Potato virus Y (PVY) is the most economically important virus affecting potato production. PVY manipulates the plant cell machinery in order to successfully complete the infecting cycle. On the other side, the plant activates a sophisticated multilayer immune defense response to combat viral infection. The balance between these mechanisms, depending on the plant genotype and environment, results in a specific outcome that can be resistance, sensitivity, or tolerance. In this review, we summarize and compare the current knowledge on molecular events, leading to different phenotypic outcomes in response to PVY and try to link them with the known molecular mechanisms. Potato virus Y (PVY) is the most economically important virus affecting potato production worldwide . It sevePotyvirus, family Potyviridae is a flexuous rod-shaped virus with a 9.7 kb positive-sense single-stranded RNA (ssRNA) genome which contains two open reading frames (ORF) encoding 11 functional proteins. One large ORF encodes a polyprotein that is cleaved by virus-specific proteases into 10 functional proteins. In addition, the protein P3N-PIPO is produced by a short overlapping coding sequence [PVY, a member of the genus sequence . The virsequence . This plsequence ,9. SimilThe complex interaction between plants and viruses includes the interplay of several mechanisms: (1) Viral hijacking of host factors required for efficient infection, (2) mechanisms of plant defense, and (3) mechanisms of viral counterdefense, to circumvent plant defense. The balance between these mechanisms defines a specific outcome that can be beneficial for the virus (compatible interaction) or for the plant (incompatible interaction). In the case of compatible interaction, the virus can multiply and spread within the plant; in susceptible plants, local and systemic symptoms appear, while tolerant genotypes develop no or very mild symptoms . In an N and PVYNTN in cvs. Igor and Nadine [N-Wilga and PVYNTN in cv. Etola [N605-GFP, PVYNTN, and PVYN-Wilga in cv. Rywal [The outcome of the interaction depends on the potato genotype, the environment, and the viral strain. Potato cultivars have different genetic backgrounds, resulting in different responses to the virus. It was shown that a single mutation can shift the outcome . On the d Nadine  and PVYNv. Etola . On the v. Rywal . There av. Rywal  and in mv. Rywal ,23,24, sv. Rywal ).Solanum tuberosum ssp. Phureja and diploid homozygous genotype Solyntus from ssp. tuberosum were sequenced . In addition, the pan-transcriptome constructed from several potato cultivars is available [Understanding of molecular mechanisms underlying those outcomes is of utmost importance for resistance breeding without growth trade-offs and adaptation of agronomical practices. The studies in the model plants uncovered several aspects of molecular mechanisms in plant immunity . Althougvailable . In the following sections, we compared the molecular responses of plants to PVY and linked them with the known molecular mechanisms of plant immunity .Ry genes and HR is conferred by Ny genes [2+ flux were detected and downstream mitogen-activated protein kinase (MAPK) cascade is activated, resulting in hormonal changes, such as increased level of salicylic acid (SA) and transcriptional reprograming, ultimately leading to pathogen arrest . There a plants .While mechanisms of pathogen arrest are well understood in Arabidopsis response to some bacteria ,43, fungBeclin1, one of the core autophagy-related genes that were upregulated by viral infection, interacts with nuclear inclusion b (NIb), the RNA-dependent RNA polymerase of turnip mosaic virus, and restricts viral infection through suppression of the viral NIb [Another resistance mechanism was found for Arabidopsis–begomovirus interaction, where translational repression blocks the spread of the virus. Activation of leucine-rich repeat receptor-like protein kinase (LRR-RLK) NIK1 leads to global translation suppression and translocation of the downstream component RIBOSOMAL PROTEIN 10 to the nucleus, where it interacts with a newly identified MYB-like protein, leading to the shutdown of global cellular and viral protein synthesis . In the iral NIb  resultinRsv4 gene encodes an RNase H protein that is able to interact with viral RNA polymerase complex and, thus, most probably degrades viral RNA when in dsRNA form [Several atypical dominant resistance genes were identified in the interaction of plants with potyviruses. For example, atypical thioredoxin (with modified active site residues) suppresses sugarcane mosaic virus RNA accumulation . This reRNA form .Resistance can also be conferred by S-genes. These are the genes the lack of which renders plants resistant. Their function is either to support viral infection cycle or to be negative regulators of immunity in plants. In contrast to R-gene mediated resistance, this type of resistance is recessive. The most studied S-gene is the alternative isoform of the eukaryotic translation initiation factor 4E (eIF4E), required for potyviral translation initiation, but dispensable for plant growth .Ny, Nc, and Nz, conferring resistance to PVYO (ordinary strains), PVYC (C strains), and PVYZ (Z strains), respectively . New vav. Rywal , Ny-2 in. Romula , and Ny-rpo Mira . None waNy-1-mediated HR to PVY where the virus was detected outside the cell death zone, that was able to reinitiate infection. This suggests that HR cell death is separated from the resistance mechanisms in this pathosystem [Ny-1 duplex, smaller lesions were developed and a lower amount of virus was accumulated, as compared to plants of cv. Rywal (Ny-1 simplex) after PVYNTN inoculation, indicating the effect of gene dosage on the efficiency of PVY restriction [Localized programmed cell death (PCD) that leads to the appearance of macroscopically visible localized tissue necrosis  is the fhosystem . Interestriction .Ny-1-mediated resistance of cv. Rywal, detection of large amounts of hydrogen peroxide and upregulation of several genes involved in redox state regulation from 1 day post-inoculation (dpi) indicated the importance of ROS signalling in an efficient HR  . In Ny-1ient HR . Specifhe virus .Ny-1-mediated resistance to PVYN-Wilga in potato cv. Rywal [SA is a phenolic compound primarily recognized for its role in local defense induced against biotrophic and hemi-biotrophic pathogens. SA biosynthesis is triggered during PTI and ETI  and is rv. Rywal , , was shown in potato in different cultivars [O was triggered by the helper component proteinase (HC-Pro) in a temperature-dependent manner in HR response of cv. Russet Burbank, carrying the so far uncharacterized Ny gene [N-Wilga [Ny-1-conferred HR [In Arabidopsis, constitutive, but not stress-related, β-1,3-glucanases were implicated in enhancing virus spread by degrading callose at plasmodesmata . Interesultivars . Moreove Ny gene   [S. stoloniferum, Rychc gene from S. chacoense [Ryadg from S. tuberosum ssp. Andigena [houRy gene was found in S. hougasii [Ny-DG gene present in the diploid potato clone DG-68 features ER-like response [phuRy(o) was identified in S. tuberosum ssp. Phureja, conferring resistance to PVYo, PVYNTN, and PVYN-Wi strains [Ry-genes is relatively low (see review [phuRy(o) and stoRy genes amplification of PVYN605 (infectious clones carrying GUS) could be detected in limited areas, while in cv. Tacna (carrying adg-Rygene) no amplification of the virus was detected [To date, several resistance genes conferring ER to PVY were identified and mapped to potato chromosomes IX, XI, and XII. They were introduced into potato cultivars from wild or domesticated Ry-fsto) ,75 are dhacoense  and RyadAndigena . The Ryhhougasii  but it hresponse . Recentl strains . The int strains ,81; howee review ). Althoudetected .stoRy gene, conferring ER response to PVY was isolated and functionally characterized [stoRy and stoRy-f alleles showed 100% homology between them. According to the authors, the elicitor of sto-Rymediated ER is PVY CP. They expressed the viral proteins in transgenic stoRy and wild type tobacco plants and only CP induced strong HR [stoRy-mediated HR in potato [Only recently, the first R gene against PVY in potato, the cterized , openingtrong HR . These rn potato ,84. FurtCapsicum annuum) [Pvr4, which confers ER to a broad range of potyviruses, including PVY [Pvr4-mediated resistance [N. benthamiana [Recently, another R gene against PVY was isolated from pepper ( annuum) . The autding PVY . It encosistance . In the thamiana .Ry genes also shed some light on the downstream signalling pathways involved in ER. Most TIR-NLRs activate resistance in a lipase-like protein Enhanced Disease Susceptibility 1 (EDS1)-dependent manner [sto-mediated resistance depends on both, EDS1 and NRG1 [The isolation of t manner ,89. Recet manner ,91. Receand NRG1 . They adand NRG1 .sto bearing resistant cultivar cv. Santé showed that cv. Santé had a higher number of differentially expressed genes at at 0.5 h post-infection (hpi), while cv. Igor exhibited the greatest response at 12 hpi. The faster perception and signaling in the ER response might enable a more efficient defense reaction [In the soybean–soybean mosaic virus pathosystem, a recent study suggested that the primary mechanism of ER against this potyvirus is the inhibition of viral cell-to-cell movement by callose deposition in an abscisic acid signalling–dependent manner . Howeverreaction . In factreaction . Howeverreaction . Recentlreaction .Glu-III, a gene that was found to be induced in response to PVY infection [Glu-III over-expression was studied in the cv. Santé. The results indicated that a transient multiplication of virus could occur in callose deficient plants; however, later the virus was blocked by Rysto gene signalling [The role of nfection ,92,94, wgnalling  . TherefF4E gene ,99. TranN with tobacco [tobacco translationally controlled tumor protein (TCTP) involved in stabilization of the ethylene receptor. The silencing of TCTP suppressed the PVY infection, whereas the over-expression of TCTP increased plant susceptibility confirming its role as a susceptibility factor [The function of S-genes is either to support viral infection cycle or to be negative regulators of immunity in plants . A S-gen tobacco . Paralley factor .In a susceptible interaction, the plant innate immune system is not able to establish an efficient defense response and the virus can replicate and invade the plant . Upon enIn contrast to plant–virus interactions, in sensitive interactions, virus infection does not trigger efficient and/or timely resistance response. Systemic disease symptoms manifest later in the infection in the upper noninoculated leaves and usually do not impede virus multiplication or its systemic movement. The disease symptoms  are a coPR genes was even stronger than in symptomless cv. Désirée and the dynamics of response was slower, compared to the resistance response, peaking two days later [NTN and PVYN, and in N. benthamina interaction with PVYO. However, the mild isolate PVYN induced a more rapid response [O [O with tomato or N. benthamiana (in both pathosystems mild symptoms develop), late response of ethylene, JA, and ROS signaling was detected, while induction of PR-1 was very weak [PVY is recognized by the plant by receptor proteins also in susceptible interaction , triggerys later ,94. The response  or a verponse [O . Additioery weak ,24.NTN in secondary infected tubers of a sensitive cv. Igor was strongly dependent on virus concentration. Viral concentration was the highest in the necrotic lesions, where a massive transcriptional response was observed. Genes for proteases with caspase-like activity that are markers of cell death were induced, as were also endoplasmic reticulum chaperones, markers of the endoplasmic reticulum stress response [PR gene induction was the strongest and correlated with detected high concentrations of the virus [An important feature of sensitive interactions is a response to the hijacked metabolism. The transcriptional analysis of responses to PVYresponse  .PR-1, PR-2, NTN, photosynthesis genes were shown to be transiently induced at early stages of infection but then rapidly repressed at the onset of virus multiplication [MIR6022 promoter region [On the molecular level, tolerant responses to PVY included attenuated expression of defense response genes . Some aPVY is the most damaging virus of potato and other Solanaceae crops worldwide. Thus, understanding the molecular mechanisms governing the outcome of the interaction is essential for sustainable production in a changing environment. During the last decade, important technical improvements have slowly improved our knowledge on general mechanisms of defense response in PVY infection. This should improve even further with the implementation of mathematical modelling to predict responses of immune signaling  and linkadgRy [With this toolkit in hand, we will be able to fill in the knowledge gaps and provide answers for remaining questions. For example, it is still not understood how PVY multiplication and/or cell-to-cell movement are blocked in resistant interaction. We know it is not the PCD that is blocking the viral movement , as was Ryadg ) viral mRyadg . So, perphuRy(o) [Another question remaining is whether, in plant, PVY is perceived by multiple receptors. This was indicated in resistant interaction governed by Ry(o)phu , where eRy(o)phu ,109. TheRelated to that, how the background genotype  affects the efficiency of resistance it is also widely unstudied. It is now generally assumed that, if the correct R-gene is incorporated into the genetic background, we will obtain the desired phenotype. The importance of the availability of certain components of downstream immune signaling was confirmed for a cochaperone involved in the maturation of RLKs  and for The next important question is: What are the mechanisms involved in the establishment of tolerance? Resistance is traditionally preferred over tolerance in breeding programs, as tolerant crops represent a virus reservoir. Nevertheless, tolerance may have an advantage over resistance for crop protection because it does not actively prevent virus infection and/or replication, therefore there is little evolutionary pressure for the virus to mutate and to evolve into more aggressive strains (, revieweThrough the design of experiments aimed to answer these questions, we will provide knowledge that will allow for the development of efficient breeding strategies as well as for improvement of agricultural management strategies.


