Genome-wide transposon expression up-regulation in host cells regardless of virus, species, and host cell tissue types occurs early during viral infection and likely contributes to promoting the host innate immune response. Virus–host cell interactions initiate a host cell–defensive response during virus infection. How transposable elements in the host cell respond to viral stress at the molecular level remains largely unclear. By reanalyzing next generation sequencing data sets from dozens of virus infection studies from the Gene Expression Omnibus database, we found that genome-wide transposon expression up-regulation in host cells occurs near antiviral response genes and exists in all studies regardless of virus, species, and host cell tissue types. Some transposons were found to be up-regulated almost immediately upon infection and before increases in virus replication and significant increases in interferon β expression. These findings indicate that transposon up-regulation is a common phenomenon during virus infection in human and mouse and that early up-regulated transposons are part of the first wave response during virus infection. Virus–host cell interactions and the spread of virus-derived material initiates host cell–defensive responses during viral infectious disease . In receHowever, there are also numerous examples of TE sequences being co-opted for use by the host. In mammals, syncytin genes are ERV-derived envelope protein genes which are expressed by cells of the placenta during embryonic development and are involved in placental cell–cell fusion to form the characteristic placental structure . Other pThe mobile ability of TEs make them a source of genomic instability, and thus, during normal cellular activities, they are mostly repressed by numerous epigenetic chromatin modifications . HoweverWe were able to identify 1,715 and 394 differentially expressed TEs (DE TEs) that were shared across several of the human and mouse infection data sets, respectively. These were enriched in the neighborhood of antiviral genes, immune response genes, and the MHC regions in both genomes. The mouse IAV infection time course analysis showed that TE expression changes are some of the earliest transcriptional events during virus infection, with up-regulation occurring even before or concurrent with interferon β expression. Together, these results suggest that DE up-regulated TEs may act as a part of an early, conserved host defense response.To investigate genome-wide transposon activity and identify DE TEs, we performed a comprehensive analysis of RNA-seq data from infected and mock-infected cells in both human and mouse. In total, we obtained 37 data sets from the GEO database: 23 human and 14 mouse virus–host data sets with at least two biological replicates per condition, consisting of 19 different human viruses and 8 different mouse viruses (Table S1). We developed a computational pipeline to assess and quantify genes, TEs, introns, as well as transcriptional readthrough (TR) levels, to determine how each changes during infection for each virus–cell pair .Tables of all human and mouse virus data sets used in the study.Table S1 + T cells. It should be noted that gene and TE expression changes for these data sets may be highly dependent on the time of infection. In addition, viruses interact with host cells via different mechanisms. It is possible that these viruses may be better adapted to circumventing detection by cellular innate immunity factors, causing lesser immune responses in the specific host cell types infected.We used two methods to assess gene and TE expression: 1) TEtranscripts to quantify gene and TE subfamily expression simultaneously for the most accurate measurements of TE expression across treatments  and S2 aAn Excel spreadsheet showing Ensembl genes quantified with TEtranscripts that are differentially expressed (DE) during infection in ≥1 virus data set in human.Table S2 An Excel spreadsheet showing Ensembl genes quantified with TEtranscripts that are DE during infection in ≥1 virus data set in mouse.Table S3 An Excel spreadsheet showing all TE and repeat subfamilies quantified with TEtranscripts that are DE during infection in ≥1 virus data set in human.Table S4 An Excel spreadsheet showing all TE and repeat subfamilies quantified with TEtranscripts that are DE during infection in ≥1 virus data set in mouse.Table S5 An Excel spreadsheet showing all TE and repeat loci quantified with featureCounts that are DE during infection in ≥1 virus data set in human.Table S6 An Excel spreadsheet showing all TE and repeat loci quantified with featureCounts that are DE during infection in ≥1 virus data set in mouseTable S7 Table summarizing transcriptional origins of top 88 shared DE TE (endogenous retrovirus and long interspersed nuclear element) loci in the human genome that have been manually annotated. Annotations are in Table S6.Table S8 Table summarizing transcriptional origins of top 60 shared DE TE (endogenous retrovirus and long interspersed nuclear element) loci in the mouse genome that have been manually annotated. Annotations are in Table S7.Table S9 Excel spreadsheets containing DE TEs at 1, 6, and 24 h post-infection with H3N2 Brisbane, Udorn, or Perth strain in human cell line time course, along with their coordinates, fold changes, relationship to genes, and their significance levels.Table S10 Excel spreadsheets showing Genomic Regions Enrichment of Annotations Tool GO term results for early, middle, late-stage TEs during the 7-d influenza A virus time course and TE enrichment results for the MHC region.Table S11 + T cells (HIVactivated CD4+T cell) and resting CD4+ T cells (HIVresting CD4+T cell), IAV, and HSV-1, which have already been shown to express HERVK during infection , indicating that particular subfamilies are sensitive to viral stress. Many of these top TE subfamilies constitute internal domains (“-int”) of LTR ERVs, which contain protein coding regions required for ERV replication  and S3C.nfection   of genes are DE when equal numbers of TEs are chosen at random . This inSome viral proteins have the ability to perturb host gene splicing and transcriptional termination, resulting in intron retention (IR) and TR of host gene transcripts . To furtTR is a common occurrence across the human genome, but cellular stress  has been shown to cause substantial increases in the number and length of gene transcripts with readthrough . To deteTo determine if TE up-regulation is primarily due to gene-related transcriptional events or is expressed autonomously, or independently of gene expression, we overlapped up-regulated DE TE loci with annotated TRs and other gene regions. We found that DE TEs originate from TR (data set range: 0–39%), IR (32–88%), upstream regions (3 kb from TSS) (0.4–10%), downstream regions  (5–31%), and intergenic regions (4–37%) . HoweverNext, we overlapped the 1,715 human DE TEs and the 394 mouse DE TEs with FANTOM 5 CAGE data to determine what fraction of the identified DE TEs have been previously shown to initiate transcription from their sequences in other data sets and under different conditions . We founGSE49933) of mouse lung tissue containing 20 time points   . This ex8–149 h) . We obseWe divided the time course into early (0–26 hpi), middle (49–75 hpi), and late (99–150 hpi) stages of infection based on the viral expression profile and performed a GREAT analysis on the DE TEs up-regulated in each stage. We found that these up-regulated DE TEs lie around genes related to interferon β response and innate immune response and observed GO terms that were unique and specific to early up-regulated TEs such as response to biotic stimulus, response to other organism, and regulation of multi-organism process .Isg15, and Irf7, a regulatory factor that activates interferon and interferon response genes  indicating that these expression changes were likely not due to changes in ISG expression. TE numbers were mostly sustained beyond 26 hpi and did not continue to increase even as the number of DE genes increased  DE TE expression, early  gene expression, and early  DE ISG expression in CPM relative to the sum of their expression changes over the time course. We also plotted the viral gene expression and the expression of an interferon stimulated gene, se genes . The exp introns , whereasncreased . TE up-rncreased . We overGSE96586, GSE61517)  infection time course in mouse RAW 264.7 cells (SE61517)   . AlthougP-value = 0.003, FDR = 0.70; not significant) and is statistically significantly differentially up-regulated by 7 hpi. Other mouse virus infections showed significant up-regulation as well, indicating that this locus is very sensitive to virus infection (2O2), and osmotic stress (KCl) from a TR study P-value =TR study , althougNext, we questioned if there is a connection between early DE TEs and the MHC regions. We performed a Fisher’s exact test on numbers of DE TEs in each MHC region during the IAV time course and found that MHC class III was significantly enriched with DE TEs beginning at 12 h during the IAV time course when compared with the genomic background or to the other MHC regions . When peAlthough many studies have examined the genome-wide response of host cells to viral infection at the level of the host’s genes, TEs have not been very well characterized during these interactions. Host ERVs, which are the remnants of ancient retroviruses, and LINEs comprise a large fraction of mammalian genomes and play important roles in host gene regulation, including of those genes involved in the innate immune response . Up to t+ B cells in latency stage III. EBV-infected CD19+ B cells showed massive down-regulation of TEs, as well as decreased IR and readthrough. We believe that this data set has opposite behavior because of either virus- and host-specific interactions or to the specific laboratory conditions in which the samples were prepared. Because EBV is able to transform B-cells, it is possible that the TE down-regulation we are observing may be due more to a changing cell state than to an infection response. HIV-infected activated and resting T-cells are another example of this; activated T-cells which is a more differentiated form of the resting T-cells showed dramatically lower levels of TEs than resting T-cells in mock samples, and also had fewer TEs changing during HIV infection than resting T cells. Thus, the TE up-regulation trends still generally hold across virus infections. In addition, beyond virus-specific and host cell differences, each virus experiment was collected at different infection time points, which will also influence the degree of TE up-regulation observed as we have already seen from multiple virus infection time courses.In this study, we have used comprehensive computational tools to explore the genome-wide expression activity of TEs from published data sets that were originally designed for gene expression profiling of human and mouse virus infections. The results have demonstrated that genome-wide TE up-regulation occurs in host cells of both mouse and human during virus infections, and that it is a common phenomenon. We observed that cell type, virus, and time of infection are all biasing factors that will influence the degree of TE up-regulation, but overall, almost all data sets showed more TE up-regulation after infection than down-regulation. Only one data set showed dramatically opposite patterns to the general trend we observed for TE up-regulation, IR, and readthrough: EBV-infected CD19We observed several intergenic full-length ERVs that became up-regulated and were shared across several human and mouse virus data sets, showing that particular loci are sensitive to virus-induced cellular stress. Moreover, we observed that up-regulated TE loci appear near genes involved in antiviral defense, interferon