In his seminal paper, Gabrielsson  distingu The distinction between emotion felt by a listener  and emotion expressed by a piece of music  has become a firmly established part of research agenda of music psychologists in the last decade. Since the seminal work of Gabrielsson  we have The inclusion criteria for the research tabulated for the review are as follows: (1) studies which made a direct comparison between external locus emotion and internal locus emotion in connection with music listening; (2) studies which use the same response regime for both external locus and internal responses; and (3) studies appearing in peer-reviewed journals in the 10-year period of 2003–2012 . Nineteen studies met all three criteria. The reason for excluding studies that have some connection with emotion locus and music are now explained.a priori label for the emotion expressed by the stimulus), as will be discussed below. Such studies were not included in the tabulated literature of this review.When discussion of locus is presented in the research literature on emotion in music, it is most frequently an acknowledgment that a locus distinction exists, but that the study limits the investigation to one locus or the other , without comparing both. Sometimes the data in each locus are not directly comparable , or comparison of physiological measures with one locus or the other  vs. expresses emotion (the cognitivist perspective). For example, Scruton . Outside such circumstances, the more purely philosophical research will be used to inform the reviewed material, rather than be part of it. Hence, a limitation of the present review will be to examine exclusively music-psychology literature  are gathered and compared. It should be noted that music psychology has been influenced by ideas about emotion locus that were primarily in the realm of philosophy and aesthetics. It is those scholars who introduced terms of emotivism and cognitivism, often quoted in the music psychology literature  in the study of emotion in music. Although this internal locus distinction is not covered in the empirical data of the literature reviewed, it will be relevant in future research , are also not covered in this review, again because empirical data investigating these distinctions are rare and mostly concerned with semantics. Instead, in this review, the terminology used to discriminate external and internal locus (rather than its semantic/linguistic utility) is reported , with the exception of one study that is included , performer(s) or other (perhaps imaginary) listener(s) are thought to be experiencing (according to the perceiver), and whether a further distinction between these other people and the music should be made when considering external locus. For example, some recent research has considered emotion ratings that others would make about a piece of music as a way of managing possible bias in external locus response. That is, instead of being asked what emotion they believe the music expresses, a participant is asked, “How would normal people feel when listening to this musical stimulus?”  accounts by music psychologists demonstrating an awareness of the distinction between felt and expressed emotion are frequent  different from the one you happen to be in? orDoes it merely intensify already existing moods?Do you merely recognize, without participating, that music represents varieties of human emotion and mood? (p. 202)From a historical point of view, and from a theory-building perspective, it is worth dwelling for a moment on the pioneering study by Lee . A questrepresents varieties of human emotion and mood?” was the real subject under examination and ought therefore to have been put first and foremost. Instead of that, and from a mistaken hope of additional clearness, it was put after the queries (intended to be supplementary to it) whether the Answerer's already existing mood could be altered or whether that mood was merely intensified by the emotion which was not merely recognized as characterizing the music, but actually participated in when hearing that music. As a result of this strategical blunder, the majority of Answerers did not notice the main question of Participation versus Recognition. What I wanted to know was whether the Answerer merely recognised that a given piece of music had  a given emotional character, e.g., was sad or cheerful, or whether hearing that piece of music made him feel sad or cheerful when he had not been so before? This fairly simple query, clearly worded as “do you merely recognize without participation that music Lee's openness and regret are explicit as she acknowledges:“Music generally substitutes new moods and emotions: if the emotion is tragic or tender, it seems that my mood becomes tragic or tender.” (This is recognition producing a sort of sympathetic imitation.)[…]“It is not that the music expressed one's own feelings, but that the feelings or mood which the music expresses awaken these very feelings in oneself. Music never intensifies existing feelings, it either awakens feelings which I haven't got or merely represents them.” Nevertheless, Lee identified participants who explicitly reported the link between felt and expressed emotion; for example, two participants, Bob and Lewis, respectively, wrote:Hence, Lee's work appears to be the first explicit attempt in English language music psychology research to collect empirical data on the distinction between locus of emotion, a pioneering effort that has received regrettably little attention in subsequent research. Her explanation of “sympathetic imitation” will be highly relevant in the theory development of this review. Aside from the data and analysis reported by Lee, it was not until 2002 when Gabrielsson acknowledged in explicit terms the possible relationships between loci . He then presented a detailed argument explaining that participants can only logically make distinctions between felt and perceived emotion in music through verbal report, as distinct from, say, physiological measurement.Gabrielsson's  publicatrelationships between the emotion loci.Gabrielsson discusses some of the reasons for the absence of direct emotion locus comparisons in music perception. For example, he recounts the belief that there were different listener types, some who focused on the music, and others who focused on their own feelings when listening to music . Music was seen by many researchers and lay people as “an object for perception and reflection” and the emotional response as a listener's reaction. As a result, Gabrielsson concluded that the distinction between emotion loci in music “is not always clearly observed, neither in everyday conversation about emotions, nor in scientific papers” , or it could be “negative” . Furthermore, felt and expressed emotions could exhibit “no systematic relationship” , or have no relationship at all . Gabrielsson traces back the presence of positive relationships to the ancient Greeks where music was thought to be able to directly affect the listener, in a manner similar to more contemporary research on mood management and regulation theory , or a sad piece of music makes the listener happy because it is relaxing, cathartic and pleasurable  emotions as identified by Scherer and Zentner  that musIn the years following Gabrielsson's influential paper, research on explicitly collected self-report felt and expressed emotion in music grew, and 16 peer-reviewed publications that met the inclusion criteria were located. Some of the publications reported more than one study that was concerned with emotion locus and music, and two reported locus data from previously published sources , and second, evaluate the emotional quality of the music regardless of the experiences it aroused in them .  is absent in all the contemporary literature reviewed, although Vieillard et al.  use the Other variants were expressed-felt Dibben, ; perceivlistener's perspective, and when the listener is the subject of the sentence, she or he feels an emotion, but perceives it (rather than expresses it) in the music—“express” does not designate external locus when the listener is the subject of the sentence (“the listener expresses happiness”). When the music is the subject of the sentence, expressed emotion is the external locus designation .In summary, no single pair of designations for each locus level was used consistently across the studies. This reflects a richness in the descriptions, subtle distinctions, but also lack of systematic classification. The recommendation of this review is to label two levels of the variable as deemed appropriate in each study: felt, induced , evoked, internal locus vs. expressed, portrayed, “in the music,” and external locus. “Perceived” is frequently used to describe external locus, however, the term could be confused because a participant can perceive many things, including their own feelings in some cases Konečni, . ContextUsing the term “locus” to describe the variable frees up labels that are commonly used to describe other variables, such as type and mode. “Perspective” is another possible term that could be used to describe the variable but was not cited with any regularity in the tabulated literature.transmission of information from one source to another, it may lead to some ambiguity .Table All included experimental studies in this review had over 25 participants in each experiment, with the Konečni et al.  study haThe bulk of music used in the studies tabulated comes from the common practice period (CPP) of Western art music, which is sometimes referred to as “classical” music. Twelve publications using experimenter-selected pieces used exclusively such music. Classical music, and in particular music from the romantic art music period, is considered particularly good for expressing and evoking emotions (Romantic(ism), Since studies by Panksepp , Blood aSome research deliberately selected music that is unfamiliar. In the two experiments investigating emotion locus by Dibben , she verTwo publications reviewed included no explicit musical stimuli  in case one rating influences the rating of the other locus? Completing the two rating items in immediate sequence has the advantage of requiring only a single pass of the music stimulus to obtain both ratings, but has a drawback if the participant (consciously or otherwise) responds to the second rating item under the influence of the first, through what is known as a “contrast” effect , leading Schubert to conclude that responding to both loci in sequence at the same time had efficiency , perhaps compensating for the possible disadvantages. The recommendation of this review, then, is that it is more efficient to collect both locus ratings together, but to counterbalance the order of loci questions, as did Konečni et al.  were happy (including happiness and very-happy) and sad (including sadness and very sad), both used in at leastTypically, the rating items were combined and manipulated to create dependent variables for statistical analysis and hypothesis testing. This transformation process is summarized in the Measure column at “DV” for each of the relevant tabulated studies in Supplementary Table When making a response via a rating item, participants are given a number of graded steps (points) along a continuum from which they are to make a single selection. The range of the available steps per rating item across the tabulated studies was from 4 to 13, with 5-point rating