Poxviridae, Reoviridae, Retroviridae, Herpesviridae, Filoviridae, and Bunyaviridae.Electron microscopy has been instrumental in the identification of viruses by being able to characterize a virus to the family level. There are a few cases where morphologic or morphogenesis factors can be used to differentiate further, to the genus level. These include viruses in the families  Papovaviridae and Picornaviridae. All other recognized viruses were listed as “Unassigned” and mostly categorized as genera [The electron microscope has been a powerful tool in the characterization of viruses. First constructed by Ernst Ruska and Max Knoll in the early 1930s, the electron microscope was soon used in the visualization of an orthopoxvirus, mouse ectromelia virus ,3. In ths genera . This was genera ,6, and is genera .etc.) onto an EM grid coated with a plastic film. The viruses that adhere to the grid are stained with a heavy metal which pools around the viruses, giving them an appearance as seen in a photographic negative, i.e., a light specimen against a dark background. Proteins on the surface of the nucleocapsid or envelope become apparent, and which allows for a morphologic differentiation among the different virus families [Electron microscopists typically use negative stain and thin section EM for diagnostic virology. Negative stain EM entails adsorbing a biological fluid , or pustule(s), on the skin and also internally on visceral organs with some species. The genera of poxviruses that can cause human disease include Although poxviruses contain DNA, the DNA replication and virus assembly do not take place in the nucleus but rather in the cytoplasm. Virus factories, or virosomes, are created and nascent crescents are formed and engulf the unit genome. Immature particles are spherical, but condense down to a dumbbell-shaped intracellular mature virus (IMV). The IMV is engulfed by Golgi vesicles, migrates to the cell surface or into microvilli and fuses with the cell membrane, releasing a particle wrapped in a single membrane known as an enveloped extracellular virus (EEV).By thin section EM, in addition to the viral factories, some poxviruses have other cytoplasmic structures know as acidophilic-type inclusions (A-type inclusions) consisting of a matrix containing the A-type inclusion protein and other proteins, with occluded intracellular mature particles ,11 FiguA. Virus Orthopoxvirus and Parapoxvirus can be distinguished by negative stain EM  with a sharp definition of the outer margin  and murine leukemia virus (MLV), respectively, were previously known as C-type particles; the cores form concomitantly with budding, and are centered in the middle of mature particles. Viruses previously known as B-type (such as mouse mammary tumor virus (MMTV)) and D-type (such as Mason-Pfizer monkey virus (M-PMV)) are now part of the genus Betaretrovirus. Both types are formed by envelopment of pre-formed cores, which are known as A-type particles, and mature into eccentrically located cores surrounded by the viral envelope. The viruses in the genus Deltaretrovirus, such as bovine leukemia virus (BLV), have a crescent-shaped budding profile which is composed of an electron-dense nucleoid and the nascent viral envelope. The cores of the mature virions are somewhat pleomorphic and fairly homogeneous, and there is often an electron-lucent space between the core and the envelope. Human immunodeficiency virus (HIV) is an example of the genus Lentivirus, and these viruses also have a crescent-shaped budding profile, which can be released from the cell to form a doughnut-shaped particle. The nucleoid then condenses into an electron-dense core that is cone-shaped, but can appear as a rod. The viruses in the genus Spumavirus, such as chimpanzee foamy virus (CFV), are seen as contaminants in some cell cultures derived from animal organs. Pre-formed cores are enveloped at cellular membranes or the plasma membrane, and the cores do not condense.The genera Cytomegalovirus (CMV) is within the subfamily Betaherpesvirinae, and infection with these viruses typically results in an increase in cell volume (cytomeglia). CMV-infected cells have nuclear inclusions, characteristic of herpesviruses, but also have cytoplasmic inclusions. The genomes of herpesviruses in general are composed of linear, double-stranded DNA. Replication takes place in the nucleus where nucleocapsids are formed, are surrounded by an “inner” tegument, and travel to the cytoplasm by budding upon the inner nuclear membrane, passing through the perinuclear space, and fusing with the outer nuclear membrane to egress to the cytoplasm. Additional tegument proteins attach to the capsid, either within the cytosol and/or at the future envelopment site on the membranes of the Golgi complex [The genus  complex  (Figure Herpesviridae, the cytoplasm in cells infected by CMV contains numerous aggregations of enveloped tegument proteins that lack capsids (Unlike the other members of the family  capsids B. These Ebolavirus and Marburgvirus genera and the recently recognized Cuevavirus genus, which is not known to cause human disease. Filoviruses are nonsegmented, negative-sense, single-stranded RNA viruses. Virions are pleomorphic, appearing as long filamentous particles, but also as branched, 6-shaped, U-shaped, or circular particles.This family includes Ebolavirus and Marburgvirus are well-known as being some of the deadliest known viruses, with case fatality rates for Ebola virus reported at 50% to 90%, and rates for Marburg virus at 24% to 88%. Early symptoms for both diseases include sudden onset of fever, headaches, weakness, muscle pains, and a sore throat. As the diseases progress, additional symptoms such as vomiting, diarrhea, impaired kidney and liver function, and sometimes a rash and internal and external bleeding may develop.The viruses in the genera s, and 805 nm, 970 nm, and 1200 nm for Ebola virus, but clearly Ebola viruses are longer [There have been differences reported in the morphologic features of Ebola and Marburg viruses. First, the lengths of the virus particles of the two genera vary, although there have been different lengths reported. For instance, there have been reports of 665 nm, 790 nm, and 860 nm lengths for Marburg virue longer ,22,23 (Fe longer  (Figure Bunyaviridae contains four genera that can infect humans—Orthobunyavirus, Nairovirus, Phlebovirus, and Hantavirus. Viruses contain single-stranded RNA with three RNA segments that are negative sense, with the exception of phleboviruses which have one ambisense segment. These are zoonotic viruses, where each virus is associated with a specific vector or natural reservoir, including mosquitoes, ticks, sand flies, and rodents. Patients will usually have a hemorrhagic syndrome which is characterized by fever, increased capillary permeability, leukopenia and thrombocytopenia.The family et al. [Uukuvirus, have a surface structure formed of subunits with a distinct central hole (In negative stain preparations, Martin, et al.  were ablral hole B. The sural hole C, and orral hole D.Hantavirus, however, have a core consisting of thin threads of ribonucleoproteins (By thin section EM, the virions of most bunyaviruses have a fairly homogeneous core and small spikes on the virus surface are visible in some preparations. The viruses mature by budding upon the membranes of the Golgi complex and can accumulate in the Golgi cisternae or extracellularly  Figure E. The viproteins F. In addEM can be an important factor in the initial diagnosis of a viral infection, in conjunction with serological, pathological, and molecular assays. At times, EM has provided identification of a virus when these other methods were unsuccessful . EM is ain situ hybridization. These techniques can be used to verify, or in tandem with, the EM examples discussed here to diagnose a virus to the genus and/or species level.Other tools are available that can be used to determine the genus of a virus. Biochemical methods consist of hemagglutination, hemagglutination inhibition, virus neutralization, and immunohistochemistry. Molecular assays include PCR, sequencing, next generation sequencing, and The analysis of virus structure has advanced greatly over the years. Negative stain EM and X-ray diffraction have long been used in the analysis of virus ultrastructure. Thin section EM is critical in the study of viral morphogenesis, and may be preferred over negative stain EM if the viruses are highly cell-associated. CryoEM using single particle analysis and 3D tomography has greatly furthered our knowledge on the architecture of viruses . In addiIn summary, EM identification of a virus in a clinical sample or a virus isolate can be instrumental for a diagnosis. Taxonomic classification for viruses by using EM has generally been at the family level, with a few exceptions. The ability of electron microscopists to discern the genus of a virus, as described in this paper, can be of great value to the clinician or to the laboratorian using cell culture for virus isolation.


