Norovirus, are a significant cause of viral gastroenteritis in humans and animals. For almost 50 years, the lack of a cultivation system for human noroviruses (HuNoVs) was a major barrier to understanding virus biology and the development of effective antiviral strategies. This review presents a historical perspective of the development of a cultivation system for HuNoVs in human intestinal epithelial cell cultures. Successful cultivation was based on the discovery of genetically-encoded host factors required for infection, knowledge of the site of infection in humans, and advances in the cultivation of human intestinal epithelial cells achieved by developmental and stem cell biologists. The human stem cell-derived enteroid cultivation system recapitulates the multicellular, physiologically active human intestinal epithelium, and allows studies of virus-specific replication requirements, evaluation of human host-pathogen interactions, and supports the pre-clinical assessment of methods to prevent and treat HuNoV infections.Noroviruses, in the genus  Caliciviridae cover many aspects of the epidemiology, host susceptibility, molecular biology, immunity, and vaccine development as summarized by Desselberger [Human noroviruses (HuNoVs) are the leading cause of epidemic and sporadic acute gastroenteritis worldwide and are the primary cause of food-borne gastroenteritis ,3,4,5,6.elberger . After HuNoVs were recognized as a cause of acute gastroenteritis and first characterized in 1972 , many atClassical cell lines used to cultivate many viruses were evaluated in early studies. In spite of some stools containing high levels of genomic copies of the virus and the evaluation of many conditions, including adding proteolytic enzymes required to grow other mucosal viruses, HuNoVs failed to replicate in vitro. This raised the following questions: (1) where does the virus replicate in infected persons and can that knowledge help predict what cell types support human HuNoV replication? and (2) are there clues from volunteer studies or studies of enteric calicivirus infections in animals that could help develop a system to cultivate the virus in vitro? Norovirus genus of the Caliciviridae family. This virus family includes four other genera  and six other proposed genera [Norovirus and Sapovirus, which cause severe acute gastroenteritis in humans and animals, with HuNoVs alone causing over 200,000 deaths annually in children <5 years of age [Sapovirus genus to be cultivated, and the addition of porcine intestinal content (IC) fluid filtrate was required for in vitro cultivation in primary porcine kidney cells [HuNoVs are members of the d genera . The moss of age . The Cowey cells ,28. Subsey cells ). In vivey cells  and a siey cells  or a HuNey cells . At lateey cells ,34,35,36ey cells . Some noey cells , suggestVolunteer studies performed between 1972 and the 1990s recognized that a subset of individuals appeared to be genetically resistant to infection with Norwalk virus, and it was suggested that a host receptor might be missing in some people . This waFor many years, cultivation of primary human intestinal epithelial cells was not possible because enterocytes, when directly cultured from human tissue, undergo apoptosis too rapidly to allow viral replication to be assessed. Progress in the fields of developmental and stem cell biology led to the ability to culture stem cell-derived epithelial only cultures  from mouse and subsequently human intestinal surgical specimens or biopsies ,45. ThesCultivation of HuNoVs requires stool samples containing infectious virus and enteroid cultures. We have established a bank of enteroid cultures primarily from adult donors. Detailed methods to produce new cultures and freeze down established cultures have been published ,53,54 anHuNoV infections are performed after 5 days of differentiation. For most experiments, we inoculate three wells of monolayers on a 96-well plate  for each time point and condition we are testing. Filtered stool suspensions are inoculated onto the monolayers. After a 1 h adsorption period, the inoculum is removed and the monolayers are washed to remove any unadsorbed virus. Three wells are then harvested to extract RNA to determine the baseline amount of virus present at 1 h and other triplicate wells are harvested at different time points to evaluate virus replication by RT-qPCR. Virus replication can also be assessed by other methods including flow cytometry to evaluate the percentage of infected cells, by immunofluorescence, by the detection of proteolytic processing of expressed proteins using Western blots, or by visualization of produced virus particles by electron microscopy . These aA variety of HuNoV strains have been successfully cultured in HIEs. Replication, as detected by increases in viral RNA, is seen by 12 h post-infection (hpi) but we routinely use 24 hpi to detect fold-increases in replication . Replica50) was ~1.2 × 102 genomic equivalents (ge) for GII.4/Sydney/2012 and ~2.0 × 104 for GII.3. Similar results were obtained when a larger number of inocula were later tested; the TCID50 was 2.1 × 103 ge/well for GII.4/Den Haag/2006b-like strain, 4.4 × 102 for GII.4/Sydney/2012-like virus and 4.0 × 103 for a GII.3 strain that were successfully cultivated [50) of 1.3 × 103 ge for secretor-positive blood group O or A persons and ~2.8 × 103 for all secretor-positive persons as determined in a controlled human infection model with GI.1 Norwalk virus [Replication of HuNoVs in HIEs is biologically relevant based on the observation that the replication of GII.4 strains is restricted to cultures derived from secretor-positive persons while GII.3 strains replicate in secretor-positive cultures and a subset of secretor-negative cultures, mimicking the findings of epidemiological studies with these strains ,61. In eltivated . These vlk virus . HuNoV replication requires differentiated HIE cultures that contain the multicellular types of a differentiated epithelium ,53,54,63Limited passaging of GII.4 virus is possible but sustained passaging (after passage 4) remains to be achieved. However, the culture system produces infectious virus, as a laboratory researcher became infected with a passaged virus that was confirmed to be the same strain as the virus in culture. This experience is a reminder that HuNoVs are highly infectious and BSL2 laboratory procedures must be used at all times. Establishment of a new cultivation system requires significant optimization and determination of what factors affect successful replication. This remains an ongoing research project as the number and range of virus strains evaluated increases. Current information indicates bacterial-free fecal filtrates containing high titer virus replicate in HIEs, and successful replication depends on viral load and genotype of the inoculum ,56. FromAlthough HuNoVs cause significant human disease, there are currently no licensed vaccines or antivirals for these pathogens. The previous inability to cultivate virus hampered the development of strategies to control and prevent HuNoV infection and to determine the effectiveness of methods to inactivate virus to prevent transmission in various settings, including in food or on contaminated surfaces. The persistence of HuNoVs in the environment, their high transmissibility, and the problem of chronic infection of immunocompromised individuals document a need for improved strategies for the treatment and prophylaxis of norovirus infections. The HIE culture system can be used to measure neutralizing antibodies. Initial studies found that virus-specific serum neutralization titers were higher compared to HBGA-blocking antibody titers, a previously described surrogate neutralization assay found to correlate with protection against clinical gastroenteritis in controlled human infection studies ,65. How Previous evaluation of control measures for HuNoVs, including disinfection measures, relied primarily on the use of cultivatable surrogate viruses . Using tCultivation of multiple strains of HuNoVs in stem cell-derived HIEs is a long-awaited breakthrough for these important pathogens. These human cultures are transformative tools to study enteric viruses, and their use has identified unique host-viral interactions, cellular tropisms as well as innate and physiologic human epithelial responses to infection. For example, studies of cell tropism show that while HuNoVs infect enterocytes ,52, rotaSeveral challenges remain to be solved to further enhance this intestinal epithelial cultivation system. First, the medium used to maintain the cultures is expensive and more complicated than standard unicellular cancer-derived tissue culture systems used for many classical virology studies. The expense and complexity of the media are based on the need to add several growth factors into the medium to maintain stem cell growth in proliferating cultures. Producing growth factors by growing three cell lines is time-consuming. It is now possible to consider alternative methods to obtain the growth factors , including producing them from a single cell line ,72 or usSecond, sustained passaging of HuNoV strains remains to be fully established. We predict that this may be related to the cellular innate response induced by an infection that likely is responsible for the plateau in growth observed between 24 and 72 hpi . An alte50 values determined for current cultivatable strains, we routinely inoculate a well of enteroid cultures with a minimum of 1 × 105 genome equivalents. As noted above, while this may seem like a high level of virus, it is not so high when considering the amount of virus needed to cause human infection and the levels of virus shed in the feces of infected persons. It may be that the stool filtrates contain noninfectious particles or that there are other factors in the stool that limit replication. Third, a question arises about whether it is possible to increase the sensitivity of the cultures to infection. Based on the IDFourth, what is the basis for the strain-specific replication requirements? To date, GII.4/Sydney strains seem to replicate more efficiently than other strains, and they can replicate in the absence of bile while other strains such as GI.1, GII.3, and GII.17 require bile. We have identified two components in bile that support replication . Are theFifth, will testing of additional cultures identify new HIE lines that are more susceptible to infection? This would not be surprising based on the range of host responses to infection. Sixth, it may be possible to increase replication by manipulating the cultures so that more enterocytes are produced in the cultures or by testing co-cultures with mesenchymal or vascular cells or the intestinal microbiome to more fully represent the intestinal milieu. Finally, identifying the cellular receptor(s) for HuNoVs may allow simplified systems to be established.It has been a long road from the discovery of HuNoVs as the first viruses to cause gastroenteritis in humans to being able to cultivate these pathogens in human intestinal epithelial cultures. This achievement was based on knowledge of genetically-encoded host susceptibility factors and the site and cell type(s) where the virus replicates in the small intestine. Animal models of infection, particularly with the porcine enteric sapovirus and bovine noroviruses, as well as studies in volunteers and chronically-infected patients, provided important information to build an understanding of how one might establish a replication system in non-transformed human cells. Cultivation in HIEs has brought on a new era for HuNoV research and has stimulated efforts to establish other models. Replication of HuNoV in zebrafish  and in o