β response genes, and the MHC region in both species with a small number of these TEs showing conservation between species. We also observed minimal overlap of virus-induced TEs with TEs up-regulated by nonviral sources of stress (6%), which may suggest that some TE loci in the mouse genome are specifically sensitive to virus infection stress, although more nonviral stress data sets will need to be analyzed for comparison.Drosophila have shown that cellular stress does change canonical TE activity . Regardless of where these TE sequences are originating from, transcribed TE sequences are capable of forming dsRNAs that can be recognized by pattern recognition receptors (PRRs) (such as RIG-1 and MDA5), which can trigger a host immune response. Although laboratory verification is still needed to support the connection between the DE TEs we have observed and host cell gene expression regulation, these results provide some valuable evidence and a catalog of DE TEs that can be used for perturbation testing in the future.Isg15 (DE at 3 h). This indicates that low levels of interferon protein are able to stimulate ISG expression. Closer inspection of early DE TE expression showed a large increase by 3 hpi, and then expression dips during the middle infection when interferon and virus levels are highest and increases again when interferon levels decrease towards the end of infection. We witnessed TE up-regulation occurring before DE interferon β expression in five time courses in two species from three independent studies, showing that these events are likely not specific to a single virus infection but is somewhat shared across viruses, cell types, and species. However, we did witness significant up-regulation of interferon genes such as Isg15 and Irf7 before interferon-β, as early as 3 and 12 h, respectively, indicating that low levels of interferon β transcript and protein may be enough to dramatically alter interferon response gene expression. Last, we observed that different TEs are turned on during early infection than later infection and early DE TEs appear to be more associated with viral defense genes than later DE TEs.Using several time course studies to determine the timing of TE up-regulation, we observed for the first time that many TEs are up-regulated very early during infection (within 3 h). More strikingly, in the IAV time course study, some TEs are up-regulated before increases in virus replication and significant interferon-β gene expression (DE at 26 h) but did show concurrent up-regulation with an interferon stimulated gene, Based on this work, we propose a new virus–host interaction model  that is http://repeatmasker.org) using the DFAM 2.0 database (hg38-Dec 2013-RepeatMasker-open-4.0.6-Dfam 2.0 & mm10-Dec 2011-RepeatMasker open-4.0.6-Dfam 2.0) (Virus data sets in Table S1 were acquired from NCBI’s GEO or the Short Read Archive database. Only virus data sets containing multiple (2+) replicates of uninfected/mock-infected cells and virus-infected cells were used for the analysis. For all data sets, only the first read pair of paired-end read data sets was used. This was performed to provide consistency across data sets because some data sets used only single-end reads and some paired-end data sets had low-quality coverage of the second read pair. The human genome version hg38 and the mouse genome version mm10 from ENSEMBL with their corresponding ENSEMBL annotations (GRCh38.91 for human and GRCm38.90 for mouse) were used for this analysis. Repeat annotations for both human and mouse were downloaded from the RepeatMasker Web site  . Virus gVirus–host RNA-seq data sets were aligned to the individual species’ genomes or a combination of the individual species genomes and the specific virus genome using the STAR aligner  with theTo determine the TE subfamily and gene expression simultaneously, we used TEtranscripts from the TEToolKit . TEtransTo determine TE loci that are DE between mock and infected cells, we quantified counts mapping to TE and gene features separately with featureCounts  from theTo compare relative coverage of infected and mock-infected samples, biological replicate bam files were merged, and their coverages were compared using bamCompare from deepTools 3.1.1  with the2(FC) > 2X) and introns (FDR < 0.05 and log2(FC) > 1X)  > 1X) . To makeTo determine TR regions, we used DoGFinder , a tool To determine if there is an association between the DE TEs that are up-regulated across multiple human virus data sets and genes that they are nearby, we took all 1,715 DE TEs (ERVs and LINEs) ≥3 human virus data sets, and determined their closest gene neighbors using bedtools closest (v 2.25.0). We calculated the percentage of gene neighbors that are DE across at least one data set. As a control, we selected equal numbers of randomly selected ERVs and LINEs from the genome  and determined how many DE genes that they are nearby.To determine if DE TEs are originating from TR regions in each virus data set, repeat annotations filtered of repeats that overlap gene exons (-a) were overlapped with the union of annotated mock and infected DoGs  from each virus data set with bedtools intersect with -wo setting. A custom in-house script categorized and quantified DE up-regulated TEs for each virus data set based on whether they overlap DoGs or not. If they did not overlap annotated DoG regions, they were further subdivided into categories such as intergenic, in introns, within 3 kb downstream of genes, or within 3 kb upstream of genes. Downstream regions were included here because we observed cases of signal downstream of genes that did not meet the standards of a DoG region with DoGFinder. In cases where a TE has multiple relationships with genes, we categorized using the following hierarchical scheme: intron > downstream > upstream.To determine if DE TEs are enriched in DoG regions, we compared DE TEs in DoGs and non-DoG regions with non-DE TEs in DoGs and non-DoG regions using a Fisher’s exact test.Shared up-regulated human and mouse DE TEs were overlapped with a comprehensive set of FANTOM 5 (October 2017) CAGE sites that represent transcriptional initiation sites found across available cell lines/tissues. We used bedtools intersect (v 2.25.0) to identify TEs from these sets whose coordinates intersect the CAGE peak coordinates by a single base pair.To determine if significantly differentially up-regulated repeats during infection are associated with any particular annotations, genomic coordinates for 1,715 individual LINE and ERV elements that are DE up-regulated in two or more human virus data sets were lifted over to the hg19 genome using the UCSC genome browser liftover tool using default settings. The features that lifted over were run through GREAT using the UCSC hg19 genome assembly. Repeat coordinates were associated with the genes whose basal regulatory domain is defined as 5 kb upstream and 1 kb downstream, plus an extended  regulatory domain up to 1,000 kb. The whole genome was used as background.GSE49933), 20-h norovirus time course (GSE96586), and three 24-h influenza A (H3N2) strain time courses  were downloaded from GEO and mapped and quantified identically to other mouse and human virus data sets .The data sets analyzed during the present study are available in the GEO repository, and the study accessions and file accessions are provided in Table S1 and also in the main text. Additional code is available on GitHub (


Rabies, for which the mortality rate is almost 100%, is a zoonotic viral disease that can be transmitted via solid organs or tissue allotransplantation. Dozens of deaths from rabies via solid organs or tissues allotransplantation (ROTA) have been documented during the last decades. In 2015 and 2016, two cases of rabies virus transmission via solid organs or tissue allotransplantation were reported in China, which further underscore the risk and importance of this special type of rabies for organ transplant recipients.From 1978 to 2017, at least 13 cases of ROTA, causing dozens of deaths, have been reported worldwide, whether in the high-risk or low-risk countries of rabies. The reported incubation period of ROTA ranges from 11 days to more than 17 months, while the historical incubation period of rabies is generally considered to range from ~ 1 week to several years. The pathogenesis of ROTA is not clear, but the use of post-exposure prophylaxis (PEP) can play a protective role in the transplant recipients. We also summarize reports about ROTA in China, combined with the actual situation regarding work on rabies surveillance and elimination, and suggest countermeasures for the prevention and control of ROTA in the future.Understanding the significance of ROTA, screening the suspected organs, assessing the risk and protecting the related population will be effective way to prevent and control further occurrence of ROTA.The online version of this article (10.1186/s40249-018-0467-7) contains supplementary material, which is available to authorized users. Please see Additional file Since the 1990s, solid organs or tissue transplantation have steadily increased, especially in developing areas such as Africa and Asia , 3. ManyLyssavirus [Rabies is an age-old infectious disease, but it remains an important public health problem today. Each year, approximately 59 000 individuals worldwide may die from an infection due to rabies virus, despite the existence of effective prophylaxis , 8. Rabissavirus . Classicssavirus . In 2015ssavirus , 12. In Most cases of rabies are caused by the bite of rabid animals, among which the most common type is the dog, followed by other animals, including cats, ferret badgers, foxes, bats, etc. Rabies virus can infect all mammals, such as cattle, pigs, sheep, wildlife and humans. As a dead-end host of rabies, humans do not typically spread rabies in a human-to-human manner , 13–16. Rabies is an infectious disease with a worldwide distribution. In Europe and other developed countries and regions, because of the large-scale use of animal vaccines, the perpetuation and spread of rabies is by wild carnivores and bats, and very few human rabies cases are reported yearly , 18. RabThe occurrence of rabies is characterized by aggregation and clustering. This is because a rabid animal may expose more than one person. Molecular biology methods can be used to find identity between the viral variants. ROTA represents the transmission of rabies virus caused by human activities. Organ transplant recipients are a population with a set of obvious medical characteristics. Different tissues and organs from one donor can be transplanted into multiple recipients, resulting in the aggregation of ROTA. In a case reported in Germany in 2004, a donor provided different organs to six recipients, resulting in the death of three patients. In a case reported in the United States in 2013, organs from the one donor were transplanted to four people , 26, 27.Humans are susceptible to rabies, but the development of the disease depends on several factors, such as the bite, severity, viral dose, wound care and PEP. Individuals with immune dysfunction or immunodeficiency are more likely to develop infectious disease. A higher incidence of rabies has been observed among organ transplant recipients infected with rabies virus, because of the use of immunosuppressive drugs, which may render them more susceptible to infection. Data from reported cases show that the incubation period of ROTA ranges from 11 days to more than 17 months. The fastest disease course in a case of ROTA occurred in 1987 in India: 11 days from surgery to the death of the patients ; whereasRabies is caused by infection with a lyssavirus, such as the type species of the genus, rabies virus, usually through skin wounds or mucous membrane damage. Rabies virus replicates in muscle tissues, followed by