In vitro studies of fibrinolysis reveal mechanisms regulating these processes and often employ fluorescent microscopy to observe protein colocalization and fibrin digestion. In this study, we investigate the effects of labeling a fibrin network with 20 nm diameter fluorescent beads (fluorospheres) for the purpose of studying fibrinolysis. We observed fibers and 2-D fibrin networks labeled with fluorospheres during fibrinolysis. We found that the labeling of fibrin with fluorospheres can alter fibrinolytic mechanisms. In previous work, we showed that, during lysis, fibrin fibers are cleaved into two segments at a single location. Herein we demonstrate that fibrinolysis can be altered by the concentration of fluorospheres used to label the fibers, with high concentrations of fluorospheres leading to very minimal cleaving. Furthermore, fibers that are left uncleaved after the addition of plasmin often elongate, losing their inherent tension throughout the imaging process. Elongation was especially prominent among fibers that had bundled together due to other cleavage events and was dependent on the concentration of fluorophores used to label fibers. Of the fibers that do cleave, the site at which they cleave also shows a predictable trend dependent on fluorosphere concentration; low concentrations heavily favor cleavage locations at either end of fibrin fiber and high concentrations show no disparity between the fiber ends and other locations along the fiber. After the initial cleavage event bead concentration also affects further digestion, as higher bead concentrations exhibited a larger population of fibers that did not digest further. The results described in this paper indicate that fluorescent labeling strategies can impact fibrinolysis results.Hemostasis is the cessation of bleeding due to the formation of a blood clot. After the completion of wound healing, the blood clot is typically dissolved through the natural process of fibrinolysis, the enzymatic digestion by plasmin of the fibrin fibers that make up its structural scaffold.  Fibrinogen, an abundant plasma protein, constantly circulates in the vasculature at normal concentrations of 1.5–4.0 mg/mL . AssociaNormally, plasmin, a proteolytic enzyme activated from its zymogen precursor, plasminogen, degrades the fibrin network after sufficient time for healing has passed, restoring normal blood flow around the injury site . Hinderein vivo is technically difficult  and leads to challenges in differentiating cause and effect due to the many molecular processes in fibrinolysis [in vitro methodologies, with fluorescent microscopy being particularly popular [Researching fibrinolysis and its complexities inolysis . Because popular . Multipl popular , fluores popular , 10, flu popular , and car popular –15. Eachin vitro fibrinolytic studies.Here we focus on investigating the unexpected fibrinolytic effects of using 20 nm diameter, carboxylate-coated polystyrene beads to label fibrin fibers during fibrinolysis studies. The beads are commonly utilized in other fibrin studies due to their photostability and low photobleaching –18, and th the fibrinogen concentration. Separately, the thrombin aliquot was diluted to 2 U/mL using HBS with CaCl2. 10 μL of fibrinogen solution was pipetted onto the ridged surface and was mixed with 10 μL of thrombin solution, giving a final fibrinogen concentration of 0.3 mg/mL and final thrombin concentration of 1 U/mL on the slide. The samples were sealed inside a petri dish along with damp paper to ensure a moist atmosphere for the sample and left to polymerize for one hour at 37°C [On a glass microscope slide a small amount of optical glue , pressed under a PDMS stamp, was cured under ultraviolet light, resulting in a ridged surface with gaps between ridges measuring 20 μm wide and 10 μm deep. Aliquots of peak 1 fibrinogen , ALEXA-488 fibrinogen , and human alpha thrombin  were thawed from -80°C. In a microcentrifuge tube, fibrinogen was diluted to 0.6 mg/mL with a HEPES Buffered Saline  and mixed with ALEXA-488 fibrinogen at 1/65 at 37°C .™ Carboxylate-Modified Microspheres, Invitrogen by Thermo Fisher Scientific/Life Technologies Corporation, Eugene, OR) (interchangeably referred to as beads) solution, at the specified dilution, was sonicated for 5 minutes and added to the sample. The bead solution was incubated with the fibrin for 1 minute at room temperature and then gently pipetted off. The sample was then washed three times with 30 μL of HBS buffer to ensure removal of any excess bead solution. Following the washes, 20 μL of HBS buffer was placed on top of the sample prior to conducting microscopy imaging.After polymerization, a pipette was used to gently remove a superficial fibrin mesh, leaving a 2-D fibrin network on the ridged surface. In experiments where beads were used, 30 μL of a 20 nm carboxy-coated microsphere , using a 63x oil-immersion objective. Using light with a wavelength of 480 nm, corresponding to the excitation wavelength of ALEXA-488 labeled fibrinogen, an imaging area was located. Sample areas containing at least 4 individual fibers, each separated by at least 1μm within the camera frame were selected for imaging to maximize the rate of data acquisition. Once an area was found, the microscope settings were changed to a wavelength of 553 nm, the excitation wavelength of the carboxylate-coated polystyrene beads (580 nm) under investigation. We located the fibers using the 480 nm wavelength to minimize fluorescent excitation of the beads while locating the fibers, because the bead excitation spectrum has a minimum ~480 nm, per the manufacturer’s data sheet. This was done because we suspected that the beads may be sensitive to light dosage. While later experiments did not bear this hypothesis out (see results), we maintained this experimental approach. To initiate lysis, plasmin  was added directly to the sample and then time-series acquisition began immediately afterwards. After the addition of plasmin, the attached microscope camera (Leica DFC9000GT SCMOS 4 Megapixel monochrome camera) captured an image every 30 seconds for one hour. The camera shutter was set to remain open during the entire imaging process, unless otherwise stated. An example time series is displayed in When the time-lapse was completed, the file was imported into FIJI image analysis software where the singular fibers were analyzed . In the C is the concentration of suspended beads in weight volume % per 100g of beads in 1 mL (0.02 for 2% solids), ρ is the density of bead material (1.05 g/mL for polystyrene), and d is the diameter of microspheres in μm. The beads used here are 0.02 μm in diameter, yielding a stock bead concentration of 4.55x1012 beads/μL, which corresponds to roughly 714 beads/fiber volume (.16 μm3) in solution, assuming a fiber length and diameter of 20.0 μm and 100 nm, respectively [The beads were provided as 2% solids, and a calculation of the bead concentration was done as follows (per the manufacturer):10 beads/μL), 1:300 (1.52x1010 beads/μL), 1:1000 (4.55x109 beads/μL), 1:2000 (2.27x109 beads/μL), 1:3000 (1.52x109 beads/μL), and 1:10,000 (4.55x108 beads/μL) were tested, corresponding to bead concentrations of: 7.1, 2.3, 0.7, 0.36, 0.24, and 0.07 beads/fiber volume, respectively. Dilution factors were chosen based on the results shown in To test the effect of bead concentration during labeling on fibrinolytic results, a dilution series of stock bead solutions was made with HBS buffer. Dilutions from stock of 1:100  to find areas with individual fibers while minimally exciting the beads. The wavelength was changed to 553 nm (corresponding to the fluorescent bead excitation wavelength (580 nm)), plasmin was added, and images were collected every 30 s for one hour. Plasmin  was aliquoted and frozen (-20° C) at concentrations that were double the desired final concentration. During an experiment, one aliquot was thawed and added to the fibrin-network containing solution at equal volumes , thereby halving the plasmin concentration. Five trials were conducted at each of the following final plasmin concentration, 0.7 U/mL, 1.0 U/mL, and 2.0 U/mL.To test the effect of plasmin concentration on the lysis of bead-labeled fibers, trials at varying plasmin concentrations were conducted. Samples were made as described previously and labeled with a bead solution at 4.55x10-5 s, was approximated from the diffusion coefficient of a 24 nm diameter bead (D≈1x10-7 cm2/s) and the 0.02 μm space step via: To test mechanisms suggested by experiments, computer simulations of 2-dimensional bead diffusion and binding to fibrin were conducted. Simulations were run with MATLAB R2016a software using custom code. The simulation domain was a 2 μm by 2 μm box with reflecting boundary conditions on the top, left, and right, and with an absorbing boundary condition on the bottom. The bottom of the domain represented a fibrin fiber, and hence the absorbing boundary condition modeled irreversible binding of the beads to the fiber. The domain was partitioned into a grid using space steps of 0.02 μm in both the x- and y-directions. In a single simulation, 100 beads (20 nm diameter) were uniformly and randomly distributed on grid nodes in the top half of the simulation domain. At each time step, each bead could remain at its current grid node or move to one of the 4 nearest neighbor grid nodes, with equal probability. The time step, 1x10timestep . Each siMuch of the data presented in the paper consists of counts of events or the percentage of one type of event occurring out of the total events. When testing differences between two results (counts or percentages), the statistical significance was determined by a two-tailed ANOVA test with a p-value cutoff of 0.05.When investigating the relationship between percentage measurements and concentration of beads or enzymes, regression analyses were performed in Rstudio . PercentThroughout the text and displayed in figures, error values or bars are representative of standard error.By analyzing the lysis of small fibrin networks labeled with fluorescent beads, we determined that multiple lytic outcomes are possible: (1) normal cleavage along the length of the fiber and subsequent digestion See , (2) cle8, 1.52x109, 2.27x109, 4.55x109, 1.52x1010, and 4.55x1010 beads/μL, using a fixed plasmin concentration of 1 U/mL. Results from these tests showed a strong correlation between the percentage of fibers cleaved and bead concentration , corresponding to only 1.2% of fibers remaining at this dilution. All non-elongated, non-cleaved fibers maintained their inherent tension present at the beginning of data collection. As bead concentration was decreased, the rate of elongation rose; 34.9% of remaining fibers elongated at 1.52x1010 beads/μL, 56.8% at 4.55x109 beads/μL, 83.3% at 2.27x109 beads/μL, and 100% at 1.52x109 beads/μL, but it is important to note that the number of uncleaved fibers also decreased, so