Ne) determines whether selection or genetic drift is the predominant force shaping their genetic structure and evolution. Populations having high Ne adapt faster, as selection acts more intensely, than populations having low Ne, where random effects of genetic drift dominate. Estimating Ne for various steps of plant virus life cycle has been the focus of several studies in the last decade, but no estimates are available for the vertical transmission of plant viruses, although virus seed transmission is economically significant in at least 18% of plant viruses in at least one plant species. Here we study the co-dynamics of two variants of Pea seedborne mosaic virus (PSbMV) colonizing leaves of pea plants (Pisum sativum L.) during the whole flowering period, and their subsequent transmission to plant progeny through seeds. Whereas classical estimators of Ne could be used for leaf infection at the systemic level, as virus variants were equally competitive, dedicated stochastic models were needed to estimate Ne during vertical transmission. Very little genetic drift was observed during the infection of apical leaves, with Ne values ranging from 59 to 216. In contrast, a very drastic genetic drift was observed during vertical transmission, with an average number of infectious virus particles contributing to the infection of a seedling from an infected mother plant close to one. A simple model of vertical transmission, assuming a cumulative action of virus infectious particles and a virus density threshold required for vertical transmission to occur fitted the experimental data very satisfactorily. This study reveals that vertically-transmitted viruses endure bottlenecks as narrow as those imposed by horizontal transmission. These bottlenecks are likely to slow down virus adaptation and could decrease virus fitness and virulence.The effective size of populations ( Ne). Ne has been estimated for several steps of plant virus infectious cycle, such as horizontal transmission by insects and the colonization of plant cells and tissues. However, although economically important, no estimate of Ne during vertical transmission of viruses, i.e. the infection of plant progenies from parental plants, is available. Here, we report that Pea seedborne mosaic virus (PSbMV), a seed transmitted virus infecting pea crops, undergoes very drastic genetic drift during vertical transmission, with an average number of infectious virus particles contributing to the infection of a seedling from an infected mother plant close to one. Such bottlenecks, as narrow as those imposed by horizontal transmission, could slow down virus adaptation and should be taken into account to improve plant protection strategies.Short generation times and high mutation rates are the hallmarks of virus. They favor their fast adaptation as illustrated by their ability to overcome natural as well as man-made barriers such as host resistance or drug treatments. However, such a fast adaptation could be slowed down when genetic drift, which introduces random sampling effects in the evolution of virus populations, is important. Whether genetic drift or selection dominates depends on the effective size of populations ( Ne, plays a core role since it determines the rate of random fluctuations of the frequency of virus variants caused by genetic drift across generations in a model population. Ne estimates the number of individuals that pass on their genes through generations. It is usually much smaller than the total size of populations: although the total size of virus populations in their host plants can be tremendous and reach 107 to 109 virus particles Ne are below 500 and most of them are actually close to one Ne is given by the harmonic mean of population sizes over generations Ne. Ne helps to predict the loss and distribution of neutral genetic variation Ne is of major interest for modeling disease emergence and can be an important issue in agriculture as illustrated by the breakdown of plant resistance genes by adapted virus variants Evolution of virus populations depends on several forces including mutation, recombination, genetic drift, selection and migration, acting concomitantly but exerting pressures that vary widely in direction and intensity. It makes therefore difficult to predict viral emergences or the durability of control strategies. The relative intensity of these forces will determine whether evolution follows predominantly stochastic or deterministic patterns. The concept of effective size of populations, i.e. plant inoculation by vectors i.e. infection of plant progenies by the parental plant(s), are available yet.It has been shown recently that plant virus populations undergo transient and recurrent bottlenecks at different steps of their life cycle, like during horizontal transmission, via the contamination of true seeds. In only a few examples, particularly stable viruses such as tobamoviruses can be retained in the seed coat and then transmitted to the seedling after germination There are three major ways of vertical transmission of plant viruses Pea seedborne mosaic virus (PSbMV)  in pea plants during vertical seed transmission and during the colonization of leaves.The goal of this work was to compare the size of bottlenecks affecting populations of The PSbMV isolate DPD1 and the variant DPD1-R only differ at codon position 116 in the VPg (Virus protein genome-linked)-coding region were used. Codon 116 is GTG  and CGA (arginine) in DPD1 and DPD1-R, respectively Pisum sativum L.) cultivar ‘Vedette’ that transmits PSbMV through seeds at high frequencies The pea  on the two upper expanded leaves . All plaAvian myeloblastosis virus reverse transcriptase  followed by polymerase chain reaction (PCR) with Thermus aquaticus DNA polymerase (Promega Corp.). Primer DPD1-VPGR  was used for RT and primers DPD1-VPGR and DPD1-VPGF (5′-AAAACACTGCAGCTTAAGGG corresponding to nucleotides 5868 to 5887) were used for PCR. The PCR program started with 3 min at 95°C followed by 35 cycles  and a final extension at 72°C for 10 min. Amplification products were sequenced directly with primer DPD1-VPGF by Genoscreen . We estimated the relative proportions of the two PSbMV variants in inocula and leaves from the height of peaks corresponding to the three polymorphic codon positions in the chromatograms. The reliability of this quantification method was evaluated with artificial mixtures of known quantities of the two PSbMV variants obtained after virus purification. As illustrated in 2 = 0.99). This chromatogram-based quantification method was also compared to another method based on the cloning of RT-PCR products obtained with primers DPD1-VPGR and DPD1-VPGF into an Escherichia coli plasmid vector. For this, 5 pea leaves with contrasted frequencies of variant DPD1-R (from 24% to 69% based on the “chromatogram” method) were chosen and, for each of them, the RT-PCR products were cloned into the pGEM-T Easy vector  and the number of clones corresponding to DPD1 and DPD1-R among a total of 40 clones per leaf was determined using the specific primers DPD1-VPgF-116V and DPD1-VPgF-116R described below. Again, the “chromatogram” and “cloning” methods provided highly similar frequency estimates , hence further validating the “chromatogram” quantification method.For inoculation, RNA extraction and enzyme-linked immunosorbent assay (ELISA), leaf tissue was homogenized in four volumes (wt/vol) of 0.03 M phosphate buffer (pH 7.0) supplemented with 2% (wt/vol) diethyldithiocarbamate. For RT-PCR, total RNA was extracted from a 150 µL aliquot using the Tri Reagent kit . To amplify the VPg coding region that contained the polymorphic codon between DPD1 and DPD1-R, reverse transcription (RT) was performed on 2 µL of each RNA extract using GTG5′-CTCGATAAACAATTGTTT) or the primer DPD1-VPgF-116R (CGA5′-CTCGATAAACAATTGTTT) corresponding to nucleotides 6336–6356 of DPD1 andDPD1-R, respectively. The PCR programs started with 3 min at 95°C followed by 40 cycles  and a final extension at 72°C for 10 min. Artificial mixtures of known proportions of RNAs of the two PSbMV variants obtained after virus purification Pods produced by the main stem  of plantNe during PSbMV colonization of upper uninoculated leaves (L1 to L6 in FST method” based on the difference between these 2 dates of Wright's FST statistics To estimate Ne = E(P)×(1−E(P))/[Var(P′)−Var(P)], where P and P′ are the random variables of the frequencies of the viral marker for each plant at the first and second sampling dates, respectively, E(P) is the expected value of P in the plant population and Var(P) its variance. In practice, E(P) and Var(P) were estimated by the sample mean and variance of the frequencies of the viral marker measured on a set of plants  of Ne = [E(P)×(1−E(P))−Var(P)]/[Var(P′)−Var(P)]. According to the FST method, Ne = (1−FST)/(FST′−FST), where FST and FST′ are values of the FST statistics of the viral populations at the first and second sampling dates, respectively  or a single leaf per plant can be considered at date 2 . In addition, different sets of plants can be considered for each date (dataset 3) to test the influence of sampling leaves at date 1 on Ne estimates (by comparing dataset 2 and dataset 3).With the nested sampling design used  and with the different plants sets available . Concerning the second step, both types of interactions were considered  virus entry into the seed  and within the plant because of spatial heterogeneity of distribution of virus variants. We considered that the relative frequencies 1f of variant 1 and 2f = (11−f) of variant 2 in the vicinity of a given seed at infection time were realization of random variables that followed Beta distributions of parameters  and , respectively, α and β varying from plant to plant. We assumed that the numbers of viral particles of each variant entering a given seed, 1N and 2N, were described by independent Poisson processes of parameters λ1×1f and λ2×(11−f), respectively, where λ1 and λ2 are the efficiencies of seed infection by variants 1 and 2. This hypothesis implies that all virus particles of a given variant have the same probability of entering a seed, and that they enter into the seeds independently of each other (i.e. there is no virus-virus interactions). Moreover, assuming that these Poisson processes are independent implies that there is no interaction between DPD1 and DPD1-R variants for entering a seed (however they can enter with different efficiencies).For the first step (virus entry into the seed), we assumed that the proportions of PSbMV variants DPD1-R (variant 1) and DPD1 (variant 2) in coinfected plants can fluctuate in time during the period of seed infection  from a Poisson distribution of parameter λc. Four alternative models were considered to describe the mechanism of seedling infection (1i.e. N+2N) strictly exceeds cN. In model M1, a variant is transmitted vertically if at least one particle of this variant has entered into the seed, meaning that the contribution to seedling infection of a virus particle of one variant does not depend on the density of the other variant: virus particles are interchangeable, whatever their type. In contrast, in model M2, a variant is transmitted vertically if its density is higher than cN or higher than the density of the other variant (when 1N = 2N the seedling becomes infected by both variants). Here, the contribution of a virus particle of one variant to seedling infection depends on the density of the other variant: virus particles are not interchangeable and model M2 assumed some inhibition between variants when one variant outnumbers the other. In model M3, there is no variant-variant interaction: the virus variants initiate seedling infection independently. A variant is transmitted vertically if the number of particles of this variant entering into the seed strictly exceeds cN. Finally, in model M4 there is no virus-virus, nor variant-variant interaction. cN is indeed set to zero: a virus variant is transmitted vertically if at least one particle of this virus variant has entered the seed.For the second step of PSbMV seed transmission (seedling infection), we hypothesized that vertical transmission occurs if a minimal number nfection , Fig. 2.r, were assumed to be independent. For a given plant, the variables describing the infection status of the seedlings, indexed by s (1≤s≤rS), were supposed to be independent and identically distributed, but potentially with different distributions, for distinct plants. The variable s issued from mother plant r. This seedling is either not infected  were considered as known parameters and have been estimated for a given plant using the proportions of the two virus variants in leaves L1, L2 and L3 at 22 dpi and in leaves L4, L5 and L6 at 61 dpi . 95% confidence intervals for model parameters were estimated using the function “profile” of the “bbmle” package.After checking that the four models were practically identifiable in our experimental conditions , model pPisum sativum cv. Vedette with two mixtures of the PSbMV variants DPD1 and DPD1-R (inocula 1 contained 38% of variant DPD1-R and inocula 2 contained 66% of variant DPD1-R) and we examined the composition of the viral populations at two time points in apical leaves of the inoculated plants, and in the plant progeny issued from the seeds collected on these mother plants. Changes in frequency of the PSbMV variants during the infection process and in the seedling progeny could be due either to genetic drift, selection or both. Since many models used to estimate Ne assume that changes of genotype frequencies in populations are due to genetic drift only, and not to selection, we tested whether the marker that allows distinguishing DPD1 from DPD1-R was neutral, i.e. if the two variants were equally competitive.To analyze genetic drift and bottlenecks affecting virus populations during leaf infection and seed transmission, we inoculated plants of a posteriori based on the sequence chromatograms . In addition, the sampling at 22 dpi had no influence on the average composition of the viral populations. Indeed, the average proportions of DPD1-R frequency in plants sampled only at 61 dpi (plants 20 to 39) were 31.4% and 58.2% for inocula 1 and 2, respectively, which is not significantly different from the DPD1-R frequencies at 22 or 61 dpi in plants that were sampled twice (plants 1 to 19)   .Consequently, the two PSbMV variants DPD1 and DPD1-R can be considered as equally competitive with regard to the colonization of leaves at the systemic level between 22 and 61 dpi.To estimate the relative competitiveness of PSbMV variants DPD1 and DPD1-R for seed transmission, we compared their relative frequencies in seedlings derived from inoculated mother plants and in leaves of these mother plants sampled at 22 and 61 dpi.n = 206 seedlings) or DPD1 only , . Accordingly, the fact that similar percentages of seed transmission were observed in single-infected or mixed-infected plants suggests independence between PSbMV variants for seed infection and justifies the Poissonian assumptions made for modeling virus entry into seeds. Finally, the seedling infection rates were similar to those observed for mixed-infected plants for which no leaves were sampled (plants 40 to 49) . All these values are in the range of seed transmission rates obtained independently with PSbMV DPD1 and Vedette pea plants . The average seedling infection rate was also similar to the infection rates observed in the seedlings of control plants inoculated by DPD1-R only  and (iii)) for inocula 1 and 2, respectively. Compared to the PSbMV variant frequencies in the leaves of the mother plants, DPD1-R seemed to be somewhat better seed-transmitted than DPD1, a difference which is significant only for inoculum 2 . Examining seed transmission results for each mother plant individually did not reveal any significant difference between the distributions of variants among the seedlings and the average proportion of PSbMV variants in leaves.For mother plants having nine or more infected seedlings, the proportion of seedlings corresponding to categories (ii) seedling infected only by variant DPD1, (iii) seedling infected only by variant DPD1-R and (iv) seedling infected by both PSbMV variants was determined . AccordiP>0.2; Khi2 tests)  (The percentages of seedlings infected simultaneously by the two PSbMV variants were similar for mother plants which leaves were sampled twice (numbers 1 to 19) and for mother plants for which no leaves were sampled (plants 40 to 49) (2 tests) . The dis2 tests) . ConsequNe. These methods are based on the differences in variance of the viral variant frequencies (“variance method”) or on the difference of Wright's FST statistics (“FST method”) between two sampling dates. For these methods, an underlying assumption is that the variance of the viral variant frequencies (or the FST statistics) increases with time. Indeed, considering that all variants are equally fit in the population, variant frequency fluctuations are due only to genetic drift, which affects both the amount and distribution of neutral genetic diversity over time (i.e. across generations) and space (i.e. between subpopulations at a given time).Since the two inoculated PSbMV variants DPD1 and DPD1-R were equally competitive during the colonization of plant leaves from 22 to 61 dpi, we used the methods described in FST statistics for the PSbMV populations at 22 and 61 dpi, suggesting very limited effect, if any, of genetic drift on viral populations during the systemic invasion of apical leaves . Overall, little difference was observed between the “variance” and “FST” methods and between the different datasets used to estimate viral frequencies at the two dates of observations (Ne estimates were comprised between 74 and 143 for dataset 3 (independent sets of plants were sampled at each date) and between 59 and 216 for dataset 2 (the same set of plants was sampled at both dates)  . DatasetNe estimates. The 95% confidence intervals were large because of the small number of plants and because the small differences in virus frequency variances or population FST between dates 1 and 2 had large impacts on Ne estimates  take into account the difference in seed transmissibility between variants and (ii) that allow to disentangle different seedling infection processes . These models showed that the mean number of PSbMV particles contributing to the infection of an individual seedling was close to one. We first checked whether our experimental design (number and nature of the data) was sufficiently informative to estimate accurately the model parameters using practical identifiability tests (The “variance” and “ty tests . Numericty tests . Moreove−17). Thus, although model M1 is supported best by the data, model M2 has also a substantial support 1 = λ2, the AIC of the models increased to 207, 210, 219 and 279 for M1, M2, M3 and M4, respectively, indicating that the mean number of viruses contributing to the infection of a seedling was significantly different for virus variants 1 and 2. Under model M1, parameter inference indicated that the mean number of DPD1-R variant infectious particles contributing to the infection of a pea seedling was 1.08  and 0.74 for virus variant DPD1 (with a CI95% ranging from 0.61 to 0.88), while the mean number of virus particles required to infect a pea seedling was 0.84 . Parameter inferences under model M2  were close to those obtained with M1, although always slightly higher: 1.52 for λ1 with a CI95% ranging from 1.08 to 2.74, 1.06 for λ2 with a CI95% ranging from 0.75 to 1.95 and 1.36 for λc with a CI95% ranging from 0.85 to 2.63.The model selection procedure applied to the experimental data set  indicatei.e. (i) healthy, (ii) infected only by variant 2 (DPD1), (iii) infected only by variant 1 (DPD1-R) and (iv) infected by both PSbMV variants) were highly correlated (R2 = 0.88) for both models. Second, between-plant variability was very well represented, as an 80% (resp. 90%) confidence interval predicted by model M1 contained 78% (resp. 83%) of the observed data. For model M2, an 80% (resp. 90%) predicted confidence interval contained 80% (resp. 89%) of the observed data.Importantly, models M1 and M2 fitted very satisfactorily the experimental data. First, the observed and predicted mean numbers of seedlings corresponding to the four categories of seedling infection  the lack of estimates of generation times for viruses Ne or of the bottleneck size corresponding to particular steps of the virus life cycle have been obtained.Estimation of Ne are quite contrasted Ne) observed during the systemic colonization of pea plants by PSbMV corroborates previous results obtained with Cauliflower mosaic virus (genus Caulimovirus) Tobacco etch virus (genus Potyvirus) Ne values were obtained by comparing the virus populations between the inoculum and apical leaves Plum pox virus  whereas a total of 33 viral variants were observed in the whole set of leaves analyzed, indicating that narrow bottlenecks acted on PPV populations during the infection of individual leaves Concerning the colonization