In some European countries, including Poland, beef production is based mostly on dairy cattle herds. Beef quality can be improved by the commercial crossbreeding of dairy cows with beef bulls. The offspring are characterized by higher fattening performance and more desirable carcass characteristics. The experiment was performed on 72 bulls produced by crossing Polish Holstein-Friesian (PHF) cows with bulls of three most popular beef breeds: Hereford (HH), Limousin (LM), and Charolais (CH), to determine the effects of genotype and age at slaughter on the content of selected minerals  and fatty acid profile in beef. The meat of PHF × LM crosses had a lower content of K and Mg, compared with the remaining crossbred bulls. The Fe content per kg of meat was higher in bulls slaughtered at 21 months of age than in those slaughtered at 15 months of age. The best sire breed for crossing with dairy cows cannot be clearly indicated based on the present findings. However, the results of this study suggest that bulls should be slaughtered at 21 months of age to achieve the optimal values of most analyzed traits and parameters, in particular the fatty acid profile.p ≤ 0.01) content of K and Mg, compared with the remaining crossbred bulls. The Fe content per kg of meat was 4 mg higher (p ≤ 0.01) in bulls slaughtered at 21 months of age than in those slaughtered at 15 months of age. The content of monounsaturated fatty acids (MUFAs) in intramuscular fat was 2.77% higher in bulls slaughtered at 21 months of age than in those slaughtered at 15 months of age. The n-6/n-3 polyunsaturated fatty acids (PUFA) ratio did not exceed 4.0, and it was most desirable in PHF × LM crosses (2.84) and in the oldest bulls (2.92).The aim of this study was to determine the effects of genotype and slaughter age on the mineral content and fatty acid profile of beef. The experiment was performed on 72 crossbred bulls produced by crossing Polish Holstein-Friesian (PHF) cows with bulls of three beef breeds: Hereford (HH), Limousin (LM), and Charolais (CH), slaughtered at 15, 18, and 21 months of age. Samples of the longissimus thoracis muscle were collected to determine their mineral  and fatty acid composition. The meat of PHF × LM crosses had a lower ( Red meat, including beef, is an important source of highly biologically active proteins, fat, vitamins, and minerals . Fat is the richest source of dietary energy, and it supplies essential nutrients such as fat-soluble vitamins and fatty acids . Bovine Mineral concentrations in meat change depending on the diet , genotypIn Poland, beef production is based mostly on dairy cattle herds. Beef quality can be improved by the commercial crossbreeding of dairy cows with beef bulls. The offspring are characterized by higher fattening performance and more desirable carcass characteristics . The mosThe study was conducted upon the approval of the Ethics Committee of the University of Warmia and Mazury (decision no. 121/2010). The experimental material comprised 72 crossbred bulls produced by crossing PHF cows with HH, LM and CH bulls. Crossbred calves were purchased at 2–3 weeks of age. The animals underwent a seven-day quarantine period and were raised until six months of age. They were fed milk replacer supplemented with hay and concentrate, followed by grass silage. The fattening period began when the calves were 181 days old. The animals were fattened in a semi-intensive system, and they were fed ad libitum a total mixed ration (TMR) composed of grass silage and concentrate .The concentrate to silage ratio, on a dry matter (DM) basis, was 25:75 . When thAt the end of the fattening period, the bulls were transported to a meat processing plant, where they were fasted for 24 h and slaughtered. The animals were weighed before slaughter, stunned, slaughtered, bled, dressed, and split into two sides that were then chilled for 96 h at +4 °C. Electrical stimulation was not applied to the carcasses. Half-carcasses were weighed within an accuracy of 0.5 kg, using an automatic in-line scale. Carcass conformation and fatness were evaluated by a trained grader, based on the EUROP system criteria . Dressin® 1577c Bovine Liver . All samples were mineralized and analyzed in duplicate. The mineralized samples were cooled and quantitatively transferred to 25 cm3 volumetric flasks. The content of selected minerals  in beef samples was determined by flame atomic absorption spectrometry (flame AAS) with the use of the AA240FS Fast Sequential Atomic Absorption Spectrometer  [In order to determine the content of selected minerals, samples of the LT muscle were homogenized, and approximately 0.5 g of the muscle tissue was wet mineralized with 7 mL of 65% Suprapur nitric acid  in the MARS Xpress 5 microwave digestion system . Each mineralization procedure involved two blank samples and two samples of Standard Reference MaterialCA, USA) ,30.2SO4 mixture  according to the modified Peisker method [Intramuscular fat (IMF) was extracted from ground meat samples by the Soxhlet method  using thr method  and Stanr method . The conijk is the value of the analyzed parameter, μ is population mean, Ai is the effect of genotype , Bj is the effect of slaughter age , (AB)ij is the genotype × slaughter age interaction, and eijk is random error. The differences between means were estimated by Tukey’s test.The data were analyzed statistically using Statistica version 13.3 software . The effp ≤ 0.01) (p ≤ 0.01) than that of PHF × CH crosses. Dressing percentage is a key indicator of carcass quality in cattle. The LM breed is known for its high carcass quality, which was confirmed by Chambaz et al. [p ≤ 0.05). Carcass dressing percentage, conformation, and fatness were significantly affected by age at slaughter. Dressing percentage was higher in heavier animals, which could result from their higher carcass fatness. In a previous study investigating young Holstein-Friesian bulls [As expected, older animals were characterized by higher BW at slaughter ( ≤ 0.01) . The finz et al.  in whosean bulls , higher p ≤ 0.01) content of K and Mg than meat from PHF × LM crosses. Fe content was higher (p ≤ 0.05) in meat from PHF × CH crosses than in meat from PHF × LM crosses. The content of Zn and Fe in the LT muscle was affected by age at slaughter. The meat of animals slaughtered at 18 months of age had the highest (p ≤ 0.05) Zn content (63.9 mg/kg of fresh meat), whereas the meat of the oldest bulls had the highest Fe content (19 mg/kg of fresh meat). The effect of cattle breed on the K and Mg content of beef was also noted by Pilarczyk [The mineral composition of the LT muscle of crossbred bulls is presented in ilarczyk  and Domailarczyk . In the ilarczyk , the Fe ilarczyk  found thilarczyk ,39 were ilarczyk , was alsilarczyk . In contilarczyk , and Kotilarczyk  demonstrilarczyk  observedilarczyk  reportedilarczyk  noted noIn the current study, neither breed nor age at slaughter exerted significant effects on the Na content of beef. The lowest Mg content of meat was noted in meat from PHF × LM crosses and bulls slaughtered at 18 months of age . In the remaining groups, Mg concentrations exceeded 200 mg/kg of fresh meat. Consumption of 100 g of cooked beef satisfied 8.1–9.6% and 6.2–7.3% of the recommended daily allowance (RDA) for Mg for women and men, respectively . Pilarczp ≤ 0.01), and on the concentrations of MUFAs (p ≤ 0.05) in the LT muscle of bulls. The IMF of animals slaughtered at 21 months of age had a significantly higher content of UFAs and MUFAs, compared with those slaughtered at 15 months of age. Age at slaughter had a significant influence on the percentage share of the following fatty acids in IMF: oleic, C15:0 anteiso (p ≤ 0.01), capric, lauric, pentadecanoic, margaric, stearic, and gadoleic (p ≤ 0.05). The IMF of bulls slaughtered at 15 months of age had the highest concentrations of capric acid and lauric acid, whereas the IMF of the oldest bulls had the lowest content of the following fatty acids: capric, lauric, C15:0 anteiso, pentadecanoic, margaric and stearic. The IMF of the oldest bulls had the highest proportion of UFAs, including oleic acid  and gadoleic acid.The concentrations of fatty acids, major fatty acid groups, and ratios in the IMF of crossbred bulls are presented in p ≤ 0.01) difference was observed between PHF × LM crosses (2.84) and PHF × CH crosses (3.3). The most desirable n-6/n-3 PUFA ratio was noted in bulls slaughtered at 21 months of age (2.92), with a significant (p ≤ 0.05) difference relative to those slaughtered at 15 months of age.The n-6/n-3 PUFA ratio was affected by genotype and age at slaughter. A significant , palmitic acid (25.17–26.24%), and stearic acid (17.35–19.38%). The predominant SFAs in beef are palmitic acid, stearic acid, and myristic acid. Lauric acid, myristic acid, and palmitic acid are associated with elevated plasma total and LDL cholesterol concentrations, which are not increased by stearic acid . Its valDifferent beef breed sires used for commercial crossing affected the content of K, Mg, and Fe in beef. The LT muscle of PHF × LM crosses had the lowest content of K, Mg, and Fe. Bulls slaughtered at a later age were characterized by higher Fe content and higher concentrations of MUFAs, in particular, oleic acid and gadoleic acid. The n-6/n-3 PUFA ratio did not exceed the nutritionally desirable value of 4.0, and it was lowest in PHF × LM crosses and in bulls slaughtered at 21 months of age. The best sire breed for crossing with dairy cows cannot be clearly indicated based on the present findings. However, the results of this study suggest that bulls should be slaughtered at 21 months of age to achieve the optimal values of most analyzed traits and parameters, in particular the fatty acid profile.