Euparkeria capensis enabled the reconstruction and three-dimensional articulation of the hindlimb. The joint mobility of the hindlimb was quantified in 3D to address previous qualitative hypotheses regarding the stance of Euparkeria. Our range of motion analysis implies the potential for an erect posture, consistent with the hip morphology, allowing the femur to be fully adducted to position the feet beneath the body. A fully sprawling pose appears unlikely but a wide range of hip abduction remained feasible—the hip appears quite mobile. The oblique mesotarsal ankle joint in Euparkeria implies, however, a more abducted hindlimb. This is consistent with a mosaic of ancestral and derived osteological characters in the hindlimb, and might suggest a moderately adducted posture for Euparkeria. Our results support a single origin of a pillar-erect hip morphology, ancestral to Eucrocopoda that preceded later development of a hinge-like ankle joint and a more erect hindlimb posture.Triassic archosaurs and stem-archosaurs show a remarkable disparity in their ankle and pelvis morphologies. However, the implications of these different morphologies for specific functions are still poorly understood. Here, we present the first quantitative analysis into the locomotor abilities of a stem-archosaur applying 3D modelling techniques. μCT scans of multiple specimens of  Archosaurs were the predominant group of large terrestrial and aerial vertebrates in the Mesozoic era and included pterosaurs, the familiar dinosaurs (including birds), crocodylomorphs and an intriguing variety of Triassic forms. Their well-documented fossil record allows retracing the evolution of the dramatic differences in morphology and locomotion in this group. By the Middle Triassic, the clade had already reached high levels of morphological and functional disparity, especially in the hip and ankle joints5 Fig. , but thiEuparkeria capensis is a small eucrocopodan archosauriform from the early Middle Triassic (early Anisian) Burgersdorp Formation near Aliwal North, Eastern Cape, South Africa; and only from a single horizon therein, the Subzone B of the Cynognathus Assemblage Zone29. Its osteology is well known from numerous well-preserved specimens30  articulation of the bones within the crus, distal tarsals and metatarsals has never been fully assessed, mostly due to the small size of the bones and the fact that most of them are still partially embedded in matrix  of limb joints has been previously quantified in Devonian and Permian tetrapods48, there have been no previous attempts to quantify hip joint mobility, or the osteological ROM of any other joint, in Triassic archosaurs (see 10). We hope that the application of quantitative computational methods to stem archosaurs could lead to new insights into the evolution of their functional morphology and hindlimb biomechanics. In particular, the combination of independent lines of evidence (e.g. see 48) allows for a more accurate reconstruction of the potential postures of extinct taxa. We quantified the ROM to determine the maximal joint excursion in the hip joint and compared it with the ankle joint rotation axes, thereby testing whether previous qualitative assessments of hindlimb posture were supported through a quantitative biomechanical analysis. We then used these results to revisit the longstanding question of whether stem-archosaurs were more sprawling or capable of a more adducted posture50.Here, we used 3D models, derived from μCT-scans of several specimens, to accurately reconstruct the 3D morphology and articulation of the pelvic girdle and ankle of Euparkeria, SAM PK 5867, and in SAM PK 6047A. SAM PK 5867 is still partially covered in matrix and the femur is overlaying most of the right ilium, thus obscuring most of the pelvic bones. However, the μCT scans revealed that the pelvis is nearly complete, missing only the left ilium, the distal tip of the left ischium, most of the left pubis and the ribs of the second sacral vertebra  hip joint spacing derived from CT scans   appears minimal (unlike in 1). We suggest that the fibulo-calcaneal joint instead assisted rotational movement of the fibula around the ankle joint (pronation and supination/LAR), in order to keep the foot steady on the ground as part of a non-parasagittal gait.The bones obtained from the µCT scans allowed virtual reassembly of the foot and ankle skeleton in its estimated natural posture Fig. . The fibThe subtriangular distal tarsal (DT) IV possesses a caudally projecting spur, which articulated with the astragalus dorsally and DT III medially and is slightly concave laterally for the articulation with the articular convexity of the calcaneum Fig. B. The arEuparkeria is oblique to the knee joint and further oblique to the orientation of the metatarsophalangeal joints , and it was even feasible to further adduct the femur medially and overstep the other limb  in sub-horizontal femur poses indicated that Euparkeria was potentially unable to engage in fully sprawling gaits which rely on external (positive) LAR of the femur54, e.g. in late swing and early stance, and/or use high degrees of femoral abduction . Additionally, this osteological limitation on LAR is absent in known taxa capable of a more sprawling gait, e.g. salamanders, Iguana or crocodylians51, and potentially further implies a less abducted limb during locomotion for Euparkeria (unlike in10). However, further studies of 3D ROM envelopes in more sprawling extant taxa; e.g. salamanders, varanids and iguanas; and the relationship between osteological and in vivo ROM in such taxa, are necessary to further characterize sprawling motion and its musculoskeletal constraints in order to draw more detailed inferences about locomotion in extinct taxa. While many poses (both sprawling and erect) could not be excluded for Euparkeria based on the osteological ROM alone, we caution against drawing conclusions directly based on these osteologically valid poses, especially those close to the border of the ROM map  for Euparkeria is in our view the most reasonable interpretation, although many postures fitting this description remain feasible in our results. Further resolution of the posture and gait of Euparkeria or other stem-archosaurs would require biomechanical methods that address how soft tissues such as muscles and the nervous system might have controlled hindlimb function.Our approach, combining 3D articulation and ROM analyses, demonstrates conflicting patterns in the hip and ankle joint. Euparkeria support a single origin of a pillar-erect hip morphology in both the Nesbitt35 and Ezcurra36 phylogenetic tree topologies, meaning that this articular morphology can be regarded as ancestral to at least Eucrocopoda, contrasting with previous hypotheses60. As we have cautioned, however, such a morphology does not necessarily suggest a ‘fully erect’ limb posture nor necessarily allow a parasagittal gait. The ancestral locomotor stance in archosaurs can thus not be inferred from the hip morphology alone. While we infer that the pillar-erect hip morphology arose prior to Archosauria (unlike in60), an ankle joint permitting a parasagittal gait appeared later on. Indicators of a pillar-erect hip morphology are laterally inclined ilia, due to the ventrolaterally projecting sacral ribs and a pronounced supra-acetabular rim, therefore allowing the ilium to completely cover the femoral head. This condition appears to be present in the basal avemetatarsallian Teleocrater rhadinus61  and the Triassic pseudosuchian ornithosuchids, e.g. Riojasuchus tenuisceps63. It thus seems likely that the pillar-erect hip morphology evolved as a precursor to the ‘buttress-erect’ morphology of both dinosauriforms64 and early crocodylomorphs65, and originated once at the base of Eucrocopoda. We infer that phytosaurs and extant crocodylians lost the erect hip morphology of their ancestors; pillar-erect in the former and buttress-erect in the latter; facilitating a more abducted hindlimb posture as a secondarily derived adaptation for their amphibious lifestyle. This is consistent with previous interpretations of the condition in crocodylians66 but novel in terms of phytosaurs (unlike in 10); thus phytosaurs show an additional level of convergent evolution with crocodylians. Depending on the interpretation of phytosaurs as either early-diverging pseudosuchians36 or stem-archosaurs61, the suchian tarsus might have evolved within Archosauria, or just outside this clade , later functionally facilitating the evolution of a hinge-like ankle joint with approximately a single degree of freedom suitable for a parasagittal gait in both pseudosuchians (suchian and ornithosuchid tarsus) and ornithodirans  and thus a fully adducted posture. These changes then further enabled the evolution of a bipedal locomotion and/or a digitigrade stance in poposaurids69 and dinosauromorphs71.Our new insights into the hip structure in 10). We caution that the hindlimb posture of stem-archosaurs cannot be determined relying solely on qualitative morphological characters , and different lines of evidence need to be combined for conclusive results. While Euparkeria constrains the origin of the pillar-erect hip morphology to the base of Eucrocopoda, the ankle structure enabling a highly adducted hindlimb posture with a parasagittal gait evolved later on within Archosauria, thus a pillar-erect hip morphology does not necessarily warrant a fully adducted ‘pillar-erect’ posture.In conclusion, we add further evidence to the homoplastic manner of postural evolution within archosaurs (see51), leaving a very wide range of possibilities in the case of Euparkeria. The remaining ROM then can only be further narrowed down to in vivo conditions using soft tissue constraints; e.g. through ligamentous ROM simulations53 and biomechanical analyses of neuromuscular control of the hindlimb joints. Incorporation of such soft tissue parameters into digital simulations will further elucidate the pattern of hindlimb postural evolution in archosaurs.Our quantitative assessment of the ROM builds the foundation for further computational investigations into the locomotor capabilities of archosaurs. While the osteological ROM is a useful tool to exclude certain possibilities in extinct species, its informational value is limited as it can only exclude osteologically impossible postures 73, was μCT scanned at the University of Cambridge, Cambridge, UK, with a Nikon Metrology XT H 225 ST; see Table High-resolution micro-CT scans of several specimens of ZC Figs. , 8A. Spehttps://www.thermofisher.com/ch/en/home/industrial/electron-microscopy/electron-microscopy-instruments-workflow-solutions/3d-visualization-analysis-software/avizo-materials-science.html.html) for segmentation and 3D model generation. Each dataset was segmented manually by tracing the bone in the individual CT slices in a single axis and corrected, where necessary, in the perpendicular planes (see47), as the inbuilt automated segmentation algorithms of Avizo could not be applied due to varying X-ray attenuation within the bones themselves and noise artefacts bleeding into the density spectrum of the surrounding matrix. Minor taphonomic artefacts; e.g. cracks within the bones; were filled in by interpolation of bordering bone and/or manual correction following Lautenschlager74. Displaced elements due to larger breaks were segmented individually and digitally rearticulated later on. High-resolution meshes of the bones were decimated in MeshLab75 2016 (https://www.meshlab.net/) using the Quadric Edge Collapse Decimation