items used most frequent . Researchers have had to balance the coarseness of completing rating items with fewer points with the greater difficulty for the participant in making a selection when a larger number of steps are offered Alwin, . While tKonečni et al. , row 9 dIn the experimental studies, response items were most frequently presented in a unipolar format , whereas bipolar labels were used for rating items in six studies . Dependent variables were generated from combinations of the rated items in 9 studies, of which 4 were unipolar  and 4 were bipolar , with one study generating an angular variable from the combination of arousal and valence ratings (row 8). Some researchers converted unipolar item ratings into bipolar dependent variables, such as Ilie and Thompson (row 18), who took differences between pairs of unipolar item ratings  to generate the dependent variable score . That study is also interesting from the point of view that it is one of the few to apply a more contemporary model of emotion dimensions to the data, with energy-arousal and tension-arousal scores generated, instead of the more common “arousal” dimension alone  are routine design matters in investigations of emotion locus. The increase in experimental efficiency of collecting a single bipolar rating item response (instead of two unipolar rating item responses) weighed against the methodological challenges of using bipolar ratings is an issue that has not been systematically addressed in the emotion locus literature but may have important ramifications.The use of bipolar vs. unipolar rating items presents some interesting challenges Yorke, . A bipolsmall number of cases had internal locus rated higher [χ2N = 178): this is shown as a fraction in the first entry in the Main Findings column of Supplementary Table n = 27) rated external locus responses in one experiment  in a separate experiment rated internal locus emotions . As shown in Figure While the tabulated literature did not allow a complete, direct statistical comparison of the relative mean magnitude of felt and expressed locus responses, a crude comparison using error bar overlapping was conducted, shown in the magnitude column of Supplementary Table In one of the two studies where mean internal locus was rated as higher than mean external locus, it was valence that exhibited a main effect, with mean felt emotions rated as above zero, and mean expressed emotions as below zero . In the Salimpoor et al. study, the method of stimulus selection mattered, with participant-selected music producing more equal ratings across loci  than experimenter-selected ratings , musical features and, as already described above, the putative emotional connotation expected in the music—for example, when the music is expected to represent negative emotion. Some studies reported relationships between interacting variables and the gap across emotion loci . With regard to personality differences, participants exhibiting high scores in trait neuroticism-anxiety and on a scale measuring the behavioral inhibition system  responded statistically with a larger GAEL score than did those scoring low in neurotic-anxiety and BIS , transforming those data into dependent variables, and the nature of the musical stimuli.and when these data are gathered via rating items Several of the reviewed studies  the listener, then we can explain inhibition as a plausible explanation for reduced felt ratings, and the inhibition may be a product of social context. Evidence from a study where internal locus emotions were rated in response to various emotion expressing film excerpts  were significant at p = 0.05, with felt emotions demonstrating larger variance than expressed emotions, with three of these being in response to one piece, “Jupiter” from The Planets by Holst . One out of 20 may have been significant by chance alone, and so the study concluded that expressed emotions are overall more stable than felt. It suggests that when all things are equal, internal locus equals external locus plus noise, a claim that requires more research .A further complication of the locus relationship debate is the variability in responses to either locus. If the properties of the artwork—in this case, the relationships among musical features over time—are consistent upon repeated exposures, then it may seem logical to assume that the emotion expressed by that stimulus is also stable, and it is the internal locus that might be more variable, depending, for example, on the mood of the listener/perceiver, as Gabrielsson points out in the “zero correlation” case of his “no-systematic relation”  but then had a negative affect evaluation of embarrassment. The Van Zijl and Sloboda study  response meant that the individual was not in a dissociated state and could, as a result, not (or no longer) enjoy the music that, in another context, he or she may have liked very much . Recent classifications of descriptive adjectives have started to separate groups of terms in ways compatible with dissociation theory. For example, Juslin and Laukka  emotions. By revising the way affect qualities are conceptualized, these may be understood as unique to induction (rather than expression) because they are affect valences . Being moved might be a result of feeling sad, or happy , or some other experience(s) which led to the affective response of being moved.The affect/emotion valence distinction is explained from the cognitive theoretical standpoint of dissociation theory, where when listening to music we are usually in a state where we “switch off” pain circuitsd Laukka  propose emotion valence of sadness, but a positive affect valence of pleasure. There is no need to view sadness and pleasure as conflicting. From a theoretical stance, the pleasure indicates that the listener is in a dissociated state, meaning that negative valence affects are inhibited, and so all emotions (negative and positive) can be enjoyed. Zentner et al. (My point is that by differentiating (dissociating) between emotion valence and affect valence, affect/emotion blends can be more simply understood than the otherwise complex responses we appear to have to music. “The music makes me sad, and that gives me pleasure” suggests a negative r et al.  present r et al. . Dissocir et al. .Gabrielsson's categories of emotion locus relationships have provided an important framework for encouraging direct engagement with and awareness of the question of emotion locus in music. The current review suggests that the categories may be reworked and calibrated to reflect the current distinctions between loci of which the research community is now aware. Table If we eliminate the no-systematic relationship category in those cases when it is due to instability of responses over time, or those that do not concern emotional relationships between expressed and felt emotions on any occasions , we end up with two broad categories: matched vs. unmatched relationships. The omission of the non-systematic and no relationships are justified by two findings (1) when no emotion is reported in both the felt and expressed loci, the categorization becomes irrelevant  than expressed emotions. According to the inhibited contagion account, these situations should not occur, but they do . Furthermore, is inhibited-contagion a satisfactory explanation for unmatched emotions across loci when the emotions are discrete ? And does the related idea of decoding theory mean that internal locus will have more variability than external locus? Such a view needs to account for the question of whether the judgment of an observed object (or piece of music) is really sensed in a fixed, stable way by the perceiver . This theory allows researchers to understand that different, apparently conflicting feelings can be experienced at the same time, without having to dogmatically attribute them to one or the other locus of emotion . Contagion and dissociation principles may work hand in hand in the perception of music: under normal, real-life, day-to-day circumstances it might be undesirable and even dangerous to acquire emotion solely through contagion transfer  of contagion circuits. Contagion circuits are the same or related to mirror circuits . More consistency in the labeling used to describe the variable in question is urged. In this review, the term “locus of emotion” or “emotion locus” has been adopted.The wide range of task wordings may be in need of some standardization, with the name of the locus variable producing little agreement across studies and therefore unnecessary confusion . While the wording of the levels within the locus variable can be quite flexible, Konečni  demonstrThe present review calls for a firmer theoretical stance to help direct future research. Interestingly, one of the rarely cited, early writers on the locus relationship had a premonition of a useful theoretical framework for understanding emotion locus in music, with Vernon Lee's idea of “sympathetic imitation,” which in contemporary literature resembles emotional contagion—the dominant theoretical framework that provides a basis for explaining a large portion of the results of the literature investigated in this review.Finally, based on an examination of the literature published in the decade after Gabrielsson's seminal work, some rearrangements and suggestions have been made that may assist future researchers investigating relationships between emotion expressed by music and emotion felt by the listener in response to music. The data examined and newly arising findings were used to rearrange Gabrielsson's categorization. Reflecting the growing interest in explaining why emotion ratings are different across locus in certain situations, the simplifying nomenclature of “matched” vs. “unmatched” emotion pairs across loci were used in this review as the basis of formulating locus relationships. There is much work to be done in understanding locus relationships, and this review, if successful, should soon become an interim report on the state of the art in the locus of emotion in music.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The PR-mediated proteolytic cleavage of Pr55gag and Pr160gag-pol, known as virus maturation, is essential for the acquisition of viral infectivity. Within the Gag-Pol, the p6gag is truncated and is replaced by a transframe domain referred to as p6* or p6pol. Removal of p6pol improves Gag-Pol autoprocessing, suggesting that p6pol is involved in regulation of PR activation [HIV-1 protease (PR) is encoded by tivation . Howeverenv-deleted HIV-1 proviral vector. Each of the constructs was transiently expressed in 293T cells, and virus assembly and processing were analyzed by Western blot. Virus infectivity was determined by a single-cycle infection assay.PCR-amplified p6pol-PR or PR fragments were inserted at the PR C-terminus of an HIV-1 mutants containing tandem repeat PR domains were severely defective in virus particle production due to enhanced Gag cleavage. Inactivation of the proximal PR affects Gag cleavage efficiency at a greater extent than inactivation of the distal PR. Placement of p6pol between the tandem repeat PR domains resulted in diminished Gag cleavage efficiency.Our study indicates that the Gag cleavage enhancement effect incurred by over-expressed HIV-1 PR is reduced following the placement of p6pol between the tandem repeat PR domains. This supports the proposal that p6pol plays a negative role in the process of PR activation.


