Arabidopsis thaliana were examined: the accumulation of small RNAs (sRNAs) using a microfluidics-based method ; and the transcript accumulation of virus-response related host plant genes, suppressor of gene silencing 3 (AtSGS3) and calcium-dependent protein kinase 3 (AtCPK3) by reverse transcriptase-quantitative PCR (RT-qPCR). The microfluidics approach using sRNA chips has previously demonstrated good linearity and good reproducibility, both within and between chips. Good limits of detection have been demonstrated from two-fold 10-point serial dilution regression to 0.1 ng of RNA. The ratio of small RNA (sRNA) to ribosomal RNA (rRNA), as a proportion of averaged mock-inoculation, correlated with known virus infection to a high degree of certainty. AtSGS3 transcript decreased between 14- and 28-days post inoculation (dpi) for all viruses investigated, while AtCPK3 transcript increased between 14 and 28 dpi for all viruses. A combination of these two molecular approaches may be useful for assessment of virus-infection of samples without the need for diagnosis of specific virus infection.To our knowledge, there are no reports that demonstrate the use of host molecular markers for the purpose of detecting generic plant virus infection. Two approaches involving molecular indicators of virus infection in the model plant  Virus infections in plants pose a major threat to agricultural, environmental, and economic security globally ,2. StricRather than detecting specific viruses, we hypothesise that molecular markers involved in host plant cellular responses to viruses may be more advantageous as they do not require viral sequence information to identify infected plant or plant material. Recently, indirect detection of viral infections through host response-related molecular markers has been explored in several human and animal studies, but not yet in plants. In humans, the use of host signatures to detect and/or discriminate viral infections, such as protein biomarkers, gene transcripts, and other molecular signals have been recently reported ,13,14,15Plants respond to viruses through a network of genetic and intracellular pathways, including RNA silencing or interference (RNAi) ,17,18. DIdentification and profiling of small RNAs using deep-sequencing methods have also been explored as an approach to diagnose and control plant virus infections in recent studies ,30,31,32Arabidopsis thaliana, suppressor of gene silencing 3 (AtSGS3) has been reported to have a role in the RNA pathway to amplify the dsRNA signal [SGS3 in Nicotiana benthamiana, was reported to cooperate with RNA-dependent RNA polymerase 6 (RDR6) in fighting against virus infection [SGS3 mRNA levels have been reported to show positive correlation with viral RNA levels in SGS3-silenced and SGS3-overexpressing leaves of N. benthamiana infected with PVA [SGS3 may be a good candidate as a molecular indicator of virus infection. Another gene in A. thaliana, calcium-dependent protein kinase 3 (AtCPK3) may also be a good candidate as it is a stress-responsive gene belonging to the most conserved group of calcium dependent protein kinases (CPKs) [AtCPK3 transcript accumulation changes in response to infection with plant viruses.In addition to sRNA, specific plant host genes may also be useful as molecular markers for virus infection. Recent studies have utilised microarray, transcriptomics, proteomics and metabolomics approaches to identify host genes and molecular signatures that differentially respond to virus infections ,40,41,42A signal  and invoA signal . The ortnfection ,45. Morewith PVA . Hence, s (CPKs) . CPKs ars (CPKs) ,52,53,54s (CPKs) ,56. WhilA. thaliana were examined: the accumulation of LMW RNA using microfluidics technology and the transcript accumulation of virus-response related host plant genes AtSGS3 and AtCPK3 using RT-qPCR. The use of these approaches to detect virus infection in A. thaliana within broad time-course of infection, from different plant virus infections, was analysed.In this study, two groups of molecular indicators of virus infection in the model plant A. thaliana RNA samples suggested an increase in accumulation of sRNAs among infected plants -infected d plants a. SampleA. thaliana LMW-RNA with that of TuMV-inoculated A. thaliana. More specifically, the region of dark banding between 10–40 nt (sRNAs) can be seen clearly in infected plans , tobacco mosaic virus (TMV), tomato spotted wilt virus (TSWV), turnip mosaic virus (TuMV), turnip yellow mosaic virus (TYMV), or inoculation buffer (mock) were loaded onto sRNA chips for analysis by the Bioanalyzer method. Each LMW-RNA component was analysed as a proportion of total LMW-RNA where the average of all mock-inoculated data at each time point was rescaled to 1.0 (proportion of averaged mock-inoculation). The standard error of the mean was calculated for all virus inoculations at the same dpi.In order to quantify separate LMW-RNA components  from Analysing LMW-RNA components as a proportion of averaged mock inoculation enables inference of LMW-RNA component accumulation irrespective of dpi and has the practical advantage of not needing to know the history of the virus infection within a plant. Since the results for the proportion of mock- and virus-inoculation demonstrated the same trend as the results for proportion of averaged mock-inoculation, only the data of each LMW-RNA component as a proportion of averaged mock-inoculated are shown. Taken together, these data suggest a great deal of variability of all components of LMW-RNA at every time point for all virus inoculations. The data for TuMV showed the most consistent profile across the infection time course with the accumulation of sRNAs increasing and the accumulation of tRNA, snoRNA and rRNAs decreasing compared to averaged mock-inoculation, at most time points. It is however notable that the sRNA proportions mostly increased, while rRNA proportions decreased, in response to virus infection.In response to most virus-inoculations, sRNAs appeared to increase in accumulation and rRNAs appear to decrease in accumulation at most time points, compared to averaged mock-inoculation. Hence, the ratio of sRNA to rRNA as a proportion of averaged mock-inoculation was calculated. The aim of this calculation was to determine whether there was a high correlation between a plant being infected and a ratio of sRNA accumulation/rRNA accumulation greater than averaged mock-inoculation rescaled to 1.0, such that this metric could be used as a predictive tool. AtSGS3 transcript accumulation across three replicate plants showed a decreasing trend for transcript accumulation in response to CaMV, TMV, TSWV, TuMV, and TYMV-inoculation at most time points from 14 dpi. While this trend was not of conventional biological significance (arbitrary fold change of ≥2.0), the same trend or biological effect was discernible in the separate biological replicates. AtSGS3 mRNA accumulation as average log transformed, mean centred and auto-scaled Q data from the three biological replicates of virus-infected plants compared to mock-inoculated plants of the same time point (dpi). Compared to mock inoculation of the same time point, AtSGS3 transcript accumulation decreased between 14 and 28 dpi for all viruses except TMV at 28 dpi. The observed fold-change of AtSGS3 was less than 2.0 in response to CaMV-, TMV-, TSWV-, TuMV-, and TYMV-inoculation compared to mock inoculation of the same time point between 14 and 28 dpi, but these data were statistically significant.AtSGS3 was specific to the biotic stress of virus infection, a further biotic stress of bacterium infection was studied. Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) was inoculated to A. thaliana plants and compared to mock-inoculated plants. Accumulation of AtSGS3 mRNA at 1, 2, 6, and 10 dpi was assessed by RT-qPCR. The data presented in AtSGS3 accumulation in response to Pst DC3000 only increased slightly, with statistical significance only at 2 dpi and not in the other time points.In order to evaluate whether the decrease in transcript accumulation of AtSGS3 transcript accumulation, the abiotic stressors of 100 mM NaCl, 200 mM NaCl, and drought stress were applied. A. thaliana plants were either (i) watered following a normal regime (control), or (ii) watered with 100 or 200 mM NaCl , or (iii) watering was withheld for 7 d prior to leaf tissue collection at 7, 14, and 21 dpi (drought stress). The data of three biological replicates presented in AtSGS3 transcript accumulation at every time point for 100 mM and 200 mM NaCl stress and at every time point for drought stress. The fold change compared to the control treatment in every case was >2.0, showing that these results are of biological significance.In order to distinguish biotic and abiotic stress effects on AtSGS3 transcript accumulation consistently decreased to a small degree (<2.0 fold) in response to the five virus infections but to a large degree (>2.0 fold) in response to the abiotic stresses investigated. AtSGS3 transcript accumulation did not significantly change in response to Pst DC3000. Thus, it is possible that a statistically significant small fold change might be indicative of virus infection whereas a large, biologically significant, fold change might be indicative of abiotic stress. However, this possibility needs to be evaluated across further viruses, including cryptic viruses, other plant species, as well as other biotic and abiotic stressors.Taken together, these results suggest that AtCPK3 transcript was assessed using the same total RNA samples. In response to the same five viruses, AtCPK3 showed different levels of increase in transcript accumulation in leaves between 14 and 35 dpi  as well as DNA genomes. Therefore, the molecular responses that are common across these infections and across time reveal basic interactions between plant viruses and their host plant. Moreover, further study examining multiple infections as well as latent infections are necessary as these may trigger different gene expression profiles. This method may prove to be a useful tool to link pathogenicity with presence of viruses and thereby complement the information gained by high throughput sequencing, which is typically bereft of biological significance of virus presence.AtSGS3 and AtCPK3 homologues that are sensitive and specific for a wide range of plant species. Moreover, as the study was only conducted on glasshouse grown test plants under controlled conditions, this method still needs to be tested on field collected samples or border security samples with infections of unknown age and from varied environments. Further studies comparing the accumulation of these host molecular indicators before and after symptom appearance, as well as comparing symptomatic and asymptomatic or latent infections will also be important.This study used only a single plant host to determine whether a broad range of plant pathogenic viruses could be detected using non-specific tests for virus infection. Further refinement to confirm that the method is widely effective includes testing the proposed method on other plant species whose genomes have been sequenced and represent major taxonomic groups and developing degenerate primers for SGS3/RDR6 pathways and can be investigated for differential expression in response to other plant virus infections. A calmodulin-like protein from N. benthamiana acts a suppressor of RNA silencing by degrading SGS3 during infection with a geminivirus; hence may be elevated in response to many other virus infections [SGS3/RDR6 bodies may also be a potential host marker of virus infection [RACK1) has also recently been reported to be downregulated by virus-induced gene silencing, and acts a s a bridge between a viral replication protein and a CPK [POX) produced in response to flagellin 22 (flg22) and has been optimised to minimise the amount of plant tissue required. It would be valuable to test this approach to viral infections. Physiological and anatomical parameters that change as part of the plant immune defence system against virus infection should also be considered for future research [Other molecular markers and approaches may also be explored, considering recent studies that have been published after we have carried out the experimental work for this study. Other genes have been reported to be involved fections . A virusnfection . On the nd a CPK . A reducnd a CPK . A micrond a CPK . This asresearch ,65,66,67Although there are many limitations and factors yet to investigate, this study is the first to put emphasis on the potential use of molecular markers for detecting generic virus infection by monitoring the host plant rather than the