Yet, current Ca2+-alginate formulations do not keep EPN vigorous and infectious for a prolonged period of time and do not allow for their controlled release upon application. Here, we introduce solid Ca2+-alginate beads which we supplemented with glycerol to better retain the EPN during storage and to ensure a steady release when applied in soil. Glycerol-induced metabolic arrest in EPN (Heterorhabditis bacteriophora) resulting in quiescence and total retainment of EPN when added to beads made with 0.5% sodium alginate and 2% CaCl2·2H2O solutions. More than 4,000 EPN could be embedded in a single 4–5-mm diameter bead, and quiescence could be broken by adding water, after which the EPN readily emerged from the beads. In a field trial, the EPN beads were as effective in reducing root damage by the western corn rootworm  as EPN that were applied in water. Although further improvements are desirable, we conclude that Ca2+-alginate beads can provide an effective and practical way to apply EPN for the control of WCR larvae.Entomopathogenic nematodes (EPN) have great potential as biological control agents against root-feeding insects. They have a rapid and long-lasting mode of action, minimal adverse effects on the environment and can be readily mass-produced. However, they have a relatively short shelf-life and are susceptible to desiccation and UV light. These shortcomings may be overcome by encapsulating EPN in CaThe online version contains supplementary material available at 10.1007/s10340-021-01349-4. The full potential of entomopathogenic nematodes (EPN) as biological control agents has yet to be achieved.Main obstacles include short shelf-life, susceptibility to desiccation and UV light, and untimely release.We formulated an alginate bead supplemented with glycerol to induce EPN quiescence.The beads can retain more than 4,000 nematodes each, and quiescence can readily be broken by adding water.In a field trial, EPN beads effectively reduced root damage by the western corn rootworm.Entomopathogenic nematodes (EPN) have been shown to be highly effective biological control agents with unique advantages that chemical and other biological pesticides do not have. EPN can kill insect pests within days after infection; tens to hundreds of thousands of new EPN may be produced from a single infested insect host and travel to infect new hosts, thus theoretically allowing them to persist in the field throughout the growing season  do not feed and use their stored energy reserves until they find an insect host. Therefore, the viability and infectivity of IJs decrease over time as energy reserves are depleted. This limits the shelf-life of EPN products to maximally a few months even when properly stored Grewal . They arConsiderable research effort has gone into the development of suitable EPN formulations with the aim to prolong storage, easy transport and handling, efficient application and enhanced post-application survival  and biomicrobial pesticides   instead2·2H2O to determine which combination would allow the optimal EPN release within 24 h. In addition, we increased the number of encapsulated EPN to more than 4,000 per bead, instead of 100–300 as previously reported , a univoltine chrysomelid beetle, whose larvae feed on maize roots and can cause tremendous yield losses , which have been repeatedly reported to be effective against WCR  larvae that were purchased from a local fishing shop as previously described  were used for the field experiment. The strain was originally obtained from the US Department of Agriculture–Agricultural Research Service (USDA-ARS) North Central Agricultural Research Laboratory in Brookings  and maintained in the USDA-ARS Plant Genetics Research Unit in Columbia .A strain of non-diapausing western corn rootworms (WCR) 2·2H2O from Fluka , glycerol from Sigma-Aldrich  and blue food dye from Schneitter . We used deionized water for all formulations.Sodium alginate, Gluco®  and xanthan were purchased from Solé Graells , CaCl2+-alginate EPN capsules were produced at room temperature as described by Kim et al. (in vacuo. IJs of H. bacteriophora were harvested from water in cell culture flasks onto a Whatman filter paper (Ø 60 mm) using a Buchner funnel and were re-suspended in the mixture of Gluco®, xanthan, glycerol and blue dye in a 15-ml Falcon tube at a concentration that ensured that each capsule would contain ca. 200 EPN. The solution was stored at 12 °C overnight and the next morning, after EPN quiescence was confirmed under an optical microscope , the EPN-containing mixture of Gluco®, xanthan, glycerol and blue dye was loaded in a 1-ml disposable syringe  from which the barrel had been cut off, resulting in an orifice of Ø 4.5 mm. Droplets of ca. 90 μl of the mixture were dropped into 20 ml of alginate–glycerol solution in a 50-ml beaker and gently and continually stirred for 20 min. The EPN capsules that were formed were rinsed in 18% glycerol, and each stored in a Ø 60-mm disposable petri dish half immersed in 18% glycerol solution at 24 °C in the dark. As controls, we also produced ten glycerol-free EPN capsules, which were stored under the same conditions, but immersed in water without glycerol. After a week, the number of EPN that had escaped from each type of capsules was counted (n = 10).Glycerol-containing Cam et al.  except t2+-alginate beads were manufactured according to the modified method of Kaya and Nelsen  were prepared and degassed in vacuo. IJs of H. bacteriophora were harvested from water in cell culture flasks onto a Whatman filter paper (Ø 60 mm) using a Buchner funnel, re-suspended in alginate–glycerol solution and stored at 12 °C overnight. The next morning, after quiescence of EPN in the alginate–glycerol solution was confirmed under an optical microscope , the EPN-alginate–glycerol solution was dripped from a 1-ml disposable syringe  into a container with CaCl2-glycerol solution. Up to 100 droplets per 100 ml of CaCl2-glycerol solution were added, and 10 min later, Ca2+-alginate beads were collected. The volume of each droplet of alginate–glycerol solution was 85.3 ± 3 μl on average (n = 20). Different from the capsules, the alginate beads floated on the surface and did not stick to each other in the solution. Stirring of the solution during bead formation was therefore not necessary and it was possible to produce as many beads in a single container as the solution surface allowed. Glycerol-free EPN beads were produced under the same conditions.Glycerol-containing Cad Nelsen . Solutio2∙2H2O solutions at concentrations ranging from 0.3 to 2% (w/v). Approximately 200 EPN were encapsulated in each bead. EPN beads were individually placed, half immersed in water, in a Ø 60-mm petri dish and stored at 24 °C in the dark. After a week, the number of EPN that escaped from each bead was counted under an optical microscope (n = 24).EPN were harvested from water, re-suspended in a series of sodium alginate solutions at concentrations ranging from 0.5 to 2% (w/v) and dripped into a series of CaCl2+-alginate beads containing on average 172, 430, 859, 1,718 and 4,295 EPN were produced under the influence of 18% glycerol as described above  and individually stored in a 3-ml glass vial half immersed in 300 μl of water with 18% glycerol. The vials were kept in a 500-ml plastic container at 24 °C in the dark. After a week, each bead was transferred into a 20-ml cell culture flask  containing 5 ml of water in order to break quiescence. An additional week later, all the EPN that were liberated from each bead were individually counted under an optical microscope (n = 10 per EPN dose).We also tested whether the nematodes indeed escape from the beads when their quiescence is broken by adding water. Twelve Ca2+-alginate capsules that stick together and sink to the bottom in the sodium alginate solution  at the Neuchâtel Platform of Analytical Chemistry . For the ICP-AES analyses, we used argon 5.0, nitrogen 5.0 and compressed air at 15 ml min−1 for plasma, 0.5 ml min−1 auxiliary and 0.65 ml min−1 for nebulizer, respectively. Samples were injected at 1.5 ml min−1. The concentration of Ca2+ ions in the samples was determined using the PlasmaCAL-SCP33MS standard solution  prepared in 2% HNO3. For comparison, concentration of Ca2+ ions was also measured in 2% and 1.5% CaCl2·2H2O solutions. One ppm manganese solution in 2% HNO3  was measured prior to each run to ensure overall suitability of the system.Different from CaHeterorhabditis bacteriophora was purchased from Arbico Organics .A series of field experiments to compare the efficacy of EPN beads produced as described above were carried out in a maize field at the Bradford Research Center of University of Missouri . Seeds of the WCR-susceptible maize cultivar P1420Hr CRM 114  were planted in the field on May 16th, 2014, and about 800 WCR eggs in 0.01% agar solution were applied in the soil next to each plant two weeks later. early application) or (ii) on June 11th in the middle of the growing season when WCR larvae were feeding on the roots (late application) or (iii) on the both dates (early and late application). In other plots, we used a conventional application of EPN in water solution on the same two dates of early and late applications. Empty beads, as the bead control, were also applied twice on the same dates. The blank plots received neither WCR eggs nor EPN, and the WCR control plots were treated only with WCR eggs.EPN beads were freshly made as described above and were applied in the soil (i) on May 22nd when the first maize seedlings started to show and WCR larvae had not yet hatched from the eggs . During application, the solution was agitated frequently to prevent EPN settlement at the bottom of the container. For the bead application, 4 K EPN beads, as well as the control beads were produced with 18% glycerol as described above a day before application, and ten beads were dropped into the two holes, five beads per hole  for normality and equal variances, and means were tested for significant differences using one-way ANOVA test or Kruskal–Wallis test, accordingly. Multiple comparisons of data were conducted using post hoc Tukey’s test when χ2 = 6.1688, p = 0.013, df = 1, n = 10, N = 20). When we embedded the EPN in solid alginate beads with 18% glycerol, the EPN were also completely retained and quiescence was maintained until the glycerol was diluted with an excess of water.After overnight treatment in an 18% glycerol solution, all EPN were found dormant the next morning. When encapsulated as described by Kim et al.  and stor2∙2H2O solutions were below 0.5% and 0.3%, respectively. We therefore manufactured alginate beads (with about 200 EPN) from solutions with concentrations of sodium alginate and CaCl2·2H2O ranging from 0.5 to 2.0% and 0.3 to 2.0%, respectively, initially without glycerol. Beads formed from 0.5% of sodium alginate solution in CaCl2·2H2O solution of any concentrations between 0.3 and 2.0% were not spherical as shown