To determine the feasibility of using radiopaque (RO) beads as direct tumour surrogates for image-guided radiotherapy (IGRT) in patients with liver tumours after transarterial chemoembolisation (TACE).A novel vandetanib-eluting RO bead was delivered via TACE as part of a first-in-human clinical trial in patients with either hepatocellular carcinoma or liver metastases from colorectal cancer. Following TACE, patients underwent simulated radiotherapy imaging with four-dimensional computed tomography (4D-CT) and cone-beam CT (CBCT) imaging. RO beads were contoured using automated thresholding, and feasibility of matching between the simulated radiotherapy planning dataset (AVE-IP image from 4D data) and CBCT scans assessed. Additional kV, MV, helical CT and CBCT images of RO beads were obtained using an in-house phantom. Stability of RO bead position was assessed by comparing 4D-CT imaging to CT scans taken 6–20 days following TACE.Eight patients were treated and 4D-CT and CBCT images acquired. RO beads were visible on 4D-CT and CBCT images in all cases and matching successfully performed. Differences in centre of mass of RO beads between CBCT and simulated radiotherapy planning scans (AVE-IP dataset) were 2.0 mm mediolaterally, 1.7 mm anteroposteriorally and 3.5 mm craniocaudally. RO beads in the phantom were visible on all imaging modalities assessed. RO bead position remained stable up to 29 days post TACE.RO beads are visible on IGRT imaging modalities, showing minimal artefact. They can be used for on-set matching with CBCT and remain stable over time.The role of RO beads as fiducial markers for stereotactic liver radiotherapy is feasible and warrants further exploration as a combination therapy approach. SBRT is a feasible and safe therapeutic option for patients with hepatocellular carcinoma (HCC) ineligible for other local treatments, with studies showing local control rates of 70–100%, comparable to that of radiofrequency ablation RFA.16The doses delivered in SBRT are ‘ablative’ and accordingly, target accuracy is crucial for liver SBRT due to the radiosensitive nature of the liver, proximity of tumours to the small bowel and significant liver motion with respiration. However, liver tumours are often difficult to visualise on radiotherapy planning computed tomography (CT) scans without the use of contrast. Image-guided radiotherapy (IGRT) is therefore typically accomplished through visualisation of a surrogate to the tumour. Surrogates can be either the whole liver, diaphragm or through the insertion of specific fiducial markers . Fiducial markers implanted close to a liver tumour have been shown to be a better surrogate of tumour position than adjacent anatomical landmarks.17,18 More recently, cytotoxic agents have been combined with microspheres in order to create drug-eluting bead TACE (DEB-TACE). The DEBs, DC Beads™ , are non-biodegradable polyvinyl alcohol microspheres, that can be loaded with calibrated cytotoxic drugs. The recent development of novel radiopaque (RO) drug-eluting beads, which can be visualised on CT or fluoroscopic imaging,19 has the advantage of providing intra- and post-procedure visualisation of the tumour vasculature. Furthermore, the durable radiopacity means that RO drug-eluting beads are visible on x-ray-based imaging after treatment.20,21 The retention of RO beads within the tumour vasculature means that RO drug-eluting beads could potentially act as a surrogate for direct image-guided tumour targeting.18TACE involves the introduction of a chemotherapeutic drug into arteries feeding a liver tumour, followed by an embolic agent that causes vessel stasis. Gelatin sponge and polyvinyl alcohol particles have been popular particulate embolic agents for TACE, whilst the embolic agent lipiodol has the advantage of being radiopaque on CT imaging. As such, lipiodol has already been shown to be a feasible surrogate for liver position during SBRT.22 The VERB is a pre-loaded drug-eluting RO bead measuring 60–160 µm diameter. An in-house phantom model was also used to assess the visibility of the RO beads in additional imaging modalities required for IGRT, including kV, MV, helical CT and CBCT.The aim of this study was to determine the feasibility of using RO drug-eluting beads as direct tumour surrogates  for IGRT in patients with liver tumours post-TACE. Specifically, this study focussed on the simulated radiotherapy planning dataset (AVE-IP images from 4D CT data) and cone-beam CT (CBCT) images acquired following treatment with a novel vandetanib-eluting RO bead (VERB), in a first-in human clinical trial .22The VEROnA study was a pilot, open-label, single-arm, phase 0, window-of-opportunity trial of VERB delivered transarterially, 7–21 days prior to surgery in patients with resectable liver malignancies . Eligibl™). Patients were instructed to breathe normally and abdominal compression was not used. The Varian Respiratory Position Management (RPM) system was used to generate a surrogate respiration signal from a marker block placed on the patient’s chest. For each patient, 1.25 mm slice thickness 4D-CT images (120kV 100mA) were acquired on a GE Lightspeed CT scanner . The images were phase-sorted into ten bins of equal time using the surrogate respiration signal from the Varian RPM. The AVE-IP data set was then primarily used for analysis. Following this, each patient was set-up in the same position, using reference skin marks and a three-point laser system on a TrueBeam linear accelerator . A free-breathing CBCT scan was acquired using the on-board imaging system . The reconstructed image volume was 17 cm along the cranio-caudal axis and 45 cm in the axial plane. A reconstructed slice thickness of 2.5 mm was used. It is important to highlight that patients in this study did not undergo radiotherapy treatment.For acquisition of the simulated radiotherapy planning scans (4D-CT), all patients were positioned supine with arms up on a wing board and immobilised using a commercial immobilisation system , that the RO bead area needed to be within 4 cm of the GTV16 and that a minimum of three discrete RO beads areas would be identified.In order for RO beads to be used as fiducial markers for liver SBRT, they need to be visible at radiotherapy treatment planning , visible on the CBCT (used for treatment verification), have the same shape and size (volume) on the simulated planning dataset and CBCT and have the same spatial separation in order to allow reliable matching, and finally show stability with negligible migration. Furthermore, in keeping with current recommendations, it was decided that the following criteria should apply: that the volume of the RO bead area needed to be >0.1 cm16 Any discrete volumes measuring <0.1 cm3 were removed  or Lipiodol. As such, in order to avoid inter-observer variation, the automated thresholding function in the Eclipse treatment planning system  was utilised for contouring regions of hyperintensity where the beads had clumped together and were more visible as conglomerates. Automated thresholding automatically detects areas above a set Hounsfield unit (HU) threshold . Firstly removed . FinallySupplementary Material.Click here for additional data file.23 This process is outlined in In keeping with our departmental practice for online matching between CBCT and AVE-IP scans, the following steps were applied offline using Varian Offline Review. Firstly, an anatomical auto-match was applied in the region-of-interest using grayscale recognition. This was visually reviewed by a radiation therapist and radiation oncologist and corrected as required to ensure that vertebral bodies were aligned. Secondly, an auto-match to the liver edge was performed. Finally, a manual match was performed based on the RO beads contoured on the AVE-IP plan using grayscale blending, split windows, and contour overlay matching . Followi™ Respiratory Motion Phantom . This is a moving platform that can be programmed to simulate internal respiratory motion in the CC direction (detected on the radiotherapy images). There is a second smaller chest wall platform that can stimulate the external motion of the chest in the AP direction. The Varian RPM marker block was placed on the chest wall phantom to generate a surrogate signal for internal respiration during the non-static helical scans, 4D-CT and treatment simulations to allow phase sorting of the 4D CT images  received the full volume (1 ml), whereas one patient received 0.4 ml and one patient 0.9 ml due to early stasis during the treatment procedure. 4D-CT and CBCT images were successfully acquired for all eight patients. For one patient, the primary radiotherapy data set was selected as the MIP as opposed to the AVE-IP, which subsequently excluded this patient from matching analysis (patient 4). Patient, tumour and treatment details are shown in RO beads were visible for all patients on the radiotherapy planning scans (AVE-IP image from 4D dataset) and CBCT imaging. The mean HU of the RO beads across all patients  was 150 HU. Areas of RO beads were then successfully auto-contoured for all patients at 150 and 200 HU on the AVE-IP and CBCT scans; below 150 HU, areas of artefact were contoured by the automated thresholding and at 250 HU and above fewer than three discrete areas were contoured. For two patients (patients 5 and 7), areas of RO beads on the CBCT were unable to be separated into discrete areas due to the distribution of the beads along the vasculature. In these cases, the whole area was contoured for matching purposes. The median number of contoured RO bead areas on the CBCT at 150 HU was 3 (range 1–4), and 1.5 (range 0–4) at 200 HU. For the AVE-IP images, median number of contoured RO bead areas at 150 HU was 3 (range 0–7) and at 200 HU 1.5 (range 0–4) .Matching of CBCT to the radiotherapy planning scan (AVE-IP) images using areas of contoured RO beads was successful in all patients . Due to After matching on RO beads, the absolute mean shift in CoM from CBCT to AVE-IP was 2.0 mm (SD 1.1) ML, 1.7 mm (SD 1.3) AP, and 3.5 mm (SD 2.4) CC . Figure Comparison of bead position on 4D-CT scans (AVE-IP and CT-50) with post-TACE CT scans showed good alignment of beads along tumour vasculature up to 29 days later .RO beads were visible on all imaging modalities in both the static and moving phantom . Using a20,21 Based on the findings of this study, we propose that a TACE-SBRT combination is likely to become a therapeutic option, using RO drug-eluting beads that can function as potential surrogates of liver tumour targeting  for IGRT.Drug-eluting beads are an effectively combined anti-cancer drug and embolisation treatment delivered directly to liver tumours during TACE treatment. The development of a novel RO drug-eluting bead, with durable radiopacity on CT scans, optimises the delivery technique by providing confirmation of bead location during and after the embolisation procedure.In this study, we have demonstrated that RO beads are visible on the imaging modalities required for IGRT, including CBCT, 4D-CT, kV and MV. Furthermore, we have shown that on-treatment matching can be performed using RO beads using CBCT scans. Due to size of the RO beads, and their varied clumping and distribution along tumour vasculature, the shape and size of high-density regions in and around a tumour are not uniform. It is also apparent from the radiotherapy images that there is a difference between the distribution of beads between HCC and mCRC tumours, with the beads being localised in HCC tumours. However, the sample size for this study was small, so definite conclusions about localisation of beads cannot be drawn comparing HCC to liver metastases from CRC. As predicted, highly selective TACE delivery is possible with hypervascular HCC tumours, whereas mCRC tumours tend to be less vascular in nature, leading to a more lobar distribution of beads during TACE delivery. Despite this, using an automated contouring method based on HU thresholding, distinct areas of RO beads can be contoured on radiotherapy planning CT scans and accurately matched to areas of RO beads on CBCT imaging for both types of liver tumours. Without any formal motion mitigation, matching on CoM of contoured areas was possible within 3.5 mm. It is likely that with the application of motion mitigation methods, as used in standard clinical practice, this translational shift will be reduced further. As shown in the phantom study using static images, matching between CBCT and AVE-IP scans is possible within 0.6 mm. However, the impact of motion on the shape, and therefore CoM, of an irregular-shaped object is evident . This is24 Furthermore, artefact did not distort the ability to visualise or contour the tumour and, for HCC patients in whom RO beads were located within the tumour vasculature, tumour contouring was enhanced.Although artefact streaking was present on CBCT, this did not impact RO bead visibility or the ability to match. Artefact streaking is a particular problem with commercial fiducials that can distort tumour contouring.17When RO bead matching was compared to matching based on liver edge or bony structures, difference in three-dimensional distances of up to 4.0 mm were seen. These findings suggest that bony anatomy and liver edge may be sub-optimal surrogates for tumour positioning in image- guided RT. This is in keeping with the findings by Yue et al, who compared matching on Lipiodol and bony anatomy; differences in three-dimensional distances were 0.9–2.6 mm (maximum 3.8 mm) in the ML direction, 1.1–2.9 mm (maximum 4.3 mm) in the AP direction, and 1.2–3.9 mm (maximum 5.5 mm) in the in the CC direction.There are limitations to our study and in the potential use of RO beads as fiducial markers. Firstly, this was a first-in-human clinical trial and, as such, our patient numbers are small and include just two patients with HCC. Furthermore, as patients did not undergo SBRT in this proof-of-principle study, radiotherapy imaging was limited to 4D-CT scans and CBCT images, and patients did not have any motion mitigation. As shown with the phantom model, translational errors and matching accuracy is improved with reduced motion. We also used an automated contouring method to contour RO beads. Although this approach was taken to reduce variation in contouring, this may not be available on all radiotherapy planning systems. However, areas of beads can be contoured manually without this approach. The use of RO beads as fiducials may also increase the complexity of RT planning and treatment delivery. Experience in contouring and matching on small areas of high intensity may take additional time. A final limitation of this study was that it did not specifically look at the impact of RO beads on dosimetry. Given the small volume of the high-density regions, it is anticipated that this will be negligible, but further evaluation would be required in a larger clinical study.In conclusion, this study has shown that RO drug-eluting beads are visible on IGRT imaging modalities required for liver SBRT, show minimal artefact, can be reliably contoured, can be used for on-set matching with CBCT, and remain stable positionally within the liver vasculature. As such, their role as potential fiducial markers is feasible and warrants further exploration in combination studies of TACE followed by SBRT for liver tumours.