Mathematical modeling of biochemical pathways is an important resource in Synthetic Biology, as the predictive power of simulating synthetic pathways represents an important step in the design of synthetic metabolons. In this paper, we are concerned with the mathematical modeling, simulation, and optimization of metabolic processes in biochemical microreactors able to carry out enzymatic reactions and to exchange metabolites with their surrounding medium. The results of the reported modeling approach are incorporated in the design of the first microreactor prototypes that are under construction. These microreactors consist of compartments separated by membranes carrying specific transporters for the input of substrates and export of products. Inside the compartments of the reactor multienzyme complexes assembled on nano-beads by peptide adapters are used to carry out metabolic reactions. The spatially resolved mathematical model describing the ongoing processes consists of a system of diffusion equations together with boundary and initial conditions. The boundary conditions model the exchange of metabolites with the neighboring compartments and the reactions at the surface of the nano-beads carrying the multienzyme complexes. Efficient and accurate approaches for numerical simulation of the mathematical model and for optimal design of the microreactor are developed. As a proof-of-concept scenario, a synthetic pathway for the conversion of sucrose to glucose-6-phosphate (G6P) was chosen. In this context, the mathematical model is employed to compute the spatio-temporal distributions of the metabolite concentrations, as well as application relevant quantities like the outflow rate of G6P. These computations are performed for different scenarios, where the number of beads as well as their loading capacity are varied. The computed metabolite distributions show spatial patterns, which differ for different experimental arrangements. Furthermore, the total output of G6P increases for scenarios where microcompartimentation of enzymes occurs. These results show that spatially resolved models are needed in the description of the conversion processes. Finally, the enzyme stoichiometry on the nano-beads is determined, which maximizes the production of glucose-6-phosphate. Such robust bioreactors can be, e.g., applied in biotechnology or drug delivery for the production of bioactive ingredients  to glucose-6-phosphate (G6P). This metabolic pathway is carried out in a microreactor consisting of a chamber separated from the surrounding medium by membranes carrying transport proteins for the input of sucrose and export of glucose-6-phosphate. Nano-beads loaded with multienzyme complexes are distributed inside the chamber. The enzymatic reactions constituting the metabolic pathway, as well as the corresponding enzymes and metabolites are listed in Table c ⊂ ℛn (n = 2 or n = 3). The space inside the chamber occupied by nano-beads, which are balls with diameter d, is denoted by Ωb, whereas the remainder, representing the domain occupied by the bulk solution, is denoted by b is strictly included in Ωc, this means that the beads do not touch the walls of the chamber. The number of beads in the reactor is denoted by bn. The boundary ∂Ω of the domain Ω is decomposed into ∂Ωc, the boundary of the chamber, and Γb: = ∂Ωb, the reactive surface of the nano-beads. Furthermore, the boundary ∂Ωc of the chamber consists of Γi, Γe, and Γ0, i.e.,i, respectively, Γe represent the boundary parts where metabolites are transported into, respectively, out of the chamber Ωc. These import/export boundaries have a complex geometric structure. They consist of fenestrations lined with lipid-membranes carrying transporters for the exchange of metabolites. On the boundary Γi the sucrose/proton cotransporters are distributed, whereas Γe contains the transporters for the exchange of glucose-6-phosphate and inorganic phosphate. In our model, the microscopic structure of the boundaries is taken into account in an averaged (homogenized) way, which makes the model amenable for numerical calculations. More precisely, we assign to each of the boundaries Γi, Γe an effective permeability for the transported metabolites denoted by iθ, respectively, eθ . These permeabilities can be calculated by an averaging approach (homogenization), assuming that the pores of the transporters are very small compared to the dimension of the reactor chamber and occur in a large number, and that the transporters are uniformly distributed within the lipid-membrane. A sketch illustrating the idea behind the homogenization approach is given in Figure The layout of the microreactor is given in Figure jy, j = 1, … , m of metabolites involved in the metabolic pathway is governed by a system of reaction–diffusion equations of the form:The spatio-temporal dynamics of the concentrations ν denotes the outer unit normal on ∂Ω with respect to Ω. Equation jD and enzymatic reactions with kinetics j. In case that enzymes are localized on beads (and thus no reactions are carried out in the bulk domain), the terms jD▽jy ⋅ ν which describes the normal component of the diffusive flux of the j-th metabolite at the boundary of the domain Ω. Thus, condition (1e) models an impermeable boundary, where the normal flux is equal to zero. Conditions (1c)–(1d) model the flux of metabolites through the import and export boundary, respectively. This flux is proportional to the permeability of the boundary, and the kinetics of the corresponding transport protein. Finally, condition (1b) describes the normal flux of metabolites at the boundary of the beads, generated by enzymatic reactions at the beads’ surface. We emphasize that the reaction rates i = 1, … , en, where en denotes the number of enzyme species involved in the reactions, holdsHere, iλ*  = 1 for some i*  ∈ {1, … en} means that all binding sites on the bead surface are occupied by the enzyme i*, whereas the case i ∈ {1, … en} means that all enzymes occupy the same amount of binding sites).(The case The existence and uniqueness of positive solutions for the model (1) can be shown by arguments similar to those in Gahn et al. (under review)e and Pie (glucose-6-phosphate and inorganic phosphate outside the chamber) are constant. This is motivated by the fact that G6Pe may be consumed in a following reaction, and Pie may be delivered at a desirable rate in the space outside the chamber. Hence, we can drop the equations for G6Pe and Pie. The constant values of the concentrations mentioned above are given by the corresponding initial concentrations . The equation for ADP can be neglected, since ADP is just a product of irreversible reactions and therefore has no direct effect on the reaction rates in equation (1). Finally, we assume that the concentrations of G6Pns Table . The sceSy). This is chosen in order to keep the notation clear.The reactions relevant for the SE-scenario are reactions 1)–(5) (Table –(5)  describes the enzymes stoichiometry on the bead. For the simulations, we choose Eλ satisfiesThe values Eλ are given in Table λ are computed.This enzyme stoichiometry leads to equal enzymatic activity for all enzyme species. The values of 2.2T] and with a given initial state.We use a finite element method in order to find an approximation to the solution of equation (1) on a fixed time interval ,The criterion for the performance of the reactor will be the total outflow of a desired product, say y(λ) denotes the numerical solution of the system . For our numerical results in Section λ(0), we either use the stoichiometry from equal activity Eλ given by equation J(0). Furthermore, we use tolX = 1.0e–06 and tolfun = 1.0e–04 for all our optimization runs.In order to determine an optimal stoichiometry θ. Combinatorial problems from  discrete parameters such as the number of beads can be solved using enumeration or, approximately, by derivative-based optimization techniques based on relaxation methods, even for time dependent parameters as in Hante and Sager 2 with L = 500 and L = 3000 μm, and different numbers of uniformly distributed beads, each of diameter of 10 μm. The outflow boundary is Γe = {L} × . The rest of the chambers’ boundary is impermeable. The parameters of the mathematical model are given in Table T varies between 0.5 and 4 h. This is appropriate, due to the fact that the outflow-membrane Γe is in general unstable and bursts after several hours. We stress that, due to the need of small time step sizes and the ratio of bead and chamber size, larger time horizons and chamber sizes lead to a higher computational complexity.We apply our simulation and optimization methods to the situation of the microreactor in the SE-scenario for realistic parameters. Hereby, we consider two-dimensional quadratic reaction chambers ΩEλ described in equation Eλ is used for the numerical simulations, whereas either of the two is used as initial parameter in the optimization, depending on which leads to a larger value of the total glucose-6-phosphate outflow.For our numerical investigations, we consider two types of hexokinase, namely HsHK2 and ScHK2. We also have two stoichiometry values which play a role in our calculations: the uniform stoichiometry 3.1c with a length of 3000 μm, a fixed run time of T = 3 h, a fixed enzyme stoichiometry λ = Eλ and a number of beads bn ∈ {02, 12, 22, …, 62, 122, 182, …, 602}, where bn = 0 refers to a scenario with enzymes distributed in the bulk solution.We consider a quadratic chamber ΩT = 1.5 h on uniform refined meshes and half of the time step size. No significant differences between the results on the coarse and on the fine meshes were observed.The meshes used for spatial discretization consist of between 15,190 triangles and 22,941 edges for 1 bead, and 93,184 triangles and 151,592 edges for 2916 beads. Detailed information about the meshes is available in the Supplementary Material. We used a time step size of 0.25 s. All calculations have been repeated for First results are concerned with the numerical computation of the spatial and temporal distribution of the metabolites. Figure t ∈ , i.e., the outflow rate at time t given byt given byj = G, … , G6P. In computing these quantities, we consider the following arrangements:(S.1)bn, where beads are completely loaded.Increase the number of beads (S.2)bn beads or in bulk. The latter case, we characterize by bn = 0.The total number of enzymes in the domain is fixed (and given by the number of binding sites on one completely loaded bead) and enzymes are distributed uniformly on Next results are concerned with the computation of quantities, which are particularly relevant for our microreactor, namely the export of G6P at time i, i ∈ {inv, hk, pgi} is calculated with [E]0 = [E]full where [E]full is the concentration of binding sites on one bead. These values are denoted by E]full. However, this concentration is now distributed on bn beads. Thus, the values for bn.Both arrangements are simulated for the two hexokinases HsHK2 and ScHK2, and the beads are distributed periodically in the bulk, like, e.g., in Figure bn ≥ 576 .The production and outflow rate for G6P are plotted in Figure E]full. We also remark that for the chosen stoichiometry Eλ, in case of arrangement (S.2), the production rate of G6P for HsHK2 is by a factor 103 to 104 larger than for ScHK2, i.e., in (S.2) the selected hexokinase has a greater influence on the conversion process as in (S.1).In case of partially loaded beads corresponding to arrangement (S.2), the production rate of G6P at the beads up to a time of 3 h first decreases with increasing number of beads and then stays nearly constant  with the hexokinase HsHK2, in Table 3.2λ* for the SE-scenario. Since our focus is on computing a variety of different scenarios with a reasonable computational effort, we have chosen the smaller chamber Ωc = (0.500 μm)2 for these experiments. The meshes used for the spatial discretization of this chamber consist of between 820 triangles and 1270 edges for 1 bead, and 2394 triangles and 3883 edges for 64 beads. Further information about the meshes is available in the Supplementary Material.We aim at finding the optimal stoichiometry (O.1)T = 1 h, we increase the number of completely loaded beads in the chamber with bn ∈ {1, 4, 9, 16, 25, 36, 49, 64}.For a fixed runtime of (O.2)T ∈ {0.5, 1, 1.5, 2, … 4} hours.For a sucrolytic chamber with one completely loaded bead, we optimize the G6P outflow for different runtimes For our study, we consider the following arrangements:We study these arrangements for the hexokinases ScHK2 and HsHK2.bn, the optimal stoichiometry λ* as well as the corresponding total outflow J* are displayed. From the arrangement (O.2), we find that the optimal stoichiometry for one bead is between 8 and 13% for invertase, between 80 and 87% for hexokinase and between 5 and 6% for phosphoglucose isomerase, again with an increase in the proportion of binding sites loaded with ScHK2 in expense of proportion for ScINV, but with an almost constant proportion of binding sites loaded with ScPGI when the runtime is successively increased. The exact results are listed in Table T, the optimal stoichiometry λ* as well as the corresponding total outflow J* are displayed.For the enzyme ScHK2, we find in case of arrangement (O.1) that the optimal stoichiometry for a runtime of 1 h is between 3 and 12% for invertase, between 82 and 94% for hexokinase and between 3 and 6% for phosphoglucose isomerase. With an increasing number of beads, the proportion of ScHK2 increases and the proportions of invertase and phosphoglucose isomerase decrease. The exact results are listed in Table In the presence of the hexokinase HsHK2, we find from the first arrangement that the optimal stoichiometry for a runtime of 1 h is between 5 and 9% for invertase, between 91 and 95% for hexokinase and approximately 0% for the phosphoglucose isomerase. With an increasing number of beads, the proportion of hexokinase increases and the proportion for invertase decreases. The exact results are listed in Table 4In this paper, we developed mathematical techniques for the simulation and optimization of metabolic processes in biological microreactors with membrane-bounded subcompartments. The results from our models will be incorporated into the design of a microreactor and will be validated by experiments with this microreactor. The microreactor is based on a microfluidic system, and consists of chambers separated by membranes which carry specific transporters for input of substrates and export of products. Inside the chambers, magnetic nano-beads carrying multienzyme-complexes are distributed, and immobilized by an outer magnetic field.We have set up a mathematical model describing the spatio-temporal dynamics of the metabolite concentrations involved in the assembled metabolic pathway, and develop efficient and accurate approaches for the numerical simulation of this model. Furthermore, we show that model-based optimization for such systems is feasible by the methods presented. These approaches are applied to the proof-of-concept microreactor carrying out the synthetic pathway for the conversion of sucrose to glucose-6-phosphate. The obtained results shed light on the following important questions linked to the design and functionality of microreactors.4.1e, and consequently G6P diffusion to the boundary Γe needs less time. We mention, however, that for longer runtimes, the higher production rate of G6P for lower numbers of beads is predominating, and the outflow rates of G6P are higher for less number of beads  the outflow rates are not reaching saturation, which implies that the activity of the transporters is not limiting the outflow for the chosen enzyme concentration [E]full.The simulation results for the arrangement (S.1) (with completely loaded beads) show that the outflow rates for G6P reach saturation, in spite of the fact that the activity inside the chamber increases with increasing number of beads. This can directly be seen for the hexokinase HsHK2  and 1% (for 64 beads) compared to the stoichiometry computed from equal activity and slightly higher compared to the uniform stoichiometry. For one bead, the improvement is also visualized in Figure To understand the optimization result obtained for the proportion of phosphoglucose isomerase , in case of the microreactor with HsHK2, namely that this enzyme should be better eliminated, we simulated the production rates for all metabolites, for one completely loaded bead Figure . We see λ(0) to λ* transverse a large part of the parameter space and are associated with a significant decrease in the costs. Moreover, we have tested various initial points λ(0) for the scenario with one bead, HsHK2 and T = 3600. In all cases, we have observed convergence to the same λ*. We conclude that the reported local maxima are reasonable candidates for an optimized stoichometry for the respective microreactor.We stress that none of the reported maxima can be guaranteed to be global optima. Nevertheless, concerning the quality of the maxima, we note that all search paths from Finally, we emphasize that the simulation and optimization approaches developed in this work can be repeated with minor adaptions for more complex biochemical microreactors and for other optimization parameters, like, e.g., other real parameters entering the reaction kinetics.MN-R and LV gave substantial contributions to the Abstract and Introduction. MG and MN-R gave substantial contributions to the conception of the Derivation of the Mathematical Model, see Section 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.