Ustilaginoidea virens is the causal agent of a serious disease of rice. Here, we report the presence of five dsRNA bands ranging from about 1.2, 1.5, 1.7, and 1.8 to 5.6 kb in an isolate of this fungus from China and the complete sequence of the largest dsRNA segment, putatively representing the genome of a novel virus, designated as Ustilaginoidea virens RNA virus 1 (UvRV1), UvRV1, which has a genome length of 5567 bp and has two consecutive open reading frames (ORFs) with a five-nucleotide overlap. Phylogenetic analysis showed that UvRV1 belongs to the genus of Victorivirus in the family Totiviridae.The online version of this article (doi:10.1007/s00705-013-1615-9) contains supplementary material, which is available to authorized users. Chytridiomycota, Zygomycota, Ascomycota, and Basidiomycota. Mycoviruses are classified into 11 families, and there are many that have not yet been assigned to a family [Totiviridae, Partitiviridae, Chrysoviridae, Reoviridae, and Megabirnaviridae, have double-stranded (ds) RNA genomes encapsidated in rigid virus particles. The type of dsRNA segments observed in fungal isolates can be quite diverse, even in the same fungal species [Mycoviruses with double-stranded RNA (dsRNA) genomes have been described in a wide range of fungi covering all four phyla of the true fungi:  species . This di species .U. virens (teleomorph: Villosiclava virens) [Rice false smut is a worldwide fungal disease, caused by  virens) –6. The d virens) –11. A ch virens) . Yield l virens) .U. virens. In this study, we report the presence of dsRNAs in a sample of U. virens isolated from rice in China.At present, there is no record of a double-stranded RNA virus infecting Ustilaginoidea virens strain JYH-ZT, used in this study, was originally isolated from rice cultivated in Hubei, China. This strain was maintained on potato sucrose agar (PSA) plates. For extraction and purification of dsRNAs, mycelia were grown on potato sucrose broth (PSB) with shaking (150 rpm) at 28 °C. dsRNA was extracted from fungal mycelia using CF-11 cellulose  column chromatography as described previously [http://www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?link_loc=BlastHomeLink), and the program PhyML 3.0 [U. virens strain JYH-ZT were termed dsRNA2 (1.8 kb), dsRNA3 (1.7 kb), dsRNA4 (1.5 kb), and dsRNA5 (1.2 kb), respectively, according to their sizes . The amino acid sequence deduced from ORF1 of dsRNA1 showed a high level of similarity to those of the capsid proteins (CP) of viruses of the family Totiviridae, particularly to that of Beauveria bassiana RNA virus 1 . The C-terminus of this putative CP had an Ala/Gly/Pro-rich region, which occurs in mycoviruses of the genus Victorivirus [Trichomonas vaginalis virus 2 (TVV2) [T. vaginalis virus 1 (TVV1) [The complete sequence of dsRNA1 was 5567 bp and had a GC content of 57 %. Sequence analysis showed that dsRNA1 had two consecutive open reading frames (ORFs) Fig. . ORF1 has slow-1  and Beauorivirus . The pro2 (TVV2) , and simVictorivirus . These conserved motifs were constructed using the COBALT web server . Abbreviations and GenBank accession numbers are as follows: Helminthosporium victoriae virus 190S ; Sphaeropsis sapinea RNA virus 1 ; Magnaporthe oryzae virus 1 ; Saccharomyces cerevisiae virus L-A (L1) ; Ustilaginoidea virens RNA virus 1 (UvRV1). (PDF 44 kb)Supplementary material 1: The eight conserved motifs of the sequences of RdRps of Below is the link to the electronic supplementary material.


Sorbus aucuparia L.), fig mosaic, rose rosette, raspberry leaf blotch, pigeonpea sterility mosaic (Cajanus cajan) and High Plains disease of maize and wheat were found to be associated with viruses that share several characteristics. They all have single-stranded multipartite RNA genomes of negative orientation. In some cases, double membrane-bound virus-like particles of 80 to 200 nm in diameter were found in infected tissue. Furthermore, at least five of these viruses were shown to be vectored by eriophyid mites. Sequences of European mountain ash ringspot-associated virus (EMARaV), Fig mosaic virus (FMV), rose rosette virus (RRV), raspberry leaf blotch virus (RLBV), pigeonpea sterility mosaic virus and High Plains virus strongly support their potential phylogenetic relationship. Therefore, after characterization of EMARaV, the novel genus Emaravirus was established, and FMV was the second virus species assigned to this genus. The recently sequenced RRV and RLBV are supposed to be additional members of this new group of plant RNA viruses.Ringspot symptoms in European mountain ash ( Sorbus aucuparia L.), fig mosaic disease (FMD), rose rosette disease (RRD), High Plains disease (HPD) of wheat and maize, pigeonpea sterility mosaic disease (PPSMD) and raspberry leaf blotch disorder (RLBD) share similarities with respect to their genomic organization and some of their biological properties. They all have segmented genomes consisting of four or more negative sense RNAs, their putative virions are double membrane-bound particles, and most of the diseases were shown to be transmitted by eriophyid mites. After elucidation of the genome sequence of the European mountain ash ringspot associated virus EMARaV [Emaravirus was established [Fig mosaic virus FMV [Emaravirus [In the taxonomy of viruses, genome organization as well as nucleic acid and deduced amino acid sequence data are the main attributes to describe a virus family or genus, in addition to particle morphology, transmission and serological typing. Today, a total number of 2,284 viruses are described, which are grouped in 18 families and 17 unassigned genera [ablished . Meanwhiirus FMV ,6 has bearavirus  might beAlthough genomic sequence information is limited for High Plains virus  and pigThis communication will resume the state of knowledge about these viruses and their associated diseases. It will give an idea about the virus characteristics as well as the relationship between each other, and to already described and classified plant viruses. Several aspects of the viruses in the group are summarized in Sorbus aucuparia), which was named ‘ringfleck mosaic’ or ‘ringspot disease’ [Emaravirus, was found to be closely associated with the disease [S. aucuparia, such as S. aria (whitebeam) and S. torminalis (service tree), have not been reported to be affected by ‘ringspot disease’ so far. Like many fruit tree species  European mountain ash belongs to the family Rosaceae. Although the economical importance of S. aucuparia in fruit production is not that high, its ecological potential as a major pioneer tree species in reforestation projects, especially at erosion-endangered areas, is remarkable [Chlorotic ringspots and mottling have been described as typical symptoms of a disease affecting European mountain ash , and closely related to EMARaV, was identified [Fig mosaic disease (FMD) was first described in the 1930s in California . Since terovirus ,25,26. Mentified ,5,6. A mentified . Rosa multiflora is highly susceptible to the disease, being a putative source of infection of cultivated hybrid roses. During several decades in the last century R. multiflora was planted for hedges and wildlife improvement in rural areas as “living fences”, but extensive natural seeding and development of the rose plant could not be controlled by farmers. Therefore, it was declared a noxious weed, and as a biocontrol means for its eradication grafting of shoots of RRD infected plants onto plants in established R. multiflora stands (augmentation) was suggested [Phyllocoptes fructiphilus as a vector of the disease [Emaravirus, suggested that it is probably causing rose rosette [Rose rosette disease (RRD), first described in the 1940s , is an iuggested . The etiuggested , the ide disease , and the disease  indicate rosette .Rubus species by the raspberry leaf and bud mite Phyllocoptes gracilis (Eriophyidae) causes a complex disease syndrome called raspberry leaf blotch disorder (RLBD). It is characterized by yellow blotching as well as twisting of the leaves and distortion of leaf margins, decrease of overall plant growth by killing the terminal growing tip and reduction of fruit quality [Emaravirus. Infestation of raspberry and other  quality ,35. Alth quality . Its genZea mays) and wheat (Triticum aestivum) with strong disease symptoms were observed in Idaho and Texas, and shortly after also in Kansas and Colorado  [Wheat streak mosaic virus (WSMV) (Potyvirus) infection, but symptoms have been