In contrast, when release is slower than ∼0.1 h−1, the simple MM accurately estimates NAI efficacy, but it can significantly overestimate the infectious lifespan , and it will significantly underestimate the total virus yield and thus the likelihood of resistance emergence. We discuss the properties of, and a possible lower bound for, the influenza A virus release rate.Mathematical models (MMs) have been used to study the kinetics of influenza A virus infections under antiviral therapy, and to characterize the efficacy of antivirals such as neuraminidase inhibitors (NAIs). NAIs prevent viral neuraminidase from cleaving sialic acid receptors that bind virus progeny to the surface of infected cells, thereby inhibiting their release, suppressing infection spread. When used to study treatment with NAIs, MMs represent viral release implicitly as part of viral replication. Consequently, NAIs in such MMs do not act specifically and exclusively on virus release. We compared a MM with an explicit representation of viral release  to a simple MM without explicit release, and investigated whether parameter estimation and the estimation of NAI efficacy were affected by the use of a simple MM. Since the release rate of influenza A virus is not well-known, a broad range of release rates were considered. If the virus release rate is greater than ∼0.1 h There are two main classes of antiviral drugs available for the treatment of influenza A virus infection: adamantanes, such as amantadine and rimantadine, and neuraminidase inhibitors (NAIs), such as oseltamivir, zanamivir, laninamivir, and peramivir. In 2005–2006, however, resistance to adamantanes dramatically increased , and theNAIs reduce the spread of influenza A virus to uninfected cells by blocking the release of progeny virus produced by infected cells. At this late step in the viral replication cycle, mature virions protrude and pinch off from the apical surface of the infected cell, co-opting the cell’s plasma membrane as their own envelope, but can remain affixed atop the cell surface. Both the virion and the cell surface, which is destined to become the virion’s outer surface, are studded with the viral proteins neuraminidase (NA) and hemagglutinin (HA), as well as the cell’s sialic acid receptors. Throughout the duration of the infection, increasing amounts of NA are expressed on the cell surface, which cleave sialic acid receptors. As the density of sialic acid receptors declines, newly budded virions are less likely to remain cell-bound due to the formation of virus-cell attachments when HA binds to the sialic acid receptors upon exit. We will refer to the transition from cell-associated, bound virus into free virus that is facilitated by NA cleavage of sialic acid cell receptors as “virus release”, though other modes of virus release might exist –6.As reviewed in , a simplIf virus release plays a significant role in the unfolding infection kinetics, the use of a MM without explicit virus release, like the simple MM, could affect the estimation of infection parameters, with broad implications. For instance, an estimate of the virus production rate can shape our perspective on the likelihood of the emergence of drug resistance –14. In aThe importance of quantifying the infection parameters from time course data of an in vitro infection with a MM has been discussed . This apThe challenge to including virus release explicitly in a MM is that the influenza A virus release rate is not well-known. From a mathematical modelling standpoint, the addition of an explicit release rate to the simple MM will result in overparameterization, such that at least some parameters would suffer with identifiability issues. To overcome this challenge, our study considers a range of possible values of the release rate of influenza A virus, and compares the simple MM against a variation of it that includes an explicit term for virus release . We idenr. Due to this small modification, the MMs have different views of virus infection, and some parameters in the two MMs require a slightly different interpretation. For example, in the simple MM, the eclipse length, τE, represents the time it takes for an infected cell to replicate virus and release it. In contrast, in the release MM, the eclipse length only represents the time it takes for the infected cell to replicate virus, but the time to viral release is handled separately with parameter r. The interpretation of the virus production rate, p, in either MM also differs: whereas in the release MM, a fraction of the virions produced will never be released if the release rate is low, in the simple MM, all produced virions are released. Thus, in the simple MM, the effects of virus release can be absorbed by either or both the virus production rate and eclipse length.We compared a simple MM (without explicit virus release) to a MM with explicit virus release . As described in the Methods, the release MM is an augmented version of the simple MM with one additional parameter, the virus release rate Vb) being released at rate r as free infectious virus (V) into the medium. When we refer to bound virus, we mean infectious virions that are still attached to the virion-producing cell, i.e., cell-associated infectious virus. Experimentally, bound virus is not measurable in a typical cell culture experiment where only the supernatant is removed and titrated for infectious virus. On the other hand, free virus here refers to infectious virus that has successfully been released from virus-producing cells into the supernatant, and corresponds to the population of virus that is typically quantified in an experiment.Another difference from the simple MM, is that the release MM explicitly describes cell-associated, bound infectious virus  before they are released, resulting in a decrease in the bound virus titer. A balance between these two processes results in the largest bound virus titer peak value.The release MM also predicts the effect of virus release on the kinetics of bound virus. In ters see . When thrf. In such a case where virus release plays a significant role in virus infections, release must be explicitly accounted for in the MM.Our results show that virus release can act as a kinetic bottleneck to significantly suppress free virus growth if the true rate of influenza A virus release is less than the critical free virus release rate, Most MMs to date have represented the virus release as an implicit part of virus replication. Here, we are interested in how parameter estimation using the simple MM compares to that using the release MM over a wide range of possible release rates.−5 infectious virus/cell, while the SC assay was performed at a MOI of 4 infectious virus/cell. In the MY assay, the virus inoculum is incubated in the absence of cells and sampled at multiple times, exhibiting the rate of loss of infectious virions . We have previously shown that a full suite of in vitro experiments—single-cycle (SC), multiple-cycle (MC), mock-yield (MY)—is needed to extract and identify the infection parameters  in the simple MM –22. BrieFor the release MM, the release rate was fixed, and the remaining five MM parameters were fitted using the simulated free virus titer from the simple MM as the data to fit (red). A set of fitted parameters was obtained for each value of the release rate explored. The release MM can reproduce the simple MM’s infection kinetics with a unique set of infection parameters, for any given value of the release rate. This shows how the parameter estimates shift under different assumptions of the release rate.r. If free virus release is rapid and effective (r > rf), the simple MM returns the same parameter estimates as predicted by the release MM, and use of the simple MM is appropriate in this circumstance. If free virus release is slow and inefficient (r < rf), the simple MM no longer correctly estimates the infection parameters.In particular, τE obtained from the fits. In r > rf), the release MM ascribes the full delay to the eclipse length which represents the viral replication time. On the other hand, if virus release is assumed to be slow (r < rf), the release MM ascribes only part of the delay to the eclipse length , while the rest is ascribed to the time required to release virus. Consequently, as the release rate decreases, less and less of the delay is ascribed to virus replication (the estimated eclipse length decreases) while the time required to release virions increases more and more. This highlights the release MM’s ability to ascribe the delay discriminately to each process, i.e., separately to virus replication and release. In the simple MM, the full delay is ascribed to the eclipse length, since parameter τE accounts for both the viral replication time and release time. Though it appears that the simple MM overestimates the eclipse length  deviates from the simple MM’s estimate in p requires a different interpretation in each MM because it represents different processes. In the simple MM, p represents the rate at which free infectious virus is produced and (instantaneously) released into the medium by an infectious cell, but in the release MM it represents the rate at which cell-associated, bound virions are produced onto the cell’s surface. If the release of these cell bound virions is slow, some will lose infectivity (at rate c) before they can be released into the medium, resulting in an effective rate of free virus production into the medium which could be much less than the rate at which cell-associated bound virus are produced onto the cell’s surface. It is easy to show (see r/(r + c) of all virions produced onto the cell’s surface will ultimately be released as free virions. This term also naturally appears in the equations for the basic reproductive number (peff = p ⋅ r/(r + c), rather than the rate of bound virus production, p, in the release MM which should be compared to the free virus production rate p which appears in the simple MM.As the virus release rate is decreased in the release MM, the production rate (show see  that onle number  and the e number . Therefoprelease = psimple(r + c)/r = (6.28 × 108(PFU/mL) ⋅ h−1)(r + c)/r. From the fitted curves in p. As the release rate decreases, the fits cannot capture the steep upslope of the SC titer. The fits performed without a constrained production rate yields the lowest Akaike information criterion , the simple MM estimates the same parameters as predicted by the release MM. When virus release is slow (r < rf), the simple MM estimates the same effective production rate as predicted by the release MM in either analysis. For the remaining parameters , the simple MM either: (1) overestimates them compared to the predictions by the release MM if there is no constraint on p; or, (2) estimates the same values as predicted by the release MM with p constrained, except for the eclipse length due to the differing definitions between the two MMs.ε, which ranges between 0 (no inhibition) and 1 . In the simple MM, virus release is modelled implicitly as a part of the virus production rate p, so the action of NAIs is represented as inhibiting the rate of virus production, i.e., (1 − εp)p [εr)r. The subscript on the symbol for efficacy denotes the infection parameter upon which the drug acts.We used the simple and release MMs to simulate infections in the absence (untreated) and presence (treated) of NAIs, and compared the two MM’s predictions for the effect of NAIs on free virus titer. In both MMs, the NAI efficacy is expressed as a fractional inhibition, 1 − εp)p –11. In tr = 0.01 h−1), r = 10 h−1), the simple MM predicts that NAI therapy results in a greater reduction and delay in the free virus peak titer than that predicted by the release MM for an equal efficacy  from the fitted parameters. The critical efficacy is defined as the minimum efficacy required to cause the suppression of infection, i.e., reduce R0 below its threshold value of 1. εcrit) from the simple MM (εcrit), indicating that a lower efficacy of NAIs is needed to achieve the total suppression of infection than predicted by the release MM. From this, we can see that the simple MM underestimates NAI efficacy if virus release is fast, where the error in estimation is largest as the virus release rate increases. Interestingly, the simple MM predicts NAI efficacies similar to those predicted by the release MM if release is slow. This is in contrast to the finding that the simple MM predicts the same estimates of the infection parameters as the release MM only if virus release is rapid.For a comparison of the differential effect of NAIs in either MM over the entire range of release rates considered, we used a proxy to concisely represent the above information. We calculated the critical efficacy (imple MM  and the imple MM  as a funp) to those that inhibit virus release (acting on r). −1, both classes of antivirals at the same efficacy will have an equal effect on the infection. On the other hand, if the virus release rate is greater than 0.01 h−1, the release MM predicts that an antiviral inhibiting virus production requires a lower dose (is more effective) to achieve suppression of infection than an antiviral that inhibits virus release. Regardless of the true influenza A virus release rate, an antiviral acting to suppress viral replication/production is predicted to always suppress free virus titer to an extent equivalent to or greater than an antiviral inhibiting virus release.The release MM provides us with the opportunity to compare antivirals that inhibit virus production  describing the kinetics of influenza A virus infections in vitro and in vivo typically represent the release of cell-bound progeny virions from the surface of productively infected cell implicitly. Specifically, the rate of virus release gets incorporated into aggregate, generic parameters which stand in for the combined kinetics of virus replication, assembly, budding, and release. Importantly, when these simple MMs are used to characterize treatment of influenza A virus infections with NAIs –11, theybound virions being released into the medium as free virions at release rate r. Unfortunately, the rate at which influenza A virus progeny is released from the surface of the cell that produces them is not well-known. Using the release MM, we explored a wide range of virus release rates to assess the impact of this additional, unknown MM parameter on infection parameter estimates, and in particular the estimation of NAI efficacy, compared to those obtained with the simple MM.Herein we developed a variant of the simple