Progressive multifocal leukoencephalopathy (PML) is a rare demyelinating disease of the white matter central nervous system occurring in immunocompromised patients particularly those with T cell deficiency such as in HIV, haematological and solid organ malignancies and those taking immunomodulatory medications. PML is caused by JC virus however in rare cases BK virus has been isolated in the cerebral spinal fluid of patients presenting with PML.3 on admission and his HIV viral load VL) was 141,000 copies/ml. Magnetic resonance imaging(MRI) showed a hypointense lesion on T1, hyperintense on T2 and FLAIR without diffusion restriction and without mass effect. A lumbar puncture was performed which confirmed JC virus was positive (PCR <50 copies/ml) and also revealed BK virus was positive . The patient was commenced on tenofovir alafenamide fumarate/emtricitabine/darunavir/cobicistat in combination with dolutegravir 50mg twice daily. On day 40 post commencement of ART the patient was readmitted with worsening of his right arm weakness and dysarthria. A repeat MRI was performed which showed the hyperdense lesion on T2 and FLAIR appeared slightly larger with some slight enhancement with gadolinium contrast but no other features suggesting PML immune reconstitution inflammatory syndrome (IRIS). The CD4 count had increased to 141(17%) and HIV VL had decreased to 85 copies/ml. A clinical diagnosis of PML IRIS was made and the patient was commenced on prednisolone 30mg BD which lead to an initial improvement in symptoms.In this case we describe a 49 year old man who presented to the emergency department with a 2 week history of progressive right sided weakness and dysarthria. His background history included HIV diagnosed in 2005, he had not engaged with care in the past 2 years and had not been taking anti-retroviral therapy (ART). Other past medical history included untreated hepatitis C. His CD4 count was 90 (11%) cells/mmInterestingly in this case, both JC virus and BK virus were detected in the CSF of this patient with the level of JC virus being too low to quantify. BK virus was not detectable on peripheral serum sampling suggesting that BK virus is replicating in the CNS independent of other body sites. There have been 5 case reports in the literature of BK virus as the cause of PML.Testing for BK virus should be considered in patients presenting with signs and symptoms of PML and encephalitis particularly when no other cause is found. It is mPolyomavirus 2 (BK virus) causes tubulointerstitial nephritis, haemorrhagic cystitis, ureteric stenosis, and to a lesser extent pneumonitis and hepatitis in renal transplant patients but also patients post ASCT, SOT, and HIV . Both JCGiven the similarities between these viruses it does appear that in some immunocompromised patients BK virus can acquire neurotropic properties and has been found in the CSF and brain tissue of patients with encephalitis, meningitis, meningoencephalitis, retinitis and even PML .1.1A 48-year-old man presented to the emergency department with a two-week history of progressive right arm weakness, right leg weakness and dysarthria. He described falling off the back of a motorbike as a passenger on acceleration from rest as he was unable to hold onto his companion driver. He had no symptoms of visual/auditory change, no bladder or bowel dysfunction and no swallowing difficulties. The patient felt that he had lost weight but was unable to quantify this. There were no symptoms of fever, chills, shortness of breath, cough, odynophagia, diarrhoea or skin changes to suggest other opportunistic infections.He provided a comprehensive history and was cachectic. Neurological exam revealed Glasgow Coma Score (GCS) of 15/15, dysarthria without focal cranial nerve deficits, decreased power in his right arm; shoulder abduction grade 4/5, elbow flexion/extension 3/5, wrist flexion/extension 2/5, MTP flexion/extension 1/5, right leg; hip flexion/extend 5/5, knee flexion/extension 4/5, and ankle flexion/extension 4/5. Babinski reflex was positive on the right side, biceps tendon, knee and ankle jerk reflexes were brisk on the right side. Sensation was globally reduced to 10% on the right arm and 30% on the right leg. Neurology was grossly intact on the left upper and lower limbs. No cutaneous, genital or palatal lesions were appreciated. Sub-centimetre nodes in the submandibular region were felt bilaterally.3. He engaged poorly with services due to complex social issues and had been off anti-retroviral therapy (ART) entirely for the previous two years. CD4 nadir was 53 (7%) in April 2020. Other history includes; chronic hepatitis C genotype 1a, pan sensitive non-cavitary pulmonary tuberculosis treated with directly observed therapy in 2011. He was homeless, prior to presentation and actively using intravenous heroin.The patients background history includes HIV which was diagnosed in 2005 on routine screening, CD4 count at time of diagnosis was 551 (17%) cells/mmA non-contrast computerised tomography (CT) brain scan showed a new ill-defined hypodensity in the left temporo-parietal region compared with a CT from April 2020. Further Magnetic resonance imaging (MRI) showed a hypointense lesion on T1, hyperintense on T2 and Fluid-attenuation inversion recovery (FLAIR) without diffusion restriction and without mass effect see .Fig. 1Pr3, HIV viral load(VL) 141,000 copies/ml, haemoglobin 8.1g/dL, lymphocyte count 0.6 × 109/L, platelet count 281 × 109/L, EBV IgG positive, serum EBV PCR <800 IU/mL, Syphilis TP assay negative, serum cryptococcal antigen negative, toxoplasma IgG negative. A lumbar puncture was performed which showed a white cell count of 3 cells/mm3Further cerebral spinal fluid showed protein 36 mg/dL, glucose 3.2 mmol/L, negative cryptococcal antigen, HIV viral load 1520 copies/ml, JC virus PCR <50 copies/ml, BK virus PCR 46,511 copies/ml and EBV virus PCR 555 copies/ml. A subsequent serum BK virus was negative.Laboratory results on admission were as follows; CD4 90(11%) cells/mmA diagnosis of PML was made and antiretroviral therapy was commenced on day 1 of admission once cryptococcal antigen was found to be negative. Tenofovir alafenamide fumarate/emtricitabine/darunavir/cobicistat once daily combination therapy plus dolutegravir 50mg twice daily was commenced. The most recent genotypic resistance profile was done in 2016, although this showed no resistance mutations this regimen was chosen due to unknown levels of subsequent integrase exposure, the gravity of the current presentation and the necessity to suppress HIV viral replication both in serum and the central nervous system to attempt to improve mortality and reduce further neurological deterioration as quickly as possible.The patient was monitored as an in-patient and by day 14 post admission no neurological progression or evolving immune reconstitution syndrome were witnessed. A ‘step-down’ bed in an off-site respite facility was arranged in the community with close out-patient clinic follow up. On day 26 post admission the patient was seen in clinic and neurological exam at this time was stable. On day 40 post admission the patient was again seen in out-patients, on this occasion he was noted to have a significant decrease in power in his right arm and leg and worsening dysarthria. Repeat lumbar puncture and routine blood tests were not concerning for a new infection as a cause for this representation. An MRI was done on 01.02.21 which can be seen in Image 1. The lesion was hyperintense on T2 and FLAIR and appeared slightly larger, and although slightly enhancing with gadolinium contrast, no other typical features of PML immune reconstitution inflammatory syndrome (IRIS) were seen. By this time CD4 count had increased to 141 (17%) and HIV VL had decreased to just 85 copies/ml. In this context prednisolone 30 mg twice daily was commenced for a clinical diagnosis if PML IRIS, paradoxical worsening of symptoms after commencement of ART. Within one week right arm and leg power and speech had improved; power 3/5 at the shoulder, 4/5 at the elbow, and 1/5 at wrist and MTP joints. Prednisolone was reduced to 20 mg once daily but unfortunately the patient deteriorated clinically and repeat MRI brain on 16.02.21 showed extension of the lesion into the corona radiata. Prednisolone was increased again to 40 mg twice daily.2In this case we describe a man with advanced HIV who developed classical PML with very high levels of BK virus in his CSF . Interestingly although JC virus was detectable in CSF, this level was too low to quantify (<50 cpm). Furthermore, BK virus was not detectable on peripheral serum sampling suggesting this CSF BKV population is replicating in the CNS independent of other body sites. In this case, BK virus testing was done as part of a full cerebral spinal fluid analysis performed by the National Virus Reference Laboratory.To date BK virus CNS infection although rare has been described. A review of the literature by da Silva RL et al., in 2011 found 24 cases of BK associated CNS infection eight of whom were self-limiting. Most of these patients had an immunocompromising factor like AIDS, malignancy or transplantation. Of the pathologies found; 9 had encephalitis, 8 meningoencephalitis, 3 meningitis, 2 PML, 2 retinitis. All patients had BK virus positivity on either CSF or brain biopsy or both . Of those with known outcomes nine passed away. Regarding typical radiological features, BK virus encephalitis has a predilection for periventricular white matter similar to HIV or cytomegalovirus (CMV) and also for pial surfaces and in general was cortex sparing . RegardiRegarding BK virus as a cause of PML, there are five cases in the literature with variable degrees of evidence to support BK PML . All patIn summary the authors would like to highlight the neurotropic properties of BK virus in immunocompromised hosts and to consider testing for this virus in the context of negative results for other known causative viruses of encephalitis and PML; JC; CMV; HIV and HSV. At present immune reconstitution of the host and mitigation of immune mediated inflammatory syndromes are at the forefront of treatment and whether PML is caused by JC or BK may be academic as our current therapeutic toolkit is not sufficiently advanced for directed therapy. Small studies have shown that potentiating the immune system with immune checkpoint inhibitors like pembrolizumab show promise for PML . SimilarNo funding was received for the preparation of this manuscript.The authors have no conflicts of interest to declare.