Measles remains a public health concern in many regions, and the imported measles cases continue to challenge the measles elimination program for most of the countries where measles was verified to be eliminated or approaching elimination. The imported measles cases have been reported since October, 2017, in Jiangsu province, China. In this study, we reported the first imported B3 genotype measles virus from Egypt and the second imported D8 genotype measles virus from Philippines through international traveling. No secondary measles cases were found after these imported cases. Our findings highlighted the importance of measles vaccination targeting international travelers in China. Measles is a highly contagious viral disease characterized by fever, cough, coryza, and conjunctivitis followed by characteristic rash . It is sJiangsu province, located in the east of China, is one of economically developed provinces in China. The number of the international travelers has been increasing in past decades. By the end of 2018, it reached 6.7 million travelers. Especially in recent years, more and more people are traveling to Africa and east-south Asia due to the low cost. Before 2017, H1a genotype was the unique virus circulating in Jiangsu province. Until October of 2017, Deng et al. identified two epidemiologically linked cases caused by D8 genotype measles virus, and the index case might be a student from Lao People's Democratic Republic who studied at college in Jiangsu province [After the local Center for Disease Control and Prevention received the report of measles case from local hospital by Epidemic Surveillance System, the epidemiology investigation was conducted within 48 hours, which included the collection of demographics, epidemiologic, and clinical data. The Chinese National Notifiable Disease Reporting System was established in 1955. In 1997, China developed a case-based, laboratory-supported measles surveillance system. The two surveillance systems were unified in 2009. Every suspected case is investigated by county-level China CDC staff members using a standardized, in-person questionnaire; outbreaks are investigated and reported by local China CDC staff members. At the same time, the specimens are collected and transported to measles network lab to conduct IgM antibody and nucleic acid test.IgM antibody and nucleic acid of measles virus test were conducted in local county and prefecture center for disease control and prevention according to standard SOP developed by Chinese Center for Disease Control and Prevention, respectively. The genotyping of Mev was conducted in Jiangsu Provincial Center for Disease Control and Prevention. The QIAamp Viral RNA Mini Kit  was used to extract measles virus (MeV) nucleic acid according to the manufacturer's instructions. Reverse transcription amplification was performed using previously described primers to amplify a 634-bp fragment of the N gene , which iThe sequences were manually edited using BioEdit version 7.2.5 software and then analyzed using MEGA 7.0 software. The reference strains include the reference strains recommended by WHO and D8 and B3 genotype sequences isolated from China. The phylogenetic tree was constructed using the neighbor-joining method with 1000-replicate bootstrap test. The two sequences were submitted to GenBank to do Blast to seek the sequences with the highest identity.th and then had fever (38°C) on November 10th, which developed into conjunctival congestion on November 12th, and cough on 13th. He was admitted to a hospital on 16th and diagnosed with measles on November 19th. WBC was 6.19 × 109/L (75% neutrophils), aspartate aminotransferase 65.0 U/L, C-reaction protein 23 mg/L, urine occult blood 2+, urine protein +, HBsAg+, HBeAb+, and HBcAb+. Nucleic acid of measles virus is positive using real-time fluorescence PCR test. Among this tour group, none except him was diagnosed as measles. He did not receive any doses of measles-containing vaccine., male, 55 years old. He traveled to Egypt during October 31st to November 10th, 2018. He had onset of maculopapular rash on his legs and back on the night of November 8th, 2019, and rash on March 8th; subsequently the rash extended to most of the body skin. He returned to China on the night of March 11th. He was diagnosed as measles in a local hospital on March 12th; the fever disappeared on March 12th. Blood and nasopharyngealswab specimens were collected on March 14th for IgM antibody of measles virus and nucleic acid of measles virus test. Results showed that both of IgM antibody against measles virus and nucleic acid of it were positive. His immunization status was unknown., male, 23 years old. He had fever of 38°C in Philippines on March 4As shown in In this study, we reported the first imported B3 genotype measles virus and the second imported D8 genotype measles virus in Jiangsu province. Two patients were infected with measles virus abroad during the international trip, and the time of onset was before they returned to China. After onset of measles, they returned to China and subsequently were diagnosed with measles at local hospitals. Case 1 was infected with D8 genotype measles virus which had higher identity with virus from Japan (Yokohama), USA (Connecticut), Brazil (Brunri-Muara), and Australia  than those isolated from China. Case 2 was infected with B3 genotype measles virus which had higher identity with virus form USA, Australia, and Hong Kong of China. During the investigation of these imported measles cases, no close contacts were diagnosed with the virus. In Jiangsu province, the average measles incidence is about 12 per million people in recent year  and vaccHerein, the evidence obviously supported that case 1 got measles virus in Egypt and case 2 in Philippines. Whether case 1 or case 2, both were infected with measles virus and had onset of measles abroad. Particularly, measles was outbreaking in Philippines when case 2 was staying there. However, it is regretful that no sequences of measles virus of these two countries were found in GenBank database. Phylogenetic analysis cannot provide direct evidence to confirm the source of measles virus in these two cases. The global distribution of measles virus has the regional specificity; the different genotype of measles viruses circulates in various regions . HoweverThere were several limitations. Firstly, the epidemiology investigation was not conducted among the people taking the same flight when case 1 returned to China. Therefore, it was not clear whether measles virus was transmitted between them. Secondly, in the tour group case 2 joined, a girl even had fever for two days and self-cured. No epidemiology investigation and lab test were conducted for her. So, it is difficult to confirm whether she had measles or not.Measles virus will continue to be imported into China if measles remains endemic around the world and international travelers increase. Our findings emphasize the importance of maintaining a high level of measles immunity in China populations  and necessity of widely conducting molecular surveillance in the world and the importance of sharing measles sequence data more widely and timely.


One year after the first autochthonous transmission of West Nile virus (WNV) to birds and horses in Germany, an epizootic emergence of WNV was again observed in 2019. The number of infected birds and horses was considerably higher compared to 2018 , resulting in the observation of the first WNV epidemy in Germany: 76 cases in birds, 36 in horses and five confirmed mosquito-borne, autochthonous human cases. We demonstrated that Germany experienced several WNV introduction events and that strains of a distinct group (Eastern German WNV clade), which was introduced to Germany as a single introduction event, dominated mosquito, birds, horse and human-related virus variants in 2018 and 2019. Virus strains in this clade are characterized by a specific-Lys2114Arg mutation, which might lead to an increase in viral fitness. Extraordinary high temperatures in 2018/2019 allowed a low extrinsic incubation period (EIP), which drove the epizootic emergence and, in the end, most likely triggered the 2019 epidemic. Spatiotemporal EIP values correlated with the geographical WNV incidence. This study highlights the risk of a further spread in Germany in the next years with additional human WNV infections. Thus, surveillance of birds is essential to provide an early epidemic warning and thus, initiate targeted control measures. Flaviviridae, genus Flavivirus) is maintained in a transmission cycle between birds as amplification hosts and mosquito vectors [West Nile virus . Phylogenetic and phylogeographic analysis showed that Germany experienced several WNV introduction events. Several virus variants circulate in the affected German regions with Austria and Czech Republic as possible origins. The majority of the WNV strains involved in the German outbreak clustered together into a distinct and dominating group (Eastern German WNV clade) comprising of mosquito, bird, horse and human-related virus variants.Since the first outbreak of Usutu virus (USUV) in Germany (2011/12), a nationwide bird surveillance network (living and dead birds) was set up to monitor for zoonotic arboviruses with a focus on WNV and USUV. In this context, a variety of dead birds and organ samples were submitted to the Bernhard Nocht Institute for Tropical Medicine and the national reference laboratory for WNV at the Friedrich-Loeffler-Institut (FLI) by the regional veterinary laboratories of the federal states of Germany, by the German Mosquito Control Association (KABS), the Nature and Biodiversity Conservation Union (NABU), citizens and independent bird clinics and zoological gardens. WNV infection in birds and horses is a notifiable disease in Germany if a recent infection is detected by a WNV-specific RT-qPCR result and/or a positive result of horses by IgM-ELISA, i.e., the detection of a fresh WNV infection. A previous vaccination of horses must be excluded. A positive IgG or neutralizing antibody detection is not notifiable in Germany.Requests for the submission of dead birds were made via press releases of involved institutes and subsequent dissemination of the information by different kinds of media, including newspaper articles, television and radio. Total RNA from homogenized tissue samples  was extracted and analyzed for the presence of flavivirus RNA by using a modified pan-flavivirus reverse transcription PCR  or WNV-sOrgan samples from affected horses were also tested by the RT-qPCR as stated above. In the case of diseased horses, often with neurological symptoms typical for WNV disease, the serum samples were screened by IgM-and/or IgG-ELISA  and positive samples were confirmed by differentiating virus neutralization tests to exclude cross-reacting flaviviruses ) . Following the first confirmed avian WNV case in the Tierpark Berlin (Wildlife Park) in 2019, mosquitoes were collected in that park by EVS  traps  equipped with dry ice as an attractant. Traps were continuously operated from mid-September to early October and emptied daily. Captured mosquitoes were morphologically identified to species or complex using the determination key by Becker et al.  and poold) of WNV were calculated with the formula −0.132 + 0.0092 × temperature [http://www.ecad.eu [d values for the subsequent days were summed up until the virus development was completed (=EIP). For each grid cell and year, EIP values were averaged for the period from 15th July to 14th August (=EIPave). The extrinsic incubation period (EIP) gives the time between ingestion of a pathogen via blood meals and the vectors’ ability to retransmit the pathogen. In contrast to other indices for transmission risk , this approach is a theoretical risk assessment using information on the temperature-dependent EIP from the literature. However, EIP values give an approximation of virus transmission risk through the