Encephalitis is a hallmark of Nipah virus (NiV) infection in humans. The exact route of entry of NiV into the central nervous system (CNS) is unknown. Here, we performed a spatio-temporal analysis of NiV entry into the CNS of hamsters. NiV initially predominantly targeted the olfactory epithelium in the nasal turbinates. From there, NiV infected neurons were visible extending through the cribriform plate into the olfactory bulb, providing direct evidence of rapid CNS entry. Subsequently, NiV disseminated to the olfactory tubercle and throughout the ventral cortex. Transmission electron microscopy on brain tissue showed extravasation of plasma cells, neuronal degeneration and nucleocapsid inclusions in affected tissue and axons, providing further evidence for axonal transport of NiV. NiV entry into the CNS coincided with the occurrence of respiratory disease, suggesting that the initial entry of NiV into the CNS occurs simultaneously with, rather than as a result of, systemic virus replication. Henipavirus genus in the family Paramyxoviridae. Like several other paramyxoviruses, Nipah virus causes encephalitis in infected individuals. During the first documented Nipah virus outbreak in Malaysia and Singapore in 1998–1999, 276 cases of Nipah virus encephalitis were observed, with 106 fatalities2467Nipah virus is a member of the Morbillivirus in the family Paramyxoviridae, has been shown to travel to the CNS via anterograde transport along olfactory neurons in the nasal cavity, and via the hematogenous pathway, probably through infected lymphocytesAlthough it is evident that Nipah virus can replicate in the CNS, it is currently unknown how Nipah virus reaches the cells of the central nervous system. Canine distemper virus, a member of the closely related genus Recently, it has been described that Nipah virus can disseminate via binding to leukocytes; however, the route of entry of virus into the CNS was not specifically addressed in this study5 TCID50 of Nipah virus, strain Malaysia resulted in an initial respiratory phase of infection after which a proportion of surviving hamsters developed neurological signs of disease5 TCID50 of Nipah virus. On 2, 4, 6, 8, 10 and 12 days post inoculation (dpi) four hamsters each were euthanized and necropsied to look at the progression of Nipah virus infection. Six hamsters developed obvious neurological signs such as paralysis, seizures, torticollis and tremors between 7 and 12 dpi and were euthanized according to approved protocol.In a previous study we determined that intranasal inoculation of hamsters with 10Beginning at 2 dpi, the nasal turbinates of every hamster in the study demonstrated inflammation and necrosis of either the respiratory epithelium, olfactory epithelium or both. The lesions were most severe in the olfactory epithelium  and incrShortly after detection of Nipah virus in the nasal turbinates, the virus entered the CNS. Single Nipah virus infected neurons extending from the olfactory epithelium through the cribriform plate into the olfactory bulb were first detected at 4 dpi, providing direct evidence for transport of Nipah virus along the olfactory neurons into the CNS .On 6 dpi the olfactory bulbs demonstrated a mild meningoencephalitis that increased in severity over time . The menNipah virus dissemination throughout the CNS started as early as 6 dpi; in addition to olfactory bulb inflammation there were multifocal areas of encephalitis . HistoloCellular pathology of the CNS was investigated by transmission electron microscopy (TEM) examination of the ventral cortex in the area of the olfactory tubercle. Examination of these tissues collected on 12 dpi revealed several ultrastructural pathological changes. Marked extravasation of plasma cells from blood vessels was observed , indicat91516In this study, we clearly showed the rapid progression of Nipah virus from the olfactory epithelium in the nasal turbinates to the CNS following intranasal inoculation, as represented schematically in Based on the observed vasculitis in the CNS of deceased patients, it was proposed that Nipah virus accesses the CNS through the hematogenous routeDisruption of the blood-brain barrier has been shown to occur in hamsters infected with Nipah virus, but only in animals with neurological signs of diseaseThe recent finding that leukocytes can transport Nipah virus without becoming infected19It is currently unclear whether CNS entry routes observed in Nipah virus animal models with a relatively large area of olfactory epithelium, such as pigs, ferrets and the Syrian hamster, can be extrapolated to Nipah virus entry into the human CNS. The increased surface area of the olfactory epithelium of these animal models may result in a higher incidence of CNS infection via the olfactory route. However, this does not exclude that the olfactory route could be used for Nipah virus entry into the human CNS. The involvement of the olfactory pathway in CNS entry by other human viruses  indicates that certain viruses can efficiently use this route to enter the human CNS23Whereas it is not clear how differences in architecture and physiology of the olfactory system affect Nipah virus CNS entry via the olfactory route, it seems logical that the intranasal inoculation used in this study does predispose the animals to efficient replication in the upper respiratory epithelium and the olfactory epithelium as opposed to either intratracheal or intraperitoneal inoculation routes used in other studies. This could result in a bias towards CNS entry via the olfactory route. However, the initiation of Nipah virus nasal shedding before oropharyngeal shedding upon natural infection via contact transmissionBy EM, we clearly observed nucleoprotein aggregates in axons in the brain of infected animals, again suggesting that Nipah virus utilizes axonal transport for rapid entry of, and dissemination through, the CNS. Although many nucleoprotein aggregates were observed, no mature virus particles could be seen. This observation is in line with several studies on brain material of patients from the Nipah virus outbreak in Malaysia that also described nucleoprotein inclusions but no virus particles916The rapid appearance of Nipah virus in the CNS coincided with the first signs of bronchointerstitial pneumonia between 2 and 4 dpi , suggestAll animal experiments were approved by the Institutional Animal Care and Use Committee of the Rocky Mountain Laboratories, and performed following the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC) by certified staff in an AAALAC-approved facility.Nipah virus  was kindly provided by the Special Pathogens Branch of the enters for Disease Control and Prevention, Atlanta, Georgia, United States and propagated in VeroE6 cells in DMEM (Sigma) supplemented with 10% fetal calf serum , 1 mM L-glutamine (Lonza), 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco).tm:AURA, Harlan Laboratories) were inoculated intranasally with 105 TCID50 of Nipah virus in a total volume of 80 µl. On days 2, 4, 6, 8, 10 and 12, one group of hamsters was euthanized. Remaining hamsters were euthanized upon displaying neurological signs of disease and processed for histopathology or electron microscopic analysis. Hamsters were anaesthetized using ketamine (80–100 mg/kg) and xylazine (7–10 mg/kg) and perfused with PBS containing 5 mM EDTA, followed by 4% paraformaldehyde. Tissues of interest were then further fixed according to BSL4 standard operating procedures for a minimum of 7 days in 10% neutral buffered formalin for histopathology or in Karnovksy's fixative  for electron microscopy.Eight groups of four 6-8 week-old female Syrian hamsters  staining and an immunohistochemical method using a rabbit polyclonal antiserum against the Nipah virus nucleoprotein (N)After fixation for 7 days with Karnovsky's fixative at 4°C, excised tissues were post-fixed for 30 minutes with 0.5% osmium tetroxide/0.8% potassium ferricyanide in 0.1 M sodium cacodylate, 1 hour with 1% tannic acid and overnight with 1% uranyl acetate at 4°C. Specimens were dehydrated with a graded ethanol series with two final exchanges in 100% propylene oxide before infiltration and final embedding in Embed-812 resin. Thin sections were cut with a Leica EM UC6 ultramicrotome , and stained with 1% uranyl acetate and Reynold's lead citrate prior to viewing at 120 kV on a Tecnai BT Spirit transmission electron microscope . Digital images were acquired with a Hammamatsu XR-100 side mount digital camera system  and processed using Adobe Photoshop v. CS5 .For immune labeling, thin sections from specimens prepared as above were collected on 200 mesh nickel grids and the following steps performed by placing grids (section side down) on 20 µl droplets of solutions unless otherwise specified. Sections were etched for 10 minutes with 4% meta-periodate, then incubated with 3% BSA in 0.1 M Tris buffer to block non-specific interactions. Primary anti-Nipah virus N antibodyHistopathology and EM were analysed by a board-certified veterinary pathologist with expertise in ultrastructural pathology.Conceived and designed the experiments: VJM, EdW. Performed the experiments: VJM, JBP, TB, DL, RR, TT, DS, ERF, EdW. Analysed the data: VJM, DS, ERF, HF, EdW. Wrote the paper: VJM, HF, EdW.Supplementary Information


Dengue virus infects approximately 100 million people annually, but there is no available therapeutic treatment. The mimetic peptide, DN59, consists of residues corresponding to the membrane interacting, amphipathic stem region of the dengue virus envelope (E) glycoprotein. This peptide is inhibitory to all four serotypes of dengue virus, as well as other flaviviruses. Cryo-electron microscopy image reconstruction of dengue virus particles incubated with DN59 showed that the virus particles were largely empty, concurrent with the formation of holes at the five-fold vertices. The release of RNA from the viral particle following incubation with DN59 was confirmed by increased sensitivity of the RNA genome to exogenous RNase and separation of the genome from the E protein in a tartrate density gradient. DN59 interacted strongly with synthetic lipid vesicles and caused membrane disruptions, but was found to be non-toxic to mammalian and insect cells. Thus DN59 inhibits flavivirus infectivity by interacting directly with virus particles resulting in release of the genomic RNA. The four dengue virus serotypes, dengue virus types 1, 2, 3 and 4, are major mosquito-transmitted, human pathogens. Currently there are no available vaccines or therapeutics. Dengue is a positive-sense RNA virus, encapsulated by a lipid membrane Ligands that mimic the structure of viral envelope components can sometimes interfere with the normal infection process and, thus, have potential as antiviral agents. For example, the T20 peptide, which is approved for treatment of HIV 50) in a FFU infection assay using mammalian epithelial cells. The infectivity of other flaviviruses  was inhibited at higher DN59 concentrations . This peptide was previously shown to inhibit the infectivity of dengue 2 virus and West Nile virus, but activity against other flaviviruses and the mechanism of action were unknown trations .Cryo-electron (cryoEM) microscopy of dengue virus