Following the flank collapse, a deep convective cloud column formed over the volcano and acted as a ‘volcanic freezer’ containing ~3 × 109 kg of ice on average with maxima reaching ~1010 kg. Our satellite analyses reveal that the convective anvil cloud, reaching 16–18 km above sea level, was ice-rich and ash-poor. Cloud-top temperatures hovered around −80 °C and ice particles produced in the anvil were notably small (effective radii ~20 µm). Our analyses indicate that vigorous updrafts (>50 m/s) and prodigious ice production explain the impressive number of lightning flashes  recorded near the volcano from 22 to 28 December 2018. Our results, together with the unique dataset we have compiled, show that lightning flash rates were strongly correlated (R = 0.77) with satellite-derived plume heights for this event.Volcanic activity occurring in tropical moist atmospheres can promote deep convection and trigger volcanic thunderstorms. These phenomena, however, are rarely observed to last continuously for more than a day and so insights into the dynamics, microphysics and electrification processes are limited. Here we present a multidisciplinary study on an extreme case, where volcanically-triggered deep convection lasted for six days. We show that this unprecedented event was caused and sustained by phreatomagmatic activity at Anak Krakatau volcano, Indonesia during 22–28 December 2018. Our modelling suggests an ice mass flow rate of ~5 × 10 A less studied mechanism is in the case of an erupting volcano where the input of heat at the surface initiates deep convection4. Intense heating at ground surface and entrainment of moist air generates positive buoyancy5, which rapidly transports volcanic gases and ash particles up to the tropopause and beyond. Here we present the first detailed account of tropical deep convection triggered and sustained by magma-seawater interactions at an island volcano.Tropical thunderstorms can be triggered in a variety of ways. Common triggering mechanisms include solar heating, convergence of surface winds and the flow of wind over topography6. On 22 December 2018, Anak Krakatau underwent a major explosive eruption after experiencing six months of intense Strombolian to Vulcanian activity. The eruption resulted in a flank collapse on the southwestern side of the volcano8, which generated a deadly tsunami that hit the coasts of Java and Sumatra at 21:27 LT (14:27 UTC)9. The flank collapse marked the beginning of sustained phreatomagmatic activity at the volcano and led to the formation of a deep convective plume recorded by satellite for about six days.Anak Krakatau (‘Child of Krakatau’) is an island volcano located in Indonesia’s Sunda Strait  between the islands of Java and Sumatra  indicative of an ice-rich cloud composed of small particles12.We analysed the eruption sequence in great detail using geostationary and polar orbiting satellite data. The initial eruption can be identified in the thermal infrared data as a rapidly expanding cloud reaching cloud-top temperatures of −80 °C before detaching from the volcano (at around 14:30 UTC) and advecting to the southwest Fig. . Our umb3/s Fig. . The ini14. The availability of salt and moisture at the surface, provided by evaporated seawater and a tropical moist atmosphere, suggest that most of the ash remaining in the plume after sedimentation was efficiently removed from the atmosphere at low levels via wet deposition and aggregation16. Low level winds were predominantly toward the east and northeast from 22 to 28 December 2018 and evidence of ash deposition on Panjang Island (east of Anak Krakatau) is clearly seen in the Senintel-2 true colour image on 31 March 2019 . Analysis of satellite radio occultation sounding data (see Methods) indicates that the mean lapse rate tropopause for December was 16.8 km ± 0.8 km . The time series, shown in Fig. −1 and a maximum of 72 min−1 (using a 1-minute average) at 21:05 UTC on 24 December. Flash rates greater than 32.9 min−1 occur in less than 0.1% of meteorological thunderstorms21. We used the ENGLN data to derive an empirical parameterization, correlating lightning flash rate (per 10 min) with Himawari-8 cloud-top heights (see Methods). For the first time for this kind of event, we show that there is a strong correlation , equating to an average flash rate of 8.7 min22. We initialised the model with 0–50 wt% water, noting that water contents ≥40% led to column collapse. Liquid water mass fractions at the vent from 0–30 wt% were capable of sustaining a convective column with a neutral buoyancy level (NBL) at ~16 km  using the 1D volcanic plume model FPLUMEUTC Fig. . It is p9 kg of ice was being maintained by the convective activity 11 and Hekla 23. The lack of ash and abundance of ice detected during this period, coupled with significant amounts of lightning, indicates that strong upward ice mass fluxes played a major role in the charge separation required to generate the lightning24. We speculate that non-inductive charging, generated by collisions between updrafts of small ice crystals and larger falling graupel26, was the main charging mechanism—a mechanism commonly attributed to lightning in meteorological thunderstorms27, but also previously invoked to explain charging mechanisms in volcanic thunderstorms29.During the period of sustained convection, satellite retrievals show that on average over six days ~3 × 1032. By considering volcanically-induced CAPE (‘volcanic-CAPE’), we find that a continual input of heat and moisture at the surface would lead to sustained, extreme convection (CAPE ~5000–6000 J/kg), but without this input, deep convection would not be expected. For CAPE of these magnitudes, parcel theory predicts maximum updrafts 35.Figure 36. We find median ice particle effective radii of 16 and 23 µm, based on retrievals (see Methods) from Himawari-8 and MODIS, respectively, in the sustained anvil cloud shown in Fig. 39. Under these conditions there is no need to consider nucleation by ash seeding in order to generate ice.Lightning activity in thunderstorms is also associated with the occurrence of small ice particles at cloud-top38. Data collected from the Support to Aviation Control Service (SACS) near real-time warning system (see Methods) indicate the presence of SO2 during our analysis period  was relatively minor  to retrieve the closest temperature (and thus height) to minimum brightness temperature measured by the satellite. This method assumes that the cloud is at thermal equilibrium with the ambient atmosphere. We estimate an error in height by this method to be ± 1 km, allowing for errors in temperature of 2 K and 3 K for Himawari-8 measurements and ERA5 profiles, respectively, and a lapse rate of ~5 K/km near the tropopause.To estimate Anak Krakatau’s cloud-top heights we took the minimum brightness temperature (one pixel) measured from the 11.2 μm channel within a 0.25° × 0.25° latitude/longitude box centered over the volcano. The minimum brightness temperature time series was then compared to ERA5 reanalysis45 that describes the change in radius with time of a spreading umbrella cloud at the neutral buoyancy level (NBL). The volumetric flow rate V (in m3/s) of the initial Anak Krakatau cloud on 22 December 2018 is computed by applying a non-linear least squares fit of the following equation to estimates of the plume radius at each Himawari-8 observation:R is the radius of the plume, λ is an empirical constant (assumed here to be 0.2), N is the Brunt Väisälä frequency  and t is the time since the plume begins spreading at the NBL (inferred to be 13:58 UTC on 22 December 2018). Only data points that correspond to times where the plume is activity feeding the umbrella are considered in the non-linear least squares fit  and take the 245 K isotherm. The uncertainty in the area using this method depends on the density of the coordinate pairs (1 pixel per pair) and the geolocation accuracy of Himawari-8. According to Takeuchi46 the geolocation accuracy is ~1 pixel or 2 km for the IR channels. For a perfectly circular umbrella cloud the resulting error is about 10% for a cloud of radius 30 km.To obtain the necessary coordinates of the enclosed loop, 22 to model the initial explosive eruption associated with the flank collapse, as a phreatomagmatic eruption assuming water mass fractions ranging from 10 to 30 wt%, a magmatic water fraction of 5% and a magma temperature of 1150 °C , noting that incandescence was observed at the onset of the eruption . To constrain our model simulations with satellite observations, we varied the mass flow rate at the vent until the NBL from the model matched our satellite-based estimate (~16 km). The NBL is defined in FPLUME as the height at which the in-plume density equals the ambient air density. For the 20 wt% simulation, the resulting ice mass flow rate at the NBL was found to be 4.8 × 106 kg/s with a total mass flow rate of 8.4 × 108 kg/s. This mass flow rate corresponds to a total volumetric flow rate of 4.2 × 109 m3/s, assuming an ice-air mixture density at the NBL of 0.2 kg/m3.We used FPLUME-1.2−2) were performed using the Himawari-849 channels with central wavelengths (λ) of 8.6 and 11.2 μm. The refractive index of ice has a strong variation at these two wavelengths50 with much greater absorption at 11.2 μm compared to that at 8.6 μm. Thus ice clouds appear colder at 11.2 μm and the brightness temperature difference (ΔT) between these two channels for the same pixel is an indication of the effect of ice particles. The amplitude of ΔT varies with the 8.6 μm brightness temperature of the cloud and is a function of both the cloud optical depth and ice particle microphysics . A model was developed to retrieve the IR optical depth and effective radius of ice particles for an assumed shape and size distribution. The required inputs to the model are the extinction efficiency (Qext), the single-scatter albedo (g). These were obtained from T-matrix calculations using the extended-precision code of Mishchenko51. The T-matrix code itself requires inputs about the shape and size distribution of the particles. The size distribution of ice in clouds has been the subject of numerous research experiments56 and likewise the shapes of ice particles have been measured and studied for many years61. Ice particles are found as roughened spheroids, bullet rosettes, hexagonal columns and plates and aggregates with complex morphology. Generally smaller and colder ice particles are more spheroidal. The size distributions of ice particles are often found to be bimodal63 and approximated by power laws64 or a modified-gamma distribution65. For the Anak Krakatau ice clouds studied here there are no in situ data on the sizes or shapes of the particles. Inspection of the ΔT curves (plots of ΔT vs T8.6 brightness temperatures) suggest the particles are small (effective radii <50 μm) and cloud-top temperatures of <200 K are found. These inferences suggest that the scattering calculations must be performed in the so-called transition region where the size parameter, x = 2πr/λ ~ O(1). For mono-dispersed spheres there are exact codes based on Mie theory that provide estimates of Qext, g for x ~ 0.1 to >100. For more complex shapes and size distributions approximate numerical schemes have been developed68. A sensitivity study was performed to assess the effects of shape and size distributions on the calculated scattering parameters. Size distribution tends to smooth out the ripples found for x > 1 and suppresses the amplitude of the extinction peak. Prolate, oblate and