exceptionally strong, leading to the assumption that a second virus might be involved in the disease [High Plains disease (HPD) was first identified in 1993, where maize  , but is all USA) . In the  disease . This se disease . Meanwhi disease , which wAvena sativa), rye , yellow foxtail (Setaria glauca), green foxtail (Setaria viridis) and downy brome (Bromus tectorum) [In maize, disease symptoms are multifold. First evidence of infection becomes visible in young plants at 30 to 45 cm height . Diseaseectorum) . As maizectorum) ,42.Cajanus cajan, Fabaceae), also known as ‘Green Plague’, leads to reduced flowering, while vegetative growth is stimulated. Furthermore, SMD-affected plants show ringspots and mosaic. Leaf size may be reduced and, if existing, seeds are often small and deformed [C. cajan, its wild relatives, as e.g., C. scarabaeoides, can also be affected by SMD, supporting a spread of the ‘Green Plague’ [Sterility mosaic disease (SMD) of pigeonpea  [C. cajan showed granular cell cytoplasm and deformation of the chloroplasts by large starch grains [C. cajan vary in susceptibility for PPSMV. Thus, PPSMV research is focused on identifying and producing resistant plants [The detection of several RNA species and filamentous structures, as can be found in the genera  (PPSMV) . Ultrathh grains . Today, t plants .Four of the six virus diseases, ringspot disease of mountain ash, FMD, RRD and HPD, were shown to be associated with the occurrence of electron-dense structures known as double-membrane-bound bodies (DMBs)  with diaTomato spotted wilt virus (TSWV). Recently, we found similar structures in partially purified fractions from EMARaV-infected mountain ash leaves [In ultrathin sections of leaves from mountain ash trees affected by ringspot disease, DMBs of about 80–100 nm in diameter were detected in the cytoplasm , suggesth leaves , obtaineh leaves  , negative-sense RNA. Double-strand RNA (dsRNA) preparations from infected plant tissue, Northern blot analyses as well as nucleic acid purification from enriched virus particle or nucleocapsid fractions indicated the presence of up to seven or eight RNA species, sized between 1.1 and 8 kb. Arenaviridae, Orthomyxoviridae and Bunyaviridae [Phlebovirus (Bunyaviridae) [EMARaV was the first virus among this group, for which full-length sequences of genomic RNAs were established . Four RNaviridae ,65 and raviridae . The secaviridae . Sequencviridae) . Furtherviridae) . RNA 3 (viridae)  and RLBVviridae) . These fviridae) ,69,70. Bunyaviridae and to EMARaV [Fig mosaic virus to the genus Emaravirus [Experimental strategies similar to the characterization of EMARaV allowed cDNA cloning and sequencing of six genomic RNAs of FMV ,6,71,72.o EMARaV . The newEmaravirus [The strategy of dsRNA-based cDNA cloning and high throughput sequencing has recently allowed the characterizing of four genomic RNA segments of rose rosette virus (RRV) . Genome aravirus . As showaravirus . P4 deriaravirus ; the autEmaravirus was present in raspberry plants exhibiting raspberry leaf blotch disorder. Therefore, RT-PCR with primers derived from the conserved terminal sequences of Emaravirus and stepwise using RNA-specific primers, followed by mass sequencing (Roche 454), led to characterization of five RNAs of the RLBV genome [N. benthamiana and N. tabacum) plants [The analysis of the genome organization of RLBV was started with random amplified cDNA derived from dsRNA . A cloneV genome . Four RN) plants . In addi) plants .Tospovirus. While the predicted proteins encoded by the (probably) two different RNA species in the RNA-M complex showed no significant sequence similarities to proteins of other viruses, RNA-S has some similarities to EMARaV and PPSMV [The RNA genome of MRSV is not fully characterized yet  Table 2Table 2. nd PPSMV . It is snd PPSMV . Partialnd PPSMV ,38,74,75Emaravirus related virus species discussed above, the genome of PPSMV was reported to consist of seven or more RNAs [In contrast to the ore RNAs . Partialore RNAs . Bunyaviridae) has been identified, as was recently reported for RRV [Bunyaviridae or the genus Tenuivirus. The role of protein P4 could not be clarified for the Emaravirus related viruses under study, and there is no remarkable sequence similarity to proteins of other virus families or genera [Taken together, comparison of the so far published deduced amino acid (aa) sequences revealed remarkable similarities among EMARaV, FMV, RRV and RLBV as well as MRSV and PPSMV, but also to bunyaviruses and tenuiviruses. In particular, P1, the putative RdRp, shows significant conserved sections due to functional domains. Thus, the RdRps of EMARaV, FMV, RLBV and RRV were described as having aa identities of 49%–68% (56%–83% similarity) to their orthologs . Neverth for RRV ,11. Thusr genera ,4,8,9. HOrthobunyavirus and Hantavirus within the family Bunyaviridae, but not with that of the plant pathogenic tospo- and tenuiviruses [Furthermore, recent studies showed that the complementary RNA termini, characterized so far, are more or less identical among EMARaV, FMV, RRV, and RLBV (5'-AGUAGUGUUCUCC … GGAGUUCACUACU-3'). The sequence of the only available MRSV RNA terminus is also similar. High similarity was also found with the RNA ends of the genera iviruses ,4. For PAs particle and nucleocapsid morphology of EMARaV, PPSMV, MRSV and FMV resembled to some extent tospoviruses and tenuiviruses, efforts were made to detect these viruses with antisera against the two genera. But no reactions were obtained, indicating a low serological relationship, which can be easily explained by the recently published sequence comparison with viruses of these genera. Today, for the specific detection of identified proteins of this group of novel viruses, several antisera are available. However, the two antisera raised against the putative glycoproteins and the N-protein (P3) of EMARaV were not recommended for routine testing, due to weak reaction and background signals in Western blot with crude protein extracts ,14. But Phytoptus pyri, collected from galls on leaves of EMARaV infected mountain ash trees [As most of the host plants infected with these novel plant RNA viruses are of high economical importance, specific and sensitive diagnostic tools are indispensable. Due to lack of sufficient sequence data for PPSMV and MRSV, primer pairs for RT-PCR, which are able to detect all RNA species and most of the existing isolates of these two putative viruses, are still rare. However, for the other four viruses, whose genomes are extensively sequenced, diagnostic primer pairs are available. Four diagnostic primer pairs were developed for EMARaV, which were shown to detect different isolates from Germany and Austria . EMARaV sh trees . RT-PCR methods for FMV detection have been reported previously, amplifying parts of the RNAs carrying the N- and glycoprotein genes . MeanwhiR. multiflora, showing typical symptoms of RRD, while RRV was not detected in 30 asymptomatic roses, used as negative controls [N. benthamiana plants, in raspberry plants inoculated with viruliferous mites, and in RNA extracted from a bulked sample of mites collected from RLBD-affected plants [Primer pairs were developed for RRV that unambiguously detected RRV in 84 of 84 samples of cultivated roses and d plants .Diagnostic primers are also available for the partially characterized PPSMV and MRSV ,42. ThusThe diseases caused by RRV, RLBV FMV, PPSMV and MRSV were all shown to be transmitted by eriophyid mites, a group of arthropods which are very host specific and also serve as vector for several potyviruses ,76,85,86Phytoptus pyri (Eriophyidae), are frequently found on EMARaV-infected S. aucuparia leaves  is the vector for both MRSV and WSMV, explaining the fact that a mixed infection with these two viruses is common. An investigation in Nebraska (USA) in 1995/96 detected both viruses in 40% of the collected samples [A. tosichella is restricted to feeding; the mite was shown to be unable to transmit transovarially [The wheat curl mite  as well as on Chenopodium sp., Datura stramonium and Vigna unguiculata [As vegetative transmission is typical for plant virus infection, PPSMV, FMV and EMARaV were demonstrated to be graft transmissible or could be passed by cuttings ,88,89,90 10%–40% . Symptomuiculata . For MRSOf the described viruses, no vertical transmission by seed or pollen has been reported so far. First efforts to detect EMARaV by highly specific and sensitive RT-PCR in plants grown