Diagnosing the cause of bovine congenital malformations (BCMs) is challenging for bovine veterinary practitioners and laboratory diagnosticians as many known as well as a large number of not-yet reported syndromes exist. Foetal infection with certain viruses, including bovine virus diarrhea virus (BVDV), Schmallenberg virus (SBV), blue tongue virus (BTV), Akabane virus (AKAV), or Aino virus (AV), is associated with a range of congenital malformations. It is tempting for veterinary practitioners to diagnose such infections based only on the morphology of the defective offspring. However, diagnosing a virus as a cause of BCMs usually requires laboratory examination and even in such cases, interpretation of findings may be challenging due to lack of experience regarding genetic defects causing similar lesions, even in cases where virus or congenital antibodies are present. Intrauterine infection of the foetus during the susceptible periods of development, i.e. around gestation days 60–180, by BVDV, SBV, BTV, AKAV and AV may cause malformations in the central nervous system, especially in the brain. Brain lesions typically consist of hydranencephaly, porencephaly, hydrocephalus and cerebellar hypoplasia, which in case of SBV, AKAV and AV infections may be associated by malformation of the axial and appendicular skeleton, e.g. arthrogryposis multiplex congenita. Doming of the calvarium is present in some, but not all, cases. None of these lesions are pathognomonic so diagnosing a viral cause based on gross lesions is uncertain. Several genetic defects share morphology with virus induced congenital malformations, so expert advice should be sought when BCMs are encountered.The online version of this article (doi:10.1186/s13028-015-0145-8) contains supplementary material, which is available to authorized users. Bovine congenital malformations (BCMs) usually occur sporadically although some herds may experience an increased number of cases over time. Farmers may in such situations seek veterinary advice regarding the cause of the malformation. Veterinarians may also be faced with the challenges of BCMs during their examination of cattle, e.g. in association with dystocia. A pathomorphological diagnosis can usually be established in cases of central nervous system (CNS) and musculoskeletal malformations as such disorders are often readily recognisable. However, farmers and their veterinarians are usually much more interested in the aetiological diagnosis, which is generally more difficult to establish under field conditions.The diagnosis of BCMs, especially virus-induced congenital malformations (VICMs), may be achieved by submitting appropriate materials to diagnostic laboratories, although this is rarely done systematically due mainly to the costs of shipment of specimens and laboratory analyses. It is tempting for the veterinary practitioner to offer an aetiological diagnosis based only on the clinical findings and gross lesions. However, the diagnosis of BCMs is a specialised field where significant knowledge of genetic and teratogenic syndromes may be required to achieve an accurate diagnosis. Detailed knowledge on BCMs is generally not a key competency of veterinarians in large animal clinical practice as they only face these disorders sporadically. Suggesting an aetiological diagnosis from limited observations and insufficient knowledge on the causes and morphology of the wide spectrum of bovine congenital syndromes, may lead to misdiagnosis.A particular challenge for veterinarians and laboratory diagnosticians is the differentiation of “true” VICMs  from BCMs where exposure to a potential teratogenic virus during foetal development has occurred but the infection did not cause the pathology observed. Precolostral calves having VICM may either have congenital antibodies against a teratogenic virus or be virus-positive, as determined by viral nucleic acid detection [polymerase chain reaction (PCR)] or culture. Being antibody- or virus-positive does not necessarily imply that the virus caused the malformation as the outcome of a foetal infection with a teratogenic virus depends on factors such as the foetal age at exposure and the teratogenic properties of the virus strain in particular –4. Some This paper aims to provide an overview of the basic knowledge required by bovine practitioners and laboratory diagnosticians on the gross morphology of VICM in cattle. This is supported by general information on the viruses, pathogenesis, etc. to achieve a better understanding. Diagnostic challenges are presented together with details on known genetic diseases of cattle sharing major features with VICMs. The most important teratogenic viruses of bovines, especially in a European context, are reviewed to assist readers to recognise and investigate VICMs in cattle, ensuring these disorders are correctly differentiated from BCMs due to other causes.http://www.ncbi.nlm.nih.gov/pubmed) using the terms “cattle, bovine, malformation, congenital” combined with viral names: Schmallenberg virus, blue tongue virus, bovine virus diarrhea virus, Akabane virus, and Aino virus. The title and abstract of the obtained hits were evaluated and articles referring to the gross morphology were obtained and assessed in detail. In addition, our own archives were used as a source of additional information. Our extensive experience with BCMs and VICMs were used to critically evaluate the literature and our personal photo archives were used to illustrate the characteristics of VICMs in cattle.This critical review is based on a search in PubMed , Schmallenberg virus (SBV), blue tongue virus (BTV), Akabane virus (AKAV), or Aino virus (AV), is associated with a range of congenital malformations of which the most prominent develop in the CNS, especially in the brain, with accompanying lesions frequently developing in the musculoskeletal system. A brief overview of the lesions is presented in Table Pestivirus of the family Flaviviridae. Based on antigenic and genetic characteristics, two genotypes (species), BVDV-1 and BVDV-2, have been identified. Within each genotype, two biotypes characterized by their ability to damage cultured cells have been found, i.e. cytopathogenic (cp) and non-cytopathogenic (ncp) biotypes [Bovine virus diarrhea virus belongs to the genus biotypes .The most commonly reported BVDV genotype is BVDV-1 and, although BVDV-2 strains in general are thought to be more virulent, both BVDV genotypes can induce the same spectrum of disease manifestations . BVDV geThe pathogenesis of BVDV infection in cattle is complex and infections with the ncp biotype belonging to either BVDV-1 or -2 genotypes occurring before and during gestation may result in a wide range of clinical and pathological presentations . The outThe most frequent brain malformation after foetal spontaneous or experimental infection with BVDV is cerebellar hypoplasia –21. MacrIn hydranencephaly, the cerebral hemispheres are transformed into large sac-like fluid-filled cavities, which are covered by thin, partly translucent leptomeninges Fig. b, c. In In cases of hydrocephalus, the lateral ventricles are slightly or markedly dilated with accumulation of clear cerebrospinal fluid Fig. d. Macrosex vacuo, i.e. the dilation of the ventricles is due loss of periventricular parenchyma. Both in spontaneous and experimental cases of bovine BVDV infection, hypomyelination of brain and spinal cord white matter has been described [Microencephaly, characterized by subnormally-sized cerebral hemispheres occurs less frequently , 23. Porescribed –28. In cCongenital ocular lesions after spontaneous or experimental BVDV infection of bovine foetuses are commonly associated with cerebellar hypoplasia known as oculo-cerebellar syndrome , 18, 19.Orthobunyavirus, characterized by three genomic ssRNA segments, and has been classified within the species Sathuperi virus of the Simbu serogroup [Schmallenberg virus is a member of the genus erogroup . SBV waserogroup .Several orthobunyaviruses of the Simbu serogroup show teratogenic properties in ruminants and transplacental orthobunyaviral infections, resulting in congenital malformations, have been reported in Asia, Australia, Africa and the Middle East –34.Culicoides spp. biting midges [Like most orthobunyaviruses, SBV is transmitted by g midges –37 and ag midges –40. Althg midges . Within g midges , 41.Hosts are infected during the vector active period and naïve adult cattle show none or only mild clinical signs such as transient fever, diarrhea, anorexia and reduced milk production during 3–11 days –43. InfeLike most viral infections, transplacental transmission of SBV does not elicit placentitis and most malformed calves are stillborn at term. The weight of malformed calves is significantly less than normal with a correlation between the body mass deficit, the severity of the malformations and the amount of skeletal muscles .Malformations of the vertebral column and arthrogryposis, which are regarded secondary to dysplastic CNS lesions, are the most conspicuous exterior gross lesions in affected calves Fig. a. The moThe axial and appendicular bones are normally founded and the vertebral malformations and arthrogryposis develop due to imbalance in foetal muscular activity (flexor vs. extensor muscles) occurring secondary to loss of neurons. In the brain and spinal cord, neuron loss is reflected in both the loss of descending tracts in the ventral spinal cord white matter and in the loss of ventral horn motor neurons in the spinal cord grey matter and in deficient ventral spinal nerve roots. Loss of spinal cord tissue, grossly visible in severely affected cases as a small dorsoventral flattened spinal cord (micromyelia), accounts for failure of normal skeletal muscle development due to denervation of the axial and appendicular musculature and hence to vertebral column malformation and arthrogryposis, respectively. Skeletal muscle tissue of arthrogrypotic limbs is hypoplastic, reflecting the extent of spinal cord dysplasia in the cervical and lumbar intumescences , 50.Occasionally, a flattened skull and brachygnathia inferior are found. Deformation of the skull is assessed in combination with brain malformations. Neuroparenchymal loss in the brain may be visible by more or less severe defects in the grey and white matter of the cerebrum, cerebellum, midbrain, brainstem and medulla. Cerebral defects range from microencephaly, porencephaly in combination hydrocephalus and lack of gyri  to hydranencephaly with only a small rim of cortical tissue left Fig. b. CerebeOrbivirus within the Reoviridae family, with a genome, consisting of 10 dsRNA segments. Currently, 26 serotypes are distinguished, determined by variations of the outer-capsid protein VP2, which induces specific virus-neutralising antibodies [Culicoides spp. biting midges, transmitting BTV between ruminant hosts. The regional presence of the various BTV serotypes is associated with particular Culicoides spp. [Bluetongue virus, the causative agent of the disease bluetongue (BT) in ruminants, is a member of the genus tibodies . The glodes spp. . BT mostdes spp. , 53. Aftdes spp. .Seasonal outbreaks of BT due to BTV serotypes 1, 2, 4, 9 and 16 have appeared since 1998 in the Mediterranean Basin at the boundaries of the temperate zone . HoweverExperimental infections with various BTV-serotypes, using direct inoculation in bovine foetuses, have demonstrated that infection early in gestation, i.e. approximately between GDs 70 and 