there were fewer fibers that could have elongated. Both trends are displayed graphically in The bead concentrations tested were, in order of increasing concentration, 4.55x10ntration . At the 10 beads/μL and 4.55x109 beads/μL at a plasmin concentration of 1.0 U/mL. Results are shown in 10 beads/μL, 134 fibers were analyzed; 85/134 were imaged with the camera shutter constantly open and 49/134 were imaged with the camera shutter only open during image acquisition. At a concentration of 4.55x109 beads/μL, 196 total fibers were analyzed; 103/196 were imaged with the camera shutter constantly open and 93/196 were imaged with the camera shutter only open during image acquisition.To test whether light dosage affected our results we performed experiments where the shutter remained open during the entire plasmin degradation process and compared the results with those from experiments where the shutter was only open during image acquisition. Images were collected every 30s with an exposure time of 200 ms. We measured the light power at the surface of the glass using a power meter (Ophir StarLite 7Z01565). Under identical conditions to those used during the experiments, 160–180 μW of power was measured. Thus, for experiments where the shutter was left open during the one-hour image acquisition period, 0.612 J of energy would be deposited into the sample, while in experiments where the light was only on during image acquisition, 0.0041 J of energy would have been deposited, 150-fold less energy. Data was collected for two bead concentrations, 4.55x109 beads/μL concentration, we varied the plasmin concentration to test its effect on these results. At the lowest plasmin concentration tested, 0.7 U/mL, 48.5 ± 19.5% of fibers initially present remained through the duration of data collection. At a plasmin concentration of 1.0 U/mL, 35.6 ± 21.8% of fibers remained (agreeing with our previous results), and at 2.0 U/mL plasmin, 34.6 ± 12.7% of initial fibers last through data collection. Elongation of persisting fibers throughout these trials at differing plasmin concentration remained roughly constant. At a plasmin concentration of 0.7 U/mL, 47.8% of fibers elongate, compared to 46.9% and 44.2% of fibers displaying elongation at 1.0 U/mL and 2.0 U/mL plasmin, respectively. This falling percentage of fibers remaining, and constant rate of elongation are shown in Using a fixed 4.55x10Often, in experiments, fiber fragments from a cleaved fiber would bundle with an uncleaved fiber, creating a thicker fiber. Sometimes the ends of two cleaved fibers would stick together and create a new fiber. For counting purposes, we count each of these situations as one bundled fiber because both situations result in a single fiber. Described in 10 beads/μL concentration, 81/85 fibers persisted throughout plasmin exposure, but only one fiber showed elongation, and it did so without bundling. Decreasing the bead concentration from 4.55x1010 beads/μL to 1.52x1010 beads/μL shows more fibers elongating without bundling: 55/86 fibers persisted, and 17/55 remaining fibers elongated without bundling. Only 3/55 remaining fibers bundled, and of those three, one elongated and two retained their tension. At a concentration of 4.55x109 beads/μL 44/103 fibers persisted. Of those 44 fibers, 20 fibers bundled and then elongated, while 18 fibers elongated without bundling, 2 fibers bundled but didn’t elongate, and 4 fibers remained tense. Thus, 90.9% of fibers that bundle at this concentration show elongation. A 2.27x109 beads/μL concentration showed an increased number of cleaved fibers, with only 12/82 fibers persisting. Of these remaining fibers, 8 fibers bundled, 7 of which elongated (87.5%), only one fiber elongated without bundling, and 3 fibers remained tensed. At 1.52x109 beads/μL, only 2/87 fibers persisted, and both of those fibers bundled with other fiber fragments and elongated. A linear regression analysis of these results indicated that elongation without bundling is independent of bead concentration, but that bundling and elongation with bundling do depend on bead concentration  of fibers that show elongation without undergoing a bundling event first. Shown in 9 beads/μL). At a plasmin concentration of 0.7 U/mL, 48/96 fibers persisted, with 23/48 observed fibers bundling, 17/23 of these elongated (73.9%), and 12/48 fibers elongated without bundling. With 1.0 U/mL plasmin, 47/118 fibers remained, with 24/47 fibers bundling, 21/24 of which elongated (87.5%), and 18/47 elongated without bundling. Finally, at a plasmin concentration of 2.0 U/mL, 25/75 fibers persisted, with 16/25 fibers remaining because of bundling, 10/16 later elongated (62.5%), and 1/25 fibers elongated without a bundling event. A linear regression analysis of these results indicated that elongation without bundling is dependent on the plasmin concentration, but that bundling and elongation with bundling are independent of plasmin concentration  of fibers bundled, and all of these occurred in areas of higher local fibrin fiber density. Importantly, all these fibers, labeled with only ALEXA-488, recoiled backwards or were digested away rather than persisting throughout image acquisition like observed in some bead labeled fibers.9 beads/μL, 97.2% of fibers had their cleavage event occur at the ridge of the optical adhesive substrate, while the remaining 2.8% cleaved at some non-specific point along the length of the fiber. Increasing the bead concentration to 2.27x109 beads/μL resulted in 90.7% of fibers cleaving at the ridge-fiber connection and 9.3% cleaving along the length of the fiber. Increasing the bead concentration again to 4.55x109 beads/μL continued this trend with 80.3% cleaving at the ridge and 19.7% cleaving along the length of a fiber. At a bead concentration of 1.52x1010 beads/μL, 62.6% of fibers cleaved at the ridge and 37.4% cleaved along the length of the fiber. Finally, at the highest tested bead concentration, 4.55x1010 beads/μL, 50.0% of fibers cleaved at the ridge and 50.0% of fibers cleaved along the length of a fiber. This correlation is shown in Next, we investigated the location of cleavage events and found that bead concentration influences the site of cleavage along a fiber. At a bead concentration of, 1.52x10Fibers that cleaved at their connection to the ridge then showed two digestive outcomes: (1) they collapsed back to the connected ridge as expected, or (2) they persisted throughout the remainder of data collection connected to one ridge and did not display signs of further collapsing. Across all fibers observed that cleaved at the ridge connection, 224 fibers displayed the expected recoil and digestion after cleaving. 248 fibers in the study were observed cleaving at a ridge connection and then showed no further signs of digestion. These digestion outcomes are shown in 10 beads/μL, the mean cleavage time was 240.0 ± 84.9 seconds after the addition of plasmin. At a concentration of 1.52x1010 beads/μL, this timeframe was reduced to 84.19 ± 20.0 s. Lowering the bead concentration from 4.55x109 beads/μL, 2.27x109 beads/μL, and 1.52x109 beads/μL, the mean cleavage times dropped to 75.31 ± 22.9 s, 34.72 ± 4.9 s, and 33.53 ± 4.8 s, respectively.Bead concentration is also a determinant of the length of time it takes for a fiber to cleave. Computer simulations reveal how the location along a fiber affects the frequency with which a bead binds  and the in vivo systems, it is vital to ensure that molecular markers added to the system do not alter the physiological processes in some way. Because of the observed fiber elongation in previously published results regarding fibrinolysis [Fibrinolysis assays have the potential to both reveal biophysical and biochemical processes that play a role in blood clot digestion and help to diagnose pathologies. In developing such assays meant to mimic inolysis , 13, we inolysis , but imp10 beads/μL, there were nearly no cleavage events observed. As the fluorescent bead concentration decreased, there was an increase in fiber cleavage events and a decrease in the percentage of fibers that remained throughout the course of imaging. These trends are in qualitative agreement with a recent report by Rimi and Helms [9 beads/μL, Rimi and Helms found that <10% of fibers were cleaved, while in our experiments > 60% of fibers were cleaved [Our results indicate that the carboxy-coated nanospheres (beads) can affect fibrinolytic outcomes via several distinct mechanisms. As shown in nd Helms , but res cleaved . These dOne possible explanation for this effect, outlined in 9 beads/μL and 1.52x109 beads/μL) often elongate could also be explained by the proposed sheathing mechanism. High bead concentrations would block plasmin binding and result in little degradation and loss of tension, while lower bead concentrations could result in small gaps in the sheath where plasmin molecules could begin the lytic process. Presumably, elongated fibers are partially digested. Plasmin digests fibrin in a specific sequence, beginning with the unstructured followed by the coiled-coil region [10–4.55x109 beads/μL, while at even lower concentrations most or all fibers are cleaved. Rimi and Helms (2022) also reported elongated fibers , however, they only reported an elongation percentage (20%) for bead concentrations at 4.5 x108 beads/μL [8 beads/μL, but often don’t report how such a dilution was obtained. Based on our results, where most fibers lyse rapidly at such a low bead concentration, we suspect that bead concentrations may often be higher than what is reported due to errors in diluting to such a small value. In this study, careful serial dilutions were employed to ensure accuracy.Also shown in l region . Perhapsbeads/μL , whereasbeads/μL . Of notebeads/μL , 16, 17,9 beads/μL concentration. Plasmin concentration was varied between 0.7, 1.0, and 2.0 U/mL. At the lowest plasmin concentration, 0.7 U/mL, the highest number of remaining fibers was observed. As plasmin concentration increased, the number of cleaved fibers increased, and the percentage of remaining fibers fell but appears to be reaching a limit ~ 2 U/mL. Changing the plasmin concentration from 0.7 U/mL to 2 U/mL increased fiber cleavage percentage by ~15%. This suggests that bead concentration was a stronger determinant of fibrin cleavage than plasmin concentration. However, the increase in fiber cleavage as a function of plasmin concentration suggests that a higher number of plasmin molecules make it more likely for plasmin to find “chinks” in the bead “armor” that it can penetrate through to initiate lysis. While the percentage of remaining fibers decreased with increasing plasmin concentration, as expected, the ratio of tensed to elongated fibers remained roughly stagnant, supporting the idea that elongation is a function of bead concentration.Bundling events in this study, 10 beads/μL, very few fibers cleaved, resulting in nearly no fibers bundling. At concentrations