mosquito vector under local temperature conditions. Therefore, daily EIP values . All sequences were confirmed as non-recombinant by the various methods for recombination detection implemented in RDP4 [A total of 39 WNV genomes from birds, humans, mosquitoes and horses were newly acquired as part of this study . The ext in RDP4 . The obt7 generations (with 10% burn-in) with subsampling every 104 iterations to achieve convergence as assessed using Tracer v1.5 [http://tree.bio.ed.ac.uk/software/figtree/). To test the hypothesis that WNV was periodically introduced to Germany, a phylogeographic analysis was conducted using a discrete model attributing state characters represented by the detection locality of each strain and the Bayesian stochastic search variable (BSSV) algorithm implemented in BEAST v1.10 [https://rega.kuleuven.be/cev/ecv/software/SpreaD3_tutorial) was used to run BSSV analysis and generate Bayes factor (BF) and posterior probability (PP) to test for statistically significant epidemiological links between discrete sampling locations. The potential transmission networks within and between countries for NS5 WNV were inferred in PopART package v1.7.2 using median joining tree method with an epsilon of zero [Genomes obtained for the German WNV strains were compared with all European complete or near complete genomes sequences publicly available. For molecular clock phylogenetics, maximum clade credibility (MCC) trees were inferred using the Bayesian Markov chain Monte Carlo (MCMC) approach available in BEAST v1.10 . Analysecer v1.5 . The MCCST v1.10 . An MCC  of zero .Culex pipiens complex mosquito pools in 2019.A total of 88 birds and 38 horses tested positive for WNV in 2018 (diagnosed between 28.8. and 9.10) and 2019 (diagnosed between 8.7. and 21.11) in Germany. In addition, five probably mosquito-borne human WNV cases were diagnosed with no history of travel to WNV-endemic countries within the last month. Except a single specimen , all WNV-positive animals originated from the eastern part of Germany with a distinct focus for the federal states Saxony-Anhalt, Saxony, Berlin and Brandenburg . In addiLow WNV activity was detected for the federal states Bavaria and Mecklenburg-Western Pomerania in 2018, which was not observed in 2019. WNV cases were found in Hamburg and Thuringia in 2019 for the first time. The number of positive birds and horses rose considerably in 2019 (76 birds and 36 horses) compared to 2018 (12 birds and two horses).Especially in 2019, a large number of different bird species was affected . A totalave (<15 days) for this area. The region along the Upper Rhine Valley (south-western Germany) was also characterized by low EIPave values, but no WNV circulation was detected in either year. Re-emergence of WNV was not observed for the most northern (Rostock) and southern (Poing) foci of WNV from 2018. This correlates with higher EIPave values for 2019  compared to 2018  for these areas, i.e., lower risk of WNV transmission.The area with highest activity of WNV circulation was similar in 2018 and 2019, i.e., central-eastern Germany with most WNV-positive samples  . In addihttps://promedmail.org/promed-post/?id=20181006.6074497). The laboratory confirmation was based on detection of an IgM response against WNV and a cross-reactive IgG response against WNV, which might be also the result of past vaccinations against TBE and yellow fever virus. The first mosquito-borne, autochthonous infection for Germany was confirmed in Leipzig, Federal State of Saxony on 20 September 2019. The 69-year-old male patient presented with WNND, received supportive treatment at the Infectious Diseases (ID) intensive care unit (ICU) between September 3rd and September 20th and was released with restitutio ad integrum. The laboratory confirmation was based on detection of WNV RNA in an early CSF, serum and urine sample and the detection of WNV IgM and IgG in serum samples. A second autochthonous case in Leipzig, an 81-year old male, was admitted to the ICU with presumptive diagnosis of pneumonia, then transferred to the ID-ICU and was found to have WNND confirmed by WNV IgM and IgG in serum samples as well as WNV RNA in CSF samples as early as from 19 September. He also recovered after 12 days of supportive care including mechanical ventilation without neurological sequelae. Both patients reported no history of travel to WNV-endemic countries and routes of non-vector borne transmission were excluded. While one patient had direct contact with the corpse of a Blue Tit (Parus caeruleus) five days before onset of fever, the other one reported no obvious contact with animals. Both patients had experienced multiple mosquito bites in the weeks before onset of illness. The third WNV infection was diagnosed on 24 September in a 46-year-old female patient from Berlin, Federal State of Berlin, who presented with West Nile Fever (WNF). The patient did not receive any treatment and recovered within two weeks. The laboratory confirmation was based on detection of WNV RNA in an early serum sample and seroconversion of WNV IgM and IgG in later serum samples. The fourth WNV infection was diagnosed retrospectively based on IgM and IgG detection in a serum sample on 16 October in a 44-year-old female patient from the district Wittenberg Federal State of Saxony-Anhalt. The patient was initially admitted to a local hospital with WNND-like symptoms on 10 September. After receiving supportive care, she was discharged on 17 September with restitutio ad integrum. The serological diagnosis was confirmed on 23 October by the detection of WNV RNA in a serum sample from the acute phase of infection. The fifth WNV infection was diagnosed on 23 October in a 24-year-old female patient from the district Leipzig, Federal State of Saxony, who presented with WNF (onset of symptoms 6 of October) and did not receive any treatment and recovered within one week. The laboratory confirmation was based on detection of WNV-specific IgM and IgG in a serum sample. As of 20 December, no further cases have been reported.In September 2018, a 31-year-old male veterinarian developed flu-like symptoms after necropsy of a WNV-positive owl  indicating that the analyzed WNV has maintained genetically stable since its first detection in 2018. The identity matrices for the genome and for individual genes were greater than 99.2%. The greatest variation was observed in the nonstructural genes coding for the NS1, NS2A, NS3 and NS5.2 = 0.31 for the “European” data set and 0.19 for the “German strains only,” p < 0.001). The coefficient of rate variation supports the use of a strict clock model for European data set and a relaxed clock for the data set “German strains only” (data not shown). The Bayesian MCC tree showed that although the WNV diversity in the “German strains only” group appears to have emerged in the last four years, the phylogeny of CEC which includes EGC suggests relative long-term circulation and evolution in Central Europe  and Bayesian MCC phylogenies of the European WNV lineage 2 data set revealed that all European strains fell into two distinct highly supported groups designated as Southeastern European clade (SEEC) and Central and Eastern European clade (CEC). All WNV strains from Germany fell into the CEC . The detl Europe .In further detail, the phylogeographic analysis suggests at least six distinct introductions of WNV into Germany from neighboring countries. It is predicted that all viral clade evolution events occurred during the last 16 years a. It shoBased on the albeit only limited Central European strain data, the tMRCA of the EGC group indicates a very recent emergence which was most likely introduced into Germany as a single introduction event. The progenitor of this Eastern European clade dates back to 2011, most probably circulating in Czech Republic  a. The EG−4  subs site−1 year−1 two times lower than for the European data set, 2.51 × 10−4  subs site−1 year−1 (3056Arg mutation from the NS5 gene was found to be common for EGC strains and some WNV from Austria (MF984341), Czech Republic (KM203862) and Germany (LR743437 and LR743434). There are several non-synonymous mutations in the nonstructural genes, which exhibit geographical structures specific of the members of the CEC , which have a higher probability of detection compared to wild animals. Furthermore, from these birds, a considerable proportion were birds of prey, which must be considered to have a higher susceptibility to WNV infection [One year after the first observed autochthonous WNV transmission to birds and horses in Germany ,13, an enfection . This inThis is reflected in the detection of five laboratory confirmed, mosquito-borne, autochthonous human WNV cases in 2019. It has to be kept in mind that less than 25% of infected humans develop noticeable symptoms . Even feWNV transmission and spread is significantly influenced by climatic conditions, e.g., shaping phenology and abundance of the vector. Temperature is one of the most important factors directly affecting the EIP in different mosquito vector species . High daThe ongoing circulation and dominance of the EGC detected in 2019 indicates successful overwintering of WNV in Germany, e.g., through WNV persistence in hibernating mosquitoes throughout the winter season . The virThere has been a comprehensive USUV/WNV monitoring system in place in Germany for over a decade which involves ornithologists, zoological gardens and bird clinics supplying thousands of zoo and wild bird samples for WNV antibody and genome analysis. Moreover, there has been an exhaustive mosquito surveillance in place in Germany since 2009. By both surveillance approaches a variety of viruses were found, such as Sindbis virus, Batai virus and USUV, but not WNV ,13,36,37Most of the singleton WNV variants in Germany do not contain the monophyletic Lys2114Arg mutation located in the C terminus of the NS3 gene, even if these strains circulate in the central-eastern part of the country with very high WNV activity and rapid expansion of the EGC. Although these singletons have circulated and evolved under the same ecological conditions as members of EGC, it seems that these variants were not able to perpetuate and establish a stable enzootic cycle leading to a similar epizootic/epidemic scenario as for the EGC group. In case of the EGC, the adaptation to naïve vector and host populations leads to the emergence of local virus variants. The most likely scenario for EGC might be enzootic maintenance similar to that observed for WNV in the United States ,39. ThisWe found evidence that the phylogenetic structure of EGC and virus genetic population growth is shaped by the geographic location and average extrinsic incubation period, which likely facilitated rapid short-distance virus dispersal in 2018/2019. This demonstrates that local ecological factors  could predict the local and regional dispersal patterns of WNV in our data sets. The purifying and negative selections observed for WNV in Germany were expected given the transmission and infection modes of arboviruses, leading to the accumulation of synonymous mutations . MutatioThis study provides a first comprehensive summary and phylogeographic analysis on the WNV epidemic in Germany in 2018 and 2019 and highlights the risk of human WNV infections causing considerable risks for transfusion and organ transplantation safety. Therefore, intensive surveillance of mosquitoes, birds, horses and humans should remain a public health priority, e.g., to monitor the occurrence and subsequent spread of WNV or to develop targeted control mechanisms. Our study also highlights the need for international cooperation in the area of WNV surveillance and monitoring, especially across national borders and as a “one-health” approach for an improved risk analysis. This should also include the generation of higher numbers of whole-genome sequences, allowing for a more precise molecular epidemiology and strain characterization.