Hepatitis delta virus (HDV) is currently only found in humans and is a satellite virus that depends on hepatitis B virus (HBV) envelope proteins for assembly, release, and entry. Using meta-transcriptomics, we identified the genome of a novel HDV-like agent in ducks. Sequence analysis revealed secondary structures that were shared with HDV, including self-complementarity and ribozyme features. The predicted viral protein shares 32% amino acid similarity to the small delta antigen of HDV and comprises a divergent phylogenetic lineage. The discovery of an avian HDV-like agent has important implications for the understanding of the origins of HDV and sub-viral agents. Deltavirus. The HDV genome is unique among known animal viruses but shares similarities with plant sub-viral pathogens named viroids  and B37-2013 [17 January 2011]). Banding was performed under Australian Bird Banding Scheme permit (banding authority numbers 2915 and 2703 [04 March 2010]). Research permits were approved by the Department of Environment, Land, Water and Planning Victoria (permit numbers 10006663 [15 February 2013] and 10005726 [20 January 2011]).Anas gracilis), Chestnut Teal (A. castanea) and Pacific Black Ducks (A. superciliosa). All the birds sampled showed no signs of disease. Fifteen samples were selected, and RNA was extracted using the MagMax mirVana Total RNA isolation Kit  and assessed for RNA quality. The ten samples with the highest concentrations were pooled using equal concentrations using the RNeasy MinElute Cleanup Kit . These ten samples comprised six Grey Teals, two Chestnut Teals and four Pacific Black Duck samples. Libraries were constructed and sequenced as per Shi et al. 2016 [Waterfowl were captured at the Melbourne Water Western Treatment Plant, Victoria, Australia, in 2012 and 2013. Combined oropharangeal and cloacal samples were collected from Grey Teal  algorithm implemented in Trinity. All contigs that returned blast hits with paired abundance estimates were filtered to remove all bacterial and host sequences. The virus list was further filtered to remove viruses with invertebrate [http://www.genome.jp/virushostdb/).Contigs were assembled, identified, and abundance calculated as per Shi et al. 2016 . Brieflyrtebrate , plant, Gallus gallus) (taxid: 9031), which has been found to be stably expressed in the Mallard (Anas playrhynchos) lower gastrointestinal tract [To compare the viral abundance to that found in the host, a blast database was created containing Ribosomal Protein S13 (RPS13) from both Mallard (taxid: 8839) and Chicken , with an expected value threshold of 1 × 10−5 We further utilised the HHpred tool [https://toolkit.tuebingen.mpg.de/#/tools/hhpred) for additional homology detection and structure prediction. Reference sequences of Hepatitis D representing all eight major clades were downloaded from GenBank. The translation of the HDAg proteins were aligned using MAFFT [http://www.atgc-montpellier.fr/phyml/).Contigs greater than 1000 bp in length were inspected (Geneious R10). Virus reads were mapped back to the HDV-like contig using the Geneious mapping function to corroborate the contig sequence and to calculate read coverage. Open reading frames were predicted within Geneious and interrogated using the conserved domain database . Avian viral transcripts were highly abundant in the library, largely representing influenza A virus. RNA from an avian HDV-like agent was ten-fold less abundant than that of influenza A virus, but was more abundant than RNA from the host reference gene RPS13 that is stably expressed in ducks  Figure B. Notabl−10). We further used the HHpred tool to detect protein homology, and the ORF matched the HDAg  with a probability of 99.79%. Critically, the conserved domain search, HHpred search, and the blast databases contain the cellular homolog of CCDC85B (previously referred to as DIPA [Boa constrictor sabogae) [The full genome of the novel avian HDV-like agent was represented by a single contig (GenBank accession MH824555), wherein the virus genome was duplicated, the result of sequencing a circular genome of 1706 nucleotides A. Unlikesabogae)  , the latter has a larger proportion of hydrophobic residues (up to 31%). Both HDV and the avian HDV-like agent have a long stretch of neutral and hydrophobic residues towards the C-terminal, and the increased hydrophobicity of human HDV may be attributed to a longer peptide and hydrophobic region .There are a number of hypotheses for the origin of HDV, including that viroid-like RNA captured host signalling mRNAs , that HD


Zernike polynomials are used to enhance the identification of mode shapes. Furthermore, the modal assurance criterion (MAC) in combination with the frequency response criterion and a data processing approach are applied in order to characterize changing modal properties of composite rotors. In addition, the developed algorithms can be further extended in order to simplify the experimental evaluation of the complex dynamic behaviour of composite structures.The analysis of the structural dynamic behaviour of composite rotor–discs by a valid description of the eigenfrequencies and mode shapes can provide significant information for action-taking before a failure occurs. Specifically, vibration-based diagnostic methods, which are able to take into consideration the interdependencies and sequential changes of the modal properties could benefit from such an analysis. Here, on the example of composite rotors, a correlation method for experimentally determined mode shapes is developed. For this purpose the  ZERNIKE polynomials and the modal assurance criterion (MAC) in combination with the frequency response criterion are applied. This method has been developed primarily to be used with circular symmetric structures, which allows us to define assumptions by which certain mode shapes can be associated to each other or excluded. For example, symmetric rotor structures with isotropic or polar orthotropic material behaviour, which correspond to those of a circular membrane, show such features.An unknown or a false understanding and subsequent description of the structural dynamic behaviour of composite rotors can lead to severe misinterpretations and false decision making. Especially in damage identification methods such as vibration-based diagnostics, a false classification of the vibration response to a damage state can result in a catastrophic failure. The goal of the current investigation is to gain information and knowledge regarding changes of the structural dynamic behaviour of composite rotors by developing a correlation method that is able to automatically identify the eigenfrequencies and the resulting mode shapes. The identification of modal properties is always influenced by structural and environmental changes, which can result in unpredicted dynamic behaviour such as mode shifting, splitting and swapping and therefore affects the correlation of the investigated mode shapes. In this paper, glass fibre reinforced composite rotors are investigated to study the influences of changes in modal properties, which where measured by a laser vibrometer. An output-only approach is used to examine the measurement data by applying a processing method that mainly focuses on the modal properties and more specifically on the eigenfrequencies and their corresponding mode shapes. To characterize changing modal properties the The aim is to develop a method that correlates the state-dependent modal properties of composite rotors and in the case of a series of several sequential states provides a dynamic response sequence. Then, multiple algorithms that provide a robust approach to identify and correlate the eigenfrequencies and their corresponding mode shapes are implemented and evaluated. A subsequent experimental modal analysis captures the dynamic behaviour of composite rotor. This is followed by the determination of the dynamic vibration behaviour in case of material alterations. Here, a multi-stage test procedure is used in which measurements and load tests are carried out under increasing loading. The resulting stresses have been determined and analysed during previous investigations . In thesIn the field of structural dynamics, it is often of importance to identify the mode shapes, extract them from the measured data and to correlate them correctly to mode shapes of different states, conditions, or numerically determined mode shapes . FurtherMany approaches have been developed over the years in order to correlate mode shapes. The most commonly used in literature is the modal assurance criterion ,9,12. Itrictions .Modal analysis provides the dynamic behaviour of a structure in the form of modal parameters, so called modes, which are represented by eigenfrequencies, mode shapes, and modal damping. If several structural conditions are considered, it is also possible to identify vibration-based changes. Appropriate correlation methods can help to detect and quantify the accordance of modal properties. In order to successfully compare experimentally or numerically determined data sets and their mode shapes respectively, and be able to validate the corresponding simulation models, a robust modal identification of the modal properties is necessary.The MAC calculates the scalar product, and thus the comparison of eigenvectors of different length is a difficulty. This problem often occurs when comparing experimentally and numerically determined mode shapes and depends on the number of degrees of freedom (DOFs), whereby numerical models usually consist of a higher amount of DOFs. Therefore, a systematic extraction or interpolation of relevant DOFs must be carried out. In  a methodTo avoid these issues, various extensions of the MAC have been developed over the years. Another possibility results from the so-called modal tracking ,14,15. En reflects the number of RNLs and m the number of CNLs. An example is given in To ease the understanding and the characterization of mode shapes, a description rule in the form of a nomenclature is introduced. The description is carried out by consideration of the established deflections perpendicular to a mode shape, based on . Lines oNote that the inner circle is considered as a CNL due to the fact that the underlying structure has been fixed in the centre. The presented description provides a simple and understandable rule, that can also be applied to rectangular structures, whereby an edge has to be defined as the reference point see  Step 1.Local material fluctuations caused during the component production or by introduced damage can lead to strong differences in the formation of a mode shape. This issue can occur also with numerical models, which are based on a number of assumptions, e.g., a uniform material distribution. Experimental or numerical modal analyses can provide data sets for example in the frequency domain in the form of the power spectral density (PSD), from which the modes are extracted. The extraction of modes such as eigenfrequencies and mode shapes from PSDs is described in detail in Zernike modes (Step 1), considering geometrical symmetry (Step 2) and the complex phase angle of the eigenvectors (Step 3). A method that associates mode shapes of changing conditions can be applied additionally and helps to reduce double modes. The method allows for the determination of frequencies that can be associated to valid eigenmodes or otherwise have to be considered invalid. The data enhancement includes several methods utilized to specifically improve the representation of the mode shapes. The data correlation can be performed between experimental or numerical data only, or in combination. In literature, the combined approach is often called mixed numerical experimental technique, which is used for example in the field of aerospace engineering [At the beginning the mode shapes are extracted from the examined PSDs. Subsequently the mode shapes are processed in three different steps that aim to improve the mode correlation, by reshaping the modes based on ineering . The maiZernike polynomials, which are orthogonal polynomials, are used to map wavefronts. The rotors investigated here exhibit mode shapes corresponding to the characteristics of such wavefronts. According to [Zernike polynomials are the product of the radius r dependent part The single complex vectors that are extracted from separate PSDs are called the eigenvectors and are usually used to correlate mode shapes utilising the MAC. For composite structures such as disc-based rotors, however, it is reasonable to take a closer look at the symmetries and derive reduced mode shapes from them. The rding to  Zernike It is therefore possible to adequately describe each mode shape by using two polynomials or two interdependent mode representations. To determine these, the rotor is conceptually cut along an RNL and unfolds, resulting in a rectangular surface with the dimensions r and Zernike decomposition is a function f which maps the eigenvector u of the length N to a matrix N;Using suitable algorithms and mathematical functions to describe the RNLs and CNLs, the vectors are transformed into polynomials for The advantage of this representation is the possibility to perform operations across one dimension, thereby eliminating disturbances. For example, in cases where the changes of mode shapes themselves are negligible, averaging along a specific dimension can be used to reduce noise and determine mode shapes more accurately.moving mode shapes or rotating mode shapes [During the