of 1.52x1010 and 4.55x109 beads/μL, a larger percentage of fibers elongated without bundling, but at 4.55x109 beads/μL roughly equal amounts elongated after bundling and elongated without bundling. At fluorescent bead concentrations lower than 4.55x109 beads/μL a decreasing number of uncleaved fibers left fewer fibers to elongate or bundle; however, those that did remain uncleaved tended to do so because of bundling, and those that bundled were likely to elongate. Statistical analysis demonstrated that the number of fibers that elongated without bundling was independent of bead concentration, while the number of fibers that bundled and elongated did depend on bead concentration. The results of When specifically looking at bundling in the bead concentration experiments, It is important to note that our experimental setup was specifically designed to isolate individual fibers. Thus, when a bundling event did occur, the fragments of one cleaved fiber often moved a distance of several microns before encountering another fiber to bundle with. In previous experiments investigating the lysis of 2-D dense networks of fibers that had also been labeled by beads, 30 ± 17% of fibers were observed to be bundled . This ag10 beads/μL, there is an even split between fibers that cleaved at the ridge and cleaved along their length, although it should be noted that there were very few fibers cleaved at all at this concentration. Interestingly, using the same ridge-based experimental setup, for fibers that weren’t labeled by beads, 39% (179/455) cleaved at the ridge, while 61% (276/455) cleaved somewhere along the fiber length.With varying bead concentration there is also a difference in the location along a fiber in which cleavage occurred . As beadThis result is somewhat surprising as intuition would suggest that as the beads become more dilute, the fiber cleavage sites should behave more like unlabeled fibers, but this was not the case. This effect could possibly be explained under the sheathing hypothesis by the assumption that beads binding in the complex angles formed between the fibers and the ridges is unfavorable, so at lower concentrations there are few beads near the ridges that would block plasmin from binding to fibrin, making those sites particularly susceptible to cleavage. At higher concentrations beads nearly completely cover the fibers, so any “exposed” plasmin binding site would be randomly located anywhere along the fiber and thus the location of fiber cleavage is more heterogeneous. The simulation results in Fibers that cleaved along their length typically had the two new fiber halves recoil backwards and collapse back onto the ridge. However, this was not always the case for fibers that cleaved at their connection to one of the ridges. When these fibers pulled away from the ridge, there were two observed outcomes: (1) the recoil and collapse/digestion of one long fiber segment, or (2) the long fiber segment showed no signs of further digestion see . Fiber sBead concentration played a major role in the length of time for plasmin to cleave a fiber. As shown in Our data provides some evidence that elongation is a function of light dosage, however statistical analysis was not indicative that this difference in means was significant see . Light dOur results have implications for future fibrinolysis studies. This study has highlighted the potential pitfalls of utilizing fluorescence labeling in a system in which beads do not exist under physiological conditions. First, the addition of fluorescent beads to a fibrin network altered how single fibers, and thus the network, digested, with roughly one-third of all fibers observed persisting through digestion and elongating. Second, the concentration of fluorescent beads played a crucial role in single fiber degradation, with almost 100% of fibers labeled with the highest bead concentration tested not cleaving, and all fibers cleaving at the lowest bead concentration tested. Third, bead concentration correlated with the cleavage location, with low bead concentrations strongly favoring cleavage locations near the stamped ridges of optical adhesive. Lastly, fluorescent bead concentration altered the amount of time for cleavage to occur, with high bead concentrations resulting in longer cleavage times. These results have arisen from the testing of one brand, size, type, and corresponding wavelength of fluorescent bead, although it is likely our results would expand to other fluorospheres, as has been recently reported . FurtherS1 FileThis supplemental file contains plots and statistical analysis referred to in the main text.(DOCX)Click here for additional data file.S2 FileThis code simulates the 2D diffusion of beads in a rectangular domain. It was used to generate the data in (M)Click here for additional data file.S1 Movie(AVI)Click here for additional data file.S2 MovieNote the taunt fiber structure in the beginning of movie (0 s) compared to the loose, “elongated”, structure present at the end of movie (60 min).(AVI)Click here for additional data file.S3 MovieNote that after bundling the newly formed fiber persists throughout the remainder of image acquisition (60 min).(AVI)Click here for additional data file.S4 Movie(AVI)Click here for additional data file.S5 MovieBeads (blue circles) diffuse in a 2 micron by 2 micron domain with reflecting boundary conditions on the top, left, and right boundaries. The bottom boundary (at y = 0) corresponds to the fibrin fiber. When a bead diffuses to a position on this boundary, it "binds" to the fiber at that location and remains there for all time. Each frame of the movie is 100 timesteps after the frame preceding it. The time step for this simulation was 0.000001 s. Beads, which have diameter 24 nm, are not drawn to scale.(MP4)Click here for additional data file.


T-cell activation is a complex process involving a network of kinases and downstream molecular scaffolds or adaptors that integrate surface signals with effector functions. One key immune-specific adaptor is Src kinase-associated phosphoprotein 1 (SKAP1), which is also known as src kinase-associated protein of 55 kDa (SKAP55). This mini-review explains how SKAP1 plays multiple roles in regulating integrin activation, the “stop-signal”, and the optimization of the cell cycling of proliferating T cells through interactions with various mediators, including the Polo-like kinase 1 (PLK1). Ongoing research on SKAP1 and its binding partners will likely provide important insights into the regulation of immune function and have implications for the development of new treatments for disease states such as cancer and autoimmunity. The Importantly, the initial contact of a T cell with an APC ligates the T-cell receptor (TCR) complex that then induces “inside-out” signals for high-avidity adhesion . This islck to human CD4 and CD8, which initiates a phosphorylation cascade and leads to the phosphorylation of the antigen–receptor complex . The earliest events induced by TCR ligation involve the induction of a tyrosine phosphorylation cascade in T cells , 11. Tyr complex , 11, 14.antigens , 16. Theantigens  while ot process .lck complexes and p56lck alone can then phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) in the CD3 and T-cell receptor ζ chains for the recruitment of a second protein kinase ZAP-70 (ζ-chain associated protein kinase-70) kinase  kinase . Both p5 nucleus , 20. p56 kinases , 21. The kinases , 23. ZAP kinases . The LAT kinases . Recentl kinases . This isIn addition to the activation of gene transcription, the TCR must activate integrins such as LFA-1 on the surface of T cells to generate stable conjugation with an APC . Integrifyn , a binding partner of SKAP1, and is abrogated by the deletion of the SKAP1 SH3 domain. Conjugate formation is also accompanied by the translocation of SKAP1 to membrane rafts  cells. Instead, the skap1-/- T cells had impaired binding to ICAM1, more transient conjugation times, and a reduced localization of TCR-CD3 micro-clusters at the IS (Early transfection and knock-down studies showed that SKAP1 promotes dwell times between T cells and dendritic cells (DCs) , 44–49. ne rafts , 47. Thevia at least two non-exclusive pathways. One pathway involves SKAP1 binding to the SARAH domain of RapL  then binds to the αL chain of LFA-1, promoting its clustering and avidity for its ligand , 61. Comvia the TCR by mechanisms that were independent of RIAM, TALIN, and beta integrin activation. Different mechanisms may therefore be at play that determine the way in which SKAP1 and its dimerization regulate adhesion or other functions. In the context of integrin activation, SKAP1 primarily regulates cluster formation and, to a lesser extent, affinity, while RIAM, KINDLINs, TALIN, and other proteins primarily mediate LFA-1 conformation and affinity changes. It is still unknown whether dimer formation influences PLK1-mediated effects on cell cycling. Overall, these findings highlight the complexity of SKAP1 cellular signaling and the need for further research to fully understand the role of SKAP1 and its dimerization in various cellular functions.One intriguing feature of SKAP1 is its ability to form dimers as mediated by the N-terminal dimerization domain (DM)  Figure, 59, 60.in vitro. Furthermore, PLK1 bound to the N-terminal residue serine 31 (S31) of SKAP1 and the interaction is needed for optimal PLK1 kinase activity in T cells  for the optimal cycling of T cells Figure 2 T cells . Further T cells .ras–extracellular signal-regulated kinase (ERK) pathway. p21ras activity is regulated by several mediators that include the guanyl releasing protein 1 (RasGRP1). RasGRP1 exchanges GDP for active GTP on p21ras in a cascade that activates ERKs (skap1-/- primary T cells had increased RasGRP1 in the trans-Golgi network (TGN) following CD3 ligation where p21ras becomes activated , we showed that itis CIA . This waitis CIA . Lakkis itis CIA . Lastly,r growth . Genome-r growth , 73. It Overall, while SKAP1 is an immune cell adaptor that regulates T-cell adhesion and optimal cell growth, much remains to be discovered about the physiological impact of SKAP1 in regulating immunity. Further research is needed to fully understand the full range of mechanisms by which SKAP1 regulates T-cell signaling and immune function as well as the downstream effects of SKAP1 activation or inactivation. This may involve assessing the impact of SKAP1 on specific immune cell subsets or its place in various affected disease states such as autoimmunity, transplant rejection, and cancer. Overall, data so far demonstrate that it is an adaptor with multiple functions associated with different regions of the protein. Continued research on SKAP1 will provide important insights into the regulation of immune function and will have implications for the development of new therapies to treat disease states.All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.