invasion of nerve endings and reproduction of a large number of virions in nerve cells; virus is then reverse transmitted to the central nervous system through axons. Numerous copies of the virus spread to the whole nervous system and glands of the host that are innervated by the nervous system, such as the salivary glands. At the late stage of rabies, rabies virus can be observed in glandular cells and epithelial cells. According to clinical manifestations, rabies can be mainly divided into two types: furious and paralytic rabies , 29–31.In organ transplant recipients, it is necessary to use immunosuppressants to prevent rejection of heterologous organs. The use of these drugs reduces the ability of the body to resist foreign pathogens, thus providing an ideal environment for the replication and transmission of pathogens in heterologous organs. There are two main views on the molecular pathogenesis of ROTA. One is that rabies virus is neurotropic: donor organs contain the virus in the nerve endings at the late stage of disease, the virus continues to replicate after transplantation, and via the nervous system, it invades the central nervous system again and causes rabies. The other theory is that the virus exists not only in nerve endings, but also in gland cells and epithelial cells, which represent a reserve of a large amount of the virus in the transplanted organs. When rabies virus enters the recipient, it invades the nervous system to cause rabies in an environment where the host immune system is depressed. Studies have shown that rabies virus from bats can replicate in epithelial cells, which are predominantly paralytic . It is aThe detailed molecular mechanism of ROTA is unclear, and the two views described above are consistent with the medical characteristics of rabies virus. The divergence between these two theories is whether the virus carried by the donor exists in nerve terminals or epithelial cells and gland cells. We believe that the two conditions are likely to occur. As a neurotropic virus, rabies virus is rare to replicate in epithelial cells.Traditionally, rabies virus is a quintessential neurotropic pathogen, with replication and transmission in the nervous system. However, rabies virus undergoes rapid replication and can replicate in selected epithelial or glandular cells. Rabies virus, especially the bat-transmitted type, can be found in epithelial cells, in which its replication and transmission has been confirmed. It has also been reported that significant replication of rabies virus replication occurs in the kidneys. The transplanted organs contain viruses in epithelial cells and nerve terminals, and, under appropriate conditions, rabies virus replicates in many locations and eventually causes the disease .In 2015, a case of ROTA was reported for the first time in China. In Beijing, two patients who received kidney transplantation were diagnosed with rabies after the organ transplantation surgery. The organs received by these two patients were from the same donor, a 6-year-old boy from Guangxi, where about ten cases of rabies are reported per year, thus representing a high-risk rabies region in China. The organ donor had not been diagnosed with rabies, but he appeared to have some symptoms of rabies, such as fever, continuous decline in neurological condition, and progression to coma with progressive loss of all physiological and pathological reflexes. The recipients of the kidney transplants were diagnosed as being exposed to rabies virus using RT-PCR techniques in the laboratory. In 2016, the second case of ROTA was reported in China’s Southern region, which is considered as a high-risk area for rabies. The organ donor was a boy under 2 years old. After treatment in the primary hospital for high fever, coma, and other symptoms, he was transferred to the municipal hospital, where the physician suspected rabies; however, there was a misdiagnosis based on a false negative ELISA test for rabies virus antibody, which is a test with poor sensitivity. Subsequently, his organs were transplanted to three patients, two of whom developed rabies and eventually died. Another recipient died from other complications and was not tested for rabies. ROTA has posed the serious threat to China’s public safety which should be a wake-up call for public health .Rabies is present in China, which is an area that has a high risk of rabies virus infection, as the number of deadly rabies cases per year in this country has been ranked second in the world. In China, the main route of transmission of rabies virus remains a dog bite, accounting for almost 90–95% of cases, followed by wildlife (about 3–5% of cases). Although the association with domestic animals is better appreciated, rabies virus transmission among wildlife is less understood. The prevalence of rabies in China has changed in recent years; the prevalence of rabies in high-incidence areas has decreased, and the occurrence of rabies in low-incidence areas has increased. Rabies remains the third major infectious disease-causing death in China. China will continue to be one of the countries where rabies is rampant for many years to come , 32.Many misdiagnoses have occurred in clinical practice because of the use of sedatives, which conceal the rabies-specific furious phenomenon, thus affecting the diagnosis of rabies. The patients’ personal habits also affect the diagnosis of rabies, such as the situation observed for a case reported in Germany in 2004. Because the organ donor had a history of drug addiction, the clinician believed that the patient’s mental problem was a delusion caused by drug abuse . A cliniIt is necessary to strengthen public and health-care professionals’ awareness of tissue and organ-transplant-related rabies, to improve the screening of organ donors, and to promote the appropriate prophylaxis of donors with suspected rabies for all types of transplant surgery. There should be awareness of the fact that any suspected animal bites are likely to cause rabies, which can also be transmitted through organ transplantation. China remains a country with a serious epidemic of rabies, and organ transplant patients and health-care professionals at all levels should recognize this possibility. Many infectious pathogens can be transmitted through solid organ transplants, e.g., West Nile virus, lymphocytic choriomeningitis virus, Balamuthia mandrill Aris amebae, and HIV . Rabies Viruses infecting the organ donors are the only source of infection in ROTA. It is necessary to strengthen the screening of donors, with the aim of rejecting solid organs and tissues that contain the virus, thus avoiding ROTA completely. The screening of organ donors should include epidemiological surveys of exposure history and laboratory rabies virus tests.Rabies has typical clinical symptoms, such as hydrophobia, aerophobia, pharyngeal muscle spasms, and progressive paralysis. Some patients with rabies present mainly with paralysis as the clinical manifestation, accompanied by symptoms of encephalitis, such as fever, headache, vomiting, and irritability. In advanced cases, coma, frequent convulsions, limb muscle flaccidity, and even paralytic symptoms appear. Therefore, donors who exhibit typical rabies symptoms must be screened for virus infection before organ transplantation. If the donor has symptoms of encephalitis, regardless of the presence or absence of exposure to rabies, screening should be performed before transplantation. Donors who have no encephalitis symptoms and for whom epidemiological studies show an absence of recent exposure to suspected sources of infection can be exempted from rabies screening , 37, 38.The screening of organ donors presents many limitations regarding social, biological, and technical aspects. First, in humans, rabies is a rare infectious disease that can be caused by organ transplantation, which is under-detected. There is currently no country or institution that requires the screening of rabies among donors before organ transplantation surgery. Second, during the incubation period, rabies virus infection does not cause symptoms; thus, rabid humans cannot be distinguished from healthy individuals. Third, patients with rabies often use a variety of treatments, including sedatives, which mask the characteristic rabies symptoms, and drug addiction can also lead to a misdiagnosis.Among the laboratory-based diagnostic methods, RT–PCR is a rapid and sensitive method that has been used widely for the detection of rabies virus nucleic acids. A study reported that in cerebrospinal fluid, saliva, urine, and tear samples collected at different times after the onset of rabies, this method did not detect 100% of rabies virus. With the progress of the disease, the specificity of the detection decreased. In samples of paralytic rabies, the detection rate was lower than that observed for furious rabies, and only one among 14 samples used for testing was positive. In addition, as the presence of rabies virus in biological fluids  is intermittent, it is necessary to repeatedly collect samples of different types and at different time points. A reliable method to improve the diagnosis of rabies in the laboratory is needed , 40.In China, many patients are in urgent need of organ transplantation. At the same time, there is a very short window of time between the announcement of brain death and transplantation surgery, which implies a limited time for testing the donors. Patients and medical personnel have a very superficial understanding of rabies virus transmission via solid organs or tissues allotransplantation and do not take rabies into account. These factors render ROTA a more serious situation in China.We strongly appeal for the improvement of screening for rabies among organ donors. We propose the establishment of a more reliable laboratory rabies diagnostic method and the addition of rabies testing to the mandatory list of organ transplant tests, to improve the detection of rabies before organ transplantation and avoid the occurrence of ROTA.In cases of donors with suspected rabies, an essential biosafety level will be required during the operation. In the course of surgery, medical personnel will come in direct contact with body fluids