extraction of the mode shapes from the PSDs, several difficulties can be encountered. For instance, the presentation of a mode shape along the node lines depends on the position at which the data extraction is initialized. Another common issue of determining mode shapes are the e shapes , which ce shapes . The modWithout consideration of the rotational symmetry of circular discs The measurement of structural conditions is always subject to external influences. Furthermore, the use of filters, window functions and averaging procedures can lead to errors that have an unintended effect on the transfer function; for example, frequency spectra can be averaged in absolute or complex terms. With absolute averaging the phase relation is lost and in this case the result is sometimes combined with the phase response of any spectrum. Since phase responses are subject to similar fluctuations as the frequency spectra themselves, there is a deviation in the transfer function. Thus, the amplitude and phase angle of a complex eigenvector are no longer in correct relation to each other. A further difficulty is the superposition of mode shapes, which arises e.g., through a combination of several successive modes caused by signal noise, signal interference, uneven excitation etc. Mode shapes with higher radial node numbers are especially more prone to be affected, since they do not necessarily have to be close to a mode shape with the same circular node number. Another issue can be closely spaced mode shapes were several mode shapes appear at nearly the same frequency. Since the appearance of this phenomena was marginal in the considered setup, solving this issue is not a goal of this work. Nevertheless, a suggestion is made in To differentiate the mode shapes and to compensate for the phase error it is necessary that the mode shapes are vectors whose elements are complex eigenvectors. These can be represented in the Euler form according to Equation (5) and form the product of a vibration amplitude The rotation of the phase angles of all eigenvectors of a mode shape can be considered as an oscillation, which is also often used for the animation of mode shapes. By rotating the angle it is possible that a mode shape becomes more noticeable, which allows for a differentiation in favour of the dominant mode shape. Mathematically the rotation of a complex vector represents a simple addition of the anglesIt should be noted that the damping part of damped vibration systems is included in the real part, and thus in highly damped systems the imaginary part can be neglected . This alAs an example, Zernike modes represent the totality of possible mode shapes of circular discs. Therefore, the mode shapes are correlated with predefined matrices of the Zernike modes. Note that only a fraction of modes have to be taken into account, since the mode shape order is restricted to half the number of sampling points of a mode shape.In case of strong deviations in the averaging, the mode shapes cannot be determined properly. Using predefined mode shape matrices ensures high matching results only for actually possible mode shapes. For instance, the The condition-dependent dynamic behaviour of composite rotors can be determined based on the frequency shifts of the mode shapes. To evaluate the specific shifts a method is developed which identifies similar modes. More specifically, it determines sequential or successive paths from the MAC matrices and the associated frequencies, which represent the tendency of the frequency shift. Because some mode shapes can occur multiple times in the spectrum, the path with the lowest frequency deviation is determined using the nearest neighbour search algorithm . Duplicai-th and j-th eigenfrequency and The resulting matrix is always diagonally symmetrical, whereby similar mode shapes lead to high correlation results off the diagonal. As an extension to the MAC, and to identify the frequency shifts, the frequency deviation correlation (FDC) is introduced, which determines the relative deviation of a frequency P and the mean value is calculated for each created path in the last step The combination of AMAC and FDC allows for a more specific association of modal datasets. First, the deviation is determined separately, always between two following data sets and afterwards the mean value is calculated. A data set consists of the matrix of the frequencies and its associated mode shapes of an analysis step. A sequence consists of two consecutive analysis steps and contains the correlation of the two matrices and the frequency deviation of the respective mode shapes. The separate treatment is necessary, because when several similar mode shapes occur their tendency shift can be bigger than the difference between the nearest modes. Mode shapes which occur several times often show a phase shift, which is noticeable in a deviation of the MAC. The MAC value decreases with increasing phase shift and decreasing image quality. For each sequence E marks the initial measurement.Paths with the same origin are then compared. Thereby those mode shapes can be sorted out by applying a threshold value for the SPs. If a mode shape occurs several times, the maximum value can be used to determine the mode shapes with the best correlation. The determined paths for two mode shapes to which the individual SPs have been assigned are shown in P of P is a simple consecutive number. For a better understanding the first digit of P in E and the second digit denotes into which parts the path has split. For instance, In Zernike modes to identify the restrained DOFs allowing for the approximation of the resulting displacement distribution. Since rotors are rotationally symmetrical structures, the transfer into matrix form by separating discs along one radius line results in symmetrical boundary conditions that are represented by the outer columns of the matrix. The amount of shifts is equal to twisting a mode shape by a specific angle around its central axis. For instance, shifting the columns of a matrix by half the total number of columns represents an angle twist of Restraining a structure causes local stiffening, thus resulting in frequency shifts and hinders the formation of mode shapes. Therefore, another method was implemented that also uses the  theorem  and the Step 3 can be used to determine the probability of each mode, whereby the correlation to Zernike modes ensures the proper mode shape recognition. A threshold value can help to distinguish whether a mode should be considered valid or be omitted. In order to determine any mode shape different from Zernike modes, the formerly described AMAC can be used to correlate all the mode shapes that were gathered by stepwise rotating the phase angle. The values of the resulting MAC matrix will form blocks or groups respectively for several similar mode shapes, easing the selection of mode shapes.Closely spaced mode shapes are mode shapes that appear to be present at the same frequency or at least within a very small range of frequencies, impeding their proper distinction. In this case, several modes may indeed be present and the method described in To evaluate the developed approach, the correlation method was applied to experimental data gathered from glass fibre reinforced rotors using experimental modal analysis . The invPeakfinder, to which a subsequent sampling was coupled. The selected peaks represent extreme values, that refer to the eigenfrequencies. On the basis of those values the relevant complex eigenvectors were read from the PSDs for each measuring point. Thus, vectors were created consisting of complex eigenvalues (Equation (5)). The procedure of this approach is also called peak-picking. It is one of the simplest methods to determine mode-shapes from PSDs [The measured data has to be processed whereby the PSDs serve as the basis for modal identification. The selection of eigenfrequencies was performed in MATLAB by the function rom PSDs . In addiEquation ). The prFibre reinforced materials exhibit a gradual damage behaviour which allows for extended operation of a structure even after initial damage has occurred . The damr and circumference or angle The layers of the investigated rotors consisted of radially and tangentially arranged glass fibres, which were placed by means of tailored fibre placement. The preform consisted of several fibre layers and was produced from so-called textile sub preforms. The fibre orientation was radial (Ra) and tangential (Ta) and thus showed a polar orthotropic alignment, resulting in a planar, polar stress distribution along the radius The identification of eigenfrequencies was carried out by analysing the PSDs, which were provided by the experimental modal analysis. The frequency extraction was performed by the former mentioned peakfinder-algorithm, which offered a threshold value to handle signal noise. For instance, a low value may have resulted in a high amount of picked peaks, whereas a high value may not have produced any results. Subsequently, a selection of frequencies was carried out by sampling the values in a specified range. A smoothed regression curve was then formed for the region of interest to minimize noise. For instance, a noisy area will result in poor regression or a large residual, which leads to the exclusion of values. Once the frequencies were identified, eigenvectors for the given frequency range were extracted from all PSD lines. Each amplitude was a complex vector, which consisted of the magnitude and its relation to the other values, in the form of the complex phase angle. The resulting eigenvector represented the mode shapes in one-dimensional form. Each element of these vectors consisted of a complex vector and thus represented the second dimension of space. Through the reference to the measuring point, a three-dimensional mode shape was created. Using a rectangular plate as an example It should be noted that the frequencies were determined from the averaged absolute values of the PSD. For a correct representation of the mode shapes however, it is necessary to extract the amplitudes in their complex form.To evaluate the efficiency of the previously described preparation and correlation methods, the gathered PSDs were analysed. An example of the frequency response and the extracted mode shapes of a rotor, determined in its initial and unaltered state, is shown in a)(Derive the MAC matrix from the unmodified mode shapes.b)(a).Shift maximum values of the mode shapes and intersect result with (c)(Zernike modes and intersect result with (b).Derive the MAC matrix from the d)(c).Derive the FDC matrix from the eigenfrequencies and intersect result with (e) can be omitted, and directly passed to (e).The evaluation was always performed according to its purpose, e.g., if the frequency deviations were not significant, step (Zernike modes, in contrast, created a much more destined result and was key in determining the mode shape notations. It should be noted that distinct shifts of the eigenfrequencies can be observed, which are dependent on certain types of mode shapes [The first MAC matrix in e shapes ,28. ThosZernike polynomials and modes to enhance and identify the mode shapes on the example of glass fibre reinforced rotors. A stepped rotational run-up test of another investigation provided rotors with altered material properties, which were examined and allow for the determination of the condition-dependent vibration behaviour of composite structures. The methods can be used to improve correlation results of circular discs. Combined with the also described frequency deviation correlation, the sequentially changing condition of a structure can be examined and provide a better understanding of the underlying interdependencies.For an improved investigation of the structural dynamic behaviour of polar-othotropic circular composite structures a new method was proposed and the capability to apply such methods to evaluate related changes in eigenmodes was investigated. Furthermore, experimental modal analysis were conducted to determine the quality of the developed methods. A satisfactory utilization of the method was found, offering the possibility to detect changing eigenmodes. This was enabled by applying an advanced correlation method, which uses Furthermore, closely spaced mode shapes should be considered in future investigations, because of its high importance in the area of identification of modal properties and its complexity regarding composite structures. A suggestion was made of how this issue may be resolved, but further research has to be carried out in order to evaluate its capability.