virus. Such a generic molecular response to virus infection may be eventually used for in-field, remote testing using technologies such as aptamers or nanoprobes linked with nucleic acid probes ,69. The A. thaliana (col-0) plants were infected separately with tobacco mosaic virus (TMV), cauliflower mosaic virus (CaMV), tomato spotted wilt virus (TSWV), turnip mosaic virus (TuMV), and turnip yellow mosaic virus (TYMV). Leaf samples were taken at 2-, 3-, 7-, 14-, 21-, 28-, 35-, and 42-days post inoculation (dpi) with three biological replicates of virus- and mock-inoculated plants for each time point. Drought experiments, fungal infection with Botrytis cinerea, and bacterial infection with Pseudomonas syringae pv. tomato strain DC3000 were also performed to determine plant responses that are unique to virus infection and not characteristic of other stress and pathogen responses. For drought, samples were taken at 7, 14, and 21 d because most plants appeared desiccated after 21 d. For fungal and bacterial infections, samples were taken at 1, 2, 6, and 10 dpi as most plant tissue had been destroyed by the infection by 10 dpi. Three biological replicates were sampled for treated and untreated plants at each time point.A. thaliana leaf tissue samples using the mirPremier™ microRNA isolation kit . This kit provided the most consistent yields of sRNA and best quality compared with two other kits, as determined by NanoDrop spectrophotometry . Procedures were completed according to the manufacturer’s protocol. All samples had an absorbance ratio (A260:A280) of between 1.8 and 2.2 where a ratio of ~2.0 is generally accepted as ‘pure’ or of good quality for RNA. Following quantification, all LMW-RNA samples were normalised to 30 ng/μL.LMW-RNA was isolated from Total RNA was extracted from the leaf or root samples collected as described above using a Spectrum TM Plant Total RNA Kit  following the manufacturer’s instructions. RNA samples were treated with DNase I  to remove any potential genomic DNA (gDNA) contamination. RNA concentration and purity were measured using a Nanodrop ND-1000 spectrophotometer  while RNA integrity was analysed using a Bioanalyzer 2100 RNA Nano LabChip 6000 . RNA Integrity (RIN) values were assigned by the Bioanalyzer software algorithm, which determines the quality of a total RNA sample from the 28S:18S ribosomal RNAs ratio and from the entire electrophoretic profile . RIN values range from 1 to 10, with 10 being a fully intact RNA and ≥7.0 being acceptable. However, the algorithm used by the software version was designed for mammalian RNA and did not consider chloroplast RNA in plants. RIN values were then adjusted with visual inspection of the peaks for 25S, 23S, and 16S RNAs. All RNA samples used for the succeeding experiments had an absorbance ratio (A260:A280) between 1.8 and 2.2, and adjusted RIN value of 7.0 or greater.® VILO™ cDNA synthesis kit  following the manufacturer’s protocol. The total amount of RNA transcribed to cDNA was adjusted to 2 µg, in a total volume of 40 µL.RNA samples were reverse transcribed into cDNA using a SuperScriptSmall RNA chips were loaded following the manufacturer’s Small RNA Kit Quick Start Guide. The chip was run in the Bioanalyzer instrument with the appropriate assay, sRNA, selected from the software menu. A successful sRNA run results in one marker peak and two regions that are defined arbitrarily by the Bioanalyzer expert B02.04 software: the LMW-RNA region from 0 to 150 nucleotides (nt), and the microRNA (sRNA) region from 10 to 40 nt. These regions were modified by selecting the region table within the software and setting regions for analysis to extend the LMW-RNA region to 200 nt and to encompass the composition of LMW-RNA components; that is 50–80 nt for transfer RNAs (tRNAs of 73–94 nt), 85–100 nt for small nucleolar RNAs  and 110–180 nt for ribosomal RNAs  in addition to the sRNA region already set (10–40 nt). The LMW-RNA component regions were set to encompass the average nt size reported for each LMW-RNA component across the literature ,71.Regression analysis was performed to estimate the relationship between each LMW-RNA component  and each virus-inoculated and mock-inoculated LMW-RNA from 120 observations. Mixed model repeated measures analyses of fixed (LMW-RNA components) and random  effects was kindly performed by Dr Nihal de Silva using the statistical analysis software system SAS/STAT 9.22 to determine if there was a difference in LMW-RNA components profile between each of the five virus-inoculations and mock-inoculation across the 2 to 42 dpi time course. To establish if the measured load on the two components differed, principal component analysis was performed on the entire data set, thus converting the complex and multidimensional data into a lower number of variables. If the load between the two calculated components differed it could be determined that the accumulation of LMW-RNA components of the five virus-inoculations can be distinguished from mock-inoculation.AtSGS3  and AtCPK3 . Primers were initially tested for their ability to amplify a product of correct size by employing an end-point RT-PCR reaction consisting of 1.0 μL cDNA synthesised from 2 µg RNA, 12.5 μL of GoTaq®Green Master Mix , 0.5 µL of each 10 µM stock forward and reverse primer, and 10.5 µL of UltraPure™ DNase/RNase-free distilled water  to a total volume of 25 µL. The following PCR conditions were employed: initial denaturation at 94 °C for 5 min, followed by amplification with 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s, followed by a final extension at 72 °C for 5 min. Negative control reactions, omitting cDNA template, were also prepared for each set of reactions.Gene specific qPCR primers were designed specifically near the 3′ end for To quantify the transcript accumulation of the target and reference genes from each sample, qPCR reactions were performed using a LightCycler 480 Real-Time PCR system . Reactions were in a 10 μL total volume containing 1 μL of primer pair (2 μM stock forward and reverse primer), 4 μL of cDNA, and 5 μL of LightCycler 480 SYBR Green I Master mix reagent (Roche). A Biomek 3000 Robot  was used to aliquot all reagents, primers, and samples into 384-well plates, with two technical replicates and three biological replicates for each sampling time point. The qPCR reaction consisted of pre-incubation at 95 °C for 5 min and amplification with 45 cycles of denaturing at 95 °C for 10 s, annealing at 60 °C for 10 s, and extension at 72 °C for 10 s. Fluorescence acquisition was set up at the end of each cycle. The amplification step was followed by a melting curve analysis, with one cycle of 95 °C for 5 s, 65 °C for 1 min, and a ramp to 97 °C at a rate of 0.11 °C/s. Five fluorescence acquisitions per °C were taken. Samples were cooled at 40° for 10s.A. thaliana [P ≤ 0.01), good (0.01< P ≤ 0.05) or weak (0.05 < P < ~0.10). Levene’s test was initially done before the ANOVA test to determine if the values have equal variance. The statistical software Minitab was used to perform all statistical tests .Fluorescence data per cycle were exported from the LightCycler 480 software into a *.csv file using Python 2.6.3 . Transcript accumulation of the targeted genes were normalised using the reference genes F-BOX family protein (FBOX), SAND family protein (SAND), and elongation factor 1 alpha (EF1 α). These have been previously validated as good reference genes for virus infection in thaliana . Baselinthaliana . Figures


Empowered by interdisciplinary collaboration, we now have the tools to identify new viruses, contain future outbreaks and broadly understand natural processes toward a global health. Never before has virology been on the public spotlight on such a prominent and global level. The ongoing pandemic, caused by the novel SARS-CoV-2, with its dire consequences for almost every country, remains as an international public health emergency. With the example of SARS-CoV-2, new and emerging viruses, their origin, genetic diversity, host range, diagnosis and prevention methodologies have attracted unprecedented interest, sometimes entailing pseudoscientific interpretations extending to conspiracy theories. Meanwhile, the field of virology has already been going through a transformation phase at least for a decade, in terms of overall capacity to identify and characterize new viruses and understanding the biodiversity of the virus world, also termed as the virosphere . The avaBasic concepts of microbiology tell us that all living cells are prone to viral infections and viruses capable of replicating in all known types of prokaryotic and eukaryotic cells, living as single-celled or multicellular organisms, are likely to exist. However, since the advent of virology as a separate field in the realm of microbiology, the majority of the well-characterized viruses have been those involved in human disease or animals and plant infections with significant public health or economical impact. This was partly due to the methodological constraints inherent to then available virus characterization and diagnostic techniques, relying mainly on virus isolation with subsequent cultivation using cell lines or susceptible animals and culture-obtained virus antigens to assess immune responses. Another widely acclaimed notion considered most, if not all, viruses as pathogenic entities, which was in stark contrast with the expected abundance of viruses in the biosphere.Major advancements for understanding viruses and their infections were provided by DNA sequencing and recombinant DNA technologies that enable antigen production from unculturable viruses. Nevertheless, the most prominent breakthrough was fueled by the dissemination and adaptation of nucleic acid amplification techniques, mostly based on PCR, facilitating a timely detection of ongoing infections as well as virus quantitation. These approaches, despite significant technical advances and automatization, still constitute the mainstay of the current diagnostic virology practices. Prior to the development of metagenomic sequencing techniques as we know today, particular approaches such as consensus PCR, representational difference analysis or sequence-independent single-primer amplification were available and have been instrumental in identifying many novel viruses, virus induced infections and diversity .The impact of metagenomic sequencing on new virus discovery is that it enables relatively sensitive and massively parallel production of target-independent sequences . Despitein silico host prediction. Incorporation of the virus genome data into the official classification scheme of the International Committee on Taxonomy of Viruses has already been under debate and analysis pipelines efficient in predicting assignments according to the current taxonomy framework are being developed [in silico predictions of viral protein structure and function, genome replication and host range. The origin of the virus genome should also be thoroughly questioned, as it may be derived from the initial host, as well as any infecting bacteria, fungus or parasite or from the environment.The description of novel viruses and the expansion of the virosphere by the metagenomic approaches come with particular shortcomings and present new challenges. First, this vast array of viruses must be properly categorized and integrated into the existing classification schemes. Traditional virus taxonomy heavily relies on phenotypic properties such as virion morphology, genome type and replication, while phylogenetic assessments provide a better guide to evolutionary relationships. Currently, genomes of the uncultivated viruses outnumber those from virus isolates many times, where phenotypic data are mostly lacking. To facilitate reporting and comparisons, the minimum information about an uncultivated virus genome standards were developed within the Genomic Standards Consortium framework . These seveloped . For uncInitial clues of the abundance and diversity of the virosphere came from bacteriophages and marine environments ,6. Then,Emerging infections pose a significant global public health threat and challenge biological and economical welfare in both developed and developing countries. Majority of the emerging viruses are of zoonotic origin, transferred between vertebrates and humans . The terHowever, it is a hardly attainable task to predict prospectively the emergence of a particular virus, as virologic and epidemiologic factors with very different dynamics and time scales intermingle in the process . ObviousViruses are often viewed as hijackers, lone-wolf pirates trying to take over the huge cellular machinery to be exploited for self-replication. However, they do have social and private lives, into which we now have the tools to peek. Virus–virus interactions not only affect the diversity and distribution of viruses but shape the way they interact with the hosts, their environment and ultimately, the ecosystem they reside in . MetagenVirus–virus interactions have particular impact and implications for potential emergent viruses transmitted by arthropod vectors. The natural virome of some invertebrate species, including mosquitoes and ticks, prominent vectors of arthropod-borne diseases, are observed to be immense, with highly-abundant RNA viruses in diverse configurations ,9. This in silico analysis tools and machine learning algorithms will intensify and provide crucial insights. As new microbial threats are expected to emerge at an accelerating rate due to the current global conditions, these will be invaluable tools to contain future epidemics and broadly understand natural processes toward a global health.The traditional approach in microbiology tends to isolate components of an environmental niche and tries to provide individual explanations. However, the current accumulation of information requires a more holistic perspective, especially on viruses and their dispersal, spillover and potential emergence. The globally acclaimed ‘One Health’ approach is an effort to establish interdisciplinary collaborations, to better elucidate the underlying mechanisms of many important public health issues, including virus-induced diseases . The nee