Brunauer–Emmett–Teller (BET) analysis demonstrated that the surface area of the CS-NAC-APTES beads was larger than that of CS-NAC beads (1.16 times). The adsorption process was well fitted by the Freundlich model (R2 > 0.95), suggesting a multilayer adsorption. The kinetic study also substantiated that the pseudo-second-order model (R2 > 0.98) was in better agreement with the experimental data. Finally, it was proved that the prepared beads can be recycled (by washing with NaOH solution) at least 5 times before detectable performance loss.A composite chitosan/nano-activated carbon (CS-NAC) aminated by (3-aminopropyl)triethoxysilane (APTES) was prepared in the form of beads and applied for the removal of acetaminophen from aqueous solutions. NAC and APTES concentrations were optimized to obtain a suitable adsorbent structure for enhanced removal of the pharmaceutical. The aminated adsorbent (CS-NAC-APTES beads) prepared with 40%  In recent years, improved living standards have led to the production, consumption, and release of pharmaceuticals and personal care products (PPCPs) into the environment. PPCPs in the environment are considered to be emerging contaminants because of their adverse effects on human health and/or ecosystems . Among pChitosan (CS) and its derivatives have gained considerable attention for their ability to remove organic pollutants from wastewater because of their low cost, availability, abundance, biodegradability, biocompatibility, nontoxicity, and high adsorption capability ,10. HoweCombining CS with other materials to prepare composite adsorbents is an effective way to overcome chitosan’s shortcomings and improve its adsorption performance . ComposiIn our previous works, combinations of CS and NACs demonstrated that synergistic effects and reductions of the drawbacks of both components can be achieved ,20. SpecMedium-molecular-weight chitosan in powder form , APTES (99% purity), sodium hydroxide (NaOH), and ACT were all purchased from Sigma-Aldrich, Saint Louis, MO, USA. These materials were used as received without further purification. Nano-activated carbon was supplied by US Research Nanomaterials, Inc.  . Glacial acetic acid, acetonitrile (HPLC grade), and formic acid (98%) were purchased from Merck .CS-NAC composite beads were prepared through a simple dropwise method. In detail, the chitosan solution was initially prepared by dissolving 1.5 g of chitosan powder in 100 mL of distilled water, followed by addition of 3 mL of acetic acid and stirring for 5 h at room temperature (25 °C) . Then, dPreparation of CS-NAC-APTES beads was conducted according to our previously described methodology . InitialField emission scanning electron microscopy  was utilized to reveal the surface morphology of the samples. Functional groups on surfaces of the samples were investigated using Fourier transform infrared spectroscopy . Zeta potential was measured with a Zetasizer Nano ZS . The surface area was calculated on the basis of the Brunauer–Emmett–Teller (BET) method using a Micromeritics ASAP 2020 surface and porosity analyzer .The prepared adsorbents were tested for adsorption of ACT from aqueous solutions in a series of batch adsorption experiments. Dried beads (50 mg) were mixed with 200 mL aqueous solutions containing different ACT concentrations. The initial pH of the ACT solution was adjusted by adding 0.10 mol/L of HCl or NaOH. Then, the solutions were shaken by an orbital shaker at 150 rpm for a certain amount of time. Process variables, including initial ACT concentration (10–200 mg/L) and contact time, were studied to determine the optimal condition for ACT removal. Moreover, adsorption kinetics and isothermal experiments were performed at different initial ACT concentrations  at 25 °C for 24 h. After these adsorption experiments, 0.5 mL of the suspensions was withdrawn at intervals of 1 h (up to a maximum of 24 h), filtered, and analyzed using HPLC  equipped with a C18 column  for the accurate determination of residual ACT.To assess the regenerability of the prepared adsorbents, adsorption experiments were conducted by mixing 50 mg of dried CS-NAC and CS-NAC-APTES beads with 200 mL of 100 mg/L ACT solution at pH 7 for 24 h. Then, the spent beads were separated, rinsed, and washed by 1.0 mol/L NaOH solution by agitation on an orbital shaker for 24 h. After desorption, the beads were repeatedly washed with deionized water to remove NaOH until a neutral pH was reached. The regenerated beads were reutilized for ACT adsorption experiments (as described above). Such adsorption cycles were performed five times.w/w (0.15 g) to 40% w/w (0.6 g) enhanced the adsorption of ACT from 12.9% to 71.5%. Moreover, the beads synthetized with a higher NAC concentration attained equilibrium earlier than those containing a lower NAC percentage. Specifically, increasing the NAC concentration from 10% to 40% led to a decrease in the time necessary to reach equilibrium from 12 to 10 h, respectively. These results clearly show the beneficial effect of NAC on the composite adsorbent. Its role is likely to improve the porosity and surface area of the beads, making functional groups more accessible for adsorption of ACT [The ACT adsorptive efficiency of CS-NAC beads, prepared with different amounts of NAC, revealed that adsorption of ACT onto the CS-NAC beads was proportional to the amount of NAC a. An incn of ACT .w/w (0.75 g) decreased the percentage of ACT removal to 49.1%. The presence of a high amount of NAC might cause a collapse of the adsorbent structure and also prevent the availability of adsorption sites in the CS matrix [w/w (0.6 g) NAC concentration, which showed the highest ACT uptake (71.5%), were chosen for the amination experiments.A further increase in the NAC concentration up to 50% S matrix . At a hiS matrix . Therefo2 groups) on the surface of the CS-NAC-APTES beads. In an aqueous environment, alkoxy groups of APTES are transformed into –OH groups and eventually form active Si–OH groups. Afterwards, the Si–OH groups can react with hydroxyl and carboxyl groups available on the CS-NAC surface. Subsequently, the number of amino groups within the structure of CS-NAC-APTES beads is increased [v/v, ACT removal reached its maximum (97.3%), while a further increase in the APTES amount resulted in reduced ACT uptake. According to our previous work  actually had the lowest removal percentages (71.5%) b. The adncreased . At an Ai et al. ), a highThe surface analysis of the CS-NAC and CS-NAC-APTES beads illustrated that amination improved CS-NAC bead surface area, pore volume, and pore size by 13.9%, 2.8%, and 4.5%, respectively . These eThe surface morphology of CS-NAC beads, before and after amination, both showed numerous tiny and irregular multilayer pores . However−1 was attributed to O–H and N–H stretching vibrations. The bands around 2940 cm−1 were ascribed to the bending vibration of CH2. In addition, the peaks at 1650, 1460, and 1370 cm−1 were due to primary, secondary, and tertiary amides, respectively [−1 corresponded to the stretching vibration of C=O [−1 were shifted to 3420, 2920, 1660, 1450, 1150, and 1015 cm−1, respectively. The increase in the intensity of these peaks could have been due to the presence of APTES molecules on the structure of CS-NAC-APTES beads. In aminated beads, peaks at 995 and 850 cm−1 had stronger intensity, ascribed to Si–OH stretching and Si–O–Si symmetric stretching, respectively [−1 appeared in the spectrum of CS-NAC-APTES. These changes confirmed the presence of APTES in the structure of the CS-NAC-APTES beads. In aqueous solution, APTES undergoes hydrolysis, and silane groups covalently bind hydroxyl and carboxyl groups of NAC [ectively . The pean of C=O . It was s of NAC . Thus, t2 groups the CS-NAC-APTES bead structure: loading by APTES molecules could make the surface charge of aminated beads more positive than that of the CS-NAC beads. At the IEP, the surface charge of an adsorbent is zero. When a solution’s pH value is lower or higher than the IEP, the adsorbent’s surface charge is positive or negative, respectively [In adsorption processes, surface charge  often plays an important role in determining adsorbent performance by influencing the adsorption rate . Zeta poectively . ObviousAn adsorption kinetics study was performed to investigate the adsorption mechanism of ACT on the CS-NAC and CS-NAC-APTES beads. Obtained results were fitted to pseudo-first-order (Equation (1)) and pseudo-second-order (Equation (2)) models:ectively .R2) and chi-square (X2) values of the pseudo-second-order model were higher and lower, respectively, than those of the pseudo-first-order model. Additionally, the adsorption capacities of CS-NAC and CS-NAC-APTES beads calculated by the pseudo-second-order model were closer to experimental adsorption capacities. This implies that adsorption of ACT onto the prepared adsorbent could be a chemisorption process.In both adsorption systems, ACT adsorption capacities gradually increased with time. The times for equilibrium adsorption were found to be 10 and 14 h for CS-NAC and CS-NAC-APTES beads, respectively . Moreoven are the Freundlich constants indicating adsorption capacity (mg/g) and the intensity of the adsorption, respectively [An isotherm study can describe the interaction between adsorbent and adsorbate under constant conditions. The obtained results were analyzed using Langmuir (Equation (3)) and Freundlich (Equation (4)) isotherm models:ectively .R2 and lower X2 values. This suggests that ACT uptake is a multilayer adsorption process that happens on a heterogeneous surface. Moreover, n values for both were found to be 1.7, indicating that adsorption of ACT is a favorable process [At lower concentrations of ACT, the curves had steeper slopes , implyin process .2 and OH) imparted polar characteristics to the surface chemistry of CS-NAC-APTES beads through physicochemical interactions with adsorbate molecules in aqueous solutions [−1 decreased and shifted to 3300, 2910, 1370, and 1020 cm−1, respectively. These changes might have been due to the interactions of ACT molecules with the functional groups on the CS-NAC-APTES beads.Experimental and analytical outcomes indicated that the prepared beads possessed heterogeneous surfaces as well as high hydrophilicity, porosity, and surface area, which are favorable for the adsorption of contaminants. The presence of a large number of functional groups (NHolutions . The mecolutions . Among tACT adsorption capacities of the CS-NAC and CS-NAC-APTES beads in the consecutive five regeneration cycles had similar trends, with a gradual performance decrease . Such a w/w NAC and 2% v/v APTES. The maximum monolayer adsorption capacities of ACT onto CS-NAC and CS-NAC-APTES beads were found to be 278.4 and 407.83 mg/g, respectively. The kinetic and isotherm experiments showed that pseudo-second-order and Freundlich isotherm models were in good agreement with the experimental results. These two models suggest that ACT adsorption is governed by chemisorption, occurring on multilayer heterogeneous surfaces. The regeneration experiment suggests that both materials have good regenerability and can be applied in the adsorption process for at least five cycles, although CS-NAC-APTES showed a less marked decrease in performance. Thus, CS-NAC-APTES beads are suitable adsorbents for the adsorption of ACT from wastewater.Aminated CS-NAC beads were successfully prepared for effective elimination of ACT from aqueous solutions. The highest enhancement in the ACT adsorption capacity of CS-NAC-APTES beads was obtained using 40%


Current virus detection methods often take significant time or can be limited in sensitivity and specificity. The increasing frequency and magnitude of viral outbreaks in recent decades has resulted in an urgent need for diagnostic methods that are facile, sensitive, rapid and inexpensive. Here, we describe and characterise a novel, calcium-mediated interaction of the surface of enveloped viruses with DNA, that can be used for the functionalisation of intact virus particles via chemical groups attached to the DNA. Using DNA modified with fluorophores, we have demonstrated the rapid and sensitive labelling and detection of influenza and other viruses using single-particle tracking and particle-size determination. With this method, we have detected clinical isolates of influenza in just one minute, significantly faster than existing rapid diagnostic tests. This powerful technique is easily extendable to a wide range of other enveloped pathogenic viruses and holds significant promise as a future diagnostic tool. Infectious diseases caused by viruses represent a huge global public health concern, causing many thousands of deaths annually. The ability to efficiently and rapidly functionalise the surface of virus particles plays a major role in their study and characterisation. Virus functionalisation is not only important for viral manipulation, isolation and quantification, but importantly, can also allow viruses to be detected and identified. Early detection of viruses helps provide targeted therapy, lengthens the treatment window, reduces treatment costs and morbidity, helps prevent transmission, and leads to more efficient disease management and control.1. Alternative nucleic acid amplification techniques such as RT-PCR are also commonly used; these assays are highly specific and sensitive, but can take several hours to obtain a result1. Antigen-based rapid diagnostic