Chebyshev-shaped particles of increasing order also tend to diminish the ripples but have a smaller effect on the extinction peak. As the radius increases above ~30 μm the extinction efficiency tends towards a constant value for all shapes and size distributions.Infrared (IR) satellite retrievals of the ice mass loading  varying from 0.1 μm to 50 μm. Refractive indices for the 8.6 and 11.2 μm channels were calculated using the data of Warren and Brandt50 after integration over the Himawari-8 filter response functions. The computed scattering parameters were then supplied to a discrete ordinates method69 (DOM) radiative transfer program with 16 gaussian angles, infrared optical depths τ varying from 0.1 to 10 in steps of 0.1 with assumed cloud-top (Tc) and surface temperatures (Ts) of 190 to 220 K in steps of 5 K and 290 to 298 K in steps of 2 K, respectively. The outputs of the DOM code are look-up tables (LUTs) of top-of-the-atmosphere brightness temperatures at 8.6 and 11.2 μm for a range of re, τ, Tc, Ts and 16 viewing angles. Selection of the appropriate LUT is based on estimating Ts and Tc through inspection of the Himawari-8 IR data and noting that the geostationary view of the scene from Himawari-8 is at a constant view angle. Interpolation of the LUTs was done by finding polynomial fits to the ΔT vs T8.6 curves for each of the modelled effective radii. Data points found lying between two consecutive radii curves were assigned radii weighted by their distance from each curve. A similar process was used to estimate the optical depth based on curve fits. Generally, the fits are excellent. The effective radii of data points lying beyond the largest radius modelled (50 μm), and with ΔT above 1 K were weighted between 50 and 200 μm—the value of 200 μm was taken as an upper limit to the size range. Only data points in the region with T8.6<240 K were included based on the premise that these are likely to represent particles in the ice phase.Based on these insights, calculations were performed for spheroids with T8.6 and T11.2 brightness temperatures were calculated and a water vapour correction applied to the ΔT based on Yu et al.70. Finally, retrievals were made by interpolating the appropriate LUT using the measurements. Retrievals that satisfied both of these conditions were rejected:Himawari-8 data were acquired at 10 minute intervals and processed for the period 22 December 2018 to 28 December 2018. The T ~ 1 K. A value of 240 K was assumed based on the expectation that hydrometeors in clouds with cloud-top temperatures <−40 °C would be in the ice phase.The purpose of these tests was to reject semi-transparent clouds with little absorption difference but allow opaque clouds which haveΔm) was calculated on a pixel-per-pixel basis (pixel area ~4 km2) from:ρice is the bulk density of ice (taken as 900 kg m−3). Total ice mass was computed from:i is the pixel number of area Ai and nj is the total number of pixels for each hour, j. The area of analysis extends from 102 °E to 106 °E and 8 °S to 5.5 °S. Within this large area there are other convective systems generating ice not associated with the Anak Krakatau cloud. In order to reject these clouds from the total ice mass calculation, the Australian Bureau of Meteorology Volcanic Ash Advisories (VAAs) text message polygons were used to delineate the area of volcanic activity around Anak Krakatau. The VAA polygons can be accessed from ftp://ftp.bom.gov.au/anon/gen/vaac/ and are based on automated numerical model and observational analyses with occasional intervention by an expert meteorologist. They are issued at times appropriate for dissemination to interested consumers (e.g. aviation industry) and so the VAAs closest to the Himawari-8 acquisitions times were used (in most cases within 10–30 minutes). The VAAs used were not post-analysed, and while in the majority of cases they appear highly accurate, it was noticed that on some occasions differences between the VAA polygon and the satellite brightness temperature indication of the cloud, differed. No attempt was made to change the VAA polygons.Ice mass loading (T’s ~0–1 K), and potential errors due to background convective activity within the VAA boundary but not necessarily associated with the Anak Krakatau cloud. To estimate this last source of error, the ice mass was calculated in a control area to the south of the volcano covering the region 105–105.9 °E and 7.8–6.8 °S. The mean ice mass found for this region was 0.16 × 109 kg ±0.14 × 109 kg compared with a mean value of 3.14 ± 2.02 × 109 kg for the Anak Krakatau cloud. The hourly uncertainty on the ice cloud mass retrievals was found to be between ~10% and ~170% with a mean value of ~60%. A data file containing the hourly ice mass retrievals, mean mass loading, mean and median effective radius and total area used is included as Supplementary Data File An error analysis was performed that included model errors (based on the sensitivity study described above), observational errors (accuracy of the brightness temperature measurements), errors due to including ice cloud not associated with Anak Krakatau, errors due to excluding Anak Krakatau ice clouds, mostly caused by cloud opacity too high for the retrieval scheme (i.e. Δ71. Nine daytime products over the Anak Krakatau area have been collected covering the period from 22 to 28 December 2018 (Supplementary Table 2) by considering the same equation used for the Himawari-8 ice mass calculation with the same ice bulk density. Also the ice mass uncertainty has been computed in a pixel-per-pixel base from the Effective Radius and Cloud Optical Thickness relative uncertainties. The same VAA polygons were used to estimate the MODIS total ice mass for consistency with the Himawari-8 retrievals. The error associated with the total ice mass is the mean error computed in the same area.The volcanic ice mass has also been computed using the level 2 cloud products derived from the measurements of the Moderate Resolution Imaging Spectroradiometer (MODIS) satellite instruments, on board the polar NASA-Terra and NASA-Aqua satellites 72 from the COSMIC Data Analysis and Archive Center (CDAAC) to analyse the tropopause altitude in the area of the volcano. The climatological values were computed using all the profiles from 2006 to 2018 in a box of 5° latitude and 5° longitude around the volcano. The climatological Coldest Point Tropopause (CPT) altitude in December around the Anak Krakatau region is 17.29 km whereas the climatological Lapse Rate Tropopause (LRT) is 16.78 km with a standard deviation of 0.8 km. In January the CPT slightly rises to 17.63 km and the LRT to 17.01 km. In December 2018, before the Anak Krakatau eruption, the tropopause altitude was consistent with the climatological values (CPT 17.24 km and LRT 17.02 km) but the eruption changed the atmospheric conditions and the CPT and LRT altitudes diverged reaching respectively 18.7 km and 15.9 km in January 2019. The RO temperature profiles during the eruption, compared to the climatological temperature profile, show a warming of the upper troposphere (15.5 km – 17 km) with a peak amplitude of 4 K and a cooling of the lower stratosphere (18 km – 20.5 km) with amplitude up to −4 K. After the eruption, the stratospheric cooling persisted for at least 1 month. Only one RO was found (01:07 UTC on 28 December) in the area of the eruption and the cloud-top altitude computed by using the bending angle anomaly73 is 18.1 km suggesting a stratospheric cloud at least 1 km higher than the climatological tropopause.We have used the Global Navigation Satellite System (GNSS) Radio Occultation (RO) profiles2 vertical columns from UV and IR polar orbiting sensors hosted by the SACS service74, with the assumption of a volcanic plume in the low stratospheric layer . The selection of SO2 pixels (considered for the mass loading) is applied using a threshold, except for TROPOMI for which the criteria is based on the ratio of SO2 measurement and its noise. For OMI and TROPOMI sensors, the estimate of SO2 mass loading is straight forward (considering surface of each footprint), while IR sensors and the composite of GOME2-A and B required a gridding to optimise the estimate of SO2 mass loading .We compiled SO76. ENTLN clusters stroke events (both IC and CG) occurring within spatial-temporal intervals of 0.7 seconds and 10 km into flashes. WWLLN is a network of lighting location sensors using time of group arrival (TOGA) techniques and operates at very low frequencies (3–30 kHz), which allows it to detect lightning from relatively large distances77. We considered all lightning flashes within a 100 km radius centred on Anak Krakatau in our analysis. To investigate the degree to which lightning from regional storms (independent of eruptive activity) affected our analysis, we reduced the search radius from 100 to 50 km and found that the number of recorded events during the time of observation reduced to 98.5% of the total. For a search radius of 30 km  continuously measures lightning stroke occurrence time, location, type (IC and CG), polarity, and peak current, at over 1,600 ground-based stations around the world. ENGLN combines data from the Earth Networks Total Lightning Network (ENTLN) and the World Wide Lightning Detection Network (WWLLN). ENTLN is comprised of wideband sensors (5 kHz – 10 MHz) and uses time of arrival (TOA) techniques to detect both the IC and CG stroke signals34. Lightning parameterizations based on cloud-top height are a commonly used method for predicting flash rate in global chemistry models78. In particular, Price and Rind34 proposed a lightning flash rate per minute, F, relationship of the following form to fit radar height, H (km), data:a and b empirical parameters, with a ≈ 3.44 × 10−5 and b ≈ 4.9. We used such a relationship to fit satellite cloud-top heights (km) as a function of lightning flash rate (per 10 minute):c and d are empirical parameters. Parameters obtained by best fitting the data gave Different studies have proposed robust quantitative relationships between lightning flash rate, cloud-top height and peak thunderstorm updraft speedentclass1pt{minima79. We note that the virtual temperature correction80 was not applied here as we did not have mixing ratio profiles from the Jakarta soundings for upper levels (>10 km). However, for large CAPE values (>4000 J/kg) this correction is expected to be small (relative errors <10%)80.To assess the convective instability of the environment prior to the convective phase of the Anak Krakatau eruption, we calculated the surface-based convective available potential energy (CAPE) for a sounding at Jakarta airport at 12:00 UTC on 22 December 2018. Figure Supplementary Video 1.Supplementary Dataset 1.Supplementary Dataset 2.Supplementary information