Wasmannia auropunctata, commonly known as the electric ant or little fire ant. Because viruses can be effective biological control agents against many insect pests, including ants, a metagenomics/next-generation sequencing approach was used to facilitate discovery of virus sequences from the transcriptomes of W. auropunctata. Five new and complete positive sense, single-stranded RNA virus genomes, and one new negative sense, single-stranded RNA virus genome were identified, sequenced, and characterized from W. auropunctata collected in Argentina by this approach, including a dicistrovirus (Electric ant dicistrovirus), two polycipiviruses , a solinvivirus (Electric ant solinvivirus), a divergent genome with similarity to an unclassified group in the Picornavirales (Electric ant virus 1), and a rhabdovirus (Electric ant rhabdovirus). An additional virus genome was detected that is likely Solenopsis invicta virus 10 (MH727527). The virus genome sequences were absent from the transcriptomes of W. auropunctata collected in the USA (Hawaii and Florida). Additional limited field surveys corroborated the absence of these viruses in regions where the electric ant is invasive . The replicative genome strand of four of the viruses  was detected in Argentinean-collected W. auropunctata indicating that the ant is a host for these viruses. These are the first virus discoveries to be made from W. auropunctata.Despite being one of the most destructive invasive species of ants, only two natural enemies are known currently for The online version contains supplementary material available at 10.1007/s11262-023-01969-1. Wasmannia auropunctata  is an invasive myrmicine tramp ant species native to Central and South America that is now well established throughout tropical and sub-tropical regions of the world, including the USA  ) indicative of helicase function and an RdRp motif characteristic of viruses in this order . Key to virus abbreviations, family, and accession numbers: AhV, Atkinsonella hypxylon virus, Partitivirdae, L39126; ALSV, Apple latent spherical virus, Secoviridae, NC030941.1; ANV, avian nephritis virus, Astroviridae, AB033998; APV, Acyrthosiphon pisum virus, Unassigned, NC003780.1; BaYMV, Barley yellow mosaic virus, Potyviridae, NC002990; BBWV 1, Broad bean wilt virus 1, Secoviridae, NC005289.1; BDRC, Bryopsis cinicola chloroplast replicon, Unclassified; BDRM, Bryopsis mitochondria-associated dsRNA; BWYV, Beet western yellows virus, Solemoviridae, NC004756; CHV1, Cryphonectria hypovirus 1, Hypoviridae, NC001492; CHV2, Cryphonectria hypovirus 2, Hypoviridae, NC003534; CHV3, Cryphonectria hypovirus 3, Hypoviridae, NC000960; CHV4, Cryphonectria hypovirus 4, NC006431; CPMV, Cowpea mosaic virus, Secoviridae/Comovirinae, NC003549.1; CPV, Cryptosporidium parvum virus-1, Partiviridae, GCA002868475; CRLV, Cherry rasp leaf virus, Secoviridae, NC006271.1;CrPV, Cricket paralysis virus, Dicistroviridae, NC003924.1; CtRLV, Carrot red leaf virus, Solemoviridae, NC006265; DCV, Drosophila C virus, Dicistroviridae, NC001834.1; DWV, Deformed wing virus, Iflaviridae, NC004830.2; EADS, Electric ant dicistrovirus, Dicistroviridae, OP518023; EAPV1, Electric ant polycipivirus 1, Polycipiviridae, OP518021; EAPV2, Electric ant polycipivirus 2, Polycipiviridae, OP518022; EASV, Electric ant solinvivirus, Solinviviridae, OP518024; EAV1, Electric ant virus 1, Unclassified, OP518025; EMCV, encephalomyocarditis virus, Picornaviridae, NC001479; FCCV, Fragaria chiloensis cryptic virus, Partitiviridae, NC009519; FCV, Feline calicivirus, Caliciviridae, GCA008767155; FGMV, Fusarium graminearum mycovirus, Unassigned, LC006128; FHV, felid herpesvirus 1, Nodaviridae, NC013590; FMDV, Foot-and-mouth disease virus, Picornaviridae, GCA008799075; GFLV, Grapevine fanleaf virus, Secoviridae/Comovirinae, NC003615.1; GGNNV, Greasy grouper nervous necrosis virus, Nodaviridae, AF318942; GLV, Giardia lamblia virus, Totiviridae, NC003555; HaRNAV, Heterosigma akashiwo RNA virus, Marnaviridae, NC005281.1; HAstV1, human astrovirus 1, Astroviridae, Z25771; HAV, Hepatitis A virus, Picornaviridae, KT229611; HcRNAV, Heterocapsa circularisquama RNA virus, Unclassified, NC007518; HRV1A, Heterocapsa circularisquama RNA virus, Picornaviridae, NC007518; IFV, infectious flacherie virus, NC003781.1; JP A, marine RNA virus JP-A, Unassigned, NC009757.1; JP B, Marine RNA virus JP-B, Unassigned, NC009758.1; KFV, Kelp fly virus, Unassigned, NC007619.1; LRV1, Leishmania RNA virus 1, Totiviridae, NC003601; LTSV, Lucerne transient streak virus, Solemoviridae, GCA000861405; MBV, Mushroom bacilliform virus, Barnaviridae, NC001633; NoV, Nodamura virus, GCA000847805; NrV, Neckar River virus, Unassigned, NC038927.1; NwV, Newbury virus, Caliciviridae, GCA000851625; OAstV1, ovine astrovirus 1, Astroviridae, Y15937, SmVB, Sclerophtora macrospora virus A, Unclassified, Go0081047; OPV, Ophiostoma partitivirus 1, Partitiviridae, NC038918; PLRV, Potato leafroll virus, GCA021461725; PnPV, Perina nuda virus, Iflaviridae, NC003113.1; PYFV, Parsnip yellow fleck virus, Secoviridae, NC003628.1; RasR1, Raphanus sativus cryptic virus 2, Partitiviridae, NC010343; RHDV, Rabbit hemorrhagic disease virus, Caliciviridae, NC001543; RiPV, Riptortus pedestris virus 1, Unassigned, NC031750.1; RsRNAV01, Rhizosolenia setigera RNA virus 01, Unassigned, NC018613.1; RTSV, Rice tungro spherical virus, Secoviridae, NC001632.1; SBMV, Southern bean mosaic virus, Solemoviridae, GCA000860745; SCPMV, Southern cowpea mosaic virus, Solemoviridae, NC001625; ScVL A, Saccharomyces cerevisiae virus L-A, Totiviridae, NC003745; SDV, satsuma dwarf virus-S58, Secoviridae, AB009958; SINV10 in EA, Solenopsis invicta virus 10 in electric ant, Unassigned, OP518026; SJNNV, striped jack nervous necrosis virus, Nodaviridae, NC003448; SmVA, Sclerophtora macrospora virus A, Unclassified, AB083060; SPMMV, sweet potato mild mottle virus, Potyviridae, NC003797; SssRNAV, Aurantiochytrium single-stranded RNA virus 01, Unassigned, NC007522.1; SV, Sapporo virus, Caliciviridae, GCA000849945; TAstV1, Astroviridae, Y15936; TEV, Tobacco etch virus, Potyviridae, NC001555; TRSV, Tobacco ringspot virus, Secoviridae/Comovirinae, NC005096.1; TrV, Triatomoa virus, Dicistroviridae, NC003783.1; TSV, Taura syndrome virus, Dicistroviridae, NC003005.1; TVV, Trichomonas vaginalis virus, Totiviridae, NC003824; WSMV, Wheat Streak Mosaic Virus, Potyviridae, NC001886. Supplementary file1 (TIF 1377 KB)Supplementary Figure 2. Mapping of RNA-Seq reads from four groups of electric ant samples collected in Argentina  to seven virus genomes. The X-axis presents virus genome length in kilobases (kb) and the Y-axis represents sequencing coverage. Arrows indicate libraries with genome-wide sequencing coverage.Supplementary file2 (TIF 2911 KB)Supplementary file3 (DOCX 23 KB)Supplementary file4 (DOCX 42 KB)Supplementary file5 (DOCX 15 KB)Supplementary file6 (DOCX 23 KB)Below is the link to the electronic supplementary material.


