The current classification of animal viruses is largely based on the virus molecular world. Less attention is given to why and how virus fitness results from the success of virus transmission. Virus transmission reflects the infection-shedding-transmission dynamics, and with it, the organ system involvement and other, macroscopic dimensions of the host environment. This study describes the transmission ecology of the world main livestock viruses, 36 in total, a mix of RNA, DNA and retroviruses. Following an iterative process, the viruses are virtually ranked in an outer- to inner-body fashion, by organ system, on ecological grounds. Also portrayed are the shifts in virus host tropism and virus genome. The synthesis of the findings reveals a predictive virus evolution framework, based on the outer- to inner-body changes in the interplay of host environment-transmission modes-organ system involvement-host cell infection cycle-virus genome. Outer-body viruses opportunistically respond to the variation in the external environment. For example, respiratory and enteric viruses tend to be associated with poultry and pig mass rearing. Ruminant and equine viruses tend to be more deep-rooted and host-specific, and also establish themselves in the vital inner-body systems. It is concluded that the framework may assist the study of new emerging viruses and pandemic risks. Animal viruses may be split into transmissible and persistent viruses. It has been proposed that transmissible viruses correlate with replication and virulence and that virus persistence instead permits a lower transmission rate . This inThe present study seeks to take these insights to the next level. Starting point in the analysis is the link between virus propagation and transmission success. Virus transmission may be considered to present the backbone of virus ecology, determining the viruses selected for. Unfortunately, the current classification of animal viruses emphasizes the importance of virus genomic architecture and the host cell infection cycle . Less atThe current analysis explores how the virus molecular world and the macroscopic dimensions of the host environment are intertwined, integral to one and the same virus transmission ecology. In vertebrate hosts the more vital organ systems are shielded off from external aggressors, small or large. It may be assumed that also the host immune defenses are structured to ensure that harmful viral pathogens remain confined to the outer-body environment, the epithelia. Epithelial viruses interface with the external environment and respond to the variation encountered here. Opposingly, infiltrative viruses establish in the inner-body environment and are expected to evolve towards a more intimate virus-host relationship. Given that the virus-host relationship changes with the position of the virus in the outer- to inner-body continuum the analysis focuses on the extent of virus host-body infiltration.The virus organ system tropism is assumed to evolve in harmony with the virus cell tropism. This may be inferred from the dichotomy in the release of viruses from epithelial cells. Apart from direct cell-to-cell transmission , virusesTaking an invasion ecology perspective, the host-body is viewed as a mosaic of organ systems. Viruses and organ systems are virtually portrayed in an outer- to inner-body fashion, based on the outer- to inner-body shifts in the virus infection-shedding-transmission characteristics which, in turn, result from the shift in virus organ system tropism. It is assumed that the nature of the virus-host interaction changes with the position of the virus in the outer- to inner-body continuum. The study describes the transmission ecology of the world main livestock viruses. The rationale for selecting the world main livestock viruses relates to the host damaging effects of these pathogens, the overt clinical signs and gross pathology, translating in prominent virus shedding and obvious virus transmission modes. Moreover, because of the major economic impact of these diseases, the causative viruses and the corresponding infection-transmission dynamics have been well studied.Placed in a wider perspective the analysis builds on the growing perception that viruses deserve to be viewed as evolving living entities ,12,13. Ap < 0.0005. It was thus found that viruses infiltrating internal organs either cause persistent infections or a combination of acute and persistent infections. Conversely, persistent viruses either colonize internal organs or a combination of internal organs and epithelia. Hence, the length of the infection period appears to present a measure for the extent of virus host-body infiltration. The analysis entailed an iterative process. As a first step, the one-to-three scores allocated to the 36 livestock viruses for the four ecological variables were examined in more detail. The scores are shown in Orthomyxoviridae and the Paramyxoviridae was found to strictly result from the involvement of the epithelia. The transmission of the viruses belonging to the Coronaviridae, the Picornaviridae and the Poxviridae was in part modulated also by the internal organ systems. The transmission of the viruses belonging to the Arteriviridae, the Flaviviridae, the Herpesviridae, plus also the single infectious bursal disease virus (IBDV), resulted from epithelial modes as well as internal organ systems involvement. Finally, the transmission of the single bluetongue virus (BTV) plus the viruses belonging to the Retroviridae family either reflected the involvement of epithelia plus internal organ systems or of just internal organ systems. A Spearman correlation of the A–D virus family specific infiltration ranking and the length of the infection period scores yielded an R = 0.71 and p = 0. The result indicates that the interrelationships among virus families may be defined in ecological terms and that the virus families may be neatly lined up in an outer- to inner-body fashion, virtually. As a next step, the eleven virus families in the study were grouped and ranked A–D on the basis of the infiltration scores allocated to the individual family viruses, see Next, the organ system tropisms of the viruses belonging to each family were collectively fitted and with the naked eye aligned with the Next, the A–D family ranking was converted into a one-to-four virus infiltration score applicable to individual viruses. Further, these scores are shown in p = 0. When the somewhat atypical, vector borne bluetongue virus was removed from the correlation, R remained 0.73 for the one-to-four scoring, became 0.74 for the A–D virus family specific ranking, and 0.62, with p = 8 × 10−5, for the one-to-three infiltration ranking. When the new, one-to-four virus infiltration scores were matched to the scores for the length of the infection period Spearman R became 0.73, and Next, all of the above findings were considered in conjunction with the literature data on the transmission ecology collated for each of the 36 viruses in For the epithelial, outer-body viruses it turned out that the length of the infection and shedding periods, as well as the virus environmental survival rate generally increased from respiratory tract to alimentary tract to skin. The respiratory viruses transmitted on the basis of aerosols, direct contact or fomites. Alimentary tract viruses were found to transmit on the basis of a fecal-oral cycle, through direct contact, contamination of feed and water, or involving fomites, persons and vehicles. Viruses infecting both respiratory and alimentary tract featured a mix of these transmission modes. Mostly, these viruses caused rather severe infections. Among the skin viruses, the more infiltrative viruses affecting all layers of the skin caused slowly healing lesions. The transmission of these deep-rooted skin viruses was found to rely on abrasion or biting flies rather than on direct touch or on indirect contact, more typical for superficial skin lesions. Some of the epithelial viruses are shed in feces over a prolonged time period, also in the absence of clinical signs, and these infections were considered to feature a systemic component. Next, the epithelial herpesviruses establishing latently in peripheral nerves and ganglia were found to cause a recurrence or persistence of the mucosal and/or skin infection, including of the distal urogenital tract and external genitalia. Virus infiltration of the inner-body environment frequently implicated the genital tract or reproductive system in general. This was found to be the case for the RNA, the DNA and for the retroviruses in the study. Virus establishment in the reproductive system translated in seminal transmission, haphazard abortion, late term abortion, stillbirth, birth of infected, yet apparently healthy offspring or, also, lactogenic transmission. The vertical transmission modes were common among the utmost deep-rooted viruses, the viruses infiltrating also the immune and circulatory systems. Some of the utmost infiltrative viruses featured an absence of epithelial transmission modes and were environmentally labile. Virus infiltration of the immune system associated with immune-suppression, severe infections, neoplasia, or instead with in-apparent, persistent infection. Virus infiltration of immune and circulatory systems associated with iatrogenic transmission modes. Virus circulation in the bloodstream facilitated arthropod borne transmission. As indicated in p = <0.0005. The indication that at least in broad terms the extent of virus inner-body infiltration correlated with a loss of virus robustness was applied in the virus ranking, along with the other factors. The finding that virus environmental survival in the outer-body environment increased from respiratory tract to alimentary tract to skin and decreased with the shift from the outer- to the inner-body environment prompted a re-examination of the relationship between the extent of virus host-body infiltration and virus environmental survival. Virus infiltration scores two-to-four, running from primarily epithelial transmission, to transmission involving also internal organ systems, to transmission primarily involving internal organ systems, were matched to the one-to-three virus environmental survival rate scores, yielding an R = −0.59 and Furthermore, the outer- to inner-body shifts in virus host tropism and virus genome were examined. Underlined in p < 0.005. From RNA to DNA to retrovirus the ruminant and equine viruses gained in prominence, with R = 0.40 and p < 0.05, and the infection severity level decreased, with R = −0.45 and p < 0.01. Moreover, the ruminant and equine viruses were found to cause less severe infections than the poultry and pig viruses, with Spearman R = −0.55 and p < 0.001. Hence, from outer- to inner-body, the virus genome type and host tropism appear to shift in concert, along with the infection-transmission dynamics. The extent of the host-body infiltration was found to increase from RNA to DNA to retrovirus, with R = 0.48 and The synthesis of the findings is presented in Implied by A diametrically opposite scenario is given by relatively stable host environments observed in ruminant and equine husbandry, with parent stock and their young grazing together in the open, not unlike wild herbivore ecologies. The viruses attracted and selected for establish in the vital inner-body systems and transmit vertically, via needles or via bloodsucking arthropods. At the molecular level, virus establishment in the vital body systems is matched by low replication rates and minor or slowly evolving host damage. The utmost infiltrative viruses in the study are the retroviruses. In addition, some of other RNA viruses are deep-rooted.The DNA viruses in the study take an intermediary position.It has been established that epithelial viruses are highly evolvable, more so than inner-body viruses . EpithelThe rinderpest virus (RPV) in cattle and buffaloes primarily colonizes the alimentary tract and transmits on the basis of direct muzzle-to-muzzle contact . In smalThe caprine arthritis-encephalitis virus (CAEV) and the Maedi-Visna virus (MVV) present an example of closely related lentiviruses establishing in the inner-body organ systems of sheep and goats. The viruses display overlap in host tropism and both transmit mainly vertically via colostrum and milk. The difference between the two viruses mainly concerns the differential inner-body virus organ system tropism.Projected on a long evolutionary timescale, inner-body viruses tend to become locked in within the host body. This internalization may turn progressive when the epithelial transmission modes are being replaced by internal organ system-based modes. Virus establishment in the reproductive system translates in vertical transmission, in turn enhancing virus-host co-evolution . Virus iThe nature of species jumps differs between generalist and specialist type viruses. For example, an opportunistic, epithelial virus of wildlife origin is likely to be found circulating in livestock before becoming first detected in humans as host. This has been the case for influenza , Henipah-infection with Aujeszky disease virus;-avian infectious bronchitis virus;-avian infectious laryngotracheitis virus;-infection