130, results in brain malformations. Infection during this time window causes hydranencephaly, occasionally combined with cerebellar defects, or leads to porencephaly and hydrocephalus . A seronIn cattle transplacental transmission with high rates ranging from 16 %  to 35 % Teratogenic BTV affects neuronal and glial precursor cells in the brain. Furthermore, vascular injury and infarction in the cerebrum may contribute to cerebral defects , 70. FoeLesions in aborted or stillborn bovine foetuses associated with spontaneous foetal BTV-8 infections mainly consist of hydranencephaly, while porencephaly is observed less frequent. This probably reflects the foetal gestational age at the time of infection as seen in experimental infections . Both leIn liveborn intrauterine BTV-8 infected calves, surviving for up to several months, the dominating lesion is hydranencephaly Fig. , althougIntrauterine BTV-8 infections do not cause significant lesions to the brainstem, medulla and spinal cord and hence are not associated with musculoskeletal disorders such as arthrogryposis and vertebral column malformations.Orthobunyavirus, and are the most pathogenic members of the Simbu serogroup in the family Bunyaviridae. Their principal vectors are Culicoides spp. biting midges, particularly Culicoides brevitarsis. AKAV was first suspected of causing congenital abnormalities in 1974 in Japan, with the distinct syndromes of hydranencephaly and arthrogryposis, sometimes accompanied by other abnormalities including abortion, stillbirth, polioencephalomyelitis and possibly microencephaly, frequently observed in parts of Australia [The arthropod-borne Akabane virus (AKAV) and Aino virus (AV) are members of the genus ustralia –75. AV iustralia , as is tustralia .AKAV is widely distributed in tropical and subtropical regions of Australia where it regularly infects young female cattle prior to gestation. Thus clinical signs are rarely observed in these endemic zones above the so-called “brevitarsis line” , Australia resulted in the identification of five groups of foetal abnormalities, corresponding to the gestational age of infection, from late (group 1) to early (group 5) . Group 1C. brevitarsis is suspected of over-wintering in protected valleys, with peak activity in the summer period causing infection with AKAV usually before or during the mating period so that heifers are immune at or shortly after joining. However, outbreaks of hydranencephaly and arthrogryposis occur when naïve pregnant animals are exposed to the virus, either by “spill-over” of the insect vectors from the endemic geographic areas of the coast to further inland where naïve cattle reside, or when naïve pregnant animals are transported from a non-vector area into AKAV endemic areas for agistment and other purposes, particularly during drought [A prospective study in the Hunter Valley coastal region of NSW in eastern subtropical Australia, enabled an understanding of the sequence of lesions induced in the bovine foetus under field conditions . In this drought , 79. ChaC. brevitarsis [In summary, AKAV outbreaks have been recorded commonly in coastal NSW in eastern Australia and occasionally beyond in eastern Australia as a result of movement of pregnant cattle into the endemic region or movement of insect vectors beyond the AKAV endemic region. Seroconversion and disease are strongly linked to the presence of vitarsis .As with other teratogenic viruses, the pathogenic outcome of infection depends on the age of foetal exposure. In cattle, infection between GDs 79 and 104 results in hydranencephaly, whereas exposure at GDs 103-174 causes arthrogryposis, with focal Wallerian-type degeneration in the brain and spinal cord  and adulCalves with congenital malformation syndromes of non-viral cause may be exposed to a potential teratogenic virus during foetal development by accident. These calves are thus born with evidence of congenital infection, confirmed by either demonstration of congenital antibodies by serology or virus infection by culture or PCR. A number of such cases have been published as observational reports and also appear in diagnostic laboratory surveys and reports. A fine example is the diagnosis of Inherited Congenital Myoclonus  in a neonatal Poll Hereford calf that presented as stillbirth in an experimental breeding herd for this disease, but the calf had also been infected by AKAV infection that resulted in severe hydranencephaly . At necrA list of similar examples is likely to be exhaustive and difficult to access as such cases do not necessarily appear in a literature search. However, some cases have been published as regular scientific articles and are summarized here to provide an overview of BCM syndromes found in calves exposed to teratogenic virus, but scientific evidence of a causal association is lacking in all cases.Perosomus elumbis This congenital syndrome is characterised by lack of the lumbar, sacral and coccygeal spinal cord and the corresponding vertebrae. The caudal half of the calf consequently consists of a soft tissue sack enclosing the abdominal organs and with dysplastic arthrogrypotic hind limbs attached to a malformed pelvis [d pelvis . A case d pelvis . The cauCyclopia The most obvious finding in cyclopia cases is the presence of a single large eye located in facial midline. This defect has been reported in a calf with congenital antibodies against BVDV [nst BVDV . Bovine nst BVDV . The prenst BVDV , so BVDVCongenital bone fragility Congenital bone fragility was reported in a calf with persistent BVDV infection. Unilateral fractures of the metaphyseal region of femur and tibia was observed, but histology revealed a generalized segmental disruption of trabecular modelling, including loss of trabecular bone [lar bone . Growth lar bone . Howeverhttp://omia.angis.org.au/home/. However, it is important to remember that even though some teratogenic and genetic causes of BCMs are known, a substantial number of at present unknown aetiologies most likely exists and remain to be diagnosed. Practitioners and diagnosticians require awareness of this dilemma as it may lead to misdiagnosis of the cause of BCMs and in case of inherited syndromes, lead to an unrecognised spread of the defect within a herd or breed. Expert advice should be sought and calves submitted for extended diagnostic investigation where surveillance programs for genetic diseases are in place.Bovine VICMs are most readily characterized by a rather uniform spectrum of lesions in the CNS such as hydranencephaly and cerebellar hypoplasia. However, neither these lesions, nor the associated localized or generalized arthrogryposis of the axial and appendicular skeleton seen in SBV and AKAV infected foetuses are pathognomonic. Malformations of the CNS and the musculoskeletal system are the most commonly recorded congenital defects in cattle  and in aA number of inherited congenital syndromes that closely resemble SBV-, AKAV- and AV-induced generalized arthrogryposis and distortion of the skeleton have been reported. The molecular background of these syndromes differs, but disturbed neuro-muscular development appears central in the pathogenesis. Inherited syndromes of tetramelic arthrogryposis and palatoschisis have been reported, especially in the Charolais and Hereford breeds e.g. –93, althA specific inherited defect belonging to the “arthrogryposis multiplex congenita syndrome” complex, originally termed “Curly calf syndrome”, has been reported in Angus cattle . AffecteArachnomelia, an inherited syndrome recognized in the Brown Swiss and Simmental breeds, share some similarities with VICMs, such as kyphosis, scoliosis and tetramelic arthrogryposis. However, the long bones of the limbs are longer and thinner than normal and the maxillae and frontal bones are deformed , 97; lesGenetic disorders resembling BVDV- and BTV-induced CNS lesions, identified as malformations mainly restricted to the CNS without associated arthrogryposis, do occur, although few have been reported. The worldwide occurrence of BVDV and its well-established association with cerebellar hypoplasia has probably out-numbered the sporadic cases of non-infectious aetiology, so cases of cerebellar hypoplasia are generally considered to be a VICM by practitioners and diagnosticians in regions where this infection is endemic. Cases of cerebellar hypoplasia in Hereford  and ShorNeuropathic hydrocephalus is an autosomal recessive defect in Angus cattle that is characterised by foetal growth retardation and an extremely severe hydrocephalus with doming of the calvarium. The malformed skull causes dystocia and drainage of the cerebrospinal fluid is required to achieve vaginal delivery . The cauOther likely genetic defects characterised by hydrocephalus such as two distinct syndromes in Hereford cattle have been reported in older literature . Howeverforamen magnum during decapitation or a domed calvarium often indicates the presence of hydrocephalus or hydranencephaly. Palatoschisis has not been reported in association with VICMs. Ocular lesions such as retinal dysplasia and retinitis in combination with brain malformation and/or encephalitis are regarded as highly indicative for foetal BVDV exposure. However, the diagnosis of VICMs in cattle based on only gross lesions is insufficient even in cases with arthrogryposis and is not recommended unless the malformed calves have characteristic lesions and occur during a well-defined outbreak, preferably supported by serological or virological evidence of recent infection.Arthrogrypotic syndromes are usually readily recognized by clinicians but can be difficult to differentiate aetiologically. However, malformations that only involve the CNS are more challenging to diagnose in large animal practice, especially if the shape of the calvarium is normal. Hydrocephalus, hydranencephaly, porencephaly, and cerebellar hypoplasia may be the only gross lesions present in bovine VICMs, especially in BVDV-, BTV- and AKAV- exposed foetuses. Clinical signs vary depending on the extent of the lesions, but are usually non-specific and affected calves may live for several months. Appropriate diagnostics requires examination of the brain and although the skull can be opened in the field, detailed examination usually requires submission to a laboratory. Oozing of larger volumes of clear fluid through the Veterinary practitioners should seek advice from experts in bovine teratology when encountering BCMs. Submission of materials should ideally consist of the entire calf, maternal blood samples and anamnesis, including herd status for viral diseases, vaccination and pedigree data. Euthanasia by the use of a captive bolt pistol should be avoided and submission of fresh, if possible cooled, rather than frozen cadavers is preferred. If submission of the carcass isn’t feasible, photos and description of gross lesions and submission of the head, lung, spleen and precolostral blood sample/pleural effusion sample may be used to confirm VICMs, but access to the entire carcass is needed to perform a thorough investigation.The geographical region may be of some use for the practitioner to rule out certain VICMs. BVDV has been eradicated in some countries and the occurrence of the vectors of SBV, BTV, AKAV and AV may be restricted to certain regions or habitats, although the recent transmission of BTV and SBV in Europe indicates that such preconceived notions can be challenged.The season at which malformed calves are delivered may also indicate if