We analyzed changes in total costs for National Institutes of Health (NIH) awarded Research Project Grants (RPGs) issued from fiscal years (FYs) 1998 to 2021 . Costs are measured in ‘nominal’ terms, meaning exactly as stated, or in ‘real’ terms, meaning after adjustment for inflation. The NIH uses a data-driven price index – the Biomedical Research and Development Price Index (BRDPI) – to account for inflation, enabling assessment of changes in real  costs over time. The BRDPI was higher than the general inflation rate from FY1998 until FY2012; since then the BRDPI has been similar to the general inflation rate likely due to caps on senior faculty salary support. Despite increases in nominal costs, recent years have seen increases in the absolute numbers of RPG and R01 awards. Real average and median RPG costs increased during the NIH-doubling (FY1998 to FY2003), decreased after the doubling and have remained relatively stable since. Of note, though, the degree of variation of RPG costs has changed over time, with more marked extremes observed on both higher and lower levels of cost. On both ends of the cost spectrum, the agency is funding a greater proportion of solicited projects, with nearly half of RPG money going toward solicited projects. After adjusting for confounders, we find no independent association of time with BRDPI-adjusted costs; in other words, changes in real costs are largely explained by changes in the composition of the NIH-grant portfolio. Inflation, defined by the United States Federal Reserve as ‘the increase in the prices of goods and services over time’ , has beeTo comprehend the nature of grant costs and trends, we define the following terms:Nominal costs: These are costs exactly as stated. Thus, the nominal total cost of a particular grant in FY2021 might be $450,000, meaning that the amounts listed in financial transactions or grant notices would add up to $450,000.Real costs: These are costs taking into account inflationary changes over time. Because of inflation $450,000 in FY2002 would have more purchasing power, that is could acquire more goods and services, than $450,000 in FY2021. Real costs are indexed against a specific year so that a comparison of real costs between two different years would reflect a comparison of purchasing power, not a comparison of costs as stated. We can think of real costs as enabling us to make ‘apples to apples’ comparisons in costs over different time periods.Logarithm-transformed costs: Grant costs followed a highly right-skewed distribution. We therefore sometimes transform costs to a base log of 10 in order to make distributions more symmetrical and less influenced by extreme values .The National Institutes of Health (NIH) issues different type of research and training awards, but by far the most common type is the “Research Project Grant (RPG)” accountiBackground inflation: Increases in prices across the economy due to increases in the money supply and/or economy-wide demand and supply stresses; these are reflected in general price indices, such as the GDP price index  and the Research-specific inflation: Increases in prices in the biomedical research and development enterprise; these are reported as the Biomedical Research and Development Price Index (or BRDPI).  The BRDPChanges in agency purchasing decisions : We might imagine an automobile-rental firm that starts one year purchasing 10 mid-size sedans. The following year, it might choose to purchase instead 10 luxury mid-size sedans; costs increase not because of background inflation because of the firm’s decisions about what it wants to buy. Alternatively, the firm may purchase two large vans, four mid-sized sedans, and four compact cars. Overall and median costs might not change (compared to the baseline of 10 mid-size sedans), but the firm’s management will be acutely aware of the costs of the two large vans. Similarly, NIH Institutes and Centers (IC’s) may choose to ‘puchase’ investigator-initiated R01 awards, R01 awards that cost more  because of use of large animals, or different size awards .We report on the distribution of nominal and inflation-adjusted prices of funded NIH RPGs since FY1998, the year that the NIH budget doubling began. We find that median and mean inflation-adjusted RPG costs have been largely stable since the doubling ended in FY2003, but that there have been changes in the distribution (variance) of costs, which largely reflect compositional effects as agency priorities have shifted over time.Most of this report will focus on real  costs of NIH RPG awards, that is total costs per RPG indexed for the FY2021 BRDPI. For context, between FY1998 and FY2021, NIH issued 827,815 RPG awards of at least $25,000 per year (BRDPI-indexed to FY2021). The number of RPGs and Principal Investigators supported on RPGs increased during the NIH budget doubling (from FY1998 to FY2003), decreased gradually between the end of the doubling and FY2015, and increased again with recent NIH budget increases , panel AMean and median real (that is FY2021 BRDPI-adjusted) total costs of RPGs increased during the doubling of FY1998-FY2003 , fell to a nadir of about $520,000 in FY2013 (the year of budget sequestration), and after a quick rebound in FY2014 has remained relatively stable at about $570,000 since . Similarthat is real, values), and human-studies only projects (Over time there have been decreases in the proportion of unsolicited awards and program (’P’) grants, while there have been increases in the proportions of R21 or R03 grants, cooperative agreements, clinical trials (since reliable data were first collected in FY2008), highly expensive projects ; these showed means much greater than medians, consistent with highly skewed distributions. The whiskers are also quite long, consistent with fat-tailed distributions. We addressed skewness by log transforming BRDPI-adjusted total costs (TC), that is calculating , Panel B).We constructed box-plot distributions of FY2021 BRDPI-adjusted total cost per RPG over time , panel Aive ends , Panel BCareful inspection of both panels  reveals What might be behind the increasing extremes (higher and lower) over the past 10–15 years? In FY1998, the top centile of RPG awards received 8% of funding, rising to 12% FY2017; this 4% absolute difference means that an additional $850 million were awarded to approximately 350 grants. There was little change in the proportion of funding going to the top decile; thus the upper extreme seems to be driven by increases in funding going to the most expensive awards .Expensive awards might be linked to agency solicitations. Before FY2010 unsolicited RPGs had a central tendency towards greater costs, but since then solicited awards were more costly , panel A, panel A) and solicited  projects show variations in whisker ranges  with BRDPI-adjusted costs of RPG projects separate from those associated with funding mechanism, solicitation (or not), involvement of human participant or animal models, or type of recipient organization. We attempted multivariable linear regressions with log-10 transformed costs as the dependent variable , but upoThe rate of inflation for NIH-funded research (that is the BRDPI) was higher than the general rate of inflation from FY1998 until FY2012; since then, the rate of inflation for NIH-funded research has been similar to the general rate of inflation. The BRDPI is determined via a sophisticated methodology; since 2005 the Bureau of Economic Analysis (BEA) uses a Fisher chain-weighted indexed methodology which is analogous to calculating compound growth on retirement portfolios over many years as the mix of stocks and bonds changes from year to year. The decrease in the BRDPI in FY2012 was likely related to an NIH-imposed salary cap ‘freeze’ in 2011. In 2012, the NIH has linked the salary cap to Executive Level II (instead of the higher Executive Level I) salaries. Since then, salary caps continue to linked to Executive Level II levels and have increased at the rate of Federal civilian salaries, which likely have risen a rate lower than academic salaries. The cap reductions in FY2011, the relatively slow rate of rise of Federal salaries which determine the NIH salary cap, along with relatively low increases in fellowship and training stipends have combined to reduce the BRDPI since FY2011 . Institunot nominal, values), and more clinical trials. The top centile of projects are receiving a substantially greater share of the overall RPG funding pool. On the lower end of cost, over time the agency has been funding more low-cost mechanism awards (R03 and R21). On both ends of the cost spectrum, the agency is funding a greater proportion of solicited projects, with nearly half of RPG money going towards solicited projects. These compositional changes likely reflect evolving priorities articulated in NIH strategic planning documents. average and median RPG costs increased during the NIH-doubling (FY1998 to FY2003), decreased after the doubling and have remained relatively stable since. Of note, though, the degree of variation of RPG costs has changed over time, with more marked extremes observed on both higher and lower levels of cost. On the higher end, over time NIH has been funding more cooperative agreements, more projects exceeding $5 million  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, including Clifford J Rosen as Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Mark Peifer (Reviewer #2).Thank you for submitting your article "National Institutes of Health Research Project Grant Inflation 1998 to 2021" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.All the reviewers agree this is a timely manuscript. But there were several issues that need further delineation and clarification. These can be summarized below; more detail about concerns are noted in the three reviews.Essential revisions:1) Clarification in the text to help the reader understand some of the terminology and methodology, rather than just a series of graphs that dominate the manuscript.2) There needs to be a clearer delineation of solicited grants and what their implications are compared to RO1s, particularly because the median grant cost is now much higher due to these grants.3) Since personnel costs dominate NIH investigator grants the issue of salary caps and the implication for overall support of investigator needs more discussion.4) Can the authors be more granular about the analysis relative to the majority of grants that are in the 250,000$ range and are well below the median level of 500k. In other words, are the results relevant for most RO1 investigators who continue to struggle with costs due to personnel and salary caps.Reviewer #1 (Recommendations for the authors):The authors provide a very comprehensive picture through modeling and graphic presentation of the relative pace at which NIH is able to maintain support for RPGs consistent with inflationary trajectories. Overall the paper is fairly well written, and the figures are illustrative, although some should probably be as supplemental so as not to overwhelm the reader.The overall message is reassuring to investigators and institutions receiving NIH support.There are several areas that require further elaboration:1 – The abstract should be clearer in respect to methodology and overall message.2 – What are the implications of larger funded, more clinically oriented projects relative to the fate of RO1s; this is paramount to investigators- particularly basic scientists.3 – Personnel costs consume much of the budgetary costs of RPGs. Are your models able to capture that and if not, how does that affect the projections?4 – Are indirect costs also keeping up with inflationary pressures? It might be embedded in