Trichovirus) has now been detected in most grape-growing countries. So far, little is known about the epidemiology of this newly emerging virus. In this work, we used datamining as a tool to monitor in-silico the sanitary status of three vineyards in Italy. All data used in the study were recovered from a work that was already published and for which data were publicly available as SRA  files. While incomplete, knowledge gathered from this work was still important, with evidence of differential accumulation of the virus in grapevine according to year, location, and variety-rootstock association. Additional data regarding GPGV genetic diversity were collected. Some advantages and pitfalls of datamining are discussed.Since its identification in 2003, grapevine Pinot gris virus (GPGV,  Trichovirus, Betaflexiviridae) has been detected in most grapevine growing regions around the world. Generally, the virus is detected using serological and/or molecular tools. In this work, we describe datamining as a potential additional method to identify grapevine infected with this virus, better estimating its distribution worldwide. While this specific work cannot be considered as an epidemiological study per se, it still unquestionably offers valuable information on the virus , providing a snapshot of the situation in three different vineyards in Italy at a specific time, giving new insight on GPGV accumulation, introduction and transmission.Since its characterization in Italy  and at a later stage of fruit development, at mid-ripening rather than pre-veraison (P < 10−4). Also, the association cultivar-rootstock seems to have its importance in virus accumulation. Indeed, GPGV seems to accumulate more in Cabernet Sauvignon cultivar grafted onto either 161–49 or Kober-5BB rootstocks, rather than in Sangiovese grafted onto 420A at either location (P ≤ 10−4). In addition, differential accumulation of GPGV was also observed according to location where grapevines were grown (P < 10−5), with GPGV accumulating more in Riccione than in Bolgheri.Our datamining study revealed that only samples from Bolgheri and Riccione were positive for GPGV. The virus was hardly detected in a few samples from Montalcino Table ; howeverST) analyses . Such segregation by location was also highlighted for grapevines grafted onto rootstock 161.49 C used exclusively in Bolgheri and grapevines onto Kober 5BB exclusively used in Riccione . Comparison of sequences obtained from the 420A rootstock also displayed statistically significant FST values; however, the values were lower than the ones mentioned above. This is most likely because 420A was used as a rootstock in both locations. Furthermore, the genetic background of the grapevine cultivar, which was also present in both locations, had no statistically significant impact on viral populations .When delving into the genetics of the virus, other information was revealed. Overall, 47 complete genome GPGV sequences  were assembled Table , all subses Fig.  confirmeIn addition to the presence/absence of GPGV in the samples, this work highlights two distinct situations at the viral genomic level. Indeed, one vineyard is infected by a single variant, identity percentage ≥ 99%, as previously defined for GPGV  (last visited 04/2021). Datamining can be also utilized to increase the number of complete genome sequences for downstream studies on the evolutionary history of specific viruses for example . We do not have the choice of the technology with which data were obtained nor the quality of the sample. However, the information being generated is still very valuable, it has already been paid for and therefore almost free , it is available to anyone and most of all, it is ever growing.Lately, datamining is becoming a very important and powerful tool to identify new pathogens, as well as new variants of known viruses, such as from the now well-known


The manipulation of superparamagnetic beads has attracted various lab on a chip and magnetic tweezer platforms for separating, sorting, and labeling cells and bioentities, but the irreversible aggregation of beads owing to magnetic interactions has limited its actual functionality. Here, an efficient solution is developed for the disaggregation of magnetic beads and interparticle distance control with a magnetophoretic decoupler using an external rotating magnetic field. A unique magnetic potential energy distribution in the form of an asymmetric magnetic thin film around the gap is created and tuned in a controlled manner, regulated by the size ratio of the bead with a magnetic pattern. Hence, the aggregated beads are detached into single beads and transported in one direction in an array pattern. Furthermore, the simultaneous and accurate spacing control of multiple magnetic bead pairs is performed by adjusting the angle of the rotating magnetic field, which continuously changes the energy well associated with a specific shape of the magnetic patterns. This technique offers an advanced solution for the disaggregation and controlled manipulation of beads, can allow new possibilities for the enhanced functioning of lab on a chip and magnetic tweezers platforms for biological assays, intercellular interactions, and magnetic biochip systems. This study reports the real‐time disaggregation and interdistance control of the magnetic beads using magnetophoretic decoupler fabricated with permalloy thin film pattern. The application of a rotating magnetic field creates a unique magnetic potential energy distribution around the decoupler at multiple points, and thus, separates the aggregated beads and transported them on the pattern to the desired direction. For example, it is possible to effectively separate nonmagnetic cells and move them to the desired position individually. This allows analysis at the single‐cell level, providing the heterogeneities in single‐cell properties with its population level.The controlled manipulation of magnetic beads or cells is recognized to be of significant importance for both fundamental and applied research, such as biomedical, diagnostics and therapeutics, drug delivery, and single‐cell analysis.4, 5 However, the irreversible aggregation or clustering of original carrier beads owing to their magnetic interactions is indeed a critical and limiting factor in the individual transportation and separation processes. This aggregation issue may prevent the quantification of the physical and biological properties of various DNA, proteins, or cells because of the gap between objective beads. In particular, the particles were transported and sorted when they were not aggregated before performing the manipulation. However, these reports do not describe thoroughly the process of manipulation or real‐time disaggregation and separation of particles that are already aggregated in a pattern. Although the aggregation can be eliminated temporarily using the z‐field, reaggregation occurs eventually when the in‐plane magnetic field is again applied to control the beads during separation. In fact, the aggregation of multiple beads occurs because the energy well created by the external field is larger than the nano‐microbeads used for labels and thus multiple beads gather at a single energy well. Here, since the size of the potential energy well created by bulk magnets or magnetic coils is too large to confine a single bead, the interparticle distance of trapped beads cannot be controlled individually.Alternatively, the energy landscape induced particle motion caused by applying the rotating magnetic field was used for colloidal particles, large molecules, and cell transportation and separation along the surfaces of the substrate, patterned magnetic surfaces, integrated circuits of overlaid patterns of magnetic films, and garnet films.12 How However, the potential well is created above the domain wall where different magnetizations meet for the control of the particle, resulting in several issues. For example, if the size of the conduit is increased to control the larger size particle, the domain wall does not scale up accordingly, and thus, becomes form a complex multidomain. As a result, the potential well is also not uniform. And, using the movement of the single domain wall along with the pattern only one particle per conduit can be manipulated. Although, using z‐field single particle can be controlled more precisely by an array of conduits but the z‐field applied for the transfer of the particle between each component also affects the potential well of all the particles located on the chip and thus it is difficult to control multiple particles parallelly.In general, the constrained domain walls in ferromagnetic nanoconduits patterned on a substrate can be used for manipulating magnetic nanocarriers on‐chip by tuning the size of the domain walls from a few tens of nanometers wide up to micrometers, where the resulting trapping potential well is of similar size, which prevents or mitigates aggregation and allows manipulation of individual nanocarriers.23 How Also, magnetic tweezers require an additional parallel tip for precisely controlling each bead; otherwise, the beads present in the total focusing area are attracted in the same direction and aggregate at one point. Therefore, it is necessary to develop a reliable 2D magnetic platform for controlling the beads on a large scale for practical magnetic tweezers platforms without any aggregation. Moreover, achieving accurate manipulation and disaggregation of particles on a 2D platform with controllable interparticle distance is crucial for monitoring the interaction dynamics between cells in on‐chip devices. To the best of our knowledge, there is no available micromagnet pattern platform that achieves both the disaggregation of beads with individual control and for interparticle distance control.Recently, combined technologies have been used for controlling the space between driving particle and fixed particle for biological applications. However, if the driving particle is magnetic, then the fixed particle has to be controlled by other methods, i.e., micropipette, acoustic and optical tweezing, and or hydrodynamic traps, which required additional processing and equipment.26 AlsHere, we propose a magnetophoretic decoupler pattern that achieves efficient magnetic bead disaggregation and interparticle distance control by applying an external magnetic field with a uniform rotational speed. The magnetic pattern was made of permalloy using conventional photolithography and lift‐off processes. The aggregated magnetic beads were consecutively detached into single ones by the successive splitting and delay of the magnetic potential wells owing to the specific shape of the magnetic patterns, demonstrating the simultaneous and accurate spacing control of multiple magnetic bead pairs. Hence, the proposed magnetic method could be a milestone achievement on the magnetic