A better understanding of when and why nudges  and other structural behavior-change measures work or fail can help avoid subsequent surprises such as unexpected political opposition. In this paper, we challenge the unilateral focus on structural interventions—which seemingly control people's behavioral decisions—as such a focus ignores the flipside—namely, attitudes or, as they are called in economics, preferences. We argue for a conceptual understanding of individual behavior that views personal attitudes and behavioral costs as its two separate compensatorily effective determinants. This classical understanding was reintroduced into attitude research as the Campbell paradigm. In the logic of the Campbell paradigm, a person's attitude becomes obvious in the face of the behavioral costs the person surmounts. Technically, individual attitudes reveal themselves in a set of cost-dependent transitively ordered performances. Behavioral costs in turn reflect the structural boundary conditions that are relevant as obstructive and/or supportive environmental forces that generically affect a specific behavior. So far, our research on people’s attitudes toward environmental protection has demonstrated that the Campbell paradigm—and thus its conceptual account of individual behavior—holds true for approximately 95% of the people in a given society. Intee, e.g., ; remembee, e.g., ). In Johe, e.g., , p. 1339conditional behavioral relevance of structural interventions  is a misconception. We will demonstrate that the driving forces behind people’s behavioral decisions are attitudes . By contrast, structural factors do not fuel behavioral decisions. Rather, they aggravate or alleviate individual performance as “behavioral costs” so to speak. Therefore, a unilateral focus on structural interventions—ignoring differences in the strength of individual attitudes—obstructs the understanding of when and why, for example, nudges work or fail.In our paper, we argue that the apparently compensatorily effective factors: a person’s attitude and the structural boundary conditions that obstruct and/or support specific behavior . With our paper, we wish to demonstrate the significance of this new paradigm to the understanding of behavioral change beyond its application as a model for environment‑relevant attitudes  that was .Science article, which presents an illustrative example about the prevalence of organ donation in societies with distinct nonaction defaults. Whereas nonaction in societies with an opt-in default denotes a refusal to donate, nonaction in societies with an opt-out default denotes an acceptance of organ donation. We argue that both these implicit connotations of people’s inaction are inaccurate as they fail to reflect people’s attitudes toward organ donation. We believe that such misunderstandings of people’s attitudes carry the risk of subsequent surprises in the form of failed communal acceptance or even public resistance.In the following section, we turn to Johnson and Goldstein’s  seminal pki) of person k becoming an organ donor  is about 10% in societies with an opt-in default and about 90% in societies with an opt-out default. Evidently, if people are nudged into one of two defaults—remaining a nondonor (with the option to opt-in) or remaining a donor (with the option to opt-out)—the probability of becoming a donor changes dramatically . Next, we describe a framework that explains individual performance  as a compensatory relation of behaviorally effective attitudes  and costs .Interestingly, by replacing the voluntary opt-out choice with a mandatory active choice—making everybody send a return-card—the likelihood of donorship can be expected to decrease from 90% to approximately 30% , we can presume that the person is highly committed to the behavioral goal implied by the attitude . That is, we can say that the person's specific attitude toward organ donation is pronounced or his/her attitude toward organ donation is extremely favorable. Thus, it seems reasonable to assume that if a person is genuinely dedicated to organ donation, he/she would presumably (1) implicitly approve of organ donation by not returning an opt-out return-card, (2) explicitly express approval for organ donation . Thus, the stronger a person's attitude is, he/she will implement more behaviors and more demanding behaviors. Recognizing an attitude therefore requires the empirical identification of a set of behaviors that represent what people commonly do to implement the strength of a specific personal attitude  that is required when a person mails a response-card using the postal service. In other words, in their first explanation, Johnson and Goldstein focused on the personal aspects that correspond with structural costs . These consist of the presumed personal expenditures that are involved when a person switches away from the default, that is, mental and physical effort.Effort refers to the mental pain that may be involved when a person makes up his or her mind or to the physical labor  has to be profitable by comparison. In other words, the departure from the benchmark has to promise some benefits or substantively fewer costs than the default; otherwise, people’s aversion to loss—through not gaining anything—results in the persistence of the default [Loss aversion, the second explanation, also refers to behavioral costs. In this explanation, a default and its presumed costs represent the cognitive reference. Any alternative . Any alIf behavioral costs are so crucial, empirically uncovering behavioral costs and detecting potentially behavior-effective boundary conditions becomes indispensable. In line with Johnson and Goldstein , we alsoSometimes nudges will not work. Children, for example, could not be nudged into replacing French fries with apples by making fries less and apples more accessible (see ). IncreaAs we presume, the behavior-effective driving forces behind people's behavioral decisions are individual attitudes . In other words, structural influences, such as nudges, depend on the driving force of supportive attitudes see  even whei.e., for large groups of people, such as societies) derive the specifically required strength of the attitude. Thus, we have to presume that persons who voluntarily opt-in . By raising the behavioral costs substantially relative to the not-opt-out condition (#1), about 80% of potential donors would be lost—the ones with a weaker attitude strength than the strength required by the specific behavioral costs involved—with the voluntary opt-in condition (#4). With fewer behavioral costs relative to the voluntary opt-in condition given by a mandatory opt-in decision (#3), only about 60% would be lost. This prediction is based on the fact that the average attitude toward organ donation in most societies is not strong enough to make the majority explicitly donate their organs—on the basis of voluntary or mandatory decisions  nor attitudes  can be understood in an absolute sense. They are relative to each other; they are interdependent. Attitudes become manifest in the behavioral obstacles they help people overcome, and conversely, costs become manifest in the strength of the attitude they require to be offset. If organ donation was inherently more important for most, we would find less resistance than the resistance represented by the opt-in conditions, resulting in more donors than only 10%–30%. Thus, with increasing costs, progressively stronger attitudes are required for a behavior to be actualized. Mathematically, the relation between behavioral costs and attitudes can be depicted by the following formula :(1)pki) that person k will engage in a specific behavior i relative to the probability that person k will not engage in behavior i (1-pki) as a function of the arithmetic difference between the strength of k’s attitude (θk) and the composite of the costs involved in realizing the specific behavior i (δi). Note that here, people are distinguishable by the strength of their attitude  and their failures, we have to be aware that the paradigm contradicts the dominant view of conjunctively effective personal and structural forces in social and much of environmental psychology.Formula 1 captures the natural logarithm of the ratio of the probability  to structural interventions such as nudges or even economic incentives. Effectively controlling behavioral decisions with structural measures, however, requires their effectiveness to be more or less uniform. Thus, the Campbell paradigm would be a promising alternative to the currently dominant conjunctive model of personal and structural forces in social and much of environmental psychology.Whereas the Campbell paradigm describes behavioral costs and, thus, structural interventions as generically effective , modernr = 0.54) in a pooled sample of N = 3338 participants  or too difficult  and, thus, their distributions remained within technically sensible boundaries for parametric statistics). Kaiser and Schultz’s findings simultaneously challenge the assumption of conditional effectiveness of either attitudes depending on structural forces or of structural forces depending on attitudes. By contrast, however, the conditional effectiveness of structural forces as depending on weak attitudes is commonplace in the literature on nudging .i.e., the attitude toward environmental protection: see e.g., [Note that any systematic influence not accounted for by the model—such as moderators, alternative attitudes , or difee e.g., ) has demIf the Campbell paradigm provides a suitable framework for understanding individual behavior, we should be able to stochastically derive the specifically required strength of the attitude from the behavioral costs that people are willing to overcome. In our own research, we expectedly found that the cost of being a vegetarian—a highly effective, but in most Western societies (with a 5%–10% likelihood), quite a costly way to protect the environment—had to be offset by progressively more favorable attitudes toward protecting the environment. As it turned out, nonvegetarians held significantly weaker environmental-protection attitudes than vegetarians (see ).i.e., costs) that come with nature preserves the further they live from any preserve. When not at risk of being affected—by living more than 30 kilometers from any preserve—people generally accept the related restrictions  unless they have comparatively weak attitudes toward environmental protection. When at risk of being affected—with increasing behavioral costs—restrictions are accepted with progressively increasing environmental-protection attitudes. Hence, people who are affected by restrictions need rather pronounced environmental-protection attitudes to accept preserve restrictions to an extent that is similar to those of people who are not at risk of being affected . NIMBY cted see .i.e., points) compared with an attitude-relevant  common resource turned out to be a function of a person’s environmental attitude. Specifically, Kaiser and Byrka [Such survey-based examples of the compensatory relation of a person’s attitude strength and the involved behavioral costs were corroborated in still another study that involved overt behavioral engagement in a laboratory experiment . Claims nd Byrka  found hiIn this paper, we challenged the unilateral focus on nudges and other behaviorally relevant structural forces—or in the language of the Campbell paradigm—behavioral costs. As we argue, ignoring the flipside of costs—namely attitudes—obstructs our understanding of why and when structural interventions generally work or fail. We advocate the conceptual understanding depicted in Effectively controlling behavioral decisions with structural measures requires their effectiveness to be more or less uniform. The Campbell paradigm describes behavioral costs and, thus, structural/environmental and personal forces as generically and jointly but compensatorily effective. As such, it provides a promising alternative to the currently dominant assumption of a person-environment interaction in social and much of environmental psychology.