in Fig. 2·2H2O solution over 2% to avoid salinity stress. Photographs of EPN beads were taken right after production, and a week later, the number of EPN that had escaped from each bead was counted    managed to escape after the beads were placed in water, prompting us to try to find a way to enhance the escape rate. We first tested if this could be achieved with larger numbers of EPN per bead, expecting that the higher numbers of EPN would facilitate movement through the matrix.About 82% of the EPN escaped from beads that had been manufactured without glycerol, as described above. In contrast, when we added 18% glycerol to similar beads, only half of the EPN , and the mean volume of a droplet of all the EPN-alginate solutions was 85.9 ± 0.7 μl (mean ± SE). Thus, the number of EPN in the beads manufactured from these solutions was estimated to be 172, 430, 859, 1718 and 4295, which we simply refer to as 0.2 K, 0.5 K, 1 K, 2 K and 4 K EPN beads, respectively. Only 0.17, 0.37, 0.33, 0.51 and 0.52% of the EPN escaped on average from these beads, respectively, during the 7 days that the beads were stored in the presence of glycerol. The 4 K EPN beads were completely filled with EPN, but the beads still maintained a solid and round form . The concentration of Ca2+ ions in 1.5% and 2% CaCl2∙2H2O solutions was 3.78 mg ml−1 and 5.21 mg ml−1, respectively. After the first production of 100 Ca2+-alginate beads in the 2% CaCl2∙2H2O solution, the concentration of Ca2+ ions decreased to 4.63 mg ml−1. After the second production of 100 beads, the concentration of Ca2+ ions decreased to 4.08 mg ml−1, and after the third production of 100 beads, the concentration of Ca2+ ions decreased to 3.68 mg ml−1. This final concentration is almost the same as the starting concentration of Ca2+ ions in a 1.5% CaCl2·2H2O solution (3.78 mg ml−1). Based on these results, we calculated that about 1.13 mg ml−1 of Ca2+ ions were consumed per 100 Ca2+-alginate beads.Different from CaTo test the efficacy of the novel bead formulation under realistic conditions, we carried out a field experiment with 4 K EPN beads and tested how their application may reduce root damage by WCR larvae in a maize field in Missouri, USA. Because it took 8–16 h for EPN to start waking up from the glycerol-induced quiescence , the beads were already transported in water to the field where they were then buried in the soil.p < 0.05; χ2 = 66.0252, df = 6, p < 0.0001, n = 16; N = 112)  Fig. .Fig. 4Ef2+-alginate EPN beads that can house and liberate as many as 4,000 EPN per 90-μl bead. EPN incorporated in these beads were stored without refrigeration in a state of glycerol-induced quiescence. It is known that that EPN can be conserved like this for at least 6 months without affecting infectivity , seeking conditions under which EPN are best retained in the formulation. We had improved the retainment of EPN inside these capsules by about 40%. However, for an effective control of root pests, the release of EPN out of the formulation into the soil is as important as retaining them inside during storage. The initial capsule formulation was also problematic because it relied on dripping droplets of sodium alginate solution into CaCl2·2H2O solution, whereby the capsule shell grows outward and the growing capsules readily stick to each other  allow the release of most of the embedded EPN. In previous studies, spontaneous escape of EPN out of Ca2+-alginate formulations was not considered , and can, with some modification, be directly applied with the seeds during sowing. Further investigation into the dynamics of EPN release from the beads under a variety of growing conditions is warranted.In summary, in this study we present a new formulation of Ca+-alginate beads over time (within 16 h) after the beads were placed in water . (PPTX 44 KB)The number of EPN that emerged from 4K EPN CaBelow is the link to the electronic supplementary material.


P = < 0.001, 95% CI = 1.55–1.77), bovine trichomonosis status of the herd , and bulls ≥3 years old  were identified as risk factors associated with Campylobacter fetus venerealis infection. We also studied the high-risk areas for circulation of the infection in extensive beef cattle herds in Spain, showing four significant clusters in “dehesa” areas in the south-western provinces of the country and a fifth cluster located in a mountain area in northern Spain. The results obtained in the present study indicate that BGC is endemic and widely distributed in Spanish beef herds. Specifically, “dehesa” herds are at greater risk for introduction of Cfv based on relatively high local prevalence of the infection and the use of specific management practices.Bovine genital campylobacteriosis (BGC) is a sexually transmitted disease that causes early reproductive failure in natural breeding cattle that are managed extensively. The aim of this study was to assess the BGC prevalence in Spain from 2011 to 2019 using data collected cross-sectionally from the diagnostic reports issued by the SALUVET veterinary diagnostic laboratory from a total of 5,182 breeding bulls from 1,950 herds managed under “dehesa” systems  or mountain systems . Infection was detected by PCR in 7.7 and 12.2% of the bulls and herds tested, respectively. The “dehesa” herd management system (OR = 2.078,  Bovine genital campylobacteriosis (BGC) is a sexually transmitted disease (STD) listed by the World Organization for Animal Health (OIE) and is considered a major cause of early reproductive failure in natural breeding cattle that are managed extensively, placing important restrictions on the international trade of animals and animal products , 2. In bCampylobacter fetus subsp. venerealis (Cfv), a microaerophilic, Gram-negative and motile bacterium with a characteristic spiral form  , which colonizes the intestine. Cff transmission occurs mainly through the fecal-oral route, followed by transient bacteraemia, during which, in pregnant ruminants, the agent can translocate to the placenta, resulting in placentitis and abortion  , and bots (Cfvi) , 13. Theabortion , 15. Theabortion , 16–18.BGC is mainly controlled by diagnostic testing, reporting and culling of infected bulls. Antibiotic treatment (streptomycin or oxytetracycline) can be successful in bulls under 3 years old, but it is often not effective in older bulls, thus culling is recommended , 19. UnfCfv detection, among breeding bulls from different areas of Spain. The prevalence of BGC was estimated at animal and herd levels, and potential risk factors for the presence of Cfv in Spanish herds were identified. The spatial distribution of the infection in the different Spanish provinces was also analyzed to highlight areas of elevated risk.The spatial distribution pattern of BGC correlates with areas where cattle are managed under extensive conditions and natural breeding of cattle is used , 20–23. The targets of this survey were bulls used for natural mating (age ≥15 months) that were subjected to BGC diagnosis by the SALUVET veterinary diagnostic laboratory and originating from farms located in the areas where more than 70% of the Spanish extensive beef cattle population is concentrated . Data weCfv and herds in which at least one bull tested positive for Cfv infection were considered positive for BGC. The apparent bull/herd prevalence of Cfv infection was established from the proportion of positive bulls/herds to the total number of bulls/herds tested during the study period. Confidence intervals of 95% (95% CI) for proportions were obtained using the exact binomial method. The true prevalence of BGC was estimated based on the sensitivity (97%) and specificity (100%) results previously described  was sent to the veterinarians to demonstrate how to perform preputial scraping to collect smegma samples, as well as to provide specific guidelines for bull sampling  and sample handling  to optimize analytical sensitivity. Collected smegma material was deposited in a 5 ml tube with phosphate-buffered saline  and sent to the SALUVET laboratory at room temperature for analysis.SALUVET laboratory provides services for bovine STD diagnosis  to private veterinary practitioners. Sampling was performed by preputial scraping of bulls in the field  by veterCfv amplification, PCR was performed as previously described  according to the manufacturer's instructions. For escribed . AmplifiCfv by PCR was determined by assaying replicates of serial 10-fold dilutions of Cfv DNA, resulting in a detection limit of 26.4 genome copies/μL. The analytic specificity of the assay was verified with a collection of 12 C. fetus strains (4 Cff and 8 Cfv) and 10 non-fetus Campylobacter species kindly provided by the Faculty of Veterinary Medicine  and the “Laboratorio Central de Veterinaria” , both Reference Laboratories for Animal Campylobacteriosis, and SALUVET laboratory collection  and BT herd status (negative or positive).Quercus spp.- woodlands and a typical landscape with a mosaic of croplands, grasslands and scrublands) are located in central, western and southern Spain  and the selected explanatory variables, both at herd-level  or at animal-level  were initially screened using Pearson's chi-square or Fisher's tests, as appropriate. Variables with P-values < 0.20 in the bivariate analysis were further scrutinized for associations using Spearman's rank correlation coefficient (r) to avoid collinearity problems. When collinearity (P < 0.05 and r > 0.4) occurred, only the variable more clearly linked to Cfv infection was retained. The next step involved a multiple logistic-regression model using a non-automatic backward selection of variables. Two independent models were constructed to determine the risk factors, one at herd-level and one at animal-level. A forward introduction of variables was used, starting with the variable with the lowest P-value in bivariate analysis. At each step, the confounding effect of the included variable was assessed by computing the change in the odds ratios (OR). Changes in the OR >30% were considered indicative of confounding. The model was rerun until all remaining variables presented statistically significant values  and a potential relationship with the response variable existed. The fit of the models was assessed using a goodness-of-fit test (Associations between the presence of fit test .P < 0.05) using a Bernoulli model  checklist .A total of 5,182 breeding bulls were analyzed between January 2011 and December 2019. Bulls younger than 15 months old and bulls from artificial insemination centers were discarded. The number of bulls tested increased from 2011 to 2013  to 2017–2019  . Data abThe bulls and herds analyzed in this study originated from 33 out of 50 Spanish provinces. The number of bulls and herds sampled was representative, corresponding to 5.5% (5182/93928) and 4.1% (1950/46761) of bull and herd population from these areas, respectively. The sampled bulls had a mean age of 4.2 (± 2.1) years old, with 74.9% of the bulls ≥ 3 years of age. There were 23 different breeds represented in the survey , with LiP = 0.124) was observed in the bull prevalence, ranging from 6.6% (50/761) in 211–2013 to 8.5% (193/2260) in 2017–2019  and 12.2% , respectively. No significant time-dependent increase , herds from the “dehesa” system (P < 0.001), herds with more than 100 cows (P < 0.001) and herds with positive BT status (P < 0.05). In the multivariate analysis, the variable “bull breed” was excluded due to collinearity with the variable “type of management” . The final multiple logistic-regression model  and in herds with a BT-positive status . Cfv infection rates were significantly higher in bulls ≥ 3 years old than in animals < 3 years old .The results obtained from bivariate analysis  showed ton model  showed tCfv-positive bulls in the different Spanish provinces is shown in Cfv prevalence , showing an endemic character, since the number of positive breeding bulls remained fairly constant throughout the study period. Information about the situation of BGC in Europe is very limited. Analysis of the veterinary laboratory data provides a potential opportunity for BGC monitoring at the national scale, but the information should be interpreted cautiously, since there are always concerns over how well the sample population reflects the true population, because of the lack of randomization of the analyzed samples. This is due to the increased likelihood that herds from which samples are submitted for testing could be herds where there is already a higher suspicion of infection (herd with reproductive disorders), which could result in overestimating BGC prevalence. However, more than the half (54.3%) of the herds submitting bulls showed no indication of reproductive problems in the herd of origin. Consequently, we consider that the error related to the voluntary participation in this sampling would be almost negligible. Moreover, although available BGC prevalence data are scarce in the literature, our data are also consistent with estimates from previous cross-sectional studies from Argentina, Nigeria or Tanzania, where the bull prevalence ranged from 2.4 to 13.3% , but Cfv was not detected by either culture or PCR . A previous study also reported that the specificity of PCR targeting the parA gene  could even increase in the future, due to the current trend  toward more extensive and traditional farming systems. In view of our results, more studies should be undertaken to investigate the situation of bovine STD in other European countries where natural breeding of beef cattle is common and there are no monitoring programmes. We also propose the adoption of validated and uniform tests for BGC diagnosis to ensure that results are fully equivalent among diagnostic laboratories.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.LO-M and EC-F conceived the study and participated in its design. PH, NP-F, and PV-A performed the sample analysis and the PCR tests. NP-F, EC-F, PV-A, DC-R, and BP-A prepared the database. DC-T and IG-B carried out statistical analyses and interpreted the results. NP-F and EC-F wrote the manuscript, with interpretation of results, material and methods and discussion inputs from DC-T, IG-B, PH, and LO-M. All authors contributed to the article and approved the submitted version.This study was supported by several research projects (RTA2017-00076-00-00 and PLATESA2-CM P2018/BAA-4370) and the grant for doctoral formation (Pre2018-086113 funded by MCIN/AEI/10.13039/501100011033 and FSE) awarded to PhD student NP-F.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Magnesium (Mg) is essential for animal health. Low Mg status (hypomagnesaemia) can be potentially fatal in ruminants, like cattle and sheep, and is widespread in Europe with economic impacts on farming. The application of Mg-rich agricultural lime products can help to ensure pasture forage consumed by animals contains sufficient Mg and, in areas of low pH, has the dual benefit of reducing soil acidity to levels best suited for grass production. This aim of this study was to determine if Mg-rich lime products could be used in a more effective manner in agricultural production systems. Potential resources of carbonate rocks  in the UK, and their Mg:Ca status were identified, using datasets from the British Geological Survey (BGS). These data were combined with the locations of agricultural lime quarries, and areas where soils are likely to be deficient in Mg and/or require liming. Areas of potential demand for Mg-rich agricultural lime include areas in south east Wales, the Midlands and North East England. Although, areas where this may be an effective solution to low soil Mg values are restricted by the availability of suitable products. Conversely, areas of low soil pH in England and Wales are often found close to quarries with the ability to supply high Ca limes, suggesting that the low rates of lime use and liming is not due to supply factors. This study provides information that can help to guide on-farm decision making for use of Mg-rich and other lime resources. This could be used in conjunction with other options to reduce risks of Mg deficiency in livestock, and improve soil pH. Hypomagnesaemia is likely to be responsible for lower-productivity and diminished well-being in more animals in a herd compared to those displaying acute symptoms, given that herds/flocks generally receive a common diet3. If Mg deficiency could be prevented, it would be of benefit to both animal welfare and economic productivity. Recent research has confirmed that whilst hypomagnesaemia is commonly reported by UK farmers, the reported use of preventative measures is low, and the use of pasture interventions is lower still4. Pasture interventions can include the application of Mg-rich fertiliser or lime products, or selection of sward species with a propensity to take-up elevated Mg concentrations5.Magnesium (Mg) deficiency (hypomagnesaemia) in ruminant livestock is a serious issue for the agricultural sector and accounts for a significant number of animal deaths annually. It is caused by a diet deficient in Mg, or due to an imbalance in the supply of Mg in comparison to other mineral cations9 and that there is little evidence for any changes in pasture soil Mg concentrations through time10. The magnesium content of soil relates to that of the bedrock, where it is high in the bedrock it is high in soil and vice versa. Thus, the composition of all pasture and farm-grown fodder will be influenced by this natural environmental endowment as well as pasture management decisions.One aspect of dietary supply is the geographic control of pasture and farm-produced fodder. It is known that total Mg and plant-available Mg concentrations in soil are controlled by geological and geographic factorsw), consistent with standard agronomic laboratory practice in the UK11. Grassland mineral soil is recommended to be maintained at pHw ≥ 6.0 in Britain13. In Ireland, where grass-clover pasture is more widely practiced, a pHw threshold of 6.5 is recommended14. However, multiple lines of evidence exist that indicate pasture soil is frequently below these pHw recommendations. Private sector on-farm sample data summaries from the UK consistently show pH typically below recommendations in pasture soil: the most recent annual data synthesis reports 57% of grassland soil with pHw ≤ 5.99, and 27% with pHw 6.00–6.498. This is consistent with systematically collected public sector data across the north of Ireland, where 84% of pasture samples were below the clover-grass recommended threshold of pHw 6.515.A key pasture management activity is that of soil pH—which here is reported as measured in water  and can also be inferred from commodity production statistics. Production can be regarded as a good proxy for consumption since due to its high bulk and low price it is not exported in significant quantities.26, which representatively samples fertiliser and lime use across the British farming sector. This captures information on lime use in three geological material categories as used in arable and pastoral systems, as well as use of sugar beet lime and ‘other’ options. Sugar beet lime use is very low on grassland ; ‘other’ categories are generally on a par with Mg-lime, but more detailed liming characteristics are not reported. Figure 27. The use of agricultural lime has continued on a declining, or flat trend, likely due to reluctance to engage in soil treatments that are seen to be costly and a lack of knowledge over its potential benefits.The BSFP, is an annual Department for Environment, Food and Rural Affairs (DEFRA) surveyFigure 15. Although this relationship is complicated by factors such as the removal of the lime subsidy in 1970s.The low use of lime on farms and the low current supply of agricultural lime suggest that currently pastoral land is being under-limed, and this is entirely consistent with the generally low pH of pasture soil reported28. Agricultural lime has a much smaller market share, less than 5% of the total sales of limestone28.Agricultural lime is any calcium (Ca) or magnesium (Mg) carbonate rich form of crushed rock  that is applied to soil in order that the high Ca, or Ca + Mg, concentration raises and neutralises the pH of the soil. Individual lime products have different neutralising values, for example, burnt lime which is ground to a fine powder will release CaO into the soil much more rapidly than coarse crushed limestone rock due to its increased solubility. However, the vast majority of agricultural lime in the UK consists of ground limestone or chalk due to the higher bulk and considerably lower prices of these materials when compared to processed products. UK limestones are principally valued for their use as construction aggregates 29.Carbonate rocks are distributed in discrete areas across England and Wales Fig. , account3) whereas dolostone is composed mainly of magnesium carbonate in the form of the mineral dolomite (CaMg(CO3)2). These carbonate rocks can contain variable proportions of other carbonate minerals and non-carbonate impurities, mainly in the form of siliciclastic, clay and other accessory minerals30. British limestones can be broadly differentiated in their physical–chemical properties according to the geological era in which they were deposited.Carbonate rocks that constitute raw materials for agricultural lime comprise limestone and dolostone. Limestone is a sedimentary rock composed mainly of calcium carbonate  is also an important carbonate rock, outcropping between Newcastle and Nottingham. Jurassic limestones also occur extensively across Dorset, Northamptonshire, Lincolnshire and the North York Moors. Older limestones of Ordovician, Silurian and Devonian ages are also present across England and Wales to lesser extents. The broad outcrop of these limestones is shown on Fig. 31.The Cretaceous Chalk is typically softer than other limestones, with lower levels of