Cryptococcus gattii in the North American Pacific Northwest (PNW) in 1999 was an unexpected and is still an unexplained event. Recent phylogenomic analyses strongly suggest that this pathogenic fungus arrived in the PNW approximately 7 to 9 decades ago. In this paper, we theorize that the ancestors of the PNW C. gattii clones arrived in the area by shipborne transport, possibly in contaminated ballast, and established themselves in coastal waters early in the 20th century.The appearance of  Cryptococcus gattii in the North American Pacific Northwest (PNW) in 1999 was an unexpected and is still an unexplained event. Recent phylogenomic analyses strongly suggest that this pathogenic fungus arrived in the PNW approximately 7 to 9 decades ago. In this paper, we theorize that the ancestors of the PNW C. gattii clones arrived in the area by shipborne transport, possibly in contaminated ballast, and established themselves in coastal waters early in the 20th century. In 1964, a tsunami flooded local coastal regions, transporting C. gattii to land. The occurrence of cryptococcosis in animals and humans 3 decades later suggests that adaptation to local environs took time, possibly requiring an increase in virulence and further dispersal. Tsunamis as a mechanism for the seeding of land with pathogenic waterborne microbes may have important implications for our understanding of how infectious diseases emerge in certain regions. This hypothesis suggests experimental work for its validation or refutation.The appearance of  Cryptococcus gattii have been identified in the North American Pacific Northwest (PNW)  –5, with t (PNW) –. In this complex  and majo complex . The PNW America ) or agriC. gattii from eastern South American port cities  to the PNW  via contaminated ballast water, due to early shipping between the regions following the 1914 opening of the Panama Canal C. gattii periods ). We nowhttps://www.ngdc.noaa.gov/nndc/struts/results?bt_0=1964&st_0=1964&type_7=Like&query_7=prince&d=7&t=101650&s=7). The Alaskan Earthquake was felt as far as 4,500 km away, with tidal effects recorded on the Hawaiian Islands. The tidal waves reported in nearby Shoup Bay, AK, were reported to be 67 m high (220 ft), causing significant shoreline devastation. Further to the south, the tsunamis caused significant water surges along western Vancouver Island, most notably in Port Alberni, where dozens of homes were destroyed or washed away. The tsunami continued south, affecting much of the coastline of western North America, even causing several deaths on the beaches of northern California .The 1964 Great Alaskan Earthquake was the largest ever recorded in the Northern Hemisphere, registering M9.2 on the Richter scale, second only to the M9.5 1960 earthquake in Chile  require a mechanism for broad dispersal from such material to the larger landscape, including the coastal forests and waters. Ocean environments might be contaminated from snow melt and rain runoff or from contamination from infected sea birds  that there has been, and continues to be, a continual presence of ea birds , althougue heron . It is iin guano . An altein guano . HoweverC. gattii comes from a case of cutaneous VGII infection in a survivor of the 2004 Indonesian tsunami in Thailand in which skin injuries presumably became infected by contaminated water  and anthropomorphic   , 13, 17.C. gattii predominantly only in coastal forests, rather than further inland, suggesting a connection to the shoreline; (iii) the presence of C. gattii in soils on the Gulf Islands between Vancouver Island and mainland British Columbia; (iv) the presence of C. gattii in humans, mammals, and the forested environment near Port Alberni in central Vancouver Island, an area of the island that was greatly affected by the 1964 tsunami; and (v) the fact that the earliest known case of PNW C. gattii occurred nearly 30 years prior to the 1999 PNW outbreak, establishing a historical record in the region that matches a terrestrial emergence in the 1960s.Multiple pieces of evidence support the ocean-first/tsunami dispersal hypothesis, including (i) evidence of phylogenetic diversification about 50 years ago; (ii) the prevalence of environmental (i) Phylogenetic evidence. Phylogenetic analysis of the PNW clones  provides fairly conclusive evidence of a single introduction event into the PNW followed by local evolution for both VGIIa and VGIIc, whereas VGIIb has a dominant PNW clade resulting from a single primary introduction with possibly one or more additional introductions, with limited evolution and spread  between the regions. While such molecular clock analyses can provide only relative dating accuracy, closer analysis of the phylogenetic trees from these studies suggests that secondary diversification events, which define much of the current population structure, appear to have initiated approximately 1 to 2 decades following the introduction of each of the clones , pr, pr(i) Pe clones . A 1964 (ii) Environmental evidence.(a) Trees and soils. The early environmental analyses of VGIIa in British Columbia identified that most C. gattii-positive environmental collections (soils and trees) were in the coastal Douglas fir forests and in coastal western hemlock forests bordering the coastal Douglas fir forest (ii) Envir forest . This is(b) Water.C. gattii from the PNW has been shown to demonstrate long-term survival (at least 1 year) in ocean water, and immediate environmental studies found multiple positive ocean samples near the Vancouver Island coastline (Cryptococcus species are adapted to and live in marine water (–C. gattii (and C. neoformans) has an intrinsic mechanism to support cell buoyancy; specifically, its ability to increase capsule production decreases cell density and supports buoyancy in marine salinity levels . These findings further support the concept of long-term ocean survival of C. gattii in the region. Alternatively, these findings may represent continued seeding of coastal waters by contaminated soil runoff and/or by dispersal of airborne yeast cells from nearby contaminated forests, as suggested previously  Geographic evidence. One of the regions on Vancouver Island to receive the most significant tsunami damage was the town of Port Alberni, tied to an inlet on the west side of the island. Several hours after the Great Alaskan Earthquake struck, multiple waves flowed up the Alberni Inlet, cresting at 8 m and striking the Port Alberni region, washing away 55 homes and damaging nearly 400 others  Patient evidence. One of the confounders for a more recent emergence of C. gattii in the PNW  is that the earliest known case of C. gattii VGIIa occurred nearly 30 years prior to the 1999 PNW outbreak, a Seattle patient in 1971  upriver, including nearly 5-foot waves at the junction of the Columbia and Willamette Rivers (C. gattii in the area. The 240-km (150-mile)-long Willamette Valley is transected from Eugene to Portland by the Willamette River, which flows north and empties into the Columbia River at Portland Harbor. Nearly 62,000 ha  of the Willamette Valley was flooded in December 1964, in a devastating “hundred-year flood”  are largely restricted to the Willamette Valley , 42, 43,r flood” , causingThe mechanisms of pathogen geographic movement and dispersal are of great interest to ecologists, epidemiologists, medical historians, and others who seek to understand the causes of infectious disease emergences. These mechanisms can be natural or anthropogenic and can include anything from displacement or migration of reservoirs to the landscape, geophysical change, and climate changes to occurrences of natural disasters (as proposed here) .Many of these are still theoretical mechanisms of dispersal; however, the point is that there are numerous biological, geological, and sociological dynamics on our planet that are likely involved in pathogen evolution and distribution and subsequent disease ecology and epidemiology. A careful study of these dynamics helps us to understand important contributors to the emergence of disease. Our hypothesis of a tsunami-driven contamination of the PNW coastal landscape certainly involves numerous other biological , geophysical , and other anthropogenic  dynamics that would lead to such an unforeseen emergence.Apophysomyces during the Joplin, MO, tornado in 2011 . For example, the tsunami hypothesis may also explain how Cygnus atratus) were eventually found in Australia, the discovery of which unraveled previous dogmas. Here, we propose a major new mechanism of pathogen dispersal not previously documented and certainly