In high-income, temperate countries, IgE to allergen extracts is a risk factor for, and mediator of, allergy-related diseases (ARDs). In the tropics, positive IgE tests are also prevalent, but rarely associated with ARD. Instead, IgE responses to ubiquitous cross-reactive carbohydrate determinants (CCDs) on plant, insect and parasite glycoproteins, rather than to established major allergens, are dominant. Because anti-CCD IgE has limited clinical relevance, it may impact ARD phenotyping and assessment of contribution of atopy to ARD.Schistosoma mansoni (Sm)-endemic communities, proximate urban communities, and importantly in asthmatic and nonasthmatic schoolchildren.Using an allergen extract-based test, a glycan and an allergen (glyco)protein microarray, we mapped IgE fine specificity among Ugandan rural Sm infection, but not with skin reactivity to extracts or sensitization to their major allergenic components. Interestingly, we discovered that reactivity to only a subset of core α-1,3-fucose-carrying N-glycans was inversely associated with asthma.Overall, IgE sensitization to extracts was highly prevalent (43%-73%) but allergen arrays indicated that this was not attributable to established major allergenic components of the extracts (0%-36%); instead, over 40% of all participants recognized CCD-bearing components. Using glycan arrays, we dissected IgE responses to specific glycan moieties and found that reactivity to classical CCD epitopes  was positively associated with sensitization to extracts, rural environment and CCD reactivity is not just an epiphenomenon of parasite exposure hampering specificity of allergy diagnostics; mechanistic studies should investigate whether specific CCD moieties identified here are implicated in the protective effect of certain environmental exposures against asthma.  Barring a few exceptions,33 anti-CCD IgE seems to have poor biological function: individuals with specific IgE to CCDs lack skin and oral reactivity to the same molecules.34The role of carbohydrate cross-reactivity in epidemiology of allergic sensitization and disease is less understood. Among the commonest sources of cross-reactivity between natural allergens and other environmental exposures are specific asparagine (N) linked glycan modifications found on some helminth, insect and plant proteins . The N-glycan trimannosyl-chitobiose core , led us to hypothesize an inverse association between anti-CCD IgE reactivity and clinical allergy, possibly indicative of CCD-mediated inhibition of allergic effector responses.In the tropics, CCDs have been reported to dominate specific IgE responses.36 and urban37 settings, and among asthmatic children and controls.38 These studies provide an unprecedented opportunity to assess anti-CCD IgE profiles, their relevance to epidemiological trends of allergic sensitization and asthma, and their association with the rural-urban environment and Sm exposure in tropical LICs.We conducted three studies in varied Ugandan settings to obtain a comparative assessment of allergy-related disease prevalence and risk factors in rural22.1The three studies comprised rural and urban cross-sectional surveys on allergy outcomes and a case-control study on asthma risk factors among schoolchildren in Uganda.Sm-endemic fishing villages of Koome islands, Lake Victoria, Uganda  was conducted in Entebbe municipality, a lower helminth exposure, urban setting37 situated on northern shores of Lake Victoria  was conducted in , Uganda . It was Victoria .38 enrolled children with doctor-diagnosed asthma and controls from schools in Entebbe and surrounding areas. All schoolchildren were screened using the International Study on Allergy and Asthma in Children (ISAAC) questionnaire.41 Children with a history of wheeze in the last year underwent a detailed clinical assessment by study clinicians, including a medical and treatment history, lung function tests, and were assessed for asthma control using the Asthma Control Test.42 Among cases, 55.5% had well-controlled asthma, 29.5% partly controlled and 15% poorly controlled asthma. Only three cases had abnormal lung function tests. All cases were seen at one time point, started on the recommended treatment and were referred for additional management.42 Children in the same class as cases, with no history of wheezing, were eligible as controls. A Stata program  was used to randomly select participants from each class register such that the number of controls was twice the number of cases.The asthma study (May 2015-July 2017)43: Dermatophagoides mix (Dpteronyssinus and D farinae), Blomia tropicalis and Blattella germanica . Visible flexural dermatitis [evaluated following Williams’ online manual44], and questionnaire-determined recent rhinitis and urticarial rash were also assessed.Recent wheeze was also assessed in the rural and urban surveys using ISAAC questionnaires. Other procedures were identical in all three studies, including assessment of skin prick test (SPT) reactivity to allergens common in our setting2.2Sm and intestinal helminths was investigated using the Kato-Katz technique45 conducted on one stool sample per participant . In the urban and rural surveys, stool was further examined for Sm, Necator americanus and Strongyloides stercoralis infections using PCR,47 and urine for Sm circulating cathodic antigen .Infection with 2.3® specific IgE test  (hereinafter “ImmunoCAP®) was used to measure plasma IgE levels to whole allergen extracts of house dust mite , peanut (Arachis hypogaea) and German cockroach (B germanica), with ≥ 0.35 kU/L defining allergic sensitization.48 The ImmunoCAP® method is described in this article’s Supplementary information.The ImmunoCAP51 was used. The ISAC microarray comprised112 allergen components from 51 sources. The binding assay is described in this article’s Supplementary information. The array comprises 67 recombinant components ; and 45 components purified from natural extracts . Previous work has shown that nMUXF3 and glycoproteins from pollen  and food (nJug r 2) are recognized by anti-CCD IgE, while other natural allergens on the ISAC are not.53 Herein, “CCD-bearing components” denotes ISAC components confirmed to carry the classical core β-1,2-xylose and/or α-1,3-fucose substituted N-glycans. We also use the term “established major allergenic components” to refer to specific proteins that bind IgE to their protein backbone and are well-recognized as the molecular drivers of allergenic potency, in specific allergen sources.55 Participants with an ISAC standardized unit (ISU) measurement of ≥ 0.3 were considered sensitized to that allergen component.57 Measurements were also reported as detectable versus undetectable (lower detection limit: 0.06 ISU).To assess IgE reactivity to established major allergenic components of these and other extracts, the ISAC (Immuno Solid-phase Allergen Chip) microarray [Thermo Fisher Scientific]59 The IgE-binding assay and the microarray image processing procedures were adapted from previous studies62 and are detailed in the Supplementary information. Median fluorescence intensities (MFIs) reported herein represent anti-glycan IgE concentrations, because fluorescence-labelled anti-human IgE was used to detect plasma IgE bound to individual glycans on the array.A noncommercial microarray of 135 chemo-enzymatically synthesized glycans with and without β-1,2-xylosylation and/or α-1,3-fucosylation  was emplSchistosoma egg [SEA]- and adult worm [SWA] antigen-specific IgE, IgG and IgG4, by ELISA [described elsewhere63].Samples were also assessed for 2.436 Differences in characteristics between rural and urban survey participants and between asthmatic and nonasthmatic children were assessed using logistic or linear regression, allowing for the survey designs (clustering and weighting). Unadjusted analysis of differences in individual N-glycan-/allergenspecific IgE levels between rural and urban participants, and between asthmatics and controls was done using the Mann-Whitney U test, correcting for multiple testing using a Monte Carlo simulation approach64 with 1000 permutations, to generate empirical p values. For analyses comparing prevalences of ISAC-determined IgE sensitization between these groups, chi-squared tests  were used.Statistical analyses were done in Stata 13.1, GraphPad Prism 7.0a  and R via the RStudio interface . Initial analyses in the rural survey investigated the impact of trial intervention on IgE profiles using a cluster-level approach, as previously described.Sm infection and survey setting were assessed using linear regression.Since anti-glycan IgE responses were strongly correlated, they were further analysed using principal component analysis (PCA). Data for participants from all studies were pooled and principal component (PC) scores generated. Unadjusted and age- and sex-adjusted associations between PC scores and atopic sensitization, asthma status, 33.1®-determined IgE sensitization were available for 780, 345 and 400 participants of the rural survey, urban survey and asthma study, respectively. For the rural survey, we present data combined from both anthelminthic treatment arms, because there was no effect of trial arm on ImmunoCAP®- or ISAC-determined IgE sensitization or on anti-glycan IgE reactivity (36Allergen- and glycan-specific IgE measurements were conducted in a subset of randomly selected samples per study . Data onactivity .36P = .001) and more likely to be male. SPT reactivity to Dermatophagoides mix (P = .003) and B tropicalis (P = .025) was higher among urban participants, while total IgE (P < .001) and allergen extract-specific IgE sensitization (ImmunoCAP® concentration ≥ 0.35 kU/L), particularly to cockroach (P < .001), was higher among rural participants. Urticarial rash was more common in rural participants (P < .001), while wheeze, rhinitis and dermatitis were rare in both settings. Rural, compared to urban participants, was more frequently infected with Sm (P < .001), T trichiura (P = .002), hookworm (P = .016) and S stercoralis (P = .001) and had higher median levels of SEA- and SWA-specific IgE, IgG and IgG4 (P < .001). Adjusting for age and sex did not impact these differences.® IgE sensitization, dermatitis and rhinitis, and higher total IgE levels  to D pteronyssinus, compared to 46.7% who did not report dust as a trigger (P = .009). This difference was not statistically significant for B germanica and A hypogaea-specific IgE sensitization.Asthmatics, compared to nonasthmatic controls, had higher prevalence of SPT reactivity, ImmunoCAPE levels . ImmunoCPrevalence of helminth infections in the asthma study was low, but concentrations of SEA- and SWA-specific antibodies were moderate, and similar between asthmatics and nonasthmatics .3.23.3® IgE ≥ 0.35 kU/L) was high  and recombinant forms of established major allergenic components of these extracts was very low among rural and urban survey participants, and among nonasthmatic schoolchildren : 0%-3% , are typified by presence of α-1,3-linked core fucose and β-1,2-linked core xylose motifs.Sm infection (®) and (d) CCD sensitization [on ISAC]  HDMon ISAC] . Howeveron ISAC] .3.6No associations were observed between glycan microarray-assessed IgE response PC scores and asthma or SPT reactivity . Assessm® IgE sensitization, as our data showed that IgE to allergen extracts reflected sensitization to environmental exposures such as CCDs, and could not accurately define allergic asthma. However, we grouped asthma cases based on SPT reactivity to at least one allergen extract, to assess associations with responses to N-glycans carrying core α-1,3-fucose  using a synthetic glycan microarray revealed strong positive associations with the rural environment and Sm infection and for the first time showed reactivity to a subset of core α-1,3-fucose substituted N-glycans to be negatively associated with asthma.We conducted an assessment of associations between IgE responses at (sub-)molecular level and allergy outcomes among Ugandan rural and urban individuals, and asthmatic schoolchildren and their controls. We found that among rural, urban and nonasthmatic participants, sensitization to extracts from common environmental allergens did not reflect sensitization to their established major allergenic components; rather, many participants recognized