from seed of infected mountain ash also gave no indication for seed transmissibility, although EMARaV is detectable in the seed itself (unpublished). FMV also repeatedly tested negative for seed transmission ,89. WhenHowever, MRSV showed a very low seed transmission rate of 0.008% . Only thTransmission by soil has not been reported for any of these viruses so far.Emaravirus. Although extensive sequence information is still lacking for MRSV and PPSMV, their well-documented biological and structural properties also argue in favor of a relationship to emaraviruses. All six viruses have a segmented RNA genome of negative polarity, and recent sequence data analyses showed significant amino acid identities of some of the encoded proteins. Furthermore, they are all associated with the occurrence of DMBs, variable in size, which might represent the virions. Whereas vector transmission has not yet been conclusively shown for EMARaV, all the other viruses are vectored by eriophyid mites. Sequence comparison indicated that these viruses are related to the family Bunyaviridae and the genus Tenuivirus, but the collected data on virus morphology, genome organization, RNA and protein sequences, as well as biological properties, did not allow assigning them into existing families or genera. Therefore, the floating genus Emaravirus has been established with EMARaV and FMV as the first members, but RRV and RLBV are promising candidates for the next viruses to be assigned to this genus.The well characterized EMARaV, FMV, RRV and RLBV were shown to share many similarities, indicating a strong phylogenetic relationship, which suggests that also the latter two belong to the genus Cercis canadensis L.) show bright yellow ringspots or blotches, and often necrotic spots. Furthermore, trees are characterized by many dead twigs and smaller branches. Very recently a virus with a genome organization highly similar to emaraviruses was shown to be associated with the disease, which was provisionally named Redbud yellow ringspot associated virus (RYRSaV) [orbus scopulina Greene (Greene’s mountain ash) was found in Alaska. Virus-like membrane-bound spherical particles could be observed and an approximately 32 kDA protein was consistently present in extracts from only symptomatic leaves. Although antiserum directed against the 35 kDa N-protein of EMARaV did not react in Western blot analyses with the enriched 32 kDa protein, circumstantial evidence, such as disease symptoms, association of the disease with gall forming eriophyid mites and particle morphology of the putative viral agent, suggest some relationship to emaraviruses [Emaravirus, might be detected. In addition to the six novel plant viruses discussed here, there remain still unassigned viruses or virus-like agents which might belong to the same group. Among them is a pathogen associated with Redbud yellow ringspot disease , which w(RYRSaV) . Furtheraviruses . It is tEmaravirus related viruses RRV, RLBV and probably also MRSV and PPSMV are the causes of the corresponding plant diseases. Although in recent years we could collect new information about this novel genus, the genomic organization of emaraviruses and their biological interaction with host plants are far from understood. Finally, it has to be considered that in no case Koch’s postulates were convincingly fulfilled. This is certainly due to the fact that no local lesion hosts have been identified yet and that the isolation of infectious virus particles was not possible so far. However, the close association of the novel viruses with typical disease patterns, the presence of virus-like particles only in symptomatic tissue, and the close relation of vector, virus and disease in almost all cases strongly support the hypothesis that the novel emaraviruses EMARaV and FMV and the


Polerovirus tentatively named Pepper yellow leaf curl virus (PYLCV). PYLCV has a wider host range than Tobacco vein-distorting virus (TVDV) and has a close serological relationship with Cucurbit aphid-borne yellows virus (CABYV) (both poleroviruses). The extracted viral RNA was subjected to SOLiD next-generation sequence analysis and used as a template for reverse transcription synthesis, which was followed by PCR amplification. The ssRNA genome of PYLCV includes 6,028 nucleotides encoding six open reading frames (ORFs), which is typical of the genus Polerovirus. Comparisons of the deduced amino acid sequences of the PYLCV ORFs 2-4 and ORF5, indicate that there are high levels of similarity between these sequences to ORFs 2-4 of TVDV (84-93%) and to ORF5 of CABYV (87%). Both PYLCV and Pepper vein yellowing virus (PeVYV) contain sequences that point to a common ancestral polerovirus. The recombination breakpoint which is located at CABYV ORF3, which encodes the viral coat protein (CP), may explain the CABYV-like sequences found in the genomes of the pepper infecting viruses PYLCV and PeVYV. Two additional regions unique to PYLCV (PY1 and PY2) were identified between nucleotides 4,962 and 5,061 (ORF 5) and between positions 5,866 and 6,028 in the 3' NCR. Sequence analysis of the pepper-infecting PeVYV revealed three unique regions (Pe1-Pe3) with no similarity to other members of the genus Polerovirus. Genomic analyses of PYLCV and PeVYV suggest that the speciation of these viruses occurred through putative recombination event(s) between poleroviruses co-infecting a common host(s), resulting in the emergence of PYLCV, a novel pathogen with a wider host range.We determined the complete sequence and organization of the genome of a putative member of the genus  Capsicumannum) is an economically important crop worldwide including Israel, where about 3,000 hectares are grown year round for the local and export markets. Since 1998, a viral disease has been found in pepper crops in the south and east of Israel, causing serious economic damage. The disease has been observed mostly in open fields and greenhouses covered with low mesh nets through which insects can easily pass. Disease incidence is higher in greenhouses in which an integrated pest management (IPM) regime or biological control methods are used. The disease symptoms include shortening of stem internodes, inter-veinal yellowing, upward curling of the leaf blade and small, discolored fruit. The disease-causing agent was tentatively named Pepper yellow leaf curl virus (PYLCV)  and with TVDV ORF2-ORF4 . Relatively low levels of shared sequence identity were calculated when these sequences were compared to the ORF0, ORF1 and ORF5 sequences of TVDV . The lowest levels of shared sequence identity were calculated for PYLCV and CABYV ORFs (ORF0-ORF5), with the exception of ORF5, for which there was a relatively high level of shared sequence identity (87%) (Polerovirus in the family Luteoviridae  and the two poleroviruses Pepper vein yellows virus [PeVYV (AB5948280)] and Tobacco vein-distorting virus [TVDV (EF529624)].Alignment of the deduced amino acid sequences corresponding to the ORF0s of Pepper (TIF)Click here for additional data file.Figure S2Pepper yellow leaf curl virus [PYLCV (HM439608)], Pepper vein yellows virus [PeVYV (AB5948280)], Pepper yellows virus [PepYV (FN600344)], Tobacco vein-distorting virus [TVDV (EF529624)], Potato leaf curl virus [PLRV (Y07496)] and Cucurbit aphid-borne yellows virus [CABYV (X76931)].Alignment of the deduced amino acid sequences of the ORF4s of the poleroviruses (TIF)Click here for additional data file.Figure S3Pepper yellow leaf curl virus [PYLCV (HM439608)], Pepper vein yellows virus [PeVYV (AB5948280)], Cucurbit aphid-borne yellows virus [CABYV (X76931)], Potato leaf curl virus [PLRV (Y07496)] and Tobacco vein-distorting virus [TVDV (EF529624)].Alignment of the deduced amino acid sequences of the N-termini of the ORF5s (CABYV-like region) of the poleroviruses (TIF)Click here for additional data file.Figure S4Potato leaf curl virus [PLRV (Y07496)], Tobacco vein-distorting virus [TVDV (EF529624)], Cucurbit aphid-borne yellows virus [CABYV (X76931)], Pepper vein yellows virus [PeVYV (AB5948280)], Pepper yellow leaf curl virus [PYLCV (HM439608)]. Each protein-coding sequence was aligned using the MUSCLE program and phylogenetic trees were then constructed for each data set using the PhyML software with 100 bootstrap replicates and PLRV, the type member of the genus Polerovirus.Phylogenetic analysis of selected poleroviruses based on the deduced amino acid sequences of the ORF5 of (TIF)Click here for additional data file.