the data but not clear to the reader.5 – With the huge increase in inflation within the past year, readers would want to know if your modeling can predict how that might project for the future.6 – Can the authors provide more detail on solicited projects since that seems to be consuming a greater proportion of the budgetary outlay?7 – If NIH is supporting larger projects that are solicited, is there evidence that this support translates into greater productivity.Reviewer #3 (Recommendations for the authors):These comments are more "food for thought" for the research team.This paper has been written at a very interesting time. Up until now, inflation has not been a problem for the US economy nor for NIH research. In fact, real funding per NIH project has fallen by about 10%. I find it both ironic and premature for the paper to be discussing Baumol's cost disease when prices per project are dropping .For me, the issue is related to the salary cap. Salaries are the largest component of research costs, and NIH has maintained a cap that essentially has controlled prices, so it follows that the changes in NIH costs are going to be driven by composition effects. NIH effectively controlled labor costs from 2012 to present. The overall labor shortage in the US economy will ultimately have an impact on the cost of research funding given the reports of people being unwilling to take postdoctoral positions (see https://www.science.org/doi/pdf/10.1126/science.add6184 and https://www.statnews.com/2022/11/10/tipping-point-is-coming-unprecedented-exodus-of-young-life-scientists-shaking-up-academia/). I raised this issue in the public comments, and I would encourage the authors to address it head-on: the cost of doing research is going to increase rapidly in the next few years as outside opportunities to have a permanent job in industry paying twice the rate of an NIH postdoc abound.This begs two questions: (A) What are the unintended consequences of this kind of these salary caps? Previous work by this research team has shown decreasing productivity of NIH funding . Could this be the result of the reduction in the real value of each NIH grant? (B) NIH is going to have to raise the stipends for NSRA postdocs. The research team should mention that this will be likely in the future. Given the demand for skilled workers and cooling relations with China, fewer students will be going to graduate school and/or taking postdocs because the opportunity cost of foregone earnings has increased significantly. While we don't have a lot of evidence of Baumol's cost disease now, I bet five years from now that will have most certainly changed. Essential revisions:1) Clarification in the text to help the reader understand some of the terminology and methodology, rather than just a series of graphs that dominate the manuscript.The paper has been extensively rewritten, streamlined, and clarified, largely following the advice of the reviewers. See italicized text.2) There needs to be a clearer delineation of solicited grants and what their implications are compared to RO1s, particularly because the median grant cost is now much higher due to these grants.We have added material on solicited grants and R01-equivalent awards. See, for example, new Tables 2 and 4, and new Figure 4.3) Since personnel costs dominate NIH investigator grants the issue of salary caps and the implication for overall support of investigator needs more discussion.We have added new text on salary caps in the Discussion. See italicized text.4) Can the authors be more granular about the analysis relative to the majority of grants that are in the 250,000$ range and are well below the median level of 500k. In other words, are the results relevant for most RO1 investigators who continue to struggle with costs due to personnel and salary caps.We have added new material on R01-equivalent awards, including on the 250K and 500K levels. See new Tables 2 and 4.Reviewer #1 (Recommendations for the authors):The authors provide a very comprehensive picture through modeling and graphic presentation of the relative pace at which NIH is able to maintain support for RPGs consistent with inflationary trajectories. Overall the paper is fairly well written, and the figures are illustrative, although some should probably be as supplemental so as not to overwhelm the reader.The overall message is reassuring to investigators and institutions receiving NIH support.There are several areas that require further elaboration:1 – The abstract should be clearer in respect to methodology and overall message.We agree with the reviewer and revised the abstract accordingly. See italicized text.2 – What are the implications of larger funded, more clinically oriented projects relative to the fate of RO1s; this is paramount to investigators- particularly basic scientists.We agree with the question. We provide additional data on R01-equivalent awards. See two new tables (Table 2 and Table 4) and one new Figure  along with italicized text .3 – Personnel costs consume much of the budgetary costs of RPGs. Are your models able to capture that and if not, how does that affect the projections?We agree with the question. We have added text in the discussion (including limitations section) on personnel costs, including noting that our data are not as comprehensive as those of others (lines 265-267).4 – Are indirect costs also keeping up with inflationary pressures? It might be embedded in the data but not clear to the reader.We agree with the question. We provide additional data on indirect costs. See new Figure 3.5 – With the huge increase in inflation within the past year, readers would want to know if your modeling can predict how that might project for the future.Our paper is not designed to be a forecasting effort, but we have added displays for projected inflation indices in Figure 1, panel B.6 – Can the authors provide more detail on solicited projects since that seems to be consuming a greater proportion of the budgetary outlay?We agree with the question. We have provided more details on solicited and unsolicited projects in Tables 3 and 4. Table 4 is a wholly new Table on R01-equivalent awards.7 – If NIH is supporting larger projects that are solicited, is there evidence that this support translates into greater productivity.This is a good question, but beyond the scope of this paper. See italicized text (lines 269-271).Reviewer #3 (Recommendations for the authors):These comments are more "food for thought" for the research team.This paper has been written at a very interesting time. Up until now, inflation has not been a problem for the US economy nor for NIH research. In fact, real funding per NIH project has fallen by about 10%. I find it both ironic and premature for the paper to be discussing Baumol's cost disease when prices per project are dropping .For me, the issue is related to the salary cap. Salaries are the largest component of research costs, and NIH has maintained a cap that essentially has controlled prices, so it follows that the changes in NIH costs are going to be driven by composition effects. NIH effectively controlled labor costs from 2012 to present. The overall labor shortage in the US economy will ultimately have an impact on the cost of research funding given the reports of people being unwilling to take postdoctoral positions (see https://www.science.org/doi/pdf/10.1126/science.add6184 and https://www.statnews.com/2022/11/10/tipping-point-is-coming-unprecedented-exodus-of-young-life-scientists-shaking-up-academia/). I raised this issue in the public comments, and I would encourage the authors to address it head-on: the cost of doing research is going to increase rapidly in the next few years as outside opportunities to have a permanent job in industry paying twice the rate of an NIH postdoc abound.This begs two questions: (A) What are the unintended consequences of this kind of these salary caps? Previous work by this research team has shown decreasing productivity of NIH funding . Could this be the result of the reduction in the real value of each NIH grant? (B) NIH is going to have to raise the stipends for NSRA postdocs. The research team should mention that this will be likely in the future. Given the demand for skilled workers and cooling relations with China, fewer students will be going to graduate school and/or taking postdocs because the opportunity cost of foregone earnings has increased significantly. While we don't have a lot of evidence of Baumol's cost disease now, I bet five years from now that will have most certainly changed.We agree with the reviewer. See new italicized text in the discussion .


Infectious bronchitis virus (IBV) has evolved through various mutation mechanisms, including antigenic drift and recombination. Four genotypic lineages of IBVs including GI-15, GI-16, GI-19, and GVI-1 have been reported in Korea. In this study, we isolated two IBVs from chicken farms, designated IBV/Korea/289/2019 (K289/19) and IBV/Korea/163/2021 (K163/21), which are two distinct natural recombinant viruses most likely produced by genetic reassortment between the S1 gene of K40/09 strain (GI-19 lineage) and IBV/Korea/48/2020 (GI-15 lineage) in co-infected commercial chickens. Comparative sequence analysis of hypervariable regions (HVRs) revealed that the K289/19 virus had similar HVR I and II with the K40/09 virus , and HVR III with the IBV/Korea/48/2020 virus (100% nucleotide sequence identity). In contrast, the K163/21 virus had HVR I and II similar to the IBV/Korea/48/2020 virus , and HVR III to the K40/09 virus (96.6% nucleotide sequence identity). The K289/19 virus exhibited similar histopathologic lesions, tissue tropism in trachea and kidney, and antigenicity with the parental K40/09 virus. The K163/21 exhibited similar pathogenicity and tissue tropism with the K40/09 virus, which were similar results with the isolate K289/19. However, it showed a lower antigenic relatedness with both parental strains, exhibiting R-value of 25 and 42, respectively. The continued emergence of the novel reassortant IBVs suggests that multiple recombination events have occurred between different genotypes within Korea. These results suggest that antigenic profiles could be altered through natural recombination in the field, complicating the antigenic match of vaccine strains to field strains. Enhanced surveillance and research into the characteristics of newly emerging IBVs should be carried out to establish effective countermeasures. Infectious bronchitis virus (IBV) is a pathogen associated with acute respiratory tract diseases in chickens. The IBV is highly contagious and can also affect multiple organs according to their pathotypes, such as the respiratory tract, kidneys, and reproductive tract. Chickens infected with IBV show respiratory signs, reduced egg production, weight loss, and decreased weight gain. Mortality may vary depending on the IBV strain, secondary bacterial infection, or coinfection with other viruses. It is economically important to control this infection because of its detrimental effects on poultry production , 2.High antigenic diversity of IBVs and the extensive emergence of variants are problematic, resulting in poor cross-protection by the available vaccine strains –5. The sAccording to the classification system of IBV lineages defined by Valestro et al. , IBVs isIn this study, we isolated two novel IBVs which are natural recombinant between the GI-15 and GI-19 lineages. We investigated their pathobiological characteristics, such as pathogenicity, antigenicity, and tissue tropism in chickens.50)  and IBV/Korea/48/2020 (B4-like subgroup of GI-15) viruses were isolated from chickens in Korea , 21. The50) .