Since the identification of this novel risk factor, scientists have conducted numerous studies to understand its relevance. Over the past few years, the body of scientific evidence supporting the reality of this risk has grown substantially. In addition, numerous papers describing actions and interventions designed to mitigate this risk have been published. Therefore, the purpose of this paper is to review the literature on the risk of feed ( In the United States, tremendous resources have been invested to reduce the risk of viral pathogens, such as the entry of porcine reproductive and respiratory syndrome virus into susceptible populations. Protocols including shower in‐shower out, transport sanitation, quarantine and testing of incoming genetics and the filtration of incoming air are commonplace throughout the US swine industry, particularly at the level of the sow farm , a surrogate for foot‐and‐mouth disease virus (FMDV), bovine viral diarrhoea Virus (BVDV), a surrogate for classical swine fever virus (CSFV), African swine fever virus (ASFV), influenza A virus of swine (IAV‐S), bovine herpesvirus type 1 (BHV‐1), a surrogate for pseudorabies virus (PRV), canine distemper virus (CDV), a surrogate for Nipah virus (NiV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine sapelovirus, (PSV) a surrogate for swine vesicular disease virus (SVDV), vesicular stomatitis virus (VSV), porcine circovirus type 2 (PCV2), and feline calicivirus (FCV), a surrogate for vesicular exanthema of swine virus (VESV) , FCV (representing VESV), BHV‐1 (representing PRV), PRRSV, PSV (representing SVDV), ASFV and PCV2 maintained infectivity as determined by virus isolation or swine bioassay, while BVDV (representing CSFV), VSV, CDV (representing Nipah Virus) and IAV‐S did not. Since ASFV had not been reported in China at the time of the study and was actively circulating in Eastern Europe, a Trans‐Atlantic model was developed involving representative feed ingredients, transport times and environmental conditions simulating shipment from Warsaw, Poland, to the port of Le Havre, France, to the port of New York City, NY, US, and then to Des Moines, IA, US  and in feed (104 TCID50) following a one‐time exposure. However, further analysis indicated that the more frequent the exposure  to ASFV in small volumes of feed or liquid, the higher the probability of infection, even in the presence of lower doses such as 102 TCID50. Another significant finding was the calculation of ASFV half‐life in feed ingredients. Original estimates based on limited (n = 2) data points derived from the trans‐Atlantic model indicated that half‐life ranged from 4.1 to 5.1 days across all nine of the virus‐positive ingredients , heat treatment, chemical mitigation and/or storage time of feed. As a complement to this work, a validated sampling method has been developed , an FDA‐approved liquid anti‐microbial used to control Salmonella contamination in poultry and swine diets. In groups of pigs fed Sal CURB®‐treated feed spiked with PEDV versus non‐treated feed, clinical signs of PEDV infection (vomiting and diarrhoea) and viral shedding in faeces were observed 2–3 days post‐consumption of non‐treated feed. In contrast, no evidence of infection was observed in pigs fed Sal CURB®‐treated feed (Dee, Neill, et al., Extensive studies have been conducted to evaluate the effect of chemical mitigation on PEDV‐contaminated feed (Cochrane et al., In regards to the elimination of PEDV from a contaminated animal feed manufacturing facility, the combined application of a quaternary ammonium‐glutaraldehyde blend cleaner, followed by a sodium hypochlorite sanitizing solution, along with a facility heat‐up to 60°C for 48 hr was effective at reducing PEDV genomic material, but did not completely eliminate it, demonstrating the residual risk of this virus at the feed mill level following purposeful contamination (Huss et al., 2.4Necessity: is importation of the ingredient an absolute necessity?Alternatives: can the ingredient be obtained from a country free from foreign animal diseases?Virus: which virus is causing the concern?Viral half‐life: is there published information on the half‐life of the virus in the designated ingredient?Transport time: what is the projected time for delivery of the ingredient from the source to its destination?Mitigation: are there safe products that can be added to the ingredient to reduce viral load during transport?Storage period: is there published information on storage time and temperature that will eliminate residual virus from the ingredient prior to use?With the generation of new knowledge on viral half‐life in feed, the application of a ‘Responsible Imports’ approach has been adapted across the US industry (Patterson, Niederwerder, & Dee, Therefore, as production companies across the United States develop storage facilities for incoming products, a new way of thinking is taking shape, one that is based on ‘feed quarantine’ that brings together information across several disciplines including feed science, microbiology and oceanic transport logistics to understand how to minimize risk. This approach is intriguing as it is non‐regulatory in nature and does not negatively impact trade.3In summary, there is a growing body of scientific evidence suggesting that contaminated feed and feed ingredients purposefully inoculated with viruses may be risk factors for the spread of viral diseases at the domestic and the transboundary levels. This information has stimulated collaborative efforts across North America between livestock and grain commodity groups, governmental agencies, and the veterinary profession in an effort to manage this risk. For example, the Canadian Food Inspection Agency has already implemented a national program using designated secondary control zones to manage the introduction of high‐risk feed ingredients, such as grains, oilseeds and meals from 44 ASFV‐positive countries into Canada. Activity in the United States includes the passage of resolutions from the National Pork Producers Council requesting collaborative efforts across North America to reduce the risk of foreign animal disease entry via the risk of feed ingredients, widespread use of feed additives as chemical mitigants and the writing of policy to guide the implementation of the Responsible Imports approach. A US feed safety task force involving representatives from governmental agencies and multi‐species stakeholder groups has been formed to develop a national plan to manage this risk. Finally, requests to restrict the importation of high‐risk feed ingredients such as soy‐based products from ASFV‐positive countries are being made to key government officials. Collectively, representatives from Mexico, Canada and the United States have begun the discussion of how to collaborate to reduce the risk of the introduction of ASFV and other foreign animal disease pathogens to North America, with focused discussion on all risk factors, including feed.However, despite all the scientific evidence, there are still differences in opinion as it pertains to the risk of feed. While a recent review concluded that the current body of scientific knowledge lacks conclusive evidence of virus contamination of imported non‐animal origin feed ingredients of commercial swine feed (Gordon et al., However, despite the progress that has been made, significant research gaps still exist regarding the risk of feed. For example, the vast majority of the published papers are based on experimental inoculation and models. Further efforts to reproduce this work using actual modes of transport, that is actual ocean freighters and commercial transport vehicles trucks, are needed. In addition, it is argued that there is a lack of evidence documenting the presence of viral pathogens in actual feed samples around the world. While the current evidence is indeed limited, viable PEDV has been detected in feed bins feeding index cases of PED on sow farms (Dee, Clement, et al., In closing, in a few short years, global agriculture has come a long way in recognizing and accepting the risk of feed and feed ingredients as vehicles for the domestic and transboundary spread of diseases, based on the research efforts cited in this writing. It is hoped that these efforts will continue to stimulate communication and collaboration between the feed and livestock industries, resulting in further research into the emerging concept of ‘global feed biosecurity’. Ideally, current and future information regarding the risk of pathogen spread in feed will enhance the accuracy of risk assessments, drive the continual development of efficacious feed‐based mitigation strategies and ultimately change the philosophy regarding the global trade of feed ingredients from one that based on price to one where the biosecurity of the feed supply chain is prioritized.The authors declare no conflict of interest.No ethical approval was required as this is a review article with no original research data.