Beauveria bassiana  Vuillemin, Metarhizium anisopliae (Metschnikoff) Sorokin, Lecanicillium lecanii (Zimmerm.) Zare and Gams, and Paecilomyces fumosoroseus (Wize) Brown and Smith, were tested for their pathogenicity, ovicidal effect, and median lethal concentrations (LC50) against exotic spiralling whitefly, Aleurodicus dispersus Russell. The applications were made at the rate of 2 × 109 conidia mL−1 for evaluating the pathogenicity and ovicidal effect of entomopathogenic fungi against A. dispersus. The results of pathogenicity test showed that P. fumosoroseus (P1 strain) was highly pathogenic to A. dispersus recording 100% mortality at 15 days after treatment (DAT). M. anisopliae (M2 strain) had more ovicidal effect causing 37.3% egg mortality at 8 DAT. However, L. lecanii (L1 strain) caused minimum egg hatchability (23.2%) at 10 DAT as compared to control (92.6%). The lowest LC50 produced by P. fumosoroseus (P1 strain) as 8.189 × 107 conidia mL−1 indicated higher virulence against A. dispersus. Hence, there is potential for use of entomopathogenic fungi in the field conditions as an alternate control method in combating the insect pests and other arthropod pests since they are considered natural mortality agents and are environmentally safe.Biological control using entomopathogenic fungi could be a promising alternative to chemical control. Entomopathogenic fungi,  Aleurodicus dispersus Russell , the native of the Caribbean region of Central America [A. dispersus in Taiwan [A. dispersus caused yield reduction up to 53% in cassava [The spiralling whitefly,  America , is a hi America . A loss n Taiwan  and A. d cassava . The nymA. dispersus management is the use of microbial biocontrol agents (MBCAs) as the natural enemies of the pest population devastate pests with no hazard effects on human health and environment. As the microbial biocontrol agents have complex mode of action, it is very difficult for a pest to develop resistance against MBCAs. The present MBCAs are viruses, bacteria, nematodes, and fungi and they are used throughout the world with great advantage and success. But fungal biocontrol agents are the most important among all the MBCAs due to easy delivery, improving formulation, vast number of pathogenic strains known, easy engineering techniques, and overexpression of endogenous proteins or exogenous toxins [50) of entomopathogenic fungi against A. dispersus under laboratory conditions.One of the potential methods in s toxins –8. Simils toxins –11 as wes toxins , 13. Wit50 of entomopathogenic fungi. The insect used for the study was A. dispersus from the culture maintained in screen house.Studies were conducted at Biocontrol Laboratory, Department of Agricultural Entomology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India. Bioassays were carried out to evaluate the pathogenicity, ovicidal effect, and LCB. bassiana , M. Anisopliae , L. lecanii (L1 strain), and P. fumosoroseus (P1 strain) were obtained from the National Bureau of Agriculturally Important Insects, Bangalore, Karnataka, India, the Department of Plant Pathology, Tamil Nadu Agricultural University (TNAU), Coimbatore, Tamil Nadu, India, and Sun Agro Biotech Research Centre, Porur, Chennai, Tamil Nadu, India, culture collections. Isolates were maintained in culture on potato dextrose agar (PDA) slants in universal bottles (30 mL) and stored at 4°C. Continuous cultures were maintained on slants with subcultures grown for 14 days at 25°C following which lids were tightly sealed and cultures stored at 4°C.The entomopathogenic fungi, A. dispersus nymphs by direct spray method in completely randomized design (CRD). Entomopathogenic fungi were sprayed with the help of automizer over the nymphs of A. dispersus with three replications. All the treated Petri dishes were maintained at 25 ± 1°C in an incubator. The nymphs were individually examined under a stereo zoom binocular microscope (Carl Zeiss Stemi 2000) at 40x magnification for verification of fungal infection. The mortality data were recorded by counting the dead cadavers and nymphs with fungal spores. Observations on the mortality of A. dispersus by entomopathogenic fungi were made at 3, 5, 7 10, 13, and 15 days after treatment (DAT). The mortality data were corrected using Abbott's formula [A. dispersus.Strains of different entomopathogenic fungi were assayed against  formula . The expA. dispersus eggs. Uniform age of A. dispersus eggs were taken from eggplant (Solanum melongena L.) leaf placed on 1.5% agar in a Petri dish. Entomopathogenic fungi were sprayed with help of automizer over the eggs of A. dispersus with three replications in CRD. All the treated Petri dishes were maintained at 25 ± 1°C in an incubator and hatchability was recorded until no change for three consecutive days. Later, the eggs were individually examined under a stereo zoom binocular microscope (Carl Zeiss Stemi 2000) at 40x magnification for verification of fungal infection. Finally, all unhatched eggs were transferred to moist chambers for three days to observe fungal outgrowth if any, as an evidence of egg mortality due to fungal infection. Observations were made at 4, 6, 8, and 10 DAT. The experiments were repeated for three times to confirm the ovicidal action of entomopathogenic fungi against A. dispersus eggs.Entomopathogenic fungi were assayed using direct spray method to evaluate ovicidal effect on 50) of four entomopathogenic fungi, namely, M. anisopliae (M1 strain), B. bassiana (B1 strain), L. lecanii (L1 strain), and P. fumosoroseus (P1 strain), against A. dispersus nymphs. Five doses (from 2 × 105 to 2 × 109 conidia mL−1) were fixed for which dilutions were prepared with double distilled water. A. dispersus nymphs were treated starting from lower to higher concentrations, whenever different test doses the of same entomopathogenic fungi were used. Uniform age of A. dispersus nymphs was taken from eggplant leaf placed on 1.5% agar in a Petri dish. Five concentrations of each respective entomopathogenic fungi were sprayed with the help of automizer over the A. dispersus nymphs with three replications in CRD. All the treated Petri dishes were maintained at 25 ± 1°C in an incubator. The nymphs were individually examined under a stereo zoom binocular microscope (Carl Zeiss Stemi 2000) at 40x magnification for verification of fungal infection. The mortality data were recorded by counting the dead cadavers and nymphs with fungal spores. Observations were made periodically at 12 h interval up to 14 days and mortality data were recorded and corrected using Abbott's formula [50) and LC95 values were estimated for A. dispersus [Studies were conducted to find the median lethal concentrations  caused significantly maximum mortality (80.4%) at 10 DAT as compared to other entomopathogenic fungi isolates (P. fumosoroseus (P1 strain) produced 100% mortality to A. dispersus nymphs. The next best entomopathogenic fungi were L. lecanii (L1 strain) (97.8%) and B. bassiana (B1 strain) (97.0%) at 15 DAT. Earlier, Avery et al. [P. fumosoroseus produced 99.5% mortality to greenhouse whitefly, Trialeurodes vaporariorum (Westwood), and this is in conformity with the present findings. Paecilomyces isolates produced over 70% mortality to T. vaporariorum as reported by Gökçe and Er [P. fumosoroseus, P. farinosus, and Beauveria bassiana) against silver leaf whitefly, Bemisia argentifolii Bellows and Perring. Eyal et al. [Bemisia tabaci (Gennadius) by B. bassiana with concentrations of 1–4 × 106 conidia mL−1. Nagasi et al. [B. bassiana was most pathogenic to first instar and adults of B. argentifolii. However, Wraight and Knaf [13 conidia (2.5 conidia mL−1) and achieved 90% control of B. tabaci nymphs on 7 DAT.All entomopathogenic fungi caused high rates of pathogenicity among ic fungi . The resisolates . P. fumoy et al.  reportede and Er . Wraighte and Er  recordedl et al.  reportedi et al.  observedand Knaf  and Wraiand Knaf  reportedM. anisopliae (M2 Strain) caused 37.3% egg mortality and the next higher egg mortality was with P. fumosoroseus (P1 strain) (22.6%) at 8 DAT (B. bassiana (B1 strain) (4.2%). The hatchability was suppressed by all the entomopathogenic fungi to some extent (L. lecanii (L1 strain) produced lesser egg hatchability (23.2%) at 10 DAT as compared to other fungi. Earlier, Pirali-Kheirabadi et al. [M. anisopliae, B. bassiana, and Lecanicillium psalliotae (Treschew) Zare and W. Gams on egg hatchability of Rhipicephalus (Boophilus) annulatus (Say). However, Malarvannan et al. [B. bassiana at 2.4 × 107 conidia mL−1 did not affect the hatchability of Spodoptera litura Fabricius.at 8 DAT . Very loe extent . L. lecahability .2% at 10n et al.  reported50 of L. lecanii (L1 strain), P. fumosoroseus (P1 strain), M. anisopliae (M1 strain) and B. bassiana (B1 strain) assessed for A. dispersus population were 3.085 × 108, 8.189 × 107, 2.197 × 108, and 3.481 × 108 conidia mL−1, respectively (95 of L. lecanii (L1 strain), P. fumosoroseus (P1 strain), M. anisopliae (M1 strain), and B. bassiana (B1 strain) assessed for A. dispersus population were 2.513 × 1013, 5.053 × 1012, 1.506 × 1013, and 3.442 × 1013 conidia mL−1, respectively. Log concentration probit mortality response of A. dispersus to entomopathogenic fungi is depicted in Figures 50 and LC95 were recorded by P. fumosoroseus as 8.189 × 107 and 5.053 × 1012 conidia mL−1, respectively, indicating higher virulence against A. dispersus. Earlier, Saranya et al. [50 value of 2.5 × 104 spores mL−1 by L. lecanii and Hirsutella thompsonii Fisher isolates against cowpea aphid, Aphis craccivora (Koch). Low LC50 value of 1.2 × 104 spores mL−1 for L. lecanii against Brevicoryne brassicae (L.) and 2.7 × 104 spores mL−1 against Aphis gossypii Glover was reported by Derakshan et al. [50 value obtained in the present study was lower than that reported by Smitha [Hirsutella sp. (5.2 × 104 spores mL−1) but higher than that reported by Liu et al. [B. bassiana (1.2 × 104 spores ml−1) and Chandler [M. anisopliae (2.45 × 106 spores mL−1). The difference in the LC50 values might be due to the difference in the virulence of fungal isolates and the host species.The LCectively . The LC9a et al.  recordedn et al.  and Karin et al. , respecty Smitha  for Hirsu et al.  for B. bChandler  for M. aA. dispersus and field evaluation of entomopathogenic fungi must be conducted before ultimate conclusions are drawn.Since they are considered natural mortality agents and are environmentally safe, there is potential for the use of entomopathogenic fungi in the field conditions as an alternate control method in combating the insect pests and other arthropod pests. Additional testing of entomopathogenic fungi with other stages of


