Recombination is a fundamental evolutionary force, but it is difficult to quantify because the effect of a recombination event on patterns of variation in a sample of genetic data can be hard to discern. Estimators for the recombination rate, which are usually based on the idea of integrating over the unobserved possible evolutionary histories of a sample, can therefore be noisy. Here we consider a related question: how would an estimator behave if the evolutionary history actually was observed? This would offer an upper bound on the performance of estimators used in practice. In this paper we derive an expression for the maximum likelihood estimator for the recombination rate based on a continuously observed, multi-locus, Wright–Fisher diffusion of haplotype frequencies, complementing existing work for an estimator of selection. We show that, contrary to selection, the estimator has unusual properties because the observed information matrix can explode in finite time whereupon the recombination parameter is learned without error. We also show that the recombination estimator is robust to the presence of selection in the sense that incorporating selection into the model leaves the estimator unchanged. We study the properties of the estimator by simulation and show that its distribution can be quite sensitive to the underlying mutation rates. Wright–Fisher diffusion with recombination   is non-singular, then the quadratic form in the exponent of the normal density of T, we recover the quadratic form appearing in (V(x)| in the diffusion, since we assume that the likelihood is with respect to a parametric form only for V would be trivial in this setting, since V is identifiable from the path via its quadratic variation.) See Basawa and Prakasa Rao  is singular since X, whose infinitesimal covariance matrix The matrix Assume  holds foX to take values in We consider the diffusion tituting  yields  aThe observed information matrix  is a keypression  for \docAs a check on the expression for entclass1pt{minimamentclasspt{minimaK possible alleles at the first locus, locus A, and L possible alleles at the second, locus B. The haplotype of an individual is denoted KL possible haplotypes in some agreed order into a vector of length We now turn to our main result, an expression for the MLE for the recombination parameter The model is completed by specifying the drift. Here it is of the formc(x) is immaterial, but for concreteness we will setk, j) then it mutates to haplotype  with probability In much of what follows the choice for Note the separate roles for the two components of the drift: here it is only Using the results of Sect. X solves a deterministic model. Setting Is nt}ρ^ in  a reasontly into  again shj):not mean that the estimator is consistent only in the stochastic setting. We have just seen that We can further describe the evolution of (summing  over j):ution to  is then2It is possible to repeat these calculations for a model with X(t) as the solution to a stochastic differential equation (SDE):W is a We can find an expression for the error associated with V. In the case of the covariance matrix of the Wright–Fisher diffusion, the Cholesky decomposition is given analytically by Sato  lies in the interior of Suppose that i, j, (i)X(t) lies in For some (ii)(iii)The integral Condition (iii) extracts the explosion of T.It is clear from the form of nt}IT in  that \doW, wheremutation only. More precisely we will compare P so that Z is accessible.  TracinThe conditions given in Theorem It is possible to use 0. Using , the appTo account for the possibility that It is possible to extend the above results to a general multi-locus model. The extension is straightforward and we omit many of the lengthy but straightforward calculations.j, haplotypes are of the form j and Y is In a multi-locus model of iℓ).From  and 14)\documented as in .Methods for inference of recombination can be confounded by natural selection  and , relative to other individuals, is parametrised by First consider the following: Suppose that, unknown to the investigator, the two loci are under selection—possibly a complicated type with epistatic interaction. What effect does this have on our estimator for ik at locus A, and similarly for From Eq. , \documed adjust  by definentclass1pt{minimac(x) to the drift leave the estimator (If (robust to the contribution of c(x). In the context of the Wright–Fisher diffusion we have the following result.Returning to the general inference problem of Sect. ed? From , its conrs as in , this rec(x) if and only ifFor a Wright–Fisher diffusion with drift coefficient nt}φ^ in  is robusWe determine =0,and  holds; bentclass1pt{minima(i)Estimation of mutation at locus A when there is selection at locus B,(ii)Estimation of genic selection at locus A when there is mutation at locus B;while the following problems are not robust: (iii)Estimation of recombination when there is mutation at either locus,(iv)Estimation of mutation at locus A when there is mutation at locus B,(v)Estimation of mutation at locus A when there is recombination,(vi)Estimation of mutation at locus A when there is selection at locus A,(vii)Estimation of genic selection at locus A when there is mutation at locus A;similarly for problems interchanging the two loci. We omit the straightforward calculations. We caution that in the estimation problems above, the likelihood, and thus the estimator S as in \documentfound in  and\docuatterson , up to aexact simulation of certain classes of Wright–Fisher diffusion (Jenkins and Spanò W(t) is the X can be approximated using Riemann sums constructed from the same set of gridpoints.In this section we conduct an empirical study of the properties of otion in . IntegraV(x) of Sato . If this occurs we set Using Euler–Maruyama simulation it is possible to obtain a realisation with In the following we posit a two-locus, diallelic , we expect it is possible to refine this result further; indeed we conjecture that T. This would provide an easy way to check whether In this article we have derived an expression for the maximum likelihood estimator of the recombination rate,  Theorem . AlthougAlthough Theorems by Ewens , p180.) T, finding the asymptotic behaviour of X.Because of the unusual behaviour of the estimator for Overbeck , §3.4. Hi, j) is small, since these regions are more informative for N, and behaviour near the boundaries is inappropriate when true frequencies can only be a multiple of 1/N. One might prefer to replace the estimator T], with one that integrates Finally, we observe that fundamental quantities appearing throughout this work are


The transmission of plant viruses from infected to healthy host plants is a process in which insects play a major role, using various transmission strategies. Environmental factors have an impact on the transmission of viruses and the subsequent development of infections or diseases. When viruses are successful, plant virus diseases can reach epidemic proportions. Many plants across different regions are vulnerable to viral infections transmitted by the whitefly vector. Begomoviruses, which are transmitted by whiteflies, represent a significant threat to agriculture worldwide. The review highlights the mechanisms of virus acquisition and transmission by whiteflies and explores the factors influencing these interactions. Understanding the impacts of these changes is crucial for managing the spread of pests and mitigating damage to crops. It underscores the need for continued research to elucidate the mechanisms driving plant–insect–virus interactions and to identify new approaches for sustainable pest management. According to recent studies, over 80% of plant pathogenic viruses are dependent upon insect vectors for their spread ,2,3. AmoBemisia tabaci) cryptic species complex stands out as a competent plant virus vector. Within B. tabaci, Middle East–Asia Minor 1  and Mediterranean  are the two most invasive members of the species complex. Both MEAM1 and MED have created havoc on major agricultural production regions of the world, including China, Africa, and the Southern United States [Among hemipterans, whiteflies are the most important group of vectors. There are more than 1500 species of whiteflies belonging to 161 genera across the world . Many pld States ,11,12. Geminiviridae is one of the largest and most important families of plant viruses [Geminiviridae is grouped into nine genera, including Curtovirus, Begomovirus, Topocuvirus, Mastrevirus, Eragrovirus, Capulavirus, Becurtovirus, Turncurtovirus, and Grablovirus. Begomoviruses, which constitute the largest genera within Geminiviridae are transmitted by B. tabaci. The genomic size of begomoviruses (monopartite or bipartite) ranges from 2.7 to 3.0 kb , SSA1 (Sub-Saharan Africa 1), SSA2 (Sub-Saharan Africa 2), and Mediterranean cryptic species [Begomoviruses can trigger cellular and molecular responses in their insect vectors, which can then affect their transmission ,46. B. t species ,48,49. G species . This gr species ,45,50. During the transmission of begomoviruses via whiteflies, the viruses undergo various interactions with insect proteins and pass through the barriers in the digestive, hemolymph, and salivary systems before transmission from the insect body ,51. The Whiteflies transmit persistent viruses through a series of steps that include ingestion, attachment, entry, translocation, circulation, retention, and release of the virus. Whiteflies acquire viral particles while feeding on infected plants. The acquired viral particles (virions) reach into the midgut and esophagus, followed by entry and translocation into the cell across the basal membrane. Following acquisition, replication, and dispersal, they enter into the hemolymph, eventually becoming located in the primary salivary glands. From the salivary glands of the vectors, the viruses are released in saliva and enter into the plant’s phloem. After injection of the virus into the plant via saliva, the virus replicates inside the plant cells and spreads to other parts of the plant, causing leaf yellowing, stunted growth, and a decreased yield ,32,52. DStudies with cucurbit leaf crumple virus (CuLCrV) and Tomato yellow leaf curl virus  have shown that the majority of ingested virions are localized in the midgut and filter chamber of the insect vector ,54. SomeThe infection cycle of a begomovirus begins when virus particles are acquired by the whitefly from the plant phloem of the infected plants through the insect’s stylets . Both thBegomoviruses are capable of replicating and building up in the salivary glands of whiteflies, resulting in alterations in their behavior and increased transmission ,71. InfeB. tabaci increases its feeding on the cucurbit chlorotic yellow virus -infected plants to increase the chance of virus acquisition and subsequent spread [B. tabaci MEAM1 under similar conditions indicated that the interactions between the begomovirus, host and vector are pathosystem-specific. For instance, studies with TYLCV-infected tomato demonstrated that B. tabaci MEAM1 that acquired no virus was attracted towards susceptible genotypes with higher TYLCV accumulation, and they accumulated higher TYLCV compared with whiteflies feeding on resistant hosts with reduced TYLCV accumulation [B. tabaci MEAM1 developmental time decreased significantly on TYLCV-infected susceptible tomato plants compared