This work presents a magnetic-driven deterministic lateral displacement (m-DLD) microfluidic device. A permanent magnet located at the outlet of the microchannel was used to generate the driving force. Two stages of mirrored round micropillar array were designed for the separation of magnetic beads with three different sizes in turn. The effects of the forcing angle and the inlet width of the micropillar array on the separating efficiency were studied. The m-DLD device with optimal structure parameters shows that the separating efficiencies for the 10 μm, 20 μm and 40 μm magnetic beads are 87%, 89% and 94%, respectively. Furthermore, this m-DLD device was used for antibody recognition and separation among a mixture solution of antibodies. The trajectories of different kinds of magnetic beads coupled with different antigens showed that the m-DLD device could realize a simple and low-cost diagnostic test. In recent years, deterministic lateral displacement (DLD)-based microfluidic methods have been proposed to effectively separate biological ,2,3,4 anThe motion of magnetic beads in microchannels driven by magnetic forces has been widely used in various applications for the manipulation of biological species, such as CTC sorting ,18,19, DIn this study, we designed a novel magnetic-driven DLD (m-DLD) separating device and demonstrated its performance. The magnetic field produced by a permanent magnet was used to drive magnetic beads to flow through a periodic array of cylindrical obstacles. No extra actuating elements such as syringe pumps were required. The effects of forcing angle and inlet width on the separating efficiency of magnetic particles of different sizes were explored. This m-DLD device with optimal parameters was further used as a simple method for the antibody recognition and separation in a mixture solution of antibodies. The trajectories of the magnetic beads carrying different antibodies were analyzed to distinguish the types of antibodies. This m-DLD separating device shows potential as a simple and portable biosensing device for recognizing and separating a mixture of multiple types of target molecules at the same time.3O4) magnetic beads (50 mg/mL) with diameters of 10 μm, 20 μm and 40 μm were purchased from Suzhou Zhiyi Microspheres . The surface of the bead was modified with carboxyl functional groups for coupling proteins. Alpha-fetoprotein protein (AFP), mouse anti-human AFP, prostate specific antigen (PSA), mouse anti-human PSA, hepatitis B surface antigen (HBsAg) and mouse anti-human HBsAg were purchased from Zhengzhou Cell To Antibody & Antigen Biotech . FITC-conjugated affinipure goat anti-mouse IgG was purchased from Proteintech Group . Sorbitol was purchased from Dingguo Biotechnology . 4-Morpholineethanesulfonic acid hydrate (MES), 1-ethyl-3-(3’-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), bovine serum albumin (BSA), and immunostaining fixative were purchased from Shanghai Yuanye Bio-Technology .Ferroferric oxide  is varied by changing the base length of the triangle at the entrance . Utilizing replica molding method, the pattern on the master was transferred to a polydimethylsiloxane (PDMS) slab. After the treatment of oxygen plasma , the PDMS slab was bonded at one end of a glass slide to reduce the distance between the magnet and the outlet of the microchannel. On the chip, hole punches were used to form the inlet and outlets, and the inlet diameter (0.4 mm) is larger than the outlet diameter (0.2 mm) to facilitate the loading of the liquid.3–104/mL) was prepared to avoid the agglomeration of magnetic beads. The viscosity and density of the solution were 26.0 mPa·s and 1.293 g/cm3, respectively. In order to avoid the influence of the pressure difference between the outlet and the inlet on the movement of the magnetic beads, the solution containing 60% sorbitol and 0.1% tween-20 were introduced into the microchannel by a capillary force before each experiment and stood for 3 min. Then, 1 mL sorbitol/tween-20 aqueous suspension containing 0.01 mg/mL of 10 μm beads, 0.02 mg/mL of 20 μm beads, and 0.04 mg/mL of 40 μm beads was introduced into the microchannel. In order to prevent the magnetic beads from adhering to the microchannel wall at the inlet, the magnet was loaded after all three types of magnetic beads sank to the bottom of the inlet.In order to minimize the effect of the magnetic force along the vertical direction, the microchannel on the chip was aligned with the center line of the magnet , which was used to generate the magnetic force. The rectangular magnet was used to construct the magnetic field, which is conducive to the stable movement of the magnetic beads see . The magThe recognition and separation of antibodies were performed by using the m-DLD chip with the optimal structure parameters. The magnetic beads were washed with MES buffer and activated with EDC/NHS. Then 30 μg/mL AFP, PSA and HBsAg were coated on the surface of 10 μm, 20 μm and 40 μm magnetic beads, respectively. Those three types of antigen-coated magnetic beads were added to a mixed solution containing 3 μg/mL mouse anti-human AFP, mouse anti-human PSA and mouse anti-human HBsAg to couple the antibodies. The antibody-coated magnetic beads were then coupled to FITC-conjugated Affinipure Goat Anti-Mouse IgG. The mixture of the as-prepared three kinds of FITC-labeled antibodies carried by magnetic beads was introduced into m-DLD device. The trajectories of magnetic beads were monitored under a fluorescent microscope . All experiments were repeated at least three times.We use a simple model to describe the trajectory of magnetic beads driven by magnetic force rounding an obstacle . In thisθ was defined as the largest forcing angle for which the particles are locked to move along the column of the array [cb. l is defined as the center to center distance between the obstacles. The cb = l sinα when the forcing angle α is equal to the critical angle θ. We show two types of particles in θ1 and large (red) ones, critical angles θ2. The critical angle of larger diameter particles is larger than that of smaller diameter particles [θ2 is assumed to be greater than the θ1 in the model. Both types of particles move along obstacles for forcing angles below the critical angles θ, showing a displacement mode  is tuned by changing the base length of the triangular structure, which is defined as a function of Dy. For example, inW1 = 2 Dy1 means that the inlet width of the first stage contains two gaps between the micropillars. The direction of the magnetic force is fixed, and we varied the forcing angles of the device by fabricating different m-DLD chips with different directions of the micropillar microarrays.Previous reports have shown that the forcing angle and the inlet width are two important parameters that affect the separating efficiency . TherefoAt the first-stage structure, we expect to collect 10 μm magnetic beads at outlets 1 and 2 and allow the 20 μm and 40 μm magnetic beads to flow into the second stage through the Joint. For each kind of magnetic bead, the separating efficiency was defined as the ratio of the number of the beads with a specific size in the desired collection outlet to its total number.inW1 of 2 Dy1. The probability of 40 μm magnetic beads flowing through the Joint is 100%. The variation of α1 from 10° to 20° does not change the probability of 40 μm magnetic beads in the outlets. This is because the critical angle for 40 μm magnetic beads is larger than the highest forcing angle considered here, and the beads move along the obstacles showing a displacement mode. At the smallest forcing angle (10°) we considered here, the probability of the smallest magnetic bead (10 μm) at the Joint accounts for half of the total number of the smallest magnetic bead in all outlets. As the forcing angle increases to 15°, the probability of the smallest beads (10 μm) at the Joint drops sharply. This is because the forcing angle is larger than the critical angle, and it is in a zigzag mode. At the maximum forcing angle, the 20 μm magnetic beads are almost evenly divided into three outlets because the forcing angle has exceeded the critical angle, and the particles change from the displacement mode to the zigzag mode. It was reported that the critical angle increased with the increase of the diameter of the particles [α1 = 15° and the sorting efficiency of 10 μm magnetic beads is 87%. The settling velocity of different magnetic beads is different, and some of the magnetic beads have entered the microarray before loading the magnet. This is the main reason for the separation efficiency of the magnetic beads to be less than 100%.articles , which iα1 = 15° , we explored the effect of different forcing angles on the separating efficiency  to recognize and separate the types of the antibodies. Furthermore, we used the m-DLD device with the optimal structure parameters (We have demonstrated the feasibility of using magnetic force to separate the magnetic beads by size in a m-DLD device. The application of external force driving DLD separating has been expanded. The effects of forcing angle and inlet width on the separating efficiency in m-DLD device have been investigated. We showed the range of critical angles of the three magnetic beads in the first stage and second stage. We find that as the inlet width increases, the separating efficiency of the magnetic beads in the displacement mode increases and the separating efficiency of the magnetic beads in the zigzag mode decreases. The separating efficiencies for 10 μm, 20 μm and 40 μm magnetic beads are 87%, 89% and 94%, respectively with the optimal parameters of forcing angle and inlet width. We illustrated that the method can be adopted to recognize the types of antibodies based on the trajectories of magnetic beads. With this method, the recognition of multiple of different antibodies in a mixture sample can be realized with only one fluorescent label instead of different labels for each antibody as in common methods.It is worth noting that this m-DLD-based method can separate and recognize more than three antibodies by increasing the number of stages of the micropillars array. In addition, it was expected to provide a new method to separate cells of the same size through coupling magnetic beads to the cells . Further