Clinical trials applying iota-carrageenan nasal spray have previously shown to reduce duration of virus-confirmed common cold. The present study pooled data of two similar clinical trials to provide further evidence for the antiviral effectiveness of carrageenan.Individual patient data were analyzed from two randomized double blind placebo controlled trials assessing the therapeutic effectiveness of carrageenan nasal spray in acute common cold. Patients with virus-confirmed common cold  were included and the following parameters were appraised: duration of disease, number of patients with relapses, number of respiratory viruses and viral titers at inclusion (visit 1) compared to days 3–5 (visit 2).Carrageenan treated patients showed a significant reduction in duration of disease of almost 2 days (p < 0.05) as well as significantly fewer relapses during 21 days of observation period (p < 0.05). The virus clearance between visit 1 and visit 2 was significantly more pronounced in the carrageenan group (p < 0.05). In both studies, virus-confirmed common cold was caused by three main virus subtypes: human rhinovirus (46%), human coronavirus (25%) and influenza A (14%) virus. Carrageenan nasal spray showed significant antiviral efficacy in all three virus subgroups, the highest effectiveness was observed in human corona virus-infected patients. The reduced duration of disease was 3 days (p < 0.01) and the number of relapses was three times less (p < 0.01) in carrageenan treated corona-virus-infected patients compared to control patients.Administration of carrageenan nasal spray in children as well as in adults suffering from virus-confirmed common cold reduced duration of disease, increased viral clearance and reduced relapses of symptoms. Carrageenan nasal spray appeared as an effective treatment of common cold in children and adults.ISRCTN52519535 and ISRCTN80148028Pooled data from  Acute viral upper respiratory tract infection, also known as common cold, is the most frequently observed infectious disease in human beings. Children get four to eight upper respiratory infections per year and adults suffer from two to four episodes per year . In the in vitro and in vivo studies have recently shown that carrageenans are potent inhibitors of papilloma virus ) of obtained real time PCR copy numbers were similar in carrageenan  and placebo  groups. A significant decrease in viral titers was observed at visit two in all groups. However, in ITT population the decrease in viral titers was significantly more prominent in carrageenan group (−2.2 log[x + 1]) than in placebo group . The data indicated that treatment with iota-carrageenan resulted in a reduction of viral titer at visit 2 which was an order of magnitude of more than 90% stronger when compared with placebo treatment. Similarly, the difference in virus titers between visit 1 and visit 2 was significantly higher in carrageenan group compared to placebo group in PP population  . Similarly, in PP population virus clearance was 0.14 ± 0.7 in placebo group versus 0.38 ± 0.7 in carrageenan group (p = 0.025). In carrageenan treated patients a significant increase of virus-free patients was observed between visit 1 and visit 2 in ITT as well as in PP population. In contrast, placebo treated patients showed no difference in virus-free individuals between visit 1 and visit 2 (Table In the two pooled studies the following viruses were detected most frequently: hRV (142 patients), hCV  and InfA (43 patients). Patients positive for different viral types were distributed evenly between carrageenan and placebo treatment arms Table .Table 5Subgroup analysis was done for the three most common virus types in the pooled population, hRV, hCV and InfA virus, respectively. In the ITT population, the duration of common cold disease was significantly reduced in all three virus subgroups treated with carrageenan. This effect was most pronounced in hCV infected patients treated with carrageenan where the reduction of duration was almost 4 days in ITT population (p < 0.01) and 3 days in PP population (p < 0.01) Table .Table 6In all three virus subgroups, a significant reduction in duration of common cold was recorded in carrageenan treated patients compared to placebo ones in ITT population. Similarly, a significant reduction was seen in PP population between carrageenan and placebo groups except for InfA patients.Relapses occurred significantly more frequently in placebo groups of hRV and hCV infected patients compared to carrageenan group, in ITT population. Similarly, in InfA infected patients a trend to more relapses was observed in placebo group (p = 0.055) Figure A. Also iLikewise, to total relapses in PP population, a higher average number of relapses per patient was recognized in all virus infected subgroups but solely in hCV infected subpopulation this difference reached significance .Overall tolerability of study products was very good and there was no difference between treatment arms in the number of patients with AEs or severe adverse events (SAEs) Table . About 8In the present study data from two randomized controlled clinical trials, investigating the effectiveness of carrageenan nasal spray during virus-confirmed common cold, were analyzed. This analysis showed that carrageenan-containing nasal spray reduced the duration of viral-confirmed common cold by almost 2 days. Furthermore, carrageenan-treated patients cleared more than twice as many viruses when compared to placebo-treated patients. Additionally, more virus-free patients were found in carrageenan-treated patients at visit 2. Another clinical benefit of carrageenan nasal spray was the significant reduction in relapses defined as recurrence of symptoms after a patient was free of symptoms for at least one day.Annually common colds account for more than 20 million doctor visits and 40 million lost school and work days in the USA alone . It is tin vitro and in vivo studies revealed that carrageenan was effective against several human respiratory viruses such as influenza A [Common cold is mainly caused by respiratory viruses, such as human rhinovirus, human coronavirus, parainfluenza, influenza, respiratory syncytial virus, adenovirus, enterovirus and metapneumovirus , 18, 19.luenza A  human rhluenza A , respiraluenza A . It was luenza A . Hence, As common cold is a benign self-limiting disease, the decrease in viral titers between visit 1 and visit 2 in carrageenan-treated and in placebo-treated patients was expected. But carrageenan-treated patients cleared more than twice as many viruses compared to placebo-treated patients leading to more virus-free patients at visit 2 in carrageenan group Table . It can The reduction of relapses and the reduction of virus titer in carrageenan treated patients might be of interest in particular patient subgroups with underlying diseases such as asthma and COPD. Numerous recent reviews discuss the presence of respiratory viruses as cause of exacerbations of asthma and COPD –22. AcutThe limitations of this study include the retrospective nature of the data analysis, while some additional clinical information of the patients would have been necessary for underlining our findings. Furthermore, the clinical benefit of using carrageenan nasally was not measured e.g. by spirometry. However, in small children included in the data set, it is not always possible to perform spirometric measurements.In conclusion, nasal application of carrageenan spray in children as well as in adults suffering from virus-confirmed common cold reduced duration of disease, increased viral clearance and reduced relapses. Therefore, carrageenan nasal spray can be regarded as a safe and effective treatment with a high potential for reducing social and economic burden caused by common cold.


Pepino mosaic virus (PepMV), a representative of the genus Potexvirus (family Alphaflexiviridae). Our results allow modeling of the CP and its interactions with viral RNA. The overall fold of PepMV CP resembles that of nucleoproteins (NPs) from the genus Phlebovirus (family Bunyaviridae), a group of enveloped (-)ssRNA viruses. The main difference between potexvirus CP and phlebovirus NP is in their C-terminal extensions, which appear to determine the characteristics of the distinct multimeric assemblies – a flexuous, helical rod or a loose ribonucleoprotein. The homology suggests gene transfer between eukaryotic (+) and (-)ssRNA viruses.Flexible filamentous viruses include economically important plant pathogens. Their viral particles contain several hundred copies of a helically arrayed coat protein (CP) protecting a (+)ssRNA. We describe here a structure at 3.9 Å resolution, from electron cryomicroscopy, of DOI:http://dx.doi.org/10.7554/eLife.11795.001 A group of “flexible filamentous” viruses can cause serious diseases in a wide variety of crops and other plants. Each virus particle contains a single molecule called ribonucleic acid (RNA), which is protected by hundreds of copies of a coat protein. The RNA and coat proteins are arranged in a helical fashion to make a flexible rod-shaped particle.The flexibility of these viruses makes it difficult to carry out in-depth studies of their three-dimensional structures. As a result, we do not know how the RNA and coat proteins interact to form the structure of each virus particle. Agirrezabala et al. used a technique called cryo-electron microscopy (or cryoEM for short) to generate a highly detailed three-dimensional model of a flexible filamentous virus called Pepino Mosaic Virus.Agirezabala et al.’s findings reveal how the virus particles assemble, and the interactions between the coat protein and the ssRNA. Unexpectedly, the structure of the coat protein from Pepino Mosiac Virus is very similar to the structure of “nucleoproteins” from a group of viruses called the Phleboviruses, which infect animals. This similarity is striking and suggests that the gene that encodes these proteins has been transferred between the two groups of viruses during evolution. A future challenge is to find out whether this similarity extends to other groups of viruses.DOI:http://dx.doi.org/10.7554/eLife.11795.002 Alphaflexiviridae, Betaflexiviridae, Closteroviridae, and Potyviridae, which have different genomic organizations (particularly different between Potyviridae and the rest of the groups) and belong to different superfamilies, but it is thought that their CPs have strong evolutionary relationships (Soybean mosaic virus (SMV) a potyvirus (from the family Potyviridae), and three different potexviruses (family Alphaflexiviridae), Potato virus X (PVX), Papaya mosaic virus (PapMV), and Narcissus mosaic virus (NMV) (Bamboo mosaic virus (BaMV), another potexvirus, was determined at 5.6 Å (Papaya mosaic virus (PapMV CP) (Flexible filamentous viruses are ubiquitous plant pathogens that have an enormous impact in agriculture . Their iionships . Structuionships . This ovus (NMV) . The fleat 5.6 Å , and theapMV CP) . The worPepino mosaic virus (PepMV) is another potexvirus which has emerged recently ssRNA genome. The NPs from phleboviruses are associated with the viral genome in loose ribonucleoproteins (RNPs)  map allows for accurate modeling of the CP, the viral RNA, and their interaction. In vivo functional studies of several CP mutants confirm the role of several residues in RNA binding and polymerization. Surprisingly, we have also found a clear structural homology between the CP of flexuous viruses and the NP of the genus s (RNPs)  protectes (RNPs) . DespiteNicotiana benthamiana plants . Attempts to purify PepMV virions were only fruitful for the wt and for the Q203A mutant , suggesti subunit interacts with a hydrophobic groove of the Ni-1 subunit and establishes the main side-by-side contact in the helical arrangement  and include our current data and previously reported structures of PapMV CP  have a similar fold in their CPs. We have, nevertheless, looked for structural neighbours within the database of Dali server (Phlebovirus (family Bunyaviridae), such as Rift Valley fever virus (RVFV), Toscana virus, and Severe fever with thrombocytopenia syndrome virus (SFTSV). These viruses infect animals, including humans, are transmitted by arthropod vectors, and pose serious public health concerns. Genera Potexvirus and Phlebovirus belong to different superfamilies of RNA viruses when these are classified based on phylogenetic relationships among RNA-dependent RNA polymerases (RdRp) and also have different