non-carbonate minerals and thus high Ca contents: this can be advantageous for liming as it is easy to work, crush and will effectively neutralise acidic soils. Jurassic limestones typically are heterogeneous with high levels of impurities, and can be associated with layers of mudrocks and siltstones, but they can also be thickly bedded and relatively soft. Permian limestones, like Jurassic limestones, are known for being soft and lithologically heterogeneous, but they are principally comprised of dolomite. The Carboniferous Limestone is formed of much more competent (stronger) rocks and these are therefore valued principally for their aggregate properties. They can be chemically very pure, or associated with interbedded muds and silts, and they can also be variably and locally dolomitised. Older Ordovician, Silurian and Devonian limestones occur in much smaller areas and have variable properties but have proved to be important resources on a local scale and are predominantly utilised for aggregate applications rather than agricultural limeThis study aims to develop simple spatial approaches, for England and Wales, to aid in the decision making process around the application of agricultural limes. Specifically, whether soil pH improvement by liming may be a cost effective solution and whether it may be beneficial to lime using high Mg materials for both pH benefits and to protect livestock against hypomagnesaemia. These approaches are fully documented to ensure they can be replicated elsewhere.This study aims to assess the potential for the supply and demand for agricultural lime in general, and Mg-rich agricultural lime in particular, through the analysis of existing data.To identify the relevant datasets the analysis was split into two aspects; supply and demand. The supply side data focuses on the current state of the industry and the location of resources. Whereas the demand side looks at geographic areas of potential need for liming materials.35. This is updated on an annual basis licensed by the British Geological Survey but active sites are available to viewer via the GeoIndex data viewer (https://mapapps2.bgs.ac.uk/geoindex/home.html). This relational database was queried using search terms for 'Active Sites' and the following end uses: 'agricultural industry end use'; 'lime' and 'agricultural lime'.Data for the location of sites producing agricultural lime was compiled using the BritPits database, a comprehensive database of mines and quarries in the UK36. This dataset, however, does not separate carbonate rocks suitable for agricultural lime from carbonate rocks that are used for aggregate or other industrial purposes. Thus the data were modified, based on a geological review, to best represent the spatial distribution of resources suitable for agricultural lime as an end use. The geological information was therefore attributed with chemical data to understand their potential as high Mg or Ca feedstock materials using Harrison et al.9 ‘Appraisal of high-purity limestones in England and Wales, part 1 resources’ which reports the average Ca and Mg values of carbonate rocks in England and Wales. This publication is focused on high specification industrial uses of limestone, but the results are also applicable to agricultural lime applications. A significant caveat with using this approach is that these sedimentary rocks can be complex and very heterogeneous. Small changes in the depositional environment during formation of the deposit can lead to large variations in rock type and the physical and chemical properties such as its neutralising value or Ca:Mg ratio, which is important when considering high Mg limes. Therefore, the chemical values used for this study represent an average for a particular formation and could vary substantially locally. All chemical values were categorised according to EU guidance of the specifications required for different grades of material29.Compositional data for lime products is not in the public domain, and generally considered confidential by the quarrying industry. Chemical data for lime products was therefore extracted from legacy data reports and geological mapping data. It was assumed that the specification of material sold would be the same as the composition of the surface bedrock geology which was being extracted using mapped information at 1:50,000 scale for England and Wales34. More problematic are limestones that are either very variable in nature (often with high siliciclastic sediment contents) or have been variably dolomitised. These units include the heterogeneous Permian aged Cadeby and Brotherton Formations which occur in a north–south band across central and northern England as well as variably dolomitised Carboniferous limestones in south-western and north-western England, as defined by BGS geological mapping data34. Generally, most other carbonate rocks, including all Cretaceous Chalk, have low-Mg values (< 2%) and primarily comprise calcium carbonate.The geological review shows that simple rules could be applied for many geological formations to categorise according to the chemical composition. Permian dolomitised limestones have some of the highest Mg values of all carbonate rocks in the study, commonly exceeding 20%. Dolostone formations from the Carboniferous Limestone have similarly high Mg contents. Both these groups could be directly attributed using BGS 1:50,000 geological mapping32 and price per tonne is the most significant factor in determining transport distances. However, in reality, the supply chain will be considerably more complex, because value can be added for lime through contracts for both soil pH testing and lime spreading, as well as supply. Transportation can also be affected by factors such as utilising return loads from other bulk haulage. A broad outline of the flows of agricultural lime from quarry to farm are outlined in Fig. Due to its low cost per tonne, transport can form a substantial part of overall costs, potentially limiting how far material is transported. No data is available for agricultural lime transport distances. Typical and maximum distances to market for aggregate was used as a proxy because they are a similar low price—high volume material. Whilst imperfect it would appear the prices for both can be similar taking price statistics for aggregates37. This will almost certainly now be higher, as can be seen by rising average transport distances for aggregates, where smaller operations have been closed and mothballed as the industry has consolidated38, and the same change is also true of the agricultural lime industry . Therefore, the average distance of 45 km38 was taken, and a maximum transport distance of 80 km was applied in this study.The most comprehensive industry consultation to date reported 60 km as the maximum transportable distance by road for aggregates39. This dataset only includes A and B roads and motorways which was judged sufficient resolution for this study and the fact that heavy goods vehicles avoid the use of minor roads. Different road types were not, however, differentiated in the analysis as no cost data was available for travel on different road types.The road network for the study area was taken from the freely available official dataset OS OpenRoads41 which we use as the framework in this study.Information on the demand for agricultural lime in different areas of the UK requires a spatial understanding of where grasslands may have pH values that indicate liming would be beneficial, and where those may coincide with low plant-available soil Mg concentrations. If pasture or arable land requires liming, how much material may be required can be calculated using a relationship between soil texture, soil pH and land use. This relationship has been used to created calculators Table  that wil33 downloaded from the UK Soil Observatory (UKSO) platform42, which records land use derived from spectral analysis of Landsat-7 imagery, reported by a 25 m × 25 m raster grid. Different categories of grassland are recorded: Acid grassland; Arable and horticulture; Calcareous grassland; Heather grassland; Improved grassland and Neutral grassland. Only improved grassland and arable and horticulture classes were selected for analysis here. In the case of other grassland categories, such as rough grasslands and upland grassland areas, it was considered unlikely that improvement via liming would be feasible or appropriate.Areas of pasture were defined using the LandCover Map Fig. , LCM201543  dataset43 Fig. . The est45. The numerous categories for soil texture were condensed into the five categories which are defined by lime requirement calculators41 as shown in Table Soil texture affects how agricultural lime will break down and deliver calcium and magnesium to the soil. It is therefore is critical to understand whether liming may be required and if so what quantity of material is needed. Data for soil texture was derived from the BGS Soil Parent Material Model (SPMM) Fig. , which wThe ‘All’ category used in the soil parent material maps was problematic as this could encompass a variety of soil types, generally either peats or siliclastic sediments, these however could be differentiated using the grain size attribute in the dataset to ensure the correct soil texture was attributed. The data from the SPMM also made it impossible to differentiate between the two classes of organic soils defined by the lime requirement calculators. These were therefore combined into one class for organic soils and peat. Very few areas (less than 1%) of arable land or improved grassland correspond to areas of organic soils and peat as defined by the LCM2015 data and the SPMM, so these assumptions make little impact to the final dataset. After the mapping exercise was complete, a subset dataset was created using the classification system used by the lime requirement calculators.6 produced spatial estimates of plant-available soil Mg and the risk of Mg deficiencies in soil based on geostatistical analysis of two soil geochemical datasets  version 10.3.1 was used for all spatial analysis. The Network Analysis Tool was used to measure distances, from quarries used as input facilities in the analysis (or subsets of these based on predicted Mg or Ca contents of products), along the road network (OSOpenRoads) used as the Network Dataset. To calculate areas which are likely to have a demand for agricultural lime datasets for land cover, pH, Mg deficiency and soil texture were linked with the spatial join function, this functionality was also used to link quarry data to geochemical properties.2 (74%) of the study area was classified as a land use with potential for pasture that may be suitable for liming, however, much of this will be arable land as opposed to land for grazing. Data for soil pH also played a critical role as no liming would be required if the soil pH was already at recommended levels. Data for pH did not cover the entire study area, some