filter and cleaned from non-manifold geometry and self-intersecting faces with the cleaning and repairing filters in MeshLab before the bones were articulated in Autodesk Maya 2017 . We then created a nearly complete composite pelvic girdle and hindlimb by combining the skeletal elements of all scanned specimens . The vertices of any imported mesh acted as point clouds to which the primitive shapes were fitted. The superimposed geometric centres of two  resulting shapes from two articulating segments represented the centre of the respective joint. A sphere and an ellipsoid were fitted to both the acetabulum and the femoral head to test the influence of different fitted primitive shapes to the articular surfaces  is insufficient to capture the complexity of the multidimensional movement in these joints and either neglects large sections of potential ROM, only enabled through combination of multiple DOFs, or erroneously increases the ROM due to the addition of all DOFs without considering their interactions and thus leads to the inclusion of ROM ‘corners’. Therefore a 3D joint sampling approach, following the methodology of Manafzadeh & Padian53 was performed to estimate the 3D ROM in the hip joint of Euparkeria.Ball and socket joints, e.g. hip and shoulder joints, are highly mobile and complex motion during locomotion results from interaction between all three degrees of freedom (DOFs)52 using XROMM MayaTools 2.2.379. A forward kinematic rig was created in Maya, following Manafzadeh & Padian53 and A. R. Manafzadeh pers. comm. (2019). The specimen was articulated in a default reference pose, with all joint rotations set to 0°  a second joint was created with a pre-set rotation of 0°/0°/90° to which the hip joint was parented (A. R. Manafzadeh pers. comm. 2019), thus ensuring desired behaviour. To test different joint setups, based on the set of different fitted geometric primitive shapes, and to accommodate for the uncertain amount of epiphyseal cartilage (e.g.81), a dynamic rig was created. Using multiple dropdown menus in Maya, the specimen and the individual primitive shapes could be selected, and the percentage of additional articular cartilage, based on the femur length, could be changed through a slider, with the JCS and joint centres automatically adjusting to the selected parameters , − 90° to 90° for abduction/adduction (ABAD) and − 180° to 180° for long-axis rotation (LAR) at 5° increments were systematically sampled. Potential mesh interpenetration, between the pelvic girdle and femur, was automatically detected using a Boolean operation in Maya and thus resulted in an inviable poseSupplementary Information 1.Supplementary Dataset 1.Supplementary Video 1.Supplementary Video 2.Supplementary Video 3.


Initial rarefaction analyses with datasets of Borna disease virus 1 human encephalitis cases revealed a stochastic behavior of virus read detection. Based on this, we transformed the Bernoulli formula to predict the minimal necessary dataset size to detect one virus read with a probability of 99%. We validated the formula with 30 datasets from diseased individuals, resulting in an accuracy of 99.1% and an average of 4.5 ± 0.4 viral reads found in the calculated minimal dataset size. We demonstrated by modeling the virus genome size, virus-, and total RNA-concentration that the main determinant of mNGS sensitivity is the virus-sample background ratio. The predicted LODmNGS for the respective pathogenic virus in the datasets were congruent with the virus-concentration determined by RT-qPCR. Theoretical assumptions were further confirmed by correlation analysis of mNGS and RT-qPCR data from the samples of the analyzed datasets. This approach should guide standardization of mNGS application, due to the generalized concept of LODmNGS.Metagenomics is a powerful tool to identify novel or unexpected pathogens, since it is generic and relatively unbiased. The limit of detection (LOD) is a critical parameter for the routine application of methods in the clinical diagnostic context. Although attempts for the determination of LODs for metagenomics next-generation sequencing (mNGS) have been made previously, these were only applicable for specific target species in defined samples matrices. Therefore, we developed and validated a generalized probability-based model to assess the sample-specific LOD of mNGS experiments (LOD It enables a relatively unbiased detection of all organisms present in a sample, including viruses, bacteria, fungi, and parasites Sensitivity is one of the major factors to assess the power of a diagnostic method. At the first glance, the sensitivity of mNGS is determined by the amount of sequenced reads. Thus, the more reads are sequenced, the further the sensitivity increases. However, the selection of the required data depth has been based mainly on economic factors and empirical and ad-hoc heuristic models, resulting in published datasets that range from 5 to 24 mio reads mNGS), as applied for other routine methods, is hampered due to the many variables influencing the sensitivity.So far, sensitivity assessments of mNGS have been made by comparison with routine methods at qualitative or semi-quantitative levels  and by spiking a collection of pathogens in serial dilutions in a specific sample matrix mNGS, which is critical for the routine application of mNGS in the clinical diagnostic context.Hence, the aim of this study was the development and validation of a pathogen/matrix independent generally applicable mathematical model to assess the detection limit of mNGS experiments. This approach should guide standardization of mNGS application. Therefore, we developed and validated a straightforward analytical tool to assess the sample-specific LOD22.1The study included 30 disease-associated samples and the respective datasets from human and animal cases , confirm2.22 ranging from 0.9998 to 1.0 and slope in the range from –3.407 to –3.348. For BoDV-1 RNA only, retrospective absolute quantification was carried out with an external calibration curve. An internal standard was used for normalization between the runs. The qPCR calibration curves for the quantification of target virus fragments in the library showed an efficiency between 100.4% and 103.2% with R2 ranging from 0.993 to 1.0 and slope in the range from –3.312 to –3.247. For this, 14 libraries, comprising nine WNV (lib03416 – lib03425), two BoDV-1 , two PGV , and one RusV (lib03123) were analyzed.Total RNA concentrations were quantified using a Nanodrop ND1000 instrument . The DNA library concentration was measured by using the Bioanalyzer 2100 . Absolute quantification of the viral RNA and the double-stranded virus cDNA (library) was performed by specific 5′ nuclease RT-qPCR and qPCR, respectively . For BoDV-1, Mix1 targeting the P gene was used 2.3Rarefaction analyses were performed initially with the five BoDV-1 metagenomics datasets only , NC_001563.2, Flaviviridae; Borna disease virus 1 (BoDV-1), NC_001607, Bornaviridae; Rift Valley fever virus (RVF), NC_014397, NC_014396, NC_014395, Bunyaviridae; Sindbis virus (SINV), NC_001547, Togaviridae; Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), NC_045512.2, Coronaviridae; Human coxsackievirus A (CV-A2), NC_038306, Picornaviridae; Measles virus (MV), NC_001498, Paramaxoviridae; Rabies lyssavirus (RABV), NC_001542, Rhabdoviridae; Rubella virus (RuV), NC_001545.2, Matonaviridae; Influenza A virus (IAV), GCA_001343785, Orthomyxoviridae; Hepatitis delta virus (HDV), NC_001653.2, Deltavirus incertae sedis.33.12 ≥ 0.9851, p ≤ 0.001), despite detection rates of <100% .As a starting point for the analyses, we performed a rarefaction analysis. We repetitively determined the detection (presence/absence) of virus reads in data subsets of different size (100 repeats per subset size). From the results of these repetitive drawings, we calculated the positivity rate, i.e. the detection rate of the virus in a given dataset size. For this, we used a set of five datasets generated from BoDV-1-positive samples covering a range of virus read percentages from 6.9E – 04 – 8.0E – 01%. The detection rate of BoDV-1 reads in subsets of these datasets differed . In subs3.2Based on the results of the rarefaction analyses (=stochastic behavior of virus read detection influenced by dataset size and virus read proportion), we sought a mathematical formula to predict the minimum required dataset size to detect one virus read with a reasonable detection rate. The Bernoulli process describes a discrete stochastic process with only two possible results (presence/absence), coupled with a statement about the probability of occurrence. The equation for the standard Bernoulli process is shown in Equation 1. The notations for the mathematical derivation can be found in Equation 1: Bernoulli processThe LOD is defined as the lowest quantity that can be detected with reasonable certainty for a given analytical procedure Equation 2: Transformed Bernoulli processValidation of the transformed Bernoulli formula was performed with ve level . We provve level . As a crve level  by the cve level . We inclEquation 3: Recovery of virus read numbers3.3As mentioned above, empirical data shows that the detection of a species depends on its abundance, the relative genome size, and the dataset size. We used R Studio Equation 4: Prediction ofApplying Eq. 2 in combination with Eq. 3 and Eq. 4, we modeled α = 0.99 . First, 0 ng/µl) a. As Fig0 ng/µl) b. This s50 ng/µl b. With a50 ng/µl b.Fig. 2TIn order to generalize the model, we investigated the influence of the genome size on the virus read numbers at a given dataset size c and thei , the theoretically necessary minimal dataset size n was calculated according to Eq. 2. The LOD is then defined as the minimal c/µl for which 1 viral read can be expected in a dataset of 5.0E + 06 reads. As shown in To assess the meaningfulness of the result obtained with a certain assay, the limit of detection (LOD) of that assay needs to be defined. Although in practice the LOD of qPCR depends on the specific assay, theoretically the LOD of qPCR is at the genome copy number of 3 c/µl but independent of the genome size. As shown above, the sensitivity of mNGS depends on both virus copy number and virus genome size. In order to investigate the limit of detection for mNGS analysis, we calculated the minimum virus genome copy number that allows for the detection of a virus in a dataset of 5.0E + 06 reads generated from a sample with 30 ng/µl total RNA. Specifically, we further examined the effect of the genome size (1.5 kb to 30 kb) on the detection limit of an mNGS analysis. For this, we calculated the LOD of an mNGS analysis as follows: For each To evaluate the sensitivity independent of the pathogen (genome size and copy number) and the total nucleic acid concentration, we calculated n  for a range of t size n f. For al3.4mNGS and RT-qPCR. To this end, we calculated RT-qPCR correlated highly significant with the mNGS . Unexpectedly, with a single exception  than RT-qPCR  in comparison with RT-qPCR . This coincided with mNGS of these libraries. However, the same libraries had an increased mNGS (median = 5.7E – 04%). Unfortunately, nine WNV libraries had to be excluded from this analysis of Library due to methodical constraints. Here, the RT-qPCR assay is located at the 5′-terminus of the genome, which is not converted efficiently during library preparation, as displayed by qPCR data and genome coverage analyses (data not shown). Hence, no reliable determination of Library was possible.As a proof-of-concept that for mNGS analysis ∼RT-qPCR b. Theref3.5mNGS using Eq. 2 and its modification  to the datasets used in this study  and compared it to the c/µl of RT-qPCR  and absolute quantitative (c/µl) RT-qPCR values were observed (mNGS) and formula-derived values , respectively. Obviously, the correlation between mNGS and formula-derived values is due to the dependency of the formula derived values from the mNGS data. None of the categories had significant correlation with the dataset size. This correlation analyses clearly shows that the calculation of LODmNGS and the necessary dataset size n is possible and yields meaningful results. These allow the assessment of the mNGS based detection limit depending on We conclusively examined the correlation between the various sample and dataset characteristics examined above. To this end, values were log-transformed prior to the calculation of spearman correlations and p-values with the rcorr function of the Hmisc package observed . As Fig.4mNGS, whereby valuable information can be derviated for the assessment of mNGS experiments and related expectations. The expression of LODmNGS in copies per microliter enables comparison with RT-qPCR derived concentrations. To the best of our knowledge, we showed for the first time a direct relationship between the ratio of viral and total RNA and its ratio after mNGS analysis.We developed a straightforward probability-based mathematical approach to test the assignment of the individual detection limit per sample for mNGS analysis. We followed a sample matrix-independent approach to preserve the advantageous non-specificity of mNGS in pathogen detection and at the same time make a statistical statement about the probability of virus detection at a certain data depth. The assessment of an mNGS result must always take into account the specific detection limit of the analysis for a certain pathogen and the analysis of specific parameters  The use of an external DNA standard ; however, we did not observe substantial differences of the low biomass lib02557 (4.1 ng/µl) to all other analyzed libraries (≥17 ng/µl; mNGS; it alters the composition of the total nucleic acids by enzymatic modifications including reverse-transcription, fragment end polishing, and adapter ligation. Of course lastly also size selection impacts the composition by removing small and large fragments from the sample during library preparation Library from a set of analyzed libraries. Although due to technical constraints only a subset of the data could be taken into account, it appears that the main difference between RT-qPCR and mNGS is introduced during library preparation. This does of course not rule out differences of viral read proportions in datasets which can derive from different sequencing platforms and their respective library preparation workflows, affecting mNGSRT-qPCR and mNGS. Therefore, further studies are needed to identify such factors to adjust our model and increase its level of precision.Deducing mNGS for every sample based on the target virus, total RNA concentration, and dataset size. While LODmNGS increased with increasing total RNA-concentrations, the impact of the dataset size was neglectable. This missing influence of the dataset size may be caused by the selected datasets, although these were randomly selected from available datasets. The accuracy of the calculated LOD remains to be assessed by systematic comparison of mNGS negative but RT-qPCR positive samples. Nevertheless, in 25/30 cases the RT-qPCR derived quantitative values were above the mNGS LOD, supporting the dependencies between sample and LODmNGS elaborated in this paper. In the remaining cases, LODmNGS was higher than the concentration derived from RT-qPCR. All these had a In mNGS seems therefore not suitable but appears rather matrix and pathogen-specific In previous studies, the detection cut-offs of mNGS have been linked to Cq ~32 and ~36 in nasopharyngeal swabs, aspirates, or sputums for different virus panels 5mNGS, considering nucleic acid concentration, genome length, and data depth. For this calculation, we define the total nucleic acid concentration as the background for modeling the LODmNGS. The results of these calculations are congruent with RT-qPCR results. This mathematical and sample matrix independent approach may guide to a more transferable and standardizable LOD for future mNGS experiments.The assessment of the detection limit is of major interest for the application of shotgun mNGS in clinical laboratories. Therefore, we developed and validated a straightforward analytical tool to assess the sample-specific LODArnt Ebinger: Formal analysis, Investigation, Methodology, Software, Validation, Visualization, Data curation, Writing - original draft, Writing - review & editing. Susanne Fischer: Conceptualization, Methodology, Validation, Writing - review & editing. Dirk Höper: Conceptualization, Funding acquisition, Methodology, Supervision, Writing - original draft, Writing - review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Alphaviruses from Africa, such as Middelburg virus (MIDV), and Sindbis virus (SINV), were detected in horses with neurologic disease in South Africa, but their host ranges remain unknown. We investigated the contribution of alphaviruses to neurologic infections and death in wildlife and domestic animals in this country. During 2010–2018, a total of 608 clinical samples from wildlife and nonequine domestic animals that had febrile, neurologic signs or unexplained deaths were tested for alphaviruses. We identified 32 (5.5%) of 608 alphavirus infections (9 SINV and 23 MIDV), mostly in neurotissue of wildlife, domestic animals, and birds. Phylogenetic analysis of the RNA-dependent RNA polymerase gene confirmed either SINV or MIDV. This study implicates MIDV and SINV as potential causes of neurologic disease in wildlife and nonequine domestic species in Africa and suggests a wide host range and pathogenic potential. Togaviridae) have been recognized as major emerging viruses. New World alphaviruses, such as Western equine encephalitis virus, Eastern equine encephalitis virus, and Venezuelan equine encephalitis virus, are traditionally associated with severe disease, such as encephalitis and a high mortality rate in humans and horses in the Americas and Australia  was discovered in the late 1950s in Identification of MIDV and SINV as possible neurologic pathogens in horses in South Africa prompted the investigation of undiagnosed neurologic and febrile disease or sudden unexplained death in wildlife, nonequine domestic animals, and birds. The purpose of our study was to investigate the host range and association of alphaviruses from Africa with neurologic disease and death, as well as to increase knowledge on pathogenesis and the zoonotic potential of these 2 viruses.This study forms part of an ongoing passive surveillance study to detect zoonotic arboviruses in South Africa. A total of 608 EDTA-treated blood or postmortem specimens from animal species other than horses that had neurologic disease, acute febrile illness of unknown origin, or sudden unexpected death during February 2010–September 2018 in South Africa were submitted to the Centre for Viral Zoonoses, Department of Medical Virology, University of Pretoria . Clinical cases were submitted by wildlife veterinarians and pathologists who identified cases that fit the case definition of febrile or neurologic signs or sudden unexpected deaths of unknown origin. EDTA-treated blood or serum samples were submitted when animals were alive, whereas tissues were submitted if the animal died. Preferred tissue type was brain matter or spinal cord and visceral organs, including lungs, spleen, and liver samples. The study was approved under Section 20 (no. 12/11/1/1) by the Department of Forestry and Fisheries and by the Animal Ethics Committee (no. V057–15 and H12/16).We performed full necropsy examination at the Section of Pathology, Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, for animals that had clinically progressive quadriparesis, apparently normal mentation, and positive results for MIDV or SINV by reverse transcription PCR. We collected a wide range of organs and tissues from all wildlife cases and preserved the samples in 10% neutral buffered formalin for histologic examination. We microscopically examined routinely prepared, hematoxylin and eosin–stained (https://www.qiagen.com) according to the manufacturer’s instructions and virus RNA from tissue samples by using the RNeasy Kit (QIAGEN). We analyzed all clinical specimens by using genus-specific nested real-time PCRs (62855′- GCAGCCTTTTGTCCGTCYAA-′36305 and reverse 66335′-GGCTTCAAGTCRTAGGTTT-3′6614) and SINV (forward 62855′-GCAACCTTYTGCCCCGCYAA-′36305 and reverse 66335′-GGGACCAAATTATRCGTCT-3′6613) nested primers to increase the RNA-dependent RNA polymerase gene region from 198 bp to 348 bp for phylogenetic analysis by using the same conditions as in the alphavirus PCR  conditions in the Department of Forestry and Fisheries compliant BSL-3 laboratory at the Centre for Viral Zoonoses, University of Pretoria. We extracted virus RNA from blood or body fluids by using a Viral Mini Kit . We analyzed sequence data by using CLC Main Workbench 8 (QIAGEN) and MEGA 6.06 software (https://www.megasoftware.net). We performed multiple sequence alignments by using MAFFT version 7 software (http://mafft.cbrc.jp) with default parameters and used them to assemble sequences. We conducted maximum-likelihood analysis by using RaxML , MycoZap CL-Plus (Lonza), 10% fetal calf serum (FCS) (Lonza) for Vero or 20% FCS for BHK-21 clone 3 cells in an Intercool Incubator  at 37°C and an atmosphere of 5.0% CO2. We subjected all PCR-positive samples to virus isolation on African green monkey kidney cells (Vero) and baby hamster kidney cells (BHK-21 clone 3). We maintained cells in Earle minimum essential medium (EMEM) containing 2. We inoculated blood or serum samples (200 μL) from animals directly onto phosphate-buffered saline (PBS)–washed cells, incubated for 1 h, and washed once with PBS, then added 2% EMEM. We cut tissue specimens into pieces (≈5 mg) and homogenized with sterile glass beads  at 100 × g for 6 min by using a TissueLyzer (QIAGEN). We then centrifuged homogenates at 1,000 × g for 10 min to collect debris. We used 200 μL of supernatant to infect PBS-washed cells, incubated them for 1 h, and then added 2% EMEM. We passaged cultures 3–4 times at 7-day intervals, observing monolayers daily for cytopathic effect. Between passages, we froze cultures at –80°C, thawed 3 times, and clarified by centrifuging at 1,000 × g for 5 min.We performed isolations by using EMEM containing 10% fetal calf serum and Mycozap CL-Plus for Vero cells and 20% fetal calf serum and Mycozap CL-Plus for BHK-21 clone 3 cells and incubated at 37°C in an atmosphere of 5% COhttps://www.cdc.gov/epiinfo/index.html) and a Fisher exact test with 95% CIs and odds ratios (ORs) to calculate the association between clinical signs and infection. We excluded animals that were found dead (n = 76) or were aborted (n = 23) or stillborn (n = 13) from OR analysis.We performed data and statistical analyses by using Epi Info version 7.2.0.1  animals and SINV in 9  . We deteThe 608 animals tested were from 99 animal species, of which 14 species were positive for MIDV or SINV . We deteAll SINV-positive animals and 20 (87%) of 23 MIDV PCR-positive animals had virus detected in postmortem specimens . MIDV waMost MIDV infections were reported during 2010  and 2015 , followed by 2017 , 2014 , 2018 , 2012 , and 2011 . No MIDV infections