with non-infected plants. In contrast, in another study, the same B. tabaci MEAM1 population that acquired no virus, avoided settling on squash infected with CuLCrV, and whitefly development on CuLCrV-infected squash did not result in any fitness benefits [Some reports suggest that ingestion of phloem sap of viral-infected plants may ultimately affect the fitness of insect vectors and their transmission capability ,73,74. Ft spread ,38,75. Ct spread . Additiot spread . Both tht spread ,75,78. Ht spread ,75. Howemulation ,69. Alsobenefits .Plant viruses frequently manipulate the insect vector’s biology, which ultimately impacts virus epidemiology ,80. MoreSome economically significant diseases transmitted by whitefly vectors include cotton leaf curl disease, cassava mosaic disease, and TYLC ,83. AbouTomato yellow leaf curl virus (TYLCV) is one of the most extensively researched begomoviruses, particularly concerning its effect on the preference of whiteflies without the virus. Several studies have reported that whiteflies tend to choose TYLCV-infected plants over uninfected ones . AccordiMixed infections with multiple viruses are prevalent ,99, but The feeding behavior of whiteflies is critical for the transmission of begomoviruses, as these insects serve as vectors for numerous viral diseases affecting crops. The resulting impact on disease epidemiology highlights the importance of manipulating whitefly feeding behavior . This maSeven articles have been published investigating the influence of virus infections on the feeding behavior of whiteflies through plant mediation . The durRegarding begomoviruses, five studies focusing on the direct effects of virus infection on whitefly feeding behavior have reported an increase in feeding on the infected host, while the other five suggest no effect ,72,102. To manipulate the behavior of whiteflies through plant viruses, outcomes may differ depending on the specific species or strain/cultivar involved in the whitefly–virus–plant combination. For instance, nonviruliferous MEAM1 species prefer to feed on the healthy tomato plants, while MED tends to feed on infected plants . On the B. tabaci MEAM1 and MED had significantly higher fitness advantage when feeding on TYLCV- and CuLCrV-infected plants compared to MEAM1 and MED individuals feeding on sida golden mosaic virus (SiGMV)-infected plants. Also, a transmission study from the same report concluded that B. tabaci MEAM1 efficiently transmitted TYLCV, CuLCrV, or SiGMV, whereas B. tabaci MED only transmitted TYLCV [B. tabaci MEAM1 and MED upon the acquisition of TYLCV, CuLCrV, or SiGMV [B. tabaci cryptic species, and two highly similar viruses  can interact very differently with B. tabaci. Thus, this signifies the context-specificity in begomovirus–whitefly interactions. Further studies are warranted to fully comprehend the events and factors that lead to positive effects of begomovirus in some systems and negative or no effects in others. On the whitefly side of these interactions, MEAM1 individuals feeding on virus-infected plants showed the downregulation of genes involved in the oxidative phosphorylation pathway and detoxification enzymes. This reduced detoxification activity probably lowered energy costs and boosted whitefly performance . Howevered TYLCV . A subseor SiGMV . Taken tCurrently, there is a lack of comprehensive case studies, making it challenging to conclusively determine the effects of viruses on whitefly biology and feeding behavior when feeding on infected plants. Typically, the modification of vector effectiveness is attributed to changes in the synthesis of phytohormones or their signaling in plants ,106,107.This diversity allows for the rapid adaptive evolution of viruses since they may already possess gain-of-function mutations that are useful for different selection pressures . PreviouBegomoviruses have been documented to infect diverse crop species in tropical, subtropical, and temperate regions. The discovery of new species of these viruses has been facilitated by recent advancements in small RNA-based deep sequencing technology . Our undThe virus can interfere with the plant’s immune system, preventing the plant from recognizing and responding to the virus infection. This allows the virus to replicate and spread throughout the plant. Begomoviruses can also cause changes in the expression of host plant genes, which can impact the plant’s physiological processes, including photosynthesis and hormone signaling. The virus may also induce the formation of abnormal structures in infected plants, such as leaf-like tissues on stems or the proliferation of roots. Symptoms of begomovirus infection differ based on various factors, such as the growth stage during initial infection, host species/cultivar, and begomovirus species, as well as weather conditions ,112. SevB. tabaci, which may or may not be carriers of plant viruses, can trigger modifications in the release of volatile organic compounds (VOCs) when interacting with different plant species. These changes in the volatile profile can either deter or attract other whiteflies [B. tabaci infestation [Begomovirus exhibit modified VOC emission profiles [Several species of iteflies ,86,87. Riteflies ,114. Begiteflies . Phytohoestation ,64,65. Zestation . Similarprofiles ,116,117.Host plant resistance is a highly effective method for managing diseases, particularly those caused by begomovirus, over a specific period . In planC2, C4/AC4, V2/AV2, Rep, and C1 (encoded by the begomovirus-associated beta satellite), which have been reported to suppress RNA silencing at the post-transcriptional level (PTGS) and/or at the transcriptional level (TGS) in different ways [Plants have implemented various defense systems to protect themselves from viral infections. Since viruses are obligate intracellular parasites, they exclusively rely on the host’s machinery for replication and mobility ,124,125.ent ways ,128.In plants, gene silencing occurs through two mechanisms: PTGS and TGS ,129,130.B. tabaci from Uganda had a higher growth rate on East African cassava mosaic virus-Uganda (EACMV-UG)-infected plants, which correlated with a significant increase in the concentration of four amino acids in the phloem sap of virus-infected cassava plants. This suggests that the favorable interaction between whiteflies and EACMV-UG may be facilitated by improved nutrition in virus-infected plants. In contrast, the MEAM1 whitefly performed better on tobacco plants with TYLCCNV infection than on plants without infection [The nutritional content of plants infected with begomoviruses may influence their suitability for whiteflies. In one study, African cassava nfection . However−4 substitutions per site per year (subs/site/year), which if accompanied by selection pressure imposed by the control strategies mentioned above, and in most cases can lead to the evolution of resistant breaking isolates of the viruses [Managing and controlling begomovirus infections has become increasingly challenging due to the rapid evolution of new viral strains. Despite significant advancements in control strategies, such as RNAi, transgenics, and markers assisting breeding for host-resistant (R) genes, begomovirus remains a major problem in commercial crops. Begomoviruses have a high mutation rate of 10 viruses . B. tabaci and tobacco. Specifically, the C2 protein of TYLCV, a devastating begomovirus that affects crops worldwide, interacts with plant ubiquitin to impede the breakdown of the JAZ1 protein. This interaction effectively hampers the JA defense pathway and suppresses the activity of terpene synthase genes regulated by MYC2; as a result, the enhanced survival and reproductive capabilities of whitefly vectors are facilitated [The relationship between begomoviruses and whitefly vectors is often indirect mutualism, as the whiteflies tend to perform better on virus-infected plants, which promotes virus spread and has significant agronomic effects. However, there is a lack of an understanding regarding how plant viruses manipulate plant defenses to promote vector performance. Recent studies have found that the C2 protein of begomoviruses lacking DNA satellites suppresses plant defenses against whitefly vectors, as demonstrated in the case of whitefly ilitated . These fThe understanding of genetic diversity in viruses is crucial for comprehending virus epidemiology and evolution. While the impact of genetic variation on virus–host interactions is well-established, its influence on virus–insect vector interactions is less understood. Mutation, recombination, and reassortment are the key drivers of genetic variation in viruses . Before Virus evolution is a dynamic process that leads to alterations in the genetic composition of viral populations over time. This process gives rise to novel viral variations, strains, and species, each possessing unique biological characteristics, including the ability to adapt to diverse host environments. This evolutionary process is influenced by various factors, such as hosts, vectors, the environment, and the viruses themselves ,148. TheBegomovirus genus, notable examples include tomato yellow leaf curl sardinia virus, tomato yellow leaf curl Axarquia virus, sweet potato leaf curl Canary virus, tomato leaf curl Mahé virus, and tomato leaf curl Yunnan virus, all of which have originated as new species through recombination. While whiteflies transmit begomoviruses to plants, the virus particles have the potential to undergo mutations or recombination events with other related viruses present in the whitefly population ,150. TheGenetic variations within viral populations, genetic drift, and selection pressures from factors, such as the host, vector, and environment, collectively shape the evolution of viruses. Viruses, with their rapid replication rate exceeding that of their hosts, undergo constant changes to elude host immune responses while preserving their functionality across diverse hosts and vectors ,152. B. tabaci complex, have spread to different countries and regions [The relationship among plants, whiteflies, and begomoviruses is crucial for understanding the population dynamics and epidemiology of these organisms ,154. Sev regions ,156,157. regions ,135. PlaBegomoviruses and whitefly species co-evolve as a result of genetic modifications in the virus genome, which call for intimate begomovirus–plant–whitefly interactions. Dynamic environmental conditions control these relationships. The manipulation issue can be confounding since some research demonstrates alterations in viruses and their vectors, while others claim that these changes may only be speculative. According to our perspective, begomoviruses affect the host and vector, respectively, making them more attractive to whiteflies and changing their feeding habits. To better understand begomovirus–plant–whitefly interactions, more research is needed because it is unclear whether these alterations are real or hypothetical. Plant virus epidemic prediction models can now be created because of recent technological advancements. Utilizing emerging technologies, like metagenomics, wisely and combining these contemporary tools with established techniques may help us better understand the intricate relationships between begomoviruses and whitefly vectors. This understanding may help us control the spread of begomoviruses and their whitefly vectors.


