Potato leafroll virus (PLRV) by the green peach aphid Myzus persicae, and the transmission of Tomato yellow leaf curl virus (TYLCV) by the sweetpotato whitefly Bemisia tabaci. These tri-trophic levels of interactions and their possible evolutionary implications are reviewed.Persistent circulative transmission of plant viruses involves complex interactions between the transmitted virus and its insect vector. Several studies have shown that insect vector proteins are involved in the passage and the transmission of the virus. Interestingly, proteins expressed by bacterial endosymbionts that reside in the insect vector, were also shown to influence the transmission of these viruses. Thus far, the transmission of two plant viruses that belong to different virus genera was shown to be facilitated by a bacterial chaperone protein called GroEL. This protein was shown to be implicated in the transmission of  It is widely accepted that the transmission of viruses to eukaryotic  hosts by arthropod vectors is coordinated between evolutionarily conserved, but highly specific, proteins. Arthropod-mediated transmission of each pathogen depends on a suite of well-coordinated interactions between evolutionarily conserved protein(s) encoded by the virus, vector and host. Viruses have evolved specific mechanisms to enable their transport across and within multiple vector/host tissues, and to evade or combat vector/host defenses. The specific types of molecules and the mechanisms that make possible such modes of virus transmission by arthropods are, however unstudied for the majority of insects and associated viral genera. This area is of particular interest as it has become more evident that the ability of a viral pathogen to be acquired and transmitted, and to evade host defenses is subject to adaptive, evolutionary change. Luteoviridae, Geminiviridae and Nanoviridae and are the most dependent upon their vector for successful transmission to new hosts and establishment of new infections. Most circulative plant viruses do not have the ability to move between plants and therefore mostly rely on their vector to be injected into the plant tissue from which they can develop a systemic infection in the xylem and, more often, the phloem. The majority of insects which are plant virus vectors are phloem feeders and all have sap sucking mouthparts [Tomato yellow leaf curl virus from Israel (TYLCV-IL) are retained for the entire whitefly life time, and are able to remain transmissible for a long time period after acquisition, some for the entire life span of the adult, indicating that a transmissible reservoir of the virus is retained at some unknown site in the insect [. TYLCV-IL has some other interesting and unexpected characteristics if compared to other TYLCV isolates. The Israeli isolate can be transferred from whitefly males to females and vice versa during mating [Squash leaf curl virus (SLCV) is another whitefly-transmitted begomovirus that is not known to replicate in its whitefly vector. However, several studies have shown that this virus can reach or “infect” several whitefly tissues and organs, other than those implicated in the translocation route of the virus inside the insect body. SLCV was further shown to provoke reproductive problems in the whitefly vector; however such studies have never been thoroughly completed for further examining the molecular nature of these interactions, and further studies must be conducted. Begomoviruses are highly genetically diverse, even within different variants of the same virus isolates. This diversity, which leads to variations on the protein level, might explain some of the differences observed on the virus-vector interactions level [Viruses that are vectored by arthropods exist in both the animal and the plant kingdoms. Plant viruses, can be generally categorized into two major groups based on their relationship with the vector; circulative and non-circulative. Circulative viruses are also termed ‘persistent’, and as more scientific evidence of their relations with their insect vector is gathered, this name is receiving more explanations for the nature of these virus-vector interactions. Circulative viruses belong to the uthparts . Circulauthparts ,3,4. Thiuthparts . Some bee insect ,6,7,8,9.g mating . Squash ns level ,12,13.Plasmodium. The virus moves along the esophagus and enters the digestive system. Luteoviruses mainly cross the aphid hindgut to the hemolymph [Tomato mottle virus (ToMoV) and Cabbage leqf curl virus (CaLCV) in B. tabaci using several light and electron microscopy techniques, as well as fluorescence in situ hybridization (FISH), immunogold and immune-flourescence methods have been studied and reviewed in detail in several research papers and reviews [All viruses that are transmitted in a circulative manner, as opposed to the non-circulative viruses, must undergo a latent time period within their vector in which they translocate. Latency occurs while translocating through various tissues along the transmission route in the insect, before the virus is injected into a new host plant. The path that the virus follows inside the insect, together with the organs crossed is generally similar for all circulative viruses transmitted in a circulative manner. The virus is acquired by the insect stylet while feeding from the plant phloem along with the ingested plant sap, similar to a blood meal ingested by a mosquito transmitting the malaria causative agent emolymph , while bemolymph . In the emolymph , begomovemolymph . Virusesemolymph . The pas reviews ,17,18,19The circulative pathway for virus transmission described above involves crossing several barriers by the virus. Some of these barriers are cellular structures such as the gut and salivary gland membranes, while others are hostile environments for the virus, especially the hemolymph. Crossing the gut and the salivary glands involves entering epithelial cells, or infecting these cells by the virus, while avoiding attack by the immune system while circulating in the hemolymph. It has been previously shown that luteoviruses enter and exit the gut epithelial cells by endo- and exocytosis ,14,20. APotato leafroll virus (PLRV) is the type member of the genus Polerovirus (family Luteoviridae) [M. persicae [viridae) . This vipersicae ,26. Genepersicae . While epersicae ,27,28. Tpersicae . Severalpersicae . Severalpersicae . Virionspersicae ,32,33. Tpersicae . In addipersicae ,34,35. Rpersicae . The barpersicae ,38. PLRVpersicae ,37.Bemisia tabaci, which specializes in plant vasculature feeding using slender mouthparts or stylets, transmits Begomovirus (Family: Geminiviridae), a plant virus group that belong to an emerging genus of ssDNA plant viruses. Begomoviruses exhibit a range of tissue tropisms in the plant with some being phloem-limited, and others not restricted to the phloem. In addition they are affiliated specifically with gut, hemolymph, and salivary organs of their whitefly vector, respectively, indicating evidence of cross-kingdom symbiosis with distinct co-adaptations to plant and animal hosts. Whether the whitefly vector derives direct benefit from this interaction is not known, but generally begomoviruses are not propagative in the vector, and so the extent of the symbiosis is not well understood. Even so, important clues suggest a possible degree of pathogenicity, or perhaps, parasitic relationship on the part of TYLCV with its B biotype whitefly vector [The phytophagous homopteran whitefly y vector ,40, basey vector ,41,42, ty vector . This isy vector ,45. Indey vector , sets TYB. tabaci sibling species group [B. tabaci biotypes differ genetically as well as in host range, insecticide-resistance and in ability to transmit plant viruses and to cause plant disorders. The most predominant and damaging biotypes are the B and Q biotypes [The whitefly vector of begomoviruses is comprised of all members of the es group ; a suitebiotypes ,50,51,52biotypes ,48, causbiotypes , the Q bbiotypes . Viral dB. tabaci sibling species group [in vivo transmission parameters, anatomical studies, and TEM localization [B. tabaci labeled TYLCV and SLCV has been observed only in the PSGs. A non-vector whitefly Trialeurodes vaporariorum (West.) has been shown to ingest begomoviruses, and in one instance evidence was provided for virus presence in the blood, however in neither instance did virus transmission occur indicating that vector specificity does not reside at the level of ingestion or at the gut interface [Hamiltonella [TYLCV infects only dicotyledonous plants and is transmitted only by the whitefly es group ,51. Begoes group . Begomoves group ,44,45. Tes group . The curlization ,44,45,56lization ,57. In tnterface ,58. Evidltonella . Additioltonella , and to M. persicae, however, this might not be the case in all aphid species [M. persicae, the Soybean Dwarf Virus (SbDV) crosses at the hindgut. Several studies have concluded that in many luteovirus-aphid systems, the most selective barrier that regulates the transmission is the ASG cells. Recently, it was shown that ASG contain two barriers: the extracellular basal lamina wrapping the ASG cells and the basal plasmalemma of the ASG cells [S. avenae acquired BYDV-MAV and later the serologically related BYDV-PAV, it transmitted the MAV variant more preferably than PAV [M. persicae whose homologue in Drosophila melanogaster was implicated in transcytosis [B. tabaci, with TYLCV, an eastern hemisphere begomovirus, it was concluded that as long as the virus is transmissible, its identity and the origin of the vector are irrelevant to the pathway the virus takes through [As detailed above, different factors may influence the virus circulative transmission pathway within its insect vector. Some viruses transmitted in a non-circulative manner (both non-persistent and semipersistent) were shown to require Helper proteins to assist in their transmission . Helper  species . It was SG cells ,63,64. TSG cells ,15. Resethan PAV ,65. Whenthan PAV ,65. Studscytosis . BWYV pascytosis . Two othscytosis . In begoscytosis ,70,71,72scytosis . Two majscytosis . When co through ,75,76,77 through ,79,80,81E. coli GroEL and GroES are the most studied proteins; and named Chaperonins [Tobacco Mosaic Virus (TMV) assembly within E. coli cells showed that the levels of TMV dropped in cells without GroEL [A. pisum endosymbionts discovered ‘Symbionin’, a protein produced by the bacteria that is essential for proper symbiotic relations [M. persicae, Buchnera. The protein is highly homologous to both Symbionin and GroEL. In the Buchnera genome, the protein operon is split into two open reading frames with 72% and 73% nucleotide homology to GroES and GroEL respectively [In vivo, the Buchnera GroEL is arranged like the GroEL from E. coli and arranged as an oligomer of 14 subunits forming two rings [Buchnera GroEL homologue from the sweetpotato whitefly B. tabaci, was later shown to be able to bind a variety of plant viruses, mediated by their capsid, mostly those whose virion is of globular or geminate shape, and whose CP has a high positive charge, a high percentage of arginine and a high isoelectric point—many of which are vectored by aphids, especially M. persicae [Buchnera, the primary endosymbiont of aphids, and the GroEL of Hamiltonella, a secondary endosymbiont of B. tabaci. Both GroEL