were detected during 2016 . SINV inNo specific macroscopic lesions could be demonstrated at necropsy for cases submitted to the Section of Pathology, Faculty of Veterinary Science, University of Pretoria. Similar to other parenchymal tissues examined, no distinct cytologic nor architectural abnormalities could be demonstrated in the CNS of examined cases. Some of these animals died after a short period (<3 days) of recumbency. In these cases, secondary factors, such as dehydration and unrelenting high levels of stress, were suspected to contribute to death. In some instances, animals with severe cases were euthanized for humane reasons or as part of disease management for elective necropsy to determine the cause of outbreaks of neurologic signs in wildlife.Phylogenetic analysis of the partial nonstructural protein 4 (nsP4) gene region (348 bp) confirmed all virus-positives cases as being infected with MIDV or SINV . MaximumWe identified a total of 32 alphavirus infections in wildlife, nonequine domestic animals, and 2 birds that had neurologic or febrile signs or unexplained death over a period of 9 years (2010–2018) as MIDV (n = 23) or SINV (n = 9). Detection of these viruses in the CNS indicates that they are able to cross the blood–brain barrier and suggests that they might cause pathologic changes, neurologic disease, and death in infected animals. Detection of viral RNA in respiratory organs and visceral organs suggest spread of these viruses throughout the body. The success rate of virus isolation from neural tissue is low because death is often the end stage of disease concurrent with a low virus titer, which can often only be detected by nested PCR. Virus isolation can also be related to the quality of clinical specimens received from wildlife and domestic animals, which were often found dead in remote areas and took some time to reach the laboratory, as compared with equine cases, which are often detected earlier by owners during the stage of clinical disease and therefore are sampled earlier.>1 infections for MIDV and SINV.All cases were accompanied by a case investigation form with clinical and epidemiologic data recorded by the submitting veterinarian. All animals that had clinical information available had neurologic signs suggesting infection with MIDV and SINV as likely etiologies of neurologic disease (p = 0.06) despite the small sample size of alphavirus-positive cases compared with alphavirus-negative cases. None of the other signs could clearly be associated with these viruses because of the small sample size of virus-positive animals. This finding suggests that these Old World alphaviruses might have similar characteristics to their New World relatives in some species  was given a diagnosis after 9 rhinoceroses from the same population had similar clinical signs. All animals were subjected to postmortem investigation over a 2-year duration at the Faculty of Veterinary Science, University of Pretoria. CNS tissues from an adult white rhinoceros (MVA007/10) from Broederstroom, North West Province, showed positive results for MIDV and equine encephalosis virus. A rhinoceros calf that was kept in the same boma (livestock enclosure) had similar signs and had died 5 days earlier. A warthog (MVA51/10) was part of a disease outbreak involving ≈50 similar cases in contact animals of the same species in Marekele National Park, Limpopo Province. All warthogs of the specific sounder (group) showed signs of ataxia that lasted 3–7 days, after which some recovered and some died. A captive-bred African buffalo calf from Bloemfontein, Free State Province, also died after paralysis developed, and an uncountable number of animals in the herd were positive for MIDV. These 3 cases of disease can be regarded as disease outbreaks affecting multiple contact animals from the same epizootic unit.Phylogenetic analyses based on the partial nsP4 gene support the monophyletic grouping of SINV with the New World viruses in the Western equine encephalitis virus complex . Attempts to amplify more gene regions, as well as virus isolation, were not successful.The results of this study demonstrate that alphaviruses might be associated with neurologic disease in wildlife, nonequine domestic animals, and birds in South Africa. Wide geographic distribution of MIDV and SINV in Africa suggests that this distribution should be investigated in other regions. Humans living near wildlife and livestock, such as domestic bovids, sheep, and porcines, might have similar exposure to mosquito vectors and should therefore also be monitored for potential zoonotic infections. SINV has been shown to have birds as its reservoir host and is a well-known human pathogen in South Africa, although most described cases are nonneurologic and associated with febrile disease or arthralgia. Our study identified SINV in neural tissue of various wildlife and nonequine domestic species that had neurologic signs of infection. The geographic range, prevalence of infection in various species, species-specific differences, and seroprevalence need to be determined to define the epidemiology of these pathogens. Serologic evidence for MIDV in humans has been demonstrated in South Africa (Reservoir hosts for MIDV are unknown. Our suggestion of a higher prevalence of MIDV relative to SINV warrants further investigation. Widespread distribution of MIDV suggest birds might play a role in the spread of this virus. However, a dove and blue crane were the only virus-positive birds identified, both submitted as postmortem specimens. Further investigation is needed regarding the potential of the identified species to function as reservoir and amplification hosts and potential for transmission of virus to vectors. Spillover of MIDV into humans through adaption to additional vectors should be monitored. Investigation of neurologic cases and deaths in wildlife and domestic animals suggested that most cases occur during January–July—from the second half of the summer months to late autumn or early winter in warmer parts of the country, where many wildlife reside—after which vector numbers usually decrease because of colder winter temperatures.Future studies into the epidemiology of SINV and especially MIDV would greatly benefit from analysis of increased sample sizes or whole-genome sequencing, combined with serologic analyses of affected species, especially in outbreak investigations. Syndromic surveillance for febrile and neurologic disease in sensitive species might act as an early warning system for outbreaks of emerging and reemerging arboviruses and predict outbreaks in humans (A limitation of our study is that all other possible infectious and noninfectious etiologies could not be excluded by comprehensive investigations for all cases, suggesting that the causative link with clinical signs still has to be regarded with caution. Also, serum samples from PCR-positive animals were not available because most animals were tested postmortem, which limited validation by serologic assays for this study. However, use of serum samples should be a focus of future investigations.Most emerging zoonotic diseases are believed to be caused by pathogens that originate from wildlife (


The extent to which employees change jobs, known as the job mobility rate, has been steadily declining in the US for decades. This decline is understood to have a negative impact on both productivity and wages, and econometric studies fail to support any single cause brought forward. This decline coincides with decreases in household savings, increases in household debt and wage stagnation. We propose that the decline could be the consequence of a complex interaction between mobility, savings, wages and debt, such that if changing jobs incurs costs which are paid out of savings, or incurs debt in the absence of sufficient savings, a negative feedback loop is generated. People are further restricted in making moves by their debt obligations and inability to save, which in turn depresses wages further. To explore this hypothesis, we developed a stylized model in which agents chose their employment situation based on their opportunities and preferences for work and where there are costs to changing jobs and the possibility of borrowing to meet those costs. We indeed found evidence of a negative feedback loop involving changes, wages, savings and debt, as well as evidence that this dynamic results in a level of wealth inequality on the same scale as we see today in the US. The US job mobility rate, describing the extent to which employees move between employers, is at an all time low after declining for decades, and this decline has important consequences. Job changing is understood to improve productivity by matching workers to more suitable employment and by promoting innovation through the inter-firm exchange of experience  of all agents in the firm. Therefore, the optimal effort for an agent will decrease as other agent efforts in the same firm increase, thus creating a free-rider problem. As free-riding increases, the utility for other firm members decreases, such that an optimal firm size that maximizes wages is not the same as one that maximizes utility. Thus agents will leave firms with high wages if they find higher utility elsewhere, even if their resulting wages are lower.9These basic microeconomic principles embodied by a group of utility seeking agents create macroeconomic conditions of “fluctuating effort and sustainable cooperation” , wages, firm productivity, loans and debts, as well as savings and total wealth. The institutional conditions represented by the costs and the costs with credit scenarios implemented at the microeconomic level have statistically significant effects on these macroeconomic measures in their respective emergent economies. Unless otherwise indicated, all simulations were run 30 times with the parameters and settings described in Table For the three scenarios described in Sect. The emergence of regions of steady state firm population stability, even though the composition and size of any given firm is in flux, is common across the scenarios. This equilibrium region emerges after roughly 100 time steps and the number of firms oscillates within a band, as demonstrated in the 30 run spaghetti time series plots for each scenario in Fig. The costs with credit scenario produces the most firms, therefore the smallest firms. The costs scenario produces the fewest, conversely the largest, firms and the number of firms in the free movement scenario falls between the two. Underlying these firm populations dynamics are employment dynamics, and the following sections explore aspects of job mobility in the contexts of free movement, costs, and costs with credit.An agent’s mobility describes its ability to make a desired change, and agents with lower mobility miss out on opportunities more often than agents with higher mobility. Figure The number of changes decreases across the scenarios and conversely the number of thwarts increases. Since the costs scenario produces more changes than the costs with credit scenario, we find the counterintuitive result that being able to borrow to make a move in aggregate results in fewer moves than overcoming the cost constraint via savings alone. As the level of costs increase, thwarts increase as well. We ran sensitivity analysis with various values for the wage multiplier, as well as modeling costs as a function of expected future wages rather than current wages. (Results for the change in cost scheme is found in “Statistical summaries of select macroeconomic