Among 89 clinical samples, 2 showed the R294K mutation. These two strains were evaluated using a neuraminidase inhibition test, which revealed that their sensitivity to oseltamivir was greatly reduced. The sensitivity and specificity of RT-dd PCR were similar to those of RT-qPCR and its accuracy was comparable to that of NGS. The RT-dd PCR method had the advantages of absolute quantitation, eliminating the need for a calibration standard curve, and being simpler in both experimental operation and result interpretation than NGS. Therefore, this RT-dd PCR method can be used to quantitatively detect the R294K mutation in H7N9.The R294K mutation in neuraminidase (NA) causes resistance to oseltamivir in the avian influenza virus H7N9. Reverse transcription droplet digital polymerase chain reaction (RT-dd PCR) is a novel technique for detecting single-nucleotide polymorphisms. This study aimed to develop an RT-dd PCR method for detecting the R294K mutation in H7N9. Primers and dual probes were designed using the H7N9 NA gene and the annealing temperature was optimized at 58.0 °C. The sensitivity of our RT-dd PCR method was not significantly different from that of RT-qPCR ( In 2013, cases of human infection with H7N9, a subtype of the highly pathogenic avian influenza virus, were first reported in Shanghai and Anhui. Since then, it has become a global public health concern . AdditioHowever, reports have emerged of influenza viruses that are resistant to NAI drugs. In 2013, a human H7N9 virus isolate (A/shanghai/1/2013) was identified as being resistant to oseltamivir . This suCurrently, the main approaches employed to detect influenza virus resistance are phenotypic and genotypic analyses. Early detection is crucial for effective drug selection and disease control. However, these methods may lack the necessary sensitivity or be slow in detecting the virus. For instance, phenotyping demands a significant amount of time and laboratory resources, whereas genotyping might not identify low-frequency mutations . Hence, The purpose of this study is to develop a highly efficient RT-dd PCR-based method for detecting drug resistance in H7N9, which will aid clinicians in selecting better drug regimens and provide essential information for controlling and treating the disease. RT-dd PCR can detect targets with very low copy numbers and quantify gene copies. Therefore, we established an RT-dd PCR platform to identify mutations in the H7N9 virus drug-resistance gene at the R294K site in NA gene. This platform can directly test clinical specimens for oseltamivir resistance. It has several advantages, such as fast analysis, simplicity, and user-friendliness. It also provides direct results for R294K-oseltamivir resistance in H7N9 viruses. Depending on the platform, the change in drug-resistance genes could be monitored over time to discover the curative effect of NAIs, such as oseltamivir, against the H7N9 virus.Eighty-nine H7N9 virus case samples were provided by the laboratory of Zhejiang Provincial Center for Disease Control and Prevention (ZJCDC). These case samples were obtained during the clinical management of patients with the H7N9 virus. All case samples were cultured successfully using chicken embryos in the BSL-3 of ZJCDC.RNA of the H1N1, H3N2, B/Victoria, B/Yamagata, H5N1, H9N2, and H7N9 virus was provided by ZJCDC. The strains used in the NAI test, GD003 R294 and GD003 R294K, were provided by the China National Influenza Center.The NA sequences of the H7N9 virus were obtained from the NCBI and GISAID influenza virus sequence databases. These sequences were aligned using Mega v.5.0 and Bioedit v.7.0 to identify the fragment within R294 and 294K. Primer Express v.3.0 was used to design the primers. Two TaqMan minor groove binder (MGB) probes  were useRecombinant plasmids of the wild type (WT) and mutant type (MT) of the NA gene were synthesized by Shanghai Sangon Company , which we used directly without extracting nucleic acid. When we received the plasmids, we divided the plasmids to ensure the same concentration of plasmids were used in the experiment.H7N9 virus RNA was extracted from 200 μL of the clinical samples, including swabs, sputum and alveolar lavage fluid, using the Qiagen Rneasy Mini Kit , according to the manufacturer’s instructions. RNA was eluted with 50 μL of Dnase- and Rnase-free water and stored at −80 °C until use.TM Supermix for Probe (no dUTP)  was used to amplify the plasmids. The volume of the PCR mixture was 20 μL, containing 1 μL of DNA template, 10 μL of supermix, 8 μL of ddH2O, and 1 μL of R294K primer/probe mix. The amplification cycling conditions included enzyme activation at 95 °C for 10 min, 40 cycles of denaturation at 94 °C for 0.5 min, annealing and extending at 58 °C for 1 min, and enzyme deactivation at 98 °C for 10 min. RNA extracted from the H7N9 virus case samples was amplified using the One-step RT-PCR Advanced Kit for Probe . The reaction mixture volume was 20 μL, including 1 μL of RNA, 5 μL of supermix, 2 μL of reverse transcriptase, 1 μL of 300 mM DTT, 10 μL of ddH2O, and 1 μL of primer/probe mix. Amplification included the following cycling conditions: reverse transcription at 45 °C for 60 min; enzyme activation at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 0.5 min, annealing and extension at 58 °C for 1 min, and enzyme deactivation at 98 °C for 10 min. A reader was used to measure the signals of the PCR products. The total number of droplets in each reaction tube was ≥10,000, and the detection results were considered to be valid. If the number of positive droplets was ≤3, the result was determined to be negative; if the number of positive droplets was >3, it was determined to be positive. The QuantSoft v.1.7.4 software  was used to determine the copy number. The results were recorded as the copy/reaction or copy/μL (copy/reaction in 20 μL of reaction).RT-dd PCR was performed using a QX200 droplet digital PCR system . The dd PCRThe annealing temperatures were set at 60.0, 59.6, 59.0, 58.0, 56.9, 56.0, 55.3 and 55.0 °C. The template at 55.0 °C was nuclease-free water as the negative control and the same concentration of plasmid nucleic acid was used as the template for other temperatures. Two groups of parallel experiments were repeated to evaluate the degree of signal differentiation and number of nucleic acid copies, and to determine the optimal annealing temperature.A ten-fold gradient was used to dilute the WT and MT plasmids three times, with the original concentration of 8146.25 copies/μL and 532 copies/μL for WT and MT, respectively, dd PCR was used to detect each dilution, and each dilution plasmid was detected in eight repeats. The mean, standard deviation (SD), and coefficient of variation (CV) were calculated to evaluate the repeatability of the dd PCR. According to the Clinical and Laboratory Standard Institute EP17-42 standard, the sensitivity of dd PCR and qPCR was evaluated using the limit of detection (LOD) of the plasmids. The reagents, reaction systems and reaction procedures used were consistent to consider the comparability of the results of dd PCR and qPCR, and the qPCR was performed on the ABI 7500. Serially dilute the plasmids 6 times at 2-fold dilution, each dilution was detected in 16 repeats using dd PCR and qPCR. The initial concentration of the mixed sample contained both MT plasmid and WT plasmid at a ratio of 1:19 with a total concentration of 10,000 copies/μL. Specifically, the WT plasmid accounted for 95% of the mixture, while the MT plasmid accounted for 5%. Further ratios were tested with the WT plasmid accounting for 97.5%, and MT plasmid for 2.5%; WT plasmid accounting for 98.75%, and MT plasmid for 1.25%; WT plasmid accounting for 99.375%, and MT plasmid for 0.625%; and finally, the WT plasmid accounting for 99.6875%, and MT plasmid for 0.3125%). Probit regression analysis was used to calculate the 95% confidence interval (CI) of the positive rate to determine the LOD. We used the RNA of influenza A (H1N1 and H3N2) virus, influenza B (Victoria and Yamagata) virus, avian influenza A (H5N1 and H9N2) virus, H7N9 virus (GD003 R294 and GD003 R294K), and nuclease-free water, as the negative control, to evaluate specificity.The WT and MT plasmids were mixed, which resulted in a sequential reduction in the final MT plasmid content to 5, 2.5, 1.25, 0.625, and 0.3125%. The MT plasmid was detected using dd PCR, and each plasmid mixture was detected in eight repeats. The positivity rate was calculated to assess the effect of low-frequency detection.RT-dd PCR was used to detect the H7N9 virus in the 89 samples. If the R294K mutation was detected, the respective samples were subjected to next-generation sequencing (NGS) to compare the mutation abundance.Extracted H7N9 virus RNA was reverse-transcribed to cDNA and amplified using a PathAmpFluA PCR kit  according to the manufacturer’s instructions. The PCR products were purified using Ampure XP beads . Quantification of the purified samples was performed using Qubit v.2.0 and agarose gel electrophoresis. Library preparation was performed using a Library Construction Kit . Sequencing was performed using the Illumina Netseq 550 platform and quality control was performed under default parameters. Sequences of the A/Anhui/1-DEWH730/2013/H7N9 virus were selected as reference databases, and in addition, the site was considered to be mutated if the frequency of variation was more than 50%.50 value (reduced enzyme activity by 50%) was calculated and compared with the standard resistant strain GD003R294K and standard sensitive strain GD003R294. According to the WHO criteria for determining influenza drug-resistant strains, an increase in the IC50 lower than ten-fold compared with the reference virus strain IC50 was considered as normal inhibition. When the increase factor was between 10- and 100-fold, the sensitivity was reduced. When the increase was >100-fold, the sensitivity was significantly reduced.The NA-Fluor Influenza Neuraminidase Assay Kit  was used to detect the inhibition of NA enzyme activity (NAI) in the H7N9 virus strains. The ICt-test, were performed using SPSS v.25.0. Statistical significance was set at p < 0.05. The IC50 values from the NAI test were