We compared the expression pattern of FgfrL1 with that of other Fgfrs. The expression pattern of FgfrL1 closely resembled that of Fgfr1, but clearly differed from that of Fgfr2–Fgfr4. It is therefore conceivable that FgfrL1 signals indirectly via Fgfr1. The mechanisms by which FgfrL1 affects the activity of Fgfr1 remain to be elucidated.Fibroblast growth factor (FGF) receptor-like protein 1 (FGFRL1) is a recently discovered member of the FGF receptor (FGFR) family. Similar to the classical FGFRs, it contains three extracellular immunoglobulin-like domains and interacts with FGF ligands. However, in contrast to the classical receptors, it does not contain any intracellular tyrosine kinase domain and consequently cannot signal by transphosphorylation. In mouse kidneys, FgfrL1 is expressed primarily at embryonic stages E14–E15 in regions where nascent nephrons develop. In this study, we used whole-mount  The fibroblast growth factor signaling system plays an important role in the development of most multicellular animals. It controls the proliferation, differentiation, migration and apoptosis of virtually all cell types. The genomes of mice and humans code for 22 different FGF ligands  that can interact with four different FGF receptors  (All FGFRs contain three extracellular immunoglobulin-like domains (Ig domains 1–3), a single transmembrane domain and an intracellular tyrosine kinase domain . AlternaThe metanephric (permanent) kidney of mammals is formed by two different tissues, the metanephric mesenchyme and the ureteric bud ,4. In miIn the year 2000, we discovered a fifth FGFR that we termed FGF receptor-like protein 1 (FGFRL1) . This reXenopus embryos, it interfered with FGF signaling and led to gastrulation defects that affected the trunk and tail of the embryos  bred at our local animal facility. For a timed pregnancy, the noon of the day, at which a vaginal plug could be detected, was counted as E0.5. All animal experiments were approved by the Swiss Federal Veterinary Office (BVET) (BE84/12).32P] dCTP. After overnight hybridization, the blot was washed at regular stringency [1X standard saline citrate (SSC)] and exposed to X-ray film .Kidneys were dissected from mouse embryos and immediately placed into RNA-later buffer . In order to obtain enough RNA, kidney rudiments of early developmental stages (E12.5–E14.5) were pooled from 20–30 individual embryos. RNA was prepared using the GeneElute mammalian total RNA kit from Sigma and separated on 1% agarose gels in the presence of 1 M formaldehyde. The resolved bands were transferred from the gel to a nylon membrane by vacuum blotting. The membrane was hybridized at 42°C with radioactively labeled cDNA probes in a buffer containing 50% formamide. These probes were labeled by the random primed oligolabeling method with [α-BamHI/XbaI site of the expression vector pSPT19 . The probe for FgfrL1 corresponded to an XbaI/BamHI fragment derived from the full-length cDNA sequence . Riboprobes were generated from cDNA sequences cloned into pSPT19 (see above) by transcription in the presence of digoxigenin-labeled UTP using the SP6/T7 DIG RNA Labeling kit from Roche as previously described (Whole-mount in situ hybridization was performed using E15.5 mouse kidneys following the protocol provided in the GUDMAP gene expression database (escribed . KidneysThe expression of the four classical receptors was analyzed by northern blotting with samples from embryonic kidneys at stage E15.5. Special care was taken so that the four hybridization probes had a similar specific radioactivity to allow the direct comparison of the resulting hybridization signals between the four probes. Moreover, the sequences of the probes were selected in a manner that the probes hybridized equally well with the b and the c splice variants of the receptors.A particularly strong hybridization signal was obtained with the probe for Fgfr1 . This reThe expression of FgfrL1 was analyzed on a separate northern blot containing RNA from kidneys at five different developmental stages . A band in situ hybridization of entire kidneys at stage E15.5 (The spacial distribution of the FgfrL1 mRNA was compared with the distribution of the four classical receptors by whole-mount ge E15.5 . The cDNThe progression of the enzymatic reaction that was used for development of the signal shown in in situ hybridization technique to show the spacial expression pattern of five different Fgfrs in the developing mouse kidney. This technique yields information about the three-dimensional distribution of the corresponding mRNAs in the cortex of the kidney, whereas in situ hybridization of thin sections would show only a two-dimensional expression pattern. Whole-mount in situ hybridization appears to be more sensitive than section hybridization as it accumulates signals from the depth of the kidney cortex, which offers an extra advantage when expression levels are very low. In fact, section in situ hybridization did not yield convincing data with probes for Fgfr3 and Fgfr4. Moreover, the signal obtained with FgfrL1, the fifth Fgfr, proved to be extremely weak by section hybridization and had to be electronically enhanced for visualization in a previous publication (in situ hybridization with FgfrL1 yielded robust signals that did not have to be enhanced.In this study, we used the whole-mount lication . By contIn this study, we demonstrate that Fgfr1 and Fgfr2 are the major receptors of the Fgf signaling system expressed in the early stages of developing kidneys. On the other hand, the expression of Fgfr3 and Fgfr4 was very low, raising doubts about the functional significance of these receptors during kidney development. This observation is in accordance with the results obtained from experiments using knockout mice . The disIn this study, we demonstrated that the spacial distribution of FgfrL1 mRNA closely resembled that of Fgfr1, but clearly differed from that of Fgfr2–Fgfr4. FgfrL1 cannot signal on its own as it lacks the intracellular tyrosine kinase domain. Since it still interacts with Fgf ligands, it is likely that it indirectly modulates the Fgf signaling of another receptor. Thus, we concluded that this other receptor may be Fgfr1, since only this receptor shows a similar distribution in developing kidneys.Originally we ,16, as wIt has been demonstrated that the developing kidneys of mice express Fgfs 1, 7, 8, 9, 10, 12 and 20 . Of thes


H5N1 highly pathogenic avian influenza virus (HPAIV) is continuously circulating in many Asian countries and threatening poultry industry and human population. Vaccination is the best strategy to control H5N1 HPAIV infection in poultry and transmission to human population. The aim of this study is to identify new temperature-sensitive (ts) mutations for developing recombinant avian live attenuated H5N1 influenza vaccine.A “6 + 2” recombinant virus C4/W1 that contained NA gene and modified HA gene from virus A/chicken/Hubei/327/2004 (H5N1) (C4), and six internal genes from virus A/duck/Hubei/W1/2004 (H9N2) (W1) was generated using reverse genetics and subsequently passaged in chicken eggs at progressively lower temperatures . The resulting virus acquired ts phenotype and one of its amino acid mutations, PA (F35S), was identified as ts mutation. Furthermore, when used as live attenuated vaccine, the recombinant virus with this ts mutation PA (F35S) provided efficient protection for chickens against H5N1 HPAIV infection.These findings highlight the potential of the new ts mutation PA (F35S) in developing recombinant avian live attenuated H5N1 influenza vaccine. H5N1 HPAIV is continuously circulating in many Asian countries and threatening poultry industry and human population. An effective vaccine to protect poultry from infections of H5N1 HPAIV is needed not only to reduce economic losses in the poultry industry, but also to minimize virus transmission from infected poultry to humans [Live attenuated influenza vaccine (LAIV) with needle-free intranasal administration and cross-protection is attractive in controlling pandemic influenza. Recombinant influenza virus with C-terminal truncated NS1, deficient