proteins were implicated in the transmission and survival of plant viruses while transmitted by their insect vectors [Several studies have shown that the process of protein folding in living cells is not a spontaneous procedure, and is mediated by a group of proteins termed ‘Chaperones’ . These pperonins . GroEL iperonins ,85. Whenperonins ,87. Chapperonins . Experimut GroEL . Complemut GroEL . A previelations ,90. The ectively . In vivowo rings . The Bucpersicae . In the  vectors ,16. et al. [M. persicae extracts. The study showed that out of five such proteins which interacted with anti-CP, four did not interact with the anti-idiotypic PLRV CP antibody (AiAb) that mimicked the surface structure of PLRV CP. One protein, however, did bind the AiAb and was the most abundant protein of the five identified in this screen [E. coli called GroEL. GroEL was previously termed ‘symbionin’ referring to its production by the endosymbionts of the pea aphid A. pisum. This protein was shown to be crucial for maintaining proper symbiotic relations between the aphid and the bacteria, and was found in all aphids including those that transmit PLRV. Immunogold labeling experiments using anti-GroEL antibody in M. persicae showed the localization of this protein within the cytoplasm of Buchnera, the primary endosymbiont of aphids. These endosymbionts reside in the hemolymph, within specialized organs named bacteriomes. GroEL was first termed MpSym (M. persicae symbionin) and later MpB GroEL (M. persicae Buchnera GroEL). Western blotting showed high levels of the protein in the aphids’ hemolymph. To ascertain whether this protein is associated with PLRV transmission through the aphid, aphids were fed with antibiotics. Although no change was recorded in the feeding behavior of the treated aphids, PLRV transmission was reduced by more than 70% [et al. [E. coli, and tested their binding to various viruses of the luteovirus subgroup II and to viruses of other families that are aphid transmissible in a non-circulative manner. They showed that all luteoviruses bind GroEL proteins (with different affinities), even thosenon-transmissible by aphids. This affirmed the previous assays in which aphids were able to acquire viruses they did not transmit. None of the non-circulative viruses tested showed any affinity to the GroEL proteins. The fact that all luteoviruses bind to GroEL proteins suggested a common binding domain found in their capsid. Various mutant of BWYV were tested for their binding to MpB GroEL. Mutants in which the RTD in their CP was completely removed did not bind GroEL, however, mutants with deletion only in the C-terminal of RTD bound as efficiently as wild-type BWYV. This indicated that the conserved N-terminal of the RTD is the part of the luteovirus capsid required for GroEL binding. Levels of GroEL non-binding BWYV mutants in their RTD that were injected directly into the aphid hemolymph declined rapidly after the injection, suggesting an essential role for GroEL in virus survival in the hemolymph [E. coli GroEL, this site was found to be conserved in all but one amino acid. Additional experiments conducted later characterized the nature of the PLRV-GroEL binding as hydrophilic. One conclusion that arose from these experiments was that the identity of the amino acids in the binding sites was not critical as long as the hydrophilic nature of the region was maintained [A study by van den Heuvel et al.  performes screen . This prthan 70% . Van den [et al.  later isemolymph . Furtheremolymph . When cointained . B. tabaci are known to harbor endosymbionts for completing their phloem unbalanced diet. B. tabaci harbors the obligatory primary endosymbiont like all whiteflies Portiera aleyrodidarum. Additionally, populations around the world were reported to harbor seven other secondary bacterial endosymbionts from different families including Arsenophonus, Hamiltonella, Wolbachia, Cardinium, Fritschea, Rickettsia and Candidatus Hemipteriphilus asiaticus) [B. tabaci biotypes may harbor different secondary symbionts as was shown in the Israeli B and Q biotype populations [et al., in which a GroEL of Buchnera was implicated in the circulative transmission of PLRV through direct physical interaction [B. tabaci [. Testing the interaction between TYLCV and the GroEL homologue from Buchnera revealed a lack of interaction. This result itself was not surprising since B. tabaci is also known to harbor endosymbionts, and thus another GroEL from a different symbiont in B. tabaci might interact with TYLCV, if the same mechanism of virus transmission occurs in this whitefly-begomovirus system. B. tabaci transmits begomoviruses in a circulative manner, similar to that of luteoviruses in aphids. Following the studies conducted in aphids, Morin et al. identified a GroEL homologue to the Buchnera protein in the B biotype of B. tabaci from Israel. Whiteflies were fed with antibiotics or with anti-Buchnera GroEL antibodies and up to 80% reduction in TYLCV transmission was observed [B. tabaci GroEL protein sequence share 80% homology with the Buchnera and E. coli GroELs [B. tabaci in TYLCV circulative transmission. Physical interaction between GroEL and TYLCV CP and virions, using western blot, yeast two-hybrid system and virus overlay assays, was then demonstrated [B. tabaci adults, using antibodies against the Buchnera GroEL showed that specific labeling was not associated with Portiera, the primary endosymbiont, but with another secondary symbiont. While the primary symbiont was termed “P”, referring to “primary” or “pleomorphic”, the secondary symbiont was termed “C” referring to “coccoid”. The specific immunogols labeling obtained in these experiments was associated with the “C” and not the “P” endosymbionts.Many insects that are phloem-feeders such as the whitefly iaticus) ,94,95,96ulations . Followieraction , a simil. tabaci . Testingobserved . No TYLCi GroELs . These rnstrated . ImmunogB. tabaci is characterized as a species complex, comprised of more than 30 different biotypes [Hamiltonella and Rickettsia, while the Q biotype was infected with Rickettsia, Arsenophonus and Wolbachia [Hamiltonella, in contrary to the Israeli Q biotype. A study conducted in Israel showed that the GroEL from Hamiltonella interacts with TYLCV CP in the Yeast two-hybrid system, immunocapture PCR and pull-down assays [et al. [Hamiltonella. As in the case with the GroEL from Buchnera, the GroEL from Hamiltonella was described as an oligomer in the hemolymph of B. tabaci, but not in its digestive system [Abutilon mosaic virus from Israel (AbMV-Is) to interact with B. tabaci GroEL, similar to the Buchnera GroEL interaction with non-transmissible viruses by aphids [Buchnera GroEL to PLRV CP, it is still likely that TYLCV binding to Hamiltonella GroEL is also based on hydrophilic nature, as is with the PLRV‑GroEL system [biotypes . The B abiotypes ,99. In Ibiotypes . A surveolbachia . The Q bn assays . Other Gn assays ,60. Thes [et al.  is a proe system . One stuy aphids . AlthougL system .B. tabaci-begomovirus system is the ability of the Q biotype that lacks Hamiltonella (from Israel) to transmit TYLCV. Although this Q biotype is a poor transmitter, its ability to transmit the virus suggests that TYLCV is able to overcome the hostile environment within the insect’s hemolymph, where the protection by GroEL is provided. The lack of Hamiltonella further suggests that the relevant GroEL required for proper circulation and transmission of TYLCV in the Q biotype is absent. No direct evidence exists to suggest a proper explanation; however, several hypotheses can be raised and are detailed in B. tabaci B biotype, which in many extents is similar to the model that exists for the aphid-luteovirus system. The transmission cycle starts by virus acquisition from the phloem of an infected plant, the virus moves along the stylet, forgut, esophagus and reaches the midgut in whiteflies and further the hindgut in some aphid-luteovirus systems. In whiteflies, the majority of the virus particles are absorbed in the filter chamber to the hemolymph while some circulate in the midgut and are absorbed along the midgut loop . TYLCV and PLRV virions attach to unknown receptors on the PSG and ASG membranes respectively, and are internalized into the glands cells, are then emptied into the glands main lumen which connects to the salivary canal in the stylet. From this canal virions are injected into the plant with salivary secretions. B. tabaci to transmit TYLCV. In this model, the midgut is pushed into the thorax of viruliferous adults, especially gravid females, when the abdomen is filled with developing eggs. Such a scenario brings the midgut very close to the PSG and significantly shortens the distance that TYLCV virions have to make before entering the gland cells (blue arrow in the figure). Such a scenario enables the virus to avoid the hostile environment in the hemolymph and rapidly enter the PSG, when protection is not available by GroEL. Pushing the midgut into the thorax of gravid females was shown to increase with age, suggesting that the more the insect is filled with eggs, the higher the chance for the midgut to be pushed into the thorax [et al. [One question that remains unanswered in the gut loop . In aphie thorax . Figure e thorax . In such [et al.  reported [et al. . M. persicae–PLRV and B. tabaci–TYLCV systems that endosymbiotic bacteria produce a GroEL protein that interacts with the transmitted virus in the hemolymph. This interaction provides proper protection for the virus and increases its probability to be transmitted. In the M. persicae–PLRV system, GroEL is produced by the primary endosymbint Buchnera. Primary endosymbionts are essential for the species persistence and complete the diet of their insect host, while the host provides the necessary protected environment for the bacteria. In the B. tabaci–TYLCV system however, GroEL is produced by a secondary endosymbiont that is not essential for the insect. An important question that arises in the B. tabaci–TYLCV system is which benefits are gained upon GroEL production by Hamiltonella, which do not provide any substantial benefit for the whitefly? Several lines of evidence lately suggested that circulative viruses may resemble propagative viruses that negatively influence the biology of the insect. This is most evident for TYLCV that has been shown to influence life span parameters of its whitefly vector including mortality, fecundity and fertility, and has been further shown to significantly alter global gene expression in the whitefly [We reviewed the factors influencing circulative plant virus transmission by arthropod vectors and focused on the aphid–luteovirus and the whitefly–begomovirus systems. Three major sites in the insect regulate virus transmission. Those sites include the gut, hemolymph and the salivary glands. In the gut and salivary gland tissues, several insect proteins, such as specific receptors that interact with the virus CP, are expected to be involved and extensive research is ongoing to identify such proteins. In the hemolymph, the transmitted virus is exposed to attack by the insect immune system, and the lack of protection in the hemolymph is expected to significantly influence the successful transmission of the virus. Indeed, it has been shown in the whitefly ,60,101.