type 2 particles incubated at 37°C for 30 minutes with 100 µM DN59 in 1% (v/v) DMSO in a 5∶1 molar ratio of peptide to E protein on the virus had lost most of their RNA genomes whereas control virus particles in the presence of 1% (v/v) DMSO showed no visible loss of RNA genome . Additio50 for the genome degradation assay to shift upwards in concentration compared to the FFU reduction assay.The release of viral RNA from the particles was consistent with the results of a genome sensitivity assay conducted by exposing peptide-treated virus particles to RNase digestion, followed by quantitative reverse transcription PCR to determine the amount of protected viral RNA. The RNA genomes of untreated particles were protected from RNase digestion, whereas the genomes of particles co-incubated with increasing concentrations of DN59 were susceptible to digestion in a dose-responsive manner . The pepThe separation of the genome from the virus particle would be expected to irreversibly destroy infectivity. Reversibility was tested directly by treating virus with peptide at a concentration expected to produce approximately 80% inhibition of infectivity, then diluting the virus:peptide mixture 10 fold to a peptide concentration expected to produce negligible inhibition. No reversibility of inhibition was observed in these experiments .The release of the virus RNA genome was confirmed by centrifuging peptide-treated, untreated, and triton detergent-treated virus particles through a tartrate density gradient, and monitoring the amount of RNA genome and E protein in each fraction. The results showed that the genome and E protein co-migrate in intact virus particles, but migrate to different fractions following peptide or detergent treatment, indicating that the genome and E protein are no longer associated after peptide treatment .To confirm that there were no other targets for the inhibitory activity of DN59, time of addition and infectivity assays in a different target cell line were conducted. There was no inhibition of infectivity when mammalian target cells were incubated with DN59 and then washed prior to the addition of virus . Nor wasBased on these experiments, DN59 appears to induce formation of holes in the viral membrane. Thus, DN59 might be expected to interact with lipid membranes and form holes or otherwise disrupt membrane bilayer structures. Consistent with this expectation, a concentration-dependent increase in the fluorescence of the tryptophan residue at peptide position nine was observed when peptide was mixed with liposome vesicles composed of either 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), or a 9∶1 molar ratio of POPC and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), indicative of strong binding . Also, aPreviously DN59 had been shown to be non-toxic to cultured cells 4 infectious virus particles, whereas in our cryoEM studies, the same concentration of DN59 causes RNA release from of 1×1010 virus particles. However, direct comparison between these two assays may not be possible. Van der Schaar et al. Schmidt et al. The most likely mechanism by which DN59 or other stem region peptides can penetrate the outer layer of E glycoproteins and gain access to the virus membrane is by way of dynamic “breathing” of the virus particle Our observations show that DN59, a 33 amino acid peptide mimicking a portion of the dengue virus E protein stem region, functions through an unexpected mechanism that involves disruption of the viral membrane and release of the viral genome.2 cells (American Type Culture Collection (ATCC), Manassas, VA, cat. no. CCL-7) 2. For the cryo-electron microscopy studies, dengue virus 2 (16681) was grown in C6/36 cells and the tissue culture supernatant was collected on day 3-4, spun at 2,704×g for 10 minutes at 4°C. 8% PEG in NTE  was added to the tissue culture supernatant and mixed. The solution was then allowed to sit overnight before the PEG precipitated virus was centrifuged at 14,636×g for 1 hr. The pellet was resuspended in 1 ml NTE buffer, loaded onto a 24% (w/v) sucrose cushion and centrifuged at 175,587×g for 90 min. Pellets were resuspended overnight in NTE before being loaded onto a 10-30% (w/v) potassium sodium tartrate step gradient and centrifuged at 175,587×g for 2 hrs. Purified virus was collected from the 20% potassium-tartrate fraction. The virus solution was then buffer exchanged to NTE buffer using an Amicon Ultra-4 centrifugal filter.Dengue virus 1 (HI-1), dengue virus 2 (NGC-2), dengue virus 3 (H-78), dengue virus 4 (H-42), and yellow fever virus (17-D) were propagated in LLC-MK2O Peptide stocks of DN59, the 33 amino acid pre-anchor domain peptide (MAILGDTAWDFGSLGGVFTSIGKALHQVFGAIY) and a randomly scrambled version of the peptide, DN59scr (YFIDTSGAIWGASHLTGVLFDFMGIQGGAVLAK) were purified and then prepared as approximately 1 mM stocks in 20% dimethylsulfoxide (DMSO): 80% H2 cell monolayers for 1 hr at 37°C, and overlaying with media containing 0.85% (w/v) Sea-Plaque Agarose . Infected cells were incubated at 37°C with 5% CO2 for 2 days (yellow fever virus), 3 days  or 5 days (dengue virus 1and 2). Infected cultures were fixed with 10% (v/v) formalin, permeablized with 70% (v/v) ethanol, and foci were detected using mouse monoclonal antibodies against yellow fever virus , dengue (E60), or polyclonal anti-Kumlinge virus rED3 antisera, followed by horseradish peroxidase-conjugated goat anti-mouse immunoglobulin , and developed using AEC chromogen substrate  as previously described FFU reduction assays were performed as previously described 2. After 72 hrs, RNA was isolated from cells using an RNeasy Mini Kit . qRT-PCR was performed as previously described C6/36 monolayers were infected with approximately 7,600 FFU of dengue virus 2 at 37°C for 1 hr before being aspirated, complete culture media added, and incubated at 37°C and 5% CO−/A2, and taken at about 4.3 to 7 µm out-of-focus. Thirty-eight DN59 treated dengue virus particles were selected for three-dimensional (3D) image reconstruction. Initial models for 3D reconstructions were generated using the program starticos in EMAN 1 mM DN59 in 10% (v/v) DMSO was mixed with 18 µl of mature dengue virus to give a final DN59 concentration of 100 µM with 1% (v/v) DMSO. The mixture was incubated at 37°C for 30 min, then 4°C for 2 hrs and frozen on holey carbon grids. Dengue virus without peptide and dengue virus incubated with DMSO only controls were also frozen. Images were collected with a Philips CM200 cryo-electron microscope using 200 KV, a magnification of 50,000, an electron dose of 25 e4 FFU of dengue virus 2 was incubated with DN59 for 1 hr at room temperature and then digested with micrococcal nuclease  for 1 hr at 37°C. RNA was extracted using the Qiagen RNeasy mini kit and qRT-PCR was performed as above using 10503F/10599R TCACCAAATCCCACGGTAGAAGCA)/3 M-2R (AGGGCATGTATGGGTTGAGAACCT), M-1F (GAGGCTGGAAGCTAGAAG)/M-1R (GAGATACGGCACCTATGG), 5 M-1F (AAGCAGAACCTCCATTCGGAGACA)/5 M-1R (AAACACTCCTCCCAGGGATCCAAA), and 5-1F (AATCCCACCAACAGCAGGGATACT)/5-1R (CGCCATCACTGTTGGAATCAGCAT) primer sets.Approximately 1.4-2.9×102 cell monolayers, the virus/peptide mixtures were diluted with serum-free DMEM to 1 ml, reducing the concentration of DN59 to 1 µM.Similar to the FFU reduction assays, approximately 200 FFU of dengue virus 2 were incubated with 0 or 10 µM DN59 in a total volume of 100 µl serum-free DMEM for 1 hr at room temperature. Immediately before infecting LLC-MK6 FFU of dengue virus 2 produced in LLC-MK2 cells and purified as described above for the cryo-electron microscopy studies, was treated with 100 µM DN59 or 1% (v/v) triton X-100 for 30 min at 37°C. Treated virus was loaded onto a 10-35% (w/v) potassium sodium tartrate step gradient and centrifuged at 175,117×g for 2 hrs. Individual fractions were collected and assayed for virus genome and E protein. Genome quantitation was carried out by qRT-PCR as described above for the RNase sensitivity assay using the 10503F/10599R primer set 2O2 stopped by adding 1% (v/v) phosphoric acid. Optical density was measured at 450 nm.Approximately 10The lipids 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) were purchased from Avanti Polar Lipids . Lipids in chloroform solution were dried under vacuum overnight followed by hydration with phosphate buffered saline (PBS). Ten cycles of freezing and thawing were used to ensure solute entrapment. Unilamellar vesicles of 0.1 µm diameter were made by extrusion of the lipid suspension through 0.1 µm polycarbonate filters The fluorescent dye 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) and its obligate quencher p-xylene-bis-pyridinium bromide (DPX) were purchased from Invitrogen . Vesicles were prepared with ANTS/DPX entrapped inside where DPX quenches ANTS fluorescence 2, BHK, or C6/36 cells for 1 hr at 37°C, the solution was removed and the cells incubated at 37°C in complete medium with 5% CO2 for 24 hrs.Cytotoxicity of DN59 was measured by mitochondrial reductase activity using the TACS™ MTT cell proliferation assay . DN59 in serum-free DMEM was added to LLC-MKSheep red blood cells (RBC)  in anti-coagulant K2-EDTA, were washed and resuspended in PBS to a final concentration of 10% (v/v). Peptide was added to 2% RBC, incubated at 37°C for 1 hr and centrifuged at 13,000 rpm. Supernatants were collected and the absorbance at 560 nm was measured. Results were normalized against treatment with 1% (v/v) triton X-100 as a control for 100% hemolysis.2 cells were carried out in a similar manner as above, except that a 1.2% solution of methylcellulose  in complete medium was used in place of agarose. Vesicular stomatitis virus eGFP-P was incubated for 24 hrs at 37°C before overlays were aspirated, rinsed with PBS, and plaques were visualized for GFP expression Plaque reduction assays in LLC-MKFigure S1Inhibitory effect of DN59 is dependent on its interaction with flavivirus particles. (A) Co-incubation of DN59 with other flaviviruses showed dose response inhibition in a focus-forming unit reduction assay with somewhat higher 50% inhibition concentrations compared to dengue virus. (B) Focus-forming unit reduction assay indicates that DN59 has no inhibitory effect on dengue virus infection when the peptide is added to LLCMK-2 cells and removed prior to the addition of dengue virus, or when DN59 is added to cells that had already been infected. (C) DN59 was co-incubated with the enveloped, negative-stranded RNA, vesicular stomatitis virus (VSV), and infectivity was assayed in a plaque reduction assay. No substantial inhibition of VSV was observed.(TIF)Click here for additional data file.Figure S2Homogeneity of virus particle preparations used for EM imaging. Lower magnification CCD images of control (A) and DN59 treated (B) dengue virus showed that the control virus particles were relatively homogenous and mature. DN59 treated particles clumped and were obscured by an electron dense material.(TIF)Click here for additional data file.Figure S3CryoEM image reconstruction of DN59 treated dengue 2 virus. (A) CryoEM image of DN59 treated particles. The particles appeared empty. (B) Reconstruction and validation of the cryoEM structure. Different starting models Click here for additional data file.