Heat shock cognate 71-kDa protein (HSC70), a constitutively expressed molecular chaperon within the heat shock protein 70 family, plays crucial roles in maintaining cellular environmental homeostasis through implicating in a wide variety of physiological processes, such as ATP metabolism, protein folding and transporting, antigen processing and presentation, endocytosis, and autophagy. Notably, HSC70 also participates in multiple non-communicable diseases and some pathogen-caused infectious diseases. It is known that virus is an obligatory intracellular parasite and heavily relies on host machineries to self-replication. Undoubtedly, HSC70 is a striking target manipulated by virus to ensure the successful propagation. In this review, we summarize the recent advances of the regulatory mechanisms of HSC70 during viral infections, which will be conducive to further study viral pathogenesis. The expression of HSPs is precisely regulated under stress to limit the detrimental consequences and facilitate cell recovery. The HSP70 family consists of molecular chaperons of approximately 70 kDa in size that are highly conserved in all organisms . All the+T cells  and acti+T cells . Due to Attachment, the first crucial step to initiate infection, depends on the interaction between virus attachment proteins and cellular receptors. Exploring virus-bound cell-surface molecules has always been the hot topic of virology. HSC70 usually distributes in the cytoplasm and shuttles between cytoplasm and nucleus to participate in many biological processes. However, the exit of HSC70 from nucleus to cytoplasm is inhibited upon stress, limiting its function to the nuclear compartment . MeanwhiDengue virus (DENV), a notorious insect-borne virus, has been reported to interact with several molecules to enter host cells, such as DC-SIGN in dendritic cells , GRP78 , and β3 After binding cellular receptors, viruses must devise strategies to cross the membrane barrier and uncoat capsid to expose nucleic acid for gene expression. Many enveloped viruses are internalized through clathrin-mediated endocytosis . After vIn addition to enveloped viruses, HSC70 also participates in transporting non-enveloped virus particles. Simian virus 40 (SV40) enters cells through caveolar internalization and accumulates in ER compartment . Then, SChenopodium quinoa, suggesting that HSC70 plays an important role in plant virus infection  is a conserved protein of murine gammaherpesvirus 68 (MHV68), which is important for latency maintenance and acute viral replication . In MHV6n system . Likewisn system . Therefon system  or via tn system  to supprn system . HSC70 uRD cells .Chaperon activity is not limited to protein–protein interactions, and HSC70 can favor virus replication through binding regulator non-coding RNA (ncRNA). Studies have reported that many viruses, such as human immunodeficiency virus (HIV) , DENV H, and WesBesides positive regulation, HSC70 also exerts a negative effect on virus replication. HSC70/HSP90 has already been confirmed to be a driving force for the RNA-induced silencing complex (RISC) assembly pathway through providing ATP to load small RNA duplexes into Argonaute proteins . DownregRenilla reporter JFH-1 HCV virus  particles through interacting with the HPD (His-Pro-Arg) motif on the E2 envelope protein of the virus. In Huh7.5 cells, HSC70, HCV core, and E2 proteins were found to colocalize at the periphery of lipid droplets, an important site for HCV assembly and release. Knockdown of HSC70 with RNA interference reduces the volume of lipid droplets and inhibits viral RNA release, without influence on virus intracellular replication level, suggesting that HSC70 might modulate HCV infectivity through contributing to assembly or budding . The folCV virus . For HBVCV virus ; howeverCV virus . Amino aCV virus . The nonCV virus . HSC70 iCV virus .638 and IRF3 on Ser386 to regulate downstream IFN expression. E1A oncogene of human adenovirus 5 restricts host antiviral response through antagonizing HSC70 and IRF3 phosphorylation  is a type of autophagy responsible for selective degradation of cytosolic proteins bearing a certain consensus amino acid motif (KFERQ) . Being arylation . Besidesma cells . HSV-2 c evasion . Howeveral trial .As a multitask chaperone protein, HSC70 acts as a main or sub-steersman, being involved in many different cellular biological processes. With virus infection, however, these physiological functions are interrupted and HSC70 is hijacked to assist virus propagation. Many studies have demonstrated that HSC70 is a major target utilized by either enveloped or non-enveloped DNA or RNA virus to participate in different infective stages. However, HSC70 is not only coerced into facilitating virus infection but also acts as a resister to eliminate virus by boosting cellular antiviral innate and adaptive immune response. Therefore, this review summarizes the diverse roles of HSC70 in virus infections , to deeprac-cryptopleurine, specifically binds to the NBD of HSC70 and stimulates ATP hydrolysis in the presence of the poly U/UC motif of HCV RNA and thus inhibits viral translation consequently (HSC70 is an important housekeeping protein, mostly responsible for maintaining protein homeostasis in non-stressed conditions and involved in rapidly inducible cell protection following stress situations. In contrast, HSC70 generally promotes, but rarely inhibits, virus infection through interacting with viral protein or viral gene, suggesting that many viruses benefit from host-defensive response to facilitate self-replication and it might be a potential antiviral target to limit virus infections. Based on the fact that constitutive deletion of HSC70 is embryonically lethal in mice  and knocequently . As an Aequently . TherefoMoreover, since HSC70 assists in the release and recycle of clathrin to form the endosome, preparing for membrane fusion, and transporting exogenous cargos to the subcellular sites, it is no doubt to play an integral role in virus invasion. It is widely believed that coronaviruses enter the host cells via two routes, the particularly important endocytic pathway and the non-endosomal pathway . HoweverAlthough being initially characterized as a chaperon to stabilize protein homeostasis, the multifunctional roles of HSC70 in cellular biological processes make it possible to participate in different stages of virus life cycle. As our understanding deepens, the underlying molecular mechanism of HSC70-regulating virus pathogenesis will be gradually elucidated, and the related antiviral drugs will be further developed.ZW and JW searched references and wrote the manuscript. YL, XY, JZ, and YC contributed to revision of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Host antiviral factor interferon-induced transmembrane proteins (IFITMs) are a kind of small-molecule transmembrane proteins induced by interferon. Their broad-spectrum antiviral activity and unique ability to inhibit viral invasion have made them a hot molecule in antiviral research in recent years. Since the first demonstration of their natural ability to resist viral infection in 1996, IFITMs have been reported to limit a variety of viral infections, including some major pathogens that seriously endanger human health and social stability, such as influenza A, Ebol, severe acute respiratory syndrome, AIDS, and Zika viruses, etc. Studies show that IFITMs mainly exert antiviral activity during virus entry, specifically interfering with the fusion of the envelope and the endosome membrane or forming fusion micropores to block the virus from entering the cytoplasm. However, their specific mechanism is still unclear. This article mainly reviews the research progress in the structure, evolution, function, and mechanism of IFITMs, which may provide a theoretical basis for clarifying the molecular mechanism of interaction between the molecules and viruses and the research and development of new antiviral drugs based on IFITMs. Human interferon-induced transmembrane proteins (IFITMs), first reported in 1984, are proteins that can be induced by interferon (IFN). Twelve years later, Alber et al. discovered that these proteins promote antiviral activity . Since tIt is reported that IFITM proteins could significantly inhibit IAV, West Nile virus (WNV), Ebola virus (EBOV), SARS coronavirus (SARS-CoV), vesicular pharyngitis virus (VSV), Rift Valley fever virus (RVFV), dengue virus (DENV), Semliki forest virus (SFV), Zika virus, Respiratory syncytial virus (RSV), human immunodeficiency virus-1 (HIV-1), hepatitis C virus (HCV), Reovirus, and other capsular or noncapsular RNA viruses –10. BesiTo date, the antiviral spectrum of this kind of protein involves more than 20 viruses from 12 families. IFITMs, as a branch of the large family of interferon stimulating genes (ISGs), have become star molecules in antiviral immune responses, especially in the study of ISGs, in recent years due to their broad-spectrum antiviral activity and unique ability to inhibit virus invasion. Numerous groups have continued to reveal that IFITMs have a restrictive effect on many viruses and made progress in their antiviral mechanism. In this article, we aim to review the latest research progress on the structure, localization, function, and mechanism of IFITM proteins, providing a reference for the further exploration of the mechanism of IFITMs and the development of their clinical application.IFITM1, IFITM2, IFITM3, IFITM5, and IFITM10 genes in humans, and these are located on human chromosome 11  in the vertebrates, among which human IFITM1, 2, and 3 with antiviral activity belong to the DSPA subfamily , arenavirus (LASV), lymphocytic choroid plexus meningitis virus (LCMV), and Crimean-Congo hemorrhagic fever (CCHFV) , 25, 62.ild mice . Althougalphavirus. It was reported that IFITM3 protein expressed in vitro had no obvious inhibitory effect on the Chikungunya virus (CHIKV) and Venezuelan equine encephalitis virus (VEEV) , than wild-type mice, and higher viral loads can be detected in multiple organs of ifitm3–/– mice, indicating that IFITM3 can limit the infection and pathogenicity of alphavirus  . Howevers (VEEV) . Ifitm3–phavirus .In recent years, progress has been made in the research on the antiviral spectrum, intracellular localization, protein post-translational modification , Jilin University Science and Technology Innovative Research Team [JLU-STIRT No. 2017TD-05], the Jilin Province Science and Technology Development Projects [No. 20200402043NC], the Science and Technology Research Program during the 13th Five-year Plan Period of Jilin Educational Committee [No. JJKH20190172KJ].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Convolvulaceae is a dicotyledonous perennial plant. Here, we conducted a comprehensive sweet potato virome study using 10 different libraries from eight regions in Korea and two different sweet potato cultivars by RNA-Sequencing. Comprehensive bioinformatics analyses revealed 10 different virus species infecting sweet potato. Moreover, we identified two novel viruses infecting sweet potato referred to as Sweet potato virus E (SPVE) in the genus Potyvirus and Sweet potato virus F (SPVF) in the genus Carlavirus. Of the identified viruses, Sweet potato feathery mottle virus (SPFMV) was the dominant virus followed by Sweet potato virus C (SPVC) and SPVE in Korea. We obtained a total of 30 viral genomes for eight viruses. Our phylogenetic analyses showed many potyvirus isolates are highly correlated with geographical regions. However, two isolates of SPFMV and a single isolate of Sweet potato virus G (SPVG) were genetically distant from other known isolates. The mutation rate was the highest in SPFMV followed by SPVC and SPVG. Two different sweet potato cultivars, Beni Haruka and Hogammi, were infected by seven and five viruses, respectively. Taken together, we provide a complete list of viruses infecting sweet potato in Korea and diagnostic methods.The sweet potato in the family  Ipomoea batatas (L.) Lam] is a dicotyledonous perennial plant and a member of the family Convolvulaceae. It is currently widely grown in tropical and temperate regions in the world1. The sweet potato is regarded as one of the most healthful foods in the world due to its high amounts of beta-carotene, carbohydrates, and dietary fiber and diverse micronutrients3. It is a very fast-growing vine plant and is usually vegetatively propagated by cutting a piece of a runner of sweet potato approximately 30 cm in length4. According to the FAO, in 2017, China was the largest sweet potato producer , followed by several African countries, such as Malawi , the United Republic of Tanzania , and Nigeria . In Korea, the sweet potato has been cultivated since the end of the 18th century, and it is the third most important crop plant in South Korea based on production 5. In general, the tuberous roots and stems of the sweet potato are used as different food materials in Korea.The sweet potato [6. The most frequently identified viruses are DNA viruses such as Sweet potato