calculated using GraphPad Prism 7.R294K mutation abundance was calculated using the mean concentration (MT/(MT + WT) × 100%). Repeatability was verified using the CV value. Statistical analyses, which included the paired samples The total number of droplets was >10,000, and adequate separation between negative and positive droplets was observed when the annealing temperature was between 55.3 and 59.0 °C. However, the highest numbers of nucleic acid copies were detected at 58.0 °C and 56.9 °C for MT and WT, respectively . The higRepeatability, sensitivity, and specificity were the main indicators used to evaluate the developed method. The CV was <10% when the concentration of the WT and MT plasmids was high and the CV >10% when the R plasmid concentration decreased to 5.30 copies/μL and the K plasmid concentration decreased to 1.12 copies/μL .t-test showed that the sensitivities of dd PCR and qPCR were not significantly different (p = 0.625).Probit regression analysis was applied to determine the LOD of the dd PCR and qPCR. Setting 95% as the positive rate, the detection limits of dd PCR were 4.00 (95% CI: 3.05–6.67) copies/μL for WT plasmids and 6.15 (95% CI: 4.59–10.47) copies/μL for MT plasmids; the detection limits of qPCR were 6.13 (95% CI: 4.87–9.13) copies/μL for WT plasmids and 2.49 (95% CI: 1.89–4.46) copies/μL MT plasmids. The paired samples Both dd PCR and qPCR detected the RNA of GD003 R294 and GD003 R294K, as well as that of the other six respiratory virus samples, including H1N1 pdm09, H3N2, B/Victoria, B/Yamagata, H5N1, and H9N2. The results showed that only GD003 R294, GD003 R294K, and the mixture of GD003 R294 and GD003 R294K were positive, while the other influenza viruses were all negative .The positive rate gradually decreased to 1.52, 0.63, 0.29, 0.09, and 0.05%, corresponding to the MT plasmid concentrations. When the positive rate accounted for 0.05%, all the plasmids were detected; in addition, the mean concentration of MT plasmids was 6.025 copies/μL , which wVerifying the clinical application of a newly established method is essential. In this study, RT-dd PCR was used to analyze the 89 H7N9 virus clinical samples. Two clinical samples exhibited the R294K mutation, named A/Zhejiang/65-1/2017 (H7N9) and A/Zhejiang/70-2/2017 (H7N9 virus); the concentrations of the mutant gene were 298 copies/reaction and 46 copies/reaction, respectively . The othNGS is a reliable method for detecting mutations; therefore, it was used to evaluate the accuracy of resistant site mutations detected by dd PCR. The WT strain A/Zhejiang/YCHH/2016 (H7N9 virus) and MT strains A/Zhejiang/65-1/2017 (H7N9 virus), and A/Zhejiang/70-2/2017 (H7N9 virus) were detected by NGS. The vaccine strain A/Anhui/1-DEWH730/2013 was used as a reference. The base G at position 882 was substituted with A in A/Zhejiang/65-1/2017 (H7N9 virus) and A/Zhejiang/70-2/2017 (H7N9 virus) compared with the reference sequences, which contributed to the substitution of the amino acid at site 294 (R294K), an essential site associated with drug resistance. According to the analysis of variation, the mutation abundance of A/Zhejiang/65-1/2017 (H7N9 virus) and A/Zhejiang/70-2/2017 (H7N9 virus) was 99.94% and 91.35% respectively, and the mutation abundance of A/Zhejiang/YCHH/2016 (H7N9 virus) was 0% .50 values of A/Zhejiang/65-1/2017 (H7N9 virus) and A/Zhejiang/70-2/2017 (H7N9 virus) were 728.3 nM and >1000 nM for oseltamivir, respectively, which were both >1000-fold higher than that of the standard strain  strain was susceptive to oseltamivir and zanamivir.Resistant phenotype analysis is the gold standard for detecting the drug resistance of the influenza virus . A/Zhejid strain . For zanProtein mutations can significantly impact viral infectivity, including the H7N9 virus . The R29p = 0.63). However, both Yan et al. and Whale et al. reported that RT-ddPCR was more sensitive than RT-qPCR [In the current study, an RT-dd PCR protocol was established to detect the R294K drug resistance mutation in the H7N9 virus, and its repeatability, sensitivity, and specificity were evaluated using plasmids. Plasmid-derived DNA standards are widely used in qPCR due to their stability and easy preparation. However, different amplification reagents and conditions are required for DNA or RNA templates in ddPCR, and the effect of reverse transcription on reproducibility and sensitivity has not been considered. Nonetheless, plasmids have been used in several studies to evaluate the sensitivity of RT-ddPCR ,15. In o RT-qPCR ,17, but  RT-qPCR . As a re RT-qPCR .In this study, 2 out of 89 H7N9 virus clinical samples showed the R294K mutation. The mutation abundances for the two strains, A/Zhejiang/65-1/2017 (H7N9 virus) and A/Zhejiang/70-2/2017 (H7N9 virus), were 99.94% and 95.08%, respectively. These two strains were also analyzed using NGS with the same RNA and showed mutation abundances of 99.94% and 91.35%, respectively. The RT-dd PCR and NGS mutation abundances for A/Zhejiang/65-1/2017 (H7N9 virus) were identical; however, the RT-dd PCR mutation abundance for A/Zhejiang/70-2/2017 (H7N9 virus) was higher than that detected by NGS. When the mutation abundance is below 10%, qPCR and Sanger sequencing results are unreliable . StrainsThe H7N9 virus can have a significant impact on the lives of infected patients and the economy of a country, especially when it involves drug-resistant mutations. It is essential to develop faster and more accurate detection methods for the early diagnosis of these mutations . qPCR ca


Aedes, Culex, Haemagogus, and Sabethes from a transition area between the Amazon, Cerrado, and Caatinga biomes in Brazil. Metagenomic high-throughput sequencing was used to characterize the virome of 20 mosquito pools. A total of 15 virus-like genomes were identified, comprising species genomically close to insect-specific viruses of the families Iflaviridae, Metaviridae, Lispiviridae, Rhabdoviridae, Xinmoviridae, and Parvoviridae and species of plant viruses of the families Solemoviridae, Virgaviridae, and Partitiviridae. However, sequences of viruses associated with human and animal diseases were not detected. Most of the recovered genomes were divergent from those previously described. These findings reveal that there are a large number of unknown viruses to be explored in the middle-north of Brazil.A wide diversity of pathogenic mosquito-borne viruses circulate in the Brazilian Amazon, and the intense deforestation can contribute to the spread of these viruses. In this context, this study aimed to investigate the viral diversity in mosquitoes of the genera  Mosquitoes (Diptera: Culicidae) are known to transmit a variety of viruses that cause diseases in humans and animals, such as Yellow fever, West Nile fever, and Japanese encephalitis . For thiFlaviviridae, Rhabdoviridae, Togaviridae, Peribunyaviridae, and Phenuiviridae, suggesting that these viruses may have an ancestral relationship [ISVs replicate exclusively in invertebrate cells and have no direct public or veterinary health impact ,4. Howevtionship .Ilheus [Rocio [Mucambo [West Nile [In Brazil, a wide variety of wild-type arboviruses circulate, mainly in the Amazon region in the northern part of the country. More than 210 arboviruses have already been registered in this area , such ass [Rocio , Mucambo[Mucambo , and Wesest Nile  viruses.est Nile . Howeverest Nile . This coJapanese encephalitis virus and Getah virus) and ISV species in mosquitoes of the genera Culex, Anopheles, Aedes, and Armigeres [In this context, mosquitoes can be good environmental samplers due to their capacity to feed on a variety of vegetable (to obtain nectar)  and animrmigeres .Aedes (Ae.), Culex (Cx.), Haemagogus (Hg.), and Sabethes (Sa.) from three locations in a transition ecosystem between the North and Northeast of Brazil.In this study, we used a metagenomic-based approach to characterize the virome of wild-caught adult female mosquitoes of the genera Collections were conducted in the proximities of Inhamum Municipal Environmental Protection Area (S 04°55′12.7″ W 043°27′37.0″), Pedras village (S 04°59′12.7″ W 043°35′59.1″), and Buriti Doce community (S 04°58′50.4″ W 043°07′51.2″), respectively located in the municipalities of Caxias, São João do Sóter, and proximities of Timon, located in Maranhão State, Brazil , in NoveAreas with extensive vegetation cover and minimal anthropic action situated near the water sources were chosen to perform the collections. These activities were authorized by the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) (grant number 68405-1).Aedes, Culex, Haemagogus, and Sabethes were selected for metagenomic analysis due to their being vectors of various viruses of medical and veterinary importance.Adult mosquitoes were collected during six days using the Center of Disease Control’s (CDC) light traps installed at 1.50 m above ground level overnight and entomological nets using human attraction techniques for the collection of diurnal insects in the periods of 8 a.m. to 11 a.m. and 3 p.m. to 6 p.m. In all the collection sites, the mosquitoes were killed by dipping them in liquid nitrogen. The mosquitoes were morphologically identified to the most specific taxonomic level possible using species identification keys ,24,25,26® kit , following the manufacturer’s guidelines.Each pool of mosquitoes was homogenized in a microtube with a solution of 500 µL of Dulbecco’s phosphate buffered saline diluent 1X (D-PBS)  with 2% penicillin and streptomycin, 1% fungizone, and 5% fetal bovine serum and a 3 mm diameter tungsten sphere using TissueLyser II equipment  at 25 Hz for 1 min based on an adapted protocol . After tTM VILOTM MasterMix  and NEBNext mRNA Second Strand Synthesis Module  kits. Subsequently, cDNA libraries were constructed using the protocol for the SureSelectQXT Whole Genome Library Prep  kit. Libraries were quantified by a Qubit® 4.0 Fluorometer  and evaluated by an Agilent 2100 bioanalyzer (Agilent Technologies) for quality control. The libraries were sequenced by the paired-end method on the NextSeq 550 platform  using the NextSeq 500/550 High Output kit v.2.5 (300 cycles).The extracted RNA was used for synthesizing the first and second cDNA strands using