space is restricted to a few sharp adaptive peaks and that covariation between different nucleotide positions occurs, as suggested by decreased fitness of chimeras of different CTV strains. Long distance movement or gene flow may have contributed in some cases to this low genetic variation.The increased number of nucleotide sequences and the availability of more sophisticated analytical tools allows a better understanding of the evolution, population genetics and epidemiology of the viruses in the family Crinivirus produce increased symptom expression and accumulation of viruses from other families in mixed infections (Karyeija et al., Although most viral isolates were composed of one major variant and a population of genetically related variants forming a quasispecies structure, some isolates had divergent variants originated from mixed infections of different strains which can affect symptom expression. Interaction between different viruses or variants from the same virus has been found in some cases. The antagonism between close variants of the same virus is the base for cross protection to control damage from severe CTV strains (Moreno et al., Changes in the population structure caused by vector transmission or host change, either by selection of viruses or genetic variants of the same virus or by genetic drift, could explain the appearance of virulent CTV isolates, or the emergence of some viruses in new areas, e.g., criniviruses, associated to their whitefly vector spread.Closterovirus and Ampelovirus, which have played an important role in their evolution by increasing genetic diversity and adaptability.Recombination between divergent genotypes have been described in the genera The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pogostemon cablin (Blanco) Benth]. The diagnostic tools used were host range, transmission studies, cytopathology, electron microscopy, serology and partial coat protein (CP) gene sequencing. Evidence from biological, serological and sequence data suggested that the causal virus belonged to genus Potyvirus, family Potyviridae. The isolate, designated as Patchouli Yellow Mosaic Virus (PaYMV), was transmitted through grafting, sap and the insect Myzus persicae (Sulz.). Flexuous rod shaped particles with a mean length of 800 nm were consistently observed in leaf-dip preparations from natural as well as alternate hosts, and in purified preparation. Cytoplasmic cylindrical inclusions, pinwheels and laminar aggregates were observed in ultra-thin sections of infected patchouli leaves. The purified capsid protein has a relative mass of 43 kDa. Polyclonal antibodies were raised in rabbits against the coat protein separated on SDS – PAGE; which were used in ELISA and western blotting. Using specific antibodies in ELISA, PaYMV was frequently detected at patchouli plantations at Lucknow and Bengaluru. Potyvirus-specific degenerate primer pair (U335 and D335) had consistently amplified partial CP gene from crude preparations of infected tissues by reverse transcription polymerase chain reaction (RT-PCR). Comparison of the PCR product sequence (290 bp) with the corresponding regions of established potyviruses showed 78–82% and 91–95% sequence similarity at the nucleotide and amino acid levels, respectively. The results clearly established that the virus under study has close homology with watermelon mosaic virus (WMV) in the coat protein region and therefore could share a common ancestor family. Further studies are required to authenticate the identity of PaYMV as a distinct virus or as an isolate of WMV.Here we have identified and characterized a devastating virus capable of inducing yellow mosaic on the leaves of Patchouli [ Pogostemon cablin (Blanco) Benth]; (Synonym Pogostemon patchouli Pellet.Vat. suavis Hook)], family Labiatae from tropical Asia is cultivated in Malaysia and Indonesia. It is now extensively cultivated in China, Indonesia, India, Malaysia, Mauritius, Taiwan, the Philippines, Thailand, and Vietnam. In India, about 30 species of patchouli have been reported. Following species of patchouli; Pogostemon cablin, P. commosum, P. hortensis, P. heyneasus and P. plectranthoides