MM, hereafter the release MM, that includes an explicit description of virus release. The release MM accounts for two distinct populations of infectious virus, bound and free virions, with cell-associated, −1. If virus release is more efficient than this, release does not significantly affect infection kinetics or free virus yield, making the simple MM a valid approximation of the release MM. If virus release is less efficient than this, it will affect infection kinetics and free virus yield. In fact, if virus release is slower than ∼0.1 h−1, the two MMs statistically significantly disagree in their estimates of some infection parameters, specifically the virus production rate, the eclipse length, and the lifespan of infectious cells . This is because although the simple and release MMs share most of the same structure, the implementation of implicit versus explicit virus release imparts onto each MM a different interpretation of the infection parameters they have in common.Mathematically, in the absence of antiviral therapy, the release MM reduces to the simple MM if the virus release rate is sufficiently large. Our work shows that for realistic parameter values in the context of an influenza A virus infection in vitro, this occurs when the virus release rate is greater than ∼4 hr/(r + c) of the bound virions produced onto the cell’s surface are ultimately released into the medium as free virions, wherein r is the virus release rate and c is the rate at which virions lose infectivity. If r is large relative to c, r/(r + c) ≈ 1, and all produced virions are released as free virions. In fact, we found that the free virus production rate (p) estimated by the simple MM agrees with the effective free virus production rate (p ⋅ r/(r + c)) estimated by the release MM. Furthermore, when we repeated parameter estimation while specifically imposing the constraint that psimple = preleaser/(r + c), the two MMs agreed in their estimation of all parameters except for the length of the eclipse phase. While the eclipse phase length in the simple MM measures the delay from the infection of a cell until free virus release, in the release MM it measures the time elapsed between infection of the cell and the appearance of progeny virions bound to the infected cell’s surface, specifically excluding the time required for their release as free virus. For a given experimentally observed delay in the appearance of free virions, the simple MM can only ascribe it to the eclipse length whereas the release MM must distribute its duration between the eclipse length and release rate. This should be kept in mind when interpreting estimates of the eclipse phase length obtained using the simple MM.Biologically, if the release of virions into the medium is slow such that the release rather than the production of virus is a kinetic bottleneck, a portion of the bound virions produced onto the cell’s surface will lose infectivity before they can be released as free virions into the medium. For the release MM, we show that only a fraction r < c) such that release rather than production limits free virus yield, the two MMs predict the same infection kinetics for the same NAI efficacy. In the release MM, if virus release is rapid such that virus production rather than release is the bottleneck, imposing the limit on free virus yield (r ≫ c), even a large reduction in the release rate has a modest impact on virus yield until it is reduced to the point where r ≈ c. For this reason, if virus release is rapid, NAIs in the release MM have a weaker suppressive effect than in the simple MM for the same efficacy. In other words, for a given experimentally observed reduction in free viral yield, the simple MM will underestimate the true NAI efficacy required to effect this reduction. This error increases with increasing virus release rates. Underestimating antiviral efficacy in turn, leads to underestimating the NAI dosage required to achieve a specific therapeutic outcome, e.g., suppression of infection. Underdosing prolongs the overall duration of infection and the number of genomes produced, which could increase the likelihood of developing resistance to NAIs. In Japan, NAI-resistant A/H3N2 strains were detected from children who received oseltamivir, and underdosing had been thought to be a contributing factor [When it comes to representing the mode of action of NAIs, the simple MM is restricted to inhibiting the aggregate free virus production rate, whereas the explicit MM can specifically inhibit the rate of virus release. If virus release is slow  virus interacting with uninfected MDCK cells. Since there are more receptors on uninfected cells, we expect that the true virus release rate would be greater than that found by Nunes-Correia et al. However, the study by Nunes-Correia et al. was performed on MDCK cells which express far fewer α-2,6 cell receptors than that found on the surface of epithelial cells lining the human respiratory tract (RT) [α-2,6 on MDCK cells, these rates likely overestimate the biologically relevant detachment rates and therefore cannot be thought of as a true lower bound. In [−1 and as high as 11 h−1. However, these were estimated with different influenza A viruses interacting in non-cell systems  virus interacting with bovine brain lipid membrane, or influenza A (H3N2) and duck influenza A (H5N3) virus strains binding to gangliosides) which might not reflect the cell receptor composition of an infected human RT epithelial cell.Our results demonstrate the need to estimate the rate of influenza A virus release in order to correctly interpret results obtained from the simple MM. However, directly measuring the virus release rate is difficult for a number of reasons. An uninfected cell will express a number of sialic acid receptors on its surface. After successful infection by an influenza A virus, virus replication gets underway and ∼1 h–3 h post-infection, the cell begins to express increasing levels of viral NA on its surface , 26. Theact (RT) . Due to ound. In , other k−1. This is consistent with the observation that favipiravir, a new antiviral which inhibits viral polymerase, is more potent and effective than NAIs [−1. In our view, the best way to obtain an estimated lower bound for the release rates, would be to repeat the attachment/detachment study performed by Nunes-Correia et al. using MDCKα2,6 cells which express α-2,6 sialic acid cell receptors at levels similar to those found on human RT epithelial cells.Our release MM predicts that at equal efficacy, treatment with an antiviral against virus production suppresses free virus yield to a greater extent than an antiviral against release if the release rate is greater than 0.01 hhan NAIs  , which could provide an accurate, independent estimate of the virus release rate. It remains to be determined whether or not viral release acts as a kinetic bottleneck in influenza A virus replication.r, and one additional equation to distinguish cell-associated bound virus from released free virus. The equations common to both MMs describes the flow of cells through the various phases of infection as followsT, are infected at infection rate β times the concentration of free, infectious virus, V (in PFU/mL). Newly infected cells enter the eclipse phase, τE and τI have elapsed, respectively. The eclipse (or infectious) phase is divided into nE (or nI) compartments such that the time spent by cells in the phase follows an Erlang distribution [Influenza A virus infections in vitro were numerically simulated with a multi-compartment ordinary differential equation (ODE) model as previously described and validated in –22. Throribution , 31, 32.V is produced directly into the medium by infectious cells (I) at a rate p, and lose infectivity at rate c. In the release MM, cell-bound virus Vb is produced onto the surface of infectious cells at a rate p, and also lose infectivity at rate c while bound to the cell. The infectious bound virus is released from the cell surface at rate r and enters the medium as free virus, which also lose infectivity at rate c. The release MM distinguishes virus release from any other processes that are encompassed by the virus production rate. Our base parameter values for the simple MM are listed in The two MMs differ only in their equations for infectious virus, wherein the release MM includes one additional variable and one new parameter to explicitly account for the release of cell-bound virus as free virusR0) and the infecting time (tinf), can be derived from the MM parameters. The infecting time represents the time it takes for one infectious, virus-producing cell to infect one other cell in a fully susceptible population of cells. It has been described in [p and β, because of the undetermined, relative relationship between infectious virus and experimental measures of virus infectivity such as 50% infectious dose  or plaque-forming unit (PFU) [Two important secondary parameters, the basic reproductive number (ribed in –33 and iit (PFU) , 34.R0 is less than the threshold value of 1, the spread of infection is suppressed. The basic reproductive number for each MM is given byThe basic reproductive number is another commonly reported quantity and it represents the number of secondary infections caused by one infectious cell in a population of fully susceptible cells. If Vb(t) into the differential equation describing free virus yields:Assuming that the fraction of productively infectious cells is unchanged over a certain timescale of interest (tinf) will deviate from the intended physical meaning for release rates that are less than 0.1 h−1. This is because the expression for tinf was originally derived under the assumption that the rate of loss of virion (approximately 0.1 h−1) could be neglected.We use the effective production rate to correct the expression of the infecting time. This corrected parameter is called the effective infecting time,ε was applied to either the production rate in the simple MM, as (1 − εp)p, or to the release rate in the release MM, as (1 − εr)r. The efficacy (εp or εr) takes on values between 0 (no efficacy) and 1 (maximum efficacy), where a more efficacious drug leads to greater reduction of either the virus production or release rate. Treatment was always applied at the start of infection (t = 0 h).When infections were treated with a neuraminidase inhibitor (NAI), the drug efficacy In each MM, treatment with NAIs is given by:εcrit) as the minimum efficacy required to reduce the basic reproductive number below 1. Re-arranging the above when R0 = 1 yields the following expressions for critical efficacies in the simple MMWe define the critical efficacy  that were infected with concentrations of the initial inoculum according to −5 PFU/cell and 4 PFU/cell, respectively. The MY assay was initiated with an initial virus inoculum of V0 = 108 PFU/cell in the absence of cells (T = 0). The free virus titer from this suite of in vitro assays served as our simulated data with which the release MM was fitted.The multiple-cycle (MC), single-cycle (SC), and mock-yield (MY) in vitro assays in Figs In nelder_mead_min from version 1.5.2 of the optim package in Octave 4.0.3). The fits minimized the combined sum-of-squared residuals per point (SSR/pt):i is the number of points of simulated free virus from the simple MM  [Simultaneous fits of the release MM to the SC, MC, and MY viral yield simulated datasets were performed with the Nelder-Mead minimization method (NMY = 4) .p, β, c, τE, τI were fitted. The remaining parameters nE, nI were kept at their base values  ⋅ h−1, enforcing the effective production rate in the release MM to be equal to the production rate in the simple MM. In this case, only four parameters were fitted  for each value of the release rate that was explored. The resultant sets of fitted parameters are shown in In another analysis, a constraint was imposed on the release MM’s virus production rate Npts = 307 is the number of simulated data points and Npar is the number of parameters fitted. In the analysis without a constraint on the virus production rate Npar = 5, and in the analysis with the constraint Npar = 4.In εp = 0.99. With the constraint on virus production (Section), the release MM was fitted to the simple MM’s untreated free virus titer, and recapitulated these kinetics with a different set of parameters for each chosen release rate, r = 0.01 h−1, 10 h−1. Using these sets of parameters, the treated infections in the release MM were simulated with NAIs applied at an efficacy of εr = 0.99 at the start of infection.To illustrate that NAIs applied at identical efficacies in both MMs had a different effect on the free virus titer, we showed a MC infection in the absence of NAIs (untreated) and in the presence of NAIs (treated), as simulated by either MM in In −1. The corresponding treated infections were simulated by applying an antiviral against production (solid) or release (dashed) at an efficacy of 0.999 at the start of infection. −1) was used to simulate the infections.In S1 Supporting Information(PDF)Click here for additional data file.S1 Figrf = 3.72 h−1 , corresponds to the variance of a mock-yield infection (dotted line). We also show the SSR/pt curve for various rates of loss of infectious virion that were explored (various colours). (Right) The peak value of bound virus titer as a function of the release rate is shown (black), where the maximum determines the critical bound virus release rate, rb = 3 × 10−3 h−1 (purple circle). The various coloured lines correspond to various rates of loss of virion infectivity to show that rb (dotted purple) strongly depends on other infection parameters.(Left) The sum-of-squared residuals per point (SSR/pt) is computed between each free virus curve in the release MM, as the release rate is varied, and the simulated MC data in the simple MM (black). The critical free virus release rate, (TIF)Click here for additional data file.S2 Figrf, weakly depends on the rate of loss of virion infectivity, production rate, infection rate, eclipse phase and infectious lifespan. The rf when all parameters are at their base values is indicated with a horizontal dotted line.The critical free virus release rate, (TIF)Click here for additional data file.S3 Figrb, depends on the rate of loss of virion infectivity, production rate, infection rate, eclipse phase, and infectious lifespan. The rb when all parameters are at their base values is indicated with a black circle.The critical bound virus release rate, (TIF)Click here for additional data file.S4 FigAs in (TIF)Click here for additional data file.