genomic organizations  for 2 additional weeks. Virions were purified from N. benthamiana systemically infected leaves following a previously described method . The mixture was then kept with constant stirring for 60 min at 4°C, and centrifuged for 15 min at 10,000 g. Virus concentration was estimated by OD readings at 260 nm, with ε0.1% = 2.9 as extinction coefficient (Virus preparations were obtained from pMV-Sp13 . For inod method  slightlyfficient . Virionstrans CP expression of selected mutants (Agrobacterium tumefaciens strain C58C1 was transformed with the different CP constructs and used in trans-complementation assays. A. tumefaciens overnight cultures (150 ml) were centrifuged at 2500 g for 10 min, and pellets were resuspended in agroinfiltration buffer  until OD600 = 0.6. The resulting suspensions, carrying plasmids pBPepGFPΔCP  using a handheld lamp . CP was detected by Western blotting in virion preparations and in protein extracts from leaves agroinfiltrated with CP constructs and pBP19. For protein extractions, 100 mg samples of N. benthamiana leaves were ground in 200 μl of protein extraction buffer . Crude extracts were mixed with 5x loading buffer and separated by SDS–PAGE followed by staining with Coomassie Brilliant Blue or by electrotransfer to nitrocellulose membranes. Blots were probed with polyclonal antibodies raised in rabbits against PepMV CP  followed by detection with anti-rabbit immunoglobulin G (IgG) coupled to horseradish peroxidase  and chemiluminescence . At 6–8 days post infiltration, N. benthamiana plants were harvested and the tissue was used to purify PepMV virions as described above.The PepMVCP gene was cloned into the binary vector pGWB2 giving raise to plasmid pGWB2-CP, and mutants were constructed based on this plasmid using standard overlapping PCR and molecular cloning methods . For fun mutants . AgrobacepGFPΔCP , the CP −/Å2 along 51 frames. Beam-induced motion correction was performed at the level of micrographs (−/Å2. Contrast transfer function parameters were estimated using CTFTILT (2 and was low-pass filtered to 3.9 Å.The PepMV samples were applied to Quantifoil R 2/2 holey carbon grids previously coated with a thin layer of carbon. Grids vitrified in a FEI Vitrobot were then transferred to a Titan Krios (FEI) electron microscope that was operated at 300 kV. Images were acquired using a Falcon II detector at nominal magnification of 59,000 and calibrated magnification of 102,967 (1.36 Å/pixel). Movie frames from the detector were recorded at a speed of 17 frames/s during 3 s. The total specimen dose was ~50 erographs  in frame CTFTILT . Selecti CTFTILT . The res CTFTILT  software CTFTILT  resulted CTFTILT . Local r CTFTILT   and the Protein Data Bank (www.rcsb.org) under accession codes EMD-3236 and 5FN1. review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and three reviewers, one of whom, Stephen Harrison, is a member of our Board of Reviewing Editors. One of the other two reviewers, Ed Egelman, has agreed to share his identity.Thank you for submitting your work entitled "The near-atomic cryoEM structure of The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.Summary:Pepino mosaic virus, a flexuous rod, like a number of other plant viruses. The structure, determined by electron cryomicroscopy, is at sufficiently high resolution to permit a proper chain trace of the capsid subunit and a clear view of the interaction of five nucleotides with each subunit. The most interesting result is the relationship of the capsid subunit to the nucleoprotein of bunyaviruses – another nice example of close relationships between plant and animal viruses, but in this case particularly striking because Pepino mosaic virus is a plus-strand ssRNA virus and the bunyaviruses are minus-strand viruses.This paper reports a structure for eLife, but it needs extensive rewriting and some substantive revision, as outlined below. All these required changes should be straightforward.The paper is appropriate for Essential revisions:1) More explicit comparison with BaMV. The authors should compare their atomic model with the BaMV model (5A2T.pdb). They should also include the BaMV sequence in Figure 2—figure supplement 4.2) 3) 4) In the subsection “CP and ssRNA interaction”: Interpretation of the CP-complementation studies  seems to be flawed. Previous studies showed CP mutants that do not interrupt CP-RNA interactions and can still assemble virions  are defective in cell-cell movement. CP mutants that do not assemble into virions can complement the defect of cell-to-cell movement. CP mutants that do not assemble into virions can complement the defect in cell-cell movement.5) In the last paragraph of the Introduction and in the first paragraph of the subsection “Structural homology between CP from potexviruses and NP from phleboviruses”: The icosahedral shell in RVFV is not a "capsid", which refers instead (in this case) to the helical structure associated with the RNA inside the particle. It is the "icosahedral shell of glycoproteins".6) The title should be changed. The data do not "reveal gene transfer" – such a revelation could come only from an observation of a transfer intermediate. We suggest: The structure of a flexuous-helix plant virus shows homology of its capsid protein with the nucleoprotein of animal bunyaviruses.7) The Abstract should be revised along the lines suggested below:Flexible filamentous viruses include economically important plant pathogens. Their viral particles contain several hundred copies of a helically arrayed coat protein (CP) protecting a (+)ssRNA. We describe here a structure at 3.9 Å resolution, from electron cryomicroscopy, of Pepino mosaic virus (PepMV), a representative of the genus Potexvirus (family Alphaflexiviridae). Our results allow modeling of the CP and its interactions with viral RNA. The overall fold of PepMV CP resembles that of nucleoprotein (NPs) from the genus Phlebovirus (family Bunyaviridae), a group of enveloped (-)ssRNA viruses. The main difference between potexvirus CP and phlebovirus NP is in their C-terminal extensions, which appear to determine the characteristics of the distinct multimeric assemblies – a flexuous, helical rod or a loose ribonucleoprotein (RNP). The homology suggests gene transfer between eukaryotic (+) and (-) SSRNA viruses. 1) More explicit comparison with BaMV. The authors should compare their atomic model with the BaMV model (5A2T.pdb). They should also include the BaMV sequence in Figure 2—figure supplement 4.The manuscript now contains a comparison between our model for PepMV CP and the reported BaMV CP (5A2T). This is shown in 2) Figure 1—figure supplement 2: The point of this figure is not clear. It shows a helical symmetry search with a peak at 9.28 units/turn, which looks even stronger than the peak at 8.75 (the correct symmetry). Did the authors choose the latter simply because of the BaMV symmetry or because the alternative did not give a plausible map? Please clarify.In the search for helical symmetry we tested all the local maxima, and selected the "best looking" reconstruction, essentially the map with discernible subunits. Then we proceed with local searches around the chosen symmetry. Of course, this is not very objective, and only the high resolution of the final map confirmed the accuracy of the parameters, and there was no need to calculate maps with alternative symmetries. It is clear that the figure legend was misleading and now it has been changed.When the map was calculated, the structure for BaMV was not available. In any case, since small structural variations in the CP might change the helical symmetry we must search the parameters in every new sample.3) resolution, but no helical grooves can be seen. The authors should show an FSC curve between that reconstruction and their atomic model.First, we calculated the local resolution using the original, unmasked and unfiltered total volume, but we displayed the output in a single, isolated CP subunit for clarity. This issue has been clarified in the corresponding figure legend.On the other hand, in the Resmap manual, it is stated that "Helical particles are not well supported". This statement is related to the fact that Resmap needs the input volume to have the reconstructed particle well centered in the box, and long helical structures might not be suitable. We calculated the resolution using a short, well centered helical particle with a few turns of the helix, so that the requirement for a central position could be fulfilled. Calculations using Resmap have been used in recent works with helical particles. For instance in:Gutsche et al. Near-atomic cryo-EM structure of the helical measles virus nucleocapsid. Science. 2015 May 8;348(6235):704-7;Clare et al. Novel Inter-Subunit Contacts in Barley Stripe Mosaic Virus Revealed by Cryo-Electron Microscopy. Structure. 2015 Oct 6;23(10):1815-26.2 as stated in Materials and methods). The FSC curve calculated between the cryoEM and the atomic model is now included in It is true that in 4) In the subsection “CP and ssRNA interaction”. Interpretation of the CP-complementation studies  seems to be flawed. Previous studies showed CP mutants that do not interrupt CP-RNA interactions and can still assemble virions  are defective in cell-cell movement. CP mutants that do not assemble into virions can complement the defect of cell-to-cell movement. CP mutants that do not assemble into virions can complement the defect in cell-cell movement.The straightforward interpretation of these results is that:"The three amino acids at the binding pocket for nucleotide at position 2  are required to complement the cell-to-cell movement of the CP-defective PepMV mutant […]“As it is first stated in the manuscript (in the revised version). The same sentence continues:“[…] suggesting a disruption of the CP-RNA interaction needed in virus intercellular transport.”Since the three amino acids are located in the binding pocket for the RNA, and the cell-to-cell movement needs a protein-RNA complex, it is reasonable to infer such a conclusion. Since we say "suggesting", it does not imply that it has been fully demonstrated, but we think that the interpretation is more than plausible and it needs to be stated. Therefore, we would like to keep it.5) In the last paragraph of the Introduction and in the first paragraph of the subsection “Structural homology between CP from potexviruses and NP from phleboviruses“. The icosahedral shell in RVFV is not a "capsid", which refers instead (in this case) to the helical structure associated with the RNA inside the particle. It is the "icosahedral shell of glycoproteins".We agree that terms such as "capsid", "shell" or "coat" can be understood in different ways when describing viral architecture. In the present comparison between potexvirus and phlebovirus the use of these words can be confusing. We have removed "capsid" and now use the suggested "icosahedral shell of glycoproteins", which is more specific and clear.6) The title should be changed. The data do not "reveal gene transfer" –such a revelation could come only from an observation of a transfer intermediate. Suggest: The structure of a flexuous-helix plant virus shows homology of its capsid protein with the nucleoprotein of animal bunyaviruses.We have changed the title to:"The near-atomic cryoEM structure of a flexible filamentous plant virus shows homology of its coat protein with nucleoproteins of animal viruses"7) The Abstract should be revised along the lines suggested below:Flexible filamentous viruses include economically important plant pathogens.Their viral particles contain several hundred copies of a helically arrayed coat protein (CP) protecting a (+)ssRNA.We describe here a structure at 3.9 Å resolution, from electron cryomicroscopy, of Pepino mosaic virus (PepMV), a representative of the genus Potexvirus (family Alphaflexiviridae).Our results allow modeling of the CP and its interactions with viral RNA.The overall fold of PepMV CP resembles that of nucleoprotein (NPs) from the genus Phlebovirus (family Bunyaviridae), a group of enveloped (-)ssRNA viruses.The main difference between potexvirus CP and phlebovirus NP is in their C-terminal extensions, which appear to determine the characteristics of the distinct multimeric assemblies – a flexuous, helical rod or a loose ribonucleoprotein (RNP).The homology suggests gene transfer between eukaryotic (+) and (-) SSRNA viruses.We have edited the Abstract following the suggestions.