of plant leaves by viruses, estimates obtained for Human immunodeficiency virus-1 (HIV-1) in utero or intrapartum transmission cases examined in these three studies  the infants harbored a single viral variant, which suggested the occurrence of narrow population bottlenecks at transmission. Note, that this percentage is very close to our own estimates for PSbMV  in Cucurbita pepo showed that 16 of 24 ZYMV variants present in the mother plant were also present in vertically-transmitted virus populations, either of the first or second plant generation On the opposite, during the same time-frame, we showed that a single infectious PSbMV particle contributed on average to the infection of an individual seedling derived from an inoculated mother plant. To our knowledge, this is the first estimate of the bottleneck size imposed by vertical transmission to a virus population. Strong bottlenecks were also observed during vertical mother-to-child transmission of Vertical transmission of PSbMV occurs through the infection of the pea seed embryos Ne when the effect of deterministic evolutionary forces, typically selection, cannot be excluded. Indeed, the temporal methods classically used to estimate Ne assume that the observed changes in allele frequency are due to genetic drift only and thus require the use of neutral genetic markers for the population of interest et al.cλ was low), indicating that each virus particle has a quite high probability of causing efficient seed transmission. However, rejection of model M4 indicates that one viral particle is not always sufficient to initiate efficient seedling infection. Model M1 being only slightly preferable to model M2, it remained unclear whether virus particles belonging to the two variants are interchangeable or not in the cumulative infection process, interchangeability meaning that the contribution to seedling infection of a virus particle of one variant does not depend on the density of virus particles of the other variant. Consequently, more data should be gathered to clearly distinguish whether frequency-dependent selection of PSbMV variants occurred (as in model M2) or not (as in model M1) during seed transmission.From a methodological point of view, the mathematical framework introduced here allowed disentangling the relative importance of selection and genetic drift in shaping the genetic composition of viral populations during seed transmission. It could be of broad interest to estimate Ne values observed for PSbMV vertical transmission are expected to impact more deeply virus evolution than bottlenecks of the same size that would be experienced during horizontal transmission The small e.g. host density, aphid population dynamics) and agronomic factors (e.g use of virus-free seeds) determine which mode of transmission is prevailing. Undoubtedly, a deeper understanding of the balance between the relative importance of these transmission modes during the course of epidemics, coupled with a deeper knowledge of the bottleneck sizes associated with these transmission modes is needed to better understand the evolution of important pathogen life history traits such as virulence, symptom severity and yield losses. Ultimately, this research could help designing more efficient strategies of plant protection relying on the knowledge and manipulation of evolutionary changes in parasites populations.Exploring to which extent such decrease in virulence or symptomatology (two life history traits that are not necessarily correlated in plant viruses) can be explained by bottleneck sizes is an important issue in parasite evolution. From an applied perspective, many vertically-transmitted plant viruses are also transmitted horizontally by vectors. For example, PSbMV is transmitted by a large number of aphid species. In the field, ecological /[H(346C)+H(346G)]+H(347G)/[H(347G)+H(347T)]+H(348A)/[H(348A)+H(348G)]], where H(zX) is the height of the peak corresponding to nucleotide X at position z of the PSbMV VPg cistron on the sequence chromatogram. DPD1 and DPD1-R possess a GTG (respectively CGA) codon at position 116  of the VPg cistron. In the graph, the relative proportion of the PSbMV DPD1-R variant is plotted as a function of the known proportion of the PSbMV DPD1-R variant in the artificial mixture for the 3 replicates realized.(PDF)Click here for additional data file.Table S1Data set used to estimate the size of bottlenecks during PSbMV seed transmission. The seedlings obtained from 12 plants (six for inoculum 1 with 38% of variant DPD1-R and six for inoculum 2 with 66% of variant DPD1-R - i.e. only from mothers plants having nine of more infected seedlings) were analyzed by ELISA and PSbMV-variant-specific RT-PCR in order to distinguishing four categories of seedlings: (i) healthy, (ii) infected by DPD1, (iii) infected by DPD1-R and (iv) infected by both PSbMV variants. In all, 828 seedlings have been analysed. For each plant, the mean and standard deviation of the relative frequency of DPD1-R variants during the flowering period were estimated using in all 6 leaves, the 3 leaves sampled at 22 dpi and the 3 sampled at 61 dpi. For each plant r (1≤r≤12), these mean and standard deviation estimated were used to calculate the parameters tion see .(PDF)Click here for additional data file.Text S1Identifiability of the virus vertical transmission models.(PDF)Click here for additional data file.


The association of mumps with peripheral facial paralysis has been suggested, but its pathogenesis remains unclear. An 8-year-old girl simultaneously developed left peripheral facial paralysis, ipsilateral cervical herpes zoster, and bilateral mumps sialadenitis. Elevated anti-mumps and anti-varicella zoster virus IgM antibodies in serological testing indicated recent infection of mumps and reactivation of VZV. Molecular studies have provided mounting evidence that the mumps virus dysregulates the host's immune system and enables the virus to proliferate in the infected host cells. This dysregulation of the immune system by mumps virus may have occurred in our patient, enabling the latent VZV infection to reactivate. A variety of virus infections have been linked to the development of peripheral facial paralysis. These viruses include herpes simplex virus-1 (HSV-1), varicella zoster virus (VZV), Epstein-Barr virus, cytomegalovirus, and mumps virus. Increasing evidence suggests that idiopathic peripheral facial paralysis , the most common clinical entity of facial paralysis, is caused by reactivation of HSV-1 . SimilarWe present here a patient who showed the simultaneous development of left peripheral facial paralysis, ipsilateral cervical herpes zoster, and mumps sialadenitis as evidenced by serological studies. Our case, as well as those in previous reports, raises the question whether the so-called mumps-associated facial paralysis is simply caused by mumps virus infection or the additional reactivation of latent VZV.An 8-year-old Japanese girl was referred to our clinic for the evaluation of a left facial nerve paralysis, left cervical zoster, and swelling of the right submandibular area. She developed cervical zoster and facial paralysis on the first and second day of illness, respectively, and was given prednisolone (1 mg/kg/day) and valaciclovir (1500 mg/day) orally by a pediatrician from the fourth day of illness. She subsequently developed a right submandibular swelling on the eighth day. She did not notice hearing loss or vertigo. She had been vaccinated against mumps at the age of 2 and had no obvious episode of mumps infection previously. Although she had also been vaccinated for chickenpox at the age of 2, she developed chickenpox at the age of 4. Another past medical history was notable for an operation for Fallot's tetralogy at the age of 2.At the first visit to our clinic on the tenth day of illness, a physical examination revealed a complete left facial paralysis (House-Brackmann grade VI). Electroneurography of the facial nerve revealed nearly complete denervation of the left facial nerve . Pure toHer sialadenitis disappeared within 1 week. Her left facial movement also gradually improved to be House-Brackmann grade 2 with slight synkinesis 7 months later. Follow-up serological tests 6 weeks after the onset of illness revealed a decrease in both IgM antimumps and anti-VZV antibodies .To our knowledge, the present report is the first to show simultaneous development of mumps sialadenitis and reactivation of VZV, both of which were evidenced by serological assays, in a case of peripheral facial paralysis. In the present case, cervical zoster and facial paralysis developed six and seven days earlier than the development of sialadenitis, respectively. The electroneurography of the facial nerve revealed nearly complete denervation on the tenth day of illness.Based on the clinical and laboratory findings, this case may be clinically diagnosed as mumps-associated facial paralysis. In fact, a small number of previous reports have suggested the possible association of mumps viral infection with peripheral facial paralysis –6. HowevAlthough it is not clear if the mumps virus infection and VZV reactivation were merely coincidental or were associated in the present case, the time lapse between the two indicates the likelihood of their relation. Assuming that they were actually associated, it is reasonable to speculate that the mumps infection enabled the subsequent reactivation of VZV, since the herpes zoster is a result of reactivation, not the initial infection, of the VZV virus. The latency period of the mumps virus is usually 2-3 weeks, suggesting that mumps infection in our case may have occurred 1-2 weeks before the development of cervical zoster and facial paralysis. Recent molecular studies have provided mounting evidence that the mumps virus may dysregulate the immune system of the host so that the virus can proliferate in the infected host cells . Such a Interestingly, besides the small number of case reports describing the development of peripheral facial paralysis in patients with mumps sialadenitis, the association of trigeminal herpes zoster and mumps parotitis has also been reported , as wellThe present case suggests that the reactivation of VZV may be associated with “mumps-induced” facial paralysis. It would be beneficial for physicians who manage facial nerve paralysis to keep this possible pathology in mind and to perform serological tests for VZV when encountering the patients with peripheral facial paralysis and mumps sialadenitis, even if they do not show signs of VZV reactivation.