are cultivated for patchouli oil. It is an herbaceous perennial plant and the oil glands are present on its leaves. Patchouli oil is a viscous liquid, yellowish green to brown in colour and has powerful and persistent fragrance. Patchouli resinoid extracted from the leaves is used in soaps and perfumes. Patchouli oil is also used in cosmetics, tobacco and incense products. The therapeutic properties of patchouli include: anti-depressant, anti-inflammatory, antiseptic, aphrodisiac, astringent, carminative, diuretic, febrifuge, fungicide, insecticide, sedative and tonic. It also possesses many antifungal and bacteriostatic properties Patchouli , South African passiflora virus NIb and CP genes and Hardenbergia mosaic virus isolate Sb-6 polyprotein gene, Hardenbergia mosaic virus isolate Sb-15 polyprotein gene, Peanut stripe virus and Patchouli yellow mosaic virus cp gene (PaYMV_CP) through multiple sequence alignment analysis. Result of sequence alignment showed that conservation of nucleotides was much higher in Bean common mosaic virus strain NL-4 coat protein gene, and Watermelon mosaic virus isolate Ard. Me coat protein gene than Cowpea aphid-borne mosaic virus and Passion fruit woodiness virus isolate SP-1 coat protein gene  PStVPaYMV, which could be useful in ELISA, Ouchterlony double diffusion and western-blot tests. The study reported here suggests that PaYMV could be a distinct potyvirus or an isolate of WMV infecting Patchouli. Complete sequencing of the CP gene would be important to define the distinctness of the virus. Using this disease indexing tools, PaYMV can frequently be detected in patchouli (Pogestemon cablin) plants collected from different parts of India or other part of the world.Earlier preliminary work has been done on viral diseases of patchouli except in three reports; Test plants: Seedlings of test plants used in host range studies were raised in shallow pots of 50 cm and 30 cm in size and transplanted into earthen pots of 30 cm and 20 cm depending upon the growth rate of the test plants. Healthy seedlings were maintained in an insect proof glass house at the temperature ranging from 25°C to 35°C.Chenopodium quinoa Wild. and maintained on Nicotiana benthamiana Domin. Inoculum was prepared by grinding the infected young leaves in 0.1 M Phosphate buffer pH 7 with 0.1% β-mercaptoethanol. Periodic checks were made on diagnostic host C. quinoa Wild. to ensure the biological purity of the virus. The infected five patchouli stocks were grafted to the healthy scions to check the transmission of the causal agent by grafting. In another experiment, Aphids (Myzus persicae Sulz.) were used for the virus transmission. Virus free aphids were fed on PaYMV infected N. benthamiana Domin. plants for 24 hours. Reverse transcription-polymerase chain reaction (RT-PCR) has been performed using potyvirus specific degenerate primers as has been described to detect PaYMV in aphids before the probing and right after the probing according to the method used earlier N. benthamiana. Twenty-four hours later, aphids were killed by spraying 0.1% Demecron in one set. In another set of the plants, aphids were transferred to another set of healthy plants. This process was repeated for 10 days to test for non-persistent or persistent mode of virus transmission.Culture of the virus isolate was initiated by a single lesion from C. amaranticolor Cost & Reyn or C. quinoa Wild.For host range studies many plants from diverse families were inoculated with the virus isolate. Sap prepared from leaves in 0.01 M Sodium phosphate buffer, pH7, was rubbed onto leaves pre-dusted with carborundum powder (600 mesh). The Leaves were then rinsed with distilled water, and plants were maintained in an insect proof glass- house for observations. Symptoms on both inoculated and upper, uninoculated leaves were recorded. Tests for latent infection were conducted by back–inoculation to either N. benthamiana and adsorbed on 400 mesh copper grid of pre-coated with carbon, which were negatively stained by 2% Uranyle Acetate. Similarly purified samples were also processed and examined under transmission electron microscope (JEOL 100 s).Leaf- dip preparation