® 2022.1.1 software (https://www.geneious.com/). A phylogenetic tree was constructed in MEGA version 10.2.5, using the neighbor-joining method with 1000 bootstrap replicates.Viral RNA was extracted from harvested allantoic fluid using the RNeasy Mini Kit  according to the manufacturer's instructions. The S1 gene of each IBV isolate was amplified using a OneStep RT-PCR Kit (Qiagen) according to the manufacturer's instructions and using two pairs of primers . The reaction was conducted in a ProFlex PCR System  at 50°C for 30 min; 95°C for 15 min, and 35 cycles of 94°C for 60 s, 53°C for 60 s, 72°C for 120 s, and a final extension step at 72°C for 7 min. The PCR products were purified using a GeneJET Gel Extraction Kit  and sequenced using Sanger sequencing . The obtained nucleotide sequences of the S1 gene were assembled and aligned with the prototype of different lineages of IBV and reference strains  shown inp-value < 1 × 10−14 were accepted (The Recombination Detection Program (RDP) 4 software (v. 4.39) was used to identify putative recombination events using several detection methods: RDP, GENECONV, BootScan, MaxChi, Chimera, SiScan, Phylpro, LARD, and 3Seq . Recombiaccepted . After iaccepted .n = 150) were randomly divided into five groups  and maintained separately in isolation cabinets . All chickens used in this study were obtained from Namduk SPF . Chickens in the treated group were inoculated with a virus with a 105 EID50 per chick via the ocular route. Chickens from the negative control group were inoculated with sterile phosphate-buffered saline (PBS) for the same volume as the virus inoculated groups. At 5 days post-inoculation (5 dpi), twenty chicks were sacrificed from each group. The upper, middle, and lower regions of tracheal and kidney tissues were collected from ten necropsied chickens from each group for histopathological examination. Ciliary loss, inflammatory response of the trachea, and severity of kidney inflammation were scored as previously described . Inactivated allantoic fluids were emulsified with Montanid ISA70  at ratio of 3:7 (v/v) to be injected into SPF chickens to produce antisera .n = 20) were randomly separated into four groups and each oil-emulsified inactivated virus (0.5 mL) was intramuscularly inoculated into each group. Two weeks after inoculation, all groups were given a booster of the same inactivated virus. Two weeks after boosting, hyperimmune antisera were collected from each group and inactivated at 56°C for 30 min. Virus cross-neutralization tests were performed using SPF embryonated eggs with the Alpha method according to the OIE Terrestrial Manual .The ciliary loss and inflammatory response scores of test groups were compared with the score of negative group using Dunnett's multiple comparisons test. Re-isolation rate of the inoculated virus among the groups was analyzed using one-tailed Fisher's exact test. Statistical significance was set at The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Konkuk University of South Korea .Phylogenetic analysis of the S1 gene revealed that the K289/19 and K163/21 viruses were genetically distinct from previously identified IBVs in South Korea. These viruses did not belong to the QX-like, KM91-like, or K40/09-like and GI-15 subgroups in the phylogeny .−14) (p < 1 × 10−14 at least five detection methods).Recombination events in K289/19 and K163/21 were identified using RDP4 software . Five di−14) . Other pThe K40/09 strain is a recombinant strain derived from QXIBV and KM91  Figure . Single The nucleotide sequence identity of the S1 gene of each recombinant was compared with the GI-15 and GI-19 IBVs identified previously in Korea. Nucleotide sequence identities of K289/19 were 81.9–87.8% with the GI-15 IBVs, and 85.9–91.6% with the GI-19 IBVs. Nucleotide sequence identities of K163/21 were 84.6–91.6% with the GI-15 IBVs, and the 78.7–86.8% with GI-19 IBVs (data not shown). Sequence analysis of the HVR I, II, and III in the S1 gene showed that the HVR I and II of K289/19 were similar to the K40/09 strain, except one substitution at 120th amino acid (S120R), but HVR III was similar to the IBV/Korea/48/2020 strain . In contMortality was not observed in the K163/21 challenged group or the negative control group . The chip > 0.05). Likewise, for the ciliary loss score of middle trachea, the scores of inoculated groups were higher than that of the negative control group, but there was no significant difference compared with negative control group. However, for the ciliary loss score of lower trachea, the score of K289/19 challenged group was the highest (p<0.05), followed by the K40/09 challenged group and the IBV/Korea/48/2020 challenged group. The K163/21 had a slightly higher score compared to the negative control group. Based on the results of the scoring of ciliary loss, it seems that the K289/19 was more pathogenic than its parental strains, but the K163/21 was less pathogenic than its parental strains.Histopathological examination  and viruP < 0.05). Chickens inoculated with each virus showed higher inflammation score than the negative control group in the middle trachea, but there was no statistically significant difference (P > 0.05). For the inflammation score in the lower trachea, the score of K289/19 challenged group was only significantly higher than the negative control group (P < 0.001). Collectively, all inoculated viruses were identified to be pathogenic in trachea, although virulence of each virus was slightly different. The inflammation score in kidney tissues of IBV/Korea/48/2020 challenged group was similar to the negative control group. However, the K40/09 group and two novel recombinant viruses showed statistically higher inflammation scores in kidney compared to the negative control group (P < 0.05).All the groups inoculated with each virus showed significantly higher inflammation score than the negative control group in the upper trachea (Similar results were also identified from the virus re-isolation test . The posCross-neutralization tests were performed using the recombinant viruses and their putative parental strains. NI values retrieved from the cross-neutralization tests were converted to R-values using the calculation method described by Archetti and Horsfall . R-valueThe IBV evolves through a variety of mutation mechanisms, not only accumulations of point mutations but also recombination events, which makes it difficult to perfectly prevent the infection of IBVs –5. In paThe HVRs of S1 gene are highly diverse and often associated with antigenicity, pathogenicity, and tissue tropism , 39. In Live attenuated vaccines derived from GI-15 and GI-19-like viruses have been used in Korea , 43. TheThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by Konkuk University IACUC.C-SS: conceptualization, validation, and project administration. D-HL, H-JK, H-CL, and AC: methodology. H-JK, Y-JC, and HL: formal analysis. D-HL, H-JK, Y-JC, and HL: investigation. H-JK and H-CL: resources. H-JK and AC: data curation. H-JK: writing—original draft preparation and visualization. D-HL, H-CL, AC, and C-SS: writing—review and editing. D-HL and C-SS: supervision and funding acquisition. All authors have read and agreed to the published version of the manuscript.


Tymoviridae, was isolated from mosquitoes (Mansonia spp.) in clone C6/36 cells, and its complete genome was sequenced. Its genome is 6494 nt in size with an organization resembling that of tymovirids. The isolated virus is phylogenetically related to two viruses isolated from Culex spp. mosquitoes: Ek Balam virus, reported in Mexico, and Culex-originated Tymoviridae-like virus, isolated in China. The results of this study suggest that this virus is a new member of the family Tymoviridae.A new virus, named Mutum virus, related to members of the family The online version contains supplementary material available at 10.1007/s00705-022-05475-x. Tymoviridae is composed of three genera of viruses with positive-sense RNA genomes: Maculavirus, Marafivirus, and Tymovirus [Tymoviridae comprised only plant-infecting viruses; however, viruses related to members of this family have been isolated from mosquitoes in recent years, such as Culex-originated Tymoviridae-like virus (CuTLV) and the Ek Balam virus (EkBV) [The family ymovirus . Initials (EkBV) –3.Mansonia sp. mosquitoes collected in 2018 in the vicinity of the Jirau hydroelectric dams in Nova Mutum Paraná, a rural village in the municipality of Porto Velho in the state of Rondônia, Brazil . MUTV was isolated from four pools of female Mansonia mosquitoes in Aedes albopictus cells (C6/36) [Here, we report genome sequencing of another distinct tymo-like virus, Mutum virus (MUTV), named after the mosquito collection area. MUTV was detected in female  (C6/36) . The virFour MUTV genomes were sequenced on an Ion Torrent PGM platform  according to the manufacturer's recommendations. A phylogenetic tree was constructed using the maximum-likelihood (ML) method with 1000 bootstrap replicates  in the RThe sequences of four MUTV isolates have been deposited in the GenBank database with the following accession numbers: MUTV-BE-AR-855909 (MT656586), MUTV-BE-AR-855928 (MT656589), MUTV-BE-AR-855911 (MT656587), and MUTV-BE-AR-855922 (MT656588). The four MUTV isolates showed nucleotide sequence identity ranging from 99.9–100% and amino acid sequence identity of 100%, indicating that they are closely related isolates of the same virus. The MUTV genome is 6,494 nt in size, with 5′ and 3′ non-coding regions of 37 nt and 53 nt, respectively. The MUTV genome has three ORFs. ORF1 is 5,331 nt  in size and encodes a 1,776-aa-long protein with an estimated mass of 201,061 kDa that is involved in virus replication. ORF2 spans 732 nt  and codes for a 243-aa-long protein with an estimated mass of 26,134 kDa identified as a viral coat protein, while the 3’-proximal ORF 3 is 285 nt long  and codes for a 10,729-kDa protein of unknown function. The total readings and the coverage of the genome reads ranged from 55,312 to 139,811 and from 16.10x to 48.47x, respectively.Searches of the Interproscan databases revealed conserved protein domains for Vmethyltransf, peptidase, helicase 1, and RdRP in the product of ORF1 and a Tymo_coat motif in the ORF2-encoded protein , distinct from the plant-infecting members of the family dae Fig. . The resTymoviridae, is yet to be determined and may involve establishment of new genera to officially classify these viruses.In summary, here we describe a new mosquito-associated virus named Mutum virus that is related to other tymovirid-like insect specific viruses (ISVs). The viruses isolated from mosquitoes are currently unclassified. The final position of MUTV and other mosquito-infecting tymovirid-like viruses within the ICTV framework of virus classification, in particular within the family Below is the link to the electronic supplementary materialSupplementary Material 1


Autism spectrum disorders (ASD) are associated with high services use, but European data on costs are scarce.Utilisation and annual costs of 385 individuals with ASD  from German outpatient clinics were assessed.Client Service Receipt InventoryAverage annual costs per person were 3287 EUR, with psychiatric inpatient care (19.8%), pharmacotherapy (11.1%), and occupational therapy (11.1%) being the largest cost components. Females incurred higher costs than males (4864 EUR vs. 2936 EUR). In a regression model, female sex (Cost Ratio: 1.65), lower IQ (1.90), and Asperger syndrome (1.54) were associated with higher costs.In conclusion, ASD-related health costs are comparable to those of schizophrenia, thus underlining its public health relevance. Higher costs in females demand further research.No significant relationships.


