A pandemic outbreak of severe acute respiratory syndrome coronavirus 2 (COVID 19) incidence data are largely available online. Until August 17, COVID 19 has hit more than 22 million individuals all over the globe. So, it is urged to get clear information about the prevalence of the virus. Therefore, one can manipulate easily a suitable mathematical model to fit these published data.We propose a mathematical model that considers the total population, in 25 countries, either infected by COVID 19 or confined (safe) during the period from November 17, 2019, to August 17, 2020. The model considers the total population as a complex number; the imaginary part is the number of infected individuals and the real part is the number of confined individuals. This classification combined with mathematical treatments leads to a transmission dynamics of the virus to be as wave-like motion. The virus can hit any country either by one wave or by successive waves (up to 11 waves).Rd0) and the median reproduction number (R0). We have found that Rd0 decreases exponentially with time from high values down to zero at the wave maximum point; and R0 varies from a country to another. For example, the virus hit individuals in Germany in R0 = 1.39 (96% CI 1.01–3.87) and in the USA R0 = 3.81 (91% CI 1.71–5.15). We have found that twice the virus has hit both the USA and Iran. The great protestation of black matter lives in the USA and the great assemblage of the new Iranian year, on March 21, 2020, have been the cause of the second epidemic attack in both countries.We find net discrimination between the 25 countries investigated in this report. The immediate response to the first attack is a substantial parameter to determine whether the epidemic attack will be in one wave or it can be in successive waves. For example, the best case was such as individuals in China hit by one wave while the individuals in the USA were attacked by nine waves; it is the worst case all over the globe. In addition, the model differentiates between the daily reproduction numbers . The Chinese strictly follow the decision-maker and therefore the virus hit by only one wave; while in the USA, the system-decision was different and the American-responses were different, therefore ten waves followed the first wave.The online version contains supplementary material available at 10.1186/s40001-021-00596-6. A pandemic outbreak of severe acute respiratory syndrome coronavirus 2 COVID 19) has lithely spread due to the deficiency of active vaccine and/or effective drugs. Therefore, the confinement scenario is still the most effective tool to resist the spread of COVID 19. It is an unexpected and unprecedented hit and strongly harms the human, economy, health-systems worldwide 9 has lit, 4.On the present, and despite the fact that some countries had passed the first epidemic attack, the virus extends to others. For example, countries such as China and Jorden have admitted a firm lockdown state; while others admit free economy positions, the virus hit the first couple by one wave while the others suffered several waves. We believe that the scenario: test-; trace-; isolate-strategy has proved to be effective when combined with good individual protection measures and social distancing . Due to I (t) occurs in one or successive waves depending on confinement conditions. (iii) The property of causing disease is termed pathogenicity, which is a nonmetric parameter (one cannot measure the pathogenicity). Here, we introduce a measurable parameter that can measure the virus strength as a measurable pathogenic value: “pathogenometric” rate (VSPR) of certain wave: VSPR = [I(t)/T]max where I(t)max is the maximum number infections of a certain wave, and Tmax is the period necessary to reach I(t)max. The VSPR depends on  the virus pathogenicity. This report proposes a mathematical model to obtain information about the prevalence of COVID 19 virus, in top-infected twenty countries. It reveals that the virus propagates in successive waves. The time delay in responding to the first wave has drastic effects on the prevalence and virus-control. In the present report, we present a mathematical model, which permits one to consider the total population as complex number; its real part is proportional to the confined individuals and the imaginary part represent the infected individuals. The model estimates the reproduction number, and the possible date to reach the maximum point in the wave.As of June 11, 2020, given the apparent net decrease in the number of infected individuals in some European countries, governments started to move away from the lockdown phase to return to a good level of economic activity. However, on August 15, there are some fears coming from the scientific community about a possible “second” viral epidemic . Here, wI(t) in the period from mid-November 17, 2019 until the date of writing the present report august 2020.To characterize the viral-prevalence dynamics in 25 countries, we fitted a proposed mathematical model to the published data available online , 4. The N, the number I(t) of infections represent infected individuals who can move freely. Conversely, B(t) = CB * log [N—I(t)] represents the number of individuals taking measures to resist the virus-propagation while being completely confined. The proportionality-constant, CB, is chosen to keep a similarity in the magnitude between I(t) and B(t) because B(t) is in the order of million individuals and I(t) about thousands. Both B(t) and I(t) differ in their phase: the model considers B(t) is completely confined and completely safe, while it considers I(t) as completely free and completely unsafe (infected). The model classifies the susceptible individuals as infected, I(t). Mathematically, to represent the phase difference between these two categories, one can write the total population as a complex number: B(t) is the real part, and [ωt. I(t)] is the imaginary part of N*(t), with ω, in days−1, is defined as the epidemic frequency, which is the ability of individuals to move from the infected phase to the confined phase  is assumed to rise exponentially from zero [δ) is important and drastically affects I(t). In terms of mathematical details, the total population N*(t) is related to the infected individuals as follows , as the imaginary part:We represent the number of daily infections, We index the rest of the symbols in Additional file Rd0, and the median reproduction number, R0 [Rd0 is the daily average number of people infected by one infectious individual; while R0 is the mean value of all Rd0’s on a certain period starting from zero time until achieving the maximum value of the wave, Tmax. To explain this, we consider that the viruses initially attack with high and positive VSPR values; however, the individuals defend by their natural immunities (NIs) [Tmax, where the slope is null. At this condition, the VSPR = NIs, and the number of infections is directly proportional to the mean number of infections that happened from the first infection. In addition, in order to take the exact value of daily reproduction number, one should take into consideration both the negative and positive slopes of the wave. These slopes represent the first derivative of I(t) that is given by Eq.   and/or an by Eq. , as follImax/Tmax) by the rate given by Eq.  is obtained as:Equation  gives a R0 is found in “More details about calculating I(t) values [I(t)|max India = 70,018 infections occur on August 20, 2020. A good fit between calculated and published data stand with the present model. Moreover, we have found a good fit between published and calculated values of I (t), in more than 20 different countries that support the validity of this model, which are shown in Additional file δ), the reproduction number R0, and the VSPR, while the fit parameter-values for these countries are reported in Additional file As seen in Fig. ) values , 4. The δ) and confinement conditions (CCs) vary from a country to another. Increasing δ and decreasing social distancing, enhance successive waves to hit any country. For example, Fig. I(t) varies as a function of time for the USA (2A) and Iran (2B), respectively. The solid red line represents the calculation values after Eq. (I(t) values [The time delay (fter Eq.  while th) values , 4. A go the USA A and IraRd0 in nine different countries. As seen, high initial values of Rd0 start at onset time, then, decrease exponentially with time until reaching minimum values, which occur at the maximum point of the wave Tmax. The inset in Fig. Rd0) as a function of time. The median reproduction number R0, estimated after Eq. R0 varies in vast range, starting from 1.39 in Germany up to 14.22 in Colombia.Figure With the present model, we have found that COVID 19 transmits, through certain population, in a successive wave-like prolongation. In general, the virus starts its first attack vigorously with high severity (virus strength). Here, we are in a real need to be more precise and replace the phrase “high severity (virus strength)” by a measurable quantity, which gives a direct value to the virus severity; because the virus pathogenicity is not a measurable one.I(t)max is the maximum value of the wave at time Tmax; thus, VSPR is proportional to the viral-severity and it can estimate how the virus is vigorous. This can be explained as following; as the VSPR increases the mitigating factors such as the social distancing and the NIs of the individuals (MFNIs) resist the viral-prevalence in such a way to decrease the VSPR. At the maximum point of the wave, one finds an equilibrium between the VSPR and the MFNIs. After attaining the maximum point, the VSPR decreases down to minimum values. However, the virus will produce several more