Rice-infecting viruses have caused serious damage to rice production in Asian, American, and African countries, where about 30 rice viruses and diseases have been reported. To control these diseases, developing accurate, quick methods to detect and diagnose the viruses in the host plants and any insect vectors of the viruses is very important. Based on an antigen–antibody reaction, serological methods such as latex agglutination reaction and enzyme-linked immunosorbent assay have advanced to detect viral particles or major proteins derived from viruses. They aid in forecasting disease and surveying disease spread and are widely used for virus detection at plant protection stations and research laboratories. From the early 2000s, based on sequence information for the target virus, several other methods such as reverse transcription-polymerase chain reaction (RT-PCR) and reverse transcription-loop-mediated isothermal amplification have been developed that are sensitive, rapid, and able to differentiate closely related viruses. Recent techniques such as real-time RT-PCR can be used to quantify the pathogen in target samples and monitor population dynamics of a virus, and metagenomic analyses using next-generation sequencing and microarrays show potential for use in the diagnosis of rice diseases. Rice, second only to corn in worldwide crop production, is produced in all Asian countries, in most of South and Central America, and in some of central and eastern Africa. More than 80% of the world’s rice is grown in South, East and Southeast Asian areas where the hot, humid climate favors not only rice but also the viruses and their vectors and about 30 rice viruses diseases have been reported .In the early years of plant virus research, detection and identification of viruses were based on symptom development on infected plants or biological indexing. However, the use of symptoms for diagnosis is not reliable because symptoms vary depending on the virus strain, the presence of any mixed viral infections, the cultivar and growth stage, growing environment, and sometimes, the resemblance of viral symptoms to those induced by environmental injury. Biological assays are one of the most widely used methods among the many diagnostic techniques for plant viruses because the assays are simple and do not require special knowledge or skill . HoweverIn addition, since almost all rice viruses are vector-borne, detection methods for rice viruses in their vectors, which show no noticeable symptom, have been needed to forecast and counter disease outbreaks. For these reasons, the development of serological methods based on an antigen–antibody reaction have been active areas of research, and highly sensitive and specific methods  based on molecular or nucleic-acid techniques have recently become available. This review introduces major methods to detect target rice viruses from the 1980s and summarizes the potential of the current technologies in contributing to diagnosis of rice diseases.Rice stripe virus , Rice dwarf virus , Rice gall dwarf virus , Rice ragged stunt virus , Rice grassy stunt virus , Rice transitory yellowing virus [RTYV, same species as Rice yellow stunt virus (RYSV), a member of Rhabdoviridae, positive sense ssRNA virus; Rice black streaked dwarf virus  and Southern rice black streaked dwarf virus . Detection of these viruses in insect vectors may be easier than in rice plants, since these viruses propagate in insect bodies and the entire insect can be tested in the procedure. The tungro-disease-associated viruses, Rice tungro bacilliform virus  and Rice tungro spherical virus  are non-propagative and transmitted in a semipersistent manner by leafhoppers, therefore, it is difficult to detect these two viruses in the insect vector using serological methods, but more sensitive methods such as real time RT-PCR and RT-LAMP can be used  is the most important causal agent of viral diseases of rice and transmitted by a planthopper in a persistent manner , is transmitted by the soil-inhabiting fungal pathogen Polymyxa graminis. The major vectors of Maize streak virus strain A  are leafhoppers  is transmissible by mechanical inoculation and insect vectors leaf beetles , and passive hemagglutination (PHA), and solid-phase examples include the enzyme-linked immunosorbent assay (ELISA), dot-immunobinding assay (DIBA). Gel-based assay  have been also reported. These serological methods are used widely to detect rice viruses at pest control stations, plant protection stations, and agricultural experiment stations in Asia. Once the serological detection system is established, highly sensitive testing of a large number of samples is simpler and cheaper even though the methods are classical and several months are required to make practical antisera against rice viruses.The overview of detection methods for viruses is shown in Escherichina coli-expressed viral coat protein are now being used . In some cases, antisera against purified virus may contain contaminating host-plant materials, which cause non-specific reactions. In addition, the antisera may cross-react with closely related viruses. For example, the antiserum against RGSV cross-reacts with the nucleocapsid protein of RSV . To reduing used .Antisera that recognize linear epitopes instead of conformational epitopes are chosen for immunodetection of denatured proteins. For example, target proteins that are boiled or treated with β-mercaptoethanol, can be separated by SDS-PAGE and detected in a Western blot by using antisera that recognize linear epitopes. In contrast, antisera that recognize conformational epitopes are preferred for ELISA and other methods that target proteins whose structure is preserved. In an ELISA to detect RDV in rice plants, an antiserum against intact RDV particles was more sensitive than that against RDV dissociated by SDS. But for Western blot analysis, the sensitivity of antiserum against the dissociated RDV was higher than that against intact RDV particles .Figure 1A). But the double antibody sandwich (DAS)-ELISA in commercially available kits has started to overtake the LAR because this technique is more sensitive, yields clear results and is inexpensive  is necessary before the target DNA sequence is amplified. Commercial kits to extract RNA and to synthesis cDNA from several companies such as Life Technologies, QIAGEN, Takara Biotechnologies, BIO-RAD and Promega are available. The variations of RT-PCR  and RT-loop-mediated isothermal amplification (LAMP) have been applied to detect rice viruses from plants or insects. The commercial kits for RT-PCR are sold by the companies mentioned, and the RT-LAMP kit is sold by Eiken Chemical.Figure 1C outlines the procedure and shows the resulting bands in the agarose gel after electrophoretic separation of the DNA fragments amplified by multiplex RT-PCR with the gene-specific primer sets (I and II), designed from coding sequences for the rice viral capsid or capsid-like proteins using the program FastPCR 6.0 . Primer set I comprises two specific primers for rice actin as an internal control and 10 specific primers for RDV, RSV, RBSDV, RTYV, and RTSV, which have been reported in northeastern and eastern Asia. Primer set II comprises two specific primers for rice actin as an internal control and 10 specific primers for SRBSDV, RGDV, RRSV, RTBV, and RGSV, which have been reported in southeastern and southern Asia. Since the RT-PCR products derived from each virus differs in size, two viruses such as RDV/RSV, which are major rice viruses in Japan can be detected in a single reaction . In rice fields of some countries where mixed infections with RRSV and RGSV, SRBSDV, and RSV and RBSDV, RSV and RBSDV, and RDV have been reported  and confirmed that our RT-LAMP system could distinguish between SRBSDV and RBSDV in infected plants . Meanwhile, The LAMP technique, developed by In real-time PCR, amplification of DNA is monitored by the detection and quantitation of a fluorescent reporter signal, which increases in direct proportion to the amount of PCR product in the reaction. This technique, combined with RT, can be used to quantify target RNA. As noted already, RT-LAMP and conventional RT-PCR techniques offer rapid, sensitive, and accurate diagnosis, but they do not provide quantitative information like real-time PCR can. The starting amount of the target nucleic acid is quantitated, and the reaction can be monitored while in progress.Figure 1E-a). A cycle threshold (Ct), calculated from an amplification curve of fluorescence, is regarded as the virus titer in the sample . In the third step, reaction reagents are incubated sequentially from 65 to 97°C at increments of ca. 0.1°C/s to obtain a melting curve for checking specific amplification of target region. The last step is cooling. Because virus titer is usually shown as the density of the virus relative to expression of a housekeeping gene in the same sample , expression of the housekeeping gene  also should be determined by real-time RT-PCR. With the quantitative approach using a real-time RT-PCR for SRBSDV, the location of the virus was revealed and changes in viral density in the rice plant and subsequent effects on symptom appearance in rice can be monitored, as can virus acquisition by vector insects . During this second step, fluorescence intensity, which corresponds to the amount of PCR product, is monitored for each cycle , the cycling conditions for this method are shorter than for standard PCR, and the detection sensitivity is generally higher than for standard PCR assays and equivalent to that of RT-LAMP . Therefo-3 to 10-5 (Advances in the technologies to detect and diagnose plant pathogens have culminated in a variety of options for researchers in laboratory and for growers. After the application of ELISA to detect plant viruses was reported , ELISA a to 10-5 .2-fold higher than dot-blot hybridization (4-fold higher than ELISA (3-fold higher than conventional RT-PCR (Since PCR was devised in the late 1980s , the PCRdization . The senan ELISA  and 103-l RT-PCR . The senl RT-PCR .Current technologies, e.g., microarrays and next-generation sequencing also hold potential for use in diagnosing rice diseases. Microarrays are suitable techniques for high-throughput detection and identification, since an almost limitless number of pathogen probes can be placed on a single array . The potTo select the most appropriate diagnostic methods, we need to focus on our objective. For large-scale analyses to evaluate incidence or for screening tests to monitor disease spread in fields, we should select methods using a user-friendly, evidence-based approach, an evaluation of cost per analysis and a calculation of post-test probability of disease . TherefoThe RT-LAMP and real-time RT-PCR techniques are too expensive for routine analysis in large surveys, but they do enable the differentiation of closely related viruses such as SRBSDV and RBSDV and very early detection of disease before symptoms are visible. Such features can help farmers and plant-health professionals to choose the best strategies to minimize potential damage. Particularly with real-time RT-PCR, applications such as screening for virus resistance and studying viral population dynamics, viral multiplication and virus–host interactions are feasible .Conventional detection methods have only provided information on the presence of target pathogens. But progress in the development of technologies to diagnose plant diseases may not only contribute to the control of rice viral diseases, but also open opportunities to analyze potential pathogens/candidate pathogens and to develop a comprehensive understanding of the ecology of microorganisms in rice fields.