tests are available for a small subset of viruses such as influenza and respiratory syncytial virus (RSV) and are relatively quick (<30 minutes), but limited by variable detection sensitivity2. Diagnostic methods based on the direct quantification and identification of intact virus particles, such as flow cytometry4, have been used to quantify a variety of viruses with differing morphologies5. These techniques make use of fluorescent dyes that bind to the viral genome6 or proteins7, all of which require a minimal 30 minute incubation period for efficient virus labelling prior to virus quantification. Negative stain transmission electron microscopy (TEM) images have been used since the 1940s8 as an important tool to image individual virus particles and quantitatively determine virus concentrations, but due to the high costs and amount of space required for a TEM instrument, this is still only available in certain facilities. Therefore, despite significant progress in the development of viral diagnostics there is still an urgent need for detection methods that meet the criteria of being easy to use, sensitive, specific, rapid and cheap.Traditionally, the gold standard for viral detection has been virus culture in eukaryotic cell lines, which takes significant time (7–10 days)2) and DNA. By adding fluorophores to the DNA, we were able to efficiently image and characterize viruses by their size in a single-particle tracking assay. This approach allowed us to detect viruses more rapidly than the currently available tests (with our results being available within one minute), and worked on all enveloped viruses tested, including viruses present in clinical samples. No amplification or purification steps were required as viruses could be detected directly in complex solutions such as cell culture media, virus transport media and allantoic fluid. We were also able to immobilise modified viruses, directly assess aggregation of fluorescent virus particles, and count labelled viruses using a fluidics-based optical approach. We therefore provide a novel tool for the rapid functionalisation, detection and quantification of enveloped viruses.In this study, we have established a novel method to label and modify enveloped viruses using calcium chloride  to reduce the background signal10  was used to give an estimate of the average diameter of the diffusing particles (dst) using the Stokes-Einstein relation , after which the sample was immediately transferred to the well of a glass slide placed on the microscope. Imaging of the sample showed the presence of multiple bright spots in the field-of-view , gave a mean diffusion coefficient log(Dm) of 1.42, which provided a Stokes’ diameter of 134 nm. It should be noted that the broad distribution of diffusion coefficients arises from the higher uncertainty in analysing short tracks and is not necessarily indicative of a broad size distribution in the particles themselves11. The overall assay time took approximately 1 minute. Our results were also consistent with the tracking of commercial, fluorescently-labelled microspheres of a similar size  strain of influenza by adding 5.25 × 10iew Fig. . Fittingize Fig. .16.In order to verify the size and shape of a typical spherical influenza strain, we stained an A/Aichi/68 (X31) virus sample and imaged it using transmission electron microscopy Fig. . We meas2 or the fluorescent DNA with water also resulted in no signal, indicating that they were both essential requirements for fluorescence labelling to occur. Interestingly, addition of 0.25 M of the calcium chelator EDTA to the sample well during observation of fluorescently labelled virus particles immediately eliminated the signal, suggesting that the labelling is reversible   Fig. .17. A small increase in the number of tracks was observed at low concentrations of virus (below 3.5 × 104 PFU/mL), followed by an approximately linear increase for the higher concentrations tested (between 3.5 × 104 and 26.2 × 104 PFU/mL) , no fluorescent particles were observed in the field-of-view; however, when PR8 virus was added, the number of fluorescent particles observed increased with increasing virus concentration Fig. . No tracmL) Fig. . The ‘li/mL Fig. .Figure 3Next, we examined clinical isolates of influenza. As we were expecting the virus concentration in clinical samples to be lower than laboratory-grown PR8, we detected viruses by adding two DNA species, labelled with either the red dye Atto647N (DNA 1) or the green dye Cy3B (DNA 2) simultaneously; this ensured that fluorescent viruses were concurrently labelled in both detection channels  of 1.06 and mean size of 171 nm, we found that a simulation movie consisting of 30% 100 nm particles, 27% 200 nm particles, 23% 300 nm particles and 20% 400 nm particles gave a mean size of 178 nm when analysed  of 0.94, which provided a mean diameter (dst) of 195 nm  of 0.26 , we obtained an approximate size of 91 nm by 536 nm for the baculovirus particles. Similarly to our experiments with influenza, over-estimation of virus size may result from aggregation. Nevertheless, our results have established the applicability of our virus detection methods to other enveloped viruses with a more complex geometry.In addition to being applicable to the detection of a broad range of influenza strains, we tested our labelling method on two other enveloped virus types, the spherical human respiratory syncytial virus (RSV) and the Autographa californica nuclear polyhedrosis virus (AcNPV), a rod-shaped baculovirus of insects. Imaging and tracking of RSV gave a mean diffusion coefficient log and illuminated using a confocal microscope with alternating red and green laser excitation  to non-specifically detect immobilized virus particles, followed by staining of viruses with red fluorescent antibodies specific to the virus strain 25. We anticipate that the detection limit of our assay can be lowered further by taking longer acquisitions, with a small trade-off in overall assay time.Our method for functionalisation of virus particles has several advantages over existing virus detection techniques. Our assay provides a simple and rapid method for quantitating whole virus particles, which is particularly important in the production of inactivated vaccines where virus titre cannot be estimated using traditional infectivity assays. The method also represents a quick way of assessing virus aggregation, and our results suggest that influenza viruses tend to aggregate independently in solution, in a strain-specific manner. We have shown that viruses could be detected directly in complex biological fluids without the need for purification, and that no fluorescent particles were observed in virus-free minimal essential media, saliva from an apparently healthy individual or allantoic fluid from uninfected chicken eggs Fig. . LabelliCompared with antibody staining of virus particles, the calcium labelling method is significantly faster, doesn’t necessitate sample preparation, requires cheaper reagents, and can be combined with alternative detection methods should specific identification of virus particles be required. Thus, we envisage that our assay has potential as a standalone technique, for example, as a rapid way of immobilizing and visualizing virus particles at the same time, by using biotinylated fluorescent DNA to pull down virus particles onto a microscope slide for observation /mL, 1.05 × 108 PFU/mL, 1.0 × 107 PFU/mL and 2.1 × 108 PFU/mL respectively. H3N2 A/Aichi/68 (X31) was grown in embryonated chicken eggs and titred by plaque assay (4.5 × 108 PFU/mL). The A2 strain of RSV was grown in Hep-2 cells and titred by plaque assay (1.4 × 105 PFU/mL). All influenza and RSV virus stocks were inactivated by shaking with 0.2% formaldehyde for 24 hours before use. Recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus was produced using the Multibac system26 and was of unknown titre. Chimpanzee adenovirus ChAdOx1-GFP was grown in HEK293 cells and titred by plaque assay (1.1 × 1012 PFU/mL)27.H1N1 A/WSN/1933 (WSN), H1N1 A/Puerto Rico/8/1934 (PR8), H3N2 A/Udorn/72 (Udorn) and B/Florida/04/2006 (InfB) influenza viruses were grown in Madin-Darby bovine kidney (MDBK) or Madin-Darby canine kidney (MDCK) cells. The cell culture supernatant  was collected and the viruses were titred by plaque assay. Titres of WSN, PR8, Udorn and InfB were 3.3 × 1029. Specimens were kept frozen at −80 °C before shipment. In total, two independent clinical samples of A(H1N1)pdm09 and one of influenza type B (Yamagata lineage) were tested by the method.For clinical samples, residual material of oropharyngeal specimens from patients with influenza-like illness, which tested positive for influenza virus by RT-PCR, were used anonymously in this study. Typing and subtyping of influenza viruses was also conducted using RT-PCR, as previously describedThe acquisition and preparation of clinical samples, which included obtaining informed consent from patients for use of the residual volume of their clinical samples for research purposes, was carried out by the National Influenza Reference Laboratory of Southern Greece, Hellenic Pasteur Institute. All samples had been previously received at the Laboratory for clinical diagnostic purposes, and they were de-identified for the purpose of this study. All methods were carried out in accordance with relevant guidelines and regulations, as approved by the Hellenic Pasteur Institute licensing committee.Fluorescent microspheres with diameters of 110 nm and 46 nm were purchased from Life Technologies. The microspheres were diluted in water and sonicated for 15 minutes on ice prior to use.30, site-specifically labelled at position 550 with ATTO647N and 744 with Cy3B.Single-stranded oligonucleotides labelled with either Atto647N, Cy3B or Cy3 dyes were purchased from IBA (Germany). Unless specified otherwise the DNA sequence used was a 64mer labelled with Atto647N (DNA 1). For dual-colour labelling a second 64mer DNA labelled with Cy3B (DNA 2) was used. The RNA was a 34mer labelled with Cy3. Short DNAs used in Fig. 31, using extrusion with a 200 nm pore size. Anionic lipid vesicles were composed of 75% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 25% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), cationic vesicles were composed of 50% DOPC and 50% Ethyl-phosphocholine, and neutral vesicles were composed of soybean phosphocholine. Vesicles at a concentration of 10 mg/mL were stored in 100 mM KCl (pH 7.5), 50 mM MOPS and 1 mM MgCl2, before being diluted, labelled and imaged.Lipid vesicles of 200 nm in diameter were prepared as described previously2 and 1 nM fluorescently-labelled DNA in a final volume of 20 µL . The sample was immediately placed in a well on a glass slide and imaged using variable angle epifluorescence microscopy (VAEM). The laser illumination was focused at a typical angle of 52° with respect to the normal, sufficiently above the surface of the glass slide to minimise background from unbound DNA settled on the surface (~3–15 µm above surface). Typical acquisitions were 1000 frames, taken at a frequency of 30 Hz, with laser intensities kept constant at 0.78 kW/cm2. In experiments where trypsin was added 0.5 µL of a 0.05x stock of recombinant trypsin  was added to the final sample volume of 20 µL. In experiments where EDTA was added, a final concentration of 0.005, 0.024, 0.048, 0.1, 0.22, 0.25 or 0.66 M EDTA was added directly to the well during imaging.Unless otherwise stated, virus stocks  were diluted in 0.65 M CaClhttps://www.oxfordni.com/). Briefly, a green (532 nm) and a red (635 nm) laser were combined using a dichroic mirror and coupled into a fibre optic cable. The fibre output was focused into the back focal plane of the objective  and, for VAEM, displaced perpendicular to the optical axis resulting in a highly oblique