Paramyxoviridae family was reported in Malaysia in 1998/1999. Due to its high mortality in humans, its zoonotic nature, the possibility of human-to-human transmission, and the lack of an available vaccine, the World Health Organization (WHO) has recognized it as a global health problem. Depending on strain specificity, neurological symptoms and severe respiratory disorders are observed in NiV infection. In most confirmed cases of NiV epidemics, the appearance of the virus in humans was associated with the presence of various animal species, but generally, bats of Pteropus species are considered the most important natural animal NiV reservoir and vector. Consumption of contaminated food, contact with animals, and “human-to-human” direct contact were identified as NiV transmission routes. Due to the lack of vaccines and drugs with proven effectiveness against NiV, treatment of patients is limited to supportive and prophylactic.Among the diseases that pose a serious threat to public health, those caused by viruses are of great importance. The Nipah virus (NiV) belonging to the  One of the consequences of the ongoing Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic is the increased interest in various aspects related to its origin, virulence mechanisms, potential sources of infection, and modes of transmission. Increasing research to develop an effective epidemic reduction strategy leads to a deeper understanding of the factors that shape its dynamics and the means of controlling it. Paradoxically, the outbreak of a global epidemic may therefore bring beneficial effects in the context of the prevention and monitoring of other infectious agents with epidemiological potential.Viruses are the cause of diseases that pose a serious threat to public health. Marburg, coronaviruses: Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome Coronavirus 1 (SARS-CoV-1), Human Immunodeficiency Virus (HIV), Hendra, Nipah virus (NiV) and those responsible for Crimean-Congo hemorrhagic fever, Lassa fever, Ebola, Influenza A virus subtype H1N1, Asian highly pathogenic avian influenza (HPAI) A(H5N1) virus or Rift Valley Fever (RVF) caused numerous epidemics in recent years. These epidemics were characterized by high morbidity and mortality, mainly in developing countries in Asia, Africa, and South America .The NiV virus, due to its high mortality in humans, its zoonotic nature, the possibility of human-to-human transmission, and the lack of an available vaccine, has been recognized by the World Health Organization (WHO) as a global health problem and included in the list of epidemic threats treated as a priority in research and development activities (R&D Blueprint) . In turnThe study aimed to characterize the NiV virus in the context of its epidemiological potential and to evaluate the effectiveness of previous preventive and intervention measures. Conclusions and observations resulting from such an analysis may constitute suggestions for the development of a scheme of action in the event of an outbreak of an epidemic on a larger than just a local scale.Paramyxoviridae family and the genus Henipavirus. Paramyxoviruses include many known types of pathogenic viruses, such as the measles virus  or mumps virus . In turn, the Henipavirus genus, in addition to NiV, also includes three species that are harmless to humans: Cedar virus, Ghanaian bat virus and Mojiang virus, and the highly pathogenic Hendra virus (HeV) (The name of the NiV derives from the name of the village of Sungai Nipah (Nipah River Village) in Negeri Sembilan state, Malaysia, where the presence of NiV [IgM antibodies in cerebrospinal fluid (CSF) against Hendra viral antigens] was first confirmed in patient serum samples with symptoms of encephalitis in 1999 . NiV firus (HeV) . The patus (HeV)  and Mojius (HeV)  are unknus (HeV) .Morphologically, NiV resembles other paramyxoviruses: it is a pleomorphic, spherical, or thread-like enveloped virus with a size of 40–1900 nm, containing a single layer of surface protrusions with an average length of approximately 17 nm .Nipah virus has single-stranded RNA of negative polarity. RNA viruses, due to their extremely short generation time and faster evolution, show an increased probability of infection of new host species, although they are already considered the main etiological factors responsible for 25–44% of recently emerging infectious diseases . NiV hasNipah virus mainly enters cells through the fusion of the virus with the cell membrane on the plasma membrane. Cellular expression of NiV glycoproteins can induce cell-membrane fusion (syncytium formation) through the interaction of G and F glycoproteins. The fusion of the virus and host cell membranes (which is pH independent) begins with the binding of G to its host receptor (ephrinB2 or ephrinB3) . This isThe first human cases of NiV were reported among pig farmers in September 1998 during an epidemic in Malaysia. Initially, they were mistaken for cases of Japanese encephalitis (JE), but both transmission through direct contact with pigs and its occurrence in adults were not typical of infections caused by this infectious agent . In May Detected in Singapore in March 1999, NiV infections involving the respiratory system and the brain affected slaughterhouse workers who received pigs from Malaysia. Of the 11 cases, 1 was fatal. The median age was 44 years (range 24–66). All patients were men; ten were Chinese, and one Indian . The epiIn Bangladesh, NiV outbreaks occurred almost annually from 2001 to 2013, mainly in the winter months. They were detected in many parts of the country, some of which appeared several times. Mortality during the first epidemic in 2001 was 69%, and in 2013 it increased to 83%. In total, from April 2001 to March 31, 2012, the number of NiV cases was 209, of which 161 (77%) were fatal . Gurley Pteropus medius) samples from the 2018 outbreak in Kerala, India, found that human NiV was 96.15% similar to the Bangladeshi strain, but 99.7–100% similar to bat virus, meaning bats were the source of the epidemic. Recent reports of NiV cases date from 2019, when a single NiV case was confirmed in the Ernakulam district, culminating in a full recovery of the patient  and the NiV Bangladesh (NiV-BD) . The anaPteropus hypomelanus, Pteropus lylei, and Pteropus vampyrus (Megaderma spasma), however, its presence was finally confirmed in fruit bats also of the genus Pteropus, e.g., Pteropus giganteus  from Malaysia, Cambodia, Thailand, and Bangladesh . The lacAlthough bats are the primary natural reservoir for the NiV virus, pigs were the source of human infection during the Malaysian epidemic . They acIn Malaysia, NiV transmission to humans has occurred through close contact with infected pigs . Pig virIn Bangladesh and India, the involvement of pigs as the virus vectors and the impact of these animals on the scale of infection have not been observed . On the Nipah Virus infection has also been found among hospital workers. NiV nosocomial transmission was reported during the outbreak in Siliguri, India . DetectiPteropus genus bats  . At the The high percentage of infections in men observed during various epidemics of NiV may be a direct result of their occupations related to close contact with infected animals or their secretions. In Malaysia, where pigs have been confirmed as the intermediate NiV hosts and contact with them has been the predominant mode of virus transmission to humans, the majority (70%) of infections were in pig farmers and piggery workers . Subsequinter alia, for this reason, pigs were not involved in the outbreaks in Bangladesh  within 3 days, is used to confirm new NiV foci. Samples from which NiV can be isolated are CSF, respiratory swabs , blood, and urine, but all testing procedures must be performed in BSL-4 laboratories .Among serological tests based on the detection of IgM and IgG antibodies, ELISA is a simple and inexpensive test . HoweverDue to the lack of an effective drug against NiV, the management of patients is limited to supportive and prophylactic treatment . The basin vitro antiviral activity of GRFT (Griffithsin) and its synthetic trimeric tandemer (3mG) against NiV and other viruses from four virus families. Researchers demonstrated that a preliminary in vivo evaluation of oxidation-resistant GRFT (Q-GRFT) showed significant protection against a lethal NiV challenge in golden Syrian hamsters. GRFT is a high mannose oligosaccharide-binding lectin that has shown a broad spectrum in vivo activity against viruses  . Yoneda The search for new vaccines is constantly ongoing. A vaccine designed by Vaccinations are an important part of the fight against the epidemic. Continued work on new vaccines against NiV is necessary, especially in view of clinical trials.Due to the limited possibilities of effective treatment and the lack of a vaccine, it seems justified to focus the efforts of scientists and institutions responsible for monitoring epidemiological threats on preventing NiV emergence and their effective supervision.Preventive strategies to limit the appearance of new and the spread of already initiated epidemic outbreaks relate primarily to avoiding direct contact with the hosts of the virus (fruit bats and pigs) and their secretions and avoiding consumption of contaminated food. On the one hand, it is recommended to carefully check and wash the fruit of the trees where bats live. On the other hand, procedures are implemented to limit their access to places and vessels used to collect date palm juice (bamboo skirt method) . It is aImplementation of actions raising public awareness of the risks associated with the outbreak of the virus and the importance of preventive measures is another factor that may help to the risk of the new outbreak. Local television, radio channels, and printed media informed local societies, for example, on the dangers of drinking contaminated date palm juice, recommending that its consumption should be limited . SimilarAn important preventive measure that can effectively reduce the scale of the spread of NiV is to prevent the spread of the virus through direct human-human contact. Washing hands, disinfecting with 70% ethanol, wearing gloves and other protective equipment, and avoiding direct contact with body fluids are standard rules for caring for a person infected or just suspected of having NiV infection . In a ho2 UV-C reduced the infectivity of NiV (≥4.3 log) to the limit of detection (LOD) in platelet concentrates, and treatment with MB (120 J/cm2) reduced NiV (≥2.7 log) to LOD in the plasma.It is important to evaluate the disinfection efficiency against NiV. Both chemical and physical methods. Like other paramyxoviruses, NiV is easily deactivated by soaps, detergents and many disinfectants. Sodium hypochlorite has been recommended for disinfection of pig farms in Malaysia . EickmanTransmission of infections to healthcare professionals can be reduced by ensuring access to hand hygiene facilities, regular and appropriate use of personal protective equipment, and isolating patients with meningitis. It is of particular importance to follow appropriate procedures during funeral ceremonies–wearing gloves