tweezers platform for performing biological interactions and extracting relevant information at the level of a single object.22.1In the magnetophoretic transportation system, the magnetic beads or magnetic fluid droplets were manipulated using the angular variation of the magnetic potential energy distribution, which is generated according to the 2D shape of the magnetic thin film under a rotating magnetic field. If the applied rotating magnetic field is in the in‐plane direction, the magnetic bead around the thin film disk slides along the boundary of the disk. This brings into focus the motion of a magnetic bead between thin films of different size of disks. The two factors that can affect the movement of the bead are the bead and pattern size, and the gap between the patterns. Based on the relative size of the bead and patterns, there could be two possible modes for the movement of the bead between the two disks in following one of the disk boundaries.Figure Db − disk =  2  × Ds − disk). It can be noted that the magnetic force increases with the bead size and reaches a maximum when the diameter of the small disk is ≈1.2 times the diameter of the bead and then decreases as the disk diameter increases further. When Ds − disk /Dbead  = 1.2 (Db − disk /Dbead  = 2.4), the forces of the small and disks attain the same value. Based on these simulations, the bead is expected to move along the boundary of the small disk, “Mode 1,” if the diameter of the small disk exceeds 1.2 times the bead diameter  and 30 µm (Db − disk /Dbead  = 6.66) with varying angles from 110° and 130° at 120 Oe of external magnetic field is shown in Figure For a bead whose diameter is 4.5 µm, the angular variation of magnetic energy and the direction of magnetic drag force on the permalloy disks of diameters 15 µm  pattern to a wave‐like DSD pattern because there is no significant difference in the potential energy variation between the disk and wave patterns for the calculated energy, where the wave‐like DSD pattern allows the bead to move along with the pattern , a horizontal potential barrier is created between two potential wells of patterns. Thus, the beads in each wave boundary cannot switch from one to another , there is still no “mode” switching because of the vertical barrier, and thus the bead cannot cross the gap and move to the next pattern . Thus, the bead moves only in one direction under one size ratio condition.Depending on the gap size (second factor), potential barriers are created or annihilated in different directions. If the gap is larger than 1.4 times the bead diameter  for further process on the next gap. However, the strength and frequency of the field play a key role for the movement of the bead on the big wave . The magnitude of the force (F) is approximately tens of pN, which is dominant compared to the dipole–dipole interaction  between each bead. Here, the ± sign of the force indicates that the force acts in the ± x‐direction, as a function of the field angle. When the origin of the position  of the bead is arranged as a triangle from the center of the gap, the calculated forces F1, F2, and F3 of each bead labeled 1, 2, and 3 are as followsAccording to the analysis of switching modes, it is more convenient to adjust the interbead forces for the performance as a decoupler, that is, decoupling and switching in one direction. θ < 90°, the three beads move together because no bead reached to the gap. When θ = 90°, the beads reach the decoupler. When angle 120°≤ θ ≤150° (Angle zone A), the forces of beads 1 and 3 are positive, but that of bead 2 is negative. This means that beads 1 and 3 move in the +x‐axis direction along the upper track while bead 2 moves in the − x‐direction. For a further rotating field, bead 2 navigates in the − x‐direction, but beads 1 and 3 reach the decoupler 2 moving along the + x‐direction after the half‐cycle of the rotating field. At the decoupler 2, two beads become new beads 1 and 2 and splits in opposite directions at the same field angle of zone A. In fact, the N‐ and S‐magnetic poles are topologically the same after rotation of the half‐cycle of the field. Therefore, we calculated only the first half‐cycle. The subsequent rotation repeats the same movement of the potential energy as the first half. During further rotation of the field, a single bead from decoupler 2 moves in the + x‐direction (upper track) and arrives at decoupler 3. Here, bead 3 switches to a lower track of a smaller wavy pattern and starts to move in the − x‐direction along the lower track  decouplers with gaps because decouplers divide the beads in the cluster into two groups. Each decoupling further divides the cluster and finally detached a single bead after (n − 1) decoupling. Further, one more decoupler is required to switch the “mode” of the last detached single bead, and thus, a total of n decouplers are required for single bead disaggregation and transfer  integer of half‐cycle of field. The decoupler, where two waves meet, is marked in green color. The bold number represents the disaggregated beads on the decoupler, as shown in Figure 5a. The aggregated beads, which are detached only in the decoupler, continue to move towards the next decoupler along the upper track until a single bead remains in the decoupler. When a single bead comes to the decoupler, it switches to the lower track and moves in the − x‐direction. Hence, this concept can clarify the bead aggregation that inevitably occurs in various experiments using magnetic bead carriers and can be controlled locally without additional tools.r Figure . Figure 2.3Figure Under the nonswitching “mode” condition, interbeads distance can be adjusted depending on the field angle Figure 6a. Figur2.4  are 0.54 and 0.89, respectively. However, according to Figure Dgap /Dbead  for the 11 µm beads lies beyond the “mode” switching range to be trapped.The proof of concept for magnetic beads with positive magnetophoresis could be extended to nonmagnetic particles using a negative magnetophoresis technology with magnetic fluid30  magnetophoretic functions because the positive magnetic pattern causes a negative cavity pattern, as shown in Figure 31 Thu3We have demonstrated a magnetophoretic technology for the decoupling and switching of magnetic beads using 100 nm thick magnetic patterning and an external magnetic field. This decoupler is realized by a unique magnetic potential energy distribution in the form of an asymmetric magnetic pattern with a nonmagnetic gap, where the decoupling performance of magnetic/nonmagnetic beads is demonstrated according to carrier size and pattern. The proposed disaggregation technique offers new possibilities for various applications, including magnetic tweezers and lab‐on‐chip processes, where the advantage of a uniform particle size configuration can be integrated with the biological assay to control the positioning of cells for studying the intercellular interactions solving the issue related to the irreversible aggregation of beads used as labels for various studies. it takes a lot of effort to repeat the process for stable results. In contrast, the proposed method can simultaneously manipulate multiple bead pairs in the 2D space with the same force and spacing, thereby reducing the time‐consuming steps to perform several sets of experiments. Here, controlling the distance of a bead pair from multiple points is accomplished easily through the angles of an external magnetic field. In addition, depending on the direction or the size of the decoupler installed in one plane, the beads located on each decoupler are adjusted in different directions and distances. However, a few limitations exist in the proposed technique, such as, if the surface is required an additional layer of thickness for immobilizing biomolecules, there is a reduction of the force on the magnetic beads depending on the thickness. Then, the difference in operating the magnetic field strength for both beads increases for the dual positive and negative magnetophoretic platforms.As most of the existing methods can control only a pair of beads at a time in turns,34 it 42 layer of 500 nm thickness. Parylene C (DiX‐C from KISCO) was used to coat the surface of the pattern.Superparamagnetic beads  and nonmagnetic polymer beads  were used in this experiment. In the experiments, the magnetic beads were diluted to 0.001% with distilled (DI) water and nonmagnetic beads were added to water‐based ferrofluid  in a ratio of 0.01% to the total volume. Ferrofluid was diluted with DI water at a concentration of 0.2%. Photoresist (AZ‐5214E) and developer (AZ‐300 MIF) were purchased from Merck Performance Materials, GmbH. The Si wafer was coated with SiO In brief, Ni80Fe20 (100 nm thickness) was deposited on the substrate in a specific shape through photolithography and DC magnetron sputtering. Then, to protect the pattern in the fluid environment and reduce the adhesion force between the bead and the substrate, parylene C (500 nm thickness) was coated with chemical vapor deposition. The external rotating magnetic field was implemented in two ways depending on its strength. Two perpendicular solenoid coils produced a rotating magnetic field of 0–200 Oe within the x–y plane. For nonmagnetic polymer bead manipulation, the required strong field strength of 900 Oe was supplied by a rotatable bulk magnet with adjustable spacing between each other. The rotational speed of this magnet was determined by the motor and the strength of the magnetic field can be controlled by adjusting the spacing.The fabrication process for detailed magnetophoretic surface patterning was described in the previous articles.36 In 3 software. Standard material parameters such as exchange stiffness Aex = 1.0 × 10−11 J m−1, saturation magnetization Ms = 800 kA m−1, damping constant α = 0.1, zero magnetocrystalline anisotropy, and cell size of 100 × 100 × 100 nm3 were used for simulation. Using the stray field created by the simulated magnetic domain, the distribution of the magnetic potential energy and magnetic force exerted on the bead was calculated using a Matlab code. The distance from the surface at which the magnetic forces are calculated for a single bead or a single layer of 2D aggregated beads is equal to the sum of the radius of the bead and the thickness of the passivation layer . All the conditions were calculated and analyzed based on the above vertical height.Micromagnetic domain simulations were performed using MuMaxThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supplemental Movie 1Click here for additional data file.Supplemental Movie 2Click here for additional data file.Supplemental Movie 3Click here for additional data file.Supplemental Movie 4Click here for additional data file.Supplemental Movie 5Click here for additional data file.Supplemental Movie 6Click here for additional data file.Supplemental Movie 7Click here for additional data file.