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila pathogen, is both a target and suppressor of RNAi. We detected viral small RNAs with a signature of Dicer-2 dependent small interfering RNAs in Nora virus infected Drosophila. Furthermore, we demonstrate that the Nora virus VP1 protein contains RNAi suppressive activity in vitro and in vivo that enhances pathogenicity of recombinant Sindbis virus in an RNAi dependent manner. Nora virus VP1 and the viral suppressor of RNAi of Cricket paralysis virus (1A) antagonized Argonaute-2 (AGO2) Slicer activity of RNA induced silencing complexes pre-loaded with a methylated single-stranded guide strand. The convergent evolution of AGO2 suppression in two unrelated insect RNA viruses highlights the importance of AGO2 in antiviral defense.RNA interference (RNAi) is a major antiviral pathway that shapes evolution of RNA viruses. We show here that Nora virus, a natural  Drosophila melanogaster as a model, we study virus-host interactions in infections by Nora virus, a non-lethal natural pathogen of fruit flies. Insects depend on the RNA interference (RNAi) pathway for antiviral defense. A hallmark of the antiviral RNAi response is the production of viral small RNAs during infection. We detected Nora virus small RNAs during infection of Drosophila, demonstrating that Nora virus is a target of the antiviral RNAi pathway. Furthermore, we show that Nora virus viral protein 1 (VP1) inhibits the catalytic activity of Argonaute-2, a key protein of the RNAi pathway. The 1A protein of Cricket paralysis virus suppresses RNAi via a similar mechanism. Importantly, whereas Nora virus persistently infects Drosophila, Cricket paralysis virus induces a lethal infection. Our findings thus indicate that two distantly related viruses independently evolved an RNAi suppressor protein that targets the Argonaute-2 protein. Altogether, our results emphasize the critical role of Argonaute-2 in insect antiviral defense, both in lethal and persistent infections.Multi-cellular organisms require a potent immune response to ensure survival under the ongoing assault by microbial pathogens. Co-evolution of virus and host shapes the genome of both pathogen and host. Using  An efficient antiviral immune response is essential for the control or elimination of virus infection and for survival of the infected host. The immune system exerts a strong evolutionary pressure that shapes the genetic makeup of viral pathogens. Indeed, viruses evolved counter-defense mechanisms to evade, suppress or inactivate host immunity. Studying these mechanisms provides important insight in the critical steps of antiviral responses and may uncover novel components and regulators of immune pathways.AGO2 mutant flies and of AGO2 knockdown mosquitoes provides genetic support for this hypothesis Plants, fungi, and invertebrate animals rely on the RNA interference (RNAi) pathway for antiviral defense The evolution of viral suppressors of RNAi (VSRs) is a testament to the antiviral potential of the RNAi pathway in plants and insects. Given the central role of dsRNA and siRNAs as initiators and specificity determinants of the RNAi pathway, it is not surprising that many VSRs sequester dsRNA. For instance, the Drosophila C virus (DCV) 1A protein binds long dsRNA and shields it from processing by Dcr-2 Drosophila melanogaster. While these viruses indeed efficiently infect Drosophila laboratory stocks and cell lines, DCV is the only natural Drosophila pathogen among these three viruses Costelytra zealandica) and field crickets (Teleogryllus oceanicus and T. commodus), respectively VSRs have been identified in dozens of plant viruses from all major virus families Drosophila laboratory stocks as well as Drosophila in the wild PicornaviralesSince viral counter-defense mechanisms co-evolve with the antiviral immune responses of the host species, it is essential to characterize a VSR within the correct evolutionary context. We therefore set out to identify an RNAi suppressor in Nora virus, a positive sense (+) RNA virus that persistently infects 1118 Drosophilaw strain that is widely used as a recipient strain for transgenesis. We isolated and sequenced 19–29 nt small RNAs from body (abdomen and thorax), thorax and head of adult 1118w flies. Sequence reads that perfectly matched the Drosophila genome were annotated and discarded. Of the remaining reads, 396.646 , 237.265 , and 1.099.496  matched the published Nora virus sequence (NC_007919.3), indicating that the 1118w strain was infected by Nora virus (Nora virus is an enteric (+) RNA virus that successfully establishes a persistent infection in flies ra virus . As RNA ra virus . We therIn all three libraries, Nora virus-derived small RNAs were predominantly 21-nt long, the typical size of Dicer-2 products. The size distribution of small RNAs derived from the (+) RNA strand, however, were noticeably wider than those derived from the (−) RNA strand . For 21-Drosophila Dcr-2 generates 21-nt duplex siRNAs in which 19 nucleotides are base-paired leaving a 2-nt 3′ overhang at each end. For each library, we collected the 21-nt RNA reads whose 5′ ends overlapped with another 21-nt RNA read on the opposite strand of the Nora virus genome. Then, for each possible overlap of 1 to 21-nt, the numbers of read pairs were counted and converted into Z-scores (Z-scores . This anZ-scores . The oriDrosophila cell culture (Renilla luciferase (RLuc) reporter plasmids and a plasmid that expresses one of the four viral ORFs. Subsequently, Fluc expression is silenced using specific dsRNA, and Fluc and Rluc activity is monitored. As expected, DCV 1A, a well characterized VSR that binds long dsRNA, efficiently suppressed RNAi, whereas the inactive DCV 1A K73A mutant was unable to do so  . Nora vi culture . In thisto do so . Cotransto do so . Howeverto do so .Next, we tested whether VP1 inhibits the production of siRNAs by Dcr-2 or a subsequent step in the RNAi pathway. To this end, we repeated the RNAi sensor assay using a synthetic siRNA that does not require Dcr-2 cleavage for its silencing activity. Also under these conditions, Nora virus VP1 suppressed silencing of the Fluc reporter. Furthermore, VP1 suppressed RNAi to a similar extent as CrPV 1A, which was previously shown to suppress the effector stage of the RNAi machinery Drosophila, the microRNA (miRNA) and siRNA pathways are separate processes, with Dcr-1 and AGO1 dedicated to the miRNA pathway and Dcr-2 and AGO2 to the siRNA pathway. Nevertheless, crosstalk between the miRNA and RNAi pathways occurs. Using miRNA sensor assays in S2 cells, in which Fluc expression is silenced by endogenous miRNAs or co-expressed primary miRNAs, we observed that VP1 does not suppress miRNA activity , out of a total of 475 aa, did not affect VSR activity. These results show that the RNAi suppressor activity of VP1 maps to the C-terminal 124 aa.VP1 is highly conserved among different Nora virus isolates . We wereted size . Deletioted size . This suin vivo using transgenic flies in which thread (th), also known as Drosophila inhibitor of apoptosis 1, can be silenced by expression of dsRNA targeting this gene . Silencing of th in the eye of RNAith flies is fully dependent on the RNAi pathway, since the phenotype is lost in an AGO2 null mutant background  in RNAith flies resulted in eyes with a normal size and a rescue of the rough eye phenotype  expressing the functional VP1ΔN351 (SINV-VP1) or GFP (SINV-GFP) from a second subgenomic promoter  induced only modest mortality in these flies with a fully functional RNAi response. After 36 days of infection, 73% of the SINV-GFP infected flies and all mock infected flies were still alive. In contrast, SINV-VP1 infection resulted in more severe mortality. SINV-VP1 infected flies died faster and only 9% of the flies survived the 36-days follow up period  and purified it from Escherichia coli. We verified that MBP-VP1 fusion proteins are fully functional in VSR assays in S2 cells to exclude the possibility that MBP interferes with VP1 VSR activity (data not shown).To further characterize the VSR activity of Nora virus VP1, we next analyzed the activity of VP1 in a series of biochemical assays that monitor individual steps of the RNAi pathway. To this end, we fused the active VP1The ability of VP1 to suppress siRNA-initiated RNAi in S2 cells  suggestsin vitro RNA cleavage assay (Slicer assay) in Drosophila embryo extract Since VP1 is incapable of interfering with the initiator phase of the RNAi pathway, we next examined the effect of VP1 on the effector phase of RNAi. For this purpose, we used an To discriminate between RISC assembly and target RNA cleavage by a pre-assembled RISC complex, we performed Slicer assays under two experimental conditions . In the Using the first protocol, cleavage of the target RNA was suppressed by VP1 , lane 3.Drosophila embryo extract and analyzed endogenous AGO2 protein levels by Western blot. Neither VP1 nor CrPV 1A affected AGO2 protein levels in embryo lysate, indicating that these two proteins do not mediate RNAi suppression through degradation of AGO2  and (−) RNA strands. During (+) RNA virus infection, (+) viral RNA accumulates in large excess over (−) viral RNA (∼50–100 fold). Cleavage of structured RNA elements by Dcr-2 is therefore expected to produce viral small RNAs that mirror this asymmetric distribution. Thus, similar to other RNA viruses, our results imply that Dcr-2 targets the dsRNA intermediates in Nora virus replication AGO2 mutants are hypersensitive to a number of RNA virus infections AGO2 phenotype is complicated by other cellular functions of AGO2, such as regulation of cellular gene transcription and control of transposon activity in vivo. Importantly, these two viruses display a strikingly different course of infection – CrPV causes a lethal infection, whereas Nora virus establishes a non-lethal, persistent infection – suggesting that the interaction between a VSR and the host RNAi machinery is not the main determinant for viral pathogenicity.The current model proposes that the antiviral RNAi response relies on dicing of viral dsRNA and on slicing of viral target RNAs using vsiRNAs as a guide. Genetic analyses support the role of AGO2 in antiviral defense: 1118w male flies using Trizol reagent (Invitrogen), and RNA quality was verified on a Bioanalyzer (Agilent). Small RNAs were then cloned using the DGE-Small RNA Sample Prep Kit and the Small RNA v1.5 Sample Preparation Kit (Illumina) following the manufacturer's instructions. Libraries were sequenced on the Illumina HiSeq platform.Total RNA was extracted from dissected heads, bodies (abdomens and thoraxes) and thoraxes from http://hannonlab.cshl.edu/fastx_toolkit/). Reads in which the adapter sequence (CTGTAGGCACCATCAATCGT) could not be detected were discarded. Only the clipped 19–30 nt reads were retained. Sequence reads were first matched against the Drosophila genome (v5.37) using Bowtie (http://bowtie-bio.sourceforge.net/index.shtml). Reads not matching the Drosophila genome were then matched against the published Nora virus sequence , allowing one mismatch during alignment. Viral small RNAs were then used to reconstitute a small RNA-based consensus genome sequence  using Paparazzi Sequence reads were clipped from 3′ adapters using fastx_clipper (Drosophila S2 cells were cultured at 25°C in Schneider's medium (Invitrogen) supplemented with 10% heat inactivated fetal calf serum, 50 U/mL penicillin, and 50 µg/mL streptomycin (Invitrogen). DCV was cultured and titered on S2 cells as described previously ΔN351 was cloned into the XbaI site of the double subgenomic pTE3'2J vector XhoI restriction, purified and used as template for in vitro transcription using the mMESSAGE mMACHINE SP6 High Yield Capped RNA Transcription kit (Ambion). In vitro transcribed RNA was purified using the RNeasy kit (Qiagen) and transfected into BHK cells. Viral titers in the supernatant were determined by plaque assay on BHK cells.RNAi reporter assays were performed as described previously using 25 ng pMT-GL3, 6 ng pMT-Ren, and 25 ng suppressor plasmid per well of a 96-well plate Wolbachia and endogenous virus infection . Virus infections of adult female flies were performed as described previously using 5,000 PFU of recombinant SINV In vivo RNAi experiments were performed by crossing GMR-Gal4, RNAi/CyOUAS-th virgins  with UAS-VSR/TM3 Sb flies. The eye phenotype was monitored in two- to four-day-old male F1 offspring lacking the CyO and TM3 Sb balancers.Flies were maintained on standard medium at 25°C with a light/dark cycle of 12 hours/12 hours. Fly stocks that were used for Sindbis virus infection and for preparation of embryo lysate were cleared of tion see . We usedE. coli as described in 2, 1 mM DTT, 140 mM NaCl, 2.7 mM KCl) Recombinant proteins were stored as aliquots at −80°C in dialysis buffer containing 30% glycerol.The GST and MBP fusion proteins were purified from Gel mobility shift assays were performed as described 5′- UCG AAG UAC UCA GCG UAA GU[mU] and passenger strand 5′- CUU ACG CUG AGU ACU UCG AUU were annealed by incubating 20 µM of each siRNA strand in annealing buffer  for 1 min at 90°C, followed by incubation for 1 hour at 37°C. For guide strand loading of RISC, embryo lysates were incubated with Fluc single-stranded guide strand RNA at a final concentration of 10 µM. Radiolabeled probes and target RNA for gel shift and Slicer assays are described in Dicer and Slicer assays were performed according to the protocol of Haley and colleagues with minor modifications, described in Figure S1VP1 is unable to suppress the miRNA pathway. A firefly luciferase (Fluc) construct containing the par6 3′UTR, a target for miRNA1 (Fluc-par6), was co-transfected with plasmids encoding Renilla luciferase (Rluc) and either Nora virus VP1 or the inactive VP1ΔC74 mutant. Fluc-par6 expression was silenced by co-transfecting a plasmid encoding pri-miRNA1, whereas a pri-miRNA12 expressing construct was used as a negative control. AGO1 or AGO2 gene expression was knocked down by co-transfection of dsRNA targeting these genes . Expression of Fluc and Rluc was induced two days after transfection, and reporter activities were measured three days after transfection. Rluc activity was used to normalize Fluc activity within each sample, and data were normalized to the pri-miR12 treated sample. Bars represent averages and standard deviations of biological triplicates. A representative graph of two independent experiments is shown. The numbers represent p-values relative to pri-miR1 treated vector control samples in a two-tailed Student's t-test assuming equal variances.