The authors would like to add the following references:63. Karlin D, Ferron F, Canard B, Longhi S (2003) Structural disorder and modular organization in Paramyxovirinae N and P. J Gen Virology:84 3239-325264. Bourhis JM, Canard B, Longhi S (2006) Structural disorder within the replicative complex of measles virus: Functional implications. Virology:344 94-11065. Longhi, S. (2003). The C-terminal Domain of the Measles Virus Nucleoprotein Is Intrinsically Disordered and Folds upon Binding to the C-terminal Moiety of the Phosphoprotein. Journal of Biological Chemistry, 278(20), 18638-18648.66. Jensen MR, Houben K, Lescop E, Blanchard L, Ruigrok RWH, Blackledge M (2008). Quantitative conformational analysis of partially folded proteins from residual dipolar couplings: application to the molecular recognition element of Sendai virus nucleoprotein. Journal of the American Chemical Society:130(25) 8055-8061)67. Longhi, S. (2009) Nucleocapsid structure and function. Curr Top Microbiol Immunol 329: 103-128


Barley stripe mosaic virus (BSMV) were neglected for more than thirty years after their basic properties were determined. In this paper, the physicochemical characteristics of BSMV virions and virion-derived viral capsid protein (CP) were analyzed, namely, the absorption and intrinsic fluorescence spectra, circular dichroism spectra, differential scanning calorimetry curves, and size distributions by dynamic laser light scattering. The structural properties of BSMV virions proved to be intermediate between those of Tobacco mosaic virus (TMV), a well-characterized virus with rigid rod-shaped virions, and flexuous filamentous plant viruses. The BSMV virions were found to be considerably more labile than expected from their rod-like morphology and a distant sequence relation of the BSMV and TMV CPs. The circular dichroism spectra of BSMV CP subunits incorporated into the virions, but not subunits of free CP, demonstrated a significant proportion of beta-structure elements, which were proposed to be localized mostly in the protein regions exposed on the virion outer surface. These beta-structure elements likely formed during virion assembly can comprise the N- and C-terminal protein regions unstructured in the non-virion CP and can mediate inter-subunit interactions. Based on computer-assisted structure modeling, a model for BSMV CP subunit structural fold compliant with the available experimental data was proposed.Virions of  The architecture of viral capsids is largely based on either icosahedral or helical symmetry Tobacco mosaic virus (TMV), one of the best studied plant pathogens first identified more than one hundred years ago, was the only helical plant virus with resolved capsid structure. Conventional X-ray diffraction analysis of “20S disks” formed by the TMV coat protein (CP) carried out in the 1970s Capsids with the helical symmetry are typical for numerous rod-shaped and filamentous plant viruses. For many years, Barley stripe mosaic virus  with rod-like helical virions In the last three decades, BSMV was the subject of detailed studies focused on the genome structure and expression, functions of encoded proteins in virus infection, and interaction with plant hosts In this paper we used sensitive methods of protein analysis to determine a number of physicochemical characteristics of BSMV and its CP and the data obtained allowed us to propose a model of the BSMV CP structure.Agrobacterium tumefaciens strain GV3101. Transformed agrobacteria were resuspended in agroinfiltration buffer  and infiltrated into leaves of Nicotiana benthamiana plants. Leaves for virus isolation were harvested 2 weeks after agroinfiltration. The leaves were homogenized in a blender with three volumes of 0.5 M borate buffer pH 9.0. The mixture was incubated with 0.5% Triton X-100 for 20 min, extracted with ¼ volume of chloroform, and centrifuged at 10000 g for 20 min. The virus was precipitated from the supernatant by incubation with 4% PEG-6000 and 1.2% NaCl for 2 hours at room temperature. The mixture was centrifuged again at 10000 g, and the pellet was resuspended in 50 mM borate buffer pH 9. After clarification, the virus was pelleted by ultracentrifugation (2 hours at 100000 g) through a 1 ml cushion of 20% sucrose. The virus was resuspended in 5 mM phosphate buffer pH 7.5 and stored at +4°C. The BSMV yield was about 30 mg per 100 g of systemically infected leaves. The homogeneity of the resulting sample was evaluated by absorption spectroscopy, electron microscopy (The cloned infectious cDNAs of BSMV (strain ND18) croscopy , and PAGcroscopy .BSMV CP was isolated by LiCl treatment of BSMV virions TMV was purified by the previously described method Unless otherwise indicated, the phosphate buffer was used as the most suitable buffer for far-UV CD measurements. The absorption spectra in the 240–340 nm range were measured in cells with an optical path of 1 cm using a Hitachi UV-2600 spectrophotometer. The CD spectra were recorded in 1–2 mm (185–250 nm) cells at 25°C using a Chiroscan CD spectrometer . The spectra were measured in 5 mM phosphate buffer pH 7.5. The sample concentration was 100 μg/ml. The CD spectra were recorded at 0.5–1.0 nm/s with a baseline subtraction. The measured spectra were smoothed with the help of the instrument software. The values of [θ] were calculated taking the mean molecular weight of amino acid residues to be 110. The intrinsic fluorescence spectra were recorded in 1 cm cells using a FluoroMax spectrofluorometer  at 25°C. Fluorescence of protein at the concentration of 0.03 mg/ml was excited in 1 mM phosphate buffer pH 7.5 at 280 nm, and the emission spectra were recorded in the 300–400 nm range.Virgaviridae was generated using ClustalW2 www.salilab.org/modeller/) using the crystal structure of the CP CGMMV (pdb ID: 1CGM). The interactions between adjacent subunits were not taken into account in the model.For homology modeling of the BSMV CP structure, a multiple sequence alignment of CPs encoded by rod-shaped viruses of the family DSC experiments were performed on a DASM-4 microcalorimeter  with capillary platinum 0.47-mL cells at a scanning rate of 1°C/min. The second heating was used as the instrument baseline because of the irreversible denaturation observed for the investigated sample. The chemical baseline was calculated and subtracted using the Origin 1.16 software.The sizes of BSMV virion and CP were determined using dynamic light scattering (DLS). All experiments were carried out on a ZetaSizer NanoZS instrument  with the laser wavelength of 633 nm. The data were analyzed using the Dispersion Technology Software version 5.10. The samples (at about 100 μg/ml) were measured in 10 mM phosphate buffer. BSMV virions were disrupted with 0.1% SDS.Nicotiana benthamiana plants were used to measure the UV spectra of BSMV virions  due to five Trp and six Tyr residues in its sequence. For BSMV virions we used absorbtion coefficient  equal to 2.4. The BSMV CP absorption spectrum was measured in 3M LiCl solution immediately after disassembly of the virions and RNA precipitation, which occurs under these conditions 260/ isolated from  virions  and BSMV virions . The BSMurbidity , thus coDynamic laser light scattering (DLS), the method making possible to reveal the size distribution of small particles dispersed in solution Potato virus X (PVX) 2*dmol−1 at 189 nm. This peak was approximately 30% higher than the peak at 208 nm and had a shoulder at 197 nm with the intensity of 58% of the 189 nm peak  spectroscopy was used to compare the structural properties of BSMV virions and the non-virion BSMV CP. The CD spectrum of BSMV virions in the far UV region (190–250 nm) had no unusual peaks and was typical for proteins with high content of α-helices . The BSM nm peak . Analysi nm peak .218 value observed for the intact BSMV virions than for the non-virion CP obtained upon virion disassembly suggested the existence of a significant fraction of β-structures in the intact virions. The [θ]218/[θ]208 ratio decreased from 0.85 to 0.60 upon the BSMV virion disruption. Additionally, the spectrum of non-virion CP exhibited a shift of the zero point from 201 to 198 nm, a considerable decrease (down to 23000 grad*cm2*dmol−1) of the main maximum at 189 nm, and the absence of the shoulder at 197 nm. All these changes in the CD spectrum of the BSMV CP could be explained by the loss of a considerable fraction of α- and β-structure elements and the corresponding increase in the proportion of unstructured elements upon the disassembly of BSMV virions in 0.1% SDS  Virgaviridae was generated using ClustalW2 Cucumber green mottle mosaic virus (CGMMV) was identified as the protein exhibiting the closest sequence relation to the BSMV CP among viral CPs with resolved structure . The CGMMV CP structure has been resolved at 3.4 Å We tried to model the BSMV CP subunit structure based on the resolved structures of tobamovirus CPs. A multiple sequence alignment of CPs encoded by rod-shaped viruses of the family Using the CGMMV CP structure as a template, we were able to build a structure model for most of the BSMV CP sequence, namely the region from residue 13 to the protein C-terminus (residue 198) . The strAccording to the proposed model, the BSMV CP subunit was characterized, as expected, by many features common with the CGMMV CP and also some significant differences. As in the CGMMV CP, the core of BSMV CP molecule was represented by a four-helix bundle . As in tFormation of ribonucleoprotein complexes (RNPs) comprising viral genomic RNA and virus-encoded proteins is essential for many steps in plant virus life cycle, such as protection of viral genome from attacks by cellular enzymes, transport of viral RNA cell-to-cell and long distance through the phloem, and interaction with different virus-encoded and cell partner proteins. The BSMV genomic RNA, in addition to virion RNPs, can be incorporated into a specialized transport RNP formed by the movement protein TGBp1 Based on general considerations, the structural features of rod-shaped BSMV virions and BSMV CP should be similar to those of TMV. A moderate degree of sequence similarity between the BSMV and TMV CPs The proposed BSMV CP model differs from the TMV CP structure in its long unstructured N-terminal sequence and the virion surface-located loop between α-helix-2 and α-helix-3 (residues 68–100). A somewhat shorter similar structure is described for the CP of PapMV, which has flexuous filamentous helical virions Based on the CD spectra of the BSMV virions and the BSMV CP, we propose that, in contrast to TMV and other plant viruses with known CP structure, the CP subunits of the BSMV virion contain a significant proportion of β-structure elements. We presume that β-structure regions, which are found by CD spectroscopy in the BSMV CP incorporated in native virions but not in non-virion monomeric CP, can be formed during virion assembly involving the CP polymerization. These β-structure regions may include the N- and C-terminal protein regions unstructured in the non-virion CP, as well as the loop between α-helix-2 and α-helix-3 located at the virion periphery. We propose that a transition from the unstructured state to the β-structure may be induced by protein-protein and protein-RNA interactions arising during the capsid self-assembly. It would be important to know whether the CP forms intramolecular β-structures or cross-β-structures involving regions of the neighboring CP subunits. The latter possibility would be reminiscent of the interaction of PapMV capsid subunits Figure S1The homogeneity of the samples BSMV virions , disks (B) and aggregates (C) was evaluated by electron microscopy  and PAGE (D).(TIF)Click here for additional data file.Figure S2Amino acid sequence alignment of the CPs encoded by different rod-shaped viruses. BSMV, Barley stripe mosaic virus (genus Hordeivirus); PSLV, Poa semilatent virus (genus Hordeivirus); LRSV, Lychnis ringspot virus (genus Hordeivirus); CGMMV, Cucumber green mottle mosaic virus (genus Tobamovirus); TMV, Tobacco mosaic virus (genus Tobamovirus); RMV, Ribgrass mosaic virus (genus Tobamovirus); HLSV, Hibiscus latent Singapore virus (genus Tobamovirus); TRV, Tobacco rattle virus (genus Tobravirus); and PEBV, Pea early browning virus (genus Tobravirus).(TIF)Click here for additional data file.Figure S33D struture of CP BSMV in the PDB file.(PDB)Click here for additional data file.