successive waves if the individuals continue to disregard the MFNIs, in particular the social distancing. We report the values of VSPR for 20 countries in Table −1 (in Norway) < VSPR < 447.513 infections day−1 (in Brazil). This vast range of variations of VSPR (VSPRBrazil/VSPR Norway is about 33.5) reflects how the individuals behave regarding the social distancing. Thus, VSPR is the maximum number of infection in a certain wave divided by the maximum period to attain the maximum point; It is a rate with a value that estimates a certain proportional average of infections per day; similarly, the first derivative R0, by simple mathematical averaging of Rd0, n is the number of Rd0 values.In addition, although the production number is an essential parameter to know the rate of viral prolongation and its contagiousness, however, it does not measure the severity of a disease. Therefore, we introduce the term “virus strength pathogenometric” rate (VSPR) of a certain wave as following: mentclass2pt{minimR0 varies in the range 1.07 (in Norway) < R0 < 13.7 (in Chili). After the considerable variations in R0 from a country to another, one confirms that COVID 19 has not a constant value of R0 and it depends on the behaviour of individuals towards the first viral attack. In particular, how they consider the MFNIs, including the social distancing, thus Rd0 drastically depends on MFNIs. The early stage infections  leads to high initial values of Rd0, these values varies in the range 10 (China) < Rd0 < about 30 in India, Russia, Peru; then it decreases exponentially down to zero at the maximum point of the wave. These high values of Rd0 have been reported in the early stages of infections in diamond Princess ship [Table Rd0 as a function of time in the onset period.For example, Chinese individuals have kept the infection level constant for a relatively long time, from March 1, 2020 until now, August 21, 2020. With the present model, we can explain this constancy state. Figure Rd0 decreases exponentially during a relatively short period, from November 17, 2019, until November 27, 2019. Ten days were sufficient to reduce the daily reproduction number from more than ten down to zero. This extraordinary decline of Rd0 is a direct result of the swift reaction of Chinese people to obey the scientific plan of the decision-maker (s) regarding the social distancing. We can confirm the presence of Rd0-exponential decay by the red line in Fig. 2 = 27.66 in Germany while VSPR|1 = 96.37. One notice that, in Germany, the ratio VSPR|2/VSPR|1 = 0.29. However, in France, VSPR|2/VSPR|1 = 46.94/106.71 = 0.44 and in Italy, VSPR|2/VSPR|1 = 41.58/380.93 = 0.11.As seen, R0 taken into account the fall and rise in daily published-infections. Table R0 combined with the corresponding tolerance.We estimated the fluctuations in As all-mathematical infectious disease models, the present one constituently faces some limitations concerning the tolerance of the published data, incomplete counts, unregistered cases, delayed and insufficient tests, etc. Specifically, for the present report, the published data at the onset period are always incomplete, dispersive and unclear. The present model shows that the delay time to respond to the first wave is an essential point and therefore we have paid particular attention to the onset-published data.It is worth noting that the present model (i) can analyse only a certain type of virus that cannot change its “genetic-imprint”. (ii) The period necessary to attain the maximum number of infections should be equal to or longer than the midpoint of the wave. (iii) The model is in direct opposition to the economical productive factors and did not present a solution to the economical defects of the virus-prevalence. (iv) Contrary to the previous point, the model can search for the optimal condition to find the most effective condition that minimize the confinement factor and maximize the economic conditions.R0 will alter due to the different infectious period and contact rate.With the present model, the recovered cases, which they are sensitive to estimate, whether they will return to normal or not? This is not a problem because the recovered cases initially are considered as confined cases. Moreover, Peirlinck et al.  have notThe decision-makers across the world have differently responded to the COVID 19 pandemic; some of them considered measures, which are highly controversial; others followed an approach towards herd immunity. However, the WHO Director-General has called “herd immunity” as unethical. In fact, suitable control-measures are, extremely, needed to control viral diversity, which increases and, then, different infectious-exposures become ordinary, in particular, when the pandemic attains its highest critical point.B(t) at a certain time for different countries. In order to get reasonable data, one should take into account several limitations:(i)B(t) to the total population N(t) in certain country at certain period.One should normalize (ii)B(t) is a function of time; therefore, one should take the same period for all countries,(iii)B(t) as a function of time has, in most cases, a maximum depending on many factors including the decision-makers response, therefore we will considered the maximum value for each country.The curve of (iv)Figure (v)B(t) is proportional to the people-safety, regardless the economic effects, B(t) represents the most suitable parameter for the decision-makers after the given published data, and after Fig. (4). One can conclude that the countries are rated as: (the first is the safest): (1) China, (2) India, (3) Iran, (4) Canada, (5) Mexico, (6) Pakistan, (7) Saudi Arabia, (8) Turkey, (9) Netherlands, (10) Russia, (11) UK, (12) Germany, (13) France, (14) USA, (15) Spain, (16) Brazil, (17) Belgium, (18) Italy, (19) Peru, (20) Chili.(vi)N) plays double-contradictory role when calculating the ratio B(t)/N: The first, it decreases, mathematically, the ratio B(t)/N, which increases the possibility to have more saving conditions; while the second it can increases the infections number I(t) which decreases the possibility to have more saving conditions. In fact, one should carry out more detailed-studies to get an optimum case between these two parameters.It is worth noting that the total population  down to zero. The intent of this preliminary work is to introduce the topic of virus-prevalence in successive waves with individual confinement considered as the specific form of mitigation. The model confirms that the confinement is an effective parameter in mitigating the damage of the virus and it shows that the number of infections is drastically affected by the delay in responding to the first wave. In addition, we have found that the second epidemic attack is by far smaller than the onset attack. We have found that the second epidemic attack of the virus. In general, to resist (or even to prevent) epidemic growth, our calculations suggest some lockdowns that be maintained for two months. However, the optimum conditions between health safety and economic damage should be achieved to get the best lockdown timing.Additional file 1: Figure S1. Model building. The model classifies the total population N* into two sections. The first class is totally confined at their domicile and they are completely safe and cannot transmit the virus to the others. The second class is totally infected, free to move, and they are completely unsafe and can transmit the virus to the others. The social, economic, physical, sociological, psychiatric, etc. functions of the two classes are different. Due to the different functions, the model mathematically represents the total population as a complex number (N*). The real part is proportional to the number of confined individuals while the imaginary part represents the number of infected individuals. After the model, the confined class is passive, cannot transmit the virus via international channels; while the infected class is active, and can transmit the virus via internal and international channels. With the present model, the susceptible, the exposed, and the infectious cases are considered as infected cases; while the recovered cases are with the confined cases.


R-BPMV gene, a major resistance gene against Bean pod mottle virus in Phaseolus vulgaris. We inoculated different BPMV constructs in order to study the behavior of the R-BPMV-mediated resistance at normal (20 °C) and elevated temperatures . Our results show that R-BPMV mediates a temperature-dependent phenotype of resistance from hypersensitive reaction at 20 °C to chlorotic lesions at 35 °C in the resistant genotype BAT93. BPMV is detected in inoculated leaves but not in systemic ones, suggesting that the resistance remains heat-stable up to 35 °C. R-BPMV segregates as an incompletely dominant gene in an F2 population. We also investigated the impact of elevated temperature on BPMV infection in susceptible genotypes, and our results reveal that elevated temperatures boost BPMV infection both locally and systemically in susceptible genotypes.In the context of climate change, elevated temperature is a major concern due to the impact on plant–pathogen interactions. Although atmospheric temperature is predicted to increase in the next century, heat waves during summer seasons have already become a current problem. Elevated temperatures strongly influence plant–virus interactions, the most drastic effect being a breakdown of plant viral resistance conferred by some major resistance genes. In this work, we focused on the  R) proteins mostly represented by nucleotide-binding domain leucin-rich repeat containing receptors (NLRs) [R genes against viruses have been identified so far, as well as the corresponding viral-encoded