Six members of the asbestinin family of marine diterpene natural products have been synthesized in an efficient and stereoselective manner from a single oxa‐bridged intermediate. Five of these natural products have not been synthesized previously and the structures of four of them have been confirmed as those proposed originally or following revisions to the original structures. The fifth natural product—asbestinin‐21—has been shown to be a diastereomer of the compound that had been proposed previously. Access to the asbestinins: The total syntheses of six members of the asbestinin family of marine diterpene natural products have been accomplished in an efficient and stereoselective manner from a single oxa‐bridged intermediate. Briareum asbestinum and Briareum polyanthes. The isolation and characterization of the first members of the family—asbestinins‐1–5—were reported by Faulkner, Clardy and co‐workers in 1980.1) and 4‐deoxyasbestinin C (2), and those that are oxidised at C‐4, such as asbestinin‐12 (3) and asbestinin‐2 (4) , 11‐acetoxy‐4‐deoxyasbestinin F (9→10), asbestinin‐10 (13→14) and asbestinin‐21 (15→16). It is likely that the structures of 4‐deoxyasbestinin G (7→8), asbestinin‐9 (11→12), asbestinin‐22 (17→18) and asbestinin‐23 (19→20) also require revision.Several of the structures that were proposed for the asbestinins upon isolation have been revised as a consequence of the acquisition of better quality NMR data and the advent of new NMR techniques.inin G 7→, asbestiThe asbestinins possess a complex fused tetracyclic core that contains two medium‐sized cyclic ethers (ring sizes seven and nine) and bears a total of 9–12 stereogenic centres. The significant synthetic challenges presented by the asbestinins combined with their biological activities and the fact that there are ambiguities about their structures in many cases, makes them highly alluring targets for total synthesis. To date, 11‐acetoxy‐4‐deoxyasbestinin D and asbestinin‐12 are the only members of the asbestinin family of natural products that have been synthesized, as reported by Crimmins and Ellis in 2005 and 2008.1) and 4‐deoxyasbestinin C (2), and prepare several other asbestinins from these natural products thereafter.1 and 2 began with the cleavage of the ester and formation of a ketone at C‐11 and removal of the C‐12 methyl substituent to give advanced tetracyclic intermediate i  protecting group, and diol 30 was produced in high yield. Selective acetylation of the primary allylic alcohol and subsequent oxidation of the free secondary hydroxyl group delivered ketone 31. Treatment of this ketone with a large excess of methylmagnesium chloride resulted in a highly stereoselective installation of the C‐3 methyl substituent and concurrent cleavage of the acetate group.32 was produced in excellent yield.Bicyclic ether 32 into the fully functionalised tetracyclic asbestinin framework was accomplished by following the route shown in Scheme 32 with triflic anhydride in the presence of 2,6‐lutidine. The primary allylic triflate was not isolated from this reaction and instead the required cyclic allylic ether 33 was obtained in excellent yield as a result of immediate intramolecular nucleophilic displacement of the triflate by the tertiary alcohol. Hydrogenation of allylic ether 33 in the presence of Adams’ catalyst was both chemoselective and stereoselective; the tetracyclic ketone 34 was obtained as a single diastereomer in high yield.34 into the natural products 1 and 2 it was necessary to install the C‐12 methyl substituent, reduce the ketone and esterify the resulting alcohol. Direct methylation at C‐12 by ketone deprotonation and alkylation was low‐yielding and non‐stereoselective, so other methods for the introduction of the methyl substituent were explored. Reaction of ketone 34 with Bredereck's reagent35, which was subjected to immediate reduction with DIBAL‐H to afford enone 36. Reduction of this enone under Luche conditions produced the crystalline allylic alcohol 37 as a single diastereomer, but the configuration at the hydroxyl‐bearing stereogenic centre (C‐11) was not the one required for the synthesis of the asbestinins, as revealed by X‐ray analysis.36 with either L‐Selectride or Stryker's reagent38 a and its C‐12 epimer 38 b, in which the latter predominated marginally. The epimeric ketones were separated and equilibration of each ketone under mildly basic conditions delivered a mixture of diastereomers in a similar ratio to that obtained by conjugate reduction of enone 36.Conversion of tricyclic diol 1) and 4‐deoxyasbestinin C (2) was finally possible  in excellent yield and reaction of alcohol 39 with butyric anhydride produced 4‐deoxyasbestinin C (2) in good yield. The NMR and other characterization data of the synthetic esters 1 and 2 matched the data reported for each natural product.Completion of the total syntheses of 11‐acetoxy‐4‐deoxyasbestinin D (e Scheme . Reducti1) in just two or three steps. Of particular interest was the synthesis of asbestinin‐20 (6) to verify its reassigned structure (Scheme 1) was first epoxidized by treatment with mCPBA to give a mixture (2:1) of diastereomeric epoxides 40 a and 40 b. The crystalline epoxides were readily separable by chromatography and their structures were determined by X‐ray crystallography.40 a and 40 b with higher levels of diastereocontrol, the epoxidation reaction was performed by the application of Shi's asymmetric protocol using both enantiomers of the fructose‐derived catalyst.40 a:40 b). In the mismatched case, in which the (−)‐catalyst was employed, the substrate bias was overturned and a 1:5.5 ratio of diastereomers (40 a:40 b) was obtained. Epoxides 40 a and 40 b were then converted into the corresponding allylic alcohols by sequential reaction with tert‐butyldimethylsilyl triflate in the presence of 2,6‐lutidine and treatment with TBAF.6 and 9 were obtained with yields of 39 and 64 %, respectively; their structures were confirmed by X‐ray crystallography.40 a, treatment with titania‐supported gold nanoparticles in dichloroethane (DCE) at 80 °C, according to the procedure described by Garcia and Stratakis,6 in 35 % yield. Comparison of NMR and other characterization data of alcohols 6 and 9 with that of natural asbestinin‐20 confirmed that the natural product is compound 6. Thus, the reassigned structure and relative stereochemistry of asbestinin‐20 published by Ospina and Rodríguez is correct.6) produced asbestinin‐10 (14) and the structure of this natural product was confirmed. Enone reduction under Luche conditions afforded the allylic alcohols in a 3.2:1 ratio favouring asbestinin‐20 (6), a result consistent with that reported by Ospina and Rodríguez when they reduced natural asbestinin‐10 (14) under similar conditions.Several other asbestinin natural products were synthesized from 11‐acetoxy‐4‐deoxyasbestinin D  under standard Upjohn dihydroxylation conditions afforded a diastereomeric mixture of 1,2‐diols 41 a and 41 b in 56 % yield, in which the crystalline diol 41 a predominated (5:1 ratio).41 a was separated from the minor isomer 41 b and oxidized to give α‐hydroxy ketone 16 by the use of the Dess–Martin protocol. Alternatively, the diastereomeric mixture of diols 41 a and 41 b was oxidized directly to produce a separable mixture of α‐hydroxy ketones 16 (67 % yield) and 42 (14 % yield).The next target to be synthesized was asbestinin‐21, which had been originally identified as the ketone 6 Scheme .6, 8 Dih41 b as the major diastereomer by performing dihydroxylation according to the Sharpless asymmetric protocol.1) resulted in the formation of diol 41 a with an enhanced level of diastereocontrol (>12:1). However, diol 41 a was obtained in only 23 % yield, and the major product was the α‐hydroxy ketone 16 (46 % yield), an unexpected product resulting from over‐oxidation. In the case where substrate control and reagent control were expected to be mismatched, dihydroxylation of alkene 1 with AD‐mix‐β produced a mixture of diols 41 a and 41 b (75 % yield combined) along with a small amount (10 % yield) of α‐hydroxy ketone 16. Interestingly, the dihydroxylation reaction proceeded with a higher level of diastereocontrol (8:1) than for the case when the reaction was performed under simple Upjohn conditions. This unexpected result presumably reflects the larger steric bulk of the osmium complex in the case of AD‐mix and the fact the reaction proceeds under substrate control rather than reagent control. Comparison of the 1H and 13C NMR data of the α‐hydroxy ketones 16 and 42 with those of natural asbestinin‐21 revealed that the natural product is 42 rather than 16, a finding which established that the configuration of the hydroxyl‐bearing the C‐7 stereogenic centre had been misassigned by Ospina and Rodríguez when performing their structural revision.Attempts were made to produce diol ocontrol :1 than f1) with borane‐THF complex followed by oxidative work‐up produced an