SuperScript−4 e-value threshold and inspected with the MEGAN6 v6.21.1 program [https://www.ebi.ac.uk/interpro/ accessed on 21 November 2022).The raw sequence data were submitted to quality control analysis and treated to remove adapters and low-quality reads using fastp v0.23.2 , and to  program  to identNucleotide sequences of extracted contigs were compared with the sequences of the closest viral members available at the NCBI by multiple sequence alignment (MSA) using Clustal Omega to determine the phylogenetic relationships. A phylogenetic tree was constructed using the maximum likelihood (MV) method  in the IAedes (N = 49), Culex (N = 18), Haemagogus (N = 1), and Sabethes (N = 26) were collected and divided into 20 pools based on collection sites and species for high-throughput sequencing. Descriptions of each pool, including specie name, number of specimens, origination, and collection date, are shown in A total of 94 adult female mosquitoes of the genera A total of 608,447,930 paired-end reads were generated by sequencing, of which 274,329,804 reads remained after quality control .Iflaviridae, Metaviridae, Solemoviridae, and Virgaviridae), negative-sense ssRNA (−ssRNA) viruses , double-stranded RNA (dsRNA) viruses (family Partitiviridae), and single-stranded DNA (ssDNA) viruses (family Parvoviridae)  viruses (families viridae) .Iflaviridae was obtained from a pool of Cx. (Cux.) spp. (AR872456). The open read frame (ORF), oriented from the 5′ to 3′ end and ranging from 585 to 9020 nt positions, encodes a polyprotein containing three structural proteins of the viral coat located at the N-terminal region and the non-structural proteins helicase, peptidase, and RdRp located at the C-terminal region  in length closely related to members of the family l region a. This sCulex Iflavi-like virus 3 (GenBank accession: MW434116), presenting 59.9% (nt) and 58.2% (aa) of identity. Furthermore, the obtained sequence’s identity ranged from 7.6% (with Culex Iflavi-like virus 4/NC_040716) to 64.7% (with Culex Iflavi-like virus 3/MW434116) by comparing their three capsid protein domains with the same region of other members of the family Iflaviridae. According to the International Committee on Taxonomy of Viruses (ICTV), amino acid sequence identity in the capsid protein below 90% is one of the criteria used to define new iflaviruses [Inhamum iflavirus (IhIV) (GenBank accession: OP918263). The polyprotein nucleotide sequence of IhIV was aligned with 46 sequences of the family Iflaviridae and four other sequences of the family Marnaviridae that represented the external group. Phylogenetic analysis provides evidence of common ancestry shared between IhIV and iflaviruses (The obtained polyprotein displayed the most significant identity with aviruses . Therefoaviruses b.Errantivirus (family Metaviridae) were identified. The first sequence was obtained from Sa. Chloropterus (AR872465), and it contains 5823 nt in length. Oriented to the 5′ to 3′ terminus, the contig has three ORFs that encode: a putative gag protein (1332 nt), responsible for encoding the domains for the capsid and the nucleocapsid; a polyprotein (3120 nt) that includes domains for the typical protease (PR), reverse transcriptase (RT) domain, integrase (INT), and ribonuclease H (RH) enzymes found in metaviruses; and an incomplete ORF encoding the gypsy protein, associated with the products of the envelope gene (Chibugado virus (GenBank accession: MN661043), an unclassified Metaviridae, presenting 62.8% (nt) and 63.7% (aa) of identity regarding the conserved region, which contained PR, RT, RH, and INT proteins. The low identity suggests that we recovered a novel species, tentatively named Inhamum errantivirus (GenBank accession: OQ779233).Two sequences closely related to members of the genus ope gene a. This gCx. usquatus (AR872498). This sequence (5014 nt) has two ORFs. The first, located in the 5′ terminus, codifies a polyprotein that contains PR, RT, RH, and INT, whereas the second, located in the 3′ terminus, codifies the gypsy protein (Chibugado virus and is considered a novel species tentatively named Buriti errantivirus (GenBank accession: OQ779240). In addition, nucleotide and amino acid alignments showed that Inhamum errantivirus and Buriti errantivirus are divergent from each other, sharing 66.6% (nt) and 69.9% (aa) of identity based on the polyprotein region.A second sequence was recovered from  protein a. This gChibugado virus differs from other members classified in the family Metaviridae. In the 5’ to 3’ direction, Chibugado virus presents a genomic organization of env-gag-polyprotein, while the two sequences obtained here present the typical organization (gag-polyprotein-env) found in other members of the family  were found in two pools (AR872461 and AR872510) of Ae. Serratus. The first is a genome of 2726 nt in length, composed of two ORFs that codify a peptidase protein and a RdRp domain  (A phylogeny based on the RdRp region placed the two sequences obtained in this study in a clade with unclassified members of the family y virus) b.Virgaviridae was identified in Sa. chloropterus (AR872486). The largest ORF (7974 nt) located in the 5′ end encodes a polyprotein predicted in the methyltransferase, helicase, and RdRp domains. A short ORF (585 nt) present in the 3′ terminus codifies capsid protein (Atrato Virga-like virus 2 (GenBank accession numbers: MN661104 and MN661105) and presents 61.2% (nt) and 59.7% (aa) of identity for both strains, suggesting that it is a novel virus, tentatively named Buriti Virga-like virus (GenBank accession number: OQ779238). The putative novel virus clustered in a distinct clade with unclassified virgaviruses based on the nucleotide sequence alignment of the polyprotein , and it is a sequence of 13,424 nt, close to Canya virus (GenBank accesion: MW434766), sharing 46.1% (nt) and 35.5% (aa) of identity. The sequence contains five ORFs, presenting a similar genome organization found in mononegaviruses in the following order of genes oriented to the 5′ to 3′ terminus: nucleoprotein , phosphoprotein , matrix protein , glycoprotein , and polyprotein (polymerase)  , mRNAcap (IPR026890), and methyltransferase (IPR039530), in the L gene, while the N, P, M, and G genes were determined based on the closest hit in the BlastX search (Pedras lispivirus (GenBank accession: OQ779241), belongs to the family Lispiviridae  a. In addX search a. The nuiviridae b.Riboviria sp. has 6397 nt, enclosing a single ORF, and was obtained from a pool of Cx. (Cux.) spp. (AR872456). It shares a closer similarity with the Stang virus (GenBank accession: MW434775), sharing 68.4% (nt) and 77.3% (aa) of identity. Just as was carried out for the previous genome, analysis of Interproscan identified the typical domains of mononegaviruses in the L gene (Rhabdoviridae (Inhamum rhabdovirus (GenBank accession: OQ779231).The other contig closely associated with e L gene a. Phylogoviridae b. This pXinmoviridae . A short and incomplete sequence of 1139 nt codifying a RdRp domain (Sa. glaucodaemon (AR872487) and presented close nucleotide (56.0%) and amino acid (52.4%) identity with Enontekio anphevirus 2 (GenBank accession: ON955256), belonging to the genus Anphevirus (Xinmoviridae). A phylogeny based on the RdRp showed that the recovered sequence has a relationship with other anpheviruses (Buriti anphevirus (GenBank accession: OQ779239).Two other sequences found were associated with the family p domain a was ideeviruses c. This sAe. scapularis (AR872521). The sequence corresponds to the nucleoprotein region (Guadeloupe mosquito mononega-like virus (GenBank accession: MN053735), an unclassified xinmovirus, with nucleotide and amino acid identities of 74.9% and 87.2%, respectively. Phylogenetic analysis showed that the recovered sequence clustered with other xinmoviruses (Pedras xinmovirus (GenBank accession: OQ779244).Another short sequence of 787 nt in length was found in n region a, and itoviruses d; howeveAe. albopictus (AR872511) had close relation to Verdadero virus (GenBank accession: MT742175), an unclassified virus of this family, with 71.4% (nt) and 80.0% (aa) of identity. The phylogenetic analysis showed that the recovered sequence clustered next to a member of the genus Cryspovirus and other unclassified partitiviruses (Pedras partitivirus (GenBank accession: OQ779243).In the present investigation, three sequences presented correspondence to the capsid proteins of different partitiviruses a. A sequiviruses b. This pSa. Quasicyaneus (AR872471-85). The first comprises 1285 nt, and it was related to Zeya Brook partiti-like virus 1 (GenBank accession: MW389559), an unclassified partitivirus with 64.0% (nt) and 66.2% (aa) of identity. As with the previous virus, this putative genome clustered with Cryspovirus and other unclassified partitiviruses, being named Buriti partiti-like virus 1 (GenBank accession: OQ779235) (Gammapartitivirus (Atrato Partiti-like virus 2 (GenBank accession: MN661058). This putative virus was tentatively named Buriti partiti-like virus 2 (GenBank accession: OQ779236).The two other sequences were recovered in a pool of Q779235) b. The otitivirus b and preSa. quasicyaneus sample (AR872471-85) (Inari deltapartitivirus (GenBank accession: OP019955), with identities of 70.8% (nt) and 81.2% (aa). Phylogenetic analysis showed that the obtained genomic fragment is related to members of the genus Deltapartitivirus (Buriti deltapartitivirus (GenBank accession: OQ779234).In addition, an incomplete genome of 720 nt codifying a RdRp domain was obtained from the same 2471-85) a. It preitivirus c. AccordDensovirinae (Parvoviridae), identified in Sa. quasicyaneus (AR872471-85). The recovered sequence has 5182 nt in length and is composed of four ORFs that codify a hypothetical protein (909 nt) and a non-structural protein 1 (NS1) helicase (1704 nt) at the 5′ terminus, and a capsid viral (VP) (2091 nt) and a hypothetical protein (693 nt) at the 3′ terminus (Atrato Denso-like virus (GenBank accession: MN661135), an unclassified Densovirinae. However, the nucleotide and amino acid alignments of the NS1 helicase domain revealed that these viruses shared 54.2% (nt) and 40.1% (aa) of identity, indicating that we recovered a novel