This article reports the results of a longitudinal study on the development of context-specific locus of control related to situations of success and failure in Polish adolescents. The participants were 90 primary school students, including 30 who learned in integrated classrooms and 60 who went to non-integrated classes in schools with and without an inclusive curriculum, located in Lublin, Poland. The students were surveyed during a three-year schooling period . The research was carried out in the years 2016–2019. The Locus of Control Questionnaire (LOQ and LOQ-R) by Krasowicz-Kupis and Kurzyp-Wojnarska measured locus of control. These instruments measure generalized locus of control and allow the assessment of context-specific locus of control related to situations of success and failure, as well as school, parent, and peer settings. At the first stage of this study, students in non-integrated classrooms in schools without an inclusive curriculum were characterized by a more internal locus of control, both generalized and in situations of failure, compared to students of non-integrated classrooms in schools with an inclusive curriculum. At seventh grade, students of integrated classes were more external in situations related to their school activity, compared to their peers from non-integrated classrooms. Moreover, we observed developmental changes in locus of control of students from non-integrated classes but only those who attended schools with an integrated curriculum. Integrated education, which was introduced in Poland under the Education System Act of 7 September 1991 ,3. TodayAn important measure that overcomes the disadvantages of integrated education is inclusive education, which has been implemented in many European countries . InclusiIn Poland, the system of integrated education follows an integrated classroom. An integrated classroom accommodates disabled learners and learners without disabilities . Entering the period of adolescence together, they take on various biological, cognitive, and social challenges that naturally come with this period of life. Although the literature on how young people with various types of disabilities experience the period of adolescence is still scarce , the keyDisabled teenagers want to have the same sort of developmental experiences and opportunities as their typically developing peers, though they usually have to face more challenges because of their disability. Often, they cannot participate in school life and social life at the same level and pace as their peers without disabilities, which leads to negative psychosocial outcomes such as stress or loneliness ,13. TheyAttitudes towards people with disabilities and towards their learning in integrated classrooms are often a source of misunderstanding and may act to the detriment of students who learn in integrated education settings. Depending on the nature of the disability, these students are perceived by others as being passive, non-self-reliant, dependent on others physically or mentally, and ill-adapted to the conditions in which they live . The basEarly adolescence is a time of formation of a young person’s character or personality. It can be assumed that the experiences of being placed in an integrated classroom can be a strong stimulus for developing openness and sensitivity to other people and their needs. Less numerous classes, a more diverse daily routine, the assistance of a support teacher, and the fact that the child stays in this supportive setting for several years are all conducive to their opening up to others and forming their own mature attitudes towards peers. The meeting of typically developing and disabled youth at school should therefore have a positive impact on both these groups of students, giving the former the possibility of interacting with their disabled classmates and opening up to their needs, and offering the latter the opportunity to interact with others in a natural social setting. As shown by Polish and international experiences with integrated education, these goals are not always achieved and so neither is real integration ,17,18. HIn Rotter’s theory of social learning, locus of control is understood as the way a person interprets the causes of various events that occur to them in their daily lives . Events With age and growing experience in distinguishing events that are causally related to one’s actions from those that are not, an external locus of control, which is characteristic of childhood, changes to a more internal locus of control, which increases in internality in adolescence. As people grow up, their competencies improve and so does their ability to influence events in their everyday lives. During this time, their locus of control undergoes intense changes under the influence of the educational environment of the family and school, in which the young person learns to take responsibility for their actions. In students’ daily lives, their locus of control allows them to decide what they want to learn and how they want to do it, and enables them to flexibly reconcile the need to fulfil their school requirements with the need to get engaged in out-of-school activities . TherefoRotter and his concept is a personality-dominant approach to locus of control. Another approach, introduced in 1977 by Stern and Manifold, treats internal locus of control as a social value. The theory of the norm of internality stresses social judgements which favour internality as a way of interpreting different events ,25. SociResearch indicates that the knowledge of students’ locus of control allows one to predict the quality of their adaptation and ability to cope with various challenges ,34,35. IBased on a literature review, Lawrence and Winschel  found thA study of visually-impaired and hearing-impaired children showed that they had a more external locus of control ,48,49 coThere is a noticeable lack of systematic research on the development of locus of control in students learning in integrated settings, a gap that the present study is intended to bridge. In these students, the locus of control may determine the way they interpret their school situation and affect the quality of their adaptation to the new environment and requirements. This applies to both disabled students and their typically developing classmates.To examine differences in the generalized and specific LOC related to success and failure in school, family, and peer situations between students in integrated and non-integrated programs; andTo investigate developmental changes in generalized and context-specific LOC related to success and failure in school, family, and peer situations in students from integrated and non-integrated programs over a three-year schooling period.A review of previous studies on the locus of control in adolescents with disabilities and the assumptions of our own research project allowed us to formulate the following aims:There are no contemporary systematic comparative studies on the specific character of the development of locus of control in students from integrated and non-integrated classes during early adolescence. We expect that the results we obtain will allow this gap in the literature to be filled and, in practical terms, will provide new conclusions and justification for further research in this area.A total of 90 students attending integrated and non-integrated classes at three primary schools in Lublin, Poland, participated in the study. The study used a longitudinal design with three waves of data collected in the following years: the school year 2016/2017—stage I; the school year 2017/2018—stage II; and the school year 2018/2019—stage III. Once each school year, questionnaires were completed by the same students who, in the sequential research stages, were in grades 6, then 7, and then 8. The study was conducted in accordance with relevant ethical principles, with particular emphasis on confidentiality, anonymity, and voluntary participation. Consent from parents or caregivers was obtained for the students to participate in this study. In addition, permission to conduct the study was sought from headmasters of the schools concerned. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee for Scientific Research of Maria Curie-Skłodowska University (No. 29). Questionnaires were completed in groups during one meeting with each class. The participants were informed about the scientific purpose of the study and the anonymity of the results.M = 12.44, SD = 0.60; stage II, M = 13.63, SD = 0.52; and stage III, M = 14.51, SD = 0.69.Students from seven classes, including three integrated classes, were surveyed. The students were divided into three same-size groups: students learning in integrated classes—30 individuals (50% girls) in schools with integrated classes; students attending non-integrated classes in schools with integrated classes—30 individuals (50% girls); and students from non-integrated classes in segregated school with a traditional general education curriculum—30 individuals (57% girls). In total, 11 students with disabilities learning in integrated classrooms took part in the study. The average ages of the participants in each stage of the study were as follows: stage I, The respondents completed a personal data sheet and the Locus of Control Questionnaire (LCQ) by Krasowicz-Kupis and Kurzyp-Wojnarska ,52; in sIn stage I of the study (school year 2016/2017), locus of control was measured using LOQ (released in 1990), the Polish version of an instrument I-E, based on the original theory of social learning and locus of control of Rooter (1966), which was available at that time . The difExamples of Success item:(a)This is my lucky day(b)I try my best to do everything efficientlyIf one day I manage to do everything that I have planned, it is because:Examples of Failure item:(a)I had bad luck that day(b)I was carelessIf one day, you lost something valuable, it is because:The LOQ consists of 46 items, 36 of which have a diagnostic value. Half of the diagnostic items refer to situations of success and the other half to situations of failure; the sum of the scores on those items is a measure of a generalized locus of control, i.e., a general tendency to attribute more internal or more external causes to various events. The minimal raw score is 0, and the maximum is 18 for each scale: Success and Failure and 0–36 for a generalized locus of control. The questions are addressed directly to the respondent and ask about situations experienced by them. External factors responsible for success or failure are situations related to the family home, peers, and school. Internal factors, on the other hand, include a respondent’s own behaviours and efforts .Examplesalpha reliability coefficients for the individual scales are as follows: global scale alpha = 0.62; Success scale alpha = 0.40; and Failure scale alpha = 0.54. Despite the low values of the coefficients alpha, we decided to administer LOQ because at the moment of conducting stage I of this research, it was not possible to measure locus of control in adolescents with another test. The LOQ has a standard ten (sten) norms for people aged 13–17.There are two versions of the LOQ sheet, one for girls and one for boys. They only differ in the grammatical form of the questions (Polish has gendered forms), the content of the questions is the same. The instrument has moderate psychometric properties. Cronbach’s Example of School item:(a)A teacher chose you(b)I decided to participateIf you participate in an important subject competition, it is because:Example of Parents item:(a)I am messy(b)They always find reasons to complainIf your parents are angry about a mess in your room, it is because:Example of Peers item:(a)He will never change his mind, independently of what I do(b)I can influence his change of opinionIf your friend has a different opinion about something than yours, it means:The LOQ-R sheet consists of 43 items, 38 of which have a diagnostic value. The test has only one form of answer sheet for both sexes, with each question containing two grammatical forms (feminine and masculine). Similarly to its predecessor, the LOQ-R allows one to calculate three basic types of score: a generalized locus of control  score and scores on the Success and Failure scales. Minimum raw score possible to obtain in Success scale is 0, maximum is 10; in Failure scale, it is 0–15, in a generalized locus of control from 0 to 38. Additionally, it can be used to measure locus of control in particular contexts: School, Parents, Peers, and Non-specific.alpha = 0.81; for the Success scale alpha = 0.63; and for the Failure scale alpha = 0.66. The instrument also has satisfactory theoretical validity. The LOQ-R has sten norms only for the Success, Failure and global scales. These norms cover the age range of 13–18 years.The LOQ-R has good psychometric properties. Reliability analyses have shown that the questionnaire has a high or satisfactory reliability. In the age range of 13–15 years, Cronbach’s reliability coefficient for the global score was High scores on each of the LOQ and LOQ-R scales are reflective of an internal locus of control, low scores point to an external locus of control, and medium scores indicate that the individual has not yet developed a locus of control. The global score is the sum of the scores obtained on the Success and Failure scales and is a measure of a person’s generalized locus of control. It provides information on a person’s generalized tendency to attribute responsibility for various events to internal or external factors. The subscale scores provide more specific information on how the respondent typically interprets the causes of events related to success and failure or the participant’s locus of control in different settings: school, parents, and peer