inseparable diastereomeric mixture of alcohols . The configurations of the stereogenic centres at C‐6 and C‐7 of alcohol 44 were determined by X‐ray crystallography,43 and 20 were established consequently. Comparison of NMR data of ketones 20 and 43 with the data of the natural asbestinin‐23 revealed that the natural product is ketone 20, which was suggested by Ospina and Rodríguez as the most likely alternative to the originally assigned structure  and 4‐deoxyasbestinin C (2) have been completed in 16 steps from the bicyclic ketone 23 and the former has been converted into abestinin‐10, abestinin‐20, abestinin‐21 and abestinin‐23 in two or three additional steps. The syntheses of abestinin‐10, abestinin‐20, and abestinin‐23 confirm the revised structures for these compounds proposed by Ospina and Rodríguez in 2006. In the case of abestinin‐21, our work has shown that this compound is the C‐7 epimer of the revised structure proposed by Ospina and Rodríguez.We have completed the stereoselective total syntheses of six members of the asbestinin family of marine diterpene natural products from the bridged‐bicyclic ether The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Shortages of personal protective equipment, including N95 respirators, during the coronavirus (CoV) disease 2019 (COVID-19) pandemic have highlighted the need to develop effective decontamination strategies for their reuse. This is particularly important in health care settings for reducing exposure to respiratory viruses, like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19. Although several treatment methods are available, a widely accessible strategy will be necessary to combat shortages on a global scale. We demonstrate that the combination of heat and humidity inactivates a range of RNA viruses, including both viral pathogens and common viral pathogen surrogates, after deposition on N95 respirators and achieves the necessary virus inactivation detailed by the U.S. Food and Drug Administration guidelines to validate N95 respirator decontamination technologies. We further demonstrate that depositing viruses onto surfaces when suspended in culture media can greatly enhance observed inactivation, adding caution to how heat and humidity treatment methods are validated. 10 inactivation of bacteriophages, and RHs greater than 25% resulted in a >3.5-log10 inactivation of MHV and IAV. Furthermore, deposition of viruses in host cell culture media greatly enhanced virus inactivation by heat and humidity compared to other deposition solutions, such as phosphate-buffered saline, phosphate-buffered saline with bovine serum albumin, and human saliva. Past and future heat treatment methods must therefore explicitly account for deposition solutions as a factor that will strongly influence observed virus inactivation rates. Overall, our data set can inform the design and validation of effective heat-based decontamination strategies for N95 respirators and other porous surfaces, especially for emerging viruses that may be of immediate and future public health concern.Supply shortages of N95 respirators during the coronavirus disease 2019 (COVID-19) pandemic have motivated institutions to develop feasible and effective N95 respirator reuse strategies. In particular, heat decontamination is a treatment method that scales well and can be implemented in settings with variable or limited resources. Prior studies using multiple inactivation methods, however, have often focused on a single virus under narrowly defined conditions, making it difficult to develop guiding principles for inactivating emerging or difficult-to-culture viruses. We systematically explored how temperature, humidity, and virus deposition solutions impact the inactivation of viruses deposited and dried on N95 respirator coupons. We exposed four virus surrogates across a range of structures and phylogenies, including two bacteriophages (MS2 and phi6), a mouse coronavirus (murine hepatitis virus [MHV]), and a recombinant human influenza A virus subtype H3N2 (IAV), to heat treatment for 30 min in multiple deposition solutions across several temperatures and relative humidities (RHs). We observed that elevated RH was essential for effective heat inactivation of all four viruses tested. For heat treatments between 72°C and 82°C, RHs greater than 50% resulted in a >6-logIMPORTANCE Shortages of personal protective equipment, including N95 respirators, during the coronavirus (CoV) disease 2019 (COVID-19) pandemic have highlighted the need to develop effective decontamination strategies for their reuse. This is particularly important in health care settings for reducing exposure to respiratory viruses, like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19. Although several treatment methods are available, a widely accessible strategy will be necessary to combat shortages on a global scale. We demonstrate that the combination of heat and humidity inactivates a range of RNA viruses, including both viral pathogens and common viral pathogen surrogates, after deposition on N95 respirators and achieves the necessary virus inactivation detailed by the U.S. Food and Drug Administration guidelines to validate N95 respirator decontamination technologies. We further demonstrate that depositing viruses onto surfaces when suspended in culture media can greatly enhance observed inactivation, adding caution to how heat and humidity treatment methods are validated. During the coronavirus (CoV) disease 2019 (COVID-19) pandemic, high demand for N95 respirators has led to interest in decontamination methods that do not compromise the effectiveness of the respirator. The application of heat, UV irradiation, and vaporized hydrogen peroxide are common decontamination treatments in medical settings, and research suggests that N95 respirators treated with these methods maintain their filtration integrity and fit 14. The U.10 inactivation , and a subtype H3N2 influenza virus  . We focuOur findings identify key parameters that drive virus inactivation on N95 respirator surfaces with heat and highlight the importance of deposition solution characteristics when validating decontamination methods. In particular, our results suggest that the common practice of depositing viruses using culture media may lead to a significant overestimation of the effectiveness of heat treatment for virus inactivation.10 inactivation, depending on the virus. Consequently, we were unable to observe the inactivation trends at elevated RHs for any of the viruses tested. We note that the dynamic ranges were between 5.5 log10 and 8.1 log10 for the bacteriophages, ∼4.0 log10 for IAV, and ∼3.7 log10 for MHV. These dynamic ranges were determined as the log10 difference in titers between the no-treatment controls and the limits of detection of the enumeration assays .To understand how temperatures and RHs impact inactivation through heat treatment, we deposited the four RNA viruses in culture media on N95 respirator coupons and treated the coupons for 30 min at 72°C and 82°C. An overall trend of increased virus inactivation was observed as temperature and RH increased see . For tre10.1128/mSphere.00588-20.4TABLE S110 inactivation of all viruses across various heat and humidity treatments and deposition solutions. Download Table S1, PDF file, 0.05 MB.LogCopyright © 2020 Rockey et al.2020Rockey et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10 and 2.0-log10 average increases in inactivation for MS2 and phi6, respectively, across different RHs  and contained different supplementary ingredients . Control10.1128/mSphere.00588-20.1FIG S110 inactivation of bacteriophages at 72°C and 13% in DMEM-A and DMEM-B. Results from independent experimental replicates (n = 2) are shown for each virus under each condition. Download FIG S1, TIF file, 0.2 MB.LogCopyright © 2020 Rockey et al.2020Rockey et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00588-20.8TABLE S5Table S5, PDF file, 0.07 MB.Compositions of DMEM-A and DMEM-B used for deposition solutions. Download Copyright © 2020 Rockey et al.2020Rockey et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10 and 2.9-log10 inactivations were observed for MS2 and phi6, respectively, when deposited in PBS. When the bacteriophages were deposited in DMEM-A and treated under the same conditions, the observed inactivations were 6.4 log10 and >6.7 log10. This is approximately a 4- to 5-log10 difference in inactivation resulting solely from the type of deposition solution. With either deposition solution, phi6 was more susceptible to heat treatment than MS2. When deposited in PBS solution, phi6 was inactivated, on average, 1.4 log10 more than MS2, although significance could not be confirmed for all temperature and RH conditions  deposition solution. We did not include MHV and IAV in these experiments due to the challenges of resuspending MHV and IAV in PBS without decreasing stock concentrations and thus decreasing the experiment dynamic ranges. For both phages, the deposition solution had a profound effect on inactivation . Bacterinditions . The dif10.1128/mSphere.00588-20.9TABLE S6t tests comparing levels of virus inactivation across different deposition solutions. Download Table S6, PDF file, 0.05 MB.Results of unpaired Copyright © 2020 Rockey et al.2020Rockey et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00588-20.10TABLE S7Table S7, PDF file, 0.04 MB.Deposition solutions used in experiments. Download Copyright © 2020 Rockey et al.2020Rockey et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10 and 3.7 log10 for MS2 and phi6, respectively. With respect to the specific effect