and masks, washing hands thoroughly with soap and water immediately after contact with the corpse .In the regions along the NiV belt, surveillance is also carried out to enable early detection of virus outbreaks, analysis of its strains, and monitoring of the relationship between environmental factors and the dynamics of epidemic development. For this purpose, specialized research teams are established to identify suspected cases of infection in humans and analyze the possibility of the emergence of new species of potential reservoir hosts of the virus responsible for its transmission to humans .The situation of the global COVID-19 pandemic, which has resulted in the deaths of nearly 5 million people, encourages scientists to conduct research that will stop the spread of the virus, reduce its effects and help to develop an effective strategy for recognizing, preventing, and managing the emergence of other pathogens with epidemic potential.One of the features indicating the viral pandemic potential is the rapid and effective transmission of the pathogen from person to person, especially in the absence of immunity in exposed people. This route of NiV transmission was confirmed to be extremely important during the epidemics in Bangladesh and India. Infection may occur as a result of direct contact with an infected person or their secretions, and an additional risk is posed by the possibility of virus transmission with saliva particles in patients with severe respiratory symptoms and accompanying cough . Many stThe airborne route is also the main mode of transmission of the SARS-CoV-2 virus, which results in the introduction of general orders to apply appropriate protective measures in the pandemic society .The high mortality characteristic of NiV is a factor that can limit the spread of a virus that does not infect another due to the death of the host. However, it is worth taking into account that the current NiV outbreaks were usually detected in sparsely populated villages . It can Amidst the known and tested strains of NiV so far, differences have been observed in transmission methods, the symptoms type and severity, and finally the size of genomes confirmed by molecular analyses. Indirectly, this indicates the need to include the risk of the emergence of mutations, e.g., facilitating the human-to-human viral transmission, in the assessment of the NiV pandemic potential. Such suggestions were made when sequencing of a NiV genome fragment from a Filipino patient (2014) showed a high degree of similarity to the Malaysian strain. However, while in the case of the epidemic in Malaysia no human-to-human transmission was recorded, this possibility has already been confirmed in the Philippines . RNA virIn addition to human-human contact, NiV infection can occur through contact with animals. The primary reservoir of NiV is bats, which are likely to have also been a source of COVID-19 infections in humans . The detDrawing conclusions from the COVID-19 pandemic, we must be prepared for the fact that any zoonotic virus, especially one with the ability to human-to-human transmission, can be very dangerous and contribute to a global pandemic. An important argument justifying the concerns about the emergence of NiV in the human population is the lack of vaccines and drugs with proven effectiveness. Attempts to develop them do not bring the expected results, and in combination with the problems in the healthcare efficiency revealed during the ongoing COVID-19 epidemic, both in the poorest and highly developed countries, they can result in serious consequences on a global scale .KS, JB-K, and KG-B: conceptualization. KG-B and MZ: formal analysis. KS, JB-K, and NW-K: writing—original draft preparation. KS, KG-B, and MZ: writing—review and editing. ZB, KG-B, and NW-K: visualization. KS and EG-K: supervision. KS and JB-K: project administration. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Dengue infection is caused by an arbovirus with a wide range of presentations, varying from asymptomatic disease to unspecific febrile illness and haemorrhagic syndrome with shock, which can evolve to death. In Brazil, the virus circulates since the 1980s with many introductions of new serotypes, genotypes, and lineages since then. Here we report a fatal case of dengue associated with a Dengue virus (DENV) lineage not detected in the country until now.The patient, a 58-year-old man arrived at the hospital complaining of fever and severe abdominal pain due to intense gallbladder edema, mimicking acute abdomen. After 48 h of hospital admission, he evolved to refractory shock and death. DENV RNA was detected in all tissues collected . Viral sequencing has shown that the virus belongs to serotype 2, American/Asian genotype, in a new clade, which has never been identified in Brazil before. The virus was phylogenetically related to isolates from central America , most likely arriving in Brazil from Puerto Rico.In summary, this was the first fatal documented case with systemic dengue infection associated with the new introduction of Dengue type 2 virus in Brazil during the 2019 outbreak.The online version contains supplementary material available at 10.1186/s12879-021-05959-2. Aedes vectors , HT = 43.3% (RR = 40–52%); peripheral blood total leukocytes = 5800/mm3 were collected from the main vital organs for molecular analysis. On microscopy, the liver presented midzonal hepatitis, with apoptotic and steatotic hepatocytes, Kupffer cell hyperplasia, sinus congestion, and hemorrhage, and mild inflammatory reaction, mainly composed of lymphocyte infiltrate , ascites, bowel and gallbladder edema  during the critical phase of the infection, with warning signs such as serosal effusions, severe abdominal pain, and vomiting, due to plasma leakage, which usually occurs on the 4–6 day of illness . GallblaViral RNA was detected on all collected tissues. This is in line with the previous detection of viral RNA in tissues, such as the kidney, heart, lung, and brain . The conSince its first report in Brazil , the AmeOur results combined with other studies , 31 suggAdditional file 1.


MicroRNAs are extremely promising candidates for early cancer diagnosis and prognosis. The levels of circulating microRNAs provide valuable information about cancer disease at its early stages. However, the levels of microRNAs that need to be detected are extremely low and difficult to discriminate from a large pool of oligonucleotides. There is the need for accurate, rapid and sensitive detection methodologies for detection of microRNAs. We developed electrochemical impedance spectroscopy peptide nucleic acid (PNA)-based sensors that can detect miRNAs in diluted serum with a limit of detection of 0.38 fM. In order to further improve the accuracy and reliability of the sensors, we developed an assay using magnetic beads for simple and rapid fishing of target microRNAs from solution and its pre-concentration prior to electrochemical detection. Our methodology utilizes magnetic beads for the capture of the target microRNA from solution and brings the concentrated sample to the sensor surface. We modify the magnetic beads with locked nucleic acids (LNA), which have high affinity and specificity to their complementary microRNA sequence. The separated and concentrated microRNA is then detected using the PNA-based sensors. By exposing the sensing electrodes only to the captured microRNAs, interferences from other nucleotides or biomolecules from the sample are eliminated. Within the context of cancer disease development and progression, epigenetic alterations are considered as one of the key drivers. Epigenetic alterations are shown to take place far more frequently than genetic mutations and often appear in early stages of tumorigenesis5. The term epigenetic here refers to all heritable changes in gene expression but eliminates the alterations in a DNA sequence. To date, miRNAs remain the most epigenetic alteration in circulation, which is a demanded property for a diagnostic and prognostic biomarker. Moreover, miRNAs are shown to be stable in blood due to their packaging into exosomes that protect them from degradation, while other circulating nucleic acids, such as DNA, are cleared from circulation rapidly.MicroRNAs (miRNAs) are a class of 19–24 nucleotides, noncoding RNAs, which regulate the expression of nearly one third of all human genes8. The levels of miRNAs in circulating blood were discovered to show fluctuations in different cancer states9, making them potential minimally-invasive diagnostic markers for cancer.MiRNAs have been a particular area of interest for oncology research since their discovery and following the evidence suggesting that they are involved in the pathogenesis of cancer, in most cases by controlling the translation of oncogenes and tumor suppressors11. In particular, miR-21 was found to be upregulated in pancreatic cancer patients13. A systematic review and meta-analysis evaluated the prognostic role of miR-21 in patients with pancreatic cancer and found that upregulation is associated with poorer overall, disease-free, and progression-free survival as well as with a higher rate of metastatic lymphnodes and poor differentiated tumors14.In pancreatic cancer, a number of miRNAs have been found to be either up-regulated or down-regulated15. Changes to specific miRNA levels in serum/plasma have been defined as up- or down- regulation between healthy controls and patients with disease state but with little literature stating the actual concentration of miRNA in healthy biological samples and the change of this concentration in disease state. This could be attributed to the lack of standardized protocols for separation of miRNAs, their processing and quantification. Hence, this makes it extremely difficult to carry out quantitative comparisons between the studies reported in literature due to the slightly different conditions of each study. Nonetheless, there are suggestions that miRNAs in blood might need to be detected in the pM to fM ranges. Other challenges are the short sequence length of miRNAs and the high sequence homology within the family of miRNAs. Hence, there is the requirement for highly specific sensors that can distinguish between miRNAs that possess a few bases mismatch.Prior to the implementation of miRNA sensing technology for clinical applications, there are number of technical challenges to be addressed. The most critical one is the low and undefined levels of miRNAs in blood to detect18 and efficient nucleic acid separation19. The adoption of magnetic beads for the electrochemical detection of miRNAs has been exploited in different studies. Bettazzi et al.20 obtained a detection limit of 7 pM for miR-222 using a multi-step approach: functionalization of streptavidin coated magnetic beads with a biotinylated DNA probe that was specific to the target miRNA; extraction of the target miRNAs from the cell samples and their labelling with biotin; hybridization of biotinylated target with the DNA capture probe on the magnetic bead; labelling of resulting biotinylated hybrid with a streptavidin–alkaline phosphatase conjugate; entrapment on to a screen-printed electrode and measurement of the enzymatic product with differential pulse voltammetry. A similar study reported detection limits as low as femtomolar levels for voltammetric detection of miRNA by designing a magnetic beads-based assay on a multi-channel screen-printed array of electrodes21. Several other multi-step studies achieved the creation of highly sensitive miRNA detection platforms26. Some of these studies are summarized on Table 27, mostly based on the amplification of the signal itself rather than on pre-concentration methods.Magnetic beads hold many applications and are a well-known solid support for electrochemical detection28 that leads to the high melting points of LNA-RNA29 and makes LNA well suited for miRNA detection and analysis for cancer diagnosis. Hence, as schematically illustrated in Fig. 31. Hence, such a methodology does not only eliminate the pre-labelling step of target but also benefits from the high affinity properties of LNA-probes for capturing low concentrations of miRNA from solution, and their quantification by highly stable PNA-probes. In essence, our approach relies on the following steps: capture of miRNAs by LNA-modified magnetic beads; move beads to sensor area; release miRNA and detect it with PNA-probe electrochemical sensor.In order to improve the current methodologies in literature, we designed a new assay that combines the high affinity and separation properties of Locked Nucleic Acid (LNA) modified magnetic beads with the high stability of Peptide Nucleic Acid (PNA) probes on gold electrode surfaces in order to develop a magnetic-bead based direct electrochemical detection of miRNAs from solution. Our methodology initially replaces the DNA/RNA capture probe that is adopted in the literature with a probe that has a higher affinity to miRNAs. LNA is known for displaying extraordinary hybridization affinity towards RNA. The enhanced affinity is a result of the modified chemical structure of LNASynthetic biotinylated LNA capture probe was purchased from Eurogentec, UK, in lyophilized form, while HPLC purified synthetic RNA sequences were purchased from Sigma-Aldrich, UK, in lyophilized from. The PNA probe was purchased from Cambridge Research Biochemical, UK. The sequences used are shown on Table All chemicals were purchased from Merck Sigma-Aldrich UK unless otherwise stated. Prior to the assay, Pierce™ Streptavidin Magnetic Beads of 1 µm nominal diameter  were washed with 25 mM Tris-buffered saline containing 0.1% Tween-20 . This was carried out by adding 30 µL (0.3 mg) of magnetic beads into a 1.5 mL microcentrifuge tube that was placed on a magnetic stand to attract the beads to the side of the tube. The supernatant was removed with a pipette and discarded. Then 1 mL of wash buffer  was added to the tube. The tube was inverted several times and a gentle vortex was applied for an even mix. The beads were then collected with a magnetic stand to the side of the tube and the supernatant was removed. The beads were stored in wash buffer until the incubation with the LNA probe was carried out.This was then followed by washing the beads within the immobilization buffer . The tube was inverted several times, and a gentle vortex was applied for an even mix and the beads were collected with the magnetic stand. The supernatant was removed, and the beads were incubated with 300 µL of 1 µM biotinylated LNA-probe solution. 