The view that satellite RNAs (satRNAs) and satellite viruses are purely molecular parasites of their cognate helper viruses has changed. The molecular mechanisms underlying the synergistic and/or antagonistic interactions among satRNAs/satellite viruses, helper viruses, and host plants are beginning to be comprehended. This review aims to summarize the recent achievements in basic and practical research, with special emphasis on the involvement of RNA silencing mechanisms in the pathogenicity, population dynamics, and, possibly, the origin(s) of these subviral agents. With further research following current trends, the comprehensive understanding of satRNAs and satellite viruses could lead to new insights into the trilateral interactions among host plants, viruses, and satellites. These satRNAs are short RNA molecules, usually <1,500 nt, that depend on cognate helper viruses for replication, encapsidation, movement, and transmission, but most share little or no sequence homology to the helper viruses [Satellite viruses and satRNAs have attracted much interest over the past decades, mainly for the following reasons –5: (1) tThis review focuses on current achievements in several important topics concerning satRNAs and satellite viruses, namely replication mechanisms, pathogenicity, and molecular evolution. Comprehensive information for all satRNAs and satellite viruses characterized previously has been provided in several reviews ,5,13,14.2.To assist in distinguishing the different categories of sub-viral RNAs, an illustrated key is provided in Tomato leaf curl virus [Another shorter, noncoding form of satellite DNAs, of about 680 nt, has been found associated with rl virus . Furtherrl virus . The focrl virus ,21.Groundnut rosette virus, GRV, [By definition, all satellites share the following features: they depend on helper viruses, at least for replication; they are not part of the helper viral genome and are not required for the infection cycle of their helper viruses ; and th2.1., which contains a single member, Chronic bee-paralysis satellite virus [Aureusvirus, Panicovirus, Tobamovirus, and Necrovirus. These satellite viruses share no sequence similarities with each other, which suggests that they might have originated from independent events in evolutionary history. Among them, only Satellite tobacco mosaic virus (STMV) has a rod-shaped virus as the helper [Tobacco mild green mosaic virus, is required for adaptation to other helper viruses, namely Tobacco mosaic virus (TMV), Tomato mosaic virus or Green tomato atypical mosaic virus [There are two subgroups in this type ,23: subgte virus ; and subte virus , Panicumte virus , Tobaccote virus , and Tobte virus ,29. Withte virus . The foue helper . Of noteic virus . Satelli2.2.in vivo and is involved in its replication [in planta replication of the satRNA [Blackcurrant reversion virus in the genus Nepovirus of the family Comoviridae also contains a satRNA, which is 1,432 nt and encodes a single protein of 402 amino acids and has unknown function [Single-stranded satRNAs are classified into three subgroups: subgroup 1, large satRNAs; subgroup 2, small linear satRNAs; and subgroup 3, circular satRNAs. Subgroup 1 contains large satRNAs of about 0.7 to 1.5 kb that encode at least one nonstructural protein. Thus, satRNAs in this subgroup are usually referred to as messenger-type satRNAs. Species in subgroup 1 recognized by the International Committee on Taxonomy of Viruses include Arabis mosaic virus large satRNA (satArMV), Bamboo mosaic virus satRNA (satBaMV), Chicory yellow mottle virus large satRNA, Grapevine Bulgarian latent virus satRNA, Grapevine fanleaf virus satRNA (satGFLV), Myrobalan latent ringspot virus satRNA, Strawberry latent ringspot virus satRNA, Tomato black ring virus (TBRV) satRNA, TBRV G serotype satRNA, and Beet ringspot virus satRNA ,33. Althlication . The P3 lication . In addie satRNA . In conte satRNA  is not ee satRNA ,39. It pe satRNA  and intee satRNA , which ffunction .,Species in subgroup 2 of single-stranded satRNAs include Cucumber mosaic virus (CMV) satRNA, Cymbidium ringspot virus (CymRSV) satRNA, Pea enation mosaic virus satRNA, GRV satRNA, Panicum mosaic virus small satRNA, Peanut stunt virus (PSV) satRNA, Turnip crinkle virus (TCV) satRNA, and Tomato bushy stunt virus (TBSV) satRNA B10, and TBSV B1 ,33. TobaSubgroup 3 includes the following species: ArMV small satRNA, Cereal yellow dwarf virus-RPV satRNA (previously Barley yellow dwarf virus satellite RNA), Chicory yellow mottle virus satRNA, Lucerne transient streak virus satRNA, Solanum nodiflorum mottle virus satRNA, Subterranean clover mottle virus satRNA (2 types), Tobacco ringspot virus satRNA, and Velvet tobacco mottle virus satRNA ,33. Spec3.Carrot red leaf virus and one of the umbraviruses, Carrot mottle mimic virus or Carrot mottle virus [Nicotiana tabacum cv. Ky 14, in an attempt to identify the origins of satRNAs [Beet black scorch virus, a necrovirus, was identified and molecularly characterized [Despite great technical advances in molecular virology, only a few new satRNA species have been identified over the past five years. These newly identified satRNAs are all associated with helper viruses in genera previously known to harbor satellites. These new satRNAs include the following. 1) A new satRNA was associated with the disease carrot motley dwarf (CMD), which is caused by a mixed infection of two viruses - the polerovirus le virus . 2) A ne satRNAs ; no new cterized ; both moThe apparent lack of new satRNAs or satellite viruses identified in other new genera of plant viruses may reflect at least two scenarios: 1) satellites associated with other genera of plant viruses cause very mild symptoms or do not cause differences in symptoms inflicted by their cognate helpers because of long-term co-evolution and thus did not attract much research attention; or 2) viruses of the other genera have developed resistance through long-term co-evolution with satRNAs. Although previous studies have provided evidence for the existence of mutual beneficial relationships between satellites and helper viruses ,22,51,524.Nicotiana benthamiana [Potato virus X (PVX)-based vector, the SPMV CP alone could cause necrosis symptoms on N. benthamiana. Omarov et al. [in trans to protect the RNAs transcribed from a Tomato bushy stunt virus-based vector harboring a GFP gene and facilitate the systemic expression of GFP, further demonstrating the multifunctional nature of SPMV CP. On the other hand, the multiple biological activities of SPMV CP are regulated separately by the N-and C-terminal regions [Several novel findings regarding SPMV in recent years have provided insights into how satellite viruses may cause symptoms. In addition to forming the protective shell for RNAs and facilitating systemic invasion, the CP of SPMV was demonstrated to elicit symptoms on a nonhost plant thamiana . When exv et al.  further v et al.  and mighv et al. . Most rev et al. . SPMV CP regions . The resConsiderable research advances have also been achieved for another well studied satellite virus system, the Satellite tobacco necrosis virus (STNV). The RNA of STNV does not contain a cap structure and poly(A) tail at the 5′- and 3′-termini, which is required for efficient translational initiation by interacting with eIF4E and poly(A)-binding protein, respectively. However, the RNA of STNV can still be efficiently translated by use of the translational enhancer domain (TED), which locates within the 122-nt region in the 3′ untranslated region of STNV RNA, to recruit the translational machinery ,59. Thusin vitro translation system to recruit translation machinery [in vitro) and tobacco protoplasts (in vivo) using serial deletion mutants as templates [et al. [in vitro and in vivo and supports the possibility that RNAs might adopt different structures for different functions, e.g., replication and translation. In addition, STNV TED was later shown to specifically interact with the eukaryotic initiation factors eIF4E and eIF(iso)4E [Barley yellow dwarf virus-like cap-independent translation element (TE) at the 3′-terminal region. Although all known members of the genus Necrovirus contain similar TE structures, the TE structure was not present in STNV [Use of the STNV system to study the early translational processes revealed that the first 38 nt at the 5′-terminal region of STNV RNA could function synergistically with the 3′ TED to enhance the translational efficiency . Later, achinery . The boaemplates . The res [et al.  establisF(iso)4E . Interes in STNV , which s5.Research on satRNAs has been a very active area in virology and has led to great insights into the molecular biology of their helper viruses and host plants. Recent advances include the following.5.1.in vivo genetic selection experiments (SELEX) [As mentioned previously, satRNAs may serve as model systems for the study of biological functions, namely replication and translation. As the non-messenger-type satRNA, satC, associated with TCV, is among the best-characterized satRNAs, both for structure and function. Although satRNAs depend on the replicases of the cognate helper viruses for replication, the required sequence or structural elements were not always found to be functionally interchangeable between the satRNAs and cognate helper viruses ,66. Desp (SELEX) . Further (SELEX) . The res (SELEX) . Togethe (SELEX) .et al. [et al. [et al. [The notion that satRNAs may adopt different replication signals and use replicase complexes different from those used by their helper viruses is also true for the messenger-type satRNAs. Annamalai et al.  and Huan [et al.  analyzed [et al.  further  [et al. ,76, conset al. [Carnation Italian ringspot virus (CIRV) in yeast cells could support the replication of several defective interfering RNAs derived from the genome of CIRV or the related CymRSV but not that of a satRNA originally associated with CymRSV. Taken together, the results suggest that satRNAs have adopted a system similar to but distinct from their cognate helper viruses for their replication and thus are not strong competitors of their helper viruses. This suggestion may explain why satRNAs share only limited or no sequence similarities to their cognate helper viruses.Comparable phenomena were observed for satellite viruses. Rubino et al.  demonstr5.2.Three types of symptom modulation are associated with satRNAs: attenuation, exacerbation, and no significant effect. Symptom attenuation is the most commonly observed effect. In most plant species, most satRNAs reduce the replication and accumulation of their helper viruses, which results in the amelioration of symptoms. In some cases, the satRNAs that attenuate the symptoms in several host plants may cause exacerbated symptoms in certain other hosts, such as lethal necrosis in tomato  and stroN. benthamiana by obstructing the Dicer-like protein DCL2/DCL4 silencing pathway [et al. [N. tabacum, greatly attenuated the severe yellowing symptoms caused by the Y satellite of CMV. The expression of self-complementary hairpin RNAs derived from the Potato spindle tuber viroid (PSTVd) in tomato plants resulted in symptoms similar to PSTVd infection. These results provide strong evidence that an RNA silencing mechanism is involved in the pathogenicity of satRNAs, whereby satRNAs direct RNA silencing against biologically important host genes. Furthermore, Wang et al. [Accumulating evidence in recent years supports that RNA-silencing mechanisms of the host plants play important roles in the pathogenicity and evolution of satRNAs. RNA silencing, also referred to as PTGS or RNA interference (RNAi), is a double-stranded, RNA-induced, sequence-specific RNA degradation process, considered one of the important anti-viral mechanisms of plants ,82. To c pathway ,83,84. T pathway ,51. Thus [et al.  showed tg et al.  demonstr5.2.1.In the past, the pathogenicity of satRNAs has been thought to be a direct interaction among the satRNAs, helper viruses and host factors by an unknown mechanism(s). Taking cucumoviruses and associated satRNAs as examples, a satRNA of CMV was originally reported to be the causal agent of lethal necrosis disease of tomato in France . Since tet al. [The determination of nucleotide sequences of a large number of biologically distinct satRNAs has facilitated the identification of specific sequences responsible for differential modulation of symptoms. For example, by inoculating tobacco plants with mutant forms of satCMV, Jaegle et al.  demonstret al. –97. For et al. . Howeveret al. . With thet al. , it is eIn the aforementioned scenarios (section 5.2.), the plant antiviral defense systems have been turned against the plants themselves by the