parvovirus that was tentatively named Buriti densovirus (GenBank accession: OQ779237).In this investigation, we also report a genome closely related to the subfamily terminus a. The coBuriti densovirus in a distinct clade where there are unclassified Densovirinae viruses and a representative member of the genus Muscodensovirus  spp. Moreover, other iflavirus species have already been reported in Ae. aegypti from the Amazon region of North Brazil [Members of  Insecta . Previou Insecta ,40,41. Hlex spp. ,43,44,45des spp. ,47, withh Brazil , showingErrantivirus (Metaviridae) co-exist in the studied area. The two genomes were identified in Sa. chloropterus and Cx. usquatus and are close to the Chibugado virus but deeply divergent between them. Errantiviruses are endogenous retroviruses that integrate into host genomes during replication [Errantivirus species, named Aedes aegypti To virus 1 and Aedes aegypti To virus 2, have already been identified in Ae. aegypti from Tocantins, North Brazil [Additionally, our data showed that two genomes related to the genus lication . This colication . In mosqh Brazil .Densovirinae (Parvoviridae). Viruses in this subfamily infect insects, decapod crustaceans, and echinoderms and are distributed into six genera [Densovirinae viruses and a representative member of the genus Muscodensovirus. Moreover, it has low amino acid identity with other densoviruses. According to the ICTV, all viruses in a genus should be monophyletic and encode NS1 proteins with amino acid identity above 30% [Buriti densovirus and the other closest unclassified viruses could form a novel genus in future revisions. However, the few available sequences related to Densovirinae limit inferences.Another ISV identified here was classified in the subfamily x genera . Howeverbove 30% . This inAnopheles gambiae densovirus, which also belongs to the subfamily Densovirinae, negatively affects the Mayaro virus (MAYV) infection in Anopheles gambiae cells and mosquitoes [It is important to highlight that the previously described ISV named squitoes . MAYV issquitoes . Then, oMononegavirales and belonging to three different families: Rhabdoviridae, Xinmoviridae, and Lispiviridae. The known ISVs, rhabdoviruses, are classified in the genera Almendravirus and Sigmavirus [Rhabdoviridae also includes arboviral species distributed in the genera Curiovirus, Ephemerovirus, Hapavirus, Ledantevirus, Sripuvirus, Tibrovirus, Vesiculovirus, and Caligrhavirus [Alpharhabdovirinae. This close relationship supports the theory that arboviruses and ISVs may have an ancestral relationship [In this investigation, we also discovered four virus-like contigs related to ISV of the order gmavirus . Howeverrhavirus . Curioustionship ,56. TherXinmoviridae, this family was established in 2018 for the inclusion of a novel genus, Anphevirus, and all species registered until now infect insects [Aedes anphevirus has important implications for the surveillance of emerging viral diseases given that it can reduce Dengue virus replication in vitro [Anphevirus species in the investigated area. In another previously conducted mosquito investigation from Brazil, another Xinmoviridae-related virus, Aedes anphevirus, was identified in Ae. aegypti from the Midwestern region. However, as with our obtained data, the authors recovered a short fragment of 552 nt associated with glycoprotein [In relation to  insects . Among xin vitro . Unfortuoprotein .Xinmoviridae, Lispiviridae were established in 2018 for the inclusion of the genus Arlivirus and encompass several viruses discovered in invertebrates [Xinmoviridae and Lispiviridae, which emphasizes the importance of metagenomic studies.As well as tebrates , but onlSolemoviridae and Virgaviridae clustered in distinct clades with unclassified species. Probably, these sequences will also form new genera in the other revisions.Four genomes identified in this study showed a relationship to plant viruses. The finding of these viruses in mosquitoes can be related to the ingested plant sap and nectar required to supply their nutritional needs . These vDeltapartitivirus (Partitiviridae) and comprises a small part of the RdRp gene. Despite being a well-conserved region and preferable for determining phylogenetic relationships, this incomplete genome could not reveal a reliable identity shared between these viruses. Additional mosquito collections in the studied area should be performed tentatively to obtain complete sequences to confirm these inferences.The other plant virus identified in this study was classified in the genus capsid gene cluster with members of genera Cryspovirus (which comprises protozoa viruses) and Gammapartitivirus (composed by fungi viruses) belong to the family Partitiviridae [Evidence of other groups of viruses was identified in this investigation. Three virus-like contigs associated with the iviridae . UnfortuIflaviridae (Inhamum iflavirus) and Rhabdoviridae (Inhamum rhabdovirus) were identified in a pool of 13 Cx. (Cux.) spp. (AR872456), while members of the families Partitiviridae  and Parvoviridae (Buriti densovirus) were identified in a pool composed by 18 Sa. quasicyaneus (AR872471-85). It Is important to emphasize that it does not suggest the co-existence of viruses given that these samples are composed of a pool of mosquitoes and the recovered genomes could be present in different individuals.In this study, we identified mutual viruses present in single samples. Viruses belonging to the families Perhaps a longer period of mosquito sampling and exploration of other transition areas would recover a greater diversity of viruses in the studied region. For this, we suggest analyzing the amplitude of time and cover area in future investigations. In addition, this research highlights the application of NGS in identifying different groups of viruses and unknown species that would not have been possible using conventional methods. Nowadays, ICTV recognizes that genomes assembled from metagenomic data contain sufficient information to be incorporated into the official classification scheme, given that this procedure is indispensable for the comprehensive characterization of the global virome . Here weThis study revealed that the virome of mosquitoes collected from transition areas in the north and Northeast of Brazil was diverse, with viral-like genomes classified into nine taxonomic families and associated with insect-specific and plant viruses. Notably, viruses associated with the invertebrate host were prevalent, and all of them are novel species, which could be of great importance to arbovirus control methods in future investigations. Furthermore, these findings expand our knowledge about the virosphere.


This study examines healthcare costs associated with adverse drug reactions (ADR) in an older population admitted acutely to an Irish tertiary hospital. Prospective cohort study involving older persons admitted to hospital with and without an ADR. Data was collected at baseline, during hospitalisation and post-discharge. Participants provided information on healthcare resource use three months before admission (baseline) and three months after discharge (follow-up). For each healthcare resource, unit costs were derived and applied. The average cost (standard deviation (SD)) associated with the hospital admission for the ADR and non-ADR are presented. In addition, baseline and follow-up care costs were compared using difference-in-difference analysis and presented with 95% confidence intervals (CI). Costs by preventability and severity of ADR are also presented.A total of n = 230 participants were included (n = 93 ADR and n = 137 without ADR). The average cost associated with hospital admission for an ADR was €9538 (SD €10442) and €9828 (SD €11770) for non-ADR. The additional follow-up costs (difference-in-difference) associated with the ADR was estimated at €2047 (95% CI: -€889 to €4983). The mean incremental follow-up cost of definite preventable ADRs was estimated at €1648 (95% CI: -€4310 to €7605), possible preventable ADRs €2259 (95 CI: -€1194 to €5712) and unavoidable ADRs €1757 (95% CI: -€3377 to €6890). The mean incremental follow-up cost associated with moderate severe ADRs was estimated at €1922 (95% CI: -€1088 to €4932) and €3580  for severe ADRs.ADRs leading to hospital admission are associated with modest incremental healthcare costs during and three months after admission. Severe and possibly preventable ADRs were associated with higher costs.The online version contains supplementary material available at 10.1007/s00228-023-03552-x. Exposure to multiple medicines, particularly in older populations with multimorbidity, may cause harm leading to poorer outcomes such as increased risk of death, falls, drug interactions, non-adherence, and hospitalization . AdverseThe economic consequences of ADRs can be considerable with most evidence based on costs associated with hospitalisation and ADRs occurring within the hospital setting , 6. HoweTherefore, the aim of this study is to estimate the healthcare costs associated with hospitalisation and post-discharge in an older aged cohort (aged 65 + years) admitted acutely to a tertiary hospital in Ireland with an ADR compared to a similar cohort without an ADR.This study uses the Adverse Drug reactions in an Ageing PopulaTion (ADAPT) cohort , 9. The The ADAPT study was a cross-sectional study of ADR prevalence in all patients aged ≥ 65 years admitted acutely to a large tertiary referral hospital in Ireland over an eight-month period (November 2016 - June 2017). A subset of those with ADRs and without ADRs (non-ADR) from this initial cross-sectional study were enrolled into a prospective cohort study of patient-reported outcomes and costs associated with ADR-related hospital admissions.There were 3091 patients initially screened for a suspected ADR-related hospital admission within the first 36 hours of admission by the research team using a previously validated screening process . ADR sevThe screening approach incorporated a multifaceted review of each hospital admission to assess the likelihood of the ADR being a reason for admission (cause of admission or contributing to admission) in the context of the medications