1 µM of biotinylated LNA-probe solution was prepared in 10 mM PB buffer (pH 7.3) prior to immobilization with magnetic beads. The incubation with LNA-probe solution was carried out at room temperature for 1 h with continuous mixing, after which the modified beads were collected by placing the tube on the magnetic stand again and removing the supernatant for further analysis. Then the modified beads were washed with 10 mM PB buffer twice and incubated with 100 µM biotin  in order to block any remaining streptavidin active sites on the probe-functionalized surface. After biotin blocking, the beads were collected with the magnetic stand, and supernatant was removed for further analysis. This was followed by a final wash of the beads in 10 mM PB buffer.32. This was followed by incubating the dry LNA-functionalized magnetic beads with the target miRNA solution for 30 min at room temperature, with continuous mixing for the hybridization to take place. In the end, the beads were collected with the magnetic stand, and the remaining solution was removed for further analysis. The beads that contained the LNA–miRNA hybrids were washed twice in 10 mM PB buffer and then a number of thermal/chemical denaturation methods were studied for the most efficient release of the target miRNA from the magnetic beads surface  and heated at 95 °C in a water bath for 5 min prior to incubation. The heating was carried out in order to prevent the formation of hairpins or secondary structures, as the evidence suggests that miRNAs have a tendency to form both hairpin and homoduplex structures in solutionsee also . FollowiThermal denaturation (release) of the miRNAs from the LNA-modified magnetic beads was performed by heating 300 µL of solution in a water bath at 95 °C for 10 min.Chemical denaturation of the LNA–miRNA duplex was performed with 1 M NaOH. The stock solution of NaOH (10 M) was prepared upon dissolving 4 g of NaOH (Sigma-Aldrich) in 10 mL of Milli-Q water. The NaOH was diluted to 1 M in Milli-Q for the assays. 300 µL of 1 M NaOH were added to the tube containing LNA-functionalized magnetic beads previously hybridized with target miRNA. The mixture was incubated at ambient temperature for 10 min prior to measurements with UV spectrophotometry.34 and these absorbance properties can be adopted for their quantification35. Hence, UV spectrophotometry was run within the region of 190–800 nm, which allows enough room to identify the LNA-probe, initial target miRNA, and the captured target miRNA. Initially, a baseline measurement was carried out by adding 80 µL of buffer solution (10 mM PB) to each of the micro-cells which sets a reference baseline prior to the actual measurements of nucleic acids within the same buffer. Following the baseline measurement, each micro-cell was emptied and re-filled with 80 µL of solutions which contained the nucleic acids prepared in the same buffer (10 mM PB) for the analysis.A Shimadzu UV-1800 with Thermal Melt Analysis System was adopted in order to validate the assay steps. The solutions of LNA-probe and target miRNA prior to their bio-functionalization and hybridization on magnetic beads surface were analyzed by UV-spectrophotometry. This was followed by the analysis of the final solution of target miRNA after its capture on the magnetic beads surface and release. 8-series micro cells with silicone plugs that fit inside the spectrophotometer were adopted in order to measure the samples; this has the benefit of allowing the user to analyze 8 different samples during one measurement. The amount of light that is absorbed by the solution is dependent on the concentration, the path length of the light through the cuvette and how well the analyte absorbs the light at a certain wavelength. It is known that single or double stranded DNA, RNA or LNA oligonucleotides, absorb ultraviolet (UV) light due to the existence of heterocyclic rings of the nucleotides. The absorbance levels are usually observed to be around 260 nm2SO4  by scanning the potential between the oxidation and reduction of gold, 0 V and + 1.5 V vs. an Ag/AgCl reference electrode , for 50 cycles until the voltammogram graphs suggest no further changes. Finally, the electrodes were rinsed with Milli-Q water and dry cleaned under a nitrogen gun.Gold working electrodes  were mechanically polished for 2 min with 1 µM diamond solution  on a polishing pad . This was followed by mechanical polishing using alumina slurry with decreasing particle sizes . Polishing was carried out for at least 2 min in each particle size. In between each polishing step, 5 min of sonication in ethanol and rinsing in Milli-Q water were performed to remove the residues. After the final polishing, 10 min of sonication in ethanol followed by 10 min in Milli-Q water was carried out. Electrodes were then rinsed with Milli-Q water and electrochemically cleaned in 0.5 M HClean electrodes were then co-immobilized with the thiolated ssPNA probe sequence and 6-mercapto-1-hexanol (MCH) in 50% dimethyl sulfoxide (DMSO) 50% ultra-pure water (v/v) immobilization solution for 16 h in a humidity chamber. After immobilization, electrodes were rinsed with excess Milli-Q water to remove any unattached thiols and were backfilled with 1 mM MCH for one hour in order to ensure complete thiol coverage. Electrodes were then rinsed with excess Milli-Q water and placed in the measurement buffer for 2 h to stabilize the SAM.6])3−/4− redox couple over a frequency range from 100 kHz to 100 mHz, with a 10 mV ac voltage superimposed on a dc bias voltage of 0.2 V vs. Ag/AgCl .The experiments were carried out with a PalmSens4 compact potentiostat  and a three-electrode cell system: Ag/AgCl (KCl) reference electrode connected via a salt bridge filled with 10 mM phosphate buffer (pH 7.3), Pt counter electrode , and gold working electrode (2.0 mm diameter). The Faradaic EIS was conducted in 10 mM PB (pH 7.3) measurement buffer containing 10 mM of the ferro/ferricyanide (3−/4− redox couple to monitor the resistance to charge transfer upon target binding events. The first assay adopted the solution of thermally released miRNA from the magnetics beads’ surface for its hybridization on complementary PNA probes. Since high temperatures led to the dissociation of the streptavidin coating from the magnetic beads surface and the release of probe-LNA, no variation of Rct was expected due to the lack of a target in the solution. The second assay eliminated the use of high temperatures and adopted a chemical denaturation method (incubation of magnetic beads containing LNA–miRNA helix in 1 M NaOH) for the release of target from the magnetic beads’ surface. Hence, EIS measurements were expected to display an increased value of Rct due to the presence of captured miRNA, that is complementary to the probe-PNA. Binding of the target to the PNA-probe is expected to increase the resistance to charge flow (Rct).Two assays were then carried out in order to observe the hybridization of the PNA-probe with the target miRNA captured by LNA-modified magnetic beads. The measurements were performed in 10 mM of [Fe(CN)Rct variation of 2.24% upon stabilization, which reflects the formation of a stable PNA-MCH SAM. Initial incubation with the blank buffer showed no significant change in Rct (∆Rct = 3.8%), which demonstrates a strong stability of the probe before its incubation with the target. This was followed by incubation with the solution that was expected to contain the magnetic beads-captured miRNA, free in solution, upon thermal release. However, the EIS results showed almost no variation in Rct,  upon target incubation, as shown in Fig. Rct variation of 32.4 ± 5.4% (average over three independent electrodes) was observed after incubation with the target, which corresponds to successful hybridization. This is significantly higher than the blank buffer incubation that had an Rct variation of 2.18 ± 0.78%. Hence, our methodology of fishing specific miRNAs from solution by use of LNA-modified magnetic beads was proven to successfully separate the target from solution and its direct electrochemical detection on a PNA-immobilized probe.The results in Fig. Rct (32.4 ± 5.4%) upon target incubation. Although further studies using clinical samples from cancer patients and healthy individuals known to express high or low amounts of miR-21 are required to ensure that the presence of miRNAs competitors does not inhibit the sensitivity and specificity of the method, initial controls with a non-specific miRNA sequence yielded a negligible signal, suggesting the applicability of the methodology. We believe such a model for detection is a crucial step towards the easy fishing of miRNAs from complex solutions, such as blood, and pre-concentration of the target prior to its direct electrochemical detection for quantification.In this work, we developed electrochemical impedance biosensors that can detect the pancreatic cancer biomarker miR-21-5p in diluted serum using PNA-modified biosensors, with a limit of detection of 0.38 fM. In order to further improve the accuracy and sensitivity of the sensors, a new method using magnetic beads was developed to separate and pre-concentrate the target miRNAs from solution. By optimizing the various assay steps, our improved methodology offers direct detection of target miRNAs with no need for pre-labelling of target sequences or enzymatic labelling on the detection probe. It is crucial to develop technologies that provide accurate results without the requirement for many amplification steps. Although the literature offers various magnetic beads-based detection methodologies with great limits of detection, there is still the need for the elimination of a number of the assay steps so that such technologies can be translated into practical point-of-care tests. For this purpose, we have investigated the feasibility of a methodology that eliminates the need for sample preparation, hence the requirement for trained labor, or for additional signal enhancement steps. Additionally, an important and neglected aspect of miRNA detection is distinguishing between the pool of miRNA sequences which can lead to false positives in the results of patients. For this purpose, the LNA-probes that were utilized have a special affinity for complementary miRNAs and offer improved hybridization efficiencies. Such a property of LNA-probes was combined with the separation properties of magnetic beads so that LNA favored capturing of target miRNA from solution was achieved. Following the capture of the target, we have carried out chemical denaturation of the hybridized LNA–miRNA helix for the release of the miRNA from the magnetic beads’ surface. PNA-immobilized gold electrodes were used for the EIS based detection of miRNA, which demonstrates a significant variation in Supplementary Information.