PRM1) is a gene or protein crucial for packaging and protecting sperm DNA until fertilization affects normal sperm function. This study analyzes the genes and proteins potential from PRM1 as fertility markers for different breeds of bulls utilized in the artificial insemination programs, expected to be an accurate tool in interpreting bull fertility in Indonesia. This study used Limousin, Holstein, and Ongole Grade bulls divided into two groups based on fertility, high-fertility (HF) and low fertility (LF). The semen quality assessment included progressive motility , viability (eosin-nigrosine), and plasma membrane integrity (HOS test). Sperm DNA fragmentation (SDF) was assessed using the acridine orange staining and the Halomax test. Sperm PRM deficiency was evaluated with the chromomycin A3 method. Moreover, PRM1 gene expression was measured using qRT-PCR, and the PRM1 protein abundance was measured with the enzyme immunoassay method. Semen quality values, relative expression of PRM1 gene, and quantity of PRM1 protein were significantly higher (p < 0.05) in HF bulls than in LF bulls. The SDF and PRM deficiency values in LF bulls were significantly higher (p < 0.05) than HF bulls. Additionally, PRM1 at the gene and protein levels correlated significantly (p < 0.01) with fertility. Therefore, PRM1 is a potential candidate for fertility markers in bulls in Indonesia.Functional genes and proteins in sperm play an essential role in bulls’ reproductive processes. They are more accurate in determining bull fertility than conventional semen quality tests. Protamine-1 ( The artificial insemination (AI) program is a proven and successful reproductive technology in increasing population and livestock production . The AI PRM) is one of the many genes or proteins reported as potential molecular markers for predicting the fertility of these bulls [Over the years, a paradigm has arisen about classical semen quality assessment, including motility, usually less valuable and reliable in predicting male fertility, particularly in bovine species , confirmse bulls ,12,13.PRM is the essential molecule in the arginine-rich nuclear protein involved in sperm DNA packing [PRM sequentially replaces histone somatic cells through a complex process during spermiogenesis [PRM until sperm development [PRM1 gene is essentially critical in normal sperm functioning. However, PRM2 and PRM3 genes have been expressed in bovine testes [PRM plays a fundamental role in protecting sperm DNA from damage such as nucleases and other factors [PRM process silences transcription in the spermatozoa and possibly imprint the paternal genome before fertilization [ packing . Sperm Pogenesis . At the ogenesis , which aelopment ,16,17. Telopment ,19,20,21e testes . PRM pla factors . Additiolization .PRM gene or protein is a potential application for determining male fertility [PRM on male fertility, causing the decline in semen quality [PRM with male infertility cases [PRM1 reduction in LF bulls, also characterized by an increase in DNA fragmentation. The PRM1 gene has been highly expressed in high-fertility (HF) Holstein bulls [PRM1 as the protein with the most potential determinant of semen quality of various bulls in Indonesia compared to PRM2 and PRM3 variants [PRM1 as a biomarker of bull fertility used for AI in Indonesia have not been reported. This study analyzes the possibility of genes or proteins from PRM1 as a marker for determining fertility in various bull breeds used for the AI program through the mRNA and protein approach, which should be an accurate tool for interpreting bull fertility.The ertility ,13. Prev quality ,27,28,29ty cases . Dogan ein bulls . It repovariants . HoweverThe Indonesian National AI center produced the frozen semen used in this study. The frozen semen sample used in this study is a commercial product from the AI center, where every procedure, from the process of collecting fresh semen to producing it into frozen semen, including the management of bulls’ maintenance, follows the operational standards of the AI center, which is supervised by a veterinarian and has complied with every principle of animal welfare. The semen was obtained from bulls at their sexual maturity and productive phase, at 4–5 years of age. This study used 18 bulls, including six Limousin, Holstein, and Ongole Grade bulls. Furthermore, each breed was grouped into two based on the fertility score, HF (n = 3) and LF (n = 3). The fertility score was based on each bull’s first service %CR; %CR > 70% are categorized as HF bulls, and %CR < 70% is classified as LF bulls . The %CRThe iSIKHNAS is an animal health information system in all regions in Indonesia, including data on AI programs accompanied by pregnancy data. The results of the analysis carried out on iSIKHNAS data, the number of cows/heifers that were inseminated in each breed of bulls in the study were 143,010 (Limousin), 81,741 (Holstein), and 98,571 (Ongole Grade) cows/heifers. Meanwhile, the number of AI services for each breed of the bull in the study were 197,588 (Limousin), 106,034 (Holstein), and 136,686 (Ongole Grade) services. The percentage of pregnant cows/heifers from the first insemination service of the total AI services (%CR) was analyzed according to Pardede et al. [A total of 90 frozen semen straws (five straws/bull) from six Limousin bulls, six Holstein bulls, and six Ongole Grade bulls were used in this stage. First, the frozen semen was thawed in a water bath at 37 °C for 30 s, then put into a microtube for further observation. Next, the sperm’s progressive motility (PM) parameters were analyzed using computer-assisted semen analysis. After that, 10 µL of thawed sperm were dropped onto a slide and covered with a coverslip. Finally, four fields were evaluated with a range of 50–250 sperm cells automatically counted using Sperm Vision  .Additionally, sperm viability was evaluated using eosin-nigrosine  staining. First, the sperm viability was evaluated by making a smear on slides from a mixture of 10 µL semen and 10 µL eosin-nigrosine (1:1). Then, the slides were dried using a heating table, observed, and counted under a light microscope with 40× objective magnification. Two hundred sperm cells per slide were evaluated, with live sperm unstained and dead sperm with damaged membranes stained (reddish) . Next, t2HPO47H2O) for 5 min in a dark room. Next, a coverslip was placed on the slide after washing with distilled water. Finally, 500 sperm cells per slide were observed and counted under a fluorescent microscope using a 490 nm excitation filter and a 530 nm emission filter. The green fluorescence denoted the sperm with mature chromatin and intact DNA integrity, whereas sperm with fragmented DNA integrity was characterized by a reddish-orange fluorescence [Sperm DNA fragmentation (SDF) was assessed using acridine orange (AO) staining and the Halomax test. A total of 90 frozen semen straws (five straws/bull) from six Limousin bulls, six Holstein bulls, and six Ongole Grade bulls were used for AO staining. First, a smear was made 5–10 µL of semen on a slide, then fixed in Carnoy’s solution ) for 2 h. Next, the slides were stained in a new AO solution . A total of 90 frozen semen straws (five straws/bull) from six Limousin bulls, six Holstein bulls, and six Ongole Grade bulls were used for this test. First, 25 µL (15–20 × 106 sperm cells/mL) was mixed with 50 µL of liquid agarose in a microtube at 37 °C. A total of 1.5–2 µL of cell suspension was dropped into marked wells on a pre-cooled slide (4 °C), and a coverslip was placed. The slide was incubated in the refrigerator at 4 °C for 5 min. The coverslip was carefully removed from the slide at room temperature (22 °C), and a lysing solution was dripped on the slide horizontally for 5 min and later dried. Next, the slides were washed with distilled water for 5 min and re-dried. Finally, the slides were dehydrated with 70% and 90% ethanol solutions, respectively, for 2 min and dried. The staining process was done by dripping 5 µL of 0.01 mm propidium iodide dye for 5 min in a dark room. Five hundred sperm cells per slide were observed under a fluorescent microscope using a 510–560 nm excitation filter and a 590 nm emission filter. Sperm cells with the fragmented DNA were then denoted by large and dispersed spotty halos, whereas tiny, dense halos represented sperm as the unfragmented DNA [Moreover, according to the manufacturer’s protocols, SDF was assessed using the Halomax test  staining method. A total of 90 frozen semen straws (five straws/bull) from six Limousin bulls, six Holstein bulls, and six Ongole Grade bulls were used in this stage. Sperm PRM deficiency was assessed by smear preparations of 5–10 µL of semen on a slide, then fixed in Carnoy’s solution ) for five min at 4 °C. Then, the slides were stained in CMA3 dye solution (0.25 mg/mL CMA3 in McIlvane’s buffer (pH 7.0) supplemented with 10 mM MgCl2) for 20 min. Next, the slide was rinsed in McIlvane’s buffer (pH 7.0) and air-dried. Finally, 500 sperm cells per slide were observed under a fluorescent microscope using a 470–490 nm excitation filter and a 510 nm emission filter. Sperm with deficient PRM (CMA3+ positive) was indicated by bright green fluorescence on the head, whereas dull green indicated sperm with intact PRM (CMA3−negative) [Sperm egative) .6 sperm cells/mL) by centrifugation at 16,000× g for 15 min. After that, the total RNA was extracted using the TRI reagent, as recommended by the manufacturer . The NanoDropTM One/OneC Microvolume UV-Vis Spectrophotometer  determined the total RNA amount and purity . According to the manufacturer’s protocols, the SensiFASTTM cDNA Synthesis Kit  was also used for cDNA synthesis. A total of 20 µL of cDNA obtained from this reaction was ready for RT-PCR. Next, the NanoDrop One/OneC Microvolume UV-Vis Spectrophotometer  was used to assess the total cDNA amount and purity. The quantitative real-time PCR (qPCR) was also used to determine the transcripts’ quantity, and the reactions were performed using the SsoFastTM EvaGreen Supermix . The genes identified in this study were PRM1  [−ΔΔCT [PRM1 gene, which were normalized to the housekeeping gene PPIA expression.A total of 144 frozen semen straws (eight straws/bull) from six Limousin bulls, six Holstein bulls, and six Ongole Grade bulls were used in this stage. Next, each frozen semen was thawed in a water bath for 30 s at 37 °C, washed three times with phosphate-buffered saline (PBS), and pelleted (about 25 × 10CCTG-3′)  with PPICCTG-3′) . The 2−Δ) [−ΔΔCT  was used6 sperm cells/mL) by centrifugation at 700× g for 15 min. The amount of PRM1 protein was then measured in sperm pellets using the enzyme immunoassay (EIA) technique, according to the manufacturer’s protocols  [A total of 108 frozen semen straws (six straws/bull) from six Limousin bulls, six Holstein bulls, and six Ongole Grade bulls were used. Each frozen sperm was thawed in a 37 °C water bath for 30 s, washed twice with PBS, and pelleted  . The preFurthermore, the reaction wells were closed and hatched in an incubator (37 °C) for 90 min. After that, the EIA plate was washed twice, then 100 µL of antibody solution was added and covered with adhesive tape on each well. Next, the EIA plate was re-hatched in the incubator (37 °C) for 60 min and then washed thrice. A total enzyme solution, 100 µL, was poured into each well, closed, and re-hatched in the incubator (37 °C) for 30 min. Washing was repeated five times on the EIA plate; then, color reagent solution was added and hatched again in the incubator (37 °C) in the darkroom for 30 min. Finally, the color C reagent mixture mixed thoroughly in each well. The analysis was conducted using an EIA reader with a wavelength of 450 nm in 10 min.PRM deficiency, the abundance of PRM1 protein, and relative expression of PRM1 mRNA in the FH and LF bulls were analyzed using the t-test method. The Pearson correlation analysis examined the overall association between the parameters evaluated and the bulls’ percentage CR. A scatter plot matrix graph presented the relationship between %CR, PRM1 protein, and mRNA. The t-test method, Pearson correlation analysis, and scatter plot matrix were obtained using the Statistical Package for the Social Sciences . The data were presented in the form of mean ± standard error.The fertility score (%CR) data for each bull breed were analyzed using Microsoft Excel (Microsoft Office Pro Plus 2019 Microsoft). The differences in the mean number of breeding, fertility score (%CR), semen quality