Benyviridae-related viruses. It can be proposed that the single-component Reclovirid genomes encode previously undiscovered transport genes. Indeed, analysis of the coding potential of these novel viral genomes reveals one or more cistrons ranging in size from 40 to 80 to about 600 codons, located in the 3′-terminal region of the genomic RNA, encoding proteins with predicted hydrophobic segments that are structurally diverse among Reclovirids and have no analogues in other plant RNA viruses. Additionally, in many cases, the possible methyltransferase domain of Reclovirid replicases is preceded by membrane-embedded protein segments that are not present in annotated members of the Benyviridae family. These observations suggest a general association of most Reclovirid proteins with cell membranes.As a continuation of our previous work, in this paper, we examine in greater detail the genome organization and some protein properties of the members of a potential group named Reclovirids and belonging to  Benyvirus [Some recent papers have highlighted a significant diversity of monopartite plant, fungal, and insect viruses encoding replicases related to those encoded by the multipartite RNA genomes of members of the genus enyvirus ,3,4,5,6.Benyvirus are most closely related to the proteins encoded by VLRAs of land plants only, and form a distinct branch of the corresponding phylogenetic tree -containing viruses” (Tecimovirids) coding for the TCMB movement gene module instead of the triple gene block (TGB) of movement genes found in members of the family Benyviridae. [Goji berry chlorosis virus (GBCV), which encodes six polypeptides and has no close relatives with similar genome organization [Kitaviridae [However, RNA-dependent RNA polymerase (RdRp) sequences from viruses of the genus ure 2 in ). This cviridae.  . In this paper, we comparatively analyzed the gene organization of Relovirid genomes and the structural properties of the encoded non-replicative “orphan” hydrophobic polypeptides as well as replicative polypeptides.In the course of recent studies on the phylogeny of benyvirus-like RNA polymerases, we found that phylogenetically close monopartite viruses include a new, previously undescribed group of viruses related to members of the family  virus 1 . In contonfirmed . Thus, ionfirmed . These dRed clover virus 1, Dactylorhiza hatagirea beny-like virus, Carrot associated RNA virus 1, and Arceuthobium sichuanense virus 3 [The NCBI non-redundant nucleotide sequence library currently contains the only four annotated Reclovirids. These viruses include  virus 3 . Public Scutellaria montana VLRA, which encodes four small proteins in addition to replicase (Gymnadenia rhellicani (family Orchidaceae)  and the shorter downstream ORF with sizes varying from 330 to 2000 nts [eplicase  (Supplemeplicase . The genidaceae) , which aidaceae) , show diidaceae)  (Supplemidaceae) .Red clover RNA virus 1 and other Reclovirids have two characteristic domains, namely, a viral helicase 1 domain  and an RdRp core motif (pfam00978). In addition, analysis of the NCBI Conserved Domain Database (CDD) has shown no viral methyltransferase domain  in the replicases of Reclovirids as well as members of the genus Benyvirus [Hubei beny-like virus 1 (HBLV1) encodes an MTR domain  in the N-terminal part of the replicase protein. The BLASTP search revealed that Reclovirid full-length replicases contain moderately comparable domains of 340–350 amino acids (Riboviria-encoded methyltransferases [Benyviridae and particularly the genus Benyvirus.We have previously reported that the RNA polymerases of enyvirus . Neverthenyvirus ) has shono acids , which yno acids . This fasferases  clearly Benyviridae . All thAstragalus canadensis VLRA ORF2 protein, contains a domain of the Mpp10 protein family (COG5384) . This family includes proteins related to Mpp10, which is part of the U3 small nucleolar ribonucleoprotein in yeast [Leontopodium_alpinum VLRA and Vicia faba VLRA) , the in yeast . Outsideba VLRA) . This reba VLRA) B.Atriplex prostrata VLRA  that encode one, two or three small proteins with hydrophobic membrane-spanning segments as well as RNA helicase. On the other hand, the Reclovirid cell-to-cell movement may be carried out by a wide variety of the hydrophobic “orphan” proteins  [The discovery of multiple plant-specific, capsidless Reclovirid VLRAs by high-throughput sequencing raises a number of questions about the evolution of the  systems . We haver virus) .Rice yellow mottle virus protein with movement and silencing suppression functions has been shown to contain two essential zinc-finger motifs [What characteristics of these proteins make them suitable candidates for viral RNA transmission from cell to cell? What are the specific features that make these proteins suitable candidates for performing cell-to-cell trafficking of virus RNA? Considering single MP-based transport systems exemplified by that of Tobacco mosaic virus, several features can be distinguished, namely, the ability to bind RNA, interact with ER/actin/microtubules, modify plasmodesmata (PD), move from cell to cell, and direct virus replication complexes to PD ,19. It sr motifs ,24,25. Or motifs . Thus, iftp://ftp.ncbi.nlm.nih.gov/refseq/release/viral/viral.1.protein.faa.gz (accessed on 20 May 2023). The resulting viral sequence hits of each SRA read were then visually explored. Tentative virus contigs were extended by iterative mapping of each SRA library’s raw reads. This strategy employed re-iterative BLAST to extract a subset of reads related to the query contig, and these retrieved reads were used to extend the contig, and then, the process was repeated iteratively using as the query the extended sequence.Reclovirid-related nucleotide and protein sequences were collected from the NCBI plant transcriptome database. Sequence comparisons were carried out using the BLAST algorithm (TBLASTn and BLASTp) at the National Center for Biotechnology Information (NCBI). The nucleotide raw sequence reads from each analyzed SRA experiment linked to the virus nucleotide sequence and TSA projects returning Reclovirid-like hits were downloaded and subjected to bulk local BLASTX searches  against a Refseq virus protein database available at https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 20 May 2023). Conserved motif searches were conducted in CDD  databases. Membrane-embedded hydrophobic protein regions were predicted using the software TOPCONS [https://topcons.cbr.su.se/pred/, accessed on 20 May 2023). Multiple sequence alignment was performed using NCBI software . Phylogenetic analysis was performed with “Phylogeny.fr”  by constructing maximum likelihood phylogenetic trees (http://www.phylogeny.fr/simple_phylogeny.cgi accessed on 20 May 2023). Bootstrap percentages received from 1000 replications were used. Genome sequences of different benyvirus viruses were downloaded from the GenBank database.ORFs were identified using the ORF Finder programs ( TOPCONS  (https:/Broom forkmoss associated tymo-like virus, Yellow horn associated tymo-like virus, Badge moss associated tymo-like virus, Polish wheat virus 1, Kava virus 1, Agave tequiliana deltaflexivirus 1, and Sesame deltaflexivirus 1 [Endornaviridae [Tombusviridae. After the initial vector transmission, the helper virus could be lost during progress of infection, for example, because of high temperatures [In conclusion, it should be noted that a number of the recently annotated plant tymovirus-like genomes may have small “orphan” hydrophobic protein ORFs and lack the well characterized CPs and MP systems. In particular, these viruses include ivirus 1 ,27. Thusaviridae . Among naviridae ,30. It ieratures .


While anaemia following liver transplant is common, anaemia in the context of BK viraemia is not a commonly recognised phenomenon.We present the case of 59-year old gentleman with severe anaemia in the context of BK viraemia and nephropathy following ABO incompatible liver transplant. Severity of anaemia appeared to correlate with high titres of BK virus in the serum. Bone marrow biopsy revealed hypocellular marrow with normal cytogenetics. Anaemia improved with treatment with cidofovir, intravenous immunoglobulin, reduction in immunosuppression and erythropoietin stimulating agent.To our knowledge, this is the first case of anaemia post liver transplant contributed to by BK viraemia. Anaemia post liver transplant is common, with rates ranging from 4.3 to 58% depending on criteria and time frame used to define anaemia , 2. CommThe human polyomavirus is known to be associated with polyomavirus-associated nephropathy and ureteric stenosis, which occurs most commonly in renal transplant patients. BK virus is known to replicate in epithelial urinary cells, and has a tropism to vascular endothelial cells , 5. BV vHere, we present a case of BK viraemia-associated anaemia post liver transplant.A 59-year old Vietnamese-born man received an ABO-incompatible orthoptic liver transplant for liver failure due to hepatitis B reactivation. Complications following transplant included acute kidney injury due to biopsy confirmed severe BK nephropathy of the native kidney with considerable interstitial inflammation. He had concurrent biopsy-proven cytomegalovirus colitis, and T-cell mediated rejection. Throughout the post-transplant course, relapsing normocytic normochromic anaemia occurred.2; and the haemoglobin was 73 g/L. After treatment of BK virus with reduction in immunosuppression and a 12-week course of cidofovir at a dose of 0.25 mg/kg, and treatment of CMV colitis with 7 days of intravenous ganciclovir followed by oral valganciclovir at a renally adjusted dose, haemoglobin improved to a baseline of 100 g/L. His renal function also improved to a creatinine of 220 mg/dL and eGFR of 27 mL/min/1.73m2, which has been sustained since.Diagnosis of biopsy-proven BK nephropathy Figs.  and 2 ocTwo months later, five doses of intravenous methylprednisolone were administered for biopsy confirmed mild acute T-cell mediated rejection, and tacrolimus was increased. Following this increase in immunosuppression, BK viraemia rose to 26 342 copies/mL and anaemia recurred with a haemoglobin of 60 g/L. His anaemia was treated with transfusion of one unit of packed red blood cells, and haemoglobin increment was sustained with a second course of cidofovir and commencement of monthly methoxy polyethylene glycol-epoetin beta. Mycophenolate was then changed to everolimus.Our patient had a stable course for ten months, during which tacrolimus was ceased and mycophenolate was reintroduced to reduce the risk of calcineurin inhibitor nephrotoxicity.Three years post-transplant, our patient was admitted after routine bloods revealed a haemoglobin of 38 g/L. There was a four week history of fatigue and shortness of breath, though no symptoms of blood loss. Examination did not show hepatosplenomegaly. The immunosuppressive regime was everolimus 2 mg bd and mycophenolate 1 g bd, and he remained on monthly erthropoietin stimulating agent. Investigations demonstrated no abnormality of other cell lines, and renal and liver function were at the patient’s baseline. BK viraemia had recurred at 12 700 copies/mL. There were no signs of haemolysis, myeloma screen was negative, CD59 + expression was normal, parvovirus IgM was negative and there was no CMV viraemia. Bone marrow biopsy showed moderately hypocellular bone marrow with moderately reduced erythropoiesis and mild dysplastic features, reduced granulopoiesis, and dysplastic megakaryocytes thought to be due secondary to either drugs, infection or inflammation. Retrospective staining for BK virus on the bone marrow aspirate was negative. The cytogenetics on the bone marrow were normal. The anaemia was treated with blood transfusion, four doses of intravenous immunoglobulin, and a third course of cidofovir, and continuation of erythropoietin stimulating agent. The haemoglobin incremented to 118 g/L and mycophenolate dose was reduced. On completion of this course of cidofovir, the haemoglobin was 121 g/L and BK titre reduced to 5980 copies/mL. BK virus quantification from transplant to present is seen in Fig. Classically, complications of polyomavirus include ureteric stenosis and polyomavirus associated nephropathy in renal transplant patients; and polyomavirus associated haemorrhagic cystitis in recipients of an allogeneic haemopoietic stem cell transplant .BK virus infection of the native kidney after liver transplant is rare, though possibly underrecognised, with only three cases previously reported to the best of our knowledge –11. RecoOur patient, who had not previously had acute episodes of anaemia, had repeated acute episodes of anaemia with contemporaneous high BK virus titres. In this case, relapse in degree of BK viraemia between periods of treatment with intravenous immunoglobulin and cidofovir appeared to proceed presentations with severe symptomatic anaemia. Anaemia also appeared to improve on treatment of BK viraemia with reduction in immunosuppression, and administration of intravenous immunoglobulin and cidofovir. While our patient’s anaemia is also contributed to by chronic renal impairment, there appears to be a temporal relationship of anaemia following periods of high BK viral load.As far as we are aware, this is the only documented case of anaemia post liver transplant with BK viraemia as a contributor.Anaemia associated with BK viraemia post renal transplant has previously been documented in a study of 72 renal transplant recipients , and two case reports .In a study by Pambrun et al., renal transplant recipients who presented for investigation of cytopenia we screened prospectively for BK virus in the blood and bone marrow. Bone marrow biopsy was performed when haemoglobin had decreased to < 11 g/dL, and/or neutrophil count was < 1000/mm3 and/or platelet count was < 120,000/mm3. BK viraemia was detected in the blood of 9 of 72 renal transplant recipients. Only 5 of these patients had BK virus replication in the bone marrow. Interestingly, BK virus replication was detected in the bone marrow of an additional three patients who had no detectable BK virus replication in the blood, therefore 8 of 72 patients (11.1%) exhibited bone marrow replication of the virus. Most of the patients who had BK virus detected in the marrow presented for investigation of neutropenia, and hypocellularity of the myelopoietic line and blockade of granulocyte maturation were the most common lesions observed in bone-marrow analyses [One case describes 17 year old renal transplant recipient who was treated for BK virus nephropathy presented with pancytopenia, with BK virus detected in blood and bone marrow at high viral loads. Graft nephrectomy and immunosuppression improved the cytopenias [Another case describes anemia after renal transplant in the context of concurrent parvovirus B19 and BK viraemia . Sharma The exact mechanism by which BK viraemia causes anaemia remains unclear. In mice with severe combined immunodeficiency, acquiring BK virus resulted in an acute haematological disorder causing haemorrhage, anaemia, thrombocytopenia and splenomegaly. Authors propose an indirect method of myeloproliferation such as secretion of growth factors or cytokines by virus-infected cells . PambrunIn summary, this is the first case of post-liver transplant anaemia, which is significantly contributed to by BK viraemia. The association of anaemia and BK virus replication in the marrow raises the need to consider bone marrow biopsy with staining for BK virus in resistant cases of anaemia post solid organ transplant, even in the absence of BK virus replication in the blood.