not predicted, and if true, it will have a disruptive impact on previous dispersal theories, thereby being a prototypical example of a black swan. It is a fact that many major disease events are black swans and therefore typically belie the possibility of prediction. In hindsight, the 2014 outbreak of Ebola in western Africa is thought to have been inevitable, given the combination of considerable human travel and migration between African countries and the pervasiveness of under-resourced health care and public health systems. However, the likely start of the outbreak . These efforts are in effect looking for black swans; however, they may fail if such events are truly unpredictable. Taleb  was cere. Taleb  argues tCryptococcus in the PNW, there was a pervasive belief that all C. gattii organisms were found in tropical and subtropical regions; we simply did not predict, nor could we predict, the possibility of the PNW emergence. However, thankfully robust health care, public health, and academic research entities quickly identified and responded to the event. The same was true for HIV, hantavirus, and SARS coronavirus, all on shrinking timescales, due to improvements in technology and communication and advancements in scientific understanding. This cross-disciplinary one-health response between public health, medicine, ecology, and biomedical research is a model that can and should be followed globally to understand and mitigate the effects of the emerging infections that we predict will continue to challenge global public health.The nearly infinite number of small and large stochastic dynamics in the natural and anthropogenic world is probably a good indicator of where we should put our health research dollars when thinking about predicting versus preparing for new emerging infections. In the case of C. gattii originated in the western region  and have been anthropogenically transported via infected animals  to other parts of the continent (One final note: Western Australia is the region of the continent most prone to tsunamis , and it ontinent . A tsunaontinent , where iC. gattii VGII reached the Pacific Northwest in ship ballast tanks from South America and then became subsequently established on land by a tidal wave suggests several lines of experimentation. Because South American ports are connected to other parts of the world by shipping lanes, other ports must also have become contaminated with C. gattii. The likelihood that C. gattii will become established in a particular locale is probably dependent on the physical and biological conditions operating in that locale. For example, amoebas are important biological control agents for C. neoformans in the environment (C. gattii, the more likely scenario is that other coastal areas are also contaminated. Hence, a sampling of water in other ports by deep sequencing may reveal C. gattii-specific genes, thus strengthening the case for shipborne transport. Similarly, sampling of waters in South American ports where rivers flow from the interior of the continent may reveal C. gattii, supporting the notion of fluvial transport by land to sea transport. A more intensive search for new South American isolates may reveal a genetically close relative of the PNW strains, which would support recent transport from that region. Finding C. gattii in other coastal waters without a concomitant increase in cryptococcosis would support the notion that something different happened in the PNW and provide powerful indirect evidence for an unusual event, such as a tsunami. In fact, if C. gattii was found in coastal waters of areas that have suffered tsunamis in recent decades, such as following the Indonesian and Japanese earthquakes of 2004 and 2011, this could allow real-time studies in those areas and perhaps forestall future outbreaks of cryptococcosis in these locales. In this regard, it is noteworthy that the PNW tsunami event was separated from the C. gattii outbreak by 3 decades, suggesting that, if our hypothesis is correct, a period of land adaptation may be required before significant numbers of human and animal cases appear. For example, land adaptation and selection by soil amoeboid predators may have been a necessary condition for increased virulence for mammal infection (C. gattii, with the expectation being that flooded areas harbor more-established sites than nonflooded adjacent lands.The hypothesis that ironment , 58, andnfection . Evidenc


B646L gene, E183L gene, CP204L gene and the central variable region of the B602L gene of ASF virus (ASFV) associated with the outbreaks in Mbala and Chipata districts was conducted. The results revealed that the ASFV detected in Mbala District was highly similar to that of the Georgia 2007/1 isolate across all the genome regions analysed. In contrast, while showing close relationship with the Georgia 2007/1 virus in the B646L gene, the ASFV detected in Chipata District showed remarkable genetic variation in the rest of the genes analysed. These results suggest that the Georgia 2007/1-like virus could be more diverse than what was previously thought, underscoring the need of continued surveillance and monitoring of ASFVs within the south-eastern African region to better understand their epidemiology and the relationships between outbreaks and their possible origin.African swine fever (ASF) is a contagious haemorrhagic disease associated with causing heavy economic losses to the swine industry in many African countries. In 2017, Zambia experienced ASF outbreaks in Mbala District (Northern province) and for the first time in Isoka and Chinsali districts (Muchinga province). Meanwhile, another outbreak was observed in Chipata District (Eastern province). Genetic analysis of part of the  African swine fever virus (ASFV) causes African swine fever (ASF), an acute, contagious and devastating haemorrhagic disease that affects swine. Because of its high mortality rates (up to ≈100%), serious socio-economic impact, high capacity for transboundary dissemination and lack of an effective vaccine or treatment, ASF is considered to be one of the most challenging animal diseases to contend with  gene, p54 (E183L) gene, p30 (CP204L) gene and the central variable region (CVR) within the B602L gene were amplified using the primer pairs p72U/p72D, PPA722/PPA89, p30-F/p30-R and CVR1/CVR2 or ORF9L-F/ORF9L-R, respectively  collected from pigs that died from the disease. Molecular diagnosis was conducted by polymerase chain reaction (PCR) using the ASF diagnostic primer set PPA1/PPA2  revealed that the p72,p54 and p30 sequences of ZAM/2017/Mbala/1 were 100% identical to those of Georgia 2007/1 (GenBank accession no. AM999764-AM999766). In contrast, BLAST showed that the p72 and p30 nucleotide sequences of ZAM/2017/Chipata/1 were 99% – 100% similar to those of ZAM/14/Chipata, which was detected in domestic pigs in the same district   and IIc  and the Zambian virus that fell into genotype VIIIa  of tetramer repeats that were 100% similar to those circulating in eastern Europe since 2007, the sequence of ZAM/2017/Chipata/1 was 100% identical to that of ZAM/14/Chipata, with 14 copies (BNABNDBTDBNAAG)  than that in Isoka and Chinsali (99.8%), it was comparable to that observed in Chipata (71.4%) where ASF is endemic. Perhaps this could be explained by the fact that since 2013 Mbala District has had prior experience with ASF associated with genotype II virus   , these f viruses .p72 genotype II ASFVs were detected in soft tick reservoir hosts in Mozambique, providing evidence for the first time of a possible sylvatic origin of these viruses in south-eastern Africa (Quembo et al. Meanwhile, reminiscent of the eastern Europe situation, the geographical extent of genotype II ASFV in south-eastern Africa appears to be expanding as this virus has been detected in Madagascar, Malawi, Mauritius, Mozambique, Tanzania, Zambia and Zimbabwe (Simulundu et al. The present study investigated the molecular epidemiology of ASF outbreaks that occurred in 2017 in Northern, Muchinga and Eastern provinces of Zambia. Genetic analysis showed that the outbreaks in the Northern region were caused by genotype II ASFV, which was highly similar to the Georgia 2007/1 isolate. Although belonging to genotype II, the ASFV associated with the outbreak in Eastern province was genetically diverse, suggesting that these outbreaks were not related. Overall, this study supports the idea that genotype II ASFV circulating in south-eastern Africa is genetically diverse, probably because of the long-term endemicity in domestic pigs and a possible sylvatic origin.