used, clinical conditions, medical history, comorbidities and investigations. In total 361 (11.7%) patients were determined to have an ADR-related admission. A random sample of patients, who were determined not to have a suspected ADR, were assigned to a non-ADR control group for comparative purposes (n = 437).Patients with an ADR-related hospital admission and the non-ADR control group were invited to complete a baseline questionnaire during their hospital stay measuring their health service use, health related quality of life (HRQOL) and medication adherence for the three months prior to hospital admission. Three months post-discharge they were asked to complete a follow-up questionnaire including the same measures. For patients temporarily unable to provide informed consent due to illness severity, their next of kin/carer provided assent to take part in the study and completed the questionnaires on their behalf. Lost to follow-up was defined as non-completion or non-return of the follow-up questionnaire by participants or their proxy.The perspective of the cost analysis was that of the Irish Health Service Executive (HSE). Publicly financed resources provided within three months before and after discharge were included. This included both accident and emergency, inpatient and outpatient hospital services, GP services, pharmaceuticals, other primary care services, home help and care in nursing homes.Age at admission, gender , medical card status , smoking status , alcohol use (Yes/No), comorbidities and number of medications were extracted from each patient’s medical record. Comorbidity was measured using the Charlson co-morbidity score  and polyHealth service use was measured for all patients with an ADR-related hospital admission and control patients using their medical record and by self-report per their baseline and follow-up questionnaires three months post-discharge. The length of hospital stay (discharge date - admission date) was recorded from their hospital record. The baseline and follow-up questionnaire completed by the individual or proxy measured the utilisation of a total of 25 healthcare items three months pre-admission (baseline) and three months post-discharge (follow-up). This included the number of: (i) GP visits; (ii) out of hours GP services; (iii) hospital visits, A&E visits, hospital inpatient (including duration of stay) and outpatient visits; (iv) use of therapies ; (v) use of services ; (vi) public health nurse and; (vii) use of day care centres and respite care.The unit costs for healthcare resources were derived at 2021 Euro (€) price level using HIQA guidelines . For hosWe compared descriptive sociodemographic and clinical variables for the different cohorts  within the study population. Descriptive analyses for length of hospital stay are provided as mean and standard deviation (SD). The main analysis was conducted using the ADR-related hospital admission cohort and the non-ADR related hospital admission cohort for individuals who consented to take part and returned both the baseline and 3-month follow-up survey. The main analysis involved comparing the costs associated with healthcare utilisation at baseline and at follow-using the completed surveys. We used Chi-squared and Fisher exact tests to explore statistical significance in group differences for the sociodemographic and clinical categorical variables and an independent samples t-test for comparison of average length of stay between the ADR and non-ADR groups.For the included pre and post-discharge resource items, we report the proportion of users of each service by the ADR and non-ADR cohorts at baseline and follow-up, and used unadjusted logistic regression to estimate odds ratios (OR) and 95% confidence intervals (CI) for variation in proportion of users at baseline and follow-up, and difference-in-difference odds ratio. We multiplied the number of contacts and the unit cost for each resource item, and summed over all items, and report the mean cost at baseline and follow-up for all individuals in the ADR and non-ADR groups. Generalised linear models with log-link function and gamma distribution were used to estimate mean cost difference-in-difference with and without adjustment for patient characteristics. We interpret these difference-in-difference estimates as the mean incremental cost at follow-up compared to baseline (pre-admission) for the ADR cohort in comparison with the non-ADR cohort.For the presentation in the main text, we have grouped the resource items at baseline and follow-up into seven cost categories defined by service providers including accident and emergency (A and E) services, hospital services, GP services, pharmacy, other primary care services, home help and nursing, and long-term care services.We further estimated the difference-in-difference costs for subgroups of the ADR-cohort which had definitely preventable, possible preventable and unavoidable adverse events, and by subgroups with moderate severe and severe ADR. As sample size for these subgroup analyses was limited, the estimation procedure using the specified generalised linear model did not converge, so we estimated these difference-in-difference estimates for aggregated costs using unadjusted ordinary least squares (OLS) regression. All data processing was done with Stata (v17) and significance levels were set at p < 0.05.There were 350 patients or proxy (carers) who consented to take part in the study at baseline which included 141 with an ADR-related hospital admission and 209 without an ADR-related hospital admission  and in the non-ADR group 11.2 (SD = 13.4). The mean difference of -0.4 days was not statistically significant (p = 0.85). The average cost associated with the hospital stay was €9538 (SD €10442) in the ADR group and €9828 (SD €11770) in the non-ADR group, with a small difference of -€290 between the ADR and non-ADR group. Admissions for patients with severe ADRs were associated with higher average costs (€9806 (SD €7193)) compared to admissions for patients with moderate severity ADRs (€9516 (SD €10693)). Admissions for possible preventable ADR was associated with higher average costs (€11147 (SD €12356)) compared to admissions with definitely preventable ADR (€7275 (SD €6110)) and unavoidable ADR (€6789 (SD €5361)).Table Table Our study findings suggest a modest difference in costs associated with an ADR-related hospitalisation and post-discharge compared to non-ADR hospitalisation with an average decrease of €290 in the costs association with the initial hospital admission and an average increase in pre and post-hospitalisation costs of €2050 per individual identified with an ADR compared to non-ADR. The majority of the differences in additional costs not associated with the index hospital stay were attributed to the increases in nursing home care, increased accident and emergency department visits and hospital services after discharge for their acute episode in the ADR cohort. The average costs associated with ADRs were highest in those with possibly preventable ADRs and those with moderate severity or severe ADRs.There have been a small number of systematic reviews of studies examining the costs associated with ADRs or adverse drug events (ADEs) , 7 whichOne recent study in the UK found that the average costs associated with hospitalisation for medication harm (including ADRs) and healthcare utilisation after 8 weeks post-discharge was approximately £550 . AnotherStrengths and Limitations – Our study provides additional unique features, including the elaborate and careful identification of an ADR on admission, the classification of ADRs using validated criteria, the comprehensive collection of healthcare resource use before and after hospital admission, and robust analysis and reporting of the healthcare resource use and costs. Other strengths are the inclusion of screened ADRs in all those admitted to the tertiary centre and the comparison to a non-ADR population.However, there are a number of limitations. Comparisons with other studies are difficult due to differences in populations, definitions used, medications included, and cost sources. In addition, our study population were much older and, therefore, at higher risk of ADRs due to underlying comorbidities and polypharmacy. In particular, a high proportion were admitted with cardiovascular medicines and one in four on antidepressants.  In additAlso, the older aged populations are likely to be associated with other contributory factors such as complex medication regimens, cognition issues, vision deficits, nutrition, mobility/falls and fracture risk, social supports. Therefore, generalisability to other populations and settings may be limited. The sample size in the ADR and non-ADR cohorts was relatively small, and only 66% of the ADR and non-ADR cohorts provided data at the 3-month follow-up. There are also limitations related to estimates of costs which are based on estimated time and units costs available.ADR-related hospitalisation is a significant burden among the older aged population and often preventable. The costs associated with ADR-hospitalisation are modest, but also depend on the severity of the ADR. However, the cost is not only financial but is often associated with costs both clinically and personally for individuals. Early intervention, where possible, is important to avoid preventable medication harm. Intervention with more targeted policies to reduce ADRs through identification of those at highest risk and more awareness among HCPs and others involved in the care of the older populations will help to reduce costly hospitalisations and avoid increased morbidity and mortality . A recenIn conclusion, ADR-related hospitalisation and post-discharge care in older individuals results in significant healthcare utilisation and costs. The costs are increased with preventability and severity of the ADR. More research is required to develop validated tools for prevention and early detection of ADRs that can be implemented in clinical practice to avoid unnecessary harm and burden, including economic burden.Supplementary file1 (DOCX 54 KB)Supplementary file2 (DOCX 53 KB)Below is the link to the electronic supplementary material.