It focuses on the work done primarily at the United States Army Medical Research Institute of Infectious Diseases during the decade of the 1980s under the leadership of Joel Dalrymple, who worked in close partnership with Ho Wang Lee. These early studies helped define the global distribution of Seoul virus and provided seminal information on its maintenance and transmission among urban rats. Other collaborations involved partners in Europe, Asia, and Latin America and resulted in the isolation of novel hantaviruses, a better understanding of their global distribution, and validation of diagnostics and therapeutic interventions for treatment of human diseases. By working in partnership, scientists from around the world made critical discoveries that led to a better understanding of the hantaviruses. “The Power of We” demonstrates that we all benefit when we work together with a shared vision, a common commitment to excellence, and mutual respect. I was fortunate to be recognized with the Ho Wang Lee Award and invited to offer a keynote address during the Xth International Conference on HFRS, HPS and Hantaviruses held in Fort Collins, Colorado in 2016. This chapter is drawn from that presentation and represents my sincere tribute to the many friends and colleagues that I had the honor of working with during the early days of discovery following the isolation of Hantaan virus by Ho Wang Lee ..15].Rattus norvegicus) having the highest prevalence [Given that we had been successful in finding hantavirus-infected rats in many locations in both North and South America, we expanded testing to include samples provided by many partners from around the world. We engaged many of our friends and colleagues from around the world in an attempt to determine the global distribution of hantaviruses in peridomestic rodents. Over 1700 sera from more than 20 sites worldwide were tested for anti-hantavirus antibodies. Antibody-positive rodents were found at almost every location sampled, with domestic rats  most frequently seropositive, with antibody-positive voles restricted to the northern two-thirds of the country and corresponding to the regions where the most human disease was seen. Through our studies, we were also able to isolate Puumala virus from the lung tissue of captured bank voles and propagate the virus in cell culture. Together with the field study results, we were able to conclude that the bank vole was the principal host of Puumala virus in Sweden and that the region near 64 degrees N was highly endemic for nephropathia epidemica in humans [A form of HFRS was known in Scandinavia and much of Europe prior to the isolation of Hantaan virus by H.W. Lee; however, the causative virus had not been isolated, and the detailed ecology and epidemiology of the disease in Sweden was lacking. In Scandinavia, the human disease was known as nephropathia epidemica and was caused by Puumala virus, a close relative to the prototype Hantaan virus. Our studies of a hantavirus in Sweden began when I was fortunate to meet a young Swedish physician, Bo Niklasson, at a meeting in the early 1980s, and we discovered our common interest in the hantaviruses. This led to a series of joint field trips and laboratory studies that included rodent collections from the southern tip of Sweden to above the Arctic Circle. We captured rodents throughout the country, attempted virus isolation from lung tissues, and tested sera for antibodies to Puumala virus. We also obtained human sera from many different parts of the country and assayed for anti-Puumala virus antibodies. The human serology found the highest prevalence rates and the highest incidence of infection in northern Sweden, while the rodent survey found the bank vole, n humans ,19.Hemorrhagic fever with renal syndrome was also known to occur in the Balkan countries prior to the isolation of Hantaan virus by H.W. Lee. A mild form of HFRS similar to nephropatia epidemica of Scandinavia was known, but in addition, a more severe disease with significant mortality existed. We were able to initiate collaborations with colleagues in both Greece and what was then Yugoslavia with the goal of determining the hantaviruses present and to further refine our understanding of their natural history.Apodemus flavicollis rodents seropositive, and we were unable to isolate a virus from the tissues of the seropositive rodents. This and other studies in Greece suggested that severe HFRS was not due to either Puumala or Seoul virus but rather a novel hantavirus more closely related to Hantaan virus of Asia. Further, it suggested that another rodent species, Apodemus flavicollis, may have been the host of this suspected new virus [In Greece, we worked with a physician-scientist and professor of microbiology at the Aristotle University of Thessaloniki, “Tony” Antoniadis. Tony had collaborated with USAMRIID scientists in the past and was eager to further his research on the hantaviruses. Jointly, we worked to enhance his laboratory capabilities with shared reagents and exchange visits for training and technology transfer and to develop research topics. In the summer of 1983, a small outbreak of severe HFRS occurred in the mountainous region of northwestern Greece involving eight patients. Those infected were shepherds who had camped in mountain pastures while tending their sheep. All were hospitalized, three were severely ill, and one died . The folew virus .Prior to the civil war in Yugoslavia, there was an initiative to foster greater collaborations between the US and Yugoslavian militaries, and, as a result, our program on the hantaviruses was selected by the US Army as a topic for joint research. Yugoslav scientists had a long history of work on viral diseases, including on smallpox virus, and at this time in the 1980s, Ana Gligic was one of the premier leaders in virology in Yugoslavia. Ana had collaborated with Carlton Gajdusek at NIH, had strong ties to the Yugoslav military academy in Belgrade, and had established collaborations with promising young scientists in Ljubljana and Sarajevo . I firstIn the spring of 1989, we set out to replicate the ribavirin antiviral drug trial of China in Sarajevo in hopes of demonstrating the drug’s efficacy in a second population and against the severe form of HFRS found there. We identified a talented team of young physicians just graduating from the medical school in Sarajevo, led by Alemka Markotic, who had been number one in her class see . As we hBy the time the war started, I had retired from USAMRIID and taken a position with the Centers for Disease Control and Prevention (CDC), with my first assignment being seconded to the World Health Organization (WHO) in Geneva as an expert on arboviruses and hemorrhagic fevers. While at WHO, I was able to communicate with Alemka in Sarajevo as the war unfolded. Realizing the dire conditions and the dangers she faced, I was able to arrange for her transport to Geneva on a UN flight, and once safely in Geneva, she was able to help as we attempted to assess health risks in the region. Even many years later, she still thanks me on the anniversary of her evacuation to safety.Apodemus flavicollis and were able to demonstrate serologically and through genomic analysis that the new virus was related to, but distinct from, all known hantaviruses.  Dobrava virus was shown to be associated with severe HFRS and is now known in many countries of Europe, fortunately at a low prevalence of human infection in most places.One of the more significant accomplishments made during this decade was the isolation of Dobrava virus by Tatjana Avsic-Zupanc and her colleagues, including scientists at USAMRIID . They suOur international collaborations yielded several very significant benefits. Using a National Research Council fellowship program, we were able to host talented young scientists to work on the hantaviruses at USAMRIID. This offered a path that allowed scientists from Asia, Europe, and other regions, as well as from the USA, to participate in extensive training and joint research that benefited everyone and truly advanced knowledge of the hantaviruses. These collaborations were critical to the development of the hantavirus community to which we all belong.After a decade of truly remarkable hantavirus discoveries, the USAMRIID “dream team” began to unravel. The greatest loss was the untimely death of Joel Dalrymple in 1992. Joel had been the driving force of the program and was very close to Ho Wang Lee. They communicated often and were more than just scientific colleagues; they were true friends. Around the time of Joel’s passing, many of the senior officer-scientists faced mandatory military retirement and were faced with few options to continue at USAMRIID. Some moved to the CDC, where options to continue work on emerging infectious diseases were greater, resulting in much of the knowledge base and critical reagents that had been painstakingly developed at USAMRIID moving with these experienced leaders. This influx of talent and skills helped rapidly build the hantavirus program as an important element of the CDC’s fledgling program in emerging infectious diseases. Finally, funding for USAMRIID was evolving, making it harder to sustain the robust hantavirus research program that had been so productive over the past decade. While critical hantavirus investigations continued at USAMRIID, and many important discoveries are still being made by the excellent staff even today, the net result was a clear erosion of what had been one of the premier centers of excellence in hantavirology anywhere in the world.When a completely new hantavirus causing hantavirus pulmonary syndrome (HPS) emerged in the Western US in 1993, the CDC was well positioned to respond, thanks in large part to the decade of productive research facilitated by USAMRIID and supported by the Department of Defense. As the HPS story evolved, “We” were able to build on our past international collaborations pioneered at USAMRIID as well as those already in place at the CDC to help address HPS in Central and South America. “We” quickly discovered novel hantaviruses that had caused serious and often fatal human infections in many locations, but whose cause had never been known. In addition, “We” discovered many new rodent hosts of these novel viruses. Today, our network continues to grow internationally as we discover yet more hantaviruses involving an ever-increasing array of animal hosts and perhaps still others causing more human diseases that have yet to be recognized.Since the original isolation of Hantaan virus by Ho Wang Lee, “We” have all contributed to and indeed benefitted from the enormous power of collaborations. It’s better to collaborate than to compete: everyone wins in a collaboration. Through mutual respect and shared vision, “We” have accomplished much and seen the tremendous rewards of working together. “We” have built a knowledge base on an important group of viruses that has been the scourge of society for ages. “We” have worked together to make fundamental discoveries and to develop interventions to mitigate the impact of infections, and “We” have shared our findings with one another through informal conversations, joint meetings, and scientific publications. Our reputations as trusted collaborators will facilitate our continued success; our networks of collaborators are our most valuable assets—we must treasure them. “We” have invested in training future generations of hantavirologists, and “We” have worked to ensure their success. The investments made in nurturing junior colleagues will pay a lifetime of dividends.“The Power of We” is all of us, working together, continuing the vision of Ho Wang Lee and Joel Dalrymple see .