leaf curl virus (SPLCV) in the genus Begomovirus in the family Geminiviridae, followed by RNA viruses in the family Potyviridae7. Examples of well-known RNA viruses infecting sweet potato include Sweet potato feathery mottle virus (SPFMV), Sweet potato virus C (SPVC), Sweet potato virus G (SPVG), Sweet potato virus 2 (SPV2), Sweet potato latent virus (SPLV), and Sweet potato mild mottle virus (SPMMV) in the family Potyviridae; Sweet potato chlorotic fleck virus (SPCFV) in the family Flexiviridae; and Sweet potato chlorotic stunt virus (SPCSV) in the genus Crinivirus in the family Closteroviridae6. SPLCV is transmitted by vegetative propagation and the insect vector whitefly (Bemisia tabaci)8, whereas potyviruses infecting sweet potato are also transmitted by various insect vectors including Myzus persicae (Sulzer)9. Many previous studies have demonstrated that multiple viruses such as begomoviruses and potyviruses frequently coinfect most sweet potato cultivars grown in fields, resulting in the reduction of sweet potato yield12. Moreover, the coinfection of potyviruses in sweet potato has been found to be common10. Vegetative propagation and insect vectors might be two main reasons for the coinfection of viruses in sweet potato.Due to their vegetative propagation, most sweet potato cultivars are severely infected by various pathogens. Of the known pathogens, more than 30 different viruses infecting sweet potato have been identified so far16. According to the previous studies, many sweet potato germplasm collections and sweet potato cultivars grown in fields in Korea are often coinfected by different viruses14. At least eight different virus species infecting sweet potato have been reported based on RT-PCR assays16. The most frequently identified viruses infecting sweet potato in Korea are two potyviruses, SPFMV and SPVC16.In Korea, several studies have examined viruses infecting sweet potato20. For instance, the NGS technique is widely applied in the study of viruses infecting sweet potato by RNA-Sequencing (RNA-Seq)21 and small RNA-Seq23. Using the Illumina MiSeq platform, two DNA viruses including Sweet potato mosaic virus (SPMaV) and Sweet potato leaf curl Sao Paulo virus (SPLCSPV) in the genus Begomovirus and two RNA viruses including SPFMV and SPCSV were identified in South Africa17. In China, 11 virus species including SPVG and SPFMV have been identified from 219 samples in 10 regions in southern China by small RNA-Seq23.With the rapid development of next-generation sequencing (NGS) technologies, NGS-based approaches enable us to facilitate the identification of viruses, virus diagnostics, and the assembly of virus genomes27. Here, we conducted a study of sweet potato viromes by RNA-Seq. For that, 10 different libraries were prepared from leaf samples collected from eight regions in Korea and two different sweet potato cultivars. Comprehensive bioinformatics analyses revealed the complexity of sweet potato viromes in different geographical regions and different cultivars. Moreover, we identified two novel viruses infecting sweet potato and two virus variants that might be ancestors of SPFMV and SPVG.Although studies associated with viruses infecting sweet potato have been intensively conducted, there has not yet been a comprehensive study using NGS techniques for sweet potato viromes. As shown previously in our studies, RNA-Seq is a very powerful tool to study viromes in diverse plantsIn order to identify viruses infecting sweet potato in Korea, we collected leaf samples from eight different geographical regions in 2016 and 2017 Table . All swede novo assembled using the Trinity program. The number of assembled contigs (transcripts) ranged from 65,866 (HN) to 187,838 (NS) (Table https://www.ncbi.nlm.nih.gov/genome/viruses/). Based on the BLASTN search using assembled contigs, we obtained a total of 646 virus-associated contigs representing 10 different sweet potato viruses from 10 sweet potato transcriptomes  Fig. . A BLASTdae Fig. . We idenus Table .Figure 3Carlavirus in the family Betaflexiviridae. We identified SPVF from the GS and IC libraries; however, the genome of SPVF isolate IC was incomplete. The phylogenetic tree using genome sequences for SPVF isolates GS and IC and three SPCFV isolates showed that two SPVF were grouped together. Species demarcation criteria for the genus Carlavirus are 72% nt identity or 80% aa identity between their respective CP or polymerase genes. RNA-dependent RNA polymerase (RdRp) and coat protein (CP) for SPVF showed 78.41% and 81.89% amino acid identity, respectively, demonstrating that SPVF is a new species in the genus Carlavirus (Table We identified two novel viruses tentatively named SPVE and Sweet potato virus F (SPVF) from the sweet potato transcriptomes Fig. . The SPVus Table .Potyvirus. In addition, we obtained several partial sequences for the identified sweet potato viruses.From sweet potato transcriptomes, we assembled genomes of several viruses infecting sweet potato. We assembled 12 nearly complete genomes covering ORFs for seven viruses including SPFMV, SPLCV, SPLV, SPVC, SPVE, SPVF, and SPVG Table . Except In order to reveal the phylogenetic relationships of the identified viruses, we generated phylogenetic trees based on genome sequences Table . The phyViruses evolve faster than other organisms and exhibit strong genetic variation within the infected host. To determine virus mutations, we mapped raw sequence reads on the 12 virus genomes in which nearly complete genomes were assembled from this study Figs.  and 6. UFirstly, we examined the mapping patterns of sequence reads on the individual virus. Many reads were mapped on the whole region of the virus without gaps, enabling us to obtain the nearly complete genome Figs.  and 6. MWe next examined the number of SNPs in each virus genome Fig. . Three SWe examined the positions of SNPs in each virus genome Figs.  and 6. Iactin gene was used as a positive control. We extracted total RNA and DNA from the same samples used for RNA-Seq. Amplified PCR products from each sample were visualized by gel electrophoresis  was identified from only one library (IS), while SPFMV  was identified from eight libraries .Research associated with viruses infecting sweet potato has been intensively conducted in many countries14. Based on multiplex RT-PCR assays, there were at least eight different viruses infecting sweet potato in Korea16. Furthermore, viral genomes of 18 isolates representing five potyviruses in Korea were determined15. As compared to previous studies associated with viruses infecting sweet potato in Korea, our sweet potato virome study provides valuable additional information associated with viruses infecting sweet potato in Korea. We revealed a total of 10 different sweet potato viromes representing eight different geographical regions and two major sweet potato cultivars in Korea. Our study demonstrated that each geographical region and cultivar displayed a unique sweet potato virome composed of different viruses, as shown in other plant viromes31. For instance, three viruses were identified from the GJ, HN, and NS regions; however, SPVC was dominantly present in GJ and HN, while SPFMV was the dominant virus in NS. The high proportion of SPLCV, which is transmitted by the whitefly, in the NS region compared to other regions suggests that insect vectors could be environmental factors determining the individual sweet potato virome. Samples for YJ-B and YJ-H were collected from the same region, Yeoju; however, the lists and proportions of infected viruses were very different, showing cultivar-specific sweet potato viromes.In Korea, several studies have been carried out to understand viruses infecting sweet potato. For example, a nationwide survey was carried out to examine viruses infecting sweet potato from 2011 to 201415. In particular, the rare similarity of the 5′ regions in SPVE and SPVF with other homologous viruses highlights the superiority of NGS techniques followed by bioinformatics analyses. Although SPVF in the genus Carlavirus was closely related to SPCFV, the BLAST results and phylogenetic analyses revealed that SPVF is a new species. Two SPVF isolates, GS and IC, in the same group were different from other SPCFV isolates. Moreover, our phylogenetic analyses demonstrated that Sweet potato yellow mottle virus (SPYMV) isolate Yeongdeok (KR072674.1) should be a member of SPCFV. Moreover, two SPVE isolates, GS and YJ-B, were closely related to SPVC. However, the phylogenetic tree using polyprotein sequences of potyviruses infecting sweet potato demonstrated that SPVE was a new species in the genus Potyvirus.Here, we identified a total of 10 different virus species infecting sweet potato. Our RNA-Seq-based approaches identified novel viruses and new virus variants more successfully than a PCR-based viral genome study, which could amplify highly conserved viral sequencesSweet potato golden vein-associated virus (SPGVaV) in the genus Begomovirus, which is rare in Korea32. Moreover, we did not identify SPCSV in the genus Crinivirus, which has been reported in many countries including China and Uganda34. Therefore, it is important to prevent the introduction of SPCSV to Korea.Our sweet potato virome study indicated that SPFMV was the dominant virus followed by SPVC and SPVE in the examined regions in Korea. In addition, these three viruses are regarded as the main viruses infecting sweet potato in the world. We identified all previously reported viruses infecting sweet potato in Korea except 35. Of course, the optimal tissues for plant virome studies depend on the plant species. In our study, the proportion of virus-associated reads in the whole transcriptome was very low, indicating that leaf tissues might not be appropriate samples for the detection of viruses infecting sweet potato. As shown in a previous study36, we carefully suggest using samples from the fibrous and tuberous roots of sweet potato for virus detection. Based on our experience studying viruses infecting plants with a large genome size such as the hexaploid sweet potato (4.4 Gb)36, selection of the proper tissue enriched with viruses is necessary for a successful virome study.Plant tissues and developmental stages are important factors for plant virome studies. Our previous studies demonstrated that viral RNA was enriched in grape fruits and lily flowers35, virus genomes with poly(A) tails can be easily assembled. It is also not surprising that genomes of DNA viruses such as SPLCV can also be assembled from transcriptome data, as shown in our previous studies26. In many cases, the number of mapped sequence reads on the viral genomes with poly(A) tails was increased from the 5′ region to the 3′ region. This result is somehow correlated with the result of poly(A) tails preferentially attaching to the transcripts close to the poly(A) tails.In this study, we used messenger RNA from total RNA for the library preparation using oligo-d(T). Eight RNA viruses in this study possessed polyadenylate (poly(A)) tails . As shown in other previous studies30. In addition, SPLCV also exhibited a high frequency of mutations of up to 4.52%. Similarly, several Korean SPLCV recombinants have been identified13. Thus, our result suggests that mutation and recombination contribute to the genetic diversity of SPFMV and SPLCV isolates. Although several potyviruses were coinfected, the mutation rate was the highest in SPFMV followed by SPVC, SPVG, and SPLV. SPLV showed the lowest mutation rate among the examined potyviruses. This result suggests SPFMV might play an important role in viral disease symptoms in coinfected sweet potato plants.We examined the mutation rates for the 12 assembled virus isolates. Of them, SPFMV showed a high mutation rate of up to 9.67%. Furthermore, a recent study has found possible recombination within the Nla-Pro, CP, and P1 genes using available SPFMV genomesHere, we examined sweet potato viromes for two popular sweet potato cultivars in Korea, Beni Haruka and Hogammi. Beni Haruka originates from Japan, while Hogammi was recently developed by the Rural Development Administration (RDA) in Korea. As they are popular for roasting, many growers cultivate both cultivars. In the case of Beni Haruka, at least seven different viruses were identified, whereas five different viruses were identified from Hogammi. Interestingly, both cultivars were cultivated in the same field in Yeoju, Korea; however, Beni Haruka was more severely infected by viruses than Hogammi. The difference in the number of infected viruses between the two cultivars might be correlated with the cultivation period. That is, Beni Haruka has been cultivated for a long time, while Hogammi was introduced to Korean growers only three years ago by the RDA.de novo assemble many viral genomes from transcriptome data37. Similarly, we obtained 12 complete virus genomes and 18 nearly complete genomes for eight viruses. A total of 30 assembled viral genomes were further used for phylogenetic analyses. Our phylogenetic analyses showed that many isolates