Vesivirus genus.The complete genome sequence of the San Miguel sea lion virus-8 (SMSV-8) was determined in this study. A comparison of this sequence to other calicivirus sequences in GenBank showed that this virus is genetically distinct from the vesicular exanthema of swine virus/San Miguel sea lion virus (VESV/SMSV) strains and belongs to a novel clade within the  Caliciviridae is composed of five recognized genera, Norovirus, Sapovirus, Lagovirus, Vesivirus, and Nebovirus. The Vesivirus genus contains viruses that primarily infect animals only, such as feline calicivirus, vesicular exanthema of swine virus (VESV), San Miguel sea lion virus (SMSV), and viruses that have not been assigned to a specific group. The first isolated SMSV type, SMSV-1, was reported in 1972 from vesicular lesions on the flipper of a California sea lion . The 3′ terminal sequence was confirmed by 3′ RACE.SMSV-8 was propagated on Vero cells and supernatant stocks stored at -80°C. To prepare for sequencing, viral RNA was purified following the treatment of the virus stock with a nuclease cocktail . The genVesivirus genus. The virus with the greatest genetic relatedness to SMSV-8 is mink calicivirus (MCV) (GenBank accession no. JX847605), at 71% identity. When the amino acid sequences of the three ORFs were compared to those of other vesiviruses, SMSV-8 was found to be distantly related. The amino acid sequences of ORF1, ORF2, and ORF3 have 73%, 63%, and 48% identity, respectively, with those of MCV. With respect to other caliciviruses, SMSV-8 has 55%, 39%, and 34% identity with the three ORFs of SMSV-1 (Genbank accession no. U15301), 55%, 38%, and 35% identity to VESV A48 (Genbank accession no. U76874), and 61%, 46%, and 48% identity to calicivirus Allston 2008/US (Genbank accession no. GQ475302). These data indicate that SMSV-8 is a novel member of the Vesivirus genus. This supports Seal et al.  sequences. This number of sequences provided an average coverage over the genome of 489×. The genome length of SMSV-8 is 8,477 bases, excluding the poly(A) tail. The genomic RNA contains 3 open reading frames (ORFs), the first encoding the nonstructural polyprotein, the second encoding the capsid protein precursor (VP-1), and the third encoding a small basic protein (VP-2). A phylogenetic analysis of the genome sequence showed that the most closely related caliciviruses to SMSV-8 are members of the KM244552.The genomic sequence of San Miguel sea lion virus-8 has been deposited in GenBank with the accession no.


Dear Sir:As with other emerging vector-borne diseases, there are many theories about the eco-epidemiology of severe fever with thrombocytopenia syndrome virus (SFTSV).In our article, we highlighted the need for further work on the eco-epidemiology of SFTSV in Korea, and use Crimean–Congo hemorrhagic fever virus as an example of the role that migratory birds may play.There is evidence that several factors might be involved in transmission, and the movement of infected ticks by birds cannot be overlooked.Other infectious diseases such as Lyme borreliosis and tick-borne encephalitis move around Europe associated with birds infested with infected ticks. Similarly, the spread of West Nile virus in North America and highly pathogenic avian influenza virus H5N1 in east Asia was facilitated partly by bird movements.We agree, however, that many factors may interact to explain the current distribution of SFTSV. One route of dissemination may not explain all incidences of disease.We welcome the comments made in this letter


Rapid influenza diagnostic tests (RIDTs) used widely in clinical practice are simple to use and provide results within 15 minutes; however, reported performance is variable, which causes concern when novel or variant viruses emerge. This study's goal was to assess the analytical reactivity of 13 RIDTs with recently circulating seasonal and H3N2v influenza viruses, using three different viral measures.50) and nucleoprotein (NP) concentration, diluted at half-log dilutions, and tested with each RIDT and real-time RT-PCR.Virus stocks were characterized by infectious dose . Influenza A viruses not detected by more than one RIDT had lower NP levels. Of the 13 RIDTs, 9 had no significant differences in reactivity across IAV when compared to NP levels.Strong correlation was observed between NP concentration and RIDT reactivity; however, only weak correlation was seen with ID50 titers, which in this study correlated only weakly with proportional amounts of viral NP in prepared virus samples. In the context of the strong correlation of RIDT reactivity with NP concentration, H3N2v was found to be as reactive as seasonal circulating IAV. While these findings may not reflect clinical performance of these RIDTs, measuring NP concentration can be useful in the future to assess comparable reactivity of available RIDTs, or to assess reactivity with newly evolving or emerging viruses.Previous reports of RIDT performance typically compare reactivity based on ID All RIDTs available in the USA during the 2012–13 season utilize lateral flow immunoassays with antibodies specific to the nucleoprotein of influenza A viruses (IAV) and influenza B viruses (IBV) for the rapid qualitative detection of each virus type. Currently available RIDTs rely on a visual colorimetric signal or require a reader to interpret reflectance or fluorescence. Previous reports note disparities between IDhttp://www.cdc.gov/flu/swineflu/h3n2v-case-count.htm) raise concerns that this virus could spread more broadly in communities.50 titer.In 2011, an influenza A variant virus was sporadically detected in human respiratory specimens. This variant carries the matrix gene from pH1N1 and the remaining genes from a triple reassortant North American swine H3N2 virus.50, cycle threshold (Ct) values, and nucleoprotein (NP) measures of virus stock dilutions to evaluate the reactivity ranges of 13 FDA-cleared RIDTs with a selection of seasonal and H3N2v viruses.This study applied ID50/ml) or MDCK tissue culture (TCID50/ml) received from the Influenza Division, WHO Collaborating Center for Surveillance, Epidemiology and Control of Influenza, Centers for Disease Control and Prevention, Atlanta, GA, USA (CDC), were used for all determinations.Virus designations with stock concentrations are listed in Figure13C- and 15N-labeled analogs of the NP target peptides . Reverse-phase separation of peptides and analysis by mass spectrometry were as described.NP concentration (μg/ml) was measured by isotope dilution mass spectrometry as described for hemagglutinin and neuraminidase proteins.−1 to 10−4. Each virus dilution was transferred into 200 μl aliquots and stored on ice or in the 4°C refrigerator until used that day.One virus stock was used each day with the real-time RT-PCR and all RIDTs described in Table10 dilution versus the corresponding Ct value were generated . All 24 viral dilution series had a linear regression r2 value above 0·95 , assuring consistent dilution series for each virus. The Ct values for the 10−1 dilution are used in analyses .The CDC Influenza Virus rRT-PCR Diagnostic (Flu A&B) Panel  was performed on each dilution as previously described.http://www.cdc.gov/flu/professionals/diagnosis/clinician_guidance_ridt.htm#Table 2. Five of the RIDTs are CLIA-waived, categorized as simple laboratory examinations that have an insignificant risk of an erroneous result.Testing with RIDTs and RT-PCR was performed between October and December 2012 at the Medical College of Wisconsin. Rapid influenza diagnostic tests are listed in FigurePositive and negative controls provided with each RIDT kit were tested upon receipt for each lot in every shipment. All aspects of the evaluation including diluent, swabs, and virus input were standardized. The manufacturer's instructions for testing a swab specimen directly (without placing the swab in transport medium) were always followed .Following virus stock dilution, 50 μl of each dilution was placed into three 1·5-ml microcentrifuge tubes and held on ice. A sterile foam swab  was used to absorb each of the 50 μl samples in the microcentrifuge tubes and used as the input. Adjustments to this procedure were used when RIDT instructions required input with liquid suspensions of swab samples. For the 3M™ Rapid Detection Flu A+B test and BD Veritor for Liquid Samples, after absorbing the sample the swab was placed into a tube containing 1 ml of UTM  and mixed prior to using the manufacturers' specified volume input for these two RIDTs. BinaxNOW requires placing the swab in an elution solution . Both FluAlert  RIDTs are only indicated for nasal wash and aspirate samples. As the required sample input for this RIDT is 250 μl, we combined the 50 μl dilution sample with 200 μl of 0·9% saline. Even though this is a CLIA-waived test, the moderate complexity protocol was used due to multiple invalid results during quality control testing with the waived protocol. Kit-provided flocked swabs were used with the Status® Flu A+B test , as instructions do not allow for foam swabs used with other RIDTs.−1·5 dilution of an A/Minnesota/03/2011 replicate, and one in a 10−1 dilution of an A/Victoria/361/2011 replicate.After the study started, Quidel issued a recall of previously used Sofia FIA lots. At that point, 1 false-positive influenza B result was recorded with a negative control during the use of over 450 Quidel Sofia tests  were omitted only from correlation analyses with ID50 due to unverified comparability of TCID50 and EID50 methods. Any virus and RIDT combination with no reactivity in the 10−1 dilution was not included in the correlation calculations; however, for subsequent analyses, the nominal value of the 10−0·5 dilution was used. To compare virus groups for each RIDT, a log transformation was applied to normalize the variances of the reactivity measures before performing one-way anovas. P-values between significantly different IAV groups for individual RIDTs were determined by Tukey's honest significant difference test. All analyses were performed in Microsoft Excel 2010.For analyses, the highest dilution reactive (HDR) was determined as the one in which two of the three replicates were positive for any one virus. Spearman's rank correlation analyses between stock ID50, Ct values, and NP concentration for the virus stocks. Correlation coefficients for the association between ID50, NP concentration, and Ct values across viruses were weak for ID50 and Ct versus NP (0·094 and −0·35) and moderate for ID50 versus Ct values (−0·75). When the viruses with TCID50 quantitation were included, the correlation was stronger for ID50 versus NP and was weaker for ID50 versus Ct values, but had no impact on interpretation of the results.Figure−1) tested  had reactivity in only seven IAVs . The reactivity of other RIDTs ranged from detection of 11 (Status) to 17  IAVs in the 10−1 dilution  and reflectance (Veritor) signals. These readers may discriminate subtle differences in reaction intensity that are not apparent in visual reads.For IAVs, patterns of reactivity are variable across all virus groups for both