HighlightsHenipavirus that has been found in shrews, bats, and rats.Langya virus is a The Langya virus’s origin is still unknown.Henipaviruses do not have any approved therapies.There is currently no evidence that the virus may spread from human to human.Dear Editor,Henipavirus that has been found in shrews, bats, rats, and other small animals. Some studies also reveal that animals such as dogs and goats have natural antibodies to the Langya virusParamyxoviridae, the Langya henipavirus (LayV) has evolutionary ties with Nipah virus and Hendra virusLangya virus is a The New England Journal of Medicine. Thirty-five persons in China’s Shandong and Henan provinces contracted the LayV between December 2018 and May 2021On 4 August, it was initially reported in Contact tracing was performed on nine of the affected persons, and after contacting 15 close contacts of each patient, the researchers found no evidence of LayV infectionScientists investigated different animals for the presence of the Langya virus to determine the origins of the infection. They discovered viral evidence in goats and dogs, but the shrew was the principal source of the Langya virus. The virus was found in 27% of the shrew samplesThe Langya virus’s origin is still unknown. However, it is most likely passed from animal to human. Because the LayV virus RNA has been identified mostly in shrews, they may be its natural hostsThose infected have reported the following symptoms: fever, fatigue, cough, anorexia, myalgia, nausea, headache, and vomiting. Furthermore, 35% of those contaminated had impaired liver function, and 8% of those infected had impaired renal function. According to doctors, the virus might cause a decrease in the number of platelets in the blood, significant damage to the kidneys and liver, and possibly deathSerum seropositivity was found in 2% of goats and 5% of dogs in a sample of domestic animalsHenipaviruses do not have any approved therapies. In animal experiments, experts have only tested a few antiviral alternatives. There is also no particular vaccination for the Langya virus. However, ribavirin may be an effective therapy. Doctors often use this medicine to treat viral infections that have no other therapeutic options. Ribavirin is effective against RNA viruses, including those that cause respiratory problems. Studies demonstrate that ribavirin is effective for both Hendra and Nipah viruses. The malaria medicine chloroquine may also be useful in treating these two conditionsThis unusual virus is poorly understood, and experts predict that the number of verified cases will rise. There is currently no evidence that the virus may spread from human to human. More study is required to determine the virus’s entire scope, its mode of dissemination, and whether it has already reached China and the surrounding region.Not applicable.There are no conflicts of interest.No funding was received.R.A.: conceptualization, data curation, writing – original draft preparation, reviewing, and editing. R.D. and H.C.: data curation, writing – original draft preparation, reviewing, and editing. T.B.E.: writing – reviewing and editing, visualization, and supervision.1. Name of the registry: not applicable.2. Unique identifying number or registration ID: not applicable.3. Hyperlink to your specific registration (must be publicly accessible and will be checked): not applicable.Talha Bin Emran, PhD, Associate Professor, Department of Pharmacy, BGC Trust University Bangladesh, Chittagong 4381, Bangladesh. Tel: +880 303 356 193, fax: +880 312 550 224. https://orcid.org/0000-0003-3188-2272Not commissioned, internally peer-reviewed.The data in this correspondence article is not sensitive in nature and is accessible in the public domain. The data is therefore available and not of a confidential nature.


Parents also reported improved access to resources and relevant information and increased confidence in using visual supports at home. The home visit model was strongly supported by the parents. Conclusion: The results provide initial evidence of the acceptability, practicality, and utility of the home-based visual supports intervention. These findings suggest that outreach into the family home may be a beneficial mechanism for delivering interventions related to visual supports. This study highlights the potential of home-based interventions to improve access to resources and information for families and the importance of visual supports in the home setting.Visual supports are an important intervention for autistic individuals and others with neurodevelopmental differences. However, families often report limited access to visual supports and lack of information and confidence in their use at home. This pilot study aimed to evaluate the feasibility and effectiveness of a home-based visual supports intervention. Methods: 29 families with children  receiving support for autism or related needs participated in the study. Parents engaged in an individualised assessment and intervention process through home visits, completing pre- and post-measures. Qualitative methods were used to explore the parents’ experiences of the intervention. Results: The intervention led to a statistically significant improvement in parent-reported quality of life (t28 = 3.09,  There is no single intervention recommended for all autistic individuals; however, some interventions are effective across several situations ,2. One sThe lack of formally published visual support resources for use by parents motivated us to complete the current study, with a gap identified in the evidence available about the use of visual supports by families at home . There iThe purpose of this study was to pilot a Home Visual Support intervention with families of autistic children and children with related neurodevelopmental differences aged 0–12. This study took place in a city in Scotland with a population of 500,000, where around 400 children, aged 0–18 years, are referred for autism diagnostic assessment annually  came to visit did I really get it.”“What I liked was the way they came back, so that we were able to tailor it to our current circumstances and then follow it up.”“We did parent groups… the experience of someone coming into your own home was quite different”“It was different from other experiences of Visual Supports.”Parents reported that the home visits allowed staff to better understand the family’s context and needs and to provide tailored support. The staff were seen as positive, supportive, and empathetic in their interactions with the families. They asked good questions, listened to the parents’ needs, and provided feedback and encouragement. As a result, the parents felt empowered and more confident in their ability to support their children.“It was better for them (staff) to actually see things (at home), it gave it all context. There are real benefits to them coming to the home.”“I had bought Visual Supports online (before) but wasn’t really using it. The [staff] helped me use it effectively.”“They asked really good questions, they could really help… made us realise that it doesn’t need to be like this.”“The [staff] workers never said you’re doing it wrong. It was so comforting to have strangers tell you that you are doing great.”The families appreciated the individualised nature of the support and the chance to participate in planning and making decisions about the visual supports. This allowed them to feel more involved and invested in the process, which helped to increase their confidence and motivation to support their children.“We came up with the ideas together.”“The [staff] team really listened and I found it encouraging. It pushed me more to help my child.”“They answered all my questions.”“Crucially, I got to choose.”Overall, the families reported that the home visits were a valuable and effective way to receive support and information about visual aids. The tailored, individualised approach was seen as much more beneficial than group settings, and the families felt that they had more control over the process. The visits provided a positive and supportive environment that allowed the families to feel listened to and encouraged.Theme 2: Individualisation, tailoring, and developmentally appropriate support is valued by parentsThe benefits of having specific and tailored support was a dominant theme among all the families. Participants were pleased with the level of support that was provided to them and appreciated the personalised nature of the support. They noted that an individualised approach helped them, as a family (including siblings), to understand and effectively utilize visual supports. Moreover, the children themselves were involved in the decision-making process to personalize the supports, which led to an increased level of engagement and ownership. Parents described the amount of support as optimal, and noted that the support was beneficial for them, for example, in reducing their anxiety.“The amount of support was just right.”“I was very anxious but this experience was so personal and focussed on my own needs, that was nice.”The participants emphasised the value of having tailored support that was focused on their specific needs, as opposed to a generic approach that was taught in a group setting. They felt that this type of support was more valuable and beneficial to them, as it was centered on their individual concerns and issues. Additionally, the participants appreciated the opportunity to have one-on-one time for specific advice, which was deemed to be more helpful than receiving advice in a group setting.“Teaching us about it individually is much better than teaching in a group. It is individualised and personalised.”“I liked how personal it was.”“Groups are not about your child but [the project] was about our child.”“Time for specific advice for me rather than in a group is so much more valuable.”An individually tailored approach supported parents and siblings to understand and use visual supports effectively. The families had children at different stages of communication and were positive about the supports that were tailored to their child’s specific stage of development. Parents appreciated that the support was set up for their child as an individual and that the progression was explained in a way that was appropriate for their child.“He didn’t have symbolic understanding, so it was tailored to his needs… It was set up for him as an individual and the progression to symbols was explained.”This individualised approach helped the families to make progress and achieve their goals, and was widely regarded as a critical aspect of the success of the intervention. Children also took part in the decision making to personalise supports, which families viewed as beneficial.“[My child made the] choice of what to focus on and symbol selection.”“My daughter has made her own visuals.”Theme 3: Timing is importantThe theme of timing was identified as a critical factor in the effective use of visual supports by parents. Three key aspects of timing were identified: early or timely access to visual supports, appropriate duration of support, and maintenance of the support over time. Participants emphasised the importance of clear expectations at the start, which helped them feel satisfied with the level of input they received. They also acknowledged the benefits of early access to expertise and the role that follow-ups play in providing support during key times, such as transitions.“The sooner you have access to expertise and from as young an age as possible, the better.”“If this had been introduced to me 2 years ago, I would have needed much more support.”“Eventually we will get better but we need lots of input.”