an insect vector transmitted virus such as SBV, BTV or AKAV may be implicated. Fullterm malformed offspring may be delivered in a wide time-frame or “window”, from around 80–220 days after foetal exposure, so the vectors must have been active at that time of gestation. Although the risk of transmission is higher during periods with high environmental temperatures, transmission may occur even during periods with temperatures of 5–9 °C , which iThe presence of maternal antibodies may indicate a specific viral aetiology in regions where the infection is not enzootic and where vaccination hasn’t been performed. As foetal lesions develop several months before parturition, sufficient time exists from the onset of infection to delivery of malformed offspring, for virus-specific antibodies to be produced. Congenital antibodies in either precolostral serum or pleural/pericardial effusion sampled during necropsy are consistent with intrauterine infection of the foetus after it has become immunocompetent, i.e. around GD 80–90 , 102. HoExamination of pedigrees for inbreeding is sometimes used as a practical tool to differentiate between inherited and non-inherited syndromes, including VICMs. However, again, this may be unreliable as although inbreeding may indicate the possibility of a genetic aetiology, it does not preclude other causes of the BCM. Furthermore, a number of described BCMs of genetic aetiology have occurred in large numbers without readily identifiable inbreeding having occurred, such as a common ancestor being present within 2–3 generations .ex vacuo), porencephaly, and cerebellar hypoplasia, although the latter lesion is rarely observed in AKAV infection. Recognition of these lesions often requires inspection of the brain, but may in some cases be associated with a domed calvarium, usually indicating the presence of hydrocephalus. As SBV, AKAB and AV also causes damage to the spinal cord leading to disturbed innervation of the skeletal musculature, these VICMs may be recognized clinically as arthrogryposis of the limbs and deviation of the vertebral column, such as torticollis and scoliosis.VICMs caused by BVDV, SBV, BTV, AKAB and AV mainly involve the CNS as the developing brain is generally vulnerable to the teratogenic properties of these viruses from around GD 60–180 but with individual viral time preferences. These viruses may cause necrosis of the neuroparenchyma that may be exacerbated by the foetal inflammatory response, leading to disturbance of tissue integrity, grossly recognized as hydranencephaly, hydrocephalus (None of the gross lesions are pathognomonic for VICMs as a number of genetic disorders resemble the congenital teratogenic syndromes. It is therefore recommended to seek advice from experts in bovine teratology and genetic diseases before diagnosing VICMs in practice and if possible, submit the animal for laboratory examination. As the association of a suspected VICM as the cause of a BCM is best confirmed by demonstration of lesions known to be associated with viral infection, supported by demonstrating the presence of congenital antibodies or virus indicative of foetal infection, the diagnosis of a congenital syndrome should be done carefully and be evidence based.


Parasites and hosts can experience oscillatory cycles, where the densities of these interacting species dynamically fluctuate through time. Viruses with different replication strategies can also interact to produce cyclical dynamics. Frequent cellular co-infection can select for defective-interfering particles (DIPs): “cheater” viruses with shortened genomes that interfere with intracellular replication of full-length (ordinary) viruses. DIPs are positively selected when rare because they out-replicate ordinary viruses during co-infection, but DIPs are negatively selected when common because ordinary viruses become unavailable for intracellular exploitation via cheating. Here, we tested whether oscillatory dynamics of ordinary viruses were similar across independently evolved populations of vesicular stomatitis virus (VSV). Results showed identical cyclical dynamics across populations in the first 10 experimental passages, which transitioned to repeatable dampened oscillations by passage 20. Genomic analyses revealed parallel molecular substitutions across populations, particularly novel mutations that became dominant by passage 10. Our study showed that oscillatory dynamics and molecular evolution of interacting viruses were highly repeatable in VSV populations passaged under frequent co-infection. Furthermore, our data suggested that frequent co-infection with DIPs caused lowered performance of full-length viruses, by reducing their population densities by orders of magnitude compared to reproduction of ordinary viruses during strictly clonal infections. Evolutionary ecology concerns interactions between biological populations and their biotic and abiotic environments. Viruses necessarily interact with host organisms , whose mde novo  that are observed in natural virus populations, and seen in clinical samples . Errors Whereas genome replication errors can cause DIP mutants to spontaneously occur during most  cellular infections , only frin vitro experiments have classically demonstrated the expected oscillations when viruses are passaged under frequent co-infection  within 24 h, on permissive cell types such as baby hamster kidney (BHK-21) cells. Serial passage of VSV under high multiplicity of infection  allows frequent virus co-infection, conditions that promote strong selection for spontaneous DIP mutants in VSV populations  as a model to study the repeatability of oscillatory dynamics, across virus populations that were independently passaged under frequent co-infection. VSV is a well-characterized arthropod-borne virus (arbovirus) in the family Rhabdoviridae, which naturally infects various species of insects and mammals, including occasionally humans . VSV hasulations .MOI. Results showed that the oscillatory dynamics of ordinary viruses were highly similar across the populations, characterized by high-amplitude oscillations between passages 0 and 10, and followed by dampened oscillations between passages 10 and 20. In addition, full-genome consensus sequencing of mixed population samples showed that parallel molecular evolution occurred across the five populations, and that most of these changes fixed early in the study. Our results suggested that negative frequency-dependent selection experienced by ordinary and DIP genotypes of VSV was highly similar across our study populations, evidenced by ordinary-virus dynamics and molecular substitutions that were repeatable and parallel across independently evolving RNA virus populations.We used a single ancestral VSV strain to initiate five replicate virus populations, which were independently passaged for 20 consecutive days on BHK cells at high L-glutamine (Gibco). Hosts for infection were baby hamster kidney (BHK-21) cells, kindly provided by E. Domingo (University of Madrid) and grown in 25-cm2 tissue-culture flasks at 37°C, 95% relative humidity, and 5% CO2 atmosphere to achieve confluent monolayers of ∼105 cells/cm2. Low passage variants of cell lines  were used for all experiments.All viruses were derived from a clonal isolate of a low-passage, laboratory-adapted strain of Mudd Summer VSV Indiana serotype that was kindly provided by E. Domingo (University of Madrid). Hereafter, we refer to this clone as wild-type (WT). Viruses and host cells were cultured in Dulbecco’s modified Eagle’s minimum essential medium  with 5% heat-inactivated newborn bovine calf serum  and 1% penicillin-streptomycin-8 plaque-forming units (PFU) per mL] to create a stock lysate that was used to found five replicate populations (L1 through L5). Confluent monolayers of BHK-21 cells were infected at an initial ratio of 10 viruses per cell (MOI ≈ 10). This MOI created a high probability for multiple virus particles to co-infect individual cells, which would selectively favor spontaneously arising DIPs to gain an intracellular fitness advantage over ordinary viruses  were estimated as averages of counts from dilutions in two separate wells.Viral populations were sequenced at three time points in their evolution: the WT virus used to found the replicates (day 0), and the five replicate populations on days 10 and 20. Genomic RNA was extracted from 140 μL of frozen viral stock using Qiagen QiAmp mini viral spin kit. cDNA was generated by reverse transcription with Superscript II (Invitrogen), using random hexamer primers. Virus was polymerase chain reaction (PCR) amplified using GoTaq polymerase (Promega), and VSV specific primers (Supplementary Table Fast Fourier Transform (FFT) analysis transforms a function or set of data from the time or sample domain to the frequency domain. This means that the Fourier transform can display the frequency components within a time series of data. By inputting the virus titer data into the FFT function in MATLAB , we obtained an estimated periodicity of the oscillations (number of passages constituting a cycle) in each virus population.10 PFU/ml data in a one-way ANOVA that included population as a model effect.To analyze the effect of evolved VSV population on the estimated titer of the ordinary virus subpopulation, we included the log3H]uridine (33 mCi/ml) in the presence of actinomycin D (10 mg/ml). Cells were harvested, and total cytoplasmic RNA was extracted and analyzed as described previously (RNA synthesis was examined by direct metabolic labeling of cells 5 h post infection. Cells were exposed for 6 h to .During the course of the experiment, all five experimental populations showed a trend of decreased titers (PFU/mL) of ordinary viruses as passage number increased. For each lineage, the average size of the full-length virus subpopulation was estimated by calculating the harmonic mean of its observed titer estimates. Hence, the estimated harmonic means equaled: L1: 4.7×1010 titer data of ordinary viruses using FFT analysis, which estimated the periodicity of oscillations (number of passages constituting a cycle) in each population . Results showed that the number of passages constituting a cycle differed somewhat across the five experimental populations: L1: 2.2 passages/cycle; L2: 2.3 passages/cycle; L3: 3.3 passages/cycle; L4: 3.6 passages/cycle; L5: 4.4 passages/cycle.To further compare population dynamics across replicate lineages, we examined the log10 titer) of full-length viruses. Alternatively, the inability of ordinary viruses to achieve high titers similar to that of WT VSV (≈108 PFU/mL) could have been driven wholly by evolved changes in the full-length viruses. In particular, evolution of ordinary virus resistance to DIPs could have coincided with reduced ordinary virus fecundity; this evolutionary growth trade-off would indicate inability of full-length viruses to simultaneously evolve DIP resistance while maintaining robust growth. If the absolute fitness of an evolved ordinary virus at MOI = 0.01  was similar to that of the WT ancestor grown at low MOI , this outcome would suggest that the observed low productivity of full-length viruses on certain passage days  was due to ecological interactions with cheating DIPs. However, if the absolute fitness of an evolved ordinary virus measured at MOI = 0.01 was less than the ancestor (≈108 PFU/mL), this result would suggest that its decreased fecundity was a trait that evolved during the course of the experiment.The observed