situations.Data obtained through the LOQ and LOQ-R questionnaires regarding the Success scale, Failure scale, and the global scale were interpreted using sten norms appropriate for the respondents’ age. Because the specific scales of the LOQ-R, defining the respondents’ locus of control in school, parents, and peer situations, do not have sten norms, the analysis of that context-specific locus of control was based on raw scores. Statistical calculations were carried out using SPSS v.25. The results are analyzed in the order in which the research questions were asked at the beginning of the article. A one-way ANOVA and a Tukey post-hoc test were used to check what statistically significant differences there were in the locus of control scores between students from integrated vs. non-integrated classes at each stage of the study. To determine developmental changes in the locus of control characteristic of each group during the three-year study period, a repeated-measures ANOVA followed by a post-hoc test with the Bonferroni correction was performed. Prior to these analyses, the Mauchly test had been used to evaluate the assumption of data sphericity This test turned out to be statistically insignificant .The results are shown in p < 0.05) and locus of control in failure situations  = 2.48; p < 0.05) in research stage I and for locus of control in school situations  = 2.48; p < 0.05) in stage III of the study. Post-hoc comparisons using the Tukey test revealed the following statistically significant differences between groups: In the first stage of the study (grade 6), the generalized locus of control and locus of control in failure situations statistically significantly (p < 0.05) differentiated students from non-integrated classes. Students who went to non-integrated classes in schools without an inclusive curriculum were characterized by a more internal locus of control, both generalized and in situations of failure. Post-hoc test data for stage III of the study (grade 7) revealed statistically significant differences (p < 0.05) in locus of control in the school setting between students from integrated and non-integrated classes. Students who learned in integrated classrooms showed a more external locus of control in situations related to their school activity compared to their peers from non-integrated classrooms.The one-way ANOVA identified differences between groups in the generalized locus of control, and in locus of control in situations of success and failure as well as situations occurring in school, family, and peer settings for each research stage. Statistically significant effects were found for the generalized locus of control  = 2.98; p < 0.05; η2 = 0.14) and generalized locus of control  = 3.32; p < 0.05; η2 = 0.10). The effect for locus of control in failure situations was strong and explained 14% of the variance in this context-specific locus of control over the three-year study period. In the case of the generalized locus of control, the effect was moderately strong and explained only 10% of the variance. For students from the school with a traditional teaching program, a statistically significant main effect was found for locus of control in situations of success  = 4.58; p < 0.001; η2 = 0.02). This effect was moderately strong and explained 2% of the variance.To determine developmental changes occurring in each of the examined groups during the three-year measurement of locus of control, a repeated-measures ANOVA was performed. This analysis revealed significant main effects for locus of control in non-integrated classes only. For students from non-integrated classes who attended a school with an integrated program, a significant main effect was found for locus of control in situations of failure  = 4.64; p < 0.05) and a more internal locus of control in situations of failure (p < 0.05). Additionally, the post-hoc test showed that there were statistically significant differences between the first and second measurements in the success-related locus of control in students from the non-integrated school. Their locus of control in situations of success was significantly weaker in the second measurement (grade 7) compared to the first measurement (grade 6). This means that these students’ locus of control in situations of success had shifted significantly (p < 0.01) towards externality.The post-hoc test with the Bonferroni correction showed that in the case of students from non-integrated classes who attended a school with integrated classes, there were significant differences in locus of control in failure situations and generalized locus of control between the first and second measurements (grades 6 and 7). Both the students’ locus of control in situations which they saw as a failure and their generalized locus of control were statistically significantly stronger in the second measurement than in the first one. Accordingly, these students had a significantly more internal generalized locus of control , the generalized locus of control and locus of control in situations of failure were statistically significant for students from non-integrated classes. Students who learned in non-integrated classrooms in schools without an inclusive curriculum were characterized by a more internal locus of control, both generalized and associated with situations of failure, compared to their peers from non-integrated classes in integrated schools. This result can be explained by the fact that in schools with integrated classrooms, students who are not in an integrated program are taught in a similar way to students from integrated classes. It is not uncommon for teachers in these schools to teach in both general education and integrated classrooms, which means they may set the same requirements and use the same educational methods in both types of classrooms, as part of their established ways of working with young people. The shift towards an external locus of control in situations of failure observed in the students from integrated schools may be the result of their receiving greater support and help from teachers in difficult and frustrating situations. In the case of schools without integrated classrooms, the requirements and teaching programs are the same for all classes. In settings in which all classes are “the same” and all students have equal intellectual and physical capacities, they are all treated in the same way, receive the same range of support, and have to fulfil the same requirements. This may explain why the students in traditional general education classes had a more internal locus of control than their peers from non-integrated classes who attended integrated schools.The second aim was related to the character of developmental changes in generalized locus of control, locus of control in situations of success and failure, and locus of control in school, family, and peer settings in students learning in integrated and non-integrated classrooms. The analyses showed that between the first and second stage of the study, developmental changes occurred in the locus of control of the participants from non-integrated classes, but only those who went to schools with an integrated curriculum. There were significant differences in these students’ locus of control in failure situations and their generalized locus of control between stage I (grade 6) and stage II of the study (grade 7). The students’ locus of control in situations of failure and their generalized locus of control were both statistically significantly stronger in the second measurement than in the first one, which meant that there had been a shift towards internality. This result is interesting and may indicate that—despite the fact that a year earlier, these students had been more likely than their peers from schools with traditional curriculum to attribute the causes of various events, including failures, to external factors—after a year, they began to show greater responsibility for their actions and choices. Perhaps, at that time , the students had already adapted to their new educational situation and felt more confident; at the same time, teachers began to be more demanding towards them, which resulted in a shift towards a more internal locus of control. This shift may have also been a natural effect of the developmental changes that people typically undergo in early adolescence .Even more intriguing is the result regarding developmental changes in the success-related locus of control in students from schools with a traditional curriculum. They had a significantly weaker locus of control in situations of success in the second measurement (grade 7) than in the first measurement (grade 6). It is worth recalling that in the first measurement, these students were characterized by a significantly stronger internal generalized locus of control and a stronger locus of control in situations of failure compared to their peers from non-integrated classes who went to integrated schools. Perhaps they focused on being responsible for their personal failures so much that they felt much less convinced that their successes were also an effect of their own activity. It is also possible that the weakening of locus of control in situations of success was a consequence of the fact that the level of work became more demanding in the seventh grade, and the youth experienced stress associated with being the first year of students continuing their education in a primary school instead of transitioning into a middle school .In stage III of the study, statistically significant differences in the locus of control were observed between students from integrated and non-integrated classes. They concerned the locus of control in school situations. Students who pursued an integrated program had a more external locus of control observed in situations related to their school activity compared to their peers from classes with traditional instruction. This means that the students from integrated classes were more likely to attribute their academic achievement to external factors instead of seeing it as an outcome of their own actions and efforts. This result is worrying, as at the time of the third stage of the study, these students were facing the prospect of choosing a secondary school and meeting the criteria set for all candidates. A lack of a sense of control over school situations, such as tests or exams, could reduce their chances of being accepted into the school of their choice. This result may also be indicative of the students’ uncertainty about their further educational career. The fact that not all secondary schools in Lublin have inclusive programs may have reduced the motivation of the students with disabilities to actualize their potential and pursue their dreams of continuing education in a school they wanted to attend. This, in turn, may have led to a feeling of alienation and uncertainty about their future. The finding that students from integrated classes have a more external locus of control is consistent with the observation made by Clark et al.  that theOne of the tasks of integrated education is to help eliminate differences in the development of locus of control between children with special educational needs and children without developmental disorders studying in one class . Our resSome authors ,63,64 poDespite the fact that this study was conducted in a relatively short longitudinal period, it was enough to determine changes in locus of control of students. The results indicate differences of locus of control development of students attending different types of classrooms and schools. In general, the results indicate the role of educational context on the development of locus of control in adolescents with and without disabilities. Among the complex underpinnings of educational influences on personality development in adolescence in the case of integrated education, the most important are a system of rewards and motivators and psychological preparation of teachers who work in this type of education.alpha coefficients in psychological tests, ranging from 0.70 to 0.95 [alpha below 0.60 (as it is in the LOQ), due to their low reliability, theoretically should not be used in the research. In spite of this, we decided to administer LOQ because, at the moment of conducting stage I of this research, it was not possible to measure locus of control in adolescents with another test. Another limitation of this study is the study cohort. It was relatively small, with 30 students from each of the analyzed groups surveyed. Moreover, the study cannot be replicated in the future because the context of the study has already changed: the new reform, which was being introduced at the time of the experiments, now is fully implemented. It is still worth further exploration of the developmental changes in the locus of control among students from integrated and non-integrated classes in the new educational reality.The conclusions of this study, though interesting, need to be interpreted with caution. Results, especially those obtained in the first stage of this study, are burdened with inaccuracy due to a low internal consistency of the LOQ . There a to 0.95 ,67. Inst