In 2008, African swine fever was introduced into Georgia, after which it spread to neighboring Armenia, Azerbaijan, and the Russian Federation. That same year, PCR and sequence analysis identified African swine fever virus in samples from 3 dead female wild boars in northwestern Iran. Wild boars may serve as a reservoir. The disease spread to the city of Khoie, located in another province (West Azarbaijan). Postmortem histopathologic investigations of tissue samples of 3 dead, female, wild boars found characteristic signs of ASF, such as disseminated intravascular coagulation with multiple hemorrhages .1,2   and showed 100% similarity to submitted sequences of Georgia isolates. A partial sequence of the isolates from Iran has been submitted to GenBank under accession no. FJ897727.Viral DNA from 6 tissue samples  from the 3 dead boars was extracted by using the JETQUICK Tissue DNA Spin Kit  according to the manufacturer’s instructions. ASFV was detected by PCR and SYBR Green real-time PCR on a Rotor-Gene 65H0  in all samples from each boar; primers used were P72 D, U (major capsid protein), and PPA,,,To confirm a specific pathogen and trace the possible sources of infection, genetic characterization of the virus strain associated with disease is crucial (Ornithodoros spp. ticks may contribute to virus persistence in the Caucasus region, including northwestern Iran. Although in our study ticks were not detected on the boar carcasses, they should be considered as potential transmission vectors. O. lahorensis ticks are found in areas with a cold climate and are mainly found near sheep and cows, so they are not likely to be isolated from an animal. In the absence of Ornithodoros spp. ticks, transmission to pigs could occur through contact with infected pigs or through feeding of virus-contaminated products (,The main obstacles to ASF eradication are wildlife reservoirs, limited ability to control movement of infected pigs and wild boars, inadequate laboratory support for rapid and accurate diagnosis, and lack of an effective vaccine (,,,–The pig industry differs among Caucasus countries. In Azerbaijan, Chechnya, and southern Russian Federation, the pig industry is not as large as it is in Georgia and Armenia (


Flavivirus genus, Flaviviridae family). They show antigenic close relationships and share many similarities in their ecology. Both are responsible for serious human diseases. The aim of this study was to investigate the presence of neutralizing antibodies to these viruses in horses from Uruguay. To do this, 425 horse sera were collected in 2007 and analyzed by plaque reduction neutralization tests. As a result, 205 sera (48.2%) were found positive for SLEV, with titers ranging between 10 and 80. Two sera remained inconclusive, since they showed low titers to WNV and SLEV (10 and 20), not allowing us to demonstrate activity of WNV in our territory. This is the first report of circulation of SLEV in horses in Uruguay.St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) belong to the Japanese encephalitis antigenic complex ( Infections caused by mosquito-borne flaviviruses are among the diseases with higher incidence in the world. Most of them are asymptomatic or present an influenza-like illness; however severe cases may occur, causing central nervous system disease, coma, and/or death , 2.Culex mosquitoes; humans and horses are dead-end hosts. These viruses can cause encephalitis in humans; only WNV infection may lead to fatal encephalitis in equines. All flaviviruses are antigenically closely related, giving frequent serological cross-reactions which are exaggerated in sequential infections. Because of this, specific etiologic diagnosis may be difficult specially in areas where two or more flaviviruses are prevalent [St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) are members of the Japanese encephalitis antigenic complex; they are usually maintained in cycles between birds and revalent .Stegomyia (Aedes) aegypti. Until today, only imported cases were diagnosed but the vector is present in several departments of the country, reinforcing the need of a close surveillance of this flavivirus [SLEV is widely distributed in the Americas. Recently, human outbreaks were reported in Argentina in 2005 and 2010 , 5 and iavivirus . More reavivirus . In 2010WNV was introduced in the Americas in 1999, in the city of New York, United States . Since tThe objective of the present study was to analyze the presence of neutralizing antibodies to SLEV and WNV in equines, in order to investigate the circulation of these flaviviruses in Uruguay.2 and a population of 3.356.584 inhabitants. It is divided in 19 departments  “Miguel C. Rubino” (Ministry of Agriculture and Fisheries) to perform epidemiologic studies. None of the horses presented any sign of disease. Blood samples were taken from the jugular vein of animals and kept at −20°C until processed. To do the present study, a minimum of 10 sera per department were tested, belonging to 18 of the 19 departments . Samples80). Positive samples were further titrated in serial dilutions beginning at 1 : 20.Detection of neutralizing antibodies to SLEV and WNV was performed using plaque reduction neutralization tests (PRNT) in VERO cells as previously described . SLEV st80 titers of SLEV seropositive horse sera ranged from 10 to 160 (data available under request).In the screening test, 207 (48.7%) of the 425 horses had neutralizing antibodies to SLEV. Positive samples were found in 17 of the 18 departments tested  . By depan = 117) were in turn screened to WNV. After these assays, eight sera were positive for WNV at a 1 : 10 dilution. These eight sera were in turn simultaneously titrated to SLEV and WNV. As result, six sera showed a fourfold or greater difference in titer to SLEV in respect to WNV and two sera remained inconclusive  , 22.To minimize the cross-reaction results in this survey, we followed the Centers for Disease Control and Prevention's (CDC) guidelines for seropositivity: when comparing sera titers to two or more viruses, a fourfold difference in PRNT is needed to identify the etiologic agent. According to this criterion, the simultaneous titration resulted in six sera seropositive to SLEV and two inconclusive. From these inconclusive sera, sample E07/204 showed a greater titer to WNV (20) than SLEV (10). These results could be due to an initial infection with WNV followed by a secondary infection with SLEV. Patiris et al. demonstrated in chickens that sequential infections with SLEV (1st) and WNV (2nd) greatly amplified antibodies to SLEV. The reverse order of infection (1st infection with WNV and 2nd infection with SLEV) produced a limited response, and titers to both viruses remained similar. In turn, titers to WNV may be considered as result of a very recent infection or a cross-reaction for an undetermined flavivirus. Lederman et al. showed that after an initial exposition to SLEV followed by a subsequent infection with WNV, detectable levels of WNV specific antibodies developed at days 9–12, with a peak at days 12–18 , 24.It is noteworthy that, a year before our sampling, WNV virus was actively circulating in the region. In 2006, the virus was isolated for the first time in South America from sick horses in Argentina, and evidence of WNV activity in birds was demonstrated in Córdoba province (Argentina) in 2005 and 2006 , 15. TheSLEV activity in horses described in this study, together with the reports of sporadic human infections and the diagnosis of three cases of SLEV meningoencephalitis (two in Montevideo and one in San José department), confirm the circulation of this virus in our country , 10. OurCulex spp. represents the 80% of mosquitoes captured in our studies, so risk of outbreaks should not be discarded.Regarding arbovirus investigation in Uruguay, our lab is conducting since 2007 the molecular detection in mosquitoes. Although we still did not detect SLEV in the vectors, it is to note that The results presented here show that SLEV is actively circulating in our country at a significant prevalence (48.2%). Seroprevalences ranged between 0 and 75%; San José and Canelones departments presented the highest values . Our results do not demonstrate the circulation of WNV in Uruguay; however recent infections with this virus or circulation of another flaviviruses cannot be excluded.Our findings, together with the sporadic SLEV infections documented in humans and the risk of introduction of dengue virus in our territory, reinforce the need of differential diagnosis of flaviviruses. With the aim of contributing to the knowledge of the ecology of flavivirus in our country, our research group is carrying out arbovirus detection in mosquitoes and initiating serologic studies in wild birds.


Flaviviridae family, transmitted by vector mosquitoes. Here, we reported the complete genome sequence of West Nile virus isolated from human samples during an acute encephalitis outbreak in Kerala, India. Phylogenetic analysis revealed that the virus genome clusters into genetic lineage 1, clade 1a.West Nile virus belongs to the  Flavivirus Japanese encephalitis antigenic complex, in the Flaviviridae family. The seroprevalence of WNV antibodies in a population indicated the presence of this virus in India (West Nile virus (WNV) is an arthropod-borne virus transmitted by vector mosquitoes. The virus belongs to the genus in India , 2. Latein India . Phylogein India . Subsequin India . In humaWe reported an outbreak of acute encephalitis syndrome (AES) from May through July 2011 in Kerala, India. Per our records, 208 AES cases, with 4 deaths, were recorded. Among 208 AES cases, 69 (33%) were in patients belonging to the pediatric age group (those aged <15 years) and 139 (66.82%) were in adults (aged >15 years). Clinically, the patients had high fever with headache and one or more of the following symptoms: neurological deficit, altered sensorium, disorientation, irritability, neck rigidity, and vomiting. No clustering of the cases was noticed in the affected area. A virus neutralization assay confirmed that the etiology of the outbreak was WNV. The virus was isolated from a serum sample (sample identification no. 1048813) from a patient with acute fever. The patient serum was diluted (1:10) in minimum essential medium (MEM) consisting of 1% fetal calf serum (FCS) and gentamicin (50 µg/ml), and it was inoculated into the Vero E6 cell line. The cell line was observed for 7 days for any cytopathic effects (CPE). WNV was able to be isolated after a second blind passage in Vero E6 cells.Total RNA was extracted from the culture supernatant using a QIAamp viral RNA minikit . Previously described primers were used for the amplification of the full genome . cDNA waThe raw sequence data were assembled using MEGA software (version 5.0). Sequence alignments and phylogenetic trees were generated using the MEGA program . The comKC601756.The complete genome sequence of the WNV Kerala strain has been deposited at GenBank under the accession no.