low productivity of ordinary viruses could be due solely to ecological interactions with DIPs, which are expected to deleteriously reduce the absolute fitness (logMOI = 0.01). We chose to examine ordinary virus samples isolated at five time points during the passage series belonging to population L2. In particular, we isolated samples from passages where the observed titers were relatively low , because these time points presumably coincided with successful cheating by DIPs that drove down the observed frequencies of ordinary viruses in the population. We used plaque purification to isolate a single clone at random from each of the five time points in population L2’s passage history. Each of these clones was used to infect BHK-21 cells at MOI = 0.01. After 24-h incubation, we quantified the resulting virus progeny using our standard plaque assay . Results  showed that all five of the ordinary virus clones had the capacity to grow to high titers (≈108 PFU/mL), comparable to results for the WT ancestor grown under identical MOI = 0.01 conditions. The data strongly implied that the observed low titers in population L2 were not due to evolved changes in ordinary virus productivity, but were rather due to ecological interactions between the ordinary viruses and the DIPs. However, we do not dismiss the possibility that some evolutionary changes occurred in the full-length viruses in our study, and we present and discuss these data below.To examine these competing hypotheses, we used plaque assays  to measure the productivity of representative ordinary viruses drawn from an evolved population, under conditions where virus particles must infect cells on their own  of the study, and to estimate whether DIPs were similar across the independently passaged populations. We first obtained a sample of the supernatant from each endpoint population. These samples were then amplified at MOI = 10 on BHK-21 cells, and subsequently subjected to RNA extraction followed by labeling with [3H]uridine . Results . These preliminary data suggested that the independently passaged VSV lineages had converged in terms of the majority DIPs present at passage 20, at least in the four populations that yielded results.Although the oscillatory dynamics in Sanger sequencing can be used to generate a consensus sequence for an evolved virus population, which usefully indicates the alleles that distinguish the evolved population from its ancestor. Although Sanger sequencing cannot determine whether observed mutations exist on a common genetic background , the method can be used to efficiently compare across replicate populations to test whether treatment conditions tended to select for parallel mutations across independently evolved lineages e.g., . We emplMOI environment – and presumably DIP presence – exerts strong enough selection for the VSV consensus genome sequence to change in the evolutionary time allowed, especially due to beneficial mutations that spontaneously appear and spread to fixation. Twenty passages in a novel environment, such as a new tissue-culture host or incubation temperature, are sufficient for the VSV consensus sequence to change via fixation of adaptive mutations  could prevent natural selection from operating efficiently, thus potentially allowing genetic changes to accrue in ordinary virus and/or DIP subpopulations through genetic drift. In summary, we expected that both adaptive and non-adaptive mutations could increase in frequency during the experiment, making these alleles detectable through consensus population sequencing.Because VSV is a single-stranded RNA virus with a typically high mutational error rate, we expected that 20 passages of experimental evolution would be sufficient to detect polymorphic loci and/or fixed substitutions that separated the evolved lineages from their common ancestor virus, and possibly from one another. Importantly, this expectation assumes that the high ns e.g., . In contns e.g., . FinallyFigure 1). In particular, we defined the ordinary virus-population dynamics occurring between days 0 and 10 as the ‘early phase,’ where oscillations showed relatively greater amplitude than between days 10 and 20, defined as the ‘late phase.’ Foremost, we compared the consensus sequence of each lineage across these two phases to test whether (i) observed molecular changes separated the evolved lineage from the common ancestor, indicating that molecular evolution via selection and/or drift occurred in the experiment, and (ii) whether different alleles dominated the population in the early versus late phase. In theory, the differing oscillatory dynamics in the early and late phases could be entirely driven by ecological interactions between full-length and DIP viruses that appeared in the early phase. If true, we would expect that a lineage’s consensus sequence at day 10 would be highly similar  to its sequence at day 20, perhaps with some early-phase polymorphic loci transitioning to fixed substitutions in the later phase. Alternatively, we might observe highly different consensus sequences in the early versus late phases, suggesting that the differing oscillatory dynamics were driven (at least in part) by both ecological interactions as well as evolutionary interactions, such as extended co-evolutionary changes between ordinary virus and DIP genotypes. Last, overall comparisons in molecular data among lineages  were used to further gage the degree of repeatability in our study, especially occurrence of parallel evolution.To examine the occurrence of molecular evolution and evidence for parallelism in our study, we focused our attention on the two phases of the experiment that clearly differed in the observed oscillatory dynamics within the evolving lineages: days 0 to 10, and days 10–20  versus appears between days 10 and 20 (late phase). In general, most of the observed mutations occurred in the early phase of the study; across all five lineages, 42 mutations were detected by day 10 and only 14 additional mutations separated the day 20 consensus sequences from their day 10 counterparts . Moreover, the vast majority of these additional mutations (11 of 14 changes) were polymorphic at day 20. Overall, these comparisons indicated that most of the molecular evolution in our study had occurred in the early phase when oscillatory dynamics showed relatively high amplitudes .Results showed that the founding VSV ancestor in our study contained 102 mutations separating it from the canonical annotated VSV Mudd-Summers strain Indiana serotype (GenBank #NC_001560.1). In itself, this result is not overly surprising because the two strains have been used in separate laboratories for decades, under unknown but presumably non-identical culture conditions. More importantly for the current study, we identified a total of 93 allele substitutions and/or polymorphisms that separated the consensus sequences of the five evolved lineages from their common ancestor. These data are summarized in Figure 2). Because VSV is an ssRNA virus, we note that synonymous mutations can affect the secondary structure of the virus and thus potentially impact fitness. One fixed, synonymous mutation occurred in the gene for the VSV phosphoprotein in all five lineages by day 10 (nucleotide position 2151), and was still present at day 20. In the VSV glycoprotein gene, all five lineages showed a non-synonymous mutation at position 3846 (D→N) and at locus 4180 (T→K); one or both of these mutations had either fixed early in an evolved lineage , late in the experiment , or remained polymorphic throughout the study. The Large protein showed the largest number of parallel substitutions across the five lineages . Here, all lineages showed the same five mutations , and most of these substitutions fixed by day 10 (early phase). Interestingly, lineage L2 fixed two of these mutations by day 10, but then reverted back to the ancestral sequence by day 20.Our molecular data also indicated that abundant parallel evolution occurred across the five lineages . However, these changes tended not to bridge the early and late phases of the experiment, and were often observed to be polymorphic. For these reasons, the minority non-parallel substitutions in our study may be considered less impactful, but this tentative conclusion warrants further investigation.Aside from the large number of parallel mutations across lineages, we observed nucleotide changes specific to a single lineage, or shared among a subset of the replicate lineages (MOI (absence of DIPs) showed that these strains retained robust reproductive ability. Changes in oscillatory dynamics (early versus late phases of the experiment) and patterns of molecular substitutions were highly similar across the five VSV lineages. Altogether, these results suggested that ecological interactions between ordinary viruses and cheaters were extremely repeatable across independently passaged lineages, and that any contribution of virus evolutionary changes to the observed dynamics within lineages was dictated by molecular substitutions occurring relatively early in virus evolution.Our study examined the repeatability of population dynamics and molecular substitutions across five independently passaged VSV lineages, cultured under frequent virus co-infection that selects for evolution of DIPs (virus cheaters). After 20 days of serial passage that facilitated negative frequency-dependent selection between ordinary viruses and DIPs, all five experimental VSV populations displayed a cycling pattern consistent with theoretically predicted effects of DIPs on full-length virus densities . A molecOur study was motivated by efforts to better understand fundamental antagonistic virus–virus ecological interactions . Only a MOI conditions that do not foster selection for cheater viruses (e.g., Previous empirical  and theoes e.g., . Our FFTAlthough consensus sequencing offers a convenient ‘snapshot’ of the allele diversity in a virus lineage, these gross changes necessarily omit key details regarding the genetic backgrounds on which mutations arise and accumulate. Nevertheless, our comparison of consensus sequences in the early versus late phases of the experiment, both within and across populations, strongly suggested that molecular evolution occurred and that it was highly repeatable. We concluded that effects of genetic drift did not generally overwhelm those of natural selection, because abundant parallel substitutions were observed and these could not have occurred by chance . Thus, tMOI on the BHK host cells. Previous work suggested that ordinary viruses could evolve specific resistance to a particular DIP (e.g., The population dynamics observed in our experimental populations were reminiscent of those observed in macro-organism predator–prey and host–parasite systems, because predator/prey and parasite/host relative densities can oscillate in a similar fashion . In thesIP e.g., , and theIP e.g., . Thus, aEW: main execution of genome sequencing and phenotypic analysis of evolved virus populations; writing and editing of manuscript. NM: design and execution of evolution experiment; writing and editing of manuscript. BW: sequence and other data analysis; editing and commenting on manuscript. VB: execution of experiments confirming presence of DIPs. SW: design of experiments confirming presence of DIPs; feedback on manuscript. PT: design and interpretation of experiments; writing and editing of manuscript.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.