Pestivirus, encompassing small positive-strand RNA viruses in the family Flaviviridae, comprises four viruses of very significant economic impact to the cattle, swine and sheep industries worldwide: bovine viral diarrhoea virus (BVDV) type 1 and type 2, classical swine fever virus (CSFV) and border disease virus (BDV). Both BVDV- and CSFV-related disease syndromes have been recognised for over 70 years and major progress has been made in elucidating the pathogenesis of these important infections of ruminants and pigs. While much research effort rightfully has gone into epidemiology, diagnostics and prevention—and continues to do so—BVDV and CSFV have also served as excellent models for understanding mechanisms in RNA-virus biology [The genus  biology , and hos biology ,5. More Viruses encompasses a range of reports on various aspects of pestiviruses, including an overview of the bovine pestiviruses with an update on the current—albeit still not widely used—nomenclature of the pestiviruses [This Special Issue of iviruses . The epiiviruses ,9,10,11.A first and fundamental step in a viral infection, is attachment to a cellular membrane molecule, a viral receptor. In this issue, two articles focus on the purported receptor for BVDV, bovine CD46 ,13, descThe interaction of pestiviruses with the host immune system has been a focus of research for decades, notably for BVDV. In this issue the effect of congenital  BVDV infection and persistence on the development of innate and adaptive immune functions is explored in an experimental infection model . MonocytOne of the characteristics of the pestiviruses, at least the ruminant pestiviruses, is the lack of strict species specificity ,20. ThisIt is the hope that this issue, which has brought together contributions from multiple disciplines—virology, immunology, veterinary clinical medicine, epidemiology and pathology—will stimulate further exploration of this fascinating group of viruses in the future. There remains many questions to be addressed even in those very same areas dealt with in this issue, including the conditions, at both the cellular and organismic level, that are conducive to events leading to biotype-switch, viral persistence, emergence of new pestivirus diseases as well as some of the discrepancies between in vitro and in vivo results in regard to immune responses—to mention just a few. Development of new approaches to the investigation of “old” and new pestiviruses are already under way ,13,29 an


Plant viral diseases are the foremost threat to sustainable agriculture, leading to several billion dollars in losses every year. Many viruses infecting several crops have been described in the literature; however, new infectious viruses are emerging frequently through outbreaks. For the effective treatment and prevention of viral diseases, there is great demand for new techniques that can provide accurate identification on the causative agents. With the advancements in biochemical and molecular biology techniques, several diagnostic methods with improved sensitivity and specificity for the detection of prevalent and/or unknown plant viruses are being continuously developed. Currently, serological and nucleic acid methods are the most widely used for plant viral diagnosis. Nucleic acid-based techniques that amplify target DNA/RNA have been evolved with many variants. However, there is growing interest in developing techniques that can be based in real-time and thus facilitate in-field diagnosis. Next-generation sequencing (NGS)-based innovative methods have shown great potential to detect multiple viruses simultaneously; however, such techniques are in the preliminary stages in plant viral disease diagnostics. This review discusses the recent progress in the use of NGS-based techniques for the detection, diagnosis, and identification of plant viral diseases. New portable devices and technologies that could provide real-time analyses in a relatively short period of time are prime important for in-field diagnostics. Current development and application of such tools and techniques along with their potential limitations in plant virology are likewise discussed in detail. Viruses are the most genetically diverse organisms that cause infections in plants, animals, and humans. In general, viruses consist of a small genome that encodes only a few proteins, which makes it difficult to control viruses with a variety of methods . In planPresently, very few treatments are available to manage or cure plant viral infectious diseases under field conditions. Therefore, early detection of the relevant causative agents represents the most crucial step to prevent the possible spread of the infectious disease . The appThe advent of next-generation sequencing (NGS) technologies has invariably led to a revolution in plant virus diagnosis ,14. ThesNumerous methods have been developed and commercialized for plant viral diagnostics. However, the practical application of each prevalent method depends on various factors, such as its cost, sensitivity, rapidity, the availability of instruments, and the stage of the disease . Plants Extensive overviews of the important diagnostic methods, from traditional visual observations of the indicator plants to advanced third-generation sequencing technologies, are presented below.Traditionally, visual inspections of infected plants and seeds were the most common method used for plant pathogen detection. At the beginning of the 20th century, symptoms of viral diseases affecting plants were proposed as a basis for the taxonomy of viruses . The chaNicotiana glutinosa L., Datura stramonium L., and Phaseolus vulgaris L. cultivated in a wet chamber [P. vulgaris were also suggested for the detection of tobacco necrosis virus and the ring spotting of tobacco, alfalfa mosaics, and common and southern bean mosaics. To identify and measure the concentration of potato viruses, it was proposed to use the leaves of Gomphrena globosa L. and white goosefoot Chenopodium album L. and, for cucumber mosaic, the leaves of Vicia faba L. (Dornburger variety) [Nicotiana glutinosa) can be used as an indicator of tomato aspermy virus, and Gomphrena globosa leaves can be used for the diagnostics of potato X-virus. These methods have many disadvantages, and their results are dependent on the ages of the leaves and the technique of rubbing the virus into the leaf. For a comparative analysis of these experiments, a method using leaf halves was proposed. The juice of control infected plants was applied to one half of the leaf, and the juice of another variant was applied to the other half. Subsequent staining of the infected leaves by iodine testing for starch, which shows externally invisible infectious zones (infected leaf cells produce a low concentration of starch), provides an improved modification that should also be considered [To measure the titter of infectious viral particles within infected plants, it is proposed to employ the method of plant infection and count the mosaic (necrotic) spots developed on the leaves of indicator plants depending on the concentration of viral particles, the number of spots that appear will vary. Empirically, it was noted, for example, that the tobacco mosaic virus titer can be investigated using isolated leaves of  chamber . Leaves variety) . Young tnsidered .In most cases, a visual inspection can be conducted by different methods, e.g., visible-light imaging, chlorophyll fluorescence imaging, hyperspectral imaging, and thermal imaging . HoweverOne of the most classic methods for visualizing viruses in plant tissues is microscopic detection using modern light and high-resolution electron microscopes ,32. CertNicotiana tabacum and Zucchini yellow mosaic virus (ZYMV) in Cucurbita pepo plants in a relatively very short period of time. The authors demonstrated use of microwave fixation in TEM sample preparation could reduce time of investigation. EM has also been utilized to investigate subcellular localization of the plant virus in the vector to understand its transmission mechanisms. A study by Deng and colleagues [As the size and ultra-structural features are specific for each group of viruses, TEM could be used for reliable diagnosis as well as ultra-structural alterations during infections in plants. Zechmann and coworkers  reportedlleagues  elucidatlleagues ,43. Planlleagues . Similarlleagues  investigVigna mungo L. Hepper) plant was determined [Many a times, other techniques such as serological are frequently combined with the electron microscopy to increase the detection sensitivity. In this context, immune electron microscopy (IEM) that basically uses specific antiserum against viral antigen has been developed. Several IEM variants like solid-phase immune electron microscopy (SPIEM) and or immunosorbent electron microscopy (ISEM) are primarily being used in viral diagnosis . IEM cantermined . ElectroAnother related application of microscopy employing a confocal microscope (CM) was used for detecting indicator compounds or molecules . In thisCurrently, cryo-electron microscopy (cryo-EM) is the most advanced microscopy technique used in the investigation of plant-virus interactions, as this technique is capable of revealing nearly the atomic-level structures of viruses . The crySerological methods, such as ELISA (enzyme-linked immunosorbent assay), which are based on the reliable detection of a protein molecule using polyclonal or monoclonal antibodies, are widely used in plant viral diagnostics. Clark and Adams  were theBadnaviruses in West African yam plants [Using a plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA), a dot enzyme-linked immunosorbent assay (dot-ELISA) was developed for the rapid detection of RDV in rice . PTA-ELIm plants . Most ofm plants .Significantly, these methods are able to detect even closely related strains of the same virus. However, there is a need for appropriate concentrations to detect viruses, where the reactions of enzyme-labeled antibodies function as the viral concentration, making the technique more applicable for the quantitative detection of viral load in the samples .https://orders.agdia.com/pathogen-tests/immunostrip-tests (accessed on 26 October 2020), with several immunostrips specific to various plant viruses, such as ImmunoComb® strips for CMV, INSV, TMV, and TSWV and ImmunoStrip® for the detection of Potato virus X and Potato virus Y (PVX and PVY).Another interesting improvement in serological methods was the development of immunostrips for the early and rapid detection of viral infections in plants. In this context, Byzova et al.  were insNucleic acid-based methods are widely employed in many fields of diagnosis, including clinical, food safety, and environmental analysis. With the rapid development of molecular and genomics techniques, the scope of such techniques has become wider in the diagnosis of infectious pathogens. Due to their higher sensitivity, nucleic acid (DNA or RNA)-based techniques have been widely accepted in the field of viral diagnostics. In general, nucleic acid-based analysis has three essential steps: the isolation of the nucleic acid (either DNA or RNA), amplification, and product analysis. The last step is the most important, as it presents the result directly . VariousPrunus species. Remarkably, authors used semi-automatic homogenizer for sample preparation, which make it ideal for screening substantial numbers of samples. In Kenya, for the first time, Tomato chlorosis virus (ToCV) was detected based on coat protein, minor coat protein, and heat shock protein 70 homolog genes specific primers using RT-PCR [Polymerase chain reaction (PCR) is one of the most important methods and is considered the gold standard for the molecular detection of various pathogens. Extensive modifications have been made for the PCR-based analysis of viral detection. PCR-based methods detect the DNA or RNA signatures of the virus using molecular primers to amplify a certain region of the viral genetic material and are among the most widely used methods for viral detection ,75. PCR-g RT-PCR . Use of g RT-PCR . In the g RT-PCR  showed sg RT-PCR . These aUse of RT-PCR was also found valuable in analyzing the viruses like Pepino mosaic virus (PepMV), which has evolved as an emerging virus to tomato crops world-wide from the last few years. PepMV, a Potexvirus that gets transmitted mechanically and has assRNA-based genome. Various studies have successfully detected and identified different genotypes of PepMV in tomato crops. Using a newly developed one-step RT digital PCR, reliable quantification of different Pepino mosaic virus genotypes has been reported and found more suitable than only RT-PCR . In anotAllexvirus [Begomovirus [Potyvirus [In the last decade, substantial developments in molecular methods that involve the implementation of multiplex detection methods have become cost-effective, as multiple viruses can be detected with increased efficiency. Using multiplex RT-PCR, it is possible to develop techniques to detect all members of a specific genus by designing new primers that target the conserved regions. Various viral genera like lexvirus , Begomovomovirus , Potyvirotyvirus  were ideotyvirus . Possessotyvirus . There iotyvirus ,102.However, there are limitations in the detection of divergent variants of known viruses implementing these techniques. Therefore, refinement of PCR, RT-PCR-based techniques for more significant sensitive results are needed. Several groups were developing such techniques simultaneously from last decades. Among them, the methods based on the principle of coupling the sensitivity of ELISA and PCR were eventually termed immunocapture PCR or immunocapture RT-PCR  were employed in detection of plant viral infections. In these techniques, the viral agents are captured by virus-specific antibodies bound to PCR tube, micro well plate which is followed by PCR or RT-PCR of the viral particles .IC-PCR methods provide two-way specificity and were also found to be convenient for detection of viruses directly from crude plant extracts, without any requirement of the purified or partially purified virus . MoreoveCucurbitaceae and Solanaceae plants. The study clearly demonstrated use of the fluorometer portable device in developing the LAMP assay to achieve real-time detection of plant viruses under field condition. An Immunocapture version of the RT-LAMP ((IC-RT-LAMP) has been successfully applied in diagnosis of severe