Animal welfare scientists have accumulated knowledge and developed interventions to improve livestock welfare, but these are poorly adopted in commercial practice. Animal welfare interventions are rarely tested for economic viability and this limits their uptake. This study employs Stochastic Partial Budgeting (SPB) to determine the viability of animal welfare improvements. Aggression between pigs is used as an example because there is a large literature base from which to draw interventions, and the problem has persisted for decades without resolution. Costs and benefits of three interventions to control aggression  were estimated by reviewing the academic and industry literature and by conducting a survey of sixteen pig farmers. The net effects were compared to farmers’ willingness to pay (WTP) for interventions to reduce aggression as identified by recent research. Results are consistent with prior research which indicates that improving animal welfare generally comes at a cost to producers. Nevertheless, pre-weaning socialisation resulted in a neutral or positive net effect 38% of the time and should be central to campaigns promoting the control of aggression in the industry. Exposing pigs to synthetic maternal pheromones did not improve profitability but the net costs were small and within the realms of WTP for a sub-group of farmers with animal welfare goals. The net costs of converting existing buildings in order to house pigs in large social groups were beyond the realms of farmers’ WTP. The approach adopted in this study, of combining SPB with WTP from the sector, should be extended to other animal welfare issues. Animal welfare scientists continue to accumulate useful knowledge identifying specific changes to animal management, nutrition and genetics which can improve the welfare of animals in agriculture. However, these interventions are poorly adopted in commercial practice e.g. , 2). In . In 2]).Although certain animal welfare standards are required to ensure productivity , in other cases measures to promote animal welfare can be more expensive than the returns gained –6. FarmeDespite general knowledge that for farmers the cost price of production is important, only a handful of studies have modelled the economic feasibility of animal welfare interventions. Specifically, researchers have estimated the economic feasibility of animal welfare improvements in broiler , 14 and This study utilises Stochastic Partial Budgetting (SPB) in order to determine the viability of animal welfare improvements. Furthermore, the net effects were compared to farmers’ willingness to pay (WTP) as identified by prior research . AggressThis study aims to determine the viability of the most promising aggression control strategies. The economic consequences of investing in three aggression control strategies are estimated. The choice of aggression control strategies included in this study is justified in the section ‘Pig management scenarios’. Cost and benefits of the interventions were estimated relative to a control situation . A key risk when estimating the costs and benefits of interventions is their over/underestimation. In order to minimise this risk a combined approach was employed whereby the costs and benefits of each pig management scenario were estimated by conducting a survey of pig farmers to gain information on the finances in practice (see section ‘Estimation of costs and benefits: farmer survey’); by reviewing the academic and industry literature (see section ‘Estimation of costs and benefits: the literature’); and by conducting sensitivity analysis using SPB . The survey was not used to facilitate the estimation of costs associated with exposing pigs to synthetic maternal pheromones as these were estimated using commercially available product information. The final SPB model is presented in Section ‘Results’.Aggression control strategies were chosen by employing a stepwise selection process which: 1) identified only effective aggression control strategies as indicated by the greatest consensus in the literature that they effectively reduce aggression  Pre-weaning socialisation of piglets. This is the most studied aggression control strategy and there is strong evidence that this social experience results in reduced aggression at weaning [ weaning  and in t weaning  under bo weaning  and comm weaning . Specifi weaning , and rem weaning , 34, 38.Housing pigs in relatively large social groups. This strategy results in reduced aggressive behaviour [ehaviour –41 likelehaviour . Thus, tehaviour –41. The ehaviour , 43. Nevehaviour , and mucehaviour , 41. Thiehaviour . FurtherExposing pigs to synthetic maternal pheromones. This strategy reduces the frequency of fights at mixing in breeding sows [ing sows , adult ping sows , and weaing sows  and comming sows . Syntheting sows . The numing sows . The pheSixteen farmers completed the paper based survey during January 2019 after piloting with two researchers and one pig farmer. Informed consent was obtained for all participants and the survey received ethical approval from the Human Ethical Review Committee at the University of Edinburgh. All responses were confidential and could not be linked to any individual. No controversial questions were asked and there were no possibilities for causing distress or embarrassment to participants. The full survey is available in Farmers (n = 16) completed the survey during one farmer discussion group (n = 8) or through contact with colleagues at Scotland’s Rural College whilst on farm visits (n = 8). Farmers were given no prior notice that they would be asked to participate in this study; therefore bias was unlikely to be introduced due to interest in the study topic. The response rate was 100%. The target population was UK pig farmers who kept pigs at the growing and/or finishing stages of production. We chose to recruit participants face-to-face with a paper-based survey in order to maintain quality of responses and due to poor response rates of farmers to previous online surveys , 50. FarIn order to estimate the costs of allowing pairs of litters to mix prior to weaning and changing existing structures in order to house pigs in large social groups (100+ pigs), farmers were asked to list the changes needed on their unit in order to implement both strategies and to estimate the monetary value of initial investment and on-going costs. Farmers were instructed to answer in hindsight if relevant. For example, those who already mix pairs of litters prior to weaning were asked to list the costs that they encountered when they made the change. If this had always been their method of production, they were asked to leave the question blank. In order to ensure that responses were interpreted correctly, farmers were asked to make it clear how costs were described . In order to make farmers’ responses comparable, all values reported were converted to reflect the costs ‘per pig produced’. Detailed information on how farmers’ responses were converted into the ‘per pig produced’ scale are available in the ‘and on-going costs were included in these analyses. For allowing litters to mix prior to weaning, n = 8 farmers fully answered the questions; for housing pigs in large social groups, n = 6 farmers fully answered the questions.The total mean costs of implementing each pig management scenario as estimated by farmers can be seen in In order to facilitate the estimation of monetary benefits, farmers were asked to indicate which positive outcomes, from a list provided, that they would expect to encounter if they saw a 50% reduction in aggression on their farm. The results are presented in A wide range of key performance indicators have been investigated in the academic and industry literature see . StudiesThe literature indicated that cross-suckling and heightened mortality were unlikely to occur when allowing pairs of litters to socialise prior to weaning . AlthougFor housing pigs in large social groups (100+), the monetary costs as estimated by farmers were included in the economic model . However2, and the diffusers will release the pheromone for up to 6 weeks following opening. A further penalty was added in order to account for the increased labour required to install diffusers into pens  is a financial tool that can support decision makers to assess the financial consequences of specific interventions, especially when detailed empirical data are unavailable. In particular, PB compares the benefits and costs resulting from implementing the proposed interventions, with respect to the current practice. Monetary units are used to quantify the costs and benefits and to provide a measure of the net benefit. The outcomes of different interventions can be compared to identify the most beneficial and, thus, the best allocation of limited resources . PB has The PB uses a single-point value for each cost or benefit element. This renders the analysis deterministic and likely to be inaccurate due to the fact that costs and benefits of an animal welfare improvement vary largely across farms . To account for the stochastic nature of the input variables for the partial budgeting model , this study uses the stochastic partial budgeting (SPB) approach instead of the PB. In addition to estimating the average net effect of a change using single-point values (i.e. mean), the SPB allows the analyst to use a range of the estimated costs and revenues  and attach to them probabilities of occurrence. A combination of the estimated values of costs and revenues and their probability distributions are used to determine the range and probability of the change’s final possible outcomes , 67. ForThe SPB was carried out as follows. First, the negative and the positive effect of implementing each of the three animal welfare interventions were computed based on the information displayed in Tables Second, a stochastical analysis was carried out to account for the uncertainty associated with the estimated costs and revenues of the three interventions. The uncertainties around the estimated negative and positive effects were modelled by fitting triangular probability distributions. The triangular distributions are commonly used in stochastic analysis when the distribution of the estimated effects is difficult to determine, and the analyst only has two data points  for each estimated effect.Third, Monte Carlo simulations were used to generate cumulative distributions of the net effect associated with each of the three animal welfare interventions. In particular, the Monte Carlo simulations were used repeatedly to draw 500 random samples from the probability distributions of the net effects. Finally, the cumulative distributions functions were illustrated as graphical forms see Figs –3. In thResults revealed that the strategy most likely to be economically viable for farmers was allowing litters to mix prior to weaning . When implementing this strategy, the cumulative distribution function (CDF) line intersects the y-axis at approximately the 62% level . This inAnimal welfare scientists have identified many changes to animal management, nutrition and genetics which can improve the welfare of livestock, but these interventions are rarely adopted in commercial practice. The economic consequences of animal welfare interventions are a primary determinant of farmers’ willingness to change their current practice –12. DespThe financial consequences of three aggression control strategies for use when regrouping unfamiliar growing / finishing pigs were estimated, to determine whether they are good investments for farmers. Results are consistent with prior research which indicates that improving animal welfare generally