Spontaneous intracerebral hemorrhage (ICH) entails significant mortality andmorbidity. Severely ill ICH patients are treated in intensive care units(ICUs), but data on 1-year healthcare costs and patient carecost-effectiveness are lacking.Retrospective multi-center study of 959 adult patients treated forspontaneous ICH from 2003 to 2013. The primary outcomes were 12-monthmortality or permanent disability, defined as being granted a permanentdisability allowance or pension by the Social Insurance Institution by 2016.Total healthcare costs were hospital, rehabilitation, and social securitycosts within 12 months. A multivariable linear regression of log transformedcost data, adjusting for case mix, was used to assess independent factorsassociated with costs.Twelve-month mortality was 45% and 51% of the survivors were disabled at theend of follow-up. The mean 12-month total cost was €49,754, of whichrehabilitation, tertiary hospital and social security costs accounted for45%, 39%, and 16%, respectively. The highest effective cost per independentsurvivor (ECPIS) was noted among patients aged >70 years with brainstemICHs, low Glasgow Coma Scale (GCS) scores, larger hematoma volumes,intraventricular hemorrhages, and ICH scores of 3. In multivariableanalysis, age, GCS score, and severity of illness were associatedindependently with 1-year healthcare costs.Costs associated with ICHs vary between patient groups, and the ECPIS appearshighest among patients older than 70 years and those with brainstem ICHs andhigher ICH scores. One-third of financial resources were used for patientswith favorable outcomes. Further detailed cost-analysis studies for patientswith an ICH are required. Despite attempts to find effective interventions, treatment options remainlimited. Consequently, patient prognosis has not improved remarkably during the lastdecade.3The one-year mortality rate is 40% to 50%, with a high proportion of survivorsremaining severely disabled.3The current AHA/ASA guidelines suggest initial treatment at dedicated neurologicalintensive care units (ICU) or stroke units, which places a significant economicburden on society, as care provided in these is expensive. Empirical evidence for intensive care’s cost-effectiveness in this patientgroup, however, remains unclear.Spontaneous (non-traumatic) intracerebral hemorrhage (ICH) accounts for approximately10% to 15% of all strokes but has disproportionately high mortality. which is also persistently implicated as a predictor of a poorprognosis.7With an aging population, the number of patients affected and severely disabled byan ICH is likely to rise in the near future, leading to increasing treatmentcosts.810 Therefore, identification ofthose patients most likely to benefit from ICU care is needed to optimize allocationof health care funds. However, the epidemiological evidence on such patientcharacteristics remains scarce. In our previous study of the treatment cost-effectiveness of patients treatedin the ICU following traumatic brain injury, subarachnoid hemorrhage, ischemicstroke, and hypoxic brain injury, spontaneous ICH was associated with the highesteffective costs per independent survivor, costing approximately €180,000 altogether. This finding prompted a more detailed analysis of the factors driving 1-yearhealthcare costs and cost-effectiveness in patients with spontaneous ICH requiringICU treatment.ICH incidence increases with age, we extracted the records of adult patients (aged ⩾18) treated forspontaneous ICH in the five university hospital ICUs from January 1, 2003through December 31, 2013. Patients with spontaneous ICH were identified basedon their Acute Physiology and Chronic Health Evaluation III (APACHE II)diagnosis, and three of the authors, blinded to knowledge of the patient’soutcome, confirmed the diagnosis by manually reviewing the patients’ admissionhead computed tomography (CT) scans. If a patient had several entries in the FICC database due to ICH, weincluded the first admission. We excluded patients with isolatedintraventricular hemorrhage (IVH) and incomplete data  database that has been collectingdata prospectively since 1994,ete data .15 were extracted from the FICC database. Three authors evaluated the hematoma volume (obtained by the ABC/2 method), the location, the extension to the ventricular system, and the midlineshift from the admission head CT scan. The bleeding location was defined assupratentorial superficial, supratentorial deep, cerebellar, or brainstem. Wealso calculated the admission ICH score for each patient to stratify the risk of death. This score is based on radiological and clinical data, with pointsranging from 0 to 6. Thirty-day mortality for those with the lowest score ispredicted to be 0% and 100% for those with scores of 5 to 6.The data on demographic and clinical characteristics, treatment and intensivecare scoring system scores (Simplified Acute Physiology Score II [SAPS II] andAPACHE II)The primary outcome was death or permanent disability at 12 months. We obtainedthe date of death from the Finnish population register  up to the end of 2016. We used a surrogate marker for permanentdisability if the patient was granted a permanent disability allowance orpension by the Social Insurance Institution in Finland by September 30, 2016. Ifthe patient did not receive disability allowance, we assumed that the patientwas independent in their daily activities. All Finnish citizens with adisability or long-term illness, regardless of personal insurance status, areentitled to a disability allowance from Kela. The criteria for granting apermanent disability allowance or pension are being unable to independentlycarry out activities of daily living or return to work due to sustained disability.:We adjusted all costs for inflation, according to the Finnish Consumer PriceIndex (CPI), to 2021 euros, as follows  The social security costs were extracted from the Kelas database andencompassed the costs of disability and sickness allowances, disabilitypensions, prescribed medication, travel expenses in connection to treatment, andexpenses for private health care (physician and physiotherapist appointments).We assessed cost-effectiveness by dividing the total 12-month cost for allpatients by the number of 12-month survivors and independent survivors to definethe effective cost per survivor (ECPS) and per independent survivor (ECPIS).We examined total 12-month costs by assessing the university hospital,rehabilitation, and social security costs for 12 months after the admission. Thehospital costs were obtained from the hospital billing departments and includedexpenses for ICU and ward stays, diagnostics , treatment received (including operations), and personnel. Therehabilitation costs were based on a report from the Finnish National Institutefor Health and Welfare (THL) and determined according to the average cost of oneward day multiplied by the duration of the rehabilitation. As the prognoses of cerebellar and brainstem ICHs are vastly different wealso considered bleeding locations in smaller subgroups . In addition, we also report results according to age quintiles .Additionally, we report outcomes separately according to SAPS II score quartiles.To identify patient characteristics associated with outcomes and costs, werepeated all outcome and cost analyzes separately for pre-defined outcomepredictors, according to the ICH score and its components.χ2) test. We tested the continuousdata for the assumption of normality, and as the data were highly skewed, weemployed the non-parametric Mann-Whitney U test or theindependent t-test to compare subgroups as appropriate. Theoutcomes are presented as numbers and percentages for the categorical variables,means with 95% confidence intervals (CI) for the cost data, and medians withinterquartile ranges (IQR) for all the other continuous variables. For thesurvival data, we also report 95% CIs. We considered p-valuesbelow 0.05 to indicate statistical significance.We used SPSS Statistics 27.0 for Mac OS  and StataStatistical software for Mac OS  for allthe statistical analyzes. We assessed differences in categorical subgroups withthe Chi-squared . Separate models were built for the supratentorial andthe infratentorial ICHs, and we correspondingly performed a subanalysis ofhospital survivors only.Further, we assessed factors independently associated with increasing costs bylinear regression, applying log transformation of the cost data to make the dataconform to normality. We present the exponentiated regression coefficients ascost ratios. which for supratentorial ICH included the following: age, the GlasgowComa Scale (GCS) score , the presence of a severe chronic comorbidity (based on the SAPS II andAPACHE II definitions),15 the modified SAPS II score , presence of IVH, the hematoma volume, and the midline shift. Theinfratentorial ICH severity of illness model was alike, except for including thehematoma location (cerebellar or brainstem) and excluding age, the midlineshift, and hematoma volume. We subsequently applied the same technique to study hospital survivorsonly.We also plotted the total costs against severity of illness. The severity ofillness models were built in accordance with our previous study on ICH long-termoutcome predictors,Supplemental Material: Methods I).The methodological appraisal followed the Consolidated Health Economic EvaluationReporting Standards (CHEERS) 2022 statement . The medianpatient age was 62 years, and most of the patients were independent in dailyliving prior to hospital admission . Most of thepatients underwent mechanical ventilation during their ICU stay. The infratentorial ICH patients had smallerhematoma volumes but were more severely ill during the first day of hospitaladmission, according to their SAPS II scores, and were more likely to undergomechanical ventilation or external ventricular drainage during their ICU stay.There were no major differences in age, severe comorbidities, treatmentintensity, or length of ICU or hospital stay between the bleeding types. Moredetailed patient baseline characteristics are shown in Altogether, 959 patients met the study inclusion criteria . Most ofp = 0.003) by ICH location: the proportion ofpatients alive and without permanently disability at 12 months was 17% forbrainstem (n = 12), 22% for deep supratentorial(n = 74), 28% for cerebellar (n = 28), and32% for superficial supratentorial (n = 144) ICHs.The unadjusted outcomes for the different subgroups are shown in n = 258). Supplementary Table 1 contain more details.The mean 12-month total cost per patient was €49,754, of which rehabilitationcosts accounted for 45%, tertiary hospital costs for 39%, and social securitycosts for 16%. All costs were higher for the supratentorial ICH patients .The mean total costs were highest for the superficial supratentorial ICHs,followed by the cerebellar, deep supratentorial and brainstem ICHs . The total costs decreased withage: the total cost was €63,607 for those aged 18–48 years compared to €37,275for those aged >70 years. Furthermore, the mean total costs were lower forthose with severe ICH compared to those with less severe ICH . Of all the summed costs,32% was spent on patients with a favorable outcome at 12 months . When considering supratentorial ICH hospitalsurvivors only, higher age was associated with lower costs, whereas GCS 5–12,ICH volume ⩾30 ml and higher SAPS II score were associated with higher costs .In the multivariable analyzes , deep ICmean = 2.62). When considering infratentorial ICH hospitalsurvivors only, a lower GCS score remained as a significant independentcontributor to high costs. Further, age higher than 70 years as associated withlower costs .The significant independent contributors to costs differed between thesupratentorial and the infratentorial ICHs. For the infratentorial ICHs, a lowerGCS score was associated with higher costs, and a higher SAPS II score wasassociated with lower 12-month total costs  curves in This multicenter study demonstrated that spontaneous ICHs treated in the ICU wereassociated with substantial 1-year healthcare costs and, in many cases, pooroutcomes. Most of the 1-year healthcare costs were associated with rehabilitation,and costs varied substantially between the different patient groups. The