However, the low efficiency of conversion of PrPC into PrPSc in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrPC may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrPC into PrPSc from ∼10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrPSc by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 1012-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrPC susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrPScin vitro.Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions  in vitro. While PMCA has become an important tool in prion research, its application is limited because of low yield, poor efficiency and, sometimes, stochastic behavior. The current study introduces a new PMCA format that dramatically improves the efficiency, yield, and robustness of prion conversion in vitro and reduces the time of the reaction. These improvements have numerous implications. The method opens new opportunities for improving prion detection and for generating large amounts of PrPScin vitro. Furthermore, the results demonstrate that in vitro conversion is not limited by lack of convertible PrPC nor by concentrations of cellular cofactors required for prion conversion.Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions  During the incubation step, small PrPSc particles grow by recruiting and converting PrPC molecules into PrPSc.PMCA reactions consist of two alternating steps: incubation and sonication. Sonication fragments PrPSc has limited its utility for structural studies. Furthermore, the efficiency of amplification in PMCA varies dramatically depending on minor variations in experimental parameters, including those that are difficult to control, such as the age of the sonicator's horn and individual patterns of horn corrosion. Previous strategies for improving the efficiency of PMCA focused on increasing the number of cycles within a single PMCA round CSc. This result also shows that the low yield observed previously has not been due to a lack of PrPC susceptible to conversion, nor has it been limited by cellular cofactors.While the discovery of PMCA has provided new opportunities for exploring the prion replication mechanism, the low yield of PrPin vitroC into PrPSc was approximately 10% as judged by Western blotting  or 2.38 mm (referred to as large or L) significantly accelerated the formation of amyloid fibrils of rPrP blotting . This amblotting .Sc amplification monitored by Western blotting revealed that in the absence of beads the newly generated PrPSc was detected by the 16th cycle, whereas in the presence of beads it already was seen by the 8th cycle (Sc amplification was observed (data not shown).The kinetics of PrPth cycle . Furtherth cycle . These rth cycle  were use4-diluted 263K brain material and subjected to amplification in the presence or absence of beads for 6 rounds of 48 cycles each. The products of each round were diluted 10-fold into fresh NBH for the subsequent round. The PMCA products from the final round were then diluted an additional 10-fold prior to inoculation of 50 µl per animal. The final dilution of the initial 263K brain material was 1010 fold. In our laboratory the concentration of 263K scrapie in the brains of hamsters in the late stages of symptomatic disease is consistently between 1 and 2×1010 Infectious Dose50/g of brain 10 dilution of 263K brain would contain 1 ID50/ml giving a probability of infection of 0.05 from a 50 µl inoculation 4-diluted 263K brain reached the endpoint by 110.5±7.5 days . None of them showed PK-resistant material on Western blotting . In the presence of beads, however, the amount of SSLOW PrPSc remained stable during serial PMCAb if the reactions were seeded with 103-fold diluted SSLOW brain homogenates (lanes 6–8), or increased if 104-fold dilutions were used for seeding (lanes 13-15). These results illustrate that the positive effect of beads is not limited to 263K and that beads improved the robustness of PMCA for a strain with poor amplification behavior.To test whether the positive effect of beads on prion amplification was limited to 263K, we used a synthetic prion strain, SSLOW, which was previously found to have a very peculiar amplification behavior in PMCA Sc in PMCA lanes 10–12). The addition of beads, however, restored the amplification rate of 263K to the level observed in the absence of rPrP and beads (lanes 13–15 to 3–5). However, this amplification level was lower than those observed in the presence of beads without rPrP (lanes 6–8).In previous studies, recombinant PrP (rPrP) was found to inhibit amplification of PrPPrion amplification in PMCA was previously shown to exhibit species specificity that faithfully reflects the transmission barrier observed in animals 5- fold diluted 263K or 104- fold diluted RML  imaging. Consistent with our previous studies Scin vitro for structural studies.The current studies demonstrated that the yield and the rate of prion conversion in PMCA can be substantially improved by including beads. Remarkably, substantial improvements in the amplification efficiency and robustness did not come at the cost of prion replication specificity. While beads were found to increase the amplification rate of two hamster strains in hamster NBHs, no detectible amplification of these strains were observed in mouse NBHs within three rounds. This shows that the species specificity was preserved . FurtherSc fragmentation. Consistent with this mechanism, beads were found to enhance significantly the amplification efficiency of SSLOW PrPSc, a strain which is deposited in the form of large plaques Sc particles but could also irreversibly damage or denature PrPSc and/or PrPC. We observed that during sonication, the beads rose from the bottom of the tubes and vibrated in the reaction mixtures. Perhaps, the presence of beads helps to redistribute the cavitation energy of bubbles into the much “softer” energy of mechanical vibration, making the conditions for breaking PrPSc particles more optimal.The effect of beads on prion amplification can be explained by several mechanisms. Using amyloid fibrils produced from rPrP, we showed that sonication in the presence of beads effectively fragmented rPrP fibrils into pieces that were substantially smaller than those observed in the absence of beads . This reSc particles, the effect of beads could be attributed to a breakage of cellular debris and an increase in the accessibility of PrPC and/or cellular cofactors essential for conversion. Considering that different strains or strains from different species might utilize a variety of cellular cofactors of different chemical natures In addition to more efficient fragmentation of PrPC could be converted into PrPSc in PMCA, which raised concerns that only a fraction of PrPC is susceptible to conversion. In an attempt to improve the conversion yield, an increase in the number of PMCA cycles C was employed CCC into PrPSc without affecting competing reactions. Remarkably, a substantial fraction if not 100% of PrPC could be converted into PrPSc in PMCAb , the differences in amplification yield between PMCA and PMCAb was approximately 10-fold .2+-free and Mg2+-free PBS, pH 7.5, supplemented with 0.15 M NaCl, 1.0% Triton and 1 tablet of Complete protease inhibitors cocktail  per 50 ml of conversion buffer. The resulting 10% normal brain homogenate in conversion buffer was used as the substrate in PMCA reactions. To prepare seeds, 10% scrapie brain homogenates in PBS were serially diluted 10- to 1014-fold, as indicated, in the conversion buffer and 10 µl of the dilution were used to seed 90 µl of NBH in PMCA. Samples in 0.2 ml thin-wall PCR tubes  were placed in a rack fixed inside Misonix S-3000 or S-4000 microplate horn, filled with 300 ml water. Two coils of rubber tubing attached to a circulating water bath were installed for maintaining 37°C inside the sonicator chamber. The standard sonication program consisted of 30 sec sonication pulses delivered at 50% to 70% efficiency applied every 30 min during a 24 hour period. For PMCA with beads, small (1.58 mm diameter) or large (2.38 mm diameter) Teflon beads  were placed into the 0.2 ml tubes first using tweezers, then NBH and seeds were added. The following beads from Small Parts  were tested in Healthy hamsters were euthanized and immediately perfused with PBS, pH 7.4, supplemented with 5 mM EDTA. Brains were dissected, and 10% brain homogenate (w/v) was prepared using ice-cold conversion buffer and glass/Teflon tissue grinders cooled on ice and attached to a constant torque homogenizer (Heidolph RZR2020). The brains were ground at low speed until homogeneous, then 5 additional strokes completed the homogenization. The composition of conversion buffer was as previously described In our experience, the amplification efficiency in PMCA depended strongly on the position of the tube within a microplate horn, i.e. distance of a tube from horn's surface to the tube and its center; and the age of the sonicator's horn. In the current studies, several Misonix sonicators were used, all equipped with horns less than one year old. The tubes were placed only in positions between 1.5 cm and 5 cm from the horn's center. Nevertheless, we experienced substantial variations in amplification efficiency in standard PMCA (no beads), which appear due to differences in the age of horns, individual patterns of horn corrosion or differences in the horizontal coordinates of tubes. In the presence of beads, the amplification was much more robust and showed only minor variations.4-fold relative to whole brain in PBS, 1% BSA. For groups 2 and 3, 10 µl of 10% 263K scrapie brain homogenate were mixed with 90 µl of PBS and subjected to a sonication procedure equivalent to a single PMCA round (48 sonication cycles) in the absence of NBH. Sonication was performed either without beads (for group 2) or with 3 large beads (for group 3). Then, the sonication products were diluted 100-fold into PBS, 1% BSA to obtain final dilution of 10−4 relative to whole 263K brain for inoculation. For groups 4 and 5, PMCA reactions were seeded with 10-4-diluted 263K brain material, then six serial PMCA rounds were conducted in the absence of beads (for group 4) or presence of 3 large beads (for group 5) using 1∶10 dilutions between rounds. After the 6th round of serial PMCA, the amplification products were diluted an additional 10-fold into PBS, 1% BSA to obtain final 1010-fold dilutions of the initial 263K brain material prior to inoculation.Weanling golden Syrian hamsters were inoculated intracerebrally with 50 µl each using the following inocula: animals of group 1 were inoculated with 263K brain homogenate diluted 10In hamsters inoculated with the 263K scrapie strain, the asymptomatic period of infection lasts 60 to 160 days followed by a stereotypic clinical progression leading to death 2 to 3 weeks later. Individual symptoms, such as wobbling gait and head bobbing, are readily recognized but their onset is subtle and subject to large inter (and even intra) observer variability. Incubation time determinations are greatly improved by an empirical determination of endpoint To analyze production of PK-resistant PrP material in PMCA, 15 µl of each sample were supplemented with 2.5 µl SDS and 2.5 µl PK, to a final concentration of SDS and PK of 0.25% and 50 µg/ml respectively, followed by incubation at 37°C for 1 hour. The digestion was terminated by addition of SDS-sample buffer and boiling for 10 min. Samples were loaded onto NuPAGE 12% BisTris gels, transferred to PVDF membrane, and stained with 3F4 or D18 antibody for detecting hamster or mouse PrPs, respectively.To analyze scrapie brain homogenates, an aliquot of 10% brain homogenate was mixed with an equal volume of 4% sarcosyl in PBS, supplemented with 50 mM Tris, pH 7.5, and digested with 20 µg/ml PK for 30 min at 37°C with 1000 rpm shaking (Eppendorf thermomixer). The reaction was stopped by adding 2 mM PMSF and SDS sample buffer. Samples were boiled for 10 min and loaded onto NuPAGE 12% BisTris gels. After transfer to PVDF membrane, PrP was detected with 3F4 antibody.To obtain calibration curves for calculating of PMCA fold amplification, 10% brain homogenate from 263K animals was sonicated for 1 min and serially diluted into 10% NBH sonicated for 30 sec. PMCA samples as well as 263K dilutions were digested with 50 µg/ml PK for 1 h at 37°C. The reaction was stopped by addition of 2 mM PMSF. All samples were diluted 10-fold in PBS, and analyzed using a 96-well immunoassay similar to those previously published Full-length hamster rPrP  was expressed and purified as previously described Figure S1Proteinase K assay of scrapie brain homogenates. Western blotting of scrapie brain homogenates from animal groups # 2, 3, 4 and 5. Two brain homogenates per group are shown. 10% brain homogenates were treated with 20 µg/ml PK for 30 min at 37C, 3F4 antibody was used for western blotting.(0.16 MB TIF)Click here for additional data file.Figure S2Scde novo. 10% NBH was subjected to a three rounds of serial PMCAb (with 3 large beads) in the absence of seeds and digested with PK. Each PMCA round consisted of 48 cycles; 10-fold dilutions were used for serial rounds. 32 independent reactions were analyzed. Undigested 10% NBH (lane 1) are showed as references.PMCAb does not produce PrP(0.56 MB TIF)Click here for additional data file.Figure S3Sc amplification fold. (A) 263K scrapie brain material was serially diluted into 10% NBH to the final concentrations of scrapie brain ranging from 10% to 0.0625%, then digested with 50 µg/ml PK for 1 h at 37°C and analyzed using a 96-well dot blot. The signal intensity was measured using a Typhoon 9200 Variable Mode Imager and was found to be linear within the concentrations of scrapie brain homogenate from 0.0625% to 1% as shown in panel B. This concentration range was used to estimate the fold amplification of PrPSc in PMCA.Quantitative estimates of PrP(0.33 MB TIF)Click here for additional data file.Figure S4Sc fold amplification in serial PMCA. 263K brain material was diluted 104-fold into 10% NBH (lane 2) and subjected to a three rounds of PMCA in the absence of beads (top panel) or presence of 3 large beads (bottom panel) and digested with PK. The material amplified in each round was diluted 10-, 20-, 100-, or 1000-fold into 10% NBH for the next PMCA round, as indicated. Each PMCA round consisted of 48 cycles. Undigested 10% NBH (lane 1) is provided as a reference.Analysis of PrP(0.44 MB TIF)Click here for additional data file.