with avian influenza virus;-infection with bluetongue virus;-bovine viral diarrhea virus;-caprine arthritis/encephalitis virus;-infection with classical swine fever virus;-infection with equid herpesvirus-1;-infection with equine arteritis virus;-equine infectious anemia virus;-equine influenza virus;-enzootic bovine leucosis virus;-infection with foot and mouth disease virus;-infectious bovine rhinotracheitis/infectious pustular vulvovaginitis virus;-infectious bursal disease or Gumboro disease virus;-infection with lumpy skin disease virus;-infection with Newcastle disease virus;-infection with peste des petits ruminants virus;-infection with porcine reproductive and respiratory syndrome virus;-infection with rinderpest virus;-sheep pox and goat pox virus;-transmissible gastroenteritis virus.A subtotal of 23 livestock viruses of global animal health significance was extracted from the OIE-Listed diseases, infections and infestations in force in 2019 . Livesto-avian encephalitis virus;-avian leucosis virus;-contagious pustular dermatitis virus;-duck virus enteritis or duck plague virus;-enterovirus encephalomyelitis or Teschen disease virus;-fowlpox virus;-Maedi-Visna virus;-Marek’s disease virus;-pulmonary adenomatosis or jaagsiekte virus;-swine vesicular disease virus.Added to the above were ten globally important livestock viruses drawn from the last, 1995, edition of the FAO-OIE-WHO Animal Health Yearbook : -avian -equine herpesvirus-3;-porcine epidemic diarrhea virus;-swine influenza virus.Furthermore, three viruses of international veterinary relevance were added:The total of 36 livestock viruses belong to eleven different families and form a mix of RNA (N = 19), DNA (N = 11), and retroviruses (N = 6).Shown in Presented in The analysis concerned an iterative process. As a first step, the one-to-three scores allocated to the 36 viruses for the four ecological variables were examined in more detail and the monotonic associations subjected to Spearman correlation. Just the scores for the virus host-body infiltration and for the length of the infection period were found to increase in value together. Next, given the coarse match between virus infiltration and persistence, it was examined how this relationship played out at the virus family level. For this, the eleven virus families in the study were grouped and ranked A–D on the basis of the one-to-three infiltration scores allocated to the individual family viruses. Also, this A–D infiltration ranking was held against the length of the infection period scores.Next, given the indication, from the above, that the virus families may be neatly lined up in an outer- to inner-body fashion, it was examined how the organ system tropisms of the family viruses aligned with it. For this, the organ system tropisms of the viruses belonging to each family were collectively fitted and with naked eye aligned with the A–D family groups. For this, the alphabetical family order within the family groups was abandoned in order to obtain an optimal visual match. The result confirms that also the organ systems may be lined up in an outer- to inner-body fashion, virtually.Next, the A–D family ranking was converted into a one-to-four virus infiltration score applicable to individual viruses, as described in Next, since the infiltration-persistence match for the individual viruses was found to be about as strong as for the virus families, the 36 viruses were individually lined up in an outer- to inner-body fashion, irrespective the family origin, strictly on ecological grounds. For this, all of the above obtained results were considered in conjunction with the literature data on the transmission ecology collated for each of the 36 viruses in The finding that virus environmental survival in the outer-body environment increased from respiratory tract to alimentary tract to skin and decreased with the shift from the outer- to the inner-body environment prompted a re-examination of the relationship between the extent of virus host-body infiltration and virus environmental survival. Virus infiltration scores two-to-four, running from primarily epithelial transmission, to transmission involving also internal organ systems, to transmission primarily involving internal organ systems, were found to match with the one-to-three virus environmental survival rate scores. The indication that, at least in broad terms, the extent of virus inner-body infiltration correlated with a loss of virus robustness was applied in the virus ranking, along with the other factors. Next, furthermore examined were the outer- to inner-body shifts in virus host tropism and virus genome. For this, the ruminant plus equine viruses were contrasted to the poultry plus pig viruses. Excluded from the host tropism correlations was the multiple host FMDV. The remaining 35 viruses all formed part of either of the two host groupings. It was found that RNA, DNA and retroviruses broadly line up in an outer- to inner-body fashion. Virus host tropism and the virus genome line-up were matched. Additional correlations concerned virus host tropism and the four virus ecological variable scores, as well as virus genome type, and the four virus ecological variable scores. It was found that from outer- to inner-body, the virus genome type and host tropism appear to shift in concert, along with the infection-transmission dynamics.The collective results above served the compilation of the predictive framework for animal virus evolution shown in https://www.socscistatistics.com/tests/spearman/Default.aspx was used for the Spearman correlations. This software has been audited by established statistics packages.The online Rho calculator


However, the pharmacokinetics of oxyclozanide in cattle are not yet clearly understood. The present study was designed to develop a sensitive method to determine oxyclozanide levels in cattle plasma using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and to study its pharmacokinetics for application in cattle.Oxyclozanide is an anthelmintic drug that is widely used to treat fasciolosis18 analytical column  at 30 °C. The mobile phase comprised 0.01% v/v acetic acid (HOAc) diluted in water:acetonitrile (MeCN) (90:10% v/v) and 5 mM ammonium formate in methanol (MeOH):MeCN  at a 10:90 ratio (v/v) and was delivered at a flow rate of 0.4 mL min− 1. A good linear response across the concentration range of 0.02048–25.600 μg/mL was achieved (r2 = 0.994). The method was validated with respect to linearity, matrix effect, accuracy, precision, recovery and stability. The lower limit of quantification (LLOQ) was 0.020 μg/mL, and the extraction recovery was > 98% for oxyclozanide. The inter- and intra-day accuracy and precision of the method showed the relative standard deviation (RSD) less than 10%. The method was successfully applied to an assessment of the pharmacokinetics of oxyclozanide in cattle plasma. In healthy cattle, a single oral dose of an oxyclozanide suspension followed the one-compartment model, with a half-life (T1/2) of 64.40 ± 30.18 h, a plasma clearance rate (CL/F) of 11.426 ± 2.442 mL/h/kg, and an average area under the curve (AUC) of 965.608 ± 220.097 h*μg/mL. The peak concentration (Cmax) was 15.870 ± 2.855 μg/mL, which occurred at a peak time (Tmax) = 22.032 ± 3.343 h.A simple and rapid HPLC-MS/MS analytical method was established and validated to quantify oxyclozanide levels in cattle plasma using niclosamide as the internal standard (IS) in negative ion mode. Chromatographic separation of the analytes was achieved using a CA reliable, accurate HPLC-MS/MS analytical method was established in our study and successful applied to study the pharmacokinetics of oxyclozanide in cattle plasma. These results will be useful for further evaluations of the pharmacokinetic properties of oxyclozanide or for monitoring therapeutic drugs in animals. Fasciola hepatica is an economically important parasite that causes the neglected tropical disease of ruminants known as ‘fasciolosis’ and affects agriculturally important ruminant animals For the standard stock solution of oxyclozanide, 100 mg of oxyclozanide was placed into a 50 mL brown volumetric flask, after which methanol was added to produce a stock solution of 2000 μg/mL oxyclozanide. A series of working oxyclozanide solutions was prepared by diluting the standard stock solution with the mobile phase to obtain the following concentrations (μg/mL): 1.024, 16.384, 32.768, 262.144, 655.360, 1024 and 1280.For the IS solution, 1.000 mg of niclosamide was placed in a 100 mL brown volumetric flask, to which methanol was added to produce a stock solution of 10.00 μg/mL niclosamide. Next, 1 mL of the niclosamide stock solution was mixed with the mobile phase in a 50 mL volumetric flask to produce a solution of 0.200 μg/mL niclosamide. All solutions were stored at 4 °C and brought to room temperature before use.Plasma calibration standards with concentrations ranging from 0.02048–25.600 μg/mL were prepared by adding 10 μL of each of the oxyclozanide standard solutions (1.024–1280 μg/mL) and 10 μL of the niclosamide (0.200 μg/mL) IS solution to 500 μL aliquots of blank plasma. Quality control (QC) samples were prepared in the same way at different oxyclozanide concentrations: 0.020 μg/mL , 0.02048 μg/mL (QC-low), 12.7612 μg/mL (QC-med), and 25.600 μg/mL (QC-high). The calibration standards and QC samples were applied for method validation in the pharmacokinetic study.Plasma aliquots (500 μL) were spiked with 10 μL of the niclosamide (0.200 μg/mL) IS solution in centrifuge tubes, after which acetonitrile (1.5 mL) was added. The sample was mixed by vortexing (30 s) and centrifuged at 3000×g for 20 min, and the supernatant was evaporated to dryness at 40 °C with a vacuum concentration system . The dry residue was reconstituted in 500 μL of mobile phase and immediately subjected to vortexing for 20 s, after which the solution was filtered through a 0.22 μm Millipore filter and injected into the LC-MS/MS system.Selectivity was examined by comparing the chromatograms of eight different batches of blank cattle plasma with those of corresponding plasma samples spiked with oxyclozanide and the IS to exclude the interfering peaks .The matrix effect was evaluated by comparing the area response of post-extraction blank plasma samples spiked with oxyclozanide at three QC levels with the equivalent concentration standard solutions that were dried directly and reconstituted with the same mobile phase .2) of at least 0.99 was required to meet the criterion.The LLOQ and the lower limit of detection (LLOD) were determined as the concentrations that produced signal/noise ratioes of 10 and 3, respectively. For the linearity of this method, a calibration curve was generated with plasma standards containing different concentrations of oxyclozanide ranging from 0.02048–25.600 μg/mL. A calibration curve was constructed by plotting the peak area ratio of oxyclozanide/IS (y) vs the nominal concentration of oxyclozanide (x) in the form of y = ax + b; the least square method was used for the linear regression analysis. A coefficient of correlation (rThe precision was determined as the relative standard deviation (RSD) of replicate measurements, and the accuracy was evaluated as the ratio of calculated vs. theoretical concentrations, as previously described . The intThree concentrations of QC samples  and a single concentration of IS (0.004 μg/mL) were analysed in six replicates to evaluate the efficiency of oxyclozanide extraction from the bio-matrix. Recovery was determined by comparing the analytical results of the extracted QC samples with the pure standards without extraction.n = 6) of the QC samples at concentrations of 0.02048 μg/mL, 12.7612 μg/mL and 25.600 μg/mL under various the sample storage and processing procedure [Stability was assessed by analysing replicates (rocedure : (1) theEight healthy breeding age Simmental cattle of both sexes aged between 1.5 and 3 years and with a mean body weight (BW) of 317 ± 24.46 kg were obtained from a commercial company  and acclimated to a standard environmentally controlled animal room  for 1 week before the experiment. Animals were orally administered oxyclozanide once, and the dose was based on live weight. All experimental procedures were approved and performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Lanzhou Institute of Animal Science and Veterinary Pharmaceutics (number: SCXK (Gan) 2014–0002). All cattle were maintained under nearly identical conditions, and adequate water and rations were provided.The animals received an oral suspension of oxyclozanide at a dose of 10 mg/kg BW. Blood samples (5 mL) were collected by jugular venepuncture at 0 h, 0.5 h, 1 h, 4 h, 8 h, 12 h, 16 h, 18 h, 20 h, 24 h, 28 h, 36 h, 48 h, 72 h, 96 h, 120 h, 168 h and 216 h. After all the blood samples were centrifuged at 3000 rpm for 10 min, plasma samples were collected and immediately stored in a − 20 °C freezer until analysis using LC-MS/MS. All the animals were alive and healthy after the experiment.max), half-life (T1/2), and peak time (T max) are presented as means ± SD.Pharmacokinetic parameters were calculated using WinNonlin Professional software version 5.2 . The best pharmacokinetic model was confirmed according to