parameters, SDF, p < 0.05) between HF and LF bulls (p < 0.05) than in LF bulls (The results indicated that the %CR obtained from the AI data from each bull breed was significantly different (LF bulls . The semLF bulls .p < 0.05) in Limousin (2.38% ± 0.77% vs. 1.25% ± 0.32%), Holstein (3.27% ± 0.63% vs. 2.34% ± 0.34%), and Ongole Grade (3.13% ± 0.79% vs. 0.94% ± 0.36%) bulls (p < 0.05) in Limousin (2.47% ± 1.11% vs. 1.31% ± 0.42%), Holstein (3.49% ± 0.71% vs. 2.58% ± 0.40%), and Ongole Grade (3.29% ± 0.99% vs. 1.13% ± 0.50%) bulls  bulls A. Howeve%) bulls B.PRM deficiency conditions were stained bright green fluorescence (CMA3+), and sperm with normal PRM were stained dull green (CMA3−) (PRM deficiency showed that sperm in LF bulls were significantly more deficient in PRM than HF bulls (p < 0.05). The mean content of sperm with PRM deficiency in LF vs. HF bulls were Limousin (2.06% ± 0.68% vs. 0.93% ± 0.33%), Holstein (2.92% ± 0.50% vs. 2.01% ± 0.37%), and Ongole Grade (3.00% ± 0.74% vs. 0.92% ± 0.42%) (Sperm with  (CMA3−) . The ass± 0.42%) A.PRM1 in each breed in HF bulls were greater (p < 0.05) than in LF bulls (PRM1 gene in HF vs. LF bulls were Limousin (4.64 ± 0.57 vs. 1.68 ± 1.23), Holstein (4.65 ± 0.61 vs. 1.42 ± 1.04), and Ongole Grade (4.95 ± 0.75 vs. 1.46 ± 1.07) (PRM1 in HF vs. LF bulls was Limousin (621.44 ± 219.37 pg/mL vs. 374.00 ± 257.71 pg/mL), Holstein (257.11 ± 54.28 pg/mL vs. 166.44 ± 38.28 pg/mL), and Ongole Grade (749.22 ± 199.30 pg/mL vs. 197.11 ± 71.20 pg/mL) (The relative gene expression and protein abundance of LF bulls . The mea ± 1.07) B. Protei0 pg/mL) C.p < 0.01), viability (p < 0.01), and plasma membrane integrity (p < 0.01) (p < 0.01) . Additionally, the results of the PRM1 assessment also confirmed it at the mRNA and protein levels, with the high relative expression of genes and protein abundance indicating linearity with an increase in the %CR (PRM1 gene expression (p < 0.01) and protein abundance (p < 0.01) was also shown in all bulls (The fertility score (%CR) in each bull breed in the study showed a positive correlation with semen quality parameters such as PM ( < 0.01) . A negat < 0.01) . The res the %CR . A closell bulls .Fertile bulls are determined by the ability of a bull to produce sperm capable of fertilizing the oocytes  and havep < 0.01)  . Moreovep < 0.05) between HF and LF bulls  in LF bulls than in HF bulls (p < 0.05) than on HF bulls  with %CR for each breed of bull  [PRM, arginine, which is rich in guanine-cytosine (CMA3−), abundant in PRM, would inhibit the dye CMA3 [3 as an indirect assay of PRM content in sperm associated with SDF and fertility is widely used [Several factors cause spermatozoa DNA fragmentation, such as hormones, age, infection, increased reactive oxygen species, chemical/toxic exposure, smoking, drugs, testicular hyperthermia, apoptosis, and protamine deficiency during spermatogenesis . As prev3 method . The gua (CMA3+) . In sperdye CMA3 ,63. Howeely used ,66,67,68PRM deficiency (p < 0.01) than HF bulls (p < 0.000) shown between CMA3+ and the %CR in all breeds of bulls was used in this study  in HF bulls than in LF bulls in all bull breeds during this study  in HF bulls compared to LF bulls in all breeds of bulls    and line< 0.009)  were alsLF bulls . AdditioLF bulls  reportedPRM deficiency or aberrant protamination of sperm resulting in LF conditions or even infertility cases is associated with several plausible causes, including disruption of transcription, translation, and synthesis processes [PRM translation control may lead to PRM inadequacy in infertile males [PRM dominates 85% of the sperm nucleus protein, is a cause of PRM aberration that causes male infertility [PRM haploinsufficiency in transgenic mice reportedly impacts changes or disruption of spermatogenesis and result in infertility [PRM, PRM2, must have a similar ratio as PRM1, and a ratio that deviates at the protein and mRNA levels, will impact infertility [PRM1 in bulls is the only PRM variant that plays a direct role in the normal function of fertile sperm [PRM1, both at the mRNA and protein levels, was the most critical PRM variant in normal sperm function in bulls.The condition of rocesses ,70,71,72le males . Additioertility . In otheertility . Howeverertility ,77,78,79le sperm ,13. Gangle sperm  and Pardle sperm  also proPRM1 in sperm remains not fully understood [PRM1 resulting from the abnormal function of transcriptional regulators, translation, or post-translational modifications affect PRM and various genes during spermatogenesis. Therefore, it is not surprising that PRM can be a “checkpoint” during spermiogenesis, and abnormal PRM expression will impact the rate of apoptosis [PRM1 in normal spermatozoa function and its relationship to fertility in bulls, especially bulls used for AI programs in Indonesia. As found in this study, PRM1 proved to be closely related to fertility, with bulls still classified as subfertile or LF, even though the semen quality was conventionally categorized as suitable for the AI program. Moreover, the combination of assessment with a gene or protein analysis approach with semen quality assessment, including SDF assessment, can be the best choice in determining the fertility of bulls.Nevertheless, specifically, the regulation and mechanism of derstood  necessarderstood  stated tpoptosis . Increaspoptosis . HoweverPRM1 shows to be a promising candidate in terms of fertility markers. Furthermore, to the best of our knowledge, this is the first work in Indonesia to demonstrate correlations in the mRNA expression and protein content of sperm PRM1 from HF compared to LF bulls. Moreover, it is not only in one breed of cattle but also represents commodity groups of cattle breeds in Indonesia, such as exotic beef cattle (Limousin), dairy cattle (Holstein), and local cattle breeds (Ongole Grade).In conclusion, inadequate or low sperm chromatin protamination at both mRNA and protein levels was associated with defects in sperm chromatin condensation, found with increased SDF, and coinciding with a decrease in the %CR (fertility score) in bulls. Therefore, considering that this research aimed to find candidate fertility markers that accurately determine bulls’ fertility and can be used in the bull selection process, especially in Indonesia,


Liquorice is a traditional medicine. Triterpenoids such as glycyrrhizin and glycyrrhetinic acid are the main active constituents of liquorice. Studies have revealed that these compounds exert inhibitory effects on several viruses, including SARS-CoV-2. The main mechanisms of action of these compounds include inhibition of virus replication, direct inactivation of viruses, inhibition of inflammation mediated by HMGB1/TLR4, inhibition of β-chemokines, reduction in the binding of HMGB1 to DNA to weaken the activity of viruses, and inhibition of reactive oxygen species formation. We herein review the research progress on the antiviral effects of glycyrrhizin and its derivatives. In addition, we emphasise the significance of exploring unknown antiviral mechanisms, structural modifications, and drug combinations in future studies. Liquorice has been documented as a herbal medicine in ancient medical books of China, India, and Greece and has been used for thousands of years . PrimariGlycyrrhizin   and glycHerpes virus is one of the most common viruses infecting animals and humans. More than 90% adults have been infected with at least one or more types of herpes viruses, and the viruses usually establish a latent infection . Some hein vivo and in vitro studies, researchers found that 500 μM CBX and 300 μM CCX have anti-HSV-1 and anti-HSV-2 (MOI = 5) activities, and thus can significantly inhibit virus replication and reduce the number of infectious virus particles by 10,000–100,000 times. Glycyrrhizin inhibits HSV-1 replication in vitro, and its IC50 is 0.5 mM  = 5], and this effect was found to be irreversible . Subsequs 0.5 mM . Sekizaws 0.5 mM .Cellular SUMOylation processes are the proposed targets for antiviral therapies. in vitro, which leads to its accumulation in the cytoplasmic vacuoles of the Golgi apparatus, changes in the intracellular transport of HBsAg, and inhibition of its sialylation in a dose-dependent manner (Glycyrrhizic acid exhibits direct anti-hepatitis virus activity and low toxicity in host cells . It has t manner . Sato et5 copies of HCV infection) by 50% at a concentration of 7 ± 1 μg/ml. Additionally, its inhibitory effect increased when it was used in combination with interferon. In addition to interferon (TM (SNMC), protects mitochondria by reducing oxidative stress during HCV treatment in transgenic mice  through the HMGB1-TLR4 signalling pathway. This protective effect is related to a substantial reduction in IL-17 and IL-22 levels rather than the direct inhibition of intracellular virus replication (A recent study reported that glycyrrhetinic acid could inhibit the release of high-mobility group box 1 (HMGB1), block the cytokine activity of HMGB1, and significantly improve the liver inflammatory injury induced by mouse hepatitis virus (1 × 10lication .In vitro experiments by The effect of glycyrrhizic acid on influenza virus was reported in the early 1980s. 50). The results revealed that compared with EW alone, glycyrrhizic acid (10 mg/kg body weight) in combination with EW  produced significant antiviral effects, which alleviated lung oedema and inflammatory cell infiltration. Use of glycyrrhizic acid in combination with ribavirin, a broad-spectrum antiviral drug, significantly inhibited lung consolidation in mice infected with H1N1 influenza virus. A combination of 50 mg kg−1 d−1 glycyrrhizin and 40 mg kg−1 d−1 ribavirin was found to provide 100% protection to infected mice (5 times LD50 of influenza H1N1 virus infection), suggesting that the combination of ribavirin and glycyrrhizin has a potential clinical value  infection after the outbreak of SARS in 2003. SARS-CoV-2 is a novel coronavirus, and the coronavirus disease 2019 pandemic caused by SARS-CoV-2 was named coronavirus disease-2019 (COVID-2019) by the World Health Organisation (WHO) . SARS-Co50 of SARS-CoV-2) by inhibiting the viral main protease Mpro  replication in Vero E6 cells in a dose-dependent manner and exhibited no significant cytotoxicity . Glycyrrase Mpro . Using c50, 1 × 10−1.5) in vitro (50), and DG was found to have a strong inhibitory effect on PPV when the virus was treated before incubation. DG also demonstrated its antiviral activity against Marc-145 cells infected with porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV could be effectively controlled through inhibition of viral replication and N gene expression and through reduction of apoptosis (Glycyrrhizin has demonstrated antiviral activity against some animal viruses. in vitro . Li et apoptosis . Studiespoptosis . By usinpoptosis . Inhibitpoptosis .Glycyrrhizin and its derivative protect against many viruses through their antiviral activity . The maiCOVID-19 has become a pandemic. Although vaccines have been approved, they have not been widely used and no specific treatment for COVID-19 is available to date . GlycyrrStructural modification of natural products is one of the approaches to discover novel drugs. Through structural modification, the potency and solubility of natural products can be increased, pharmacokinetic properties such as absorption and distribution can be improved, and side effects can be eliminated or reduced . Many arin vivo results and clinical evidence, Isbrucker et al. recommended that the acceptable daily intake of glycyrrhizin is 0.015–0.229 mg/kg body weight/day (The United States Food and Drug Administration, Council of Europe, and Joint FAO/WHO Expert Committee on Food Additives have allowed the use of liquorice extract and glycyrrhizin in food . Althougight/day . This ra