effectors that are subsequently referred to as avirulence (Avr) factors [Among all plant diseases, viruses account for about half of all known pathogens  and conss (NLRs) . NLR recs (NLRs) . Many R  factors ,15. Othe factors  or ‘rece factors .R genes [R genes have been shown to be overcome at high temperatures, usually around 28–30 °C, allowing viral infection and spreading at the whole-plant level. This is the case for the N-mediated resistance that occurs only at temperatures below 28 °C in Nicotiana tabacum [Tsw-gene-mediated resistance that is overcome between 25 and 30 °C in Capsicum species [R genes, resistance is still efficient at temperatures above 28–30 °C, such as, for example, the Rx-mediated resistance that is not compromised against Potato virus X  at temperatures up to 32 °C [R genes. Indeed, the resistance phenotype can be modified, meaning that local necrotic lesions such as hypersensitive reaction  is a major pulse crop of agronomic importance cultivated as a dry grain or fresh vegetable. Indeed, it is the most important grain legume for human consumption worldwide, especially in developing countries such as Central and South America and Southeastern Africa [Common bean , Bean common mosaic necrosis virus (BCMNV), Bean yellow mosaic virus (BYMV), and Clover yellow vein virus (ClYVV) affect the quality and quantity of bean productions  in P. vulgaris genotype BAT93 [In the main production areas of common bean, several viruses including iewed in ). The we I locus ,40. Thispe BAT93 .R-BPMV-mediated resistance in P. vulgaris. Does the R-BPMV-mediated resistance to BPMV depend on temperature? If so, at which temperature does the phenotype switch occur? What is the inheritance of the R-BPMV gene? Here, we report that R-BPMV induces HR lesions at 20 °C. Further analysis revealed that at 25 and 30 °C, R-BPMV-mediated resistance still induces HR lesions, whereas at 35 °C, local HR lesions are replaced by chlorotic lesions but resistance is not overcome at the whole-plant level. Finally, we also investigated the impact of elevated temperature on BPMV infection in a compatible context , and our results highlighted that rising temperature boosts BPMV infection.The aim of the current study was to address several questions concerning the Phaseolus vulgaris were used in this study: BAT93 (Mesoamerican breeding line), JaloEEP558, Black Valentine (both Andean landraces), and two Near-Isogenic Lines for the I locus [I/I, resistant to BCMV and BCMNV) and Black Turtle 2 . The inheritance of R-BPMV was studied using 60 F2 individuals derived from a cross between BAT93  and the Andean landrace JaloEEP558 .The following genotypes of  I locus , Black TGrowing conditions from sowing to virus inoculation were followed as described in Pflieger et al.  with somAn infectious BPMV cDNA clone derived from isolate IA-Di1 was provided by C. Zhang and S. Whitham  and described previously . BrieflyP. vulgaris plants was performed as described previously [P. vulgaris cv. Black Valentine with a mortar and pestle in the presence of a mock buffer  [Viral rub-inoculation of eviously ,44. Briew/v trypan blue) in universal tubes and heated in a boiling water bath for 2 min. After cooling, the solution was replaced with chloral hydrate (2.5 g/mL), and samples were shaken until leaves were fully destained. For observations, the chloral hydrate was replaced with 60% glycerol.For HR assays, the leaves of 7 days post-inoculation (dpi) plants were stained with trypan blue in lactophenol solution (lactic acid:glycerol:liquid phenol:distilled water (1:1:1:1), 0.067% GFP fluorescence in whole plants was detected by using a Black Ray long-wave UV lamp . Higher-magnification fluorescence detection was performed with an epifluorescence microscope for temperature assays at 20 and 25 °C  equipped with a fluorescein isothiocyanate–tetramethyl rhodamine isothiacyanate multiband filter. The GFP fluorescence specter was checked using a confocal microscope . For temperature assays at 30 °C, we used an Axio Zoom V16 fluorescence stereomicroscope . Specific GFP fluorescence was detected using a short-pass filter . To detect all UV-fluorescent cell components including chlorophyll and GFP, a long-pass filter  was used. Images were obtained using a digital video camera (Axiocam 506 mono) coupled to the Axio Zoom microscope.TM enzyme  and Oligo-dT  according to the manufacturer’s protocol and finally diluted 2.5 times in Milli-Q H2O.For BPMV RNA detection, inoculated and systemic leaves from BPMV-inoculated plants were sampled at 7 and 14 or 21 dpi, respectively. Total RNA was extracted using the NucleoSpin RNA kit . RNA concentrations were determined by measuring the absorbance at 260 nm on a NanoDrop 8000  and integrity was checked by electrophoresis on a 1% agarose BET gel. cDNA was synthesized from 1 µg of total RNA using Reverse Transcription (RT) ImProm-IIPvUBIQUITIN , and PvINSULIN-DEGRADING ENZYME (PvIDE) , as described previously [PvUBI and PvIDE, PCR products were analyzed by electrophoresis on a 2% agarose BET gel.Semi-quantitative RT-PCR was performed on 1 µL of diluted cDNA using GoTaq G2 Flexi  and using primers specific to BPMV RNA1, RNA2, eviously . After ePvIDE mRNA, in four biological and three technical replicates (unless otherwise stated), in order to obtain a ratio of virus RNA to plant RNA. RT-qPCR protocol and analyses were performed as described in Richard et al. [® 96 instrument in a volume of 15 µL reaction containing 2 µL of diluted cDNA, each specific primer with a final concentration of 0.1 µM, 7.5 µL of SYBR Green , and distilled water. The program used consisted of initial denaturation at 95 °C for 5 min and 50 cycles of 15 s of denaturation, 15 s of hybridization, and 15 s of elongation at 95, 60, and 72 °C, respectively. The results were analyzed using LightCycler® 96 software version 1.1.BPMV virus titer was estimated using a method based on a quantitative RT-PCR (RT-qPCR) analysis by determining the quantity of BPMV RNA1 and plant d et al. . BrieflyR-BPMV [R-BPMV gene  xJaloEEP558 (susceptible to BPMV). Each F2 plant was inoculated with the construct BPMV-GFP, and resistant/susceptible phenotypes were scored at 7 to 10 dpi. The observed segregation ratio was 56 resistant: 4 susceptible. The goodness-of-fit test indicated a significant deviation  from the expected Mendelian ratio (3:1) with a deficit of susceptible phenotypes. Thus, the R-BPMV gene segregates as an incompletely dominant gene.To determine the inheritance of R-BPMV is closely linked to the I gene at one end of chromosome 2 of P. vulgaris [I-mediated resistance is associated with a systemic necrosis that may kill the host when infected by either BCMV above 28 °C or BCMNV regardless of the temperature [I gene in response to BCMV infection is temperature-dependent and switches at 28 °C from extreme resistance to systemic necrosis. In order to check if the resistance mediated by R-BPMV is dependent on temperature, we studied the resistance phenotype at 25 and 30 °C. We first performed inoculation assays using BPMV-WT on the resistant genotype BAT93. We observed that the resistance phenotype is expressed as local HR lesions in the inoculated leaves at all tested temperatures but that HR lesions become more expanded as the temperature increases  or Axio Zoom microscope (30 °C). JaloEEP558 was used as a susceptible genotype . As expeR genes against viruses are overcome at elevated temperatures  [R-BPMV-mediated resistance was still efficient at temperatures up to 30 °C, but we wanted to test whether higher temperatures (35 °C) could break the resistance conferred by R-BPMV. For this purpose, we inoculated BAT93 plants with BPMV-WT and BPMV-GFP and placed the plants at constant 35 °C. As expected, at 7 dpi, HR lesions developed on inoculated leaves of BAT93 + BPMV-WT at 30 °C, and GFP fluorescence was macroscopically detected on inoculated leaves of BAT93 + BPMV-GFP (It is known that some dominant for Tsw) ,23,49, mBPMV-GFP A. By conBPMV-GFP A. QuantiBPMV-GFP B,C.R-BPMV is heat-stable up to 35 °C. The R-BPMV-mediated phenotype is expressed as chlorotic lesions in the inoculated leaves, and no symptoms are detected in the systemic parts.To ensure that no virus moved systemically, we studied the systemic leaves of BAT93 plants grown at 35 °C and inoculated with either BPMV-WT or BPMV-GFP. No viral RNAs were detected in systemic leaves at 7 dpi, and no GFP fluorescence was visible D,E, atteIn the resistant genotype BAT93, elevated temperatures  promoted viral replication and cell-to-cell movement in the inoculated leaves A. TherefFurthermore, the effect of temperature on the rate of systemic spreading of BPMV-GFP was investigated by scoring the date of appearance of GFP foci on systemic leaves of Black Valentine plants at 20, 25, and 30 °C between 6 and 14 dpi. From 6 to 10 dpi, the systemic spreading is faster at 30 °C compared to 25 °C, and at 25 °C compared to 20 °C C. At 10 R-BPMV gene is closely linked to I, a famous locus conferring a broad-spectrum resistance to at least ten different Potyviruses  and BT-2  are two near-isogenic lines created to study the I locus [R-BPMV gene at the tissue and cell levels, as done for the I locus [The iewed in ) and sev I locus . We test I locus ,51,52. WR-BPMV-mediated resistance at the cellular level.At 7 dpi, local HR lesions developed on inoculated leaves of BT-1 and BT-2 at 20 and 25 °C . As in BR-BPMV-mediated resistance to BPMV in P. vulgaris at normal (20 °C) and elevated temperatures . Strikingly, we found that R-BPMV-mediated resistance is heat-stable up to constant 35 °C at the whole plant level  are not [Rx1 gene was reported to confer a temperature-insensitive resistance to PVX in Nicotiana benthamiana that is not overcome