Flaviviruses of public health concern. However, the extent of human exposure to these viruses and associated disease burden in Kenya and Africa at large remains unknown. We assessed the seroprevalence of Yellow fever and other Flaviviruses in human populations in West Pokot and Turkana Counties of Kenya. These areas border Uganda, South Sudan and Ethiopia where recent outbreaks of Yellow fever and Dengue have been reported, with possibility of spillover to Kenya.Yellow fever, Dengue, West Nile and Zika viruses are re-emerging mosquito-borne Human serum samples collected through a cross-sectional survey in West Pokot and Turkana Counties were screened for neutralizing antibodies to Yellow fever, Dengue-2, West Nile and Zika virus using the Plaque Reduction Neutralization Test (PRNT). Seroprevalence was compared by county, site and important human demographic characteristics. Adjusted odds ratios (aOR) were estimated using Firth logistic regression model.n = 87/413) than in West Pokot . Zika virus seroprevalence was significantly higher in West Pokot (7.11%) than in Turkana County . A significantly higher Yellow fever virus seroprevalence was also observed in Turkana (10.7%) compared to West Pokot . A high prevalence of West Nile virus was detected in Turkana County only (10.2%) while Dengue was only detected in one sample, from West Pokot. The odds of infection with West Nile virus was significantly higher in males than in females . Similarly, the risk of Zika virus infection in West Pokot was twice higher in males than females .Of 877 samples tested, 127 neutralized with at least one of the four flaviviruses , with a higher proportion in Turkana  contains supplementary material, which is available to authorized users. Flaviviruses that have a potential to cause severe disease and mortalities in humans with economic and ecological consequence [Yellow fever virus (YFV), Dengue virus (DENV), West Nile virus (WNV) and Zika virus (ZIKV) are important mosquito-borne sequence , 2. Apprsequence . EpidemiThere have been no cases of Yellow Fever (YF) in Kenya since the first and last ever documented outbreak in 1992–95 , 5. HoweZika virus was first discovered in Uganda in 1947 , and sinFlavivirus diseases in this region remain poorly understood. West Pokot and Turkana Counties are located in areas that border countries that have had and reported outbreaks of these Flaviviruses in recent times and therefore the risk of virus spread from neighboring countries remains high. This study sought to determine the seroprevalence of Yellow fever, Dengue, West Nile and Zika viruses among the human populations in the border locations of West Pokot County bordering Uganda and Turkana County bordering South Sudan and Ethiopia to the north.There is limited epidemiologic knowledge about the presence and spread of arboviral diseases in northern Kenya. Surveillance capacities are lacking, with most resources for study and control of these viruses being focused on epidemic periods. As such, the magnitude of human exposure and the burden of these important This study was approved by Kenya Medical Research Institute (KEMRI) Scientific and Ethics Review Unit (SERU) (under protocol number KEMRI-SERU 2787) and University of Pretoria. All adult participants provided written informed consent and assent for child/minor participants. Only those who consented or assented were included in the study.This was a cross sectional population based study conducted in Turkana and West Pokot Counties of Kenya. Six sites each were selected and sampled in West Pokot County: Kacheliba sub-county  and Turkana County, Lokitoung sub-county  Fig. . TurkanaThe human populations in West Pokot are mainly nomadic pastoralists although there is increasing tendency for them to move to market centers and become sedentary. In Turkana, they are mainly nomadic pastoralists and tend to look for pasture in areas surrounding the lake, and some engage in fishing. As of 2009, the population of Turkana was 855,399 and West Pokot 512,690 . The livicipe) Duduville Campus in Nairobi, where they were stored at − 80 °C until tested.Human blood samples were collected through a cross-sectional study designed into village clusters based on administrative boundaries, in West Pokot and Turkana Counties, Kenya Fig.  in Febru90. Respective virus isolates were diluted to a standard concentration that gave 20–50 plaques. Appropriate 10-fold dilutions of serum samples were prepared in maintenance media  supplemented with 2% heat-inactivated fetal bovine serum (FBS), 2% L-glutamine, 2% penicillin/amphotericin B , starting with a seropositivity threshold of 1:10. The serum dilutions were added to microcentrifuge tubes containing the standard concentration of the diluted virus and incubated for 1 h at 37 °C. The virus–antibody mixture was inoculated on a 24-well plate containing confluent Vero cell monolayer (VERO E6) and incubated for 1 h for virus adsorption. After adsorption process, an overlay of methylcellulose (2.5%) (Sigma) mixed with 2X Minimum Essential Medium (Sigma), was added in to the wells. Between 6 and 14 days depending on the virus tested, the plaques were fixed using 10% formalin (Sigma), stained with 0.25% crystal violet (Sigma) diluted in absolute ethanol and the plaques counted manually as described previously [Samples were retrieved from the freezer, aliquoted in volumes of 30 μl, heat inactivated at 56 °C for 30 min and then tested for neutralizing antibodies to YF (YFXSMB), Dengue-2 008/01/2012), West Nile (AMH005348) and Zika (MR766) viruses in two-fold serum dilutions from 1:10 to 1:320 using PRNT12, West eviously . Endpoineviously .Flaviviruses tested. Reported prevalence was compared by county, site and important demographic characteristics such as sex, age, occupation and YF vaccination status. Comparisons of proportions and evaluation of heterogeneity of the seropositive proportions across the independent variables listed above were performed using Chi Square test. The 95% confidence intervals (CIs) for the proportions positive for a virus were estimated using the Agresti-Coull method [Flaviviruses with a frequency of six or more positive samples , a multiple logistic regression model with covariates as site, sex, age in years, occupation, whether the participant was a herdsman, and whether the participant reported to have or have not received YF vaccination. As the data were sparse, Firth’s logistic regression [Field data captured were stored in a password-protected database linked to the laboratory results, and were analyzed using R version 3.3.1 . First, l method . Next, wgression  was chosgression . All tesTable Flaviviruses. This proportion was, however, higher in Turkana (21.1%) than in West Pokot . Figure Flaviviruses between the two counties. Whereas DENV neutralizing antibodies were detected in one sample  from West Pokot only, WNV antibodies were detected in Turkana only . While YFV and ZIKV antibodies were detected in samples from both counties, YFV was significantly more prevalent in Turkana (10.7%) than West Pokot . The converse was true for ZIKV.Of 877 samples tested, 127  were positive for at least one of the four Flavivirus prevalence across the sites within as well as between the counties. Yellow fever prevalence ranged from 0% in Kokipei, Shaba, Longarkau, and Pkotong to about 4% in Kanyerus in West Pokot. In Turkana, YF prevalence varied from about 7% in Kare Edome to about 20% in Elelea. For West Nile virus antibodies, which were detected in Turkana only, the prevalence varied from 8% in Prigan to 11.1% in Elelea. Also samples from only two sites had neutralizing antibodies to three viruses detected. These were Sangakai in West Pokot  and Elelea in Turkana .Table Flaviviruses  varied by age group in both counties  and lowest among the age group 20–29 years. Zika virus antibodies were only detected in the 50+ years age group in Turkana County. In general, variation between seroprevalence to Flaviviruses was quite limited between age groups.Prevalence of the three most prevalent ies Fig. . In WestFlaviviruses that had more than 30 samples neutralizing with either of the three most prevalent viruses for each county ; Turkana: YFV (n = 44), WNV (n = 42) by demographic characteristics are presented in Additional file Table Flaviviruses in West Pokot and Turkana counties of northern Kenya. Overall, we found that 14.5%  of the samples were positive for at least one flavivirus . However, there were variations in the rates of human exposure for the individual viruses between the counties and across sites. Such variation in prevalence by site or even region is not uncommon in Kenya [The present study reports on the serological evidence of human exposure to in Kenya . At the in Kenya . Such vaCulex mosquitoes, the virus having been previously isolated in some Culex species in Kenya [Culex spp. abundance and diversity varies between the areas. Grossi-Soyster [Culex species, which are important determinants of the epidemiology of WNV [Culex spp. in this ecology [West Nile virus was detected in Turkana , but not in West Pokot. This difference could relate partly to ecological variation between the two areas. West Nile virus is associated with in Kenya –33. It i-Soyster  found hiy of WNV . This alP < 0.0001). Site specific variation in exposure rates was also evident ranging from 7% in Kare Edome to about 20% in Elelea in Turkana County. It has been about three decades since the last documented outbreak of YF in Kenya in the neighboring Rift Valley [Neutralizing antibodies against YFV were detected in samples from both counties, although significantly higher in Turkana than West Pokot  and Yellow Fever, West Nile (Turkana County) by demographic characteristics. 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Using metagenomic analysis, we identified a novel picornavirus in young preweaned lambs with neurologic signs associated with severe nonsuppurative encephalitis and sensory ganglionitis in 2016 and 2017 in the United Kingdom. In situ hybridization demonstrated intralesional neuronotropism of this virus, which was also detected in archived samples of similarly affected lambs (1998–2014). In 2016 in Scotland, and in 2017 in Wales, progressive neurologic signs were observed in young lambs. These cases were associated with nonsuppurative encephalomyelitis predominantly involving gray matter, including neuronal necrosis/neuronophagia and ganglionitis consistent with lesions caused by a neuronotropic viral infection. The target sites included cerebellar roof nuclei in the Purkinje molecular layer of the cerebellum, caudal brainstem nuclei (red nuclei and vestibular complex), and all levels of spinal cord examined  located mainly in the ventral horns and dorsal root ganglia  assay (q] 18–21), where the most severe lesions were also observed. We detected lower loads in the cerebrum (Cq 30), ileum (Cq 29–32), tonsil (Cq 32–36), and mesenteric lymph node (Cq 34–36). Lung and spleen samples were negative, as were CNS samples from 2 additional lambs from the Scotland flock with compressive spinal cord lesions and no evidence of nonsuppurative encephalitis  assay . Using tR) assay . We detephalitis .Picornaviridae (genera Enterovirus and Sapelovirus), and several unclassified species with relatively low sequence identities (64.8%–96.8%). Several reads were classified only after translation into amino acid sequences and comparison to the protein database. We found no evidence of other pathogens that could have resulted in the neuropathological manifestations.We subjected spinal cord samples from selected lambs affected in the 2017 lambing season to metagenomic analysis , which yPicornaviridae generated a complete genome of a novel virus, tentatively named ovine picornavirus (OvPV). The obtained 7.5 kb OvPV genome is only very distantly related to known picornaviruses, with nucleotide sequence identities of 59% with a bovine picornavirus    Table. Werovirus  Figure. We attempted virus propagation from selected samples of affected animals on various mammalian cell lines ; however<3–4 weeks of age with neurologic signs; the earliest case identified dated back to 1998  material proved to be challenging for sequencing and amplification, we identified several additional cases of OvPV infection using reverse transcription PCR, and we generated whole-genome sequences from selected cases . We dete to 1998 . CompareWe performed in situ hybridization on OvPV PCR-positive FFPE tissues, with a subset of neurons within the brain and spinal cord as hybridization targets . In the >20 years, we suspect that OvPV could be present in the intestinal tract commonly, similar to porcine teschovirus and porcine sapelovirus (Complete clinical data were not available for all of the archived cases; however, information was available for 5 flocks. In 4 of these flocks, the affected lambs were being fed artificially, usually because of maternal death or insufficient colostrum. Therefore, insufficient colostral antibody intake may predispose to development of neurologic disease similar to that proposed for porcine teschovirus, but this remains unproven. On the basis of the finding that OvPV has already been present in Wales, Scotland, and England for >20 years, affecting sheep in Scotland, Wales, and England in a range of sheep breeds and management systems, but solely young lambs to date. These findings could suggest that OvPV infection is usually subclinical and self-limiting, particularly in older animals. For some cases, particularly those involving multiple affected lambs in a single flock over 1 lambing season, the clinical histories of snatching at birth or artificial feeding of orphan or pet lambs indicated likely failure of transfer of colostral antibodies. The resulting colostrum deficiency may have acted as a predisposing factor. Further research is needed to determine geographic distribution, disease burden, transmission route, and other factors; a serologic assay is also needed to further analyze the distribution in the field. However, OvPV should be considered in the differential diagnosis of ovine nonsuppurative encephalomyelitis oriented on gray matter, particularly in young lambs with sensory ganglionitis.In this study, we report identification of a novel neuroinvasive picornavirus associated with severe nonsuppurative encephalomyelitis and sensory ganglionitis in sheep. The virus has been associated with encephalomyelitis for Discussion of the materials and methods in study of picornavirus in lambs with severe encephalomyelitis.