the minimum Akaike Information Criterion (AIC) value principle and utilized for data fitting and parameter estimation . Plasma − ions at m/z 401.46 and m/z 327.12 for the IS. The quantification of oxyclozanide was carried out with MRM mode for high selectivity and sensitivity of acquisition data. The ion transitions of MRM were selected as m/z 401.46 to 176.2 for oxyclozanide and m/z 327.12 to 171.1 for the IS , and mobile phase B, 5 mM ammonium formate in MeOH:MeCN 75:25, v/v), at a ratio of 10:90 v/v for mobile phases A and B. A 75 mm column subjected to isocratic elution of the mobile phase for 5 min at a flow rate of 0.4 mL/min was used for chromatographic separation. Under optimized LC and MS conditions, oxyclozanide and the IS were separated with retention times of 2.10 min and 2.46 min, respectively, and endogenous substances in the plasma did not interfere with analyte detection. Figure 5, v/v, aThe specificity of the method was assessed by analysing each blank plasma sample from eight different cattle using the aforementioned chromatographic conditions. None of the samples exhibited endogenous substance-mediated interference in the retention time of oxyclozanide or the IS.In an evaluation of the effect of the plasma matrix on oxyclozanide and IS levels in eight diverse blank cattle plasma samples, the mean matrix effect on oxyclozanide was 96.3 ± 2.1%, while the matrix effect on the IS was 95.7 ± 1.6%.2) least-square linear regression analysis. The calibration curves are shown in Fig. 2 = 0.994) for concentrations ranging from 0.02048–25.600 μg/mL.The LLOQ and LLOD of oxyclozanide were 0.020 μg/mL and 0.010 μg/mL, respectively. The calibration curve for oxyclozanide was linear over the concentration range of 0.02048–25.600 μg/mL according to the results of a weighted  and LLOD (0.020 μg/mL) samples are presented in Table Table The results of stability for oxyclozanide under different storage conditions was shown in Table max, Tmax and T1/2 of oxyclozanide were 15.870 ± 2.855 μg/mL, 22.032 ± 3.343 h, and 64.40 ± 30.18 h, respectively. The AUC(0-∞) for oxyclozanide was 965.608 ± 220.097 h*μg/mL.The well-validated method described above was successfully applied to quantify oxyclozanide levels in plasma samples after the oral administration of an oxyclozanide suspension to cattle at a dose of 10 mg/kg. The plasma concentration-time curves for oxyclozanide were adequately fitted using a one-compartment model. The mean plasma concentration vs. time curves for oxyclozanide are shown in Fig. Full ESI scans were performed in both ESI (±) detection modes to optimize the mass spectrometry parameters for oxyclozanide and the IS. Better MS responses were achieved in ESI (−) mode compared with ESI (+) mode, consistent with previous reports , 18, 19.In the validation study, the LC-MS/MS method was validated in terms of selectivity, linearity, matrix effect, LLOQ, recovery, accuracy and precision and stability. These variables were assessed according to guidelines established by the US Food and Drug Administration for bioanalytical method validation . A good 1/2 were 21.74 h for sheep and 18.71 h for goats, the AUC(0–∞) values in sheep and goats were 488.70 μg.h/mL and 309.33 μg.h/mL, respectively, and the Cmax in sheep and goats were 11.01 μg/mL and 6.68 μg/mL, respectively. However, the T1/2 of oxyclozanide in cattle plasma reported here was 64.40 h, which is a much longer time than the period in sheep and goats. The AUC(0–∞) (965.608 h*μg/mL) of oxyclozanide in cattle is larger than in sheep and goats, and the Cmax (15.87 μg/mL) of oxyclozanide is higher than in sheep and goats. The reasons for these differences may be due to the differences in animal species, the selectivity of the analytical methods or the formulation and preparation techniques. However, following the oral administration of an oxyclozanide suspension, the drug is slowly eliminated from cattle plasma, and this property may contribute to the therapeutic effect in vivo. To our knowledge, this study is the first to report the pharmacokinetics of oxyclozanide in cattle, and our results will provide a basis for further research on the administration of oxyclozanide suspensions in animals.The pharmacokinetics of oxyclozanide in cattle were assessed using the LC-MS/MS method and the pharmacokinetic properties were obtained in this study. A previously published study reported the pharmacokinetics of oxyclozanide in sheep and goats following the administration of a single oral dose . The T1/A simple and sensitive LC-MS/MS method has been completely validated, displayed excellent sensitivity, linearity, precision and accuracy, and was successfully applied to an evaluation of the pharmacokinetics of oxyclozanide in cattle plasma after oral administration. This study is the first to report the pharmacokinetic parameters of oxyclozanide in cattle and will be useful for further evaluations of the pharmacokinetic properties of oxyclozanide or for the monitoring of therapeutic drugs in animals.


Hospitals should monitor the costs of all direct and indirect processes in order to achieve efficiency and safeguard financial sustainability. One neglected process with significant costs is the processing of reusable medical devices and their packaging performed in the central sterilisation supply department and the operating room. The objective of this research is to analyse and compare processes and costs of four different packing alternatives, i.e. non-woven sterilisation wrap with two sheets, one-step wrap, sterilisation container with inner wrap and sterilisation container without inner wrap.We defined sub-processes that are directly related to the packaging options and measured them through a comprehensive time study. For all sub-processes and the total processes a distribution fitting and a Monte-Carlo-Simulation were performed. We calculated the costs for all sub-processes, i.e., costs for personnel, variable costs and the respective share of fixed and jump-fixed costs (e.g. depreciation of containers) associated with each packaging option. All results are discussed through various scenarios to evaluate the advantageousness of all packaging options.The four packaging options are associated with different costs. “Sterile container without inner wrap” causes 2.05€ per use. The options “sterile container with inner wrap” (3.24€), “one-step sterilisation wrap” (3.44€) and “two sheets sterilisation wrap” (3.87€) cause higher costs. With regard to personnel costs the option “sterile container without inner wrap” clearly causes the lowest costs. In addition, variable costs are lower in case of sterile container. Sterile container only cause higher costs in the aspect of fixed and jump-fixed costs per packaging.The analysis shows that even under a broad set of scenarios the “sterile container without inner wrap” is the most cost-effective alternative. The evaluation of the options “sterile container with inner wrap” and “one-step sterilisation wrap” remains particularly interesting as they often yield comparable results. Both options cause approximately the same personnel costs, so the decision appears to be more dependent on the material prices for wrap or the frequency and duration of use for container. It turns out that the personnel time and consequently the personnel costs significantly influence the rational choice of the packaging options. Since diagnosis-related groups were introduced in German hospitals (G-DRG) as a compulsory financing scheme in 2003, the management of these institutions has to assess the financial impact of all components of in-patient care. Before, hospitals could claim a refund from the health insurance funds for their actual expenditure with higher costs resulting in higher revenues. At that time, German hospitals could hardly generate a deficit and cost containment was rather irrelevant . After aA clinical pathway is a series of interdependent processes and respective sub-processes which are required to treat a patient from admission to discharge. They are developed primarily in order to increase the effectiveness and quality of medical treatment. In addition, clinical pathways are proven instruments to reduce costs and increase cost-effectiveness of health care delivery.  For thisOne of these service processes which is not perceived by the patient and usually outside the focus of hospital managers is the reprocessing of medical devices in the Central Sterilisation Supply Department (CSSD). It is obvious that the provision of sterile materials by the CSSD is of high relevance for the quality of the entire treatment process . At the This paper intends to close this research gap by analysing the processes and resource consumption of different packaging options of sterile goods in CSSD sub-processes. In the next section we present background information on different packaging systems in CSSDs. Afterwards the methodology of this study is presented consisting of a detailed process analysis of the entire sterile supply cycle, a comprehensive time study and the financial assessment. As the supply cycle does not only comprise CSSD, the processes of the operating room (OR) are included in the analysis. The fourth section will present the results. The analysis does not only present the processes and the respective resource consumption, but also compares sub-processes of the four main process options. The paper closes with a discussion of the results by presenting various scenarios and provides recommendations for hospital managers which are relevant not only to German health care providers.The supply and disposal of sterile goods is of high importance for maintaining functional services in hospitals, in particular for surgical units , 12. Abo2 for two layers of wrap. Further distinction can be made between a sequential wrap (two sheets of wrap per set) or a one-step wrap made of two sheets [The non-woven sterilisation wrap is a one-way material covering the sterilised set of instruments or other products . After the set is used, the wrap is disposed. Different types and qualities of non-woven sterilisation wrap are available on the market and have to be chosen based on the size and weight of the set. Typical wrap qualities range from about 70 g to 140 g per mo sheets . Sterilitwo sheets non-woven sterilisation wrap (TSW): This is the traditional way of wrapping sterilised sets. The set is wrapped with one sheet of wrap. Afterwards it is wrapped again in another sheet. It is important to note that the two wrapping processes must be separated, i.e., it is not common practice to wrap a set within two sheets in a single step unless a special one-step wrap is used.one-step non-woven sterilisation wrap (OSW): A special wrap is used so that the set can be wrapped in one step. However, it requires a material consisting of two pieces of wrap specially attached to each other.sterilisation container with inner wrap (SCW): A sterilisation container with single layer wrapped surgical sets inside the container is commonly used in Germany. Lower quality wrap material is typically used in this case.sterilisation container without inner wrap (SC): According to DIN EN ISO 11607-1 container are rigid sterile barrier systems and thus inner wrapping in a sterile container is not obligatory so most hospitals worldwide  use sterilisation containers without additional wraps.Wrapping and container packaging can be combined resulting in the before mentioned packing alternatives used in Europe and Northern America:Instrument cycle: A sterile container is part of every single step of the surgical instrument cycle, i.e. (1) disassembling and pre-cleaning, (2) cleaning and disinfection, (3) inspection and maintenance, (4) preparation for sterilisation, (5) sterilisation, (6) transport and storage, (7) use and (8) transport back to the CSSD. The use of surgical wrap starts with step (4) and ends with step (7). In addition, it needs a disposal sub-process after (7) not required by containers .Reprocessing: Most containers are made of anodised aluminum and have to be re-processed under special conditions separate from surgical instruments.Space requirement: The containers are voluminous, i.e., containers will need more space than wraps in particular for special reprocessing. This is especially challenging if hospitals are facing space constraints.Waste: As wraps are one-way materials they lead to a high volume of waste. This induces costs and environmental challenges. Some manufacturers have established recycling programs but still recycling costs and the associated challenges are much higher for wraps than for containers .Capital and running expenditure: Sterilisation container are investment goods (except of single use filters and locks depending on the container series used), wraps are disposables. Thus, using containers entails high but unique investment costs which are usually written-off in 8–10 years. Using sterilisation wrap incurs a steady flow of funds for consumables. The variable cost per sterilisation set is constant for the wrap but decreases for the container solution if the work load increases. Wraps bear the risk or chance of increasing or decreasing future material prices.All four alternatives are used in Europe and North America and show