at temperatures up to 32 °C, a temperature at which the virus was no longer infectious [R-gene-mediated resistance has also been reported for pathogens other than viruses. For instance, the tomato Mi-9 gene confers a heat-stable resistance to root-knot nematodes (Meloidogyne sp.) [Mi-1, which is heat-sensitive [R genes against Meloidogyne species, named Me, have been reported [Solanum species, stoRy and chc RRy genes present in S. stoloniferum and S. chacoense, respectively, confer extreme resistance to the Tobacco veinal necrosis strain of PVY (PVYN) and are functional at both low (16–20 °C) and elevated temperatures (above 24 °C) [This is in agreement with results obtained for other plant–virus pathosystems for which heat-stable  are not . More refectious . Additioyne sp.) ,55, despensitive . In peppreported ,58. In Se 24 °C) ,60.N is unable to confer resistance to the Tobacco mosaic virus (TMV) above 28 °C [Capsicum sp. NLR Tsw fails to trigger resistance to the Tomato spotted wilt virus (TSWV) at 32 °C and above [R genes conferring HR to PVY including Ny in Solanum sparsipilum and S. sucrense, or Ny-1 in S. tuberosum cv. Rywal, confer resistance only at low temperatures (16–20 °C), whereas at higher temperatures (24–28 °C), resistance is inhibited and PVY infects plants systemically [Mi-1 in tomato , for thcatoria, ), the no fulvum, ,65), as yringae, ; RPS2 anyringae, ; RPS4/RRacearum, ; and SNCasitica, ,70,71). N gene, resistance was effective at elevated temperature, thus suggesting that these mutations may prevent the temperature-sensitive conformational loss of function of the NLR N [R-BPMV and I genes, and one way to confirm this hypothesis would be to clone their corresponding gene sequences. Other putative mechanisms of temperature sensitivity have been proposed such as homeostasis of R-interacting chaperons, reduced R protein amounts resulting in a lower R activity, and deregulation of nucleo-cytoplasmic localization of R proteins . More riewed in  and dereiewed in  have alsR-BPMV in P. vulgaris genotype BAT93 confers resistance to BPMV by inducing local HR lesions at 20 °C, where BPMV is able to multiply in a first step (Turnip crinkle virus (TCV) was impaired in Arabidopsis eds5 and sid2 SA-deficient mutants without affecting HR cell death [Potato virus Y  spread is even faster in SA-depleted plants (NahG-potato transgenic plants) with rapid lesion expansion. Interestingly, the expression of SA-dependent responses was reduced at elevated temperature in tobacco and potato plants infected with Cucumber mosaic virus (CMV) and PVY, respectively [Ny-1 R gene against PVY, temperature shift assays from 22 to 28 °C induced the detection of PVY in infected cells outside the cell death zone [Our results show that the major gene rst step . HR lesirst step . Interesrst step  and (ii)rst step B,C. Consll death . Lukan ell death  showed tectively ,80. Whetectively ,85,86,87ath zone . Neverthath zone .R-BPMV-mediated resistance remains functional at the whole-plant level (Glycine max) infected by strains G1 and G7 of Soybean mosaic virus (SMV) [R genes Rsv1 and Rsv1-n. The STN in genotype V262 induced by strain G1 and in genotype V94-3971 by strain G7 developed at 10, 15, 20, 25, and 30 °C, on 44, 41, 14, 8, and 8 days after inoculation, respectively. By contrast, at 35 °C, no STN reaction and, consequently, no cell death was induced by any strain. Interestingly, cell death reduction at elevated temperature has also been reported for plant–bacteria interactions. AvrRpt2-mediated cell death in Dex-AvrRpt2 plants was significantly reduced at 28 °C and was almost completely abolished at 32 °C [WORKY46 transcription factor acting as an upstream regulator of SA metabolism was also temperature-sensitive with a peak at 16 °C and a decrease at temperatures above 28 °C. In the same way, AvrRpm1- and AvrB-mediated cell death was significantly attenuated at elevated temperatures (32 °C) [R-gene-mediated response.Interestingly, at 35 °C, HR lesions that developed on BPMV-inoculated leaves of BAT93 were replaced by chlorotic lesions, while the nt level . We confnt level . Thus, int level E. The reus (SMV) . Indeed,at 32 °C . Interes (32 °C) . Thus, tR-BPMV is present in genotype BAT93 of P. vulgaris and that this gene is genetically linked to the I locus on chromosome 2 [I locus is a broad-spectrum resistance locus that confers resistance to BCMV, BCMNV [Watermelon mosaic virus-2 [Cowpea aphid-borne mosaic virus [Soybean mosaic virus [Peanut mottle virus [Zucchini yellow mosaic virus, Thaïland passiflora mosaic virus, and Passionfruit woodiness virus-K [R-BPMV and I could correspond to the same gene with pleiotropic effects and if so, one important question would be: do R-BPMV- and I-mediated resistance have the same behavior regarding temperature?We previously showed that mosome 2 ,90. The V, BCMNV ,92, and  virus-2 , Cowpea ic virus , Soybeanic virus ,97, Peanle virus ,99, Zucc virus-K , BYMV, a virus-K ,102). ThI was reported to be temperature-sensitive for BCMV resistance [R-BPMV that occurs between 30 and 35 °C  and I segregate as incompletely dominant genes [R-BPMV in an F2 population of 60 plants, we found a deficit of susceptible phenotypes (56 resistant: 4 susceptible) and thus, a significant deviation from the expected Mendelian ratio (3:1). A similar segregation deviation was reported for the I gene in an F2 population obtained from the cross Calima (susceptible Andean genotype) × Jamapa (resistant Mesoamerican genotype) [Finally, it is worth noting that both nt genes ,51,91. IP. vulgaris-BPMV compatible interaction, we performed experiments on two BPMV-susceptible genotypes P. vulgaris cv. Black Valentine and cv. JaloEEP558. We reported that in both genotypes, the area of primary infection sites at 7 dpi was larger on BPMV-GFP-inoculated leaves at 25 and 30 °C compared to 20 °C, and these results were correlated with a higher virus titer in these leaves (Turnip mosaic virus (TuMV) in Chinese cabbage. It took 48 days for systemic infection to occur at 13 °C but only 6 days at 22–33 °C. Likewise, in Solanum tuberosum, plants became 100% systemically infected at 24 and 28 °C, while at 20 °C, only 20% of the plants were systemically infected [Barley yellow dwarf virus . Infected plants grown under an elevated temperature developed virus symptoms earlier and had higher virus titers than plants grown at an ambient temperature. In potato, susceptibility to PVY was dramatically increased in systemic infected leaves at higher temperatures [N. benthamiana, plants infected with the potyviruses PVY and Potato virus A had fewer symptoms and reduced CP protein accumulation at 20 °C compared to 30 °C [N. benthamiana infected with PVY and PVX and showed attenuated symptoms for PVY and no symptoms for PVX at 30 °C compared to 25 °C. Aguilar et al. [Plum pox virus (PPV) in N. benthamiana. Importantly, the RNA silencing machinery was shown to be more active at elevated temperatures in several plants including cassava and N. benthamiana [P. vulgaris-BPMV.To study the effect of elevated temperatures in a e leaves A,B. Moree leaves C. Overalinfected ,27. Nanceratures . In casseratures . Neverthto 30 °C . In the r et al.  describethamiana ,106, sug2 for example) to analyze whether their effects are additive or antagonistic. In C3 plants like P. vulgaris, high CO2 improves photosynthesis and activates hormonal pathways, whereas high temperatures decrease the efficiency of Rubisco and the solubility of CO2. Consequently, combined high CO2 and elevated temperatures may induce antagonistic effects. At the international level, little work has been published on the combined effects of elevated temperatures and high CO2 on plant–virus interactions (reviewed in [2 level (970 versus 405 ppm)) by studying CMV, PVY, and PVX in the model plant N. benthamiana. They showed that viral titers in systemic leaves under CCC showed no significant differences at 7 and 12 dpi for CMV. Conversely, viral titers of PVY and PVX were significantly lower under CCC compared to the standard condition at both 7 and 12 dpi. In the case of PVY, viral titer was lower at 12 dpi compared to 7 dpi under CCC, and the contrary was observed for PVX [S. tuberosum), Chung et al. [Potato leaf roll virus (PLRV) RNA accumulated at higher levels, and larger numbers of potato plants were infected by PLRV under combined high CO2 levels (940 ppm) and elevated temperature (30 °C). More recently, Aguilar et al. [A. thaliana (compatible context) and N. benthamiana plants (incompatible context). They demonstrated that infection by the PVX/PPV virus combination induced resistance to the bacteria P. syringae pv. tomato and to drought in both compatible and incompatible host–bacteria interactions. However, combined high CO2 levels (970 ppm) and elevated temperature (30 °C) negatively affected resistance to Pst and to drought induced by a virus infection, and this correlated with diminished H2O2 production, decreased expression of defense genes, and a drop in virus titers.Global warming leads to rising temperatures, which alter plant–virus interactions , potentiiewed in ). Del Toiewed in  focused  for PVX . In potag et al.  demonstrr et al.  studied To conclude, our study gives a first insight into the impact of elevated temperature on the level of resistance/susceptibility to viruses in common bean. Indeed, natural genetic resistance is often the most effective and environmentally friendly way of controlling plant diseases, in particular against a virus where no chemical alternative is available. This research needs to be continued in order to decipher the underlying cellular and molecular mechanisms of heat tolerance and to engineer robust thermostable resistances in the context of global warming.