Total virus removal increased with sediment concentration whereby the removal rate depended on the virus used , suggesting a different interaction strength with the sediment. Moreover, we observed that the adsorption of viruses to sediment is a reversible process, and that desorbed viruses are still able to infect their respective hosts. Nonetheless, the addition of sediment to infection experiments with the Arctic prasinovirus MpoV-45T substantially delayed host lysis and the production of progeny viruses. We demonstrate that glacier-derived fine sediments have the potency to alter virus availability and consequently, host population dynamics.Over the past few decades, the Arctic region has been strongly affected by global warming, leading to increased sea surface temperatures and melting of land and sea ice. Marine terminating (tide-water) glaciers are expected to show higher melting and calving rates, with an increase in the input of fine sediment particles in the coastal marine environment. We experimentally investigated whether marine viruses, which drive microbial interactions and biogeochemical cycling are removed from the water column through adsorption to glacier-delivered fine sediments. Ecologically relevant concentrations of 30, 100 and 200 mg·L Coastal marine systems in the Arctic typically contain high concentrations of suspended sediment particles which enter the water column via melting and calving of tide-water glaciers, and via streams and rivers that originate from melting land ice (glaciers) and snow. Glacier-derived sediments contain especially high concentrations of fine clay and silt particles (resp. <8 μm and 8–63 μm), and are produced by abrasion  of the underlying bedrock. They can remain suspended in the water column for a very long time and extend far off the coast ,2. This −1 and higher, for example in [−1 e.g., [Colwellia psychrerythraea, with ambient clay concentrations of 10 mg·L−1. Hewson and Fuhrman [−1 of cleaned and dried sediment from the same site. However, these losses may be influenced by the presence of dead cells and organic matter, including transparent exopolymeric particles (TEP) as the samples were not filtered. Finally, Drewes et al. [Marine microbial populations are strongly controlled by lytic viruses resulting ultimately in the death of these unicellular hosts ,9,10,11;ample in ,26. Suchample in . Typical−1 e.g., . As far −1 e.g., ,29,30. W Fuhrman  did obseThe focus of this study was to investigate whether: (i) the loss of viruses by adsorption to (settling) sediments could potentially take place in tide-water glacier influenced marine waters, and (ii) whether the sediments reduce the impact of the viruses on their phytoplankton hosts. The removal (i) was tested by in vitro “adsorption-settling” experiments whereby sediment was added to virus suspensions in glass tubes and allowed to sink out while the concentrations of viruses at the surface of the tubes were monitored. Hence, this simulated the natural export of sediment with adsorbed viruses from surface waters. The possible effects of sediments on the actual interaction of viruses with their phytoplankton hosts (ii) were investigated with one-step and two-step infection experiments, with and without the presence of glacier-derived sediment , by which the speed of lysis and production of new virus progeny was monitored. The sediments were collected from inner Kongsfjorden, a tide-water glacier influenced fjord system in North Western Svalbard (79° N). Fresh virus lysates of temperate and polar virus cultures, as well as a community of natural temperate viruses (53° N) were used.Micromonas polaris, M. commoda and Phaeocystis globosa. These lytic phytoplankton viruses were maintained by fortnightly inoculation of an exponentially growing axenic host culture with 0.2 μm filtered  lysate. Algal cultures were maintained on Mix-TX medium [M. polaris) and 15 °C (M. commoda and P. globosa) and under 80 μmol quanta m−2·s−1  at a light:dark cycle of 16:8 . Before experimental use, the phytoplankton viruses were filtered through 0.2 μm Minisart polyethersulfone membrane filters  and diluted to ecologically relevant abundances of 0.5–1 × 105 mL−1 [The adsorption-settling experiments were carried out with 3 different phytoplankton viruses  taken at the same location in 2008 (R/V Lance cruise NP08-16). Sample preparation and calculation of clay:silt:sand content follows [Sediment material used for the experiments was collected from the inner fjord in Kongsfjorden, Svalbard in 2009, during a R/V Lance cruise NP09-13 at multicore station NP09-13-26. A Fritsch A22 laser-diffraction particle size analyzer (VU Amsterdam) was used to measure the grain-size distributions of samples obtained from multicore NP08-16-63 . The av−1 final concentration sediment was added to 10 mL virus suspension in triplicate glass tubes (15 mm diameter). The sediment was pre-weighed and added as a 1 mL suspension in Mix-TX medium (the controls without sediment received only Mix-TX). Right before sediment addition, samples for viral abundances were taken from the tubes as a control for the effects of dilution and handling (T0 h). After sediment addition, the tubes were mixed by gently turning the tube upside down once after covering the tube with parafilm. Samples were kept stable with minimal movement so the sediment with and without viruses could sink out for 36 h (incubation conditions were similar to culturing). As soon as possible , virus samples were taken from the upper half of the tube to investigate the initial loss upon sediment addition. Additional surface samples were taken after 6 and 36 h (T6 and T36). The samples for flow cytometric virus enumeration were directly fixed with 25% glutaraldehyde  to a final concentration of 0.5%, placed in the fridge at 4 °C for 30 min, flash frozen in liquid nitrogen and stored at −80 °C until analysis [5 mL−1 for the lysates of the algal viruses and approximately 1 × 108 mL−1 for the natural virus communities (in situ abundances). To test the maximum removal capacity of the sediment, an additional test was done with higher algal viral abundances (approximately 108 mL−1 versus the diluted ones of 0.5 to 10 × 105 mL−1 in the diluted tests).Adsorption and removal of viruses with different sediment concentrations was tested by sediment-settling experiments for which 0, 30, 100 or 200 mg·Lanalysis . For ecog) or filtered  to remove sediment. The supernatant or filtrate containing the remaining viruses was then fixed for flow cytometry as described above. Mixing and sampling was repeated at T6 and T36 h. In contrast to the settling approach, centrifugation and especially filtration resulted in substantially higher loss of viruses from the controls . Although the differences between the controls and sediment treatments were still measurable for the centrifugation experiments, the reproducibility was lower than with the original method. Furthermore, this approach has more ecological relevance, i.e., in a sediment-influenced water column, adsorbed viruses are expected to sink down with the sediment out of the photic zone.We chose the adsorption-settling method described above as it was found to be the most reproducible compared to centrifugation filtration as a method for determining adsorption rates of viruses to sediment. In short, sediment was added to the lysate in 50 mL Erlenmeyer flasks and mixed well. A sample of 2 mL was then either centrifuged  produced from a dense Phaeocystis globosa culture (1 × 106 cells mL−1) was added to 4 g·L−1 sediment The high density of the lysate assured saturating abundances of viruses and cell debris from the lysed cells. The lysate with added sediment was split into 2 batches, of which one batch (“detritus only”) was sonicated  for 3 × 60 s at the highest amplitude to destroy all viruses. For the other batch (“detritus + viruses”) the viruses were left intact. The sediment suspensions were then centrifuged at 3500× g for 5 min, after which the supernatant was removed. The sediment was washed once again with medium and centrifuged in the same way, after which the sediment was resuspended in medium (such that a working stock of 2 g·L−1 sediment was obtained) and added to undiluted MpoV-45T lysates for the adsorption-settling experiments with final concentrations of 200 mg·L−1.An additional experiment was done with the Arctic phytoplankton virus MpoV-45T to test whether sediment pre-exposed to detritus or other viruses (before addition to the MpoV-45T lysate) can occupy potential binding sites and consequently reduce the adsorption of MpoV-45T. PgV lysate  and N(0) respectively stand for the number of viruses at the second and first time point and t stands for the past time in days. Statistics were calculated in the program SigmaplotTM 14 . Differences between the treatments were tested with one-way ANOVA and Holm-Šídák pairwise comparisons. Significant differences (p = 0.05) with the control are depicted in the tables and graphs.The obtained viral abundances were corrected for potential loss in the control treatment (no sediment) after which they were used to calculate the absolute and relative losses of viruses at each time point and the loss rates over time between the time points. Each virus loss calculation for a specific time point was corrected with the abundances of the control treatment at that time point, and each calculation per time interval was corrected for loss in the controls over the specified time period. Because the adsorption dynamics seemed to follow exponential decay , specifi−1 final concentration sediment added to determine the virus growth characteristics, and (ii) a two-step infection experiment (0.5:1 virus:host ratio) with 