strains of Citrus tristeza virus (CTV) that cause quick decline and stem pitting in citrus [Due to its feasibility to work under field condition, LAMP has emerged as a robust, rapid and sensitive method for point-of-service detection and diagnosis of plant viruses. In general, LAMP works on mechanism of strand displacement and auto-cycling activity of Bst DNA polymerase, where amplification is done under isothermal conditions that do not require expensive devices like thermal cyclers . Moreoven citrus . In the n citrus . LikewisBefore the emergence of next-generation sequencing technologies, first-generation sequencing (Sanger sequencing) dominated the field of biological research. Even after the emergence of high-throughput sequencing technologies, these methods remained popular for analysis due to their higher throughput and relatively low cost . NGS tecMetagenomics is being developed for the detection of viruses. In these approaches, total nucleic acid (DNA or RNA) is extracted from the infected plant samples and sequenced using NGS platforms, and viral sequences are identified using bioinformatics tools. Blawid et al.  describemarafivirus (Grapevine Syrah virus-1) was identified in vines affected bysyrah decline along with other RNA viruses infecting grapevine [Second-generation sequencing technologies evolved based on high-throughput sequencing with the fast delivery of results at a reduced cost. Various platforms like GS FLX by 454 Life Sciences/Roche diagnostics; HiSeq, MiSeq, and NextSeq by Illumina, Inc.; SOLiD by ABI; and Ion Torrent PGM, Proton, S5, and BGISeq-500 are commercially available . These srapevine . It was rapevine . Furtherrapevine .potyvirus (Moroccan watermelon mosaic virus) and carlavirus, including the Cowpea mild mottle virus (CpMMV) and two other putative carlaviruses related to Cucumber vein-clearing virus (CuVCV) from papaya leaves in Kenyan fields [Ampelovirus, family Closteroviridae) along with three viruses of the family Betaflexiviridae , Grapevine virus B, and Grapevine rupestris stem pitting-associated virus (GRSPaV). Crucially, this study clearly showed multiple distinct strains of three viruses  in the diseased grapevine [begomoviruses, a potyvirus, and a crinivirus in individual plants could be identified without initial knowledge by employing Illumina sequencing platforms [begomoviruses together play a significant role in development of the disease.Using Illumina MiSeq sequencing, Mumo and colleagues marked the genome sequences of four different viruses, a n fields . This strapevine . Similarlatforms . These rVigna unguiculata L.) using 454 (GS FLX), Palanga and colleagues identified three novel plant virus species  [mycotymovirus, which likely affects cowpea plants and associated fungal species from Burkina Faso, West Africa. Plant viral diagnoses were also reported using Ion Torrent PGM, which is a competitive technique in metagenomics due to its low sequencing cost and similarly efficient operation.From the last decade, Illumina platform-based plant viral diagnosis has been applied to virome studies by analyzing the diseases of various important crops worldwide. Simultaneously, other available platforms like 454 (GS FLX) and Ion Torrent have been used for plant viral disease analysis . Howeverspecies) . This sttobamovirus isolate in Israel that infects tomato varieties harboringTm-22 resistance grown in protected structures [Using Ion Torrent, the genetic diversity of the Little cherry virus 1 (LChV1) in sweet and sour cherry species was studied . This structures . This unructures . These tSecond-generation sequencing transformed the field of biological research and is routinely used in environmental studies of microbial diversity, the human-associated microbiome, plant genomics, etc. During the last decade, third-generation sequencing technologies, also called single-molecule sequencing technologies, have gained much attention ,165,166.The SMRT platform from PacBio has been used widely in various types of research as it can sequence single molecules. This platform uses hairpin adaptors to form a closed ssDNA template, SMRTbell, which is placed in a zeptoliter-sized chamber, with the zero-mode waveguide (ZMV) featuring an attached single polymerase molecule at the bottom of the chamber. With the system, the addition of fluorescent-labeled nucleotides in the phosphate group can be detected in real-time . The SMRCurrently, Oxford Nanopore MinION has proven to be effective for real-time analyses with an average sequence length of more than 15 kb . MinION Solanum tuberosum L.), and high genetic diversity of this virus represents a challenge for its detection and classification. Della Bartola and colleagues [Begomovirus species (family Geminiviridae) are known to cause economically important diseases in major crop plants. Nanopore sequencing has also been applied to detect, and for the genome sequencing of Begomovirus species. The single-strand DNA Sri Lankan cassava mosaic virus (SLCMV) was found to be a major emerging pathogen in Southeast Asia. Leiva and colleagues sequenced the complete genome of this virus using nanopore technology [begomovirus (family Geminiviridae) was also detected in cowpea (Vigna unguiculata) plants exhibiting bright golden mosaic symptoms on their leaves under field conditions in Brazil [The portable nanopore sequencer has been reported to rapidly distinguish and accurately detect widely spreading plant viruses of the various agricultural plants. Potato virus Y (PVY) is the most economically important virus infecting cultivated potato  in Brazil [Physalis angulata using the rolling circle amplification method via ONT sequencing [P. angulata by ToSRV makes one believeit has an expanding host range in tropical and subtropical weeds. Surprisingly, in a study of a wheat plant containing the Wheat streak mosaic virus (WSMV) resistance gene (Wsm2), the plant showed characteristic symptoms of WSMV. Serologically, WSMV was detected in all four samples. The infected plant samples were used to isolate RNA, and the single-strand cDNA was sequenced using Oxford Nanopore sequencing technology (ONT). In this study, nanopore technology confirmed the presence of WSMV and was also found effective for the detection of TriMV and BYDV in most of the samples. Deep sequence analysis of the single-strand WSMV revealed variation within the WSMV Sidney-81 reference strain, indicating new variants with Wsm2 resistance. The result of the study showed that nanopore technology can more accurately identify causal virus agents and has sufficient resolution to provide evidence of causal variants [Plant viruses are typically known to have expanded host range. The weeds of the n Brazil . Tomato quencing . This stvariants .Badnavirus genus), Yam mild mosaic virus , and Yam chlorotic necrosis virus  in the yam plants. The validation of the genome sequence of Yam mild mosaic virus obtained using MinION and the Sanger sequencing approach indicated that MinION could, in the near future, reliably enhance research on, and the monitoring of, plant viruses. Such studies reveal that third-generation sequencing technologies, especially MinION nanopore techniques, are becoming popular among plant virologists for effective viral detection and diagnosis. Due to its long read lengths, fast run times, portability, reasonable cost, and possibility to be used in every laboratory, the nanopore platform will, in the near future, benefit plant viral diagnosis for the early protection of the crop plants.Most of the studies performed inplant viral diagnostics showed that Oxford Nanopore sequencing can remain a general method for diagnosis. To validate the use of nanopore sequencing for general diagnosis, Chalupowicz and colleagues infected the plants of several families with diverse pathogens like bacteria, viruses, fungi, and phytoplasma . The totThe management of plant viral diseases requires an effective diagnosis method that can generate early results for analyzing and preventing diseases. To date, various methods have been developed to identify and diagnose viral diseases among crop plants. The traditional diagnostic methods for plant viral detection, however, are time-consuming, bulky, and expensive, requiring cell culture and sample preparation. In recent years, sequencing technologies have been used in many fields, including metagenomics, medicine, plant physiology, and pathology. Therefore, advanced NGS-based techniques that offer large advantages in the diagnosis of viral diseases are becoming popular in analyzing the infectious diseases of bacterial, fungal, and viral species . FurtherHowever, there is a need for these revolutionary technologies to rapidly and accurately diagnose both identified and unidentified viral pathogens, as well as new variants of well-known pathogens . Due to The development of innovative techniques should also satisfy anticipated needs more effectively than previous techniques. These techniques should be competitive in terms of their accuracy and cost-effectiveness and should involve all on-chip processing steps from sample preparation and enrichment to amplification. In this regard, the portable devices developed by Oxford Nanopore are more promising. Due to their ability to remain functional under an enormous range of operating conditions and their portability, these devices can be operated directly by growers and producers in the fields. Although sequencing techniques have experienced great advancements and are now able to provide a large number of datasets, there is still a need for the development of computer-intensive data processing steps like assembly, mapping, and alignment tools. The most fundamental problem in identifying novel viruses is associated with the requirements of iterative rounds of assembly and mapping with specialist knowledge of viral genomes. In this context, nanopore sequencing technology is a promising technology for viral tracriptome, structural variation, and genome re-sequencing studies, which can be performed directly in the field . Nanoporbegomovirus (family Geminiviridae) from cowpea (Vigna unguiculata) plants showing golden mosaic symptoms on leaves under field conditions in Brazil using MinION nanopore sequencing techniques [begomovirus species previously reported infecting cowpea around the world which has resulted in low identity. Phylogenetic analysis revealed that the closest relatives with 85–87% nucleotide sequence identity were three legume-infecting begomoviruses; Macroptilium common mosaic virus, Macroptilium yellow vein virus, and bean golden mosaic virus [More focused research on the development of such tools in the future will undoubtedly provide a more thorough understanding of the various components of viral pathosystems for future sustainable crop management. However, little progress has currently been made regarding the use of MinION sequencing in analyzing plant viruses. Possibly, most of these more innovative techniques that rely on the isolation of total DNA or RNA from the infected samples have difficulties in eliminating the host plant DNA or RNA, which makes sequencing more complex when interpreting the results. In a few of the cases, total double-stranded RNA (dsRNA) from pathogen-infected tissue was partially eliminated by hybridization to nucleic acid isolated from healthy plants to enrich the virus sequences in infected plant material before sequencing. Naito and colleagues detected the new bipartite chniques . The autic virus .Third-generation sequencers produce sequence reads with hitherto unprecedented lengths and will help to strongly increase the quality of genome assemblies. Further, the speed of sequencing and ease of sample preparation will facilitate sequencing in the field. The ONT device is pocket-sized, portable, and controlled by a laptop, thus offers immense advantage for in-field diagnostics. The first in-field use of ONT was demonstrated for human diseases during the 2014 Liberia ebola outbreak . TherefoIn this specific context, a case study was performed using an effective point-of-need field diagnostic system with MinION and MinIT mobile sequencing devices. In the field, Boykin and colleagues  developeMonitoring plant health and quickly detecting pathogens are essential to reduce viral disease spread and facilitate effective management practices. Diagnostic methods commonly used to detect plant pathogens have limitations, such as the requirement of prior knowledge of the genome sequence, low sensitivity, and a restricted ability to detect several pathogens simultaneously. The development of advanced DNA sequencing technologies has enabled the determination of total nucleic acid content in biological samples. The possibility of using omics techniques, including third-generation sequencing platforms, such as Oxford Nanopores, as a general method for the diagnosis of plant diseases, is most readily applicable to viral diagnostics. However, there is still much to be discovered to advance the current state of viral diagnosis in plant diseases. Despite the great successes in the field of pathogen diagnosis tools, widely applicable, inexpensive, and simple approaches are still missing. Moreover, mixed infections remain a hurdle in disease diagnosis. The shortcomings need to be eliminated in all methods, including cell culture methods, molecular-based methods, immunological methods, and other advanced methods. Choosing the correct target site is vital for success in molecular detection, but finding such targets is not an easy task. Metagenomics has revealed many new viral entities recently and seems to be a promising approach. Further developments in bioinformatics tools using NGS methods could facilitate potential technological advancements in disease diagnosis that will help reduce crop losses due to emerging diseases.