Whereas the protein composition and overall shape of several giant virus capsids have been described, the mechanism by which these large capsids assemble remains enigmatic. Here, we present a reconstruction of the capsid of Cafeteria roenbergensis virus (CroV), one of the largest viruses analyzed by cryo-electron microscopy (cryo-EM) to date. The CroV capsid has a diameter of 3,000 Å and a Triangulation number of 499. Unlike related mimiviruses, the CroV capsid is not decorated with glycosylated surface fibers, but features 30 Å-long surface protrusions that are formed by loops of the major capsid protein. Based on the orientation of capsomers in the cryo-EM reconstruction, we propose that the capsids of CroV and related giant viruses are assembled by a newly conceived assembly pathway that initiates at a five-fold vertex and continuously proceeds outwards in a spiraling fashion. However, whereas rapidly advancing DNA sequencing methods facilitate genomic analysis of giant viruses, structural studies of large viral capsids are lagging behind. In contrast to small and medium-sized capsids, the >200 nm isometric capsids of giant DNA viruses still pose a significant technical challenge for high-resolution methods such as X-ray crystallography and cryo-EM12. Other techniques, including atomic force microscopy, scanning electron microscopy, and X-ray free electron lasers, have been used to study giant virus structures, but are unable to provide near-atomic resolution16. During the last decade, cryo-EM has become an increasingly powerful tool to determine the structure of virus particles, circumventing the need for crystallization20. The resolution of cryo-EM reconstruction of viruses has gradually improved from sub-nanometer to near atomic levels30. In addition, combined with X-ray crystallography of purified capsid proteins, it is possible to fit the atomic structures of individual components into the cryo-EM reconstruction map and determine a pseudo-atomic structure34. Examples for the successful combination of X-ray crystallography and cryo-EM to determine large DNA virus structures are Paramecium bursaria Chlorella virus 1 (PBCV-1)36 and Chilo iridescent virus (CIV)37.Viruses with long dsDNA genomes (>200 kilobases) and large particles (>0.2 µm) are a common occurrence in nature and several new families of giant viruses have been reported in recent years38. The giant virus CroV infects the widespread marine zooplankter Cafeteria roenbergensis, a single-celled eukaryote and ecologically important bacterivore39. Phylogenetically, CroV is a distant relative of the giant Acanthamoeba polyphaga mimivirus (APMV)1 and the sole member of the genus Cafeteriavirus in the family Mimiviridae. With a diameter of 3000 Å, the CroV particle is smaller than the 5000 Å wide capsid of APMV40 or the 1.5 × 0.5 µm ovoid particles of Pithovirus sibericum4, which renders it more easily accessible to cryo-EM. Nevertheless, obtaining a close-to-nanometer cryo-EM resolution for an intact virus capsid of these dimensions is still problematic. The thickness of the vitreous ice that embeds the viral capsid in cryo-EM is one of the most important factors limiting the resolution. Multiple and inelastic scattering of electrons increases as the ice becomes thicker, which reduces the signal-to-noise ratio. Unlike APMV, the CroV capsid is not covered by a dense layer of 125 nm-long surface fibers that would complicate cryo-EM analysis41. Thus, CroV is ideally suited to advance the limit of structural studies of giant viruses. Although crystallization is not required for cryo-EM, thousands of homogenous particles need to be imaged and analyzed to achieve a high resolution structure. In this study, we observed that the CroV particles were homogenous and could be averaged to high resolution. Such detailed structural information of an intact giant virus capsid may help to shed light on their assembly mechanism. Based on our cryo-EM reconstruction of the CroV capsid and by comparison with other giant viruses, we propose a new spiral assembly pathway for the formation of large icosahedral virus capsids.Here, we push the limits of cryo-EM application to large virus particles by reconstructing the capsid of Cafeteria roenbergensis virus (CroV)Cafeteria roenbergensis culture, we obtained enough CroV sample for cryo-EM data collection  exist in one icosahedral asymmetric unit. By tracing the capsomers in the hexagonal array from one 5-fold vertex to the neighboring one along axes h and k, which follow the center of the MCP   Fig. . Previou45. The CIV virion has short fibers on most capsomers37, while PBCV-1 and Phaeocystis pouchetti virus (PpV01) capsids have fibers only on certain capsomers46. In contrast, the CroV capsid is not decorated with fibers , and the biggest trisymmetron consisting of 231 capsomers. Based on the orientation of capsomers around 5-fold axes, we propose that the capsids of CroV and related icosahedral giant viruses are assembled in a spiral mechanism, rather than from preformed MCP arrays, as has been generally accepted for giant icosahedral viruses. It is noteworthy that our assembly model is based on the observation that MCP capsomers are oriented in an identical fashion around the pentasymmetron in several giant icosahedral viruses. Although our model lacks direct experimental support, it is consistent with the continuous capsid assembly observed in cryo-tomography studies52. CroV samples were loaded on Quantifoil S7/2 grids , blotted manually and frozen using a guillotine style plunging device. The cryo-EM images were recorded on Kodak SO-163 film in a FEI CM300 field emission gun microscope at a calibrated magnification of 20,629 and a dose level of approximately 25 e-/Å2 at the Purdue University Cryo-EM facility. Micrographs were scanned on a Nikon Coolscan 9000 with a final pixel size of 6.156 Å. The cryo-EM reconstruction was calculated using the program FREALIGN53. Most computation was performed at Texas Advanced Computing Center (TACC). Resolution of the reconstruction was determined using Fourier Shell correlation (FSC) for the capsid part with threshold of 0.333.CroV was grown and purified as previously describedAll data associated with the manuscript are available to readers on request.Supplementary s01Supplementary s02


Aedes albopictus as a potentially invasive species.Zika virus is an emerging pathogen of global importance. It has been responsible for recent outbreaks in the Americas and in the Pacific region. This study assessed five different mosquito species from the temperate climatic zone in Australia and included 50) for CPE positive samples. Additionally, the presence of Wolbachia endosymbiont infection was assessed by qPCR and standard PCR.Mosquitoes were orally challenged by membrane feeding with Zika virus strain of Cambodia 2010 origin, belonging to the Asian clade. Virus infection and dissemination were assessed by quantitative PCR on midgut and carcass after dissection. Transmission was assessed by determination of cytopathogenic effect of saliva (CPE) on Vero cells, followed by determination of 50% tissue culture infectious dose (TCIDCulex mosquitoes were found unable to present Zika virus in saliva, as demonstrated by molecular as well as virological methods. Aedes aegypti, was used as a positive control for Zika infection and showed a high level of virus infection, dissemination and transmission. Local Aedes species, Ae. notoscriptus and, to a lesser degree, Ae. camptorhynchus were found to expel virus in their saliva and contained viral nucleic acid within the midgut. Molecular assessment identified low or no dissemination for these species, possibly due to low virus loads. Ae. albopictus from Torres Strait islands origin was shown as an efficient vector. Cx quinquefasciatus was shown to harbour Wolbachia endosymbionts at high prevalence, whilst no Wolbachia was found in Cx annulirostris. The Australian Ae. albopictus population was shown to harbour Wolbachia at high frequency.Aedes species triggering large Zika epidemics in the southern parts of Australia is low. The potentially invasive Ae. albopictus showed high prevalence of virus in the saliva and constitutes a potential threat if this mosquito species becomes established in mainland Australia. Complete risk analysis of Zika transmission in the temperate zone would require an assessment of the impact of temperature on Zika virus replication within local and invasive mosquito species.The risk of local  Aedes mosquitoes, and primarily Aedes (Stegomyia) africanus, was suspected as the main sylvatic vector following direct isolation of Zika virus in 1956 [Stegomyia subgenus showed vector competence such as Aedes aegypti, an important vector in Yellow Fever transmission. Aedes (St.) luteocephalus has also been incriminated in Nigeria [Ae. aegypti in Malaysia [Aedes (St.) albopictus whose local presence and assumed role in rural dengue transmission made it another possible candidate. More recently, populations of Ae. aegypti [Ae. albopictus [Ae. (St.) hensilli, instead of Ae. aegypti was suspected of being responsible for the outbreak, and was shown experimentally capable of infection and dissemination [Aedes (St.) polynesiensis was found not able to transmit Zika virus [Ae. aegypti was shown to be a vector, both by molecular detection in field-collected mosquitoes [Culex quinquefasciatus, with conflicting results which either showed it as an efficient experimental vector [Zika virus was first isolated in Uganda in 1947 from a febrile rhesus monkey.  in 1956  and West in 1956  demonstrbopictus  in Singabopictus , then Frbopictus  in 2014.mination . In Frenka virus . In 2014squitoes . The roll vector  or not [l vector , 18, 19.Ae. albopictus is exotic to the mainland and with Ae. aegypti’s limited distribution, the dengue risk is limited to the Northern tropical regions. Therefore, local populations of Ae. aegypti and potential tropical vectors from Queensland, such as Ae. (Ochlerotatus) vigilax, Ae. (Rampamyia) notoscriptus, and Culex. quinquefasciatus, have been tested for Zika virus vector competence [Ae. aegypti was confirmed as the main suspected vector species, with no other local major or associated species demonstrating virus transmission or considered to play any role. Much of Australia is out of the current dengue risk zone, however the temperate southern zones do harbour dense urban populations and can experience warm summer temperatures. The climatic conditions of these zones is close to those of Southern and Western parts of Europe [Aedes camptorhynchus, or populations of Aedes notoscriptus and Culex annulirostris have been implicated in the circulation of these viruses. Incursion of invasive species, such as Ae. albopictus has been detected in the state of Victoria and successfully controlled [Culex quinquefasciatus has been well established in Victoria and can be captured during summer. Similar to European researchers who have begun to worry about local Zika transmission [Wolbachia, infection is known to potentially impact on virus infection in insects [Wolbachia infection screening for tested mosquito populations. To get a full view of the Zika risk assessment for temperate zones of Australia, we have included Ae. albopictus collected from the top northern Torres Strait Island as a potentially invasive species to