individual viruses Figure and for 50 titer ranges were similar and overlapped considerably across each of the virus groups as did the Ct value ranges at the 10−1 dilution for the IAVs. The range of stock NP concentrations was narrower for IBVs, yet HDRs varied widely across RIDTs.Differences in mean stock NP concentrations between IAV that were reactive in all RIDTs and those that were not reactive in more than one RIDT suggest a link between NP concentration and test reactivity . Those I50, and 10−1 dilution Ct values. For IAV, the correlation is strong (−0·86) between stock NP concentration and the mean HDR for all test kits. On the other hand, correlation between stock ID50 and HDR is practically zero (−0·015) and weak between Ct values and HDR (0·24).Figureanovas showed no significant difference  between any of the IAV groups for nine individual RIDTs. These nine RIDTs include those reactive with all IAVs (n = 4). While individual RIDTs showed some variation between IAVs, no individual IAV subtype was significantly less reactive across all RIDTs, when compared with NP concentrations by anova as shown in FigureP-value <0·05) for any one RIDT.As NP concentration versus mean HDR had a strong association across all RIDTs for IAV, the mean NP  was plotted against each RIDT Figure. anovas IAVs n = . While iIAVs n = . While iIAVs n = . While i50 titers and Ct values) for this small group of viruses were notably uniform, yet HDRs varied widely across individual RIDTs.FigureThe primary goal of this study was to determine whether RIDTs are as reactive with H3N2v IAVs as with other influenza viruses. Aggregate reactivity results for all RIDTs with each virus in Figure50 titers, a virus measure known for variability between laboratories and methods, warrants caution with assessing RIDT analytical reactivity with propagated influenza viruses characterized solely by ID50 titers.The strong correlation of the virus stock NP concentrations with mean HDRs in this evaluation is not unexpected given that all of the RIDTs are designed to detect viral nucleoprotein using different antibodies to capture and detect influenza A and B viruses, and this association was also observed in a previous study.In this study, the BD Veritor RIDT for liquid specimens  showed a decrease in reactivity when compared to the Veritor RIDT for swab specimens (tested directly). Previous testing with an RIDT using two sets of virus samples (50 μl adsorbed onto a swab and 50 μl added to 1 ml diluent prior to testing) showed a consistent decrease in reactivity for the set added to diluent (data not shown). A major advantage with RIDTs is the rapid time to results (if specimens are tested at the time of collection); placing swabs into transport media for RIDT may offset the benefit with rapid results if NP levels are lowered by dilution.r2 = −0·86) in this study, it is unable to explain all of the variability in reactivity due to other potentially contributing factors. Such factors can include proprietary differences between individual RIDTs, sequence variations affecting epitope-binding sites, or differences in virus replication properties within infected host or culture cells and the potential for multiple virus quasispecies. Observations from this study, specifically that the (A/Montana/05/2011) virus with the lowest NP concentration was not reactive in the least number of RIDTs and  virus with the highest NP concentration was not reactive in all of the RIDTs, suggest that there must be factors other than NP concentration contributing to reactivity. Furthermore, several RIDTs showed significant differences in reactivity between IAV groups, suggesting that antibody design may not be optimal for all IAV and amino acid variations between IAV groups could be a factor. A previous reportAlthough the correlation between NP concentrations and IAV reactivity is strong  for individual RIDTs. Our observation that H3N2v viruses as a group produced less NP in virus culture may be indicative of their growth in mammalian cells. Further characterization of these viruses in both virus culture and respiratory samples would be important for better understanding of these observations, for improving RIDT performance, and for use of RIDTs in clinical practice. This evaluation reinforces that negative RIDT results are more likely when any virus samples have low NP concentrations, regardless of IDM. E. Bose contributed to data analysis and interpretation and manuscript preparation. A. Sasman contributed to the RIDT, data compilation, and manuscript preparation. H. Mei and K. McCaul performed the RIDT and data compilation. W. Kramp and R. Shively contributed to the concept and design of the study, data analysis and interpretation, and manuscript preparation. E. T. Beck and K. J. Henrickson contributed to the concept and design of the study and manuscript preparation. L. Chen prepared the viruses used in this study. T. L. Williams managed the isotope dilution mass spectrometry quantitation of NP for these viruses. All authors have approved the final version of the manuscript.


For the first time, the complete genome sequences of the six serotype O foot-and-mouth disease viruses from persistently infected carrier cattle are reported here. No consistent amino acid changes were found in these viruses obtained from persistently infected cattle compared with the complete genome of the parent virus that was used to infect the cattle. Aphthovirus in the Picornaviridae family. FMD virus (FMDV) can persist in the oropharynx of ruminants for years following the resolution of acute infection (Foot-and-mouth disease (FMD) is a highly contagious acute viral disease of cloven-hoofed animals caused by a single-strand positive-sense RNA virus of the genus nfection . As liveFour FMDV vaccine challenge experiments were previously conducted in the animal isolation unit (biosafety level 3 [BSL3] containment) at The Pirbright Institute to study FMDV infection in vaccinated animals –6. The p–The complete genomes of the challenge virus and the persistent viruses are 8,183 nucleotides in length. The genomes contain a 1,091-nucleotide-long untranslated region (UTR) at the 5′ end, followed by a 6,999-nucleotide-long polyprotein-coding region and a 94-nucleotide-long UTR at the 3′ end, excluding the poly(A) tail. The poly(C) within the 5′ UTR and the poly(A) tail at the 3′ end of FMDV are highly varied; 10 nucleotides of C were substituted during sequence analysis and no As were included. The nucleotide identity between the challenge and carrier viruses ranged between 98.90% and 99.77%, and the amino acid identity ranged between 99.18% and 99.96%. There were no consistent changes found in the amino acid sequences between the challenge virus and the viruses from persistently infected cattle. This observation indicates that there is no viral determinant that plays a role in persistently infecting cattle.KR265072, KR265073, KR265074, JX947859, KR401161, JX947858, and KR265075.The complete genomes of the FMD viruses recovered from persistently infected cattle and the virus used to challenge cattle have been deposited in GenBank under the accession numbers


Natural populations of pathogens are frequently composed of numerous interacting strains. Understanding what maintains this diversity remains a key focus of research in disease ecology. In addition, within‐host pathogen dynamics can have a strong impact on both infection outcome and the evolution of pathogen virulence, and thus, understanding the impact of pathogen diversity is important for disease management.Spodoptera exempta nucleopolyhedrovirus (SpexNPV) isolated from the African armyworm, Spodoptera exempta. NPVs are obligate killers, and the vast majority of transmission stages are not released until after the host has died.We compared eight genetically distinguishable variants from NPV variants differed significantly in their virulence and could be clustered into two groups based on their dose–response curves. They also differed in their speed of kill and productivity  for S. exempta. The mixed‐genotype wild‐type (WT) SpexNPV, from which each variant was isolated, was significantly more virulent than any individual variant and its mean mortality rate was within the fastest group of individual variants. However, the WT virus produced fewer new infectious stages than any single variant, which might reflect competition among the variants.The WT virus over any single variant. Spodoptera exempta larvae infected with WT SpexNPV were predicted to die 2·7 and 1·9 times faster than insects infected with isolates from either of the two clusters of genotypes.A survival analysis, combining the mortality and speed of kill data, confirmed the superiority of the genetically mixed Theory suggests that there are likely to be trade‐offs between pathogen fitness traits. Across all larvae, there was a negative linear relationship between virus yield and speed of kill, such that more rapid host death carried the cost of producing fewer transmission stages. We also found a near‐significant relationship for the same trend at the intervariant level. However, there was no evidence for a significant relationship between the induced level of mortality and transmission potential (virus yield) or speed of kill. Pathogens (micro‐parasites) are ubiquitous in animal and plant populations and often cause acute infections that can result in high levels of mortality and devastating epizootics  diversity could be maintained by a variety of processes, including genotype × genotype interactions and negative frequency‐dependent selection, trade‐offs between virulence traits or differential selection  and its NPV (SpexNPV). Spodoptera exempta is found predominantly on the eastern side of the African continent, where it is a major pest of various graminaceous crops such as maize, sorghum, millet and also pasture grasses  causing irregular pest outbreaks during the rainy season. This species is highly migratory and can travel thousands of kilometres during a field season, while undergoing numerous generations, and crop damage can be extensive. NPV is frequently associated with high‐density populations, particularly later in the season  of covert NPV is very high, approaching 100%  WT virus was collected from S. exempta populations in Tanzania in 1972. A preliminary study of this isolate, using restriction fragment profiling of the DNA, revealed numerous submolar bands, indicating the presence of multiple ‘variants’ (genotypes). The variants were separated by in vivo cloning in S. exempta. In vivo cloning involves challenging individual larvae with a pathogen dose that will result in low levels of mortality , under the assumption that infections at this level will result from a single virus OB , and it is possible that more than a single clone could be present within each variant. However, if present, these would be at very low levels and are unlikely to produce biological effects over one round of infection.The original sample of . et al. ]. We choS. exempta culture was initiated from pupae collected in Arusha, Tanzania, in 2002. It