CCDs. By contrast, a high proportion of asthmatics were sensitized to both HDM extract and its major components, lending substantial support to increasing evidence that much asthma in tropical LICs is atopic, contrary to earlier perception.Sm infection among our urban and rural survey participants were initially reported in our recent publication.70 Here we present data from an allergen extract-based test (ImmunoCAP®) and an allergen (glyco)protein microarray (ISAC) to confirm these associations and to explore associations with allergic sensitization and asthma.Positive associations between antibody reactivity to classical CCD epitopes and the rural environment and 71 as well as cross-reactive protein and carbohydrate components that are conserved in other environmental antigens such as those from schistosomes.72 This obscures the identity of the molecular drivers, and hence interpretation, of a positive response in vitro to extract-specific IgE blood immunoassays . Our results show that this has important implications for diagnosis of atopy, and phenotyping of asthma  as “atopic”, in tropical settings and for the use of population attributable fractions to assess the contribution of atopy to allergy-related disease in tropical LICs.Allergen extracts contain a mixture of allergenic and nonaller-genic components,Sm-transmission rural setting is reminiscent of previous findings by Hamid et al among Indonesian schoolchildren from areas endemic for soil-transmitted helminths (STH) but not schistosomes.4 These results imply that cross-reactive protein motifs and/or CCDs from environmental antigens other than schistosomes play an important role in allergy epidemiology. Exposure to STH might theoretically contribute; however, as discussed in our previous article on associations between helminth infections and N-glycan-reactivity,70 expression of “classical” CCD epitopes in most STH is not yet well mapped, with STH glycomes understudied compared to the Schistosoma glycome. However, absence of significant associations between anti-CCD IgE and infection with any STH (reported here and in our previous study70) opposes the notion that STH express “classical” CCDs.The high prevalence of sensitization to CCDs and to allergen extracts but low sensitization to established major allergenic components in our high-Schistosoma antigens and peanut, pollen or insect venom allergens is predominantly caused by CCDs.74 It is likely that previously observed positive associations between Sm and HDM extract-specific IgE in our rural setting40 are partially attributable to CCD sensitization. In the same population, prevalence of clinical allergies is low, supporting the hypothesis that helminths may protect against allergic effector responses. The role of CCDs in protection against allergy, if any, is unclear. Alpha-1,3-gal has been implicated in meat allergy33; however, our studies did not find any evidence of this, despite elevated α-1,3-gal-specific IgE concentrations. Other than α-1,3-gal-specific IgE, carbohydrate-specific IgE rarely, if ever, translates into clinical allergy.35 We postulated that elevated IgE responses to specific immunogenic glycans during chronic Sm infection might result in reduced allergic effector responses. Many native allergens occur as glycoproteins; therefore, prior high exposure to specific CCDs (perhaps resulting from Sm infection or other exposures) may prime initial and recall IgE responses to preferentially target-specific CCD rather than the protein epitopes of allergens. Indeed, rural-urban comparisons of responses to components on the ISAC array imply that in our Sm-endemic rural setting, IgE may be less effectively induced against common protein allergens than against CCDs.There is evidence showing that cross-sensitization between 75 offer great promise for availability of tailored glycan epitopes for initial testing in animal models. The observed inverse associations between asthma and core α-1,3-fucosylated N-glycans are unlikely to represent an “epiphenomenon” (merely reflective of inverse Sm-asthma associations) since, from our data, Sm prevalence and concentrations of SEA- and SWA-specific antibodies were similar between asthma cases and their controls.In our setting, ISAC-determined anti-CCD IgE was not inversely associated with SPT reactivity or asthma, and PCA of microarray-determined anti-glycan IgE responses suggested no overall association (inverse or otherwise) between asthma and anti-glycan IgE. However, analysis of levels of IgE to individual glycans revealed that reactivity to a subset of core α-1,3-fucose substituted N-glycans was lower among asthmatic schoolchildren compared to controls, and this was consistent for all N-glycan structural variants on the array bearing core α-1,3-fucose only or in combination with α-1,6-fucose. It is plausible that responses to specific glycans are not merely a smokescreen that obscures readouts for assessment of atopy. Mechanistic investigations in animal and human studies are required to investigate whether specific glycans might be involved in protection against asthma, or whether they merely represent surrogate markers of protective environmental exposures. Current efforts to reconstruct specific glycoproteins (including those carrying N-glycan core substitutions) in plant systems37 the prevalence of wheeze  in our rural setting was very low (and some of it probably nonasthma related). Second, the urban community had considerable exposure to light helminth infections, while rural participants had received three years of mass anthelminthic treatment at the time of the survey. Nonetheless, the two settings were still quite distinct with regard to helminth prevalence, providing an interesting sample for assessing urban-rural differences in fine specificities of IgE responses to allergen extracts, their major allergenic components and “classical” CCD motifs.Our study had some limitations. First, it would have been valuable to have a comparison group of asthmatics from the rural setting, to ascertain the generalizability of our findings and their interpretation. However, as shown in ® assays) reflects sensitization to a myriad of environmental exposures (absent in high-income countries), such as CCDs, and does not accurately define allergy-related disease phenotypes. In fact, our findings call for studies to investigate whether specific CCD epitopes have a mechanistic role in the protective effect (against asthma) of certain environmental exposures.Overall, our data indicate that, in tropical LICs, IgE to allergen extracts (detected in standard ImmunoCAPAdditional supporting information may be found online in the Supporting Information section.Supplementary information


The metabolism of Chinese hamster ovary (CHO) cell lines is typically characterized by high rates of aerobic glycolysis with increased lactate formation, known as the ”Warburg” effect. Although this metabolic state can switch to lactate consumption, the involved regulations of the central metabolism have only been partially studied so far. An important reaction transferring the lactate precursor, pyruvate, into the tricarboxylic acid cycle is the decarboxylation reaction catalyzed by the pyruvate dehydrogenase enzyme complex (PDC). Among other mechanisms, PDC is mainly regulated by phosphorylation–dephosphorylation at the three sites Ser232, Ser293, and Ser300. In this work, the PDC phosphorylation in antibody‐producing CHO DP‐12 cell culture is investigated during the lactate switch. Batch cultivations were carried out with frequent sampling (every 6 h) during the transition from lactate formation to lactate uptake, and the PDC phosphorylation levels were quantified using a novel indirect flow cytometry protocol. Contrary to the expected activation of PDC  during lactate consumption, Ser293 and Ser300 phosphorylation levels were 33% higher compared to the phase of glucose excess. At the same time, the relative phosphorylation level of Ser232 increased steadily throughout the cultivation . The intracellular pyruvate was found to accumulate only during the period of high lactate production, while acetyl‐CoA showed nearly no accumulation. These results indicate a deactivation of PDC and reduced oxidative metabolism during lactate switch even though the cells undergo a metabolic transition to lactate‐based cell growth and metabolism. Overall, this study provides a unique view on the regulation of PDC during the lactate switch, which contributes to an improved understanding of PDC and its interaction with the bioprocess. PDC is  subunit , 21. The subunit , 23. Fur subunit , 26, 27. subunit . HoweverChanges in cell metabolism in mammalian producer cell lines are characterized by ineffective glucose metabolism with high lactate formation. Furthermore, cells may utilize lactate as a secondary substrate and shift their metabolism (lactate shift). Nonetheless, the role of the pyruvate dehydrogenase complex (PDC) regulation during the metabolic shift from lactate formation to lactate uptake has not yet been sufficiently investigated. With this study, the interaction of oxidative metabolism with the PDC regulation by phosphorylation–dephosphorylation was descriptively studied for the first time during the lactate shift. Contrary to the general assumption of increased PDC activity  during lactate uptake, the results indicate that PDC is deactivated  during lactate consumption compared to the glucose excess phase. These results contribute toward the recent efforts in understanding this metabolic switch, as it is important for both mammalian cell culture and human diseases.Based on the current understanding of PDC regulations, an increased phosphorylation of PDC  could be expected during the exponential growth phase with high lactate formation . During α was determined in batch cultures with frequent sampling (every 6 h). Furthermore, cell death effects  activity), the intracellular concentrations of pyruvate and acetyl‐CoA, and the changes of extracellular components  were analyzed during the lactate switch.In this work, the regulation of PDC by phosphorylation and dephosphorylation was experimentally investigated during the transition from lactate production to lactate consumption in antibody‐producing CHO DP‐12 cultures. The relative phosphorylation status of the regulating sites Ser232, Ser293, and Ser300 on PDC E11.1PDCE1α sites through four PDKs, which exhibit differences with respect to site‐specificity and phosphorylation rate. Two different PDP isoforms  activate PDC by dephosphorylation of the phosphorylated Ser PDCE1α sites (pSer‐ phosphorylated Ser), which have the same degree of activity towards the regulating sites [PDCE1α are schematically shown in Figure Each of the Ser residues .Suspension‐adapted CHO DP‐12 cells  producing an interleukin‐8 antibody were used in all experiments conducted in this work. The cell line was cultivated in chemically‐defined, animal component‐free TC‐42 medium, available with or without glucose . The medium was supplemented with variable concentrations of glucose, 6 mmol 2.242mmolL−1 glucose, 6 mmol L−1 of glutamine and 200 nmol L−1 methotrexate . The conditions inside the shaker  were 37∘C temperature, 5% CO2 and 85% humidity, with a shaking frequency of 200 rpm (25 mm shaking diameter).The cells were precultured in medium containing 2.320mmolL−1 glucose, 6mmolL−1 glutamine and 200 nmol L−1 methotrexate (same as in Subsection 0.3·106cellsmL−1. Air was supplied to the headspace at a constant rate of 10mlmin−1 throughout the culture duration. The