areas had no values attributed to them, these were, however, mainly urban areas so did not have a large impact on the spatial analysis (only 0.2% of land use suitable for liming had no pH data that could be associated with it). The CEH pH dataset showed soil pHs in land suitable for liming ranges from 6–8 with the majority around or just over 6. After integration of all datasets spatial analysis shows that 18% of the study area may be both suitable for and require liming with Ca liming material and 8% with Mg liming material.A critical component of this study was to identify areas of demand for liming materials and if these areas could also benefit from increased Mg soil concentrations. This was done by defining areas where the application of agricultural lime was predicted to have a beneficial impact on crop yields. This required continuous data for England and Wales for land use, soil pH, soil texture and soil Mg levels as described in the “46 and Carboniferous dolostones principally caused by post diagenetic alteration from hydrothermal fluids47. The geological history is relevant here as it indicates that the former will be almost entirely dolomitised (Mg-enriched) and the latter only partially dolomitised.The supply factors considered for agricultural lime included the location of production sites, composition of the products and market penetration. In total 96 potential supply sites were identified . Of these only 14 have the potential to produce high Mg lime and 29 have the potential to produce high Ca lime. These sites, like limestone resources, are widely spread across England and Wales. High Mg sites were restricted to the Permian Magnesian Limestone Formation due to pervasive dolomitisation which occurred in these rocks soon after formation, likely due to recirculation of fluids in a restricted basin environmentFigure The result of the network analysis using the supply and demand information is shown in Fig. 5. This is the case for many areas that have Mg deficient soils, as highlighted by areas that may be benefited by the application of high Mg limes in Fig. This spatial analysis shows that for many areas the distance from source to market for Mg-rich liming material is substantial so other methods of Mg intervention need to be considered, for example by application of processed Mg additives or planting of Mg-rich forageThis spatial analysis also shows areas where soil Mg vales are already high and, if liming were to be used to treat soil pH, the use of low Mg, high Ca material may be beneficial to avoid deleterious Mg levels, which can, in some circumstances, inhibit the uptake of other soil nutrients. For example around the outcrop of the Permian magnesian limestone in the Midlands and North East England. For some of these areas the nearest quarry supplying high Ca limes may be up to 80 km away, making sourcing limes of the appropriate chemistry a concern. There are very few areas in England or Wales that are outside the potential supply range of quarries that can supply high Ca liming materials. This is due to the widespread nature of limestones and a well-developed extractive industry, as shown by Fig. 25. These types of land cover may be protected, or may be valued for their specific biodiversity as a unique function of soil chemistry. For these reasons the inclusion of upland grass areas was not considered useful for an accurate representation of areas that may benefit from liming and justified specifically excluding them. Although in some cases the application of lime to upland areas can also have positive environmental impacts48 and these will need to be assessed on a case by case basis. The exclusion of upland grassland areas was a simple process as they are separately classed, "Heather grassland" and "Acid grassland", in the land use dataset used.This study only considers improved grassland and arable land with low pH. In many upland grass areas with acidic soils soil treatment is impractical, may cause harm to delicate grassland ecosystems and is uneconomic as a low maintenance/low yield approach is more appropriate19. It is also important to consider negative environmental effects of the use of liming products. There is little data regarding different footprints of crushed rock agricultural lime vs more processed products, however lime quarries are generally small operations, with minimal processing requirements. The CO2 emissions of the use of agricultural lime are relatively small due to reactions during the breakdown of lime, which sequester CO249.This study only deals with crushed and ground liming products that comprise untreated quarried and crushed geological material. There are also a wide range of other products that have been processed in some form, i.e. pelletised lime as well as products derived from non-geological materials, such as sugar beet waste. The market dynamics of these products are in no way comparable to ground lime due to higher costs, due to processing, although the overall effects of these products to soil parameters are broadly similarDue to limited data availability, transport distance used in this study rely on many assumptions. In reality market factors may lead to a very variable end price where transport distances may exceed those of the raw material. How far the material will travel is dependent on a complex interplay of local markets (e.g. utilising return loads from other freight traffic) and the nature of contracts between end users and suppliers which may include testing and mitigation for a range other soil improvement measures. There are examples of Mg limes being transported, by road, several hundred miles from northern England to Scotland, however these are exceptional . Although not factored into figures used in this study, due to lack of data, there is evidence that Mg limes will have greater transport distances when compared to low Mg alternatives due to its ability to increase soil Mg indices increasing its value .It is labour and resource intensive to identify and treat areas of low pH and Mg and so there would seem to be substantial benefits in tools for preliminary assessments of soil improvement interventions. The simple tools developed by this study show how integration of the many already available and easily accessible spatial datasets and legacy survey data can be developed into such decision making toolkits. Such integration of datasets can also add to the understanding of the supply and demand dynamics of agricultural lime in England and Wales, ensuring that the most realistic and appropriate resource management strategies are applied. It is hoped that provision of such toolkits can aid in ensuring that soil treatment techniques are used to their maximum potential for enhancing productivity.High Mg and Ca agricultural limes may have wider market use than is currently realised, as shown in farm-practice and trade statistics data. This analysis specifically shows that, in certain areas, the use of high Mg lime for soil treatment is a viable possibility for the duel benefit of altering pH and increasing soil Mg contents. Also shown is the wide distribution and existing supply chain for high Ca limes, these products are readily available for the vast majority for lowland grasslands in England and Wales. This approach also opens up the opportunity to compare the likely usefulness of liming as a tool to reduce the risks of hypomagnesaemia, in conjunction with the assessment of other approaches which can be used on-farm. Pasture management options should always be selected after analysis of soil and/or forage to ensure an appropriate course of action is taken given local conditions. This analysis shows how it is possible to create useful relevant new datasets that can benefit new users from the huge range of existing, publicly available, legacy datasets, that may not always appear accessible to potential users outside their immediate domain, e.g. geological sciences.


Grapevine virus T (GVT) is a recently described foveavirus, which was identified from a transcriptome of a Teroldego grapevine cultivar in 2017. Recently, we surveyed vineyards and rootstock plantations in Hungary using small RNA (sRNA) high-throughput sequencing (HTS), at a time when GVT had not yet been described. A re-analysis of our sRNA HTS datasets and a survey of grapevines by RT-PCR revealed the presence of GVT in most of the vineyards tested, while at rootstock fields its presence was very rare. The presence and high variability of the virus in the country was confirmed by sequence analysis of strains originating from different vineyards. In this study, we demonstrate the presence of GVT in Hungary and show its high diversity, suggesting that GVT presence may not seriously affect grapevine health and that it could have been present in European vineyards for a long time as a latent infection. Grapevines serve as the basis of the wine industry, and the table grape is an agronomically important plant. Many chemicals are used to protect these vines from pathogens, but without an efficient plant protection method against viruses they will still be exposed to them. Grapevines are able to be infected by more than 80 viruses or viroids .While traditional serological tests and RT-PCR can only detect the presence of the investigated virus, new high-throughput sequencing-based metagenomics methods can disclose the presence of all pathogens in a sample. High-throughput sequencing (HTS)-based virus diagnostics of grapevines is widespread and could lead to the characterization of the viromes of vineyards  and vineGrapevine virus T (GVT) is one of the viruses discovered by HTS in recent years. The original description of this new foveavirus dates back to 2017, when re-analysis of published grapevine transcriptome data revealed its presence in an Italian Teroldego cultivar . The avaRecently, we surveyed Hungarian vineyards and rootstock fields for the presence of viruses using small RNA (sRNA) HTS ,14. WhenOur deposited datasets in the NCBI GenBank GEO, GSE106240 and GSE130994, contain sRNA sequence data from 18 samples representing 15 vineyards, and samples representing 17 certified rootstock fields and two rootstock collections, respectively . We usedFrom the sequenced sRNA reads we built up longer contigs using a de novo assembler of CLC Genomic Workbench  software , and searched for GVT-specific contigs. For BLASTn analysis, we used the sequence of the retracted reference genome .Small RNA reads (both without and with redundancy) were directly mapped to the GVT reference genome. In the latter case, we calculated a normalized RPMR (number of redundant virus specific reads/million sequenced reads). Consensus sequences of the sRNA reads were prepared (extract consensus function of CLC) and, with the classical sequence analysis function of CLC, the coverage of the genome by virus-specific sRNAs was calculated as a percentage .The cDNAs were synthetized using random primer and the