ECPIS washighest among the patients with older age, brainstem ICH, and more severe illness.The patient characteristics independently associated with costs also appeared todiffer somewhat between the supratentorial and the infratentorial ICHs. It remainsto be seen whether better triage and a focus on the appropriate patient groups couldimprove cost-effectiveness. However, ICH is also associated with a high rate of lifelong disability,which creates substantial economic expenses due to productivity losses. Furthermore, although recovery is more rapid in the first few weeks, it maycontinue for many months after ICH. Therefore, assessing outcomes and costs from alonger time perspective would provide a more comprehensive estimate of thecost-effectiveness and the total economic burden of ICH on society. In our population, 51% of the patients who survived for 12 months were leftseverely disabled, and only 27% of all the patients had a favorable outcome at12 months. Our findings are in line with previous studies, as van Asch et alreported in a systematic review that the independence rate after ICH was 12–39%.Previous studies have assessed the hospital-wide costs and mortality of spontaneousICH, but data on a selected ICU cohort remain scarce. In a retrospective study onICU-treated intracranial hemorrhage, Fernando et al. found the mean total costs forthe hospital stay to be $53,491  for ICH. However, as only one-third of all costs was allocated to the patients with afavorable functional outcome, the resource allocation may not be optimal, andfurther studies on optimizing resource use are needed. Our previous study reportedsimilar estimates with a mean total 12-month cost of €47,661 for ICH (costs shown ineuros at the 2013 rate). However, compared to other critical illnesses, the mean total 12-month costof ICH appears high.27We observed a mean total 12-month cost of €49,754, of which rehabilitation costsformed the biggest part. As we found the total costs to be substantially lower forthose with a favorable outcome compared to those permanently disabled at 12 months, this emphasizes the importance and efficacy of investment inrehabilitation to improve and maintain functional ability, which is also in linewith the current ICH guidelines. Furthermore, some previous studies have shown that approximately half of thedeaths occur within the first two days.28 Due to the devastatingprognosis of brainstem ICH, many patients require only a short ICU and hospital stayand receive fewer investigations and treatments before their inevitable death,leading to lower average total costs compared to other ICH subgroups. Interestingly,age over 70 was also associated with lower total costs, in line with the findings byFernando et al. This could be explained in two ways. First, do not attempt resuscitation(DNAR) orders are significantly more common among older patients compared to youngerage groups. Therefore, the oldest patients in the cohort likely had a favorableprognosis, as patients with poor prognoses may not have been referred to ICUs due tothe presumed futility of care. Also, as previous studies have showed, early DNARorders are a proxy of less active management and therefore might haveself-fulfilling prophecies of poor outcomes and, accordingly, lower total long-termcosts.30 In line with this hypothesis, we found the unadjusted indexhospital costs and the ECPS to be lower among the older subgroups although mortalityrates were similar to that of the younger age groups, indicating that the costs werelower because aggressive treatment was withheld. Further, elderly patients with ICHadmitted to the ICU probably represent a very selected cohort of patients that havebeen admitted with a perceived high likelihood of recovery. Therefore, age-relatedcosts should not determine the selection of patients for the ICU but should beconsidered when assessing total health care spending for ICH.We found the lowest mean costs for the patients with a brainstem ICH, an ICH volumeof >30 ml, an ICH score of 4–5, and a high SAPS II score (quantile 4), which allindicated severe ICH. As our previous study on ICH long-term outcome predictorsshowed, brainstem ICH was a strong predictor of mortality and was associated with asharp drop in survival immediately after the insult. Furthermore, the mean costs were highest among the young (ages 18–48 years)among all bleeding locations, although for infratentorial ICH the associationbetween costs and age was statistically significant when considering hospitalsurvivors only. Together with a greater likelihood of an active treatment plandespite the severity of the illness discussed above, the expenses caused by alifelong disability are higher among younger subgroups, which explains theage-related cost difference.Among all the subgroups, we found the mean costs to be highest among those with a GCSof 5–12, no IVH, and an ICH score of 2, indicating that most resources are spent onsubgroups with a moderate prognosis. Among these subgroups, recovery tends torequire longer ICU and hospital stays, increased numbers of hospital interventions,and more intensive rehabilitation compared to those with a milder disease picture,hence leading to higher total costs.We also found that the ECPS and the ECPIS were highest for the patients with a GCSscore 3–4, an ICH volume of >30 ml, an ICH score of 3, and a high SAPS II score(quantile 4). As these subgroups are associated with poor functional outcome despitehigh costs, it makes the cost-effectiveness of these subgroups questionable, whichshould be considered in resource allocation. Although a low GCS score associated with any location is a marker of poorprognosis, a patient with cerebellar ICH and impaired consciousness is more likelyto undergo hematoma evacuation, which increases both survival and costs. In contrast, a low GCS score in patients with supratentorial ICH is often asign of notable mass lesion and intracranial hypertension, which is seldomlytreatable by for example surgical evacuation.The costs associated with supratentorial and infratentorial ICHs differed somewhat.Low GCS scores were independently associated with lower costs among thesupratentorial ICH patients, yet the opposite was true for infratentorial ICH.Similarly, high SAPS II scores were independently associated with higher costs amongthe survivors with supratentorial ICHs but with lower costs among all theinfratentorial ICH patients. These findings reflect the differences in the diseasepicture and treatment options among the bleeding types. Overall, infratentorialbleeding is a well-known independent marker of poor prognosis, and as the very sickpatients  die early, the costs remain low. This implies that functional ability is a determinant of the costs of ICHover 12 months, and, therefore, research should concentrate on treatments that couldhave a positive effect on functional outcome to reduce costs. However, thosepatients who are very sick die early and are therefore associated with lowercosts.Like the study by Lekander et al., we found a minor correlation between ICH severityand costs when considering hospital and 12-month survivors only. The cost data were gathered with a wide perspective from three resourcescovering rehabilitation and social security costs and costs of hospital care,offering a comprehensive estimate of the total economic cost of intensive carefor ICH.Our study has several strengths. Given the multicenter study design that includedall the tertiary hospitals in Finland and the tax-based Finnish healthcaresystem, the cohort is considered to capture population-based ICHs with noselection bias due to socioeconomic or insurance status. Additionally, afteridentifying cases by APACHE III diagnosis, all admission CT scans were screenedto ensure the validity of the diagnoses and minimize misclassification bias. Thedatabases used were high-quality, ensuring no loss to follow-up, and the data oncost status were almost 100% (99.6%). However, as a common intensive care database, FICC does not includeneuro-specific data, and, hence, we used a surrogate marker for permanentdisability for functional outcome, which was based on granted permanentdisability allowances and pensions by the Social Insurance Institution. However,as all Finnish citizens with a disability or long-term illness are entitled to adisability allowance from Kela regardless of personal insurance status, webelieve that the surrogate marker describes the functional outcome in ourpopulation well, although the surrogate marker carries a theoretical risk ofboth over- and underestimation of the patients’ functional status. A directtranslation of our surrogate marker to the modified Rankin Scale is notpossible, but it seems likely that a favorable outcome as defined by oursurrogate outcome marker would translate into a mRS of 0–3 in most cases andinto 0–4 in some instances. However, 1 year is a rather short time to assessfunctional outcome, and, in addition to the difference in reporting functionaloutcome, this should be considered when interpreting the results. Third, due tothe data-derived restrictions, we used the ECPIS to measure cost-effectivenessinstead of a quality-adjusted life year (QALY). However, the ECPIS is a roughestimation, and in addition to having a non-existent commonly described range,it cannot be directly translated to QALYs to make inferences. Nevertheless,there are similar studies in critical care in which cost-effectiveness has beendescribed similarly, although there are no current guidelines on what can beconsidered as cost-effective ECPS or ECPIS values.27 More cost studies usingsimilar metrics for other diseases are needed in order to establish suchthreshold values. For example, the ECPIS of ICU-treated ICH patients is lower than that of ICU-treated patients with acute ischemic stroke  but higher than that of ICU-treated patients with traumatic brain injury  and subarachnoid hemorrhage . However, ECPS and ECPIS do not account for indirect future costs, suchas loss of ability to work. Fourth, most spontaneous ICH patients are treated atsecondary and tertiary hospital stroke units in Finland instead of an ICU. Asthis study was based on patients treated at tertiary hospital ICUs only, thestudy cohort represents the patients comprising the more severe ICH cases thatstill were admitted to the ICU. For example, assuming an annual ICH incidence of25 per 100,000 inhabitants, approximately 15,000 ICHs should have occurredduring the 11-year study period in a population of 5.5 million inhabitants (ref). Thus, the included study population constitute approximately 6% of allICH patients, which should be considered when interpreting these results. Fifth,the infratentorial ICH population was notably smaller than the supratentorialICH population (786 vs 173), and, hence, the subgroup analyzes are potentiallyunderpowered and prone to generating false-negative results. Sixth, it shouldalso be acknowledged that the patients were treated during 2003–2013. There mayhave been changes in care even though guidelines on the management of ICH haveremained largely unchanged.35 Finally, given thediversity in organizing healthcare, these findings might not be readilygeneralizable to countries with completely different healthcare systems.Some limitations also need to be acknowledged. First, due to the study’sretrospective nature, we were restricted to the data available in the databases.Therefore, we were unable to account for the effects of treatments, earlytreatment restrictions and withdrawal of care on costs. Hence, some patients inthe study cohort might have been admitted to the ICU under consideration aspotential organ donors. As early withdrawal of care might lead to decreasingtotal costs over a longer time perspective, withdrawal of treatment could be animportant confounder in the association of illness severity and long-term costs.However, as we conducted a sensitivity analysis on hospital survivors only, thedifferences between supratentorial and infratentorial ICH remained. As themedian duration of hospital care was 6 days and did not differ significantlyamong the supratentorial and the infratentorial ICH patients, the impact ofwithdrawal of care at an early phase is considered to have been covered in thissubgroup analysis. Second, the modified Rankin Scale is the most common metricto estimate neurological outcome post-stroke.Costs associated with ICHs treated in ICUs varied substantially between differentpatient groups. The ECPIS was highest among the patients older than 70 years,brainstem ICH, and more severe illness. The patient characteristics independentlyassociated with costs differed somewhat between supratentorial and infratentorialICHs. With better triage and a focus on the appropriate patient groups,cost-effectiveness could increase. As only one-third of all costs were allocated tothose with a favorable functional outcome, further detailed cost-analysis studiesfor patients with ICH are required.Click here for additional data file.Supplemental material, sj-docx-1-eso-10.1177_23969873221094705 for One-yearhealthcare costs of patients with spontaneous intracerebral hemorrhage treatedin the intensive care unit by Marika Smeds, Markus B Skrifvars, MattiReinikainen, Stepani Bendel, Sanna Hoppu, Ruut Laitio, Tero Ala-Kokko, SamiCurtze, Gerli Sibolt, Nicolas Martinez-Majander and Rahul Raj in European StrokeJournal