subcritical incident angle on the sample to decrease background fluorophore excitation. Fluorescence emission was collected by the objective, separated into two emission channels and imaged onto a sCMOS camera .Single-particle tracking experiments were performed using wide-field imaging on a commercially available fluorescence Nanoimager microscope . The exclusion radius was taken asN is the average number of particles per unit area, calculated by averaging the number of particles per unit area in each frame of a movie. The channel with the largest number of localisations was used to calculate the exclusion radius. In cases where the exclusion radius was smaller than the maximum distance, in samples with a high density of fluorescent particles for example, both parameters were set to the maximum distance. In the case of multicolour tracking, a particle observed in both channels was considered to be co-localized if simultaneous localisation in the red and green channels was separated by less than 200 nm.For single-particle tracking analysis, the NanoImager software suite was first used to localize the fluorescent molecules in each frame by finding intensity peaks that were significantly above background, then fitting the detected spots with a Gaussian function. The NanoImager single-particle tracking feature was then used to map trajectories for the individual virus particles over multiple frames, using a pre-defined maximum step distance (the maximum distance that a particle may travel between frames) and nearest-neighbor exclusion radius. These parameters were used to maximise the track count; the maximum step distance was used to exclude particles that were diffusing much more quickly than the predicted size of virus particles (e.g. free dye) and the exclusion radius was used to exclude tracks which crossed over, which could lead to ambiguity in trajectory assignment. The 110 nm and 46 nm microspheres were used to calibrate the optimum parameters as a function of the expected distance travelled between frames and the spatial density of localisations. The maximum step distance was taken asD is the diffusion coefficient, <(Δx)2> is the mean square displacement, Δt is the change in time and ϵ is a correction factor to account for a non-zero exposure time32. A histogram of the natural log diffusion coefficient for each track was then plotted using a custom-written MATLAB script. The histogram was fitted with a Gaussian function and the diffusion coefficient which corresponded to the curve peak was taken to be the sample mean (Dm). The Stokes’ diameter (dst) was then calculated using:m is the mean diffusion coefficient and η is the viscosity of the solution. The sample temperature was obtained from the microscope sensors and the viscosity of the solution was corrected for the presence of calcium chloride33.The trajectories were then used to calculate a diffusion coefficient for each track using the equation:34, with a number of modifications. For each virus particle, an initial position and time at which the particle entered the field-of-view was generated randomly, with the bounds of diffusion set to beyond the field-of-view to simulate unconfined diffusion. The position of each particle was then calculated at 100 sub-steps per frame to simulate the effects of motion blur, using track lengths that followed an exponential distribution. Point-spread-functions were calculated for each localisation and added to a matrix that formed the final movie, before random noise was added to simulate fluctuations in the background photon intensity. The simulated movies were then analysed using custom-written MATLAB software.Movies simulating the unconfined diffusion of viruses were produced using existing software35. Briefly, they were fixed with 4% paraformaldehyde, adsorbed onto grids, negatively stained with 2% aqueous uranyl acetate and imaged in a Tecnai 12 transmission electron microscope  operated at 120 kV.H3N2 A/Aichi/68 (X31) virus was visualized by transmission electron microscopy at the Bioimaging Facility of the Sir William Dunn School of Pathology, University of Oxford. Virions were prepared as described previously38. Fluorescent microspheres with a diameter of 110 nm or fluorescently-labelled A/Puerto Rico/8/1934 virus was flowed through a microslide channel  using a syringe infusion pump  and illuminated using alternating red (647 nm) and green (532 nm) laser excitation at a modulation frequency of 10 kHz. Bursts of fluorescence corresponding to the passage of each virus particle through the confocal volume were split spectrally onto two avalanche photodiodes detecting red and green fluorescence. Fluorescence data recorded by the avalanche photodiodes was acquired using a custom-written LabVIEW virtual instrument into the single molecule (.sm) format, and converted to plain text using a second virtual instrument. Analysis of the raw time series data was performed using custom-written MATLAB code, which implemented a threshold-based sliding window algorithm to detect events, combined with a Gaussian peak fitting process to identify the height, width, and location of individual peaks. Using a trigger level, re-arming level, minimum event duration, maximum event intensity, and baseline input by the user, the procedure first subtracted the baseline. The trace was then scanned point-by-point, building a map of time points for which the fluorescence intensity exceeds the trigger level without re-crossing the re-arming threshold. To minimise errors in the Gaussian peak fitting process for short-lived events, data were automatically extracted from either side of these events to ensure that a minimum of 10 data points were available to the peak fitting algorithm. Data from each event were fitted using a Gaussian peak fitting procedure, outputting the peak intensity, duration, and position within the trace of each fluorescent event. In rare instances, where the expansion of the event window caused overlap with neighbouring events, fitting errors may occur. To address this issue, the procedure identified overlapping regions, which were then merged. The peak fitting process then identified only the largest peak, and discarded the smaller peaks. Error checking was then performed, removing events that exceeded the maximum expected event duration, and intensity. Descriptive statistics for detected peaks were output for further analysis and presentation in Origin.A custom-built confocal microscope was used for single-particle virus counting experimentsA/Puerto Rico/8/1934 (H1N1) virus particles were fluorescently labelled by incubation with 1 nM biotin-conjugated DNA labelled with Atto647N, before being immobilised on the surface of a pegylated glass slide treated with neutravadin. The slide surface was imaged using TIRF  with the laser illumination focused at a typical angle of 53° with respect to the normal. Intensity profiles from 20 pixel slices of the fields of view were taken using the built-in ‘plot profile’ feature on ImageJ. The intensity peaks were fitted using the ‘fitpeaks’ function in MATLAB, the local background was subtracted, and the peaks were integrated to give an estimate of the intensity of the peak. Multiple particles were analysed and the mean or median peak area of the resulting histogram was taken as the average particle intensity, which was compared between virus particles and free DNA.2 and 1 nM biotinylated Cy3B-labelled DNA before being immobilized via neutravidin on the surface of a pegylated glass slide. Viruses were fixed in 4% paraformaldehyde and permeabilised with 0.5% Triton-X-100, before being incubated with primary and secondary antibodies. A/Udorn/72 virus was stained with an anti-NA primary antibody and Alexa647-labelled secondary antibody and A/WSN/33 virus was stained with an anti-NP primary antibody and Alexa647-labelled secondary antibody. Localisations in each channel were identified using the NanoImager software suite.Virus was incubated with 0.65 M CaCl6 PFU/mL. The virus was incubated with 0.65 M CaCl2 and 1 nM Atto647N-labelled DNA before being added to the cells. The cells were incubated for 1 hour at 37 °C to allow viruses to adhere before being fixed for 15 minutes in 4% paraformaldehyde and mounted onto glass microscope slides with Mowiol containing DAPI. Cells were viewed using an Olympus Fluoview FV1200 microscope and images processed with ImageJ.MDCK cells were grown on 13 mm coverslips in 24-well plates before being infected with A/Puerto Rico/8/1934 virus at a multiplicity of infection of ~20 × 1017. The LOB was defined as the highest concentration found when replicates of a blank sample containing no virus were tested, and was calculated using:The Limit of Blank (LOB) and Limit of Detection (LOD) were calculated as previously described4 PFU/mL).The LOD was defined as the lowest virus concentration reliably distinguished from the LOB and at which detection was feasible, determined by utilizing both the measured LOB and test replicates of a sample known to contain a low concentration of virus (0.6 × 10Supplementary InformationMovie S1


West Nile (WN) virus infection of humans is frequently asymptomatic, but can also lead to WN fever or neuroinvasive disease. CD4 T cells and B cells are critical in the defense against WN virus, and neutralizing antibodies, which are directed against the viral glycoprotein E, are an accepted correlate of protection. For the efficient production of these antibodies, B cells interact directly with CD4 helper T cells that recognize peptides from E or the two other structural proteins (capsid-C and membrane-prM/M) of the virus. However, the specific protein sites yielding such helper epitopes remain unknown. Here, we explored the CD4 T cell response in humans after WN virus infection using a comprehensive library of overlapping peptides covering all three structural proteins. By measuring T cell responses in 29 individuals with either WN virus disease or asymptomatic infection, we showed that CD4 T cells focus on peptides in specific structural elements of C and at the exposed surface of the pre- and postfusion forms of the E protein. Our data indicate that these immunodominant epitopes are recognized in the context of multiple different HLA molecules. Furthermore, we observed that immunodominant antigen regions are structurally conserved and similarly targeted in other mosquito-borne flaviviruses, including dengue, yellow fever, and Zika viruses. Together, these findings indicate a strong impact of virion protein structure on epitope selection and antigenicity, which is an important issue to consider in future vaccine design. West Nile (WN) virus is a mosquito-borne flavivirus that has caused repeated epidemics in the Americas since its first appearance in 1999, with more than 48,000 documented human cases and ~2,300 deaths . DespiteLike flaviviruses in general, mature WN virus particles consist of a nucleocapsid, which is composed of the single-stranded RNA genome and multiple copies of the capsid (C) protein, as well as a lipid envelope with two membrane-associated proteins, the membrane (M) protein, and the envelope (E) protein . The M pThe mechanisms influencing peptide presentation are incompletely understood, but a number of factors have been shown to affect the selection of epitopes during different stages of antigen processing. These include protein sequence-related factors, such as protease cleavage sites and MHC binding affinity . FurtherAn analysis of CD4 T cell epitopes in the context of the atomic structures of viral protein antigens may therefore provide important clues about how the dominance of certain epitopes is controlled by structural features. Recent studies examining immunodominant epitopes in the context of three-dimensional protein structures have led to the identification of critical CD4 T cell epitopes