Drosophila. Using sequence data from these new viruses, and the previously described sigma viruses, we show that they have switched between hosts during their evolutionary history. Our results suggest that sigma virus infections may be short-lived in a given host lineage, so that their long-term persistence relies on rare horizontal transmission events between hosts.A diverse range of endosymbionts are found within the cells of animals. As these endosymbionts are normally vertically transmitted, we might expect their evolutionary history to be dominated by host-fidelity and cospeciation with the host. However, studies of bacterial endosymbionts have shown that while this is true for some mutualists, parasites often move horizontally between host lineages over evolutionary timescales. For the first time, to our knowledge, we have investigated whether this is also the case for vertically transmitted viruses. Here, we describe four new sigma viruses, a group of vertically transmitted rhabdoviruses previously known in  Verticase flies , Blochmater ants  and Blatlbachia, , Rickettckettsia  and Spirbacteria  and theibacteria .Drosophila—Drosophila melanogaster (DMelSV) [Drosophila obscura (DObsSV) and Drosophila affinis (DAffSV) [Diptera, and use a phylogenetic approach to show that sigma viruses have switched between host species during their evolution.In contrast to bacterial endosymbionts, little is known about the evolutionary history of vertically transmitted viruses. Sigma viruses are vertically transmitted rhabdoviruses previously known from three species of (DMelSV) , Drosoph(DAffSV) . These v(DAffSV) ,12. Here2.(a)Drosophila tristis in Derbyshire, UK; Drosophila immigrans in Marktredwitz, Germany; Drosophila ananassae in Kilifi, Kenya; and Muscina stabulans in Cambridge, UK. Infected flies were detected by exposing them to pure CO2 at 12°C for 15 mins. Uninfected flies recover after approximately 30 mins while infected flies remain paralysed [COI and/or Cytb genes. Additional species were also collected and tested with the CO2 assay, but we only report those species from which we were able to amplify a sigma virus.We collected aralysed . RNA wasaralysed ), and amaralysed ). To obt(b)lustalW. Alignments were trimmed to contain only a conserved region of the RDRP that could be robustly aligned. Phylogenies were inferred using maximum-likelihood (ML)  (PAUP ) and Bay [rBayes ) methodsdel Test . Node-su(c)4 random viral topologies to that derived from the posterior sample of viral topologies.To detect topological incongruence between host and parasite phylogenies, we used a Shimodaira–Hasegawa test (SH-test) , which c3.Drosophila—D. tristis, D. immigrans and D. ananassae—and one member of the Muscidae, Muscina stabulans. We have tentatively named these new viruses as DTriSV, DImmSV, DAnaSV and MStaSV, respectively. This brings the total number of sigma viruses described to seven, and for the first time extends their distribution outside the genus Drosophila.We detected novel sigma viruses in four dipteran species, including three species of We sequenced 1845–3006 bp of the RDRP gene from the four viruses (accession-numbers: JF311399–JF211402). The sequences are highly divergent from one another, with the most closely related pair, DAffSV and DTriSV, having an amino acid sequence identity of 0.73. The other five genes of sigma viruses have previously been shown to have even greater divergence , the sigΔlnL = 328, p < 0.005). For the Bayesian trees, we also found that the viral phylogeny differed significantly from the host topology, with none of the topologies in the posterior sample of trees matching that of the hosts. Therefore, both methods suggest these viruses have switched between host species. Incongruence is owing to two factors: first, the presence of DImmSV in a clade of viruses with hosts from a different subgenus of Drosophila to D. immigrans [D. obscura is much more closely related to D. tristis than D. affinis [To test whether the sigma viruses have exclusively cospeciated with their hosts, we compared the host and virus phylogenies. The phylogeny of these host species is extremely well resolved ,18. We f affinis , yet a vHowever, although the trees were not congruent, we found that the inferred virus topology was more similar to the host topology than expected by chance. Only 2 per cent of random viral topologies were closer to the host topology than the posterior sample of actual virus topologies were to the host topology . This ma4.D. ananassae, D. immigrans, D. tristis and M. stabulans. Together with the three existing sigma viruses in other Drosophila, they form a clade of Dipteran-infecting rhabdoviruses. It is probable that these viruses are vertically transmitted as not only are all of the previously known sigma viruses vertically transmitted [2 sensitivity—the hallmark of sigma virus infection—is known from other Diptera [We have discovered four new sigma viruses in nsmitted , but als Diptera ,24. The D. melanogaster populations, there has been a recent selective sweep of a gene conferring resistance to DMelSV [Sigma viruses have highly dynamic interactions with their hosts. In o DMelSV , and thio DMelSV . DObsSV o DMelSV . Such rao DMelSV . If so, o DMelSV –8, and go DMelSV  and homio DMelSV . Althougo DMelSV , and arto DMelSV ,29,30.Drosophila suggests that these viruses may be widespread in Dipterans, if not insects as a whole. Unlike bacterial endosymbionts, the rapid evolution of the sigma virus genome makes it impossible to design a single pair of diagnostic PCR primers that can be used to test for new strains of the virus. In the course of this study, we encountered CO2-sensitive individuals of other species of flies from which we were unable to amplify virus using our primers, and these may harbour other sigma-like viruses. CO2 sensitivity has also been reported in 13 other Drosophila species [Culex mosquitoes [A sigma-like virus outside of the genus  species , and in squitoes . Additiosquitoes  and rhabsquitoes . The nonsquitoes . The dis


Bunyamwera virus NSs protein is involved in the inhibition of cellular transcription and the interferon (IFN) response, and it interacts with the Med8 component of Mediator. A spontaneous mutant of a recombinant NSs-deleted Bunyamwera virus (rBUNdelNSs2) was identified and characterized. This mutant virus, termed mBUNNSs22, expresses a 21 aa N-terminally truncated form of NSs. Like rBUNdelNSs2, mBUNNSs22 is attenuated in IFN-deficient cells, and to a greater extent in IFN-competent cells. Both rBUNdelNSs2 and mBUNNSs22 are potent IFN inducers and their growth can be rescued by depleting cellular IRF3. Strikingly, despite encoding an NSs protein that contains the Med8 interaction domain, mBUNNSs22 fails to block RNA polymerase II activity during infection. Overall, our data suggest that both the interaction of NSs with Med8 and a novel unidentified function of the NSs N-terminus, seem necessary for Bunyamwera virus to counteract host antiviral responses. Bunyamwera virus (BUNV) is the type species of both the genus Orthobunyavirus and the family Bunyaviridae, members of which are important human pathogens causing diseases such as febrile illness and haemorrhagic fever. Bunyaviruses possess a tri-segmented, negative-sense RNA genome that encodes four structural proteins: the viral RNA-dependent RNA polymerase (L protein) on the large (L) segment, two glycoproteins (Gn and Gc) on the medium (M) segment, and the nucleoprotein (N) on the small (S) segment. In addition, orthobunyaviruses encode one or two non-structural proteins, NSm on the M segment and NSs  antagonist involved in evading host innate immune responses . During The N and NSs proteins are translated from overlapping open reading frames (ORFs) in a single mRNA transcribed from the S segment. To examine the role of NSs in infection, a recombinant virus lacking NSs (rBUNdelNSs2) was created in which NSs expression was abrogated by changing the tandem AUG initiation codons to ACG codons and converting codon 3 (serine) to a stop codon. In addition, codons 4 and 5 were changed from leucine to proline  leads to accumulation and therefore more sensitive detection of NSs . Whereas5 p.f.u. ml−1 vs 1.5×106 p.f.u. ml−1; Fig. 6 p.f.u. ml−1 vs 7.6×105 p.f.u. ml−1; Fig. Multicycle virus growth in BHK cells was analysed as described previously . Interesβ in response to virus infection  from the AUG at codon 30, but this virus had the same phenotype in BHK cells as the subsequently made rBUNdelNSs2 virus ] or at tThe fact that mBUNNSs22 arose spontaneously suggests a selective pressure for some function of NSs to be regained by the virus. Our results indicate that although mBUNNSs22 expresses a large part of the viral IFN antagonist protein, this mutant virus behaves indistinguishably from rBUNdelNSs2 in its inability to inhibit IFN induction. The selective pressure must thus be for another function of NSs, which could be to inhibit protein translation (The results presented in Figs