mainland Australia.With a lack of approved vaccines and antivirals, vector control is a key measure for decreasing the public health burden and risk for Zika virus. Identifying vectors and understanding the transmission mechanism is the first step in designing the best suited vector control policy. Despite the presence of Zika virus in the Pacific region (Micronesia and Polynesia), and that, early in the outbreak, travellers returning to Australia have been found to carry the virus , no locampetence . From thf Europe . Endemicntrolled . Culex qsmission –27, in o insects , we have- Aedes (Och.) camptorhynchus samples were collected as larvae in coastal Victorian region of Gippsland (Wellington)  Fig. . Followi- Aedes (Ram.) notoscriptus samples were collected as larvae in the Bellarine (Geelong - Highton), and the Melbourne regions  conditions in the insectary at the Australian Animal Health Laboratory.KU955593) [Ae. aegypti, Ae. albopictus, Ae. camptorhynchus, Ae. notoscriptus, Cx quinquefasciatus, and Cx annulirostris. It belongs to the Asian/Pacific/American clade [Cambodia 2010 (Genbank U955593)  Zika viran clade  and was 50 105.6/mL) through membrane feeding using chicken blood and skin. Uninfected chicken blood and skin were provided by the Small Animal Facility  from chicken bred in the laboratory without any arboviral infection. The procedure was conducted with approval from AAHL Animal Ethics Committee. The blood was spiked with Zika virus just before mosquito blood-feeding. For control, media supernatant was added to the blood before feeding. After one hour, the mosquitoes were anaesthetised with CO2 and blood fed females were sorted and kept in a 200 mL cardboard cup at 27.5 °C, 65% humidity and 14:10 day:night photoperiod. The blood-fed specimens were kept for 14 day extrinsic incubation period with 10% sugar solution provided ad libitum.Five to eight days old females were starved the day before being challenged with an infected blood meal  was tested in duplicates on Vero cells (2 × 25 μL) at Day 5 post inoculation. For most samples showing CPE, the TCIDescribed .50 / mL. Negative control was a cDNA preparation from Vero cell culture infected with Chikungunya virus. Samples were considered positive if both duplicates had melting temperatures within the positive control range a CT value lower than 40.Molecular testing: After homogenisation of mosquito tissues by bead beating, RNA was extracted using either MagMax (Thermo Fisher) or RNeasy RNA isolation kit  as manufacturer’s protocol. Ten microliters of RNA was used to prepare cDNA using random hexamers and Superscript-III reverse transcriptase  as manufacturer’s protocol. A SYBR Green two step real-time PCR assay was designed for detection of Zika viral partial coding sequence (107 bp) of non-structural protein 5 (NS5) . Screening was conducted using the SYBR™ PreMIX Ex Taq™ II  and run on a QuantStudio™ 6 Flex Real Time PCR System (Applied Biosystems). Cycling conditions were as follows, 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 30s, before performing melting curve analysis. The theoretical melting point of a Zika positive sample was 85 °C. However the discriminant melting point with our positive control (Cambodia 2010 strain) was fixed to 80.5 + −0.5 °C. Dilution curve showed positive signal up to the fourth 10-fold dilution, indicative of a concentration of ~100 TCIDWolbachia was first assessed for all species except Ae. albopictus, using pools of 5 individual cDNA samples obtained from mosquito carcass of the same species. Screening was performed using a SYBR™ Green based quantitative PCR adaptation of the protocol from Mee et al. [Wolbachia surface protein (wsp) [The presence of e et al. . Positivin (wsp) .Wolbachia infection was identified by testing several individuals of each mosquito population, including Ae. albopictus with the two conventional 16S and wsp PCR assays.In a second step, prevalence of 50 values were respectively compared by Kruskall-Wallis two-tailed tests.Zika infection rate was defined by the number of midguts found positive for viral nucleic acid by qPCR. Similarly, the dissemination rate was calculated by the number of carcasses found positive by qPCR for viral nucleic. Transmission rate was defined by the number of saliva samples showing CPE in Vero cells over the number tested. These different rates were compared by Fisher exact two-tailed test. Average CT and TCIDVector competence of six mosquito species for transmission of Zika virus have been assessed.Ae. aegypti as the most efficient vector, with a high rate of midgut infection and dissemination and high virus prevalence and loads in saliva  was also tested for Zika virus competence and showed high prevalence (>75%) of virus in the saliva at day 14. The TCID50 of virus in the saliva of Ae. aegypti was found to be significantly higher than Ae. albopictus (p < 0.0001) and Ae. notoscriptus (p = 0.0002) pti Fig. .Fig. 2QuAe. notoscriptus and Ae. camptorhynchus, could be infected by Zika virus and deliver virus in their saliva (Table Ae. notoscriptus (42%) and Ae. camptorhynchus (13.5%) by assessing the cytopathogenic effect of the saliva on Vero cells. The prevalence of virus in the saliva was lower than that of Ae. aegypti (87%) , by qPCR. All samples were positive for the presence of Zika virus RNA, with low CT values  (data not shown).The results show that two local temperate Aedinae, va Table . The molrhynchus 3.5% by aCx quinquefasciatus, and Cx annulirostris. In addition, no midgut or carcass sample of any Culex species tested positive for viral nucleic acid 14 days post-infection using the Cambodia strain , our results showed moderate infection rates and low dissemination rate for these Aedes species. However, in contrast to Hall-Mendelin et al., our results showed presence of virus in the saliva of these mosquitoes’ Victorian populations: 42% and 13.5% of Ae. notoscriptus and Ae. camptorhynchus, respectively. The prevalence of virus in the saliva was lower than that of Ae. aegypti (87%) , as a relative measure of the initial virus load.The results of this study demonstrate that two local Aedinae of the temperate zone, n et al.  for Ae. aegypti 8%  cDNA shows a very low prevalence of Wolbachia for the temperate zone population of Ae. notoscriptus and absence of positive molecular signal on pools of 5 cDNAs by qPCR. In contrast, a high prevalence of Wolbachia natural infection has previously been described in tropical Ae. notoscriptus populations either from a colony or field samples collected in Brisbane [Wolbachia infection did not influence the dengue virus competence of infected Ae. notoscriptus populations [Wolbachia infection on Ae. notoscriptus for Zika virus infection and could justify further research. It is to be noted that, despite the distance, Ae. notoscriptus has been described as potentially invasive for USA [Aedes notoscriptus from Brisbane has been shown to transmit yellow fever virus better than Ae. aegypti [Ae. notoscriptus of unknown origin failed to develop infections with the same virus [Aedes notoscriptus from Brisbane area has been previously shown as a poor vector for the four serotypes of dengue virus [Ae. aegypti. The contact between human populations and Ae. notoscriptus has been documented in Queensland but not in temperate regions. Aedes notoscriptus has been shown to bite humans 19% of the time for blood meals in residential areas of Brisbane [Ae. aegypti and identify it as a potential vector in urban areas. Although it is known to be a backyard pest in the southern zones, much of its biology remains to be well defined in order to properly assess risk for transmission. The case of Ae. camptorhynchus is quite different: its ecology is restricted to coastal areas, or around inland brackish zones [Ae. notoscriptus, but to a lesser degree, Ae. camptorhynchus showed intermediate levels of vector competence for Zika virus. Both are probably not primary vectors, meaning they may be unable to sustain large outbreaks on their own, unlike Ae. aegypti in tropical areas, however they could be potential candidates to trigger few secondary cases and reinforce outbreaks in the presence of primary vectors. Aedes notoscriptus is known as a backyard pest, due to its abundance and taste for human blood. Aedes camptorhynchus has been shown to be responsible for Ross River virus outbreaks, here too displaying a close proximity with humans. However, a big gap in our knowledge remains: the experiments were performed at 27.5 °C. Whilst the state of Victoria can experience very high temperatures and heat waves, on average, summer temperatures are much lower than 25 °C. Performing vector competence experiments at lower temperatures would be useful in establishing a proper risk assessment for local transmission of Zika virus, both by local and potentially invasive species.Beyond the different methodological approaches, the different results between the vector competence assessments performed on two different populations of y et al.  hypothesBrisbane , with thBrisbane . Althougulations , our stu for USA . Aedes n aegypti , howeverme virus . Aedes nue virus . Indeed,Brisbane . Its disBrisbane  are simish zones , with a sh zones , with losh zones , and epish zones . This insh zones . Except Ae. albopictus, from Australian Torres Strait island, is a competent vector for Zika virus transmission. This is concordant with Wong et al. [Ae. albopictus from Singapore and used the dissection of salivary glands and virological method. Beside the methodological approach, with dissection of salivary gland instead of saliva collection, the main differences between the studies are the temperature of rearing (29 °C versus 27.5 °C) and the Zika virus strain . Aedes albopictus is considered as an invasive species with a large threat potential [Ae. albopictus [Ae. albopictus populations. With our results, this demonstrates that distant populations of Ae albopictus could show large difference of vector competence. Consequently the risk analysis for this species cannot be carried across different regions, even within comparable climate [Aedes albopictus population coming from Torres Trait Islands seems to harbour Wolbachia endosymbionts. The results do not show them protected from being infected and transmitting Zika virus. However, a more quantitative experimental assessment could answer this question more fully.Our results demonstrated that g et al. , who tesotential  at a glootential  and Austbopictus . Studiesbopictus  or Ameribopictus  Ae. albo climate  (Italy aCulex species are devoid of proof of potential vector competence for Zika virus. Conversely, the potential invader for inland Australia, Ae. albopictus, is shown to be a competent vector, with a potential threat at a similar level as found in Ae. albopictus population in Singapore and very different from European populations. Two native Aedes species show an intermediate level of vector competence, whereby they are probably unable to sustain large outbreaks themselves, but are potential candidates to trigger some secondary cases. A gap in our knowledge exists in lower temperature assessment of vector competence to better mimic local climatic conditions.The experimental assessment of vector competence for Australian mosquitoes focusing on the temperate region shows different degrees of risk.