Arteriviridae: simian hemorrhagic fever virus (SHFV)-krc1 and SHFV-krc2, infecting a single wild red colobus (Procolobus rufomitratus tephrosceles) in Kibale National Park, Uganda. Nearly nothing is known about the biological properties of SHFVs in nature, although the SHFV type strain, SHFV-LVR, has caused devastating outbreaks of viral hemorrhagic fever in captive macaques. Here we detected SHFV-krc1 and SHFV-krc2 in 40% and 47% of 60 wild red colobus tested, respectively. We found viral loads in excess of 106–107 RNA copies per milliliter of blood plasma for each of these viruses. SHFV-krc1 and SHFV-krc2 also showed high genetic diversity at both the inter- and intra-host levels. Analyses of synonymous and non-synonymous nucleotide diversity across viral genomes revealed patterns suggestive of positive selection in SHFV open reading frames (ORF) 5 (SHFV-krc2 only) and 7 (SHFV-krc1 and SHFV-krc2). Thus, these viruses share several important properties with some of the most rapidly evolving, emergent RNA viruses.Key biological properties such as high genetic diversity and high evolutionary rate enhance the potential of certain RNA viruses to adapt and emerge. Identifying viruses with these properties in their natural hosts could dramatically improve disease forecasting and surveillance. Recently, we discovered two novel members of the viral family  Certain RNA viruses have biological properties that make them particularly likely to emerge Arteriviridae that have only recently been detected in wild primates Macaca assamensis, M. arctoides, M. fasciularis, M. nemestrina, and M. mulatta) between 1964 and 1996. Erythrocebus patas), grivets (Chlorocebus aethiops), and Guinea baboons (Papio papio) – could persistently harbor SHFV-LVR in captivity without signs of disease The simian hemorrhagic fever viruses (SHFVs) are a poorly understood group of single stranded, positive-sense RNA viruses within the family Procolobus rufomitratus tephrosceles) in Kibale National Park, Uganda , which we named SHFV-krc1 and SHFV-krc2 Cercopithecus ascanius) from the same location In 2011, we discovered two highly divergent simian arteriviruses infecting a single wild red colobus (Arteriviridae family: porcine reproductive and respiratory syndrome virus (PRRSV), equine arteritis virus (EAV), and lactate dehydrogenase-elevating virus of mice (LDV). Previous publications regarding SHFV have treated the naming of these additional ORFs inconsistently. For clarity, we have adopted the nomenclature scheme presented in SHFV genomes contain a duplication of four open reading frames (ORFs) relative to the other viruses in the All animal use in this study followed the guidelines of the Weatherall Report on the use of non-human primates in research. Specific protocols were adopted to minimize suffering through anesthesia and other means during capture, immobilization, and sampling of the non-human primates. These included use of anesthesia during capture , minimization of immobilization time and the use of an anesthetic reversal agent (Atipamezole) to reduce recovery time, and conservative limits on blood sample volumes (<1% body weight), as previously described 2 semi-deciduous park in western Uganda  known for its exceptional density of primates belonging to diverse species. Blood was separated using centrifugation and plasma was frozen immediately in liquid nitrogen for storage and transport to the United States. Samples were shipped in an IATA-approved dry shipper to the USA for further analysis at the Wisconsin National Primate Research Center in accordance with CITES permit #002290 (Uganda).Red colobus were sampled between 2/5/2010 and 7/22/2012 in Kibale National Park, Uganda, a 795 kmSamples were processed for sequencing in a biosafety level 3 laboratory as described previously 5′-ACACGGCTACCCTTACTCC-3′ and 5′- TCGAGGTTAARCGGTTGAGA-3′) or SHFV-krc2 (5′-AACGCGCACCAACCACTATG-3′ and 5′GCGTGTTGAGGCCCTAATTTG-3′). The SHFV-krc1 probe (5′-Quasar 670-TTCTGGTCCTCTTGCGAAGGC-BHQ2-3′) and SHFV-krc2 probe (5′-6-Fam-TTTGCTCAAGCCAATGACCTGCG-BHQ1-3′) were also virus-specific. The fluorophores used do not produce overlapping spectra, so no color compensation was required. Viral RNA was reverse transcribed and quantified using the SuperScript III One-Step qRT-PCR system  on a LightCycler 480 . Reverse transcription was carried out at 37°C for 15 min and then 50°C for 30 min followed by two minutes at 95°C and 50 cycles of amplification as follows: 95°C for 15 sec and 60°C for 1 min. The reaction mixture contained MgSO4 at a final concentration of 3.0 mM, 150 ng random primers , with all 4 amplification primers at a concentration of 600 nM and both probes at a concentration of 100 nM.We developed a multiplex quantitative RT-PCR (qRT-PCR) assay to quantify plasma viral RNA of both SHFV-krc1 and SHFV-krc2 in each sample. Taqman assays were designed with amplification primers specific for either SHFV-krc1  within a co-infected animal. Briefly, total reads from a co-infected animal were mapped to the SHFV-krc1 consensus sequence generated from de novo assembly and “unmapped reads” were collected, then mapped to the SHFV-krc2 consensus sequence obtained from de novo assembly. The resulting SHFV-krc2 consensus sequence was then used as the reference for mapping and collecting unmapped reads to map to the SHFV-krc1 consensus sequence generated from de novo assembly. This process was repeated until changes between the reference and the consensus sequences were not observed for either virus. Using this method, reads corresponding to SHFV-krc1 and SHFV-krc2 were reliably segregated in co-infected animals, with less than 0.2% of SHFV-specific reads mapping to both viruses. The average coverage per genome was 5,654×  for SHFV-krc1 variants and 2,264  for SHFV-krc2 variants. For intra-host genetic analysis, sequencing reads were mapped to the corresponding consensus sequence for each variant. Single nucleotide polymorphism (SNP) reports were generated in Geneious, with a minimum coverage threshold of 100 reads and a minimum frequency threshold of five percent.Sequence data were analyzed using CLC Genomics Workbench 5.5  and Geneious R5 . Low quality (<Q25) and short reads (<100 bp) were removed and the full genome sequences for each virus were acquired using S) and the non-synonymous nucleotide diversity (πN) were estimated for each ORF individually from SNP reports generated by mapping sequencing reads to their corresponding consensus sequence. We estimated πS = ns/Ls and πN = nn/Ln, where ns is the mean number of pairwise synonymous differences; nn is the mean number of pairwise synonymous differences; Ls is the number of synonymous sites; and Ln is the number of nonsynymous sites. Ls and Ln were estimated by the method described in S and πN separately for each ORF in each virus from co-infected animals, we used a factorial analysis of variance to test for main effects of the virus (SHFV-krc1 vs. SHFV-krc2) and the ORF, and for virus-by-ORF interactions. In the case of πS, there were highly significant main effects of virus  and of ORF , but there was not a significant virus-by-ORF interaction . In the case of πN, there were significant main effects of virus  and of ORF , and there was a highly significant virus-by-ORF interaction . Sliding window analysis involved estimating πS and πN in a sliding window of 9 codons, numbered according to the numbering in the sequence alignment of the first codon in the window.The synonymous nucleotide diversity . Twenty-four animals (40.0%) were infected with SHFV-krc1, and 28 animals (46.7%) were infected with SHFV-krc2. Twenty-one animals (35.0%) were co-infected with both SHFV-krc1 and SHFV-krc2  (4–4.1×107 copies/ml) and significantly lower than SHFV-krc1 with an average plasma titer of 7.5×106 vRNA copies/ml plasma . Although instances of mono-infection were scarce relative to co-infection, mono/co-infection status did not impact the load of either virus to a statistically significant extent  . SHFV-kri.e. “mutant swarm”), the consensus sequence may not actually be present in the within-host viral population i.e. inter-host diversity). For SHFV-krc1, percent pairwise nucleotide identity between variants ranged from 86.9%–99.5%. A highly related core group  with pairwise nucleotide identities ranging from 94.5%–99.3% comprised 63% of the variants , indicating greater overall nucleotide diversity in SHFV-krc1 than in SHFV-krc2 .To examine the genetic diversity of SHFV-krc1 and SHFV-krc2 within individual monkeys, we calculated the non-synonymous and synonymous nucleotide diversity, πHFV-krc2 . A positS and πN in ORFs in both viruses. In general, 3′-proximal ORFs displayed more non-synonymous diversity than 5′-proximal ORFs, suggesting that the proteins encoded by 5′-proximal ORFs may be more functionally constrained than those encoded by 3′-proximal ORFs. However, the extent to which underlying RNA structures may have affected this analysis is unknown N in SHFV-krc1 and among the highest in SHFV-krc2  can facilitate speciation, and related yet distinct viruses can recombine e.g. tissue tropisms), possibly resulting in distinct aspects of infection that could contribute to the observed differences in infection frequency  N-glycosylation sites in GP5 had the additional effect of altering the tissue tropism of these “neurotropic” LDV strains N-glycosylation sites were variably found in association with each peak of non-synonymous nucleotide diversity identified ORF5/GP5 of both SHFV-krc1 and SHFV-krc2 .Glycans in this region of the GP5 ectodomain – in addition to aiding viral attachment through the binding of host molecules  including long-term observation of this red colobus cohort in particular – and perhaps studies of SHFV in captive primates – should provide deeper insights into the biology of SHFVs and primate host-virus interactions in general.Finally, these discoveries may facilitate research into many aspects of SHFV biology that remain poorly understood such as host range, tissue tropism, pathogenesis, immunity, and the question of persistence. Further characterization of these viruses in their natural free-living hosts (


AbstractOsornophryne from the Amazonian slope of the Ecuadorian Andes. Morphologically, the new taxon is distinguished from all others species in Osornophryne by having the Toes IV and V longer than Toes I–III, a short and rounded snout with a small rostral papilla, and conical pustules on flanks. The new species previously was confused with Osornophryne guacamayo. A taxonomic key is provided for all known species of Osornophryne.Combining a molecular phylogeny and morphological data, we discovered a new species of  Osornophryne is endemic to the Andes of Colombia and Ecuador, where it occurs in mountain forests and paramo at elevations between 2100 and 4000 m , and Museo Ecuatoriano de Ciencias Naturales (DH-MECN), all based in Quito, Ecuador. Specimens examined are listed in Appendix I. Fingers are numbered preaxially to postaxially from I–IV to facilitate comparison with previous literature dealing with anurans; however, we stress that in an evolutionary perspective anuran fingers should be numbered from II–V, consistent with the hypothesis that Digit I was lost in anurans , anurans . Morphol anurans , 1993, a anurans , 1993; bPageBreakmens, including all the species in Osornophryne, except Osornophryne talipes, and three outgroup taxa. In addition, sequences were downloaded from GenBank (NCBI). Sequences were initially aligned in Clustal X . To amplify the mitochondrial gene 12S, we used the primers MVZ59 and tRNA-val, developed by lustal X  and adjulustal X . Best-filustal X  under thp > 70% are considered to indicate strong support  may be over-estimates of confidence , Maximum Likelihood (ML), and Bayesian Analyses (BA). Parsimony analyses were performed in PAUP  using henfidence .Osonophryne and three species of Atelopus, we obtained a total of 800 bp from the mitochondrial marker 12S rRNA , Reserve Ankaku-Zona, Río Challuwayacu, Provincia de Pastaza, Ecuador, by Elicio Tapia on 21 October 2009.QCAZ 49774 , 3, an aQCAZ 48781, 49777, 45899 obtained with holotype.1°20'57.87"S, 78°09'31.37"W, 2250 m.a.s.l.), Parroquia Río Negro, Cantón Baños, Provincia de Tungurahua, Ecuador, by MYM, M. Urgiles y A. Laguna on 17 May 2008; QCAZ 39769 also obtained near Reserva Biológica Río Zuñac, by DJP, A. Narváez, and J. P. Reyes-Puig on 21 January 2009.DH-MECN 5660 adult female, DH-MECN 5261, 5263, 5258–59, adult male obtained near Reserva Biológica Río Zuñac  by having Toes IV and V longer than Toes I–III , and light brown to orange conical pustules on the flanks ; the venter of female Osornophryne guacamayo is mostly whitish to yellowish with brown marks, whereas that of female Osornophryne simpsoni is