RevertAid First Strand cDNA kit  from the RNA of individuals or the pooled RNAs representing the vineyards (the same RNA which was used to prepare the sRNA sequencing libraries). The PCR amplification was done by Q5 DNA polymerase (New England Biolabs), using two different sets of primers . PrimersTo investigate the phylogenetic correlations of the GVT variants present in the country, we cloned the amplified 904 bp long product to a pJET vector . One clone from each positive vineyard was Sanger sequenced and their sequences were deposited into GenBank , both with and without redundancy.To determine whether GVT is present in Hungary, we re-analyzed our sRNA HTS data generated in surveys of vineyards and rootstock plantations ,14. To oDuring our previous surveys, we found that the presence of the virus could be validated later if, besides the presence of a virus-specific contig, the RPMR was higher than 200 and the coverage of the genome was higher than 60% ,14. In tDuring our previous surveys, we found that detection of another foveavirus, grapevine rupestris stem pitting-associated virus (GRSPaV), the closest relative of GVT, failed using sRNA HTS in most cases. The number of GRSPaV-specific sRNAs was very low in the infected plants ,14, whilThe results of the amplification using the RNA pools of the vineyards revealed the presence of GVT in more samples than the sRNA HTS did  Figure .In addition to 9_SZHT, 13_BV, and 18_MK7, we detected a virus-specific product in 1_TK, 2_PH, 4_PP, 5_CS, 8_ET, 10_EH, 12_DF, 14_MK1, and 15_TC. Results with different primers correlated quite well, but in 12_DF and 18_MK7 we obtained the product only when amplifying a longer product .To reveal how widespread the infection is at different vineyards, sampled vines were individually tested for the presence of GVT . This teThe low coverage of the GVT genome did not allow us to use the sRNA datasets to see how different the isolates were, and is why cloned, amplified RT-PCR products from each field were sequenced. The variants present in Hungary showed high variability, 82–89% nucleotide and 92–96% amino acid identity with the Italian Teldorego isolate, suggesting their different origins . PreviouDF, EH, and MK1 are vineyards in Tokaj, in the northeast part of the country, planted with Furmint, an ancient Hungarian cultivar . The DF Re-analysis of RNA samples from our previous surveys revealed high prevalence of GVT in the country. Based on studies on GRSPaV, foveaviruses have no identified vectors  and coulVitis sylvestris species in the wild flora could survive. Their virome can reveal information about the presence of endemic viruses. Investigation of the virome of a Vitis sylvestris population in Switzerland revealed the presence of a foveavirus, grapevine foveavirus A (GFVA–MN553040) [GVT was only described recently, and it can be present in grapevines without any visible symptoms. Its European origin is highly probable, as it has been identified only in European cultivars. The vineyards of Europe were almost totally replanted after phyloxera epidemics. However, N553040) . GFVA isHigh-throughput sequencing techniques offer the possibility of gaining sequence information about previously unknown viruses. A recent paper, investigating the biological information in publications describing new viruses of fruit trees, revealed a big gap in their biological data, including host range studies and symptom descriptions, which could affect their correct risk assessment . This isLatency and the symptomless nature of GVT infection, together with high variability of the genome, result in uncertainty in the viral diagnosis. The question of regulating GVT is open, but more information is needed about how GVT affects its grapevine host to make a sensible decision.


Temporo-mandibular joint (TMJ) joint and the condyle of mandible are observed in the radiographs of the skull and the jaw. Therefore, it is of interest to assess the predictability of four different shapes of condyle in skeletal class I, II and IIImalocclusion. The four commonly visualized shapes are oval, bird beak, diamond and crooked were assessed using an ortho pantomogram (OPG). Each of the malocclusion was visualized for different shapes of the condyle. 987 OPGs were radiographically evaluatedand the morphology of 1974 condylar heads was visualized. The shapes of the condyles were grouped under four different types. Data shows that oval shaped condyle was most common followed by bird beak. There was variability in the diamond and crooked shape andwas lesser than the other types. Thus, the shapes of the condyle are useful predictable guide in deciding the nature of the occlusion. Temporomandibular joint (TMJ) is a ginglymoarthroidal joint that is formed by articulation between two bones, the condyle part of mandible and glenoid fossa at the base of the skull . The variation and the pattern noticed among individuals in the morphologicalvariations in the condyle and the fossa has now become an area of research . The treIn literature there are various studies that have assessed the various condylar morphology. There is difference in condyle morphology in various malocclusions. There are variations in male and female with men having a larger condyle than woman. The malocclusionthat has a major change in the condyle is the transverse malocclusion. Another important finding Tadej et al had pointed out was the medio-lateral dimension as compared to anterior- posterior  . SeymouThe predictability of condylar morphology is of significant importance. Data showed that all three types of skeletal malocclusion I, II and III, the oval shaped condyle was found to show the maximum occurrence. The next common shapes are diamond and bird beakand the crooked shaped condyle is the least common. There was no significant difference between the left and right side of the condyle. Results of the study suggest that reasonable predictability of condylar morphology with nature and any deviation from the ovalshape would require the need for further investigation with relevance to any clinical significance.


The use of short oligonucleotide or peptide molecules as target-specific aptamers has recently garnered substantial attention in the field of the detection and treatment of viral infections. Based on their high affinity and high specificity to desired targets, their use is on the rise to replace antibodies for the detection of viruses and viral antigens. Furthermore, aptamers inhibit intracellular viral transcription and translation, in addition to restricting viral entry into host cells. This has opened up a plethora of new targets for the research and development of novel vaccines against viruses. Here, we discuss the advances made in aptamer technology for viral diagnosis and therapy in the past decade. While usually in the range of a hundred nucleotides, the most effective size is found to be around forty nucleotides. The term “aptamer” was first coined by the Szostak lab in the year 1990 and is derived from the Latin word “aptus”, which means “to fit”, and the Greek expression “meros,” which means “part” . In SELEIn this regard, various aptamers with applications ranging from catalysis to cancer therapy have come into existence. Their increased use and development have been noted for the detection and therapy of a variety of diseases, including cancer, diabetes, auto-immune diseases, bacterial and viral infections ,4,5,6,7.Addressing this problem, aptamers have shown substantial efficacy in both diagnosis and treatment, with minimal or no side effects. In addition, aptamers do not encounter the issue of drug resistance that traditional antiviral medications inevitably run into. Owing to these exceptional qualities, numerous research papers on the diagnostic abilities, and both practical and potential therapeutic properties of aptamers have come into existence over the past two decades.In this review article, we elucidate in detail the advancements that have been made in the past decade in the field of aptamers, specifically as they pertain to viral diagnosis and therapy. We also discuss the nature of these viral infections, by detailing their cellular and molecular machinery involved in causing infection. Furthermore, a comprehensive review of aptamers developed for each of these cellular mechanisms involved in viral infection is described. By reviewing the most recent forms of aptamer modifications implemented by researchers for improving their efficiency, we also delve into the recent, more advanced developments that have been reported in the field of aptamer detection and treatment for HIV, hepatitis, influenza and HSV. Retroviridae family, which causes acquired immunodeficiency syndrome (AIDS) in humans, a lethal disease that kills millions of people worldwide. The structure of HIV has been studied closely, and multiple treatment models have been presented in the past. However, with the advent of aptamer technology, scientists are adopting a multifaceted approach to curing this life-threatening disease that is safer than other alternatives. The molecular details of entry, including the added information on the structure of the virus, play a key role in designing the perfect aptamers for prevention, inhibition, and recovery from a viral infection. Here we briefly discuss the various receptors, proteins and organelles involved in viral infection, while highlighting those that have been used for the design of aptamers. (HIV) is a member of the HIV structure, cell binding and host cell entry were described in detail in a 2012 report . HIV, li+ lymphocytes; however, monocytes, macrophages, and dendritic cells  have all been shown to get infected [HIV famously infects CD4infected . The proinfected . When ininfected . The formation of the fusion pore assists in the entry of viral RNA and HIV-RT into the infected cell. The HIV-RT is responsible for the conversion of viral RNA into DNA that is consequently integrated into the host genome . The virIn summary, gp120, gp41, viral Env, HIV-RT, CD4 receptor, CCR5 and CXCR4 co-receptors play a significant role in HIV infection. They remain potential targets for the development of aptamers in the successful inhibition of HIV infection. Owing to their conformational flexibility, RNA oligonucleotides have been used conventionally in the selection of aptamers for HIV therapy. However, multiple studies on DNA aptamers for the therapy/inhibition of productive HIV infection have also been described. In recent years, the focus was placed on developing aptamers for CD4, CCR5, gp120/gp41 and HIV-RT. Other notable aptamer targets include HIV-gag, thrombin, human cyclin T1, P24 antigen, aspartyl protease and nucleolin. d) of 47.91 nM. Continuing this body of work, in 2013, the same group reported the in vivo delivery of the same aptamer-siRNA chimera using a chemically synthesized sticky bridge [Small interfering RNAs (siRNA) have gained a reputation for disrupting undesirable