of BSA in the deposition solution, MS2 was inactivated 1.3 log10 more when the PBS deposition solution contained BSA. Two out of four of the temperature/RH conditions exhibited statistically significant differences   . On avers in PBS . For phin in PBS . These r10 on average) for both MS2 and phi6 in all deposition matrices (P = 0.0061) or saliva (P = 0.029). Although the average inactivation was higher for phi6 deposited in DMEM-A than for phi6 deposited in PBS and saliva, the differences were not statistically significant.To assess whether virus inactivation under ambient conditions was also affected by deposition solution, we tested bacteriophage inactivation at room temperature (20°C) and 36% RH using different deposition matrices. This RH and temperature were selected at the lower end of standard thermohygrometric ranges for health care facilities . After 2matrices . MS2 ina10 inactivation of viruses, specifically those related to SARS-CoV2 , along with ≥6-log10 inactivation of either mycobacterial or bacterial spores. Our results demonstrate that heat treatment (72°C and 82°C) with moderate-to-high RHs (above 25%) for 30 min can inactivate at least 3 log10 of MS2, phi6, IAV, and MHV. However, the USFDA guidance was replaced with recommendations to demonstrate ≥6-log10 inactivation of three nonenveloped viruses and ≥6-log10 inactivation of two Gram-positive bacteria and two Gram-negative bacteria    and at aus 65°C) , 22, 23.us 65°C)  and in aus 65°C) , 42. Pruus 65°C) . Overallus 65°C) , 44, 45,10.1128/mSphere.00588-20.2FIG S210 virus inactivation as a function of absolute humidity for MS2 bacteriophage (A), phi6 bacteriophage (B), IAV (C), and MHV (D). Open faded symbols indicate virus inactivation beyond assay detection limits. Results from independent experimental replicates (n = 2) are shown for each virus under each condition. Download FIG S2, TIF file, 1.1 MB.LogCopyright © 2020 Rockey et al.2020Rockey et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Virus inactivation improved as RH increased from 1% to 48% in our experiments at 72°C and 82°C . A similar RH effect was observed previously for elevated heat treatment of IAV deposited on steel coupons . The imp10 inactivation of MS2 and phi6 bacteriophages on N95 respirators was observed, regardless of the deposition solution used. Other studies observed ≤1-log10 inactivation of MS2 and phi6 at ambient temperature and RH over 24 h  suggest that laboratory-made solutions  are not fully representative of respiratory droplets and aerosols , 35, 56.10 virus inactivation at 70°C under “dry heat” conditions for 60 min  RH treatments at the same temperatures were not as effective. We note that research to validate the inactivation of bacterial and fungal pathogens in addition to viral pathogens must be completed to entirely satisfy the USFDA’s guidelines for N95 respirator decontamination technologies for all pathogens. We also observed that inactivation was strongly influenced by the deposition solution. Dried virus droplets in cell culture media were inactivated significantly more than in any other deposition solution . These findings suggest that virus inactivation may be vastly overestimated when using culture media as the deposition solution in surface disinfection studies. We suggest the use of deposition solutions more similar to human saliva or respiratory fluid in virus inactivation experiments to ensure representative results.Our work demonstrates the virus inactivation efficacy of heat and humidity treatments for N95 respirator decontamination. The USFDA’s recommended 6-logHospitals and other health care settings can expect extensive virus inactivation of N95 respirators through heat treatment for at least 30 min at 72°C or 82°C and RH above 50%. High-humidity heat treatment is particularly appealing, as it can readily be adapted and scaled to a range of settings, from health care facilities to private residences. Further, implementation is equally suitable for health care systems or individuals without access to specialized equipment, including those in low- to middle-income countries. These results provide timely and useful information for efficacious N95 respirator decontamination, enabling reuse when necessary due to shortages.Escherichia coli host were obtained from the American Type Culture Collection (ATCC 15597). Bacteriophage phi6 and its Pseudomonas syringae pv. phaseolicola host were provided by Linsey Marr at Virginia Tech. MHV strain A59 and its murine delayed brain tumor (DBT) host cell line were provided by Julian Leibowitz at Texas A&M Health Science Center College of Medicine. For IAV, we used a recombinant virus that expresses the luciferase reporter in infected cells. This system allows for rapid titering based on light emission in infected cells. The virus is a 6+2 reassortant, in which the genomic segments encoding the surface hemagglutinin (HA) and neuraminidase (NA) are derived from A/Wisconsin/67/2005 (H3N2) and the remaining six segments are derived from A/WSN33 (H1N1). In this case, the segment 3 RNA encodes a polymerase acidic (PA) protein that is fused to the NanoLuc reporter  at 37°C and 5% CO2. Cells were infected at an MOI of ∼0.01 when they were 75% confluent and then incubated with virus for 24 h. The cell suspension was frozen at −80°C, thawed, and centrifuged at 3,000 × g for 15 min at 4°C, and then the supernatant was recovered. The resulting virus stock (∼106 PFU/ml) was filtered with a 0.22-μm PES membrane and stored in single-use aliquots at −80°C. For plaque assay enumeration, samples were diluted in DMEM-B  and applied to confluent DBT cells washed with 1× PBS . After 1 h of incubation at room temperature with rocking, the virus suspension was removed from monolayers and an overlay of 1.6% agar mixed 1:1 with a 2× minimum essential medium  containing 5% horse serum, 10 mM HEPES , 1× MEM nonessential amino acids , 2% l-glutamine, and 2% penicillin streptomycin was applied. Infected cells were then incubated for 48 h at 37°C and 5% CO2 before being stained with neutral red solution  diluted in 1× PBS to a 0.01% final concentration. Plaque assays were conducted in triplicate, and a negative medium control was performed with each assay.For MHV propagation and enumeration, DBT cells were grown in DMEM  supplemented with 10% horse serum , 1% penicillin streptomycin , and 1% IAV propagation was performed in DMEM-A  with 25 mM HEPES, pH 7.2 to 7.5, 0.1875% fraction V bovine serum albumin , 1% penicillin and streptomycin , and 2 μg/ml TPCK . IAV stocks were stored as single-use aliquots in 0.5% glycerol at −80°C. IAV cells were enumerated in MDCK-SIAT1 cells by endpoint dilution using IAV titer medium, which contained 1% BSA but otherwise had the same components as DMEM-A. Eighteen hours postinfection, media were aspirated and replaced with IAV titer medium containing 7.5 μM ViviRen live-cell substrate . Light emission was measured using a BioTek Synergy HTX luminometer using the following settings: a 3-min dark adapting hold, emission hole, top optics position, gain of 160, integration time of 1.00 s, and read height of 2.24 mm. The room temperature A well was considered positive for infection if the relative light units (RLU) were greater than or equal to twice the average background RLU from eight mock-infected wells.10 PFU/ml in various deposition solutions. These deposition solutions . Lamp intensity was measured using chemical actinometry (6 PFU/ml) in DMEM-B (7 TCID50/ml) in DMEM-A  a control experiment to test the potential impact of drying time on virus reduction and (ii) methods for assessing virus recovery from N95 respirator coupons. Download Copyright © 2020 Rockey et al.2020Rockey et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The temperature- and humidity-controlled oven (TestEquity 123H temperature/humidity chamber) used in all experiments was calibrated for temperature to be accurate within 2°C and for RH to be accurate within 5%. A second external instantaneous hygrometer probe  rated to be accurate to within 0.2°C and 1.5% RH was also used to monitor the oven. The experiments were designed to test regular intervals of RH using the humidity chamber’s readout ; however, due to the lower error associated with the external hygrometer, external hygrometer values  were used in the data analysis.The heat- and humidity-controlled oven was set to the desired RH and temperature for at least 30 min prior to use. Dried coupons were immobilized on a coated metal test tube rack with metal binder clips. The metal rack with coupons was then transferred to the oven at the predetermined temperature and humidity settings. Treatment times were started when the oven RH was within 1% of the target RH for 13 to 71% RH and within 5% of target RH for 1% and 89% RH. The oven reached these conditions within 5 min. Heat decontamination experiments with phi6 and MS2 deposited in PBS and DMEM-A were conducted at 72°C and 82°C and 1%, 13%, 25%, 36%, 48%, 71%, and 89% RH. Experiments with IAV and MHV were carried out in duplicate for a subset of experimental conditions . Experiments with additional deposition solutions, including human saliva and PBS with BSA, were carried out for MS2 and phi6 at 72°C with 13% and 25% RH and at 82°C with 1% and 13% RH. These additional experiments were not conducted with MHV and IAV due to issues with resuspending them in saliva and PBS without further reducing the dynamic range of the experiments. DMEM-A and DMEM-B contained different supplementary ingredients; to assess the possible effects of these differences on observed virus inactivation, duplicate control experiments