Bemisia tabaci Middle East-Asia Minor 1 (MEAM1) to transmit BaCV, its morphological and cytopathological characteristics, and assess the incidence of BaCV across bean producing areas in Brazil. Our results show that BaCV is efficiently transmitted, in experimental conditions, by B. tabaci MEAM1 to bean cultivars, and with lower efficiency to cowpea and soybean. Moreover, we detected BaCV RNA in viruliferous whiteflies but we were unable to visualize viral particles or viroplasm in the whitefly tissues. BaCV could not be singly isolated for pathogenicity tests, identification of the induced symptoms, and the transmission assay. BaCV was detected in five out of the seven states in Brazil included in our study, suggesting that it is widely distributed throughout bean producing areas in the country. This is the first report of a whitefly-transmitted rhabdovirus.The knowledge of genomic data of new plant viruses is increasing exponentially; however, some aspects of their biology, such as vectors and host range, remain mostly unknown. This information is crucial for the understanding of virus–plant interactions, control strategies, and mechanisms to prevent outbreaks. Typically, rhabdoviruses infect monocot and dicot plants and are vectored in nature by hemipteran sap-sucking insects, including aphids, leafhoppers, and planthoppers. However, several strains of a potentially whitefly-transmitted virus, papaya cytorhabdovirus, were recently described: (i) bean-associated cytorhabdovirus (BaCV) in Brazil, (ii) papaya virus E (PpVE) in Ecuador, and (iii) citrus-associated rhabdovirus (CiaRV) in China. Here, we examine the potential of the  Rhabdoviridae) are a group of negative-sense, single-stranded RNA viruses that infect plants, vertebrate animals, and invertebrate animals. They cause harmful diseases in humans and animals and can cause high yield losses in crops. Plant-infecting rhabdoviruses are currently taxonomically assigned to six genera , and 4 healthy cowpeas [Vigna unguiculata (L.) Walp. ‘BRS Imponente’] plants for an inoculation access period (IAP) of 7 days. Likewise, as controls, non-viruliferous flies were placed in 2 healthy plants of each plant species for 7 days. After this period, the leaves inside the polyester voile bags were detached from plants to avoid nymphal maturation, and plants were sprayed with imidacloprid (32 mg/L) to remove any possible remaining whiteflies [To further refine the transmission tests, two three-week old ‘BRS FC 401 RMD’ plants infected with BaCV-Luz (and CPMMV) were removed from the cage, sprayed with imidacloprid (32 mg/L) to eliminate any whiteflies present in the plants, transferred to a bugdorm tent, and kept in a greenhouse at room temperature for 10 days. f 7 days . As a coiteflies . Plants act11) [RbcS) [RpL9) [v-ATPase) subunit A [To identify the presence of BaCV-Luz in bean plants and whiteflies, the total RNA was treated with TURBO DNase  to eliminate any DNA trace from the RNA preparation as described by Cao, et al. . The cDNact11)  and the ) [RbcS)  genes fr) [RpL9)  and Vacuubunit A  genes frSmall leaf pieces were cut from ‘BRS FC 401 RMD’ bean plants that were experimentally inoculated with whitefly and tested positive for BaCV by RT-PCR. Leaf sections were fixed overnight with Karnovsky modified fixative . The samples were post-fixed with 1% osmium tetroxide (in 0.05 M cacodylate buffer) for 1–2 h. Tissues were dehydrated, embedded in low viscosity epoxy Spurr resin , and sectioned in a Leica UC6 ultramicrotome equipped with a Diatome diamond knife. Ultrathin sections (70–100 nm thick) were transferred onto 300 mesh copper grids, stained with 3% uranyl acetate and Reynold’s lead citrate, and examined in a JEOL JEM 1011 transmission electron microscope. Healthy bean leaves were used as controls. Images were digitally recorded. Whiteflies were reared and collected from BaCV-Luz-infected bean plants, dipped into a NaCl 0.9% solution, and dissected at the thorax region in two parts: head/thorax and abdomen. For each whitefly, either the head/thorax or the abdomen parts were used for BaCV detection by RT-PCR, and the other part was fixed in a cold solution of 2.5% glutaraldehyde and 1.8% sucrose in 0.1 M cacodylate buffer. For the whiteflies that one part of the body was positive for BaCV presence by RT-PCR, the other part was processed to be examined by electron microscopy, as described above for plant tissues. Three pairs of head and abdominal parts of presumably viruliferous whiteflies were examined.BaCV was identified for the first time in Brazil in 2014 in bean plants collected in Santo Antônio de Goiás, Goiás State (GO) . In JuneGiven the high infection rate, we further investigated the occurrence of these viruses in newly collected (2016–2018) and archived samples (2007–2016) from Central, Southeast, and Northeastern Brazil, comprising six states and the federal district . In totan = 22), BGMV (n = 1) or with CPMMV and BGMV (n = 62)  . Mixed iB. tabaci (B. tabaci TSA 2 (AKC57270.1), confirming that these viruses belong to the species Papaya cytorhabdovirus. Moreover, yerba mate chlorosis-associated virus clustered with B. tabaci TSA 1 (AKC57275.1), as a sister group of papaya cytorhabdovirus strains. Furthermore, assuming that the viral surface envelope glycoprotein G interacts directly with receptors in the vector cells, a sequence similarity network (EFI–EST webserver) was generated using the glycoprotein amino acid sequences encoded by the 28 cytorhabdoviruses. Interestingly, the analyses show three distinct clusters  and two singletons (leafhopper and an undescribed vector) with a high degree of interconnectivity.The complete BaCV-Luz genome was determined by PCR of six overlapping fragments and was 13,467 nt in length. As expected, the genome presented the seven ORFs originally described in BaCV-GO , flanked by two non-transcribed leader and trailer regions . As show. tabaci . The phy. tabaci . As prevThese results, together with the detection of BaCV in concomitant infection with the whitefly vectored BGMV and CPMMV in all plants , and witThe known vectors of cytorhabdoviruses are insects belonging to the families Aphididae (aphids), Delphacidae (planthoppers), and Cicadellidae (leafhoppers). In general, when the vectors are known, there is a highly specific relationship, and only one (or a few related) types or species of a vector are capable of transmitting a given virus. Thus, it is possible to establish a strong correlation between viral detection and the presence of its vector in the field ,49.B. tabaci MEAM1, the prevalent whitefly species in Central Brazil [n = 48) of the bean plants ‘Pérola’, ‘Jalo’, and ‘BRS FC 401 RMD’ tested positive for the presence of BaCV RNA by RT-PCR. Whitefly adults collected at the same time as bean leaves were also positive for BaCV and CPMMV , criniviruses (family Closteroviridae), torradoviruses (family Secoviridae), ipomoviruses (family Potyviridae), and the carlaviruses CPMMV and melon yellowing-associated virus (MYaV) (family Betaflexiviridae [Luteoviridae) were also shown to be transmitted by B. tabaci [Furthermore, the whiteflies’ ages were not synchronized in the initial trials, and the insect stage of the life cycle may influence the efficiency of BaCV transmission. Soybean is an economically important cash crop in Brazil and is also susceptible to CPMMV  and BGMVymbionts ,54,55. Biviridae ,57). Rec. tabaci ,59.B. tabaci MEAM1, are vectors of the cytorhabdovirus BaCV in Brazil, highlighting the importance of whiteflies as plant virus vectors and emphasizing their designation as super vectors [Our transmission results demonstrate that whiteflies, in this case the species  vectors . MoreoveB. tabaci MEAM1 was very efficient in transmitting BaCV, especially to bean plants. B. tabaci MEAM1 predominates as a vector of various plant viruses in Brazil [B. tabaci Mediterranean (MED) and B. tabaci New World (NW) are also present in more restricted areas of Southeastern and Southern States. Whether whiteflies MED and NW can transmit BaCV to beans or other crops remains to be investigated.Despite whiteflies being successful plant virus vectors, the transmission efficiency may vary depending on the virus, virus isolate, host plant, whitefly species and biology, and virus and whitefly population’s geographical origin . In our n Brazil . HoweverB. tabaci MEAM1 is