was made by crushing the infected leaves patchouli and 2 were vacuum infiltrated with chilled 2.5% gluteraldehyde in 0.01 M Cacodylate buffer, pH 7.2 (v/v). After fixation in cold (about 2°C) for 1 h; 1 mm strips were cut avoiding tissue bordering on previously cut edges. The strips were blotted and washed in 0.01 M cacodylate buffer (pH 7.2). After blotting these pieces were transferred to cold 1% osmium tetraoxide (prepared in cacodylate buffer) till they become a Z-black. Thereafter leaf strips were processed for dehydration after thorough washing in distilled water in 30, 50, and 70% alcohol series for 30 minutes each at room temp. Dehydration was carried out for 15 min. at 90% alcohol and for 2×15 minute in 100% alcohol. A treatment of propylene oxide was given for 15 minutes to the dehydrated leaf strips. Infiltration was started taking a mixture of araldite and propylene oxide in 1∶1 ratio and thereafter into pure araldite. Araldite mixture was used as; Araldite-6005-5 g, DDSA –4.35 g.Young and vigorously growing leaves of patchouli plants with typical yellow mosaic symptoms were harvested when highly infectious and comparable leaves from healthy plant were also sampled. Pieces of leaf 1 cmThe resin was prepared as reported Sections were cut with LKB ultra-tome Nova using glass knives. Only silvery sections were collected on 400 mesh grids.Sections were stained with saturated aqueous uranyl acetate at room temperature for 6 hours and then rinsed in triple distilled water. All the sections further stained by 2% lead citrate N. benthamiana at about 15–20 days after inoculation and grinded in 20 mM HEPES buffer (1∶3 wt/vol.), pH 7.2, containing 1 M Urea, 1% Triton X-100, 10 mM Di-ethyl Dithiocarbamate, and 0.1% β-mercaptoethanol. The extract was passed through cheesecloth and the filtrate was clarified with ice - cold 10% chloroform. The virus was precipitated by adding 6% polyethylene glycol 6000 and 0.2 M Nacl. The precipitate was suspended in resuspension buffer (0.02 M HEPES pH 7.2 containing 0.5 M urea and 0.02 M sodium sulfite), pelleted at 120,000 g for 3 h at 4°C in Sorvall ultracentrifuge Pro-80. The pellets were resuspended in resuspension buffer (20 mM HEPES pH 7.2 containing 0.5 M Urea and 0.02 M Sodium Sulfite) and loaded onto 10 to 40% preformed sucrose density gradient prepared in the same buffer and subjected to centrifugation at 92,500 g for 2.5 h in a SW28 rotor. A light scattering zone which appeared at about 1.4 cm from the bottom of the tube was collected and subjected to centrifugation at 120,000×g for 3 h. The final pellet was resuspended in a minimum volume of resuspension buffer and absorbance was taken in 260 and 280 nm. The purified virus preparation was observed under JEOL 100 s transmission electron microscope.Young leaves with typical symptoms were harvested from the infected Antiserum was produced in rabbits against the coat protein of PaYMV separated by SDS gel electrophoresis, by giving a series of intradermal injections. 500 µg of the virus was used in complete adjuvant for the first injection and 250 µg of the virus in incomplete adjuvant for three subsequent injections at 1 week intervals. The presence of PaYMV specific antibodies in the serum was tested by direct antigen coating – ELISA (DAC- ELISA) and western blotting, which confirmed the presence of respective antibodies in serum.PaYMV was used as antigens which were pre-coated with100 ng/well in Polystyrene microtitre plates. Pepper vein banding virus (PVBV), which was a well characterized potyvirus specific polyclonal antibodies PVBV was used in serial double dilutions of 1∶100, 1∶1000, 1∶2000, and 1∶4000 up to 1∶164000. The binding of antibodies in the serum was assayed with goat anti-rabbit immunoglobulin (Ig) conjugated to alkaline phosphatase (sigma), the absorbance were read at 405 nm in a Bio- Tek auto reader EL311.The virus was initially identified serologically by direct antigen coating  using a standard protocol N. benthamiana leaves sap inoculated with the virus and M. persicae Sulz. (just before the acquisition feeding and right after the acquisition feeding) using TRIzol® reagent (Invitrogen #15596-018 