of SPVC, SPLV, SPV2, and SPLCV in this study were grouped together with other known isolates from Korea. In general, it is likely that virus genomes are highly correlated with geographical regions. However, two isolates of SPFMV and a single isolate of SPVG were genetically distant from other known isolates. For example, two SPFMV isolates, YJ-H and YJ-B, were revealed as common ancestors of other known SPFMV isolates. Furthermore, SPVG isolate IS in this study and SPVG isolate WT325 from Taiwan were genetically distant from other SPVG isolates, suggesting they are new variants of SPVG.It is now possible to 38. Unfortunately, most sweet potato cultivars in Korea were severely coinfected by many viruses14. There are several possible reasons explaining how viruses infect sweet potato plants. The first is vegetative propagation. Most growers purchase sweet potato sprouts from seedling markets, and they are often already infected by diverse viruses. Surprisingly, a recent study showed that most sweet potato germplasm (83.8%) in the Bioenergy Research Center of the RDA in Korea, which is used to develop new cultivars or provide sweet potato cuttings, was already infected by multiple viruses14. Furthermore, a recent study demonstrated that SPLCV can be transmitted by seeds, suggesting the newly developed sweet potato cultivars might also be highly infected by viruses8. Thus, it seems that the sweet potato virus control in Korea should be conducted from the early stages of breeding.As shown in other previous studies, the coinfection of multiple viruses in sweet potato resulted in the dramatic reduction of sweet potato production by up to 50%38. For that, knowledge of the viruses infecting sweet potato and diagnostic methods are needed. In this study, we provide a complete list of viruses infecting sweet potato in Korea and diagnostic methods, which could be valuable information for the development of a virus-free sweet potato in Korea in the near future. Moreover, we suggest that the development of a virus-free sweet potato in Korea should be carried out by not only national institutes such as the RDA but also universities and companies. We have to provide various reasonable choices to growers to promote competition among different developers of virus-free sweet potatoes. Dependence on a single national institute does not guarantee the production of a high-quality virus-free sweet potato, as we have seen.Based on the above evidence, it is necessary to develop virus-free sweet potato cultivars in order to prevent the damages caused by viral diseases, as suggested previouslyWe collected sweet potato leaf samples from eight different geographical regions in Korea in May 2016 and May 2017 Table . The eigLeaf samples were pooled and then frozen in the presence of liquid nitrogen. The frozen leaf samples were ground with a pestle and mortar. We used the RNeasy Plant Mini Kit  to extract total RNA for RNA-Seq based on the manufacturer’s instructions. The quality and quantity of extracted total RNA were measured by gel electrophoresis followed by using an Agilent 2100 Bioanalyzer . Using the extracted total RNA, we generated 10 different RNA-Seq libraries using the NEBNext Ultra RNA Library Prep Kit for Illumina in accordance with the manufacturer’s instructions . Briefly, poly(A)-tailed mRNA was extracted by using poly-T oligo-attached magnetic beads. We synthesized the first strand of cDNA from the extracted mRNA followed by a second strand of cDNA. The 3′ ends of the DNA fragments were adenylated. After adapter ligation, we performed PCR amplification to selectively enrich DNA fragments with adapters and amplify the large amount of DNA in the library. We again measured the quality and quantity of each library using the 2100 Bioanalyzer. The six prepared libraries from 2016 were paired-end sequenced by Theragen  using the HiSeq2300 platform, and the four libraries from 2017 were paired-end sequenced by Macrogen Co.  using the HiSeq2000 platform.de novo transcriptome assembly and BLAST search, a workstation with two 20-core CPUs and 256 GB of RAM installed with Ubuntu 16.04.4 LTS was used. Based on the results of our previous studies31, we only used the Trinity program  with default parameters for de novo transcriptome assembly39. Raw sequence reads from each library were de novo assembled using Trinity. We generated our own plant viral genome database by selecting only viruses infecting plant species from the viral reference database of the NCBI (https://www.ncbi.nlm.nih.gov/genome/viruses/). To identify virus-associated contigs, the assembled contigs (transcriptome) from each library were blasted against the plant viral genome database using MEGABLAST40 with a cutoff E-value of 1e–6. The obtained virus-associated contigs were again blasted against NCBI’s NT database to remove sweet potato host sequences and other contaminated sequences. Ultimately, only virus-associated contigs were used for the study of sweet potato viromes. To calculate the number of virus-associated reads for identified viruses, the BBMap program was used (https://sourceforge.net/projects/bbmap/).In the bioinformatics analyses including the 41. Several nearly complete viral genome sequences were obtained without any further alignment. Poly(A) tail sequences at the 3′ terminal were deleted. We again aligned raw sequence reads on the identified virus genome sequences to fill the missing gaps of the virus genome using a Burrows–Wheeler Aligner (BWA) program with default parameters42. To predict the ORFs in each virus genome, we used the ORF Finder program (https://www.ncbi.nlm.nih.gov/orffinder/). In addition, we manually checked the identified ORFs and the 5′ and 3′ untranslated regions (UTRs) by comparing the corresponding reference virus genome. Twelve complete viral genome sequences covering whole ORFs were deposited in NCBI’s GenBank database with respective accession numbers  files. The SAM files were converted into Binary Alignment Map (BAM) files using SAMtools43. After that, the sorted BAM files were used to generate the Variant Call Format (VCF) file format using the mpileup function of SAMtools for SNP calling. Finally, we called SNPs using BCFtools implemented in SAMtools. The positions of identified SNPs and mapped reads on each viral genome were visualized by the Tablet program44.For virus SNP identification, it is important to use the assembled virus genome sequences in each library as reference virus genome sequences to increase SNP specificity. We analyzed single SNPs for the 12 assembled virus genomes as described previouslyactin gene of sweet potato was used as a positive control. In the case of SPVG, two different primer pairs, SPVG and SPVG_IS, were designed. The SPVG_IS-specific primer pair can amplify the variant of SPVG identified from the IS region. Detailed information of designed primer pairs can be found in Table 31, the RT-PCR conditions were 50 °C for 30 min, 95 °C for 15 min, followed by 30 cycles at 95 °C for 20 sec, 50 °C to 56 °C for 40 sec , and 72 °C for 1 min, with a final extension at 72 °C for 5 min. We checked the amplified RT-PCR products by gel electrophoresis followed by EtBr staining. Furthermore, we cloned the amplified RT-PCR product in the pGEM-T-Easy Vector  followed by Sanger sequencing to confirm the sequences of amplified PCR products.In order to confirm the results of RNA-Seq, we carried out RT-PCR. For that, we designed 12 RT-PCR primer pairs. An To confirm complete genome sequences of SPVE and SPVF, we carried out RT-PCR using newly designed primers Table . The ampSupporting information.Table S1.Table S2.Table S3.Table S4.


Furthermore, LSM images of mortar blended with intermediate L-to-VA blend ratio ≈0.55 (L: 6% and VA: 5%) exhibit compact packing of sand grains, indicating strong adhesion and higher cementing value. The results of the study are promising and encourage using the treated dune sand in engineering construction even with a low percentage use of lime (2%) and volcanic ash (1–3%) as stabilizers.This study focused on evaluating dune sand stabilized with lime and volcanic ash as base course materials in engineering construction. Dune sands are found in Saudi Arabia in huge quantities. Due to the high demand for construction materials, this makes them highly suitable for construction. A testing program was designed to investigate the effect of adding different percentages by weight of lime  and volcanic ash  on the engineering properties of the stabilized mixture. Unconfined compressive strength (UCS) and California bearing ratio (CBR) tests were conducted. In addition, Raman spectroscopy and laser-scanning microscopy (LSM) tests were performed to explore the chemical characteristic, packing, and structure of the mixture. The results showed that the UCS, CBR, and the Young’s modulus (E Vast growth in industry and population necessitate searching for more and different resources of good quality earth materials for construction work. The extensive use of quarried construction materials for the construction of building and roads have led to severe damage to the environment and increased pollution levels. Governments and environmental agencies place colossal pressure on the construction industry to look for new materials and assimilate sustainability. Extensive research works were carried out to serve sustainability and to stimulate construction process and development. Most of the research work directed toward using different additives to improve the engineering properties of materials not meeting the required standards. In arid regions where dune sands are abundant in huge quantities, many researchers have investigated their use as construction materials. Since dune sands are almost classified as SP or SP-SM according to USCS (A-1 to A-3 according to AASHTO classification) . Over thMany other studies have been conducted on dune sands to explore their characteristics. Studies on chemical and mineralogical compositions showed that dune sand is mainly composed of quartz ,4,5. StuMany researchers performed several studies for improving the mechanical and microstructural properties of dune sand used in road construction by incorporating a different binder such as cement, lime, ground granulated blast furnace slag, municipality waste, fly ash, silica fume, metakaolin, bentonite, etc. ,18,19,20Due to the current trends in infrastructure development, the demand for construction material is also increasing. Nowadays, the research is focused on using locally available materials for the construction of the road network due to their economic and sustainability benefits. Dune sands are available in huge quantities all over Saudi Arabia, and therefore, their uses in the construction industry can be thrifty. The locally available due sand is an attractive material to be used for the construction of roads and foundations. However, due to the low bearing capacity of these soil, it is necessary to improve their properties by using different stabilizers ,25,26. Is, and CBR value as a measure of the bearing capacity of the stabilized dune sand for practical use in roadways and foundations construction.Therefore, in this study, lime and locally available volcanic ash (VA) were used as stabilizing materials to improve the engineering properties of the dune sand for its potential use as a base course material for roads and foundations construction. Different percentages of lime  and of volcanic ash  and their blends were selected. Initially, all materials were analyzed and characterized by performing the different conventional tests. In the second step, mechanical properties such as UCS, Young’s modulus, and CBR tests were performed for all samples with and without lime and volcanic ash and its blends. In the third step, the effect of optimized quantities of lime and VA on the improvement of microstructure was investigated by performing laser scanning microscopy and Raman microscopy analysis. Finally, useful and practical relationships were developed between the UCS, E50 = 0.4 mm). Dune sand used in this study was collected from one of the stockpiles of construction materials located along Dammam road (the eastern region of Saudi Arabia). 2 [2), Aluminum Oxides (Al2O3), and Ferric Oxide (Fe2O3) are the most abundant oxides that form the chemical composition of the used volcanic ash with 73.6% as a total sum by weight.Basaltic volcanic ash was collected from the western part of Saudi Arabia, near the city of Jeddah. Millions of years ago, the western part of the country was subjected to volcanic activities, which led to the formation of basaltic flows called “Harrat” which cover an area of about 90,000 km2 . Table 290 and D50 of the used ash are 25 μm and 3 μm, respectively [X-ray diffraction analysis of the volcanic ash powder sample was performed using Rigaku MiniFlex II and XRD intensity peaks as shown in ectively .2, a minimum of 90%, and the particle size analysis shows that 90% of the grain size is less than 90 micron. When lime is added to silicon and aluminum oxides (within soils or added volcanic ash) with the presence of water, pozzolanic reactions occur and lead to forming calcium silicate hydrate (C-S-H) and calcium aluminate hydrate (C-A-H) gels that, after crystallization, will substantially contribute to connecting the soil particles to a relatively strong structure [The hydrated lime used in this study was procured from a Saudi lime factory. The hydrated lime has a purity level of Ca(OH)tructure .