In this study, we investigated the effect of initial concentrations of PNP and glucose and applied voltage on PNP reduction in biocathode BESs and open-circuit biocathode BESs (OC-BES). The PNP degradation efficiency of a biocathode BES with 0.5 V (Bioc-0.5) reached 99.5 ± 0.8%, which was higher than the degradation efficiency of the BES with 0 V (Bioc-0) (62.4 ± 4.5%) and the OC-BES (59.2 ± 12.5%). The PNP degradation rate constant (kPNP) of Bioc-0.5 was 0.13 ± 0.01 h-1, which was higher than the kPNP of Bioc-0 (0.024 ± 0.002 h-1) and OC-BES (0.013 ± 0.0005 h-1). PNP degradation depended on the initial concentrations of glucose and PNP. A glucose concentration of 0.5 g L-1 was best for PNP degradation. The initial PNP increased from 50 to 130 mg L-1 and the kPNP decreased from 0.093 ± 0.008 to 0.027 ± 0.001 h-1. High-throughput sequencing of 16S rRNA gene amplicons indicated differences in microbial community structure between BESs with different voltages and the OC-BES. The predominant populations were affiliated with Streptococcus (42.7%) and Citrobacter (54.1%) in biocathode biofilms of BESs, and Dysgonomonas were the predominant microorganisms in biocathode biofilms of OC-BESs. The predominant populations were different among the cathode biofilms and the suspensions. These results demonstrated that applied voltage and biocathode biofilms play important roles in PNP degradation. The useIn this study, we investigated the kinetics of PNP degradation and PAP formation in biocathode BESs. We analyzed the effects of applied voltage and the initial concentrations of glucose and PNP on PNP degradation. We also explored the biocathode microbial communities using high-throughput sequencing of 16S rRNA gene amplicons.We used two-chamber BES reactors consisting of glass bottles separated by a cationic exchange membrane . The working volume of each chamber was 300 mL. Carbon brushes  were used as the electrodes. Prior to use, the membrane and electrode brushes were pretreated as previously described . The Ag/-1 of NaAC, trace minerals, vitamins, and 50 mM of phosphate buffer solution (PBS)  to determine the impact of biocathode microbial communities on PNP degradation. The reactors and the medium were autoclaved at 121°C for 15 min. All experiments were operated in replicated cycles for consistency, and all experiments were conducted at 25 ± 2°C.The reactors with biocathode were inoculated in fed-batch mode as previously described . PNP degon (PBS) . The catThe concentrations of PNP and PAP were determined, as previously reported, and the production was analyzed by a high performance liquid chromatography mass spectrometer (HPLC-MS) . We used-3) was calculated based on the cathode volume (300 mL). PNP degradation efficiency (DEPNP) was calculated based on the difference between the influent and effluent PNP concentrations. The kinetics of the PNP reduction and PAP formation were assumed to follow the first-order reaction models C = C0-kte and C = C0(1--kte), respectively (C represents the PNP or PAP concentration (mg L-1) at time (h) and C0 is the initial PNP concentration or maximum PAP concentration); the rate constant k (h-1) of PNP and PAP was calculated by Origin 8.0 software. The half-life time (t1/2) of PNP was calculated using the equation t1/2 = 0.693/k.Current density  with the same instrument as the CV tests with a frequency range from 100 KHz to 10 mHz using a 10 mV sine wave.We conducted cyclic voltammetry (CV) on the cathode using an electrochemical workstation ; the anode was the counter electrode, and Ag/AgCl was the reference electrode (+0.197 V vs. SHE). We measured CV for 30 mg LSamples of cathodic biofilms  and suspended growth cultures  were removed aseptically from the corresponding reactors. DNA was extracted using the PowerSoil DNA Isolation Kit . DNA samples were stored at -20°C before analysis. Polymerase chain reaction (PCR) amplifications of bacteria were sequenced using the universal primers 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 533R (5′-TTACCGCGGCTGCTGGCAC-3′) for the 16S rRNA gene V1–V3 region (length of approximately 455 bp). We conducted 454 GS-FLX pyrosequencing using the method described previously . The abukPNP) and PAP formation rate (kPAP) were fitted with first-order kinetics  (Supplementary Table kPNP) of Bioc-0.5 was 0.13 ± 0.01 h-1 (R2 = 0.996), which was five times greater than that of Bioc-0 , and almost 10 times greater than that of OC-BES . The PAP formation rate (kPAP) followed the same trend and the values were 0.11 ± 0.01 (R2 = 0.983), 0.051 ± 0.012 (R2 = 0.967), and 0.038 ± 0.011 (R2 = 0.972) for Bioc-0.5, Bioc-0, and OC-BES, respectively. The PNP degradation efficiency (DEPNP) of Bioc-0.5 at 36 h was 99.5 ± 0.8%, which was significantly higher than the DEPNP of Bioc-0 (60.9 ± 0.05%) and OC-BES (37.7 ± 5.2%) . The concentration of VFAs of Bioc-0.5 was lower than the VFA concentration of OC-BES and higher than the VFA concentration of Bioc-0. Abundant cell biomass was present in the suspension of cathodic chamber , and the cell biomass of the effluent was different among three modes. The cell biomass of Bioc-0.5 increased in the first 12 h, then decreased from 12 to 24 h, and the cell biomass was stable at the final stage (from 24 to 36 h). The cell biomass of Bioc-0 and OC-BES increased gradually, and the cell biomass was greatest in the OC-BES.In all experiments, glucose was consumed quickly and the concentrations were less than 11.4 ± 2.2 mg LFigure 3). The current density of Bioc-0.5 reached 2.49–2.26 Am-3, which was much higher than the current density of Bioc-0 (approximately 0.057 Am-3). After reaching the maximum, the current density of Bioc-0.5 decreased and stabilized at approximately 0.5 Am-3 after 20 h; the current density of Bioc-0 was approximately 0.028 Am-3. These results showed that applied voltage can significantly increase the absolute cathodic potential and the current density.The cathode potential decreased and stabilized after 20 h at approximately -1.0 V for Bioc-0.5, which was lower than the cathode potential of Bioc-0 (-580 mV) and OC-BES (-493 mV) , 0.086 ± 0.009 h-1 (R2 = 0.987), and 99.8 ± 0.35%, respectively (Table 1). A higher glucose concentration (>0.5 g L-1) resulted in PNP degradation (0.070 ± 0.009–0.084 ± 0.008) decreased and a lower glucose concentration (<0.5 g L-1) resulted in decreased PAP formation (0.0391 ± 0.00945–0.0509 ± 0.0111). All samples had similar cathode potentials  . Maximum peak current density was at 0.5 g L-1 glucose (3.1 Am-3) . The peak current density of other glucose concentrations decreased to <2.9 Am-3. These results showed that 0.5 g L-1 was an optimum glucose concentration for PNP degradation.Different initial concentrations of the exogenous carbon source substantially influenced PNP reduction. With the addition of 0.5 g LkPNP) decreased from 0.098 ± 0.011 to 0.022 ± 0.001 . We found that kPNP decreased from 0.093 ± 0.008 to 0.027 ± 0.0001, kPAP decreased from 0.086 ± 0.00914 to 0.0231 ± 0.005, and DEPNP decreased from 99.8 ± 0.35 to 64.1 ± 2.4% when the initial concentration of PNP increased from 50 to 130 mg L-1 . As the PNP concentration increased from 50 to 130 mg L-1, the cathode potential and the current density increased and then decreased . A lower initial concentration of 30 mg/L showed a higher kPNP (0.13 ± 0.01 h-1) . These results showed that the initial PNP concentration influenced the rate of PNP reduction and its efficiency.Five initial concentrations of PNP were used to assess PNP reduction with 0.5 g LTotal operational taxonomic units (OTUs) of 440–545 were obtained from the clean reads of 7833–11121 . The predominant phyla of Bioc-0.5-C was Firmicutes (60.4%) and Bacteroidetes (30.9%); Proteobacteria (43.4%) and Firmicutes (35.7%) were the predominant phyla in Bioc-0.5-S. By contrast, the predominant phyla were Proteobacteria (72.8%) for Bioc-0-C and Bacteroidetes (58.3%) and Proteobacteria (33.1%) for Bioc-0-S. In OC-BES, the main phyla were Bacteroidetes (47.6%) and Proteobacteria (46.0%) for OC-BES-C and Proteobacteria (71.8%) and Bacteroidetes (23.6%) for OC-BES-S. At the family level, Streptococcaceae was predominant in Bioc-0.5-C (58.4%) and Bioc-0.5-S (29.6%); Porphyromonadaceae was predominant in OC-BES-C (45.2%) and Bioc-0-S (52.5%); Enterobacteriaceae was dominant in Bioc-0-C (65.9%) , Lactococcus (15.6%), and Bacteroides (11.4%). Streptococcus (22.2%), Comamonas (18.1%), and Citrobacter (11.2%) were the predominant populations in Bioc-0.5-S . Bioc-0-C was mostly Citrobacter (54.1%) bacteria, and Bioc-0-S was mostly Dysgonomonas (52.3%) bacteria. By contrast, the relative abundance of Dysgonomonas and Comamonas were 45.2 and 24.1%, respectively, in OC-BES-C and 19.7 and 52.4%, respectively, in OC-BES-S. These results indicated that power supply greatly affected the community composition of the electrode biofilms and suspensions.Most populations of bacteria in Bioc-0.5-C were affiliated with kPNP (0.093 ± 0.008 h-1) of BESs fed with glucose was approximately three times greater than the kPNP of BESs fed with sodium bicarbonate (0.02978 ± 0.00339 h-1)  . More imFigure 2A). The VFAs were then used as the electron donors for PNP degradation after the glucose was consumed (6 h), which was called a syntrophic interaction in a previous study (-1). PNP degradation in the OC-BES further indicated that partial PNP reduction was by glucose and not by the cathode.In the cathodic chamber, glucose was first transformed by the bacteria to relatively lower molecular VFAs through a fermentation process (us study . Syntrop-1, further demonstrating the advantage of PNP tolerance in biocathode BES for PNP degradation. The biofilm removed from the biocathode of BESs, the PNP degradation and the PAP formation decreased (Supplementary Figures A previous study indicated that PNP can inhibit PNP biodegradation because it is toxic to bacteria . A high p-Nitrophenol-degrading