In the Netherlands, screening for tick-borne pathogens is performed using real time PCR on DNA extracted from ticks, followed by sequencing of positive samples, thereby disallowing identification in case of double-infections. Until recently, testing was performed with several reverse line blots (RLBs), each containing ~10 different probes. Advantages of RLBs are the capabilities to differentiate between bacterial species and to detect double infections, disadvantages are that RLBs are fastidious, elaborate, and time-consuming.The aim of this work was to 'test drive' bead-based suspension arrays for the rapid detection and identification of tick-borne Borrelia species. The platform used is the flow cytometry-based xMAP technology of Luminex, in which fluorescent nanoparticles (beads) are used to build multiplex assays simply by mixing different bead sets that are covered with specific probes. Each bead set contains a different ratio of red and infrared fluorophores, which enables identification of beads that pick up a third fluorophore by hybridization to a labelled target molecule.Using standard chemistry, aminated oligonucleotides were covalently linked to carboxylated Luminex beads, resulting in bead sets with general and specific probes for the 23S-5S intergenic spacer (IGS) of Borrelia species. Presence of probes on the beads was verified by hybridization of complementary biotinylated oligonucleotides in presence of a mixture of 1000 beads per bead set, using a 30 minute incubation at 37°C in a NaCl/Tris buffer, followed by labelling with streptavidin-phycoerythrin. Of each mixture, a minumum of 100 beads per bead set were investigated by flow cytometry on a dedicated Luminex instrument.~20% were found to carry one or more Borrelia species (resp. 15 and 5%). The most prevalent species was B. afzelii, followed by B. burgdorferi sensu stricto, B. garinii, B. sensu lato (unidentified) and B. valaisiana. These results show the viability of bead-based suspension arrays for (relatively) rapid detection and identification of multiple species, but at this stage need verification by a 'gold standard'. Currently, confirmation of these results is being performed by next generation sequencing.To evaluate the suspension array, DNA of nearly 1000 ticks was isolated and used to generate amplicons with a universal PCR targeting the 23S-5S IGS of Borrelia spp. All PCR reactions were then used in a direct hybridization assay in presence of a mixture of probe-containing beads. Out of 981 ticks, this work was partly funded by the European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement Number 222633 (WildTech).


Hepatic glycogen storage diseases (GSD) underscore the intimate relationship between carbohydrate and lipid metabolism. The hyperlipidemias in hepatic GSD reflect perturbed intracellular metabolism, providing biomarkers in blood to monitor dietary management. In different types of GSD, hyperlipidemias are of a different origin. Hypertriglyceridemia is most prominent in GSD type Ia and associated with long-term outcome morbidity, like pancreatitis and hepatic adenomas. In the ketotic subtypes of GSD, hypertriglyceridemia reflects the age-dependent fasting intolerance, secondary lipolysis and increased mitochondrial fatty acid oxidation. The role of high protein diets is established for ketotic types of GSD, but non-traditional dietary interventions (like medium-chain triglycerides and the ketogenic diet) in hepatic GSD are still controversial and necessitate further studies. Patients with these rare inherited disorders of carbohydrate metabolism meet several criteria of the metabolic syndrome, therefore close monitoring for cardiovascular diseases in ageing GSD patients may be justified. To date there are 14 types of glycogen storage diseases (GSD), which are classified according to enzyme or transporter deficiency and organ distribution  complex  are associated with fasting ketotic hypoglycemia and considered relatively mild compared to GSD I because GNG is intact  lactose free formula feeds enriched with maltodextrin. In 1974 continuous nocturnal gastric drip-feeding (CNGDF) via a nasogastric tube was introduced. It was demonstrated that normoglycemia could be maintained  long-term complications Moses . For GSDIn GSD I patients, triglyceride concentration are considered the most useful parameter for chronic metabolic control. Like blood glucose concentrations, lactate concentrations fluctuate rapidly, liver enzymes are only slightly increased before initiation of dietary management and rapidly normalize, and uric acid concentrations can be influenced by allopurinol treatment. In our personal clinical experience with GSD Ia patients, increasing triglyceride concentrations can reflect decreased metabolic control, for instance when young patients outgrow their diet, by decreased compliance, during increased growth in puberty or co-morbidity like hypothyroidism. Based on an expert meeting as part of the European Study on GSD I (ESGSD I), follow-up guidelines for the frequency of routine laboratory investigations (including lipid profiles) are age dependent: age 0–3 years every 2 month; 3–20 years every 3 months; adults every 6 months  and GSD Ib (n = 4) patients hypercholesterolemia was noted in 43 % versus 25 %, whereas 98 % versus 75 % displayed hypertriglyceridemia. In this cohort, complications associated with hyperlipidaemia were rare; pancreatitis was reported in three patients and cholelithiasis in two patients. In the management guideline paper originating from 2002, serum triglyceride concentrations <6.0 mmol/L was included as a biomedical target , the rate-controlling step of mFAO. Pharmacological inhibition of the glucose 6–phosphate transporter by S4048 induces hypoglycemia in wild type mice, increased hepatic glycogen and triglyceride content, and a markedly enhanced hepatic lipogenic gene expression . Examples include the type of nocturnal management (UCCS versus CNGDF), the timing of the introduction of UCCS (at which age?), the methods by which exogenous carbohydrates are given and the extent of dietary restrictions of fructose, lactose and saccharose. Recent data from a meta-analysis of three studies suggest that UCCS was associated with significantly higher plasma cholesterol concentrations compared to CNGDF  and overtreatment . In the European Study on GSD I, about 25 % of the GSD Ia and 33 % of the GSD Ib patients under 20 years of age display BMI above the 90th percentile, interestingly highest prevalence was observed in young patients between 2 and 5 years of age.Figure From a practical point of view, increasing one macronutrient (carbohydrates) without affecting the second (protein) inevitably lowers fat intake. Given the carbohydrate deficiency and hyperlipidemia in GSD I and GSD III, increasing dietary fat is somewhat counterintuitive. However, there are two positive reports about high-fat diets in GSD III patients. The combination of synthetic ketone bodies, 2:1 ketogenic diet and high-protein was associated with reversal of hypertrophic cardiomyopathy in a 2-months-old GSD IIIa patient  in GSD I and III . For all methods, the potential risk of glycerol overload towards impaired GNG in GSD I patients is unknown. Triheptanoin is a medium-chain triglyceride of 7-carbon fatty acids observationally studied in patients with long-chain mitochondrial fatty acid oxidation disorders. Besides the ease of use, the anapleurotic effect of triheptanoin GSD Ia could be beneficial.The role of dietary protein in GSD I has not systematically been studied and has recently been reviewed for GSD III  interventions and generated new hypotheses. Future experimental animal studies and well-designed collaborative dietary intervention studies are necessary to improve our understanding about manipulations in macronutrients, like the restrictions of simple sugars, traditional versus heat-treated modified starch, the role of protein, MCT-treatment, triheptanooin and the ketogenic diet in GSD type III. International patient registries will be necessary to study outcome and the effects of (elements of) dietary management.In the next decades, dietary management of GSD patients will be individualized more than ever, integrating personal metabolic and genetic data to optimize metabolic control. Continuous glucose monitoring systems are used in GSD patients in the real life situation to improve dietary management and thereby parameters of metabolic control, including the lipid profile (Hershkovitz et al The definition of the metabolic syndrome and their criteria has been revised several times, leading to multiple definitions in current use. However, the general concept is that patients have a combination of conditions, among which are insulin resistance or hyperglycemia, hyperlipidemia, hypertension and obesity. Regardless of which definition, GSD I patients meet some of the criteria applied. Currently there are conflicting experimental data (Ubels et al