pathways, in turn inhibiting viral infections. However, siRNAs innately lack the ability to target the desired site and need to be transported via a molecular carrier. In 2011, Zhou et al. reported RNA aptamers targeting HIV’s viral gp120 . By conjy bridge . The stiy bridge . This apy bridge . Howevery bridge . d = 0.15 nM) and elicited an IC50 (50% inhibitory concentration) in the nanomolar range. The group reported the synergistic effect of the aptamer alongside gp41- and CD40-inhibiting antibodies. UCLA1-based protection against HIV-mediated cardiomyopathy (HIVCM) was reported by [In 2012, Khati et al. showed significant modifications to an earlier-reported gp120 RNA aptamer named UCLA1, which was a shortened synthetic version of its predecessor [orted by . They suorted by . The groIn the last decades, many synthetic G-rich oligonucleotides have been identified as promising anti-HIV candidate drugs ,24. BrieThe first G-quadruplex-forming oligonucleotide identified as a potent anti-HIV agent was the 8-mer phosphorothioate TTGGGGTT (ISIS 5320) , which eIn 2014, Romanucci et al. prepared a number of novel analogs bearing different hydrophobic tails at the 5′-ends of Hotoda’s sequence for the inhibition of HIV . AccordiAlthough all the techniques and aptamers mentioned above are effective in reducing HIV infections, they do not elicit immunity in an individual who is being treated with them. In 2012, Burke et al. reported a robust model to inhibit HIV infection, where HIV-RT-inhibiting RNA aptamers were produced intracellularly by modified cells . They shIn 2013, the group showed a high-throughput sequencing (HTS)-based SELEX process in which different populations of RNA were evaluated for their binding capacity to the HIV-1 virus . HTS anaAlthough non-conventional HIV-detection kits have not hit mainstream markets, they harbor an enormous potential to reduce the burden on diagnostic labs and change the landscape of the patient population visiting diagnostic labs if they become available. Aptamer technology represents one such way to improve the diagnostic capability regarding HIV infections. Multiple models currently exist for the detection of HIV infections, of which the optical and fluorescent models remain popular. Rahim et al.  developeIn 2011, Liang et al. developed a direct visualization technique by conjugating fluorescent beacons to HIV-RT targeting aptamers . Anotherd values simultaneously [d between nucleocapsid protein 7 of HIV and the stem-loop 3 of an RNA aptamer. The nanopores were 6 nm in diameter and were made of silicon nitride. In a separate study, an atomic-force microscope (AFM) was used to carry out direct and label-free detection of gp120 HIV type-1 envelope glycoprotein as a target protein [In an ergonomic model, Niedzwiecki et al. demonstrated that by using nanopores that are smaller than the largest aptamer-target conjugate, one can screen for successful aptamers and determine their Kaneously . In this protein . The met protein , who useVarious other aptamers that bind to the CCR5 and gp41 regions have been reported and are noted in Viral hepatitis is caused by 5 well-known viruses, namely hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis E virus (HEV). Of these, HBV and HCV are the most studied with respect to their structure and mode of entry into a host cell. Despite the similarity in their names, HBV and HCV belong to two different virus families, with profoundly distinct features. Hepadnavirus family, which consists of a spherical, partially double-stranded DNA inside an icosahedral nucleocapsid core protein. Similar to HIV, HBV contains DNA polymerase, which has reverse transcriptase activity. The outer lipid membrane consists of three kinds of HBV surface antigens (HBsAg)—“S”, “M” and “L” HBsAg, which are important for virus-cell fusion by targeting the surface receptors of the host cell [HBV belongs to the ost cell ,73. HBV attaches to host hepatocytes through the sodium/bile acid co-transporting peptide SLC10A1, with the help of HBsAg. This is followed by a clathrin-mediated endocytosis step that allows for the internalization of the virus. HBV DNA and core proteins are then released into the cytoplasm and are then transported by the cellular machinery into the nucleus, through a nuclear pore . HBV DNA undergoes a transformation from partially double-stranded to a covalently closed circular double-stranded DNA (cccdsDNA) inside the nucleus. At this stage, cccdsDNA is converted into a long mRNA using host RNA polymerase, which is then transported into the cytoplasm for translation. The viral mRNA not only codes for core proteins and other structural components but also for the genome of the newly born virus. The virion P protein, which is known to be synonymous with reverse transcriptase, is responsible for the synthesis of viral DNA . Flaviviridae family. It is known to showcase two surface glycoproteins, “E1” and “E2”, on its envelope that envelop a single strand of the viral RNA, encompassed by core proteins [Unlike HBV, HCV is a positive-strand RNA virus that belongs to the proteins . The comHCV circulates in the blood, where it encounters the basolateral surface of hepatocytes. The surface E1 and E2 glycoproteins attach to the surface receptors, which assist in the entry of the virus into the cell. Evidence is also available that HCV hybridizes with lipids and cholesterol in the blood to form “lipo-viral particles” (LVP) that give HCV immunity against antibody neutralization before entering the host cell . Five suVia translation of the viral RNA, a polyprotein is produced that is cleaved by both viral and cellular proteases. Each polypeptide chain of the polyprotein encodes for structural proteins, core proteins, glycoproteins and non-structural (NS) proteins. Non-structural proteins, namely, p7, NS2, NS3, NS4A, NS5A and NS5B, are not incorporated into the virus but help coordinate HCV replication, viral RNA synthesis and the assembly of virus particles. HCV particles are formed by budding into the endoplasmic reticulum (ER), followed by the sequential assembly of glycoproteins, core proteins and the viral RNA. It has been shown that post-assembly, HCV particles follow a series of subcellular compartments (secretory pathway) to the exit cells . From the collected data, it was evident that DNA aptamers were predominantly used for the treatment of HBV and HCV. It is also interesting to note that most of the research conducted in recent years is an extension of previously reported aptamers, as opposed to novel oligonucleotide sequences. This could be due to well-defined, commercially available aptamers through reputed sources. It is also worth noting that studies have predominantly concentrated on HCV diagnosis and therapy rather than HBV or other hepatitis viruses. In 2011, Feng et al. demonstrated a proof-of-concept by constructing an RNA aptamer that was able to bind and suppress the HBV P protein . As mentBerzal-Herranz et al. demonstrated the development of RNA aptamers for the inhibition of the cis-acting replication element (CRE) located on the 3′ end of the NS5A coding region . They shA study by Lee et al. reported the selection of RNA aptamers and their consequent 2′-hydroxyl, 2′-fluoropyrimidine modification against the NS5B replicase ,89. By bIn 2013, Yang et al. reported the production of aptamers that target surface E1 and E2 proteins of HCV . PurifieA competitive binding assay using RNA aptamers was developed by Suh et al.  that helUsing an Octet optical biosensor, Roh et al. were able to detect the viral NS5B protein with the help of RNA aptamers . The bioOrthomyxoviridae family and are sub-divided into three major types: influenza types A, B and C viruses. Of these, A and B primarily cause infections in humans and are known to cause flu pandemics around the world. All three influenza virus types have been thoroughly studied and their life cycles are well documented. To address the significant public-health cost and threat caused by these viruses, aptamer technology has seen rapid progress to provide cost-effective solutions to influenza infections. Here, we will review the structure and life cycle of the influenza-A virus and its sub-types, while highlighting some key anti-influenza aptamers. The information regarding the structure and lifecycle of the influenza-A virus can be found in much greater detail in the Elsevier article report on rapid-reference influenza [Influenza viruses belong to the nfluenza . The influenza-A virus is characterized by the presence of two major surface glycoproteins, namely, “hemagglutinin” (HA) and “neuraminidase” (NA). There are 16 HA subtypes and 9 NA subtypes. Based on the nature of these proteins, various influenza-A virus subtypes have been discovered and, of those, only H1N1, H2N2 and H3N2 viruses are considered mortally harmful to humans. The complete lifecycle of a typical influenza virus is depicted in The influenza virus consists of 8 negative-sense RNA segments, nucleoprotein, and polymerase inside a lipid membrane envelope, which is coated by M1 proteins on the inner surface and M2 proteins on the outer surface. As mentioned earlier, the envelope is also encompassed by rod-shaped HA and mushroom-shaped NA, present on the outer surface in a 4:1 composition ratio. HA consists of two major subunits, namely, HA1 and HA2, which are bound by disulfide bonds. The HA2 subunit consists of a conserved hydrophobic amino acid chain, known as a fusion-peptide, which conducts the membrane fusion process between the virus and host cells. Conversely, NA has enzymatic activity that cleaves the bonds between surface receptors and the virus, assisting in viral egress . The influenza virus enters a host body through both the enteral and parenteral routes. When it encounters the epithelial cells of the mucous membrane, the virus attaches to sialic acid residues, via HA binding . Post-biThe viral genome traverses through the cytosol to enter the host nucleus, where the viral nucleoprotein transcriptase  converts the negative-sense viral RNA to messenger RNA (mRNA) . The mRNMost studies done on developing aptamers against influenza viruses have concentrated on H1N1, H3N2, H5N1, H5N2, H9N2, and PERTH sub-strains. It is worth noting that DNA aptamers are more commonly used in recent years, as opposed to RNA aptamers. The common target in SELEX (for anti-influenza aptamer isolation) was hemagglutinin, specific for the influenza virus. d values, which was indicative of their use as effective inhibitors of viral entry. Musafia et al. reported a DNA aptamer—BV02—that had high specificity toward hemagglutinin [d of 3.7 nM and was able to bind to the virus in serum samples extracted from clinical patients. RNA aptamers against the hemagglutinin of the H1N1 virus were developed by Gopinath S.C.B. and Kumar P.K.R. in 2013 . The aptglutinin . They deglutinin . The strd values as well as an IC50 concentration as low as 10 nM, making them very exciting additions