The Bio-Plex® system utilizes xMAP technology to permit the multiplexing of up to 100 different analytes. Multiplex analysis gives researchers the ability to look at analytes simultaneously providing more information from less sample volume in less time than traditional immunoassay methods. Similar to ELISA, xMAP utilizes an antibody sandwich for detection but differs from ELISA in capture substrate and detection method. Rather than a flat surface, Bio-Plex®assays make use of differentially detectable bead sets as a substrate capturing analytes in solution and employs fluorescent methods for detection. These bead sets identify the analytes and detection antibodies are used to measure the quantity of analyte. The use of differentially detectable beads enables the simultaneous identification and quantification of many analytes in the same sample. For more than 50 years, immunoassays have allowed sensitive and highly specific detection of analytes of interest in biological samples in life science research and clinical diagnostics. Immunoassays provide informa-tion to researchers on the roles proteins and other bio-molecules play in a myriad of biological processes and diseases. The first immunoassay was developed by The Bio-Plex® suspension array system utilizes xMAP technology, licensed from Luminex Corp., to permit the multiplexing of up to 100 different assays within a single sample. This technique involves 100 distinctly coloured bead sets created by the use of two fluorescent dyes at distinct ratios. Beads are conjugated with a reagent specific to a particular bioassay. The reagents may include antigens, antibodies, oligonucleotides, enzyme substrates or receptors. The technology makes use of multiple assays whereby one antibody to a specific analyte is attached to a set of beads with the same colour and the second antibody is used to quantify the bound antigen. The use of different coloured beads enables the simultaneous detection of many other analytes in the same sample. Imaging or laser excitation is then used to determine the different assays by bead colours, and determine analyte concentration by measuring the reporter dye fluorescence .For laser excitation detection (Bio-Plex 200 and Bio-Plex 3D) the contents of each microplate well are drawn into the array reader and precision fluidics align the beads in single file through a flow cell, where two lasers excite the beads individually. The red classification laser excites the dyes in each bead, identifying its spectral address. The green reporter laser excites the reporter molecule associated with the bead, which allows quan-titation of the captured analyte. High-speed digital signal processors and software record the fluorescent signals simultaneously for each bead, translating the signals into data for each bead-based assay .The Bio-Plex MAGPIX system employs low-cost light-emitting diodes (LEDs) and a charge-coupled device (CCD) imager to illuminate and image immobilized magnetic beads Similar to ELISA, a majority of assays are designed accord-ing to a capture sandwich immunoassay format. Briefly, the capture antibody-coupled beads are first incubated with antigen standards or samples for a specific time. The plate is washed to remove unbound materials, followed by incu-bation with biotinylated detection antibodies. After wash-ing away the unbound biotinylated antibodies, the beads are incubated with a reporter streptavidin-phycoerythrin conjugate (SA-PE). Following removal of excess SA-PE, the beads are passed through the array reader, which measures the fluorescence of the bound SA-PE .The substrate for the antibody sandwich is the bead. Bead characteristics define instrument compatibility and workflow and can be classified into two categories, non-magnetic and magnetic. The non-magnetic beads are smaller in size ; they can be used with the magnetic workflow as well as vacuum workflows. These magnetic beads are compatible with all currently available life science instruments from any Luminex partner. In the magnetic workflow, the beads are washed in the well with dispense and aspiration washing.xMAP assays may contain non-magnetic or magnetic beads as substrates. Magnetic COOH beads are the new-est core components of xMAP assays. They are unique in that they exhibit both fluorescent and magnetic properties. The beads are stained with a fluorescent dye formulation proprietary to Luminex . 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Flavivirus–reactive primers and yellow fever virus–specific primers. Nucleotide sequencing of the amplicons obtained with the genus Flavivirus oligonucleotide primers confirmed yellow fever virus as the etiologic agent. Isolation attempts in newborn mice and Vero cells from the samples yielded virus isolates from five patients. Rapid and accurate laboratory diagnosis enabled an interagency emergency task force to initiate a targeted vaccination campaign to control the outbreak.In May 2003, the World Health Organization received reports about a possible outbreak of a hemorrhagic disease of unknown cause in the Imatong Mountains of southern Sudan. Laboratory investigations were conducted on 28 serum samples collected from patients in the Imatong region. Serum samples from 13 patients were positive for immunoglobulin M antibody to flavivirus, and serum samples from 5 patients were positive by reverse transcription–polymerase chain reaction with both the genus  Flavivirus of the family Flaviviridae, is the etiologic agent of yellow fever, a viral hemorrhagic fever that occurs in South American countries and much of sub-Saharan Africa , an arthropod-borne virus in the genus ,YFV has been successfully isolated from acute-phase serum and liver samples by injection of suckling mice and cell cultures  office for southern Sudan a suspected disease outbreak in Imatong, a mountain range located in the southern part of Eastern Equatoria, southern Sudan. The range extends northwards to the town of Torit at 3,000 m above sea level. The mountain is covered by tropical rain forest with rich flora and fauna. Though the civil war has had a negative effect on the ecosystem, the Lango people continue to live on the eastern slopes of this mountain range. The total population in Imatong is estimated to be 24,387 people . Their primary means of livelihood is agriculture, although they also keep some livestock. According to the report by WHO’s Early Warning and Response Network (EWARN), which was established in southern Sudan in 1999 to monitor disease outbreaks, and the Norwegian Church Aid, the disease has clinical signs and symptoms of a viral illness and has caused the deaths of seven adults >30 years of age. On the basis of clinical symptoms, a viral hemorrhagic fever was suspected. On May 12, 2003, a team led by WHO arrived in southern Sudan to investigate and verify the cause of the outbreak. We report on laboratory investigations performed and the identified causative agent.A case definition was established as follows: an illness in a patient of any age with high fever, severe headache, neck and back pain, possibly accompanied by vomiting, abdominal pain, diarrhea, hematemesis, bloody diarrhea, jaundice, and epistaxis. A total of 28 serum samples were collected: 18 from patients with acute disease who met the working case definition, 8 from patients with possible disease with headache and joint pain , and 2 from convalescent patients (patients 020 and 028). Patient details, including their locations, are shown in the Table. Blood was collected in sterile evacuated clot activator Vacutainer tubes. Thick and thin smears were made on microscope slides for 17 patients as indicated in the Table, and the serum samples were then separated in the admission facility and stored in a liquid nitrogen dry shipper for transportation to the WHO Collaborating Center for Arbovirus and Viral Hemorrhagic Fever Reference and Research (CCAVHFRR) Laboratory at the Kenya Medical Research Institute (KEMRI) in Nairobi, where they were frozen at –70°C before testing.,All the serum samples were heat-inactivated at 56°C for 30 min and tested for antibodies specific to flaviviruses by MAC-ELISA . RT-PCR specific for amplification of a 267-bp region of NS5 gene of flaviviruses was performed with 50 pmol of oligonucleotide primers FU1 (TAC AAC ATG ATG GGA AAG AGA GAG AA) and CFD2 (GTG TCC CAG CCG GCG GTG TCA TCA GC) (Flavivirus-reactive primers were purified by excising the 267-bp fragment from the agarose gel and purifying the fragments with Qiagen gel purification kit (Qiagen) for nucleotide sequence determination.All 28 serum samples were subjected to RT-PCR by using a universal pair of primers for members of the genus Gel-purified PCR products were quantitated, and the partial nucleotide sequences of amplicons with a minimum of 60 ng DNA/µL were determined by using Big Dye terminator sequencing ready reaction kits with AmpliTaq DNA polymerase FS . A cycle sequencing reaction was performed with each of the primers CFD2 and FU1 in a final volume of 20 µL with 30 ng of PCR product, 3.5 pmol of primer, and 4 µL of BigDye Terminators premix, according to the manufacturer’s protocol. Briefly, the tubes were heated to 96°C for 2 min, and the reaction mixture underwent 25 cycles of 30 s at 96°C, 30 s at 50°C, and 4 min at 60°C. Excess Big Dye Terminators were removed by precipitation with absolute ethanol. The partial nucleotide sequence was obtained with an Applied Biosystems 377 sequencer, according to manufacturer’s instructions.A search performed on Basic Local Alignment Search Tool (BLAST)  suggested that the sequence obtained was amplified from the yellow fever genome. This finding was confirmed by aligning the sequence data with data from other known flaviviruses retrieved from GenBank.Aliquots of each of the serum samples were injected intracerebrally into litters of eight newborn (24–48 hours after birth) Swiss albino mice. The mice were observed daily for signs of illness. Sick mice were euthanized, and a preparation was made of 10% of the harvested mouse brain suspension made in Eagle’s Maintenance Media with 2% serum albumin, 2% glutamine, and 1% antibiotics  and centrifuged at 3,000 rpm for 10 min. The clarified supernatant fluid was filtered with a 0.45-µm syringe filter and injected intracerebrally into a litter of suckling mice to confirm the isolation.2 culture flasks. Flasks were incubated at 37°C and observed daily for evidence of cytopathogenic effect (CPE). After CPE was evident, the supernatant fluid was clarified by centrifugation at 3,000 rpm for 10 min. Viral RNA was extracted from both the 10% brain suspension from sick mice and from the clarified cell culture media and screened for flavivirus and YFV viral nucleic acid RNA by RT-PCR as described previously.All the sera were diluted 1:10 in sterile phosphate-buffered saline pH 7.4 , and viral isolation attempts in Vero cell cultures were made by injecting 100 µL of the diluted sample onto confluent monolayer of Vero cells in 25-cmAlphavirus and Bunyavirus. For 17 patients, thick blood smears were checked for Borrelia spp. and thin blood smear slides were checked for malarial parasites.IgM antibody tests were performed on all the samples to exclude recent exposure to Ebola virus, Marburg