variables including changes and thwarts across the scenarios are given in Table Wages are an employee’s share of firm output, as defined in Eq. The mean firm productivity values for the three scenarios are 2.77, 4.33 and 1.93, with the costs scenario producing the most productive firms and the costs with credit scenario the least productive firms. This is a consequence of the cost scenario firms being larger, so fewer of them, and wages highest, while the lowest wages and the most firms are produced by the costs with credit scenario.One of the effects of agents setting effort according the utility function described in Eq. a, b and Firm sizes and wages emerge out of the interactions of utility maximizing agents who have differing preferences for income over leisure, given by Wage and size are slightly correlated in the free movement scenario, less correlated in the costs scenario, and uncorrelated in the costs with credit scenario Table . Wage anar Table . The corAll agents have a non-zero savings rate, so will save a percentage of their wage. The costs with credit scenario allows agents with insufficient savings to pursue utility improving opportunities by taking out a loan. Debt quickly becomes pronounced, increasing superlinearly for lending rates above 0, as demonstrated in Fig. The aggregate loan amounts are so great they overwhelm the positive wealth values. We therefore consider a variant of the wealth metric, net wealth, which is the difference between the sum of all agent savings and all agent loans. Figure The model restricts agents who can make loans to those who don’t currently have one, which is a simplification of lenders’ risk avoidance. We conducted a sensitivity analysis with a variant of the model whereby agents can take out loans up to ten times their current wage, which amounts to agents being able to make roughly ten job changes or being self-employed five times. The model behaviour is the same as the base model in that thwarts exceed changes, though this occurs later than in the base version, and the quantity of loans grows superlinearly. Once the interest rate for loans rises as well, the thwarts exceed changes in the same timeframe as the base model. (Results for these alternative loan schemes are provided in “We noted in Sect. l is the lending rate, then To explore the dynamics underlying this superlinearity in aggregate loan value, we consider s is an agent’s savings rate and w his wage, and b the set of agents with outstanding loans, then loan payments areb. New loans are a function of the number of singleton loans, firm move loans, the costs for these two activities and the wages of the borrowers. If s any given borrower’s rate, and mean costs multiplier c represents both singleton and move costs, and Loan payments are a function of the wages and savings rates of borrowers. If The plot in Fig. The discrete second derivative of total loan and wage values for the two simulation are shown in the right hand plot in Fig. In the free movement and costs scenarios, all 600 agents have some amount of savings, while in the costs with credit scenario only 156 agents on average have savings greater than 0 at Inequalities within a population are canonically represented by Lorentz curves, thus Fig. 14The EF model demonstrates that adding cost constraints to free movement of workers, along with the ability to borrow to make a move, produces a negative feedback loop described by decreasing job mobility, savings and increasing debt, as hypothesized. As job mobility decreases in this costs with credit scenario, wages and savings fall and debt rises. As debt rises, job mobility and savings continues to fall and debt continues to rise, imitating the observed qualitative behaviours described in Sect. In the free movement scenario, agents are free to join firms until the optimal firm size is formed Axtell , at whicExploring the costs with credit scenario at the subpopulation level is highly informative. The debt-free subpopulation, the most mobile class, has the highest average firm r et al. , agents a, b and a, such that the two counteract each other in determining firm production. A future implementation could correlate these values for each agent, particularly the production parameters, to perhaps strengthen the productivity related correlations and further explore the emergence of agent homophily within firms under different scenarios.In this model, as in the Axtell model, the firm production parameters Agent characteristics in this and the Axtell model are not dynamic, and network edges, effort preferences and productivity parameters are fixed. Currently, costs are modeled as a linear function of current wage, with the intent that an agent’s current situation is a representation of their network or capacities, but this means that lower wage agents incur lower mobility costs. Just as networks could evolve and become dynamic, the cost function could become nonlinear such that a low-wage agent could incur large mobility costs, representing an improving action such as upskilling, which would in turn update the agent’s productivity parameters.In the costs with credit scenario a subset of agents are further impeded in making changes because they have outstanding loans they must pay off before they could either begin saving for a future move or borrow again. The superlinear growth in total loans exhibited in this scenario has multiple causes, all of which results in an agent not being able to save because they cannot pay off debt, and cannot move to an improved situation where they could earn higher wages in order to pay off debt. Savings rates are heterogenous in the EF model so there will be agents who make a loan and will not be able to repay that loan because their savings rate is lower than the lending rate. In another case, a perpetually indebted agent may have a saving rate equivalent to or higher than the lending rate, but may have chosen an opportunity that increased utility but decreased wage, again resulting in insufficient payments. Alternatively, an agent my have chosen a situation with a higher wage, but the decisions made by other agents eventually cause the firm’s productivity to decrease and the wage becomes insufficient to repay the loan. In each of these cases, the amount that borrowers owe will continue to grow over time. Unlike the costs scenario where there are two classes of agents, those with sufficient funds and those with temporarily insufficient funds, three classes of agents emerge in the costs with credit scenario: agents with sufficient savings who move at will, agents with loans who will pay off that loan and either borrow again to make a move or accrue savings before an opportunity arises, and agents who are hopelessly indebted and will never make a move. Changes are rare in the costs with credit scenario, and since fewer agents are able to place themselves in superior situations both wages and productivity are depressed.The superlinear growth behavior in aggregate loan values is an intriguing result as the total amount of loans will exceed the total wealth in the model in the situations where a significant number of agents are unable to repay loans. The purpose of credit is to allow agents to complete contracts otherwise not obtainable, thus expanding markets, or in this case, permitting an employee to make a beneficial change and therefore increasing wages and productivity. Yet clearly too much available credit has significant negative consequences Turner . Russo dWhile the above suggests the possibility of a sustainable credit regime, it is in no way prescriptive. The EF model is highly stylized, and not stock-flow consistent. Credit does not come from another agent nor are costs and interest paid to another agent, so the total wealth in the system is greatest for free movement, where it is equal to the cumulative output over the simulation run, and least for the costs scenario. This current formulation means any growth in output is the result of more efficient combinations of workers. If we were to apply an endogenous rather than exogenous lender then the debts held by agents would show up as credits held by others. In these cases of negative wealth the Gini index can be greater than 1, and inequality even more widely spread (Chen et al. N decreases the options available to other agents become increasingly limited. Thus changes are fewer in the costs with credit scenario not only because perpetually indebted agents won’t make moves, but also because there are fewer opportunities for improvement for the mobile agents. Van Bavel describes this lack of access and diminished participation as a hallmark of the downward trend in historic cycles of the rise and fall of market economies (Despite its stylized nature, the model demonstrates that if pursuing the best productive opportunity incurs costs, then having financial capital matters. Without that capital, and especially in the presence of debt, agents are unable to participate in the economic activity of finding the most efficient uses of their labor. As the effective conomies . Our curconomies . Studiesconomies .15 The tconomies .16 As meHayek observed that when dealing with complex systems ‘the aspects of the events to be accounted for about which we can get quantitative data are necessarily limited and may not include the important ones’ . PerhapsWe have developed a stylized model to explore the hypothesis that the observed decline in job mobility could be the consequence of a complex interaction between mobility, savings, wages and debt. We indeed found evidence of such a negative feedback loop, as well as evidence that this dynamic results in a level of wealth inequality on the same scale as we see today in the US. The EF model serves as a qualitative experiment that can generate testable hypothesis and justify efforts to generate datasets describing this dynamic which could be tested empirically. Before expanding the EF model in scale,