Bats are known to be reservoirs of several highly pathogenic viruses. Hence, the interest in bat virus discovery has been increasing rapidly over the last decade. So far, most studies have focused on a single type of virus detection method, either PCR, virus isolation or virome sequencing. Here we present a comprehensive approach in virus discovery, using all three discovery methods on samples from the same bats. By family-specific PCR screening we found sequences of paramyxoviruses, adenoviruses, herpesviruses and one coronavirus. By cell culture we isolated a novel bat adenovirus and bat orthoreovirus. Virome sequencing revealed viral sequences of ten different virus families and orders: three bat nairoviruses, three phenuiviruses, one orbivirus, one rotavirus, one orthoreovirus, one mononegavirus, five parvoviruses, seven picornaviruses, three retroviruses, one totivirus and two thymoviruses were discovered. Of all viruses identified by family-specific PCR in the original samples, none was found by metagenomic sequencing. Vice versa, none of the viruses found by the metagenomic virome approach was detected by family-specific PCRs targeting the same family. The discrepancy of detected viruses by different detection approaches suggests that a combined approach using different detection methods is necessary for virus discovery studies. With more than 60 million years of evolution they belong to the oldest mammals we know today6. Furthermore, bats are often discussed as being special in the way they react to infections. Bat immunology is aiming to shed light on the very special way bats and pathogens coevolve9. Numerous publications and studies are focusing on virus discovery in this very distinct mammalian order and accelerate the publication of thousands of novel viral sequences. While mainly family-specific PCR approaches were used to detect novel virus strains during the first years of virus discovery in bats, next generation sequencing approaches have gained in significance in recent years. The metagenomic view on viruses (virome) supports the simultaneous detection of viruses in individual or pooled samples within a very short time. However, for samples with a high host nucleotide background it became apparent that some sort of purification procedure might be necessary for NGS approaches to increase significantly the number of obtained viral sequences from biological samples. Various protocols have been published to address this problem12.Bats have been recognized as potential reservoir host of several highly pathogenic viruses like Hendra virus, Nipah virus, Marburg virus and SARS-CoV viruses25  sequence is described here for the first time. No arenaviruses, filoviruses, nairo- and phenuiviruses, hantaviruses and flaviviruses were detected by PCR.The PCR screening resulted in the detection of several novel viral sequences for paramyxoviruses, adenoviruses, herpesviruses and coronavirus Table . Sequenc.35. Bat number E210/09 (Pipistrellus pipistrellus) was found to be positive for CoV in the intestinal sample. Phylogenetic analysis revealed that the sequence was most closely related to alphacoronaviruses of group 1  or shared 100 percent identity to sequences obtained before (possible cross-contamination). Supplementary Table 38, MEGAN6 and further analysis. In Fig. Sequencing of the nine bat pools via Illumina HiSeq revealed 127,338,644 reads after trimming. In Fig. Parvoviridae, Picornaviridae, Totiviridae, Mononegavirales, Reoviridae, Bunyavirales, Tymovirus, Retroviridae and phages) were confirmed within the bat samples by virome sequencing and further analysis. For each family, selected viruses were retested by specific PCRs in the individual bats’ organs. For the viruses of highest interest, phylogenetic reconstructions were calculated as described in the methods section.Viruses of nine different families and orders  and Parvovirinae . The virus sequences were named after the order of appearance and in relation to reference strains: Blatella Germanica densovirus-like virus 1, Blatella Germanica densovirus-like virus 2, Blatella Germanica densovirus-like virus 3, Parus major densovirus-like virus and Bat bufavirus .Parvoviruses (n = 5) were found in pools 1, 2, 3 and 9 belonging to the subspecies Picornaviruses (n = 7) were found in pools 1, 2, 3, 4, 5, 7 and 8 , all of which were related to King virus UWV1, King virus UWV2, Tetnovirus 1 or Hubei Picorna-like virus. The virus sequences were named after the order of appearance and in relation to reference strains: King virus UWV1-like virus, King virus UWV2-like virus 1, King virus UWV2-like virus 2, King virus UWV2-like virus 3, King virus UWV2-like virus 4, Tetnovirus 1-like virus and Hubei Picorna-like virus. Obtained sequences are available under the following accession numbers: MN851294, MN851280, MN851281, MN851282, MN851283, MN851284 and MN851286.One totivirus was found in pool 2  which appears to be most closely related to Eimeria tenella RNA virus 1. The virus sequence was named in relation to the reference strain: Picorna Eimeria tenella virus 1-like virus. The obtained sequence is available under the following accession number: MN851287.Mononegavirales is displayed in . The obtained sequence is available under the following accession number: MN851288. Phylogenetic reconstruction of 665 nt (polymerase) Wenzhou tapeworm virus-like virus in comparison to the reference strains of 30. Obtained sequences are available under the following accession numbers: MN851289, MN851290 and MN851291. Phylogenetic reconstruction of orbiviruses (780 nt) and rotaviruses  in comparison to the two novel sequences is displayed in Figs. Reoviruses n = 3) were found in pools 2, 5 and 8  and include related reoviruses of three distinct genera orbivirus (related to Bat orbivirus China—unpublished AccNo. MH144554.1), rotavirus (related to Human rotavirus A-like virus) and orthoreovirus (Bat orthoreovirus T3/Bat/Germany/342/08). Bat orthoreovirus T3/Bat/Germany/342/08 had been isolated before from the same sample set; no further analysis was conducted here were fouNairoviruses (n = 3) were found in pools 3, 5 and 6 (8132 reads). The found virus sequences share the highest identity with Issyk-Kul virus, Sapphire II virus and Avalon Bres virus. Obtained sequences are available under the following accession numbers: MN851301, MN851292 and MN851296. Phylogenetic reconstruction of Sapphire II-like virus and Issyk-Kul-like virus with other members of nairoviruses  is displayed in Figs. Phenuiviruses (n = 3) were found in pools 3 and 8 (8061 reads). The found virus sequences share the highest identity with the strains Laurel Lake virus (genus Laulavirus) (n = 2) and Malsoor virus/SFTS (n = 1) (genus Banyangvirus). Obtained sequences > 200 nt are available under the following accession numbers: MN851297 and MN857542. Phylogenetic reconstruction of Malsoor-like virus with other members of phenuiviruses  is displayed in Fig. Tymoviruses (n = 2) were found in pools 3 and 5 (423 reads). The found virus sequences share the highest identity with the strains Bombyx mori latent virus and Grapevine Red Globe virus. The virus sequences were named after the order of appearance and in relation to reference strains: Bombyx mori latent-like virus and Grapevine Red Globe-like virus. Obtained sequences are available under the following accession numbers: MN851298 and MN851299.Myotis brandtii endogenous retrovirus, Feline endogenous virus FERV1, and Myotis lucifugus endogenous retrovirus. The virus sequences were named after their host and in relation to reference strains: Pipistrellus pipistrellus endogenous retrovirus, Myotis myotis endogenous retrovirus, Myotis daubentonii endogenous retrovirus. Obtained sequences are available under the following accession numbers: MN851300, MN851302 and MN851303. Phylogenetic reconstructions for Myotis brandtii endogenous retrovirus-like sequence and Myotis lucifugus endogenous retrovirus-like sequence are available on request.Retroviruses (n = 5) were found in pools 1, 6 and 9 (234 reads). The found viral sequences share the highest identity with the strains Myoviridae and Podoviridae, followed by the Siphoviridae.Numerous phages were found in all pools . Most of the phages belong to the Of all viruses identified by family-specific PCR in the original samples, none was found by metagenomic sequencing. Vice versa, none of the viruses found by the metagenomic virome approach was detected by family-specific PCRs targeting the same family. The initially isolated reovirus T3/Bat/Germany/342/08 was identified by virome sequencing within the original sample.39. Table Virome sequencing of German bat carcasses of 16 bat species resulted in a high variety of confirmed viral sequences of parvoviruses, picornaviruses, totiviruses, reoviruses, nairoviruses, phenuiviruses, tymoviruses, retroviruses and several phages. In addition to the confirmed viral sequences, several sequencing reads were identified that shared a high homology and identity to other viruses. These high-identity reads were excluded as they were very likely false-positive results (i.e. poxviruses)  and Gossas virus (KR534878)41. Other nairoviruses of bats have been described and form two monophyletic genogroups within the nairoviruses, Keterah and Kasokero41.Six Bunyaviruses were identified in this study (nairoviruses and phenuiviruses). Eptesicus nilssonii bats in pool 3 were infected with a yet undescribed Issyk-Kul virus strain , Sapphire II virus  and Avalon Bres virus . Nine out of twelve Pipistrellus pipistrellus bats from pool 5 and confirmed predominantly in lung and spleen tissues  after the origin of the corresponding bats.Sapphire II-like virus was detected in eleven Pipistrellus pipistrellus bats of pool 6  after the origin of the corresponding bats.Avalon Bres virus-like sequence was detected in Bunyavirales40. The majority of the ten phenuivirus genera is mosquito-borne; however, some genera are transmitted by ticks (i.e. Banyangviruses) and are capable of causing severe diseases in humans and animals. Three phenuiviruses (genus phlebovirus) have been reported to be identified from bats: Malsoor virus, Rift Valley virus and Toscana virus43. Malsoor virus was isolated from Rousettus leschenaultii in India and is by phylogenetic reconstruction monophyletic with viruses of the genus Banyangvirus, being related to Huaiyangshan banyangvirus (former SFTS) and Heartland virus which are capable of causing severe diseases in humans48. Here we describe the detection of three phenuiviruses related to Laurel Lake virus (Id 43% aa) (genus Laulavirus) and Malsoor virus /SFTS  (genus Banyangvirus).Phenuivirus is a family within the order Eptesicus nilssonii bats from pool 3 were tested positive for Malsoor-like virus in liver, spleen, lungs and intestines  virus, Heartland virus and Malsoor virus  after the origin of the corresponding bats. We named the Laurel Lake-like virus 2 Munich bat lalavirus (MblV) after the origin of the corresponding bats.Laurel Lake-like virus 1 and Laurel Lake-like virus 2 (genus Laulavirus) were identified in bats from pool 8  and are capable of replication in several arthropod and vertebrate hosts51. In pool 2 an orbivirus related to the yet unpublished Bat orbivirus from China (AccNo. MH144554.1) (Id 81% aa) and Sathuvachari virus was identified in Nyctalus noctula bats (Supplementary Table 52. There have been two further orbiviruses identified from bats (Myotis rickettsi and Rhinolphus ferrumequinum) in China (Acc. No. KX343070.1 and KX161703.1). We named the Sathuvachari-like virus Irlbach bat orbivirus (IboV) after the origin of the corresponding bats.Three