to the field. In 2013, a G-Quadruplex-based DNA aptamer against the NS-1 protein of the influenza H5N1 virus was developed . While NTwo RNA aptamer studies conducted in 2014, targeting the hemagglutinin of the H5N2 virus, were reported by Kim et al. ,123. TheDetection of the influenza virus during the past five years has predominantly concentrated on the H5N1 virus. This can be attributed to the rising numbers of reported deaths caused by the highly pathogenic H5N1 virus in many parts of Asia and the Middle East. Avian influenza is still a persistent public health threat in many Asian countries. Of the various sensors used for the detection of the H5N1 virus, electrochemical impedance-based sensors were the most used. This could be due to the ease of building one in a small amount of time. Lui et al. have reported multiple studies on the detection of the H5N1 virus ,130,131.All the studies used DNA as the nucleic acid of choice. Using a surface plasmon resonance-based setup (SPR), biotinylated aptamers were used for the rapid detection of a virus. The detection limit in this report was noted as 0.128 hemagglutinin units (HAU). A similar study that used quartz crystal monitors instead of an SPR setup showed a lower detection limit of 0.0128 HAU. In a different study, an SPR-based SELEX process was used to elucidate the binding affinity of the aptamer in targeting the hemagglutinin of H5N1. The group proposed this technique as a potential novel system for the screening of aptamers. The same group showed a detection limit of 0.0004 HAU, using an enzymatic sandwich assay with concanavalin A, glucose oxidase, and Au nanoparticles. Using the models described above, another study described a detection methodology that used an aptamer bio-nanogate that could selectively respond to target molecules and control enzymatic reactions for electrochemical measurements. The setup had a detection limit of 2–9 HAU and the capacity to sense within 1 h of activation. In a follow-up report, the same group revealed a microfluidic setup, consisting of biotinylated aptamers that could sense H5N1 viruses, with a detection limit of 0.0128 HAU within 30 min of activation. In 2011, Liu et al. revealed an electrochemical sensing assay with a detection limit that was the lowest of all the detection setups mentioned earlier . The mulA G-Quadruplex DNA aptamer-based SPR sensor was also reported in 2015, which was able to detect H1N1 hemagglutinin with a detection limit in the nM range . It was In 2011, Negri et al. used a surface-enhanced Raman (SERS)-based aptamer assay for the detection of viral nucleoproteins . The stuHerpesviridae family. HSV has two serotypes, namely, HSV-1 and HSV-2, which predominantly cause orofacial and genital lesions, respectively. It is estimated that upwards of 70% of American adults suffer from HSV infection, and it is the main cause of infectious blindness in the USA. HSV is infamous for recurring infections leading to herpetic lesion outbreaks, owing to a significant decrease in host immunity or high stress levels, which makes the virus a serious global public health threat [Herpes simplex virus (HSV) is a double-stranded DNA virus that belongs to the h threat ,148,149.HSV consists of single linear double-stranded DNA, enclosed within an icosahedral capsid that consists of the four capsid proteins—VP5, VP26, VP23, and VP19C . The capViral gB and gC surface glycoproteins interact with Heparan sulfate proteoglycans (HSPGs) to facilitate the viral attachment to the host cell membrane . Using tThe viral capsid follows a trans-microtubule network to reach the nuclear pore, where the viral DNA is injected into the nucleus . SimultaClosely regulated expression and transcription of all genes result in the replication and assembly of the viral proteins and genome to form virus particles. A significant contribution in HSV studies was conducted by our group, showing that heparan sulfate, required for the attachment/fusion of the virus to the host cell, is also responsible and implicated in holding newly budded virus particles to the host cell membrane. Heparanase, a host enzyme, is required and upregulated in infected cells, which cleave surface heparan sulfate, aiding viral egress from the cells . There are two interesting studies that have been published in the field of aptamers treating HSV infections. While one study considered aptamers for HSV-1, the other study explored aptamers targeting HSV-2. Both the reports used RNA aptamers in their studies. Potent aptamers with an IC50 range of 20–50 nM were reported to block infection pathways that were dependent on both major entry receptors, Nectin1 and HVEM of HSV-2, by Moore et al. in 2011 . The stud value approximately constant. The final aptamer had a Kd value of 40 nM, with an IC50 value of 0.8 μM. This was done by closely monitoring the structural changes that occurred with the removal of non-essential domains from the aptamer. In a similar study, Gopinath et al. developed RNA aptamers against the gD of HSV-1 in 2012 . The inid of 50 nM with an IC50 of 2 µM when used in PBS solution. This is the first and only DNA aptamer that has been studied in detail with respect to its therapeutic efficacy, both ex vivo and in vivo. This body of work has the potential to change the current landscape of perspectives on topical antivirals in treating HSV-1 keratitis. Topical antivirals have lower systemic toxicity-based side effects, so using anti-inflammatory and biocompatible drugs such as DNA aptamers can improve a disease prognosis significantly when it comes to HSV-1 keratitis.Our group has reported the use of a DNA aptamer for the treatment of an HSV-1 infection in the eye . The aptA list of aptamers that have been used for the treatment and diagnosis of other viruses, such as apple stem pitting virus, coronavirus, norovirus, etc., have been tabulated in As aptamers are characteristically short nucleic acid sequences, any development in the field of nucleic acid research directly influences them. In this regard, the advent of newer and more sophisticated DNA sequencing systems has greatly helped the production of aptamers itself. The SELEX process heavily relies on understanding the repeatability of an oligonucleotide sequence in successive runs. With every successive round of selection, the aptamers that are bound to the target molecule are produced in higher quantities. Prior to 2011, the selected aptamer sequences would then be cloned into bacteria, and the colonies with the highest repeatability would be taken for further consideration. With the advent of next-generation sequencing techniques, a lot of these processes require no cloning or post-selection processing. This has hugely affected aptamer development globally in the past decade. In the last five years, the price for evaluating the sequence of a human-sized genome has fallen further to half its price, resulting in more researchers opting for this technique over older ones. Additionally, the production/commercialization of instruments that have next-gen sequencing capabilities have given researchers access to new tools that they did not have a decade ago .As SELEX comprises a repeated selection process spanning anywhere between 10 and 20 cycles, robotic protocols with minimal human intervention have facilitated an error-free, simple and extremely efficient evolution of aptamers . Combine3COO) that can easily be procured by researchers or users and are readily available. This has broadened the scope for aptamers, as researchers with no prior experience in using aptamers now have access to purchase and experiment with them in developing sensors and diagnostic assays for metabolites and infectious diseases.The advent of these technological advances, along with growth in global connectivity and sharing, has given rise to companies that commercially produce the aptamers of your choice, based on a given target. SELEX commercialization, combined with academic proficiency, has given rise to numerous commercially available aptamers for a plethora of targets. All these aptamers have been target-tested and include several readily available modifications . The device used in the study is Aptamex, a product of Achiko’s DNA aptamer platform technology—a gold nanoparticle-based test that targets the S1 spike protein on the surface of SARS-CoV-2 and is reported to take approximately 15 min to use . Multiple studies in the recent past have been conducted in an effort to develop diagnostic systems for the detection of SARS-CoV-2. A handful of studies also show the therapeutic benefit of using aptamers against SARS-CoV-2 and COVID-19. Yang et al.  have shoPramanik et al.  developeThe detection and treatment of viral infections using antibodies and conventional drugs have seen success in the past. As described in this review, aptamers unequivocally provide similar if not better diagnostic and therapeutic efficiencies, with lower production costs when compared to their predecessors, and without running into issues like microorganism resistance. This can be attributed to their simple structural features, non-immunogenicity, biocompatibility, non-toxicity, and ease of production. However, their selection using the SELEX process remains a major hurdle in developing aptamers for viral infections. The aptamer industry has come a long way since its humble inception in the early 1990s. Today, we have access to numerous aptamers that are not only competitive with the antibody industry but are also paving the way for novel, complication-free modes of treatment. However, the research on aptamers and their prospects has only begun and will need far greater amounts of funding and focused research to unleash their true potential and results. From the previous sections, it is evident that some infectious agents, such as HIV and HCV, have received massive attention in the past decade, while others such as HBV and HSV have received less or no attention at all. While all these pathogens have been well studied, and their structures and lifecycles have been clearly charted out, there is disproportionate funding/research being provided/conducted for tackling these issues. Although this depends on the research focus of the funding agencies that support a research group, new reforms in research to eliminate non-lethal diseases also need to be created. In addition, further research has to be focused on translational research in the field of antiviral aptamers, for studies to reach clinical trials. This will shift the current research and healthcare industry paradigm toward using more adaptive, cost-effective and targeted technologies. With the advent of new companies that commercialize the aptamer generation process, there is space for fruitful collaborations between industry and academia to develop novel aptamers for the diagnosis and therapy of various diseases, such as HSV, that continue to remain evasive while carrying out tremendous damage to public health.