the vector of PpVE to papaya plants in Ecuador and if this virus infects papayas in Brazil. Cornejo-Franco, et al. [B. tabaci is a major pest of papayas in Ecuador and is the vector of papaya virus Q (an umbra-like virus). Thus, this whitefly species may also be the vector PpVE to papayas. We have tested a limited number of papaya plants (n = 27) collected in the Distrito Federal and the State of Espirito Santo for the presence of BaCV with negative results. Nine whitefly species, including B. tabaci MEAM1, were already identified on papaya trees in Brazil, but Trialeurodes variabilis is the primary species associated with this fruit crop, and B. tabaci MEAM1 has a limited occurrence in papayas in the country [B. tabaci MEAM1 or if a helper virus is required.It is also necessary to investigate if , et al.  mention  country . ConsideB. tabaci is still unknown [B. tabaci complex in a persistent circulative manner. However, at least for one begomovirus, tomato yellow leaf curl virus (TYLCV), there is evidence that it replicates in the whitefly [Depending on the taxonomic group they belong to, viruses are transmitted by their whitefly vectors by different transmission modes . The car unknown ,57. Begowhitefly ,63,64,65whitefly .Plant rhabdoviruses are transmitted by their arthropod vectors in a persistent, circulative-propagative manner. After the acquisition, viruliferous insects transmit plant rhabdoviruses for their entire lives. In plants, rhabdoviruses infect, replicate, and accumulate in a variety of tissues, including leaf epidermis and mesophyll, phloem tissues, and roots ,67. In tact11 and RbcS were only amplified from bean cDNA, and v-ATPase and RpL9 from whiteflies  to visualize BaCV accumulation in bean and whitefly tissues. Electron microscopic examination of BaCV-infected bean leaves revealed bacilliform particles typical of rhabdoviruses in parenchymal cells. Longitudinal and cross-sectioned particles were located in the cell cytoplasm, commonly at the periphery of an electron-lucent mass of coiled filamentous material, believed to be the viroplasm, where the virus replicates a–d 5,68,685,68. By contrast, feather-like aggregates of CPMMV particles were readily recognized in all examined bean tissue samples. These in situ observations of BaCV and CPMMV virions presence in dually infected bean leaf corroborate the previous HTS sequencing study in which 8.2% of the total sequence reads from multiple virus-infected bean leaves corresponded to CPMMV whereas only 0.01% mapped to BaCV . In someThe spatial and temporal distribution of BaCV within the whitefly body could have hindered the localization of particles by TEM. Moreover, plant rhabdoviruses appear to replicate and accumulate at lower levels in insect cells when compared to plant cells ,6,69. BaB. tabaci MEAM1 to three bean cultivars grown in Brazil, and with lower efficiency to cowpea and soybean. It remains to be determined whether BaCV replicates in whiteflies, as observed for other plant-infecting rhabdoviruses in their arthropod vectors. BaCV could not be singly isolated for pathogenicity tests, identification of the induced symptoms, and the transmission assay. Moreover, BaCV was detected in five out of seven Brazilian states evaluated. Besides BaCV in Brazil  and PpVs study, , similars study, , beans fs study, , and cits study, , implyin


For improving the effective concentration of berberine hydrochloride (BH) in the gastrointestinal tract, a series of pH-responsive hydrogel beads were prepared based on carboxymethylstarch-g-poly (acrylic acid)/palygorskite/starch/sodium alginate (CMS-g-PAA/PGS/ST/SA) in the present work. The developed hydrogel beads were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and thermogravimetric analysis (TG). Effect of palygorskite (PGS) content on the swelling properties of hydrogel beads and BH cumulative release were discussed. The pH responsiveness of hydrogel beads was also investigated in different media. Results illustrated that swelling of hydrogel beads and BH cumulative release from hydrogel beads were obviously affected by PGS content. The swelling ratio and BH cumulative release of composite hydrogel beads remarkably slowed down with PGS content increasing in the range from 10 to 40 wt%. The composite hydrogel beads were pH-responsive. At pH 7.4, the swelling ratio and BH cumulative release from composite hydrogel beads were the fastest among the dissolution media of pH 1.2, pH 6.8 and pH 7.4. The BH cumulative release from hydrogel beads was related to the swelling and relaxation of composite hydrogel beads and could be fitted better by the Higuchi model. The obtained composite hydrogel beads could be potentially used for the development of BH pharmaceutical dosage forms. Traditirdifolia ,3. In rerdifolia , anti-turdifolia  and antirdifolia , have alrdifolia . These nrdifolia ,9, whichrdifolia . To effeet al. [in situ gel system, which was able to prolong the residence time on the ocular surface after topical administration. Massoumi's group obtained novel pH-responsive PEGylated hollow nanocapsules, which had excellent potential for cancer chemotherapy [N-isopropylacrylamide) [et al. [Drug carriers are an essential part for preparing new medicines in the field of pharmaceutics, which are receiving increasing attention owing to the advantage of responding to the pH value of the external environment changes, improving patient compliance, increasing drug-residence time and ensuring drug released at the desired site –14. For et al.  preparedotherapy . The druotherapy , chitosaotherapy , alginatotherapy , κ-carraotherapy  and polyylamide) , have grylamide) ,23. Currylamide) ,25. For ylamide) –29. Palyylamide) ,31. For  [et al.  report t2+ to prepare the CMS-g-PAA/PGS/ST/SA composites hydrogels beads. The schematic of CMS-g-PAA/PGS/ST/SA composite hydrogel beads is given in Carboxymethyl starch (CMS) attracts increasing attention as an excipient in drug delivery systems due to its low price, good compaction for tablet preparation and pH-responsive properties, but pure CMS is apt to solubility causing a high burst effect of drug –35. Base2.2.1.N, N′-methylenebis acrylamide (MBA) was obtained from Drug Group Chemical Reagent Co., Ltd, China. The reagents including potassium persulfate (KPS) and acrylic acid (AA) were from Tianjin Kaixin Chemical Industrial Co., China. All solutions were prepared with distilled water.BH raw powder (purity > 98.2%) was purchased from Nanjing Zelang Pharmaceutical Technology Co., Ltd, China. PGS powder was obtained from Linze Colloidal Co. of Gansu province in China. CMS was purchased from Shanghai Maclean Biochemical Co., Ltd, China. Sodium alginate (SA) was from Shanghai Chemical Co. Ltd, China. Soluble starch (ST) was from Tianjin Hedong Hongyan reagent factory, China. 2.2.CMS-g-PAA/PGS composites with different PGS contents were prepared as our previously reported method ,37. CMS 2.3.2 solution to form CMS-g-PAA/PGS/ST/SA composite hydrogel beads (SA could be cross-linked with Ca2+ in the CaCl2 solution immediately). The obtained hydrogel bead products were screened and washed with distilled water several times to remove unreacted the CaCl2 on surface, and then dried at 60°C in an oven to obtain the final product.The CMS-g-PAA/PGS/ST/SA hydrogel beads were prepared as follows. Firstly, BH (0.10 g) and distilled water (100 ml) were put in a 500 ml beaker equipped with a mechanical stirrer and dissolved. CMS-g-PAA/PGS (1.0 g) microparticles were then added into the mixture solution and stirred for 3 h. After that, SA (2.0 g) and ST (5.0 g) were added and stirred for 4 h at 1000 r.p.m. Subsequently, using a 1 ml syringe the slurry was dropped into a 5 wt% CaCl2.4.2.4.1.W0 represents the weight of BH in hydrogel beads, W1 indicates the weights of BH-loaded composite hydrogel beads.The BH-loaded CMS-g-PAA/PGS/ST/SA hydrogel beads were soaked in pH 6.8 phosphate buffer solution  for 12 h. Afterwards, the swollen beads were crushed and transferred into a beaker. Subsequently, the crushed hydrogel beads were soaked in fresh PBS (20 ml) again and sonicated for 30 min to extract BH from hydrogel beads. The BH solution was centrifuged at 5000 r.p.m. for 20 min to remove the polymeric debris. UV spectrophotometer was used to analyse the BH content. The drug loading (%) was calculated using the following equation:2.4.2.M0 and