bacteria have been isolated from enrichment cultures to reveal whether the same microorganisms are responsible for PNP degradation in the natural environment (Pseudomonas (Arthrobacter (Flavobacterium (Achromobacter (Sphingomonas (Burkholderia (Stenotrophomonas (ironment . Most PNudomonas , Arthrobrobacter , Flavobaacterium , Achromomobacter , Sphingongomonas , Burkholholderia , and Stephomonas  are PNP-Streptococcus was highest in the biocathode biofilms of Bioc-0.5-C. Streptococcus, a biofilm-forming pathogen, has been studied (Dysgonomonas was present in the suspension and the OC-BES cathode, and it was the predominant genus detected in the BESs (Comamonas, enriched in the biofilms and suspension of OC-BESs, can use phenol, 4-nitrobenzoate, 4-chlorophenol, and nitrobenzene (Streptococcus, Dysgonomonas, and Comamonas should be investigated in the future. Revealing functional genes related with PNP degradation and syntrophic interaction between different populations using metagenomic technology is still important to understand PNP degradation in future study on BES.The relative abundance of  studied , but itsthe BESs . The eleobenzene . The PNPp-Nitrophenol degradation was enhanced in the biocathode BESs with glucose and an applied voltage of 0.5 V. PNP degradation efficiency of the BES was much higher than that of OC-BES. The initial concentrations of glucose and PNP influenced PNP degradation and PAP formation. The microbial communities of the biocathode biofilm and suspension were different in OC-BES and BES with different voltages, implying that the differences in microbial communities and BES resulted in different PNP degradation. These results demonstrated that voltage and biocathode biofilms contribute to PNP degradation.DX and NR designed the experiments. XW performed the experiments. XW, DX, XM, and BL contributed the data analysis and wrote sections of the manuscript. All authors contributed to manuscript revision and approved the submitted version.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.


Anguilla marmorata) in Taiwan. The virus has been characterized as being related to Japanese eel endothelial cell-infecting virus (JEECV), with a large T-antigen-like protein. The sequence of the marbled eel virus displays low homology to the JEECV.We report here the complete genome sequence of a virus isolated from a diseased marbled eel ( Anguilla marmorata) is an important emerging species aquaculture in Taiwan (Anguilla marmorata virus (AMV) was named marbled eel polyoma-like virus (MEPyV) or Anguilla marmorata polyoma-like virus (AMPyV), with a polyomavirus large T-antigen-like (LTL) gene (–Marbled eel (n Taiwan . RecentlTL) gene . The parTL) gene –5. JEECVL) gene –. AdditioL) gene –, 4. Herede novo using ABySS (MEPyV (AMV-6) was proliferated in EK-1 cells, and the DNA was isolated and purified by a modification of the Hirt method  and deling ABySS . All obtThe circular genomic DNA comprised 16,930 bp and displayed very low similarity to known viruses, including JEECV. A BLASTN search showing the sequence fragment at positions 14627 to 14713 and at 3223 to 3295 was respective homologous to some polyomavirus large T-antigen (LT) genes and certain phycodnaviruses, baculoviruses, and herpesviruses. The genome was suggested to encode 10 proteins, including an LTL, a DNA polymerase, an adenain-like protein, five capsid proteins, and two unknown proteins. Alignment revealed that the LTL amino acid sequence was closer to the LT of giant guitarfish polyomavirus (KX781210.The genome sequence of AMV-6 has been deposited in GenBank under the accession no.


The addition of 1 mg L-1 sodium hypochlorite resulted in an immediate free chlorine residual of 0.16 mg L-1, which inactivated 3.5 log10 units of Ebola virus in 20 seconds. Further inactivation was not evident due to the rapid consumption of the chlorine residual. Elevating the pH to 11.2 was found to significantly increase viral decay over ambient conditions. These results indicate the high susceptibility of the enveloped Ebola virus to disinfection in the presence of free chlorine in municipal wastewater; however, we caution that extension to more complex matrices (e.g. bodily fluids) will require additional verification.Concerns have been raised regarding handling of Ebola virus contaminated wastewater, as well as the adequacy of proposed disinfection approaches. In the current study, we investigate the inactivation of Ebola virus in sterilized domestic wastewater utilizing sodium hypochlorite addition and pH adjustment. No viral inactivation was observed in the one-hour tests without sodium hypochlorite addition or pH adjustment. No virus was recovered after 20 seconds (i.e. 4.2 log Ebola virus infected individuals may generate up to nine liters of potentially infectious liquid waste per day. Previous recommendations were to directly dispose of this waste into a sanitary sewer or latrine; however, release of infectious virus raised the concern of environmental transmission through unintentional contact with contaminated wastewater. One possibility to reduce or eliminate the release of infectious virus is disinfection of Ebola virus contaminated liquid waste. A hurdle to making recommendations for liquid waste disinfection is the lack of data on disinfection efficacy. Here we demonstrate that Ebola virus in municipal wastewater is highly sensitive to disinfection in the presence of free chlorine. In addition, elevating the pH to 11.2 significantly increased the rate of decay over neutral pH conditions. These results provide a basis to develop recommendations for the disinfection of Ebola virus contaminated wastewater. The conmacaques .90 (time for 90% inactivation) of Ebola virus in sterilized wastewater to be 2.1 days [ad hoc liquid waste disinfection approaches prior to disposal [The World Health Organization initially recommended that liquid waste from Ebola patients be directly disposed into the sanitary sewers or latrines without disinfection . The rec2.1 days , which i2.1 days . Additio2.1 days . Previou2.1 days . In respdisposal . The Wordisposal .Currently, the disinfection kinetics of Ebola virus in liquid is unknown. In a previous evaluation of Ebola virus disinfection on surfaces, sodium hypochlorite at 0.01% and 0.1% was found to be ineffective but 0.5% and 1% sodium hypochlorite removed viable virus in five minutes . AdditioThe overarching study goal was to determine the disinfection of Ebola virus in municipal wastewater, of direct relevance to wastewater management in an outbreak scenario. Our scope was limited to municipal wastewater and did not consider the disinfection of Ebola virus in concentrated human waste . It should be noted that disinfection under high organic load , which is not the focus of the current manuscript, would require hyper-chlorination, which has been suggested to inconsistently achieve adequate disinfection and would require additional experimental verification . In the 7.3 TCID50 mL-1) [5 TCID50 mL-1 for both Ebola virus disinfection experiments and pH inactivation experiments. All experiments were completed in triplicate at 20°C. Ebola virus titration and cultivation were performed as previously described [50 mL-1.Wastewater samples were collected from a municipal wastewater treatment plant as described previously  and ship50 mL-1)  was diluescribed . The lim-1. Samples were then taken at the indicated time points and chlorine demand immediately quenched by the addition of sodium thiosulfate. The ‘time zero’ sampling point was taken approximately 20 seconds following the addition of chlorine to enable sample mixing.For disinfection experiments, sodium hypochlorite (Acros Organics) was added to two milliliter vials of the wastewater/virus suspension at initial doses of 0, 1, 5, and 10 mgLThree pH values were evaluated for pH inactivation experiments: 6.9 (intrinsic), 4.3, and 11.2. pH values were found to be stable for the time period evaluated. The tested pH values were chosen to be below the previously recognized Ebola virus glycoprotein stability down to pH = 4.8  and to b0 was the modeled initial chlorine residual. The concentration-time exposure was then calculated for each sampling time point by integrating the area under the modeled chlorine residual curve at each time point.Chlorine residuals in both the untreated and the gamma-irradiated wastewater were experimentally determined outside of the Biosafety Level 4 facility using a Hach Free Chlorine test kit (method 10069) in triplicate. Chlorine residual was experimentally found to be dose dependent . To deteStatistical analyses and graphing were completed with Prism 7.0a and Microsoft Excel 2011.p < 0.05); however, the chlorine decay was more rapid in irradiated wastewater compared to the wastewater without irradiation, suggesting that testing in the irradiated wastewater would demonstrate less rapid viral inactivation than would be observed in the non-irradiated wastewater at the same applied dose. Chlorine residual was found to be dose-dependent . Data for the 0 and 1 mg L-1 conditions from the 105 TCID50 mL-1 virus concentration is shown in 10 TCID50 mL-1 in 20 seconds), no further viral removal was observed in the 1 mg/L condition, likely due to the low concentration of free chlorine  of viral particles remain not particle associated , 30.Despite the end of the most recent Ebola virus epidemic, concerns remain regarding the potential transmission of emerging enveloped viruses via water , highligS1 Table50 mL-1. Limit of detection for each replicate was 0.75 log TCID50 mL-1.All values log TCID(DOCX)Click here for additional data file.S2 Table50 mL-1. Limit of detection for each replicate was 0.75 log10 TCID50 mL-1.All values log TCID(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 FigNo residual detected at later time points.(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.S5 Fig(DOCX)Click here for additional data file.