30 and 200 mg·L−1 sediment added to mimic more natural conditions and allow testing of the potentially interfering effect of sediments on virus-to-host adsorption. The virus:host ratios were calculated after quantification of the total viruses and algae by flow cytometry.The effect of sediment on virus-host interaction was tested with two different experiments: (i) a one-step infection experiment (14:1 virus:host ratio) with 200 mg·L−1 final concentration) was added either to the host directly after the addition of lysate or first to the virus lysate prior to the addition to the host culture . For the two-step infection experiment, the sediment was added to the host directly after lysate addition. At the day of the experiments, triplicate 100 mL Erlenmeyer flasks with 50 mL exponentially growing algal culture were inoculated with 0.2 μm filtered  virus lysate. Samples for algae and viruses from these flasks, and from uninfected control cultures (with Mix-TX instead of virus lysate) were taken at T0 (between 5 and 10 min after lysate/sediment addition) and subsequently at regular time intervals until the cultures were visibly lysed (cleared). Cultures were gently mixed every 8 h. Phytoplankton samples (1 mL) were fixed with 0.5% final concentration formaldehyde  and virus samples (0.5 mL) with 0.5% final volume of glutaraldehyde . Samples were fixed for 30 min at 4 °C, followed by flash freezing in liquid nitrogen and storage at −80 °C until analysis. The differences in viral abundances between treatments were tested with one-way ANOVA and Holm-Šídák pairwise comparisons as described above.For the one-step infection experiments, sediment suspension  control cultures, the same protocol was used but with sediment that was not exposed to viruses. Lysis was monitored as optical clearance of the cultures in comparison with the controls.To test whether sediment-attached viruses can still infect a host after desorption from sediment, sediment was added to 50 mL fresh 0.2 μm filtered lysates of MpV-08T, PgV-07T and MpoV-45T, and left for 24 h in which the lysate was gently mixed 3 times. The lysate with sediment was then gently filtered over a 47 mm GF/F filter  and rinsed 3 times with 2.5 mL Mix-TX medium. The filter was placed in an Erlenmeyer flask, covered with 5 mL Mix-TX and vortexed to resuspend the sediment. Samples were taken for viral abundances and the resultant sediment suspension was added to the respective host cultures in 10:1, 1:10 and 1:100 dilutions in final concentrations of 30 and 200 mg·L−4 of the SYBRGreen I stock  for 10 min at 80 °C and then quantified on a BD FACSCalibur with the trigger on green fluorescence [TM C6 cytometer , and triggered a red autofluorescence on chlorophyll [TM C6 software.Thawed virus samples were 100× to 10,000× diluted in TE buffer (pH 8.2), stained with a final concentration of 0.5 × 10rescence . The virorophyll . Algal d−1 sediment, respectively  did not significantly differ between the treatment tubes . Sedimenectively . In geneest rate . The exp23–4 min .6 and 105 mL−1 for MpoV-45T and MpV-08T, respectively), the total percentage of viruses lost over the 36 h incubation . Likewise, PgV-07T and NVC showed similar total relative losses , whereas the starting abundances were respectively 105 and 108 mL−1. The effect of higher virus concentrations on the removal capacity of the sediment was specifically tested using undiluted algal virus lysates with starting abundances of 0.1–2.4 × 108 mL−1. Absolute total losses .The additional experiment testing the effect of lysate/cell detritus exposure of the sediment clearly showed mitigated adsorption of MpoV-45T compared to the standard non-treated sediment treatment . Virus l−1 sediment addition strongly affected the growth of the Micromonas polaris TX-01 (6 mL−1), the starting virus:host ratio was still 9:1, thus, all hosts could still be infected. Sediment addition did not affect the maximal number of viruses produced (1.4 ± 0.06 × 107 mL−1) and consequently the virus burst sizes were comparable . These findings were independent of the Micromonas host and virus used, as equivalent results were obtained for an experiment in which M. commoda LAC38 (grown at 3 °C) was infected with MpoV-44T, with and without 50 mg·L−1 kaolinite clay addition (p = 0.422). The addition of the same type and concentration of sediment to a filtered lysate of MpoV-44T led to relatively gradual losses of up to 50% of total free viruses over time.The one-step infection experiment to test the direct effects of sediment on virus-host interaction, showed that 200 mg·Lis TX-01 A. The viis TX-01 B,C. Althaddition . Host lyM. polaris TX-01 control culture was also strongly reduced by the addition of 200 mg·L−1 sediment , the growth of the uninfected sediment A. Howeve culture B. Lysis  culture . While soduction D. Analog5 mL−1 for MpV-08T, MpoV-45T and PgV-07T, respectively). Within a week, all 3 virus suspensions led to lysis of their respective host  at all tested dilutions, showing that at least a fraction of the viruses was released and still infective. The controls with sediment that was not exposed to viruses showed normal growth.Approximately 2 h after resuspension of the virus-exposed and rinsed sediment in culturing medium, a significant proportion of the viruses from the sediment was countable by flow cytometry  during storms [−1 extend many kilometers off the coast, and thus, have a larger zone of influence [Virus losses increased with higher sediment concentrations and were especially high (up to 88%) with 100 and 200 mg·Ler up to  g·L−1 dunfluence . Our obs8 viruses mg−1 sediment. Hewson and Fuhrman [7 viruses mg−1 sediment. The difference is possibly explained by the type of sediment used. Hewson and Fuhrman [The increased losses with increased sediment concentration must be the result of increased contact rates between sediment particles and viruses . This is Fuhrman  showed a Fuhrman  only use Fuhrman ,22, and  Fuhrman ,43, our The viruses we used showed differences in removal dynamics, i.e., the Arctic virus MpoV-45T showed the most instantaneous and highest total loss, whereas MpV-08T showed the slowest and PgV-07T the lowest total loss in viruses. Since the sediments used were the same for all viruses and the algal viruses were similar in particle size, it is likely that the viruses differ from each other in characteristics such as hydrophobicity or isoelectric point, resulting in different binding strengths or only binding to a specific type  of the sediment particles . The cau−1 sediment addition by 31% . Theoretically, organic matter may thus counter virus losses by glacier-derived sediments in the natural pelagic system. However, for preparation of the detritus-treated sediment we used high concentrations of host cells, resulting in very high organic matter concentrations . Under μg୼·mL−1 ,46. A beμg୼·mL−1 ,47, furtM. polaris TX-01 as well as M. commoda LAC38), but also the production of the respective lytic viruses, MpoV-45T and MpoV-44T. The reduced growth of the host after 200 mg·L−1 sediment was added seems to be the logical result of increased turbidity, and the consequent reduced light availability [−2·s−1 for 0, 30 and 200 mg·L−1 sediment, respectively). Furthermore, the photosynthetic efficiency  of M. pusilla TX-01 was not affected by sediment treatment, indicating that photophysiology was not affected by turbidity. Microscopic observations revealed that some of the M. polaris TX-01 cells were attached to sediment particles on the bottom of the chamber, but this was not enough to explain the difference in abundances between the 0 and 200 mg·L−1 sediment treatment. We speculate that possibly a milder mechanical effect due to collision with particles may have caused the reduction in growth rate [Sediment addition delayed not only the growth of the algal host , which may have resulted from the sediment-induced stress of the host cell. Alternatively, anti-virus effects of elevated DMS concentrations [−1) and virus production (both 30 and 200 mg·L−1). These experiments indicate that removal of viruses by sediment can strongly reduce the chance of successful infection and virus proliferation.In the one-step infection experiments, lysis of the host was slightly delayed by the addition of sediment (200 mg·Ltrations ,53 may htrations . The expAlthough the total number of produced viruses did not vary amongst the treatments, the observed slower production can be expected to have ecological consequences in the natural environment. Furthermore, continuous input and settling of glacier-derived sediment particles  will cauWe found that glacier-derived sediments led to substantial losses of algal and bacterial viruses, and that the losses increased with higher sediment load and viral abundance. Organic matter prevented virus removal, but the ultimate effect on host-virus dynamics is likely to be limited as the attached viruses will still be restricted in successfully encountering their host. Sediment delayed host lysis and virus production, also under ecologically relevant conditions. Our study shows that glacier derived sediments have the potential to strongly impact microbial interactions by reducing host mortality through diminished virus infection as well as declined host growth. Continued global warming is anticipated to further enhance virus loss by adsorption to glacier melt-induced sediments. Additionally, melt-induced vertical stratification and the consequent reduction in mixing may accelerate settling of sediments with viruses attached away from suitable hosts. These findings indicate the need for more direct in situ investigations of the effects of glacier-derived fine sediments on virus removal and viral lysis.