dissolved oxygen level was maintained above 40% via sparging of pure oxygen into the culture medium if required and the pH was maintained at 7.1 (sparging with CO2 or addition of 0.5molL−1Na2CO3). The temperature was maintained at 37∘C and the impeller speed was set to 400 rpm. The sampling interval was 24 h from t=0−72h and then reduced to 6 h until the end of the cultivation. The offgas composition was measured using an analyzer .Two batch cultivations were performed in the benchtop bioreactor Vario 1000 . One cultivation targeted the PDC phosphorylation and is discussed in the main manuscript, whereas the second cultivation was performed to validate the intracellular pyruvate fluxes and is shown in Supporting Information Subsection 2.3. In both cultivations, the initial working volume was 200 mL fresh medium containing 2.4300g, 3min), the supernatant was frozen (−20∘C) for metabolite analysis, and the cells were suspended in phosphate‐buffered saline . The viability was determined with DNA staining using the DAPI  method. Therefore, cells were suspended in 4∘C PBS with 1μgmL−1 DAPI and immediately measured with flow cytometry . The 405nm laser and 450/50nm filter signal were used. Debris and doublets were excluded with side scatter‐A vs. forward scatter (FSC)‐A and FSC‐H vs. FSC‐A gating, and non‐stained cells were gated as viable .Total cell concentration and cell size distribution were measured with the Z2 particle counter  as explained in . The cel2.5105 cells in 100μL of 1x binding buffer were labeled using 5μL of V450 annexin V followed by 5μL of propidium iodide solution (50μgmL−1), and incubated at room temperature (≈20∘C) for 15 min in the dark. Then, the cells were analyzed using flow cytometry . Debris and doublets were excluded and the V450 annexin V fluorescence was detected using the 405nm laser and 450/50nm filter signal. The 488nm laser and 690/50nm filter signal were used for the propidium iodide signal. 30 000 events were recorded (CytExpert). Please see Supporting Information Subsection 1.1 for more information about gating.The proportion of healthy and apoptotic cells in the culture was quantified by annexin V and propidium iodide  staining. 2.6The LDH activity in the culture medium was quantified to determine the degree of cell lysis using a colorimetric assay kit . The assay was carried out as per the manufacturer's protocol.2.72.7.1cGlc), glutamine (cGln), and lactate (cLac) were quantified with the YSI 2900D  biochemistry analyzer. The ammonium concentration (cAmm) was determined with an enzymatic test kit . The antibody titer (cAb) was determined using bio‐layer interferometry  with protein A biosensors . Amino acid concentrations were determined from pre‐column derivatized supernatant  by reversed‐phase chromatography using a Poroshell HPH‐C18 column  with fluorometric detection [Concentrations of glucose (etection .2.7.2cPyr) and acetyl‐CoA (cAcCoA) concentrations were quantified with assay kits . The cells harvested from the bioreactor (≈2·106 cells) were washed with PBS  and the cell pellets were snap‐frozen and stored in liquid nitrogen . For the quantification of intracellular metabolites, the snap‐frozen cell pellets were resuspended in assay buffer  and incubated for 5 min on ice with intermediate vortexing. Then, the suspension was centrifuged at 21100g for 5 min at 4∘C to obtain debris‐free lysate . Deproteinization was carried out using a 10 kDa spin filter  with centrifugation at 14000g for 10 min at 4∘C. Subsequently, the assays were carried out according to the manufacturer's protocol.Intracellular pyruvate  at two consecutive time points  and the measured viable cell concentration (Xv) as follows:The cell‐specific uptake/formation rates were calculated using the measured metabolite concentrations (2.7.4qOUR) was calculated using data averaged over t=12h intervals. Therefore, the oxygen input flow rate and the oxygen offgas flow rate were averaged over t=12h intervals and the difference between the average values were considered to be the total oxygen consumed (OURavg) in the t=12h interval. qOUR was calculated based on the oxygen density at 37∘C (ρO2=1.24gL−1), the molecular weight of oxygen (MO2), and the liquid bioreator volume (Vl):The cell‐specific oxygen uptake rate  for 20 min and washed with PBS [0.5·106cellsmL−1 using PBS. The assay was carried out in 96‐well flat‐bottom black plates  with 200μL of the fixed cell suspension per well. Subsequently, the cells were treated with different buffers . These steps were repeated twice (centrifugation time reduced to 10min) and they together constitute the washing sequence.The phosphorylation of the with PBS . The finα‐related antibodies can be found in [α subunit. Then, the cells were divided into four groups: one group without anti‐pSer antibodies , three groups stained with anti‐pSer antibodies, separated as one group for each pSer residue . The incubation with primary antibodies was carried out for 16 h and with secondary antibodies for 20 min, and the cells were washed and analyzed by flow cytometry .First, the cells were permeabilized using the permeabilization buffer for 20 min. Then, the cells were incubated in a blocking buffer for 2 h after which staining with antibodies was performed. The antibodies used are listed in Table found in . The antE1α positive‐IE1α∼ and pSerE1α positive‐IpSer,E1α∼, respectively) were determined and the relative phosphorylation P was quantified:From the flow cytometer data, the median of the individually gated positive intensities  measurements, and pSer (relative) = PP0 was used for data analysis.Considering fluctuations in biological samples and data measurements, 3PDCE1α phosphorylation is investigated during the metabolic switch from lactate formation to lactate uptake. Antibody‐producing CHO cells were cultivated in pH‐controlled bioreactor experiments and frequent sampling was carried out during lactate switch. In the beginning, the changes in the medium concentrations, LDH and apoptotic fractions are shown, followed by the amino acid profiles. Then, the PDCE1α phosphorylation levels and the intracellular pyruvate  and acetyl‐CoA concentrations  are presented, followed by a separated discussion of their role in lactate switch. Based on the cell growth and lactate profile , lactate consumption phase  and death phase .In this study, the e Figure  and B, te Figure , 3, 4, 520mmolL−1  to maintain steady cell growth and lactate uptake. Please see Supporting Information Subsection 2.1 for detailed information about the pre‐experiments for the development of the experimental design.Experimental settings were chosen to avoid two commonly overlaying effects. First, due to the pH control, constant lactate export was ensured , 41. Hen3.1Xv=10.6·106cellsmL−1 at t=120h  was observed. Simultaneously, the apoptotic fraction had increased to 45%. Additionally, LDH activity , as described in Subsection 12.78±0.14μm (average ± standard deviation) and it decreased only by a negligible extent to 12.40±0.17μm during lactate consumption.The changes of the metabolites are shown in Figure 3.2.1cGlc=18.6mmolL−1 and glucose was fully depleted at t=84h. By this time, the lactate concentration reached a maximum of cLac=29.6mmolL−1 , without any further lactate depletion. After t=120h, cell death  between t=72h−84h, qGlc was drastically reduced and was ≈0pmolcell−1h−1 upon glucose depletion at t=84h.The cell‐specific glucose consumption rate , isoleucine (cIle), leucine (cLeu), methionine (cMet), phenylalanine (cPhe), threonine (cThr) and valine  were constantly consumed during the entire cultivation, but were not depleted [cGly) was consumed only during the lactate formation phase, and the concentration changes were rather small during lactate consumption , lysine (cLys) and serine (cSer) showed very small changes in concentration during the lactate formation phase and are seen to be consumed once lactate consumption begins , aspartate (cAsp) and glutamate (cGlt) were produced during lactate formation phase 3.4α phosphorylation levels were quantified by indirect flow cytometry . The changes in pSer293 and pSer300 show similar trends, which are different from the pSer232 changes. As can be seen in Figure t=84h). After the switch to lactate consumption, the phosphorylation state was constant, varying slightly between 1.6 ‐ 1.7. At t=114h, the levels of pSer293 and pSer300 began to simultaneously decrease until ≈1.2 at t=126h. A higher phosphorylation level indicates a deactivation of PDC and vice versa [The relative phosphorylation of Ser232 Figure  increasece versa . Here, i3.4.2cPyr=0.8fmolcell−1 (t=0h−24h), an accumulation of cPyr up to 4.9fmolcell−1 is observed at t=48h. Then, the pyruvate concentration decreased below the detection limit and was consistently low. This prominent spike was confirmed in two independent cultivations  the phosphorylation levels of Ser293 and Ser300 were rather constant, at a 33% higher level when compared to glucose excess phase. Such an increase in PDC phosphorylation and steady cell growth and productivity at the same time could hint that pyruvate is metabolized in alternative pathways , as observed in primary cancer cells [The er cells , 57.α subunit of PDC leads to the deactivation of the enzyme complex [Phosphorylation of Ser232 is seen to behave very differently compared to Ser293 and Ser300, with the phosphorylation level increasing even during lactate consumption.  used a m complex , pSer293 complex , 59. Mor complex  suggest  complex . The her3.5.3qLac is comparatively lower than that during lactate formation, and the observed lack of pyruvate accumulation during lactate uptake indicates that the activity of PDC is high enough to metabolize the pyruvate formed from lactate or the pyruvate is metabolized by alternative pathways as previously stated. This indicates a reduced pyruvate flux into the TCA cycle, which is also confirmed by the reduced qOUR  and the regulation of pyruvate metabolism may be involved. It can be stated that PDC regulation is much more complex than so far understood, even in the field of well established and characterized CHO processes. To our knowledge, this is the first study which provides data on PDC regulation by phosphorylation under dynamic process conditions. This knowledge could help gauge the importance of PDC and pyruvate better when designing genetic engineering or feeding strategies to regulate lactate metabolism. Further experimental and model‐based studies are needed to enable a more systematic and quantitative understanding of the dynamic regulations of pyruvate metabolism and PDC, which have important implications for both mammalian cell culture and human diseases.For the first time, the interaction of oxidative metabolism with PDC phosphorylations is investigated in this study during the metabolic shift to lactate consumption in antibody‐producing CHO cells. Contrary to the general assumption of reduced phosphorylation levels during lactate uptake , the relative phosphorylation of all three phosphorylation sites was higher during lactate consumption compared to the exponential growth phase. The relative phosphorylation levels of Ser293 and Ser300 were found to be constant during the initial phase of lactate consumption and to decline in the late lactate consumption phase. The phosphorylation of Ser232 increased steadily. These findings suggest that the oxidative metabolism was reduced in CHO DP‐12 cells during lactate uptake, also confirmed by the decreasing Supporting InformationClick here for additional data file.