−2) and less than 3000 ppm of sulfates (SO4+2).Water used in the testing program was tapped water. According to AASHTO T 26 specifications , it has 3 and optimum water content of 7.8%.An experimental program was performed to attain the outcomes of this study. The program concentrated on the investigation of the engineering properties and behavior of the stabilized dune sand using various percentages by weight of hydrated lime  and volcanic ash  mixed at the maximum unit weight and optimum moisture content of dune sand samples compacted by the standard Proctor test, ASTM D698-07 method A . The preA testing program was designed to investigate the morphologic, cement chemistry, and pozzolanic reactions between sand, volcanic ash, and lime. The hydration of lime and volcanic ash is performed in different ratios, as shown in −1. All Raman spectra were recorded using the confocal microscopic method keeping a hole size fixed at 50 A.U.  and a spectral accuracy of 0.5 cm−1.Macro Raman spectra were recorded from Horiba Labram (Evolution) spectrometer using a He-Ne laser excitation source . The spectrometer was calibrated by a Si standard using the Raman peak at 520.7 cmWe have deployed the laser scanning microscopy (LSM) method with confocal data acquisition capabilities to perform the morphological studies of sand, lime, ash, and their mixtures in the various ratios under consideration. We collected the morphologies using both the 50× and 5× objectives lenses for high resolution imaging and large area imaging respectively. The scan-areas of 200 μm and 2 mm were used for the two said morphologies. All the imaging data were collected by deploying a confocal hole of a size of ~1 A.U. and 402 nm diode laser for the sample illumination.CH and SH represent calcium hydroxide CSH, is a pozzolanic reaction compound CSH formation on the mortar strengths has been explained in detail for lime hydration in the recent literature [Raman spectroscopy is a simple and effective technique to explore cement chemistry ,37. The terature ,46.−1 representative of different pozzolans present in volcanic ash. The peak at 481 cm−1 is broad and represent bending of various Si-O-Si linkages in the silicates and silica [−1 originates from the Al-O-Al stretching mode of a C3A-type pozzolan [4AF) peak is also detected at 670 cm−1 in the Raman spectrum of volcanic ash indicating the presence of calcium iron aluminates (Ettringite phases) in the pozzolan [−1 peak also represents iron (III) oxide minerals (Magnetite) presence [−1 peak of the AlO4 vibration [−1 corresponding to the C2S-type silicate (Belite phase) present in the ash [−1 also indicates the presence of aluminum silicates  in volcanic ash [The Raman spectra in d silica . The secpozzolan . Slowly pozzolan . In addipresence . Howeveribration . Anotheranic ash ,51.−1 and portlandite modes at 355 and 719 cm−1 [−1. The pozzolanic activity of the blend and thus the binding performance of the blend are directly related to the size of the yellow box, which signifies the presence of new pozzolanic reaction compounds. The box area is found to increase with the increasing content of ash, pointing towards the requirement of silicates and aluminates to complete the pozzolanic reactions. One can see that the largest number of pozzolanic reaction compounds form at the lime-to-blend ratio ≈0.55. Nevertheless, on further decreasing the ratio ≈0.44, the pozzolanic activity decreases, indicating a deficiency of lime, which is required to complete the reaction.As shown in 719 cm−1 ,53. The 2S and C4AF) present in the blends are also proven from the Raman studies.Microscopic images of grains of sand dune particles, volcanic ash, and lime are shown in The laser scanning microscope analysis performed on hydrated volcanic ash mortar with sand is shown in Hydrated lime mortar’s surface morphology is depicted in LSM images of the blended mortar with a lime-to-blend ratio of 0.67 are shown in In the hydrated and lime-to-blended mortar ratio ≈0.55, all grains are very well connected forming larger lumps due to increased binding properties, as shown in The results of UCS of the treated dune sand with lime content and for different percentages of volcanic ash are shown in o), are calculated for both the effects of lime and VA on the UCS response. The statistically calculated Plime and PVA values for the UCS data are 0.001 and 0.0103, respectively. The P value for the lime is by an order of magnitude smaller than the traditional cutoff value for the meaningful statistical significance (≈0.05) and thus the UCS results are undoubtedly dependent on lime content. Whilst the PVA value is smaller but comparable to the order of the statistical significance, implying a weak dependence of the UCS on the volcanic ash content.Two factor multi-level statistical analysis were performed to find out the statistical significance of the results of the effects of lime and volcanic ash on UCS . Coeffics) of the treated dune sand was also investigated at different percentages of lime and volcanic ash. The modulus was evaluated as the slope of the linear elastic region of the stress-strain curve. The results in s with the increase in lime content is insignificant. Besides that, there is almost no improvement in Es for lime content greater than 2%. In addition, the results show that, with the absence of lime, there is no increase in Es with the addition of volcanic ash. Ramon spectroscopy results in s with the increase in lime content. Interestingly, by using just 2% of lime and 1% of volcanic ash, sand Es of the treated sand increases significantly from almost zero to 50 MPa. The slopes of the Es curves in s is more significant than the increase in the effect lime content.Young’s modulus  pointing towards the dependence of the modulus of the mix more so than on the volcanic ash.Coefficients of variance calculated for lime and volcanic ash treatments, using the 9 test samples, on the Young’s modulus response are 5.1 and 6.0, respectively. The statistical analyses show that the calculated PCBR tests b,c were lime and PVA values for the CBR are 0.0077 and 0.0059, respectively, which are much smaller than the traditional cutoff value for the meaningful statistical significance ≈0.05. Since the difference between the P values are also small, therefore the CBR of the mix equally depends on both the lime and VA.Coefficients of variance calculated for lime and volcanic ash treatments, using the 9 test samples, on the CBR response are 7.6 and 8.2, respectively. The statistical analyses show that the Ps of the dune sand treated with lime and volcanic ash, as presented in 2 = 0.88. s. In this figure, the Es increases with the increase in the UCS and the relationship can be strongly expressed as a second-degree polynomial equation with correlation factor, R2 = 0.95. The relationship between the Es and CBR value is presented in s also increases with the increase in the CBR value. The relationship can be expressed as a second-degree polynomial with a correlation factor, R2 = 0.90.For engineering practice and to minimize the efforts of assessing the properties and parameters needed for the design of foundations of buildings and roadways, useful relationships were developed among the CBR value, UCS, and EThe Raman spectroscopy analysis of the samples having hydrated lime and volcanic ash with lime to blend ratio of 0.55 (VA = 6% and L = 5%) showed better Raman peaks as compared to all other samples. The high Raman peaks demonstrated high pozzolanic reactivity between lime and volcanic ash and ultimately formed a large number of reaction products for better strength properties;Laser scanning microscopy (LSM) results showed that dune sand treated with hydrated lime and volcanic ash with lime to a blend ratio of 0.55 were more compacted and had better binding properties than other mixtures. High pozzolanic reactivity of both lime and volcanic ash resulted in the formation of reactive compounds which improved the adhesion between the sand particles and are ultimately responsible for the compact packing of sand particles;The UCS and CBR value of the treated dune sand increased with the increase in the percentages of volcanic ash and lime content. The maximum value of strength was observed at a lime-to-blend ratio of 0.55. The UCS and CBR results are consistent with the results observed from Raman and laser microscopic tests for a lime-to-blend ratio of 0.55;s of the treated dune sand increased with the increase in lime content. Interestingly, using just 2% of lime and 1% of volcanic ash, the Es increased significantly from almost zero to 50 MPa;As the percentage of the added volcanic ash increased, the Es of the dune sand treated with lime and volcanic ash. These relations can helpfully be used for the design of foundations of roadways and structures using dune sands stabilized with lime and volcanic ash.Practical and useful relationships were developed among the CBR value, UCS, and EThe testing program was conducted to investigate the effect of using lime and volcanic ash as stabilized materials on the engineering properties of dune sand used in the construction of foundations of roadways and buildings. The program consists of two major parts. The first part investigates the engineering properties of the treated sand, including UCS, Young’s modules, and CBR values. The second part focused on the chemical characteristic of the interaction between the additives using Raman spectroscopy and morphologic analysis of the lime-ash-sand mortar using laser-scanning microscopy (LSM). The results of the testing program of the second part were very important and strongly supported the findings obtained from the testing programs of the first one. The following conclusions were derived:The current study concluded that the combined use of volcanic ash and hydrated lime were more effective in the stabilization of dune sand by improving its mechanical and microstructural properties. However, to further investigate the effective use of locally available VA as a potential dune sand stabilizer, future research is recommended by using the blend of VA with cement and other highly reactive materials such as silica fume and metakaolin for the stabilization of dune sand. To get a better understating of the properties of the treated dune sand with VA and reactive materials, other aspects may need to be considered such as the effect of water soaking in conjugation with curing time. This would help the researcher and designer in selecting the best stabilization materials for stronger, economical, and sustainable construction. The main challenge facing the use of treated dune sand as construction materials for roadways in Saudi Arabia is to set proper standards and codes that cover the entire processes, including the design phase, construction procedures and methods, monitoring techniques and tools, and remedial measure processes. This can be solved and improved with close cooperation between researchers in this field and the decision makers of the roadways industry.


As population’s age and the need for long term care (LTC) increases, so too does the focus on the costs to provide that care. Providing food, oral nutrition supplements and meals, can be a considerable expense to a home. The objective of this research was to develop a valid foodservice costing tool (FCT), to calculate the real cost of providing foods and meals in LTC. Current costing methodologies are not specific to LTC and do not account for all costs of a foodservice, including staff, procurement and nutrition supplements. An initial tool was developed using the systems approach in conjunction with literature and professional knowledge. This was piloted in real world contexts, using volunteer LTC homes. Four iterations of the tool were completed to assess its feasibility in calculating costs and useability. Managers were interviewed after completing the tool to gather an understanding of how the tool was interpreted and to refine completion. Following feedback, the resulting tool consists of nine sections, measuring both costs incurred in meal production and service as well as analysis of staff workloads. Preliminary results show consistency between homes within Australia, indicating that the true cost is much higher than that reported in the literature to date. The development of a comprehensive, usable tool which captures the total cost of foodservice allows homes to accurately report and understand costs from a systems level. This information can be used to demonstrate cost effectiveness of a foodservice and the potential to justify and plan future system changes.