was reared for six generations on wheat seedlings at the University of Stirling with no signs of overt NPV infection and then transferred to NERC Centre for Ecology and Hydrology in Oxford. The culture was reared on a semi‐synthetic wheat‐based diet at 28 °C with an 10 : 14‐h light : dark cycle. At each generation, eggs were surface‐sterilized in 1% hypochlorite for 10 min and then 5% formalin for 30 min, in order to remove any potentially contaminating pathogens. Larvae were separated at the second instar and reared individually from this point. Pupae were sexed and mated in groups of around 50 pairs in cylindrical rearing cages and were fed with a 10% honey solution.The An experiment was set up to measure three fitness parameters of the virus: mortality and speed of kill, both of which can be used to describe the virulence of the variants, and the number of offspring produced (virus yield) as an estimate of the virus transmission potential. As is standard practice in insect pathology, these metrics were estimated over a range of pathogen doses, because bioassays performed at a single dose can be misleading as they give no indication of the shape or slope of the dose–response curve. The eight variants were compared with the mixed‐genotype, parental WT SpexNPV from which they were derived.S. exempta larvae were challenged with five SpexNPV doses: 50, 100, 500, 1000, 5000 OBs per individual. Each dose was given in 1 μL distilled water pipetted onto a small (c. 1 mm3) plug of artificial diet to individual larvae in square 25‐well plates and left for 24 h. Twenty‐five larvae were infected per dose, except for the lowest two doses where 50 larvae were used in order to produce sufficient numbers of cadavers for the analysis of pathogen yield. An additional 25 larvae dosed with distilled water were used as controls. The virus treatments were replicated twice. All the larvae that had eaten the diet plug in 24 h were transferred into individual 12‐mL pots containing sufficient diet to maintain them through to pupation. Larvae were maintained at 28 °C and were monitored every 8–16 h to produce an estimate of the time taken to kill the larva (infection duration), in addition to virus‐induced mortality. Speed of kill was estimated from the time that the larvae were exposed to the viral dose to when they were observed to be dead. NPV infections are usually obvious as virtually the whole body is converted to virus and the larva becomes pale and flaccid and releases a thick milky fluid when the cuticle eventually ruptures. Any ambivalent deaths were confirmed using Giemsa staining and oil immersion microscopy (×1000). Virus‐killed cadavers were frozen individually at −20 °C in 1·5‐mL Eppendorf tubes until further analysis. Ten cadavers per dose for each virus isolate were selected to estimate the number of progeny OBs produced (virus yield). Only cadavers that could be transferred whole were used for yield analysis. Each dead larva was macerated in 1 mL of sterilized water with a plastic pestle and vortexed thoroughly. OBs from two subsamples were then counted using a 0·1‐mm improved Neubauer haemocytometer and a light microscope at ×400. Four virus‐killed cadavers from each genotype treatment were randomly selected to confirm that the genetic identity of the virus remained constant after the bioassay using RFLP profiling with EcoRV  and so both were analysed using linear regression. Survival was modelled using a parametric survival model, with an exponential distribution with data censored at 160 h (longer times are likely to be secondary infections). Survival curves were visualized using Kaplan–Meier plots.Mortality data were analysed using generalized linear models using a binomial error distribution and logit link function. As no control larvae died of virus infection, no correction factors needed to be applied. Non‐viral deaths, predominantly bacterial, did occur at a very low level (<5%) late in the assay, that is after any viral deaths would be expected to occur. Cadavers were stained with Giemsa and checked using a light microscope in case a mixed bacteria–NPV infection was involved; this was not found to be the case, and therefore, these deaths were not included in the virus‐killed total. Speed of kill data (1/number of hours until death) were normally distributed, as was log10) viral dose  and differed significantly between the virus genotypes , even after excluding the WT virus from the analysis . The interaction between virus dose and virus genotype was non‐significant in both cases , indicating that all SpexNPV variants responded similarly to viral dose. All SpexNPV genotypes analysed at the end of the bioassay matched those of the virus used for dosing, verifying the stability of the single genotype. As expected, larvae killed by the WT virus treatment produced a profile with multiple submolar bands, indicative of a mixed isolate (data not shown).Host survival was severely reduced by baculovirus infection. NPV‐induced mortality increased with . Across all variants (including the WT virus), there was a sixfold variation, and within the eight SpexNPV single‐genotype variants, there was a threefold difference , which had consistently lower LD50s in the range of 484–825 OBs per larva. The LD50 of the parent WT SpexNPV was significantly lower (i.e. more virulent) than any of the individual variants, 240 OBs per larva. After grouping the genotypes in this way, the interaction between virus genotype and dose remained marginally non‐significant . When the eight single‐genotype variants were combined and compared against the WT parental virus treatment, there was a highly significant difference between the two types of virus treatments, that is multiple‐genotype WT vs. single‐genotype variants , with the slope of the relationship between virus dose and virus‐induced mortality being steeper for the WT virus .This variation in virulence of the virus treatments is best illustrated by comparing their LDnce Fig. a. Model F8,787 = 7·24 P < 0·0001; Fig. F7,720 = 7. 39, P < 0·0001). However, the speed at which larvae died of NPV infection also increased with increasing viral dose , and there was a significant interaction between viral dose and virus variant , such that the slope of the relationship between viral dose and speed of kill was steepest for the WT virus and shallower for each of the single‐genotype variants .Mean time to death across the variants ranged between 89·9 h (variant B) and 105·2 h (variant H); the WT virus was intermediate and took an average of 90·9 h to kill its host. Speed of kill (1/time to death) varied significantly between variants . Mortality rate also increased with increasing viral dose , but all variants responded in a similar manner to viral dose . Model simplification revealed that the variants could be grouped into the same two clusters, as described for the mortality analysis (see above), with no loss in explanatory power . Thus, larvae infected with the WT virus are predicted to die 2·7 times faster than those infected with variants from Group I  and 1·9 times faster than those challenged with Group II variants .A survival analysis was conducted to determine the compound effects of virus‐induced mortality and time to death. Overall, there was significant variation between variants in their mortality rates , which yielded fewer OBs than any of the single genotypes . Variants E and H differed significantly in the yield produced from the other variants, with E producing significantly fewer and H significantly more , and there was no change in yield with increasing virus dose .The number of transmission stages produced varied significantly between variants , with the WT virus yielding fewer OBs for a given speed of kill than any of the single genotypes, significantly so for all variants except E (as determined by comparison of the coefficients and their standard errors). The interaction between speed of kill and NPV genotype was non‐significant , as was virus dose . The results remained largely unchanged after excluding the WT parental virus from the analysis .As predicted, we observed that across all larvae, production of virus OBs  was significantly negatively related to speed of kill . There was no evidence for a relationship between speed of kill and mortality (LD50) , although the non‐significant trend was in the direction of a faster speed of kill being associated with a lower LD50 . However, an analysis of virus yield vs. LD50 indicated a non‐significant positive relationship when the WT was included  and a near‐significant negative relationship when it was removed  .Across genotypes, there was also evidence for a genetic trade‐off between virus yield and speed of kill, but the correlation was marginally non‐significant , in which virus yield plateaued at slower speeds of kill  and differing background mortality within the system . The number of virus occlusion bodies produced was taken as a surrogate for transmission potential, in common with other studies. There was a clear linear relationship between the duration of infection and the production of virus transmission stages for individual genotypes, with OB production steadily increasing as the duration of the infection increased Fig. a. This lS. exempta might benefit from more rapid rounds of infection, although not if the host is continually moving to different areas. This relationship could, however, be modulated by vertical transmission of the virus  pathogen, Nosema whitei, which is an obligate killer requiring host death for spore production and transmission. In contrast to other studies  trends suggesting that higher mortality is positively associated with faster speed of kill and greater yield. However, these relationships, if present, are clearly not strong, and their exploration would require a greater number of virus clones. Few studies have investigated trade‐offs with mortality in obligate killers. In an experimental evolution study, Bérénos, Schmid‐Hempel & Wegner  investig50 between the least and the most virulent individual virus variants. However, what was most striking was that the WT virus was significantly more virulent than any of the individual virus variants, with an LD50 that was half that of the most virulent variant. This clearly indicates that infections with multiple virus genotypes are much more virulent. All the SpexNPV variants compared in the experiment here were derived from the WT source; however, more than these eight variants are present within the WT mixture  and the winter moth (O. brumata) show that WT isolates can be both fast to kill and retain high productivity (Raymond et al. Mixed‐genotype infections and their impact on virulence have recently received a lot of attention (e.g. Alizon, de Roode & Michalakis http://dx.doi.org/10.5061/dryad.427m6 (Redman, Wilson & Cory Data available from the Dryad Digital Repository: Fig. S1. Effect of the interaction between viral group and viral dose on virus‐induced mortality.Fig. S2. Effect of the interaction between virus variant and viral dose on speed of kill.Click here for additional data file.