with MS2 and phi6 at 72°C and 13% RH were carried out in both DMEM-A and DMEM-B.Twenty-four-hour experiments were conducted with MS2 and phi6 to assess whether the virus deposition solution also had an effect on virus inactivation when N95 respirators are stored under ambient conditions in health care settings. MS2 and phi6 suspended in DMEM-A, PBS, PBS with 0.1785% BSA, and saliva were deposited on N95 respirator coupons as described above for the elevated temperature experiments. Coupons were then incubated in the temperature- and humidity-controlled oven at 20°C and 36% RH for 24 h. Infective virus concentrations on coupons following 24 h of incubation were compared to the infective virus concentrations on control coupons that were determined immediately after droplets were dried for 1 h. Three independent replicates of the experiment were conducted.To recover viruses from control and treated coupons, the coupons were cut into 4 to 6 pieces with sterilized scissors and suspended in 1.3 ml elution medium. For phi6 and MS2, the elution medium consisted of 1.3 ml of 1% BSA  in PBS. For MHV, the elution solution was DMEM-B. For IAV, the elution solution was IAV titer medium . The coupon suspensions were vortexed at medium speed for 1 min. Viruses extracted in the elution buffer were enumerated as described above. To assess recovery from the coupons, 50 μl of the virus deposition solution was suspended into 1.3 ml of 1× PBS with 1% BSA, DMEM-B, or IAV titer medium for the phages, MHV, or IAV, respectively. Virus recovery was determined as the ratio of the control coupon virus titer to that of the suspended virus solution and were greater than 7% for all viruses and conditions .t tests were performed to determine differences in virus inactivation for different treatment conditions and viruses using GraphPad Prism 8 software. Statistical significance was considered a P value of <0.05.Unpaired


Subacute sclerosing panencephalitis (SSPE) is a late-onset, intractable, and fatal viral disease caused by persistent infection of the central nervous system by a mutant strain of the measles virus. Ribavirin intracerebroventricular therapy has already been administered to several SSPE patients in Japan based on fundamental and clinical research findings from our group, with positive therapeutic effects reported in some patients. However, the efficacy of this treatment approach has not been unequivocally established. Hence, development of more effective therapeutic methods using new antiviral agents is urgently needed. This review describes the current status of SSPE treatment and research, highlighting promising approaches to the development of more effective therapeutic methods. Despite the availability of effective measles vaccination programs, measles remains a major cause of child mortality worldwide. The WHO Global Vaccine Action Plan aims to achieve measles eradication in at least five World Health Organization regions by 2020 . HoweverSubacute sclerosing panencephalitis (SSPE) is a progressive and fatal neurodegenerative encephalitis caused by persistence of the measles virus in the central nervous system (CNS). Since the growth of SSPE virus is thought to be directly involved in neuropathy in SSPE, treatment with antiviral drugs and immunostimulants that suppress virus growth has been attempted. This review discusses various prevalent approaches and new avenues in SSPE antiviral therapy.SSPE is a slow viral infection caused by a mutated measles virus (SSPE virus). This disease develops approximately 2 to 10 years after the onset of measles, causing diminished intelligence, personality changes, bradykinesia, etc. Subsequently, cerebral function is progressively impaired, leading to severe dementia, vegetative state, and eventually, death . The diaSince the prognosis of SSPE is poor, the best means of controlling this disease is by preventing its occurrence. The number of SSPE patients is positively correlated with the number of measles patients , and theMoreover, individuals with SSPE are more likely to have had measles during infancy. In a study of 350 SSPE patients in the United States, 292 patients 83.4%) had a history of spontaneous infection of measles and were more likely to have had measles under the age of 2 (24% under 12 months and 22% at 12–24 months). Of the 58 patients with no history of measles, 40 had been vaccinated with the live measles vaccine % had a h. BelliniIn 1933, a report by Dawson suggested that SSPE is caused by a virus . SubsequParamyxoviridae and the order Mononegavirales. This enveloped virus produces pleomorphic virus particles with size up to 900 nm, and an average size of 150–300 nm [N-terminus, followed by seven repeating domains complementary to the N-terminus and C-terminus, respectively [The measles virus belongs to the Morbillivirus genus within the family 0–300 nm . Its gen0–300 nm , and the0–300 nm ,25,26,27ectively ,29. The ectively ,30,31,32ectively . On the ectively . Moreoveectively ,35. Wildectively ,37,38. Tectively ,40. HoweOn the contrary, some reports suggest the involvement of host factors in the onset of SSPE. For instance, children infected with measles before the age of two are at an increased risk of developing SSPE . It is sSince the incidence of SSPE is extremely low in developed countries, randomized controlled trials on SSPE with large sample sizes have not been reported. Nevertheless, therapies involving oral administration of inosine pranobex and intraventricular administration of interferons have been attempted and proven to be effective in a relatively large number of cases.p < 0.01), as the 8-year survival rate of 98 patients who received IP was 61% compared to the survival rate of 8% seen in patients who did not receive this treatment [Inosine pranobex (IP), a combination of inosine, acetamidobenzoic acid and dimethylamino isopropanol, is a drug that exerts both antiviral and immunostimulatory effects . The exareatment .Interferons are naturally produced by animal cells as a defense mechanism, and are used in the treatment of SSPE due to their antiviral activity. One to three million units of interferon (IFN) (α or β) is typically administered intrathecally or intraventricularly one to three times a week to treat SSPE. IFNs have also been reported to be effective when used in combination with IP. Yalaz et al. reported improvement of symptoms in 50% of patients (11/22) when intraventricular IFNs were delivered with IP , while GIn search for alternatives, we screened for drugs showing anti-SSPE effects in vitro and identified the nucleic acid analogs pyrazofurin, 6-azauridine, 3-deazaguanine, and ribavirin as candidate drugs . RibavirHowever, antiviral effects of ribavirin against SSPE have been observed in animal experiments. Daily intracerebral administration of ribavirin to hamsters that ingested a lethal dose of SSPE virus improved survival rate in a dose-dependent manner  and incrHence, high-dose intravenous administration of ribavirin was attempted in human SSPE patients as well, based on the report that blood ribavirin is transferred to the central nervous system via the blood-brain barrier . The minTherefore, administration of ribavirin directly into the ventricle, similar to IFN, was attempted to maintain concentrations of ribavirin in the brain sufficient for eliciting its inhibitory effects on the SSPE virus. Ribavirin was administered intraventricularly using an Ommaya reservoir. The concentration of ribavirin in CSF was maintained at a concentration that completely suppresses the growth of SSPE virus by administering 1–3 mg/kg per day of ribavirin directly into the ventricles one to three times a day ,79. WhilHowever, it was difficult to achieve the effective concentration of ribavirin in CSF in some cases by the intraventricular administration method through the Ommaya reservoir  Figure A. Such d®, Codman, Germany) was attempted. This pump had been previously used for intrathecal administration of baclofen as treatment for spastic paraplegia [To maintain effective concentrations in CSF and to avoid frequent puncture during bolus administration (single repeated administration), continuous administration of ribavirin into the ventricles (continuous infusion therapy) by a subcutaneous implantable continuous infusion pump , which is a viral RNA synthesis inhibitor, and its analogs AS-136A and 2O (ERDRP-00519) show high anti-measles virus activity in vitro ,93,94. I-Phe Gly  and AS-4-Phe Gly ,98. Furt-Phe Gly ,100. ThuSince the incidence of SSPE is low in developed countries, evaluating the efficacy of drugs against SSPE using comparative studies with a control drug is difficult. In addition, the clinical course of SSPE varies from case to case, and therefore, the effectiveness of antiviral therapy and changes in prognosis must be carefully determined by long-term observation in a larger number of cases. Our studies suggest that intraventricular administration of ribavirin resulted in a clear improvement in symptoms in patients who started treatment in the early stages, indicating the importance of early diagnosis and treatment of SSPE. Clearly, drug development for SSPE is an urgent issue since none of the currently available drugs can cure SSPE. Hence, improving the measles vaccination rate and eradicating measles are the best strategies for controlling and preventing the onset of SSPE. However, new treatment approaches using molecules that display potential against SSPE must also be explored, with special focus on developing oral or intravenous drugs that can penetrate the blood-brain barrier and show desired anti-SSPE activity.