invading virus/satRNA combinations. In response, plants have adapted mechanisms of defense. For example, one of the key proteins in the antiviral RNA silencing pathway, DCL4, can target the satRNAs of CMV at novel secondary structures, preferably T-shaped hairpins, instead of the classic double-stranded hairpins, to produce small interfering RNAs (siRNAs) against satRNAs . The resN. tabacum [et al. [et al. [Furthermore, helper viruses may also exploit the RNA silencing mechanisms of host plants to control the accumulation of parasitic satRNAs. The accumulation of a parasitic satRNA of CymRSV strongly depends on the presence of the RNA silencing suppressor P19 of the helper virus, which suggests the involvement of an RNA silencing mechanism in the competition of CymRSV and satRNA. As well, siRNAs derived from CymRSV targeted satRNA with higher efficiency than did the siRNA derived from a distantly related helper virus, CIRV . Thus, C tabacum . The red [et al.  and Chen [et al.  reported5.2.2.Recent studies indicating the involvement of RNA silencing pathways in the pathogenicity of satRNAs have provided new insights into the underlying mechanisms of satRNA-mediated symptom modulations. Clearly, the pathogenicity of satRNAs is the result of a complex interaction among satRNAs, helper viruses, RNA silencing mechanisms, and the physiologically important genes of the host plants  A, which5.3.A long-standing question regarding satRNAs is the origin of these small RNAs. Considering the infection cycle of satRNAs, at least three natural sources are the origin of satRNA segments: the genomes of the helper viruses or other co-infecting viruses/satellites, the host organisms, and transmission vectors. Although many reports claimed the “spontaneous” occurrence of satRNAs under experimental conditions, previous attempts to locate the sequences with significant similarity to satRNA in the genomes of hosts, helper viruses and vectors have in general been unsuccessful ,110. Howin vivo [et al. [Previous research found that 3′-terminal deletions of an satRNA of TCV, satD, could be repaired in vivo . The satin vivo . The aut [et al.  extendedIn the natural plantation, sometimes more than one species of viruses/satellites may infect the same plant. Thus, the genomes of other co-infecting viruses or satellites have also been suggested to be another possible origin of satRNAs. For example, the sequence of the nonstructural protein P20 of satBaMV was found to share 46% identity with the CP of SPMV and could be folded into a similar “jelly-roll” structure ,115. On Arabidopsis thaliana contains stretches of nucleotide sequences with significant similarity to satCMVs [The genome of  satCMVs . The pos satCMVs ,117. Thede novo from the genomes of CMV and host plants after the activation of RNA silencing mechanisms, which would generate a large quantity of small RNA fragments for assembly into the initial form of satRNAs. That satRNAs were not detected when CMV-LS was used for serial passages suggested the main involvement of viral genomes in the emergence of satRNAs. Although the experimental conditions did not completely eliminate the possibility of contamination, as with performing the whole experiments in a new laboratory environment never exposed to satRNAs [Recently, a novel satRNA of CMV was reported to have emerged naturally following serial passages of CMV-Fny . Great e satRNAs , as ackn satRNAs , this wo satRNAs ,52.In view of the above notion, the sequence similarities between satRNAs and viruses or host plants may reflect the genes targeted in the host or viral genomes by use of siRNAs derived from satRNAs. As mentioned previously, satRNAs and viroids may direct the RNA silencing mechanism against biologically important genes of the cognate host plants . Similar5.4.Because most satRNAs lack any significant coding capacity, they are expected to express their biological functions through direct interaction of the structure or nucleotide sequences of satRNAs with the respective host factors. Thus, the structures of satRNAs are under the constraint of their biological functions in evolutionary history. In addition, the conservation of certain structural elements may contribute to the stability and/or efficient accumulation of satRNAs.et al. [et al. [SatRNAs and viroids have evolved and maintained secondary structures that are considerably more resistant to RNA silencing-mediated degradation . Sun andet al.  performe [et al.  develope [et al. . These r6.in vivo stability, and iii) high expression level. First, because of the relatively small size of satRNAs and satellite viruses, they are simpler systems to use for cloning, sequencing, genetic modification, and regular maintenance. Second, most satRNAs are highly structured and thus significantly more resistant to degradation by nucleases in vivo than are other viral RNA-based vector systems. For instance, about 49% of the genome of one of the most thoroughly characterized satCMVs participates in the formation of base-paired structures [in planta. And finally, most satRNAs and satellite viruses can accumulate to high levels in host plants, which leads to the increase in level of proteins translated from the messenger-type RNAs of the satellites. However, despite the aforementioned potentials, most attempts to develop vectors based on coding or non-coding satRNAs for applications in biotechnology have failed, probably because of the strict requirement for the maintenance of certain secondary structures, which severely limited the sequence alterations that could be introduced. The current applications of satRNAs are demonstrated only for the following rare cases:in planta expression of foreign genes:As vectors for Chenopodium quinoa and demonstrated that satBaMV RNA could be useful as a satellite-based expression vector.The practical application of satBaMV RNA was demonstrated more than ten years ago , and remAs vectors for functional studies:Foxtail mosaic virus were expressed by use of satBaMV RNA-based vectors to complement the functions of green fluorescent protein-tagged, movement-defective BaMV with mutation(s) in the matching gene(s). The results revealed the requirement for species-specific interactions among TGBps for cell-to-cell movement of BaMV and possibly other potexviruses. In addition, a satBaMV-based vector system has been used in analysis of promoter sequences to generate potexvirus subgenomic RNAs (sgRNAs) [in vivo. Such applications in functional analysis further demonstrated the additional advantages of satRNA-based vectors: i) Applicable for mutational studies of essential genes or regulatory sequences: satRNAs are physically separated from the viral genomes and are not essential for the infection cycle of the helper viruses; thus, they can be used in mutational or complementary assays of regulatory sequences. ii) Convenient for combinational studies on multiple genes: two or more satRNA-based vectors harboring different genes may co-exist in one plant and be supported by the same helper virus, although they might be not expressed at the same level.satBaMV RNA has been used for the expression of individual TGBps in a complementary experiment for study of cell-to-cell movement of BaMV . To diss(sgRNAs) . InsertiAs vectors for gene silencing:N. tabacum of a variety of genes involved in different biochemical pathways . Most of the silenced phenotypes could be observed 10–12 days postinoculation, which indicates the efficiency of STMV-based vectors in the induction of gene silencing [Gossele and Metzlaff  describeilencing .Apart from the contribution of satRNAs in basic research into molecular virology, satRNAs have also been used in application-oriented studies. One of their potential applications lies in the development of satRNA-based vector systems for the expression of foreign genes in plants. In comparison with using other types of vectors for foreign gene expression, such as plasmids and viral vectors, using satRNAs and satellite viruses as vectors has the following advantages: i) ease of manipulation, ii) high ructures , which iIn addition, the symptom-attenuation features of satRNAs have been successfully exploited in developing satRNA-based disease-management systems, either directly, with satRNAs used as biological control agents ,126, or 7.et al. [With significant advances in research into satRNAs and satellite viruses, our understanding of the pathogenicity, evolution, and molecular biology of satRNAs and satellite viruses is now gaining momentum. Further breakthroughs can be expected soon. Although the detailed mechanisms remain to be elucidated, clearly, RNA silencing mechanisms are involved in these processes: satRNAs may use RNA silencing mechanisms of the host plants to target biologically important genes to exacerbate symptoms , and viret al. . The autConsiderable progress has been made in the comprehensive understanding of satRNAs and satellite viruses; however, several intriguing questions still remain. The following topics, for example, those concerning the involvement of RNA silencing mechanisms in the pathogenicity, population dynamics, and possibly the origin(s) of satRNAs and satellite viruses, are expected to continue to attract the attention of researchers in the coming years. (1) How do viruses, satellite viruses, or satRNAs actually “harness” the RNA silencing mechanism of their host plants for their own benefits? (2) What are the actual targets of RNA silencing mechanisms in host genomes directed by satRNAs and satellite viruses that eventually lead to the development of symptoms? (3) Given that satRNAs depend on helper viruses for major biological functions, why do certain satRNAs observed in laboratories or specific field conditions evolve to compete with or reduce the expression of their helper viruses, instead of evolving toward a mutually beneficial situation, as has been observed for TCV and satC ,49? Clea


Numerous studies have investigated cospeciation (or cophylogeny) in various host-symbiont systems, and different patterns were inferred, from strict cospeciation where symbiont phylogeny mirrors host phylogeny, to complete absence of correspondence between trees. The degree of cospeciation is generally linked to the level of host specificity in the symbiont species and the opportunity they have to switch hosts. In this study, we investigated cophylogeny for the first time in a microalgae-virus association in the open sea, where symbionts are believed to be highly host-specific but have wide opportunities to switch hosts. We studied prasinovirus-Mamiellales associations using 51 different viral strains infecting 22 host strains, selected from the characterisation and experimental testing of the specificities of 313 virus strains on 26 host strains.All virus strains were restricted to their host genus, and most were species-specific, but some of them were able to infect different host species within a genus. Phylogenetic trees were reconstructed for viruses and their hosts, and their congruence was assessed based on these trees and the specificity data using different cophylogenetic methods, a topology-based approach, Jane, and a global congruence method, ParaFit. We found significant congruence between virus and host trees, but with a putatively complex evolutionary history.Mechanisms other than true cospeciation, such as host-switching, might explain a part of the data. It has been observed in a previous study on the same taxa that the genomic divergence between host pairs is larger than between their viruses. It implies that if cospeciation predominates in this algae-virus system, this would support the hypothesis that prasinoviruses evolve more slowly than their microalgal hosts, whereas host switching would imply that these viruses speciated more recently than the divergence of their host genera. Micromonas, Ostreococcus and Bathycoccus[Mamiella, Mantoniella, Dolichomastix and Crustomastix). The genomes of several species in the former three genera and their viruses have been entirely sequenced  Manton and Parke 1960, and 3 Ostreococcus species: O. tauri[O. lucimarinus[O. mediterraneus[Micromonas, Guillou et al. distinguished 3 major genetic clades [Ostreococcus, Rodriguez et al.