(TIF)Click here for additional data file.Figure S2Alignment of VP1 sequences from different Nora virus isolates. Alignment of VP1 sequences of Nora virus isolate Umeå 2007 (accession number GQ257737) and Nora virus sequences from infected fly stocks from our own laboratory . We analyzed VP1 sequences in a total of eight Nora virus infected fly stocks. Five VP1 sequences were identical to NL1, one was the NL2 sequence, and two stocks contained a mixed population of Nora virus sequences. These eight stocks were obtained from five different laboratories or stock centers. However, they have been maintained in our laboratory before we tested them for Nora virus infection, and we cannot exclude the possibility that they became infected in our laboratory. Although we therefore cannot infer overall virus diversity from these data, they do indicate that VP1 is a conserved protein. The FR1 isolate is the Nora virus genome that was reconstituted from small RNA sequences from wildtype 1118w flies from a laboratory based in France (GenBank accession number JX220408).(TIF)Click here for additional data file.Figure S3Nucleotide and protein sequence of full-length and VP1 mutants fused to V5-His tag at the N-terminus. Nucleotide and amino acid sequence of V5 epitope and Histidine (His) tagged full-length VP1 sequence (VP1FL). A linker sequence between the His tag and VP1 was created to facilitate cloning. Start and stop sites of the respective N- and C-terminal deletion mutants of VP1 are indicated. The VP1 deletion mutants were fused to the V5-His tag in an identical way as the VP1FL construct.(TIF)Click here for additional data file.Figure S4Nora virus VP1 is unable to bind long dsRNA or to interfere with Dcr-2 activity. (A) Mobility shift assay of suppressor proteins with long dsRNA. Uniformly radiolabeled long dsRNA was incubated for 30 minutes with buffer (lane 1) or recombinant MBP-VP1ΔN284 (lanes 2–4), MBP (lanes 5–7), GST-DCV 1A (lanes 8–10) or GST (lanes 11–13). Ten-fold dilutions of recombinant protein were used starting from the following concentrations: MBP-VP1ΔN284 , MBP , GST-DCV 1A , and GST . RNA mobility shifts were analyzed on an 8% native polyacrylamide gel. (B) Dicer activity in S2 cell extract in the presence of viral suppressor proteins. Uniformly radiolabeled long dsRNA was incubated in S2 cell extract for 3 hours with buffer (lane 3) or the indicated recombinant proteins. Two-fold dilutions were used for MBP-VP1ΔN284  and MBP . Two independent preparations of GST-DCV 1A were used . GST was used at a concentration of 1.2 µM (lane 14). As size markers, dsRNA input (lane 1) and end-labelled siRNAs (lane 2) were used. Dicer products were analyzed on a 12% denaturing polyacrylamide gel.(TIF)Click here for additional data file.Figure S5ATP depletion during Slicer assay. (A) Schematic representation of the protocol used to deplete (−ATP) or to regenerate ATP after initial depletion (+ATP) for Slicer assays of Drosophila embryo lysate was incubated with an siRNA for 30 minutes under standard conditions. Subsequently, N-ethylmaleimide (NEM) was added in both conditions to inhibit the ATP regenerating activity of creatine kinase. After incubating the reactions for 10 minutes on ice, DTT was added to quench the NEM in both conditions. Hexokinase, glucose, and milliQ water (MQ) were added in the –ATP protocol to deplete the pool of ATP. For the +ATP condition, Hexokinase was substituted by hexokinase buffer, and MQ was substituted for Creatine kinase to restore the ATP regenerating activity. Subsequently, the reactions were incubated for 30 minutes after which recombinant protein (rec. protein) was added. Following another 30 minutes incubation period, the 32P-cap-labelled RNA was added to the reaction, after which the incubation was continued for another 2 hours. Subsequently, reactions were analyzed on a polyacrylamide gel. (B) ATP concentrations before and after the Slicer assay under –ATP and +ATP conditions. ATP levels were measured at the moment of target RNA addition (0 hrs) or after 2 hours of incubation with target RNA. For ATP concentration measurements, recombinant protein was substituted for protein storage buffer, and target RNA was substituted for MQ. ATP levels were measured using the Celltiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer's protocol.(TIF)Click here for additional data file.Protocol S1Extended and supplemental methods for molecular cloning, miRNA sensor assay, clearance of Wolbachia and endogenous viruses from fly stocks, production of recombinant proteins in E. coli, production of radio-labeled probes and target RNA, Dicer and Slicer assays.(DOC)Click here for additional data file.Text S1Nora virus VP1 is unable to suppress the miRNA pathway.(DOC)Click here for additional data file.


C. aurantium, sour orange; C. sinensis, orange; C. limon, lemon; and C. aurantifolia, lime) were collected and genotyped using 20 nuclear SSR markers and four indel mitochondrial markers. Multi-locus genotypes (MLGs) were compared to references from French and Spanish collections.Citrus represents a substantial income for farmers in the Mediterranean Basin. However, the Mediterranean citrus industry faces increasing biotic and abiotic constraints. Therefore the breeding and selection of new rootstocks are now of the utmost importance. In Tunisia, in addition to sour orange, the most widespread traditional rootstock of the Mediterranean area, other citrus rootstocks and well adapted to local environmental conditions, are traditionally used and should be important genetic resources for breeding. To characterize the diversity of Tunisian citrus rootstocks, two hundred and one local accessions belonging to four facultative apomictic species (The differentiation of the four varietal groups was well-marked. The groups displayed a relatively high allelic diversity, primarily due to very high heterozygosity. Sixteen distinct MLGs were identified. Ten of these were noted in sour oranges. However, the majority of the analysed sour orange accessions corresponded with only two MLGs, differentiated by a single allele, likely due to a mutation. The most frequent MLG is shared with the reference sour oranges. No polymorphism was found within the sweet orange group. Two MLGs, differentiated by a single locus, were noted in lemon. The predominant MLG was shared with the reference lemons. Limes were represented by three genotypes. Two corresponded to the 'Mexican lime' and 'limonette de Marrakech' references. The MLG of 'Chiiri' lime was unique.The Tunisian citrus rootstock genetic diversity is predominantly due to high heterozygosity and differentiation between the four varietal groups. The phenotypic diversity within the varietal groups has resulted from multiple introductions, somatic mutations and rare sexual recombination events. Finally, this diversity study enabled the identification of a core sample of accessions for further physiological and agronomical evaluations. These core accessions will be integrated into citrus rootstock breeding programs for the Mediterranean Basin. Phytophthora sp., parasitic nematodes and viruses. Rootstocks are also significant in the adaptation of the tree to several abiotic constraints affecting the Mediterranean citriculture. These include water resource scarcity and soil salinity. Citrus rootstocks are generally apomictic and seed-propagated. Therefore, both scions and rootstocks are clonally propagated. Sour orange (C. aurantium L.), one of the most important citrus rootstocks in the world, is still the predominant rootstock on the southern edge of the Mediterranean Basin. Sour orange is tolerant to limestone, alkalinity and salinity and is resistant to Phytophthora sp. Furthermore, sour orange is widely compatible with scion varieties and confers good fruit quality. Unfortunately, combining sour orange with commercial citrus varieties results in trees that are sensitive to Citrus Tristeza Virus (CTV), causing the rapid decline and death of the trees grafted on sour orange rootstocks. As CTV spreads throughout the Mediterranean basin, citrus production on sour orange rootstock will soon be uneconomic. On the other hand, rootstocks selected for their resistance to CTV are not well adapted to other local constraints [Worldwide production of citrus in 2009 reached greater than 120 million tons [Citrus are those of Swingle and Reece [C. medica L. (citron), C. reticulata Blanco (mandarin) and C. maxima (Burm.) Merr. (pummelo). Recent molecular studies have confirmed the central role of these three taxa [C. micrantha Wester [C. aurantium L. (sour orange), C. limon (L.) Burm.f. (lemon), C. aurantifolia (Christm.) Swing. (lime), C. sinensis (L.) Osb. (sweet orange) and C. paradisi Macfad. (grapefruit), were derived from hybridization amongst the true species [Citrus species and several related genera are sexually compatible [Citrus taxonomy and systematics remain controversial [th century, and the citrus industry was established in the beginning of the 20th century. Since 1934, export trade has undergone great expansion, and producers began producing the 'Maltaise demi-sanguine' orange. Today, citrus orchards occupy approximately 20,400 hectares, with citrus production oscillating between 210,000 and 300,000 tons during the last decade. Sour orange is still the most widely used traditional rootstock of Tunisian citriculture. Despite clonal propagation, the morphological variation exhibited by local accessions of sour orange is remarkable. Additional rootstock varieties and non-grafted citrus trees are still used, particularly in the oasis areas, and these trees are related to C. sinensis, C. limon and C. aurantifolia. The traditional Tunisian rootstocks are well adapted to the adverse regional soils and climatic conditions. Consequently, these rootstocks constitute a very important germplasm to be employed as an abiotic stress tolerance source for future rootstock breeding projects. Therefore, the activities of investigation, collection, preservation and characterization of this germplasm are a priority. The assessment of genetic diversity within the rootstock germplasm is a prerequisite to the optimization of its management at the national and regional level.The most widely accepted taxonomic systems for nd Reece  and Tanand Reece , who recnd Reece  and Barrnd Reece  indicateree taxa -15 and ca Wester ,8. The s species ,9,10,16.mpatible ,18 and cmpatible . This haoversial . Citrus Simple sequence repeat (SSR) markers have gained considerable importance in plant genetics due to their many desirable genetic attributes, including high polymorphism, wide genomic