Bovine viral diarrhoea (BVD) is an important disease of cattle, with significant impacts on animal health and welfare. The wide host range of the causative pestiviruses may lead to formation of virus reservoirs in other ruminant or wildlife species, presenting a concern for the long-term success of BVD eradication campaigns. It is likely that the quasispecies nature of these RNA viruses contributes to their interspecies transmission by providing genetic plasticity. Understanding the spectrum of sequence variants present in persistently infected (PI) animals is, therefore, essential for studies of virus transmission. To analyse quasispecies diversity without amplification bias, we extracted viral RNA from the serum of a PI cow, and from cell culture fluid after three passages of the same virus in culture, to produce cDNA without amplification. Sequencing of this material using Illumina 250 bp paired-read technology produced full-length virus consensus sequences from both sources and demonstrated the quasispecies diversity of this pestivirus A genotype 1a field strain within serum and after culture. We report the distribution and diversity of over 800 SNPs and provide evidence for a loss of diversity after only three passages in cell culture, implying that cultured viruses cannot be used to understand quasispecies diversity and may not provide reliable molecular markers for source tracing or transmission studies. Additionally, both serum and cultured viruses could be sequenced as a set of 25 overlapping PCR amplicons that demonstrated the same consensus sequences and the presence of many of the same quasispecies variants. The observation that aspects of the quasispecies structure revealed by massively parallel sequencing are also detected after PCR and Sanger sequencing suggests that this approach may be useful for small or difficult to analyse samples. Pestivirus A sequences used in this article are as shown in Bovine viral diarrhoea (BVD) viruses are globally important cattle pathogens, which impact performance due to acute infection and BVD-induced immunosuppression. Eradication of BVD in cattle is widely pursued, but is hampered by the production of persistently infected calves – the offspring of cows infected in early pregnancy – which shed virus constantly and drive the spread of infection. Genetic variation in BVD viruses is an important feature of their biology, allowing them to adapt to changing conditions and to infect different hosts. Inaccurate virus replication produces a population of viruses with slightly different sequences, a quasispecies, some of which may grow better in other hosts or in culture. Analysing virus sequence variation may help us understand how the virus evolves within and between its hosts. In this paper, we show that a BVD virus strain loses quasispecies diversity quickly when cultured and that these changes can be detected even in small diagnostic samples, implying that cultured viruses do not perfectly represent the field strains they were isolated from and, therefore, may not provide reliable molecular markers for source tracing or transmission studies.www.ebi.ac.uk/ena) with the GenBank/EMBL/DDBJ accession numbers as follows. Consensus genome sequences: MRI103 serum NGS, LR699799; MRI103 culture NGS, LR699800; MRI103 serum Sanger, LR699801; MRI103 culture P3 Sanger, LR699802; MRI103 culture P5 Sanger, LR699803. NGS raw data: serum dataset, ERR3624580; culture dataset, ERR3624581.Sequence data associated with this paper have been submitted to the European Nucleotide Archive  and BVDV type 2 (Pestivirus B) are considered distinct virus species . Th. Th47]. Library generation used the TruSeq CHiP protocol (Illumina) because of the small amounts of cDNA available (<15 ng DNA per sample) and each library was tagged with barcoding primers to allow multiplex analysis of all six samples in a single MiSeq run with 250 base paired-end reads. Data was returned as FastQ format files.bwa .Examples of sequencing traces from polymorphic positions (including two that represent consensus base changes between serum and cultured viruses) are shown in Overall, in the serum PCR sequence, 58 positions were called as polymorphic, including 10 that were not seen in the serum NGS dataset. Among the remaining 48 positions, the corresponding SNP frequency in the serum NGS dataset varied from 4.9 to 46 %. These included 68 % of serum NGS SNPs with frequency >10 %. Within the P3 culture PCR sequence, 33 positions were called as polymorphic, all of which were variable in the NGS data, with SNP frequency ranging from 5 to 80 %. These positions included 80 % of P3 culture NGS SNPs with frequency >10 %.For the P5 culture PCR sequence, 57 positions were called as polymorphic. In the absence of a P5 NGS dataset, these were compared with the P3 NGS data. This showed that 12 SNPs were not found in the P3 culture NGS data. Corresponding SNP frequencies for the remaining 45 SNP positions in the MRI-P3-dT dataset varied from 1.4 to 91 %, including 73 % of P3 culture NGS SNPs with frequency >10 %. The P5 PCR dataset was clearly distinct from the serum and P3 sequences, and contained 17 SNPs at positions that had variant frequencies below 5 % in the P3 culture NGS dataset. This may reflect further changes in quasispecies structure between P3 and P5, with some variant bases becoming more highly represented in the P5 virus population.www.ebi.ac.uk/ena). Of these, most were sequenced by Sanger methods after cloning or amplification of genome segments, while many of the more recently determined sequences were defined by NGS approaches. In almost all cases, however, only the consensus sequences have been published and quasispecies variation has seldom been examined. For example, Workman and co-workers [The number of complete genome or polyprotein sequences for pestiviruses that are publicly available now exceeds 100 for each of pestivirus A and B  in the serum virus, raises the question of their potential function. Genomes carrying stop codons may be defective or may express truncated gene products. Genomes with polyprotein truncation may enhance the survival of the population as a whole, or be rescued by translational frameshifting, the use of alternative start codons or by complementation in cells infected with multiple viruses. Some stop codon mutations may benefit persistent infection by reducing the expression of viral antigens , althougin vitro . The obsrns  and E2  and in each case the substitutions lay within the exposed domains of the proteins , which has RNase activity, and the C-terminal region (residues 167–227), which mediates membrane retention and processing. These residues have not been recorded as being associated with any recognized antibody epitope [rns structure/function.The four consensus base changes in the cultured virus sequence were also those that had the greatest change in SNP frequency across the entire genome (Table S2). The changed consensus bases encoded amino acid changes to the virus glycoproteins Erns, the substitutions in E2 appeared to be conservative in nature and may not cause any clear change in structure or function of E2. However, the observation that mutations in the Erns glycoprotein-encoding gene can lead to failure of diagnostic assays implies that serum-derived and culture-derived samples of the same virus could give different results in such assays [In E2, the substitutions were in the two outermost  domains that possess immunoglobulin-like folds, and the substitution in the outermost domain (V61I) was close to a recognized neutralizing motif in E2 of classical swine fever virus (residues 64–76), which has analogous structure . As seenh assays .in vitro on sites that are not under such pressure in vivo.Although the overall degree of variation within the cultured virus was lower than in the serum virus genome, it was clear from analysis of shared SNPs that culture passage did not lead to a consistent reduction in the degree of variation of individual SNPs, suggesting that culture may select for increased frequency of some SNP variants but not others. In this context, we also note that the 12 variant positions within the NADL genome, with SNP frequencies of 2.1–6.3 %, did not appear to correspond with SNP positions in the MRI103 genome, when the two genome sequences were aligned (Table S2). This may suggest that extended culture exerts selective pressure Drivers that may act to fix sequence changes include selection pressures resulting from infection of new cell types within a host, of new individuals or of new species. Where available, data on cross-species infection suggest that success may be facilitated by the presence of ‘pre-adapted’ variants that could be selected and rapidly established in the new host . The appSanger sequencing of MRI103 RT-PCR products produced the same virus consensus sequences as the NGS analysis and the trace data identified many polymorphic sites , suggestThe PCR-based Sanger sequence data obtained here reflected the quasispecies variation detected by NGS, but SNPs with frequency<10 % in the corresponding NGS dataset appeared to be more difficult to detect, reflecting the more qualitative nature of Sanger sequencing. The application of NGS analysis to PCR amplicon sequencing could be an easy and cost-effective way to gather information on quasispecies variation and allow evolution within chains of infection to be monitored. Such a forensic use of sequencing could also be applied in situations such as the late stages of BVD eradication campaigns, where single locus sequencing may not distinguish similar viruses shared across multiple premises from true transmission events. However, it is clear that for such forensic epidemiology we will also need to improve our understanding of virus sequence variation within a chain of infection and between animals infected by the same source.rns and E2 glycoproteins, were found after only three passages in culture, while fifteen novel variant sites were observed in the virus after five passages. This was accompanied by a clear reduction in quasispecies diversity with culture, as defined by the number of variant sites and total entropy, suggesting that genome sequences derived after virus isolation may not present an accurate picture of the quasispecies variation in vivo or the true virus consensus sequence. The very low total entropy of variant sites in the NADL genome analysed here suggests that extensive culture works to maintain a genome sequence that is well-adapted to the tissue-culture environment.This work provides evidence that initial culture of a pestivirus A strain drives virus evolution, producing changes in the virus consensus sequence. Here, four clear changes in the MRI103 virus consensus sequence, encoding amino acid sequence changes in the Ein vivo, although previous work suggests such reversion might occur [While this study has demonstrated clear changes in quasispecies structure after only three passages in culture, additional experiments could enhance the future value of this work. Repetition of the culture passages and quasispecies analysis would provide useful information on the reproducibility of the changes induced by culture, while infection of BVD-naive animals with the cultured virus could help define whether the changes were reversible ht occur . FurtherWe also showed that PCR amplification of viral RNA produces amplicons in which many viral polymorphisms appear to be conserved. Further quantitative analysis, with and without PCR amplification, will help define how closely genome sequence diversity in PCR amplicons reproduces that obtained directly from viral RNA. However, the data produced here suggest that even Sanger sequence analysis of PCR-amplified virus genome fragments can identify quasispecies variation in the source material, at least for more frequent variants. The combination of PCR amplification and NGS analysis of the quasispecies structure of virus genomes may be a powerful approach to allow investigation of samples that would otherwise be difficult or impossible to study.1. Russell GC, Zadoks RN, Willoughby K, Bachofen C. European Nucleotide Archive (www.ebi.ac.uk/ena/) accession numbers: consensus genome sequences, LR699799–LR699803; NGS raw data, ERR3624580 and ERR3624581 (2019).Click here for additional data file.