[Bathycoccus strains recorded so far form a more homogeneous evolutionary group[Viruses are recognized as important players in marine microbial ecosystems, but whithycoccus (as wellced (see). Numeroced . In MicB and C) while otB and C)-24 furthB and C) suggestiz et al. have desz et al., and alsz et al.. All of ry group, but recry group. A similry group. Many gery group,27. HencWe hereby investigate the cophylogenetic pattern in this host-virus system using molecular phylogenies, by experimentally assessing the specificity of many virus strains on several host strains, and we use dedicated numerical methods to assess the level of cospeciation. Understanding how hosts and viruses coevolve, taking into account the observed pattern of host specificity, is crucial for predicting the possibility of viral host-switching and for understanding speciation processes. For example, strict host specificity and limited capacities for viruses to switch hosts should be reflected in a strong cospeciation pattern, whereas strict host specificity with no cospeciation suggests a high speciation rate in viruses.We show that these viruses are generally highly host-specific and display a significant, while complex, cophylogenetic pattern with their hosts. This may have important implications for the ecology and the dynamics of planktonic ecosystems.Micromonas viruses, and 4 out of 18 (22.2%) in Ostreococcus viruses). Host range (i.e. specificity) varies from 1 to 6 in virus strains studied here, and 18 viruses out of 51 (35.3%) are strict specialists, infecting only 1 host strain.Experimental tests of host-specificity suggest that virus strains are specific to their host genus Table , and infMicromonas.The susceptibility of host strains to viruses, i.e. the number of virus strains that can infect a given host strain, varies from 1 to 14. The most highly susceptible strains are found in Sequences ranged between 609 and 624 bp (DNA polymerase gene or dpo) for viruses and 1996 and 2238 bp (rDNA 18S (1620–1635 bp) + ITS2 (366–607 bp)) for hosts. Respective alignment lengths were 624 bp (208 AA) and 2289 bp. Unpublished sequences were deposited in GenBank  for hosts (18S + ITS), and CpREV + G + F  for the alignment of virus protein sequences.http://purl.org/phylo/treebase/phylows/study/TB2:S15522). The host phylogeny obtained is clearly coherent with the phylogeny of Mamiellophyceae published by[Ostreococcus and Bathycoccus form the Bathycoccaceae and Micromonas strains cluster separately. Three distinct clades are observed in Ostreococcus, clustering strains in the clades A, C and D defined by[Micromonas. Bathycoccus is much more homogeneous, and the only two strains retained are closely related. However, the 18S sequence from RCC1105 (Bathycoccus) included an intron that was removed for the phylogenetic analysis.Whatever the methods (Bayesian inference (BI) or maximum likelihood (ML)) and datasets (DNA or AA for viruses), phylogenetic trees were the same for hosts and very similar for viruses, therefore only BI trees are presented  between host and parasite trees for the Micromonas dataset (78/111 significant links) and the Bathycoccus and Ostreococcus dataset (12/26 significant links) but some individual host-virus links were found non-significant. For the Ostreococcus dataset, the global fit was not significant . In Jane analyses, the global congruence was significant for the Micromonas dataset (P = 0.001), but not for the Bathycoccus-Ostreococcus dataset (P = 0.1) and the Ostreococcus dataset (P = 0.46). Note that the results were similar with the trees obtained from BI or ML trees, and using a virus tree where the monophyly of the viruses of Micromonas, Ostreococcus and Bathycoccus were each enforced  prasinoviruses are specific to their host genus (2); within a genus, their viruses are generally specific to a clade (i.e. they can infect different host strains that belong to the same clade) (3); the cophylogenetic analysis using ParaFit and Jane revealed significant patterns of associations between host and virus phylogenetic trees and then suggests the existence of a common macroevolutionary scenario between Mamiellophyceae and their viruses.Ostreococcus strains and their viruses[Micromonas viruses[While viruses can often infect several host species or genetic clades, a phylogenetic specificity is nevertheless clearly observed, i.e. prasinoviruses tend to infect related host strains, as observed in  viruses. This ha viruses,32, and  viruses. In the  viruses,21,22. B viruses allow th viruses. We hypo viruses,35.Micromonas RCC2484 or Ostreococcus tauri RCC745) are susceptible to a wider range of viruses than others (e.g. Micromonas RCC804 or Ostreococcus RCC1108). This may be related to lower resistance of these Mamiellale strains to viruses, as previously observed by Thomas et al.[Micromonas perhaps reflects the higher number of host and virus strains found in this genus.Some host strains . The differing results obtained by ParaFit and Jane for Bathycoccus-Ostreococcus and Ostreococcus only might also arise because the genetic distances (used by ParaFit) do not always correlate with phylogenetic (patristic) distances as used in Jane. Thus genetically close viruses tend to colonize close hosts but this may not be always the case at a phylogenetic point of view supporting the hypothesis that viruses can switch to different, but not too distantly related, host strains. The global significance observed for the complete dataset with all methods confirms the genus-specificity of viruses: Bathycoccus, Ostreococcus and Micromonas have their own viruses that do not cross the genus boundaries with detectable frequencies. Within each genus, even if viruses tend to be clade-specific, several strains possess a wide intrageneric, and probably interspecific, host range. This is especially true in Micromonas viruses where several strains can infect hosts from the three clades, while this pattern is much less frequent in Ostreococcus viruses. However, this is not strong enough to break the significant cophylogenetic congruence between Micromonas strains and their viruses (P = 0.01), while between Ostreococcus-Bathycoccus and their viruses the cophylogenetic signal is not significant, as well as between Ostreococcus and their viruses. The different results obtained with ParaFit and Jane for the Ostreococcus-Bathycoccus dataset may in part be due to the different ways these methods works: ParaFit relies only on distances and the influence of the tree topology on the outcome is far less important than in a method such as Jane. However, this issue exists for the Micromonas as well as for the Bathycoccus-Ostreococcus and Ostreococcus datasets , and the cophylogenetic congruence in the Bathycoccus-Ostreococcus dataset is only slightly below the significance threshold with Jane. Duplication and sorting probably also play a role here to explain the lack of topological congruence between trees. However, while viruses seems currently unable to switch from a genus to another, the absence of match between host and virus phylogenetic trees at the genus level suggests an early host-switch from Bathycoccus to Micromonas whose colonization by prasinoviruses would then be more recent. That could explain the more general pattern of association between Micromonas strains and their viruses with strains displaying a cross-clade specificity.A significant global signal of cospeciation was found with all methods used, suggesting that Prasinovirus evolution is in part driven by the evolution of their hosts, or at least that related viruses tend to use related hosts. When partitioning the dataset into In most previous cophylogenetic analyses on host-virus systems, a significant cospeciation signal was found,50,52-64The presence of a cospeciation signal does not necessarily imply real cospeciation, i.e. a significant amount of concomitant speciation in hosts and their parasites. For example, a host switch to a sister host species followed by a speciation of the parasite produces a false cospeciation pattern,65,67. IThe data and analyses provided in this paper support that prasinoviruses, while generally highly host specific, sometimes display a wide host range, with some strains able to infect hosts from different species. This can have important consequences when considering the role of viruses in microbial ecology.A significant cospeciation signal between prasinoviruses and their hosts has been found in the cophylogenetic analyses performed in the present study, but their joint evolutionary history is complex, certainly involving host switches, duplication and losses, in addition to cospeciation events. Because it has been shown in a previous study that host genomes diverge more than corresponding viruses, additional data and analyses are needed to identify cospeciation events and to estimate the timing of these events, in order to be able to compare evolutionary rates in prasinoviruses and their hosts.Ostreococcus lucimarinus (CCE9901) and part of the viruses are referred to by their RCC (Roscoff Culture Collection) numbers . Viruses from Bathycoccus, Micromonas, Ostreococcus are respectively named BatV or BpV, MicV or MpV, and OtV, OmV or OlV with numbers and letters corresponding to strains and the clade containing the host strain used for isolation. For example, MicAV31, refers to a Micromonas virus (strain 31) isolated from a clade A host.Hosts and viruses were isolated from environmental samples and kept in culture collections in Banyuls-sur-Mer and Roscoff. All hosts except 4  and conserved at 4°C. This technique ensures the presence of active viral particles in the isolate.Virus isolation and purification were obtained by a plating technique,13. ThisIsolation and growth of host strains was performed as in. Briefly7 cells/ml of a growing host culture and a 1.5% solution of agarose) and we added 2 μl of virus on the top of these plates. They were cultured  inside a transparent plastic box to maintain humidity for 10 days. Plates that were not lysed 10 days after viral inoculation were considered not to be susceptible to infection by the virus.We first assessed the pattern of host specificity, i.e. the host range of each viral strain investigated. Prasinoviruses infect hosts that can be cultured on plates, allowinEach test was performed in duplicate, to obtain a precise picture of the global pattern of host specificity in prasinoviruses. The specificity of 313 virus strains was tested on 26 host strains.2, 30 pmol of each primer and 0.5 U of Taq DNA polymerase (Promega). PCR bands were purified directly by using a nucleospin kit (Macheray-Nagel company) and DNA fragments were sequenced  of the Station Biologique de Roscoff, France). To control for PCR or sequencing errors fragments were sequenced in reverse and forward directions and all nucleotide differences were checked visually.Prasinoviruses are typically characterized by analyzing the sequence of a portion of the DNA polymerase gene (or dpo),72. This2 solution , 2 μl of deoxynucleoside triphosphate , 0.5 μl of each primer , 0.125 μl of Taq Polymerase , sterile water and 1 μl of extracted DNA. PCR products were cloned using the TOPO TA cloning kit  following the protocol provided by the manufacturer. The 18S and ITS2 were concatenated (named here 18S + ITS) after their homogeneity was established using a partition homogeneity test[Algal hosts were characterized via the sequencing of the full 18S rDNA (SSU) and Internal Transcribed Spacer 2 (ITS2). DNA was extracted by a modified cetyltrimethylammonium bromide (CTAB) protocol, and celity test.We combined the results of the host specificity experimental assessment with molecular data to select a non-redundant virus dataset, and we kept the 51 virus strains differing in term of host specificity or dpo nucleotide sequence and the 22 host strains differing in their susceptibility to viruses or with differences in sequences. The full cross-infection dataset (313 virus strains on the 26 host strains) is available on request to the authors.6 generations, trees sampled every 100 generations, and burnin value set to 20% of the sampled trees. In BI, coding DNA sequences (dpo) were considered with an evolutionary model designed for coding sequences taking the genetic code into account[Sequences alignments were performed with MAFFT v5,77, and  account-84, and  account. We chec account,86 and vMicromonas is the basal lineage) and[No outgroups were used: trees were oriented using previous knowledge from for hostage) and for viruhttp://sites.google.com/site/cophylogeny, and was used here to draw the tanglegram on Figure Several methods have been published to study cophylogenetic patterns between hosts and their symbionts,41, whicGenBank accession numbers for the sequences used in this study are provided in Tables Sequences newly submitted for this study have GenBank accession numbers KF500985 to KF501037.The authors declare that they have no competing interests.LB, CC, RE, EF and NS carried out the molecular biology and virology experiments. YD and LB performed the data analyses. YD, LB, NS and NG wrote the manuscript. All the authors read, edited and approved the final manuscript.