in other flaviviruses, including Zika, yellow fever (YF) and tick-borne encephalitis (TBE) viruses –24. CrysHere, we exploited this model to obtain insights into the selection of CD4 T cell epitopes in WN virus infection. By measuring responses in 29 individuals with either West Nile virus disease (WND) or asymptomatic infection, we identified 67 peptides in the viral capsid and envelope proteins that were recognized in the context of multiple different HLA II alleles. Although WND patients and asymptomatic subjects displayed differences with respect to the recognition of certain epitopes associated with specific HLA alleles, the overall epitope patterns between these groups were similar. Both in C and E proteins, immunodominant epitopes clustered in structurally conserved regions that have also been identified as frequent targets in other mosquito-borne flaviviruses. These data are consistent with an important influence of conserved conformational features in the structural proteins of flaviviruses that shape the specificities and immunodominance of CD4 T cell responses.A total of 29 WN virus-infected subjects were enrolled in this study . The cohAt the time of diagnosis, 25 out of 29 (86%) individuals had detectable WN virus RNA in blood samples, and 29 out of 29 (100%) had detectable WN virus IgM . WN viruThe study was performed in accordance with the recommendations of the Declaration of Helsinki, and all study participants provided written informed consent. The ethics committee of the Medical University of Vienna, Austria, approved the study protocol . Patient samples were processed in accordance with biosecurity regulations and institutional safety procedures.WN virus PCR was performed as described previously . BrieflyWN virus-specific IgM antibodies were detected in a capture ELISA using purified formalin-inactivated WN virus  and a WN50 (50% tissue culture infective dose) of WN virus strain NY99 for 1 h at 37°C. Vero cells (ECACC) were added and incubation was continued for 4–6 days at 37°C. The presence of infectious  virus was assessed by microscopic evaluation of cytopathic effects. Virus NT titers were expressed as the reciprocal of the serum dilution required for hundred percent protection against virus-induced cytopathic effects. Virus NT titers ≥20 were considered positive. Zika virus-specific neutralizing antibodies were determined in Vero cells (50 Zika virus (strain H/PF 2013) for 1 h at 37°C. Vero cells were added and incubation was continued for 3–4 days. Virus NT titers were expressed as the reciprocal of the serum dilution required for hundred percent protection against virus-induced cytopathic effects. Virus NT titers ≥20 were considered positive  to confirm WN virus infection were performed with follow-up samples from all individuals, as described previously . Brieflyro cells . Duplicapositive . YF and positive , 34. Duppositive . Virus Npositive . Virus NDepletion of cross-reactive antibodies and dengue IgG ELISAs (Euroimmun) were performed to rule out previous DEN virus infection . Broadly2. For use in ELISPOT assays, cells were resuspended at a final concentration of 2 × 106 cells/ ml in AIM-V. The purity and viability of CD8-depleted PBMCs in each sample was assessed using anti-CD8-APC, anti-CD3-PE, anti-CD4-PacificBlue™, and 7-aminoactinomycin D (BD Bioscience) and flow cytometry  were separated from whole-blood samples using Ficoll-Paque Plus™  and were cryopreserved in liquid nitrogen, as previously described . PBMCs wytometry . After dAGX89731). Three master pools covered the C, prM, or E protein sequences. Matrix pools of C (n = 10) and E (n = 22) contained up to 14 peptides with each peptide present in two distinct pools. All positive results obtained with the matrix pools were confirmed by testing the samples with single peptides in independent experiments.Peptides were purchased from JPT Peptide Technologies . Peptide purity was >70%. Peptides were quantified and verified for correct sequence by JPT, using high-performance liquid chromatography. The average purity was >85%. Lyophilized peptides were dissolved in dimethyl sulfoxide and diluted in AIM-V medium . A total5/ per well) were incubated at 37°C and 5% CO2 for about 45 h with peptides , AIM-V medium (negative control) or phytohemagglutinin  . After washing, spots were developed with 0.05 μg biotin-conjugated anti-IL-2 antibody , streptavidin-coupled alkaline phosphatase , and 5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium . The plates were analyzed using a Bio-Sys Bioreader 5000 Pro-S/BR177 and Bioreader software generation 10. Data were calculated as spot forming cells (SFCs) per 106 CD8-depleted PBMCs after subtraction of the spots from the negative control (mean spot number from three to four unstimulated wells). The response to a single peptide was defined positive if the corresponding master pool, matrix pool as well as single-peptide testing yielded >20 SFCs per 106 CD8-depleted PBMCs . For comparisons, all epitopes were derived from ex vivo ELISPOT assays obtained with human CD4 T cells and peptides that span the entire protein sequences  using Clustal Omega and manually refined, as described previously  , the Kunequences , 40, 41.equences , DEN-2 sequences , and YF equences . For comeviously –44. Thiseviously , 46, as eviously .Structural similarity of the identified epitope regions was assessed using crystallographic structures of the WN virus (PDB 2I69), YF virus (PDB 6EPK), Zika virus (PDB 5LBV), and DEN virus sE proteins (PDB 1OAN) lacking the stem-anchor region. For selected WN virus epitope regions E41, E149, E245, and E381, as well as for the corresponding regions in YF, Zika, and DEN viruses, structural superposition of WN virus sE domains I , II (E245), or III (E381), respectively onto those of YF, Zika and DEN viruses was performed using the PyMOL algorithms “align” and “super” , 49. RooHLA-DRB1, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1 alleles from all study participants were genotyped by sequencing of the whole gene. Genotyping of HLA-DRB3/4/5 allele genotyping was done from exon 2 to the 3′UTR by next generation sequencing as described previously , 50. Briwww.iedb.org) and “NetMHCIIpan” (AGX89731) were entered separately. Predictions were done for all experimentally tested peptides and the HLA class II alleles from all individuals (Predictions were done in July 2019 using the Immune Epitope Database (IEDB) analysis tools “IEDB recommended”   . Overallr = 2.8) . Howeverr = 2.8) .Since the structural context of an epitope has been proposed to influence immunodominance , 57, 58,ex vivo ELISPOT testing of human CD4 T cells and overlapping peptides covering the entire protein sequences  using the NetMHCIIpan algorithm provided by the Immune Epitope Database (IEDB). Approximately 1,400 peptide-MHC II pairs yielded IC50 values of < 500 nM, indicating likely binders  was performed for each of the 29 donors . First,  binders , 53. Ana binders . These f binders , 62.www.iedb.org). In this analysis, 82% of all experimentally identified peptides had an IEDB percentile rank of ≤20 for the individual HLA II alleles. For the illustration of the results in In addition to this population-based approach, we assessed specifically for each individual which of the HLA alleles had the best prediction value (best percentile rank) for the peptides detected in ELISPOT assays, using the Immune Epitope Database (IEDB) analysis tool “IEDB recommended” , 75% (9/12) were associated with two to four different HLA molecules  were detected experimentally only in asymptomatic but in none of the WND subjects and are indicated by asterisks in To assess the strength of association between a particular HLA allele and the epitope response in each individual, we calculated odds ratios (ORs) for all combinations of HLA alleles and dominant peptides shown in olecules . These folecules , 64. Theolecules  which maolecules . The OR ex vivo T cell epitope repertoire, defining the structural features and the host genetic factors that underlie the preferential selection of epitopes. Toward these objectives, we examined the CD4 T cell epitope specificities in 29 WN virus-infected individuals, using a library of overlapping peptides covering the entire virion proteins. We focused on CD4 T cells directed against epitopes within the virus structural proteins, because these can mediate help to B cells by direct cell-cell interactions and are therefore essential for the production of neutralizing antibodies, the major protective correlate in flavivirus infections  cells with at least as many CD4 T cells secreting IL-2 as compared to IFN-γ  which have been shown to be important sites for potent neutralizing antibodies against WN virus in humans , 66, 67.In contrast to the other WN virus epitopes identified, the E417 epitope appears to be less accessible in the context of mature virions, because it is sandwiched between the external protein shell and the lipid membrane . Howeverin silico predicted peptide-MHC II affinities provide evidence that most immunodominant peptides were predicted as strong binders and could potentially be presented in combination with multiple different MHC II molecules. Our results are consistent with recent studies (ex vivo samples by peptide-matched tetramers, which included a number of different alleles. Comparing the characteristics of WN virus-specific CD4 T cells in individuals with either neuroinvasive disease or asymptomatic infection, the same study found that individuals with neuroinvasive disease had higher numbers of virus-specific CD4 T cells (In addition to protein structure-specific factors that are responsible for characteristic immunodominance patterns, our ELISPOT data revealed considerable individual variation in epitope specificities, indicating additional influences of individual-specific factors that control epitope recognition. Evaluations based on  studies , demonst T cells . This is T cells . Apart f T cells , we obseDespite the overall similarities of epitope responses between asymptomatic subjects and WND patients, we did observe certain peptide-HLA combinations which only occurred in asymptomatic individuals. It is unclear at present whether these differences relate to the presence of alleles that protect from WND. An HLA-specific association for both protection and susceptibility to WND was suggested in previous studies –77. AddiIn summary, we have demonstrated that WN virus infection elicits CD4 T cell responses to peptides that cluster in specific regions of the viral structural proteins and are frequently predicted as strong binders for individual HLA alleles. The immunodominant epitopes locate to structurally conserved antigen regions which are also frequently targeted in other clinically relevant flaviviruses, including yellow fever, dengue and Zika viruses. Together, these data provide novel insights into a key aspect of flavivirus immunity, namely how virion protein structure shapes CD4 T cell responses, which is an important issue to consider in rational vaccine design.All datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by the Ethics committee of the Medical University of Vienna, Austria. The patients/participants provided their written informed consent to participate in this study.MK, JA, FH, SA, KS, and SM performed experiments, reviewed data, and planned experimental strategy. BK, MK, FS, and DF performed bioinformatic analysis. GF and IF performed and coordinated HLA typing. FB, IC, HL, and CJ collected samples and provided clinical information. JA and FH conceived the study. JA wrote the first draft. FH and MK contributed to the writing of the manuscript. All authors critically read and edited the manuscript.BK was employed by company Symptoma GmbH, Vienna, Austria. The remaining 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.