comes at a cost to producers , 13–18. Nevertheless, allowing litters to mix prior to weaning  resulted in a neutral or positive net effect 38% of the time, with a maximum net benefit of £0.22 per pig produced. For the majority of surveyed farmers, implementing this strategy required only small labour costs associated with the removal of barriers between adjacent farrowing pens. Pre-weaning socialisation is also the most studied and promising aggression control strategy identified by the academic literature . EvidencA variety of factors influence farmers’ willingness to improve animal welfare in practice  whereby as many as 45% of 122 surveyed pig farmers reported using large social groups to reduce aggression, and they found them to be moderately useful. The monetary consequences of slowed growth rates were estimated based on the assumption that farmers would increase group size from 50 pigs per pen to 100 pigs. However, large variation in group size, and thus associated costs, exist in the industry. This is illustrated by the finding that the sixteen farmers surveyed in the current study housed growing / finishing pigs in groups of 64 on average  but this ranged from 15–400. Furthermore, in the study of Peden et al. (2019b) farmers kept growing / finishing pigs in groups of 85.6 on average  but this ranged from 12–600. The costs of increasing group size rely heavily on initial and target group size; therefore advice should be tailored on an individual farm basis using the methodology described in the current study. Furthermore, although the costs are not justified if converting existing buildings, it is likely that the cost / benefit ratio will be different when building new sheds. When building new sheds, large social groups would be associated with substantial reductions in construction costs related to buying fewer pen divisions and water and feed equipment. Large social groups also allow for automatic sorting technology and other precision livestock developments that enable better individual nutrition and care . TherefoFarmers’ costs of production vary widely with feed prices, seasons and trading cycles . Pig farThe aggression control strategies investigated in the current study were selected based upon published evidence of their effectiveness and the possibility to implement them being in the farmers’ control, which is not the case for strategies that require alternative diet formulations or breeding strategies. Only three aggression control strategies met this criterion. This illustrates that, although many aggression control strategies have been identified by research, very few can be practically implemented and managed by farmers under commercial conditions. Therefore, researchers should concentrate on developing practical solutions. EU legislation requires the control of aggression but does not specify what management interventions are needed to achieve this ; all thrThis study provides a valuable initial exploration into the costs and benefits of aggression control strategies. However, an important limitation of the current study is that many of the estimates regarding costs and benefits were based on assumptions which contain a high degree of uncertainty. This is due to the limited sample size of farmers included in this survey; the limited availability of monetary cost and benefit data; the large variation in farm size and structure observed in the industry; and the costs and benefits experienced by farmers varying widely over time and country. For example, it was assumed that the lifetime of structural investments would be ten years. However, in reality, the lifetime of structural changes will vary widely depending on the type and quality of construction. Nevertheless, a major strength of the current research is the collaboration with industry stakeholders at all stages. Farmers’ knowledge and experience of pig management mean that the estimations included in the current study are more accurate than could have been estimated by researchers without surveying farmers. Nevertheless, in depth interviews to replace or complement surveys would have been valuable and should be considered in future similar research. Furthermore, uncertainty was accounted for by considering the ‘optimistic’ and ‘pessimistic’ scenarios, and this should be conducted in future economic analysis of welfare interventions.Farmers who participated were all based in Scotland and it is likely that their estimations are not representative of farmers in other countries . NeverthWhen estimating the monetary benefits of reducing aggression, a linear relationship between financial benefits and the extent to which aggression is reduced was assumed. Specifically, farmers’ estimated the monetary benefits of an ‘optimistic’ reduction in aggression of 50%, and their response was scaled in order to account for the ‘most likely’ scenario of a 25% reduction. The linearity of this relationship has never been studied. Nevertheless, it is not expected that farmers would be able to sensitively take into account any non-linearity in their monetary estimations should it exist.Farmers are heterogeneous in their willingness to pay for animal welfare, and their motivations for doing so . A majorThis research did not consider the role of consumers. Under animal welfare and organic quality assurance schemes, the additional costs of certain animal welfare improvements can be passed to consumers through product labelling , 75. ForFinally, when using Monte Carlo simulation it is important to account for correlations between parameters . HoweverThe economic feasibility of animal welfare improvements have rarely been estimated despite being fundamental in determining their adoption in practice. This study conducted stochastic partial budgeting of strategies to reduce aggression between pigs, in order to identify economically viable solutions. Results revealed that pre-weaning socialisation is the most economically viable aggression control strategy. The strategy resulted in a neutral or positive net effect 38% of the time. When negative net effects did arise, they were small and within the realms of willingness to pay for a sub-group of farmers with animal welfare goals. Exposing pigs to synthetic maternal pheromones did not improve profitability; nevertheless, the economic costs were also small and within the realms of willingness to pay for a sub-group of farmers with animal welfare goals. The costs of converting existing buildings in order to house pigs in large social groups were high and beyond the realms of farmers’ WTP. The approach adopted in this study, of combining stochastic partial budgeting with willingness to pay from the sector, should be extended to other animal welfare issues.S1 FileThe survey completed by farmers.(PDF)Click here for additional data file.S2 FileDetailed information on how farmers’ cost estimations were converted into the ‘per pig produced’ scale.(DOCX)Click here for additional data file.S3 FileDetailed information on how the costs of lowered growth performance with increasing group size were calculated.(DOCX)Click here for additional data file.S4 FileDetailed information on how the costs of synthetic maternal pheromones were calculated.(DOCX)Click here for additional data file.S5 FileDetailed information on how the benefits of large social groups were calculated.(DOCX)Click here for additional data file.S6 FileDetailed information on how farmers’ willingness to pay was converted to reflect ‘per pig produced’.(DOCX)Click here for additional data file.


Tobacco bushy top disease (TBTD) is a devastating tobacco disease in the southwestern region of China. TBTD in the Yunnan Province is often caused by co-infections of several plant viruses: tobacco bushy top virus (TBTV), tobacco vein distorting virus (TVDV), tobacco bushy top virus satellite RNA (TBTVsatRNA) and tobacco vein distorting virus-associated RNA (TVDVaRNA). Through this study, two new poleroviruses were identified in two TBTD symptomatic tobacco plants and these two novel viruses are tentatively named as tobacco polerovirus 1 (TPV1) and tobacco polerovirus 2 (TPV2), respectively. Analyses of 244 tobacco samples collected from tobacco fields in the Yunnan Province through RT-PCR showed that a total of 80 samples were infected with TPV1 and/or TPV2, and the infection rates of TPV1 and TPV2 were 8.61% and 29.51%, respectively. Thirty-three TPV1 and/or TPV2-infected tobacco samples were selected for further test for TBTV, TVDV, TBTVsatRNA and TVDVaRNA infections. The results showed that many TPV1 and/or TPV2-infected plants were also infected with two or more other assayed viruses. In this study, we also surveyed TBTV, TVDV, TBTVsatRNA and TVDVaRNA infections in a total of 1713 leaf samples collected from field plants belonging to 29 plant species in 13 plant families and from 11 provinces/autonomous regions in China. TVDV had the highest infection rates of 37.5%, while TVDVaRNA, TBTV and TBTVsatRNA were found to be at 23.0%, 12.4% and 8.1%, respectively. In addition, TVDV, TBTV, TBTVsatRNA and TVDVaRNA were firstly detected of co-infection on 10 plants such as broad bean, pea, oilseed rape, pumpkin, tomato, crofton weed etc., and 1 to 4 of the TBTD causal agents were present in the samples collected from Guizhou, Hainan, Henan, Liaoning, Inner mongolia and Tibet autonomous regions. The results indicated that TBTD causal agents are expanding its host range and posing a risk to other crop in the field. It was reported that the TBTD in China was caused by tobacco bushy top virus (TBTV)2, tobacco vein distorting virus (TVDV)3, tobacco bushy top virus satellite RNA (TBTVsatRNA)4 and tobacco vein distorting virus associated RNA (TVDVaRNA)5. The occurrence of TBTD in Yunnan Province, China, was first recorded in 19936. Later, TBTD was found in multiple tobacco growing regions such as Chuxiong, Baoshan and Dali in Yunnan Province7. The overall incidences of naturally infected tobaco plants were 91%, 32.5%, 35.7%, 100%, 42.5% and 100% in 1993 to 1998, respectively. The total infected crop was approximately 51,300 ha and harvest losses were estimated to exceed US$ 33 million till 20017. The devastating disease has become a limiting factor in the production of tobacco in the province.Tobacco bushy top disease (TBTD) is one of the important tobacco diseases and is caused by multiple causal agents. TBTD was first reported in Zimbabwe, based on its disease symptoms and vector transmission way8. In other two studies, TBTVsatRNA and TVDVaRNA were identified in tobacco plants with TBTD symptoms in China through double stranded RNA (dsRNA) analysis and sequencing5. Tobacco bushy top virus is a member in the genus Umbravirus, family Tombusviridae and Tobacco vein distorting virus is a member in the genus Polerovirus, family Solemoviridae9. In a recent study, TBTD was found in several tobacco species in Ethiopia and these diseased plants were infected with Ethiopian tobacco bushy top virus, a new member of genus Umbravirus, Potato leafroll virus, a member in the genus Polerovirus, family Solemoviridae, and an Ethiopian satellite RNA (ETBTVsatRNA)10. To date, how TBTV, TVDV, TVDVaRNA and TBTVsatRNA co-infecttobacco and other natural plants in fields and whether TBTD plants have other unidentified viruses are remain unknown.In 2002, TBTV and TVDV were found in diseased tobacco plants and aphid vectors through RT-PCR using degenerate primers for umbraviruses or polerovirusesUmbravirus) are a group of positive-sense single-stranded RNA viruses. Umbravirus RNA does not have a 5′ cap and a 3′ poly(A) tail, and does not encode a capsid protein (CP)9, and thus doesn’t form typical virion structure. For successful aphid