Recent transmission of dengue viruses has increased in tropical and subtropical areas and in industrialized countries because of international travel. We describe a case of nosocomial transmission of dengue virus in Germany by a needlestick injury. Diagnosis was made by TaqMan reverse transcription–polymerase chain reaction when serologic studies were negative. Aedes mosquitoes in tropical regions worldwide. The global incidence of epidemic and endemic dengue fever has increased substantially and is estimated at 50–100 million cases per year. International travel leads to imported cases in countries of the Northern Hemisphere (Dengue viruses are transmitted by i), a nurse sustained a needlestick injury with a hollow needle that had been used for drawing blood from the index patient. The needlestick resulted in a bleeding puncture wound that was immediately treated with an antiseptic. The index patient did not report any high-risk activity for HIV or hepatitis B virus, and the nurse had been immunized against hepatitis B virus. Therefore, no specific postexposure prophylaxis was performed. The nurse had previously been in good health and had not traveled outside Germany in the preceding 12 months.The index patient, a 26-year-old woman, was admitted to the infectious disease ward of a university hospital with a temperature of 40°C and myalgias 3 days after she returned from a 3-week trip to Cambodia and Thailand. Dengue virus infection was subsequently diagnosed, and mild hepatitis and a rash developed. She was discharged in good condition after the fever subsided. On the day of admission of the index patient (day 0n). Physical examination showed bilateral cervical lymphadenopathy. On day 2n, she visited our service, where dengue virus infection was diagnosed by using a Light Cycler  polymerase chain reaction (PCR) method. Her symptoms lessened gradually over the course of 4 weeks, and she was on sick leave for 5 weeks. The time frame of the respective clinical presentation and the virologic results of the index patient and the nurse are shown in the Four days after the needlestick, headache, myalgias, and arthralgias developed in the healthcare worker, for which she took ibuprofen. Seven days later, when she was experiencing an intense headache and noticed a macular rash on her trunk, she sought treatment from a local doctor  M capture enzyme-linked immunosorbent assay (ELISA) and PanBio dengue indirect IgG ELISA  (–This is the fourth reported case, to our knowledge, of nosocomial dengue virus transmission (The diagnosis was confirmed in both cases by both seroconversion and detection of dengue viral RNA by TaqMan RT-PCR; the latter gave positive results in both cases 3 and 6 days, respectively, before serum specimens were shown to contain antibody. Dengue viremia is known to correlate well with the presence of fever (


We analyzed the virus shedding of an oseltamivir-treated patient who had been infected with the pandemic swine-origin influenza A (H1N1) virus which had an oseltamivir-sensitive neuraminidase. The virus was isolated from the pharyngeal swabs of the patient using MDCK cells, and the virus genome RNA was detected in the same samples both by real-time RT-PCR and RT-PCR. The virus was isolated until 44 h after oseltamivir administration although the virus genome was detected until one day after oseltamivir treatment was stopped. Due to their high sensitivity, RT-PCR and real-time RT-PCR may cause misdiagnosis by detection of viral genome which does not infect, and classical virus isolation and clinical symptoms are recommended for the evaluation of oseltamivir treatment. The novel swine origin influenza A virus (S-OIV) H1N1) caused pandemic in 2009-2010 N1 caused. Here, wA 24-year-old female in Shanghai, China was infected with the pandemic S-OIV in September 2009. She had received a seasonal flu vaccination (Sanofi Pasteur) the previous year. Her first symptom was a sore throat, and on the 4th day after the onset of her sore throat, she had a high fever (>38.5°C). The next day (the 5th day after onset), she visited a clinic. Her blood cell count was normal. Her chest X-ray did not show bronchitis or pneumonia. Oseltamivir (75 mg × 2) was administered at 36 hours after the onset of her high fever for five days. On the second day of oseltamivir administration, her high fever disappeared, and all eight segments of the S-OIV genome were cloned from her pharyngeal swabs, and the sequences of the pandemic S-OIV were confirmed. Its neuraminidase (NA) contained an oseltamivir-sensitive sequence (275H). Since then, pharyngeal swabs were collected everyday for eight days and subjected to virus isolation and examined for virus genome RNA (from the 6th to the 13th day). Each pharyngeal swab was aliquoted into three fractions: for virus isolation, for RT-PCR, and for real-time RT-PCR, and each experiment was performed in a different lab. For virus genome detection, RNA was extracted from the pharyngeal swabs using Trizol (Invitrogen). The RNA was then analyzed by real time RT-PCR for S-OIV haemagglutinin (HA), which was performed in a SmartCycler II System (Cepheid) using a QuantiTect Probe RT-PCR kit (Qiagen) with the primers (HA primer 1 and 2) and HA probe listed in μm filter (Millipore) and then incubated in DMEM with 2 μg/ml trypsin (DIFCO), 1 mg/ml BSA (Sigma), 50 units/ml of penicillin (Invitrogen), 50 μg/ml of streptomycin (Invitrogen), and 0.1 mg/ml kanamycin (Invitrogen) at 37°C for the detection of cytopathic effects (CPEs). When CPEs were detected, the pandemic S-OIV genome segments were also identified by RT-PCR of the culture supernatant.Her pharyngeal swabs were also inoculated into MDCK cells after filtration through 0.22 Oseltamivir is accepted as the standard treatment for S-OIV , 4. AlthBy 36 hours of oseltamivir administration in previous cases, the influenza A/Texas/91 (H1N1) virus had disappeared from the patients' nasopharyngeal swabs . The +++The classical virus isolation and clinical symptoms are recommended for the evaluation of oseltamivir treatment although RT-PCR and real-time RT-PCR are convenient.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no conflict of interests.L. Weng and Q. Wang collected samples and performed RT-PCR, W. Wang, P. Ren, and V. Deubel performed virus isolation and real-time RT-PCR, and L. Weng and T. Toyoda wrote the paper.