Enterocytozoon bieneusi in three wild rabbits and Encephalitozoon intestinalis in one wild rabbit and three Iberian hares. However, none of the 462 sampled wild lagomorphs showed Encephalitozoon hellem nor Encephalitozoon cuniculi infection. This is the first report of E. intestinalis infection in wild rabbits and Iberian hares. The presence of E. bieneusi and E. intestinalis in organ meats from wild lagomorphs can be of public health concern. Additional studies are required to determine the real prevalence of these parasites in European wild rabbit and Iberian hare.A cross-sectional study was carried out to assess the presence of zoonotic microsporidia in organ meats of European wild rabbits and Iberian hares consumed by humans in Spain. Between July 2015 and December 2018, kidney samples from 383 wild rabbits and kidney and brain tissues from 79 Iberian hares in southern Spain were tested by species-specific polymerase chain reactions (PCRs) for the detection of microsporidia DNA. We confirmed the presence of Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, and Encephalitozoon cuniculi) in organ meats of European wild rabbit (Oryctolagus cuniculus) and Iberian hare (Lepus granatensis) consumed by humans in Spain. Between July 2015 and December 2018, kidney samples from 383 wild rabbits and kidney and brain tissues from 79 Iberian hares in southern Spain were tested by species-specific PCR for the detection of microsporidia DNA. Enterocytozoon bieneusi infection was confirmed in three wild rabbits  but not in hares , whereas E. intestinalis DNA was found in one wild rabbit  and three Iberian hares . Neither E. hellem nor E. cuniculi infection were detected in the 462  lagomorphs analyzed. The absence of E. hellem and E. cuniculi infection suggests a low risk of zoonotic foodborne transmission from these wild lagomorph species in southern Spain. To the authors’ knowledge, this is the first report of E. intestinalis infection in wild rabbits and Iberian hares. The presence of E. bieneusi and E. intestinalis in organ meats from wild lagomorphs can be of public health concern. Additional studies are required to determine the real prevalence of these parasites in European wild rabbit and Iberian hare.Microsporidia are obligate intracellular protist-like fungal pathogens that infect a broad range of animal species, including humans. This study aimed to assess the presence of zoonotic microsporidia ( Oryctolagus cuniculus) and the Iberian hare (Lepus granatensis) are two endemic species in the Iberian Peninsula. Both lagomorph species play a key role in the ecology of Mediterranean ecosystems [Lynx pardinus) and the Spanish imperial eagle  [The European wild rabbit  . These lalberti) . Public alberti) . In thisalberti) ,7,8.Enterocytozoon bieneusi, Encephalitozoon intestinalis, E. hellem, and E. cuniculi are the most common species reported in humans [The phylum Microsporidia comprises more than 1500 obligate intracellular spore-forming parasites classified as fungi that can infect a wide range of vertebrate and invertebrate hosts through the fecal–oral route . Among tn humans ,11. Micrn humans .Sylvilagus floridanus), the European brown hare (Lepus europaeus), and the European rabbit [E. bieneusi, E. intestinalis, E. hellem, and E. cuniculi in organ meats from European wild rabbit and Iberian hare, the most frequent wild lagomorph species consumed by humans in Spain.Foodborne transmission has previously been evidenced as a zoonotic route of microsporidia infection . In thisn rabbit ,15,16,17n rabbit ,19. Howen rabbit . Hence, 2) between July 2015 and December 2018. The sample size for wild rabbits was calculated assuming a prevalence of 50%  with a 95% confidence level (95% CI) and a desired precision of ±5% [A cross-sectional study was carried out on wild lagomorphs in Andalusia (southern Spain) , and 1.25 U of Taq polymerase. Conditions for PCR reactions were as follows: denaturing at 94 °C for 30 s followed by 35 cycles of annealing at 45 °C for 30 s for E. intestinalis primers or 55 °C for 30 s for the remaining microsporidia species and extension at 72 °C for 90 s. PCR products were analyzed by electrophoresis in 2% agarose gel stained with ethidium bromide and examined under UV light.Genomic DNA was extracted from the kidney and brain samples using the G-spin™ total DNA extraction kit , following the manufacturer’s instructions. PCR amplifications were performed using the GenAmp kit  to detect small-subunit rRNA (SSU rRNA) coding regions of the microsporidia using species-specific primers previously described  in a GenE. bieneusi infection was detected in wild rabbits  but not in Iberian hares . E. intestinalis was confirmed in one wild rabbit  and in two brain and one kidney samples from three Iberian hares . Co-infections with E. bieneusi and E. intestinalis were not observed. None of the 462  wild lagomorphs showed positive results for E. hellem or E. cuniculi infection in the examined organs.Microsporidia infection was detected in 7 of the 462  wild lagomorphs analyzed. The distribution of prevalence according to species, province, year, age, and sex is shown in E. bieneusi, E. intestinalis, E. hellem, and E. cuniculi, in wild rabbits in the Iberian Peninsula and also the first report of microsporidia infection in Iberian hares worldwide. The presence of E. bieneusi infection in wild rabbits is consistent with that previously documented [E. bieneusi infection has also been detected in farmed rabbits [E. bieneusi in wild lagomorphs in the study area. In Spanish Mediterranean ecosystems, E. bieneusi infection has been detected in different sympatric species, including wild boar [Vulpes vulpes), beech martens (Martes foina), and European badgers (Meles meles) [E. bieneusi isolates obtained from humans, wild boar, and wild carnivores provides evidence of the zoonotic potential of wildlife [E. bieneusi infection has been reported in HIV/AIDS patients, organ transplant recipients, and immunocompetent individuals in Spain [This is the first large-scale study on zoonotic microsporidia species, including cumented  and conf rabbits ,28,29. Tild boar , red foxs meles) ,27, somewildlife ,30. In tin Spain ,32,33. FE. intestinalis infection in wild rabbits and Iberian hares, which increases the number of animal species susceptible to this microsporidian species. There is only one previous report of E. intestinalis infection in wild lagomorphs, particularly European brown hares [E. intestinalis infection has been reported in humans in several European countries [E. intestinalis circulation in this country and, therefore, the risk of transmission of these food-borne zoonotic pathogen cannot be ruled out. Based on the number of animals infected with E. bieneusi or E. intestinalis found in the present study and hunting bag records for wild lagomorphs [To the best of our knowledge, this is the first report of wn hares . E. inteountries ,35,36. Egomorphs , around E. hellem or E. cuniculi DNA was not found in any of the 383 wild rabbits or 79 Iberian hares analyzed. These results are consistent with previous studies that also failed to detect these parasites in feces in wild and domestic rabbits [E. hellem was confirmed in the kidneys of a free-living European brown hare with chronic interstitial nephritis [E. cuniculi infections have been confirmed in kidney and brain tissues of free-living Eastern cottontail rabbits [E. cuniculi exposure has also been detected in wild rabbits in other European countries, with seroprevalence values ranging between 3.9% in France and 44.7% in Slovakia [E. cuniculi infection in Iberian hares in our study is in accordance with previous observations in other hare species [E. hellem and E. cuniculi circulation in the wild lagomorph populations in Spain. rabbits ,27,28,29 rabbits . In contephritis . In addi rabbits  as well  rabbits ,38. SeroSlovakia ,39. The  species ,40. Like species ,41. FurtE. intestinalis DNA was detected in brain samples from Iberian hares, whose kidney tissues tested negative. Unfortunately, brain samples could not be collected from wild rabbits in the present study. While kidney tissue has been shown to be suitable for detection of microsporidia infection in wildlife species [E. intestinalis obtained in wild rabbits in the present study, as well as those previously reported in other species, may be underestimated. To increase the sensitivity of microsporidia detection in organ meat samples, kidney and brain tissues should be tested in parallel.Our study has some limitations that should be noted. Even though our objective was focused on assessing the risk of zoonotic foodborne transmission of the selected microsporidia species, fecal or duodenal tissue could be more appropriate samples for detecting infection of some of these pathogens. On the other hand,  species , includi species , previou species ,41,42. OE. hellem or E. cuniculi denotes a limited role of wild rabbits and Iberian hares in the zoonotic transmission of these microsporidia species in southern Spain. The detection of E. bieneusi in wild rabbits and E. intestinalis in both wild lagomorph species could be of public health concern. Further studies are warranted to elucidate E. bieneusi and E. intestinalis infection levels in meat and products derived from wild lagomorphs and the risk of transmission of these food-borne zoonotic pathogens.The absence of positivity to