“Follow ups are crucial at key times .”Establishing clear expectations from the outset provided a sense of comfort for parents, as they felt informed about the level of involvement required. The participants observed that although support was essential, they did not expect to require it indefinitely. Theme 4: Parents report multifaceted benefits to visual supports at homeA range of benefits were reported with regards to understanding the full range of what visual supports were capable of doing. Parents particularly reported increased knowledge and understanding of the capabilities of visual supports, beyond their initial perception of them as ‘just timetables’. They found that visual supports provided more than just a visual representation of routines, and the intervention gave them a deeper understanding of the possibilities for visual aids.“I didn’t realise the scope of [visual supports] before. A lot more info would be useful to parents.”“Visuals helped immediately–it was like, really? That’s all it takes?”“The [visual supports] has helped, it’s so much more than just timetables.”“The Nursery was always supportive in using visuals but it was really timetable based. I found using these difficult as home is different than school. However now I understand that it’s about so much more than timetables. I have a much broader understanding.”In addition, families reported increased independence in their children, with some children setting routines for themselves and taking more control over their schedules. This was seen as a positive outcome, as it not only helped with consistency, but also encouraged independence.“One morning my child got up and set the timetable himself.”“We wanted our son to be more independent and the [visual supports] has helped.”“It can help behaviour but also encourages independence.”“I do believe it has helped keep his behaviour more consistent at home.”Furthermore, the use of visual supports provided families with access to resources and information, making them feel informed and equipped to tackle new challenges. Sharing information with other families and learning from each other was also seen as a benefit, with parents feeling inspired by the experiences of others.“I want to share my story to inspire others.”“It felt good to know that other families were experiencing the same thing.”“I’m thinking creatively now and I have ideas for tackling other issues.”Finally, visual supports were seen as beneficial in terms of helping children develop skills, particularly in communication, through consistency in words and routines. Parents saw the use of visual supports as a tool that provided numerous benefits, helping them to tackle difficult challenges and improve the skills and independence of their children.“It has helped me tackle hard things.”“We became more consistent with words we used and the order we did things, his speech has really improved.”Theme 5: The challenges of visual supportsWhile the majority of parents expressed positive views towards the use of visual supports, they were also aware of the limitations and challenges associated with this approach. Parents recognised that visual supports are not a one-size-fits-all solution, but rather a tool that needs to be tailored to each individual child and situation. Parents noted that visual supports were not always practical, especially for those with busy schedules, and that not all approaches were effective for all children. However, they emphasised that the challenges were not significant enough to outweigh the benefits of using visual supports, and that they gained valuable insights into new ways and places to use visual supports effectively.“It’s not been magical but it helps.”“Visual support is great but is does have its limitations.”“If you are busy it’s not always practical.”The overall conclusion from the parents was that visual supports were a useful tool, but one that had limitations, and that continued experimentation and adaptation were necessary to find the most effective approach.“Not everything worked but I have ideas of other ways and places to use visual supports.”Regarding the home visit mechanism, parents reported feeling listened to and positively engaged with during visits from support staff. They valued the opportunity to receive information and support in their own homes, as it provided context and a personalised experience compared to previous methods of receiving information. They appreciated the opportunity to participate in the planning process and receive positive feedback from the staff, leading to feelings of empowerment and confidence. The individualisation and tailoring of support was also highly valued. Parents appreciated the personal and focused approach that was tailored to their own needs and those of their children. They reported positive outcomes in terms of understanding and effectively using visual supports, as well as their children being involved in the decision-making process to personalize the supports. Parents were also positive about supports being tailored to their child’s stage of communication.Timing was another key theme that emerged, with parents reporting the importance of early and timely access to visual supports, the appropriate duration of support, and the need for follow-up maintenance. They emphasised that the sooner they had access to support, the better, and that follow-ups at key times, such as during transitions, were crucial. Parents also reported a range of wide benefits from using visual supports at home. They reported increased knowledge and understanding of the capabilities of visual supports, and appreciated the support being broader than just timetable-based.In a pilot study involving families of 29 children, a novel visual supports intervention was found to be feasible and effective. Experienced staff were able to deliver the intervention in three to five home visits, using resources that were relevant and accessible to parents. The approach required individualisation for each family through assessment. Pre-prepared packs and guidance were time saving for staff and the assessment structure facilitated comprehensive discussions. The home visit mechanism was reported to be very beneficial by parents.The families completed the QoLA, a measure of quality of life, which revealed a statistically significant improvement after the intervention. Families also reported an increase in their confidence and knowledge to use visual symbol supports, as well as improved access to visual support resources. However, they did not view visual supports as a panacea, but rather as a useful tool to increase predictability, understanding, and desirability of home and community activities.Families gave feedback in relation to types of visual supports that are useful at home and highlighted that visual timetables are more difficult to use at home, which is less structured than school. A range of visual supports were found to be helpful in planning routine and non-routine events, such as such as shopping trips, holidays, transitions or new activities. Notably, the individual visual supports introduced in the study were not novel. Rather, the addition provided by this approach was a framework that structured the process of assessment and decision-making, combining professional knowledge of the approaches and developmental expectations with parent knowledge and experience.Overall, the intervention created access to the supports most useful at a point in time, and knowledge about how to use them, with support to start and adapt their use. To our knowledge, this is the first intervention study to use the QoLA as an outcome measure, showing positive results that are reflected in the qualitative data. Our findings suggest that this intervention has the potential to improve the quality of life for families, and warrants further investigation in larger studies.Our study adds to an emerging body of home and community visual supports research . In prevStudies consistently highlight the need for visual supports to be integrated alongside parent and child goals . Good paFinally, working in a naturalistic context (the home) is cost-effective . FamilieFrom a professional’s perspective, visual supports are unlikely to be used as the only support or strategy in place, and it is important to ensure they are meaningful and desirable to the individual and to integrate their use well with other approaches in place . WorkingTime taken to learn to use a visual support varies as a result of many factors. The challenge of ‘teaching’ individuals to use visual supports at home is reduced when these are at the right developmental stage and are meaningful; however, just putting the visual supports near the child might not lead to them knowing what to do. Some learners may need adults to use coaching methods  to modelIn terms of limitations, this is the first study of this intervention and the sample size is relatively small. We cannot be certain which elements of the intervention were effective, and there was no control group. Replication studies would improve confidence in the effectiveness of the intervention. There are, as of yet, no long-term outcome measures to evaluate the effects of the intervention over time. The inclusion of children undergoing assessment, as well as after diagnosis, could be seen as a limitation and may prompt further debate; however, professionals working across health and education are now encouraged to offer early intervention and to support need, not diagnosis. This is certainly a message strongly received from families, who indicated that they do not want these environmental supports to be kept from them until diagnosis is made; therefore, the findings could be seen as being relevant to and respectful of parents of children who do not fall neatly into categories.Based on the parent consultation and the review of the outcomes of the study, recommendations are made below for the application of visual supports intervention with home visiting.Home visits can be used as an effective way of providing brief interventions focussed on visual supports;Parents can take positives, even when things do not work—so several symbols and methods should be tried;Professionals should listen to families and their needs and set appropriate expectations about the level of input and likely benefits of visual supports;Professionals should offer visual support as early as possible, and help parents find it, understand it, and use it;Parents report positively when they have access to a range of resources relevant to a range of developmental levels;Parents should be involved in the assessment process and selecting the symbols used from an early stage;It is helpful when staff model how to use visual supports in context ;Individualisation and tailoring are viewed positively by parents, particularly around the child’s individual developmental level;Follow up visits are needed with parents with the chance to review and adapt, and maintenance options for symbols and other technology are helpful, though these should not be too complex;Enhanced knowledge and skills to assess needs and implement visual supports are a core practice for specialist educational professionals and some allied health professionals . Such professionals would be well placed to apply home visual support interventions.We make a number of recommendations for further study. First, a replication study with a larger sample would be beneficial. Second, the resources can be made available online, with feedback gathered on the utility of this delivery mechanism. Third, we aim to develop and evaluate professional learning for staff delivering visual support interventions for families in the home setting. Lastly, further consideration is required of neurodiversity affirming  use of vVisual supports are an efficient, low-cost intervention. This study describes development and piloting of a novel visual supports interventions for families of autistic children and those with related neurodevelopmental differences. The study identified that parents can effectively be included in a visual supports intervention at home. A structured approach to implementing visual supports is effective in increasing parent-reported QoL, and need not be time intensive when pre-prepared resources are available.