Orthonairovirus) has been isolated from the sera of Egyptian rousette bats  captured in Uganda and unengorged Ornithodoros (Reticulinasus) faini ticks collected from the rock crevices of ERB colonies in South Africa and Uganda. Although evidence suggests that KASV is maintained in an enzootic transmission cycle between O. (R.) faini ticks and ERBs with potential for incidental virus spillover to humans through the bite of an infected tick, the vertebrate reservoir status of ERBs for KASV has never been experimentally evaluated. Furthermore, the potential for bat-to-bat and bat-to-human transmission of KASV is unknown. Herein, we inoculate two groups of ERBs with KASV; one group of bats is serially sampled to assess viremia, oral, fecal, and urinary shedding and the second group of bats is serially euthanized to assess virus-tissue tropism. Throughout the study, none of the bats exhibit overt signs of clinical disease. Following the detection of high KASV loads of long duration in blood, oral, fecal, and urine specimens collected from ERBs in the serial sampling group, all bats seroconvert to KASV. ERBs from the serial euthanasia group exhibit high KASV loads indicative of virus replication in the skin at the inoculation site, spleen, and inguinal lymph node tissue, and histopathology and in situ hybridization reveal virus replication in the liver and self-limiting, KASV-induced lymphohistiocytic hepatitis. The results of this study suggest that ERBs are competent, natural vertebrate reservoir hosts for KASV that can sustain viremias of appropriate magnitude and duration to support virus maintenance through bat-tick-bat transmission cycles. Viral shedding data suggests that KASV might also be transmitted bat-to-bat and highlights the potential for KASV spillover to humans through contact with infectious oral secretions, feces, or urine.The human-pathogenic Kasokero virus (KASV; genus  The devastating health, economic, and social consequences resulting from the coronavirus 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)10, a virus with an ancestral bat origin11, underscores the need to preemptively characterize RNA virus infection and transmission dynamics in their natural reservoir bat hosts to assess virus spillover risk and develop appropriate risk-reduction strategies.Bats have been implicated as natural reservoir hosts for many high-consequence, zoonotic RNA viruses, including coronavirusesOrthonairovirus (family Nairoviridae) comprises 48 viruses, including the highly human-pathogenic Crimean Congo hemorrhagic fever virus, that are maintained in enzootic cycles involving tick and vertebrate hosts12. Eight of the orthonairoviruses, including Estero Real13, Gossas14, Issyk-Kul18, Kasokero20, Keterah21, Leopards Hill22, Uzun Agach24, and Yogue25 viruses, have been isolated from bats and/or their argasid tick ectoparasites. A mouse model of Leopards Hill virus infection demonstrated an acute, lethal disease resembling Crimean Congo hemorrhagic fever in humans22, and Issy-Kul17 and Kasokero19 viruses are known human pathogens that cause febrile illness, often progressing to prolonged systemic disease. Despite their public health importance, the infection and transmission dynamics of orthonairoviruses in their bat hosts are poorly understood. Of particular interest, is whether orthonairovirus-infected bat hosts can shed virus and represent direct virus spillover risks to humans.The genus Rousettus aegyptiacus), cave-roosting frugivores with a geographic range extending north into Turkey, west into Senegal, south into South Africa, and east into Pakistan26, are natural reservoir hosts for Marburg 29, Ravn 5, and Sosuga 30 viruses. Kasokero virus (KASV) was first described in 1977 after scientists at Uganda Virus Research Institute isolated infectious virus from 2/74 (2.7%) serum samples collected from ERBs captured at Kasokero Cave, Uganda and detected virus-specific antibodies in 50/74 (67.6%) of the bat serum samples19. During initial virus characterization studies at the institute, 3 laboratory staff and 1 support staff acquired KASV infection, and presented with manifestations ranging in severity from mild febrile illness to prolonged systemic disease characterized by fever, headache, myalgia, arthralgia, abdominal pain, nausea, diarrhea, chest pain, coughing, and hyperactive reflexes19. The 4 KASV-infected staff seroconverted, and KASV antibodies were detected in 10 additional staff. KASV was later isolated from both engorged and unengorged argasid ticks (Ornithodoros (Reticulinasus) faini) collected from rock crevices of ERB colonies at Lanner Gorge Cave, South Africa in 1994–1995 and Python Cave, Uganda in 201720. Consistent with the behavior of other tick species in the family Argasidae31, O. (R.) faini ticks emerge from rock crevices to rapidly feed (≤ 30 min) on roosting ERBs before retreating into the crevices. As a result, O. (R.) faini ticks have been observed only occasionally on wild-caught ERBs. In line with their rapid feeding ability, a previous blood meal analysis of O. (R.) faini tick pools collected from rock crevices of Python Cave revealed that 28.2% of the pools included ticks that had recently fed on ERBs (authors’ unpublished data).Egyptian rousette bats  faini ticks and ERBs with potential for incidental virus spillover to humans through the bite of an infected tick20, the vertebrate reservoir status of ERBs for KASV has not been experimentally evaluated. Herein, we determine the potential for KASV to be transmitted tick-to-bat, bat-to-tick, bat-to-bat, and bat-to-human by experimentally inoculating groups of ERBs with KASV and testing blood, oral, fecal, and urine samples collected through 18 days post inoculation (DPI) for the presence of KASV. We then evaluate health status and virus-tissue tropism by performing clinical chemistry panels and assessing 14 tissue types collected from KASV-inoculated ERBs serially euthanized at regular intervals through 20 DPI.Although evidence suggests that KASV is maintained in an enzootic transmission cycle between 27 were used in this study. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the United States Centers for Disease Control and Prevention (CDC) and performed according to the Guide for the Care and Use of Laboratory Animals32. The CDC is an Association for Assessment and Accreditation of Laboratory Animal Care fully accredited research facility. Reporting for this study was carried out in compliance with the ARRIVE guidelines. Although the Biosafety in Microbiological and Biomedical Laboratories Manual33 recommends that KASV work be performed under biosafety level (BSL) 2 containment, all procedures conducted with KASV or KASV-inoculated bats were performed under BSL-4 containment. All bats were housed in a climate controlled BSL-4 animal area, with a 12 h day/12 h night cycle. Bats were provided daily with their body mass in fresh fruit supplemented with protein/vitamin powder  and received water ad libitum.A total of 24 captive-bred juvenile ERBs (5–7 m of age) from the ERB breeding colony10 tissue culture infectious dose 50 (TCID50) of the UGA-Tick-20170128 strain of KASV  prepared in 0.1 mL of sterile phosphate buffered saline (PBS), and 2 negative control (NEG CO) bats  received 0.1 mL of sterile PBS. The KASV inoculum dose was selected based on the estimated volume of saliva secreted by feeding Ornithodoros spp. ticks (10 µL)34 and the KASV titer of a pool of 5 unengorged O. (R.) faini ticks /10 µL). The KASV-inoculated bats were housed in a single flight cage maintained within a bio-flow isolator with HEPA-filtered inlet and exhaust air supplies , while the NEG CO bats were housed in a non-human primate-sized cage maintained within a second bio-flow isolator unit. Temperatures, blood, duplicate oral swabs, rectal swabs, and urine  were collected from all bats daily  to the end of the study, while weights were taken on a weekly basis. At 18 and 20 DPI, the bats were euthanized by an overdose of isoflurane followed by cardiac exsanguination.The 24 ERBs were divided into 2 study arms: 9 bats were assigned to the serial sampling arm and 15 bats were assigned to the serial euthanasia arm. Bats in the serial sampling arm were acclimated to the BSL-4 laboratory for 5 days before beginning the study. To mimic a tick bite, all bats were inoculated by the intradermal route in the subcaudal abdominal region under isoflurane anesthesia at 0 DPI. 7 bats  received 4.0 logBlood was taken from the cephalic vein using a sterile lancet , polyester-tipped applicators  were used to swab the oral mucosa, and rectal swabs were obtained opportunistically at the time rectal temperatures were taken by repurposing the plastic thermometer probe cover . Opportunistic urine collection was attempted by allowing a single bat to hang in a sterile mouse cage fitted with an inverted wire top  and later collecting the accumulated urine. Aliquots of whole blood were used to monitor for viremia (KASV RNA by quantitative reverse transcriptase-polymerase chain reaction [qRT-PCR]) and antibody responses (anti-KASV IgG by indirect enzyme-linked immunosorbent assay [ELISA]). Oral swabs, rectal swabs and urine were used to detect virus shedding (KASV RNA by qRT-PCR).10TCID50 of the UGA-Tick-20170128 strain of KASV prepared in 0.1 mL of sterile PBS and 3 NEG CO bats  received 0.1 mL of sterile PBS. The KASV-inoculated bats were housed in primate-sized cages according to the DPI of euthanasia  that were maintained within a bio-flow isolator unit, while the NEG CO bats were housed in a non-human primate-sized cage maintained within a second bio-flow isolator unit. All bats were euthanized by an overdose of isoflurane followed by cardiac exsanguination. Temperatures, weights, and blood were taken from all bats at 0 DPI and at euthanasia. Aliquots of whole blood collected at 0 DPI and at euthanasia were used to assess viremia and antibody responses. Serum collected at euthanasia was used to perform clinical chemistries and attempt virus isolation (if corresponding whole blood specimen was positive for KASV RNA). Tissues  collected at necropsy from all bats in the serial euthanasia arm and randomly-selected bats euthanized at 18 (n = 3 bats) and 20 DPI (n = 3 bats) from the serial sampling arm were tested for KASV RNA by qRT-PCR to determine virus-tissue tropism. Following necropsy, bat carcasses and remaining tissues were immersed in 10% neutral buffered formalin for a minimum of 7 days in BSL-4 containment, with a complete formalin replacement occurring at least 3 days prior to processing for histopathology.Bats in the serial euthanasia arm were acclimated to the BSL-4 laboratory for 10 days before beginning the study. All bats were inoculated by the intradermal route in the subcaudal abdominal region under isoflurane anesthesia at 0 DPI. 12 bats  received 4 logRNA was extracted on the MagMAX Express-96 Deep Well Magnetic Particle Processor  from blood, oral, rectal, and urine specimens using the MagMAX Pathogen RNA/DNA Kit (Thermo Fisher Scientific) and from homogenized tissues using the MagMAX Total RNA Isolation Kit (Thermo Fisher Scientific).FAM-AGCAGTCAT-ZEN-CGCAGCCACCAGAAA-IABkFQ) and the