advantages and disadvantages. There are discussions on the sterility of the packaging during the entire logistic process. However, compliance with DIN EN ISO 11607-1 is normally proven by all manufacturers for all four alternatives. Thus, we assume that all necessary basic functions especially sterility up to the point of use can be taken for granted if one can ensure compliance with the instructions for use. In other words: In absence of evidence from the literature, this paper assumes that the quality of packaging is similar for all four systems. However, the choice of the sterile barrier system has a strong influence on the different sub-processes of the sterile supply cycle. In particular the following differences occur:The differences between the systems shown are obvious. However, no detailed analysis of the economic dimension of the packaging system can be found in the literature. This paper focusses on the resource consumption and the respective costs of the four different packaging systems described above while assuming that the quality of sterility is identical for the respective systems.In order to analyse each packaging option, process information was required and therefore two German CSSDs were chosen that primarily produce sterile goods for maximum care hospitals. While the first CSSD mainly uses non-woven sterilisation wrap for the transport, reprocessing and storage of sterile goods, the second CSSD mainly uses sterilisation containers for this purpose. As a first step, main processes of the sterile supply cycle were defined. Due to the fact that main processes in hospitals take place at multiple locations and multiple professional groups contribute to the workflow, various department locations were taken as a basis for identifying main processes. In particular, processes of CSSD and OR were strictly differentiated.As a second step, the main processes were subdivided into sub-processes thus achieving a more precise subsequent allocation of costs. For this purpose a team of researchers observed and documented all processes and sub-processes for several days in both hospitals. In addition, interviews with the leadership of the respective departments and other CSSD experts were conducted. Furthermore, the results of a literature analysis were included in the process analysis of the CSSDs and ORs. This analysis included the review of standard operating procedures of those particular institutions as well as DIN EN ISO standards and German laws related to the reprocessing of medical devices. The decisive factor for determining sub-processes were those procedure steps that required personnel and time for producing sterile instrument trays while peel packed instruments were not part of the analysis. Interim results of defining processes were discussed in several workshops with key-informants in terms of sterilisation and supply of medical devices.As a third step, we determined the time consumption per service unit for each sub-process by measuring the time based on a so-called “elapsed time measurement”.  ConsequeThe total process times of all sub-processes of the entire sterile supply cycle is equal to the minimum overall time which one single instrument tray needs to pass through the CSSD and OR. This alone is an important information, but it does not allow to compare the different packaging alternatives. This is due to the fact that the infrastructure of both hospitals is slightly different which might bias the results. For this reason, only selected sub-processes were considered for further analysis. We focus only on those sub-processes that differ strongly between packaging options. Furthermore, we neglect sub-processes that are associated with transporting sterile goods. Consequently, the relevant sub-processes for a container comprise the packaging activities, the preparation of containers at the CSSD as well as the opening and closing activities in the OR. The respective sub-processes of non-woven sterilisation wrap consist of packaging activities, the additional process for wrap disposal at the CSSD and OR sub-processes. With regard to the chosen focus of one single reprocessed unit, we adjusted collected sub-process times belonging to multiple units. This was necessary to ensure that personnel times and costs would not be overestimated when multiple objects were processed at a time. For those cases sub-process times were divided through the amount of objects, e.g. multiple containers at one loading trolley.In order to determine a distribution of time consumption and costs of the alternatives it was necessary to perform a distribution fitting and a simulation. In a first step, outliers were identified by elimination of all process times outside the range of the fourfold standard deviation under and above the average. The methodology of elimination of outliers is based on the Chebyshev’s theorem which states that for unknown distributions at least 93.75% of all data is situated between the two bounds of four standard deviations in both directions from the mean value . In sum To calculate the theoretical distribution behind the gained data the software tool EasyFit Professional Version 5.6 by MathWave Technologies was used. A lower bound of zero and an unknown upper bound were assumed in order to test all input samples to fit on the following distributions: Beta, Johnson SB, Kumaraswamy, Pert, Power Function, Reciprocal, Triangular, Uniform, Burr, Chi-Quadrat, Dagum, Erlang, Exponential, Fatigue Life, Frechet, Gamma, Generalized Gamma, Inverse Gaussian, Levy, Log-Gamma, Log-Logistic, Lognormal, Nakagami, Pareto, Pareto 2, Pearson 5, Pearson 6, Rayleigh, Rice, Weibull, Generalized Extreme Value, Generalized Logistic, Generalized Pareto, Log-Pearson 3, Phased Bi-Exponential, Phased Bi-Weibull and Wakeby.To decide which distribution fits best the Anderson-Darling (AD) test was used as defined in EasyFit Professional 5.6. Subsequently, the best-ranked distribution based on the AD-test was chosen for further analysis. Significant results at a = 0.01 based on AD-testing are marked with “*”. Furthermore, “°” shows significant distributions based on the Kolmogorov-Smirnov (KS) test. Due to the circumstances of some sub-processes being quite short (range of a few seconds) and the time study only measuring integer second-values, some distributions do not generate significant results. Consequently, the P-P-plot was also checked in order to decide if the highest ranked distribution could be used for simulation. As an example Fig. Based on the fitted distributions a Monte Carlo simulation was performed. In this case, 1000 random numbers were taken from the distribution of each sub-process. The results of individual simulation runs were combined in an additive manner, so as a result a determination of the overall distribution for the CSSD, the OR and the entire process was possible. The following assumptions had to be made in the context of the four defined procedural options. All assumptions can be regarded to as a correction factor of the process time, as some collected sub-processes are only partially allocable to the overall process. Table An example of a process with a weight of less than one is “dispose of all collected wrap” (W02S) for OSW and TSW. The correction factor results from the ratio of the process “remove and dispose wrap” (W01S) and the process “dispose of all collected wrap” (W02S). While process W01S was measured 1360 times, process W02S could be measured 66 times. That results that process W02S has to be performed once per 17.12 units and the correction factor is 0.05840708.In addition, staff costs per minute as well as costs for consumables and allocated costs were calculated. Table According to these assumptions, the respective minute rate are 0.45 € (CSSD) and 0.60 € (OR).The variable costs include wrap, trayliner, labels, container filter, container seal and indicator tape. The price information is based on the considered units. Since prices are also subject to strong fluctuations, scenarios are formed in the discussion section of this article. Table The acquisition costs of containers and transport baskets were attributed to the process by defining a lifetime and the annual turnover. Assuming a lifetime of ten years and a turnover rate of 120 per year, the total usage frequency is 1200. It should be noted that the life time and frequency of circulation are variable. As part of the sensitivity analysis in the discussion, alternative scenarios are formed.Annual total maintenance costs for the containers were allocated according to the number of containers and the annual turnover. It should be noted that the resulting repair costs of 0.80 € per container per year are not attributable exclusively to wear and tear. The costs also include the repair of damages due to handling errors and can therefore be considered as conservative. For the waste disposal, the costs per litre are 0.04 €. The survey found that the volume of waste per one layer wrap is around 3.504 l. Consequently, a volume of 7.009 l is assumed for the disposal of two sheets (TSW). Since OSW also exists on two firmly connected layers, we also assume 7.009 l. As part of SCW, only one sheet of trash is produced. Disposal costs for container filters and container seals are included in the area of variable costs.Finally, the additional costs per unit produced were determined for the large-scale washing facility. The costs of the large-scale washing system result from the costs of a cleaning process divided by the load. Since, in addition to the containers, other materials are being cleaned in the large-capacity washing system, it is assumed that containers will take up 70% of the load per run. Thus, for the cleaning of containers, each run costs 5.19 €. Within the study, an average load of 22.45 containers per cycle was recorded. Due to the fact that the mean of loading was at 22.45 containers and the maximum loading was 33, the assumption that 70% of the costs are allocated to container cleaning can be considered as conservative.Furthermore, it has to be taken into account that the options SCW and SC in certain circumstances require a higher capacity for container cleaning. In the base scenario, it is assumed that the use of containers does not result in full utilization of the large-capacity washing system. Acquisition, maintenance and repair costs are therefore not relevant to the decision. Two further scenarios are considered in the discussion. The first assumes that a larger large-capacity washing system will be needed to clean containers with additional acquisition costs of 17,500 €. Maintenance and repair costs are not relevant to the decision in this scenario, since it is assumed that the size of the machine has no influence on the absolute maintenance and repair costs. In the second scenario it is assumed that the CSSD requires an additional large-capacity washing system, which causes 167,500 € acquisition costs. Furthermore, 9500 € per year are taken into account for maintenance and repair. For the large-scale washing system a lifetime of 13.5 years and an annual production volume of 82.500 container is assumed. It should be noted that large-capacity washing systems are not used exclusively for cleaning containers. As previously stated, a value of 70% is assumed for the cleaning of containers and 30% for the cleaning of other products .As a result of observation, expert interviews, literature analysis and key-informant interviews we determined the processes as described in Figs. Table Table In order to answer the question of which distribution can be assumed for the CSSD, the OR and the overall process, the theoretical distribution behind the simulation data was determined. Table Based on the distributions from Table Figure A similar picture can be observed in case of the processing times in the OR. According to the determined distribution, the option SC is dominant in about 97.7%. However, the OSW option dominates the SCW option in only about 37.33% of cases. At Q1, SCW causes about 7.1% higher process times than OSW, while OSW causes 4.6% higher times at Q3. The distribution function is shown in Fig. The determination of the personnel costs per packaging option was carried out by formation of a minute set. As a result, average personnel costs amount to 2.05 € for TSW, 1.58 € for OSW, 1.60 € for SCW and 1.09 € for SC based on the times shown. The variable costs show a value of 1.42 € for TSW and a value of 1.46 € for the OSW option. The SCW option costs 0.87 € and the SC option 0.32 €.In terms of additional costs, it becomes clear that the acquisition costs of a container cause a total of 0.40 € per use (Option SCW and SC). There is also a value of 0.01 € for repairs. The costs for the transport baskets are 0.13 € per use (Option TSW and OSW). Disposal costs in the context of TSW and OSW result at around 0.27 €. The disposal costs for SCW are around 0.13 €. The costs of the large-capacity washing maschine are 5.19 € per run. Within the study, an average load of 22.45 containers per cycle was recorded. Consequently, per container about 0.23 € are attributable (SCW and SC).For the material costs as well as the allocated costs results show 1.81 € for TSW, 1.86 € for OSW, 1.64 € for SCW and 0.96 € for SC. In comparison to the personnel costs, a partially changed ranking shows up. The previously dominant option SC remains dominant. The option OSW  is now dominated by all other options.Table Figure Rank 1 - SC (Sterile Container without inner wrap) - mean: 2.05 €Rank 2 - SCW (Sterile Container with inner wrap) - mean: 3.24 €Rank 3 - OSW (one-step non-woven sterile wrap) - mean: 3.44 €Rank 4 - TSW (two sheets non-woven sterile wrap) - mean: 3.87 €In the overall result we clearly see that the option SC dominates all other options. Rank two is now taken solely by SCW. The OSW option dominates the TSW option by approximately 100%. The significant right shift of the OSW option is caused by the material costs of this option. Based on the presented input values, the following sequence of options can be displayed.