distribution, co-dominant inheritance and reproducibility allowing networking activities, multi-allelic nature and chromosome-specific location. In citrus, nuclear SSR development from genomic libraries -24, ESTsThe main objective of this study was to assess the genetic diversity of Tunisian citrus rootstock germplasm. Specifically, the following questions were addressed: (i) What are the extent and the structure of the Tunisian rootstocks genetic diversity? (ii) What are the origin and the extent of the genetic diversity within facultative apomictic species? (iii) How is the Tunisian germplasm diversity related to reference genotypes of the same varietal groups of certified Citrus collections? For this purpose, 201 individual from different regions of Tunisia were analysed, and a set of reference cultivars from Spanish and French germplasm banks were genotyped for 20 nuclear SSR markers and four Indels mitochondrial markers.mCrCIR06B05) to ten (mCrCIR03C08) with an average of six .All 201 accessions collected were analysed using the 20 nuclear SSR markers. A total of 120 alleles were scored from 20 loci. The number of alleles per locus varied from three . Interestingly, five of the other sour orange MLGs, each corresponding to one accession , displayed approximately half of the heterozygosity of the primary sour orange MLG without additional alleles. These five accessions may have arisen from zygotic seedlings following self-pollination. The two last sour orange MLGs (G and I) displayed intermediate proportions of heterozygous loci  and a high number of 'new' alleles  not shared with the sour orange groups A, B and C.Among the 201 analysed accessions, only 16 distinct MLGs were obtained for the 20 loci studied yielding a The four 'Chiiri' limes displayed the same genotype for the 20 analysed loci. The two 'Sweet' limes shared the second lime MLG, the third lime MLG being represented by 'Arbi' and 'Beldi lemon'. Eleven of the 12 lemon accessions were identical, and the last one differed only at a single locus.To assess the relationship of local accessions with reference genotypes of the same varietal groups, the 16 Tunisian MLGs and 23 citrus reference cultivars were analysed together using the same 20 SSR markers using capillary electrophoresis. The results were double checked to detect possible discrepancies with patterns observed with radio-labelled primers. They were found consistent both for the allele differentiated and the genotyping of all individuals. Accordingly, a NJ tree was established based on simple matching dissimilarity from allelic data  were found to be shared with 'Willow leaf' mandarin. Sour orange MLG I showed 12 alleles that were not observed in the sour orange references, although these 12 alleles are present in sweet oranges. These results suggest that G and I are hybrids of sour orange and mandarin, and of sour orange and sweet orange, respectively.C. deliciosa reference and with Pummelo 'Pink' included as the C. maxima reference.The sweet orange MLG from Tunisia and all sweet orange reference cultivars were identical. Furthermore, the MLG clustered with the 'Willow leaf' mandarin included as a All acid citrus (limes and lemons) clustered together. The 'lemon A' MLG, representing 11 of the 12 Tunisian lemon accessions, was identical to the two lemon references. The 'Lemon Beldi'/lime 'Arbi' MLG was identical to the 'Mexican' lime reference. Lime 'Chiiri' was closely related to these genotypes  but differed from all the reference genotypes included in this study. Indeed, 'Chiiri' lime displayed 14 loci (50% of them were homozygous), which varied from the Mexican lime reference. This included nine alleles not observed in the Mexican lime, with eight (88.9%) of these being common to Corsican citron Table . The 'swC. webberi, C. macrophylla and C. excelsa. 'Rangpur' lime, 'Rough' lemon and 'Volkamer' lemon constituted a third cluster. The mandarin 'Willow leaf' and 'Corsican' citron represented the two last mitotypes.The 39 accessions analysed with nuclear SSR markers were also genotyped with rrn5/rrn18-, nad2/4-3, nad5/2-1 and nad7/1-2 mitochondrial markers. Five mitotypes were observed . These results evidenced the important differentiation between the four varietal group analysed  and the limitation of gene flow between these groups.et al. [citrus species are sexually compatible [The set of 20 SSR markers used in this study was sufficient to obtain genetic aggregations, enabling the clear identification of varietal groups of citrus and producing results consistent with previous studies ,9,15,16.et al.  and Gulset al.  proposedet al. ,8. The nmpatible ,18. HoweThe observed heterozygosity within each varietal group is very high. This heterozygosity is associated with low inter-cultivar differentiation within species (low MLG/accession ratios). These results are consistent with the generally accepted inter-specific origin of these varietal groups ,10,14,15Arbi' and 'Souri' shared identical MLG A. It is suggested that the phenotypical variants observed between and within these three MLGs may be somatic mutants of the ancestral sour orange. The seven other sour orange MLGs corresponded to single accessions. All of them shared the same mitotype as the reference sour orange. Five of these MLGs exhibit reduced heterozygosity compared with the reference sour orange, as well as an absence of additional alleles when compared with the three predominant MLGs. It is highly probable that these MLGs are zygotic plants resulting from self-pollination within the predominant sour orange groups. The two remaining sour orange MLGs are likely sour orange × mandarin and sour orange × sweet orange hybrids. Six of the seven sour orange MLGs of zygotic origin were collected in Sbikha (Kairouan) from an orchard of mother trees used for seed propagation. All sour orange trees in this orchard were grown from seed. The other 22 plants analysed from this orchard genetically conformed to the predominant sour orange MLG. Thus, a rate of 21.4% zygotic plants can be estimated in this population. Facultative apomixis of citrus results from adventitious nucellar polyembryony in seeds also containing one zygotic embryo. The frequency of apomictic plants among seedlings is determined by the competition between nucellar embryos and the zygotic embryo during seed development and seed germination [Genotypic analysis of the 169 Tunisian sour orange accessions, at 20 microsatellite loci, revealed ten unique genotypes with important redundancies (G/N = 0.059). These redundancies were expected, as standard vegetatively-propagated rootstock varieties are distributed throughout the country. Thus, 162 accessions were found to correspond to three highly heterozygous, very closely clustered MLGs that differed only in a single allele. The most represented MLG (128 accessions) was identical to the three reference genotypes originating from other countries in the Mediterranean basin . Within this group, different accessions were collected and named according to peculiar morphological traits. For example, the accessions 'sour orange Chiiri', 'sour orange with flattened fruits', 'sour orange with very small fruits and leaves', and sour oranges 'mination , as wellmination ,32. Frosmination  reportedmination  did not The sweet orange accessions collected in Tunisia, although identified by phenotypic characters of the fruit  or reported for their resistance to salinity (cv Meski), were identical for all analysed markers and highly heterozygous. Moreover, the sweet orange accessions also exhibited the same molecular profile as the five reference cultivars from the IVIA collection. This narrow genetic basis of the sweet orange accessions has been recognized in previous studies ,31,35,36Lemons were highly heterozygous for the SSR loci analysed. The Tunisian accessions displayed two MLGs. The main one (A) was identical to 'Eureka' and 'Lisbon' lemon reference accessions, and the second displayed only one different SSR allele, along with a marked mammiform apex of a pyriform fruit. The latter type most probably resulted from mutations of the reference lemons.C. aurantifolia Swing. (Tanaka classification). The sweet lime accessions did not differ from 'limonette de Marrakech', a lime from Morocco and are classified in C. limetta Risso (Tanaka classification). The very close phenotypic relationship of the Tunisian sweet lime and 'limonette de Marrakech' was mentioned by Hodgson [C. maxima mitotype is shared with 'Marrakech limonette' and 'Palestine sweet lime', the C. micrantha mitotype is shared with 'Mexican' lime, and the acid mandarin (C. reshni) mitotype is shared with 'Rangpur' lime. In our analysis, the mitochondrial data confirmed the identities found at the nuclear level between limonette de Marrakech and Tunisian sweet limes as well as between Mexican lime and Beldi' lemon and 'Arbi' lime. 'Chiiri' lime also shared the same cytoplasm with 'Mexican' lime, which is consistent with Mexican lime being its maternal parent.Three different MLGs were identified for lime in Tunisia. Although easily identified by the farmers due to their phenotypic differences, the Tunisian 'Arbi' and 'Beldi' limes exhibited the same genotype as the 'Mexican lime' reference accession, of which they may be phenotypic variants. These two Tunisian limes are therefore part of  Hodgson . For thi Hodgson  suggestede novo variation in Tunisia. These results show that, although rare, sexual reproduction occurs, and the progeny of self or inter-specific crossing may be selected by growers. However, it appears that mutation or epigenetic variation were the major factor of diversification within the different varietal groups, as previously underscored for other apomictic species [The analysis of Tunisian germplasm using microsatellites confirmed that the intra-specific diversification of the four apomictic species has resulted from three processes: (1) multiple introductions from diversified material, (2) mutation of local material, and (3) sexual recombination. The contributions of each of these processes varied depending on the species considered. For sweet oranges and lemons displaying very high heterozygosity and no or very little intra-group genetic diversity, sexual recombination can be discarded. For sour orange, sexual recombination was at the origin of seven accessions among 169 analysed (4%). For lime, the identities of two Tunisian MLGs with references from other countries points to a minimum of two introductions. The 'Chiiri' lime potentially arose from sexual events in Tunisia. However, it is difficult, without traceability of plant origin, to distinguish between multiple introductions of pre-differentiated materials or a  species ,40. The  species , olives  species , yams [4 species  and cass species , has als species .In Tunisia, sour orange is found in nearly all citrus growing areas and is well adapted to environmental conditions. Lemon and lime represent other species traditionally used as self-rooted trees or as rootstocks, mainly in southern Tunisia, where rainfall is only 120 to 200 mm per year and most irrigation water is brackish. In this region, irrigation water can contain up to 4-7 g/l of salt, especially during the summer. These rootstocks are well-adapted to local conditions and bear full flavoured fruits. Farmers consider the 'Chiiri' lime variety to be the best adapted citrus rootstock to local bioclimatic conditions  and report that it has better water uptake ability in comparison to sour orange. This study revealed that Tunisian citrus rootstock diversity has resulted from multiple introductions, mutations and residual sexual recombination of apomictic genotypes, as well as the impact of human selection. Original germplasm has been identified, particularly mutants and zygotic plants (mostly from selfing) of sour oranges, phenotypical variants of Mexican limes and a probable hybrid between a Mexican lime type and citron ('Chiiri' lime). This variability may be exploited either directly by nursery men and growers or by breeders to combine resistance to biotic and abiotic stresses with improvements in horticultural behaviour through conventional breeding or biotechnological approaches, such as somatic hybridization ,46. ThisC. maxima, C. medica and C. reticulata) from the IVIA (Spain) and INRA/CIRAD (France) germplasm banks.The rootstocks used in this study were collected from different regions of the country Figure , includiTwenty nuclear SSRs Table  were selet al. [Four mitochondrial indel markers , developed by Froelicher 33P prior to PCR amplification. The PCR reaction for SSR analysis was performed in a total volume of 20 μl containing 25 ng genomic DNA, 0.2 μM of each primer, 200 μM of each dNTP, 2 μl of 10X PCR buffer , and 0.75 units of Taq DNA polymerase. PCR reactions were performed using a MJ research thermal cycler model PTC-100™ and the following parameters: an initial denaturation at 94°C for 5 min, 35 amplification cycles of 30 s at 94°C, 1 min at 50-55°C (depending on the primer pair), 30 s at 72°C, and a final elongation at 72°C for 4 min. PCR products were visualized by running 6 μl of each sample on 5% polyacrylamide denaturing gels. Gels were dried and exposed to autoradiographic film. To minimize genotyping errors and for further comparison, all gels included a control individual (sour orange 'Granito') in four gel lanes and a ladder as a size standard. Unclear genotypes were systematically reanalysed.Initially, the 201 collected accessions were genotyped using 20 polymorphic SSR loci at CIRAD . Primers were radio-labelled with 2 and 0.5 U/μl of Taq DNA polymerase (Fermentas). The PCR conditions were as follows: 5 min at 94°C; 40 cycles of 30 s at 94°C, 30 s at 55°C (or 50°C depending on the primer) and 1 min at 72°C; and 4 min at 72°C.Using the same 20 nuclear SSR markers and four Indels mitochondrial markers, the different multi-locus genotypes (MLGs) observed in the initial analysis were reanalysed along with the reference set of 23 accessions. These analyses were conducted using capillary electrophoresis  at IVIA  and repeated two times. The PCR reaction was performed using 2 ng/μl of template DNA, 0.2 mM of dNTPs, 0.2 μM of WellRED dye-labeled forward primer, 0.2 μM of reverse primer, 10X PCR buffer (Fermentas), 1.5 mM of MgClFor each primer, bands (PAGE) or peaks (capillary electrophoresis) were scored as allelic data. The allelic pattern of each accession was cross-checked using two readers.ij) between pairs of accessions (units) [These data were used to calculate a genetic dissimilarity matrix using the simple matching dissimilarity index (d (units) .ij represents the dissimilarity between units i and j, L represents the number of loci, and ml represents the number of matching alleles between i and j for locus l. From the dissimilarity matrix, a Neighbor-Joining tree [http://darwin.cirad.fr/darwin[where ding tree  was compfr/darwin. Branch http://www.genetix.univ-montp2.fr/genetix/genetix.htm[Ho and He, respectively). The number of distinct MLGs in each varietal group was identified from the whole set of markers.GENETIX software 4.05.2 netix.htm was usedThe authors declare that they have no competing interests.HS collected the germplasm for this study, carried out the molecular characterization using radio-labelled markers at CIRAD, participated in the data analysis and drafted the manuscript. MFD participated in the design of the study, coordinated the data analysis and helped draft the manuscript. AGL carried out capillary electrophoresis analyses at IVIA. ZB participated in the design of the study and coordinated the collecting part. YF provided the primers used in this study and participated in the data analysis. AMR supervised the molecular characterization at CIRAD. XP and JPJC supervised the statistical analyses. LN furnished the reference germplasm accessions from IVIA collection and participated to the manuscript. MH contributed to the collecting part of the study. PO conceived the study, coordinated the project and contributed to draft the manuscript. All authors read and approved the final manuscript.Additional file 1aTunisian local germplasm sampled for diversity analysis (201 accessions) from different varietal group. Additional file Click here for file


Better understanding of transmission patterns will enhance control and elimination programs. To determine the origin of the virus associated with a measles outbreak in Menglian County, Yunnan Province, People’s Republic of China, in 2009, we conducted genetic analyses. Phylogenetic analyses based on nucleoprotein (N) and hemagglutinin (H) gene sequences showed that these Menglian viruses were not closely related to sequences of any World Health Organization (WHO) reference strains representing the 23 currently recognized genotypes. The minimum nucleotide divergence between the Menglian viruses and the most closely related reference strain, genotype D7, was 3.3% for the N gene and 3.0% for the H gene. A search of the databases of GenBank, WHO, and the Health Protection Agency Measles Nucleotide Surveillance showed that the Menglian viruses, together with the 2 older non-Menglian viruses, could be members of a new proposed measles genotype, d11. The new genotype designation will allow for better description of measles transmission patterns, especially in the Southeast Asian and Western Pacific regions. Paramyxoviridae, genus Morbillivirus. Infection with this virus typically causes high fever, maculopapular rash, conjunctivitis, cough, and coryza . Outbreaks of measles continued to occur because of increased numbers of susceptible children, especially in areas with low routine vaccination coverage. At the end of 2009, a measles outbreak in Menglian County was reported to the National Measles Laboratory of the Chinese Center for Disease Control and Prevention. Menglian County is situated on the western side of Yunnan Province and shares a border with Myanmar. As part of routine surveillance activities, the measles outbreak was investigated. Cases were confirmed through detection of immunoglobulin (Ig) M, and virus isolates were obtained for genetic analysis.Although deaths attributed to measles have declined by 78% worldwide, from ≈733,000 deaths in 2000 to ≈164,000 in 2008, risk for illness and death from measles still exists in countries with poor routine vaccination coverage, and outbreaks are a threat in most of these countries (Staff members from the Yunnan Center for Disease Control and Prevention collected serum and throat swab or urine specimens from patients during the outbreak (14 from Myanmar and 16 from Menglian). Serum samples were collected from 9 Myanmar and 7 Menglian case-patients. Throat swabs or urine specimens, from the first 6 Menglian patients, were obtained according to the WHO procedures for laboratory diagnoses of measles and rubella virus infections (www.mbio.ncsu.edu/BioEdit/BioEdit.html). Phylogenetic analyses were performed and trees were generated by using MEGA4 (www.megasoftware.net). The robustness of the groupings was assessed by using bootstrap resampling of 1,000 replicates.Measles virus was isolated by using the Vero/hSLAM cell line . No IgM against rubella virus was detected .Two measles virus isolates, MVi/Menglian.Yunnan.CHN/47.09 and MVi/Menglian.Yunnan.CHN/51.09, were obtained from urine and throat swab specimens of 2 persons from Menglian. Positive RT-PCR products of the partial N gene were obtained from clinical specimens from 5 of 6 case-patients, and the entire H gene was amplified from the 2 isolates . The seqThe sequences of these viruses were not closely related to the sequences of any of the WHO reference strains that represented the 23 currently recognized genotypes. These sequences were closest to the sequence of the genotype D7 reference strain and the contemporary genotype D7 strains available from GenBank. The minimum nucleotide divergence between the Menglian viruses and the D7 WHO reference strain (Victoria.AUS/16.85) was 3.3% for the N gene and 3.0% for the H gene . Bootstrwww.who-measles.org) identified only 2 closely related sequences. The most closely related sequence was from a measles virus isolate, MVi/WA.AUS/12.01 (GenBank accession no. AF481492), previously designated as genotype D7, which had been imported from Myanmar into Australia in 2001  was chosen as the reference strain because an isolate is available, it grows to high titer in cell culture, and the sequence is representative of the Menglian strains. Although designation as a genotype is not permanent until after the new genotype has been acknowledged and formally designated by WHO, the percentage sequence divergence between the N and H gene sequences of the Menglian isolates and the sequences of the reference strains exceed the recommended threshold for designation of a new measles genotype, 2.5% and 2.0%, respectively  that are circulating in neighboring countries. Therefore, the Menglian viruses are providing baseline genetic information about viruses endemic to Myanmar and possibly neighboring countries. The new genotype designation will enable better description of measles transmission patterns, especially in the Southeast Asian and Western Pacific regions of WHO.–Molecular epidemiologic data, when analyzed in conjunction with standard epidemiologic data, can help document viral transmission pathways, identify whether a virus is endemic or imported, and aid in case classification, thus enhancing control and elimination programs (


Cowpox with a severe, generalized eruption was diagnosed in an atopic 4-year-old girl by electron microscopy, virus isolation, polymerase chain reaction, and immunoglobulin (Ig) M and low-avidity IgG antibodies. The hemagglutinin gene of the isolate clustered with a Russian cowpox virus strain, and more distantly, with other cowpox and vaccinia virus strains. The patient’s dog had orthopoxvirus-specific antibodies, indicating a possible transmission route. Orthopoxvirus genus , and monkeypox viruses within the A 4-year-old girl from a small farm in eastern Finland was hospitalized in September 2000 because of umbilicated vesicopapules, which developed over the previous 5 days , and unrThe girl was treated in isolation at the hospital with chlorhexidine washings and wound dressings with fusidic acid. Intravenous dicloxacillin was administered to prevent secondary bacterial infections. On day 12, skin lesions progressed to deep-seated, hard, black eschars. At the same time the patient’s general condition improved. Two months later, all lesions were healed with scars.Orthopoxvirus genus; some strains cluster with vaccinia viruses and others (including the reference Brighton strain) with variola virus  to measure specific immunoglobulin (Ig) G, IgM, and avidity of IgG antibodies .The serum samples from the patient’s pets were collected later and tested with IFA. A hunting dog had antibodies with a titer of 320; another dog had a titer of 20. Thus, a dog might have transmitted the infection from a wild rodent to the patient, although cats are thought to be the main source of human infection ((Clethrionomys glareolus). The two seropositive horses were from a region near where the patient lives in eastern Finland, and the three seropositive cats were free-roaming and found in southern Finland. In addition, seroprevalence rates of 50% (7/14) and 1.4% (1/73) have been found in foxes and lynxes, respectively, from a limited geographic area in Finland  were 0% to 92%, depending on the trapping time and the location . In VärtSera collected at a Finnish Veterinary meeting in 2001 showed that every person >50 years had orthopoxvirus antibodies, as measured by IFA. The seroprevalence decreased gradually for younger age groups , reflectAlthough cowpox virus infection usually causes a single, painful, ulcerated vesicopustule and local lymphadenopathy, immunocompromised patients and children, especially those with atopic eczema, are susceptible to a generalized, even lethal, smallpoxlike infection (Cowpox virus (orthopoxvirus) infection was diagnosed by electron microscopy, PCR, virus isolation, and serologic testing (positive IgM or low avidity of IgG antibodies). Cowpox virus strains show considerable genetic variations with different positioning in the orthopoxvirus phylogenetic tree. Circulation of cowpox virus in wild and domestic animals, together with decreased immunity in humans, may lead to increased occurrence of human cowpox, especially in atopic and immunocompromised persons who are at risk for generalized infection. The described case further suggests atopy to be a contraindication to smallpox vaccination.