From November 2015 to July 2017, two successive episodes of H5 highly pathogenic avian influenza viruses (HP AIVs) infections occurred on poultry farms in France, mostly in domestic ducks raised for foie gras production in southwestern France. During the two epizootics, epidemiological investigations were carried out on infected farms and control and biosafety measures were implemented in association with surveillance in order to stop the spread of the viruses. Effluents are known to be an important factor in environmental dissemination of viruses, and suitable effluent management is needed to help prevent the spread of epizootics to other farms or pathogen persistence at the farm level. The present study was therefore designed to assess how long infectious A/H5 HP AIVs can persist in naturally or experimentally contaminated fecal slurry samples from ducks, with or without sanitization by lime treatment. Infections by A/H5 and A/H7 avian influenza viruses (AIVs) can cause acute disease and are therefore notifiable in poultry and wild birds. During winter 2015-2016, several cases of infection caused by highly pathogenic (HP) AIVs belonging to the A/H5N1, A/H5N2, and A/H5N9 subtypes were detected in southwestern France. Throughout winter 2016-2017, several cases of infections caused mainly by A/H5N8 HP AIV  were detected across Europe. On both occasions, the viruses were widely detected on palmiped farms in France. This study was designed to evaluate the persistence of A/H5 HP AIV in slurry from various duck productions. This was achieved (i) in the laboratory setting by artificially spiking four AIV-free slurry samples with known amounts of A/H5N9 HP AIV and monitoring virus infectivity, with or without lime treatment to achieve pH 10 or pH 12, and (ii) by sampling slurry tanks on five naturally A/H5N8 HP-contaminated farms. Experimental results in artificially spiked slurry suggested virus survival for 4 weeks in slurry from Muscovy or Pekin duck breeders and for 2 weeks in slurry from ducks for foie gras production during the assisted-feeding period, without lime treatment. Persistence of infectious A/H5N9 HP AIV in all slurry samples after lime treatment at pH 10 or pH 12 was less than 1 week. The A/H5N8 HP AIV persisted in naturally contaminated untreated slurry for 7 weeks. The results obtained provide experimental support for the 60-day storage period without treatment or the 7-day interval after lime treatment defined in French regulations for slurry sanitization.IMPORTANCE From November 2015 to July 2017, two successive episodes of H5 highly pathogenic avian influenza viruses (HP AIVs) infections occurred on poultry farms in France, mostly in domestic ducks raised for foie gras production in southwestern France. During the two epizootics, epidemiological investigations were carried out on infected farms and control and biosafety measures were implemented in association with surveillance in order to stop the spread of the viruses. Effluents are known to be an important factor in environmental dissemination of viruses, and suitable effluent management is needed to help prevent the spread of epizootics to other farms or pathogen persistence at the farm level. The present study was therefore designed to assess how long infectious A/H5 HP AIVs can persist in naturally or experimentally contaminated fecal slurry samples from ducks, with or without sanitization by lime treatment. Anseriformes and Charadriiformes) are the natural reservoir of all currently described AIV subtypes, including 16 different hemagglutinin (H1 to H16) and 9 neuraminidase (N1 to N9) subtypes. These birds usually shed the virus through respiratory aerosols or contaminated feces, without developing symptoms (–Avian influenza virus (AIV) infections are widespread in many different species of wild and domestic birds. Wild waterfowl .Infection of terrestrial poultry, occurring by direct spillover from wild birds or indirect transmission of AIV, is usually asymptomatic or results in mild respiratory disease or a drop in egg production . HoweverFrom November 2015 to July 2017, two successive episodes of A/H5 HP AIV infections occurred on poultry farms in France, mostly in domestic ducks raised for foie gras production in southwestern France. During winter 2015-2016, 81 outbreaks were reported on poultry farms (mostly domestic waterfowl without overt disease) due to one of the three detected subtypes: A/H5N1, A/H5N2, and A/H5N9 HP AIVs. No infection was detected in wild birds. These A/H5 HP AIVs belonged to an H5 genetic lineage significantly different from the A/goose/Guangdong/1/1996 lineage and therefore do not have an assigned clade number (clades have been defined within the A/goose/Guangdong/1/1996 lineage only  of 2) as those detected in untreated slurries. Following lime treatment to achieve pH 12, only slurry C allowed the isolation of A/H5N9 HP AIV, on the day of spiking only, whereas slurries A, B, and D did not allow virus reisolation. The pH 12 condition induced a strong decrease in M gene detection, as the M gene was mostly detected on the day of spiking only and was undetectable  afterwards.For each slurry, sequences encompassing the cleavage site in the H5 gene of each A/H5N8 HP AIV corresponding to the initial isolates were obtained. Regarding the 201 common nucleotides, sequencing data from slurries 1 to 5 were found to be identical, with only a nucleotide change that did not change the corresponding amino acid. All sequences are available in the GISAID or GenBank database (accession numbers are presented in Table S2).n = 5) was necessarily limited, due to practical laboratory constraints and the labor intensity of the study protocol, and this precludes any statistical evaluation of the duration of virus survival. Furthermore, for biosafety reasons , only one initial sample was collected on each farm instead of weekly samples .This study was initiated during the 2015-2016 French H5 AIV epizootic, when the A/H5N9 virus was most prevalent . As thisThe persistence of infectious AIV in the environment is influenced by various physical and chemical parameters, such as temperature, exposure to UV, pH, presence of chemical agents or detergents, salinity, the nature of the matrix, and the presence of organic material 4–19–, 27–46. 27–In complex environments with a high content of biological material, such as manure or chicken and duck feces, influenza viruses appear to retain infectivity for shorter periods than in water 19–35–, 46, 48.http://www.meteofrance.com/climat/france/nouvelle-aquitaine/regin09/normales), which is the largest duck-producing region in the country and which was severely affected by two A/H5 HP AIV epizootics in the winters of 2015-2016 and 2016-2017  which was either naturally infected and collected from an A/H5HP outbreak or experimentally spiked in the laboratory and stored at 5°C under biosafety level 3 (BSL3) controlled conditions. Analyses were performed on weekly subsamples of the stored slurries. This temperature of 5°C was selected as it is representative of the mean winter temperature in southwestern France . Consistently with previous findings obtained with ducks and chickens , 36, 48,TC values obtained in rRT-PCR for the M and H5 genes under natural or experimental conditions demonstrate stable persistence of the genome in untreated slurries throughout this period. As a result, whereas AIV genome detection demonstrates the previous presence of AIV, it cannot be used to assume the actual presence of infectious AIV, which must be confirmed with another method such as virus isolation.In contrast to virus isolation, which was possible for only 4 (A/H5N9) to 7 (A/H5N8) weeks, the AIV genomes of both viruses could still be detected for 2 to 5 weeks after extinction of infectious virus . The Our experimental study also investigated two protocols to sanitize duck slurry: lime treatment at pH 10 and at pH 12. Both treatments strikingly reduced virus persistence. Live virus could be isolated only on the day of spiking at pH 10 and at pH 12 in 3 of 4 and 1 of 4 assays, respectively. In addition, increasing pH directly increased the degradation of the virus genome in the treated slurry: at pH 10, the genome could be stably detected, whereas at pH 12, the genome could no longer be detected as early as 1 week after lime treatment. These results extend previous knowledge on the effects of basic pH on AIV survival in allantoic fluids, as 15 min of treatment at pH 10 or pH 12 was previously shown to have no detrimental effect on AIV hemagglutinating activity . SimilarInterestingly, naturally contaminated slurries presented coinfections with hemagglutinating viruses other than A/H5N8 HP AIV  that were adventitiously detected upon inoculation of our slurry samples on embryonated eggs. Coinfections are not uncommon and have been found previously on duck farms, making it unsurprising to detect other AIVs or avulavirus . Reassor4–It is very likely that other viruses that undergo digestive excretion were also present in the studied slurries but went undetected, as they are possibly difficult to isolate using the allantoic route in embryonated eggs. Further studies with different isolation methods would therefore be necessary to more fully assess the role of slurry as a vector contributing to the spread of duck viral diseases.The physicochemical composition of slurries could significantly influence the persistence of infectious AIV particles , 36, 48.28–In conclusion, this study was aimed at evaluating the efficiency of specific sanitization practices implemented in France. Whereas HP AIVs can be disseminated by different routes , this study was designed to better assess the risk of AIV persistence associated with slurry, a common way to store duck feces under farming conditions. To minimize the risk of AIV dissemination through slurries from infected farms, international guidelines have been published , 55. In (i) AIV-negative farms. Four AIV-free farms were selected based on their AIV-negative status, determined by testing duck flocks with nucleoprotein (NP)–enzyme-linked immunosorbent assay  on 20 blood samples and with M gene real-time RT-PCR (M-rRT-PCR) (described below) on 20 cloacal swabs and 20 tracheal swabs, according to the OIE manual  Naturally infected mule duck farms. Five contaminated farms carrying out foie gras production, during or before the assisted-feeding period, were selected in collaboration with the district animal health authorities , based on their positive A/H5N8 HP AIV status, as determined according to the OIE manual . All disposable equipment and materials used for sampling were sealed in a plastic bag and destroyed at the farming site by an approved company.The slurry tanks where samples were collected had not previously been homogenized, in order to keep a possibly higher virus concentration close to the feces adduction duct. No previous sanitizing treatment had been carried out in the tanks in order to keep the titer of possibly infectious virus as high as possible.For each slurry tank, two 1-liter samples were collected nearest to the adduction duct delivering the fresh droppings into the tank. The sampling location was selected so as to possibly sample the virus last delivered to the slurry tank . One liter was taken from the surface of the tank and one liter at a 2-m depth. For surface sampling, a 4-meter long telescopic pole was used, with a 1-liter plastic beaker attached to the end. A bag with a capacity of 1 liter, weighted and equipped with a nonreturn valve , was used to collect deep samples.The surface and deeper slurry samples were transferred to separate screw-cap sterile glass bottles with a capacity of 1.5 liters each. The bottles were sealed, and their outside surfaces were cleaned with soapy water and then disinfected via a spray of Virkon. The bottles were then packed in double plastic bags and transferred with paper towels to a water-tight cooler containing frozen ice packs. The samples were directly transferred to an approved AIV-diagnostic laboratory and immediately shipped to the BSL3 containment facility of the French National Reference Laboratory, without any intermediate freezing step.One slurry sample per farm  was sent to the laboratory for storage at 5°C before processing (see below).(i) Physicochemical characterization. The characteristics and composition of slurry samples used for artificial contamination were determined using official methods from the International Organization for Standardization (ISO) or European norm (EN): pH (EN 12176), electrical conductivity at 20°C and 25°C (EN 27888), density and dry matter content (percent) (EN 13040), organic matter and ash content (percent) (EN 13039), organic carbon (percent) (ISO 10694), total nitrogen (percent) (Kjeldahl method) (EN 13654), ammoniacal nitrogen  , organic nitrogen , nitrate nitrogen  (colorimetry), nitrite nitrogen  (colorimetry), calcium (EN 11855), potassium (EN 11855), sodium (EN 11855), phosphorus (EN 11855), sulfur, and carbon/nitrogen ratio.