reovirus sequences  were identified in the virome data of pools 2, 5 and 8  after the origin of the corresponding bats.The bat rotavirus identified in this study was detected in pool 5 which comprises tissues from Mononegavirales was identified in intestinal samples of Nyctalus noctula bats in pool 2  and Midway virus55. Phylogenetically the strain was grouped distinct from Midway virus and was more closely related to Borna disease virus and rabies virus. The similarities on nt level are quite low and the tree has to be interpreted with caution (SF2). However, these findings are interesting as bats are often discussed as potential reservoir host of many viruses of the order Mononegavirales . We named the Wenzhou tapeworm 1-like virus Berlin bat mononegavirus (BbmV) after the origin of the corresponding bats.A sequence related to other members of the order 32. The respective tissues were subjected to virus purification and virome sequencing within pool 4, but no paramyxoviruses were detected in the virome data. A possible explanation could be that the purification method we have used, TUViD-VM, did not sufficiently purify paramyxoviruses from tissues; but this is exactly what this protocol was designed for10. Actually, the whole protocol was validated and tested with a range of viruses , with the most remarkable results from paramyxovirus (Sendai virus)10. In addition, we have tested the reproducibility and consistency of the TUViD-VM protocol numerous times and found it to be very stable56. Furthermore, it has to be considered that we have used pooled tissues for our examinations instead of i.e. single organs. It is possible that this may have affected the detectability of the different viruses. However, we have used pooled tissues for all methods compared and therefore results should be comparable. Taking this into account, we propose that nothing is more important in virome studies than the ratio between targeted and untargeted sequences. For instance, if we apply a family-specific PCR to a mixture of cDNA containing 100 genome equivalents of the targeted paramyxovirus, we will most probably be able to obtain a clean and sufficient amplification of the targeted sequences, although the cDNA contains only the 100 genome equivalents of paramyxovirus over millions of other sequences. In comparison, the approach of virome sequencing differs significantly. When millions of other sequences compete (e.g. host genome) the likelihood of detection of an underrepresented number of viral sequences would be very low. To address and circumvent this obvious fact, the TUViD-VM protocol was applied in this study, as it aims to decrease the amount of background (host) sequences and amplify the viral sequences to increase the detection ratio and likelihood. One accompanying effect we observed was that high numbers of viral reads of other viruses  impeded the sequencing of low amounts of other viral amplicons. Similarly, the analyses of PCR-negative results revealed a surprising outcome. All bats were screened for the presence of bunyaviral sequences, especially for phenuiviruses and nairoviruses, by family-specific PCR with negative results. Interestingly, virome sequencing revealed the presence of six novel strains of bunyaviruses with identities ranging from 70 to 95 percent identity on nt level to known virus sequences.The screening of 375 bats with numerous family-specific PCRs resulted in several novel sequences Table . The inoThis leads to the conclusion that the outcome of virus discovery studies, whether approached by PCR, metagenomics or cell culture, provides only a fraction of the viruses truly present in a sample. The methods should be used complementarily to give a more complete picture of virus diversity rather than to replace each other—as has often been described for the advantages of metagenomics over classic PCR approaches. The combined data of existing individual studies investigated by different virus discovery protocols with their conclusions are further used to extrapolate and predict outbreak risks and hotspots. As long as we use single virus detection protocols in studies only, the very true diversity of viruses will stay hidden, unrecognized by our insufficient methodological approaches. It might be time for common approaches, where scientists doing virus discovery and modeling work join forces and design studies in a more comparable and meaningful manner.https://ec.europa.eu/environment/nature/legislation/habitatsdirective/) and the Agreement on the Conservation of Populations of European Bats (www.eurobats.org), investigative research on bats was permitted by local government bodies38. We named the manuscript Virome of German Bats as the bats were found in Germany. However, bats are migrating animals, many of them covering long distances throughout Europe. Therefore, our results could also be expected in other parts of Europe. We herewith confirm that all permits to investigate carcasses of deceased bats and all experimental protocols on the dead bats were approved by the respective local governmental authorities . We herewith further confirm that all experiments were performed in accordance with relevant guidelines and regulations. As part of a study on diseases in native bats38, 375 bats of 18 vespertilionid bat species were examined . Animals were found dead, injured or moribund near roosting sites or human habitations38 in urban and suburban areas of different regions in Italy (n = 1) and Germany (n = 374) . All bat carcasses were kindly provided by bat researchers and bat rehabilitation centers from the different geographic regions. In case bats died in care or had to be euthanized for medical reasons, the carcasses were handled as described before59. Aliquots of the individual organs were divided between two tubes: one tube with RNAlater®  for RNA extraction and one tube native frozen at − 80 °C and sent to the Robert Koch Institute for virological examination  PCR screening, (2) Virus isolation via cell culture and (3) Virome analysis. Every section had its own difficulties and individual steps for sample preparation explained in the following.32. The pool containing 240 µl of organ homogenate was then extracted using the PureLinkTM Viral RNA/DNA Mini Kit . Extracted RNA was transcribed to cDNA by using the TaqMan® Reverse Transcription Reagents . The cDNA pools of 375 bats were screened by differing family-specific PCR systems for the detection of paramyxoviruses60, arenaviruses61, coronaviruses35, filoviruses62, flaviviruses63, hantaviruses64, nairo- and phleboviruses65 and influenza viruses66.30 µl of each of the eight individual organ homogenates (in RNAlater®) of one respective bat were pooled . RNA extraction from RNAlater®-stabilized samples and cDNA synthesis was performed as described before33. The DNA pools of 375 bats were screened by differing family-specific PCR systems for the detection of adenoviruses67, herpesviruses68 and poxviruses70.30 µl of each of the eight individual organ homogenates (native frozen at − 80 °C) of one respective bat were pooled . DNA was extracted using the PureLinkTM Viral RNA/DNA Mini Kit (Invitrogen) as described before31. For cell culture isolation different cell culture systems were used; these are listed in Supplementary Table Virus isolation in cell culture was performed from pools containing all eight individual organs of the respective bats natively frozen at − 80 °C as described before71. Several bats were found to have alterations that may be related to viral infections . Due to the large number of individual samples, organs were allocated to nine pools for virus purification with TUViD-VM and sequencing10. Pools were established using the following criteria: (a) Pools should consist of similar sample numbers, (b) Pools should have only one bat species and (c) If number of bats is not sufficient to build a single pool, species are mixed. Table Selected single organs (native frozen at − 80 °C) of 118 of the 375 individual bats were subjected to further investigation. These 118 bats and their corresponding 189 native frozen organ tissues were chosen by analysis of the pathological findings73. The original TUViD-VM purification protocol used in this study comprises the following steps: (1) homogenization, (2) clearing centrifugation, (3) ultracentrifugation through sucrose cushion, (4) ultracentrifugation, (5) digestion, (6) extraction, (7) cDNA and double strand synthesis and (8) random amplification and size selection via agarose gel electrophoresis10. Instead of using 200 µl of tissue homogenate as prescribed in the original protocol, 3 ml of organ tissue was used and the protocol was upscaled with small modifications10: in most pools the homogenate volume exceeded 3 ml; however, in case the amount of homogenate was too low, the volume was adapted with PBS to 3 ml; for ultracentrifugation, the rotors and pace were adapted accordingly by using the SW32 rotor  also for the first ultracentrifugation step. From step (4) of the original TUViD-VM protocol onward, the protocol was performed without any further modifications.200 µl of each individual organ from the 118 bats was pooled into nine differing pools, depending on histopathological results and according to the species. The composition of the individual pools is given in Table 10. The sequencing reaction was performed on the Illumina HiSeq Sequencer in high throughput mode using 150 cycles.After size selection by excision of the area between 500 and 1000 nt following agarose gel electrophoresis, samples were prepared for sequencing. Libraries were built using the NexteraXT protocol as described before74. Contigs were then compared to the viral protein database (nr NCBI) via the DIAMOND tool and results were visualized in MEGAN6 (–sensitive)38. Selected results from MEGAN6 were blasted using BLASTx on the NCBI GenBank database. Full genomes (if available) of the most similar sequences were downloaded to serve as a reference. The initial trimmed reads were then mapped to the reference sequence using Bowtie2 to identify more matching reads and check the correct allocation visually for quality75. Virus genomes were also assembled using Bowtie2. Sequences were checked for plausibility by analyzing the closest related virus. In the case of i.e. retroviruses the closest related sequences were also from bat species, proving that they are possible to occur. Sequences of highest interest were extended by spanning PCRs and primer walking followed by Sanger sequencing. For all sequences of interest specific primers were designed to confirm the sequences in cDNA by conventional PCR of the individual extracted pools and organ samples eventually. Cycling conditions and primers are available on request. Any bands of targeted height were purified, Sanger sequenced and compared to the original sequence obtained from Next Generation Sequencing (NGS).Raw data was processed as follows Fig. : Adapter76. Model of evolution was predicted via jmodeltest and the model with the best AIC score was picked77. The actual reconstruction was performed via the Bayesian MCMC approach using MrBayes with the following settings 78. Reconstructed trees were visualized using FigTree and posterior probabilities were depicted for each node (http://tree.bio.ed.ac.uk/software/figtree/).Final sequences obtained from data analysis and Sanger sequencing were used to reconstruct phylogenetic trees. Sequences were aligned to type species (whenever possible) of the respective viral family (ICTV database) and other related viruses of interest via ClustalW. Alignment quality was checked with the online tool T-CoffeeSupplementary Information.