NA, cleavage-site modified HA or cytoplasmatic-tail deleted M2 has been reported as LAIV . Besideset al. developed an avian H9N2 LAIV by successively passaged a wild-type H9N2 influenza virus A/Chicken/Korea/S1/03 in embryonated chicken eggs at progressively lower temperatures [N265S, PB1K391E/E581G/A661T and NPD34G) were similar to the ts mutations of the MDV A/Ann Arbor/6/60 (H2N2). However, transferring these ts mutations  into the corresponding internal genes of an avian H9N2 influenza virus A/Guinea Fowl/Hong Kong/WF10/99 could not sufficiently result in an avian live attenuated H9N2 influenza backbone, further introduction of an HA tag into PB1 gene was needed [LAIV has been widely used in humans, while rare LAIV was reported to be used for poultry. Lee eratures . Althougs needed . This stTherefore, the aim of this study was to acquire new and simple ts mutations within an avian H9N2 influenza backbone for developing recombinant avian live attenuated H5N1 influenza vaccine. All experiments with H5N1 HPAIV were performed in ABSL-3 containment facility. We initially generated a “6 + 2” recombinant virus C4/W1 that contained NA gene and modified HA gene (cleavage site was changed from PQRERRRKKR↓G to low pathogenic characteristic PQIETR↓G)  from H5N3 EID50 of the low-temperature passaged virus (0.2 ml/egg) and incubated at 37°C or 41°C for 48 hours, the parent virus C4/W1 was used as comparison. Allantoic fluids were collected and viral titers were determined by 50% egg infectious dose (EID50) at 37°C. Virus could be considered ts if it displayed 100-fold or greater reduction in viral titers at 41°C compared with that observed at 37°C. As shown in Figure 10 EID50/ml, respectively). Nevertheless, at 41°C, the virus C4/W1 still grew up to high viral titers (9.0 log10 EID50/ml), while the low-temperature passaged virus was highly impaired in growth (under limited detection). These results indicated that the low-temperature passaged virus acquired ts phenotype and was named ts-C4/W1.The ts phenotype of this low-temperature passaged virus was evaluated. Six 10-day-old specific-pathogen-free (SPF) chicken eggs were inoculated with 1010 EID50/ml) in chicken eggs, but was highly impaired in growth at 41°C(under limited detection). These results indicated that PA (F35S) was the ts mutation.Subsequently, eight gene segments of the virus ts-C4/W1 were sequenced and compared with that of virus C4/W1. Five amino acid mutations were identified in the genome of the virus ts-C4/W1. They were PB1 (K698N), PA (F35S), HA (H119Y), M1 (R174K) and NS1 (K108N) (data not shown). To determine which amino acid mutation or mutations contributed to the ts phenotype of the virus ts-C4/W1, the five amino acid mutations were individually introduced into virus C4/W1 and five single-mutation recombinant viruses were generated. The ts phenotype of these single-mutation recombinant viruses was evaluated. As shown in Figure 6 EID50 of the single-mutation recombinant virus C4/W1-PA (F35S), ts-C4/W1 or C4/W1, At 3 and 5 days post-inoculation (p.i.), four chickens in each group were sacrificed, tracheal swabs, cloacal swabs and lungs were collected in 1 ml phosphate buffered saline (PBS) containing penicillin and streptomycin, lungs were homogenized. Viral titers were determined by EID50 at 37°C. Virus C4/W1 grew efficiently in chickens and could be detected in tracheal swabs, cloacal swabs and lungs of inoculated chickens with high viral titers at 3 and 5 days p.i.; virus ts-C4/W1 couldn’t be detected in any swabs or lungs of the inoculated chickens; while virus C4/W1-PA (F35S) could only be detected in the tracheal swabs of inoculated chickens at 3 days p.i. with much less viral titers (1.8 log10EID50/ml) (Table To determine whether recombinant virus with the ts mutation PA (F35S) was attenuated in chickens, pathogenicity and growth property of the single-mutation recombinant virus C4/W1-PA (F35S) was assessed, viruses ts-C4/W1 and C4/W1 were used as comparison. Animal studies were performed according to protocols approved by the Hubei Provincial Animal Care and Use Committee. The intravenous pathogenicity index (IVPI) was performed  and indi50) was determined as described previously [50 > 8.0 log10EID50/50 μl) in mice than virus C4/W1 (MLD50 = 6.5 log10EID50/50 μl). This phenotype of attenuation could also be observed from body weight loss of mice infected i.n. with 50 μl 106 EID50 of virus C4/W1-PA (F35S), ts-C4/W1 or C4/W1. Virus C4/W1 caused maximum 20% of initial body weight loss, while no apparent body weight loss was observed when mice infected with virus C4/W1-PA (F35S) or ts-C4/W1 (Data not shown). Their growth ability in mice was also determined. Groups of 6-week-old female BALB/c mice were inoculated i.n. with 50 μl 106 EID50 of virus C4/W1-PA (F35S), ts-C4/W1 or C4/W1. At days 3 and 6 p.i., three mice in each group were sacrificed, and lungs were removed and homogenized in 1 ml PBS containing penicillin and streptomycin. Viral titers were determined by EID50 at 37°C. The results showed that viruses C4/W1-PA (F35S) and ts-C4/W1 grew up to lower titers than that of virus C4/W1 at 3 or 6 days p.i. . The fifty percent mouse lethal dose  [et al., 2010) [et al., 2009) [The PA subunit was reported to be critical in modulating ribonucleoprotein (RNP) activity under thermal stress  . Effects., 2010) . The RNP6 EID50 of the virus C4/W1-PA (F35S), ts-C4/W1 or 0.2 ml PBS. Three weeks post-vaccination, six chickens in each group were challenged with 100 50% chicken lethal dose (CLD50) of either virus C4 (clade 9) or virus A/Duck/Hubei/hangmei01/2006 (H5N1) (D6) (clade 2.3.4)  [To determine the protective efficacy of the indicated vaccines in chickens, groups of twelve 3-week-old White Leghorn chickens were inoculated i.n. with 0.2 ml 10., 2009) , tracheaet al., 2000) [Different sublineages of avian H9N2 influenza virus showed different pathogenicity to chickens or mice. Virus of A/Quail/Hong Kong/G1/97 sublineage with internal genes similar to the highly pathogenic Hong Kong/97 H5N1 influenza virus killed mice and spread to mouse brain, while virus of A/Duck/Hong Kong/Y280/97 H9N2 (Y280) sublineage were nonpathogenic to chickens or mice  . ChoosinIn light of rare LAIV used for poultry, we highlighted the potential of the new and simple ts mutation PA (F35S) in developing recombinant avian H5N1 LAIV with avian H9N2 influenza virus as backbone.The authors declare that they have no competing interests.WZ and JT performed the whole experiments. ZZ and HC analyzed the data. MJ designed the study and supervised the experiments. All authors read and approved the manuscript.


N-glycosylation sites that affect viral pathogenesis. Diversity of rabies virus might have public health implications in Bangladesh.Arctic/Arctic-like rabies virus group 2 spread into Bangladesh ≈32 years ago. Because rabies is endemic to and a major public health problem in this country, we characterized this virus group. Its glycoprotein has 3 potential  Rabies virus causes severe encephalitis in a wide range of mammals, including humans. Conservative estimates suggest that 55,000 persons worldwide die of rabies each year  in 2010 . A portiFull-length nucleoprotein (N) and glycoprotein (G) gene sequences from samples were determined. Nucleotide identities of N and G genes were 98%–100%. Amino acid identities of N and G genes were 100% and 98%–100%, respectively. Complete genomic sequencing  of strain BDR5 was also conducted.Evolutionary analysis was performed by using full-length N gene. We created a maximum clade credibility phylogenetic tree using the Bayesian Markov chain Monte Carlo method available in BEAST version 1.6.1 (4 substitutions/site/year (95% highest posterior density [HPD] 1.4–3.1 × 104 substitutions/site/year). This rate is consistent with that of a previous study (The mean rate of nucleotide substitution estimated for the N gene was 2.3 × 10Approximately 397.0 years ago (95% HPD 273.5–589.5 years), AAL and cosmopolitan rabies virus segregated from their most recent common ancestor . ApproxiN-glycosylation site was predicted by using the NetNGlyc 1.0 server (www.cbs.dtu.dk/server/netnglyc). With the exception of BDR6, the G gene of all strains had potential glycosylation sites at position 37, 146, and 319.Compared with the AAL2 strain from India (AY956319), BDR5 had several amino acid substitutions . Sizes oGenetic analysis and phylogenetic studies can contribute to understanding the epidemiology of rabies virus in disease-endemic countries. Molecular analysis of animal rabies viruses showed that AAL2 appeared in central Bangladesh only 32 years ago. A close association between N genes sequences from rabies viruses in Bangladesh and Bhutan indicates that they originated from a common ancestor. If one considers the ease of human movement between countries, AAL2 most likely entered Bangladesh from India rather than from Bhutan.Circumstantial evidence suggests that rabies virus spread from India to Bhutan . TherefoThe G protein is the major factor responsible for the pathogenesis of rabies virus and contains 2 glycosylation sites (AAL viruses could have moved southward from Siberia or other northern regions of the former Soviet Union into Nepal, India, and other countries in Asia by a species jump from fox to dog at some point (The timeline of divergence of different lineages determined in this study was similar to that previously reported (