orange-brown. Osornophryne cofanorum differs from Osornophryne simpsoni by having its vertebrae and urostlyle coosified with the overlying skin (not co-osified in Osornophryne simpsoni) and vertebral neural spines that are visible dorsally (not visible in Osornophryne simpsoni); also, males of Osornophryne cofanorum have yellow pustules on the tip of the snout, upper eyelid, limbs, and dorsolateral pustular clusters (absent in Osornophryne simpsoni). Finally, Osornophryne simpsoni is distinguished from its sister species, Osornophryne occidentalis, by having a rounded snout in lateral view , brown dorsum with some lighter patches , orange-brown venter , and by inhabiting in the Amazonian slopes of the Andes .es I–III . Morpholn = 1); males small ; outer edge of the eyelid delineated by a continuous row of warts, which are more conical in males than in female; canthus rostralis straight; loreal region slightly concave, with small warts in males and female; pale brown lips; eyes with oval horizontally pupil; infraorbital and postorbital regions with some prominent tubercles of variable size in males and females. Skin of dorsum highly tuberculate, with discontinuous row of conical tubercles starting at level of posterolateral edge of cranium and ending at level of sacrum in males and females; in males, ventral skin with several small pustules and few conical tubercles on gular region and toward the flanks, pustules much denser on chest and abdomen and less conical; in females, ventral skin smooth, with small, non-conical isolated pustules, pustules more numerous on abdomen.Ten adult males and one adult female. Females of medium size  of SVL in males and 28.5% in female; extensive webbing between fingers . The osteological description of females was not possible because only one female is known.The following osteological description of Shape and proportions. The skull is widest posterior to the orbit at the level of the articulation of the maxilla with the quadratojugal. The braincase is broad; at the level of the midorbit, the width of the braincase is about 41.2% of the greatest width of the skull and 26.2% of the medial skull length.Cranium. The neurocranium is formed by five bones—the sphenethmoid, and the paired prootics and exoccipitals. Anteriorly, the neurocranium is completely ossified. A minute septomaxilla is embedded in the anterior nasal capsule cartilage. In dorsal aspect, the cartilaginous planum antorbitale has a perpendicular orientation in relation to the longitudinal axis of the skull. In lateral and ventral views, a broad cartilaginous separation between bony sphenethmoid and prootic is evident. The frontoparietal fontanelle is partially exposed medially between the frontoparietals. Distally, the otic capsules are cartilaginous. Medially, the exoccipitals are slightly separated from one another. The dorsal surface of each prootic is smooth. The epiotic eminences are prominent.The stapes and tympanic annulus are absent. The operculum is oval and cartilaginous.PageBreak. The anterolateral and posterolateral processes of the hyoid are absent. The bony posteromedial processes are slightly expanded proximally; each process has a bony flange along the posteromedial margin. The hypoglossal sinus is broadly U-shaped. The hyalia are simple and lack any processes .There are six prepresacral vertebrae. Presacrals I and II are not fused and are notably shorter than Presacrals III–VI. The vertebral profile in decreasing order of overall width of bony parts is: Sacrum > III > IV > V > VI > II > I. Presacral I, or the atlas, lacks transverse processes. All presacrals are non-imbricate. The transverse processes of Presacral II have a anterolateral orientation, Presacrals III–V have a slightly posterolateral orientation, and Presacral VI is approximately perpendicular to the longitudinal axis of the body. The bony sacral diapophyses are broadly expanded; posteriorly, the sacrum is broadly fused with the urostyle, which is greatly expanded laterally. The urostyle bears a well-developed dorsal crest throughout most of its length .PageBreakPageBreakthat is separated from the pars glenoidalis; the leading and posterior edges of the scapula are slightly concave. The suprascapula is mostly cartilaginous, but it is mineralized at both ends, with the ossified cleithrum apparent as a slender bone along the leading edge of the suprascapular blade and with a proximal end that is wider than its distal end. The sternum is small and completely cartilaginous; it contacts the epicoracoid cartilage, which is extensive, and the posterior margin of the coracoid. The omosternum is absent.The clavicles have a slight orientation, with the medial tips distinctly separated from one another and located at about the same level of the anterolateral end of the clavicle, which articulates with the pars acromialis of the scapula . The corThe long, slightly concave, and slender ilial shafts bear small dorsal crests, which extend from the anterior third to the posterior end of the shafts . The iliThe phalangeal formulae for the hand and foot are standard—i.e., 2-2-3-3 and 2-2-3-4-3, respectively; however, the distal phalange of Finger I, Toe I, and Toe III are greatly reduced and formed mostly by cartilage . Relativsimpsoni is a patronym for Dr. Nigel Simpson in recognition for his continual efforts in protecting the Andean cloud forests of Ecuador. Dr. Simpson is a collaborator of two of the most important conservation NGOs in Ecuador, EcoMinga Foundation (www.ecominga.net) and Jocotoco Foundation (www.fjocotoco.org). As the common name of the species, we suggest “Simpson’s Plumb Toad.” In Spanish, we suggest the name “Osornosapo de Simpson.PageBreakPageBreak”The specific name Osornoprhyne simpsoni is only known from the type locality and surrounding areas, Reserva Zuñac  and Reserve Ankaku-Zona  criteria, we consider Osornophryne simpsoni as Data Deficient; however, it is likely that Osornophryne simpsoni has a restricted distribution, as observed in other Osornophryne species.Osornophryne simpsoni represents a good example of such approach. The phylogeny presented in Osornophryne angel and Osornophryne bufoniformis; similarly, Osornophryne antisana and Osornophryne puruanta are not reciprocally monophyletic, although they have conspicuous morphological differences . Last, within Osornophryne guacamayo, there are two genetically distinctive populations that might represent evolutionary species.It has become increasingly evident that lineage independence is not always accompanied by morphological change when ecological conditions remain similar . Therefo


Arenaviridae, Menekre and Gbagroube virus, were detected in Hylomyscus sp. and Mus (Nannomys) setulosus, respectively. Arenavirus infection of Mus (Nannomys) setulosus was also demonstrated by serological testing. Lassa virus was not found, although 60% of the captured animals were Mastomys natalensis. Complete S RNA and partial L RNA sequences of the novel viruses were recovered from the rodent specimens and subjected to phylogenetic analysis. Gbagroube virus is a closely related sister taxon of Lassa virus, while Menekre virus clusters with the Ippy/Mobala/Mopeia virus complex. Reconstruction of possible virus–host co-phylogeny scenarios suggests that, within the African continent, signatures of co-evolution might have been obliterated by multiple host-switching events.This study aimed to identify new arenaviruses and gather insights in the evolution of arenaviruses in Africa. During 2003 through 2005, 1,228 small mammals representing 14 different genera were trapped in 9 villages in south, east, and middle west of Côte d'Ivoire. Specimens were screened by pan-Old World arenavirus RT-PCRs targeting S and L RNA segments as well as immunofluorescence assay. Sequences of two novel tentative species of the family  Arenaviridae are negative strand RNA viruses hosted by rodents of the superfamily Muroidea. Previous comparisons of arenavirus phylogeny with rodent host phylogeny provided several examples in which virus–host co-speciation was potentially occurring during evolution Hylomyscus sp. captured in Menekre, Prefecture Gagnoa, and three samples  originated from Mus (Nannomys) sp. captured in Gbagroube, Prefecture Divo. The six virus RNA-positive animals were genotyped by sequencing the mitochondrial cytochrome B and 16S rRNA genes. Phylogenetic analysis indicates that the rodents from Menekre presumably constitute a new Hylomyscus species (details to be reported elsewhere), while the rodents from Gbagroube are Mus (Nannomys) setulosus. The virus RNA-positive Hylomyscus species had been trapped in cocoa farm and forest, and the positive Mus (Nannomys) setulosus in banana plantations and forest. Both are usually forest species living in zones with high rainfall. Additional testing of all Hylomyscus sp. (n = 3) and all Mus (Nannomys) setulosus (n = 47) that were negative in the initial screening using RT-PCRs specific for the new virus sequences did not increase the number of positive animals. Thus, virus prevalence was 100% (3/3) in Hylomyscus sp. from Menekre and 12% (3/25) in Mus (Nannomys) setulosus from Gbagroube  setulosus constitute another new arenavirus, called Gbagroube virus. Attempts to isolate the viruses in cell culture or mice were not successful  revealed that all strains from sful see .Hylomyscus sp. were antibody-negative, 7 of 50 Mus (Nannomys) setulosus were positive for anti-OWA IgG antibodies. All seven animals had been captured in Gbagroube in March, August, and November 2005, resulting in an overall seroprevalence of 28% (7 of 25) in this area. Thus, serological testing confirms arenavirus infections in Mus (Nannomys) setulosus in Gbagroube. In addition, 12 animals of other species, including Crocidura spp. (1/61), Lophuromys sikapusi (2/58), Mastomys natalensis (5/729), and Mus (Nannomys) minutoides (4/49), were seropositive  to Lassa virus in NP (14.1%) slightly exceeds the maximum intra-species genetic distance in OWA complex (12.1%). However, the minimum distance in GPC (9.8%) is smaller than the maximum intra-species difference within the Mopeia and lymphocytic choriomeningitis virus (LCMV) clades . Menekre virus is virtually equidistant to Lassa, Gbagroube, Mopeia, Mobala, and Ippy virus (25.4–29.7% in NP and 22.2–25.5% in GPC)  setulosus, respectively, and in none of the other animal species strongly suggests that the virus sequences were recovered from the natural host species. The high prevalence of anti-OWA antibodies in Mus (Nannomys) setulosus from the Gbagroube village lend further support to this conclusion.Genetic distance analysis revealed that Gbagroube virus is closely related to Lassa virus . The min in GPC) . We consIn the phylogeny, Gbagroube virus has a sister relationship with Lassa virus in GPC, NP, and L gene trees , which iThe identification of arenaviruses in rodents so far not implicated as OWA host species led us to investigate how the new data fit with current hypotheses on arenavirus–host co-evolution −4 substitutions×site−1×year−1 with good statistical precision: the smallest interval that contains 95% of the posterior probability density was 3.6–6.8×10−4 site−1×year−1. Nevertheless, it may be interpreted with caution, given that the difference in date between the samples is not large compared to the overall time scale of the reconstruction. The rate estimate for the OWA clade is in the same order of magnitude than estimates for other RNA viruses Another piece of information on the evolutionary history of arenaviruses in Africa may be obtained by estimating the nucleotide substitution rate. The alignment of 122 partial NP sequences from OWAs, mainly collected between 1969 and 2008, contained sufficient temporal structure to estimate a global rate for the clade. The BEAST program calculated a rate of 5.1×10Hylomyscus sp. and Mus (Nannomys) setulosus, respectively. The serological data are consistent with the circulation of Gbagroube virus in Mus (Nannomys) setulosus around the Gbagroube village. No evidence for the spread of this virus in a larger geographical area was found. In addition, the data from the other animal species do not suggest the presence of major hot-spots of arenavirus circulation in the study area. Only few animals have been found serologically positive. Noteworthy is a shrew (Crocidura sp.) that was captured on the same trapping line like the virus-positive Mus (Nannomys) setulosus, which raises the hypothesis of a cross-species transmission of Gbagroube virus from the rodent to the shrew. The arenavirus antibodies in Mus (Nannomys) minutoides, reaching a prevalence of nearly 10%, may point to the circulation of Kodoko virus, which has been found in this rodent species in the neighboring Guinea Mastomys natalensis suggest that