Drosophila melanogaster, the neuropeptide prothoracicotropic hormone (PTTH) is a modulator of metamorphic timing and aids in adaptation to environmental challenges . The ptth gene can be found in most insect species, with the exception of Apidae , suggesting conservation of structure and function. PTTH binds to its receptor Torso, a receptor tyrosine kinase, to activate a MAPK signaling pathway, which ultimately regulates transcription of the ecdysone biosynthetic genes and the production of ecdysone . Trunk, a paralog of PTTH, also binds Torso, but unlike PTTH, Trunk signals in the early embryo to specify termini cell fates . It has been shown that the pro-domain of Trunk is cleaved intracellularly by Furin 1 and Furin 2 at amino acids K75,R76 and that processing is essential for terminal patterning . While prior in vivo and in vitro experiments in Antheraea pernyi (Sauman and Reppert 1996), Manduca sexta , and Helicoverpa armigera  demonstrated that the recombinant mature fragment of PTTH has the same activity as brain extracts, in each of these cases the negative control was not the pre-propeptide. Only in Bombyx mori has it been shown that mature PTTH, and not the pre-propeptide, is the active species . Based on these observations, it has been generally accepted that pro-domain processing of PTTH is essential for function. For this reason, we wished to determine if, like Trunk, Drosophila PTTH also requires proteolytic cleavage of the pro-domain to be active.In pUAST attB, and integrated by phiC31 at the attP2 site on the third chromosome. The putative processing sites were mutated to two alanines (HR-AA and KR-AA); additionally, the KR site was mutated to tetra-basic RRKR (“KEX”) to better match an optimal Furin cleavage site. The 8K1Jptth mutation was built into each of the UAS-PTTH lines, and the 120F2Aptth mutation was also introduced to the fat body driver line r4-Gal4.Two dibasic residues in PTTH, H34,R35 (HR) and K89,R90 (KR), were predicted to be candidates for proteolytic processing . Wild type PTTH cDNA, as well as mutated versions of the putative processing sites, were 3XHA tagged at the C-terminus, cloned into ptth mutants. We find that when either of the candidate processing sites are mutated, rescue of the timing defect still occurs, suggesting that proteolytic processing at these sites is not required for PTTH activity. Interestingly, when the KR site is converted to a more optimal Furin recognition sequence RRKR (KEX), timing is not only rescued, but accelerated approximately one additional day relative to wild type.The assay for PTTH activity involved looking for rescue of the one-day developmental timing delay of In order to identify the PTTH species found in hemolymph and fat body, samples were obtained from wandering third instar larvae and analyzed on Western blots. We observe the predicted full length 24 kDa protein in wild type PTTH and the three mutants, predominantly in the fat body, as well as a minor 20 kDa fragment in all lines and a new strong 14 kDa fragment in the KEX mutated PTTH line. These results suggest that efficient cleavage is not occurring at the two computer identified sites, which are likely not biologically relevant. However, if one of those sites, KR, is mutated to make a more optimal Furin cleavage site resulting in a new processed protein species, then the biological activity of PTTH is significantly enhanced as evidenced by the acceleration in time to pupation. Using a new prediction algorithm , we find another possible Furin cleavage site at D42,R43 which could produce the minor 20 kDa fragment seen in all lanes. Future work will entail mutating D42,R43 in order to test if Furin cleavage at this site is required for PTTH rescue. Taken together these results show that proteolytic processing of PTTH at either of two tested dibasic sites is not essential for activity. Perhaps cleavage at D42,R43 is required for wild type activity, since we find that engineering a new tetra-basic cleavage site results in higher PTTH activity, which is consistent with the idea that proteolytic processing is required.w1118 ; ptth120F2A/CyO, P{w[+C]=ActGFP} ; r4-Gal4/TM3, P{w[+mC]=ActGFP}Ser[1]w1118 ; ptth8K1J ; {UAS-PTTH 3XHA WT} attP2w1118 ; ptth8K1J ; {UAS-PTTH 3XHA KEX} attP2w1118 ; ptth8K1J ; {UAS-PTTH 3XHA KR-AA} attP2w1118 ; ptth8K1J ; {UAS-PTTH 3XHA HR-AA} attP2yw ; ptth120F2A yw ; ptth8K1J P{w[+mC]=r4-GAL4}3 BDSC 33832anti-HA C29F4 goat anti-rabbit IRDye680 Developmental timing curves. Culture conditions and timing curve derivations are described in Shimell et al. with these modifications. Newly ecdysed, GFP negative L1 larvae were transferred to food vials at a density of ~35 larvae/vial. Pupariation data from 4 individual vials was pooled and analyzed in GraphPad Prism using non-linear regression curve fit. Developmental time is that time at which 50% pupariation occurs or logIC50 of the regression curve.Hemolymph and fat body collection. Hemolymph (HL) from wandering L3 larvae, cultured identically to the timing curves, was harvested by tearing open 5-6 larvae, aspirating the HL with a P2 pipette and placing in 25 μL ice cold PBS. Between 2 and 4 μL of HL were pooled in each of 4 biological replicates. Samples were spun at 10K 10 minutes at 4°. The supernatant was removed using a gel loading tip, sample buffer was added to a 2X concentration  glycerol, 4% (w/v) SDS, 100 μg/mL bromophenol blue, 10% (v/v) β-mercapto-ethanol), heated at 95° 5 minutes and then stored at -40 until use. Additional 2X sample buffer was added to HL samples prior to loading gel to ameliorate bulleting of the sample.Fat body (FB) was teased away from the carcasses of dissected wandering L3 larvae, making sure to avoid salivary glands, Malpighian tubules, and guts. Four biological replicates of the combined FBs of 3-4 larvae in 25 μL PBS were brought to 2X in sample buffer, heated at 95° 5 minutes and stored at -40 until use.The four biological replicates of HL and FB were analyzed by Western blot, and the best sample was chosen for the composite Western.Western blots. NuPAGE™ 4-12% Bis-Tris gels were run in MES SDS running buffer, blotted onto nitrocellulose, pre-hybridized and hybridized to antibodies under conditions recommended by Life Technologies and Li-Cor. Membrane was scanned using the Li-Cor Odyssey instrument with scan parameters of 84 μm resolution, 5.0 intensity and medium quality.