Reducing health care costs requires the ability to identify patients most likely to incur high costs. Our objective was to evaluate the ability of the Charlson comorbidity score to predict the individuals who would incur high costs in the subsequent year and to contrast its predictive ability with other commonly used predictors.We contrasted the prior year Charlson comorbidity index, costs, Diagnostic Cost Group (DCG) and hospitalization as predictors of subsequent year costs from claims data of fund that provides comprehensive health benefits to a large union of health care workers. Total costs in the subsequent year was the principal outcome.th and 10th percentile of costs, the comorbidity index, prior costs and DCG achieved almost identical explained variance in both adults and children.Of the 181,764 predominantly Black and Latino beneficiaries, 70% were adults . As the comorbidity index increased, total yearly costs increased significantly (P<.001). At lower comorbidity, the costs were similar across different chronic diseases. Using regression to predict total costs, top 5The comorbidity index predicted health costs in the subsequent year, performing as well as prior cost and DCG in identifying those in the top 5% or 10%. The comorbidity index can be used prospectively to identify patients who are likely to incur high costs.NCT01761253ClinicalTrials.gov  Health costs in the U.S. have been rising at an unsustainable pace In this analysis, we operationally define multiple chronic diseases, using the Charlson comorbidity index Our objective was to evaluate whether comorbidity, as assessed by the Charlson comorbidity index during one year, could be applied to a large population to identify those patients who would incur high health care costs in a subsequent year. The performance of the comorbidity index in predicting future costs was compared to prior year total costs, a usual benchmark; to the Diagnostic Costs Groups (DCG), one commonly used population-based risk adjustment strategy The study was reviewed and approved by the Weill Cornell Internal Review Board. Patient information was anonymized and de-identified prior to analysis; individual consent was not obtained.The population is a union of health and hospital workers in the Northeast, whose self-insured trust fund administers comprehensive health benefits to the members, their spouses and children. The fund covers all medically necessary hospital, medical, maternity, behavioral health and pharmacy services, and maintains their own claims data repository. There were 226,157 individuals who were eligible for benefits for at least 11 months between January–December 2009; of those, 185,294 (86.2%) remained eligible for at least 11 months from January–December 2010. Overall 18.1%  did not remain eligible for reasons such as retirement or dependent children reaching the maximum age of eligibility. This analysis focuses on the 181,764 beneficiaries who were consistently eligible for benefits over at least 22 months in 2009 and 2010, who also received DCG codes. Beneficiary age and gender were available. Comorbidity was assessed through the Charlson comorbidity index The comorbidity index was assessed from claims data for services provided between January and December 2009; each claim had at least one primary ICD-9 diagnosis code and up to three secondary codes. The comorbidity index was assessed using the Deyo strategy The prospective DCG model utilizes claims data and age, sex, diagnoses and their interactions to predict future costs; the 2009 prospective DCG was used to predict 2010 cost Claims data was used to document the cost and utilization of services among these consistently eligible beneficiaries. The type and place of service were available. The prior year hospitalization was the actual number of hospitalizations. Total amount paid for all services including inpatient, outpatient, emergency room, laboratory tests, behavioral health and prescription drugs for January–December 2010 were evaluated.and other comorbid diseases, an adjusted comorbidity index was calculated by subtracting the Charlson comorbidity weight for that disease from the total comorbidity score. For example, patients with congestive heart failure (CHF) who have an adjusted index score of zero have only CHF, while those with an index of one or more have other illnesses as well.To compare the costs for those with a given chronic disease versus those with that disease Four predictors of subsequent yearly costs  were compared using four different analytic approaches: a two part regression modeling strategy often used in econometric analysis; quantile regression to predict the upper 5% and 10% of the cost distribution; logistic regression to predict whether a specific individual would be in the upper 5% or 10% cost strata using positive predictive value; and receiver operating characteristic (ROC) analysis to evaluate sensitivity and specificity. Since cost data was skewed by both high cost patients and by those with zero costs, standard regression could not be used. Age, gender and mental health were controlled for in all regressions. There was no data on race/ethnicity in the claims; however, the beneficiaries are predominately Black and Latino. The data did not include the specific location where the beneficiaries received services.The first part of the two-part model is a binary outcome model that describes the distinction between non-users (zero cost) and users of services (non-zero cost), while the second part is a linear regression that describes the distribution of total health care cost for patients who used services see  [25]. AsQuantile regression was employed to assess the relationship between predictors and the upper tail of the cost distribution, controlling for age, gender and mental health diagnoses 2 is the measure of model fit 2 in quantile regression is calculated as 1 – (sum of weighted deviations about estimated quantile)/(sum of weighted deviations about raw quantile) . We used this definition of pseudo-R2 for each of the models. The pseudo-R2 is analogous to the classical R2 = 1 – (error sum of squares)/ from multiple regression.Positive predictive value was assessed through logistic regression which was used to build a model that predicted whether an individual would fall into the top 5% and 10% highest predicted cost groups Receiver operating characteristic (ROC) analysis evaluates the sensitivity and specificity at various threshold settings of prior year costs, prior year comorbidity, prior year hospitalizations and prior year DCG to predict membership in the top 5% and 10% cost groups Overall, 69.3% of the predominantly Black and Latino beneficiaries were adults , while 30.7% were children less than 18 years of age . With regard to the adults, 75.3% were union members; 20.5%, their spouses; and 4.2%, their covered adult children (mean age 19.5±1.2 years). 99.2% of the beneficiaries live in the New York tristate area where they receive care through many different physicians, practices and hospitals.The total health care costs in 2010 were $670 million. Beneficiaries with a comorbidity index ≥4 constituted only 6.5% of the population, but accounted for $154 million, or 23.0% of total 2010 costs. As expected, adults and children differed significantly in comorbidity (p<.001); only 0.2% of children had a comorbidity index ≥4 in comparison to 9.2% of the adults.Overall 6.4% of adults and 2.0% of children were hospitalized. The only mental or behavioral health conditions in the comorbidity index are depression and dementia. 0.9% of the beneficiaries had dementia, and only 2.9% of adults (average age 45.2) and 1.0% of children (average age 13.8) had a diagnosis of depression. In total, 4.9% of adults (average age 45.2) and 1.0% of children (average age 11.3 years) had another mental health diagnosis. Overall, 5.9% of beneficiaries who had a mental health diagnosis (other than depression or dementia) accounted for 11.0% of the costs (p<.001). The frequency of a mental health diagnosis increased with increasing levels of comorbidity. For those with a comorbidity index of 0–1, 5% had a mental health diagnosis; comorbidity of 2–3, 8.8%; comorbidity of 4, 9.4%; comorbidity 5–7, 10.2% and those ≥8, 14.1%. At each level of comorbidity, those with mental health diagnoses had significantly increased costs (p<.001).Overall, 9.7% of beneficiaries had zero costs in 2010. Those with lower comorbidity (p<.001), without a mental health diagnosis (p<.001), men (p<.001), and those with zero costs in 2009 (p<.001) were more likely to have zero costs in 2010. In adults, prior year costs explained 31% of the variance, while the comorbidity index and prospective DCG each explained 20%; prior hospitalization explained only 11%. In children, prior costs explained 20% of the variance, and the comorbidity index explained 17% of the variance, while prospective DCG and prior hospitalization explained 7%. Each model in th and 10th percentile of costs for both adults and children. Prior year costs, prior year comorbidity, prior year DCG, and prior year hospitalizations were all evaluated as predictors of upper 5% and upper 10% of subsequent (2010) costs in separate models controlling for age, gender and mental health diagnosis. In adults, the comorbidity index was equivalent to DCG and prior cost in predicting the top 5% and 10% of cost, while prior hospitalization had much lower ability to identify such patients. In children, the comorbidity index, prior costs, and DCG also had similar predictive ability for the top 5% and 10% of costs. Table S3 in Another approach to classification assessed how likely it was for an individual to be in the top 5% and 10% of 2010 costs based on prior year characteristics. Table S4 in In this study, multiple chronic disease was operationally defined using the Charlson comorbidity index. Cited in more than 9,500 publications, the comorbidity index is the most extensively validated measure of the prognostic impact of multiple chronic illnesses. The prior year comorbidity index was compared to prior year cost, prior year DCG and prior year hospitalization as a predictor of subsequent year costs using two part regression and quantile regression as well as several classification approaches.2 for prior year costs was 4.9%; DCG, 7.2%, and the comorbidity index, 3.6%; however, the explained variance was low because costs were estimated base on units of service  and not on actual expenditures th and 10th percentile. One study that used the Medical Expenditure Panel Survey data to predict the top 10% of costs found that the pseudo R2 for DCG was identical to our study, 20% With two part regression the comorbidity index explained 20% of the overall variance in adults vs. 31% for the benchmark prior year costs, 20% for DCG and 11% for prior hospitalizations. The comorbidity index did almost as well as prior cost in children (17% vs. 20%), better than DCG (7%), or hospitalizations (7%). A similar analysis evaluating predictors of costs among VA patients enrolled in an ambulatory care quality improvement trial found that the adjusted RTwo classification approaches positive predictive value and ROC analysis were also used to classify patients according to their likely membership in high cost groups. Prior year comorbidity had a slightly higher positive predictive value than prior hospitalization and both were better than prior costs and prior DCG score. With respect to the ROC analysis, the comorbidity index achieved almost identical area under the curve as prospective DCG and prior cost in predicting the top 5% and 10% of subsequent costs.These results were obtained from analysis of a specific population and cannot be directly compared to the explanatory power of other published cost models, in part because any analysis of costs, including this one, encompass differentials in provider pricing. However, contrasting the different models in this population provides a context for evaluating the predictive ability of prior comorbidity, prior cost, prospective DCG and prior hospitalizations to predict subsequent costs in the same population. The comorbidity index could not be calculated for the 10% who did not have claims in the first year.In addition, the comorbidity index has not been independently validated in children, although it has been used in some pediatric studies To be successful in reducing health care expenditures, interventions have to identify the patients who are most likely to have sustained high costs. If not targeted precisely toward such patients, interventions will cost more than they will save. This was the experience with disease management interventions Focused on adults, a comorbidity index ≥5 identifies 5% of patients who will have 17.5% of costs; or comorbidity index ≥4 identifies 9.3% of patients who will incur 25.6% of costs. A recent study using the VA criteria found that 32% of patients had 65% of costs This study used the comorbidity index calculated for patients from claims data from the prior year to predict subsequent year costs and contrasted it with commonly used predictors of cost including prior hospitalization, prior costs, and DCG. Prior costs and prior DCG usually require 12 months of claims data, which generally creates a lag time of 6–18 months Importantly, the Charlson comorbidity index can also be documented prospectively by a short questionnaire as well as through claims or chart data. For more than 20 years, we and others have ascertained the comorbidity index in less than 10 minutes through interview in person or by telephone Among 181,764 beneficiaries, the Charlson comorbidity index from a prior year predicted high health costs in the subsequent year. The index performed as well as prior cost or DCG in identifying those who would have higher costs, and those who would fall in the top 5% or 10% of costs in both adults and children. The comorbidity index also predicted subsequent hospitalization and repeated hospitalization, the largest component of costs. The comorbidity index provides a reproducible and prospectively applicable method of identifying patients at high risk of incurring high subsequent costs. As a reproducible, clinically validated and widely used method that has commensurate face validity for clinicians, the Charlson comorbidity index shows where the cost curve bends Data S1(ZIP)Click here for additional data file.File S1Contains the description of the two-part regression.Table S1, Yearly costs for adults and children according to each rank of the comorbidity index. Table S2, Number of patients with specific chronic diseases according to the adjusted comorbidity score. Table S3, Percent of patients falling into the upper 0.5%, 1%, 2%, 5% and 10% of 2010 costs based on 2009 comorbidity index. Table S4, Classification of individual's likelihood of membership in the top 5% or top 10% of subsequent costs according to different prior year characteristics.(PDF)Click here for additional data file.