○ staff costs increase by 10%○ staff costs increase by 20%○ personnel costs are not relevant to the decisionPart A (personnel costs)○ material and special costs increase by 10%○ material and special costs increase by 20%○ material costs decrease by 10%○ material costs decrease by 20%○ acquisition costs for containers increase by 10%○ acquisition costs for containers increase by 20%○ acquisition costs for containers increase by 50%○ material cost for wrap decrease by 10%○ material cost for wrap decrease by 20%○ material cost for wrap decrease by 50%○ material cost for wrap (TSW and OSW) increase by 10%○ material cost for wrap (TSW and OSW) increase by 25%○ material cost for wrap (TSW and OSW) increase by 50%Part B :○ turnover rate of containers and transport baskets increases by 10%○ turnover rate of containers and transport baskets increases by 25%○ turnover rate of containers and transport baskets increases by 50%○ turnover rate of containers and transport baskets decreases by 10%○ turnover rate of containers and transport baskets decreases by 25%○ turnover rate of containers and transport baskets decreases by 50%○ transport baskets are not used○ containers are used only 5 years○ containers are used for 15 years○ containers are used for 20 years○ larger large-capacity washing system needed○ additional large-capacity washing system neededPart C (changes in usage)○ personnel costs are not relevant to the decision and material costs for wrap decrease by 75%○ material costs for wrap decrease by 50% and turnover rate of containers and transport baskets increases by 50%○ material costs for wrap decrease by 91% and transport baskets are not used○ additional large-capacity washing system needed, material costs for wrap decrease by 50% and transport baskets not used○ personnel costs not relevant to the decision, material costs for wrap decrease by 40%, transport baskets not used and additional large-capacity washing system neededPart D (extreme scenarios)The results of this analysis show that the SC option causes the least costs. For the decision maker however, the question arises how stable this result is if major assumptions are subject to change. Consequently, we have to assess the sensitivity of these results if major parameters change. The following 33 scenarios analyse the consequences of the following changes of parameters:Part A: As part of the scenario building process, personnel costs were varied. The changes + 10% and + 20% can have two meanings. Either 10% or 20% increase in personnel costs or 10% and 20% increase in personnel time consumptions. It turns out that the order of the results does not change. The reason is that the ranking of the personnel time consumptions follows the overall ranking. Another situation arises when personnel costs are not relevant to the decision. This situation occurs when the differences in time consumptions do not require additional personnel. Consequently, this situation only occurs if – regardless of the packaging option – the staff is working within its capacity limits. In this scenario, TSW (now in rank three) costs less than OSW (now in rank four). In all scenarios, SC causes the least costs and SCW the second lowest costs. The changed values are shown in Table Part B: Within the scenarios, the material or contribution costs were varied. It turns out that a change in the variable costs by + 10% and + 20% and the special costs by + 10% and + 20%, does not result in any changes in the ranking. The same applies to the increase in container price by + 20%. Only if the container price is increased by + 50% is the option OSW beneficial compared to SCW (0.004€). An identical picture is found in the change in price of the sterilisation fleece. A change of - 20% will not influence the original ranking. Only if the price for wrap is reduced by 50% does the OSW option benefit SCW. TSW will not be advantageous over SCW. This is due to relatively high staff costs for TSW. Furthermore, the effects of a higher price for non-woven wrap in the TSW and OSW options were investigated. The reason for this is that with a higher weight of the sterile goods, expensive nonwovens must be used, while there are no changes in the container options. It turns out that with a 50% increase of prices for wrap, TSW and OSW options can cost more than twice as much as SC. In reality, large price fluctuations can exist. On one hand side, discounts for high sales volumes lead to strong benefits, on the other hand side, low sale volumes or high weights of the instrument tray (requiring stronger wrap) significantly affect the costs. Consequently, the price fluctuations assumed here are rather realistic. The result shows that the price of wrap  is the relevant cost driver. In the baseline scenario, they cause about 38.6% of the packaging related costs in the TSW option. In the option OSW it is about 44.7% and in the option SCW about 21.0%. Table Part C: The third part examines changes in usage. This includes changes in the turnover rate and the non-use of transport baskets in the TSW and OSW option. Furthermore, changes in the period of usage of containers is examined. It turns out that an increase in the turnover rate has no influence on the ranking of the results. The increased usage of containers and transport baskets, however, leads to a decrease in the packaging related costs per piece produced. Another result shows in case of the reduction of the turnover rate. While the results are unchanged at a − 25% reduction, a 50% reduction makes OSW less expensive than SCW. The reason for this are the acquisition costs for containers. With a lifetime of 10 years and a circulation of 60 per year, the containers are used only 600 times in total. This shows that the circulation rate has a significant impact on costs. Furthermore, the changes in the non-use of transport baskets were examined. It turns out that the option OSW now dominates the SCW. However, this does not take into account the additional measures required to store the sterile goods in order to compensate for the disadvantage of the missing transport baskets. Thus, the option will only be beneficial if the additional cost for storage is less than 0.04 € per piece. It should be noted that option SC is still dominant in this case. Finally, the influence of the usage time of the containers was examined. While an extension to 15 or 20 years increases the advantage of the SCW and SC options, in case of a reduction to a 5-year usage, the OSW option becomes dominant over SCW. However, it should be noted that a useful lifetime of 5 years with a circulation frequency of 120 per year is rather pessimistic. On the basis of surveys of 36 CSSDs in Germany, the average period of usage was 13.25 years, with a standard deviation of 6.86 years and a median of 12 years. It should be noted, that even with a usage time of only 5 years, the SC option is dominant. This is due to the low personnel time consumption of the option. As part of the large-capacity washing system scenarios, it becomes clear that a larger washing system has only a small impact on the costs per item (0.01 €). The reason for this is that the depreciation per year is very small, since only the difference of the acquisition costs and no maintenance and repair costs have to be considered. If an additional system is required, the costs of the SCW and SC options increase significantly. The cost of the SCW option is then almost identical to the OSW option. However, it turns out that the SC option still causes the least cost. Table Part D: The last three scenarios can be regarded as extreme scenarios. It turns out that the replacement of the SC option as best option requires massive changes. The SC option becomes third-rate, if personnel costs are not relevant to the decision and the price for wraps decreases by 75%. If personnel costs are included, the option SC even remains optimal if the wrap price decreases by 50% and the turnover rate is reduced by 50%. The third scenario shows that the TSW option would only become dominant over the SC option by 0.01 € if the wrap price were to fall by 91% and transport baskets would not be used. It turns out that personnel costs have a massive impact on determining the best option. Scenario number four includes a 50% reduction in wrap price, the non-use of transport baskets and the need for an additional washing system for container use. Even in this scenario, the SC option dominates. The last extreme scenario shows under which circumstances both wrap options dominate the container options. If personnel costs are not relevant to the decision, the price for wrap is reduced by 40%, transport baskets are not used and an additional washing system for containers is required, the options TSW and OSW dominate. Table The scenarios underline that the option SC is dominant and favorable in almost all constellations. Furthermore, the option SCW causes the second lowest costs. Often the option SCW and OSW are close together. The reason for this is that the higher material costs of the OSW option are almost compensated by higher special costs (contribution costs for containers and large-capacity washing system). However, this does not apply if stronger and more expensive fleece must be used as protective packaging. In some cases significantly higher costs result in the option OSW. The option TSW causes the highest costs in most cases. This is due to the higher process times and thus higher personnel costs.This study has a number of limitations. Firstly, it focuses purely on the economic dimension assuming that all the four options achieve the same level of hygiene-quality, which is in agreement with the fact that compliance with DIN EN ISO 11607-1 is normally proven by all manufacturers for all four alternatives. Meaning that we did not have to compare cost-effectiveness ratios but could concentrate on a cost-comparison alone. However, further research must follow to give more evidence to this assumption.Secondly, our study calculates the actual costs based on the real-life situation in two hospitals. We do not assess whether the processes are efficient but assume that the time consumption reflect the real situation. It might be worthwhile to produce a standard-costing for “best” processes instead of “actual” processes. Thirdly, the analysis took a strategic perspective, i.e., we assume that all costs are relevant. If we take a short-term perspective, depreciation charges are to be neglected as they are sunk costs in the short-run. This will not change the ranking of the alternatives, but has an impact on the comparative advantages of the options container versus wrap. Finally, our analysis is based on two German hospitals with respective laws and regulations, personnel costs, processes and financing systems.This analysis shows that the costs of different packaging alternatives do differ – a result of high relevance to hospital managers worldwide. This result is not a consequence of structural differences of the two hospitals analysed as we concentrated only on those processes which are comparable. Instead, differences in time and costs are consequences of the packaging alternatives. However, this result should not be overestimated. Instead, each CSSD or hospital should analyse its own situation, requirements and circumstances. For some institutions, for instance, capital is the scarcest agent of production as these institutions have no access to loans while relying on (declining) government funded grants to purchase containers. For these institutions, leasing of containers or using wraps might be the most rational solution. For other institutions, rising costs of personnel or a shortage of personnel are the most pressing influencing factors. In this case, the alternative with the lowest consumption of personnel time should be sought. Although an advantage of about 98 s per sterilisation set (SC vs. TSW) appears low, this difference accumulates to more than one full-time position in the CSSD if we assume an annual output of 49,000 units. From the economic point of view, it becomes clear that factors such as personnel time consumptions, turnover rates, material costs and acquisition costs should always be taken into account.It will be challenging to see whether an analysis in another country will reproduce the same results. Thus, this paper calls for more research in the economics of hospital management and in particular the economics of hygiene management.