Despite the fact that the chikungunya viral infection is a neglected disease, complications such as hemorrhagic fever, arthritis, and lymphopenia remain a health concern. The aim of this study was to determine the prevalence of the chikungunya virus in the Southern Region, Saudi Arabia. Enzyme immunoassay and polymerase chain reaction have been compared between samples.Forty samples from two southern hospitals in Saudi Arabia were collected between December 2019 and February 2020 and screened for chikungunya virus IgG antibodies and for viral RNA. Selection criteria were based on hematological parameters and rheumatological profiles such as rheumatoid factor, c-reactive protein, anti-nuclear antibody, and anti-cyclic citrullinated peptide (anti-CCP) of out-patients.One confirmed case of chikungunya virus was detected using the ELISA test. However, no viral RNA was detected in any of the samples. This suggests that the virus is cleared rapidly in patients.Chikungunya is a neglected viral disease in Saudi Arabia. Future work should focus on detailed investigation of this viral infection and its vectors. Togaviridae family and alphavirus genus that is transmitted by mosquitoes, and is considered a neglected tropical disease [Arboviruses are arthropod-borne viruses that can be transmitted from ticks and mosquitoes to humans and animals. Chikungunya virus belongs to the  disease . The Jaz disease .Flaviviridae and Togaviridae, cause hemorrhagic fever which is known as “bone-breaker fever” [Arboviruses, which include the families r fever” . In Saudr fever” . Moreover fever” . IgM andr fever” . Other rr fever” –11.Aedes species have not been reported at the highlands of the Asir region. Nevertheless, the endemicity of dengue virus has been confirmed only in the Tihamah part of the Asir region (Tihāmat Asīr) which is similar to the Jazan region in terms of climate conditions [Aedes species in this area. The most significant human viral diseases including dengue, chikungunya, and zika are mainly transmitted globally by Aedes aegypti and Aedes albopictus. Therefore, controlling the Aedes species using insecticide and providing risk maps with vector populations will help to reduce the arboviruses transmission [Mosquito diversity and abundance vary between the Asir and Jazan regions due to the huge climatic and geographical variation between the two regions , 13. In nditions , 15, whismission . To the smission , 17. TheAedes aegypti mosquitoes thrive. The collection was conducted in the winter season between December 2019 and February 2020 where cases of hemorrhagic fever dominate. At Asir Central Hospital, 30 samples were selected after they had been sent to the laboratory based on the physicians’ requests for rheumatological profiles  of out-patients). Those patients were clinically diagnosed with rheumatoid arthritis. At Baish General Hospital, samples from patients with nasal bleedings who were admitted to the hospital were selected.To increase the chance for detection of an arbovirus that has not been extensively studied, inclusion criteria should focus on proper sample selection. The most common clinical presentations of arboviruses including the chikungunya virus are joint pain and bleeding. Abnormal platelet and white blood cells (WBCs) are other important laboratory profiles. Serum samples from arthritis patients and patients with hemorrhagic fever were collected from different altitudes: 30 serum samples from Asir Central Hospital and 10 patients from Baish General Hospital in the Jazan Region  was then added. After one hour of incubation time, chikungunya virus biotinylated antibodies were added, incubated, and washed, followed by the addition of streptavidin peroxidase conjugate. Tetramethylbenzidine (TMB) is catalyzed by the peroxidase enzyme. Absorbance was measured using a spectrophotometer at 450 nm wavelength using FLUOstar Omega , and the color is directly proportional to the captured anti-chikungunya virus IgG antibody. Data and figures were presented using GraphPad Software, version 8, .Viral nucleic acid was extracted using ABIOpure Viral, version 2.0, DNA/RNA Extraction kit, #M561VT50, AllianceBio, USA. This uses advanced silica-binding technology to purify viral RNA that binds to the silica membrane, and finally the eluate was collected in a clean tube. The protocol was performed using 10 μl proteinase K, 200 μl sample, 7 μl carrier RNA to enhance binding of viral nucleic acid to the spin column membrane, 200–400 μl of the intended buffer, and 20 μl nuclease-free water.The following materials were obtained from meridian Bioscience™, or otherwise stated. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the genome of chikungunya virus in blood samples. The forward primer AAGCTCCGCGTCCTTTACCAAG and the reverse primer CCAAATTGTCCTGGTCTTCCT  were synAedes mosquito thrives. They both showed abnormal low WBC and platelet counts  in which ten samples were collected with abnormal CBC, and the second group was from Asir General Hospital  who suffered from arthritis, in which 30 samples were collected. Only one sample was positive for the anti-chikungunya IgG antibodies, and one equivocal sample of duplicate tests. These two samples were from the first  group in which the All samples were screened to detect chikungunya virus RNA following the viral extraction protocol, cDNA synthesis, and PCR. Gel electrophoresis was then prepared at a concentration of 2% agarose gel using UltraPure™ Agarose, Invitrogen, 1X TAE buffer, and ethidium bromide (Sigma). Samples were loaded with DNA loading buffer and with HyperLadder 100 bp . No bands were observed for any samples. This suggests that the viremic state of the chikungunya patient is very short.During the winter season, patients were provisionally diagnosed with dengue fever virus, but no detection kits specific for arboviruses were available at the hospitals’ laboratories that time. Patients with low WBC and low platelet count were selected from Baish General Hospital. Another cohort from Asir Central Hospital was included in the screening, using samples from patients who suffered from arthralgia. Another criterion for inclusion was the positivity of anti-CCP and rheumatoid factor (RF). These markers aided in the diagnosis of rheumatoid arthritis patients .Aedes mosquito breeding; the vector of the dengue and chikungunya virus. Although hemorrhage is considered a rare complication in chikungunya infection, the upsurge of hemorrhagic fever cases in Jazan during the breeding period of mosquitoes led us to investigate the prevalence of chikungunya infections in arthritic and hemorrhagic fever patients.While the dengue virus caused severe morbidity and mortality in Saudi Arabia, the Southern Region is at risk of other arboviruses due to outbreaks in neighboring Yemen –24. DengPatients’ sera were screened for the presence of specific IgG antibodies to chikungunya virus. Platelet and WBCs count of seven patients from the first group were below the normal range. However, only one patient was found to be positive for anti-chikungunya IgG antibodies. In addition, sera were tested for the presence of the viral E gene. However, results were all negative using RT-PCR. We believe that the virus was rapidly cleared by the host immune system. Having negative results for PCR suggests that the virus is characterized by a short viremic state. Infected patients clear the virus after one week of chikungunya infection .Finally, it is important to note the limitations. Selecting specific samples that fit the inclusion criteria at the peak of infection from different locations would be challenging. Viral hemorrhagic cases are soared only in certain periods throughout the year. This drawback limited our ability to satisfactorily increase the sample size. Because the likelihood of detecting the chikungunya virus was low, and because PCR results were negative, the control sample size was moderate. Following initial chikungunya infection, the virus is shed after approximately one week. Therefore, the detection probability of viral nucleic acid would be restricted. The study is limited by the small sample size; however, samples from two different hospitals were collected. Patients might have arrived at the hospital after a week of the infection, and therefore, molecular detection of chikungunya was challenging and not possible, at least during our study. It was not possible to test IgM. Instead, molecular detection of the viral RNA was attempted to detect the acute phase of the chikungunya virus. While the background noise of the ELISA test seems somewhat high, the cut-off value (COV) was set as a basis for the results interpretation. COV average  was higher than the negative control (0.287). Most samples were within the negative control range.Aedes species within selected areas. To avoid a large impact of any conclusion drawn from the small samples, a large sample size should be collected for future investigations.Out of 40 samples, one confirmed case of chikungunya virus was found after detecting anti-chikungunya IgG antibodies, but PCR showed negative results in all tested samples. Detection methods for the chikungunya virus are not applied routinely, and the virus might be circulating in regions where mosquitoes are abundant. Arboviruses such as the chikungunya virus should be considered a priority with health implications. There have been reports of death due to bleeding. A call to conduct extensive studies on such neglected infections is indeed required. Chikungunya is a neglected viral infection, and future work should focus on investigating the endemicity of arboviruses within the vector by performing a field work collection of


Alouatta) and led the state to declare a Public Health Emergency of State Importance, despite no human cases reported. In this study, near-complete genomes of yellow fever virus (YFV) recovered from the outbreak were sequenced and examined aiming at a better understanding of the phylogenetic relationships and the spatio-temporal dynamics of the virus distribution. Our results suggest that the most likely sequence of events involved the reintroduction of YFV from the state of São Paulo to RS through the states of Paraná and Santa Catarina, by the end of 2020. These findings reinforce the role of genomic surveillance in determining the pathways of distribution of the virus and in providing references for the implementation of preventive measures for populations in high risk areas.The 2021 re-emergence of yellow fever in non-human primates in the state of Rio Grande do Sul (RS), southernmost Brazil, resulted in the death of many howler monkeys (genus  Yellow fever (YF) is a viral hemorrhagic fever caused by the Yellow Fever Virus (YFV), the prototype of the genus Flavivirus, family Flaviviridae . In SoutIn Brazil, the area of YFV occurrence extends from the Amazon region, northern Brazil, to the state of Rio Grande do Sul (RS), the southernmost region of the country. The latter is sporadically affected when the virus overflows from the endemic North Region to the Southeast and South Regions in expansion waves with irregular intervals of time ,4,5,6,7.Among South American NHP, the genus Alouatta is particularly important for YF surveillance, since its members are most severely affected by YFV ,10,11,12MG/SP) and the “Yellow Fever Virus Minas Gerais/Espírito Santo/Rio de Janeiro” (hereafter YFVMG/ES/RJ) [Altogether, that YF outbreak affected ten states plus the Federal District outside the Amazon region, leaving behind about two thousand human cases and countless NHP deaths. That outbreak was monitored by real-time genomic surveillance , and theG/ES/RJ) .Alouatta guariba clamitans) within RS. In the present study, we report the complete sequence of YFV genomes recovered from NHP in the state of RS and establish its phylogenetic relationship with viral lineages YFVMG/SP and YFVMG/ES/RJ recovered from outbreaks reported in other regions of Brazil.Since the virus entered Santa Catarina, RS’s neighboring state, in 2019 , RS statThis study comprised analysis of routinely collected surveillance data performed by the state and municipalities health departments and followed the guidelines of the Ministry of Health of Brazil and the Brazilian National Committee for Ethics in Research. All samples were obtained from dead NHP. This study followed the guide to epizootic surveillance in non-human primates and entomology applied to yellow fever surveillance  and InstAll NHP samples were collected in municipalities in the Northeast Region of the state of Rio Grande do Sul, Brazil. Samples from liver, kidney, lung, spleen, and heart were collected in the field from dead animals and kept refrigerated (4 °C) or frozen in dry ice (−78 °C) and dispatched to the central office of the State Health Department, where they were stored at −80 °C. All collections followed biosafety protocols and were in accordance with the state YF surveillance strategy carried out by the Environmental Health Surveillance Division, State Center of Health Surveillance, State Health Department of RS, and the Ministry of Health of Brazil . Data co® classic filter paper . A hole punch was used to excise a single dried 6 mm diameter circle