(ii) Validation of AIV-free status. The AIV-free status of each slurry used for the artificial contamination study was checked in two steps. First, cloacal swabs were collected from the ducks present in the farms and were tested with M-rRT-PCR. Second, the collected slurry samples were tested again with M-rRT-PCR and were then analyzed by inoculation into embryonated eggs to check for a lack of embryo toxicity . Slurries selected for the study had negative results for all these tests .All experiments involving infectious A/H5N9 HP AIV were performed under BSL3 containment conditions. Our BSL3 laboratory has been approved by the French National Agency for Medicines and Health Products Safety (ANSM) to use specific zoonotic avian influenza viruses (no. ADE-077572017-1).(i) Artificially spiked slurries. AIV-free slurry samples were artificially contaminated with the A/H5N9 HP AIV A/duck/France/150236b/2015 produced as contaminated allantoic fluids derived from specific pathogen-free (SPF) hen eggs as described previously  Subsequent lime-treatment of artificially contaminated slurry samples. The second and third portions of the spiked samples were treated by addition of 45% milk of lime , at room temperature for less than 10 min, under continuous stirring. pH was controlled with pH universal indicator strips , so as to achieve pH 10 and pH 12 in the second and third portions. Artificially contaminated slurry samples, either untreated or treated with lime, were stored at 5°C ± 3°C for the rest of the study. A positive control consisting of PBS artificially contaminated with A/H5N9 HP AIV, at the same dose as the tested slurries, was prepared and stored similarly. Negative controls consisting of noncontaminated PBS, noncontaminated slurry without lime treatment, and noncontaminated slurry with lime treatment at pH 10 and pH 12 were prepared. All negative and positive controls were stored as described above and were collected and tested according to the same protocol as the contaminated slurries.For naturally contaminated slurry samples, the first day of the kinetic study (D0) was estimated as the last day when fresh fecal material from infected ducks could have been delivered into the slurry tank at the farm level. D0 therefore corresponded to the date when slurry was collected, or to the date when the ducks were depopulated . For spiAll slurry samples, both naturally contaminated and artificially spiked, either lime treated or untreated, were subsampled weekly (5 ml), after rehomogenization by 5 min stirring and were then analyzed by virus isolation in embryonated eggs, as described below, until the virus had not been isolated for two consecutive weeks. All slurry subsamples and allantoic fluids extracted at the end of each isolation assay were also tested using M-rRT-PCR. For the study of naturally contaminated slurry only, and in order to confirm the identity of the possibly detected virus, H5-rRT-PCR was also performed from the same slurry subsamples and allantoic fluids samples.(i) RNA extraction. A 1-ml portion of each collected subsample and allantoic fluid was clarified by centrifugation . Then, 200 μl of supernatant was collected, and viral RNA was extracted using the RNeasy minikit (Qiagen) following the manufacturer’s protocol. Briefly, 200 μl of supernatant was mixed with 600 μl of lysis buffer (RLT) containing 6 μl of β-mercaptoethanol (Sigma) and left to stand for 10 min at room temperature. After addition of 700 μl of stringent washing buffer  and 400 μl of 70% ethanol followed by mixing, the liquid was applied to a spin column in two steps. Finally, washing and elution (50-μl volume) were performed.(ii) M- and H5-rRT-PCR. M-rRT-PCR was carried out using a commercial kit  and the Applied Biosystems (ABI) 7500 Fast real-time PCR system. M-rRT-PCR primers, probe, and cycle conditions were as described by Spackman et al.  of the same viruses. The standard deviation of the M gene rRT-PCR was estimated to be a TC value of 1.2 , and that of the H5 gene rRT-PCR was a TC value of 1.3  using a standard RNA in a series of 20 repeated assays.Analytical protocols used for extraction and rRT-PCR were first validated using this unusual slurry matrix: equivalent results of detection with M-rRT-PCR and H5-rRT-PCR were obtained with both PBS and slurry contaminated with the same dose of H5 virus. All negative controls in PBS were confirmed as negative by M-rRT-PCR in all experiments based on naturally contaminated or experimentally spiked slurries. In addition, each sample tested was analyzed with M-rRT-PCR or H5-rRT-PCR using an internal positive control, to confirm the absence of amplification inhibitors. Each real-time RT-PCR run included a dilution series of an appropriate common RNA standard, controlling for the fidelity of the methods and allowing the comparison of results between different runs. The analytical sensitivity of the M gene rRT-PCR was previously estimated to be 200 and 700 RNA copies per reaction for the A/H5N8 and A/H5N9 viruses, respectively, corresponding to 1,000 and 100 50% egg infective doses   Virus isolation was performed according to the official method recommended for AIV  of 4 ml of each subsample, 1,200 μl of supernatant was treated for 15 min at 4°C with/ml of penicillin, 10 mg/ml of streptomycin, 0.25 g/ml of gentamicin, and 0.25 mg/ml of amphotericin B (Fungizone). Then, 200 μl was used to inoculated the allantoic cavity of 9- to 11-day-old SPF embryonated chicken eggs . Five eggs were inoculated for each studied sample. Eggs were incubated at 37°C ± 1°C with daily candling. After 5 to 6 days of incubation or when embryo mortality was observed, allantoic fluids were collected and their hemagglutinating activity (HA) was tested. The HA assays were carried out with 1% chicken red blood cells in PBS, according to the standard protocol  Confirmation of the identity of the isolated viruses. Samples of fecal material from conventional poultry may contain a variety of infectious hemagglutinating viruses that could be reisolated in embryonated eggs; it was therefore important to confirm the identity of each virus isolated throughout the kinetic study.(a) Virological identification. Hemagglutinating viruses were identified through standardized hemagglutination inhibition (HI) assays  assays , using a(b) AIV molecular identification by sequencing. The HA2 domain of the HA gene was amplified with a pan-HA RT-PCR  on an automated DNA sequencer ABI 373XL (Applied Biosystems). Sequencing data were assembled and inspected with Mega software. Subtype determination was performed by BLAST analysis on the influenza virus resource database .(iii) Detection of non-AIV contaminating viruses.(a) Molecular identification of non-AIV contaminating viruses. AAvV-1 occasionally detected during the course of the study was pathotyped by sequencing the region encoding the cleavage site of the F protein, using in-house RT-PCR with the following primers: NDV F (sense), TAGAAAAAACACGGGTAGAAGA, and NDV R (antisense), TTGGTWGCRGCAATRCTCTC. Sequencing was performed as described above. The sequence of the cleavage site is correlated with the virulence of AAvV -1  Neutralization of viral contaminants. During the study of naturally A/H5N8 HP AIV-contaminated slurry, some samples yielded viruses different from A/H5 HP AIV. Moreover, their in ovo propagation was still associated with significant detection of the H5 genome by H5-rRT-PCR. This situation suggested coculture of A/H5 HP AIV with another virus, the latter being more easily reisolated than A/H5 HP AIV. In order to check whether infectious A/H5 viruses were in fact present in such cases, a confirmatory isolation assay was implemented, preceded by viral neutralization of the non-A/H5 contaminating virus.These viral neutralizations were performed by incubating an equivolume mixture of the studied allantoic fluid with a hyperimmune antiserum, monospecific for the virus to be neutralized , for 75 min at 4°C. Sera used for neutralization belonged to the same panel of AAvV and AIV monospecific antisera as those used in HI (see above). After this neutralization step, virus isolation in embryonated eggs was performed as described above .


Hepatitis B virus (HBV) is a life-threatening virus that causes very serious liver-related diseases from the family of Hepadnaviridae having very rare qualities resembling retroviruses. In this paper, we analyze the effect of antiviral therapy through mathematical modeling by using Liao’s homotopy analysis method (LHAM) that defines the connection between the target liver cells and the HBV. We also examine the basic nonlinear differential equation by LHAM to get a semi-analytical solution. This can be a very straight and direct method which provides the appropriate solution. Moreover, the local and global stability analysis of disease-free and endemic equilibrium is done using Lyapunov function. Mathematica 12 software is used to find out the solutions and graphical representations. We also discuss the numerical simulations up to sixth-order approximation and error analysis using the same software. Hepatitis B virus (HBV) infection is one among the very serious infections that threatens the people worldwide, less than 300 million people live with chronic hepatitis B infection worldwide. It directly or indirectly affects the liver, causing diseases affecting also infants and young children. This virus is genetically from the family of Hepadnaviridae having very rare qualities resembling retroviruses . HepatitApplication of the mathematical models to different parameters shows that some patients get cleared of the virus quickly due to drug therapy, whereas in case of other patients the drug therapy works much slower . TherefoThis paper is divided into eight parts. The first part of the paper forms an introduction which deals with the existing literature and proposed work. The second and third parts talk about mathematical modeling and analytical study of the HBV model. In these parts we analyze the local and global stability of HBV. The fourth part explains the LHAM method, and the fifth part is applications that have been used to find out the solutions using LHAM. The sixth part of the paper contains numerical experiments which deal with numerical simulations obtained up to sixth-order approximations using Mathematica 12 software. Error analysis forms the seventh part of the paper, the eighth part is discussion, and final part of the paper is conclusion.X represents the target uninfected cells (uninfected hepatocytes), Y represents the infected cells (infected hepatocytes), and Z represents the HBV virus. This model represents the target cells that are infected at rate β, and infection occurs by contact between the target cells and the virus. It also considers that the infected cells die at a rate of δ and production of new virus happens at a rate of v. The rate of dead virus is c. The new target cells introduced in the liver may die before getting infected. The constant production rate is denoted by s, and the natural death rate is denoted by We consider the mathematical model for the basic virus dynamics . This momentclass2pt{minimThese assumptions lead to the following model: We consider three cells as the target cells, the infected cells, and the hepatitis B virus cells . This moLetIfthentinthen the solution isTo prove this for of model  are posiLetwhereThe solutions of system , , 5.15),  by Liao , 26. FigHepatitis B virus is proved to be a dangerous disease affecting people enormously. Though there has been vaccine to cure this disease, the antiviral therapy is recognized as the best method to eradicate this disease utmost at the root level. In this paper, the reproduction number epatitis . This re