Dengue virus and Zika virus coexist in tropical regions in Asia where healthcare resources are limited; differentiating the 2 viruses is challenging. We showed in a case–control discovery cohort, and replicated in a validation cohort, that the diagnostic indices of conjunctivitis, platelet count, and monocyte count reliably distinguished between these viruses. Aedes mosquito. Dengue is endemic in Singapore with >10,000 case notifications annually (Zika virus and dengue virus (DENV) are arboviral infections transmitted by the Co-circulation of both viruses poses challenges to healthcare providers in distinguishing between the 2 infections. These infections have similar clinical features, including fever, rash, and myalgia. Because most patients enter the primary healthcare setting with nonspecific symptoms, we sought to determine if either infection had distinguishing symptoms, signs, or basic laboratory findings.We conducted a case–control study at the National University Hospital with ethics approval from the hospital’s Institutional Review Board. Patients infected with Zika virus and DENV who were seen at the hospital in 2016 constituted the discovery cohort. We confirmed Zika virus infection through testing for viral RNA in serum or urine, as described by Lanciotti et al. (http://www.R-project.org).We compared clinical characteristics of both infections by univariate logistic regression against dichotomous symptomatology and continuous laboratory parameters. We selected predictors that could differentiate Zika virus and DENV infection as input for subsequent multivariate regression models and computed the area under the receiver operating characteristic curve (AUC) to compare model performance. We validated the results in a separate cohort of Zika virus and DENV patients from Tan Tock Seng Hospital, Singapore  were male and 19 (55.9%) female; 57 (65.5%) DENV patients were male and 30 (34.5%) female. Thirty-one Zika patients (91.1%) were PCR positive by urine test and 3 (8.9%) by plasma.Zika patients sought treatment earlier in their illness than did DENV patients. Whereas viral symptoms including fever and arthralgia were common to both, differences were discernible . Conjunc9/µL, range 15–386 × 109/µL) and monocytosis , whereas Zika patients tended to have normal platelet  and monocyte  counts. The odds of Zika virus infection increased 2% with every unit (109/µL) increase in platelet count   . Lower mPatients with DENV had biochemical evidence of liver injury with hepatic alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels >2 times the upper reference limit . The reference range for ALT is 10–70 U/L, and for AST, 10–50 U/L. In contrast, Zika virus patients did not have pronounced abnormalities in albumin, ALT, AST, or alkaline phosphatase levels.Our findings point to conjunctivitis, platelet, monocyte, ALT, and AST levels as candidate markers to differentiate Zika virus patients from DENV patients. Conjunctivitis alone had an AUC of 0.79 in identifying Zika virus patients; normal platelet count in addition to conjunctivitis increased the AUC to 0.92, and adding a normal monocyte count further improved the AUC to 0.95 . The useWe applied these 3 indices to a validation cohort consisting of 25 Zika virus and 70 DENV patients , resultiZika virus and DENV coexist in many developing nations in equatorial South America and Southeast Asia, where there is limited accessibility to health resources and virus-specific diagnostics are not readily available. Differentiating Zika virus and DENV infections early is important in the prognostication and subsequent monitoring and follow-up of these patients. Although Zika virus infection is self-limiting, concerns about its sequelae in pregnant women and birth defects are well established (We applied both definitions from the US Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) for suspected Zika cases . We therOur results highlight the utility of conjunctivitis and normal platelet and monocyte counts to distinguish Zika virus infection. We found conjunctivitis to be already a strong predictor of Zika virus infection. The study by Waggoner et al. in Nicaragua had reported conjunctivitis and rash in association with Zika virus infection . The accuracy of our derived indices exceeds that of WHO’s and CDC’s definitions for Zika case identification, notwithstanding that performance may differ with disease prevalence or population factors. Distinguishing Zika virus from DENV infection on clinical grounds remains daunting, and it will be ideal to validate these derived indices in a prospective patient cohort. Until then, these simple clinical assessments using conjunctivitis and basic blood count parameters will be helpful in regions of the world where both Zika virus and DENV are endemic.


Zika virus was first discovered in 1947. For a long time afterward, no large-scale outbreaks occurred. However, more recently, in 2007 and 2016, there were two episodes of ZIKV outbreak that have produced serious public health problems. By analyzing the evolution of the viral genome, we can understand the potential for its outbreak. In this study, we constructed a maximum clade credibility (MCC) tree for the ZIKV non-structural protein 5 (NS5) gene using the Bayesian method. A total of 108 whole-NS5 sequences were retrieved from the GeneBank. We carried out an analysis of potential glycosylation and phosphorylation sites of the ZIKV virus NS5 gene and dynamic analysis of the evolutionary characteristics of the gene. Phylogenetic analysis revealed the presence of two sequence lineages: African and Asian. The sequence of the strains obtained from GeneBank has high homology of 85% to 100%. There are 35 potential phosphorylation sites and glycosylation sites in the ZIKV-NS5 sequences. This article analyzes the possible causes of ZIKV virus outbreaks from the perspective of genetic evolution and analyzes the dynamic trends of virus outbreaks to provide a theoretical basis for predicting the outbreak of the virus. Flaviviridae and genus Flavivirus and is a mosquito-transmitted virus. The virus particles are spherical, with diameters of about 40 ~ 70 nm. Zika virus is a type of single-stranded, positive-sense RNA virus. The whole-genome length is about 10.8 kb, and its single ORF encodes three structural proteins and seven non-structural proteins   was first isolated from a monkey in Zika forest, Uganda. . ZIKV isand NS5) . The nonand NS5) .Aedes mosquito bites, perinatal transmission, sexual contact, and blood transfusion  is based on using the evolutionary model of sequence evolution to reconstruct the statistical method of the system tree. The resulting tree not only reflects the best estimate of the phylogenetic relationship but also provides the exact support for the branch . BecauseThe total of 108 NS5 gene sequences that had been added to GenBank before October 2017 were downloaded for Bayesian analysis. These gene sequences are the complete NS5 sequences. The detailed sequence information is listed in Additional The nucleotide sequences of Zika virus NS5 were analyzed using DNASTAR Lasergen 7.0 software to compare their homology.http://www.cbs.dtu.dk/services/NetOGlyc/) (http://glycomine.erc.monash.edu/Lab/GlycoMine/).We used the NetOGlyc 4.0 Server (tOGlyc/)  to estimtOGlyc/) . GlycoMiThe Bayesian analysis method was used to study the present evolution rate and evolution model of the epidemic ZIKV strain. The complete NS5 sequence alignment of the ZIKV was disposed carefully with the Clustal W program in MEGA. The RDP3 recombination package was used to detect the recombination of all the sequences. The saturation monitoring was also tested by screening sequences with DAMBE software. If ISS < ISS.c, it means that the sequence substitution is not saturated and meets the requirements for building a phylogenetic tree using Bayesian methods. Finally, the best evolution model was selected with jModelTest software. BEAST v1.8.0 was employed under the GTR+I+G model of nucleotide substitutions and with the Relaxed clock:Uncorrelated Log-normal setting to perform 80 million MCMC runs to construct a maximum clade credibility tree (effective sampling size >200). The analysis was sampled at every 8000 states. Posterior probabilities were calculated with a burn-in of 8 million states. The analysis of the sampling data was output by Tracer v1.6, and the Tree Annotator program was employed to output the results of the MCC tree model. FigTree program was then used to plot the MCC molecular evolutionary tree.Flaviviridae and genus Flavivirus and is a mosquito-transmitted virus. In the phylogenetic tree, it is close to Dengue virus, Japanese encephalitis virus, and West Nile virus;, the closest virus is Spondweni , the National Natural Science Foundation of China (#81871699), and the Foundation of Jilin Province Science and Technology Department (#172408GH010234983).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.