However, other defective genomes were also detected. Since these additionally produced defective particles are non-infectious, they might be still useful in antiviral therapies. In case they would interfere with quality of the final product, we examined the impact of virus seeds and selected process parameters on DI244 yield and contamination level with other defective particles. With a DI244 fraction of 5.5%, the yield obtained was 1.7 × 108 vRNA copies/mL but now without additional defective genomes. Although the DI244 yield might be decreased in this case, such controlled manufacturing conditions are not available in chicken eggs. Overall, the application of these findings can support design and optimization of a cell culture-based production process for DIPs to be used as antivirals.Defective interfering particles (DIPs) lack an essential portion of the virus genome, but retain signals for replication and packaging, and therefore, interfere with standard virus (STV) replication. Due to this property, DIPs can be potential antivirals. The influenza A virus DIP DI244, generated during propagation in chicken eggs, has been previously described as a potential candidate for influenza antiviral therapy. As a cell culture-based manufacturing process would be more suitable to fulfill large-scale production needs of an antiviral and enables full process control in closed systems, we investigated options to produce DI244 in the avian cell line AGE1.CR.pIX in chemically defined suspension culture. With a DI244 fraction of 55.8% compared to STV, the highest DI244 yield obtained from 50 million cells was 4.6 × 10 Orthomyxoviridae family with a segmented genome that consists of eight negative-stranded RNA segments. Influenza viruses can cause respiratory illness known as flu. Annual flu epidemics worldwide are estimated by the WHO to result in about 3 to 5 million cases of severe illness, and about 250,000 to 500,000 deaths. The most effective way to prevent flu is vaccination, but influenza vaccines do not always match the strains that are currently circulating. Two classes of antiviral drugs against influenza are currently available, to which however, virus resistance has become more and more frequent (Mc Mahon and Martin Loeches Influenza viruses are enveloped viruses of the The use of defective interfering particles (DIPs), which reduce infectious load and stimulate the adaptive and innate immunity  or HA assay . Based on small-scale experiments, we propose a cell culture-based process for DIPs generation, specifically DI244, for manufacturing of this new class of antivirals.In this study, we characterized specific properties of a virus seed used for DI244 production in AGE1.CR.pIX cells and the impact of cultivation conditions on DI244 yield and contamination level with additional defective genomes, i.e., the impact of MOI and trypsin activity. In addition, we have established a RT-qPCR assay which enables the quantification of DI244 and STV copy numbers based on segment 1, which cannot be monitored by other titration methods, i.e., TCID2. The chemically defined CD-U3 medium (Biochrom/Merck) was supplemented with 2 mM glutamine (Sigma), 2 mM alanine (Sigma), and 10 ng/mL recombinant insulin-like growth factor . Cells were passaged twice a week by dilution to the starting cell concentration of 8 × 105 cells/mL using fresh medium.The cell line AGE1.CR.pIX was provided by ProBioGen AG and cultivated in 125 mL vented and baffled shaking flasks  at 185 rpm, 37 °C, and 5% CO50 assay (see v/v) fetal calf serum (PAN Biotech) and 1% (v/v) peptone (Lab M) at 37 °C in a 5% CO2 atmosphere.Adherent MDCK cells , used for the innocuity test see  and TCIDssay see , were ma−6 U/cell. The first passage was conducted by directly infecting cells with 50 μL of DI244/STV. The resulting virus showed a TCID50 titer of 7.2 × 104 infectious virions/mL and a HA titer of 1.37 log HAU/100 μL at 12 hpi. For the second passage, MOI 10−3 (based on TCID50 assay) was used and the virus was harvested at 24 hpi. The third passage was performed at MOI 10−6 and resulted in the DI244 working virus seed, which showed a TCID50 titer of 5.5 × 105 infectious virions/mL and a HA titer of 1.45 log HAU/100 μL at 24 hpi. The fourth passage was conducted at MOI 10−8 and resulted in the DIP-free STV working virus seed, which showed a TCID50 titer of 2.1 × 106 infectious virions/mL, a HA titer of 1.82 log HAU/100 μL, and 6.8 × 109 STV vRNA copies/mL by RT-qPCR at 48 hpi. No defective genomes including DI244 were detected by RT-PCR. In addition, a STV-free DI244 seed was produced by UV irradiation (see below) of the DI244 working virus seed.The egg-derived DI244/PR8 virus, previously described  of the DI244 working virus seed was exposed for 8.5 min to UV light (254 nm) in the PCR Workstation Pro (VWR Peqlab) with a distance of about 65 cm to the two UV tubes (each 25 W).UV irradiation to destroy the full-length segments of STV , based on the TCID50 assay  peptone (Lab M) and 5 U/mL trypsin . Virus samples were collected at 72 hpi. Samples were centrifuged at 300g for 5 min and the supernatant was stored at − 80 °C until HA assay.For the innocuity test, confluent cultures of adherent MDCK cells were washed twice with PBS. 1 mL of UV-irradiated DI244 working virus seed was added to the cells in 50 mL serum-free GMEM (GIBCO) containing 1% (50) assay by titration of supernatant samples on MDCK monolayers as described previously (Genzel and Reichl Infectious virus particle concentrations were measured using a tissue culture infectious dose 50 (TCID10 HA units per test volume (log HAU/100 μL).Total virus particle concentrations were determined using a hemagglutination assay as described previously  according to the manufacturer’s instructions.The reverse transcription (RT) of isolated RNA to cDNA as well as the amplification of all eight influenza virus genome segments with segment-specific primers for detection of defective genomes was performed as described previously  copy numbers, a reverse transcription quantitative PCR (RT-qPCR) was performed using RNA reference standards for the targeted RNA as described previously , the egg-derived virus material showed the highest DI244 vRNA concentration with 20.5 × 10−5) to reduce the risk of de novo generation of additional defective genomes and increased MOI stepwise. The addition of trypsin at time of infection is required for hemagglutinin cleavage to facilitate virus infection. A trypsin activity of 10−6 U/cell was previously shown to be the most effective in influenza virus production in AGE1.CR.pIX cells . The highest copy number was achieved 12 hpi with only a slight decrease over the next 36 h . The specific inactivation rate of this first order reaction was 3.1 min−1 (linear range 0.33 to 2.0 min). Complete loss of infectivity (TCID50 titer below the detection limit) was observed for UV exposure times of 4.5 min and above  and the same concentration of UV irradiated (8.5 min) DI244. At 36 hpi, an increase in vRNA copy numbers was observed for STV and DI244 by RT-qPCR , the egg-derived virus material showed with 20.5 × 1050 titers of DI244 preparations are relatively low, the calculated volume for inoculation of a bioreactor with a specific MOI could be overestimated. Adjusting the virus input by DI244 copy numbers would be a more reliable method for DI244 virus seed characterization and for monitoring of production.Although Dimmock and Easton specified DI244 copy numbers required for protection of mice, they used a specific DI244 virus protein mass for inoculation  of replication. Furthermore, it was shown that up to 90% of influenza A virus-infected cells can fail to release infectious progeny at low MOI  particles and allow better designed studies to elucidate the mechanisms of DIP replication, not only in cell cultures, but also in animals and humans to explore the full potential of DIPs as antivirals. However, while such an approach would eliminate the need to use infectious STV for seed generation as well as avoid the establishment of inactivation processes, scale-up, and, in particular, optimization of cell-specific DIP yields could be issues to be addressed.