ERB B2M gene , respectively. To account for variation in weight among the tissue specimens, KASV qRT-PCR cycle threshold (CT) values were normalized using ERB B2M qRT-PCR CT values. Relative KASV TCID50eq/mL  or g (tissue specimens) were interpolated from standard curves generated from serial dilutions of the titrated KASV UGA-Tick-20170128 strain spiked into appropriate biological specimens.Reverse-transcribed KASV and ERB beta-2-microglobulin (B2M) RNA were detected on the ABI 7500 Real-Time PCR System (Thermo Fisher Scientific) using the SuperScript III Platinum One-Step qRT-PCR Kit (Thermo Fisher Scientific), with amplification primers and reporter probes targeting the nucleoprotein gene , the roller bottles were incubated at 37 °C. At 6 DPI, ≥ 90% of cells exhibited evidence of KASV infection by IFA.KASV antigen lysate for the anti-KASV IgG indirect ELISA was prepared by inoculating monolayers of Vero E6 cells in 10, 850 cmAt 7 DPI, glass beads were added to each roller bottle, the bottles were gently swirled to release virus-infected cells from tissue culture monolayers, and the cellular media was centrifuged at 8000 rpm for 10 min at 4 °C. Following centrifugation, the supernatant was discarded, cell pellets were resuspended in borate saline, and centrifuged at 12,000 rpm for 10 min at 4 °C. The cell pellets were resuspended in 28 mL of a 1% Triton X-100 borate saline solution, gamma-cell irradiated with a 5 megarad dose, and then sonicated for 15 min using a setting of 2 s on/2 s off. The sonicated KASV lysate was centrifuged at 12,000 rpm for 10 min at 4 °C and the supernatant was transferred to a fresh conical tube. Uninfected Vero E6 cell control lysate was prepared using the same procedure.Wells of 96-well ELISA plates were coated (100 µL) with the dilution of KASV lysate  that was found to result in optimal reactivity with blood pooled from KASV-inoculated bats euthanized at 20 DPI (1:2000) and corresponding wells were coated with an equivalent dilution of uninfected control lysate (1:2000). After incubation overnight at 4 °C, the plates were washed with PBS containing 0.1% Tween-20 (PBS-T) and 100 µL of serum diluent (PBS containing 5% skim milk and 0.1% tween-20) was added to each well of the plate. After 10 min, 33 µL of a 21:521 dilution of gamma-irradiated bat serum pre-diluted in masterplate diluent  was added to the first well of the plate and fourfold serial dilutions, ranging from 1:100 to 1:6400, were performed. Following a 1 h incubation at 37 °C, the plates were washed with PBS-T and 100 µL of a 1:20,000 dilution of goat anti-bat IgG conjugated to horseradish peroxidase  in serum diluent was added to the plates. The manufacturer product specification sheet indicates that this antibody reacts specifically with bat IgG and with light chains common to other immunoglobulins. After incubation for 1 h at 37 °C, the plates were washed with PBS-T, 100 µL of the Two-Component ABTS Peroxidase System  was added, and the plates were allowed to incubate for 30 min at 37 °C. The plates were then read on a microplate spectrophotometer set at 410 nm. To negate non-specific background reactivity, adjusted optical density (OD) values were calculated by subtracting the ODs at each fourfold dilution of wells coated with uninfected control antigen lysate from ODs at corresponding wells coated with KASV lysate. The adjusted sum OD value was determined by summing the adjusted OD values at each four-fold serial dilution.The cut-off value for seropositivity (0.27) was determined by calculating the mean adjusted sum OD value plus 5 standard deviations (SDs) of 38 KASV-naïve bats from the ERB breeding colony. A bat with an adjusted sum OD value 0.27 was considered to have been infected with KASV and seroconverted with a confidence level > 99.99999%.2 tissue culture flasks were inoculated with 100 µL sera plus 500 µL maintenance media  and incubated for 1 h at 37 °C/5% CO2. Following the addition of 7 mL maintenance media, cultures were incubated at 37 °C /5% CO2. At 13 DPI, tissue culture monolayers were scraped to release virus-infected cells. Part of each cellular medium (100 µL) was transferred to 500 µL MagMax lysis buffer solution for RNA extraction and qRT-PCR, and a second part of each cellular medium (1.5 mL) was suspended in 8 mL borate saline. After pelleting the cellular suspensions by centrifugation, the borate saline was decanted, the cells were resuspended in 500 μL borate saline, and 12-well spot slides were spotted with 25 μL of the cellular resuspensions. The slides were fixed in acetone before receiving 2 megarads of γ-irradiation.Virus isolation was attempted on the serum fraction of all KASV RNA positive whole blood specimens collected at euthanasia. Monolayers of 85% confluent Vero E6 cells  in 25 cmSix spots on each slide were incubated with 25 μL of a 1:100 dilution of KASV mouse immune ascitic fluid , and the other 6 spots were incubated with normal mouse ascitic fluid for 30 min at 37 °C. After incubation, the spot slides were rinsed 2 times with PBS, incubated with 24 μL of a 1:40 dilution of goat anti-mouse fluorescein isothiocyanate  for 30 min at 37 °C, rinsed with PBS, stained with Eriochrome Black T, rinsed with PBS, and then observed under a fluorescence microscope.Histopathology was performed on 18 KASV-inoculated bats euthanized in groups of 3 at 3, 6, 9, 12, 18, and 20 DPI, as well as 3 NEG CO bats. A full set of tissues from each formalin-fixed carcass was collected for histologic examination, including representative sections from the brain, eye, heart, lungs, trachea, thymus, esophagus, thyroid glands, liver , stomach, small intestines , large intestines, spleen, kidney, adrenal glands, salivary gland, submandibular lymph node, tongue, axillary, inguinal, sublumbar and mesenteric lymph nodes, skin from the inoculation site, patagium, skin from the chest region and pectoral muscle. Tissue sections were placed into cassettes and processed routinely for histological analyses at the University of Georgia Histology Laboratory. Representative formalin-fixed paraffin-embedded (FFPE) tissues were sectioned at 4–5 μm, mounted on glass slides, and stained with hematoxylin and eosin. Slides were reviewed in a blinded manner without knowledge of time point or infection status.In situ hybridization was performed on FFPE liver tissues from a subset of KASV-inoculated bats  using the RNAscope® 2.5 HD Reagent Kit—RED  according to the manufacturer’s instructions. A custom anti-sense probe targeting the complimentary RNA sequence of the nucleoprotein gene of KASV (GenBank Accession Number: MT309090) was designed. To confirm adequate RNA integrity of the FFPE samples, an ERB-specific, medium-expressing housekeeping gene  probe was used as a positive control. A probe targeting the dapB gene  was used as a negative control.Serum collected at euthanasia from 18 KASV-inoculated bats euthanized in groups of 3 at 3, 6, 9, 12, 18, and 20 DPI, as well as 3 NEG CO bats, were analyzed on Piccolo Xpress chemistry analyzers  using the General Chemistry 13 Panel.35. Analyte data were assessed for conformation to a normal Gaussian distribution using the Shapiro–Wilk test (two-tailed P > 0.05). For normally distributed analytes , 2.5% and 97.5% reference intervals were estimated using the mean plus two SDs. For non-normally distributed analytes , data were log10-transformed to achieve normality, 2.5% and 97.5% reference intervals were estimated using the mean plus two SDs, and the reference intervals were back-transformed to their original units. Reference ranges could not be calculated for gamma-glutamyl transferase (GGT) and amylase (AMY), as these analytes were not previously measured in serum collected from the 13 NEG CO bats.ERB-specific reference ranges were established for glucose (GLU), blood urea nitrogen (BUN), creatinine (CRE), calcium (CA), albumin (ALB), total protein (TP), alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and total bilirubin (TBIL) using serum collected at euthanasia from 13 NEG CO bats used here and in previous studiesThe number of bats used in this study was based on the reproductive capacity of the ERB breeding colony. An effort was made to sex-match bats according to group. Investigators were not blinded during the study. No bats or individual data points were excluded from any analysis. Prism 9.0.0 software  was used to perform all data management tasks, figure constructions, statistical analyses, and measures of central tendency .35. Like the serial sampling arm of the study, weights  had detectable viremias Fig. a or anti10TCID50eq/mL (mean peak load = 5.6 log10TCID50eq/mL) and occurring from 4–5 DPI (mean day of peak load = 4.3 DPI)  and occurring from 6–7 DPI (mean day of peak load = 6.1 DPI)  and occurring from 4 to 8 DPI (mean day of peak load = 5.4 DPI)  of the KASV-inoculated bats Fig. . The intPI) Fig. a. The inPI) Fig. b. The in36. Cumulative KASV RNA loads varied among the 7 KASV-inoculated bats, with sum log10TCID50eq/mL loads ranging from 5.4 to 6.3  for blood specimens, 3.2–5.8  for oral specimens, and 2.4–4.0  for rectal specimens. The Lorenz curves for viremia  and together accounted for 93.1% of the total KASV oral shedding.Viremia and viral shedding are measures of infectiousness and were calculated for each KASV-inoculated bat by summing KASV RNA loads detected in the blood, oral and rectal swabs throughout the studyAll KASV-inoculated bats (7/7) seroconverted to KASV by 14 DPI Fig. a; anti-KPrior to inoculation, none of the bats in the serial euthanasia study arm (n = 15) had detectable viremias or anti-KASV IgG Fig. b, indica10TCID50) were observed in the skin at the inoculation site (through 9 DPI), spleen (3 and 9 DPI), and inguinal lymph node (20 DPI).Blood collected from all bats euthanized at 3 and 6 DPI were KASV RNA positive and infectious KASV was isolated from serum collected from all bats euthanized at 3 DPI Table . All 14 Gross examination at necropsy revealed a liver hemorrhage in one bat euthanized at 9 DPI 909,659). In the liver, KASV infection was associated with mild to moderate lymphohistiocytic hepatitis, which was histologically evident beginning at 3 DPI  liver enzymes  faini ticks to experimentally evaluate bat-to-tick transmission of KASV, we demonstrated that ERBs experimentally inoculated with a tick isolate of KASV at a dose (4.0 log10TCID50) similar to that detected in a pool of unengorged O. (R.) faini ticks, mounted viremias that are likely of sufficient magnitude (mean peak load = 5.6 log10TCID50eq/mL) and duration (mean duration = 7.7 days) to infect feeding O. (R.) faini ticks. In line with a previous study that recovered infectious KASV from 2/74 (2.7%) serum specimens collected from ERBs captured at Kasokero Cave, Uganda19, we isolated infectious KASV from all serum specimens collected from bats euthanized at 3 DPI. Here, all KASV-experimentally infected ERBs in the serial sampling group seroconverted by 14 DPI and in the previous study at Kasokero Cave, virus-specific antibodies were detected in 50/74 (67.6%) of the ERB serum specimens19, indicating cumulative exposure of ERBs to KASV in nature. These data are consistent with the expectations of a natural vertebrate reservoir host of a virus.The results of this experimental infection study combined with those of a previous ecological investigation of KASV circulation in ERBs at Kasokero Cave, Uganda10TCID50eq/mL; mean duration: 8.6 days), fecal , and urinary  shedding was detected in all KASV-inoculated bats at some point during the study. Although KASV infection following mucosal virus inoculation of ERBs and/or experimental transmission of KASV from KASV-inoculated donor ERBs to naïve contact ERBs will be required to definitively demonstrate horizontal bat-to-bat transmission of KASV, our data indicate the potential for horizontal transmission of KASV between ERBs in nature through direct or indirect contact with infectious bodily fluids. KASV oral shedding dynamics were strikingly similar to those previously observed in MARV-subcutaneously inoculated ERBs 28. Additionally, both KASV- and MARV-inoculated ERBs exhibited comparable heterogeneities in cumulative oral shedding, with 20% of the KASV and MARV study populations responsible for 89.8% and 95.4% of the viral