Totiviridae currently contains five genera Totivirus, Victorivirus, Leishmavirus, Trichomonasvirus, and Giardiavirus. Members in this family generally have a set of two-open reading frame (ORF) elements in their genome with the 5′-proximal ORF (ORF1) encoding a capsid protein (CP) and the 3′-proximal one (ORF2) for RNA-dependent RNA polymerase (RdRp). How the downstream open reading frames (ORFs) are expressed is genus-specific. All victoriviruses characterized thus far appear to use the stop/restart translation mechanism, allowing for the expression of two separate protein products from bicitronic genome-sized viral mRNA, while the totiviruses use a −1 ribosomal frame-shifting that leads to a fusion product of CP and RdRp. We report the biological and molecular characterization of a novel victorivirus termed Alternaria alternata victorivirus 1  isolated from Alternaria alternata in Pakistan. The phylogenetic and molecular analyses showed AalVV1 to be distinct from previously reported victoriviruses. AalVV1 appears to have a sequence signature required for the −1 frame-shifting at the ORF1/2 junction region, rather than a stop/restart key mediator. By contrast, SDS–polyacrylamide gel electrophoresis and peptide mass fingerprinting analyses of purified virion preparations suggested the expression of two protein products, not a CP-RdRp fusion product. How these proteins are expressed is discussed in this study. Possible effects of infection by this virus were tested in two fungal species: A. alternata and RNA silencing proficient and deficient strains of Cryphonectria parasitica, a model filamentous fungus. AalVV1 showed symptomless infection in all of these fungal strains, even in the RNA silencing deficient C. parasitica strain.The family  Totivirus) and members of other genera in the Totiviridae [T = 1 capsids as shown for totiviruses [There are many double-stranded RNA (dsRNA) viruses with a two-open reading frame (ORF), nonsegmented genome organization, one of the simplest genome types, reported from plants, fungi, protozoa, and insects, as exemplified by totiviruses , are characterized by undivided dsRNA genomes with two ORFs encoding a CP and an RdRp or RdRp domain of a fusion protein. Among them, most members of which (belonging to genera Totivirus and Victorivirus) are isolated from fungi. Most totiviruses (members of the genus Totivirus) characterized thus far have been reported from yeast as exemplified by Saccharomyces cerevisiae virus L-A (ScV-L-A). However, recently totiviruses were reported from obligate filamentous phytopathogenic fungi [T = 1 capsids with 120 homodimers was proposed [Members of the family ic fungi  and planic fungi   [UGAA” or “AUAUG” [AUAUG is also used by the well-studied prototypic hypovirus Cryphonectria hypovirus 1  [UGAA motif combined with an upstream pseudoknot RNA structure, as the necessary sequence elements for the stop/restart translation of victoriviruses [Like totiviruses, victoriviruses form icosahedral particles of ~40 nm in diameter with a lecules) . Victorilecules) , while tlecules) ,8. The r “UAAUG” ,8, in whA virus) . Li et aHelminthosporium victoriae) and an RNA silencing mutant of Cryphonectria parasitica [Rosellinia necatrix  causes no overt phenotypic alterations in the natural host and the standard strain of C. parasitica [Almost all victorivirus show symptomless infections. Exceptions include Helminthosporium victoriae virus 190S (HvV190S) that induces debilitation in the natural fungal host . Surprisingly, the virus lacks a typical stop/restart signature sequence for the translation of RdRp, thought to be the rule for victoriviruses, but instead likely has a sequence feature often found for the −1 frame-shifting at the ORF1 and ORF2 junction. This paper represents the second report on a fungal virus from the Indian subcontinent.Here we report the molecular and biological characterization of a victorivirus strain, which was isolated from a field strain, A-16, of The fungal strain designated as A-16 was isolated from a wheat grain at the National Agricultural Research Centre (NARC), Islamabad, Pakistan by a standard blotter method as described by the International Seed Testing Association) ISTA .A. alternata based on morphological characteristics of spores and on the sequence of the internal transcribed spacer (ITS) regions of the fungal ribosomal DNA (rDNA) amplified by using a primer pair ITS1 (TCCGTAGGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGATATGC) [A. alternata f. sp. lycopersici [C. parasitica and its mutant ∆dcl2 [The strain was identified as GATATGC) . The culGATATGC) . A virusopersici , was pront ∆dcl2  were a gTotal nucleic acid was extracted from the mycelia of the fungal strain A-16 cultured in 20 mL PDB while the dsRNA fraction was isolated from mycelia grown on PDA overlaid with cellophane (PDA-cellophane) until it covered the plate . HarvestA. alternata strain A-16 and unidentified fungi were pooled (namely “pool A1”) and 5.9 µg of dsRNA (91 ng/µL) was subjected to next generation sequencing. Viruses detected from the other two strains of the fungi will be reported elsewhere. The cDNA library construction and subsequent deep sequencing on the Illumina platform  were carried out by Macrogen Inc. . After sequencing, the adaptor sequences are trimmed and then the adaptor-trimmed reads  were assembled de novo into 14,209 contigs (~488–6785 nt in length) using CLC Genomics Workbench . These contigs were subjected to local BLAST searches against the viral reference sequence (RefSeq) dataset of the National Center for Biotechnology Information (NCBI). The complete nucleotide sequence of the virus genome in this article has been deposited with the EMBL/GenBank/DDBJ Data Library under Accession No. LC477336.An equal amount of dsRNA samples prepared from three virus infected strains of different fungi including Potential ORFs were identified using the EnzymeX version 3.3.3  SequencePhylogenetic analysis was based on a maximum likelihood (ML) method as described previously  with min2, 10 mM dithiothreitol, 40 U of moloney murine leukemia virus reverse transcriptase , and 20 U of RNase inhibitor . One µL of the reaction mixture was subjected to PCR with sequence-specific primer sets, AJ-19F (1)/AJ-19R (1) and AJ-19F (2) and AJ-19R (2), respectively (Reverse transcription (RT)-polymerase chain reaction (PCR) was carried out to confirm that contig (A1-19) was derived from the genome of a victorivirus. Approximately 10 µg of purified dsRNA along with 10 pmol random hexamer (N6) was denatured by dimethyl sulfoxide (DMSO) and reverse transcribed at 37 °C for an hour in a 20-µL reaction mixture containing 50 mM Tris-HCl (pH 8.3), 50 mM KCl, 4 mM MgClectively .4-GATCCAACTAGTTCTAGAGCGG-NH2-3′) was ligated to the 3′-end of the dsRNA using T4 RNA ligase (Takara) at 16 °C for 16 h. The ligated dsRNA was then denatured and used as the template in the first strand cDNA synthesis at 42 °C for one h in which an oligo-deoxynucleotide , complementary to the adapter, and Superscript II RNase H-Reverse Transcriptase (Thermo Fisher Scientific) were used. Resulting cDNA was used for PCR with sequence-specific primer  was done according to the procedure described by Suzuki et al.  with slic primer  along wiViral particles were purified as described by Shamsi et al. . This prThe protein bands were silver-stained and subjected to in-gel tryptic digestion followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis as described by Shamsi et al. .C. parasitica was performed as described previously [C. parasitica Δdcl2 strain were prepared using the method of Eusebio-Cope et al. [C. parasitica Δdcl2 spheroplasts using polyethylene glycol (PEG). After regeneration of the spheroplasts, mycelial plugs from multiple positions were transferred onto new PDA plates and propagated. Transfectants were analyzed by agarose gel electrophoretic analysis of their dsRNA-enriched fractions.Transfection of eviously ,45. Sphee et al. . A-16  from strains Ally-12 and A-16 were mixed and fused in the presence of PEG. Fungal colonies showing a HygR phenotype were tested for virus infection.Protoplasts of the Japanese i et al.  and tranAlternaria alternata morphologically and molecularly. The nucleotide sequences of the internal transcribed spacer (ITS) regions of A-16 were compared using [A. alternata .A Pakistani fungal strain A-16 A which ced using , and werE-value, 0; nt identity 68.3%) and Phomopsis vexans RNA virus  , which are unclassified dsRNA mycoviruses that belong to the genus Victorivirus. The nucleotide sequence of this putative mycovirus, which we called “Alternaria alternata victorivirus 1”  has been deposited in the GenBank/EMBL/DDBJ databases under accession number LC477336.The raw next generation sequencing (NGS) reads obtained by the HiSeq sequencing were assembled into different contigs. A local BLAST analysis showed the presence of a virus-like contig (A1-19), with an average coverage of 2680 , and a size of 5159 bp in length C, which GUUGAUC-3′, bold letters) were noted in AalVV1 and several other victoriviruses (Totiviridae in having an Ala/Gly/Pro-rich region predicted near their C-termini [RNA ligase-mediated amplification of cDNA ends (RLM-RACE) and RT-PCR analyses revealed that the AalVV1 genomic dsRNA was 5120 bp long and contained two overlapping open reading frames (ORFs) on its plus-strand D. ORF1 wiviruses . One of iviruses , boxed. -termini . The C-tUGAA or AUAUG facilitate it together with pseudoknot structures located at immediately upstream of the facilitators [Victoriviruses generally utilize stop/restart strategies to translate the RdRp-encoding ORF2 whose in which litators ,8. Visualitators , was fouE-value, 0.0), to the putative CP of PvRV , to the putative RdRp of NoVV1 (Totiviridae (Victorivirus) and