transmission, umbraviruses need the presence of their helper viruses, mostly a polerovirus or a enamovirus. For instance, aphid transmission of groundnut rosette virus (GRV) depends on the presence of groundnut rosette assistor virus  and groundnut rosette virus satellite RNA (GRVsatRNA)11. Aphid transmission of pea enation mosaic virus 2 (PEMV-2) requires the presence of pea enation mosaic virus 1 13.Umbraviruses (members in genus Polerovirus) are also positive-sense single-stranded RNA viruses. The genome size of poleroviruses range from 5.6 to 6.0 kilobases (kb). Polerovirus RNA does not have a Poly(A) tail and a tRNA-like structure at its 3′ end. Polerovirus, Polemovirus, Sobemovirus and Enamovirus are the four genera in the family Solemoviridae, there were 5 (Enamovirus), 20 (Sobemovirus), 1 (Polemovirus), 26 (Polerovirus) species in these four genera. Polerovirus genomic RNA contains 7 ORFs. Three ORFs  are translated from the 5′ half of the genomic RNA while ORF3a, ORF3, ORF4 and ORF3-ORF5 are expressed from the subgenomic RNA transcribed from the 3′ half of the genomic RNA. Most poleroviruses have limited natural host range, mainly in family Solanaceae, and some in family Amaranthaceae and family Cruciferae14. While some poleroviruses have a broad host range, for example, beet western yellows virus (BWYV) infects more than 150 plant species in over 20 families9.Poleroviruses  and tobacco polerovirus 2 (TPV2). We then tested 1713 samples from 29 plant species in 11 provinces/autonomous regions in China by RT-PCR using primers specific for TBTV, TVDV, TVDVaRNA or TBTVsatRNA. The results presented in this paper should allow us to better understand TBTD, and the potential risk of TBTD outbreak in many crop species. These knowledge should benefit the development of an effective management strategy for this diseases.A recent study showed that many plants without typical TBTD symptoms were also infected with one to three TBTD causal agents, while the tobacco plants infected with all the four causal agents didTwo tobacco plants (referred to as YBSh and YKMPL) showing TBTD symptoms were collected from two different tobacco planting fields in Baoshan and Kunming Cities, Yunnan Province, China in 2015 and 2016 respectively, and were stored in insect-proof greenhouse. To survey the occurrence of TBTV, TVDV, TBTVsatRNA and TVDVaRNA in fields, 1550 virus-like leaf samples were collected from plants belonging to 29 species in 13 families in Yunnan Province from 2013 to 2018. And 65 pepper plants with virus-like symptoms were collected from Guizhou, Liaoning, Henan, Hainan, Shandong, Zhejiang, Hubei Province and Tibet, Inner Mongolia Autonomous Region, China, and 83 tomato plants with virus-like symptoms were collected from Liaoning, Henan, Hainan, Shandong, Shaanxi, Zhejiang Province, and Tibet, Inner Mongolia Autonomous Region. In addition, 11 crofton weed leaf samples were also collected from Guizhou, 1 purple perilla and 3 dahlia were collected from Liaoning  for High throughput sequencing (HTS) RNA-Seq sequencing after depletion of the rRNAs with Epicentre Ribo-ZeroTM kit, which was then sequenced using the Illumina HiSeq X-ten platform with PE150 bp . Sequence data were analyzed using CLC Genomic Workbench 9.5  as describedThe sequence gaps between the aligned contigs were filled by RT-PCR using virus-specific primers. The 5′- and 3′-end sequences of TBTV, TVDV, TBTVsatRNA and TVDVaRNA were determined by the rapid amplification of cDNA ends (RACE) technique using SMARTer RACE 5′/3′ Kit .https://www.ncbi.nlm.nih.gov/orffinder/) was used to predict their ORFs. Pairwise comparisons were performed using the EMBOSS Needle Pairwise Sequence Alignment software available at the http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html. Phylogenetic relationship between the two new viruses and the other known poleroviruses was determined by the MEGA 5.0 software. The sequences were all linearized at the start of the RdRp gene and then aligned using MEGA 5.0. The alignments were used to infer Neighbor joining trees in MEGA 5.0 with P-distance model and 1000 bootstrap replicates as described20.The genomic sequences of the viruses were assembled using the DNASTAR 7.0 package , and then submitted to the GenBank database in NCBI. To characterize the two newly identified viruses, ORF finder software  for reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR reactions were performed using specific primers based on the two new poleroviruses sequences . All samples were stored in the Virus Laboratory .All the protocols involving plant adhered to relevant ethical guidelines.Nicotiana tobacum) in field include small leaves, irregular necrotic lesions on leaves, yellowing or chlorosis, internode shortening and stunting  as described15. The results showed that the PCR products representing the six viruses were indeed present in the YBSh and YKMPL samples, but not in the sample from healthy plant . Based on the results of sequence alignment, we tentatively named these two new poleroviruses as tobacco polerovirus 1 (TPV1) and tobacco polerovirus 2 (TPV2). To validate the reliability of HTS results, the total RNA samples isolated from the YBSh, YKMPL samples as well as healthy tobacco sample were analyzed by RT-PCR. TPV1 and TPV2 detection primers were designed according to the virus contigs identified through HTS, while the primers and multiplex one-step RT-PCR used to detect TBTV, TBTVsatRNA, TVDV, TVDVaRNA were described previouslyt Figure A–C.To obtain the full-length genome sequences of TBTV, TVDV, TVDVaRNA and TBTVsatRNA of YBSh and YKMPL isolates, overlapping amplicons cloning strategy was used in a series of sequential RT-PCR with virus specific primers designed according to the virus sequences from HTS. The primers, amplification strategies (position in the virus genomes), size of the amplicons, and specify chemistry for sequencing were add to the supplementary materials Fig. . At leashttps://www.ncbi.nlm.nih.gov/orffinder). The genomic organization and structure of TPV1 and TPV2 is typical of poleroviruses when comparing with PLRV, and both TPV1 and TPV2 contain seven ORFs: ORF0, ORF1, ORF1-ORF2, ORF3a, ORF3, ORF4 and ORF3-ORF5 , TPV1-YKMPL (GenBank accession number: MW579553) and TPV2-YBSh (GenBank accession number: MW579554), TPV2-YKMPL, (GenBank accession number: MW579555) were confirmed to be 5722nt, 5725nt and 5907nt, 5912nt, respectively, by series of sequential RT-PCR and SMARTer®RACE 5′/3′ kit  with virus specific primers based on the HTS data followed by Sanger sequencing. Pairwise comparison results of the nearly complete sequences showed that TPV1-YBSh shared 99.7% nt identity with TPV1-YKMPL, and TPV2-YBSh shared 98.9% nt identity with TPV2-YKMPL, respectively. The genomic sequences of TPV1-YBSh and TPV2-YBSh, therefore, were used in the subsequent sequence analysis. The genomic nucleotide sequence identity between TPV1 and TPV2 is 54.2%, suggesting they are two distinct species. Blast search results indicated that TPV1 and TPV2 had the highest nt sequence identity with the known poleroviruses. The genome structures of TPV1 and TPV2 were predicted using the ORFfinder software  to 79.1%  among the 19 poleroviruses , 67.0% (ORF1), 71.7% (ORF1-ORF2), 94.1% (ORF3), 88% (ORF4) and 77.7% (ORF3-ORF5) with other poleroviruses. In Table 9, suggesting that TPV2 could be a distinct member in genus Polerovirus.TPV2 shared the nt sequence identity from 50.2%  to 70.4%  among the 19 poleroviruses  and 59 samples were single infected with TPV2 (24.18%) Table . In addiThen 33 TPV1, TPV2, or TPV1 + TPV2 infecting samples were selected and tested for TBTV, TVDV, TBTVsatRNA and TVDVaRNA infections by RT-PCR with virus specific primers. The results showed that TPV1 and TPV2 always co-infected field plants with two to four TBTD casual viruses Table . For exaAmorphophallus konjac), Araliaceae (Sanqi), Dioscoreaceae (Yam), Liliaceae (Garlic) and Amaranthaceae (Alligator weed) were infected with at least one of the four assayed viruses  are associated with this disease in field are still unknown. With the development of HTS and bioinformatics in recent years, discovery of new TBTD causal virus(es) or TBTD associated causal agents becomes possible22. In virus infections plants, large numbers of virus-derived siRNAs (vsiRNAs) will be generated along with the viral genomic RNAs, and these vsiRNAs can be identified and assembled into virus contigs or even full-length viral genome25. This technology can also help us to identify new virus(es) associated with TBTD.Several viral agents have been reported to cause TBTD in some countries. For example, an early study had suggested that TBTD in Zimbabwe was caused by a co-infection of TVDV and TBTVPolerovirus. Sequence alignment result also showed that TPV2 shares the highest nucleotide sequence identity of 70.4% with TVDV. The predicted amino acid sequences of TPV2 proteins, except P3, share less than 90% identities with that of viruses in the family Solemoviridae. Therefor, we conclude that TPV1 and TPV2 are two novel poleroviruses.In this study, HTS was used to analyze two tobacco samples showing typical TBTD-like symptoms in two different locations. Based on the assembled sequences, we have determined two new near full-length polerovirus  sequences. Sequence alignment result showed that TPV1 shares the highest nucleotide sequence identity with TV2 (79.1%). The deduced amino acid sequences of the TPV1 P4 protein and the read-through protein (P3-P5) share less than 90% identities with that of viruses in the genus 10. Recent study showed that ETBTV can complete its vector transmission assist by cowpea polerovirus 1 (genus Polerovirus) besides PLRV26, and the results of our group also proved that TBTV can complete its aphid transmission with the assistance of barley yellow dwarf virus GAV . It can be inferred that there may be other polerovirus could assist TBTV acomplish its aphid transmission. Our survey results also revealed that 0.35% and 0.88% field samples infected TBTV or TBTV + TBTVsatRNA do not coinfected with TVDV (Table Ethiopian tobacco bushy top disease symptoms are similar to that of TBTD in China, and is also caused by several different polerovirus and umbravirusDV Table . That me18. In this study, we have determined that except tobacco, a total of 21 plant species in 12 families can be infected with at least one of the TBTD causal viruses. In addition, we have found TVDV + TVDVaRNA + TBTV + TBTVsatRNA co-infection in crofton weed in Guizhou Province, tomato plants in Hainan Province, and tobacco, sticktight, broad bean, pea, oilseed rape, pumpkin, tomato as well as black nightshade plants in Yunnan Province, respectively. This new finding indicates that the TBTD causal viruses can infect weeds and many other cash crops, resulting in a large virus reservoir in nature. It is noteworthy that many plants co-infected with these four causal viruses did not show typical TBTD-like symptoms. Taken together, we conclude that the causal agents of TBTD are a group of different viruses, these viruses have much broader host ranges and distributions than what have been reported, and these infected plants can serve as the key overwintering or intermediate hosts for the six causal viruses. These three points should be considered when developing an effective control strategy for TBTD in fields.Earlier studies on TBTD were done mainly in tobacco plants in Yunnan Province, China. In recent years, several reports have indicated that TVDV and TBTV can also infect other plant species, including pepper, Dahlia, tomato and crfton weedSupplementary Information 1.Supplementary Figures.Supplementary Tables.Supplementary Information 2.