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. Average 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.The online version contains supplementary material available at 10.1007/s10803-021-04955-4. Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder with a prevalence of up to 1%, which usually manifests in early childhood  are scaGiven the lack of real-life user data from European countries on service use and related costs in individuals with ASD—especially in adults, this study aimed to estimate health service use and associated costs in a validly diagnosed sample of German children, adolescents and adults with ASD with and without intellectual disability.This research study was conducted within the framework of the ASD-Net, a German clinical and research network that focuses on key challenges in ASD diagnostics, therapy and health service research . All participants had been diagnosed by experienced clinicians using the current diagnostic gold standard in ASD, including the Autism Diagnostic Observation Schedule (ADOS), and the Autism Diagnostic Interview-Revised (ADI-R)  and health service utilisation was assessed through a questionnaire which included a modified version of the Client Service Receipt Inventory (CSRI). The CSRI is a well-established semi-structured interview, in which participants are asked about health services used over the preceding 12 months . Patients and caregivers, respectively, received the questionnaire via postal mail and were asked to consent both to the study, and to the pseudonymised linkage of study data with their clinical data . Specific data on socioeconomic status and race/ethnicity was not recorded. For participants under 18 years of age, parents/legal guardians filled in the questionnaire. The questionnaire for adults with ASD was directly addressed to these. We assumed that the majority of adult participants with an IQ < 85 were assisted in filling in the questionnaire. 418 persons returned the questionnaire including a signed written consent form (response: 32.3%), of which 385 questionnaires could be evaluated. Characteristics of survey responders vs non-responders are reported in the results section.Within the German health insurance system  created in OpenClinica® (OpenClinica Enterprise Version: 3.3), and was checked by another person.Based on the CSRI data, annual service use costs for inpatient and outpatient medical care were calculated, and stratified by sex, age group, diagnostic category, ADOS symptom score, intellectual functioning (IQ < 85 vs. IQ ≥ 85), and educational level. The level of education was defined in accordance with the International Standard Classification of Education (ISCED) UNESCO, , 2012, awww.pedz.de). Finally, daily drug costs for long-time therapy drugs were multiplied by 365.25.Direct costs were calculated by multiplying each resource use item by an appropriate unit cost (macro-costing). All costs were assessed from the perspective of the social security system and were calculated in Euros based on prices of the year 2016, obtained from a number of sources : Where possible, medical services, including consultations of medical practitioners and non-physician practitioners , as well as hospital stays and rehabilitation were valued by unit costs according to the calculation of Bock et al. . Costs fBaseline data were analysed using descriptive statistics. Mean values and standard deviations for healthcare service use, stratified by type of service , were calculated separately for children (0–11 years), adolescents (12–17 years) and adults (≥ 18 years). To account for non-normality of cost data, average annual costs per individual and by type of service were estimated with 95% confidence intervals (95% CI), using the bias-corrected accelerated bootstrap method Barker,  based onThe baseline characteristics of the sample are presented in Table Health services use by age and type of service are shown in Table The average annual costs for health services per individual are displayed in Table Costs for children with ASD (4177 EUR) were higher than for adults (3466 EUR) or adolescents (2510 EUR). Patients with an IQ < 85 yielded higher costs than those with an IQ ≥ 85 (4475 EUR vs. 2484 EUR). Costs did not differ very much by diagnosis, with costs for individuals with Asperger syndrome being only marginally higher. Higher ADOS comparison scores were not associated with higher costs.The different cost components are shown in Table In the regression model, female sex (Cost Ratio (CR): 1.64), lower IQ (CR: 1.90), and Asperger syndrome CR: 1.54) were significantly associated with higher costs  , accounting for 36.0% of total costs in adults with ASD. At least for psychiatric healthcare services in children with ASD, the preponderance of inpatient service cost has also been shown by other researchers  and occupational therapy (11.1%) are similar is somewhat surprising, given the shaky evidence for occupational therapy for ASD-related symptoms   were not. Regarding the association of lower IQ with higher costs, this result is in line with previous studies. The study of Buescher et al. , for exaThe association of female sex with higher ASD health costs is a novel finding. One possible explanation are statistical effects—often, ASD studies suffer from low numbers of females , thus leading to lower service use.Interestingly, in our study age was not associated with costs. This stands in clear contrast to other studies which reported higher costs in older individuals , and a significant proportion of females. To ensure high quality diagnoses, the diagnostic assessment of participants employed the current gold standard; which is key given the significant rates of over- and misdiagnoses in ASD , age, male/female ratio, IQ, and level of ASD symptom severity), it only contains those service users with a formal ASD diagnosis.Moreover, the above-average portion of individuals with Asperger syndrome in our sample may have led to a bias in health costs distribution.Another limitation is the lack of information on co-occurring health conditions, on adaptive functioning , other cost types, e.g. educational costs, can be more important cost drivers in ASD-related societal costs Below is the link to the electronic supplementary material.


Morbillivirus genus includes measles virus, canine distemper virus (CDV), phocine distemper virus (PDV), peste des petits ruminants virus, rinderpest virus, and feline morbillivirus. We detected a novel porcine morbillivirus (PoMV) as a putative cause of fetal death, encephalitis, and placentitis among swine by using histopathology, metagenomic sequencing, and in situ hybridization. Phylogenetic analyses showed PoMV is most closely related to CDV (62.9% nt identities) and PDV (62.8% nt identities). We observed intranuclear inclusions in neurons and glial cells of swine fetuses with encephalitis. Cellular tropism is similar to other morbilliviruses, and PoMV viral RNA was detected in neurons, respiratory epithelium, and lymphocytes. This study provides fundamental knowledge concerning the pathology, genome composition, transmission, and cellular tropism of a novel pathogen within the genus Morbillivirus and opens the door to a new, applicable disease model to drive research forward.Morbilliviruses are highly contagious pathogens. The  Paramyxoviridae encompasses a group of large (300–500 nm in diameter), enveloped, pleomorphic viruses with RNA genomes of 14.6–20.1 kb. The family comprises of 4 subfamilies and 17 genera that contain >70 species and includes global human and animal viral pathogens of concern , rinderpest virus (RPV), peste des petits ruminants virus (PPRV), canine distemper virus (CDV), phocine distemper virus (PDV), cetacean morbillivirus (CMV), and feline morbillivirus (FeMV)  protein, phosphoprotein (P), matrix (M) protein, hemagglutinin (H) protein, fusion (F) protein, and large polymerase (L) protein , we identified a novel morbillivirus in swine as the putative cause of an outbreak of reproductive disease characterized by fetal mummification, encephalitis, and placentitis. Paramyxoviruses known to naturally infect swine include porcine rubulavirus, Menangle virus, Nipah virus, and porcine parainfluenza virus  received 22 porcine fetuses from 6 litters (A–F) that originated from a commercial breeding herd in northern Mexico for routine diagnostic investigation . The breAt necropsy, we recorded the condition, including neonatal morality, mummified fetus, moderate autolysis, or stillbirth, and the crown-to-rump length (CRL) of each fetus or piglet in the case record . We prochttps://acdbio.com) to perform RNA ISH according to the manufacturer’s instructions for formalin-fixed paraffin-embedded samples  specific for PoMV by using the MBLV-900F and MBLV-988R primers and the MBL-959P probe Table 2.https://www.illumina.com) to sequence the libraries by using the MiSeq 600-Cycle Reagent Kit v3 (Illumina). We preprocessed raw sequencing reads and classified reads by using Kraken version 0.10.5-β (https://blast.ncbi.nlm.nih.gov), SeqMan Pro (https://seqman.software), and integrated genomics viewer for visualization  to generate multiplex sequence alignments. We constructed phylogenetic trees based on whole-genome sequences and amino acid sequences of the L protein from aligned sequences by the maximum likelihood model in MEGA version X (https://www.megasoftware.net). We used L protein sequences because paramyxoviruses currently are classified based on the sequence comparison of L protein, the RNA-dependent RNA polymerase. We evaluated the robustness of the phylogenetic tree by bootstrapping using 500 replicates. We used interactive Tree of Life  to display, manipulate, and annotate bases of the whole-genome sequence and L protein amino acid sequence trees . We used 1 pair of primers to close the genome gap and 14 pairs to confirm the genome sequence Table 2.Among 22 porcine fetuses from the 6 litters (A–F) submitted for diagnostic investigation, CRL length varied from 7 to 29.5 cm . We noteLeptospira sp. by qPCR or quantitative RT-PCR (qRT-PCR) in