We validate the hypothesis that phosphorylation at Ser262 is necessary for Tau-dependent learning deficits and a “facilitatory gatekeeper” to Ser238 occupation, which is linked to Tau toxicity. Importantly we reveal that phosphorylation at Thr245 acts as a “suppressive gatekeeper”, preventing phosphorylation of many sites including Ser262 and consequently of Ser238. Therefore, we elucidate novel interactions among phosphosites central to Tau mediated neuronal dysfunction and toxicity, likely driven by phosphorylation-dependent conformational plasticity.Hyperphosphorylated Tau protein is the main component of the neurofibrillary tangles, characterizing degenerating neurons in Alzheimer’s disease and other Tauopathies. Expression of human Tau protein in Drosophila CNS results in increased toxicity, premature mortality and learning and memory deficits. Herein we use novel transgenic lines to investigate the contribution of specific phosphorylation sites previously implicated in Tau toxicity. These three different sites, Ser However238 and Thr245, as essential for its toxic effects on mushroom body (MB) integrity , yielded animals with structurally normal but profoundly dysfunctional MBs, as flies accumulating the mutant protein exhibited impaired associative learning  and Elavc155-Gal4;Elav/CyO , were constructed by standard crosses. The hTauR0N4 (0N4R) transgenic flies were a gift from M. Feany .Drosophila crosses were set up d before . The ElaR0N4 cDNA , was subcloned into the pUAS.attB vector (BglII/XbaI fragment. The mutants were generated by replacing the indicated Ser and Thr residues with Ala or Glu using the QuickChange XL site-directed mutagenesis kit (Agilent) according to the manufacturer’s instructions. The complementary mutagenic oligonucleotides pairs (5’ to 3’) for each mutant are shown below. The silent restriction sites introduced for effective screening of positive clones appear underlined in italics whereas the amino acid substitution is shown in bold.To generate the transgenics carrying point mutations, the hTauB vector  as a BglPvuII): 5’ CCACCCAAGTCGCCGTCAGCTGCCAAGAGCCGCCTGCAGACAGC.S238A (TCGACGGTTCTCGGCG GACGTCTGTCG 5’.GGTGGGTTCAGCGGCAGXbaI): 5’ CCAAGTCGCCGTCTGAGGCCAAG TCTAGACTGCAGACAGCCCC.S238E : 5’ CTTCCGCCAAGAGTCGACTGCAGGCAG CCCCCGTGCCCATG.T245A : 5’ CGTCTTCCGCCAAGAGTCGACTGCAG GAGGCCCCCGTGCCCATG.T245E (CAGCTGACGTCCTCCGGGGG CACGGGTAC 5’.GCAGAAGGCGGTTCTBglII): 5’ GCCAGATCTGAAGAATGTCAAGTCCAA GATCGGCGCCACTGAG.S262A (TCTAGACTTCTTACAGTTCAGGTTCTAGCCGCGG TGACTC 5’.CGGBglII): 5’ GCCAGATCTGAAGAATGTCAA GTCCAAGATCGGCGAGACTGAG.S262E (TCTAGACTTCTTACAGTTCAGGTTCT AGCCGCTCTGACTC 5’.CGGPvuII): 5’ CCAAGTCGCCGTCAGCTGCCAAGAGCC GCCTGCAGGCAGCCCCCG.STA (GTCGAC GGTTCTCGGCGGACGTCCGTCGGGGGC 5’.GGTTCAGCGGCAThe sequence of the mutants was confirmed by sequencing (VBC-biotech). Transgenic flies were generated by phiC31-mediated transgenesis by BestGene Inc. . DNAs were injected into genomic landing site 53B2 on the second chromosome (BDSC #9736).To obtain flies for experiments detailed herein, virgins from the driver strains were crossed to males bearing the hTau transgenes and experimental genotypes were selected from the progeny as appropriate and as detailed below:II or w1118 (for controls).Female Elav X 0N4R or 0N4RII: Select male and female non-CyO progeny.Female Elav;Elav/CyO X 0N4R or 0N4RII: Select male and female non-CyO progeny.Female Elav;Ras2 × 0N4R or 0N4RS238A or 0N4RS238E or 0N4RT245A or 0N4RT245E or 0N4RS262A or 0N4RS262E or 0N4RSTA: Select male and female progeny.Female Elav;Ras2 × 0N4R1118: Select male and female progeny for controls.Female Elav;Ras2 X wImmunohistochemistry on paraffin sections of Drosophila adult heads were performed on paraffin sections as previously described  using th202/Thr205-Pierce Endogen, 1:1,000), AT100  and the polyclonal antibodies pS396  and pS262  were used. We generated a novel polyclonal antibody against phospho-Ser238 of hTau, raised in rabbits (Pocono Rabbit Farm and Laboratory) against peptide TPPKSPSpSAKSRLQTAPVPMP and affinity purified before use. In order to normalize sample loading, anti-Syntaxin  was used at 1:3,000. Secondary antibodies were applied at 1:5,000.Total Tau levels and occupation of particular phosphosites were determined in 2–5 adult female head homogenates in 1x Laemmli buffer . The monoclonal antibodies T46 , AT8 , 1–2 day-old males per genotype were maintained at 30°C transferring to fresh vials every 3 days until they expired  with the electric shock unconditioned stimulus (US). The odors used were benzaldehyde (BNZ) and 3-octanol (OCT) diluted in oil (5% v/v for BNZ and 50% v/v for OCT). One hour before training flies were transferred to fresh food vials. For training, a group of 50–70 flies were transferred into a tube lined with electrifiable grid and presented with air (500 mL/min). Flies were first exposed to an odor for 30 s paired with 90 V shock  and then 30 s of air. Subsequently, flies were exposed to the second odor for 30 s without shock and then 30 s of air. Two groups of animals of the same genotype were trained simultaneously such as, one to avoid BNZ and the other to avoid OCT, while the complimentary odorant was used as control odor. The animals were transferred to a T-maze apparatus immediately and flies were tested simultaneously for preferential avoidance of the conditioned odorant allowed to choose between the two odors for 90 s. A performance index (PI) was calculated as the fraction of flies that avoided the CS+ minus the fraction that avoided the CS− odors divided by the total number of flies in the experiment. A final PI is the average of the scores from the two groups of flies trained to complementary conditioning stimuli and ranges from 0 to 100. Because all new transgenes are inserted in the same site we used one of the cognates as heterozygous controls.Behavioral assays were performed under dim red light at 24–25°C and 70-75% humidity. All flies were 2–5 days old, collected under COQuantification of all Western blots was performed by densitometry and the ratio of Tau relative to that of Syntaxin (Syx) was calculated. The ratio of the control genotype was set to 1 and all experimental ratios were reported as relative to that. The means and SEMs were compared following an initial, significant differences- indicating ANOVA (positive ANOVA), using Dunnett’s tests relative to the designated control. Similarly, learning performance indices were calculated for each genotype as indicated above and following positive ANOVA, means and SEMs were compared to that of controls using Dunnett’s tests.Survival data were examined for differences at each assessment day using Wilcoxon/Kruskal-Wallis tests. If significant differences were uncovered then the means and SEMs from each genotype for that day were compared relative to controls using the Steel with control tests. Means and SEMs of survival upon oxidative stress toxicity were compared to that that of controls or the variants as designated using Dunnett’s or planned multiple comparisons tests following an initial positive ANOVA. All statistical details are presented in STA was generated in the 2N4R isoform  (II) did not result in MB ablation , or the pan-neuronal Elav driver on the X with the ubiquitous adult CNS driver Ras2  , pan-neuablation , as prevablation . We hypover Ras2  as detaiII levels relative to those of the original 0N4R revealed significant differences under Elav, Ras2, but not the double driver Elav;Elav, or Elav;Ras2  compared to those in animals expressing 0N4RII under Elav;Ras2 (0.1268 mm2) did not yield significant differences . This indicates that the structural deficits of the MBs are highly sensitive to the amount of hTau present during their development , on the 0N4R isoform suppresses hTau toxicity measured as elevated age-dependent mortality as did when on the 2N4R isoform , supports the notion that its occupation is required for subsequent Ser238 phosphorylation, the opposite might be expected for the phospho-mimic 0N4RS262E. The fact that Ser238 is under-phosphorylated on the 0N4RS262E protein argues that the single charge- bearing Glu is not an efficient substitute for phosphorylated Ser262 in mediating conformational changes promoting phosphorylation at proximal and distant sites  samples from Alzheimer’s disease (AD) patients that show significant enrichment in Ser238 and Ser262 occupation , Thr212/Ser214 (AT100), but also the distant Ser396 at the far carboxy-terminus of the protein (Domains such as the highly conserved microtubule binding repeats and the amino terminal region of the protein appear functionally specialized . Notablycupation . Moreovepatients . We prop protein , which w protein . As all 245 phosphorylation appears to be inhibitory not only to Ser262 and consequently to Ser238 occupation, but also on Ser202/Thr205 and Thr212/Ser214 (245 appears exclusively in physiological human brain lysates (205 (262 phosphorylation as an enabler of phosphorylation at many additional sites, probably a lot more than we have tested herein (238. To that end ongoing efforts aim at generating an anti p-Thr245 antibody, which along with either pSer238and pSer262 may be useful biomarkers in monitoring progression of Tauopathies.Importantly, Thr2/Ser214  and perh2/Ser214 . Hence,  lysates . To our tes (205 . This cod herein , includi245 and others like pSer262 that enable them. This is reflected in the schematic on 245 phosphorylation removes the effect of the toxicity and dysfunction “suppressing gatekeeper,” enabling occupation of the “facilitatory gatekeeper” phospho-Ser262 (Collectively therefore, there appear to be two types of “gatekeeper” phosphosites, those that when occupied suppress additional phosphorylations such as pThro-Ser262 . In agreo-Ser262 , we posi238 (262 (One notable result from the work herein is that the single charge contributed by Glu replacing the relevant Serines and Threonines does not mimic the double charge of the phosphate group in the case of Ser238 , 5C and 238 (262 , 5E. Thi238 (262 . Interes238 (262 , 5C,E, p245, whose phosphorylation appears to function as “suppressive gatekeeper” to subsequent pathology-linked phosphorylations, the therapeutic challenge is to maintain its occupation. In addition, development of an anti-phospho Thr245 antibody for early detection of its dephosphorylated state may be a good disease prognostic biomarker.Although they are obvious pharmaceutical targets, it remains a challenge to identify and interfere with the phosphatases and kinases that target specific phosphoepitopes in part because of overlapping consensus sequences and participation in many other physiological cellular processes . For ThrThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.IK and EV performed research and prepared the manuscript. EV wrote the manuscript initial draft. KP performed research and guidance. ES directed the research and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A total of nine positive isolates , based on real-time reverse transcriptase PCR targeting nucleocapsid (N) gene, were used for further genetic characterization