from the dried paper to avoid carryover contamination, the punch was disinfected with bleach, water, and ethanol 100% [YFV RNAs were extracted from NHP tissues (liver and kidney) samples spotted on Whatman FTAnol 100% . The matnol 100% . Positiv® dsDNA System assay kit on a Quantus™ Fluorometer . DNA library preparation was performed using the Ligation sequencing kit SQK-LSK309  and the Native barcoding kit . The sequencing library (22 samples and a negative control per run) was loaded onto a R9.4 flow cell  and sequenced between 6 to 18 h using MiNKNOW software . The RAMPART  package was used to monitor coverage depth and genome completion. The resulting Fast5 files were basecalled and demultiplexed using Guppy . Variant calling and consensus genome assembly were carried out with Medaka  using the sequence JF912190 as the reference genome.All samples that met the previous criteria were submitted to cDNA synthesis protocol using LunaScript™ RT SuperMix Kit  following the manufacturer’s instructions. Then, a multiplex tiling PCR was performed using the previously published YFV primers  and 40 cn = 359, sequences > 8 kb excluding sequences from vaccine and patents). A subset of sequences belonging to recent (2015 to 2021) extra-Amazonian region waves, including clades YFVMG/ES/RJ and YFVMG/SP , were selected. Metadata such as sample collection date and geographic coordinates were retrieved from GenBank files or gathered manually from genome associated publications. The genomes recovered here (n = 22) combined with YFV-set-1 were aligned with MAFFT v.7.480 [https://nextstrain.org/ncov, accessed on 23 August 2021). The new genome sequences were sent to the NCBI GenBank database under accession numbers MZ712127 to MZ712149.To perform phylogenetic analysis, we selected from NCBI all near-complete YFV sequences  and epizootics  of YF in Brazil were provided by the Ministry of Health of Brazil . The totAlouatta guariba clamitans and it became evident that, at the date of collection of the last sample sequenced, the virus remained in circulation in the state for at least 10 weeks, with epizootics occurring mainly in the Northeast Region of RS, near to the border with Santa Catarina state (Between January and 08 March 2021 (date of collection of the last sample whose sequence was recovered here), 78 epizootics were notified to the RS State Health Department and Ministry of Health of Brazil surveillance, with sample collection performed in 42 cases (54%). From these, 34 (81%) tested positive for YFV by RT-qPCR assay. All confirmed YFV-positive NHP were na state .Twenty-two near-complete YFV genomes were generated from liver samples from dead NHP collected in eight municipalities of RS. Sequenced samples displayed median Ct values of 12 (range 8–20) at RT-qPCR . PhylogeMG/SP sub-lineage, named Yellow Fever Virus Minas Gerais/São Paulo/Rio Grande do Sul (hereafter YFVMG/SP/RS), revealing that the origin of these isolates is São Paulo. Furthermore, the time-scaled phylogenetic tree  and sequenced.MG/ES/RJ, which circulated in the east of Minas Gerais in 2016, entering the west of Espírito Santo and following to the north of the state of Rio de Janeiro. From there, in 2017, the virus spread southwards, arriving at the border with São Paulo in 2018 [MG/SP, circulated in the west of Minas Gerais and the northwest of São Paulo in 2016–2017, from where it advanced towards the south and east of that state, reaching the most densely populated region of the country between 2017 and 2018 [MG/SP . These findings suggest that this sub-lineage had moved through the three states of the South Region of Brazil.In Brazil, over the past six years, previous genetic analyses have indicated the circulation of at least two distinct sub-lineages, which spread in two different transmission routes. The first one was YFV in 2018 ,30,37,38 in 2018 . Meanwhiand 2018 ,40. Betwand 2018 ,22,23. DPrevious records of occurrence of YF in RS showed that the northwestern border of RS with Argentina has historically been the first affected area for YF cases in NHP, as happened in 1947, 1966, 2001, 2002, and 2008/2009 ,36,41,42Interestingly, the sequences obtained here represent virus samples of the farthest place reached by YFV dispersion in Brazil, since the spread from the endemic area, approximately in 2014, in this expansion wave. Rio Grande do Sul is the southernmost limit for NHP distribution in the Americas  and the Several environmental and ecological issues must be addressed to better understand the dynamics of virus dispersion. Epidemic outbreaks in the sylvatic cycle of YFV demand a high density of competent vectors and the presence of susceptible NHP, acting as amplifiers for the virus ,14,36,44In the present report, we examine the likely origin of YFV strains that were recovered from NHP in RS, the southernmost state of Brazil, in the current YFV wave which was found to be heading south within the country, as opposed to previous outbreaks in the state that had been introduced via Argentinian border. Phylogenetic analyses revealed that such sequences are related to the lineage that occurred in São Paulo during YFV circulation in 2017–2018. However, further studies of the virus dispersion including ecological, climatic, and anthropogenic factors associated with the disease cases are needed to improve the predictive power, allowing rapid decisions into surveillance and prevention efforts. Additional studies shall be conducted to compare eventually available YFV genomic sequences from specimens collected in the states of Paraná and Santa Catarina to provide further evidence of the dissemination of the virus within the country. Real-time genomic surveillance must be continued in order to allow deeper understanding of the factors driving the transmission and dispersal of the Yellow Fever Virus in NHP.In Brazil, YF vaccine is freely offered by the Brazilian Unified National Health System , as part of routine immunization, but the adherence in the target population (from 9 months to 59 years of age) is unequal, mainly when not accompanied by special communication of risk to society and health professionals, which leads to a very heterogeneous vaccine coverage (VC) across the country . The areThis report highlights the importance of capacity building for inter-institutional exchange of data and human resources, to strengthen epidemic surveillance and outbreak management during pandemics. Additionally, our study represents a model for YFV response that reinforces the need to bring together epidemiology, surveillance, vaccination, and genomic tracking.


Through the motion of the layers and the surface tension in the liquids, reagents move from top chambers to bottom chambers and mix homogeneously. A tip in the top layer with a mobile permanent magnet moves the immune complexes comprising the magnetic beads, virus particles, and QD2 between the bottom chambers. In this way, our automated device achieves a highly sensitive magnetic bead-based sandwich immunoassay for the influenza A H1N1 virus within 32.5 min. The detection limit of our method is 5.1 × 10−4 hemagglutination units, which is 2 × 103 times more sensitive than that of the conventional hemagglutination method and is comparable to PCR. Our device is useful for the rapid and sensitive detection of infectious diseases in point-of-care applications and resource-limited environments.Preventing the rapid spread of viral infectious diseases has become a major concern for global health. In this study, we present a microfluidic platform that performs an immunoassay of viral antigens in a simple, automated, yet highly sensitive manner. The device uses silica particles embedded with highly bright quantum dots (QD Virus detection on microfluidic platforms can be useful to prevent and control large outbreaks of infectious disease . Nucleic2), which has 500 times stronger photoluminescence than individual QDs , two linear stepper motors  were used. To control the rotation of the bottom layer, a stepper motor  was used. The layer motions were coded in open-source software  and uploaded on a microcontroller  to control the three stepper motors automatically. For the excitation of QD2 fluorescence, a UV LED  with a wavelength of 380 nm was positioned with an aspheric lens  at the side of chamber B5 . A filter with wavelength of 620 nm  was fixed on the top layer as a detector filter and its center wavelength was designated depending on the emission wavelength of QD2s fluorescent (625 nm). Details of the device components can be found in To control the vertical motion of the top layer and the permanent magnet [3 (φ) × 15 (L) mmamber B5 . The QD26-fold dilution) by comparison with the negative control group without virus. In viral hemagglutination assay, one HAU is the amount of virus that will agglutinate 50% of the red blood cells in the standard agglutination assay  [B) of the blank solution plus three times the standard deviation (SD): IB + 3SD. The blank solution was PBS solution. We used CdSe@ZnS QD to make QD2 for high sensitivity immunoassay platform. MBs and QD2 were fabricated according to the protocol of our previous study [Virus samples comprising influenza A  and influenza B (FluB/Yamagata: Massachusetts/02/2012 Yamagata lineage) were kindly provided by the Korea Institute of Radiological & Medical Sciences. The amount of virus was quantified using a hemagglutinin assay. We added the FluA/H1N1 virus in an erythrocyte solution with twofold dilution sequentially. The titer value was determined to be 64 hemagglutinating unit (HAU) (2f virus) . The buff virus) . LOD evaus study . More de2 were formed in B3. First, the tip transferred the complexes of MB–virus from B2 to B3. Next, the antibody-conjugated QD2s were moved from T2 to B3 by the contact between T2 and B3, and the complexes of MB–virus–QD2 were formed in B3. By its repeated vertical motion, T2 enhanced the formation of the complexes in B3. In Step 4, the complexes were washed in B4 to reduce the background signal. For this step, the tip moved the complexes from B3 to B4. Subsequently, instead of using the tip to enhance the washing process of the complex in B4, we additionally used T3, which is a hollow cylinder. This is because T3 showed better mixing efficiency than the tip , where DT and DB are the diameters of the top and bottom chambers, respectively. After the contact of the two chambers, liquid remained in the top chamber when DT/(DB − DT) < 1.9. However, for DT/(DB − DT) ≥ 1.9, the liquid was completely transferred to the bottom chamber (FL/FU) at the moment when the top chamber rises up after the contact, where FL (FU) is the surface-tension force that attracts down (up) the liquid meniscus at the bottom (top) side of the top chamber  and agitation only for approximately the first 6 s to ensure an initial homogeneous suspension (Case I2). 2 to chamber B4, the tip agitated chamber B4 only for 6 s to homogenize the suspension. When we used the viral suspension, the agitation slightly decreased the fluorescence intensity in Step 5 by 4% regardless of the washing conditions  and MBs was successfully applied on a device with movable layers. The movable-layer device enabled multiple fluid manipulation steps including fluidic transport, mixing, and MB manipulation without any on-chip valves and pumps in a simple and precise manner. With the use of QD2, the automated device had a high level of sensitivity and specificity for influenza A H1N1 virus detection within 33 min. The proposed device, which provides a high level of sensitivity and specificity for influenza A H1N1 virus detection in a fully automated manner, could help in the implementation of point-of-care testing. Currently, users need to apply reagents to the device. However, with the improvement of the device design, reagents could be preloaded and stored in the device to facilitate its use. With the improved level of sensitivity and turnaround time of the immunoassay, the proposed fluid manipulation technique can be applied to a wide range of immunoassay applications.Sandwich immunoassay using highly bright multi-QD embedded particles (QD


Escape variants can cause new waves of COVID-19 outbreaks and put vaccination strategies at risk. To prevent or delay the global spread of these waves, virus mobility needs to be minimised through screening and testing strategies, which should also cover vaccinated people. The costs of these strategies are minimal compared to the costs to health, society and the economy from another wave.