any litter.To address differential diagnoses, we used quantitative PCR (qPCR) to detect known swine viral and bacterial reproductive pathogens. We did not detect PCV2, PCV3, PRRSV, PPV1, or Morbillivirus. The 2 shortest contigs had <40% nt identity to PDV (GenBank accession no. KC802221) and CDV (GenBank accession no. AF014953) at the 3′ end. The 2 longer contigs had >60% nt identity to PDV and CDV at the 5′ end. We propose this paramyxovirus be named porcine morbillivirus (PoMV).We pooled samples of fetal thoracic tissue from litters A and B (A–B), both of which had encephalitis noted histologically. We also pooled fetal thoracic tissue from litters D and E (D–E), which had leukocytes in the epicardium noted histologically. We performed NGS on the 2 pooled samples by using the MiSeq platform (Illumina). After using an in-house bioinformatics analysis pipeline, we detected and identified 693 paramyxovirus-like reads in A–B and 118,772 in D–E. No reads of other pathogens were identified. De novo assembly obtained 2 contigs with 4,869 and 10,456 nt from pooled sample A–B and another 2 contigs from pooled sample D–E. The nucleotide sequences of the contigs from A–B and D–E were 100% identical but the contigs assembled from pooled sample D–E were slightly longer, 5,042 and 10,705 nt. Sequence analysis of the 4 contigs suggested the presence of a previously undescribed paramyxovirus of genus A complete genome sequence of PoMV (GenBank accession no. MT511667) was obtained by using RT-PCR to fill the gap between the 2 contigs. We designed 14 pairs of primers according to the obtained genome sequence and sequenced the RT-PCR products again, confirming the accuracy of the whole-genome sequence. The genome size of PoMV is 15,714 bases and has a G+C content of 45.19%. The 3′ leader sequence of the PoMV is 55 nt with 13/20 initial nt being highly conserved among morbilliviruses  Figure 1Morbillivirus L in morbilliviruses containing the sequence MASL in the nucleoprotein (N)  Figure 2Paramyxoviridae  value of 19.7; the Cq for litter E was 19.4. Fetal thoracic tissues from litters B and D had a lower viral load values; B had Cq of 23.4 and D had Cq of 20.2. PoMV was not detected by real-time RT-PCR in the fetal thoracic tissues of 2 litters of unaffected fetuses selected as negative controls. In addition, porcine rubulavirus was not detected in any sample by RT-PCR.Fetal thoracic tissues from litters A, B, D, and E were further subjected to real-time RT-PCR to approximate the viral load by litter. All were positive for PoMV with fetal thoracic tissues from litters A and E having a higher viral load. Litter A had a quantification cycle  and amino acid (26%–75.2%) identities with members in the genus  L genes . Phyloge L genes , panel A L genes , panel BParamyxoviridae that infect swine include porcine rubulavirus , Menangle virus , Nipah virus , and porcine parainfluenza virus 1 . Among these, only porcine rubulavirus and Menangle virus are thought to cause fetal mummification and stillbirths, as observed with PoMV , which is expressed on activated lymphocytes, dendritic cell subsets, and macrophages and cells in the alveolar lumen and lining the alveolar epithelium (Morbillivirus genus, but much is left to learn. The geographic distribution and species susceptibility of PoMV currently is unknown. Virus isolation to facilitate research, in vitro studies evaluating cell entry receptors, and in vivo studies to further elucidate pathogenesis and generate samples of known status for diagnostic assay development and evaluation are needed. Nonetheless, this discovery opens the door to a new and possibly more applicable model of disease to drive research forward (Our study provides essential information about a newly discovered pathogen within the Additional information on novel porcine morbillivirus causing fetal death and encephalitis among piglets.


Only ejaculates that fulfill minimum quality requirements are processed and eventually used for artificial inseminations. We examined 70,990 ejaculates from 1343 Brown Swiss bulls to identify bulls from which all ejaculates were rejected due to low semen quality. This procedure identified a bull that produced 12 ejaculates with an aberrantly small number of sperm . The analysis of testis transcriptomes from 76 bulls showed that the transcript with the premature termination codon is subject to nonsense-mediated mRNA decay. The 1-bp deletion resides in a 675-kb haplotype that includes 181 single nucleotide polymorphisms (SNPs) from the Illumina BovineHD Bead chip. This haplotype segregates at a frequency of 5% in the Brown Swiss cattle population. Our analysis also identified another bull that carried the 1-bp deletion in the homozygous state. Semen analyses from the second bull confirmed low sperm concentration and immotile sperm with multiple morphological abnormalities that primarily affect the sperm flagellum and, to a lesser extent, the sperm head.The genome of this bull was sequenced at a 12× coverage to investigate a possible genetic cause. Comparing the sequence variant genotypes of this bull with those from 397 fertile bulls revealed a 1-bp deletion in the coding sequence of the QRICH2 gene likely causes low sperm concentration and immotile sperm with multiple morphological abnormalities. Routine sperm analyses unambiguously identify homozygous bulls for this allele. A direct gene test can be implemented to monitor the frequency of the undesired allele in cattle populations.A recessive loss-of-function allele of the bovine The online version contains supplementary material available at 10.1186/s12711-022-00710-0. Semen quality and male fertility are complex traits with low to moderate heritabilities . These tGenome-wide association testing revealed quantitative trait loci (QTL) for semen quality and male fertility in various breeds of cattle, many of them expressing non-additive effects –6. The rImpaired semen quality and low fertility in healthy bulls can be caused by deleterious alleles at genes that show testis-biased or testis-specific expression –10. SuchQRICH2 gene that, in the homozygous state, likely causes male infertility due to immotile sperm with multiple morphological abnormalities. Using historic and current semen quality data from two Brown Swiss bulls, we show that this mutation remained undetected although phenotypic consequences started to be observed almost two decades ago. Our findings offer the opportunity to implement direct gene testing to monitor the frequency of this undesired allele in cattle populations.Here we report a 1-bp deletion inducing a frameshift in the bovine We assessed records for 70,990 ejaculates that were collected from 1343 Brown Swiss bulls between January 2000 and March 2018. All ejaculates were examined by laboratory technicians employed by Swissgenetics immediately after semen collection as part of routine quality assurance during semen processing. The parameters recorded were semen volume (mL), sperm concentration (million sperm per mL) that was quantified using photometric analysis, and the percentage of sperm with forward motility. The proportion of sperm with head and tail anomalies was quantified for each ejaculate with scores ranging from 0 to 3 . Subsequently, we considered 35,785 ejaculates that were collected from 1258 bulls aged between 330 and 550 days to identify those for which all ejaculates were discarded due to insufficient semen quality. Ejaculates that contained less than 300 million sperm per mL, less than 70% motile sperm, more than 10% sperm with head and tail abnormalities, or with a volume smaller than 1 mL were deemed to be unsuitable for artificial inseminations.https://github.com/broadinstitute/picard). Alignments were sorted by coordinates using the Sambamba tool ) for QRICH2 in the testis tissue of six heterozygous (green) and 70 homozygous wild-type (grey) bulls. Red asterisks indicate exons that were differentially expressed  between heterozygous carriers and wild type bulls. (b) Integrative Genomics Viewer coverage tracks from RNA sequence read alignments overlapping the frameshift-inducing 1-bp deletion (red arrow) in six heterozygous bulls. The identifiers of the coverage tracks refer to accessions from the sequence read archive.Additional file 6: Figure S2. DNA sequence alignment of SAMEA6272098. Output from «samtools tview» centered on BTA19:55436705TC>T representing DNA sequence read alignments from a bull (SAMEA6272098) that carries the 675-kb haplotype in the heterozygous state, but was genotyped as homozygous for the reference allele at the position of the BTA19:55436705TC>T variant. The asterisk within the only read that overlaps BTA19:55436706 indicates that the bull carries the 1-bp deletion.Additional file 7: Figure S3. Phase-contrast images of sperm from a bull homozygous for the 1-bp deletion. All images display sperm with major sperm head and/or flagellar defects. Very short, shortened or absent flagella were the most prevalent flagellar abnormalities: very short flagella , shortened flagella , and absent flagella . Short doubled  or short thickened  were apparent too. Some flagella were strongly folded  or coiled (y left sperm). Many sperm showed defective heads as well as defective flagella: pyriform , round , abnormal contour (p), and diadem defect/vacuoles . Underdeveloped sperm with the flagella strongly folded around the sperm head are considered the most severe sperm defects in bull . Scale bar: 5 µm.Additional file 8: Figure S4. TEM and SEM images of sperm from a bull homozygous for the 1-bp deletion. TEM cross-section of an underdeveloped sperm with multiple flagellar structures next to the nucleus (black) enclosed by a cell membrane (a). Underdeveloped sperm with the flagellum curled on the head visualized using SEM (b). Longitudinal TEM cross-section (c) and SEM (d) of sperms with a thickened vesicularized structure at the sperm neck and multiple disorganized flagellar structures at the mid-piece. Longitudinal TEM cross-section of the neck region of a normal sperm from a control bull (e). Scale bar: 1 µm.