Lassa virus is either absent (i.e. the antibodies result from infection with another arenavirus) or circulating at extremely low level in south, east, and middle west of Côte d'Ivoire. In 2002, the study sites were selected in areas with high rainfall in Côte d'Ivoire based on the assumption that rainfall is an important determinant for Lassa virus circulation . However, recent risk mapping using a larger set of environmental conditions and sophisticated mathematical modeling predicts Lassa virus circulation rather in the west and north of Côte d'Ivoire Screening of about 1200 small mammals of various genera from Côte d'Ivoire provided genetic evidence for the existence two new arenavirus species, Menekre and Gbagroube virus, in Mastomys natalensis, they are associated with less abundant rodent species, which do not live as close to humans. However, Hylomyscus sp. and Mus (Nannomys) setulosus may have contact to humans as they occur in cultivation areas. Thus, provided they can cause disease, cases might be observed rarely.Related studies in Africa found two new arenaviruses (F4-8/TZA and TZ22285/TZA) in Tanzania by genetic screening of 600 rodents From a technical point of view, our previously described screening PCR assay targeting the L gene has proven a powerful molecular tool for discovery of new arenaviruses While the phylogenetic position of Menekre virus is somewhat ambiguous — it may stabilize with the availability of sequences of new arenaviruses related to Menekre virus — the position of Gbagroube virus is quite clear and interesting. This virus is now the closest relative of Lassa virus. In the GPC amino acid sequence, Gbagroube virus shows nearly the same genetic distance to some Lassa virus strains (9.8% between Gbagroube virus and Lassa virus Nig08-04) than some Lassa virus strains among themselves (9.2% between Lassa virus strains CSF and Nig08-A47). However, Gbagroube virus probably is not a spillover infection of Lassa virus in Côte d'Ivoire or vice versa. In the phylogeny, it is basal to the Nigerian Lassa virus strains (lineages I to III) and phylogenetically far from the Lassa virus strain circulating in the area of Côte d'Ivoire (strain AV in lineage IV). Assuming that the dating of evolutionary events as shown in Reconstructing the evolutionary history of viruses and timing of diversification events has become an intensively studied issue in recent years. Early hypotheses for evolution of rodent-borne viruses such as arena- and hantaviruses assumed that these viruses co-evolve with their hosts −4 to 10−3 substitutions×site−1×year−1How old viruses are, is a controversial issue. Estimates of the molecular clock rate based on sequences of contemporary RNA viruses are typically in the range of 10Permission to sample small mammals has been granted by the Ministère Délégué auprès du Ministre de la Solidarité Chargé de la Santé, République de Côte d'Ivoire .Traps were placed in the fields and the houses of 9 villages in south, east, and middle west of Côte d'Ivoire . TrappinMus (Nannomys) 3×5 µl) and RNA was prepared by using the E.Z.N.A. Blood RNA Kit (PEQLAB). Screening was performed by using two pan–OWA RT-PCRs. The first assay targeting the large (L) gene was performed as described GCGCACCGGGGATCCTAGGC) and OWS-1000A-rev (AGCATGTCACAAAAYTCYTCATCATG), and the QIAGEN OneStep RT-PCR Kit (Qiagen). All pools were also tested by a Lassa virus-specific RT-PCR as described GTTTTTGCTGCAAGATAAAGGCATGGTCAC) or Gbagroube virus (GbaS-339-rev GTTTTTCGTACAAGAGAGCGGCATGGTCAT). Samples were tested individually rather than pooled in the virus-specific RT-PCRs. For sequencing the complete S RNA segment, nucleoprotein (NP) gene fragments were amplified using the SuperScript One-Step RT-PCR Kit (Invitrogen) and the pan-OWA primer mixtures (i) OWS-2165A/B-fwd (TCTTCAGGTCTCCCTTCWATGTCNATCCANGT+TCTTCAGGTCTCCCTTCWATGTCNATCCA) and OWS-2840A/B-rev (AAYAAYCAGTTTGGGACNATGCCAAG+AAYAAYCAGTTTGGGACNATGCC) or (ii) OWS-2380A/B-fwd (GATGTYCTTGATGCWATGTATGGCCANCC+GATGTYCTTGATGCWATGTATGGCCA) and OWS-2840A/B-rev. PCR fragments were sequenced on both stands. Remaining gaps were closed after designing ≈50 virus-specific primers. S RNA sequences (excluding the conserved 19 nucleotides at the termini) were assembled using SeqMan software (DNASTAR).Blood of 3 animals was pooled  identified the general time-reversible model of sequence evolution with a gamma distribution of among-site nucleotide substitution rate variation (GTR+gamma) as the substitution model that best describes the data in the alignments; it was used in all phylogenetic analyses. The fraction of invariant sites was not considered because estimates for this parameter are very sensitive to the number of taxa. Phylogenies were inferred by the Bayesian Markov Chain Monte Carlo method implemented in BEAST software 7 steps with sampling every 105th step; and two independent runs combined . For complete genes, the tree topology was reexamined by a maximum likelihood approach with bootstrap testing implemented in the PhyML program Phylogenetic analysis included the novel sequences as well as OWA sequences available from GenBank by January 2011. Amino acid sequences of GPC, NP, and L gene were aligned by using MacVector software (MacVector). The alignment was refined manually and used for calculation of 7 steps and sampling every 105th step were combined .As partial NP gene sequences were available for a large number of OWA strains isolated over a period of more than 70 years, the respective alignment  contained sufficient temporal structure to estimate substitution rates and divergence times. The list of taxa with GenBank accession numbers and references for the collection dates is provided in Co-phylogeny scenarios were reconstructed by using CoRe-PA 0.3 CGCCTGTTTAACAAAAACAT) and 16S-Hm (AGATCACGTAGGACTTTAAT) and cytochrome B (∼1190 nucleotides) using primers L7 (ACCAATGACATGAAAAATCATCGTT) and H15915 (TCTCCATTTCTGGTTTACAAGAC) http://blast.ncbi.nlm.nih.gov/) or aligned with rodent sequences from GenBank of the superfamily Muroidea. Phylogenetic reconstruction was performed by using the PhyML program Rodents were genotyped by amplification and sequencing the mitochondrial genes for 16S rRNA (∼530 nucleotides) using primers16Sar L . If serum was not available in sufficient quantities, rodent organs (heart and spleen) were vigorously washed in 100 µl PBS and the wash fluid was used for the assay. Organ wash fluid had previously been validated for antibody detection and shown to be equivalent to serum . Diluted serum or organ wash fluid was incubated with the cells and bound rodent IgG was detected with anti-mouse IgG-fluorescein isothiocyanate (Dianova). Signals were visualized with a fluorescence microscope.3 of spleen was homogenized in 1 ml cell culture medium using a bead mill. The homogenate was cleared by centrifugation. African green monkey kidney (Vero) cells and mouse fibroblast cells (L cells) in culture vials were inoculated with blood and cleared homogenate, respectively. Cells and supernatant were passaged 3 times. Potential virus growth was monitored in supernatant by pan-OWA RT-PCR (see above), and in cells by immunofluorescence using a mixture of OWA-specific monoclonal antibodies Virus isolation was attempted with blood and spleen specimens of rodents CIV839, CIV843, CIV1227, CIV608, CIV674, and CIV1290. About 8 mmTable S1Old World arenavirus strains included in the study. The table provides GenBank accession numbers for all sequences included in the phylogenetic analyses as well as collection dates with references for the NP gene sequences.(PDF)Click here for additional data file.Abstract S1Translation of the abstract into French by Elisabeth Fichet-Calvet.(PDF)Click here for additional data file.


This review describes the current knowledge on the emergence, epidemiology, clinical signs, molecular virology and diagnosis of SBV infection.After the unexpected emergence of Bluetongue virus serotype 8 (BTV-8) in northern Europe in 2006, another arbovirus, Schmallenberg virus (SBV), emerged in Europe in 2011 causing a new economically important disease in ruminants. The virus, belonging to the O Introduction2. Timeline of SBV infection in Europe3. Molecular virology3.1. Genome and structure3.1. Life cycle3.1. Pathogenicity3.1. Phylogeny4. Clinical signs5. Transmission6. Origin7. Diagnosis8. Surveillance9. Seroprevalence10. Impact11. Prevention12. Conclusions13. Competing interests14. Author’s contributions15. Acknowledgements16. ReferencesOrthobunyavirus genus from the Bunyaviridae family. Douglas, Sathuperi and Shamonda viruses were later identified as closer relatives of SBV [Culicoides variipennis larvae cells (KC cells) caused fever and mucous diarrhoea, providing experimental evidence that SBV might be responsible for the clinical signs observed [At the end of the summer and in the autumn 2011, hyperthermia and drop in milk production were reported in adult dairy cows in north-west Germany and The Netherlands -5. In Nos of SBV . A speciobserved .This paper reviews current knowledge on the emergence, molecular virology, clinical signs, diagnosis and seroprevalence of SBV and is based on data published up to the end of January 2013 in peer-reviewed journals, internet-based reporting systems such as the Program for Monitoring Emerging Diseases (proMED-mail), communications from research institutes and official reports from governmental and European institutions such as the European Food and Safety Authority (EFSA).nd of January 2012. France reported its first case of SBV on the 25th of January 2012 after the virus genome was detected by RT-qPCR in brain samples from malformed lambs born on farms located in the territorial divisions of “Moselle” and “Meurthe et Moselle” in north-eastern France [th of February [th of February, SBV was confirmed in a malformed goat in north-east Italy [th of March, in Spain , in a newborn lamb [SBV was first detected in Germany and The Netherlands in 2011 . In Decen France . The preFebruary . On the st Italy  and on torn lamb .By the end of April 2012, SBV had been detected in 3628 herds in Europe . SBV-infEarly 2012, the development of assays to detect anti-SBV antibodies, as discussed later in this review, provided a useful tool to show evidence of SBV infection since viraemia is transient ,15.th of June, Denmark reported the presence of antibodies against SBV in two cattle from southern Jutland [th of July, Switzerland confirmed its first cases of acute SBV infection in cows from two farms in the canton of Berne [On the 5 Jutland  and on tof Berne .By August 2012, more than 5500 cases of SBV infection in ruminants had been recorded across northern Europe .In mid-September, anti-SBV antibodies were detected in Austria in cattle and sheep . At the By the end of October 2012, SBV infection was confirmed by RT-qPCR and/or serology in approximately 6000 holdings in Europe .In November 2012, antibodies against the virus were detected in milk from cattle herds in Norway  and an oAll these reports show that SBV has spread rapidly in vast parts of Europe.Bunyaviridae family and the Orthobunyavirus genus. The Bunyaviridae family is composed of 350 viruses that are divided into 5 genera: Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus and Tospovirus. Viruses from this family infect vertebrates with the exception of tospoviruses that are plant viruses. Viruses such as Rift Valley fever virus (Phlebovirus), Akabane virus (Orthobunyavirus) and Nairobi sheep disease virus (Nairovirus) are important pathogens in veterinary medicine. Other viruses, such as hantaviruses responsible for hemorrhagic fever with renal syndrome and cardio-pulmonary syndrome or Crimean-Congo hemorrhagic fever virus (Nairovirus), can cause serious disease in humans. The Orthobunyavirus genus is composed of more than 170 viruses divided into 18 serogroups. SBV belongs to the Simbu serogroup that also includes Simbu virus, Oropouche virus, Akabane virus, Douglas virus, Sathuperi virus, Aino virus, Shamonda virus, Peaton virus and many others.Analysis of viral sequences has led to the classification of SBV in the The bunyavirus genome consists of 3 segments of negative-sense single-stranded RNA: the L (large), M (medium) and S  segments  Figure A. PhleboVirions from bunyaviruses are enveloped, spherical and have a diameter of approximately 80 to 120 nm. They acquire their membrane when budding at the Golgi apparatus lumen -39. ElecSince SBV was discovered recently, very little is known on its life cycle and knowledge accumulated on other bunyaviruses can only be discussed.The replication cycle of bunyaviruses is exclusively cytoplasmic. It begins with the recognition of the cellular receptor, unknown for the majority of bunyaviruses, by the Gn/Gc heterodimers present