tM are the weight of hydrogel beads before and after soaking in the swelling medium of pH 6.8 at time t, respectively. The swelling ratio under various pH conditions was tested by the same procedure. The various buffer solutions were made by combining NaH2PO4, Na2HPO4, HCl and NaOH solutions. And pH meter (PHS-3E) was used to determine pH values. All swelling experiments were carried out thrice carefully under the same conditions and the average values were reported.BH-loaded CMS-g-PAA/PGS/ST/SA hydrogel beads swelling properties were studied in pH 6.8 PBS. The 0.20 g of hydrogel beads were put into the baskets of intelligent disintegration instrument  at 37 ± 0.5°C. At the set time intervals, the hydrogel beads were taken out from the swelling medium of pH 6.8 and weighed after removing residual liquid on the surface of hydrogel beads. The swelling ratio is calculated using the equation given below.2.4.3.In vitro release study of BH from BH-loaded CMS-g-PAA/PGS/ST/SA hydrogel beads was carried out as follows: 0.20 g of dried BH-loaded CMS-g-PAA/PGS/SA hydrogel beads were placed in 100 ml of the release medium, and incubated at 37 ± 0.5°C under 100 r.p.m. The release medium  was made by combining HCl, KH2PO4 and NaOH solutions properly referring to the Chinese Pharmacopoeia 2015. At predetermined time intervals, 5 ml of the release medium was withdrawn and replaced by an equal amount of fresh release medium to keep a constant volume. The BH concentration in the release medium was assayed by UV spectrophotometer. For release medium pH 1.2, the BH concentration was assayed at 343 nm, and for release medium pH 6.8 and pH 7.4, the BH concentration was assayed at 224 nm. The BH cumulative release per cent was obtained using equation (2.3). All cumulative release results were done in triplicate.M and tM represent the BH initial amount and cumulative release amount of at time t, respectively. All release experiments were carried out thrice carefully under the same conditions and the average values were reported.2.4.4.tM/∞M represents the fraction of drug released in time t, n is the release exponent characterizing release mechanism and K is a constant. If n ≤ 0.43, it represents that BH release from hydrogel beads is Fickian release. When 0.43 < n ≤ 0.85, it is defined as the non-Fickian release (both swelling-controlled release and diffusion controlled release). If n > 0.85, it indicates that BH release is mainly related to the relaxation (or swelling ratio) of hydrogel beads, and BH release is case II transport. It was worthwhile to note the tM/∞M in the Korsmeyer–Peppas model was below 60% as reported in the literature [The data of BH release kinetics from hydrogel beads was fitted using zero-order model, Higuchi model and Korsmeyer–Peppas model.terature .2.5.θ range of 3–80° at a scan speed of 5° min−1; an America TA Company Instruments (TGA-Q100) was used for the thermogravimetric (TG) analysis of samples.Samples of FTIR spectra were taken in KBr pellets using a FTIR-FTS3000 spectrophotometer; an Ultra Plus scanning electron microscope (SEM) instrument (Carl Zeiss AG) was used to analyse micrographs; X-ray diffraction (XRD) was carried out on a Rigaku D/Max-2400 diffractometer in the 23.3.1.a, CMS presented three characteristic peaks, one at 1606 cm−1 and one at 1421 cm−1 (attributed to vibration of –COO–) [−1 (corresponding to the vibration of –C–O–C), which almost vanished in the spectra of CMS-g-PAA, and the peak at 3425 cm−1 (ascribed to the vibration of –OH) weakened in the spectra of CMS-g-PAA. Meanwhile, compared with the spectra of CMS, some new peaks appeared at 2850, 1714 and 1588 cm−1 in the spectra of CMS-g-PAA. However, the peak at 1588 cm−1 shifted to 1626 cm−1 in the spectra of CMS-g-PAA/10% PGS. Also, by comparison with the spectra of PGS, the peaks at 1039 cm−1 (corresponding to the vibration of Si–O) and 879 cm−1 (assigned to the vibration of Al–O), almost vanished in the spectra of CMS-g-PAA/10% PGS. In addition, the characteristic peaks of BH  disappeared in the spectra of BH-loaded CMS-g-PAA/10% PGS/ST/SA in figure 1b. However, the peak at 1506 cm−1  , another of –C=C ) still e3.2.a, swollen BH-loaded CMS-g-PAA/10% PGS/ST/SA hydrogel beads appeared as yellow spheres with smooth surfaces. After they were dried, the beads showed a rough surface and the size was approximate 2.5 mm (figure 2b). Additionally, CMS-g-PAA composite displayed a flat and tight surface (figure 2c), while CMS-g-PAA/10% PGS exhibited a relatively loose, undulant and coarse surface (figure 2d). Moreover, some pores could also be seen in the surface of CMS-g-PAA/10% PGS. These results indicated that the introduction of PGS could affect the specific surface area of composites, and might eventually cause the changes of swelling behaviour and drug release property [The digital photos of the BH-loaded CMS-g-PAA/10% PGS/ST/SA hydrogel beads, and the SEM images of CMS-g-PAA and CMS-g-PAA/10% PGS are shown in figure 2property .Figure 3.3.θ = 31.52° and 45.3°. While in the XRD profile of BH-loaded CMS-g-PAA/10% PGS/ST/SA, the two characteristic peaks were weakened. It could be concluded that BH crystal structure was changed in the BH-loaded CMS-g-PAA/10% PGS/ST/SA hydrogel beads, which might be due to the interaction between BH and CMS-g-PAA/10% PGS/ST/SA hydrogel beads.The XRD patterns of BH, CMS-g-PAA/10% PGS/ST/SA and BH-loaded CMS-g-PAA/10% PGS/ST/SA are shown in 3.4.The thermal degradation behaviour of BH, CMS-g-PAA/10% PGS/ST/SA and BH-loaded CMS-g-PAA/10% PGS/ST/SA was studied under a nitrogen atmosphere between 34 and 790°C. The results are shown in 3.5.As shown in 3.6.3.6.1.Clay content has a very important effect on the swelling of polymer/clay composites because clay could affect the network structure of polymer/clay composites . Thus, t3.6.2.− groups converted to −COOH groups in the acidic solution, which strengthened the hydrogen-bonding interaction of the polymeric chains in CMS-g-PAA/10 wt% PGS/ST/SA hydrogel beads causing the small swelling ratio. At pH 6.8, the electrostatic repulsion of –COO− within the test hydrogel beads weakened the hydrogen-bonding interaction of the polymeric chains, which made the hydrogel beads swollen. With further increase of pH to 7.4, the hydrogen-bonding interaction of hydrogel beads disintegrated and the electrostatic repulsion of –COO− within the test hydrogel beads strengthened, which led to the further increase of the swelling ratio. The above information indicated the hydrogel beads had a benign pH-responsive behaviour with an external pH solution, which was also similar to the previous research reports in the literature [terature ,51.Figu3.7.3.7.1.3.7.2.et al. [The effect of pH on BH release from BH-loaded CMS-g-PAA/10% PGS/ST/SA hydrogel beads is shown in et al. .Figure 3.7.3.R2 = 0.9216–0.9433) better than the zero-order equation (R2 = 0.8467–0.9332) in pH 6.8 PBS according to the respective correlation coefficients (R). For the relationship between BH cumulative release from hydrogel beads and the relaxation as well as erosion of hydrogel beads, the Korsmeyer–Peppas model showed all the n values were between 0.9451 and 1.397, which means that BH release mechanism was mainly related to the swelling and relaxation of composite hydrogel beads.The results of model fitting are shown in 4.In this paper, a series of new pH-responsive composite hydrogel beads were successfully developed in order to improve the effective concentration of BH in the gastrointestinal tract. The incorporation of PGS could influence the amount of BH loading in hydrogel beads. The swelling ratio of hydrogel beads and BH cumulative release decreased with the increase of PGS content in hydrogel beads. The swelling ratio of hydrogel beads and BH cumulative release from hydrogel beads at pH 7.4 were faster than that at pH 6.8. BH cumulative release from hydrogel beads was related to the swelling ratio and relaxation of composite hydrogel beads and could be described better by the Higuchi model. These obtained results showed that the incorporation of PGS in composite hydrogel beads could regulate drug load and release, which would facilitate the preparation of novel drug carriers. The composite hydrogel beads is useful for improving the concentration of BH in the gastrointestinal tract. The method of preparation for hydrogel beads was also proved to be simple.