ATG1 belongs to the Uncoordinated-51-like kinase protein family. Members of this family are best characterized for roles in macroautophagy and neuronal development. Apoptosis-induced proliferation (AiP) is a caspase-directed and JNK-dependent process which is involved in tissue repair and regeneration after massive stress-induced apoptotic cell loss. Under certain conditions, AiP can cause tissue overgrowth with implications for cancer.Atg1 in Drosophila (dAtg1) has a previously unrecognized function for both regenerative and overgrowth-promoting AiP in eye and wing imaginal discs. dAtg1 acts genetically downstream of and is transcriptionally induced by JNK activity, and it is required for JNK-dependent production of mitogens such as Wingless for AiP. Interestingly, this function of dAtg1 in AiP is independent of its roles in autophagy and in neuronal development.Here, we show that dAtg1 in autophagy and neuronal development, we report a third function of dAtg1 for AiP.In addition to a role of The online version of this article (doi:10.1186/s12915-016-0293-y) contains supplementary material, which is available to authorized users. Autophagy-related gene 1 (Atg1) in yeast, dAtg1 in Drosophila, uncoordinated-51 (unc-51) in C. elegans, and Unc-51-like kinase 1 and 2 (ULK1/2) in mammals are members of the evolutionary conserved Uncoordinated-51-like kinase (ULK) protein kinase family that play critical roles in macroautophagy (referred to as autophagy) and neuronal development  . To. Toey > dAtg1 as a strong AiP suppressor of ey > hid-p35 by RNAi  animals  DEts > hid AiP model. When hid expression is induced for 12 h in the dorsal half of the eye imaginal disc, the dorsal half of the eye disc is severely ablated [DEts > hid control discs regenerate a normal ELAV pattern 72 h after hid-induced tissue ablation  antibody. Although apoptosis is blocked by p35 expression, the cCasp3 antibody still labels undead cells  causes strong overgrowth of the wing imaginal disc compared to nub > p35 control discs  activation.We also characterized the involvement of scs Fig. . dAtg1 Ract Fig. . To furtdAtg1 relative to JNK in the AiP pathway. The JNK activity reporter puc-lacZ is strongly induced in AiP models compared to controls , another marker in the AiP pathway [wg expression is induced compared to controls . Inisc Fig. ’. In addwth Fig.  and normern Fig. ’. Togethhid and p35 in either wildtype, dronc, or dAtg1 mutant clones (by MARCM) and examined for JNK activity  and Wg K marker . The inders Fig. ’. Importted Fig. ’ suggestdAtg1 is required for wg expression in AiP, we tested if dAtg1 was also sufficient for expression of AiP markers including wg, dpp, and kekkon1 (kek), the latter being a marker of EGFR activity [hid in the DEts > hid model is sufficient to induce wg, dpp, and kek expression  is not  and regenerative (hhts > hid) wing imaginal discs. In both AiP models, dAtg1 is transcriptionally induced , is a strong induction of dAtg1 expression detectable  [bsk RNAi does not affect dAtg1 expression in control discs  for a short pulse of 6 h with 6 h recovery at 18 °C (TS6hR6h) is sufficient to induce dAtg1 expression in wing imaginal discs  cannot induce dAtg1 expression  , 74. Indscs Fig. ’, it supscs Fig. ’, j. Conscs Fig. . Howeverion Fig. . CombinedAtg1 acts upstream in the autophagy pathway and its activation can induce autophagy [dAtg genes, including dAtg1, as well as autophagy in midgut and fat body cells [ey > hid-p35-expressing tissue accumulates large quantities of GFP-mCherry-dATG8a-containing particles. However, it is unclear if these particles are classical autophagosomes. While the GFP signals are weaker compared to the mCherry signals, which may be an indicator of autophagic flux, there are clearly GFP-only particles which do not display mCherry fluorescence  [Gal4 driver as in the ey > hid-p35 AiP model. Therefore, tissue-specific and/or Gal4-dependent differences do not account for the failure of these RNAi stocks to suppress AiP.Because of this unexpected result, we tested other AiP Fig. . Most notion see  [3, 19–2sq,lola)  conditiodAtg13 and dAtg7 mutants which can survive to pupal or adult stages, respectively, for suppression of AiP. dAtg13 encodes a component of the ATG1/ULK protein complex, while dAtg7 encodes the E1-conjugating enzyme for autophagosome maturation. However, dAtg13 and dAtg7 mutants fail to suppress the abnormal morphology of ey > hid-p35 discs as visualized by ELAV labeling and the ectopic Wg expression  [dAtg1 [In addition to targeting essential autophagy components by RNAi, we also tested homozygous ion Fig. . These r(TORTED) , which a) [dAtg1 , both ofdAtg1 uses the same mechanism in AiP that it uses during neuronal development. However, RNAi targeting unc-76, which is an important mediator of the function of dAtg1 during neuronal development [ey > hid-p35 AiP model , which are controlled by JNK activity. This observation suggests a direct way of wg expression by JNK-dependent AP-1.It is generally assumed that the secreted mitogens Wg, Dpp, and Spitz promote the proliferation of surviving cells during AiP , 45. Theneration . AP-1 isdAtg1 acts downstream of or in parallel to JNK. Furthermore, we placed dAtg1 genetically upstream of wg expression. Therefore, dAtg1 may act in at least two different ways in the AiP network. It may directly modulate the activity of the AP-1 transcriptional complex. An indirect mode of action is also possible in which dATG1 provides a permissive environment for AP-1 activity. However, dAtg1 does not control all AP-1 activities. Expression of puc-lacZ and MMP-1 are not affected by dAtg1RNAi and dAtg1 mutants, respectively , dAtg1 affects only wg expression. Future work will address the mechanistic role of dATG1 for the control of AiP.How does dATG1 fit into the AiP network? Our genetic data suggest that ly Figs.  and 4. IdAtg1 is required for AiP, it is not sufficient. Overexpression of dAtg1 using DEts-Gal4 for 12 h followed by 24 h recovery does not trigger AiP markers such as wg-lacZ, dpp-lacZ, or kek-lacZ  encode subunits of the ATG1/ULK complex [dAtg1, inactivation of any of these genes does not suppress the overgrowth phenotype of ey > hid-p35 animals. Furthermore, although we detect the formation of ATG8a-containing particles in undead eye imaginal discs, these particles are not dependent on dAtg1 and do not contribute to AiP and overgrowth  [droncI29 [dAtg1Δ3D [dAtg13Δ7 [dAtg7d14, dAtg7d77 [dAtg8a::GFP-mCherry-dAtg8a [eyeful  [puc-lacZE69, wg-lacZ, dpp-lacZ, kek-lacZ, ey-Gal4, hh-Gal4, nub-Gal4, GMR-Gal4, tub-Gal80ts, UAS-p35, UAS-hid, UAS-hepCA, UAS-GFP, and UAS-TORTED were obtained from the Bloomington Stock Center. UAS-based RNAi stocks of the following genes were obtained from Bloomington, VDRC or NIG-FLY stock centers: bsk , dAtg1 (BL26731), dAtg3 , dAtg6 , dAtg8a , dAtg8b (V17097), dAtg9 (V10045), dAtg17 (V106176), Vps15 , Vps34 (V100296), raptor , and unc-76 . Comparable results were obtained from multiple RNAi lines targeting the same gene. Functionality of BL26731, V101364, V43097 and V17097 was tested on inhibition of starvation-induced autophagy [ey > hid-p35 is either UAS-hid; ey-Gal4 UAS-p35  or UAS-hid; ey-Gal4 UAS-p35 , moderate (M) and severe (S), were used as previously described [ey > hid-p35, with scoring more than 50 ey > hid-p35/(RNAi or mutant) adult flies.negative . Either DE-Gal4) , droncI2droncI29 , dAtg1Δ3dAtg7d77 , dAtg8a:y-dAtg8a , and eyepsqueak)  were as escribed . Each scDEts > hid ; (2) DEts > dAtg1RNAi ; (3) DEts > hid-dAtg1RNAi ; (4) DEts > dAtg1DN ; (5) DEts > hid-dAtg1DN  were raised at 18 °C. Expression of UAS-constructs  was induced by a temporal temperature shift to 29 °C for 12 h. After a 72 h recovery period at 18 °C, late third instar eye discs were dissected and analyzed as indicated in the panels . For the developing wing tissue  was used to temporally control expression of UAS-constructs in the posterior compartment of wing discs.Larvae of the following genotypes (1) 80ts/+);  DEts > eviously . At leasAtg1DN);  DEts > hid and p35 co-expressing “undead” clones: hs-FLP tub-GAL4 UAS-GFP/UAS-hid; UAS-p35/+; tub-GAL80 FRT80B/FRT80B. (2) Generation of hid and p35 co-expressing dronc mutant clones: hs-FLP tub-GAL4 UAS-GFP/UAS-hid; UAS-p35/+; tub-GAL80 FRT80B/droncI29FRT80B. (3) Generation of hid and p35 co-expressing dAtg1 mutant clones: hs-FLP tub-GAL4 UAS-GFP/UAS-hid; UAS-p35/+; tub-GAL80 FRT80B/dAtg1Δ3DFRT80B. (4) Generation of dAtg1 mutant clones in GMR-hid eye discs: ey-FLP/+; GMR-hid/+; dAtg1Δ3DFRT80B/ubi-GFP FRT80B. The mosaic assay in starving fat body  was used as a template to generate digoxigenin-labeled sense and antisense RNA probes (Roche). Labeled probes were detected with a TSA Cy3 kit (PerkinElmer) as previously described [For in situ hybridization to detect ey-GAL4 or ey-GAL4 UAS-Atg1RNAi (ey > dAtg1RNAi) third instar larvae using the TRIzol Reagent (Thermo Fisher Scientific). cDNA was then generated from 1 μg of total RNA with the GoScript™ Reverse Transcription System (Promega). This is followed by the real-time PCR using the SensiFAST SYBR Hi-Rox kit (BIOLINE) with a ABI Prism7000 system (Life technologies). dAtg1 mRNA levels were normalized to the reference gene ribosomal protein L32 (RPL32) by using the ΔΔCt analysis. Three independent biological repeats were analyzed. The following primers suggested by the FlyPrimerBank [dAtg1 Fw, CGTCAGCCTGGTCATGGAGTA; dAtg1 Rv, TAACGGTATCCTCGCTGAG; RPL32 Fw, AGCATACAGGCCCAAGATCG; RPL32 Rv, TGTTGTCGATACCCTTGGGC.Total RNA was isolated from 100 eye discs collected from either the control imerBank  were use