oral shedding, respectively28. Based on the ability of the subcutaneous inoculation route to generate infection profiles that recapitulated MARV infection in ERBs in nature27 and the frequently observed altercations over roosting space in densely populated sub-Saharan African ERB colonies38, it was speculated that biting represents a primary route of bat-to-bat transmission of MARV27. As ERB bites presumably reach the intradermal and/or subcutaneous skin layers, horizontal transmission of KASV between ERBs through biting likely represents a viable transmission mechanism. Like KASV-experimentally infected ERBs, SOSV-experimentally infected ERBs inoculated through the subcutaneous route exhibited fecal  and urinary  shedding30. ERBs roosting in a densely populated colony at Python Cave, Uganda  have been observed urinating and defecating just before and during flight, often when positioned directly above other bats, including virus-naïve juveniles, that frequently roost on vertical cave walls or the cave floor which is covered in guano5. Based on these observations and experimentally determined virus shedding routes, it was hypothesized that SOSV is horizontally transmitted between ERBs through direct or indirect contact with virus-contaminated urine and/or feces30. As KASV and SOSV exhibit comparable fecal and urinary shedding dynamics, it is likely that KASV can also be transmitted ERB-to-ERB through direct or indirect contact with urine and feces. Due to an unreported − 80 °C freezer failure, duplicate oral swab and urine specimens collected during this study for virus isolation attempts on KASV qRT-PCR positive specimens were lost. Nonetheless, persistently high KASV RNA loads in oral swab and urine specimens that occurred prior to the development of a robust virus-specific IgG response28 is consistent with virus replication in the host and indicates that virus isolation attempts would have likely resulted in the recovery of infectious virus.In this study, KASV oral  faini ticks, KASV has the potential be transmitted between ERBs and from ERBs to humans and wildlife through direct or indirect contact with infectious bodily fluids. Given that the known geographic range of KASV circulation and spillover risk extends from Uganda to South Africa, future investigations are needed to: 1) detect and isolate KASV from oral, rectal, and urine specimens from wild-caught ERBs and 2) determine if peaks of active KASV infection occur in young juveniles coincidental with the loss of maternal immunity similar to MARV infection5. Given that a mouse model of Leopards Hill virus infection generates a lethal disease resembling Crimean Congo hemorrhagic fever in humans22, and the extensive geographic range , outbreak history and disease profile of Issyk-Kul virus18, these two orthonairoviruses and the five other understudied bat-associated orthonairoviruses merit further investigation. Finally, the KASV-ERB system could serve as an experimental natural reservoir host model for studying the zoonotic potential of other bat-borne orthonairoviruses.In conclusion, the results of our study suggest that in addition to transmission by


Recent reports suggest that cell-surface and intracellular immune receptors function synergistically to activate robust defence against pathogens, but whether they co-evolve is unclear. Here we determined the numbers of cell-surface and intracellular immune receptors in 350 species. Surprisingly, the number of receptor genes that are predicted to encode cell-surface and intracellular immune receptors is strongly correlated. We suggest this is consistent with mutual potentiation of immunity initiated by cell-surface and intracellular receptors being reflected in the concerted co-evolution of the size of their repertoires across plant species. Comparative genomic analysis of 350 plant species reveals that cell-surface and intracellular immune receptor gene families co-expand or co-contract. This suggests an evolutionary relationship between the two branches of the plant immune system. Cell-surface pattern-recognition receptors (PRRs) recognize apoplastic and usually conserved pathogen-associated molecular patterns (PAMPs) and activate pattern-triggered immunity (PTI). Virulent pathogens secrete effector molecules into plant cells that suppress PTI and promote infection. Intracellular nucleotide-binding leucine-rich repeat (NLR) receptors recognize effectors and activate effector-triggered immunity (ETI). Although PTI and ETI were envisaged as two independent immune systems1, emerging evidence suggests they are inter-dependent and share multiple signalling components6. Thus, PTI and ETI function synergistically to provide robust immunity against pathogens. As PRRs and NLRs are functionally inter-dependent, in this Brief Communication, we investigated whether the sizes of these two receptor gene families are correlated.Plants have evolved a two-tier immune system that recognizes and activates defence against pathogens7. LRR-domain-containing RLKs (LRR-RLKs) and RLPs (LRR-RLPs) are the largest RLK- and RLP-gene families in plants9. LRR-RLKs can be further classified into 20 subgroups, with each subgroup involved in different biological processes10 . Members of LRR-RLK-XI are involved in recognition of self-peptides13. Members of LRR-RLK-XII, such as FLAGELLIN-SENSITIVE 2 (FLS2), EF-TU RECEPTOR (EFR) and Xa21 (refs. 7), are involved in detecting pathogen-derived molecules  or receptor-like proteins (RLPs). RLKs carry extracellular ectodomains and cytosolic kinase domains, while RLPs lack cytosolic kinase domains. RLKs carry multiple types of extracellular domains, such as leucine-rich repeats (LRRs), lectins and lysM motifs (LysMs)To investigate expansion or contraction of genes that encode PRR and NLR proteins, we identified these gene families in annotated proteomes from 350 publicly available genomes. These genomes include 26 algal species, 5 bryophyte species, 10 gymnosperms and 300 angiosperms   of LRR-RLKs, LRR-RLPs, LysM-RLK, LysM-RLP and NB-ARCs from each genome (number of identified genes/number of searched genes × 100) , suggesting that NB-ARC and LRR-RLP gene families expand together . On the other hand, %LysM-RLKs and %LysM-RLPs show weak or no correlation with %NB-ARC . We propose that PRRs involved in pathogen recognition are more likely to co-expand with NB-ARC gene families. This is consistent with the observation that characterized LRR-RLPs are usually involved in pathogen recognition, while LRR-RLKs and LysM receptors can be involved not only in immunity but also in development, reproduction or establishing symbiosis12.Next, we determined the correlation between the percentages of PRRs  and NB-ARCs in angiosperms. Surprisingly, %NB-ARC and %LRR-RLPs show a strong positive linear correlation  compared with LRR-RLK-VIII_1, LRR-RLK-VIII_2 and LRR-RLK-Xa   . On the other hand, %LRR-RLP combined with %LRR-RLK-XII show strong positive correlation with %NB-ARC (Pearson’s r = 0.859)  Fig. .23 for 238 species and tested whether percentage receptor distances correlate with each other while taking into account the phylogenetic distances with a partial Mantel test. Again, %LRR-RLK-XII and %LRR-RLP show strong positive correlation with %NB-ARC . These include Genislea aurea, Utricularia gibba, Utricularia reniformis, Zostera marina, Zostera muelleri, Lemna minor, Wolffia australiana and Spirodela polyrhiza  in these groups is similar to other plant species, as are most other LRR-RLK subgroups  and can survive losing 95% of cellular water25. Despite its small genome, O. thomaeum has a similar number of predicted proteins as other Poales species such as Ananas comosus, Oryza longistaminata and Triticum urartu, suggesting that the contraction of immune receptor families could be independent of the reduced genome size.Some other species and genera also show lower %NB-ARC, %LRR-RLK and %LRR-RLP. For example, the Cucurbitaceae show far fewer immune receptors than the phylogenetically close Malpighiales or Fagales clades Fig. . Remarkaent Fig. . O. thomOryza and Triticum genera  underpinning these observations. As functionally inter-dependent genes often co-expand/contract together, it is likely that the functional relationship between cell-surface and intracellular immune receptors is conserved across plant species.As parasite pressure drives the retention of sexual reproduction that reshuffles immune receptor alleles each generation32). The Arabidopsis sequences that were previously classified into 20 LRR-RLK subgroups10 were filtered likewise for the presence of LRR and kinase domains. Eleven sequences were removed because they did not pass the threshold filter for the kinase (two sequences) and LRR (nine sequences) domain searches. To classify all candidate sequences according to the Arabidopsis subgroups, the highest-scoring kinase domain region of each candidate was extracted and aligned to the Arabidopsis reference sequences using diamond33 .Protein sequences from all 350 plant proteomes were first filtered for the primary gene model. Sequences shorter than 250 amino acids (AA) were removed as they are unlikely LRR-RLKs. The remaining proteins were searched for the presence of a protein kinase domain (PFAM PF00069.26) and an LRR domain  with hmmer . Trees were rooted with gotree37 (v0.4.2) using the sequences belonging to the most basal species as outgroup ).The phylogeny of each subgroup was inferred using the kinase domains. Sequences were aligned with FAMSAArabidopsis LRR-RLPs38. The domain was trimmed manually, starting from the conserved Y in the C2 domain . Candidates were finally filtered for the presence of a transmembrane domain using tmhmm40 with default settings (version 2.0).LRR-RLPs were identified similarly but filtering for proteins of a minimal length of 150 AA first. Proteins were then searched for the presence of LRR domains and the absence of a kinase domain (hmmer options as above), as well as the presence of a C3F domain . The hmmer profile for the C3F domain was obtained from a multiple alignment of NB-ARCs were identified using the set of proteins with a minimal length of 150 AA. Proteins were then searched for the presence of NB-ARC (PF00931.23) domains (hmmer option -E 1e-10 for NB-ARC).40 . Candidates were split into LysM-RLKs and LysM-RLPs by searching for presence/absence of a kinase domain .LysM-RLKs and LysM-RLPs were identified using the set of proteins with a minimal length of 150 AA. Proteins were filtered for the presence of a LysM domain  and a transmembrane domainn and group B (NB-ARCs) with m genes. The observed distance was calculated as the average closest distance between genes in group A and genes in group B. A distribution for the expected distance was obtained by randomly sampling m genes and calculating the average closest distance of genes in group A to the genes in the random set . Genes were sampled from the list of genes that was used to search for the genes in group B  with https://www.ncbi.nlm.nih.gov/Taxonomy/CommonTree/wwwcmt.cgi). Phylogenetic tree of the 350 species is generated by phyloT (https://phylot.biobyte.de/) based on NCBI taxonomy database. Phylogenetics trees were visualized and figures were generated by iTOL41. The tree used for testing the relationship between the fraction of candidates found and the phylogenetic distances were obtained from ref. 23. The latter contained 238 out of the 350 genomes analysed.The taxonomic tree was obtained from NCBI  found per species correlated with the phylogenetic relationships, we converted the fractions and the phylogenetic tree to distance matrices and tested for correlation with mantel tests . Analogously, we also tested for correlation between distance matrices obtained for two different sets of proteins . https://www.originlab.com/) and R (version 3.4.4).Statistical analyses were performed with OriginPro (version 2022; Further information on research design is available in the Supplementary InformationSupplementary Figs. 1–7.Reporting SummarySupplementary TablesSupplementary Table 1. List of species included in this study. Supplementary 2. Number of receptor genes in each species. Supplementary Table 3. Percentage of receptor genes in each species. Supplementary 4. Statistical analyses in this study.