USA) according to the manufacturer’s instructions. After precipitating with ethanol total RNA was resolubilized in RNase free water. Reverse transcription reaction for cDNA synthesis was carried out in 20 µl reaction mixture using Revert Aid™ H Minus, Fermentas kit using Oligo dT primer according to the manufacturer’s protocols and reagents. cDNA samples were tested for the presence of PaYMV using potyvirus specific degenerate primers designed to amplify the coat protein gene. Forward primer (U335) 5′- GAATTCATGRTNTGGTGYATHGANAAYGG 3′ and reverse primer (D335) 5′-GAGCTCGCNGYYTTCATYTGNRHDWKNGC 3′ Total RNA was extracted from test plant 5′-CGACTCACTATAGGGAG AGCGGC-3′ using the Sanger’s dideoxy chain termination method The amplified fragments were cloned in pJET 1.2/blunt Cloning Vector using the PCR Cloning Kit,  and transformed into XLI blue competent cells. Clones containing the PCR product of the expected size (based on primer locations) were identified by digestion with restriction enzyme –Bgl II and colony PCR (data not presented). This restriction site was present at both corners of blunt ends where insert was ligated and visualized by agarose gel electrophoresis and isolated by modified alkaline lysis method www.ncbi nlm.nih.gov/) Closely related homologs were identified by pairwise sequence alignment studies. The partial coat protein gene sequences of Patchouli virus were compared with non-redundant database sequences of nucleotides and proteins by using Basic Local Alignment Search Tool (BLAST) program viz., ClustalW v2.1  After BLAST analysis, a data set of potential homologs was prepared by considering those database sequences which had >98% sequence identity to the query sequence. Phylogenetic analysis and nucleotide/protein conservation of the data set sequences were studied through multiple sequence alignment program


To the Editor: Rabies is a fatal encephalitis caused by a neurotropic RNA virus of the family Rhabdoviridae, genus Lyssavirus. The predominant rabies virus reservoir hosts are bats and carnivores. Among these, rabid dogs represent a substantial public health problem, particularly in developing countries , paralysis of superior and inferior left limbs, shaking, and hallucinations. On May 4, 16 days after clinical manifestations began, the patient died; the cause of death was registered as a cerebral vascular accident. One month later, the body was exhumed to obtain a sample from the central nervous system (CNS), which was sent to Instituto Pasteur, São Paulo, registered as sample 5341 M/04 and tested by FAT, MIT, and RT-PCR.In total, 8 smears were prepared from the sample to be analyzed by FAT according to the method of Dean et al. (Fluorescent inclusions were observed in 6 of the 8 slides prepared for the FAT. The RT-PCR of the RNA sample resulted in amplicons of the correct size (249 bp), as did the positive control sample, CVS strain rabies virus. No bands were observed in the reaction corresponding to the negative/reagent control (ultra-pure water). The MIT results were negative. Because the virus could not be isolated, antigenic typing with monoclonal antibodies could not be performed.http://www.ebi.ac.uk/clustalw) and Bioedit software . The phylogenetic tree was produced by using the neighbor-joining DNA-distance method and the Kimura 2-parameter model with 1,000 bootstrap replicates in Mega 2.1 (version 2.1) (available from http://www.megasoftware.net/). The sequence was segregated in the variant 3 cluster (Desmodus rotundus–related variants), which suggests that D. rotundus is the most probable source of infection  in a MegaBACE DNA sequencer (Amersham Biosciences) and resulted in a 165-nucleotide sequence. The final sequence was aligned with homologous sequences from GenBank by using the ClustalW . Rabies should be suspected when early clinical symptoms, for example, itching and paresthesia, are demonstrated at the local site of infection. In addition, the laboratory investigation showed that molecular methods such as RT-PCR and sequencing were sensitive assays for nucleic acid detection and determination of the rabies virus variant in this unusual case from an exhumed human.