virus, Crimean-Congo hemorrhagic fever virus, West Nile virus, dengue virus, chikungunya virus, Sindbis virus, and Rift Valley fever virus. In addition, Ebola virus was excluded by using an antigen capture ELISA. PCR was performed to exclude the following pathogens: Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, Ebola virus, Marburg virus, rickettsia, leptospira, brucella, and members of the genus On May 17, 2003, serologic tests and RT-PCR performed at the WHO CCAVHFRR at KEMRI identified YFV as the causative agent of the outbreak in southern Sudan. The diagnosis was supported by subsequent gene sequencing of the amplicons and isolation of the virus. The results obtained are summarized in the Table. Duplicate samples submitted to the Special Pathogens Unit at the National Institute for Communicable Diseases, South Africa, confirmed an outbreak of yellow fever.Samples from five patients were positive for flavivirus viral nucleic acid and subsequently were positive for yellow fever viral nucleic acid with primers specific for YFV. Analysis of the nucleotide sequences obtained with the flavivirus primers confirmed that the amplicons were from the NS5 region of the yellow fever genome (phylogeny has been performed separately). The serum samples that were positive by PCR were collected from patients on day 1 after onset of illness except for one sample collected on day 8 from a patient with a fatal infection.YFV was isolated, in both suckling mice and cell cultures, from five patients from whom PCR products were obtained. Virus isolation attempts on the remaining 23 serum samples from both suckling mice and Vero cell culture were negative.Alphavirus and Bunyavirus. The thick smears were negative for Borrelia by Giemsa staining. However, malaria parasites were demonstrated in thin smears from 7 of 17 patients tested; 6 of the patients with malaria also had evidence of YFV infection.The sera were all negative by using various tests as described for Ebola virus, Marburg virus, Crimean-Congo hemorrhagic fever virus, West Nile virus, dengue virus, chikungunya virus, Sindbis virus, Rift Valley fever virus, rickettsia, leptospira, brucella, and members of the genus Samples from 28 patients were collected from various villages in the Imatong region as follows: 10 were from Locomo village, 3 from Lofi, 4 from Tarafafa, 3 from Imatong, and 1 each from Itohom, Lofulung, Ogolok, Lotodo, Lofong, and Itede; 2 were from unknown locations. A total of 18 patients had possible YFV infections demonstrated by detection of IgM antibody, while 5 patients were confirmed as positive for YFV by RT-PCR and virus isolation. Antibody was not demonstrable in the five patients who had positive RT-PCR results; in 4 patients, the specimens were collected too early (day 1) for antibody to be detectable. In patient 001 , the spePlasmodium, the causative agent of malaria, was demonstrated in six patients who were positive for YFV. This finding demonstrates that co-infection with malaria and YFV is possible. Therefore, malaria should not be assumed as the etiologic agent of a disease outbreak solely based on positive screens, without first excluding other causes.YFV maybe be enzootic in the Imatong Mountains, yet outbreaks have not been previously recorded in this region. Political unrest and population movement of highly susceptible (unvaccinated) people within the Imatong Mountains might have been a predisposing factor for the YFV outbreak in this region. During the civil unrest, large populations of monkeys were reportedly seen. As they became more dependent on human crops for food, the monkeys came closer to human habitations. Weather factors might have also contributed to the timing of the outbreak. The months of March to May 2003 were the rainy season, and heavy rains were reported around the mountain region. The persistent downpours may have resulted in high vector populations. With no history of human yellow fever in the outbreak zone, the monkey population must be adequate to support continued circulation of the virus in the sylvatic cycle. A combination of these factors likely contributed to the timing of the outbreak.The early detection of this outbreak was due to EWARN, a WHO-facilitated network that brings together several healthcare providers and laboratories. One of the objectives of EWARN is the early detection and response to outbreaks, such as this one. The rapid response to this outbreak of YFV and subsequent laboratory-based diagnosis clearly demonstrate that an efficient surveillance system can lead to quickly detecting outbreaks and appropriate intervention, potentially saving many lives. At the time of this writing, 80% of the population at risk in Imatong had been vaccinated, with the campaign extending to Torit County and neighboring counties. Vaccination in Torit County had covered 51% of the population at risk .


Enveloped viruses utilize cellular membranes to bud from infected cells. The process of virion assembly and budding is often facilitated by the presence of certain conserved motifs within viral proteins in conjunction with cellular factors. We hence examined the West Nile Virus (WNV) Envelope protein for the presence of any such motifs and their functional characterization.461PXAP464 and 349YCYL352 motifs in the WNV envelope glycoprotein bearing resemblance to retroviral late domains. Disruptive mutations of PXAP to LAAL and of the highly conserved Cys350 in the YCYL motif, led to a severe reduction in WNV particle production. Similar motifs in case of retroviruses are known to interact with components of host sorting machinery like PXAP with Tsg101 and YXXL with Alix. However, in the case of WNV, siRNA mediated depletion of Alix or Tsg101 did not have an effect on WNV release. Molecular modeling suggested that while the 461PXAP464 motif is surface accessible and could potentially interact with cellular proteins required for WNV assembly, the 349YCYL352 motif was found to be internal with Cys350 important for protein folding via disulphide bonding.We identified conserved 461PXAP464 and 349YCYL352 motifs in the WNV envelope are indispensable for WNV particle production. Although these motifs bear sequence similarity to retroviral late domains and are essential for WNV assembly, they are functionally distinct suggesting that they are not the typical late domain like motifs of retroviruses and may play a role other than Alix/Tsg101 utilization/dependence.The conserved  West Nile Virus (WNV) is a single stranded positive sense RNA virus of the genus Flavivirus. The 11Kb RNA genome is translated in the cytoplasm as a polyprotein and processed to yield 3 structural  and seven non-structural  proteins .Following the synthesis of viral genome and proteins, enveloped viruses utilize cellular membranes to bud from infected cells. This is often facilitated by the presence of certain conserved motifs within viral proteins and their ability to interact with the cellular processes/machinery. The best known example of this process is the interaction of retroviral late domain motifs with components of the ESCRT  sorting machinery to promote budding. Three types of consensus late domain motifs have been identified thus far: (i) the PT/SAP motif recruits the ESCRT-1 component Tsg101 (Tumor Susceptibility growth factor 101) ,10, (ii)The process of WNV budding into the lumen of the ER is topologically similar to the process of MVB biogenesis in that both occur in a direction that is away from the cytosol. MVB biogenesis is mediated by the family of ESCRT proteins namely ESCRT-0, -I, -II and -III and other associated proteins like Alix/AIP1. The membrane associated ESCRT-III complexes are finally disassembled and recycled by the ATPase Vps4. A number of enveloped viruses via the conserved late (L) domain motifs that mimic similar motifs in cellular proteins are able to recruit the ESCRT machinery to the site of virus budding . Disrupt461PXAP464 and 349YCYL352 motifs in the E protein  in the Flavivirus structural E proteins and determine whether they play a role in virus assembly and release. Sequence analysis of different Flavivirus structural proteins and different WNV isolates revealed the presence of conserved We next aimed towards conducting a functional analysis to determine if WNV may utilize the above conserved motifs for virus assembly and release. To this end we developed a rapid renilla luciferase (ren-luc) based virus release assay and compared it to the classical radioimmunoprecipitation based assay Figure . This woAs mentioned above, the conserved motifs identified in the WNV envelope resembled the late domain like motifs of retroviruses. We hence asked whether some of the well characterized inhibitors of ESCRT pathway previously used to study retrovirus budding would affect WNV assembly and release. To inhibit Tsg101 we utilized either Tsg-5’ expression vector that prevents HIV Gag-Tsg101 interaction or Tsg-F and TSG-3’ that have been shown to inhibit HIV release by globally disrupting the endosomal sorting machinery ,49. We aTo further examine the relevance of these conserved PXAP and YCYL motifs in WNV assembly and release, we constructed mutations in the PAAP residues to either LAAL or PSAP Figure A using sIt has previously been shown in context of HIV-1 that the PAAP motif interacts poorly with Tsg101 in in-vitro binding assays using purified proteins ,21,55. SOur findings that Tsg-5’ expression inhibits WNV release suggests a role for the ESCRT pathway in WNV budding. However, in other enveloped viruses that bear late domains  these motifs are located on the cytoplasmic side of the membrane and thus would be able to interact with ESCRT proteins to facilitate budding and particle release. The Flavivirus E protein on the other hand is translated into the lumen of the ER and hence these conserved motifs in WNV E protein would only be minimally exposed to the cytoplasmic side of intracellular vesicles or the plasma membrane. Hence in order to confirm the role of Tsg101 and/or Alix in WNV assembly and release we used a siRNA based approach. For this 293T cells were knocked down of endogenous Tsg101 and Alix expression