at the surface of the virion membrane ,42. ViriAssembly of bunyaviruses is thought to occur mostly in tubular virus factories containing both cellular and viral components situated around the Golgi complex ,46,47. FOrthobunyavirus genus and the Bunyaviridae family, also contributes to viral pathogenesis and seems to be a major virulence factor. This non-structural protein inhibits protein synthesis and the host cell antiviral response by interfering with RNA polymerase II-dependent transcription, IFN-I production and apoptosis mediated by IRF-3 [The pathogenicity of orthobunyaviruses is dependent on multiple viral factors encoded by the three genomic segments. For example, the neuroinvasive ability of La Crosse virus, another orthobunyavirus from the California serogroup, is determined by the polymerase and/or the glycoproteins whereas the host immune response is inhibited by NSs that antagonises the expression of type I interferon (IFN-I) and the transcription mediated by RNA polymerase II -59. NSs by IRF-3 -65.Although there is no conservation of sequence, NSs from other bunyaviruses are also involved in pathogenesis and inhibition of the host cell antiviral response. For example, NSs from Rift Valley fever virus suppresses host transcription by interfering with the subunits of the transcription factor II H (TFIIH) complex, degrades dsRNA-activated protein kinase (PKR) and represses the activation of the promoter IFN-β via its association with a subunit of the Sin3A repressor complex -70.However, little is known about the viral factors involved in the pathogenicity of viruses involved in veterinary medicine, such as Akabane virus, Shamonda virus and SBV. Recently, it was shown that IFN-I receptor (IFNAR) knock-out mice  are suscShamonda virus , species Sathuperi virus (Douglas virus and Sathuperi virus), species Shuni virus (Aino virus and Shuni virus), species Akabane virus (Sabo virus) and species Simbu virus (Simbu virus)) [Sathuperi virus species. This conclusion is also supported by a serological investigation showing that Douglas and Sathuperi viruses, but not Shamonda virus, are neutralised by anti-SBV serum [Initial phylogenetic analysis of the SBV genomic segments indicated that SBV displays 69% identity with Akabane virus for the L segment, 71% identity with Aino virus for the M segment and 97% identity with Shamonda virus for the S segment . After aSheep and goats seem to be very mildly affected by SBV infection. Symptoms are more apparent in adult cows, and include loss of appetite, hyperthermia and diarrhoea, which can lead to a 50% reduction in milk production -78. SympIn December 2011, The Netherlands reported a teratogenic effect of SBV in sheep with manifestations comparable to those observed for Akabane and Aino viruses -82. InfeIn case of twin gestation, one twin can suffer from arthrogryposis and the other from neurological disorders. One twin can also be born malformed and the other one viable or only affected by a delayed growth.Newborns suffer from severe neurological disorders that generally lead to death of the animal several hours to several days after birth. It was reported that a SBV-positive one-week old calf born at term showed severe central nervous system lesions, severe dysfunctions of the cerebral cortex, basal ganglia and mesencephalon, severe porencephaly or hydranencephaly but no arthrogryposis . InteresNecropsy has revealed some cases of hydranencephaly , hydrocephaly, cerebral and cerebellar hypoplasia and porencephaly ,86-88 F.Histological studies have revealed lymphohistiocytic inflammation in the central nervous system and glial nodules in the mesencephalon and hippocampus in ovine species. Astrogliosis and microgliosis were detected in both calves and lambs and some cases of myofibrillar hypoplasia of skeletal muscles were reported for both species . Histoloth and 36th day of gestation in sheep, the 30th and 50th day in goats and the 76th and 174th day in bovines [In the case of Akabane virus, infection of the foetus occurs between the 28 bovines . The sev bovines ,81,90. D bovines . A study bovines . No visi bovines .Orthobunyavirus genus. However, SBV infection has not yet been reported in these species. Most of the viruses from the Simbu serogroup are not considered to be zoonotic, with the exception of Oropouche virus, which can infect humans and provoke severe flu-like symptoms. To date, no evidence of SBV infection in humans has been reported and no SBV-neutralising antibodies have been detected in sera from persons (farmers and veterinarians) exposed to the virus [SBV can infect bison as reported in Germany . The virhe virus ,96.Aedes and Culex genus and by several species of ticks [C. obsoletus complex, C. chiopterus and C. dewulfi) trapped from July to October 2011 in Belgium [C. obsoletus group trapped in Denmark during the same period also contained SBV RNA [C. obsoletus complex and C. chiopterus collected in August-September 2011 in the Netherlands where the prevalence of SBV among Culicoides at this period was estimated to be around 0.25% [The majority of bunyaviruses are transmitted by arthropod vectors and, in particular, mosquitoes, phlebotoms, culicoides, ticks and thrips, with the exception of hantaviruses which are transmitted by rodents. Studies have shown that viruses within the Simbu serogroup are mostly transmitted by culicoides, but also by mosquitoes from the of ticks ,98. Rece Belgium . Culicoi SBV RNA . Furthernd 0.25% . The virnd 0.25% -104. Thend 0.25% -108. To culicoides species are present inside farm buildings during the winter and are able to complete their life cycle in animal enclosures [Cases of acute SBV infection were recorded following the start of the 2012 vector season in France, the United Kingdom, Switzerland, Germany and Italy ,17,29,31closures ,110. It The history and geographical origins of SBV raise numerous questions. As for the introduction of BTV-8 in The Netherlands and Belgium in 2006, the causes of SBV emergence in northern Europe remain unknown. The closest relatives to SBV are Sathuperi, Douglas, Shamonda, Akabane, Aino, Peaton or Sango viruses but, to date, they have not been identified in Europe . These vOrthobunyavirus genus [As discussed above, Shamonda virus is thought to be a reassortant containing the S and L genomic segments from SBV and the M segment from an unclassified virus . Such reus genus . In the us genus  and thatus genus .Orthobunyavirus belonging to the Bunyamwera serogroup, in populations of Anopheles maculipennis trapped in the south-western part of Germany in 2009 illustrates the importance of this type of surveillance [SBV emergence is a reminder that bunyaviruses and particularly orthobunyaviruses are a potential threat for European livestock. Akabane and Aino viruses are already present in the Mediterranean region and might be introduced into Europe. Consequently, the surveillance of livestock and vector populations is critical to monitor the emergence of such viruses in Europe. The identification of the Batai virus, an eillance .It is surprising that SBV has emerged in the same region of Europe as BTV-8 in 2006 and later BTV-6 and −11, and may suggest that the viruses followed the same route of introduction. SBV might have been present before in a region of the world where no and/or rare clinical signs were manifested or reported in the native population. Considering the increase in international trade of animals and products of animal or vegetal origin, it is of no doubt that Europe will face more and more frequently this type of emergence.Diagnosis of SBV infection relies on the detection of the viral genome by RT-qPCR, as mentioned above. It is a duplex assay that was initially developed by the FLI ,116. TheCulicoides variipennis larvae) cells with brain, serum or blood samples.Viral isolation requires the inoculation of Vero , BHK-21 (baby hamster kidney fibroblast) or KC  and a plaque reduction neutralisation test were developed to detect antibodies present in the serum of infected animals ,119,120.Interestingly, in a study looking at anti-SBV antibodies in foetal blood samples from calves and lambs, it was shown that 74% of the samples tested positive for SBV infection by ELISA whereas only 27% were SBV-positive by RT-qPCR . Using aSharing knowledge and tools has allowed affected and neighbouring regions to efficiently and rapidly monitor the progression of SBV infections in Europe as shown in Figure th of January 2012 in consultation with the French epidemiological surveillance platform for animal health. Veterinarians were asked to send samples  from stillborns or malformed ruminant offspring showing clinical signs such as arthrogryposis, torticolis, scoliosis, brachygnathia and/or hydranencephaly. RT-qPCR was performed to detect SBV RNA and virus was isolated using the protocols kindly provided by Dr Martin Beer and Dr Bernd Hoffmann from the FLI, as described above [st of May for ovine and caprine ruminants and on the 31st of August for bovines. As of the 31th of August 2012, 3197 SBV infected farms had been reported in France , including 1143 sheep farms, 2019 cattle farms and 35 goat farms  [th of November 2011 and the 4th of April 2012) and a within-herd seroprevalence of 40.68% was found in goats (142 goats from 8 flocks sampled during the same period) [Antibodies against SBV have been detected in 91% of cows sampled within 250 km of the location where SBV emerged in Belgium in February-April 2012 [ch 2012) . A betwe period) . These rPreliminary seroprevalence studies were also undertaken by the Alfort ANSES laboratory in farms from different French territorial divisions using ELISA and VNT . A high The high seroprevalence of SBV found in The Netherlands, Belgium and in several French farms, indicates a widespread exposure to SBV during the biting insect season in 2011. Retrospective and prospective serological studies on ovine, bovine and caprine livestock will help to elucidate the period of SBV introduction in Europe. Similarly, it would be interesting to study the seroprevalence of SBV in wild species to evaluate their involvement as a reservoir of the virus. A study has reported the presence of anti-SBV antibodies in roe and red deer from southern Belgium sampled from October to December 2011 . An averSBV has a low or limited impact on animal health. Reports submitted by different European countries affected by SBV have estimated morbidity and mortality rates of less than 3% . The higFew data are available on the impact of SBV within herds. A preliminary survey investigating the impact of SBV infection in sheep flocks in France has been conducted recently. This study showed that in SBV RT-qPCR-positive flocks, an average of 85% lambs were healthy while 13% were born dead or died rapidly after birth and 2% were born with malformations but survived for more than 12 h (data from 363 flocks and 64 548 lambs) . HoweverConsidering SBV has a limited impact on animal health, trade restrictions have not been advised and are regarded as unjustified by the European Union (EU) and the World Organisation for Animal Health (OIE) . SBV is Vaccination is a preventive measure that could reduce the impact of SBV . The devThe emergence of SBV at the end of 2011 in Europe is a reminder that the introduction of new diseases remains a threat for European countries. The rapid response to the emergence of SBV established by affected European countries has shown that an efficient network of laboratories is in place to face the emergence of new animal viruses. SBV was able to re-circulate after the winter season and is still circulating in Europe. A certain level of protection exists within the ruminant populations but it is likely that a significant percentage of animals remains susceptible to SBV in areas where no or few cases of SBV have been reported. This suggests that new congenital cases of SBV infection will occur during the winter 2012–2013. Maintaining an efficient surveillance would be essential to further describe the progression of the epidemic and its impact on the breeding industry. More studies are needed to determine the regions in which SBV is present, to understand its geographical and genetic origin and to identify its putative reservoirs. A better understanding of the pathogenesis associated with SBV infection and the ability of SBV antibodies to protect animals against the disease will also be useful to control the disease.The authors declare that they have no competing interests.VD structured the review and prepared, along with DV, EL and SZ, the draft of the manuscript. CS, GB, EB, JR, CV, MD, PH, DC, AD, JL, LC, PP, JE, BE and PM helped with the French epidemiological, serological and clinical data presented in this paper. All authors read and approved the final manuscript.