To estimate annual direct costs of early RA by resource component in an inception cohort, with reference to four distinct treatment strategies: no disease modifying antirheumatic drugs (DMARDs), synthetic DMARDs only, biologic DMARDs in the first year , and biologic DMARDs from the second year after inclusion ; to determine predictors of total and non-DMARD related costs.The ESPOIR cohort is a French multicentric, prospective study of 813 patients with early arthritis. Data assessing RA-related resource utilisation and disease characteristics were collected at baseline, biannually during the first two years and annually thereafter. Costs predictors were determined by generalised linear mixed analyses.Over the 4-year follow-up, mean annual direct total costs per treatment strategy group were €3,612 for all patients and €998, €1,922, €14,791, €8,477 respectively for no DMARDs, synthetic DMARDs only, FYB and LYB users. The main predictors of higher costs were biologic use and higher Health Assessment Questionnaire (HAQ) scores at baseline. Being a biologic user led to a higher total cost (FYB Rate Ratio (RR) 7.22, [95% CI 5.59–9.31]; LYB RR 4.39, [95% CI 3.58–5.39]) compared to non-biologic users. Only LYB increased non-DMARD related costs compared to all other patients by 60%.FYB users incurred the highest levels of total costs, while their non-DMARD related costs remained similar to non-biologic users, possibly reflecting better RA control. Rheumatoid arthritis (RA) is an autoimmune disease causing chronic inflammation of the joints, often resulting in severe functional disability. The prevalence of RA in most world regions is under 1% of the population, In France, the mean annual direct cost of RA was estimated  cohort is a multicentric, prospective study of French patients with early arthritis (EA). The The ESPOIR cohort recruited 813 patients in France between 2002 and 2005. Our analysis included only direct RA costs from the French health system perspective, all three payers combined , complementary health insurance, and patients), divided between the cost of RA-specific drugs (either synthetic or biologic DMARDs) and costs of other healthcare resources. The cost of DMARDs was defined as the cost of the drugs themselves plus costs related to administration . Other healthcare costs included consultations with physicians and other health professionals, symptomatic treatments using non-DMARD drugs, imaging , magnetic resonance imaging (MRI), gastric endoscopies, colonoscopies), transportation, hospitalisations  and clinical workups including blood tests. Because costs associated with the DRG coding version used were available only until 2007, costs were expressed using €2007 For both specialists and general practitioners, an average consultation fee was estimated using 2007 SHI tariffs and average extra billing. Clinical workups were assigned their 2007 values based on expert opinion for inclusion, monthly and annual blood tests. Drugs were grouped according to their international nonproprietary names (INN), and their costs were calculated by estimating mean tariffs per milligram or per dose, weighted for the volumes sold in France in 2007 based on claims data. Hospitalisation costs were estimated according to diagnosis-related groups (DRG) using 2007 2-test.Patient characteristics were described by the mean (SD) or number (%) and costs by the mean ± SD (median). Group comparisons were made using the Student t-test, the Wilcoxon Mann Whitney-test or the χThe clinical impact of the time of biologic initiation among biologic users was assessed by comparing the proportion of patients with low disease activity (DAS-28≤3.2), the proportion of patients needing assistance from other personnel, the proportion of patients with RA caused disability, mean HAQ and mean EuroQol Five Dimensional Questionnaire (EQ-5D-3L) throughout the study period in groups of FYB and LYB users matched on baseline characteristics using a logistic regression propensity score the variables of which were selected using the stepwise selection method.A regression analysis was conducted to identify determinants of total direct costs in the first four years of illness for patients who had attended all follow-up visits. To assess the timeliness of biologic initiation, another regression analysis investigated determinants of other (non-DMARD) healthcare costs that reflect the patients’ healthcare needs and level of disease activity. Because of the skewed distributions of both primary outcomes and the hierarchical structure of the data by center, we used generalised linear mixed models, which have been recommended for this type of cost data analysis. Variables used to investigate potential cost factors and to calculate propensity scores included patient sociodemographics, disease severity and RA management characteristics. Sociodemographic variables included age, sex, marital status, income and health insurance coverage. Disease severity indicators included hospitalisation at baseline or in the preceding six months (yes/no), functional ability at baseline expressed in four mutually exclusive groups , Dropouts and missed study visits may be linked to patients’ disease severity, and thus missing data may lead to bias in cost analyses. To address this issue and assess the robustness of the models, sensitivity analyses were conducted using a Markov chain Monte Carlo (MCMC) multiple imputation (24 cycles) method based on a multivariate normal distribution. Patients who missed all study visits and those who died during the study period remained excluded. Imputations concerned log-transformed costs per period (12.3% of missing data) as well as the variables FYB (4.5%) and LYB (22.5%), which were imputed without rounding. Statistical analyses were performed using SAS 9.3 .Two-thirds (n = 548) of the 813 patients in the ESPOIR cohort attended all follow-up visits and were included in the baseline analysis . The majAmong the 265 excluded patients, baseline HAQ and DAS-28 scores were not different from the study population’s , nor was sex distribution (p = 0.80). However, excluded patients were slightly younger , fewer were positive for ACPA  and IgM rheumatoid factor (IgM RF)  and fewer met the 1987 American College of Rheumatology (ACR) RA criteria .The study population for the regression sensitivity analyses (n = 777) included the 813 cohort patients, less the seven who died during the study period and the 29 patients who attended no study visits following inclusion.A comparison of groups of FYB and LYB users matched on baseline characteristics  with p = 0.93 and p = 0.68 respectively after matching) shows that over the entire study period, FYB users compared to LYB users display lower proportions of patients with low disease activity (DAS-28≤3.2) see , lower pThe variables retained in the propensity score model were: satisfaction of ACR criteria, HAQ level and home ownership status.The breakdown of costs by resource component is shown in For patients receiving biologics, DMARDs constituted the highest share of total costs. For FYB patients the mean annual cost per patient was more than five times higher than that of the rest of the population, nearly twice that of LYB users and nearly eight times that of patients receiving only synthetic DMARDs.Other healthcare costs were one-third lower among FYB patients compared to LYB patients, representing an average annual offset of €1,042. Healthcare costs of FYB patients remained comparable to patients who received no biologic treatment . This cond year after inclusion), €7,592  and €5,384  respectively), while their other mean annual costs remained comparable . Inferential statistics were not produced due to the small size of these groups.Grouping LYB patients by year of biologic initiation revealed that their total mean annual costs decreased with later initiation of biologics , analgesics (<2%) and transportation (<1%). There was a substantial degree of missing values, with 33% of patients missing at least one follow-up visit. However, sensitivity analyses found the models to be robust. In addition, one may question the impact memory bias In our statistical analyses, the multiple imputation method we used to address the problem of missing data in the sensitivity analyses may have resulted in bias. We undertook log transformations of costs per period prior to implementing the multiple imputation in order to approach normality and obtain exclusively positive imputed costs following back transformation. Although some experts recommend such transformations, Our analysis suggests that early use of biologics may reduce other healthcare costs by slowing the evolution of the disease. We do not know the extent to which the high direct costs incurred from early use of biologics costs may be balanced in part by other health care cost offsets due to the reduction in the need for surgery or other interventions beyond the fourth year of the disease. Moreover, recent price reductions for biologics could make them more cost-effective,