using specific siRNAs and WNV release determined using both Ren-luc based and immunoprecipitation based virus release assays. As shown in Figure Our siRNA mediated depletion studies above suggested that WNV may not rely on the ESCRT host cell sorting machinery for assembly and release. Thus, it is plausible that these motifs may interact with other host factors to facilitate the assembly of the virion particles. In fact our structural analysis shows that the PXAP motif is surface accessible and could participate in protein interactions with yet unidentified cellular factors Figure A. In theIn this study, we have developed a rapid assay to study WNV assembly and release and identified conserved motifs in the viral envelope (E) that have functional relevance. These motifs bear sequence homology to late domain like motifs described in retroviruses. Experiments aimed at elucidating their role demonstrated that while expression of Tsg-5’ and Alix-V domain modestly inhibited WNV particle production, expression of Vps4EQ had no effect on WNV release. These data combined with the fact that siRNA mediated depletion of Alix or Tsg101 did not affect WNV release argues against their utilization or the ESCRT pathway by WNV. For instance, it has been documented that HSV possesses PT/SAP and YXXL motifs in several of its proteins but virus particle production is independent of Alix or Tsg101 expression . LikewisOur data are also reminiscent of the effects of Alix V domain expression versus Alix depletion on HIV particle production. While siRNA depletion of Alix does not affect HIV release, dominant negative inhibition via Alix V domain expression does ,53. MoreSequence analysis and information based on other viruses showed the presence of PXAP and YXXL conserved motifs in the E protein of Flaviviruses and different WNV strains, motifs that resemble the retroviral late domain-like motifs. It is worth mentioning that sequence analysis of a large portion of several different Flavivirus E proteins showed only 18% conservation in the amino acid residues, although the number does reflect the maximum diversity across the whole Flavivirus family . This coFurther studies are needed to determine the precise mechanism via which these motifs, specially the PXAP domain, regulates WNV assembly and release and whether it functions via interaction with certain host factors or merely play a structural role in regulating virus assembly and release.293T cells were cultured in DMEM supplemented with 10% FBS. All transfections were performed using Lipofactamine2000™ reagent (Invitrogen) as per the manufacturer’s instructions. In cases where transfections involved multiple DNAs, efficiency of co-transfection was carefully controlled by using an equal amount of plasmid expression vectors for each well and adjusting the total input DNA in each well to be constant by using pUC DNA.461PAAP464 and 349YCYL352 motifs to PSAP, LAAL, ACYA and AAAA were constructed by site directed mutagenesis (Stratagene) using specific primer pairs. The full-length HIV-1 proviral clone pNL4-3 [The WNV CprME and Ren/Rep plasmids have been described previously  and weree pNL4-3  and its e pNL4-3 . The HIVe pNL4-3 . Expresse pNL4-3 . The EIAe pNL4-3 .35S]Met/Cys. Culture supernatants were filtered and subjected to ultracentrifugation at 100,000 x g for 45 min. Cell and virion samples were lysed in cell lysis buffer . Thereafter, they were immunoprecipitated either with HIV-Ig (Kindly provided by the NIH AIDS research and reference reagent program) or anti-WNV serum  coated Protein A beads. Immunoprecipitated cell lysates were washed three times in RIPA buffer and once with SDS-DOC wash , resolved by SDS-PAGE followed by PhosphorImager analysis. Virus release efficiency was calculated as ratio of virion associated versus total cell plus virion associated HIV-1 Gag or WNV E protein.The protocol for radiolabeling and immunoprecipitation of cell and virus lysates has been described in detail previously . BrieflyThe overall strategy for this assay is summarized in Figure Selected Flavivirus proteins were downloaded from NCBI . The NCBhttp://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=11082.The strain designations as listed in the alignment were taken from the NCBI taxonomy on West Nile viruses: Several of these strains like Sarafend belong to the pathogenic lineage 2. These are: West Nile virus H442, West Nile virus SA381/00, West Nile virus SA93/01, West Nile virus SPU116/89. Please note that the Kunjin virus has been recognized as WNV strain which is also visible by the identical sequences in the 2 displayed patterns.HIV: Human immunodeficiency virus type 1; WNV: West Nile virus; EIAV: Equine infectious anemia virus; JEV: Japanese encephalitis virus; YFV: Yellow fever virus; SLE: St. Louis encephalitis virus; C: Capsid; prM: Pre membrane; NS: Non-structural; VLPs: Virus like particles; ESCRT: Endosomal sorting complex required for transport; Tsg101: Tumor susceptibility growth factor 101; MVB: Multi vesicular bodies; ER: Endoplasmic reticulum.The authors declare they have no competing interests.HG and AJ designed the study, performed experiments, analyzed data and wrote the manuscript. RL, OT and SM performed sequence analysis, analyzed data and wrote the manuscript. All authors read and approved the final manuscript.


Severe fever with thrombocytopenia syndrome, which results in severe illness and has a high case-fatality rate, is caused by a novel bunyavirus, severe fever with thrombocytopenia syndrome virus. We found that samples from 2/237 (0.8%) healthy persons and 111/134 (83%) goats in Yiyuan County, Shandong Province, China, were seropositive for this virus. Severe fever with thrombocytopenia syndrome (SFTS) is a serious infectious disease with 12% case-fatality rate that has been documented in 6 provinces rural in northeast and central China. SFTS is caused by a novel bunyavirus, SFTS virus (SFTSV) , has an area of 1,636 kmMost farm families have a herd of goats, and some of them have dogs. We found that goats and dogs in this county were heavily infested with ticks, with several hundred ticks found on each goat and dog. A recent serosurvey of domestic animals in Jiangsu Province found an SFTSV antibody positive rate of 57% in goats, 32% in cattle, 6% in dogs, 5% in pigs, and 1% in chickens ; 150 (63%) were female. No volunteer was <20 or >81 years of age. The age and sex distribution of the study population may have resulted from the migration of men and young people from rural areas to cities or lower participation among older and younger person.Serum samples were tested for total antibodies (IgG and IgM) to SFTSV by using a double-antigen sandwich ELISA kit, provided by Jiangsu Province Center for Disease Control and Prevention (>2.1× the absorbance of the negative control (provided by the manufacturer), which was 3 SD above the mean optical density at 450 nm for the persons sampled. The ELISA had similar specificity and sensitivity to the microneutralization assay and exhibited no cross-reactivity with hantavirus or dengue virus antibodies . The goat serum samples were tested for total antibodies to SFTSV by using the double-antigen sandwich ELISA kit as described human samples. Of the 134 goats sampled, 111 (83%) had antibodies to SFTSV according to the established cutoff of  all >32 .We found a high seroprevalence of SFTSV among goats in Yiyuan County but a low seroprevalence among humans. The seropositive persons were from 2 different villages and were not linked to each other; only 1 was involved in herding goats and neither in milking or butchering. Thus, the exposure route was not clear. Neither of the seropositive persons reported clinical illness; however, this finding is limited by the possibility of recall bias.A previous study of 200 serum samples collected from SFTSV-endemic areas showed no subclinical infection with SFTSV (


Cucumovirus 2b protein inhibits the systemic movement of RNA silencing by either binding dsRNA/siRNA or inhibiting the slicer activity of AGO1. Potato virus X P25 protein also inhibits the systemic movement of RNA silencing , a specialized evolutionarily conserved gene module involved in the movement of viruses. The TGB-based transport system exploits the co-ordinated action of three polypeptides to deliver viral genomes to plasmodesmata (PD) and to accomplish virus entry into neighboring cells. TGB-encoded proteins are referred to as TGB1, TGB2, and TGB3 , which also exploit this protein as silencing suppressor . Furthermore, the molecular masses of TGB1 in filamentous viruses range from 24 to 26 kDa and the NTPase/helicase domain comprises the entire sequence, whereas TGBp1s of rod-shaped viruses are substantially larger – from 39 to 63 kDa – and contain the additional long N-terminal domains  which codes for polymerase related to that of family Virgaviridae (Melzer et al., Benyvirus representing a distinct group within the alpha-like supergroup of positive-strand RNA viruses (Koonin and Dolja, Further evolution of such virus genomes may result in origination of the TGB. The main driving force of this evolutionary step could be dependence of helicase activity on the membrane binding. Indeed, positive-sense RNA viruses are known to exploit host cell membranes to facilitate RNA replication. Formation of these replication sites often involves virus-induced membrane synthesis, changes in fatty acid metabolism, and viral recruitment of cellular factors to membrane subcellular domains serving as a physical scaffold for replication complexes (Sasvari and Nagy, n Figure . Moreoven Figure . Althougn Figure . The TGBn Figure . Thus, tn Figure . This isIn conclusion, new data on interrelation of RNA silencing and virus movement and, on the other hand, recently published sequences of new TGB-containing viruses allowed us to put forward the hypothesis of a three-step TGB origin in virus evolution. These steps include autonomization of a second virus RNA helicase initially possessing the function of silencing suppression, gaining the virus movement function by this protein, and acquisition of accessory membrane proteins.