The coronavirus disease (COVID-19) pandemic has increased pressure to develop low-cost, compact, user-friendly, and ubiquitous virus sensors for monitoring infection outbreaks in communities and preventing economic damage resulting from city lockdowns. As proof of concept, we developed a wearable paper-based virus sensor based on a molecular imprinting technique, using a conductive polyaniline (PANI) polymer to detect the lentivirus as a test sample. This sensor detected the lentivirus with a 4181 TU/mL detection limit in liquid and 0.33% to 2.90% detection efficiency in aerosols at distances ranging from 30 cm to 60 cm. For fabrication, a mixture of a PANI monomer solution and virus were polymerized together to form a conductive PANI sensing element on a polyethylene terephthalate (PET) paper substrate. The sensing element exhibited formation of virus recognition sites after the removal of the virus via ultrasound sonication. A dry measurement technique was established that showed aerosol virus detection by the molecularly imprinted sensors within 1.5 h of virus spraying. This was based on the mechanism via which dispensing virus droplets on the PANI sensing element induced hybridization of the virus and molecularly imprinted virus recognition templates in PANI, influencing the conductivity of the PANI film upon drying. Interestingly, the paper-based virus sensor was easily integrated with a wearable face mask for the detection of viruses in aerosols. Since the paper sensor with molecular imprinting of virus recognition sites showed excellent stability in dry conditions for long periods of time, unlike biological reagents, this wearable biosensor will offer an alternative approach to monitoring virus infections in communities. The global health threat from COVID-19 has raised attention regarding the need for low-cost, rapid, sensitive, compact, and selective detection platforms for viruses. In particular, keeping viral diseases from spreading into the community relies heavily on the detection of such viruses at the initial stages of an outbreak. Individual personal virus testing and aerosol virus detection can trace the spread of infection, minimizing virus outbreaks and preventing city lockdowns. Currently, virus detection relies on conventional approaches such as serological . SerologIn an effort to reduce sensing cost, improve convenience, and provide a more compact design, various new virus sensing approaches have been put forth. Electrochemical detection through low-cost paper substrates is one of the most popular for detecting viruses ,7. ReferTo replace the costly biological reagents, molecularly imprinted electrodes for electrochemical detection have also been suggested. Instead of the metal or carbon working electrode, a conductive polymer electrode with target molecule templates was used to detect the Zika virus ,16. In tThe present research demonstrated a low-cost MIP lentivirus sensor that requires no biological reagent and enables virus detection in liquid and aerosol phases without bulky equipment. We previously developed glucose and perfluorooctanesulfonic acid (PFOS) conductive molecularly imprinted paper sensors ,24,25 anWe established a dry measurement technique for virus detection that includes the process of the virus and molecularly imprinted cavity hybridization and sensor element drying. The electrical resistance of the sensor, fabricated from the conductive polymer, enabled the passive measurement of the resistivity ratio over time through the use of a portable multimeter. With this approach, we were able to demonstrate detection of the lentivirus on the sensing element (i) within 1 h by simply dispensing liquid, and (ii) within 1.5 h after spraying a virus solution. This simple and low-cost approach can potentially detect viruses from liquid and aerosol samples in a short period of time. To the best of our knowledge, this is the first attempt to demonstrate the detection of viruses directly in aerosols. The stability of the molecularly imprinted structure in the air  will posAniline and ammonium persulfate (APS) acting as the monomer and the oxidant, along with phosphate-buffer saline (PBS) for adjusting the virus concentration, were purchased from Sigma-Aldrich . Acidic stock solutions including hydrochloric acid  and acetic acid were acquired from Macron . Methanol was ordered from VWR Chemicals . Polyethylene terephthalate paper and silver conductive paste (Cat# 125-15) were provided by Xerox  and Creative Materials , respectively.5 TU/mL, where TU denotes the transducing units of viral particles. Retro-CMV-GFP retroviruses , polystyrene latex beads , and human whole blood  were used as selectivity references. Phosphate-buffered saline (PBS) for adjusting the virus concentration was diluted from concentrated PBS (10×) purchased from Sigma-Aldrich .The generation of lentiviruses was described previously , and the5 TU/mL) as the molecular imprinting template were blended into 1 M HCl with a final volume of 5 mL as the monomer solution. The final virus concentration in the polymer solution was 1.1 × 104 TU/mL. Paper strips (1 cm × 0.5 cm) were dipped into the monomer solution for 5 min to saturate the solution on the paper. The oxidant solution prepared by mixing 409 mg of APS with 5 mL of 1 M HCl was then added drop by drop to initiate polymerization for 10 min. After the bulk polymerization, the PANI strips were taken out from the solution and rinsed with deionized water until the eluent showed no excess PANI particles. The virus templates were removed by sonicating the strips in acetic acid/methanol solution (vol/vol = 1:6) for 4 h, followed by rinsing the strips until the pH value of the eluent reached to 7. The resulted lentivirus molecularly imprinted (MIP) PANI paper strips were air-dried at ambient temperature overnight. The monomer solution without lentiviruses was synthesized as non-molecularly imprinted (NIP) control devices. NIP preparation without virus followed the same treatment as MIP.The synthesis of the virus-imprinted PANI followed the protocol detailed in the previous study . BrieflyTo fabricate the duplex sensor device, one NIP and another MIP strip were integrated with a plastic stencil substrate (2 cm × 2.5 cm) using double-sided tape, with copper tape (1 cm × 0.635 cm) as the contact electrode. The strip and electrode were separated by a 1 mm gap which was then filled with silver conductive paste. The silver paste was cured at 25 °C for at least 12 h to ensure optimal conductivity, and the resulting device was used to measure the resistivity responses of NIP and MIP to lentiviruses on a single device. The surfaces of NIP and MIP PANI were investigated by scanning electron microscopy, and the result is shown in 5 TU/mL. Then, an aliquot of 30 μL virus solution was dispensed in the middle of the PANI sensing element (0.5 cm × 0.5 cm) and saturated for 30 min, followed by gentle aspiration and air-drying at 25 °C for 30 min. For virus detection in aerosol, virus contained in DMEM solution was sprayed onto the PANI sensing element, as described in detail in To test the detection efficacy of virus MIP sensor, virus samples with different concentrations were prepared. Specifically, for the selectivity test, the virus concentration of lentivirus and Retro-CMV-GFP retrovirus was set at 1.65 × 10To obtain the virus concentration calibration curve, the resistance of the PANI electrode was measured using a multimeter  in direct current mode after a drying time of 30 min. This was the time needed to air-dry the sensor surface after 30 µL of virus solution was dispensed and aspirated until no visible moisture was found. Resistance was converted to resistivity as shown in Equation (1).A and L are the cross-sectional area and the longitudinal length of the PANI sensing element, respectively. In addition, the resistivity after virus exposure was divided by the resistivity before virus exposure to determine the resistivity ratio which is the output signal of the sensor calculated using Equation (2) [In this equation, tion (2) .(2)ResiTo investigate the effect of non-specific bonding, the resistivity ratio of NIP were subtracted from that of MIP as shown in Equation (3).LoD) was estimated using Equation (4), where the slope (m) was obtained from the linear regression fit of specific binding. σ is the standard error of blank samples that were exposed to PBS [The limit of detection (d to PBS .(4)LoD=To study the stability of the PANI electrode, PANI was first synthesized on paper specimens (1 cm × 1 cm) using the same synthetic protocol above. The resistance was then measured weekly at room temperature (25 °C) for 13 weeks after the fabrication , and the normalized resistivity was calculated.The resistivity ratios of the MIP and non-molecularly imprinted polymer (NIP) electrodes . At high lentivirus concentrations (1.65 × 105 TU/mL and 2.2 × 105 TU/mL), there was a slight increment in the NIP resistivity ratio. However, this extremely small signal increment (attributable to non-specific binding) was removed from the overall sensor signal . To eliminate such bias from the total sensor response, the subtracted  data were essential, accounting for the specific recognition of the lentivirus by the MIP sensor. The significant difference in resistivity ratio between the MIP and NIP responses supports the strategy of molecular imprinting technology.The lentivirus served as an effective template for fabricating the molecular imprinting virus sensor. The sensor’s virus-capturing efficiency and detection limit could be further improved by increasing the concentration of the virus used for fabrication, which would amplify the number of MIP cavities. Further surface investigation is needed to visualize the functional groups and intermolecular interactions between the virus particles and MIP polyaniline matrix. Studies of the viability and possible structural transition of the lentivirus during synthesis are also necessary to reveal the detailed mechanisms of molecular imprinting associated with microorganisms.We investigated the selectivity of the MIP sensor by comparing the resistivity ratios of the lentivirus, retrovirus, latex beads, and human blood. The results are shown in 5 TU/mL virus concentration), in addition to the lentivirus, since the resistivity ratios on the MIP sensors were similar. Because the lentivirus and retrovirus belong to the same virus family and their viral envelope structures are almost identical, the retrovirus was also captured by the MIP structure synthesized with the lentivirus template. This shows the ability of the sensor to capture the same family of viruses. Future investigations will compare the selectivity of different families of viruses.The MIP sensor detected the Retro-CMV-GFP retrovirus , no difference from the control in terms of resistivity ratio was observed. This confirmed that the MIP electrode did not respond to the beads, even though they were the same size as the virus. Additionally, no significant MIP resistivity change was found with the human whole blood sample containing potential contaminants such as plasma and blood cells. The resistivity ratio for the blood sample on the MIP electrode was close to that of the NIP control. Even though the geometric shapes of polystyrene nanoparticles and blood proteins are roughly spherical, similar to the lentivirus shape, they may lack the surface interactions found in the lentivirus. The absence of surface interactions with the molecularly imprinted cavities may have prevented nanoparticles and blood proteins from being captured on the molecularly imprinted sensor.Moreover, the detection of 100 nm polystyrene latex beads roughly the same size as the lentivirus were investigated at the same concentration of 1.64 × 10Due to the presence of MIP cavities for virus capturing, this sensing device exhibited more substantial responses to the lentiviruses than did the NIP device. Although the strongly acidic condition of the monomer solution remains a concern with regard to preventing the lentiviruses from maintaining viability, the geometric structure may still be maintained during the molecular imprinting process, since it is of a short duration. More characterizations are needed to acquire further evidence and visualize the formation related to the molecularly imprinted structures.The resistivity ratio of the MIP sensor for the lentivirus in aerosol form was measured from multiple spray distances (between 30 cm and 60 cm) to investigate the feasibility of the sensor . While the importance of aerosol virus detection is enhanced by the urgent need to slow down and prevent the respiratory transmission of infectious viruses such as COVID-19 , there iWe designed a low-cost paper-based virus sensor with a detection limit of 4181 TU/mL using the molecularly imprinted polymer technique. We also demonstrated virus detection in aerosols using our paper-based MIP sensor, as well as a wearable sensor application in the form of a face mask, which will be critical in future public health and safety. In the aerosol experiments, we detected the lentivirus at distances ranging from 30 cm to 60 cm away from the virus sprayer. The sensor showed a virus detection efficiency of 0.33% to 2.90% from the lentivirus present in the aerosol. The dry measurement of the virus was obtained within 1.5 h after spraying the virus aerosol solution onto the sensor electrode. Furthermore, our paper-based MIP sensor was easily integrated with a wearable face mask for the future detection of viruses in aerosols.A few limitations regarding this MIP sensor still exist. It took 1.5 h to detect the lentivirus in aerosol form. In the future, the drying time of aerosols on sensing electrodes could be reduced by using a convection evaporation process, in addition to drying using diffusion ,34 resulFurther improvements could be made by integrating this sensor with artificial intelligence (AI) and machine learning. With a humidity sensor and the addition of an AI algorithm, the virus detection time could be significantly reduced. Lastly, the MIP and NIP sensing electrodes were connected to a multimeter via electrical wires, limiting their real-world use. This could be overcome by wirelessly connecting the wearable MIP and NIP sensing electrodes to a smartphone app or cloud server, employing the Internet of things (IoT) in healthcare.