Background. Rabies virus (RABV) has circulated in Madagascar at least since the 19th century. Objectives. To assess the circulation of lyssavirus in the island from 2005 to 2010. Materials and Methods. Animal (including bats) and human samples were tested for RABV and other lyssavirus using antigen, ribonucleic acid (RNA), and antibodies detection and virus isolation. Results. Half of the 437 domestic or tame wild terrestrial mammal brains tested were found RABV antigen positive, including 54% of the 341 dogs tested. This percentage ranged from 26% to 75% across the period. Nine of the 10 suspected human cases tested were laboratory confirmed. RABV circulation was confirmed in 34 of the 38 districts sampled. No lyssavirus RNA was detected in 1983 bats specimens. Nevertheless, antibodies against Lagos bat virus were detected in the sera of 12 among 50 Eidolon dupreanum specimens sampled. Conclusion. More than a century after the introduction of the vaccine, rabies still remains endemic in Madagascar. Lyssavirus (Rhabdoviridae family), including the rabies virus (RABV), the most common [Rabies is a zoonotic disease caused by 11 viral species belonging to the genus t common . These vt common . There it common .http://www.who.int/rabies/rabies_maps/en/index.html). Rabies virus has circulated in Madagascar at least since the 19th century. The son of one administrator of the former French Colony was reported dead of rabies in 1896, and his death was one of the reasons of the establishment of the Institut Pasteur in Madagascar in 1898. The first rabies postexposure treatment using rabies vaccine was implemented in 1902. Since that period, several reports have described the rabies situation in the island [Lyssaviruses are present in all continents with the exception of Antarctica. RABV is the most widespread, widely distributed across the globe, with only a few countries (mainly islands and peninsulas) being free of the disease. Madagascar, an island in the south-western part of the Indian Ocean, does not belong to these exceptions  exposed to these animals. Human samples consisted of postmortem brain biopsies or postmortem skin biopsies taken from the nape of the neck, sent generally at +4°C by hospital staff. Upon reception at the NLR, brain biopsies were kept at +4°C and processed within 48 h. Skin biopsies were kept at −80°C till processing.Furthermore, samples collected from bats were also tested. They were obtained during a survey looking for virus associated to bats. Samples consisted of sera, blood clots, and pharyngeal swabs kept in viral transport medium (VTM). They were sent within 12 hours to the laboratory and then stored at −80°C at their arrival. When the field was far from the laboratory, they were stored in liquid nitrogen and then transported to the laboratory. When tested, each clot was grinded at a 1 : 10 dilution in cell culture medium (DMEM) containing 30% foetal calf serum and centrifuged at 3,000 rpm for 10 min at +4°C. Then pools of up to 10 supernatants or 10 pharyngeal swabs VTM were constituted before testing.Rabies nucleocapsid detection was performed by fluorescent antibody test (FAT) using rabbit IgG against RABV nucleocapsid  and performed on the brain postmortem biopsy as the standard .RNA was extracted from skin biopsies according to the procedure described by Dacheux and colleagues . RNA wasLyssavirus RNA detection was performed using a reverse transcription and a heminested PCR targeting a conserved region of the polymerase genes of lyssaviruses .Virus isolation was performed to confirm the negative result of the rabies virus antigen detection in animal samples tested routinely for rabies. From 2005 through 2007, virus isolation was performed in newborn mice , then isVirus isolation in new-born mice was also used for the samples collected from bats.Antibodies against RABV, Lagos Bat Virus (LBV), European Bat Lyssavirus type 1 (EBLV-1), EBLV-2, Mokola virus (MOKV), and Australian Bat Lyssavirus (ABLV) were detected in bat sera using lyssavirus rapid fluorescent focus inhibition test .n = 409, 90.9%), including dogs  and cats . We noticed that lemurs, an endemic primate from Madagascar, counted for 2% of the animal samples. All lemurs sampled were reared as pets. Brain was available for all animals. Eleven human suspected rabies cases were also laboratory investigated. Human samples consisted of skin biopsy for 6 cases, brain for 4 cases, and cerebrospinal fluid for one case. Fourteen samples were inadequate and could not be tested, mostly because of inadequate storage  were found positive using FAT. All the samples from lemurs were tested negative. Cattle and pigs, not frequently sampled, were often found positive. More than half of the dogs tested were found infected . This peDuring the 6-year period, the 447 samples tested were received from 38 of the 111 administrative districts of Madagascar. Most of these samples  were received from Antananarivo province. Rabies circulation was confirmed in 34 of the 38 districts . The virCryptoprocta ferox), the largest mammalian carnivore of Madagascar, and one roof rat (Rattus rattus). They tested negative.Brain samples from only two wild terrestrial nonflying mammals were received: one fossa (Eidolon dupreanum)  . No lyssPteropus rufus) and from 50 Malagasy straw-colored fruit bats (Eidolon dupreanum) were tested for antibodies against lyssaviruses. Antibodies against EBLV-1 and LBV were detected in five and one Malagasy flying fox, respectively. Antibodies against LBV were detected in 12 Malagasy straw-colored fruit bats (24%), titers ranging from 35.2 to 65. No antibodies were detected against MOKV, EBLV-2, and ABLV.Sera from 28 Malagasy flying foxes  in Ihosy district, in 2007, and the strain obtained from this case was confirmed as a lyssavirus of the species RABV, phylogenetically closely related to those circulating in Malagasy dogs (data not shown). Consequently, the question of a possible vector in the wild terrestrial carnivorous mammals remains unanswered. This question is of importance considering a rabies control programme targeting the eradication of the rabies in the island. Several endemic or (few) introduced carnivorous mammals (Families Viverridae and Herpestidae) are present in Madagascar . So far,Eidolon dupreanum) and Dakar bat virus from the Peters's wrinkle-lipped bat (Mormopterus jugularis) (unpublished data). Low prevalence of active infection (detection of virus) has been observed in North American and European bats colonies (0.1 to 2.9%), especially in clinically normal bats [Chaerephon pumilus) [Eidolon helvum), the second of the two species in this African genus in various countries of Africa [Our extensive survey in bats failed to detect any lyssavirus associated to these mammals. The molecular technique we used to detect lyssaviruses was demonstrated to be sensitive, reproducible, and repeatable . Furthermal bats . Becausepumilus) . Interesf Africa . We isolf Africa . These tf Africa , 23. Thef Africa .We recently showed that a heminested PCR targeting a conserved region of the polymerase genes of lyssaviruses and applied to antemortem or postmortem skin biopsy (a specimen easier to collect than a piece of brain) was a successful procedure to perform rabies diagnostic . We raisSo far, for economic reasons, there are rabies postexposure prophylaxis centres in only 26 of the 111 administrative districts of Madagascar. We received samples of rabies-suspected cases from only 13 of them, and rabies virus circulation was confirmed in all of them. There is a need to confirm repeatedly its circulation in all of these 26 districts, especially in two islands (Nosy Be and Sainte Marie), where there is no recent report of rabid animals. Sampling should be promoted in the 13 other districts to evaluate the pertinence of these centres.More than a century after the introduction of the vaccine against rabies in Madagascar, rabies remains endemic in the island. So far, preventing human rabies through dog rabies control and eventual elimination has been limited to local initiative. Madagascar, like other countries, is facing numerous public health issues. Because of the low incomes of the country and the lack of epidemiological data, this disease has not been prioritized, and a control program could not reasonably start. However, Madagascar is an island, and the elimination of rabies and its sustainability should be facilitated by the limited risk of introduction of rabid animals Therefore, the collection of such data  should be promoted at first on pilot scale in order to validate the tools used. Afterward, data collection should be expanded to the rest of the country, while a pilot rabies control program  should start on pilot sites and then extended to the rest of the country.


A biosensor that relies on the adsorption immobilization of the 18-mer single-stranded nucleic acid related to dengue virus gene 1 on activated pencil graphite was developed. Hybridization between the probe and its complementary oligonucleotides (the target) was investigated by monitoring guanine oxidation by differential pulse voltammetry (DPV). The pencil graphite electrode was made of ordinary pencil lead (type 4B). The polished surface of the working electrode was activated by applying a potential of 1.8 V for 5 min. Afterward, the dengue oligonucleotides probe was immobilized on the activated electrode by applying 0.5 V to the electrode in 0.5 M acetate buffer (pH 5.0) for 5 min. The hybridization process was carried out by incubating at the annealing temperature of the oligonucleotides. A time of five minutes and concentration of 1 μM were found to be the optimal conditions for probe immobilization. The electrochemical detection of annealing between the DNA probe (TS-1P) immobilized on the modified electrode, and the target (TS-1T) was achieved. The target could be quantified in a range from 1 to 40 nM with good linearity and a detection limit of 0.92 nM. The specificity of the electrochemical biosensor was tested using non-complementary sequences of dengue virus 2 and 3. Infection with DENV may cause an acute “influenza-like” febrile disease called classic dengue fever (DF), or progress at the time to dengue hemorrhagic fever (DHF) characterized by bleeding and plasma leakage [Dengue is a disease caused by four serologically related viruses called dengue virus (DENV) type-1, -2, -3 and -4. They are members of Aedes aegypti (Diptera: Culicidae) is responsible for the spread of diseases such as yellow fever and dengue among humans because of its role as a vector [The mosquito a vector . Yellow a vector , and thea vector –7.Dengue virus is difficult to control due to massive urbanization, overpopulation, continually increasing travel, and failure to maintain effective control programs against the mosquito vector . To dateDue to their high sensitivity, small dimensions, low cost, and compatibility with microfabrication technology electrochemical transducers are often used for detecting hybridization events ,15,16. SA technique using direct guanine oxidation signal on carbon paste electrodes in which PCR amplicons of specific genotypes of the factor V Leiden mutation were identified has been previously reported . In this2.2.1.Probe: Dengue virus type specific 1 (TS-1P)_ 5′CGTCTCAGTGATCCGGGG 3′; Target: Dengue virus type specific 1 (TS-1T)_ 5′ CCCCGGATC ACTGAGACG 3′; Non-complementary: Dengue virus type specific 2 (TS-2 NC)_ 5′ CTGTTCATGG CCCTTGTGG CG 3′; Dengue virus type specific 3 (TS-3 NC)_ 5′ GCTCTGTCTCATGATGATGTT A 3′; PolyG-NC_ 5′ GGGGGGGGGGGGGGGGGGGG 3′. The oligonucleotide stock solutions and dilute solutions were prepared with 0.5 M acetate buffer (pH 4.8) and kept frozen.Tris base was obtained from Promega (USA), and sodium acetate was obtained from Sigma (USA). UltraPure distilled water was purchased from Invitrogen (USA), and reagents were of analytical grade. All DNA oligonucleotides  were synthesized by Integrated DNA Technologies (Brazil). 