Bunyaviridae family comprises viruses causing diseases of public and veterinary health importance, including viral haemorrhagic and arboviral fevers. We report the isolation, identification and genome characterization of a novel orthobunyavirus, named Wolkberg virus (WBV), from wingless bat fly ectoparasites (Eucampsipoda africana) of Egyptian fruit bats (Rousettus aegyptiacus) in South Africa. Complete genome sequence data of WBV suggests it is most closely related to two bat viruses (Mojuí dos Campos and Kaeng Khoi viruses) and an arbovirus (Nyando virus) previously shown to infect humans. WBV replicates to high titres in VeroE6 and C6-36 cells, characteristic of mosquito-borne arboviruses. These findings expand our knowledge of the diversity of orthobunyaviruses and their insect vector host range.The  CPE, cytopathic effect; EMEM, Eagle’s minimum essential medium; KKV, Kaeng Khoi virus; L, large; M, medium; MDCV, Mojuí dos Campos virus; NP, nucleocapsid protein; RdRp, RNA-dependent RNA-polymerase; RER, rough endoplasmic reticulum; S, small; TCMV, Tacaiuma virus; WBV, Wolkberg virus.Bunyaviridae family contains over 530 members and has an extensive host range, including arthropods, rodents, large mammals and plants. The typical bunyavirus genome comprises single-stranded RNA separated into small (S), medium (M) and large (L) segments with complementary terminal sequences unique to specific viral genera. The majority of bunyavirus genomes are negative sense, but some viruses use ambisense strategies to express genes from the S segment. The S segment encodes the nucleoprotein, the M segment encodes the glycoprotein precursor and the L segment encodes the RNA-dependent RNA polymerase [The lymerase .The orthobunyavirus genus comprises more than 170 known viruses assembled into 48 species and 19 serogroups . VirusesEucampsipoda africana), which were collected from wild-caught Egyptian fruit bats (Rousettus aegyptiacus) in South Africa. The virus has been named Wolkberg virus (WMV), after the location of the cave harbouring the Egyptian fruit bats from which these ectoparasites were collected.Several orthobunyaviruses have been detected in South Africa  , but non5.0 TCID50 ml−1 in Vero cells by the third passage. A standard curve (R2=0.999) generated by triplicate testing of a dilution series of stock virus enabled us to establish TCID50 equivalents relative to RT-PCR Ct values  between HEK293 and the other cells lines ranged from 1 log to 3 logs in some cases, especially with the lowest virus input. The ectoparasites from which the viruses were isolated were morphologically identified as nycteribiid bat flies, Eucampsipoda africana Theodor (Diptera: Nycteribiidae) [From a total of 273 bat ectoparasite pools tested, 11 caused a non-characteristic cytopathic effect (CPE) by day 5–7 post inoculation (from the third passage onwards) in the form of small, rounded, refractory detached cells in the presence of a mostly intact Vero monolayer. All ectoparasite pools originated from apparently healthy Egyptian fruit bats. The 11 isolates replicated to titres in excess of 1×10t values . This en 2 weeks . Howeveribiidae) .n=100). The envelope appearance varied from indistinct and fuzzy, to fairly regularly spiked, in which projections measured up to 8 nm , but with an average diameter of 87 nm . All 11 isolates contained contigs that had matches for an S, M and L segment of an orthobunyavirus.Identification as an orthobunyavirus was confirmed by using an unbiased next-generation sequencing approach that resolves the 5′ and 3′ termini . Assembled contigs were aligned to the nt sequence database , show the placement of the 11 WBV isolates amongst other orthobunyaviruses in the family. Orthobunyavirus genus [Maximum-likelihood trees, constructed with nucleic acid sequence data for the L, M and S segments of representative viruses from the different genera within the us genus .Orthobunyavirus genus  and ORF-deduced amino acid sequences of the L and M segments , but closest to MDCV based on an S-segment ORF amino acid sequence (65.6 %). The ICTV species demarcation in the Orthobunyavirus genus is complicated by the lack of biochemical characterization of named viruses, and species are thus demarcated based on serological cross-reaction criteria [Orthobunyavirus genus. However, this needs to be further investigated by evaluating serological cross-reactivity to closely related viruses. The sequences of the 11 WBV isolates are not identical. There appear to be three sub-lineages of the virus based on the ORF-deduced amino acid sequences of all three segments  to 4.1 % (L-segment ORF), 28.8 % (M-segment ORF) or 1.3 % (S-segment ORF). Based on the M-segment ORF, the WBV isolates 2763 and 2824 seem to be closely related (0.4 % p-distance) but quite distinct from the other nine isolates (between 27.9 and 28.8 % difference). The close-relatedness between all 11 isolates is based on L- and S-segment ORFs, but clear divergence in two isolates in the M-segment might represent evidence of re-assortment with another variant of WBV that is yet to be isolated, and thus possibly evidence of higher undiscovered divergence of the new virus. Based on limited data which suggest that re-assortment can only occur between viruses in the same species [The original 11 homogenates from which the strains of WBV virus were isolated were subsequently tested by RT-PCR to determine viral loads. Similarly, bat serum pools from around the period of virus isolation were also tested by RT-PCR. The results are summarised in Orthobunyavirus genus (Bunyamwera virus RdRp 2238 aa) [The deduced 2253 aa size of the WBV RdRp  corresponds to the size of the same protein of other members of the 470-EA, which corresponds to other orthobunyaviruses [www.cbs.dtu.dk/services), of which one is in the G2, three in the NSm and two in the G1 protein.The 1422 aa WBV polyprotein  is likely co-translationally cleaved into the 951 aa G1 , 285 aa G2  and 166 aa NSm . Cleavage of the signal peptide is predicted at position 18 relative to the first methionine . The G2 contains the conserved arginine at position 303, which likely represents the cleavage site from the downstream NSm . SignalPOrthobunyavirus genus to regard it as a separate, new species [125, G131, Y158 and I231) are all conserved in WBV [The WBV-deduced nucleocapsid protein (NP) is 234 aa in length  and corresponds to other orthobunyaviruses . It satid in WBV . Contrard in WBV . The prod in WBV .Anopheles mosquito in South America, has been shown to cause febrile illness in humans and has been associated with other vertebrate hosts  [in vitro and in vivo studies. The decreased replication of WBV in HEK293 cells versus Vero cells might indicate that, without the expression of NSs, the virus is not able to replicate as efficiently in the presence of an interferon response, but this requires further in-depth investigation in in vivo interferon-competent versus interferon-deficient mouse models.Viruses from the Tete, Anopheles A and Anopheles B serogroups also do not express an NSs protein . It has d horse) . The abiin vitro in a mosquito cell line might indicate its ability to be disseminated by mosquitoes, but this needs further investigation through vector competence studies.Based on sequence divergence of the NP from other known orthobunyaviruses, it is likely that WBV represents a new species in the genus. Evidence of re-assortment of the M segment in two of the 11 isolates described here, which are quite divergent from the other nine isolates, likely indicates that the diversity of viruses in this proposed new species is higher than that indicated by the data. The high percentage of isolates from a rather low number of ectoparasite pools  might indicate a very high prevalence in this specific bat colony, or may indicate that the timing of collection represents a period of high transmission rates. All isolates were made from ectoparasites collected during the dry winter period in South Africa when fruit is scarce, and also overlaps with the period when young adult bats have lost their maternal immunity. Aspects which need further investigation include whether WBV infects bats and what is the seroprevalence of bats collected from Mahlapitsi cave. Whether this new virus has any public or veterinary health importance is yet to be determined. Antibodies to the virus most closely related to WBV, KKV, have been detected in humans who suffered a mild disease . The disRousettus aegyptiacus) were sampled between March 2013 and March 2014 at Mahlapitsi cave in the Mahlapitsi Valley, Limpopo province, South Africa, as described before [2. Culture medium was removed and the monolayers in individual wells inoculated with 200 µl of ectoparasite pool homogenates (one pool representing parasites from one bat). After 1 h adsorption at 37 °C, the inoculum was removed and fresh EMEM containing antibiotics and 2 % fetal calf serum added. Parasites were morphologically identified, and confirmed by amplification and amplicon sequencing of a region of the cytochrome oxidase 1 subunit gene, as belonging to the Nycteribiidae family, genus Eucampsipoda [2 tissue culture flasks to prepare stocks. Stock virus titres were determined by standard TCID50 titrations on 96-well microtitre plates.Egyptian fruit bats .Stock virus culture supernatant was added to Trizol-LS (Life Technologies) at a ratio of 100 µl supernatant to 300 µl Trizol-LS. RNA was extracted using a column based kit . To increase sensitivity, rRNA was depleted using the method described by Morlan et al. . Sampleset al. . Sequencet al. , qualityet al.  and readet al. . Resultamega (version 6) program was used to prepare alignments  of nucleic acid segment sequences, deduced amino acid sequences, phylogenetic trees and pairwise distance calculations [Bunyaviridae family were used to prepare a maximum-likelihood tree showing the placement of WBV in the family based on complete sequences of all three segments . Maximum-likelihood trees were prepared using amino acid sequences of all ORFs from all segments, showing the placement of WBV in the Orthobunyavirus genus relative to other viruses in this genus for which sequence is available on GenBank. Virus sequence accession numbers are summarised in mega6 [The ulations . The pubin mega6 . ORFs weIn vitro replication of WBV was evaluated in three cell lines: Vero E6 (source: African green monkey kidney), HEK293 (source: human embryonic kidney) and C6-36 . Cells were grown to 50–70 % confluence in 25 cm2 flasks, supernatant removed and respective flasks inoculated with 1 ml of 10−1, 10−2, 10−3 and 10−5 dilutions from stock virus (1×106.75 TCID50 ml1 determined by standard 50 % tissue culture infectious doses titration) in EMEM. After 1 h adsorption at 37 °C (Vero E6 and HEK293) or 28 °C (C6-36), the inoculum was removed, cells washed with 5 ml PBS and fresh EMEM, antibiotics and 2 % fetal calf serum (Hyclone) added. Cultures were incubated for 13 days at 37 °C (Vero E6 and HEK293) or 28 °C (C6-36) while 0.5 ml aliquots of supernatant were collected from each flask directly after inoculation and addition of fresh medium (day 0), followed by days 1, 4, 5, 7, 11 and 13. RNA was extracted from 140 µl of the supernatant collections from respective days  and subjected to TaqMan real-time RT-PCR. A TaqMan real-time RT-PCR was developed to target the S segment of WBV. Primers and probe sequences are: forward MAOBV_783F (5′-TTGGCTTTCTTTGCATTCAG-3′), reverse MAOBV_869R (5′-ATGGTTTCAACCCTGAGGAA-3′) and probe MAOBV_831P (FAM-TCTTCACAAGTGGCAATGC-BHQ), with the number in the oligonucleotide name indicating the nucleic acid position in the S segment. Real-time RT-PCR was performed on the extracted RNA using the Qiagen One-step RT-PCR kit (Qiagen) on a SmartCycler (Cepheid) with the following parameters: reverse transcription (50 °C for 30 min), hot-start Taq activation (95 °C for 15 min) and 50 cycles of amplification . RNA extracted from diluted stock WBV  was used as a qualitative positive control in each run. A standard curve was prepared by testing a dilution series of stock virus (1×106.75 TCID50 ml1) in triplicate and correlating Ct values to TCID50 equivalents.