Although social behaviour can bring many benefits to an individual, there are also costs that may be incurred whenever the members of a social group interact. The formation of dominance hierarchies could offer a means of reducing some of the costs of social interaction, but individuals within the hierarchy may end up paying differing costs dependent upon their position within the hierarchy. These differing interaction costs may therefore influence the behaviour of the group, as subordinate individuals may experience very different benefits and costs to dominants when the group is conducting a given behaviour. Here, a state-dependent dynamic game is described which considers a pair of social foragers where there is a set dominance relationship within the pair. The model considers the case where the subordinate member of the pair pays an interference cost when it and the dominant individual conduct specific pairs of behaviours together. The model demonstrates that if the subordinate individual pays these energetic costs when it interacts with the dominant individual, this has effects upon the behaviour of both subordinate and the dominant individuals. Including interaction costs increases the amount of foraging behaviour both individuals conduct, with the behaviour of the pair being driven by the subordinate individual. The subordinate will tend to be the lighter individual for longer periods of time when interaction costs are imposed. This supports earlier suggestions that lighter individuals should act as the decision-maker within the pair, giving leadership-like behaviours that are based upon energetic state. Pre-existing properties of individuals such as their dominance will be less important for determining which individual makes the decisions for the pair. This suggests that, even with strict behavioural hierarchies, identifying which individual is the dominant one is not sufficient for identifying which one is the leader. Dominance hierarchies could offer interacting animals a quick way to settle disputes without having to use too much effort. However, individuals may pay a price for acknowledging their position within the hierarchy, which could influence how they choose to behave within the group. Consequently, the actions of the group may be shaped by the effects of the hierarchy on each of the group’s members. I consider the behaviour of a pair that consists of a dominant and a subordinate individual, where the subordinate pays an energetic cost when it interacts with the dominant. I show that having to pay this cost affects the behaviour of the pair. I also demonstrate that, although a social hierarchy is imposed, the behaviour of the pair is not determined by the dominance relationship, but is instead influenced by the energetic reserves of the pair, where the decision-maker may just be whoever is the hungriest. Animals can gain many benefits from associating in groups Differing effects of dominance upon the behaviour of interacting individuals have been integrated into a number of theoretical studies. Some have concentrated upon how dominance affects the order of access to resources, where dominants may have priority or exclusive access to a foraging resource et al. As well as effects upon foraging ability and group composition, living in social hierarchies can impose differing costs on individuals The way in which this socially mediated interference cost was implemented within the spatially-explicit individual-based framework of et al. The model followed here builds on the dynamic foraging game described by Rands both individuals in the pair. If an individual choses to rest during a period, it incurs a low risk of being predated. If it forages at the same time as its colleague, it incurs a moderate risk of being predated . If it forages on its own (whilst its colleague rests), it incurs the greatest risk of being predated. Therefore, there is a trade-off within this framework between being predated when foraging, and starving whilst resting. Rands et al. et al. As well as the risk of starvation if an individual doesn’t forage, there is also the risk of predation, which depends on the actions of et al. The model I describe here builds further on this framework. Although Rands when both it and the individual who does not pay a cost (which I refer to as the ‘dominant’) are foraging together ;when both the dominant and the subordinate are resting ;when the subordinate is foraging on its own ;when the subordinate is resting on its own .iii) and (iv), I assume the dominant individual is conducting the opposite behaviour to the subordinate.In , which defines the probability that an individual of type i who is alive at the start of time step t, in state ix (>0), paired with a living colleague of corresponding type j in state jx (>0) who follows a strategy defined by the candidate strategy π, will survive until the start of the final time step T, if it adopts action iu and its colleague adopts action ju in the current time step . Note that, as with the model described in π encompasses the candidate responses of both subordinate and dominant individuals within the population .Where the current model differs from that presented in iH function described in s, and terms relevant to dominants are denoted with a subscript d.In order to consider the effects of dominance on behaviour, I replace the If the individual is dominant, I assumeIf the focal individual is subordinate, I instead assumea has a probability aRm of being predated if it is resting, aAm if it is foraging alone, and aTm if it is foraging with its colleague. The function aW is the fitness at time t of an individual of type a with energy reserves ax, and whose colleague has energy reserves bx, assuming that both individuals follow policy π from that point forward in time. Λa(x) defines a ‘chop’ function for an individual of type a as defined in a(x)  =  min), and aS is the maximum state value possible for an individual of type a. aκ denotes the probability that an individual of type a spends ac state units of energy during a period if it conducts action u, and aγ(ag) denotes the probability that it gains ag state units of energy during the period if it forages. Both energetic costs and gains were represented within the current model using the same functions described in Apart from the novel interference cost terms (described below), terminology here follows dTm  =  sTm, etc.). The only difference considered between them was in the extra cost paid by the subordinate individual when it was conducting a paired behaviour that incurred extra energetic costs. These energetic costs are denoted dRsRD, dFsRD, dRsFD and dFsFD, which represent the extra energetic cost (in state units) incurred for the four possible pairs of behaviours . The dominant is not expected to pay any extra costs for its actions dependent upon the behaviour of the subordinate.For simplicity, both dominant and subordinate individual were assumed to have identical probabilities of incurring given gains and costs, and share identical predation risks when conducting particular activities , where both members of a pair rested during a period (RRp), where the dominant foraged and the subordinate rested (FRp), or where the dominant rested and the subordinate foraged (RFp).Similarly, knowing stable paired states and policies also allowed us to calculate the proportions of the population where both members of a pair foraged during a period (RRp ⋅ FFp - RFp ⋅ FRp)/(RRp ⋅ FFp + RFp ⋅ FRp). Note this term is referred to in D′, but I avoid using this notation here to avoid confusion with the cost of dominance used in the current model.Having calculated the proportions of the population conducting the four types of paired behaviour, I quantified the amount of behavioural synchronisation during a period as , decreases in resting together. The paired interactions when the members of the pair were conducting differing behaviours were mostly non-significant, although there were increases in cases where the dominant rested and the subordinate foraged when the costs experienced by the subordinate occurred when the pair differed in their behaviour.S statistic) does not discriminate between resting together and foraging together. Therefore, an increase in only one of these paired behaviours may not lead to a corresponding increase in the general level of synchronisation within the pair.As would be expected, pairs become more synchronised when there are costs involved with not being paired, and become less synchronised when there are costs to conducting the same action as each other . ConsideS statistic decreased in response to a dominance cost when foraging together, and decreased when resting together . Most of the interactions shown for the subordinate individual also indicate a positive trend. Paired behaviours were also repeated more often when these costs were incurred. These increases are likely to be due to the increase in synchronisation behaviour seen when there is an extra cost to being non-synchronised.As would be expected, incurring an extra cost of dominance to the subordinate meant that its energetic reserves tended to be reduced . This wavice versa. This trend was seen, but was only significant for the situations where the subordinate’s costs were paid for conducting the opposite behaviour to the dominant individual.The length of time that the subordinate remained heaviest (when it managed to reach that state of being) was in most cases reduced by imposing a cost of dominance . The excThis model demonstrates that if a subordinate pays energetic costs when it interacts with a dominant individual, this has distinct effects upon the behaviour that it shows, and subsequently it affects the behaviour of the interacting dominant individual. Considered independently, both individuals tended to increase the amount of foraging behaviour they conducted when there were interaction costs. Considered together, the behaviour of the pair was driven by the subordinate individual. Costs imposed when the subordinate forages tend to increase paired foraging behaviour.e.g.e.g.In the model presented here, the subordinate individual tended to be the lighter individual for longer periods of time when interaction costs were imposed. This lends support to the suggestion that the lighter individual acts as the decision-making ‘pace-maker’ of the group et al. et al. et al.Therefore, imposing direct energetic costs of dominance should lead to effects upon the paired behaviour of foragers, lending support to the rules proposed for larger groups by Rands Furthermore, the current model makes a simplifying assumption by assuming that the subordinate paid additional costs . We could conceivably see a situation where the dominant individual also pays additional costs for being dominant. If these costs are less than those paid by the subordinate, these could simply be subsumed into the general metabolic costs paid by individuals, giving us a similar model structure to that described. However, if the dominant and the subordinate individual paid different levels of cost for different behaviours such that both paid more than the other for at least one of the four behavioural pairs, then this would be a situation not covered within the current model. For example, we could imagine a situation where the dominant paid the higher metabolic cost when foraging at the same time as the subordinate , whilst the subordinate could show a higher metabolic cost than the dominant when it was foraging on its own . In this hypothetical example, the current model is not sufficient, and an extended version would need to be considered where costs to the dominant individual are also modelled. I would suggest that this exercise might be useful if exact predictions are needed for a well-defined system , but investigating a more general model would be unlikely to yield more tangible results than described in the simpler model I present here.As well as behavioural interactions leading to subordinates gaining less energy during an interaction, physiological processes may also mean that they spend more energy, and could be mediated hormonally, such as through stress responses by individuals. Studies on many species have demonstrated that social stress and dominance interactions have effects upon body mass and composition Table S1F values and significance terms for results presented in F1,11566 value obtained from a fully-crossed ANOVA model that the fifteen terms given. The second number (in parentheses) is the critical Fp<0.05)Click here for additional data file.Table S2F values and significance terms for results presented in (DOCX)Click here for additional data file.