2.2.The complete genomes of dengue virus types 1, 2 and 3, corresponding to GenBank_ accession numbers U88536.1, U87411.1 and AY099336.1, respectively, were obtained from the National Center for Biotechnology Information (NCBI) database. The probe specific for dengue type 1 (TS-1P) was constructed based on Duebel , and the2.3.Electrochemical analysis was carried out with the Autolab PGSTAT apparatus . Voltammetric signals were measured using a system consisting of two electrodes  Pencil g2.4.2. Afterwards, the graphite electrode was washed with ultra-pure water to remove possible contaminants on the surface of the electrode. The polished surface of the working electrode was then activated by applying a potential of 1.8 V for 5 min [Pencil lead  typically composed of natural graphite was used as the pencil graphite electrode (PGE). The graphite was polished with an emery-impregnated disc to obtain a smooth surface. The body of the pencil was coated well with silicone, resulting in a free area for immobilization of 28.50 mmor 5 min . The wor2.5.The TS-1P probe was immobilized on the activated electrode by applying 0.5 V  to the e2.6.The working electrode with the immobilized probe was immersed in a solution containing the target oligonucleotides in acetate buffer, pH 4.8, and incubated at 57 °C for 3 min. The electrode was then washed with Tris-HCl (pH 7.0) to remove non-hybridized sequences. The same protocol was applied for the interaction of the probe with non-complementary sequences.2.7.To obtain electrochemical signal data, the biosensor surface with hybridized probe and target nucleic acid was immersed in an electrolytic cell containing Tris-HCl (pH 7.0), and attached to the Autolab potentiostat, equipped with GPES 4.9 software. Differential pulse voltammetry was applied to detect electrochemical signals, whereby a potential sweep was applied between 0.5 and 1.2 V, at a pulse amplitude of 50 mV and scan rate of 20 mV/s. The raw data were treated using the GPES software with a moving average baseline correction using a “peak width” of 0.01.2.8.Experimental data were analyzed using the Statistica 8 software by non-parametric tests. To evaluate statistical significance, the Mann–Whitney U test was used for the comparison of two unpaired groups. The Kruskal-Wallis test was used for the comparison of multi independent group data. A level of p < 0.05 was considered significant.3.3.1.vs. Ag/AgCl reference electrode) of 1.8 V for 5 min. The pretreatment of the carbon surface increases its roughness and hydrophilicity, facilitating the adsorption of the probe on the electrodes [Research has demonstrated the utility of graphite electrodes  in the cectrodes .The covalent immobilization of the probe is more stable, but the electrode surface may contain groups capable of reacting with and fragmenting the oligonucleotide molecule, damaging and changing its original structure . The immThe influence of the pretreatment of the PGE at 1.8 V was investigated. 3.2.The immobilization of the probe on the activated PGE involved the application of a potential of 0.5 V for 5 min ,23,29,30et al. [The influence of the number of guanine in the probe was investigated immobilizing 1 μM of TS-1P (with seven guanines) and TS-1T (with five guanines) on different working electrodes. et al.  with a bThe TS-1T sequence was chosen as a target sequence, because it is present in the dengue virus genome. TS-1P is the sequence complementary to the RNA present in the virus, and thus used as the probe for construction of the biosensor. The data obtained were analyzed using the CLC Combined Workbench v 3.6.1 and NCBI Software.3.3.Probe immobilization is a crucial step for fabricating an electrochemical biosensor . In Figu3.4.The effect of probe concentration is shown in The results showed that oxidation peak currents increased from 0.1 μM up to 1.0 μM with TS-1P. However, since the 1 μM the signal is stabilized, it was the chosen concentration to carry on further experiments. The results, analyzed with STATISTICA 8.0 using non-parametric tests , showed that 1 μM–10 μM results were statistically identical. Thus, the concentration of the 1 μM and 5 min were considered the best experimental conditions for electrode preparation. Next, the study sought to build a working electrode, with an area completely filled with the oligonucleotide sequence (probe).3.5.The electrodes modified with nucleic acid (polynucleotide) identify the sequence of complementary bases, through the formation of a double helix. This identification is effective and specific showed in the presence of other non-complementary sequences. When the target oligonucleotide sequence corresponds to the probe (based on the complementarily principle stating that G pairs with C and A with T or U), a hybrid is formed (probe-target).The development of biosensor DNA or RNA consists of three steps: (a) adsorption of the oligonucleotide probe; (b) hybridization between the probe and its complementary sequence (the target) to form the hybrid; and (c) transduction . The hybThere have been various reports on label-free oligonucleotide biosensors for hybridization detection ,22. The The hybridization experiments were performed in a microtube (0.2 mL), containing the probe-modified PGE, which were immersed in acetate buffer containing a known amount of complementary target oligonucleotides at a specific annealing temperature. Detection of target oligonucleotides was monitored with the formation of a guanine current peak on the probe immobilized on PGE with or without the target sequence.The results are shown in the voltammogram for guanine oxidation  on the probe-modified and activated PGE, before and after hybridization in solutions of complementary and non-complementary oligonucleotides . The curThe current peak of the hybridization at the concentrations 0.5, 1, 10, 20, 30, 40 and 100 nM was statistically different from that observed with the TS-1P sequence at the concentration of 1.0 μM. The results showed that the complementary sequence could produce hybridization, causing a decrease in the guanine oxidation signal.3.6.The highest guanine oxidation signal was observed with TS-1P immobilized on PGE, which has seven guanine bases present in the oligonucleotides. Experiments with non-complementary oligonucleotides including TS-2 NC, TS-3 NC and PolyG-NC  were perThese results confirmed that the electrochemical detection in the present study successfully distinguished complementary sequences from non-complementary sequences, using the annealing temperature to perform the hybridization. The selectivity of the biosensor was also examined in a sample containing both complementary and non-complementary sequences in similar proportions. The mixture showed a peak current similar to immobilized TS-1P after hybridization with complementary sequence. This demonstrates that the presence of non-complementary samples did not interfere in the specificity of the biosensor. These results are important for the construction of the device, because the system can be better controlled by regulating temperature than by other means such as the application of a potential.3.7.The detection limit was determined using the synthetic target sequence. The difference in the guanine oxidation signal of the probe-modified PGE in the absence and presence of complementary sequence (ΔI) increased when the target concentration increased and leveled off at a concentration of 40 nM. As seen in the inset of LOD = 3s/m and yLOQ = 10 s/m, respectively [The detection limit and quantitation limit values were calculated using the following equations: yectively , where sCompared with other nucleic acid biosensors obtained under the same immobilization conditions (adsorption), ours displayed good sensitivity with a hybridization time of 3 minutes. In 4.The importance of oligonucleotide biosensors for hybridization reactions via guanine oxidation has been demonstrated. Oligonucleotide biosensors utilizing pencil graphite for the detection of hybridization are useful for identifying specific nucleotide sequences (dengue virus) with selectivity, providing a convenient and rapid electroanalytical method. The hybridization of oligonucleotides on graphite was used in combination with ADP to obtain information about the hybridization reaction using a guanine oxidation signal. The detection limit for this biosensing electrode with complementary DNA target is found to be 0.92 nM within a hybridization time of 3 minutes.Therefore, we anticipate that modification of the sequences could be used for the diagnosis of other acute infectious diseases and the utility of new electrochemical hybridization utilizing annealing temperature in the detection of hybridization of oligonucleotides using a biosensor for the diagnosis of diseases is promising. The detection of guanine oxidation eliminates the use of some compounds such as methylene blue, metal complexes and toxic substances as hybridization markers in electrochemical DNA biosensors. Breakthroughs in biosensor techniques and prudent design of diagnostics at the molecular level may contribute to future advancements in type-specific dengue diagnosis.


Phlebovirus and Orbivirus genera. Both Phlebovirus and Orbivirus genera include many economic important viruses or serious human pathogens. Quick and accurate identification of microbial pathogens is essential for both diagnosis and response to emerging infectious diseases. The advent of next-generation sequencing technology offers an unprecedented platform for rapid sequencing-based identification of novel viruses. We have developed a customized bioinformatics data analysis pipeline, VirusHunter, for the analysis of Roche/454 and other long read Next generation sequencing platform data. To illustrate the utility of VirusHunter, we performed Roche/454 GS FLX titanium sequencing on two unclassified virus isolates from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA). VirusHunter identified sequences derived from a novel bunyavirus and a novel reovirus in the two samples respectively. Further sequence analysis demonstrated that the viruses were novel members of the  Quick and accurate identification of microbial pathogens is essential for both diagnosis and response to emerging infectious diseases. High-throughput sequencing has recently emerged as a powerful approach to identify both known and novel viruses in clinical specimens. For example, high throughput Sanger sequencing combined with bioinformatic analysis lead to the discovery of human bocavirus , KI polyThe World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) maintains a large collection of virus isolates and provides reagents and support for investigations of virus outbreaks throughout the world . This NIAethomys medicatus) collected in 1971 in Salanga, Central African Republic [Bunyaviridae (acronym SGAV) while the other is an unassigned species in the Poxviridae (acronym SGV) [Salanga virus (strain AnB 904a) was originally isolated from a rodent (Republic  by reseaRepublic . Currentnym SGV) . Howeverth, 1965 from the blood of a bat (Myotis macrodactylus) collected in a mine at Heramatsu, Kagoshima, Japan. The initial isolation was made by intracerebral inoculation of newborn mice. KY-663 virus was found to be partially resistant to treatment with sodium deoxycholate and did not produce a hemagglutinin. Intracranial inoculation of KY-663 into suckling mice causes lethality in 4-5 days, but it is not pathogenic to adult mice [KY-663 virus was originally isolated on July 18Phlebovirus genus in the Bunyaviridae while KY-663 was determined to be a novel member of the Orbivirus genus in the Reoviridae. We tentatively named KY-663 Heramatsu virus (HERMV) in this manuscript.In order to identify and taxonomically classify these two viruses, we used NGS technology to obtain the genomic sequences of those viruses and our customized bioinformatics pipeline, VirusHunter, to analyze the sequences. Furthermore, traditional virologic methods, including electron microscopy and serology, were also employed to provide additional modes of characterization of these viruses. Based on these analyses, Salanga virus was classified as a novel member of the Bunyaviridae. In BHK (baby hamster kidney) cells infected with Heramatsu, virus groups of particles ~55 nm in diameter with dense core typical for Reoviridae were found either in host cell cytosol or inside membrane-bound vacuoles  and then sequenced on the 454 FLX Titanium platform. 