Day-to-day observations reveal numerous medical and social situations where maintainingphysical distancing is either not feasible or not practiced during the time of a viralpandemic, such as, the coronavirus disease 2019 (COVID-19). During these close-up,face-to-face interactions, a common belief is that a susceptible person wearing a facemask is safe, at least to a large extent, from foreign airborne sneeze and cough droplets.This study, for the first time, quantitatively verifies this notion. Droplet flowvisualization experiments of a simulated face-to-face interaction with a mask in placewere conducted using the particle image velocimetry setup. Five masks were tested in asnug-fit configuration : N-95, surgical, cloth PM2.5, cloth, and wetted cloth PM 2.5. Except for the N-95 mask, the findings showed leakageof airborne droplets through all the face masks in both the configurations of (1) asusceptible person wearing a mask for protection and (2) a virus carrier wearing a mask toprevent the spreading of the virus. When the leakage percentages of these airbornedroplets were expressed in terms of the number of virus particles, it was found that maskswould not offer complete protection to a susceptible person from a viral infection inclose  face-to-face or frontal human interactions. Therefore,consideration must be given to minimize or avoid such interactions, if possible. Thisstudy lends quantitative support to the social distancing and mask-wearing guidelinesproposed by the medical research community. Recent research on face masks shows that they help control the spreading of the respiratorydroplets when the wearer sneezes or coughs into the mask.µm in size orimportantly, from the perspective of a virus carrier wearing a mask to contain the spreadingof the virus.µm indiameter).It can be observed that most of the research conducted so far on face masks has been eitherqualitative or on respiratory droplets that are >5 µm–10 µm) and droplet nuclei, the droplet nuclei canlinger in the air much longer.μm-sized droplets or nuclei can escapethe pores of face masks more easily than the large droplets. It was found that particles thatare <200 nm in size are often captured by filters  due to theBrownian motion , socialdistancing is also suggested by the CDC and WHO for controlling the spreading of respiratorydroplets and nuclei from a virus carrier. However, day-to-day observations reveal numerousmedical and social situations where maintaining physical distancing is either not feasible ornot practiced, e.g., either in closed spaces, such as hospitals, homes, gymnasiums, publictransportation, and schools, or physically close interactions in indoor and outdoor spaces,such as in crowded gatherings at organized events and political campaigns. People willeventually start to socialize and travel to combat depression. Restaurants will allow indoordining, and airliners will eventually allow middle-seat occupancy; all of these will increasethe number of close-up human interaction scenarios even more when the wearer sneezes or coughs into it?3.Can a facemask be made more effective by wetting it with water?In many such close face-to-face or frontal interaction scenarios, a common belief appears tobe widely pervading that a susceptible person wearing a face mask is safe, at least to a largeextent, from foreign sneeze and cough droplets. This study verifies this notion using particleimage velocimetry (PIV)-based counting of particles. Accordingly, this study quantitativelyanswers the following questions:An experimental setup was built to answer these questions, and the particle image velocimetry(PIV) setup was employed to visualize and count the number of simulated droplets . The setTwo 0.25 in.-thick transparent acrylic glass square tubes, 34 in. and 12 in. long, of 3 in. ×3 in. internal cross section were joined with a mask snugly fit between them. Air-tightsealing of the junction with the mask was achieved using duct tape. An aluminum stand wasbuilt to fix the tubes above the ground and allow space to position a laser under the tubesfor visualization. The camera lens was placed in parallel to the laser sheet plane such thatthe masks are located at the center of the captured images. The PIV system is composed of aNd:YAG laser with a wavelength of 532 nm, a charge-coupled device (CCD) camera, an externaltrigger, and DaVis 8.2 software for image processing. A temporal resolution of 3 Hz was usedfor the image acquisition, and 100 pairs of images were secured for each mask experiment forcapturing (and counting) the particles on both sides of the mask.To avoid saturation of brightness and suppress reflection and boundary glare, the squaretubes’ top and back walls (from the perspective of the camera) were completely blackened. Thebottom wall was also blackened except for a thin slot for allowing the laser sheet to pass. Aflow generator was attached to one end of the longer tube for generating airflow with thesimulated droplets of water. A centerline velocity of 3.2 m/s was used in all the experiments.This value, as an average, captures most of the typical sneeze and cough scenarios in close(<6 ft) frontal exposure situations.µm.μm-sized droplets inall the experiments.Given the scope of this study, the size of the particles is an important parameter. It wasobserved in extensive independent analyses of various human exhalations that the size of thedroplets—that are more prone to be airborne and thereby travel with moving air to a personnearby—is on the order of 1 For ease of interpretation, the post-processed data are provided in combination with the flowvisualization images in For providing the readers with an understanding of the practical impact of EP, the resultsare also presented in terms of the average number of foreign sneeze and cough droplets thatcan escape a mask in the considered close (<6 ft) face-to-face interaction scenario. Thesenumbers are based on multiple studies that stated that a typical sneeze and a cough couldcontain 40 000 and 3000 droplets, respectively.6virus particles depending on the virus concentration (or load) of the carrier.6 virus particles per one cough or sneeze.Studies have shown that the viral infection threshold, e.g., for COVID-19, for a susceptibleperson is 1000 virus particles, inhaled either at once or in batches.Finally, another set of experiments was also conducted to measure the mask effectiveness if avirus carrier is wearing a mask (assuming a snug fit) and sneezes or coughs into it. The maskdirection was reversed to simulate these experiments to measure the difference, if any, in theEP. The EP values are as follows: N-95: 0.98% ± 0.69% SE, Surgical: 0.19% ± 0.06% SE, Cloth PM2.5: 0.19% ± 0.06% SE, Cloth: 2.7% ± 0.2% SE, and Cloth PM 2.5 Wet: 0.07% ± 0.06% SE. Sincethe mask in this scenario in real life will be closer to the ejection or exhalation source(mouth or nose), the velocity could be 3–4 times larger than 3.2 m/s.1.Without a face mask, it is almost certain that many foreign droplets will transfer tothe susceptible person. Wearing a mask will offer substantial, but not complete,protection to a susceptible person by decreasing the number of foreign airborne sneezeand cough droplets that would otherwise enter the person without the mask.2.Consideration must be given to minimize or avoid close face-to-face or frontal humaninteractions, if possible. If the relevant social distancing guidelines are compromised,the study shows that foreign airborne sneeze and cough droplets could pass through allthe masks tested (except for the N-95 mask) even when assuming a 100% snug fit.3.6 virusparticles depending on the carrier’s virus load. In close-up, face-to-face interactions,factors that could dilute the virus concentration [such as diffusion of the droplets orsome of the large (>5 µm–10 µm) droplets falling tothe ground] will be less important. Even when the concentration of the virus particlesin a single sneeze or cough is less than 10 × 106, the results show that noneof the masks tested would be able to offer protection to the susceptible person.Depending on the mask’s type, they appear to be effective only when the susceptibleperson is exposed to virus concentrations of less than 5 × 106 per sneeze orcough for surgical and cloth PM 2.5 wet masks. This cutoff is as low as less than 100000 for the dry cloth masks. If the edge leakage factor of the masks is taken intoaccount, these cutoffs could be even lower. Such low concentrations could be possibleonly when there is sufficient and rapid dispersion of a sneeze or cough in thesurrounding environment, or if the virus load of the carrier is very low. In a studythat modeled airborne droplet transmission, it was found that saliva droplets (with adecrease in the droplet concentration and size) could travel as far as 19 ft–20 ft inthe wind direction for wind speeds varying from 1.1 m/s to 4.1 m/s.Studies show that a single sneeze can usually contain 10–200 × 104.The cloth PM 2.5 mask wetted with water has exhibited a better performance in blockingthe airborne droplets than all the other masks tested (except for the N-95 mask),including the surgical mask. If a mask is wetted, the material’s fibers will swell,reducing the pore size available for the droplets to penetrate through the mask. The wetmask might have to be disposed or occasionally washed to replenish with freshwater toaddress the virus saturation and water evaporation concerns. The wet mask approach couldprove useful in medical environments where it could be disposed of after eachinteraction with the virus carrier.5.The experiments that simulated the scenario of a virus carrier wearing a mask show thatmany small sneeze or cough droplets can still escape a mask even when the mask is snuglyfit. Therefore, both the virus carrier and the susceptible person could consider wearinga mask to reduce the transmission and spread of the virus.6.If close-up contact cannot be avoided, such as in frontline worker activities, someideas could be: using wet masks or turning the face away from the sneeze or cough orboth. Such additional measures could at least lower the risk to some extent by avoidinga direct frontal exposure to the droplets and possibly providing a longer time for thediffusion/dilution of the airborne droplets to occur. Some medical experts and priorresearch also suggest the second option  by early nextyear, and close-up interactions and the inconsistent use of masks are cited as some of thepossible reasons.J.A., A.L.G., and L.S. have worked on building the experimental setup, statisticalpost-processing of the data, and setting up the micro-PIV experiment, respectively. Allthree of them have equally contributed to conducting the experiments and obtaining the data.S.K., F.S., and K.K. have participated in the analysis of the data. S.K. and K.K. havecontributed to writing the paper, and F.S. has reviewed it. F.S. and K.K. have conceived theexperiments. K.K. has initiated the work.


We used metagenomic analysis to identify Tacheng tick virus 2 infection in a patient with a history of tick bite in northwestern China. We confirmed the virus with reverse transcription-PCR, virus isolation, and genomic analysis. We detected viral RNA in 9.6% of ticks collected from the same region. Emerging pathogenic tickborne viruses have attracted much attention because of the increasing incidence of tickborne viral diseases and their effects on human health , an increased protein level (0.99 g/L), and decreased levels of CSF glucose (2.3 mmol/L) and chloridion (116.0 mmol/L). The patient was given intravenous ceftriaxone for 12 days, but still experienced headache, nausea, and vomiting, and his erythema was not decreasing.On June 24, the patient was admitted to the First Affiliated Hospital of Medical College of Shihezi University in Shihezi. Physical examination showed erythema at the bite site , panel ABorrelia burgdorferi sensu lato, Anaplasma, Babesia, Rickettsia spp., Tacheng tick virus 1, TcTV-2, Tacheng tick virus 5, BlTV-1, and Bole tick virus 4  for potential tickborne pathogens, including severe fever with thrombocytopenia syndrome virus, tickborne encephalitis virus, On July 18, the patient was admitted to the hospital again. He was given intravenous acyclovir for 12 days and his clinical symptoms and erythema vanished without any sequelae  Table 3.To isolate the virus, we inoculated human hepatocellular carcinoma (SMMC-7721) cells, African green monkey kidney (Vero) cells, baby hamster kidney cells, and human foreskin fibroblasts with the serum samples collected during early illness onset  Figure 2We isolated total RNA from infected cells and used the isolates to amplify the L and small (S) gene segment sequences by using primers based on our metagenomic analysis , D. marginatus (14.8%), H. asiaticum (11.9%), and D. nuttalli (5.5%). We obtained the partial fragments of the S segment of TcTV-2 in ticks and phylogenetic analyses showed that sequences from TcTv-2 in ticks were closely related to the isolate from the patient  segment of TcTV-2 by designing a set of primers based on the conservative sequences of M segments from 15 typical phleboviruses  Table 6.Among currently known emerging tickborne phleboviruses, severe fever with thrombocytopenia syndrome virus and Heartland virus have been reported to infect humans and cause multiple organ damage, including to the liver and kidneys (Transmission electron microscopy showed that TcTV-2 might harbor glycoprotein encoded by the M gene segment. The lack of M sequence data on homology-based approaches could indicate that insufficient homology exists between these viruses to detect the M gene in this manner. Sequencing methods that obtain a greater depth of coverage might help obtain the missing M sequences. To increase the virus titer and the likelihood of obtaining the M sequence, we recommend performing deep sequencing on the isolated virus.D. marginatus ticks from China (H. marginatum ticks from Turkey (D. nuttalli, D. silvarum, H. asiaticum ticks and in blood, urine, and throat swab samples from a patient with febrile illness. Our findings suggest that person-to-person transmission might be possible through direct contact with body fluids or by droplet transmission. In addition, we noted more tick species found in northwest China that could act as TcTV-2 vectors (TcTV-2 previously was identified in Additional information on Tacheng tick virus 2 isolated from a patient in China.