The detection of pathogens in clinical and environmental samples using high-throughput sequencing (HTS) is often hampered by large amounts of background information, which is especially true for viruses with small genomes. Enormous sequencing depth can be necessary to compile sufficient information for identification of a certain pathogen. Generic HTS combining with in-solution capture enrichment can markedly increase the sensitivity for virus detection in complex diagnostic samples.A virus panel based on the principle of biotinylated RNA baits was developed for specific capture enrichment of epizootic and zoonotic viruses (VirBaits). The VirBaits set was supplemented by a SARS-CoV-2 predesigned bait set for testing recent SARS-CoV-2-positive samples. Libraries generated from complex samples were sequenced via generic HTS (without enrichment) and afterwards enriched with the VirBaits set. For validation, an internal proficiency test for emerging epizootic and zoonotic viruses  was conducted.The VirBaits set consists of 177,471 RNA baits (80-mer) based on about 18,800 complete viral genomes targeting 35 epizootic and zoonotic viruses. In all tested samples, viruses with both DNA and RNA genomes were clearly enriched ranging from about 10-fold to 10,000-fold for viruses including distantly related viruses with at least 72% overall identity to viruses represented in the bait set. Viruses showing a lower overall identity (38% and 46%) to them were not enriched but could nonetheless be detected based on capturing conserved genome regions. The internal proficiency test supports the improved virus detection using the combination of HTS plus targeted enrichment but also points to the risk of cross-contamination between samples.The VirBaits approach showed a high diagnostic performance, also for distantly related viruses. The bait set is modular and expandable according to the favored diagnostics, health sector, or research question. The risk of cross-contamination needs to be taken into consideration. The application of the RNA-baits principle turned out to be user friendly, and even non-experts can easily use the VirBaits workflow. The rapid extension of the established VirBaits set adapted to actual outbreak events is possible as shown for SARS-CoV-2.Video abstractThe online version contains supplementary material available at 10.1186/s40168-020-00973-z. Disease control is encompassing several fields like powerful diagnostics for early detection, efficient therapy, and disease prophylaxis. The present study focused on broad and powerful diagnostics for viral pathogens including evolved, newly emerging or unrecognized viruses. The detection of the latter can be challenging for conventional virus diagnostics relying on specific quantitative PCR assays until their improvement or development of new assays , 2. SequPrior to sequencing, an enrichment of certain pathogens using capture enrichment methods can help to increase the target sequence information considerably, along with avoiding extensive sequencing efforts, as recently reviewed by Gaudin and Desnues , and conFor the purpose of improved virus diagnostics, we used RNA baits for an in-solution capture assay with subsequent HTS Fig. . In a piAnas platyrhynchos GCF_000355885.1, Anser cygnoides GCF_000971095.1, Bos taurus GCA_000003055.4, Camelus dromedarius GCF_000767585.1, Canis familiaris GCA_000002285.2, Capra hircus GCF_001704415.1, Cervus elaphus GCA_002197005.1, Chlorocebus sabaeus GCA_000409795.1, Enhydra lutris GCF_002288905.1, Equus caballus GCA_000002305.1, Felis catus GCF_000181335.3, Gallus gallus GCA_000002315.3, Homo sapiens GCA_000001405.15, Mus musculus GCA_000001635.2, Ovis aries GCF_000298735.2, Rattus norvegicus GCA_000001895.4, Sus scrofa GCF_000003025.6, Vicugna pacos GCF_000164845.2, Vulpes vulpes GCA_003160815.1) to remove those oligonucleotides that were likely to hybridize and capture host nucleic acids. Finally, redundancy was decreased by removing oligonucleotides that were > 95% identical to each other. Based on the final set, biotinylated RNA baits were manufactured. The tailored custom myBaits kit (VirBaits) for 48 hybridization reactions including the RNA bait set comprising 177,471 baits and adapter blockers fitting with Ion Torrent sequencing adapters was obtained (Arbor Biosciences). All baits including the GenBank accession number of the respective virus genomes are provided in Additional file The aim of the study was to design a diagnostic bait set for epizootic and zoonotic viral pathogens. For this purpose, virus genomes were compiled based on a curated list of complete virus genomes from the viralzone website  provided five samples . In brief, hybridization reagents including baits were thawed, collected, and mixed with each other preparing hybridization (HYB) and blocker master mixes. The HYB mix was incubated for 10 min at 60 °C to clear the solution. After adapting to room temperature for 5 min, the HYB mix was aliquoted to 18.5 μl per reaction. The blocker mix was aliquoted to 5 μl each and subsequently combined with up to 7 μl of the sequencing library . The LIB mix of each library was denatured for 5 min at 95 °C using a thermal cycler. Both the LIB and HYB mixes were afterwards incubated for 5 min at 65 °C. Subsequently, 18 μl of the HYB aliquot was mixed with the LIB mix aliquot each (12 μl) using pipetting up and down and spinning down, and then incubated for about 16 h at the suggested hybridization temperature (65 °C). We tested three variations from the protocol recommended in the manual. (i) The rabies virus (RABV)-infected sample L03007 was hybridized with only the half amount of the reaction volume. (ii) For IAV-infected samples L02651 and L03706, the baits in the HYB mix were diluted 1:5 with ultrapure water. (iii) The Kotalahti bat lyssavirus (KBLV)-infected sample L02374 was incubated at 62 °C instead of the default hybridization temperature of 65 °C as recommended in the manual when the expected bait-target divergence exceeds 5%. After hybridization, the samples were purified three times at the hybridization temperature using binding beads contained in the kit and finally eluted with 30 μl 10 mM Tris-Cl, 0.05% Tween-20 solution (pH 8.0–8.5). An aliquot (16 μl) of the enriched samples was amplified using the GeneRead DNA Library L amplification Kit (Qiagen) according to manufacturer’s instructions performing 14 cycles and purified using solid-phase paramagnetic bead technology. Quality check using a Bioanalyzer 2100 (Agilent Technologies) and quantification of the enriched libraries using the KAPA Library Quantification Kit Ion Torrent (Roche) were performed as described  to map1 map2) while the previously determined whole genome sequence was set as reference. In addition, the variant analysis tool integrated in Geneious Prime (2019.2.3) was applied for further confirmation and the acquisition of variant frequencies for each variant . Positions of identified potential SNVs were visually inspected in Geneious Prime.The VirBaits panel covered 35 viral pathogens of notifiable epizootic and zoonotic diseases Table . The resThe VirBaits performance was assessed via direct comparison of datasets generated without and with enrichment. The portion of virus reads extracted from the metagenomics datasets using reference mapping for both datasets per sample and the enrichment factor are shown Table , Fig. 3.Case one, for all tested viruses that are represented in the VirBaits set, we could reach an enrichment between 16-fold for sample L02726, slightly loaded with West Nile virus (WNV), and 9200-fold for the RABV-infected sample L03015  was enriched 26-fold, and sample L03038 infected with Usutu virus  as the genome assembly of the VirBaits dataset. The variants in each genome assembly were found to exhibit nearly unaltered frequencies per SNV Fig.  indicatiIn the proficiency test blind samples, high-impact transboundary or zoonotic viruses detected in the samples X1 to X4 were ASFV, FMDV, RVFV, NiV, EOV, and Marburgvirus . Altogether, an increase of 9-fold to 9200-fold of the target sequence reads was achieved. The obtained enrichment factors were neither dependent on the bait abundance within the bait set nor on the target genome size  compared with panels for single species applying a tiling density of 3×  or 4× 33. This iA general benefit of capture approaches is that unlike for targeted assays like qPCR or serologic assays no prior decision is necessary for which pathogen (virus) is sought after, i.e., which specific test system needs to be applied. Of course, this requires that substantial knowledge and effort be put into the design of the bait set and that the used bait set fits the specific purpose. This saves time and money by avoiding application of many laborious diagnostic techniques successively until a causative agent can be suspected and confirmed. Moreover, a negative result of a PCR approach can mask an undetected viral pathogen when PCR primers do not fit anymore with the genome of evolved pathogens e.g., , 35). Ot. Ot35]).The results of the internal proficiency test corroborate the applicability of the VirBaits workflow combined with HTS as all-in-one approach for different pathogenic viruses without the necessity to do laborious diagnostic tests for at least each single virus or small groups of viruses. The datasets generated were relatively small but resulted in all cases in enough reads to determine the respective pathogen without any doubt .Additional file 2: Table S1. Coverage statistics of genomes of the viruses in all samples generated in this study with enrichment approach (VirBaits) after reference mapping using the Genome Sequencer software suite, including used references, number of baits in the VirBaits panel and enrichment factor of virus reads.Additional file 3: Figure S1. Coverage of genomes of selected viruses generated without (generic HTS) or with enrichment (VirBaits) after reference mapping with the Genome Sequencer software suite; displayed using Geneious Prime (2019.2.3).


Cortical laminar necrosis comprises ischemic neuronal changes and glial reaction. Despite fewer reports in the pediatric population, we encountered a case of cortical laminar necrosis with influenza virus A infection in an infant. Cortical laminar necrosis comprises ischemic neuronal changes and glial reaction. Despite fewer reports in the pediatric population, we encountered a case of cortical laminar necrosis with influenza virus A infection in an infant. Although cortical laminar necrosis is a classic presentation related to conditions of energy depletion in adults, reports concerning children are few.Herein, we report cortical laminar necrosis with influenza virus A infection in an infant.Cortical laminar necrosis comprises ischemic neuronal changes and glial reaction. Ischemic neuronal changes involve the third layer of cerebral cortex, and laminar deposition of fat‐laden macrophages accompanies glial reaction.A 1‐year‐old boy with a medical history of ventricular septum defect developed status epilepticus and shock. Blood chemistry at admission revealed aspartate transaminase, 239 U/L; alanine transaminase, 57 U/L; lactate dehydrogenase, 1063 U/L; and ferritin 3381 ng/mL. These results indicated that the patient may have hypercytokinemia. Real‐time polymerase chain reaction using nasopharyngeal swab samples was positive for the influenza A virus; he was clinically diagnosed with influenza virus–associated acute encephalitis/encephalopathy. Oseltamivir phosphate and methylprednisolone pulse therapy was administered. Head computed tomography (CT) at admission showed loss of gray‐white differentiation. Follow‐up CT performed 2 weeks after admission revealed overall hyperdensity in the cerebral cortex and hemorrhage in the bilateral basal ganglia (Figure These results enabled the diagnosis of cortical laminar necrosis with bilateral deep gray nuclei involvement as a manifestation of influenza virus–associated acute encephalitis/encephalopathy and anoxic‐ischemic brain injury.The authors declare that there is no conflict of interest regarding the publication of this article.KY: made substantial contributions to the study's conception and design, and acquisition, analysis, and interpretation of data. MW, YS, KS, KH, and MH: were involved in drafting or revising the manuscript critically for important intellectual content.