20,893 and 8,042 total reads were obtained from Salanga virus DNA and RNA sample preparation respectively. The cell lysate and fluid of Heramatsu virus cell culture were sequenced independently yielding 19,379 and 30,116 reads respectively. Phlebovirus of the family Bunyaviridae were identified. No viral sequence was identified from the DNA sample of Salanga virus. From sequencing of total nucleic acid extracted from the cell lysate and fluid of Heramatsu virus cultured in BHK cells, VirusHunter identified 234 and 186 unique viral reads that shared 28.8-86.1% sequence identity to viruses of the Orbivirus genus in the family Reoviridae. We used a customized data analysis pipeline, VirusHunter, to identify viral sequences from the raw sequences of each sample. VirusHunter classifies each sequence based on the best BLAST similarity to sequences in reference databases , Ngari virus (NRIV), Hantaan (HTNV) and Rift Valley fever virus (RVFV). Most viruses in the Bunyaviridae family are carried and transmitted by arthropods . Many bunyaviruses are classified as emerging pathogens due to their recent increased incidence in new vertebrate hosts and in new geographical locations. Bunyavirus genomes contain three negative-sense, single-stranded RNA segments, designated as large (L), medium (M), and small (S) segment. The family We used all individual reads with detectable similarity to bunyaviruses as well as all sequences that did not share detectable sequence similarity with any sequence in Genbank databases as inputs for assembly using the Newbler assembler. Four contigs of length 555-3853 bp were identified. These contigs were most closely related to Durania virus, Ariquemes virus, Leticia virus (Phlebovirus sp. Co Ar 171616) and Sandfly fever sicilian virus with 61.0, 44.3, 51.7 and 26.3% aa identity respectively. Of the 4 contigs, 2 mapped to the L segment, 1 mapped to the M segment and 1 mapped to the S segment.Using RT-PCR amplification followed by Sanger sequencing we closed the gap between the two initial L segment contigs. Additional RACE (Rapid Amplification of cDNA Ends) reactions extended the initial contigs and yielded final contigs of length 6513 bp (L segment), 4412 bp (M segment) and 1795 bp (S segment). Over-lapping RT-PCR amplification and Sanger sequencing were employed to confirm the sequences with at least 2 fold coverage for L segment nucleotide 174-6225, M segment nucleotide 280 - 4312 and the S segment nucleotide 51-1675. Using standard gene prediction tools, open reading frames were predicted in each segment. No further effort was made to completely sequence the 5’ or 3’ termini of the viral genome segments. Phlebovirus genus [We obtained 6513 bp of the Salanga virus L segment . A singlus genus , with 59We obtained 4412 bp of the Salanga virus M segment and a single open reading frame of 1398 aa was predicted. The predicted ORF was most closely related to the polyprotein of Arbia virus , with 40% aa identity . The M segment of all bunyaviruses encodes the glycoprotein precursor which is subsequently processed to yield two glycoproteins, Gn and Gc. However, the M segments of orthobunyaviruses, phleboviruses and tospoviruses encode an additional protein, the nonstructural protein NSm. The NSm protein is an integral membrane protein localized in the Golgi complex and it is important for virus assembly and morphogenesis . InteresWe obtained 1795 bp of the Salanga virus S segment. Two open reading frames of 269 and 245 aa were predicted. One ORF was predicted on the negative sense genomic strand while the other was encoded on the anti-genomic strand. The predicted ORF positioned at the 5’ end of the genomic strand was most closely related to the nonstructural protein (NSs) encoded in the S segment of Durania virus, an Aguacate group phlebovirus , with 26Phlebovirus genus within the Bunyaviridae family. Both Neighbor-joining and maximum parsimony trees  and at least 160 different serotypes (strains) [The family strains) . The orbstrains) .th, 2012) using BLASTx to identify all viral contigs. Fourteen putative viral contigs with length 131-3835 bp were identified that were most closely related to orbiviruses. The contigs shared 26.7- 71.2 % aa identity to Chuzan virus, AHSV, or BTV, each of which belongs to the Orbivirus genus of the Reoviridae. Chuzan, also known as Kasba virus, is a strain (serotype) of Palyam virus.To obtain the best genome assembly, all sequences with detectable similarity to orbiviruses as well as all sequences that did not share detectable similarity with any sequence in the databases were pooled together and assembled using Newbler assembler. Assembled contigs were compared with NCBI nr database . Segment 2 of Chuzan virus encodes the outer capsid protein VP2. Reassortment of genome segments can contribute to virus diversity and is known to occur in orbiviruses ; the disOrbivirus genomes consist of 10 RNA segments. For most of the Heramatsu virus segments, single contigs were assembled that were approximately the same size the corresponding segment of the most closely related orbivirus in Genbank. The exceptions to this were segment 3 for which we obtained four contigs of length 869, 428, 260 and 886 bp and segment 9 for which we had two contigs of length 131 and 606 bp. These gaps were closed by PCR followed by Sanger sequencing of the cloned PCR amplicons. For segment 3, a final contig of 2603 nt was generated. Overlapping RT-PCR amplification and Sanger sequencing were employed to sequence the segment 3 contig nucleotide 21-2559 with at least 2-fold coverage. For segment 9 a final contig of 738 bp was obtained, of which nucleotides 8-723 were confirmed with at least 2-fold coverage. While these contigs did not represent the complete sequences of the genome segments, they sufficed for taxonomic classification and thus no effort was made to obtain the terminal sequences of any of the segments. Orbivirus genus within the Reoviridae family. Both Neighbor-joining and maximum parsimony trees  collected in September 1971 at Salanga, Central African Republic. The Heramatsu virus (strain KY-663) used in this study was originally isolated from the blood of a bat (Myotis macrodactylus) collected in a mine at Heramatsu, Kagoshima, Japan in July 18th, 1965. Salanga virus (strain AnB 904a) was originally isolated from a rodent . Ultrathin sections were cut on Leica EM UC7 ultramicrotome , stained with lead citrate and examined in a Philips 201 transmission electron microscope at 60 kV. For ultrastructural analysis in ultrathin sections, infected Vero or BHK cells were fixed for at least 1 h in a mixture of 2.5% formaldehyde prepared from paraformaldehyde powder, and 0.1% glutaraldehyde in 0.05 M cacodylate buffer pH 7.3 to which 0.03% picric acid and 0.03% CaClAntigens used in serologic tests were infected newborn mouse brains prepared by the sucrose/acetone extraction method . SpecifiHemagglutination inhibition (HI) tests were done in microtiter plates, as previously described , using fTotal nucleic acid was extracted from Heramatsu virus infected BHK cell culture supernatant and cell lysate independently with a Qiagen DNeasy kit according to the manufacturer’s instructions. DNA and RNA were extracted from Salanga virus infected culture of Vero E6 cells cell separately. Both cell lines were obtained from the American Type Culture Collection . Nucleic acid from each sample was reverse-transcribed to enable subsequent detection of both RNA and DNA viruses and was amplified as previously described ,30. AmplBecause the nucleic acid used for sequencing contained a mixture of host cell DNA and virus RNA, we used a customized informatics pipeline VirusHunter to identify viral sequences. The VirusHunter pipeline is controlled by a master Perl script VirusHunter.pl, which executes each step in the pipeline. The input to the pipeline is a directory path. The directory holds sequencing data from different samples. Each sample has its own directory with a single file containing FASTA format sequence reads. The workflow of the pipeline is shown in 1Remove redundant sequences. Identical or nearly-identical sequences are frequently present in NGS data, either due to the sheer depth of NGS or because many of the pre-sequencing sample preparation methods involve PCR amplification. To reduce the computing cost of downstream analysis, CD-HIT  is first2http://www.repeatmasker.org) is used to mask interspersed repeats and low complexity DNA sequences. A sequence fails the quality control criteria if it does not contain a stretch of at least 50 consecutive non-“N” nucleotides  or if greater than 40% of the total length of the sequence is masked . These sequences are removed from further analysis. Remaining sequences are “good sequences”.Mask repetitive sequences and sequence quality control. Many eukaryotic genomes contain stretches of highly repetitive DNA sequences which cause problems in BLAST-based similarity searches and result in high rates of false-positive alignments. RepeatMasker  for simplicity; for Heramatsu virus, which was cultured in the BHK (hamster) cell line we used the golden hamster Mesocricetus auratus genome as the reference (GenBank Assembly ID: GCA_000349665.1).Filtering host sequences. Sequences are subjected to BLASTn  alignmen4BLASTn against NCBI nt database. Sequences retained from the previous step are queried against the NCBI nt database using BLASTn. Sequences with significant hits  are broadly classified as human, mouse, fungal, bacterial, phage, viral or other based on the taxonomy identity of the best BLAST hit. The NCBI gi – taxid data for nucleotide sequences are uploaded to a MySQL database. The gi number of the best BLAST hit is used to query the database to obtain the taxonomy ID which is in turn used to retrieve the full taxonomy lineage using BioPerl. In some instances, one query has two or more hits with the same e value. If a sequence aligns to both a virus and a sequence derived from another organism type (e.g. bacteria or fungi) with the same e value it is classified as “ambiguous”. All eukaryotic viral sequences are further classified into viral families based on the taxonomy ID of the best hit. Sequences without hits progress to the next step.5BLASTx against NCBI nr database. Sequences retained from the previous step are queried against the NCBI nr database using BLASTx . BLASTx output files are parsed and sequences are phylotyped as described in the previous step. Sequences without any significant hit are placed in the “unassigned” category. 6Report of the FindingsThe final output of the VirusHunter pipeline is a single file summarizing all the viruses identified in each dataset in the input directory. The pipeline can be customized to generate similar outputs for other bacterial, fungal or parasitic sequences. VirusHunter is written in Perl. It uses shell scripting, the BioPerl library, MySQL database, CD-HIT, RepeatMasker and NCBI BLAST suite. The pipeline is fully automated and high-throughput. All components are organized in a hierarchical set of readily modifiable scripts, and multiple copies of the pipeline can be run in parallel. The pipeline is designed for running on a high performance computing cluster using GridEngine as the job scheduler. It can be easily modified to use other job management software. The pipeline can be easily customized, for example to replace the human genome database with that of a different host. Installation and configuration of VirusHunter requires basic knowledge of Perl, MySQL database and Linux system administration. Distribution and source code for VirusHunter 1.0 are available at (http://www.ibridgenetwork.org/wustl/virushunter). Sequences identified as viral as well as sequences that had no significant hit to any sequence in the NR and NT databases were assembled using Newbler  with default parameters. ORFs were predicted and annotated using Artemis . MultiplThe viruses analyzed included the following genera, species, and strains: Aguacate virus , Arbia virus , Arumowot virus , Durania virus , Leticia virus , Odrenisrou virus , Rift Valley fever virus , Rift Valley fever virus , Salehabad virus , Toscana virus , Uukuniemi virus .African horsesickness virus serotype 1 , African horsesickness virus serotype 9 , Bluetongue virus Serotype 10 , Bluetongue virus Serotype 12 , Chuzan virus , Epizootic hemorrhagic disease virus serotype 1 / strain New Jersey , Epizootic hemorrhagic disease virus serotype 7 / strain CSIRO 775 , Great Island virus . St Croix River virus , Yunnan orbivirus . The sequence of the Salanga virus and Heramatsu Orbivirus (HERMV) KY-663 genomic fragments has been deposited in the NCBI database under GenBank accession number KC669549 - KC669551 and KC669539 - KC669548 respectively. Raw data is deposited in the Sequence Read Archive (SRA) under accession number SRR959764.File S1VirusHunter summary output file of the Salanga virus RNA sequencing data.(TXT)Click here for additional data file.File S2VirusHunter summary output file of the Heramatsu Orbivirus (HERMV) KY-663 cell culture lysate sequencing data.(TXT)Click here for additional data file.File S3VirusHunter summary output file of the Heramatsu Orbivirus (HERMV) KY-663 cell culture fluid sequencing data.(TXT)Click here for additional data file.