was separated from the members of the genera Leishmaniavirus, Trichomonasvirus and Totivirus in the family Totiviridae, and it showed the closet homology with PvRV and NoVV1 amongst all the known victoriviruses (BLASTp searches of the deduced amino acid (aa) sequences of ORF1 of the contig A1-19 dsRNA revealed the highest identity, 69.8%  fraction where the AalVV1 dsRNA was enriched, nonenveloped spherical particles of ~40 nm diameter were observed with transmission electron microscope A. ProteiSequences of peptide fragments obtained after in-gel tryptic digestion of the major 80 kDa protein band of A-16 was predicted by liquid chromatography/tandem mass spectrometry (LC-MS/MS). As a result, the band contained the peptides derived from both ORF1 and ORF2 of the AalVV1 genome . FurtherA. alternata Pakistani A-16 strain was grown on PDA plates amended with different concentrations of cycloheximide  and then single spore isolation was done from the mycelial plugs. However, our attempts to cure A-16 were unsuccessful. Thus, we took a protoplast fusion approach which was used for different viruses.In order to determine the effect of the virus on the host, attempts were made to create virus-free isogenic line and for that purpose, treatment of a translation inhibition drug cycloheximide, which has been previously used to cure fungus of a mycovirus infection , along wA. alternata was transformed with a HygR gene cassette as described by Shahi et al. [First, a virus-free Japanese strain (Ally-12) of i et al.  and usedC. parasitica, as an experimental host, which proved useful for studying virus/host and virus/virus interactions [C. parasitica strain EP155 and an RNA silencing-deficient strain, ∆dcl2, derived from EP155 were transfected by semipurified AalVV1 particles. Infected colonies were readily obtained in both the EP155 and ∆dcl2 background and showed an unaltered phenotype which was indistinguishable from virus-free EP155 or ∆dcl2  [A. alternata, and contributes to the growing database of viruses of Alternaria spp. To our knowledge, this study represents the second report on a fungal virus from the Indian subcontinent next to the paper by Shamsi et al. [A. alternata.A number of RNA viruses have been reported from rnavirus , a partirnavirus , botybirrnavirus ,53,54, crnavirus , and vicrescens) ; positivrescens) ,58, a furescens) , and an rescens) ; and othrescens) . This sti et al.  that repTotiviridae use −1 frame-shifting , −2 frame-shifting (trichomonasvirses), presumable +1 frame-shifting or ribosomal hopping (leishmaniaviruses) and stop/restart strategies (victoriviruses) [UGAA reinitiation facilitator and an H-type pseudoknot structure immediately upstream of the tetra-nucleotide is necessary and sufficient for the stop/restart translation. Similar translational strategies are used in other fungal and animal viruses with (+)ssRNA genomes, such as hypoviruses [AalVV1 has interesting features similar and dissimilar to previously characterized victoriviruses. For the RdRp translation from bicistronic genome-sized viral mRNA, members of the family viruses) ,62  ,8 in whioviruses  and calioviruses ,64, wheroviruses . The sliUGAA or AUAUG. Simultaneously, the virus has a signature-like sequence “C CaA AAU” for possible −1 frame-shifting, slippery sequence conforming to the consensus sequence (X XXY YYZ) at map positions 2555–2561 immediately upstream of the stop codon, UAG, of ORF1, and a predicted stem-loop structure downstream of the stop codon  of the detected proteins. If the virus uses termination-coupled reinitiation, there must be an unidentified noncanonical start codon downstream the H-type pseudoknot structure and upstream of the nucleotide sequences (map positions 2593 to 2622) encoding amino acids TASDLDLYLK detected by peptide mass fingerprinting  from A. arborescens, the tomato pathotype of A. alternata [Both the phylogenetic trees with the CP and RdRp sequences showed two unclassified victoriviruses, Nigrospora oryzae victorivirus 1 (NoVV1) and Phomopsis vexans RNA virus (PvRV), to make up a sister clade to the one containing AalVV1 A,B. NoVVlternata . This islternata ,26,27. Tlternata ,66,67,68R. necatrix and a model filamentous fungus, C. parasitica [H. victoriae) [A. alternata strain (A-16) was shown to cause symptomless infection in wild type strains of a Japanese A. alternata (Ally-12) and the RNA silencing-proficient standard strain of C. parasitica (EP155). Moreover, unlike RnVV1 and HvV190S [C. parastica, ∆dcl2, in spite of the enhanced accumulation of AalVV1 in ∆dcl2 , and RnPV6 [dcl2 or EP155 [C. parasitica, belonging to a class, Sordariomycetes, different from another one, Dothideomycetes, which include the original host, A. alternata. The previous and current studies suggest broad host ranges of victoriviruses in contrast to the relatively narrow host ranges of two other C. parasitica viruses, a chrysovirus, Cryphonectria nitschkei chrysovirus 1 [Victoriviruses are ubiquitous in filamentous fungi . Howeverrasitica . Subsequrasitica . To our ctoriae) . In this HvV190S ,26, AalVin ∆dcl2 , indicat viruses ,32,70,71nd RnPV6 ,72. A caor EP155 . The occovirus 1  and a miovirus 1 .


Medicago sativa L.) samples collected in Southern France. The nucleotide sequence of the viral genome was determined by de-novo assembly of VANA reads and by 5’/3’ RACE with viral RNA extracted from enriched viral particles or with total RNA, respectively. The virus shares the greatest degree of overall sequence identity (~78%) with Medicago sativa marafivirus 1 (MsMV1) recently deduced from alfalfa transcriptomic data. The tentative nucleotide sequence of the AVF coat protein shares ~83% identity with the corresponding region of MsMV1. A sequence search of the predicted single large ORF encoding a polyprotein of 235kDa in the Pfam database resulted in identification of five domains, characteristic of the genus Marafivirus, family Tymoviridae. The AVF genome also contains a conserved “marafibox”, a 16-nt consensus sequence present in all known marafiviruses. Phylogenetic analysis of the complete nucleotide sequences of AVF and other viruses of the family Tymoviridae grouped AVF in the same cluster with MsMV1. In addition to 5’ and 3’ terminal extensions, the identity of the virus was confirmed by RT-PCRs with primers derived from VANA-contigs, transmission electron microscopy with virus-infected tissues and transient expression of the viral coat protein gene using a heterologous virus-based vector. Based on the criteria demarcating species in the genus Marafivirus that include overall sequence identity less than 80% and coat protein identity less than 90%, we propose that AVF represents a distinct viral species in the genus Marafivirus, family Tymoviridae.Viral infections of alfalfa are widespread in major cultivation areas and their impact on alfalfa production may be underestimated. A new viral species, provisionally named alfalfa virus F (AVF), was identified using a virion-associated nucleic acid (VANA) metagenomics-based approach in alfalfa ( Medicago sativa L.) is the most extensively cultivated forage legume in the world and the fourth most widely grown crop in the U.S. Alfalfa productivity has often been limited by various biotic and abiotic components in the ecosystem [Alphaflexiviridae discovered in alfalfa samples exhibiting chlorosis and stunting [Alfalfa  [sativa marafivirus 1 (MsMV1) was deduced from alfalfa transcriptomic data by Kim et al., [Marafiviruses are positive strand RNA viruses transmitted by leafhoppers in a persistent manner. Their genome has a large open reading frame (ORF) of about 6.3–6.8 kb in size, encoding a precursor polypeptide composed of the replication-associated proteins and one of the two forms of the coat protein (CP) found in the virions . The typs (MRFV) . Until v et al., . These a et al., . The repde-novo assembly of VANA reads and by 5’/3’ RACE with total RNA extracted from infected plants. The identity of the virus reported in this study was also confirmed by RT-PCRs with primers derived from VANA-contigs, transmission electron microscopy observations, and transient expression of the viral coat protein gene using a heterologous plant virus-based vector. Based on the current species demarcation criteria, the virus, provisionally named alfalfa virus F (AVF), represents a distinct species in the genus Marafivirus, family Tymoviridae.In this work, we report identification and the complete genomic sequence of a new alfalfa marafivirus, obtained by in vivo in insect proof growth chambers at Cirad, International Campus of Montpellier, France.From 2010 to 2016, 33 alfalfa leaf samples were collected in Southern France to explore their virome using metagenomics approaches. The owners of the alfalfa fields where the plants were sampled gave permission to conduct the study on their sites. Samples were collected randomly, regardless of any potential symptoms. Two alfalfa plants, from which leaf samples were collected, were maintained https://cutadapt.readthedocs.io/en/stable/) and de-novo assemblies were generated using SPAdes 3.6.2 software (http://cab.spbu.ru/software/spades/).Thirty-three alfalfa samples were processed using the VANA-based 454 pyrosequencing approach exactly as described in François et al. . PCR ampFor 5’ and 3’ RACE systems and RT-PCR assays, total RNA was extracted from one alfalfa leaf sample collected in a commercial alfalfa field at Prades-le-Lez, Southern France (GPS location: 43°42'16.44"N 3°51'48.31"E) using TRIzol RNA isolation reagent as described by the manufacturer . RT-PCRs were performed with total RNA employing the SuperScript RT-PCR system per the manufacturer’s directions (ThermoFisher Scientific). RT-PCR products were either sequenced directly or cloned into the pCRII-TOPO vector with dual promoter (ThermoFisher Scientific) for sequencing.The 5’terminus of the virus was determined using the SMARTer® RACE 5'/3' Kit  with the genome-specific primer LN529 . The 3’ Marafivirus was assembled. These sequences were aligned and phylogenetic tree built with the CLC Genomics Workbench software (Qiagen Inc) using neighbor-joining algorithm, Jukes-Cantor distances and 1000 bootstrap replicates.A dataset consisting of the complete nucleotide sequences of alfalfa virus F and 12 ICTV-approved and tentative members of the genus EcoRV, gel-purified and sub-cloned into the EcoRV-linearized PVX-based vector pP2C2S [SpeI, and capped transcripts were generated from cDNA clones using Ambion’s T7 mMessage Machine kit (ThermoFisher Scientific). The transcripts were mechanically inoculated onto fully expanded leaves of Nicotiana benthamiana.RT-PCR products amplified with primers LN568-LN569 were cloned into the pCR TOPO II vector (ThermoFisher Scientific), digested with r pP2C2S , , viral particles from infected alfalfa tissues were partially purified using a protocol developed for ic virus . For TEMribed in . Virus cDe novo assemblies of VANA reads revealed the presence of contigs sharing similarities with marafiviruses in four alfalfa samples, including one plant sample from Prades-le-Lez (Montpellier region) and three plant samples from the Rhône delta region. Interestingly, one of these three plants, originating from the Rhône delta region, has been maintained at the Cirad laboratory and is not exhibiting any visible symptoms that would differentiate it from healthy plants . The next closest species from the genus Marafivirus, per PASC tool [Marafivirus. Multiple nucleotide sequence alignment using the MegaAlign tool and ClustalW algorithm of the DNAStar package  generated even lower identity between the complete genomes of AVF and MsMV1 (75.3%); identity with other viruses of the genus was at the 50% level or less . The next closest species was Nectarine marafivirus M . The Pfam database search and analysis of the AVF polyprotein (http://pfam.xfam.org/) resulted in five significant Pfam-A matches corresponding to five viral domains characteristic for the genus Marafivirus, family Tymoviridae: viral methyltransferase , tymovirus endopeptidase , viral (superfamily 1) RNA helicase , RNA dependent RNA polymerase  and tymovirus coat protein .AVF encodes a single precursor polyprotein 2130 amino acids (aa) in length. In BLASTP query, the AVF polyprotein was 82% identical to the polyprotein of MsMV1  ,18. Howe Ser1936 .Marafivirus [The nucleotide sequence of the AVF CP25 displays 80% identity with the MsMV1 polyprotein gene . On the amino acid level, AVF CP25 shares 83% identity with the polyprotein of the MsMV1 . Noteworthy, less than 90% aa sequence identity among coat protein sequences is another criterion demarcating species in the genus 5’ GAGGGTGAATTGCTTC 3’). The possible 5’ end of the subgenomic RNA encoding the viral CPs starts at the conserved adenine position 10 nt downstream of the core promoter sequence (A5759), [The AVF genome contains a conserved “marafibox”, a 16-nt consensus sequence of the subgenomic RNA (sgRNA) promoter that is present in all known marafiviruses, at positions 5734–5749 nt ((A5759), ,18.Interestingly, the marafivirus-related contigs, assembled from three alfalfa plants collected from the Rhône delta region, shared 75–97% identities with the AVF genome , indicating that these plants were most likely infected with the virus.A complete genome of the AVF obtained via VANA-based Illumina MiSeq and 5’/3’ RACE was used to design primers for RT-PCR amplification of viral sequences encoding the minor CP25 in order to confirm the virus identity and accuracy of the nucleotide sequence. Using primer pair LN568 (forward) and LN569 (complementary)  in an RTRoutine sample preparation for negative staining  did not N. benthamiana plants via a PVX vector [N. benthamiana, an experimental host species commonly used in plant virology. Two weeks after inoculation, plants developed symptoms such as stunted growth, vein clearing and chlorotic mosaic (not shown). TEM of crude leaf extracts revealed characteristic PVX rods surrounded by isometric virus-like particles (VLPs) ~ 30 nm in diameter resembling typical marafivirus virions  was transiently expressed in latforms .Marafivirus in alfalfa, MsMV1, was described only recently [Marafivirus [Tymoviridae study group.The first proposed species of the genus recently  and befoafivirus . Notablyafivirus . In addiPrimary research by Zhang et al. , whose tBeyond identification and confirmation of AVF as a new viral species infecting alfalfa, the RT-PCR assay with virus-specific primers developed in this work may be of practical use for detection of the virus.Marafivirus, family Tymoviridae and a more evident relation to the unclassified Medicago sativa marafivirus 1. The genome sequence of AVF was deposited in NCBI GenBank on 12/24/2017 as accession № MG676465.Notwithstanding, we conclude that AVF represents a unique virus species with a close resemblance to the members of the genus S1 FigThe plant is not exhibiting any visible symptoms that would differentiate it from the healthy plants.(TIF)Click here for additional data file.


In September 2018, African swine fever in wild boars was detected in Belgium. We used African swine fever–infected spleen samples to perform a phylogenetic analysis of the virus. The causative strain belongs to genotype II, and its closest relatives are viruses previously isolated in Ukraine, Belarus, Estonia, and European Russia. Asfarviridae. ASF is endemic in sub-Saharan Africa countries and has become more prevalent in the Caucasus region since its spread from eastern Africa to Georgia in 2007. The epizootic then spread to the surrounding countries, including the Russian Federation, and further to Belarus and Ukraine. In 2014, ASFV reached the European Union member states of Estonia, Latvia, Lithuania, and Poland; in 2016, Moldova; and, in 2017, the Czech Republic and Romania (African swine fever (ASF) is a devastating disease of domestic pigs and wild boars caused by a DNA arbovirus, African swine fever virus (ASFV), belonging to the family On September 13, 2018, authorities in Belgium reported that ASF had been confirmed in 2 wild boars near the village of Étalle , in the province of Luxembourg, which is located 12 km from the border with France and 17 km from the country of Luxembourg. ASFV appears to have jumped a considerable distance from previously affected countries: ≈500 km from the border with the Czech Republic, 800 km from Hungary, and 1,200 km from the border with Romania. Since then, ≈75% of the wild boars found dead near the primordial spot have been found to be ASFV positive; a total of 96 positive results had been recorded as of October 16, 2018. B646L gene by PCR as described by Gallardo et al. (https://www.geneious.com). We performed phylogenetic analysis using MEGA7 (http://www.megasoftware.net) and the Kimura 2-parameter substitution model, using the neighbor-joining method, as determined by a model selection analysis (To investigate the virus, we performed initial genetic characterization using standard genotyping procedure on virus DNA directly extracted from homogenized spleen or kidney tissues of each animal. First, we obtained a segment of the analysis , panel AI73R and I329L genes, according to Gallardo et al. (To further define the most likely origin of this strain, we performed PCR targeting a ≈350-bp fragment in the variable intergenic region between the o et al. , panel B


Macaca fascicularis) trapped in Peninsular Malaysia in 2009, 2010, and 2016 for Zika virus RNA and antibodies. None were positive for RNA, and only 1.3% were seropositive for neutralizing antibodies. Long-tailed macaques are unlikely to be reservoirs for Zika virus in Malaysia.We tested a sample of 234 wild long-tailed macaques ( Macaca mulatta) in the Zika Forest in Uganda, reemerged in the Pacific Islands and Americas in 2015 and caused unprecedented outbreaks associated with serious congenital syndromes  and vervet monkeys (Chlorocebus pygerythrus) , the most common macaque in Peninsular Malaysia, which is also widespread throughout Southeast Asia.In Malaysia, Zika virus seropositivity has been reported in residents  traps monkeys foraging in human-populated areas and relocates them to deep forest areas (https://www.qiagen.com), we tested samples with sufficient serum volume (n = 228) for Zika virus envelope gene by real-time PCR (50) on Vero cells (50 titers ≥20 (50), and samples had titers identical to or within 1 dilution of the PRNT50 titer.After extracting viral RNA from samples with a QIAamp Viral RNA Mini Kit  or if the Zika virus PRNT50 titer was ≥20 and 4-fold greater than the DENV-1 and DENV-2 FRNT50 titers (1 sample). Only 3 of 6 samples fulfilled these criteria; the remaining 3 contained detectable Zika virus, DENV-1, and DENV-2 antibodies, indicating past flavivirus infection of an indeterminate type. Thus, 3 (1.3%) of 234 samples were Zika virus seropositive, although we did not test for other flaviviruses.Because flavivirus antibodies are known to cross-react, these 6 samples were further examined for antibodies specific to the major known circulating flaviviruses in Malaysia, dengue virus serotypes 1 (DENV-1) and 2 (DENV-2), by FRNTThe 3 Zika virus–seropositive monkeys were captured 35 km away , 77 km away , and 164 km away  from Bentong (Pahang), where Zika virus was first isolated outside of Africa in 1966  in macaques is unknown. This information is arguably needed before an animal can be designated a reservoir (Research methods for determining neutralizing titers against Zika and dengue viruses in serum samples from long-tailed macaques in Peninsular Malaysia, 2009, 2010, and 2016.