and evolutionary analysis. Genetic characterization of the partial spike (S1) gene revealed the clustering of the reported isolates into three different genotypes, whereas four additional isolates were grouped within 4/91 genotype, two isolates within IS/885 genotype, one isolate was closely related to IS/1494/06, and two isolates were grouped within classic serotype (vaccine-like strains). Phylodynamic revealed clustering of four isolated viruses within GI-13 lineage, three isolates within GI-23 lineage, and two isolates within GI-1 lineage. Results indicate that there are high evolutionary distances between the newly identified IBV strains in this study and the commercially used vaccines (GI-1), suggesting that IBV strains circulating in the KSA are under constant evolutionary pressures. Selective pressure biostatistics analyses consistently demonstrate the presence of a higher positive score which highlights the role of natural selection, a mechanism of virus evolution on sites located on the protein surface, within or nearby domains involved in viral attachment or related functions. Recombination analysis revealed emergence of two isolates through recombination events resulting in new recombinant viruses. Taken together, these finding demonstrate the genetic and evolutionary insights into the currently circulating IBV genotypes in KSA, which could help to better understand the origin, spread, and evolution of infectious bronchitis viruses, and to ascertain the importance of disease monitoring as well as re-evaluation for the currently used vaccines and vaccination programs.In the last 5 years, frequent outbreaks of infectious bronchitis virus (IBV) are observed in both broiler and layer chicken flocks in the Kingdom of Saudi Arabia (KSA) in spite of extensive usage of vaccines. The IBV is a widespread avian coronavirus affecting both vaccinated and unvaccinated chicken flocks and is attributed to significant economic losses, around the globe. In the present study, 58 ( Gammacoronavirus within the family Coronaviridae [Infectious bronchitis virus (IBV) is a highly contagious and emerging viral disease-causing organism of chickens and belongs to genus aviridae . Infectiaviridae . IBV is aviridae ,3. The saviridae ,5. Owingaviridae ,10,11,12Continuous evolution of IBV variants in various regions remains a major concern for chicken production, around the world . Till noThe clinical samples used in this study were collected in strict accordance with the guidelines for Animal Ethics Committees, King Faisal University, Saudi Arabia. Samples of tracheas were collected from diseased and/or dead chickens, stored at −80 °C till used, for diagnosis and virus isolation. The diseased chickens examined in this study were received at Department of Clinical Studies, College of Veterinary Medicine, King Faisal University, Al Hofuf, Saudi Arabia and approved by the poultry farm owners. Diagnosis and experimental protocols in this study were approved by College of Veterinary Medicine, King Faisal University Animal Care Committee.During 2019, 58 samples were collected from different chicken flocks in 4 Saudi Arabian districts . The collected samples were originated from poultry flocks experiencing clinical forms of the disease as respiratory symptoms or problems with egg production associated with high mortality and morbidity rates. Epidemiological information of the studied isolates is summarized in RNA was extracted directly from the allantoid fluid using a QIAamp Viral RNA Mini Kit  according to the manufacturer’s instructions. Detection of IBV was conducted using a real-time reverse transcriptase PCR based on the highly conserved N gene, as described previously . For spihttp://www.ncbi.nlm.nih.gov/WebSub/?tool=genbank), and the accession numbers were obtained.To further characterize the biology and evolution of the newly identified IBV strains, full-length S1 gene sequencing was carried out using BigDye Terminator v3.1 Sequencing Kit  and an automated sequencer . The quality of obtained S1 gene sequences were checked, assembled, and edited using BioEdit software version 7.0.4.1  and submhttp://tree.bio.ed.ac.uk/software/figtree/).Phylogenetic analysis was conducted to explore the phylogenetic relationships with high-level of clustering pattern dependent on the full-length S1 gene between the reported isolates and recently described strains from the Middle East including Saudi Arabia and other parts of the world. Alignments of the obtained nucleotide sequences of full-length S1 genes were performed using the Multiple Alignment using Fast Fourier Transform (MAFFT) method in Geneious software, v11.1.3 . The alignments were then exported to the MEGA program, v7.0.26 . Maximumhttp://www.pymol.org/).Previous studies have reported that most of the neutralizing epitopes and receptor-binding domain of S1 protein were distributed in the three hypervariable regions (HVRs), including HVR I (38 aa–67 aa), HVR II (91 aa–141 aa), and HVR III (274 aa–387 aa), which were usually employed to classify IBV genotypes ,29. Dedup-value was calculated below 1.0 × 10−30. Selective pressure for the putative S1 protein was predicted using synonymous nonsynonymous analysis program (SNAP) services (http://hcv.lanl.gov/content/sequence/SNAP/SNAP.html). The ratio of nonsynonymous to synonymous substitutions (dN/dS) for each amino acid site in the S1 coding region was used to scan for evidence of positive or negative selection.Sequence alignments were conducted, in barrel with different available viruses resembling different genotypes; GI-1, GI-13, and GI-23. The RDP4 software (v4.97) was used to detect any recombination events in the obtained sequences . The S1 We present the isolation and integrative genetic analysis that map the evolution of IBV in Saudi Arabia during 2019. Typical IBV lesions such as embryo curling and dwarfism were detected in embryonated chicken eggs (ECEs) after three successive passages. Using real-time RT-PCR assay that targets the N gene, only nine samples were detected positive (9/58) . FurtherPhylogenetic relationship among IBV strains has been established based on the analysis of the full-length S1 gene. Previous studies for virus characterization are based on the sequence of either HVR I-II or the HVR-III regions only. However, S1 gene sequences including three HVRs should be carried out for both differentiation between the vaccine and field strains and for accurate genotyping ,7,8. OurThe isolated IBV strains were genotyped based on sequencing and subsequent sequence analysis for the S1 gene. The obtained nucleotide and corresponding amino acid sequences were aligned and compared with parental origin and vaccine strains. Sequences of the IBV strains detected in this study were submitted to the GenBank database and the following accession numbers MT270486–MT270494 were assigned. The characterized viruses in this study shared different levels of nucleotide and amino acid sequence identities compared to currently used vaccines in Saudi Arabia, which were between 75% and 90%. Out of the nine reported isolates, two isolates (SA/8/2019 and SA/9/2019) were closely related to GI-1 (vaccine strain) and four identified isolates  were closely related to 4/91 genotype. These isolates showed distinctive amino acids mutations in the HVRs compared to their parental origin and CR88 vaccine strain, which is commonly used in Saudi Arabian poultry . LikewisCompared with the 3D capsid structure of reported isolates in the PDB database (PDB ID: 6CV0), prominent amino acid substitutions were located in the major receptor-binding domain (aa 19–aa 253) a and adjMoreover, residues at positions 38, 43, 63, and 69 are critical for binding of the IBV spike protein to the receptors in the chicken respiratory tract [p < 0.01 and recombinant score >0.6. The recombinant IBV strain IBV/CH/SA/7/2019 could be as a result of recombination between two strains, major parent is belonging to GI-23 lineage, while the minor parent is closely related to GI-13 lineage, which is a vaccine like strain (CR88)  a, wherean (CR88) b. To furn (CR88) c. Additin (CR88) c.Notoriously, IBV strains can undergo many genetic modifications generated both by recombination and mutations such as substitutions, deletions, and insertions, which could lead to the emergence of new variants. There are characteristic features of coronaviruses in the genome structure (large single-stranded RNA) and virus biology  and immunological pressure caused by the vaccines that create favorable conditions for such mutation events . It is wCurrently, several new genotypes or serotypes have been reported worldwide as a result of mutations within the S1 gene ,18,19. AIn this study, we focused on the molecular characterization of IBVs within the Saudi Arabian commercial poultry flocks. Genotyping was accomplished by phylogenetic analysis of the S1 coding region sequences against reference strains representing all genotypes and lineages ,18,19 foPhylogenetic analysis indicated that IBV strains in this study were of three distinct genotypes: GI-1, GI-13, and G-23 ,18,19. OHypervariable regions (HVRs) in the S1 gene showed distinct patterns with prominent amino acid substitutions among different viruses compared to the parental origin for the isolated viruses in this study. Some of these amino acid residues are responsible for the virus tropism and pathogenesis at positions 38 (T/Q/S), 43 (Y), 63 (G/Q), and 69 (S/A) in the receptor-binding site. Likewise, using the protein 3D modelling prediction, there are three amino acid substitutions near the HVR I/II, which might help in the virus evolution leading to change in the antigenicity and pathobiology especially due to localization of these mutations within the RBD.IBV of GI-13, GI-16, and GI-23 lineages have been circulating in the Middle East region for the previous 20 years and are also detected in Saudi Arabia ,36,37,38The role of vaccination in driving viral evolution has been reported for different diseases affecting both animals and human beings . When imIn conclusion, continuous monitoring and surveillance for IBVs is required not only in commercial poultry flocks but also in wild birds which will elucidate sequence characteristics of prevailing strains to help revising and updating the vaccination strategies to develop and or choose a vaccine that can give cross-protection against majority of the circulating genotypes. Transmission of IBVs through wild birds has been reported in neighboring countries to Saudi Arabia such as Israel, Jordan, and Egypt, highlighting that wild birds might play a vital role in the virus epidemiology. The current study revealed the cocirculation of multiple IBV genotypes within Saudi Arabia owing to high mutation rate and subsequent residue substations leading to the evolution of IBV variants and potentially, vaccination failure. Likewise, further studies are required to understand the genetic and biological characteristics of IBV variants in relation to antigenicity, pathogenicity, tropism, and shedding as well as evolution-driven failure in vaccine efficacy.