To the Editor: In 2010, detection of henipavirus (Hendra or Nipah virus) and rubulavirus (Tioman or Menangle virus) antibodies in fruit bats in Papua New Guinea (PNG) was reported , New Britain (1997), and Lae (1999 ) . The 20 Of the 20 samples from Madang, 2 (10%) reacted in the Hendra virus ELISA. Of the 147 samples from New Britain and Lae that yielded definitive VNT results, 11 (7.5%) yielded neutralizing antibodies to Hendra virus and 5 (3.4%) to Nipah virus. All samples with antibodies against Nipah virus also had antibodies against Hendra virus; titers against Hendra virus were greater (4 samples) or equivalent (1 sample) to those against Nipah virus. Reciprocal titers against Hendra virus were 5–160 (median 10) and against Nipah virus, 5–80 (median 10). None of the 103 samples from Lae had antibodies against Menangle virus .Pteropus conspicillatus) from proximate locations (Lae and Madang), the significant differences in antibody prevalence remained: 0 (95% CI 0–23%) in 1999 and 65% (95% CI 50%–78%) in 2009.The common and contrasting findings between the study of Breed et al.  that had a neutralizing antibody titer to Nipah virus had a higher neutralizing titer to Hendra virus , suggesting that the circulating henipavirus was more similar to Hendra virus than to Nipah virus. These findings are supported at a regional level by those reported for nearby Indonesian islands by Sendow et al.  associated with negative ecologic effects  (More robust interpretation of the serologic findings of both studies is constrained by the lack of henipavirus sequence data from PNG and neighboring countries. We concur with Breed et al. (


Pipistrellus pipistrellus bats, which represents the only chiropteran virus isolate found in Europe besides lyssavirus (rabies virus). Evidence was found for adenovirus transmission between bats.We tested 55 deceased vespertilionid bats of 12 species from southern Germany for virus infections. A new adenovirus was isolated from tissue samples of 2  Since the recent discoveries of Ebola virus, Henipavirus, and severe acute respiratory syndrome–associated coronavirus infections, interest in the role of bats as hosts for pathogens has markedly increased  were collected at certified bat rehabilitation centers in southern Germany and were investigated macroscopically, bacteriologically, and histologically.We performed an extensive search for unknown viruses in 55 German vespertilionid bats based on both generic PCR assays and virus isolation techniques, as part of a broader study investigating histopathologic changes in German bats in association with infectious pathogens. Dead or moribund bats of 12 species (cytB) gene, a standard technique for species identification . A cytopathic effect was detected in Vero E6/7 cells after the second passage, indicating the presence of virus in the cell culture. Purified supernatant of these cell cultures was subjected to negative-staining electron microscopy, which showed numerous adenovirus-like particles (Adenoviridae was verified by the first reaction of a generic adenovirus-specific nested PCR (Mastadenovirus and was tentatively named bat adenovirus 2 (bat AdV-2) strain P. pipistrellus virus 1 (PPV1).Of the tested samples from 55 bats, virus was initially detected in only 2 adult common pipistrelles   , panel Byeyamae) .On the basis of newly acquired sequence information, we designed a specific real-time TaqMan PCR to detect bat AdV-2 . Cycler conditions were as follows: predenaturation (95°C for 10 min), 45 amplification cycles , and final extension (72°C for 10 min).After screening all 55 bats from Germany of 12 species, comprising an additional 11 common pipistrelles, an identical adenovirus was detected in 1 additional common pipistrelle. Moreover, the tissue tropism of bat AdV-2 was investigated in all 3 infected bats . Of all In contrast to Maeda et al.  mammalian species  with the available sequence information of a ≈550-bp fragment of the DNA polymerase gene showed their distant relationship. This strict separation reflects either the co-evolutionary development between the 2 adenoviruses  and their host families Pteropodidae and Vespertilionidae or a host switch of the virus originating from a yet-undetermined vertebrate host. To elucidate this problem, further research will be necessary.Various adenovirus types of the genus In conclusion, we isolated a new virus from free-ranging vespertilionid bats, which represents the only chiropteran virus isolate besides lyssavirus (rabies) found in Europe. Moreover, the detection of this chiropteran virus can be connected with its transmission between individual bats living in close proximity to other bats.


During infection, many viruses induce cellular remodeling, resulting in the formation of insoluble aggregates/inclusions, usually containing viral structural proteins. Identification of aggregates has become a useful diagnostic tool for certain viral infections. There is wide variety of viral aggregates, which differ by their location, size, content and putative function. The role of aggregation in the context of a specific virus is often poorly understood, especially in the case of plant viruses. The aggregates are utilized by viruses to house a large complex of proteins of both viral and host origin to promote virus replication, translation, intra- and intercellular transportation. Aggregated structures may protect viral functional complexes from the cellular degradation machinery. Alternatively, the activation of host defense mechanisms may involve sequestration of virus components in aggregates, followed by their neutralization as toxic for the host cell. The diversity of virus-induced aggregates in mammalian and plant cells is the subject of this review.  Increasing evidence suggests that the assembly of many mammalian viruses occurs at specific intracellular sites, which have been termed “virus factories”. The ultrastructure of the factories has been determined for a number of RNA and large DNA viruses that assemble in the cytoplasm, at the microtubule organizing center (MTOC) ,2,3. In In general, mammalian and plant viruses make use of aggregates as scaffolds for anchoring the replication complex, increasing the local concentration of viral and host components required for replication and assembly, and shielding the process of replication from host defense. Alternatively, these aggregates may be part of an innate cellular response that recognizes virus components and targets them for storage and degradation. To understand the aggregation processes accompanying virus infection, it is important to discover the origin of the cellular components that gives rise to the virus‑induced inclusions and smaller aggregates, and to identify the molecular motors that are involved in their trafficking from the site of origin to the final destination. Virus aggregates often result in rearrangement of cellular membrane compartments and/or cytoskeleton. The functions of these organelles are carefully regulated in cells. Changes in cellular architecture may constitute responses to the stress associated with virus infection. Throughout this review we suggest that the line that separates viral aggregates as storage of dead-end material from a functional viral factory is rather artificial. Viruses may target key stages in the regulatory pathways that control organelle structure and function to generate sites that are essential for replication and assembly. The same structures can be associated with cellular defenses against infection and cell stress. Given the co-evolution of viruses with their host cells, changes in cell structure induced during infection are likely to involve a combination of the two strategies.Numerous viruses assemble and replicate in large insoluble inclusion bodies. In the case of mammalian viruses, these inclusions, called “virus factories” or “viroplasm”, are generally localized near the MTOC and maintained by dynein microtubule motor proteins (reviewed in ). Virus Once in the cell, viruses may appear to the host as foreign or misfolded proteins, stimulating an aggresome response. Many viral core particles have a size 60 to 100 nm) similar to the aggregates that are transported to aggresomes by dynein motors . In the 00 nm simPotato leaf roll virus (PLRV) developed large aggregates in cells treated with the 26S proteosome inhibitor clasto-lactacystin B-lactone [Tobacco mosaic virus (TMV) 30-kDa MP was shown to become polyubiquitinated during virus infection and, subsequently, to enter the 26S proteasome degradation pathway [MTOCs are not found in plant cells. Therefore, viral proteins are not gathered according to the aggresome-like pathway for microtubule dependent aggregation of toxic or misfolded proteins. Nonetheless, several studies demonstrated the possible involvement of an aggresome-like pathway in movement protein (MP) accumulation during RNA virus infections. Plasmid encoded protein MP17 of -lactone . Tobacco pathway . TMV inf pathway .Potato virus X (PVX) multiplication. PVX replicase was detected in large membrane bound containers that developed from the ER (approximately 375 nm in diameter) together with the virus MP TGBp3 [The association of positive-stranded RNA viruses’ replicating machineries with intracellular membranes is a feature common in animals, plants and insects (reviewed in ). IntracMP TGBp3 . TGBp3 cMP TGBp3 . ConsequMP TGBp3 . In geneMP TGBp3 ).Herpes simplex virus type-1, Adenovirus type 5 (Ad5), and SV40 accumulate at nuclear sites named promyelocytic leukemia nuclear bodies  ,18,19,20Evidence has accumulated showing that not only DNA viruses, but also RNA viruses belonging to several different families relate to PML/ND10s. Human foamy virus, Human T-cell leukemia virus type 1, Human immunodeficiency virus type 1 and many other mammalian RNA viruses were found to interact with the subnuclear structure PML/ND10s . It is iCajal bodies (CBs) could be considered as a part of the nuclear inclusion family . CBs areViruses often disrupt host cell gene expression. On the other hand, cellular anti-viral response may involve inhibition of viral gene expression via RNA silencing or translational arrest. Degradation, inhibition and storage of mRNA and mRNA-protein complexes (mRNP) are important for cellular homeostasis. While translation of mRNPs takes place in polyribosomes, degradation and storage often occurs in cytoplasmic aggregates . Hence iBrome mosaic virus RNAs accumulate in PBs, confirming the importance of PBs in the formation of replication complexes [Processing bodies (PBs) are cytoplasmic foci in eukaryotic cells that are involved in mRNA decapping, degradation and storage. They contain RNPs, decapping proteins and proteins involved in RNA-induced silencing such as Argonaute (AGO) . Plant Pomplexes .In mammals, upon a large diversity of stress including viral infection, cells inhibit translation by converting active translation initiation complexes into inactive mRNPs. These complexes are shuttled to cytoplasmic stress granules (SG) for storage . Indeed,In plants, SGs were first observed in the cytoplasm of tomato cells upon heat stress and were termed heat shock granules (HSGs) . In addiIndian cassava mosaic virus AV2 protein implicated in viral movement was shown to form cytoplasmic and nuclear inclusion bodies [Abutilon mosaic virus [Cauliflower mosaic virus (CaMV) firstly produced small electron-dense inclusion bodies (EDIBs), which were later exported to a single massive ELIB in each infected cell [The appearance of large inclusion bodies containing virions in geminivirus-infected plants is a characteristic known for many years . The kinn bodies . Similaric virus . Other pted cell ,54,55. Cted cell . It mustted cell . Moreoveted cell . In tomated cell . The molted cell ), howeveted cell . In the ted cell . ResistaThe similarity between TYLCV cytoplasmic large inclusions with perinuclear virus factories or similarity of virion-containing nuclear inclusions with nuclear aggresomes or PML/ND10s is debatable. The absence of MTOC in plants, a required virus factory’s component, has been discussed already; PML/ND10s have not been detected so far in plant cells. At present, we are unable to name the TYLCV-induced inclusions, but their abundance  correlates with efficient virus multiplication. Alternatively, the maintenance of small aggregates containing TYLCV CP is a characteristic of TYLCV-resistant tomatoes .For a long time, electron microscopy studies revealed virus particles arranged in aggregates of different sizes in infected cells. In the current review, we have summarized the multiplicity of virus‑induced aggregates, their function, localization, size and content.Virus-induced aggregation in plants is much less known than aggregation of mammalian viruses, even though cylindrical inclusions induced by the potyvirus Tobacco etch virus , and cryVirus-induced aggregates play a dual role in virus propagation in the infected cells. The recruitment of host cellular proteins into cytoplasmic and nuclear inclusions to facilitate virus replication has been described for many viruses (see above); on the other hand, the same viruses could be captured in cellular protective aggregative structures. For example, replication sites of DNA Vaccinia virus and ASFV can be targeted by SG components and Mx proteins, respectively ,69. Mx pIn the case of TYLCV, two different types of virus-induced aggregates have been described . Large n